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Immunofluorescent studies on antibodies directed to a buried membrane structure present in lymphocytes and erythrocytes.

Brief digestion of human peripheral blood lymphocytes by vibrio cholera neuraminidase (VCN) revealed hidden components of the membrane. Autologous human serums contained antibodies directed to these components that were readily demonstrated by immunofluorescence. Antibodies of similar specificity were found in all normal serums. The antibodies were principally of the IgM variety with lesser amounts of the IgG class present. They were equally active at 4 degrees C and 37 degrees C. The VCN revealed membrane determinants were present in normal B and T lymphocytes, monocytes, lymphocytes of patients with chronic lymphatic leukemia and cells of lymphoid lines. The newly revealed determinants slowly disappeared upon culture of the lymphocytes. These hidden components were similarly demonstrated in erythrocyte membranes and represent the T antigen long known for the red blood cells. Absorption by either VCN treated autologous lymphocytes or erythroyctes removed all of the antibodies capable of reacting with both cell types. Absorption by VCN digested isologous lymphocytes removed all reactivity with autologous lymphocytes.

Complement-induced histamine release from human basophils. I. Generation of activity in human serum.

The incubation of zymosan, endotoxin, or immune aggregates with normal human serum activates a factor which induces release of histamine from autologous basophils. The reaction can be divided into two steps: in the first, complement must be activated and in the second, the histamine-releasing factor interacts with basophils. The generation of histamine-releasing activity in serum occurs at 17 to 37 degrees C but not at 0 degrees C, is inhibited by heating the serum at 56 degrees C for 30 min, or by the addition of EDTA to the serum. Once generated, the histamine-liberating activity is stable to heating at 56 degrees C for 30 min. Gel filtration of the activated serum demonstrated that this factor eluted in the same region as a factor with chemotactic activity. Both factors have a molecular weight of about 16,000 daltons and their activities were inhibited by antibody to human C5. This is therefore a pathway for histamine release by C5a where the activation of the basophil is unrelated to the membrane bound IgE.

Role of the eosinophil in the allergic reactions. I. EDI-an eosinophil-derived inhibitor of histamine release.

An inhibitor of histamine release was found to be associated with the human eosinophilic leukocyte. This eosinophil-derived inhibitor (EDI) was released from eosinophil-rich fractions upon sonication or interaction with immune reactants (specific allergens or anti-IgE). EDI was found to exert its inhibitory action at the target cell level by increasing the intracellular levels of cyclic-AMP. Preliminary electron microscopic studies show the presence of IgE on the eosinophilic leukocyte and it is suggested that the allergen or anti-IgE-induced release of EDI might be due to a direct interaction of these immune reactants with the eosinophil-bound IgE antibody. The results also suggest that by virtue of liberating a histamine release inhibitor such as EDI, the eopsinophil assumes a modulating role in the allergic inflammatory reaction.

Macrophage-lymphocyte interaction. II. Antigen-mediated physical interactions between immune guinea pig lymph node lymphocytes and syngeneic macrophages.

The effect of specific antigen on the development of physical interactions between lymph node lymphocytes (LNL) obtained from animals which had been immunized to that antigen and macrophages was examined. We found that the presence of antigen, either limited to the macrophage () or free in the medium, profoundly increased the degree of ) or free in the medium, profoundly increased the degree of Mphi-LNL interaction observed. This enhanced interaction was dependent on the coincidence in the cultures of Mphi bearing antigen and LNL from animals specifically immunized to that antigen. Although antigen-independent interactions developed equally well between syngeneic and allogeneic combinations of lymphocytes and macrophages, antigen mediated interactions required that macrophages and lymphocytes be syngeneic. Prolongation of antigen-mediated Mphi-LNL interactions resulted in the induction of LNL DNA synthesis, initially involving those lymphocytes physically associated with antigen-bearing Mphi. These studies are interpreted to indicate that physical interaction between immune lymphocytes and antigen-bearing Mphi represents a morphological correlate of the functional activation of immune lymphocytes. Further, it is suggested that the physical events involved in lymphocyte proliferation may proceed sequentially from antigen-independent reversible binding of lymphocytes by macrophages to prolonged antigen-stabilized interaction eventuating in the triggering of specifically immune lymphocytes.

The specificity of cellular immune responses in guinea pigs. I. T cells specific for 2,4-dinitrophenyl-o-tyrosyl residues.

Guinea pigs immunized with the hapten 2,4-dinitrophenyl (DNP) coupled directly to Mycobacterium tuberculosis of strain H37Ra (DNP-H37) show a variety of cell-mediated immune responses to DNP coupled to protein carriers. The cells responsible for this specific response are thought to be T lymphocytes for the following reasons: Guinea pigs immunized with DNP-H37 displayed delayed hypersensitivity reactions to several DNP-proteins and contact sensitivity to dinitrofluorobenzene. Peritoneal exudate lymphocytes (PELs) obtained from DNP-H37 immune animals respond to DNP-proteins with DNA systhesis and cause inhibition of macrophage migration. PELs are highly enriched in T lymphocytes and contain few immunoglobulin-bearing cells. Further depletion of immunoglobulin-bearing cells from this population does not diminish the in vitro proliferative response to antigen. Nitrophenyl conjugates of proteins lacking a paranitro group stimulated DNA synthesis poorly or not at all, indicating the importance of the paranitro group of DNP in antigen recognition by T cells in this system. In this respect, the specificity of T cells resembles that of DNP-specific antibody from the same animals. On the other hand, DNP conjugates of copolymers of glutamic acid and lysine and DNP conjugated to proteins via an interposed beta-alanyl-glycyl-glycyl spacer failed to stimulate DNA synthesis, although such compounds bind very efficiently to anti-DNP antibody. By contrast, DNP conjugates of synthetic polypeptide carriers containing as little as 7% tyrosine strongly stimulated DNA synthesis in DNP-H37 immune PELs. That the determinant responsible for this stimulation was DNP coupled to the hydroxyl group of tyrosine was shown by selective removal of DNP from tyrosine by thiolysis with 2-mercaptoethanol, which abolished their ability to stimulate T cells.

Structural, functional, and idiotypic characteristics of a phosphorylcholine-binding IgA myeloma protein of C57BL/ka allotype.

An IgA phosphorylcholine (PC)-binding myeloma protein with IgCH allotypic determinants different from those of BALB/c mice is characterized. The myeloma, CBPC 2, was induced in the CB-20 strain of mice which is congenic to BALB/c but differs from it by carrying the A15 allotypic determinant of C57BL/ka mice. Sequence analysis of the CBPC 2 light chain through the first hypervariable region, as well as isoelectric point analysis, show that this chain is indistinguishable from that of T15, a PC-binding myeloma protein of BALB/c origin. The heavy chains of CBPC 2 and T15 differ by only two amino acids (positions 14 and 16) through the first hypervariable region. As measured by inhibition of precipitation, both CBPC 2 and T15 have the same specificity for PC, glycerophosphorylcholine, acetylcholine, and choline. In addition, CBPC 2 possesses the binding site-associated idiotypic determinant which is present on T15. However, like normal or induced C57BL/6 anti-PC antibody, it does not possess the nonbinding site idiotypic determinant.

Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H.

DNA polymerase was purified from a cloned isolate of Moloney murine leukemia virus (M-MuLV). Purified M-MuLV DNA polymerase, upon analysis by polyacrylamide gel electrophoresis, showed one major polypeptide of mol wt 80,000. Estimation of molecular weight from the sedimentation rate of the purifed enzyme in a glycerol gradient was consistent with a structure containing one polypeptide. M-MuLV DNA polymerase could transcribe ribopolymers, deoxyribopolymers, and heteropolymers as efficiently as did purified DNA polymerase from avian myeloblastosis virus (AMV). M-MuLV DNA polymerase, however, transcribed native 70S viral RNA less efficiently than did AMV DNA polymerase. Addition of oligo(dT) enhanced five to tenfold the transcription of 70S viral RNA by M-MuLV DNA polymerase. Purified enzyme also exhibited nuclease activity (RNase H) that selectively degraded the RNA moiety of the RNA-DNA hybrid. It did not degrade single-stranded RNA, single-stranded DNA, double-stranded RNA, and double-stranded DNA. M-MuLV DNA polymerase-associated RNase H acted as a random exonuclease. When [3-H]poly(A)-poly(dT) was used as a substrate, the size of the M-MuLV DNA polymerase-associated RHase H digested product was larger than the size of the digestion products by AMV DNA polymerase. The oligonucleotide digestion products could be further digested to 5'-AMP by snake venom phosphodiesterase, indicating that the products were terminated by 3'-OH groups. Alkaline hydrolysis of the oligonucleotide digestion products generated pAp, suggesting that M-MuLV DNA polymerase-associated RNase H cleaves at the 3' side of the 3',5'-phosphodiester bond. The ratios of the rates of DNA polymerase activity and RNase H activity were not significantly different in the murine and avian enzymes.

Periampullary carcinoma.

In the majority of instances, periampullary tumors include adenocarcinomas of the pancreatic head, duodenum, ampulla of Vater, and lower bile duct. Diagnosis is based mainly on a history of jaundice or is made by endoscopic duodenoscopy with retrograde pancreatography or by cholangiography or both. The best treatment for these tumors is pancreatoduodenectomy or palliative bypass if the tumor has spread beyond the region encompassed by resection. In experienced hands, resection can be accomplished with a mortality rate of less than 10 per cent and is followed by a 5-year survival rate of 30 to 40 per cent in carcinomas of the ampulla, duodenum, or lower bile duct and of about 10 to 15 per cent in carcinomas of the pancreatic head. Palliative surgical, chemotherapeutic, and radiotherapeutic procedures as yet do not prolong life appreciably.

Diuretic-induced oedema.

Diuretics are now commonly prescribed for women with so-called idiopathic oedema. Two patients with idiopathic oedema, one of whom was oedematous on diuretics, had the diuretics stopped. In one the oedema became much worse, and in the other oedema developed. However, this oedema disappeared spontaneously in both patients and they have remained free of oedema on no treatment. It is suggested that in some patients treated with diuretics compensatory mechanisms to retain sodium and water may be stronger than the diuretic-effect itself and may lead to perpetuation of unnecessary treatment.

Hazards from simian herpes viruses: reactivation of skin lesions with virus shedding.

A new simian herpes virus with biological properties similar to herpes simplex and to simian "B" virus has been used as a model system for studying virus latency in dorsal root spinal sensory ganglia. Following intradermal injection, virus is present in the skin lesions and corresponding ganglia only, during the acute stage of the disease. By organ-culture techniques, latent virus was rescued from ganglia up to 2 years later. No latent virus was ever found in skin organ cultures of the primary site. Treatment with cortisone up to 18 months later reactivated virus latent in the ganglia, and virus returned to the skin where it produced small but typical herpes lesions which shed virus. Reactivation of Herpesvirus tamarinus was achieved after 28 months. This is believed to be the first report of a model system for the study of herpes latency in which skin lesions are found to recur, and provides an opportunity for more detailed investigations of the mechanisms of virus latency in man. The presumption that reactivation of skin lesions will also be possible in rhesus monkeys seropositive for "B" virus points to a possibly grave and largely unsuspected hazard for those engaged in primate research.

Chronic progressive panencephalitis due to rubella virus simulating subacute sclerosing panencephalitis.

Late-onset chronic progressive panencephalitis developed in a 12-year-old boy with congenital rubella syndrome from whose brain rubella virus was isolated. Progressive dementia began at eight, and ataxia, choreiform movements, myoclonic seizures, and fine perimacular pigmentation appeared at 11 years of age. The cerebrospinal fluid was minimally pleocytotic and had a total protein of 156 mg per deciliter, of which 52 per cent was gamma globulin. Electroencephalography demonstrated generalized medium and occasional high-voltage slowing without burst suppression. The antibody titer to rubella virus (hemagglutination inhibition) was 1:8192 in serum and 1:256 in cerebrospinal fluid. Antibody titer to measles virus (complement fixation) was less than 1:8 in serum. Microscopical study of biopsied brain tissue at the age of 11 disclosed panencephalitis similar to subacute sclerosing panencephalitis, but with perivascular deposits and without inclusion bodies. Rubella virus was isolated from the brain by cocultivation with CV-1 cells.

Inhibition of oncornavirus functions by 2'-azido polynucleotides.

The 2'-azido analogs of poly(U) and poly(C), poly(dUz) [poly(2'-azido-2'-deoxyuridylic acid)], and poly-(dCz [poly(2'-azido-2'-deoxycytidylic acid)], were found to inhibit the RNA-directed DNA polymerase (reverse transcriptase) activity of murine leukemia (Moloney, Rauscher) and sarcoma (Moloney) virus, and feline leukemia (Theilen) and sarcoma (Gardner) virus, while under the same conditions the unsubstituted parent compounds failed to do so. In addition, poly(dUz) and poly(dCz) inhibited the replication of exogenous murine sarcoma virus (Moloney) in nontransformed cells (as assessed by an infectious center assay), but poly(dUz) failed to suppress the formation of endogenous sarcoma and leukemia viruses in transformed cell lines (MO-P, JLSV5). In these same cells, poly(dUz) failed to inhibit the multiplication of vesicular stomatitis virus. These data add further strength to the contention that reverse transcriptase is necessary for the productive infection and transformation of normal cells by oncornaviruses but is not essential maintenance of this transformed state and the continuous production of new viruses particles by these transformed cells.

Origin of the ATP formed during the light-dependent oxygen uptake catalyzed by Rhodospirillum rubrum chromatophores.

The oxygen uptake which is observed when Rhodospirillum rubrum chromatophores are illuminated under air and in the presence of reduced 2, 6-dichlorophenolindophenol (DCIP), 2, 3, 5, 6-tetra-methyl-P-phenylenediamine (diaminodurene, DAD) or N, N'-tetramethyl-p-phenylenediamine (TMDP) depends on the electron-donor concentration according to the equation of Michaelis-Menten. The apparent Km for the donor is lowered by the electron-transfer inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) which causes therefore a stimulation of the rate of the reaction at non-saturating concentrations of the donors. In contrast, the ATP formation which takes place simultaneously to oxygen uptake does not show an enzyme-like dependence on donor concentration. Moreover it is inhibited by HQNO to a variable extent, depending on the particular donor present and on its concentration. Therefore it appears that the HQNO-sensitive phosphorylation is coupled to a cyclic flow which coexists and competes with the non-cyclic flow from donor to oxygen. In the presence of HQNO, substrates and uncouplers of ATP formation accelerate somewhat the rate of the oxygen uptake supported by reduced DCIP and DAD. Thus part of the HQNO-resistant phosphorylation seems to be associated with the non-cyclic flow from those tow donors to oxygen. The lack of stimulation by phosphorylation or by uncoupling of the TMPD-supported oxygen uptake does not permit a conclusion as to whether this reaction is coupled to ATP formation or not. Another part of the HQNO-resistant ATP formation is independent of the presence of oxygen and appears to be associated to cyclic flows which bypass the HQNO site. This type of phosphorylation is most important in the presence of TMPD.

