E.s.r. of spin-trapped radicals in aqueous solutions of peptides. Reactions of the hydroxyl radical. The reactions of hydroxyl radicals with 30 dipeptides and several larger peptides were studied in aqueous solutions. The OH radicals were generated by U.V. photolysis of H2O2. The short-lived peptide radicals were spin-trapped using t-nitrosobutane and identified by e.s.r. For dipeptides containing the amino terminal residues glycine, alanine and phenylalanine, abstraction of the hydrogen from the carbon adjacent to the peptide nitrogen was the major process leading to the spin-adducts. Such radicals will be referred to as backbone radicals. Dipeptides with a carbonyl terminal serine residue and also glycylglutamic acid form both backbone and side-chain radicals, with the latter being formed in larger quantities. For dipeptides, side-chain radicals were detected on either the carboxyl or amino terminal residues of both. The effect of pD on the e.s.r. sectrum of the spin-adducts of glycylglycine was studied and the pK of the carboxyl group of this radical was determined to be 2.5. For (Ala)3 and (Ala)n, with an average value of n = 1800, backbone and minor side-chain radicals were observed. For ribonucleases-S-peptide, containing 20 amino acid residues, both backbone and side-chain radicals were detected.


E.s.r. study of spin-trapped radicals formed during the photolysis of aqueous solutions of acid amides and H2O2. Free radicals formed by the reactions of OH radicals with amides and their N-methylated derivatives in aqueous solutions have been studied. The OH radicals were produced by U.V.-photolysis of H2O2, and the short-lived amide radicals were converted to more stable nitroxide radicals by addition to a spin-trap, tert-nitrosobutane. The spin-trapped radicals were identified by e.s.r. spectroscopy. For acetamide, chloroacetamide, malonamide, succinamide and propionamide, the observed radicals were formed by H-abstraction from the carbon atoms attached to the carbonyl group. The H atom attached to the carbonyl group was abstracted in formamide. For N-methyl acetamide, N,N-dimethyl acetamide and the corresponding formamide derivatives, H-abstraction occurred only from the N-methyl group. The non-equivalency of the amide protons was observed in the spin-trapped radicals for acetamide, formamide, malonamide, succinamide and propionamide. The identification of the site of OH attack on N-methyl amides is helpful for the study of radical formation in peptides and proteins.


Modification of rat thymocyte membrane properties by hyperthermia and ionizing radiation. Thymocytes are one the most widely used cell models for the study of radiation-induced interphase death. This cell-type was chosen for the study of hyperthermic and radiation effects on two membrane-related processes implicated in the interphase death of cells: Na+-dependent 2-aminoisobutyric acid (AIB) transport and cyclic 3'-5' adenosine monophsophate formation. The response of AIB transport to heat is dose-dependent, but the biphasic thermal response curve (AIB uptake versus time) differs fom the sigmoidal radiation response curve. Heating thymocytes for 20-30 min at 43 degrees C stimulates AIB uptake. Additional heating at 43 degrees C, however, markedly reduces AIB uptake. Despite the immediate stimulating effect of heat (30 min at 43 degrees C), the thymocyte has already developed irrepairable impairments, as demonstrated by the fractionated heating experiments. The heat-induced impairment of AIB uptake is mainly on the Na+-dependent component of neutral amino-acid transport, affecting primarily the maximal rate of uptake, i.e. Vmax. Additional evidence for heat-induced plasma membrane damage is the alteration in cAMP levels. Heating thymocytes for 30 min or longer at 43 degrees C causes a massive rise in cAMP level within the cell. This differs from thymocytes exposed to radiation where no rise in cAMP is observed.


The effect of prednisolone on antibody-dependent cell-mediated cytotoxicity and the growth of type I herpes simplex virus in human cells. Treatment of human skin and corneal fibroblasts with prednisolone-21-phosphate did not increase the capacity of these cells to replicate type I herpes simplex virus (HSV). The steroid however was found to (1) inhibit human lymphocytes from mediating antibody-dependent cell-mediated cytotoxicity (ADCC) against HSV-infected fibroblasts and (2) suppress the replication of virus in PHA-stimulated human lymphocytes. The data suggest that the exacerbation observed when patients with dendritic keratitis are inadvertently treated with prednisolone may be due to the steroid suppressing ADCC and not by promoting the growth of virus in the corneal cells.


Histochemical demonstration of cyclic guanosine 3',5'-monophosphate phosphodiesterase activity in retinal photoreceptor outer segments. A technique for the histochemical demonstration of cyclic guanosine monophosphate phosphodiesterase in retina is described. Enzyme activity was identified on photoreceptor outer segment lamellae, a finding in agreement with previous biochemical data on isolated outer segment preparations. The distribution of phosphodiesterase activity for cyclic guanosine monophosphate was similar to that found previously in rod outer segments for cyclic adenosine monophosphate, suggesting that the same enzyme may hydrolyze both nucleotides.


Involvement of the sympathetic nervous system in the urinary bladder internal sphincter and in penile erection in the anesthetized cat. Male cats were anesthetized with pentobarbital. In one series of experiments a Foley catheter was placed suprapubically in the urinary bladder and physiologic saline, under a constant head of pressure, was allowed to flow at a constant rate through the bladder. Alpha-adrenergic stimulants such as ephedrine, norepinephrine, or methoxamine all caused a decrease or cessation in urinary flow. This effect was reversed by the alpha-adrenergic blocking agent, phentolamine. In addition, phentolamine administration caused penile erection. Erection also occurred after administration of the alpha-adrenergic blocking agent, phenoxybenzamine, or the beta2-adrenergic stimulants terbutaline or salbutamol. In each case the erection could be reversed, and usually terminated completely, by administration of the beta-adrenergic blocking agent, propranolol. Hypotension per se was not the required event to initiate erection because administration of acetylcholine, histamine, or prostaglandin E2 did not result in penile erection. In two of five experiments nitroprusside caused an erection. It was concluded that a predominance of beta2 activity in the penile vasculature is involved in erection.


