Regulation of lipolysis in adipose tissue. The normal human or animal has a series of finely coordinated and overlapping control mechanisms that regulate the storage or release of fat according to the overall energy requirements of the body. We have examined only one side of this system, the part responsible for the release of fatty acids from adipose tissue. Considerable information is available on the extracellular factors that control lipolysis on a minute-to-minute or day-to-day basis, but less is known about long-term regulation or about the precise role of intracellular factors that almost surely condition the response of the fat cell to external stimuli. When one poses the question of what determines the overall fat mass or adiposity of an individual, even less is known. Psychogenic factors regulating food intake, circulating hormonal signals, and local regulators of metabolism in the adipose tissue may all play a role. This remains an intriguing but elusive problem to be answered by future research.


The effect of attapulgite and charcoal on enterotoxicity of Vibrio cholerae and Escherichia coli enterotoxins in rabbits. Vibrio cholerae and certain strains of Escherichia coli produce heat-labile enterotoxins which play a significant role in the pathogenesis of the intestinal disease. Activated attapulgite, a heated magnesium aluminum silicate, was previously shown to prevent the toxic effects of endotoxin. The present study has revealed that this drug inhibits the toxic effects of cholera and E. coli enterotoxins in the intestinal loop of rabbits, when toxin and attapulgite are pre-incubated prior to injection. Up to 50 to 100 minimal effective doses are inhibited. Attapulgite is effective also when injected separately, albeit simultaneously, into the intestinal loops, but not when administered after the toxin. Since supernates of toxinattapulgite mixtures are non-toxic, it is postulated that attapulgite acts by adsorption and that the attached enterotoxin is no longer toxic to the rabbit intestine. The previously reported effect of charcoal on V. cholerae enterotoxin paralleling that of attapulgite, was confirmed. In contrast to the effects of these absorbents on isolated toxin, both failed to prevent enterotoxicity in the rabbit model of an enterotoxin-producing strain of E. coli.


Acute encephalopathy with liver dysfunction, chylous ascites and cytomegalovirus infection. A child with acute encephalopathy and liver dysfunction subsequently developed acute chylous ascites. Titers for cytomegalovirus increased from less than 1:2 to 1:32 during the illness, and cytomegalovirus was isolated from the urine. The case is the first one possibly linking cytomegalovirus and acute encephalopathy and liver dysfunction in a child. In our patient, enlargement of the abdominal lymph nodes, as seen on a lymphangiogram, resulted in a severe obstruction of abdominal lymphatic flow, producing a transudation of lymph into the peritoneal cavity. The acute chylous ascites associated with mesenteric lymphadenitis was likely caused by the cytomegalovirus infection.


Transformation of human cells by temperature-sensitive mutants of simian virus-40. Conditions necessary for the establishment and maintenance of transformation of human cells by wild type and temperature-sensitive mutants of SV40 were examined. For both early and late mutants, the frequency of transformation was found to be up to 5-fold higher, and virus yield 100-fold lower, at 39 degrees than at 33 degrees. No such effect was observed with the wild type virus under the same conditions. This observation is apparently at variance with previously published work, but may be explained by the semipermissive nature of the cells that we used. Increasing the temperature to 40.5 degrees caused cells transformed by the early mutant, tsA30, to lose T-antigen as detectable by staining, and also to lose the ability to grow to high density, while it produced no effect on cells transformed by wild type virus.


Virus shedding by SV40-transformed human cells. Cultures of human cells transformed by SV40 were found to release infectious virus, even after several passages in vitro. Virus shedding by these cultures did not depend on propagation of virus from cell to cell, as it was not affected by anti-SV40 antiserum that could effectively block virus propagation in acutely infected cells. Cells transformed by the 'early' temperature-sensitive mutant tsA30, and maintained at the restrictive temperature of 39 degrees, shed virus in reduced amount. Finally, xeroderma pigmentosum cells transformed by SV40 were also found to release virus, indicating that the enzymes of excision and repair of UV-induced damage to DNA probably were not involved in the molecular mechanism underlying virus shedding.


Factors affecting the interferon sensitivity of human cytomegalovirus. Several factors affected the interferon sensitivity of human cytomegalovirus in human foreskin fibroblast cultures. An inoculum of infected cells was up to 300-fold less sensitive than a cell-free inoculum of equivalent input multiplicity. A 10-fold increase in the dose of infectious units of either type of inoculum was associated with a 10-fold or greater decrease in interferon sensitivity. Several aspects of the virus-cell interaction were examined and parameters indicative of cell infection were less inhibited by interferon than were those for virus replication.


A simple method for concentration of enteroviruses and rotaviruses from cell culture harvests using membrane filters. Organic compounds in cell culture harvests known as membrane-coating components (MCC) prevent virus adsorption to membrane filters. Blending cell culture harvests with fluorocarbon removed the MCC and permitted adsorption of virus in acidified harvests to epoxy-fiberglass filters. Subsequent elution with high pH buffer resulted in recovery of greater than 90% of the virus with concentrations of up to 100-fold.


Epidemic of hand, foot and mouth disease associated with enterovirus 71 infection. Viruses isolated from patients with hand, foot and mouth disease in widespread outbreaks in Japan in 1973 were identified as enterovirus 71. Although cases with aseptic meningitis were observed concurrently, the main clinical symptom associated with enterovirus 71 infection was hand, foot and mouth disease.


Ribosomal proteins in normal simian cells, SV40-transformed simian cells, and simian cells infected with SV40, adenovirus 5, and vesicular stomatitis virus. The protein patterns of monosomes and polysomes isolated from the T-22 line of SV40-transformed GMK cells and from uninfected CV-1 cells and CV-1 cells infected with SV40, adenovirus 5, or vesicular stomatitis virus were analyzed by two-dimensional PAGE. All gel patterns were similar except for the presence of one additional protein associated with T-22 monosomes.


The structural polypeptides of equine infections anemia virus. The structural polypeptides and glycoproteins of equine infectious anemia virus were identified following electrophoresis in SDS-PAGE. The major non-glycosylated polypeptides had molecular weights of 25,000, 14,000 and 11,000 daltons. Two glycoproteins of 80,000 and 40,000 daltons were also detected. The relationship of these components to the structural elements of mammalian C-type oncoviruses is discussed.


A new lymphoid cell line, Reh 6, with characteristics of non-T and non-B cells, lacking the Epstein-Barr virus genome and derived from human acute lymphoblastic leukemia. A new lymphoid cell line, Reh 6, has been established from the peripheral blood of a patient with acute lymphocytic leukemia. Reh 6 cells were assayed for the presence of Epstein-Barr virus (EBV) DNA by nucleic acid hybridization and for Epstein-Barr nuclear antigen (EBNA) by the immunofluorescence test. Reassociation kinetics between in vitro [3H]-labeled EBV DNA and Reh 6 cell DNA indicated the absence of detectable amounts of EBV DNA in Reh 6 cell DNA. In addition, attempts to detect EBNA by the immunofluorescence test in Reh 6 cells were unsuccessful. Thus, this new lymphoid cell line apparently lacks the EBV genome.


Chicken embyro lethal orphan (CELO) virus DNA present in hamster cell lines derived from CELO-induced hepatomas. The kinetics of renaturation of the isolated DNA fragments of chicken embryo lethal orphan (CELO) virus generated by the restriction enzyme EcoR1 was measured in the presence of DNA extracted from two lines of CELO virus-induced hamster hepatoma cells and from control hamster cells. One hepatoma cell line (CILT-2), which did not produce the CELO virus tumor (T) antigen, lacked sequences from about one-half the virus genome while the other hepatoma cell line (CEHEP), which did produce the CELO virus T antigen, lacked sequences corresponding to one-fifth of the viral genome.


Effect of thymic humoral factor on intracellular levels of cyclic AMP in human peripheral blood lymphocytes. The exposure of human peripheral blood lymphocytes to thymus hormone causes an increase in the intracellular cyclic AMP level in these cells. Studies of the thymus-hormone-induced changes in cellular cyclic AMP levels revealed that cord blood lymphocytes contain significantly more target cells for the hormone than adult peripheral blood lymphocytes. The data obtained also suggest that the interaction between the hormone and its target cell is not readily reversible and that only a certain proportion of target cells in a given population of lymphocytes can be activated by a constant and limited amount of thymus humoral factor.


[Hormones and hair growth in man].

[Hormones and hair growth in man]. A literature review tries to diminish the ambiguity between hormones and hairs. Therefore the hormonal action in general (regulation of the protein synthesis indirectly by enzymatical regulation of the AMP-system or directly by hormones as active metabolites) and the methods to explore hormones-hair-interaction are discussed. Hormones pertaining to the pituitary-adrenal-gonadal axis are regarded as the paramount hormones; therefore the results of research in testosterone, 5-alpha-dihydrotestosterone, estrogens, progesterone, glucocorticoids, the hypophysis and its tropins are recapitulated. The main disorders of hair-growth, pattern baldness and "idiopathic" hirsutism, which would be dependent on a similar disturbance of androgen metabolism, are discussed. Pathology in hair-growth may arise in any point of the cascade of hormone action.


Isolation of two new polymyxin group antibiotics. (Studies on antibiotics from the genus Bacillus. XX). Two new members of polymyxin group antibiotics, polymyxins S1 and T1, were isolated from the culture broths of strains identified as Bacillus polymyxa Rs-6 and Bacillus polymyxa E-12, respectively. These antibiotics are strongly basic substances, their hydrochloric acid salts are soluble in water and methanol. They are primarily active against Gram-negative bacteria in vitro and in vivo though polymyxin T1 exhibits higher activities against Gram-positive bacteria than other polymyxin group antibiotics.


The structure of polymyxin S. (Studies on antibiotics from the genus Bacillus. XXI). Amino acid analysis on the acid hydrolyzate of polymyxin S1 revealed its amino acid composition. Isolation of the constituent amino acids and measurement of their optical activities clarified their chiralities: Dab(5L), Thr(3L), Ser(1D) and Phe(1D). The constituent fatty acid was identified with anteisononanoic acid by gas chromatography and mass spectrometry. By the action of polymyxin acylase, deacyl polymyxin S was obtained. Successive EDMAN degradation reaction on deacyl polymyxin S revealed the amino acid sequence. Further evidence for the structure of polymyxin S1 was obtained by partial acid hydrolysis on tetra-(DNP)-polymyxin S1.