A comparative study of predictive criteria in the predisposition of homicidal adolescents.

The authors evaluated the criteria that are cited in the literature as predictive of homicidal predisposition. They applied three categories of criteria--clinical, developmental, and environmental factors--to a study group of 10 adolescents who had committed homicide, 10 who had threatened or attempted homicide, and 10 hospitalized controls. Their findings did not support the presence of a well-crystallized predisposition for homicidal behavior in this population, but they did show that the adolescents who committed homicide were psychotic-regressive and those who threatened or attempted homicide were organic-impulsive. The study strongly suggests the importance of environmental factors in reinforcing homicidal behavior.

On basic proteins in bovine peripheral nerve myelin.

1. Myeline proteins in bovine peripheral nerve migrated as two main band-(BF and BR protein) and one faint middle band (BM protein) on sodium dodecyls sulfate-polyacrylamide gel electrophoresis. The relative mobility of these two main bands differed from those of myelin proteins in the central nervous system. 2. The acid extract of the myelin fraction from bovine peripheral nerve was separated into one main peak and two minor peaks on a Sephadex G-75 column. The major component of the second minor peak was the BM protein; the major component of the main peak was the BF protein. The BR protein was not extractable by acid solution. 3. Molecular weights of the BF, the BM and the BR protein were determined as around 13 000, 20 000 and 28 000, respectively, by sodium dodecylsulfate-polyacrylamide gel electrophoresis. 4. The amino acid composition of the BF protein was quite different from the encephalitogenic protein and the Folch-Lees type proteolipid protein in the central nervous system. However the BM protein showed similar amino acid composition to the encephalitogenic protein. 5. The tryptic peptide maps of the BF protein and of the encephalitogenic protein were quite different. The results suggested that the amino acid sequences of these two proteins are different and that they contain no common tryptophan-containing peptide.

Histological evidence for direct connections between the optic lobes of the cockroach Leucophaea maderae.

Heretofore, descriptions of direct interconnections between insect optic lobes have been based on histological examinations of normal brains or on inference from electrophysiological or behavioral data. We present here what we believe to be the first demonstration of such monosynaptic connections by techniques of experimental neuroanatomy. Twenty-four to 39 h after extirpation of the left optic lobe, degenerating axons and axon terminals, as silvered by a modified Nauta technique, were abundant in the central portion of the medulla of the right optic lobe. The periphery of the medulla was free of argyrophilic debris as were the lobula and lamina. The distribution of neuronal somata with processes terminating in the the left optic lobe was established by retrograde axonal transport of horseradish peroxidase injected into the left lobe and by the development of distinctive perinuclear rings of RNA (a 'chromatolytic' reaction) by some cells within 1-2 weeks following amputation of the left lobe. Both techniques revealed distinct clusters of cells in the anteroventral and posterior regions of the right optic lobe, and in the medial portion of the right protocerebrum. The cells which interconnect the two optic lobes may be involved not only in the bilateral representation of visual information, but also in the coordination of optic lobe pacemakers which control a circadian rhythm of locomotory activity.

Studies on recovery from chemically induced damage in mammalian cells.

The survival of plateau-phase or nondividing Chinese hamster ovary cells (in vitro) is reduced to a greater extent by treatments with nitrosourea compounds than are cells treated in the exponential phase of growth. The greatest decrease in the survival fraction occurred following treatments with 1-trans-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea where approximately 128 times more cells were killed in plateau phase than in the dividing state (at the 10 mug/ml-for-1-hr dose). Only 5 times more cells were killed in plateau phase than in exponential growth when cells were treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Cells treated with either nitrosourea compound failed to recover from potentially lethal damage and sublethal damage. The breakdown products of the nitrosourea compounds are known to inhibit DNA repair and may explain the failure of mammalian cells to recover from sublethal damage and potentially lethal damage induced by these chemicals. Both dividing and nondividing cells were able to recover from bleomycin-induced potentially lethal damage but not from sublethal damage. The recovery from bleomycin-induced potentially lethal damage by nondividing cells was twice as great as that exhibited by dividing cells; however, potentially lethal damage recovery was suffieiently high for cells in both growth states to conceal the true response to sublethal damage.

The interaction of actinomycin C3 and actinomine with DNA. A small-angle x-ray scattering study.

Small-angle X-ray scattering was applied to solutions of calf thymus DNA and calf thymus DNA complexed with various amounts of actinomycin C3 or actinomine in phosphate-saline buffer at pH 6.9 and I equals 0.2. From the measurements of DNA in the absence of dye, two cross-section radii of gyration of R-c equals 0.875 plus or minus 0.015 nm and R-c2 equals 0.81 plus or minus 0.02 nm, and a mass per unit length of M/l equals 1906 plus or minus 43 daltons/nm resulted. The investigation of DNA complexed with dye revealed a decrease of the cross-section radii of gyration as compared to those for the DNA in the absence of dye and a relatively low increase of the mass per unit length on binding of actinomycin and a slight decrease of M/l on binding of actinomine. The latter results are interpreted on the basis of a length increase of the DNA double helix by 0.47 plus or minus 0.03 nm per actinomycin molecule and by 0.355 plus or minus 0.03 nm per actinomine molecule bound. The results for R-c and R-c2 obtained for the various samples of complexed DNA were extrapolated to the limiting binding ratio where each dye molecule is associated with a minimum of six nucleotide pairs. According to this extrapolation, the cross-section radii of gyration of such a complex would amount to (R-c)b equals 0.805 plus or minus 0.015 nm and (R-c2)b equals 0.76 plus or minus 0.015 nm for the complex with actinomycin, and to (R-c)b equals 0.77 plus or minus 0.015 nm and (R-c2)b equals 0.75 plus or minus 0.01 nm for the actinomine complex. On the basis of a core and shell model for solvated DNA, these results can be understood as to indicate a decrease of the radial dimensions of both the core and the shell when the dye is bound. The experimental results are compared with theoretical data calculated from the atomic coordinates of the detailed intercalation model for the actinomycin - DNA complex as recently proposed by Sobell and Jain. The model proves to be consistent fairly well with our data on the length increase of the double helix, but it appears to be unable to explain the experimentally observed decrease of R-c2 on binding of dye.

Studies of the inhibition of C56-initiated lysis (reactive lysis). IV. Antagonism of the inhibitory activity c567-INH by poly-L-lysine.

The stable intermediate complex C56 can initiate the lysis (reactive lysis) of unsensitized erythrocytes (E) by the membrane attack machanism of complement. Certain serum constituents designated C567-INH inhibit reactive lysis by preventing the C567 complex, once formed, from attaching to a membrane surface. It is shown here that microgram quantities of poly-L-lysine (PLL), a synthetic polycation of molecular weight 180,000, can reverse the effests of C567-INH, and thereby potentiate formation of EC567 by erythrocytes, C56 and C7 in whole serum. Erythrocytes exposed to PLL in a preincubation step did not show either increased susceptibility to C567 or resistance to C567-INH, and reversal of C567-IHN by given amounts of PLL was not diminished as cell concentrations were greatly increased, indicating that the effect of PLL was predominantly directed against fluid phase rather than against erythrocyte membrane substrates. The effects of PLL and C567-INH were quantitatively reciprocal. Thus, PLL-induced potentiation of C56-induced lysis is a solute effect which seems to involve direct neutralization of naturally occurring serum inhibitors of the C567 trimolecular complex of complement. The use of PLL thus provides a suitable antagonist for C567-INH in reaction mixtures, and allows evaluation of the role of C567 and C567-INH in a variety of situations involving C-mediated lysis.

Ontogeny of lymphoid cell surface determinants in the chicken.

Five antigenic lymphoid cell surface determinants (LCSD) were detected in hatched chickens using specific antisera. These LCSD were: thymus-specific surface determinants, bursa-specific non-immunoglobulin determinants, IgM-specific determants, IgG-specific determinants, and IgA-specific determinants (ASD). Viable cell suspension of embryonic yolk sac, bursa, thymus and spleen were tested by means of indirect or direct immunofluorescent staining procedures for the presence and frequency of LCSD during maturation. Experiments performed with liver cells. brain cells and red blood cells of embryos confirmed the specificities of the antisera used for determinants present on cells of lymphoid tissues. The results showed LCSD to occur on yolk sac cells on the 5th to 7th embryonic day (ED). This suggests the presence of a stem cell pre-committed for the lymphoid cell line already in the yolk sac. Furthermore, findings are reported indicating the presence of distinct lympoid stem cell populations or maturation stages in the yolk sac, which may be responsible for either populating the thymus or the bursa. The finding of ASD-bearing cells early in ontogenesis of the lymphoid system suggests the presence of two specificities in anti-chicken IgA sera, one of which may be directed against an antigenic site on a rudimentary immunoglobulin molecule, which becomes lost or hidden in later maturation. Studies on the bursa and the thymus show that covering, hiding, or loss of antigenic determinants plays an important role in lymphoid cell differentiation. Furthermore, the spleen is reached by B-determined stem cells as early as the bursa, but these stem cells seem not to proliferate in the former to any considerable extent until hatching. Finally, the sequence of the appearance of immunoglobulin classes as proposed by other authors is confirmed with reservaitons concerning IgA, and it is suggested that immunoglobulins are detectable earlier on cell surfaces than intracytoplasmatically.

Mammary tumor virus induction by glucocorticoids. Characterization of specific transcriptional regulation.

Dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), a potent synthetic glucocorticoid, stimulates mouse mammary tumor virus expression 10- to 20-fold in tissue culture cells. This hormone effect was observed at concentrations as low as 1 times 10-10 M and was maximal at 10-7 to 10-8 M. The time course of induction indicated that detectable increases in extracellular viral DNA polymerase were first noted 18 to 24 hours following the addition of dexamethasone, and cells produced the highest polymerase levels at the time monolayers approached confluence. Steroid responsiveness was associated with specific increases in type B murine mammary tumor virus structural polypeptide (gp52(sl) expression and murine mammary tumor virus RNA that quantitatively paralleled the increase in extracellular virus production as measured by electron microscopy and supernatant RNA-dependent DNA polymerase activity. Another virally transformed murine cell line, KA 31, did not contain detectable levels of murine mammary tumor virus gp52(sl) or RNA before or after dexamethasone stimulation; thus induction was noted only in murine cells with pre-existing murine mammary tumor virus expression. No increase in basal levels of type C murine leukemia viral proteins or RNA was detected in dexamethasone-treated mammary cell lines which were producing increased levels of murine mammary tumor virus. Therefore, increases in murine mammary tumor virus gene products are specific for murine mammary tumor virus DNA sequences under these conditions.

Immunological characterization of hypoxanthine-guanine phosphoribosyl transferase mutants of mouse L cells: evidence for mutations at different loci in the HGPRT gene.

A large collection (105) of mouse L cell mutants lacking hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT; E. C. 2.4.2.8) were analyzed for the presence of serologically cross reacting material (CRM). Antibody directed against highly purified mouse liver HGPRT was used for detecting DRM activity by two methods: (1) the standard precipitation-inhibition assay; and (2) a radioimmune-precipitation assay. The latter assay proved to have far greater sensitivity for the detection of altered forms of HGPRT. Approximately 40% of the HGPRT- cell lines contain CRM activity (i.e., were CRM+). This indicates that a minimum of 40% of the HGPRT- clones arose as a result of mutations in the HGPRT structural gene. The CRM+ cell lines were shown to contain different levels of CRM activity. Measurements of the heat sensitivity of CRM in the different HGPRT- cell lines showed a broad spectrum of CRM heat inactivation kinetics. These latter two observations provide strong evidence that the mutations giving rise to the HGPRT-CRM+ phenotype occurred at different sites in the HGPRT structural gene.

Organochlorine content of milks, dairy products and animal feed ingredients: Ireland 1971-1972.

Milks (bovine and human) and dairy products (butter, cheese, skim and whey powders, calf-replacer, casein, butter-oil and dietetic food) were collected during 1971/2 throughout Ireland together with a more limited samples of the 10 major animal feed ingredients, and analysed for organochlorine insecticide residues using electron-capture gas chromatography. The different materials contained low or negligible levels of chlorinated insecticides. Apart from some of the animal feed ingredients the DDT residues were generally the predominant contaminants detected together with lower levels of gamma-BHC (lindane), aldrin/dieldrin and heptachlor/heptachlor epoxide. The maximum levels of these insecticides in the bovine milk and dairy products (511, 100, 62 and 21 mug/kg fat respectively) constitute only 50% or less of the Codex Tolerance Limits. The correspondingly low residue levels in the human milk (maxima of 128, 1, 1, and 5 mug/kg fat respectively) which at most represent insecticidal ingestion by infants equivalent to 13, 0-05, 5 and 5% respectively of the WHO/FAO acceptable daily intake for DDE, gamma-BHC, aldrin/dieldrin and heptachlor/heptachlor epoxide again pose no obvious health hazards and are strongly indicative of negligible organochlorine contamination in the general diet. The samples of animal feed ingredients examined also contained trace levels of ogranochlorines (maxima of 0-9, 0-1, 1-6 and 1-0 mug/kg respectively). More extensive monitoring of the residues in animal feed ingredients (the most probable source of milk contamination is advocated, and the desirability of tolerance limits for insecticides in animal feeds discussed.

Reaginic antibody formation in the mouse. V. Adoptive antihapten IgE antibody response of dinitrophenyl-keyhole limpet hemocyanin-primed spleen cells cultured with dinitrophenyl heterologous carrier conjugates.

DBA/1 mice were primed with dinitrophenyl-keyhole lympet hemocyanin (DNP-KLH) included in aluminum hydroxide gel, and the adoptive anti-DNP IgE antibody response of their spleen cells was studied by transfer into irradiated syngeneic mice. As expected, depletion of T cells by anti-theta antiserum and complement abolished the response of the spleen cells to homologous antigen. If the same T-depleted spleen cells were cultured with DNP-KLH for 24 hr and then transferred into irradiated mice, anti-DNP IgE antibody response was obtained. It was also found that DNP-KLH-primed spleen cells were triggered for IgE antibody response if they were cultured for 24 to 48 hr with DNP heterologous carrier conjugate, such as DNP-bovine gamma-globulin or a copolymer of D-tyrosine, glutamine, and lysine. Transfer of the cells cultured with the antigen into irradiated recipients resulted in the formation of anti-DNP IgE antibody. The DNP-KLH-primed cells cultured for 24 hr in the absence of antigen and then treated with DNP-BGG at 0 degrees C, also gave an adoptive IgE antibody response. If the same DNP-KLH-primed cells were treated with the DNP heterologous carrier conjugate at 0 degrees C or injected into irradiated recipients together with the antigen, no IgE antibody response was obtained. The results indicated that T cell dependency of the IgE antibody response is diminished by culture of DNP-KLH-primed cell for 24 to 48 hr.

Modulation of cyclic AMP in purified rat mast cells. II. Studies on the relationship between intracellular cyclic AMP concentrations and histamine release.

Changes in intracellular and extracellular rat mast cell adenosine 3':5' monophosphate (cAMP) concentrations during stimulation of histamine release by 48/80 were studied. There was a rapid and progressive fall in intracellular cAMP beginning within 10 sec after the addition of 48/80. The lowest cAMP values were obtained at 10 min, with return to control levels by 30 min. The fall in cAMP was dose-related with progressive decreases in 10-min cAMP measurements as the 48/80 concentration was increased from 0.25 to 1.00 mug/ml. There was a graded increase in histamine release over the same concentration range. Attempts to demonstrate significant amounts of cAMP in the medium during 48/80 stimulation were unsuccessful, indicating that the changes in cAMP intracellularly are not due to altered cellular permeability. There was a general correlation between the ability of pharmacologic agents to sustain high intracellular levels of cAMP in the presence of 48/80, and inhibition of histamine release. Theophylline (20 mM) which increased cAMP levels 2- 3-fold prevented a detectable decrease in cAMP after 1 mug/ml 48/80 (measured at 10 min) and almost completely inhibited histamine release. Prostaglandin E1 (27 muM) also raised cAMP levels, decreased the 48/80-induced fall in cAMP (by 42%). Epinephrine increased mast cell cAMP levels, but did not prevent the subsequent 48/80-induced decrease in cAMP and did not inhibit histamine release. Carbamylcholine (1 nM), adenine (1 muM), and diazoxide (10 muM) lowered mast cell cAMP and potentiated 48/80 induced release. In view of previous studies from this laboratory indicating that 48/80 stimulates mast cell phosphodiesterase, it seems likely that the 48/80-induced fall in cAMP is due, at least in part, to increased cAMP destruction. Since agents which prevent the fall in cAMP inhibit histamine release, it is apparent that cAMP is an important part of the control mechanism of histamine secretion. On the other hand, it cannot be concluded that a decrease in cAMP alone is sufficient to produce a response since carbamylcholine, diazoxide, and adenine which lower cAMP do not alter histamine release unless 48/80 is also present.

Abundant nuclear rods in adult-onset rod disease.

Abundant, highly organized, rod-shaped particles have been found in skeletal-muscle nuclei of two patients with adult-onset rod disease. They were usually single in affected nuclei. Like myofibrillar rods, the nuclear rods consisted of bundles of long, parallel, apparently cross-linked filaments. On longitudinal section the rods had an axial periodicity and in transverse section a wire-mesh appearance. Average periodicities were 189A (nuclear) and 178A (myofibrillar) axially, 180A (both) transversely between the longitudinal rod-filaments on longitudinal-section and cross-section of the rods. Minor differences were that the nuclear rods were slightly lighter stained after osmium-uranyl acetate-lead citrate, lacked thin filaments protruding from their ends, and often were broader. It is proposed that nuclear rods may have a contractile-protein composition and pathokinesis similar to that of myofibrillar rods. Their formation may reflect an epitomization of a newly recognized general biological capability of exogenous, or perhaps endogeneous, nuclear protein alteration.

Studies on myocarditis in childhood, with special reference to the possible role of immunological process and the thymus in the chronicity of the disease.

Twenty-one cases of idiopathic myocarditis were studied. Fulminant form 1 case, acute fatal form 2 cases, acute benign form 4 cases, recurrent form 1 case. Chronic dilated form 2 cases progressive hypertrophy form 2 cases and asymptomatic and sudden death form was 10 cases. Clinical signs and symptoms of these cases were manifold. Also laboratory findings were nonspecific. Ten cases of latent myocarditis which showed sudden death syndrome were found among 47 cases, progressive hypertrophy form 2 cases and children. Early detection of latent myocarditis is considered to be necessary for preventing sudden death syndrome in school children. The relationship between cardiomyopathy and idiopathic myocarditis was discussed. Two cases of idiopathic myocarditis simulating cardiomyopathy were presented. The pathogenesis of chronicity in idiopathic myocarditis was discussed. Bound gammaglobulin in heart muscle was detected in a case showing progressive hypertrophy of the ventricles. Autopsy results showed a previously undescribed finding that the cases having normal thymus/body weight ratio tended to have markedly increased heart weight but the cases having increased thymus/body weight ratio tended to have near normal heart weight. These results are considered to suggest the possible role of immunity in the chronicity of idiopathic myocarditis.

Neuropsychological dysfunction in children with chronic low-level lead absorption.

To investigate the relation between low-level absorption and neuropsychological function, blind evaluations were under-taken in forty-six symptom-free children aged 3-15 years with blood-lead concentrations of 40-68 mug. per 100 ml. (mean 48 mug. per 100 ml.) and in seventy-eight ethnically and socioeconomically similar controls with levels greater than mug. per 100 ml. (mean 27 mug. per (100 ml). All children lived within 6-6 km. of a large, lead-emitting smelter, and in many cases residence there had been lifelong. Mean age in the lead group was 8-3 years and in the controls 9-3. Testing with Wechsler intelligence scales for schoolchildren and preschool children (W.I.S.C. and W.P.P.S.I.) showed age-adjusted performance I.Q. to be significantly decreased in the group with higher lead levels (mean scores, W.I.S.C. plus W.P.P.S.I., 95 v. 103). Children in all ages in the lead group also had significant slowing in a finger-wrist tapping test. Full-scale I.Q., verbal I.Q., BEHAVIOUR, AND HYPERACTIVITY RATINGS DID NOT DIFFER.

Prostaglandins and obesity.

In metabolic obesity energy in triglyceride stores is not readily accessible, and lipolysis to free fatty acid and glycerol seems to be somehow restrained. In the normal situation, there is a balance between a forward reaction via cyclic A.M.P. ending in lipolysis and a negative-feedback mechanism in which prostaglandins participate. In metabolic obesity there may be a biochemical error leading to overproduction of prostaglandins; as a result the forward reaction is overwhelmed and lipolysis does not take place. Since prostaglandin antagonists and inhibitors of prostaglandin synthesis are known, this hypothesis is not without therapeutic interest.

Arteriography and infusional chemotherapy in localized trophoblastic disease.

Despite recent advances in the systemic chemotherapy of patients with malignant trophoblastic disease, there remains a small group of patients who fail to achieve remission with this type of treatment. The present study presents 19 patients with malignant trophoblastic disease who underwent a total of 25 arteriographic studies for localization of persistent malignant lesions and 8 patients who underwent arterial infusional chemotherapy after they had failed to achieve remission by standard administration of systemic methotrexate or actinomycin D. Patients underwent pelvic, pulmonary, carotid, and multiple abdominal selective arteriograms with a high correlation of positive findings and the later documented presence of persistent malignant disease. Findings include prominent uterine arteries, arteriovenous shunts, hypervascularity, irregular vessels, and tumor staining within the tumor. Arterially infused chemotherapy with methotrexate or actinomycin D was used in 8 patients whose disease was resistant to systemic chemotherapy. Technics of arterial infusion are discussed.

Effect of bilirubin on the distribution, elimination and anticoagulant action of dicumarol in Gunn rats-1-2 (38547).

Jaundiced Gunn rats were found to have a significantly larger apparent volume of distribution and higher rate constant for elimination of dicumarol than their nonjaundiced littermates. Anticoagulant effect-log plasma dicumarol concentration lines were parallel in the two groups of animals, with the line for the jaundiced rats shifted to a lower concentration range. In vitro studies showed that bilirubin can displace dicumarol from serum protein binding sites. There are indications of a genetic influence on the distribution and anticoagulant effect of dicumarol in Gunn rats.

[Risk of chronic carbon monoxide poisoning in automobile garages. Results of a study in the Lausanne region].

Clinical and physiologic data on chronic carbon monoxide poisoning are reviewed and the results of an investigation involving 7 garages in the Lausanne (Switzerland) area are reported. The aim was a practical approach to the relationships between carbon monoxide level in the air, COHb and clinical picture. The study covered working conditions (especially ventilation) but did not take into account of other factors (stresses) which may effect the parameters investigated. CO was measured by continuous recording with an MSA Carbon Monoxide Alarm and the hourly and daily averages were determined. The garage personnel replied to a questionnaire and underwent a brief clinical examination including taking of digital blood samples for measurement of hematocrit and carboxyhemoglobin level by the method of COMMINS and LAWTHER as modified by BUCHWALD. One of the garages did not meet present health requirements. Statistical analysis revealed a significant correlation between carbon monoxide and carboxyhemoglobin. The incidence of complaints was highest in poorly ventilated garages. On the basis of COHb level in the total group of employees, together with data from individual histories, it is possible to evaluate the risk of carbon monoxide poisoning in a given garage.

Effects of major abdominal operations on human blood rheology.

Serial blood rheological measurements were systematically carried out in 14 patients undergoing major elective abdominal operations for a period up to 10 days after operation. The measurements included blood viscosity over a wide range of shear rates (0.01 to 208 sec.-minus 1), hematological data, and plasma protein concentrations. Significantly elevated plasma viscosity preoperatively accounted for a normal blood viscosity in the presence of anemia. After an initial drop due to hemodilution lasting for 1 to 3 days after operation, blood viscosity returned to normal by the tenth postoperative day. This was the result of an increased plasma viscosity, primarily due to rise in plasma fibrinogen and alpha2- and beta2-globulin concentrations. The red cell deformability of these patients was normal except for one patient who received seven units of transfused blood. The results of this investigation suggest that abnormal rheological properties of blood and plasma should be considered in therapeutic attempts to improve microcirculatory flow and oxygen delivery to tissues.

The effect of progestogens on vaginal cytology.

Various progestogens were given to postmenopausal and premenopausal women. The first group consisted of 46 postmenopausal patients and in the postmenopausal atrophic smear, 17 acetoxyprogestogens produced no effect. However, the administration of two 19 nortestosterone derivatives, ethynodiol diacetate and norethisterone increased the Karyopyknotic Index and degree of proliferation of the epithelium, presumably due to their estrogenic metabolites. Low grade hypotrophic smears, consisting of parabasal and intermediate cells, reacted by a decrease in the size of the intermediate cells associated with clumping, and a disappearance from the smear of parabasal cells. In the case of ethynodiol diacetate, its estrogenic effect of elevating the Karyopyknotic Index occurred before the progestogenic effect of clumping and fusing of the intermediate cells. The second group consisted of 370 women using an oral form of sequential contraception, based on administration of 0.05 mg ethinyl estradiol daily for three weeks followed by a combined oral contraceptive containing the same amount of estrogen with varying progestogens for a further week. The anti-estrogenic effect of the various progestogens was then compared by their ability to lower the Karyopyknotic Index. This levelled out after four or five tablets. The most potent progestogen was d-norgestrel in the lowest dose of 0.25 mg and this compound exerted a greater effect than 4 mg norethisterone, 1 mg ethynodiol diacetate or 4 mg megestrol acetate.

Characterization of spermatozoal auto-, iso- and allo-antigens.

Three approaches are utilized to study and characterize spermatozoal antigens. An immunological approach has demonstrated the presence of spermatozoal auto-, iso- and allo-antigens. Spermatozoal auto-antigens studies by several authors are able to induce the whole spectrum of immune reactions (delayed hypersensitivity, complement-fixing antibodies and anaphylactic antibodies0 as well as of autoimmune aspermatogenic orchiepididymitis (AIAO). Different extraction procedures result in various preparations and even in different independent autoantigens (at least four), one protein, one membrane-linked antigen and at least two glyco-proteins. Spermatozoal iso-antigens stricto sensu are determined by the Y chromosome and present on at least 50% of the spermatozoa. Spermatozoal allo-antigens are also present at the surface of spermatozoa, especially blood group antigens (ABO and MNS systems), transplantation antigens (HL-A, H-2) and also some other unidentified ones. A biochemical approach has mainly been directed towards spermatozoal enzymes that have been directed towards spermatozoal enzymes that have been shown to be antigenic even in the species of origin. This is the case for lactic dehydrogenase LDH-X (a mid-piece enzyme) and for acrosomal enzymes, e.g., hyaluronidase, possibly sorbitol dehydrogenase and trypsin-like acrosomal proteinase (the auto- and allo-antigenicity of the latter having not been established). At least three of these enzymes are known or supposed to play a role in the process of fertilization. A clinical approach has described the presence of spermatozoal-coating antigen(s), such as transferrin or blood group substances from secretors obtained following the admixture of the secretions of the seminal vesicles. Indications were also obtained for the existence of antibodies directed against defined antigens. Several types of localization of antibodies on spermatozoa were described: acrosome (front part), equatorial segment, post-nuclear region, mid-piece and tail. Attempts at fractionation of human psermatozoal antigens are still at a preliminary stage. Whatever the approach, the main interest of these antigens is that they are able to induce, in the species of origin or in a related species antibodies capable of interfering with the normal process of reproduction, especially fertilization..

Acute viral hepatitis: factors possibly predicting chronic liver disease.

A number of clinical, biochemical, immunological and morphological variables were recorded at first admission of 500 consecutive patients with biopsy verified acute viral hepatitis in the period February 1969-June 1972. In February 1973, 28 of these patients had a morphologically documented chronic liver disease: 4 cirrhosis of the liver, 15 chronic aggressive hepatitis, and 9 chronic persistent hepatitis. 74 patients were followed up until morphological normalization took place. The initially recorded variables in the two groups were compared, and the following factors were significantly higher in the group with subsequent development of chronic liver disease:--frequency of drug addicts, median of the highest gammaglobulin, ANA, SMA, partial destruction of the limiting membrane, incidence of piecemeal necrosis, and pronounced plasma cell infiltration in the portal tracts. These preliminary results suggest that factors in the initial phase of acute viral hepatitis can be helpful to some extent in predicting the course and prognosis of the disease.

The symbolic context of Chinese medicine: a comparative approach to the study of traditional medical and psychiatric forms of care in Chinese culture.

This article explores a distinctly different aspect of Chinese medicine, and of health care in Chinese culture, from that receiving most attention and serious study at present in this country. It examines the symbolic structure and significance of illness and care in the Chinese context by (a) applying concepts developed in anthropology and the cross-cultural study of medicine and psychiatry; (b) examining recent studies of folk and popular forms of health care in contemporary Chinese communities; and (c) raising questions about the congnitive structure, cultural background, and bio-social significance of traditional Chinese healing beliefs and practices. An attempt is made to place this analysis in a comparative framework, so that Chinese cases can be related to health care systems in other cultural settings.