The structure of polymyxin T1. (Studies on antibiotics from the genus Bacillus. XXII). Amino acid analysis of the acid hydrolyzate of polymyxin T1 revealed the amino acid composition. Isolation of the constituent amino acids and measurement of their optical activities clarified their chiralities. These were 2,4-diaminobutyric acid (6L), Thr(1L), Leu(2L) and Phe(1D). The constituent fatty acid was identified as anteisononanoic acid by gas chromatography and mass spectrometry. Deacylation with polymyxin acylase afforded deacyl polymyxin T. Successive EDMAN degradation on deacyl polymyxin T revealed most of its amino acid sequence. The chemical cleavage reaction for fragmentation of threonyl peptide amino acid sequence. The chemical cleavage reaction for fragmentation of threonyl peptide on penta(DNP)-polymyxin T1 cleaved it at the C-terminal side of the Thr residue to afford a DNP-octapeptide, whose sequence was clarified by EDMAN degradation. Thus, the structure of polymyxin T1 was determined.


Disruption of the Escherichia coli outer membrane permeability barrier by immobilized polymyxin B. One of the apparent roles of the outer membrane system in gram-negative bacteria is to function as a selective permeability barrier. A number of antibiotics active against gram-positive bacteria are relatively ineffective against gram-negative bacteria presumably because of the implied barrier function of the outer membrane. This interpretation has been strengthened by studies demonstrating synergism between outer membrane perturbing agents such as EDTA or polymyxin B and specific antibiotics. In the case of polymyxin B, it is not totally clear that synergism with other antimicrobials is due to disruption of the outer membrane permeability barrier or to interactions with the inner membrane. In order to resolve this question, polymyxin B was covalently attached to agarose in order to limit interactions with the outer surface of E. coli. These studies demonstrate that immobilized polymyxin B acts synergistically with bacitracin, rifampicin, or lysozyme. It is proposed that synergistic effects exhibited by polymyxin B are due to its interaction with the outer membrane system.


Electron spin resonance studies on the lipoxygenase reaction by spin trapping and spin labelling methods. The rate of oxygenation and that of trapping linoleic acid free radicals in the lipoxygenase [EC 1.13.11.12] reaction were measured in the presence of linoleic acid, oxygen, and nitrosobenzene at various concentrations, with a Clark oxygen electrode and ESR spectroscopy. The results were interpreted under the assumption that the free radical of linoleic acid, an intermediate of the lipoxygenase reaction, reacts competitively with oxygen or nitrosobenzene. The oxidation of the iron in the active site of lipoxygenase caused by the spin label reagent, 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl, was also observed by ESR- and fluorescence-spectroscopy.


Energy transduction and adenine nucleotides in mitochondria from rat liver after hypoxic perfusion. The characteristics of mitochondria isolated from perfused livers of rats under hypoxic or oxic conditions were studied. The electron transfer activity was about 60% of normal after hypoxic perfusion for 3 h, but respiratory control was abolished almost completely. These parameters recovered considerably on subsequent oxic perfusion. The adenine nucleotide contents and their net uptake decreased in hypoxia, closely correlated with the energy transduction. Energy-dependent nicotinamide nucleotide transhydrogenase activity and NAD reduction by succinate in submitochondrial particles were most severely inhibited after hypoxic perfusion and were also correlated with adenine nucleotide contents in the particles. These results are discussed in terms of the involvement of adenine nucleotides in energy-transducing systems in mitochondrial membranes.


Studies on nitrate reductase of Clostridium perfringens. Purification, some properties, and effect of tungstate on its formation. Nitrate reductase (NaR) linked to reduced methyl viologen from Clostridium perfringens was purified by ammonium sulfate precipitation. DEAE-cellulose chromatography, disc electrophoresis on polyacrylamide gel, and triple DEAE-Sephadex chromatography. The specific activity was increased 1,200-fold with a yield of 9%. The purified preparation was nearly homogeneous in disc electrophoresis. It was brown, and its spectrum showed a slight shoulder near 420 nm as well as a peak at 280 nm. The molecular weight was found to be 90,000 based on s020,w (5.8S) and 80,000 by Sephadex G-100 gel filtration. In SDS-polyacrylamide electrophoresis, it showed only a single band with a molecular weight of 90,000; it had no subunit structure. The isoelectric point was pH 5.5, and the optimum pH was 9. Mn2+, Fe2+, Mg2+, and Ca2+ stimulated the activity. Km for nitrate was 0.10 mM, and nitrate was stoichiometrically reduced to nitrite in the presence of 2 mM Mn2+. Ferredoxin fraction obtained from extracts of the bacterium was utilizable as an electron donor at pH 8. Cyanide and azide inhibited the enzyme. The formation of NaR was induced by nitrate and inhibited by 0.5 mM tungstate, but recovered in the presence of 0.1 mM molybdate; NaR of C. perfringens appears to be a molybdo-iron-sulfur protein.


GLC analysis of the hydroxy and oxo compounds produced by the Smith degradation of reducing Di- and trisaccharides. Reducing di- and trisaccharides were oxidized with periodate under conditions minimizing overoxidation, and the hydroxy and oxo compounds produced by subsequent reduction and hydrolysis were analyzed by GLC after trimethylsilylation. The present study has demonstrated that GLC analysis of the hydroxy and oxo compounds produced by Smith degradation is useful for linkage analysis of reducing di- and trisaccharides. In addition, it is suggested that the rate of periodate oxidation of the internal hexopyranose residue of trisaccharides depends markedly upon their structures.


Mechanism of the reaction catalyzed by carbamyl phosphate synthetase. Binding of ATP to the two functionally different ATP sites. Application of the pulse-chase procedure to study of the binding and utilization of ATP by glutamine-dependent carbamyl phosphate synthetase from Escherichia coli showed that the enzyme binds the two molecules of ATP used in this reaction at the same time, and that the two ATP-binding sites are functionally different. Thus, ATP bound to the first ATP site is used for carboxy phosphate formation, and ATP bound to the second ATP site is used for phosphorylation of carbamate. The present and previous findings support a mechanism that involves intermediate formation of two highly unstable intermediates: carboxy phosphate and carbamate. It is proposed that the presence of all of the reactants on the enzyme at the start of the catalytic cycle allows immediate utilization of these labile compounds in the carbamyl phosphate synthesis reaction.


The selection of a stable rat hepatoma variant with concomitant increase in ploidy and permeability to glycerol. By repeated selection for longer survival in an isotonic solution of glycerol, a stable subline of Novikoff rat hepatoma cells has been isolated. The cells exhibit markedly increased resistances to osmotic lysis  in isotonic solutions of glycerol. They are twice as large and have twice as many chromosomes as cells of the parental line. It is suggested that the osmotic stress procedure can be extended for the selection of numerous kinds of mutants and can be used as a method of analysis of membrane properties.


The role of adenosine 3',5'-cyclic monophosphate in the density-dependent regulation of growth and tyrosinase activity of B-16 melanoma cells. Incubation of cultured B-16 melanoma cells with 1-methyl-3-isobutyl xanthine (MIX) produced a sustained rise in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) which preceded an increase in the specific activity of tyrosinase (EC 1.10.3.1). Cultures of two clones of melanoma cells, one having a mean population doubling time twice that of the other, showed density-dependent inhibition of growth. The tyrosinase activity of each line increased progressively during logarithmic growth, reaching maximal values shortly after the cultures achieved confluence. Intracellular cAMP levels fell during logarithmic growth, being minimal in confluent cultures. The stimulatory effects of MIX and confluence on tyrosinase activity were additive. Cells plated at high density had a lower tyrosinase activity than cells allowed to achieve a similar density by successive division from sparsely planted cultures although the intracellular cAMP levels of such cultures were not different. We support the observations of other investigators that agents which increase intracellular cAMP concentrations can both inhibit cell division and stimulate tyrosinase activity. There are, however, mechanisms for increasing tyrosinase activity and inhibiting cell division which are expressed as B-16 melanoma cells approach confluence and which are not mediated by an increase in intracellular cAMP concentrations.


Increased membrane transport of 2-deoxyglucose and 3-O-methylglucose is an early event in the transformation of chick embryo fibroblasts by Rous sarcoma virus. Transformation of chicken embryo fibroblasts with Rous sarcoma virus results in cells with an enhanced rate of hexose uptake. We have examined transport of the glucose analogs 2-deoxyglucose and 3-O-methylglucose in cells infected with a temperature sensitive variant of the virus. In cells shifted from restrictive to permissive conditions for transformation, increased transport of the non-phosphorylatable analog 3-O-methylglucose occurs at the same time as that of 2-deoxyglucose, a phosphorylatable analog. This enhanced rate of transport can be observed within three hours of the temperature shift. There is a corresponding decrease in the transport rate of both analogs following shift to the restrictive temperature. These results suggest that increased transport is likely to be the primary event in causing transformation-specific changes in sugar metabolism. We have also examined uptake into the internal pools of both the phosphorylated and non-phosphorylated forms of 2-deoxyglucose in normal cells and in cells transformed by the wild-type virus. These data indicate a corresponding increase in the rate of accumulation of the free sugar in transformed cells and point to transport as the rate limiting step in the accumulation of 2-deoxyglucose in both normal and transformed chicken embryo cells.


A fluorescence enhancement assay of cell fusion. Two probes were synthesized which consist of fluorescent molecules conjugated to saturated hydrocarbon chains, 18 carbons long, to ensure their localization into cellular membranes. There is an overlap between the emission spectrum of one probe (donor) and the absorption spectrum of the other probe (acceptor). By the use of appropriate wavelengths it is possible to specifically excite the donor probe and record the fluorescence of the acceptor probe. Two cell populations, each labelled with one of the probes, were infected with a virus that causes cell fusion, mixed in equal proportions, and the fluorescence of the acceptor probe measured as a function of time after infection. An increase in fluorescence was observed beginning at the time of onset of cell fusion indicating a mixing of the fluorescent membrane molecules. An investigation of the distance dependence indicated that the increase in fluorescence was mainly due to resonance energy transfer and not to photon emission and reabsorption. Resonance energy transfer requires that the 2 probes be close together and that there be an overlap of the emission spectrum of the donor probe and the absorption spectrum of the acceptor probe. The possible application of this assay to other types of membrane fusion is noted.


Endotoxin-induced platelet aggregation and secretion. I. Morphological changes and pharmacological effects. Endotoxin lipopolysaccharide (LPS) from Acinetobacter 199A induced aggregation of blood platelets from immune adherence-positive species (rat, rabbit) but not from immune adherence-negative species such as pig and man. Aggregation occurred in 2 phases: the first was not accompanied by secretion of platelet constituents, was apparently a consequence of C3 activation, and was selectively inhibited by EGTA. The second phase of aggregation was associated with secretion of platelet granule contents, and with a lesser amount of cytoplasmic leakage. Secondary aggregation was abolished by the sulphydryl alkylating agent N-ethylmaleimide, and by agents which increased the level of cyclic AMP in platelets, such as prostaglandin E1 (a stimulator of adenylate cyclase) and methyl xanthines (inhibitors of phosphodiesterase). Secondary aggregation was partly inhibited by agents which block platelet prostaglandin biosynthesis (e.g. aspirin, indomethacin). Primary aggregation was unaffected by these inhibitors at concentrations which blocked secondary aggregation.