Effects of large doses of intravenous atropine on heart rate and arterial pressure of anesthetized patients.

A bolus of 3 mg. of atropine was given intravenously (I.V.) TO 123 ANESTHETIZED PATIENTS. Increases in heart rate were seen in 109 patients (88 percent), while 7 patients (5.7 percent) had no change. A mean increase in systolic arterial blood pressure of 14 mm. Hg was noted. A certain pattern in increases in heart rate and systolic blood pressure was observed. Ninety-six percent of the patients under cyclopropane, fluroxene, ether, ketamine, or regional (spinal or epidural) anesthesia had heart-rate increases, compared with only 77 to 89 percent of the patients anesthetized with halothane, enflurane, or neuroleptanalgesics. Rise in systolic blood pressure was seen in 40 to 50 percent of the former, but only in 31 to 40 percent of the latter group. The arrhythmias observed were transient A-V junctional tachycardias in three instances and bigeminal rhythm in one patient under cyclopropane anesthesia, The administration of a vagolytic dose of atropine to anesthetized patients appears not to be as arrhythmogenic as previously considered.

Comparison of the specificity of human and bovine tuberculin PPD for testing cattle. 2. South-eastern England.

A tuberculin testing trial was carried out in eight counties of south-eastern England to compare the specificity for bovine tuberculosis of Weybridge human PPD with that of Rotterdam bovine PPD. The matching of these two tuberculins for potency in naturally infected cattle had already been established, the bovine PPD being approximately one-and-a-half times more potent than the human PPD per unit of weight. In 1110 cattle in 25 herds with histories of long-standing freedom from tuberculosis and in which non-specific tuberculin sensitivity was present, cross reactions were less to the bovine PPD than to the human PPD, showing that in the environment of this trial the bovine PPD was more specific than the human PPD. Induration diameter was a satisfactory alternative to skin thickening as a measure of tuberculin reactions in cattle under field conditions. Due to the steep slope of the dose-response curves of the avian PPD in the different groups of non-tuberculous cattle, the discriminating power of the comparative test, using avian and mammalian tuberculins, was less at lower doses of tuberculin. Concentrations of 1-0 mg per ml of bovine PPD and 0-5 mg per ml of avian PPD are recommended for use in a comparative tuberculin test.

Exposure of histone antigenic determinants in chromatin.

The exposure of antigenic determinants of histones present in "native" chromatin was studied by: (1) testing their ability to elicit anti-histone antibodies and (2) measuring their ability to interact with anti-histone sera. To this end, antisera specific to purified histone fractions and to purified rat liver chromatin were elicited in rabbits. The anti-chromatin sera did not react with pure histone fractions and pure histone fractions F2b, F3, F2a1, and F2a2 failed to inhibit the complement fixation resulting from the binding of anti-chromatin to chromatin. These results suggest that in native chromatin, determinants in these histones are not immunogenic. Histone F1, however, inhibited the reaction between chromatin and anti-chromatin. Antisera elicited by histone fractions reacted weakly with "native" chromatin. The maximal complement fixations (obtained with 5-10 mug of chromatin DNA) were as follows: 60% with anti-F2b, 20% with anti-F1 and anti-F3, and less than 5% with either anti-F2a1 or anti-F2a2. Studies of the interaction between anti-histone antibodies and chromatin in which chromatin was used as an immunoadsorbent indicated that antibodies against different histones were adsorbed to a different degree by the same amount of chromatin. Differences in the immunoadsorbing capacity between sonicated and nonsonicated chromatin were found. Quantitative adsorbtion studies revealed that in the "native" chromatin structure, antigenic determinants of F1 and F2b were more available to interact with homologous antibody than those of F3 and F2a1 and that determinants in F2a2 were the least available. It could be calculated that the "equivalent antigenicity" of the histones in chromatin was 9.6% for F1, 3.2% for F2b, and 0.90% for F3 and F2a1. Upon sonication these values did not change for F1 but increased two-, three-, and fourfold for F2b, F3, and F2a1, respectively. Digestion of chromatin with trypsin totally abolished the ability of chromatin to adsorb anti-histone antibodies.

A partial reaction in photosystem II: reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition.

Silicomolybdate functions as an electron acceptor in a Photosystem II water oxidation (measured as O2 evolution) partial reaction that is 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) insensitive, that is, reduction os silicomolybdate occurs at or before the level of Q, the primary electron acceptor for Photosystem II. This report characterizes the partial reaction with the principal findings being as follows: 1. Electron transport to silicomolybdate significantly decreased room temperature Photosystem I side of the DCMU had no effect on the fluorescence level, consistent with silicomolybdate accepting electrons at or before Q. In the absence of DCMU, silicomolybdate is also reduced at a site on the Photosystem I side of the DCMU block, prior to or at plastoquinone, since the plastoquinone antagonist dibromothymoquinone (DBMIB) did not affect the electron transport rate. 3. Electron transport from water to silicomolybdate (+ DCMU) is not coupled to ATP formation, nor is there a measurable accumulation of protons within the membrane (measured by amine uptake). Silicomolybdate is not inhibitory to phosphorylation per se since neither cyclic nor post-illumination (XE) phosphorylation were inhibited. 4. Uncouplers stimulated electron transport from water to silicomolybdate in the pH range of 6 to 7, but inhibited at pH values near 8. These data are consistent with the view that when electron flow is through the abbreviated sequence of water to Photosystem II to silicomolybdate (+ DCMU), conditions are not established for the water protons to be deposited within the membrane. Experiments reported elsewhere (Fiaquinta, R.T., Dilley, R.A. and Horton, P.(19741 J. Bioenerg. 6, 167-177) and these data, are consistent with the hypothesis that electron transport between Q and plastoquinone energizes a membrane conformational change that is required to interact with the water oxication system so as to result in the deposition of water protons either within the membrane itself or within the inner oxmotic space.

[Re-entry mechanism of ventricular tachycardias in the syndrome of nonhomogeneous delayed repolarisation (author's transl)].

The syndrome of Jervell and Lange-Nielsen with its characteristic combination of Q-T-(U) abnormalities and recurrent episodes of ventricular tachycardia (VT) or ventricular fibrillation can serve as a model for establishing recycling excitation (re-entry) as the likely cause of VT. The ECG abnormality in the Q-T-(U) segment indicates the underlying asynchronous repolarisation, probably localized in the His-Purkinje system. Asynchronous delayed repolarisation facilitates re-entry excitations. A personal case is reported which demonstrated two different patterns of inducing premature beats and VT, indicative of two different fascicular pathways of circus movement. The inducing extrasystole determines the circuit pathway and thus the ECG pattern of VT. The VT shows a fixed time relation depending on conduction delay and refractory period, which is a further indication for an underlying re-entry mechanism. Spontaneous changes in time relation or morphology point toward alterations in circuit pathways and can terminate the VT. Clinical findings in three further cases of the syndrome are reported. Furthermore, the possible general significance of these findings is highlighted by the occurrence of the described phenomena in seven patients in whom Q-T-(U) abnormalities exist merely as transient symptomatic disturbances.

Cellular proliferation and renewal in the various zones of the hamster epididymis after colchicine administration.

Cellular proliferation was examined in the various regions of the excurrent ducts of the reproductive system of the male golden hamster after colchicine administration. Each of the two zones of the ductuli efferentes displayed only infrequent mitotic figures. Similarly, the basal cell population of the epididymis as well as the epithelium of the vas deferens exhibited too few mitotic figures to allow meaningful evaluation of cellular proliferation. The following observations were reported. 1. The principal cells of the entire epididymis are characterized by a singular diurnal cycle of proliferative activity reaching its maximum about 3 P.M. and its nadir about 9 A.M. 2. A circadian rhythm of cell division is also found in the principal cells of each of the six zones of the epididymis, although the time of maximal activity may vary from zone to zone. 3. The most actively proliferating region of the excurrent duct system is zone 3 of the epididymis, whereas the least active region is the ductuli efferentes. 4. The renewal rates of the principal cells vary considerably at various points along the excurrent duct system, ranging from cells which renew themselves as many as 10 times to cells which fail to renew themselves at all during the normal life-span of the animal.

Immunologic and virologic aspects of secretory immune system in human respiratory tract.

Many external mucosal surfaces in man are replete with immunoglobulin containing plasma cells and thymic dependent (T) lymphocytes. Immunization with viral vaccines administered via different routes have indicated that viral specific secretory immunoglobulins particularly of gamma A class are synthesized locally in the external mucosal surfaces. Local availability of viral antigens especially after local mucosal immunization appears to be the most effective means of inducing viral specific secretory antibody responses in the respiratory tract. Similarly, local induction of specific cellular immune responses in the respiratory tract and tonsilar lymphoid cells has been demonstrated after intranasal immunization with viral vaccines. Generally, the locally induced secretory antibody and cell-mediated immune responses in the respiratory tract appear to be independent of the immune response in the systemic sites, with little or no contribution from circulating immunoglobulins and T-cells. Information obtained after natural or vaccine induced infections with polio, influenza, measles, rubella and other viruses suggest that the outcome of a reinfection challenge in the respiratory and alimentary tracts is determined to a major extent by the presence and level of pre-existing secretory antibody. Although the precise role of locally induced cellular immunity in protection against viral infection remains to be determined, these observations suggest that the mechanism of immunologic defence in external surfaces may be mediated largely through specific secretory immunoglobulin and cellular immune response.

Plasma proteins in canine gastric lymph.

Because it has not been possible to collect gastric lymph by cannulation of lymph vessels, the methods of renal micropuncture were adapted to collection and analysis of 0.5- to 3-mu samples obtained by puncturing lymph vessels on or immediately adjacent to the ventral surface of the dog's stomach. Samples of gastric lymph collected immediately after completion of surgical exposure of the stomach were subjected to acrylamide gel electrophoresis, and they were found to show all the protein bands exhibited by simultaneously collected samples of plasma. Quantitative analysis of similar samples by cellulose acetate electrophoresis gave the following ratios of protein concentration in gastric lymph to plasma: total protein, 0.51; albumin, 0.68; total globulins, 0.42; alpha1-globulin, 0.49; alpha2-globulin, 0.62; beta-globulin, 0.35; fibrinogen, 0.39; and gamma-globulin, 0.46. When samples were collected over 4 hr the concentrations of all proteins in lymph increased, and an argument is presented that this was not the result of increasing small pore size as the result of injury. When the mucosa was irrigated with solutions of dithiothreitol, a substance causing plasma shedding, the concentrations of proteins in gastric lymph over a 4-hr period were similar to those found in control experiments. Gastric lymph mixed with 100 mN HCl was found to have proteolytic activity.

Demonstration of a unique antigenic specificity for the collagen alpha1 (II) chain from cartilaginous tissue.

The rabbit antibody response to native collagen (chain composition [alpha1(II)3) from cartilaginous tissue, has been examined by agglutination assays, gel diffusion, haemagglutination-inhibition studies, and immunoadsorption. The results show that the rabbit antibody response to the cartilage-type collagen is characterized by considerable reactivity to both helical [alpha1(II)]3 as well as alpha1(II) chains. This is in contrast to the rat antibody response to the same antigens where titres are generated to largely helical antigenic determinants. Similarly to the rat response, rabbit antibodies to [alpha(II)]3 exhibit no strong cross-reaction with the genetically distinct [alpha(I)]2ALPHA2 collagen or its component chains. Strong cross-reactions were, however, observed between bovine and chick alpha1(II) chains. One of the major antigenic sites on [alpha1(II)]3 collagen appears to reside in the sequence represented by CB-11, a peptide derived from the helical portion of the [alpha1(II)]3 molecule after cyanogen bromide cleavage. The data, however, are compatible with the presence of other antigenic determinants which are probably located in the amino- and carbocy-terminal portions of the molecule.

Expression of virus-associated antigens and immune cell functions during spontaneous regression of the Friend viral murine leukemia.

Spontaneous regression and/or remission of Friend virus (FV)-induced splenic erythroblastic leukemia was observed in CD-1 mice infected with several isolates of FV. Regression of splenic tumor was accompanied by the loss of both specific FV-induced cell membrane antigen (FVMA) and virus group-specific antigens (gsa) from the spleen cells. The frequency (percentage) of immunoglobulin-positive cells (B) and thetapositive cells (T) in the spleen was markedly decreased during leukemia progression, but there was a subsequent increase during regression. The appearance of gsa-positive (gsa+) cells in peripheral blood correlated well with the early progressive and regressive phase of leukemia (up to 7 weeks after infection). Later, the presence of these cells became unpredictable in regard to status of disease. Gsa+ blood cells reappeared in most mice with regressed splenic tumors, suggesting persistence of the virus complex in the animals. Antibody responsiveness as determined by the numbers of hemolytic plaque-forming cells, PFC, after a single immunization with sheep red blood cells (SRBC), was suppressed in leukemia progression and recovered, spontaneously, during regression of leukemia. However, hemolytic PFC elicited by antigen in both progressors and regressors expressed the specific virus-induced membrane antigen, FVMA, detectable by the PFC-inhibition test with specific antiserum and complement. Recovery of immunological responsiveness also included the spontaneous appearance of virus-neutralizing antibody to FV. However, this was not paralleled by the appearance of antibody to FVMA. Traces of anti-FVMA antibody activity were occasionally detectable in serum of both progressors and regressors and did not correlate with virus neutralization, in individual mice; This may explain the susceptibility of regressors to secondary relapse and to reinfection.

Tannic acid-stained microtubules with 12, 13, and 15 protofilaments.

Subunit structure in the walls of sectioned microtubules was first noted by Ledbetter and Porter (6), who clearly showed that certain microtubules of plant meristematic cells have 13 wall protofilaments when seen in cross section. Earlier, protofilaments of microtubular elements had been described in negatively stained material, although exact counts of their number were difficult to obtain. In microtubular elements of axonemes, some success has been achieved in visualizing protofilaments in conventionally fixed and sectioned material (8, 10); much less success has been achieved in identifying and counting protofilaments of singlet cytoplasmic microtubules. By using glutaraldehyde-tannic acid fixation, as described by Misuhira and Futaesaku (7), Tilney et al. (12) studied microtubules from a number of sources and found that all have 13 protofilaments comprising their walls. These authors note that "...the number of subunits and their arrangement as protofilaments appear universal...". Preliminary studies of ventral nerve cord of crayfish fixed in glutaraldehyde-tannic acid indicated that axonal microtubules in this material possess only 12 protofilaments (4). On the basis of this observation, tannic acid preparations of several other neuronal and non-neuronal systems were examined. Protofilaments in microtubules from these several cell types are clearly demonstrated, and counts have been made which show that some kinds of microtubules have more or fewer protofilaments than the usual 13 and that at least one kind of microtubule has an even rather than an odd number.

Olivocochlear and vestibular efferent neurons of the feline brain stem: their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry.

Anterograde degeneration studies have shown that the cochlear and vestibular receptor organs receive an efferent innervation from neurons in the brain stem. This pathway may provide a mechanism by which the CNS could modulate its own afferent input. The neurons which provide this innervation have so far escaped positive identification with methods which depend on retrograde cell changes after axotomy. In the present study, horseradish peroxidase (HRP) was injected into the labryinths of kittens and after allowing 24 hours for the retrograde axonal transport of this tracer, its presence in neurons of the brain stem was demonstrated histochemically. Because there is evidence that the efferent innervation of the labyrinth is cholinergic, acetylcholinesterase (AChE) was also demonstrated histochemically in the same or in adjacent tissue sections. Neurons labelled with HRP were found bilaterally in most periolivary cell groups of the superior olivary complex (cochlear efferents) and in the parvocellular reticular nucleus lateral to the abducens nucleus (vestibular efferents). Counts of labelled neurons yielded estimated totals of 1,700-1,800 cochlear and 400-500 vestibular efferent neurons. Approximately 60% of the neurons in each total were located on the side ipsilateral to the injection. The distribution of HRP-labelled neurons was virtually identical to that of AChE-positive neurons found in adjacent sections, and in those regions with predominantly ipsilateral or contralateral projections, there was an approximate correspondence in number of HRP- and AChE-positive neurons. In tissue sections processed successively for demonstration of HRP and AChE, virtually all HRP-labelled neurons were found to be AChE-positive. These findings suggest that a number of current conceptions regarding labyrinthine efferent systems may need revision.

Conformation-dependent antigenic determinants in the toxic lectin ricin.

The major part of the ricin-precipitable antibodies in sera produced by immunizing rabbits with formaldehyde-treated ricin is precipitated also by the isolated ricin A and B chains. In contrast, in antisera produced by immunizing with formaldehyde-treated ricinus agglutinin only a small part of the antibodies cross-reacting with ricin can be precipitated by the isolated A and B chains, or bound to immunoabsorbents containing the isolated ricin chains. In immunodiffusion studies with anti-ricinus agglutinin sera, a star-shaped precipitate was formed when isolated A and B chains recombined to form intact ricin. Both anti-ricin and anti-ricinus agglutinin sera neutralized effectively the ability of ricin to inhibit protein synthesis in HeLa cells. Anti-ricin serum also neutralized the inhibitory effect of the isolated A chain on protein synthesis in a cell-free system and the ability of the isolated B chain to induce indirect hemagglutination. In contrast, antiricinus agglutinin serum did not neutralize the biologic activities of the isolated ricin A and B chains. Anti-ricinus agglutinin serum formed a precipitate with the hybrid ricin A chain/abrin B chain, and protected against the toxic effect on HeLa cells of this hybrid, indicating conformational changes of ricin A chain upon binding to the B chain. It is concluded that the anti-ricinus agglutinin serum contains antibodies directed against conformational determinants present on intact ricin, but not present or exposed in the isolated A and B chains. At least part of these conformational determinants appears to be carried by the A chain.

The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex.

Isolation of the C5b-9 complex from inulin-activated whole human serum was effected by molecular sieve column chromatography employing Biogel A-15 M, preparative Pevikon block electrophoresis, and removal of low density beta-lipoproteins by flotation in CsCl. The final product was homogeneous upon cellulose acetate strip electrophoresis and analytical ultracentrifugation. Ouchterlony analyses indicated that the complex reacted with antisera to C5, C6, C7, C8, and C9 to form a continuous, circular precipitin line without spurs. The C5b-9 complex was dissociated by sodium dodecyl sulfate (SDS) in the absence of reducing agents, and analytical SDS-polyacrylamide gel electrophoresis revealed seven protein bands after straining with Coomassie Blue. Bands 1, 2, 3, and 6 were identified as C5b, C7, C6, and C9, respectively. Bands 4 and 7 were identified as two noncovalently bound subunits of C8. Molar ratios among C5b, C6, C7, C8, and C9 dissociated from the complex by SDS were estimated to be 1:1:1:1:3. Band 5 protein, which had an estimated mol wt of 88,000 and was found to occur with a molar ratio of 3, has not yet been identified. Its nature and possible biological functions are discussed.

Recognition by pregnancy serums of non-HL-A alloantigens selectively expressed on B lymphocytes.

A group of alloantibodies are found in pregnancy sera which react with antigens present on B lymphocytes and monocytes but are not detectable on the vast majority of unstimulated T cells. This specificity distinguishes them from HL-A antibodies which react with both cell types. They were readily recognized through indirect fluorescent antibody analysis by employing the combination of B-cell lymphoid lines and normal peripheral blood T cells. Different sera gave a variety of patterns of reactivity with a panel of 11 lymphoid lines. Similar differential patterns were also observed with normal B cells from different individuals particularly after concentrating the B cells. The antibodies were also cytotoxic to B cells and this procedure gave parallel results to the fluorescence method. The pattern of reactions obtained indicated a very heterogeneous system similar to that for HL-A. Special study of certain of the sera provided evidence that the lymphocyte-defined determinants of the mixed lymphocyte reaction system were involved. For convenience the term HL-B has been employed for these antigens.

The in vitro induction of immunological tolerance in the B lymphocyte by oligovalent thymus-dependent antigens.

B-cell tolerance has been induced by oligovalent thymus-dependent antigens in an entirely in vitro system. Dissociated spleen cells from congenitally athymic (nu/nu) mice were preincubated for 24 h with 0.1 -- 1 mg/ml of either fowl gamma globulin (FGG) of DNP-human gamma globulin (DNP-HGG). After washing, the cells were tested for the ability to mount in vitro, thymus-independent responses against FGG and DNP. A state of specific responsiveness to either FGG or DNP was thus demonstrated. Features of this wholly in vitro system that paralleled previous findings on the in vivo induction of B-cell tolerance in nu/nu mice were the kinetics, 24 h being required for tolerance induction in either case, the abrogation of tolerance induction by the presence of POL both in vivo and in vitro, and finally the observation that in neither case was there a requirement for the antigens to be deaggregated. It was shown that DNP-(Fab) 2 fragments prepared from HGG induced DNP-specific tolerance indicating that the Fc piece was not required for tolerance induction in this in vitro system. DNP-bovine serum albumin was less effective than DNP-HGG or DNP-(Fab)2. Preincubation with subtoxic concentrations of DNP-lysine of DNP-epsilon-capric acid had only a marginal effect on DNP responsiveness. Since nu/nu mice, lacking in detectable T-cell function, were used as spleen cell donors, this work provides further evidence that B-cell tolerance to thymus-dependent antigens can be induced without the participation of T cells. It is suggested that B-cell tolerance to thymus-dependent antigens occurs when the antigen in a sufficient concentration and over a sufficient period of time has direct access to the B cell. This contact with antigen must be in the absence of an additional influence provided either by adjuvants like endotoxin or POL, or by activated macrophages, which may be stimulated by activated T cells; otherwise not tolerance but B-cell activation will occur.

H-2 compatability requirement for T-cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus. Different cytotoxic T-cell specificities are associated with structures coded for in H-2K or H-2D;.

Use of syngeneic, allogeneic, F1, AND H-2 recombinatn mice has shown that animals injected with lymphocytic choriomeningitis (LCM) virus generate T cells which are cytotoxic for H-2K or H-2D compatible, but not H-2 different, virus-infected target cells. Three separate lines of evidence are presented which indicate that these immune T cells are sensitized to "altered-self," the self antigens involved being coded for in the H-2K or H-2d regions. Firstly, cytotoxic activity associated with mutuality at H-2D iy, lysis mediated by immune T cells from F1 or H-2 recombinant mice is specifically inhibited only by presence of unlabeled, virus-infected cells that are H-2 compatible with the targets. Thirdly, LCM-immune F1 and H-2 recombinant T cells inoculated into irradiated, virus-infected recipients proliferate only to kill target cells that are H-2 compatible with both the donor and the recipient. All of these experiments establish that there is a dissociation of T-cell activities between parental haplotypes in F1 mice, and between H-2K and H-2D in recombinants. It would thus seem that there are at least two specificities of tlcm-immune T cells in homozygotes, associated with either H-2K or H-2D, and four specificities in F1 hybrids. The significance of these findings, with respect both to gene duplication and to the marked polymorphism in the H-2 system, is discussed.

Regression of warts. An immunological study.

Altogether 173 patients with warts were under observation for at least 3 and in most cases 6 months. In 80% of the patients wart-virus antibodies were present and could be measured by immunodiffusion (I.D.) and in 20% also by complement-fixation (C.F.) techniques. The mean duration of the warts in patients with C.F. antibodies was 0-6 years and in the others 1-9 years. The occurence of C.F. antibodies (IgG) was associated with rapid healing; 75% of these patients were cured during the first 2 months of the observation period. In contrast, of the patients with antibodies measurable only by the I.D. technique (IgM and/or low titres of IgG), only 16% were cured during the first 2 months and they had a fairly constant cure-rate (approximately 9% per month) during the 6 months' observation period. The results indicate that the cure of warts is partly connected with immunological phenomena, especially with the presence of C.F. antibodies. In other cases wart regression may be mainly a non-immune process, perhaps due to a limited lifespan of wart cells.

Elective induction of labour. A randomised prospective trial.

In a prospective, randomised trial, 111 obstetrically normal pregnant women, who had elective induction of labour performed between 39 and 40 weeks, were compared with 117 controls who were managed expectantly until 41 weeks. Compared with the controls, the patients who had elective induction of labour had significantly less meconium staining in labour and a smaller blood-loss after delivery. The mean length of labour, the amount of pethidine used, and the Apgar scores at 1 minute were similar in the two groups. In the electively induced group, the caesarean-section rate was lower and the use of epidural analgesia more common than in the controls, but the differences were mot statistically signficant. The hour of delivery was similar in the two groups, suggesting that convenience to medical and nursing staff would not be greatly changed by elective induction of labour. There was no evidence that the hazards to mother and child were increased by elective induction, and its use might improve perinatal mortality by reducing the number of unexplained mature stillbirths.

Controlled trial of repeated halothane anaesthetics in patients with carcinoma of the uterine cervix treated with radium.

39 patients with carcinoma of the uterine cervix who were treated with radium and required repeated general anaesthetics were randomised to halothane and control groups. Their serum-alanine-aminotransferase (S.G.P.T.) levels were measured before each general anaesthetic, and those patients whose S.G.P.T. levels rose above 100 I.U. per litre were freed from the restriction determined by the initial allocation and treated as indicated clinically. None of the 21 patients in the control group had S.G.P.T. levels rising above 100 I.U. per litre. 4 out of 18 patients in the halothane group developed S.G.P.T. levels above 100 i.u. per litre before their third radium treatment. None of these had any symptoms or alteration in other liver-function tests, but liver biopsies in 2 of these patients showed changes characteristic of Hepatitis. Arbitrary selection of 18 out of the 39 patients would only give rise to the degree of abnormality observed in the halothane-treated group with a probability of about 0-02. In the patients studied who required repeated general anaesthetics at short time intervals, the monitoring of S.G.P.T. levels before each operation was useful screen for liver damage and may have reduced postoperative hepatic necrosis by preventing further anaesthetics with halothane when the liver was already damaged.

Hormonal pattern of relapse in hyperthyroidism.

22 patients with Grave's disease were followed up for up to a year after antithyroid drug therapy was discontinued. Clinical assessment and serum T3, T4, and thyroid-stimulating-hormone (T.S.H.) estimations were done serially and simultaneously. Serum T3 or T4 concentrations may be elevated briefly in the first few weeks after antithyroid drugs are stopped, as a rebound effect not necessarily indicative of subsequent relapsf. Clinical relapse of hyperthyroidism with subsequent improvement on antithyroid drugs occurred in 13 patients. Of these 13, serum T3 concentrations became elevated before serum T4 concentrations in 5, thus predicting the subsequent development of clinical hyperthyroidism. In the remaining 8 patients who relapsed, serum T4 was elevated a month before the serum T3. Hyperthyroidism was diagnosed clinically after elevated serum T3 concentrations in 11 patients and at the same time in 2 patients. The mean period of "biochemical hyperthyroidism" in these 11 patients was 12 weeks, with a range of 1 to 56 weeks. During this period 9 of the 11 had minor clinical changes attributable to hyperthyroidism. It is concluded that serial estimations of serum T3 provide the most reliable method of monitoring relapse in hyperthyroidism.

Formalin infiltration of the ductus arteriosus. A method for palliation of infants with selected congenital cardiac lesions.

To circumvent the decreased pulmonary blood flow associated with closure of the ductus arteriosus in newborns with heart defects, we infiltrated buffered formalin solution into the wall of that structure to delay its closure. In four infants with pulmonary atresia in whom shunts had been unsuccessful or were technically not feasible, this procedure produced rapid improvement of arterial oxygen tension that was maintained in three infants for one to nine months. The other died of complications of attempted shunt procedures. In an infant with interrupted aortic arch and a large ventricular septal defect, formalin infiltration of the ductus and pulmonary arterial banding alleviated cardiac failure and improved lower-body perfusion. Formalin infiltration of the ductus is an effective palliative technic for treating certain congenital cardiac defects. No adverse effects have been noted.

Immunological escape mechanism in spontaneously metastasizing mammary tumors.

Immunological and biochemical studies of spontaneously metastasizing and nonmetastasizing rat mammary carcinomas and their plasma membranes indicated that: (i) all spontaneously metastasizing tumors have little or no demonstrable glycocalyx, while all nonmetastasizing tumors have a thick glycocalyx; (ii) there is a direct relationship between the glycocalyx and immunogenicity, and an inverse relationship with the metastasizing capacity of tumor cells, properties which can be quantitated by levels of the plasma membrane marker enzyme 5'-nucleotidase (EC3.1.3.5;5'-ribonucleotide phosphohydrolase) activity; (iii) the absence of glycocalyx from the metastasizing tumor cell surface seems to result from its dissociation from plasma membranes, for solubilized cell surface antigen is readily found in the blood of metastasizing tumor bearing rats, while there was no detectable tumor cell surface antigen in the blood of the nonmetastasizing tumor hosts tested; (iv) both metastasizing and nonmetastasizing mammary tumors appear to have a common soluble cell surface antigen; (v) in addition to this common antigen, there is another membrane-bound antigen in the nonmetastasizing, immunogenic tumor cell surface which presumably is the tumor specific transplantation antigen; and (vi) this antigen is immunobiologically unique, but seems to be immunochemically related to the common soluble antigen. It is postulated that the lack of an immunogenic coat and/or the presence of solubilized tumor cell surface antigen in the blood may provide an immune escape mechanism for tumor cells by interfering with cell-mediated immune response of tumor hosts, leading to their dissemination.