Isoelectric focusing of viral polypeptides in urea. A methodological study on poliovirus. The applicability of isoelectric focusing in urea to the analysis of viral polypeptides is reported, using poliovirus as a model. Experimental techniques for the dissociation of virus particles, for isoelectric focusing in urea-containing polyacrylamide gels (rods, flat bed and slab gels) and for pH measurement and two-dimensional analysis are described and their results, as applied to poliovirus polypeptides, discussed. Special attention is given to problems of recovery of all of the proteins and the incidence of artifacts. The influence of reagents, dissociation conditions, focusing procedures and preparation and storage of virus material on the occurrence of charge modifications of the polypeptides has been investigated. Some recommendations are made for the application of the method to other viruses of particles.


Interaction of polymorphonuclear neutrophils with Escherichia coli. Effect of enterotoxin on phagocytosis, killing, chemotaxis, and cyclic AMP. Enterotoxigenic Escherichia coli are associated with noninflammatory diarrhea and stimulate adenylate cyclase activity of mammalian cells, thereby increasing intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP). Increased concentrations of cyclic AMP in polymorphonuclear neutrophils (PMN) inhibit phagocytosis, candidacidal activity, granule discharge, and chemotactic responsiveness. We examined the effect of enterotoxin on the interaction of human PMN with E. coli. Enterotoxigenic and nonenterotoxigenic strains, including serotypes of E. coli identical except for the presence or absence of the plasmid coding for enterotoxin production, were utilized. Enterotoxigenic and nonenterotoxigenic E. coli, tumbled with PMN, were phagocytized and killed (>97%) equally well, and these strains stimulated PMN hexose monophosphate shunt activity equivalently.However, a chemotaxis assay under agarose demonstrated that filtrates of 10 enterotoxigenic strains were less chemotactic for PMN by 15+/-2% total migration or 46+/-1% directed migration, when compared with 6 non-enterotoxigenic strains (P < 0.001). Inactivation of the enterotoxin by heat (65 degrees C for 30 min) or antibodies formed to E. coli enterotoxin eliminated the inhibitory effect of the enterotoxic filtrates for PMN chemotaxis. Addition of purified E. coli enterotoxin directly to the PMN decreased chemotaxis to E. coli filtrates by 32+/-2% (P < 0.001). These data suggest that the effect was due to the heat-labile enterotoxin. The phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (0.1 mM), which potentiates effects due to an increase in intracellular cyclic AMP, further decreased total PMN migration (random plus directed) toward enterotoxic filtrates to 46% of that to nonenterotoxic filtrates (P < 0.001). Addition of cholera toxin (1 mug/ml), which is similar to E. coli enterotoxin, to the PMN inhibited total migration toward nonenterotoxic filtrates by 16+/-2% (P < 0.001). Exogenous dibutyryl cyclic AMP (2 mM) inhibited total PMN migration toward E. coli filtrates by 32% (P < 0.001). PMN intracellular cyclic AMP levels increased by 220% after 2 h of incubation with purified E. coli enterotoxin. The decreased chemotactic attractiveness of enterotoxic E. coli filtrates appears to be related to the ability of enterotoxin to increase cyclic AMP in PMN. Enterotoxin production by E. coli may be advantageous to the microbe by decreasing its chemotactic appeal for PMN.


Protein degradation in normal and beige (Chediak-Higashi) mice,. The beige mouse, C57BL/6 (bg/bg), is an animal model for the Chediak-Higashi syndrome in man, a disease characterized morphologically by giant lysosomes in most cell types. Half-lives for the turnover of [(14)C]bicarbonate-labeled total soluble liver protein were determined in normal and beige mice. No significant differences were observed between the normal and mutant strain for both rapidly and slowly turning-over classes of proteins. Glucagon treatment during the time-course of protein degradation had similar effects on both normal and mutant strains and led to the conclusion that the rate of turnover of endogenous intracellular protein in the beige mouse liver does not differ from normal. The rates of uptake and degradation of an exogenous protein were determined in normal and beige mice by intravenously injecting (125)I-bovine serum albumin and following, in peripheral blood, the loss with time of phosphotungstic acid-insoluble bovine serum albumin and the parallel appearance of phosphotungstic acid-soluble (degraded) material. No significant differences were observed between beige and normal mice in the uptake by liver lysosomes of (125)I-bovine serum albumin (t((1/2)) = 3.9 and 2.8 h, respectively). However, it was found that lysosomes from livers of beige mice released phosphotungstic acid-soluble radioactivity at a rate significantly slower than normal (t((1/2)) = 6.8 and 3.1 h, respectively). This defect in beige mice could be corrected by chronic administration of carbamyl choline (t((1/2)) = 3.5 h), a cholinergic agonist which raises intracellular cyclic GMP levels. However, no significant differences between normal and beige mice were observed either in the ability of soluble extracts of liver and kidney to bind [(3)H]cyclic GMP in vitro or in the basal levels of cyclic AMP in both tissues. The relevance of these observations to the presumed biochemical defect underlying the Chediak-Higashi syndrome is discussed.


Control of 3-hydroxy-3-methylglutaryl-CoA reductase activity in cultured human fibroblasts by very low density lipoproteins of subjects with hypertriglyceridemia. Very low density lipoproteins (VLDL) and low density lipoproteins (LDL) from human normolipemic plasma, and the VLDL, the intermediate density lipoprotein (IDL), and LDL from patients with Type III hyperlipoproteinemic plasma were tested for their abilities to suppress the activity of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase in cultured human fibroblasts from normal subjects and a Type III patient. Regulation of cholesterol synthesis in the fibroblasts of a patient with Type III hyperlipoproteinemia appears to be normal. VLDL from normal subjects, isolated by angle head ultracentrifugation (d < 1.006) or by gel filtration on BioGel A-5m, were about 5 times less effective than LDL in suppressing HMG-CoA reductase activity, based on protein content, in agreement with previous reports with normal fibroblasts. Zonal centrifugation of normal VLDL isolated by both methods showed that the VLDL contained IDL. Normal VLDL from the angle head rotor, refractionated by the zonal method, had little, if any, ability to suppress the HMG-CoA reductase activity in either normal or Type III fibroblasts. VLDL, IDL, and LDL fractionated by zonal ultracentrifugation from Type III plasma gave half-maximum inhibition at 0.2-0.5 mug of protein/ml, indistinguishable from the suppression caused by normal LDL. Type III VLDL did not suppress HMG-CoA reductase in mutant LDL receptor-negative fibroblasts. Zonally isolated VLDL obtained from one Type IV and one Type V patient gave half-maximal suppression at 5 and 0.5 mug of protein/ml, respectively. Molecular diameters and apoprotein compositions of the zonally isolated normal and Type III VLDL were similar; the major difference in composition was that Type III VLDL contained more cholesteryl esters and less triglyceride than did normal VLDL. The compositions and diameters of the Type IV and Type V VLDL were similar to normal VLDL. These findings show that the basic defect in Type III hyperlipoproteinemia is qualitatively different from the cellular defect found in familial hypercholesterolemia, since the regulation of HMG-CoA reductase activity is normal in Type III fibroblasts. The metabolic defect in hypertriglyceridemia is related to the triglyceriderich lipoproteins which, free of other lipoproteins, have an enhanced ability to interact with cultured fibroblasts to regulate HMG-CoA reductase activity. These studies suggest that, in hypertriglyceridemia, there is a mechanism for direct cellular catabolism of VLDL which is not functional for normal VLDL.


Developmental aspects of the pituitary-adrenal axis response to hemorrhagic stress in lamb fetuses in utero. Plasma ACTH and corticosteroid concentrations were measured by radioimmunoassay in chronically catheterized fetuses of 32 pregnant sheep. Fetal plasma ACTH levels 38+/-5 pg/ml (means+/-SEM) were slightly (P < 0.05) lower than maternal 54+/-4 pg/ml levels. No general rise in fetal plasma ACTH concentration was noted before 140 days gestation; however, fetal plasma corticoid levels began to increase after about 125 days. This suggested that an increase in fetal adrenal responsiveness to endogenous ACTH occurred during gestation. Hemorrhage of 15% of estimated blood volume decreased mean arterial pressure from 54+/-3 to 36+/-3 torr and increased plasma ACTH from 30+/-5 to 130+/-30 pg/ml in fetuses older than 0.80 gestation. In fetuses younger than 0.67 gestation, 15% hemorrhage caused no change in plasma ACTH levels despite a significant fall in mean arterial pressure. This suggests that system(s) subserving the ACTH response to mild hemorrhage are either absent or nonfunctional in the younger fetuses. The hemorrhage-induced increase in plasma ACTH levels was associated with a small rise in plasma corticoids in fetuses younger than 0.94 gestation. In older fetuses, a similar increase in plasma ACTH was associated with a pronounced increase in plasma corticoid levels. This also suggests that an increase in adrenal responsiveness to endogenous ACTH occurs during gestation. No detectable changes in maternal plasma ACTH or corticoids were found in response to fetal hemorrhage, thus the fetal pituitary-adrenal axis can autonomously respond to stress.


Differences in oxygen metabolism of phagocytosing monocytes and neutrophils. The oxidative metabolism of monocytes and polymorphonuclear leukocytes from human peripheral blood was studied in resting and phagocytosing cells. Monocytes, like neutrophils, showed an increase in oxygen consumption during phagocytosis with a concurrent release of superoxide anions and hydrogen peroxide. Both oxygen products are highly reactive agents with potential bactericidal activity. Neutrophils consumed two and a half times as much oxygen, generated about twice as much superoxide, and released five times as much hydrogen peroxide as monocytes did. Monocytes generated superoxide and hydrogen peroxide at equivalent rates.Antimycin A, a specific mitochondrial respiratory chain inhibitor, depressed the oxygen consumption of monocytes by congruent with70% but had no effect on neutrophil respiration. Therefore, the oxygen consumed by phagocytosing monocytes appeared to be metabolized in two distinct processes: congruent with30% of the oxygen is converted to hydrogen peroxide, whereas the remaining 70% is metabolized via the mitochondrial respiratory chain. The release of superoxide and hydrogen peroxide was unaffected by antimycin in either cell type. Phagocytosis of zymosan particles by monocytes was nearly abolished by antimycin, whereas no effect was noted with neutrophils. Thus, phagocytosis appears to be highly dependent on oxidative phosphorylation in monocytes but not in polymorphonuclear leukocytes. Moreover, in monocytes treated with antimycin, an addition of opsonized zymosan particles induced stimulation of the oxidative metabolism without occurrence of ingestion.


Rat intestine secretes discoid high density lipoprotein. High density lipoprotein was isolated from pooled rat serum and mesenteric lymph of lymph fistula rats. In most experiments, 5,5'-dithionitrobenzoic acid, an inhibitor of the enzyme lecithin:cholesterol acyltransferase, was added during the collection of lymph to prevent modification of the lipid composition of newly secreted lipoproteins. Negative staining electron microscopy of lymph high density lipoprotein revealed discoidal particles (190+/-3 x 55+/-1 A) which tended to form rouleaux, smaller spherical particles were also present. Serum high density lipoprotein contained only spherical particles (diameter 93+/-4 A). Lipid analysis showed that lymph high density lipoprotein was enriched in phospholipid and deficient in cholesterol esters when compared to serum high density lipoprotein. The phospholipid to cholesterol esters ratio was greatest in basal lymph high density lipoprotein when compared to fatty lymph and serum high density lipoprotein. From analysis of the lipid compositional data and direct particle measurement by electron microscopy, it could be determined that congruent with50% of basal lymph high density lipoprotein and 30% of fatty lymph high density lipoprotein was discoid. Basal lymph high density lipoprotein was enriched in apoA-I and deficient in the arginine-rich peptide, and the apoprotein composition of fatty lymph high density lipoprotein more closely resembled serum. These observations demonstrate that intestinal lymph contains two types of high density lipoprotein particles, a discoid nascent particle deficient in cholesterol ester and rich in apoA-I, and spherical high density lipoprotein derived from plasma. A significant amount of lymph high density lipoprotein appears to be secreted by the intestine.


Effects of d-amphetamine and strychnine on cycloheximide- and diethyldithiocarbamate-induced amnesia in mice. Groups of C57BL/6J mice were administred cycloheximide (CYC) 30 min before or immediately after training on a passive avoidance task and tested 72 hr later. Some CYC-pretreated groups were given strychnine or d-amphetamine (d-amp) immediately after training and others were given d-amp 1 hr after training. Other groups were given diethyldithiocarbamate (DDC) at various times before or after training. Some DDC-pretreated groups were gived-amp or strychnine as described above for CYC groups. Immediate posttraining administration of 5 mg/kg d-amp, but not strychnine, prevented amnesia in CYC-pretreated mice. The DDC induced an apparent amnesia when administered from 30 min before training to 3 hr after training.  Posttraining administration of d-amp or strychnine did not prevent DDC-induced amnesia. These results are discussed in relation to previous suggestions that CYC- and DDC-induced amnesia may be the result of a functional impairment of catecholamine neurotransmitter systems by these drugs.


An attempt to separate mononuclear cells fused with human red blood cell-ghosts from a cell mixture treated with HVJ (Sendai virus) using a fluorescence activated cell sorter (FACS II). Nucleated cells (Ehrlich ascites tumor cells or L strain cells) and human red blood cells (RBC)-ghosts were mixed and fused by ultraviolet-inactivated HVJ (Sendai virus). The cell mixture was stained with FITC conjugated anti-RBC ghost antiserum and then applied to FACS II apparatus. The apparatus sorted mononuclear cells fused with RBC-ghosts from the cell mixture on the basis of both the light scattering and fluorescence profiles. When the same procedure was carried out on a mixture containing cells and intact human RBC, the cells sorted by this method were cells into which hemoglobin had been injected. The sorted cells were capable of forming colonies in culture. This sorting method may be useful for collecting cells in which macromolecules have been injected artificially by fusion of RBC-ghosts enclosing macromolecules.


Dose-response relationships in a microneutralization test for foot-and-mouth disease viruses. Two-dimensional quantal microneutralization tests on foot-and-mouth disease viruses, in which neutralizing antibody activity was titrated against a serial range of virus doses, demonstrated a variety of dose-response curves some of which were rectilinear, others clearly curvilinear. Moreover, in the case of the non-linear responses obtained with some antisera, the shape of the curve was such that antibody titres recorded with doses of virus ranging from 10(3)-10(5) TCD50 were closely similar. Studies were carried out on the effect of varying the conditions of the test on the shape of the dose-response curve: significant differences were obtained after treatment of the antiserum-virus mixtures with anti-species globulin, and when the test was assayed in cells of differing susceptibility to infection.


The genetic mapping of a defective LPS response gene in C3H/HeJ mice. The expression of a defective LPS response gene Lps and the major urinary protein (Mup-1) are concordantly inherited in backcross (C3H/HeJ x C57BL/6J)F1 x C3H/HeJ mice, indicating genetic linkage of these loci. Mup-1 is known to be linked to the brown coat color locus on chromosome 4 in mice; thus Lps can now be assigned to chromosome 4. A value of 0.06 +/- 0.02 has been estimated for the recombination frequency between Mup-1 and Lps. We have used the polysyndactyly (Ps) mutation further to localize Lps on chromosome 4. Lps is located between the Mup-1 and Ps loci.


Correlation between the ability of tumor cells to resist humoral immune attack and their ability to synthesize lipid. Agents that increase (certain metabolic inhibitors, chemotherapeutic agents, and x-irradiation), decrease (hormones), or have no effect (hyperthermia) on the susceptibility of line-1 and line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of these tumor cells to synthesize macromolecules. A correlation was found between the drug-induced increase in sensitivity of these cells to antibody-C mediated killing and the loss of their ability to incorporate fatty acids into complex cellular lipids. Similarly, the hormone-induced increase in resistance of the cells to killing was accompanied by an enhancement in complex lipid synthesis by these cells was also observed after the cells were exposed to physical means of insult (x-irradiation or hyperthermia). No correlation was found between the sensitivity of the cells to antibody-C mediated killing and their ability to synthesize DNA, RNA, protein, or complex carbohydrate, or their capacity for de novo lipid synthesis as measured by incorporation of acetate and glycerol into cellular macromolecules. The assembly of free fatty acids into complex lipid moieties is therefore proposed to be of fundamental importance for the ability of the tumor cells to resist humoral immune killing.


Identification of lipids associated with the ability of tumor cells to resist humoral immune attack. Certain metabolic inhibitors or chemotherapeutic agents that increase the susceptibility of line-1 or line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of the cells to synthesize lipids. A direct correlation was found between the drug-induced increase in sensitivity to antibody-C mediated killing and the inhibition of the ability of the cells to incorporate acetate, glycerol, and fatty acids into complex cellular lipids. Drug-treated cells recultured in drug-free medium regained their resistance to antibody-C mediated killing; these cells recovered their ability for complex lipid synthesis at this time. Thin layer chromatography of CHCl3:CH3OH lipid extracts from these cells indicated that the drug-induced increase in susceptibility to humoral immune attack correlated with the inhibition of acetate, glycerol, and fatty acid incorporation into cardiolipin and triglyceride in line-10 cells and the inhibition of incorporation of these compounds into cardiolipin alone in line-1 cells. No direct correlation was found between the sensitivity of the cells to humoral immune attack and the ability of the cells to incorporate precursors of lipid synthesis into other lipid moieties (sphyngomyelin, phosphatidyl serine, phosphatidyl choline, phosphatidyl glycerol, or cholesterol esters). The synthesis of cardiolipin and triglycerides, therefore, appears to be associated with the mechanism whereby these tumor cells resist antibody-C mediated killing.


In vitro mitogenic stimulation of murine spleen cells by herpes simplex virus. Spleen cells of B6 mice not previously immunized were induced to DNA synthesis by supernatants from HSV-infected tissue culture. The stimulatory principle could be passed through a 45-micrometer filter and sedimented at 100,000 x G. It was abolished by UV light, heating at 56 degrees C, and by an anti-HSV serum. The possibility that the observed stimulation was caused by LPS was therefore excluded, and there was a-so no indication of mycoplasma contamination. Partial purification of spleen cells from macrophages resulted in an increased stimulation by HSV. From experiments with nylon columns, anti-theta antibody, and nude mice it was concluded that HSV acted as a B cell mitogen. Strains of both HSV types 1 and 2 were stimulatory for B6 spleen cells. Of nine freshly isolated HSV strains with identical passage history (twice in HEF) four were strongly stimulatory, three showed a moderate stimulation, and two did not stimulate. Spleen cells from A/J and DBA/2 mice were stimulated to the same extent by HSV (WAL) as spleen cells from B6 mice. No viral replication was demonstrable in B6 spleen cell cultures stimulated for DNA synthesis by HSV. Thus our study demonstrates induction of cellular DNA synthesis in B lymphocytes by HSV which is abolished by inactivation of the virus.


Characterization of a fetal calf serum-derived molecule reactive with human natural antibodies: its occurrence in tissue culture-grown type C RNA viruses. By means of a sensitive radioimmunoprecipitation (RIP) assay, simian sarcoma virus-simian sarcoma-associated virus (SSV-SSAV), purified from culture fluids of infected normal rat kidney (NRK) cells, was shown to acquire a surface antigen from serum used in the tissue culture medium. This antigen, which was acquired when serum from either fetal calf, horse, swine, rabbit, or chicken origin was used, accounted for a substantial portion (but not all) of the total precipitating activity exhibited by natural human antibodies for membrane-associated antigens of these viruses. By 1) alcohol precipitation, concanavalin A chromatography, and Sephadex G-150 filtration of fetal calf serum (FCS) proteins or 2) chromatography of serum proteins over a human IgG-containing immunoaffinity column, a glycoprotein of approximately 55,000 daltons has been identified which is a minor constituent of FCS (less than 0.1% of total protein) and has the antigenic capacity of whole FCS.


Labeling of high density lipoproteins with [3H] acetic anhydride. Rat serum HDL was labeled by reaction with [3H] acetic anhydride at pH 7.2 for 30 min at room temperature by a modification of the method of Montelaro and Rueckert (1975. J. Biol. Chem. 250: 1413). Protein specific activities of 60 dpm/ng were achieved. Seven percent of the label was in lipid, of which 92 percent was recovered in phospholipid. The labeled HDL migrated as a single band as seen by electrophoretic or column chromatographic analysis. When the labeled HDL was injected into rats without re-isolation, the biological half-life was not significantly different from HDL labeled in vitro with 125I or in vivo with amino acids. All of the apoproteins were labeled; their specific activities were closer to one another than those obtained with 125I. For some applications, acetylation may provide a useful alternative to the 125I labeling procedure.