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Human globulin messenger RNA, purified by oligo(dT)-cellulose column chromatography, is reproducibly separated into two bands by polyacrylamide gel electrophoresis in the presence of 99% formamide. The more rapidly migrating (fast) band is somewhat more abundant than the slow band in normal (nonthalassemic) total reticulocyte globin messenger RNA. In alpha-thalassemic (Hb H disease) messenger RNA, the slow band is 6.5 times more abundant than the fast band, whereas in beta-thalassemic messenger RNA the fast band is three times more abundant than a second band, which has a slightly greater mobility than the slow band of normal and alpha-thalassemic RNA. The RNA bands of nonthalassemic globin messenger RNA were eluted from the gel and efficiently transcribed into DNA copies by use of the RNA-dependent DAN polymerase of avian myeloblastosis virus. Hybridization of these copy DNAs to fast and slow band RANs and to nonfractionated normal, alpha-thalassemic, and geta-thalassemic messenger RNAs revealed that the eluted fast band RNA contains predominantly alpha-chain specific sequences, whereas the eluted slow band RNA contains predominantly beta-chain specific sequences. Nucleotide sequence analysis of 32-P-labeled RNA transcribed from the slow band copy DNA also indicated that the slow band RNA is beta messenger RNA.

Correlation of the critical micelle concentrations of surfactants with their effects on a bacterial demethylase.

The activity of a sarcosine dehydrogenase isolated from a strain of Pseudomonas is enhanced by the addition of Triton X-100, Brij 35, and Tween 80, and is inhibited by deoxycholate and Sarkosyl NL-97. 2,6-Dichlorophenolindophenol, which is used as the oxidant in the dehydrogenase assay, has also been employed as an indicator in the spectrophotometric determination of the critical micelle concentrations (CMC) of both the nonionic and anionic detergents under conditions optimal for the enzyme analyses. A correlation between the activation or inhibitory activities of the surfactants and their CMC values has been established.

The functional state of the thyroid gland of the mother and fetus in the prenatal development of rabbits.

The functional state of the thyroid gland of the mother and fetus was studied in different periods of intrauterine development of rabbits, as well as in newborn rabbits according to the level of protein-bound iodine (PBI) in the blood plasma and thyroid gland tissue. Similar studies were conducted after a thyroidectomy of females on the 10th-12th day of pregnancy in order to demonstrate the possibility of mutual compensation of the hormonal function under pathological conditions. The level of PBI in the blood plasma of the mother clearly increases in the second half of pregnancy and decreases sharply after birth. The content of PBI in the fetal blood plasma increases continuously beginning with the 22nd day of intrauterine development. The level of PBI in the thyroid gland tissue both of the mother and the fetus increases sharply at the end of pregnancy. In fetuses of thyroidectomized females the amount of PBI in the blood plasma and thyroid gland tissue on the 22nd day of pregnancy considerably exceeded that in normal fetuses.

[Synchronization of estrus in heifers by means of gestagenic preparations].

Studied were the synchronizing effect, the course of treatment, the combination of the gestagens CAP and MGA with PMS as well as their effect on the conception rate in heifers. A total of 143 heifers of the Bulgarian Brown breed (89 test and 59 control), aged 17--18 months, weighing 350--360 kg on an average, were used in the experiment, divided into three test and three control groups. I test group. Twenty-five animals were given daily one tablet of Synchrorosyn-Peach, the tablet containing 10 mg active chloromadinoacetate. The course of treatment lasted 15 days. II test group. Thirty-six animals were treated daily with 5 g of the premix preparation MGA-100 in the course of 20 days, the heifers being divided into two subgroups of 18 animals each. Each heifer of the first subgroup was injected with 2500 IU PMS. III test group. Twenty-eight animals were given MGA-100 at the same rate as in the II test group, but in the course of 14 days. Half of the test heifers were injected with 2500 IU PMS each on the day when the treatment with MGA-100 was discontinued. It was found that the oral administration of Synchrosyn-P for fifteen days and MGA-100 for 20 days results in a synchronized estrus in 84, resp. 44 per cent of the heifers, with a total conception rate of 76 and 94 per cent, respectively. The combined use of MGA-100 and PMS (2500 IU) enhanced the synchronizing effect. The shorter period of feeding with MGA-100 (14) days lowered both the synchronizing effect and the total conception rate, regardless of its combining with 2500 IU of PMS.

Vagally mediated suppression of premature ventricular contractions in man.

Twelve patients with PVC's were studied to assess the possible role of the vagus nerves in suppressing PVC's. All were without significant heart disease and under forty years of age. A series of five autonomically active drugs, including vagotonic and vagolytic agents, was administered intravenously, each drug being given after the effects of the previous one had abated. Two of the patients did not have PVC's at the time of study. Of the remaining ten patients, five showed vagally mediated suppression of PVC's. Phenylephrine (40 to 60 mug per minute) reduced HR, from an average of 63.2 bpm to 48.5 bpm by a vagally mediated reflex, and decreased PVC incidence in all five patients. The per cent of ventricular heart beats which were PVC's (per cent PVC) decreased from an average of 18.2 per cent to 3.2 per cent in these patients (p smaller than 0.005 in each case). Edrophonium (10 mg.) produced less bradycardia and less reliable PVC suppression. In two of these five patients, atropine (1.5 mg.) increased PVC incidence markedly, although the per cent PVC did not change significantly because of the concomitant tachycardia. These data suggest that strongly increased vagal tone can suppress PVC's in a significant percentage of such patients. This finding in man extends previous animal work which has shown a protective role of the vagus against ventricular arrhythmias under certain conditions.

Models of ionic transport in biological membranes. Raman spectroscopy as a probe of valinomycin, gramicidin A', and rhodopsin conformations.

There is evidence that membrane proteins can serve as the functional units of ionic transport in biological membranes. Laser Raman spectroscopy has been used to probe specific molecular interactions inside two models of transport membrane proteins, valinomycin and gramicidin A. Conformational changes of these molecules, as well as specific interactions with ions, can be detected and may help elucidate how membrane transport proteins such as Na+ minus K+ ATPase and rhodopsin function. Resonance Raman spectroscopy has also been used to study conformational changes and protein-chromophore interactions in rhodopsin, the membrane protein that acts as the primary unit of visual excitation in the eye.

Clinical evaluation of priming solutions for pump oxygenator perfusion.

Various hemodilution agents are now used routinely to prime heart-lung machines for cardiac operations. Hemodilution has resulted in considerable conservation of blood as well as diminution of plasma and corpuscle damage by decreasing the concentration of these elements in blood during extracorporeal circulation. Controversy has existed regarding the relative efficacy of various hemodilution solutions. This study covers 68 patients, divided into three groups, for whom hemodilution was done as follows: (1) the pump was primed with a 5% dextrose solution containing no colloid; (2) Ringer's lactate solution containing approximately 1% low-molecular-weight dextran was used; and (3) a new plasma expander, hydroxyethyl starch, was used as the colloid component of an electrolyte solution. Evaluations and comparisons were carried out for flow rates, blood pressure, urine volume, hematocrit, BUN, blood loss, clotting factors, and the patient's clinical course with regard to pulmonary and neurological complications. We conclude that a colloid is beneficial, especially with longer perfusions.

[On the sex chromatin and sex chromatin-like nuclear structures in human Purkinje-cells].

Animal experiments have shown that in the course of chromatolysis the nuclear sex chromatin of nerve cells of female mammals changes its customary location at the nucleolus and migrates to the nuclear membrane; moreover, in the nuclei of nerve cells of males there occur under similar conditions nuclear structures which resemble the sex chromatin. It was, therefore, thought of interest to see if similar findings also apply to human Purkinje-cells.

Acute myelogenous leukemia of the Wistar/Furth rat: establishment of a continuous tissue culture line producing lysozyme in vitro and in vivo.

A transplantable myelogenous leukemia of an inbred Wistar/Furth rat has been established in tissue culture and cloned. The resulting transplantable leukemia line demonstrates in vitro doubling time of 20 hr, colony-forming efficiency of 5% in liquid and methylcellulos-containing medium, and a saturation density of 3.0 x 106 cells/sq cm in liquid medium. Following intraperitoneal inoculation, newborn rats developed solid tumors, ascities, and leukemia with ld50 of5 x 103 cells and mean latency of 60 days. The tumor cell morphology was consistent with that of acute myelogenous leukemia. Histochemical staining for myeloid enzymes revealed no evidence of myeloperoxidase, esterase, or leukocyte alkaline phosphatase; however, fluorescent antibody staining for lysozyme was markedly positive. Serum, urine, and ascitic fluid from rats with transplanted leukemia also contained elevated levels of lysozyme. There was no detectable type-CRNA virus production by this cell line after as long as 100 days in vitro. This inbred rat myelogenous leukemia should provide a useful model for studies of chemotherapy and immunoltherapy of human acute myelogenous leukemia.

Mercaptoethanol-resistant human serum antibodies reacting with endotoxin from Neisseria gonorrhoeae.

Sera from fifty patients with gonorrhoea, thirty with non-specific urethritis, and eighty blood donors were treated with mercaptoethanol (ME) and examined by the indirect haemagglutination test for antibodies against endotoxin from gonococci. Erythrocytes sensitized with determinant a of endotoxin from Strains 8551, V, and VII, or determinant b from Strain V were used. The percentage of sera active in the haemagglutination test was much higher in the gonorrhoea group than in the controls. The geometric mean titre was also significantly higher in the gonorrhoea group. This applied for all four antigens used. Results obtained in an anti-globulin test indicated that the titre of ME-treated serum was determined by IgG antibodies against the endotoxin. Many sera had titres which varied according to the strain origin of the antigen used in the test. The sensitivity of tests for antibodies was increased by using endotoxin from several different strains of gonococci for the examination of each serum. A simplified procedure for determination of antibodies against endotoxin from different strains of gonococci was elaborated.

Virologic and immunologic studies of human prostatic carcinoma.

Morphologic, tissue culture, immunologic, and biochemical methods have been used in an attempt to detect and characterize oncogenic viruses or their subviral components in cells derived from human prostatic carcinoma (PrCa) or benign prostatic hyperplasia (BPH). Electron microscopy was used to characterize the ultrastructural features of normal and neoplastic prostatic tissue. Examination of specimens of prostatic tissue from 34 patients with PrCa, ten patients with BPH, and three patients with bladder tumor (BT) revealed the presence of particles resembling type-C virus in three cases of PrCa and structures resembling budding type-C virus particles in one case of BPH. Fifty human prostatic tissue specimens have been set in tissue culture, of which 30 have been successfully grown for varying periods of time. Of 20 currently active cultures, nine consist primarily of epithelial cells. Immunofluorescence and mixed hemadsorption tests of cells derived from benign and malignant prostatic tissue and sera derived from patients with PrCa, BPH, BT, and other types of tumors, and from normal donors revealed that sera from patients with PrCa, BPH, or BT contain antibodies to antigens in cells derived from PrCa, BPH, or BT. The nature of these antigen-antibody reactions is under study. Initial biochemical studies have not detected reverse transcriptase in the tissue culture fluid from a small number of sparsely growing PrCa cultures nor specific gene sequences homologous to murine leukemia virus-Rauscher genomic RNA in preparations of either normal or malignant prostatic cell DNA. The results of these preliminary studies have demonstrated the applicability of the techniques employed to the study of the relationship of viruses to human PrCa and have provided a number of promising leads for further investigation.

The binding of carcinoembryonic antigen by antibody and its fragments.

In order to assess the potency of antigenic fragments of carcinoembryonic antigen (CEA) in the radioimmune assay; it is necessary to know whether the high affinity of goat anti-CEA antibody (which makes possible the detection of as little as 10--11M CEA) is due to bivalent binding of the CEA molecule. Immunoglobulin G and the F(ab')2 and Fab fragments derived from it were prepared from an anti-CEA serum and tested for their abioity to bind CEA. Equivalent concentrations of binding sites of the bivalent F(ab)2 and univalent Fab fragments of anti-CEA were identical to the immunoglobulin G fraction in the standard inhibition curve. Fragments of CEA obtained by trypsin digestion produced equivalent inhigition curves when tested with either immunoglobulin G, F(ab')2, or Fab". This, increased avidity due to bivalent binding to a single antigen molecule cannot be invoked to explain the sensitivity observed in the CEA assay. This high sensitivity implicates the protein rather than the carbohydrate as an important part of the antigenic determinant(s) of CEA.

Echocardiography of the intra-atrial baffle in dextro-transposition of the great vessels.

Twelve patients with dextro-transposition of the great vessels (age eight months to four years) were studied by echocardiography following Mustard's procedure. Nine of them had also been studied preoperatively. Postoperatively all patients demonstrated structural echoes in the atrial cavity behind the pulmonary root. In ten, the motion pattern generally resembled that of a stenotic atrioventricular valve iwth a sharp anterior movement followed by flattening in diastole and rapid posterior excursion in systole. The maximum amplitude of motion ranged from 4 to 9 mm (average 6.6 mm). In the remaining two cases, the anterior diastolic movement was attenuated. Similar moving, linear echoes with larger amplitudes of motion (10-14 mm) were observed behind the tricuspid valve in four patients while poorly moving, multiple or thick conglomerate echoes (2-11 mm wide) were detected in seven cases. Echocardiographic contrast studies performed by injecting indocyanine green via catheters placed on either side of the intra-atrial baffle identified it as the source of these echoes. Following operation, coarse diastolic undulations of the mitral valve (ten cases) and the tricuspid valve (nine cases) were noted. Also, fine flutter of both atrioventricular valves, not present before, appeared after operation in three patients. These findings may be related to the altered pathway of blood flow and turbulence resulting from the insertion of the baffle in the atria. Echocardiography appears useful in delineating the character and movement pattern of the intra-atrial baffle and this may have potential in evaluating its long-term functional status.

Synovial fluid analysis.

Synovial fluid analysis plays an important role in the differential diagnosis of various forms of arthritis. The results of macroscopic, biochemical, bacteriological, and microscopic techniques of evaluating synovial fluid must be correlated to establish the diagnosis of each of the various forms of arthritis. The principle of polarized microscopy as applied to the differential diagnosis of crystalline arthritis is currently of special interest.

Mechanisms of chromosome banding. IV. Optical properties of the Giemsa dyes.

A thorough understanding of the mechanisms of R-, C- and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral absorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. - Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not necessary component for banding. - The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their absorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.

Detection and localization of specific antigens in the reproductive tracts of cycling, pregnant, and ovariectomized hamsters.