Metabolsim of apoB and apoC lipoproteins in man: kinetic studies in normal and hyperlipoproteininemic subjects. The kinetics of apolipoproteins B and C were studied in 14 normal and hyperlipoproteinemic subjects after injection of exogenously (125)I-labeled very low density lipoprotein (VLDL) particles. Plasma radioactivities of apoB and apoC were determined over a period of 4 days in VLDL (d < 1.006) and total radioactivity in intermediate (IDL) (1.006 < d < 1.019), low (LDL) (1.019 < d < 1.063), and high (HDL) (1.063 < d < 1.21) density lipoproteins. The data were analyzed by the use of a model, developed mostly from these data, with the following results. The VLDL particle undergoes a series of incremental density changes, most likely due to a number of delipidation steps, during which apoB stays with the particle until the density reaches the IDL range. There is, however, a loss of apoC associated with these delipidation steps. In our normal subjects, all IDL apoB eventually becomes LDL. In our hyperlipemic subjects some of the apoB on IDL is also degraded directly. The apoC lost by VLDL and IDL recycles to HDL, and most of it is picked up again by newly synthesized VLDL. There is a slowdown of the stepwise delipidation process in all hyperlipemic individuals studied. Three additional features became apparent in the type III subjects. First, there is a significant increase (a factor of 2 compared to normal) in the apoB synthesis rate by way of VLDL; second, there is an induced direct apoB synthesis pathway by way of IDL (and/or LDL); third, a bypass of the regular stepwise VLDL delipidation pathway is induced by which VLDL particles lose apoC but none of their apoB, thereby forming a new particle with metabolic properties similar to LDL, but with a density still in the VLDL density range. Two type III patients treated with nicotinic acid and clofibrate showed a sharp decrease in their VLDL apoB synthesis rates. This was somewhat compensated by an increased IDL apoB synthesis rate. A type I patient on a medium chain triglyceride diet also showed a number of metabolic changes, including reduced VLDL apoB synthesis and the induction of considerable IDL and/or LDL apoB synthesis.


A comprehensive evaluation of the heparin-manganese precipitation procedure for estimating high density lipoprotein cholesterol. The accurate quantitation of high density lipoproteins has recently assumed greater importance in view of studies suggesting their negative correlation with coronary heart disease. High density lipoproteins may be estimated by measuring cholesterol in the plasma fraction of d > 1.063 g/ml. A more practical approach is the specific precipitation of apolipoprotein B (apoB)-containing lipoproteins by sulfated polysaccharides and divalent cations, heparin-Mn(2+) being the most commonly used combination. The present heparin-Mn(2+) procedure was found to be reasonably specific and not often subject to large errors; however, 9% (primarily hypertriglyceridemic samples) of the 966 plasma samples treated with heparin-Mn(2+) had obvious supernatant turbidity, indicating incomplete sedimentation of apoB-associated lipoproteins. Furthermore, 48% of the nonturbid supernates contained more than 1 mg/dl (mean 2.5 mg/dl) of apoB-associated cholesterol when measured by a radial immunodiffusion procedure, indicating slight overestimation of HDL cholesterol. Determination of the extent of the unprecipitated apoB-associated lipoproteins by sensitive radioimmunoassay and of the amount of precipitated high density lipoprotein by radial immunodiffusion assay of apolipoproteins A-I and A-II at various heparin and Mn(2+) concentrations indicated that the usual heparin level (approximately 1.3 mg/ml) was adequate. However, a twofold increase in Mn(2+) concentration to 0.092 M improved precipitation of the apoB-associated lipoproteins without excessive precipitation of high density lipoprotein from plasma. This increased Mn(2+) level also provided improved sedimentation of the apoB-associated lipoproteins from hypertriglyceridemic plasma. Additional observations suggested that, for convenience, the heparin and Mn(2+) can be added simultaneously as a combined reagent, that samples can be incubated for 10 minutes at room temperature before centrifugation, and that turbid supernates from hypertriglyceridemic samples can usually be made free of apoB-associated lipoproteins by centrifugation at 12,000 g for 10 minutes.


Effect of serum on ganglioside uptake and choleragen responsiveness of transformed mouse fibroblasts. NCTC 2071 cells, transformed mouse fibroblasts, did not respond to choleragen when grown in chemically defined medium. When grown in medium containing 10 percent fetal calf serum, however, the cells accumulated cyclic AMP upon exposure to the toxin. Gangliosides isolated from the fetal calf serum were as effective as whole serum in inducing choleragen responsiveness in the cells. The putative choleragen receptor, the monosialo-ganglioside GM1, could not be detected by chemical analysis in cells exposed to serum. 3H-Labeled GM1 was detected in these cells, however, following sequential exposure to galactose oxidase and sodium borotritide. Thus, uptake of minute amounts of GM1 from serum by these cells sensitized them to choleragen.


Ionic mechanism for the generation of horizontal cell potentials in isolated axolotl retina. The ionic mechanism of horizontal cell potentials was investigated in the isolated retina of the axolotl Ambystoma mexicanum. The membrane potentials of both receptors and horizontal cells were recorded intracellularly while the ionic composition of the medium flowing over the receptor side of the retina was changed. The membrane potential of the horizontal cell is highly depender side of the retina was changed. The membrane potential of the horizontal cell is highly dependent on the extracellular concentration of sodium. When the external ion concentration of either chloride or potassium was changed independently of the other, there were shifts in the membrane potential of the horizontal cell which could not be explained by changes in the equilibrium potential of these ions. If the external concentrations of both potassium and chloride ions were varied so that the product of their external concentrations did not change, the shift in the membrane potential of the horizontal cell was in the direction predicted by the Nernst equation. The results are consistent with the suggestion that in the dark the receptors release a synaptic transmitter which increases primarily the sodium conductance of the horizontal cell postsynaptic membrane.


Appearance of early and late components of Epstein-Barr virus-associated membrane antigen in Daudi cells superinfected with P3HR-1 virus. This study investigated the synthesis of membrane antigen (MA) as well as virus capsid antigen (VCA) and early antigen (EA) in Daudi cells which had been superinfected with the P3HR-1 strain of Epstein-Barr virus (EBV) and then treated with trypsin to remove initially absorbed MA-positive material from the cell surface. Synthesis of MA, VCA and EA was completely inhibited by puromycin. A marked reduction in the frequency of MA positive cells was observed in superinfected cells cultured in the presence of either cytosine arabinoside (Ara-C) or phosphonoacetate (PA); however, a small fraction of MA synthesis occurred, suggesting an inhibitor insensitive component in MA, A differential absorption of EBV antibody-positive human serum with the Ara-C treated or untreated infected cells detected two antigenically different components in MA: early (Ara-C insensitive) and late (Ara-C sensitive) MA.


Expression of early virus functions in human cytomegalovirus infected HEL cells: effect of ultraviolet light-irradiation of the virus. Ultraviolet (u.v.) light-irradiation of human cytomegalovirus (HCMV) resulted in differential inactivation of virus capacities, e.g. induction of cell rounding, early antigens (EA), nuclear inclusion, HCMV DNA synthesis, cellular DNA synthesis, HCMV-specific DNA polymerase, cellular DNA polymerases and plaque production, while the capacity of HCMV to penetrate cell nuclei was not critically impaired. These results indicated that the virus-coded functions expressed after infection were responsible for sll these events except for HCMV-induced stimulation of cellular RNA synthesis which was enhanced by irradiation of the virus at a low dose of u.v. light (6600 ergs/mm2). In these experiments phosphonoacetic acid was effectively utilized to detect EA formation by immunofluorescent staining and to differentiate cellular DNA synthesis from virus DNA synthesis.


Multiple interactions between murine cytomegalovirus and lymphoid cells in vitro. Spleen cultures from various strains of mice were infected in vitro with murine cytomegalovirus (MCMV). Infectious centres were established in a small proportion (not greater than 1%) of the cells. Virus could be rescued from these cells by co-cultivation with syngeneic or allogeneic fibroblasts, but the frequency of rescue could not be altered by incubation with cyclic nucleotide analogues, iododeoxyuridine, cortisol, or allogeneic spleen cells. In addition a smaller fraction of the cell population, possibly a sub-population of the infectious centres, replicated virus spontaneously. The presence of mitogens did not affect these interactions qualitatively or quantitatively. A third response to infection was an inhibition in DNA synthesis, which was suffered by unstimulated cultures and by cells transformed by concanavalin A and bacterial lipopolysaccharides, although overall cell viability was maintained. This response was also mediated by u.v.-inactivated virus.


The isoelectric point of the p30 polypeptide as a marker of mouse endogenous viruses. The isoelectric point (PI) of the p30 polypeptide of members of the three known classes of mouse C-type endogenous viruses was determined both by column and by thin-layer gel isoelectric focusing. Each class was found to be characterized by a particular variant of p30 (isop30), with pI values of 6.1 for class I (ecotropic), 5.7 for class II (xenotropic), and 5.5 for class III (NZB, NIH, ATS124, also xenotropic). The 6.1-isop30 was found as a minor component of rat-grown NZB virus and of a number of laboratory strains of mouse C-type viruses.


Characterization of snap-back RNAs in vesicular stomatitis defective interfering virus particles. VSV defective interfering particles of various sizes and from several independent sources frequently contain plus and minus strand RNA. In many cases some of the complementary strands are covalently linked as snap-back molecules. Infectious particles on the other hand package little or no plus strands. Snap-back molecules from the three different sources examined so far vary in size but appear to conform to the same overall linear duplex structure with cross-links at the ends only. They each contain a base sequence which is a subset of the next larger one and appear to correspond to unique sequences in the L cistron of the genome. Possible origins for these snap-back molecules are discussed.


Inverted complementary terminal sequences in single-stranded RNAs and snap-back RNAs from vesicular stomatitis defective interfering particles. Complementary single-stranded RNAs from three independent VSV defective interfering particle (DI) sources examined can anneal and give rise to monomeric and multimeric circular and linear double-stranded structures observable by electron microscopy under aqueous conditions. When the RNA from the shortest of these DI is spread from 80% formamide solutions, as many as 32% of the molecules are circular, suggesting that the single-stranded RNAs contain inverted complementary terminal sequences. This is strongly supported by the isolation of the putative terminal sequences which rapidly become RNase resistant base-paired structures after melting and quick-cooling the RNA. RNase digestion yields a major and a minor component, 60 to 70 and 135 to 170 nucleotides long respectively. Snap-back DI RNAs also contain inverted complementary sequences at both ends of the plus and minus strands of the duplexes since nicking these at the ends gives rise to double-stranded molecules which can form monomeric and multimeric circular and linear molecules. Thus, snap-back molecules most likely contain a covalent linkage between or near complementary terminal sequences on the two complementary strands as schematically shown in Fig. 5D.


4-aminopyridine--a new drug tested in the treatment of Eaton-Lambert syndrome. A 67 year old man with the myasthenic syndrome associated with small cell bronchogenic carcinoma was treated with a new drug, 4-aminopyridine. The muscle weakness showed marked improvement and electrophysiological examinations demonstrated restoration of neuromuscular transmission.


Accessory nerve palsy.

Accessory nerve palsy. After apparently uncomplicated excision of benign lesions in the posterior cervical triangle, two patients had shoulder pain. In one, neck pain and trapezius weakness were not prominent until one month after surgery. Inability to elevate the arm above the horizontal without externally rotating it, and prominent scapular displacement on arm abduction, but not on forward pushing movements, highlighted the trapezius dysfunction and differentiated it from serratus anterior weakness. Spinal accessory nerve lesions should be considered when minor surgical procedures, lymphadenitis, minor trauma, or tumours involved the posterior triangle of the neck.


Occupational exposures to cyanide in Baltimore fire fighters. A group of 479 fire fighters were investigated for routine exposure to hydrogen cyanide in the fire atmosphere using measurements of serum thiocyanate (SCN-). Our findings indicate that fire fighters were exposed to levels of hydrogen cyanide sufficient to raise their mean serum thiocyanate (SCN-) levels above that of controls. This elevation of serum SCN- occurred independently of smoking habits. Exposure to cyanide occurred much less frequently than previously studied exposures to carbon monoxide. Difficulties of correlating acute symptoms and the effectiveness of mask use with serum thiocyanate are discussed.


Ectopic thyroid gland. A clinical study of 30 children and review. Of 108 children being treated at our Institute for primary (nongoitrous) hypothyroidism, tests with radioactive iodine 131I uptake showed that 26 of them (24%) had an ectopic thyroid gland. Four euthyroid children also had anterior swellings of the neck which, in each case, proved to be an ectopic thyroid gland. Of the 30 children studied, 20 were girls and 10 were boys. Nine patients were diagnosed within the first year of life. Growth retardation, manifest in 20 patients, was the most common clinical finding at the time of diagnosis. Delayed bone age was a feature in all of them. Growth, after diagnosis was within normal limits in 83% of the infants who were treated within the first two years of life; only 50% of the children diagnosed later grew within normal limits. Similarly, mental function was best preserved in those patients in whom treatment was initiated within the first two years of life.


Rickets in children of rural origin in South Africa: is low dietary calcium a factor? Studies of nine children 4 7/12 to 13 years of age who had rickets are presented. No evidence of renal abnormalities, vitamin D deficiency, or of the inherited varieties of rickets was found. The salient features were their rural origins, mild hypocalcemia with evidence of secondary hyperparathyroidism, and improvement with a normal diet that contained an average of 944 mg calcium/24 hours. It is proposed that the etiology of the rickets is related to low calcium intake with or without a high oxalate concentration in the diet.


Kinetics and mechanism of oxidation of promazine and promethazine by ferric perchlorate. The equilibrium constants, kinetics, and mechanism of promazine and promethazine oxidation by ferric perchlorate were investigated at different temperatures and acidities using a stopped-flow spectrophotometric technique. The overall reaction can be represented as follows: (formula: see text) where P+ represents the radical cation corresponding to the phenothiazine derivative. The equilibrium quotients were evaluated at 1.00 M HClO4, 25.0 degrees, and ionic strength 1.0 M. The kinetics of reaction follow the equation: -d[P] divided by dt = k1[Fe3+][P]-k-1[Fe2+][P+] The rate constants k1 and k-1 are independent of acidity and are related to the corresponding equilibrium quotients.


In vitro uptake of oral contraceptive steroids by magnesium trisilicate. Some steroids used in oral contraceptives were adsorbed significantly by magnesium trisilicate. The adsorption affinity followed the sequence: ethindrone greater than mestranol greater than norethindrone greater than ethinyl estradiol. Adsorption data obtained at relatively low initial concentrations fitted a Langmuir plot; the values for monolayer adsorption ranged between 0.24 and 0.32 mg/g. At higher concentrations of the steroids, multilayer adsorption occurred. The results of desorption experiments made at 37 degrees in water and 0.05 N HCl suggested that desorption was incomplete and depended on the amount of steroid adsorbed. During the dissolution testing of a brand of contraceptive tablets containing norethindrone acetate, the presence of 0.5% (w/v) magnesium trisilicate in the medium resulted in almost complete reduction in the amount of the steroid remaining in solution after 1 hr.


Antihypertensive activity in rats for SQ 14,225, an orally active inhibitor of angiotensin I-converting enzyme. SQ 14,225 (D-3-mercapto-2-methylpropanoyl-L-proline) markedly lowered the blood pressure of the renin-dependent aortic-ligated and two-kidney Goldblatt hypertensive rat and failed to reduce blood pressure in the one-kidney Goldblatt hypertensive rat. In the two-kidney Goldblatt rat, SQ 14,225 (p.o.) was about 10 times as potent as teprotide, the nonapeptide SQ 20,881 (s.c.). Oral doses of SQ 14,225 moderately reduced the blood pressure of the Wistar-Kyoto spontaneously hypertensive rat but not that of the normotensive Wistar-Kyoto rat. Bilateral nephrectomy abolished the antihypertensive activity of SQ 14,225 in the spontaneously hypertensive rat. SQ 14,225 and SQ 20,881 elicited parallel dose-response curves in the two-kidney renal hypertensive rat. Post-treatment of spontaneously hypertensive rats with either agent failed to augment the antihypertensive effect produced by effective doses of the other agent. The results suggest that SQ 14,225 acts primarily by inhibiting the renin-angiotensin system to reduce elevated blood pressure, especially in presumably renin-dependent models of hypertension.


Purinergic inhibition of adrenergic transmission in rabbit blood vessels. The effect of ATP and its congeners on the adrenergic neuroeffector transmission was evaluated in isolated blood vessels of the rabbit. ATP, ADP, AMP and adenosine inhibited the contractile response of the portal vein to adrenergic nerve stimulation, with a threshold concentration of the order of 0.1 muM and ED50 of about 1 microM. These agents, but not papaverine, inhibited the nerve stimulation-induced response in preference to the norepinephrine- or serotonin-induced response in the portal and saphenous veins and pulmonary and ear arteries. In the portal vein labeled with [3H]norepinephrine, ATP diminished the nerve stimulation-induced efflux of tritiated material. This nucleotide also reduced the KCl-induced tritium efflux but not the tyramine induced-efflux in the [3H]norepinephrine-labeled thoracic aorta. ATP had no significant effect on the uptake of [3H]norepinephrine in the portal vein, ear artery and thoracic aorta. Indomethacin and theophylline partially blocked the inhibitory action of ATP on the neurogenic constrictor response in some of the ear artery and saphenous vein preparations. Desipramine, atropine, propanolol, haloperidol and 2,2'-pyridylisatogen, a blocking agent against ATP in the taenia coli, were without such antagonistic effect. The results are consistent with a proposed negative feedback modulator role of ATP or a related purine compound in adrenergic transmission.


Compensatory renal hypertrophy in hypophysectomized rats. 1. After hypophysectomy, both body and kidney weights fall, but at different rates. The rate at which the kidney decreases in weight is faster than that of the whole body.2. Seven days after unilateral nephrectomy, the dry weight of the remaining kidney of hypophysectomized rats, with the exception of rats which had been hypophysectomized for 2 days only, was always heavier than the kidney of control hypophysectomized rats of similar body weight.3. The difference between the dry weight of kidneys of unilaterally nephrectomized hypophysectomized and control hypophysectomized rats increased from 15% in early hypophysectomized (9 days) to about 35% in late hypophysectomized animals (23 days).4. The implantation of renal cortical cells from 2 day hypophysectomized rats into unilaterally nephrectomized control litter-mates inhibited compensatory renal hypertrophy in the latter. When a similar operation was made using kidney cells from animals which had been hypophysectomized for 23 days, there was no significant inhibition of compensatory renal hypertrophy.5. The renal contents of adenosine-3',5'-monophosphate (cyclic AMP) and of guanosine-3',5'-monophosphate (cyclic GMP) in rats hypophysectomized for 2 days were of the same order as those in normal rats, but were markedly lower in rats hypophysectomized for 23 days.6. In contrast to what had been observed in normal rats, in hypophysectomized (2 or 23 days) rats, unilateral nephrectomy did not affect significantly the levels of cyclic nucleotides in the remaining kidney.7. Cross-circulating anephric normal rats with 2 day hypophysectomized animals resulted in an increase of cyclic GMP content in their kidneys. The cross-circulation between anephric normal rats and 23 days hypophysectomized rats had no effect on the level of renal cyclic GMP of the latter.8. When rats hypophysectomized for either 2 or 23 days and which had been nephrectomized were cross-circulated with normal rats, there were no changes in the content of cyclic GMP in the kidneys of the latter.


Neuromuscular transmission in arterioles of guinea-pig submucosa. 1. Intracellular recordings were made from arterioles lying in the submucosa of guinea-pig small intestine. 2. Low frequency perivascular nerve stimulation evoked subthreshold excitatory junction potentials which facilitated. 3. Higher frequency stimulation caused summation of excitatory junction potentials and the initiation of muscle action potentials. 4. Arteriolar constriction was only observed following the initiation of a muscle action potential.


Permeability of the post-synaptic membrane of an excitatory glutamate synapse to sodium and potassium. 1. The changes in permeability of the post-synaptic membrane at the insect skeletal neuromuscular junction caused by the excitatory transmitter and L-glutamate have been studied using the voltage clamp technique. 2. The reversal potential (ER) of the excitatory post-synaptic current and the glutamate current was +3 and +4 mV respectively. 3. ER of the synaptic current did not change when external K was altered between 0 and 20 mM, but did show a small positive shift in 40 mM external K. Reducing external Na to 1-10 mM changes ER by 12-18 mV. Reducing external Cl to to zero caused no change in ER. 4. It is proposed that the transmitter and L-glutamate cause an increase in permeability to Na and K, but not to Cl. 5. In normal saline, the ratio of the permeability increase to Na and K (delta PNa/delta PK) is 0.9. 6. The changes in ER caused by altering external K were similar to those predicted by the Goldman-Hodgkin-Katz equation, assuming delta PNa/delta PK stays constant. 7. The changes in ER caused by alterations of external Na are much less than those predicted by the Goldman equation. 8. No glutamate current could be recorded in Na- and Ca-free saline either at the resting potential or at depolarized or hyperpolarized membrane potentials. 9. It is proposed that the outward K current is dependent upon the inward Na current, and that the increase in K permeability is abolished in zero external Na.