A systematic search was made for components specific to the female reproductive tract in golden hamsters. Antisera produced in rabbits against saline homogenates of hamster uteri (collected on the night of estrus) cross-reacted extensively with extracts of 12 other tissues in agar gel double-diffusion assays. Absorption of the antisera with small intestine, lung, and liver rendered the immune sera specific for uterine and oviductal antigens (within the limits of the sensitivity of the precipitin assays). Immunoelectrophoretic analysis resolved 12 uterine antigens, many of which were similar to components in several other tissues. Absorbed antisera specific for reproductive tract antigens formed one postalbumin arc with uterine and oviductal extracts in immunoelectrophoretic studies. No reactions were detected between specific antisera and five other organ extracts or plasma. An indirect immunofluorescent antibody technique was used to detect changes in the distribution of specific antigens in reproductive tracts of cycling, pregnant, and ovariectomized hamsters. The gamma-globulin fraction of anti-uterus sera (absorbed with small intestine, lung, and liver), shown to be specific for reproductive tract tissues in precipitin tests, was used to localize antigens. Appropriate controls indicated that the fluorescence observed was due to antigen-antibody interactions. During the cycle, specific antigens were usually confined to the ampullary lamina propria, except during estrus, when they were prominent in the lamina propria and luminal epithelium of the ampula. Specific antigens were never abundant in the isthmus of nonpregnant hamsters. On day 1 postcoitum, the components were found throughout the ampullary and isthmic regions. By day 2 postcoitum, ampullary antigens were usually confined to the lamina propria. The specific components were not prominent in the oviduct on day 3 postcoitum, but were conspicuous in both ampulla and isthmus on day 4. Specific antigens in the uterus were confined to endometrial glands in nonpregnant animals during proestrus, estrus, and (occasionally) metestrus. Diestrous uteri contained no specific antigens. During the first 2 days of pregnancy, antigens were not abundant and were usually confined to the glands and stroma. On days 3 and 4 of pregnancy the specific antigens were prominent in the endometrial glands and stroma and along the apical borders of some luminal epithelial cells. By day 5, these components were less conspicuous in all areas of the endometrium. Uteri of spayed animals receiving no hormones or estradiol alone lacked the specific antigens. However, progesterone (after estrogen priming) promoted the appearance of these components, and the distribution resembled that seen in uteri of 3- and 4-day pregnant animals.

Importance of short-lived lymphocytes in the immune response.

Lymphocytes are heterogeneous with respect to their life-span. Typical B cells, bearing on their membranes immunoglobulin receptors, easily detectable by immunofluorescence, belong mainly to the long-lived population: this can be observed using combined autoradiography and immunofluorescence. However, when primed mice receive (-3H) thymidine before a boosting injection of tobacco mosaic virus (TMV), many plasma cells appearing in the spleen during the secondary response are labelled. In irradiated recipients repopulated with spleen cells from donors primed with TMV and injected with tritiated thymidine 2 hours before killing, the majority of plasms cells appearing in the spleen after antigen injection were labelled. If irradiated mice were repopulated simultaneously with spleen cells from donors primed with TMV and injected with (-3H) thymidine, and from donors primed with haemocyanin, most of the anti-TMV plasms cells were labelled, while most of the anti-haemocyanin plasma cells were unlabelled. These results allowed us to exclude non-specific reutilization of labelled thymidine as the main reason of our observations. It is concluded that either plasma cells derive from shortlived precursors or they receive material from a labelled cell able to co-operate specifically with plasma cell precursors.

Inhibition by serum of encephalitogenic activity of myelin basic protein.

Basic protein of myelin from bovine brain (B-BPM) in Freund's complete adjuvant (FCA) is highly encephalitogenic for the guinea pig. However, when B-BPM is mixed with serum from various species it loses its encephalitogenic effect but not its immunogenic properties. When the synthetic tryptophan peptide matching residue 115-126 of human BPM is mixed with normal human serum it loses both its encephalitogenic and immunogenic effects. The time of exposure to serum necessary for complete inhibition of encephalitogenic activity of B-BPM varied: rat, horse, sheep and human sera produced their inhibitory effect immediately after mixing, whereas guinea pig serum required 8h. The capacity of serum to abrogate the encephalitogenic effects of B-BPM was not due to complete degradation of the molecule by proteinases in serum, because all animals injected with the B-BPM serum mixture gave cutaneous delayed hypersensitivity reactions to B-BPM. The inhibitory effect could be attributed to a factor in serum which is non-dialysable, thermostable at 56 degrees C, for 1 h, and present in high concentration in fetal calf serum. It may be an alpha2 macroglobulin which can act selectively on the main encephalitogenic determinant, around or within residues 115-126 of the basic protein of myelin.

Locus ceruleus in rhesus monkey (Macaca mulatta): a combined histochemical fluorescence, Nissl and silver study.

The locus ceruleus (LC) of the rhesus monkey (Macaca mulatta) was investigated using the histochemical fluorescence method and Nissl and ammoniacal silver stains. Caudally, a few fluorescent cells were observed in the lateral wall of the ventriculus quartus near the velum medullaris superior. Rostrally, the fluorescent cells were compactly clustered and reached their greatest density medial and, to a lesser extent, lateral to the tractus mesencephalicus n. trigemini at the level of the decussation of the nervus trochlearis. In the most rostral plane, fluorescent cells were more diffusely scattered ventral and medial to the pedunculus cerebellaris superior with a few cells situated near the nervus trochlearis. Nissl staining of tissue previously used for histochemical fluorescence showed that fluorescent cells were largely found in a region labelled LC in two rhesus monkey brain atlases and erroneously labelled nucleus tractus mesencephalicus n. trigemini in one brain atlas. Ammoniacal silver staining resulted in a dense accumulation of silver granules in the cells found to display a positive reaction for monoamines. The silver stains therefore offer an alternative to the histochemical fluorescence method for identifying the monoamine-containing LC neurons.

Esterase zymograms of Proteus and Providencia.

The intracellular esterases of 80 strains of Proteus and Providencia were analysed by the acrylamide-agarose zymogram technique using several synthetic substrates. The esterase bands were classified in five main groups. The alphaA-esterase bands hydrolysed alpha-naphthyl acetate and were resistant or relatively insensitive to di-isofluoropropyl phosphate (DFP). The alphaB-esterase band hydrolysed both alpha-naphthyl acetate and alpha-naphthyl butyrate and were very sensitive to DFP. Both groups of esterase bands were inactivated by heat. The betaA- and betaB-esterase bands hydrolysed beta-naphthyl acetate and were sensitive to DFP; these were distinguishable by the difference in their relative activity towards beta-naphthyl butyrate and in their relative stability to heat. The alpha-beta-esterase bands hydrolysed alpha- and beta-naphthyl acetates and alpha- and beta-naphthyl butyrates; they were inactivated by heat and were sensitive to DFP. The distribution of these esterase bands among the strains of Proteus and Providencia and their electrophoretic patterns established esterase profile types which correlate with the classification based on traditional bacteriological tests. The degree of inter-strain similarity in esterase pattern varied highly among species. The homogeneity of Proteus mirabilis and especially of Providencia stuartii contrasted with the heterogeneity of other species. This disparity suggests that the bacteria of the tribe Proteae have not the same degree of intra-specific differentiation in physico-chemical properties of esterases.

Preoperative laparoscopy in diagnosis of acute abdominal pain.

During 1973, 56 patients on one of three general surgical services at the Peter Bent Brigham Hospital, Boston, Massachusetts, who were judged to require hospital admission for acute abdominal pain were dividied into two groups. This division was determined by whether or not the physician responsible thought a definite diagnosis could be established on clinical grounds. 27 patients were thought to have a definite diagnosis and underwent laparotomy without preoperative laparoscopy; at laparotomy, 6 of these patients (22%) had no operable lesion. An additional 29 patients had severe abdominal pain and required observation in hospital. An exact diagnosis could not be clinically established in these patients, and many would in the past have required exploratory laparotomy. These 29 patients underwent laparoscopy resulting in all but 1 (4%) having the presence or absence of intra-abdominal disease requiring operative intervention definitely established. At laparoscopy, diagnosis was made in 18 patients who did not require laparotomy while 11 had disease requiring laparotomy after laparoscopy. No complications resulted from laparoscopy. The difference in the median length of stay and hospital charges resulted in a saving of one and a half days in hospital and $87 when laparoscopy rather than explatory laparotomy determined that acute abdominal pain was caused by a condition not requiring surgical intervention.

Sunlight and hypercalciuria.

Urinary calcium and magnesium excretion was measured in two groups of soldiers leaving the temperate climate of the united Kingdom for service in the Persian Gulf. In one group urinary calcium levels and magnesium/calcium ratios were similar, ten days after arrival in the Gulf during the "cold season", to those found in the U.K. The other group went to the Gulf in the "hot season", and calcium excretion rose immediately to levels comparable with those found in the first group after eight months. Mg/Ca ratios fell to levels seen in stonformers, and 2 of 91 soldiers followed up for three years have had urinary calculi. Increased exposure to sunlight seems to be the most likely cause of the hypercalciuria.

Rubella-virus infection in juvenile rheumatoid arthritis.

Antibody activity against mumps, measles, polio, and rubella viruses was determined in patients with juvenile rheumatoid arthritis (J.R.A.), rubella-vaccine associated arthritis, adult rheumatoid arthritis, other chronic systemic disorders (e.g., systemic lupus and dermatomyositis), and in a matched population of normal, non-rheumatoid (control) children. The antibody levels against mumps, measles, and poliovirus were similar in all patients. Rubella-antibody levels in rheumatoid arthritis and other systemic disorders were similar to those observed in controls. The mean rubella-antibody levels in rubella-vaccine arthritis were 4 times higher than in controls. The IgM and IgG rubella-antibody levels in J.R.A. were found to be 4-6 times higher when compared to titres observed in the controls. Highest antibody levels were seen in younger children with J.R.A. Detection of rubella-virus antigen was attempted by immunofluorescence in the sediment smears of synovial fluid of patients with J.R.A., adult rheumatoid arthritis, and other non-rheumatoid joint diseases. Specific staining for rubella virus antigen was observed in the synovial fluid of 33 percent of patients with J.R.A. No antigen was detected in the synovial fluid from other patients. These observations suggest a possible role of rubella-virus infection in J.R.A.

Survival after 40 minutes; submersion without cerebral sequeae.

Cardiopulmonary resuscitation and rewarming were successful in a 5-year-old boy who had been submerged for 40 minutes in ice-cold fresh water. Severe metabolic acidosis was corrected by intravenous infusion of sodium bicarbonate solution before spontaneous circulation could be re-established. Fulminant pulmonary oedema developed after re-establishment of spontaneous circulation. This was efficiently reversed by positive-end-expiratory-pressure ventilation. During 2 days of treatment of a respiratory the patient gradually regained consciousness; the endotracheal tube was then removed and the patient immediately started talking intelligently. The patient went through a period of slow cerebration and motor dysfunction but recovered rapidly, and on examination 13 months after the accident all findings were normal.

Congenital cytomegalovirus infection after maternal renal transplantation.

Congenital cytomegalovirus infection was found in an infant whose mother had a successful renal transplant and was treated with immunosuppressant therapy before and during pregnancy. Although so far not experiencing any untoward infections, the child had impaired T-lymphocyte function and subnormal serum-IgA.

Two antigenically distinct species of human interferon.

Rabbit antisera prepared against interferon produced in human fibroblast cell cultures stimulated with poly(1).poly(C) neutralized the activity of interferon preparations produced in various human fibroblast cultures timulated either with poly(1)poly)C) or with viruses. However, these antisera showed no detectable neutralizing activity against interferon produced in cultures of human leukocytes. On the other hand, most rabbit antisera against the human leukocyte interferon were active in neutralizing both homologous interferon and fibroblast interferons. A preparation of antiserum against leukocyte interferon, active against both leukocyte and fibroblast interferons, was shown by affinity chromatography to have two distinct antibody populations, one of which was specific for the fibroblast interferon. We conclude that the heterologous neutralizing activity of sera from rabbits immunized with leukocyte interferon is liekly to be due to the presence of two antigenic species of interferon. The major antigenic species of leukocyte interferon preparations (designated "Le") is distinct from huamn fibroblast interferon. The minor species of leukocyte interferon ("F") is either identical with, or closely related to, interferon produced in human fibroblast cultures.

Complement components in normal serum and plasma quantitated by electroimmunoassay.

Sjöholm, A. G. Complement Components in Normal Serum and Plasma Quantitated by Electroimmunoassay. Scand. J. Immunol. 4, 25-30, 1975. The concentrations of C1q, C1s, C3, C4, C5, C3 proactivator, and C1 inactivator in serum and EDTA plasma from 100 normal adults were determined by electroimmunoassay. The normal range of each of the proteins is given. The C1q values varied more closely with the C1s values than with the levels of the other complement components. C3, C5, and C3 proactivator seemed to form a fairly interdependent group. The reproducibility of double determinations (interplate variation) was 4.9% to 7.9%. The variation of the complement component levels on repeated sampling from normal individuals was investigated. Also, repeated freezeing and thawing and storage at room temperature of serum and plasma were studied for their effect on the quantitation of the complement components. C3 and C4 values obtained by electroimmunoassay were in agreement with the values obtained by single radial immunodiffusion.

The possible role of i region determined cell surface molecules in the regulation of immune responses.

An hypothetical model has been presented by which I region determined cell surface molecules (Ia antigens) mediate the collaboration between T cells and B cells leading to control of the humoral immune response. The model proposes an analogy between Ia antigens and the constant regions of Ig chains. The absolute requirements of this model are: a) On the B cell the Ia antigen is closely associated with the Fc receptor; b) On the T cell the Ia antigen is closely associated with the product of a linked variable region gene which functions as a specific T cell binding site; and c) The interaction between the T cell Ia molecule and its B cell counterpart leads to B cell activation. By the proposed interactive model no additional Ir gene products are required to explain current concepts of Ir gene function. The experimental evidence from our own laboratory and elsewhere upon which this model is based has been reviewed and a variety of consequences and predictions of the model have been examined. There are numerous aspects of the model which, because of a lack of hard data, are open to alternative explanations. The possible usefulness of this model should lie in its ability to suggest further experiments to elucidate the mechanism of B cell activation and control of the immune response.

A study of complement components C3, C5, C6, C7, C8 and C9 in chronic membranoproliferative glomerulonephritis, systemic lupus erythematosus, poststreptococcal nephritis, idiopathic nephrotic syndrome and anaphylactoid purpura.

In a comparative study the hemolytic activity of C3, C5, C6, C7, C8, C9 and the C3 proactivator (C3PA) were measured in sera of 22 patients with chronic membrano-proliferative glomerulonephritis (CMPGN), 15 patients with idiopathic nephrotic syndrome, 10 patients with systemic lupus erythematosus, 7 patients with anaphylactoid purpura and 10 patients with acute poststreptococcal nephritis. In CMPGN, C3, C5, C6, C7 and C8 were low in the majority of the patients, whereas C9 and C3PA were depressed only in 21% and 11% of the patients, respectively. By contrast, C3PA and C8 showed striking depressions in the idiopathic nephrotic syndrome. In lupus erythematosus, all the C factors, including C3PA were found to be low with the exception of C9, which was normal in 80% of the patients studied. C3, C5, C6 and C7 were found to be depressed in acute glomerulonephritis; C8 and C9 titers were normal. In all patients studied with anaphylactoid purpura, CH50 and C3 titers were elevated markedly.