Action of brown widow spider venom and botulinum toxin on the frog neuromuscular junction examined with the freeze-fracture technique. 1. Structural changes which normally accompany transmitter release at frog neuromuscular junctions are visualized with the freeze-fracture technique. The effects of brown widow spider venom and botulinum toxin were evaluated in terms of their ability to block or produce these structural changes. Changes produced by these neuropoisons were correlated with their known effects on neurotransmitter release. 3. Fusion of synaptic vesicles with the presynaptic plasmalemma, normally evoked by electrical stimulation, was abolished at neuromuscular junctions from frogs treated with botulinum toxin. 3. The concentration of large intramembranous particles in the presynaptic plasmalemma, an indication of the excess of synaptic vesicle fusion over recovery of synaptic vesicle membrane, was increased by treatment with brown widow spider venom, even in the presence of botulinum toxin. 4. When external calcium was present, sites of vesicle fusion induced by brown widow spider venom, as well as by electrical stimulation, were located mainly in the active zone. In the absence of external calcium, many plasmalemmal deformations, also though to be sites of vesicle fusion, were more evenly dispersed over the presynaptic surface of nerve terminals. 5. Botulinum toxin decreased the number of vesicle fusion sites in the active zone induced by spider venom in the presence of external calcium but had little effect on the number of fusion sites induced by spider venom in the absence of external calcium. 6. Nerve terminals soaked in a sodium-free Ringer solution were partially depleted of vesicles. Addition of spider venom to this Ringer did not cause additional depletion of vesicles. 7. Formation of cation-permeable channels in the presynaptic membrane could account for these effects of spider venom on the frog neuromuscular junction. Botulinum toxin blocks vesicle fusion by some means which is not yet understood.


Ultrastructural observations of experimental Naegleria meningoencephalitis in mice: intranuclear inclusions in amebae and host cells. Primary amebic meningoencephalitis was experimentallly produced in mice through intranasal instillation of pathogenic Naegleria fowleri. Experimental animals had a 64% mortality with average time of onset of symtoms of death occurring on the 7-8th day following inoculation. Ultrastructural studies of the olfactory lobes from brains of dead (or sacrificed) animals revealed major concentrations of amebae in the perivascular regions; amebae were also seen to be under attack by host polymorphonuclear leukocytes, and in the lumina of blood vessels. Amebae in brain tissue contained 30 nm intranuclear particles arranged in clusters. In the brains of some mice, dead presumably as a result of amebic meningoencephalitis, particles and crystalloids were observed in the nuclei of degenerating cells of the central nervous system. Some alternatives are examined to explain a possible relationship between ameba intranuclear particles and mouse brain cell intranuclear inclusions.


Effects of factors released from eggs and other agents on cyclic nucleotide concentrations of sea urchin spermatozoa. Factors released from eggs (FRE) of the sea urchin, Strongylocentrotus purpuratus, caused up to 20-fold increases in sperm cyclic AMP levels after a 1-min incubation. Putative cyclic nucleotide phosphodiesterase inhibitors such as theophylline acted in a synergistic manner with FRE to cause even greater increases in sperm cyclic AMP levels. This effect appeared to be specific for egg factors since various hormones (triiodothyronine, norepinephrine, histamine), nucleosides (adenosine, guanosine), nucleophiles (axide), anaesthetics (procaine), ionophores (X537A, A23187), metals (Mn2+) and neurotransmitters (acetylcholine) did not increase sperm cyclic AMP levels. Various mammalian tissue extracts (serum, uterus, adrenal, ovary, lung) also had no effect. We suggest that the activity which elevates the cyclic AMP of sea urchin spermatozoa is specifically associated with sea urchin eggs.


Immunocytochemical localization of binding 'sites' for LH and hCG in preimplantation mouse embryos. By using an immunoperoxidase antibody method, mouse blastocysts were found to bind specifically hCG and ovine LH but not FSH or the beta unit of hCG. Brown peroxidase reaction products were present in the morula and increased with the formation of the blastocoele. The LH/hCG binding 'sites' may be related to the initiation of steroidogenesis in the embryo.


Preovulatory changes in ovarian cyclic AMP and prostaglandins in immature rats injected with PMSG. In 28-day-old rats injected with PMSG, ovarian cyclic AMP and PGE and PGF increased 2 and 3-4 h respectively after the LH peak.


Microbiological investigation of meat wholesale premises and beef carcases in Johannesburg. Microbiological surveillance by swabbing meat wholesaler premises revealed ineffective cleaning and build-up of bacteria. Proper cleaning, sanitation and handling resulted in a vast improvement during 1975-77. Beef samples from the neck of carcases in the wholesale trade were investigated by microbiological methods. Excessive total bacterial counts were obtained from numerous carcases. Most carcases carried coliform organisms. Roughly 90% of carcases were contaminated with E. coli I; counts exceeded 10(3)/g in 18% of carcases tested. Twenty serotypes of Salmonella were identified. Salmonella contamination decreased from nearly 5% in 1975 to less than 0,5% in 1977, and S. aureus contamination from 52% to 36% during the same period. Approximately 30% of carcases revealed contamination with unidentified clostridial species. The results indicate the need for stricter control over the production and slaughter of animals and over the handling of carcases in the wholesale trade.


5-O-Alkylated derivatives of 5-hydroxy-2'-deoxyuridine as potential antiviral agents. Anti-herpes activity of 5-propynyloxy-2'-deoxyuridine. Alkylation of 5-hydroxyuridine or 5-hydroxy-2'-deoxyuridine with various activated alkylating agents in the presence of 1 equiv of NaOH gave a series of new nucleoside analogues which were evaluated for antiviral activity against vaccinia virus, herpes simplex-1 virus, and vesicular stomatitis virus in both primary rabbit kidney cells and human skin fibroblasts. One of these analogues, 5-propynyloxy-2'-deoxyuridine, was a potent inhibitor of herpes simplex virus. Structure-activity considerations suggest that the anti-herpes activity is dependent on the integrity of the acetylene group since substitution of phenyl, p-nitrophenyl, vinyl, carboxamido, or carboxyl for the triple bond led to diminished antiviral activity.


The role of breast-feeding in the prevention of rotavirus infection. Breast-fed infants are less susceptible to gastroenteritis than bottle-fed infants. Antibodies against rotavirus, the major pathogen of infantile gastroenteritis, were sought in human sera, colostrum and milk specimens by immunofluorescence. An experimental murine-rotavirus model was established by infecting the second litters of dams 4 weeks after infecting their first litters. Antibodies were absent from human and murine colostrum and milk specimens despite being present in virtually all sera, and the second mouse litters were as susceptible as the first. The inability of rotavirus to infect adult human beings and mice may prevent the formation of gut-derived antibody-secreting lymphocytes in milk, and thus prevent transmission of passive immunity. The association of bottle-feeding with rotavirus gastroenteritis appears to be the result of increased opportunity for spread of infection rather than of the absence of specific protective antibody.


Cyanamide mediated syntheses under plausible primitive earth conditions. I. The synthesis of P1, P2 dideoxythymidine 5'-pyrophosphate. When an aqueous solution (pH 7.0) of deoxythymidine 5'-phosphate, 4-amino-5-imidazolecarboxamide and cyanamide was dried and heated for 18 h at 60 degrees C, P1, P2-dideoxythymidine 5'-pyrophosphate (I) was formed in a 58% yield. Oligonucleotides were not detected in the reaction product. Under conditions employed in the above reaction, (I) was shown to be stable. In prebiotic polymerization reactions employing deoxythymidine 5'-triphosphate as the polymerizing species, (I) could therefore function as a primer and minimize the formation of cyclic nucleotides.


Effect of dietary protein on the response of rainbow trout (Salmo gairdneri) to aflatoxin B1. Diets containing either 49.5% or 32% casein or fish protein concentrate (FPC) were fed to young rainbow trout (Salmo gairdneri) for 12 months. Five levels [0, 2, 6, 18, and 54 parts per billion (ppb)] of aflatoxin B1 (AFB1) were given in each of four different diets. A 30-fish sample was taken at 6, 9, and 12 months to determine the influence of diet on the carcinogenicity of AFB1. Both levels of casein produced similar hepatoma incidences at each level of AFB1. The diet high in FPC produced more tumours than did the casein diets at 2, 6, and 18 ppb AFB1, whereas fish fed the diet low in FPC had a significantly (P less than 0.05) lower hepatoma incidence than did the other three groups. The liver size (percent body wt) was smaller at higher toxin levels in all instances. The growth of fish given 32% casein was less than that of the other groups.


Carcinogenic effect of N-nitroso-2,6-dimethylmorpholine in Syrian golden hamsters. N-Nitroso-2,6-dimethylmorpholine was intragastrically administered to Syrian golden hamsters at four different dose levels [74 mg/kg body wt = 1/5 median lethal dose (LD50); 37 mg/kg body wt = 1/10 LD50; 18 mg/kg body wt = 1/20 LD50; and 9 mg/kg body wt = 1/40 LD50]. Up to a 71% rate of pancreatic tumors was induced. These tumors were adenomas or adenocarcinomas of ductal origin. In addition, neoplasms developed in different percentages in the nasal cavities, larynx, trachea, lungs, liver, gallbladder, kidneys, and forestomach. The number of tumors induced was not significantly dose-dependent.


Rescue of a transforming virus from a spontaneous nonproducing osteosarcoma in BALB/c mice. Cells from spontaneous osteosarcoma V793 that originated in a 19-month-old female BALB/c mouse were cultured. They did not produce a C-type oncovirus as determined by extracellular reverse transcriptase assay and cytoplasmic immunofluorescence. After cocultivation with Balb/3T3 cells chronically infected with a murine leukemia virus (MuLV), a focus-forming principle that transformed 3T3 cells, secondary BALB/c mouse embryo and WAG/Rij rat embryo fibroblasts were rescued. The transformation could be inhibited by antiserum to MuLV.


Glucan: attempts to demonstrate therapeutic activity against five syngeneic tumors in guinea pigs and mice. Animals with established syngeneic tumor transplants were treated with glucan to study the therapeutic potential of this agent under well-defined experimental conditions. The tumors used were a guinea pig hepatoma, 2 murine fibrosarcomas, a murine melanoma, and a murine adenocarcinoma. All tumors were syngeneic to the host. Living BCG, administered directly into guinea pig tumors, cured all animals, whereas glucan, administered under the same conditions, had no significant antitumor activity. Neither BCG nor glucan, when administered iv, was active against the guinea pig hepatoma. An emulsion prepared with endotoxin, a fraction of mycobacteria related to cord factor, and mineral oil when administered intratumorally was also effective in treatment of line 10 tumor. A similar emulsion, in which glucan was substituted for endotoxin, was inactive, intralesional, ip, or iv administration of glucan was ineffective against the murine tumors. Previous reports of glucan-induced activity against a B16 murine melanoma were not confirmed. BCG was tested against the 2 murine fibrosarcomas and, when given either intratumorally or iv, was found to be effective against one of them.


Incidence, latency, and morphologic types of neoplasms induced by simian virus 40 inoculated intravenously into hamsters of three inbred strains and one outbred stock. The incidence, latency, and morphologic types of neoplasms induced in hamsters of the three inbred strains LSH/SsLak, LHC/Lak, and MHA/SsLak, inocuated iv at 3 weeks of age with 10(7.5) median tissue culture infective dose (TCID50) of simian virus 40 (SV40). were determined and compared with those of the outbred stock LVG/Lak. Although the incidence and latency were approximately the same in hamsters of the three inbred strains, hamsters of the outbred stock exhibited almost complete resistance to tumor induction under identical experimental conditions. The morphologic types of neoplasms, i.e., lymphocytic leukemia, reticulum cell sarcoma, osteogenic sarcoma, and anaplastic sarcoma, induced in inbred hamsters were similar to those induced in outbred hamsters inoculated iv with 10(8.5) TCID50 SV40. The lymphocytic leukemias that developed in the 2 LSH/SsLak inbred hamsters were established as tumor transplants in vivo and as permanent cell lines in vitro.


Oncogenicity of murine mammary tumor virus produced in tissue culture: brief communication. Murine mammary tumor virus (MuMTV), produced from a glucocorticoid-stimulated C3H mouse mammary adenocarcinoma cell line, was free of murine leukemia virus and oncogenic for weanling BALB/c mice. Adenocarcinomas were induced by MuMTV as early as 136 days post inoculation and with as low as 5 X 10(3) virus particles/mouse. Tumor incidence did not correlate directly with virus dose; rather, it was low at higher MuMTV concentrations (1.2 X 10(8) particles/mouse), reached and optimum at 1.3 X 10(5) particles/mouse, and decreased with virus dilution.


Toxicity of vinylidene chloride in mice and rats and its alterations by various treatments. The toxicity of vinylidene chloride (VDC) was studied in mice and rats exposed to various concentrations of the vapors for 23 hr/day. In addition, the ability of various compounds to alter parameters of toxicity was evaluated. Mice were more sensitive than rats to the lethal, hepatotoxic, and renal toxic effects of VDC. Disulfiram protected mice from these toxic effects of inhaled VDC and reduced the levels of covalently bound radioactivity in the liver and kidney after the ip administration of [14C] VDC. Diethyldithiocarbamate and thiram also protected mice from the acute lethal effects of VDC.


Theoretical considerations of local neuron circuits and their triadic synaptic arrangements (TSA) in subcortical sensory nuclei. Certain sequential models of the so-called synaptic triadic arrangements (TSA) occurring in sensory nuclei are analyzed. The considerations are addressed mainly to the lateral geniculate body. The model is based on the assumption that the neurons establishing these triads consist of conditionally coupled decision-making subunits. It might be concluded that this and similar arrangements could play a role in the discrimination between stationary and moving retinal inputs and in the detection of temporal correlations in certain input channels.


Neurochemical effects of cocaine following acute and repeated injection. Following repeated injection in the rat, cocaine decreased the concentration of serotonin in the septum-caudate and increased the metabolism of hypothalamic norepinephrine and also striatal dopamine to a lesser extent. Furthermore, cocaine significantly decreased the activity of the rate-limiting enzyme, tryptophan hydroxylase. In a comparative study d-amphetamine and methylphenidate were found to exert an effect opposite to cocaine in the activation of tryptophan hydroxylase. These findings indicate that cocaine may lower central serotonin function by decreasing its availability for neural transmission. This could account for the stimulation of locomotor activity observed after cocaine administration.


The effect of antibiotics, primary and secondary closure on clostridial contaminated open fracture wounds in rats. In a study of experimentally induced open tibial or femoral fractures in rats, after either closing or leaving the wounds open, the animals were given: no antibiotic, cephalothin (Keflin), or penicillin. The rats with wounds closed primarily and receiving no antibiotics had the highest mortality rate (11 of 25) from experimentally produced clostridial myonecrosis. The lowest overall mortality rate (5 of 99) was found in the penicillin-treated groups. The higher mortality rate in the femur fracture groups was probably because of the large muscle mass of the thigh. The importance of ideal anaerobic conditions for producing experimental clostridial myonecrosis is emphasized.


Some adenovirus DNA is associated with the DNA of permissive cells during productive or restricted growth. We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Similar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency integration.


Virus-specific DNA in the cytoplasm of avian sarcoma virus-infected cells is a precursor to covalently closed circular viral DNA in the nucleus. Three principal forms of viral DNA have been identified in cells infected with avian sarcoma virus: (i) a linear duplex molecule synthesized in the cytoplasm, (ii) a covalently closed circular molecule found in the nucleus, and (iii) proviral DNA covalently linked to high-molecular-weight cell DNA. To define precursor product relationships among these forms of viral DNA, we performed pulsechase experiments using 5-bromodeoxyuridine to label by density the linear species of viral DNA in the cytoplasm during the first 4 h after infection. After a 4-to 8-h chase with thymidine, a portion of the density-labeled viral DNA was transported to the nucleus and converted to a covalently closed circular form. We conclude that linear viral DNA, synthesized in the cytoplasm, is the precursor to closed circular DNA observed in the nucleus.


Identification of a 30S RNA with properties of a defective type C virus in murine cells. A novel species of 30S RNA has been detected in a variety of mouse cell lines. The 30S RNA is specifically packaged by helper-independent type C viruses propagated in such cells. Nucleic acid hybridization detects no homology between the 30SRNA and the genomic RNA of helper-independent mouse type C viruses. The properties of the 30S RNA suggest that it is a defective endogenous mouse type C virus and that it is analogous to a previously described class of defective endogenous rat type C virus, which has been shown previously to be the progenitor of Kirsten and Harvey murine sarcoma viruses.


Effect of cordycepin triphosphate on in vitro RNA synthesis by picornavirus polymerase complexes. Cordycepin triphosphate inhibited in vitro [3H]GMP incorporation by pricornavirus-specific polymerase complexes isolated from infected HeLa cells. The inhibition of [3H]GMP incorporation could be reversed with ATP added to the reaction mixture along with the inhibitor, but not with GTP so added or with ATP added 10 min after the inhibitor. Products synthesized in vitro in the presence of cordycepin triphosphate lacked full-length single-stranded viral RNA. These results support RNA chain termination by specific competition with ATP as the mechanism of inhibition of picornavirus-specific RNA synthesis by cordycepin triphosphate.


Cytofluorometry of lymphocytes infected with Epstein-Barr virus: effect of phosphonoacetic acid on nucleic acid. DNA synthesis in Epstein-Barr virus (EBV)-infected lymphocytes was inhibited by phosphonoacetic acid (PAA) as measured by [3H]thymidine incorporation. PAA, at a concentration of 200 microgram/ml, inhibited [3H]thymidine incorporation by human umbilical cord lymphocytes infected with EBV strain P94 but had little effect on DNA synthesis in mitogen-stimulated cells. Transformed cell lines did not develop from infected cord cell cultures treated with 100 microgram of PAA per ml. Cytofluorometric analysis showed marked increases in cellular nucleic acid content (RNA plus DNA) as early as 9 days after infection of cord cells in the absence of PAA and before significant enhancement of [3H]thymidine incorporation became apparent. Moreover, EBV led to increases in cellular nucleic acid even when 200 microgram of PAA per ml was added to cell cultures before infection. The apparent discrepancy between results obtained by [3H]thymidine incorporation and cytofluorometry is explained either by significant inhibition of cellular DNA polymerases by PAA or by a block at the G2 + M phase of the cell cycle. The data suggest that EBV initiates alterations in cellular nucleic acid synthesis or cell division without prior replication of viral DNA by virus-induced DNA polymerases.


Glucocorticoids induce focus formation and increase sarcoma viral expression in a mink cell line that contains a murine sarcoma viral genome. Dexamethasone (3 X 10(-10) to 3 X 10(-6) M) induced foci of morphologically transformed cells in a small proportion of a mink cell line that contains the Moloney murine sarcoma viral genome (S+L-). The induction was glucocorticoid specific, since other steroids with glucocorticoid activity (prednisolone, cortisol, and aldosterone) induced foci with an efficiency that paralleled their glucocorticoid activity, and steroids lacking glucocorticoid activity (17B-estradiol, testosterone, and progesterone) failed to induce foci. Viral antigen, as measured by specific immunofluorescence, was localized to the foci. The induction of foci by dexamethasone (3 X 10(-7)) was accompanied by an approximately 10-fold increase in intracellular Moloney murine sarcoma virus-specific RNA and viral p30 antigen. Removal of dexamethasone was followed by the disappearance of foci and a decrease in viral RNA and p30. In this cell system, therefore, glucocorticoids can affect the intracellular levels of type C viral RNA and protein.


Comparison of nuclease digestion of polyoma virus nucleoprotein complex and mouse chromatin. We digested polyoma virus nucleoprotein complex, isolated from disrupted virions, with micrococcal nuclease and DNase I. The results were compared with digestions of chromatin from mouse nuclei. The nucleosome "core" structures were similar, but the spacing of the nucleosomes in the isolated polymoma nucleoprotein complexes was irregular, whereas in mouse chromatin it was regular. The average nucleosome repeat length in each case was 190 to 200 base pairs. This figure suggests that, unless there are substantial stretches of free DNA, the polyoma nucleoprotein complex contains about 26 nucleosomes. The commonly used method of preparing the nucleoprotein complex by disruption of virions at pH 10.2 may lead to significant damage to the structure. Such damage may be more clearly revealed by the susceptibility of the DNA to nuclease digestion than by the usual criteria of sedimentation velocity and buoyant density.


Electron microscope study of the base sequence homology between simian virus 40 and human papovavirus BK. The base sequence homology between the genomes of simian virus 40 (SV40) and human papovavirus BK (BKV) was studied by the heteroduplex method of Ferguson and Davis (J. Mol. Biol. 94:135-149, 1975). When mounted for microscopy in 30% formamide (Tm-35 degrees C), BKV/SV40 heteroduplexes were an average of 92% double-stranded and contained only two small nonhomologous regions that mapped near the junctions between the early and late regions of the SV40 Genome. At higher formamide concentrations, the fraction of duplex DNA in the BKV/SV40 heteroduplexes decreased, indicating  significant base mismatching in the homologous regions. The strongest regions of homology were located in the late region.


Host function-dependent induction of defective interfering particles of vesicular stomatitis virus. Suppression of host cell function by treatment with actinomycin D prior to infection prevented the induction of defective interfering particles of vesicular stomatitis virus, which had been cloned and propagated in cell pretreated with actinomycin D. Replication of defective interfering particles already present in an infecting virus stock, however, was not affected by pretreatment of cells with actinomycin D. Thus, the induction, but not the replication, of defective interfering particles appears to be a host cell function-dependent phenomenon. The implications of this phenomenon for host defense mechanisms against virus infections are discussed.