Fine structural and cytochemical identification of microperoxisomes in developing human erythrocytic cells.

An alkaline diaminobenzidine (DAB) medium has been used to identify peroxidase activity in small granules (0.09 to 0.2 mu in diameter) present in all forms of maturing erythrocytic cells with the exception of erythrocytes. These granules, which were more frequent in proerythroblasts (from two to seven by thin section), were distinct from pleomorphic granules present in the close proximity to the Golgi apparatus. They were also distinct from ferritin molecules which were seen as aggregates in siderosomes of polychromatophilic erythroblasts. They often appeared in close association with the smooth membrane of the nuclear envelope. Optimal conditions for the visualization of these granules by incubation in alkaline DAB were obtained when the peroxidase activity of hemoglobin was reduced by addition of low concentrations of potassium cyanide. Lack of hydrogen peroxide in the incubation media completely inhibited the staining reaction of hemoglobin, while the positive reaction persisted in the granules. Aminotriazole in the incubation media prevented the staining of these organelles. These findings suggest that small granules seen in maturing erythroblasts contain catalase and that they correspond to microperoxisomes described in other tissues. The mechanism of their disappearance during reticulocyte maturation is unknown. The relationship between particulate catalase of erythroblasts and soluble erythrocytic catalase has not been elucidated.

Correction of tricuspid atresia.

Although Fontan and Baudet in 1971 described a physiological correction procedure for tricuspid atresia, very few successful operations have been reported. Two patients corrected 20 and 10 months ago at the Brompton Hospital are presented. These two patients exhibit many of the problems in the management of tricuspid atresia. The first patient aged 20 had undergone three previous palliative operations, a Blalock-Taussig shunt, a Glenn procedure, and an infundibular resection, and therefore presented a number of operative problems. In contrast the second patient, aged 8, whose condition had deteriorated considerably over the previous year, had had no previous surgical treatment. At operation he was found, in addition, to have a partial atrioventricular canal. Details of the operative procedures and the patients' postoperative course are described. The criteria for selection of patients for the Fontan operation are discussed as are the possible long-term hazards of homograft failure, atrial dysrhythmias, and hepatic dysfunction. The successful outcome of these two patients suggests that palliative surgery in infancy should allow for this form of correction in later life.

Basophil counting with a new staining method using alcian blue.

Difficulties in obtaining reproducible and accurate enumeration of circulating basophils with existing techniques have hampered investigation of this infrequent cell population. A new basophil staining method is described that employs alcian blue dye for staining of heparin within basophils at low pH and in the presence of lanthanum ions. Basophil recognition is facilitated by reducing nonspecific nuclear staining. This objective is achieved because of the differences in stability of alcian blue-heparin, alcian blue-nucleic acid, lanthanum-heparin, and lanthanum-mucliec acid complexes. Reduction of pH after staining also favors solubilization of leukocyte cytoplasmic proteins, providing greater contrast between stained and unstained cells by reducing light scattering of the unstained leukocytes . The alcian blue staining method is suitable both for chamber basophil counting and automated basophil counting using continuous-flow sampling and electro-optical detection. The new staining method was evaluated by comparing it with the chamber counting method using toluidine blue in a triple-blind study in which the results of basophil counting by the alcian blue chamber method, alcian blue automated instrument method, and the toluidine blue chamber method were analyzed for reproducibility and compared with an indirect basophil count obtained from a 1000-cell leukocyte differential and a total leukocyte count. Both alcian blue staining methods gave greater reporducibility that toluidine blue and were more accurate, as evidinced by a significantly higher correlation with the indirect basophil count. The improved reproducibility, accuracy, and convenience of this method over existing methods should facilitate the collection of more meaningful information about circulating basophil levels in health and disease.

Multiform ventricular ectopic rhythm. Evidence for multiple parasystolic activity.

Six patients whose standare electrocardiograms showed multiform ventricular ectopic rhythm were studied. All patients had advanced organic heart disease and a significant intraventricular conduction defect (left bundle branch block in five and right bundle branch block plus left anterior hemiblock in one). The ventricular arrhythmia was generally resistant to antiarrhythmic therapy. Five of the six patients died after 2 to 6 months form the period of observation from terminal heart failure. None died suddenly. The ventricular arrhythmia did not seem to be directly related to mortality in any patient. Critical analysis of several long rhythm strips in each case revealed that discharge from multiple ventricular parasytolic foci shared in the multiform ventricular activity. The concurrent discharge of a minimum of three parasytolic foci and a maximum of six foci was found in the same case with a total of 24 parasystolic foci in the six patients. There was a remarkable constancy of the QRS configuration of all parasytolic foci over periods of observation of up to 16 months. However, 22 out of 24 parasystolic rhythms showed significant variation in the apparent rhythm or the administration of drugs. Fourteen parasytolic foci showed evidence of exit block, some of which were exaples of a rapid parasystole with a high degree of exit block. The study suggests that multiform ventricular ectopic rhythm may, in part, be due to the concurrent discharge of multiple parasystolic foci.

Rapid effects of single small doses of L-thyroxine and triiodo-L-thyronine on growth hormone, as studied in the rat by radioimmunoassy.

The effects of thyroid hormone deprivation and restitution on growth hormone (GH) economy have been studied in the rat by means of a specific radioimmunoassay. The pituitary GH content and the plasma GH levels before and during stimulation with pentobarbital ("PB-test") were studied in male rats at different intervals after surgical thyroidectomy (T), and in T rats at different time intervals after the ip injection of 0.20, 1.75, and 5.0 mug thyroxine (T4) or 0.05, 0.10, 0.20 and 1.0 mug triiodothyronine (T3), all doses being referred to 100 g body wt. Pituitary GH content decreased very rapidly after T, a difference being shown at the end of the shortest time interval studied (24 h); 24 days after T, pituitary GH content was 0.3 percent or less of the pre-T level, the basal plasma GH was lower than in intact controls and an increase in plasma GH during PB-stimulation was no longer observed. When rats T for 30 days or longer were injected once with T4 or T3, pituitary GH content increased; basal plasma GH levels increased also and a positive response to PB was observed. An effect on pituitary GH content could be observed as soon as 2 h after the ip injection of 1.0 mug T3, or 6 h after 5.0 mug T4. The "latent period" was somewhat longer when lower doses of the hormones were used. Effects of a single 0.10 mug T3 dose could be detected within 12 h L-T3 appeared to be at least 9 times more potent in vivo tha T4, as assessed from the effect on pituitary GH. The mea-urement by RIA of changes in GH content of the rat pituitary may thus provide the most adequate parameter available at present (other than suppression of TRH-induced TSH release) for a biological effect in vivo of single doses of the thyroid hormones, a measurement clearly related to an important physiological role.

Multiple specificities of mammalian blood group substances comparatively studied with human isoagglutinins and fractionated anti-H lectins.

Purified blood group-active substances derived from different pig, horse, baboon, Rhesus monkey and human tissues were quantitatively studied for their haemagglutination inhibiting potency with: (1) human IgM anti-A and anti-B; (2) human anti-Lea and anti-Leb; (3) Ulex europaeus extracts separated into lectin fractions with respective L-fucose-inhibitable ('anti-HF') and chitobiose-cellobiose-inhibitable ('anti-HC') combining sites. Irrespective of species origin, A and B blood group activity per milligram of purified material tended to be strikingly higher in substances low in, or devoid of, Lewis blood group activity. Most of the blood group substances displayed variable but about equally balanced amounts of Ulex anti-HF and anti-HC inhibiting activity. In contrast, pig submaxillary gland mucins displayed strikingly high levels of Ulex anti-HC inihibiting activity, even in the complete absence of Ulex anti-HF inhibiting activity. These serological findings are consistent with current biochemical concepts regarding the heterosaccharide microheterogeneity of blood group-active glycoproteins.

Nonoxidative fungicidal mechanisms of mammalian granulocytes: demonstration of components with candidacidal activity in human, rabbit, and guinea pig leukocytes.

Granulocytes from the peripheral blood of normal subjects and a patient with hereditary myeloperoxidase deficiency were homogenized in 0.34 M sucrose. A granule-rich fraction, prepared by sedimentation at 27,000 x g for 20 min, contained components that killed C. parapsilosis in vitro. These were extractable with 0.01 M citric acid and were shown by micropreparative polyacrylamide electrophoresis to be multiple. The candidacidal activity of these neutrophil components was heat stable and they were somewhat more active at pH 5.0 than at pH 7.0. When rabbit or guinea pig heterophils were obtained from sterile peritoneal exudates and similarly fractionated, they also were found to contain components that killed C. parapsilosis in vitro. These were primarily associated with a group of lysosomal cationic proteins lacking direct counterpart in human neutrophils. Among the candidacidal components of the human neutrophil was a protein, more cationic than lysozyme, that exhibited naphthol-ASD acetate esterase activity.

The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hydroxytryptamine in whole mounts.

The reactions of glyoxylic acid with peripheral stores of noradrenaline and 5-hydroxytryptamine to provide a fluorescence histochemical method for their localization have been investigated. Incubation in glyoxylic acid, followed by drying and heating of whole mount preparations gives an intense and well localized reaction. For incubation, a concentration of 2% glyoxylic acid, buffered to pH 7 at room temperature for 30 minutes gives ideal results. The method is equally good if the pH is varied in the range 6 to 9 or if the tissue is stored in the incubation mixture for up to 6 hours. Ideal development of the fluorophore requires an initial excess of moisture in the tissue, that this moisture is driven off during development, and that the tissue is protected from further moistening. A suitable method of achieving these ends is to heat partially dried tissue at 100 degrees C for 4 minutes and then cover it with paraffin oil. 5-hydroxytryptamine can be readily distinguished from noradrenaline because it forms a fluorophore after reaction at pH 3.5, whereas noradrenaline does not. Both amines can be visualized after incubation at neutral pH. Comparison with the formaldehyde vapour technique reveals three main advantages (and no disadvantages) of the glyoxylic acid method: (1) it gives a finer localization with higher fluorescence yield, (2) the glyoxylic acid method is less susceptible to variations in procedure and, (3) it is both simpler and quicker to apply.

Radiocopper in L-tryptophan 2,3-dioxygenase isolated from Pseudomonas acidovorans grown in the presence of 64Cu (II).

L-Tryptophan 2,3-dioxygenase (EC 1.13.11.11), isolated from L-tryptophan-induced Pseudomonas acidovorans, ATCC 11299b, which has been grown in a medium containing 64Cu(NO3)2, has been shown to contain radiocopper. At several stages of purification of the enzyme samples were taken, and these were subjected to disc acrylamide gel electrophoresis in the presence of 10 mM L-tryptophan. After electrophoresis the position of the yellow heme band, corresponding to tryptophan oxygenase, was visually located, and the gels were sliced and counted. A large peak of radioactivity was seen to occur at the location on the gel of tryptophan oxygenase no matter what the stage of purification. Treatment of each sample before electrophoresis for 30 min at 37 degrees with gamma-globulins prepared from rabbits sensitized to homogeneous pseudomonad tryptophan oxygenase greatly reduced this peak of radioactivity, whereas treatment of each sample with rabbit preimmune gamma-globulin did not. This direct demonstration of the presence of coper in pseudomonad tryptophan oxygenase, using 64-Cu, avoided the problems and artifacts inherent in the usual techniques of copper analysis and unequivocally refutes the recent contention of Ishimura and Hayaishi ((1973) J. Biol.Chem. 248, 8610-8612) "that copper is not an essential component of L-tryptophan 2,3-dioxygenase of Pseudomonas." The presence of copper in pseudomonad and rat liver tryptophan oxygenases, previously reported by us (Brady, F. O., Monaco, M. E., Forman, H. J., Schutz, G., and Feigelson, P. (P. (1972) J. Biol. Chem. 247, 7915-7922), is reaffirmed by the experiments reported herein.

The pattern of microtubules in the axonemes of Gymnosphaera albida sassaki: evidence for 13 protofilaments.

The axonemes of Gymnosphaera albida radiate from a central axoplast. Their proximal ends form a shell around the axoplast. Within the shell each axoneme is enveloped by a fibrillar sheath and the microtubules are interconnected by electron-dense linkages, which sometimes appear to be double. In nearly transverse sections the microtubules and their linkages form hexagons of 2 irregular types arranged in alternating rows. The shapes of the hexagons vary from one axoneme to the next. The variation is caused largely by the inclination of the axonemes to the line of sight, but also by distortion occurring during the preparation, observation and photography of the sections. Calculations show that, of a number of likely basic patterns (as would be seen in strictly transverse section), only one is compatible with measurements made on 9 of the axonemes. This involves only one type of hexagon oriented in 2 directions to form a 'parquet-floor' pattern. The hexagon is bilaterally symmetrical and its 6 microtubules all have the same set of angles between their linkages, namely an unpaired angle of 138 degrees 28' and paired angles of 110 degrees 46'. Because these angles are in the ratio of 5:4:4, it is deduced that the microtubules have 13 protofilaments forming their walls. Morphogenetically the lateral growth of the pattern is governed by 2 rules: (1) there must be one, and only one, direction of 2-step zig-zagging of the linkages, and (2) linkages forming opposite sides of a hexagon must be in parallel.

Cell-mediated immunity in systemic lupus erythematosus: alterations with advancing age.

Cell-mediated immunity (CMI) was tested in young patients with systemic lupus erythematosus (SLE) (mean age 30.9 years), in elderly patients with SLE (mean age 70.8 years), and in young and elderly control subjects. Pre-existing CMI as evaluated by skin test response to PPD and mumps antigens and the response of peripheral blood lymphocytes in culture to the mitogen phytohemagglutinin was not significantly different in thefour groups. However, the mean area of induration to Candida antigen was significantly less in both groups of elderly patients, suggesting a diminution of CMI with advancing age. Elderly control subjects and elderly patients with SLE had a significant decrease in incidence and severity of skin-test response when sensitized with 2-4-dinitrochlorobenzene (DNCB), suggesting that the capacity to respond to a new antigen is impaired in the elderly, with and without SLE. Young SLE patients were easily sensitized to DNCB and developed strong skin-test responses to Candida antigen suggesting normal CMI in the young SLE group. SLE is a milder disease in the elderly. If, as seems likely, cell-mediated immune injury is of importance in the pathogenesis of SLE, then these data are consistent with the possibility that an altered state of CMI in the elderly modifies the clinical expression of SLE.