Angiotensin-converting enzyme in macrophages and Freund's adjuvant granuloma. Low angiotensin-converting enzyme (ACE) activity was found in rat and mouse peritoneal macrophages, in rat and rabbit pulmonary alveolar macrophages, in cultured mouse peritoneal macrophages stimulated i.p. for four days by thioglycollate, and in rabbit pulmonary alveolar macrophages stimulated in culture by 0.1 to 100 microgram/ml Salmonella typhosa endotoxin for four days. Rat subcutaneous Freund's adjuvant granulomas induced with Mycobacterium butyricum or M. tuberculosis H37 Ra contianed low ACE activity which was generally lower than that present in control tissues. Increased ACE activity in sarcoidosis and Gaucher's disease lesions does not reflect a universally high synthesis of ACE in mammalian macrophages or granulomas, but may be due to specific mechanisms.


Distribution of ACTH-like immunoreactivity in rat brain and gastrointestinal tract. Immunocytochemistry reveals ACTH-like immunoreactivity to reside not only in the pituitary but also in central nerves and in central nerves and in antral gastrin cells. In all probability, the central nerves store a peptide identical with or closely resembling true ACTH. Their pattern of distribution is, in some regions, similar to that of enkephalin-immunoreactive nerves. The antiserum demonstrating ACTH-like immunoreactivity in central nerves and in antral gastrin cells is directed towards the COOH-terminal part of the hormone. A peptide corresponding to this part, the corticotrophin-like intermediate peptide (CLIP) is manufactured by the pars intermedia of the pituitary. CLIP is devoid of adrenocortical activity, but has recently been shown to possess insulin-releasing activity. The occurrence of CLIP-like peptides in antral gastrin cells may indicate a role for such peptides in the gastrointestinal regulation of insulin release. The simultaneous occurrence of enkephalin-like and ACTH-like immunoreactivity in the antral gastrin cells is of particular interest since a large precursor molecule, containing both the enkephalin and the ACTH sequence has recently been identified.


Ventilatory and waking responses to hypoxia in sleeping dogs. We examined  waking and ventilatory responses to acute hypoxia in four dogs during natural sleep. Progressive hypoxia was induced by a rebreathing technique in which alveolar CO2 pressure (PACO2) was held at the eucapnic level. Arterial O2 saturation (SaO2) was measured with an ear oximeter, and sleep stage was determined by electroencephalographic and behavioral criteria. Arousal from eucapnic hypoxia occurred at a SaO2 of 87.5 +/- 2.6% (mean +/- SE) during slow-wave sleep (SWS), and at a SaO2 of 70.5 +/- 3.4% during rapid-eye-movement (REM) sleep (P less than 0.005). The irregular pattern of breathing typical of REM sleep persisted during hypoxia. However linear regression analysis of breath-by-breath instantaneous minute volume of ventilation (VI) against SaO2 revealed regression coefficients in REM sleep that were similar to those found in SWS and wakefulness. This finding contrasts with earlier observations of a decreased response of VI to CO2 during REM sleep. The results indicate that although waking responses to hypoxia are delayed in REM sleep, ventilatory responses remain intact and therefore may be of importance in maintaining adequate ventilation during this stage of sleep.


Physical activity and hypophysectomy on the aerobic capacity of ligaments and tendons. Traditionally, ligaments and tendons (L and T) have been regarded as metabolically inert structures. However, sufficient biochemical evidence on the metabolism of collagen has indicated that such a concept is no longer tenable. To determine whether L and T respond to increased or decreased levels of chronic exercise, studies were undertaken to measure their aerobic capacities. For comparative purposes, similar measurements were obtained from liver and skeletal muscles secured from normal and hypophysectomized male rats. Oxygen consumption and cytochrome oxidase (CO) activity was recorded from cell suspensions that had been prepared with the inclusion of collagenase and with elastase added to the medium. The O2 results showed that L and T had values that were approximately 10 times lower than liver tissue and 7.5 times less than the means from skeletal muscles. Hypophysectomy caused marked reductions in O2 uptake of liver and muscle tissues; but had no impact on L and T. When CO activity of these connective tissues were evaluated, immobilization and hypophysectomy caused significant reductions that ranged from -36% to -59% respectively. Training, on the other hand, resulted in increases of less than 10% in the activity of this enzyme within L and T while being elevated in muscle tissue by 58%. It was concluded that the metabolic activity of L and T was lowered with decreased levels of physical activity but it was unclear why chronic exercise did not produce the opposite effect.


Epizootic of concurrent cutaneous streptococcal abscesses and swinepox in a herd of swine. Concurrent cutaneous abscesses and swinepox were observed in a herd of swine. An ungroupable hemolytic streptococcus was isolated from the abscesses. Tests indicated that streptococci isolated from different swine were similar if not identical. Swinepox lesions were observed on swine only after weaning. Lice (Haematopinus suis) were numerous on the suckling and weaned swine and appeared to be resistant to lindane. Abscesses, swinepox, and lice were eliminated after the swine were treated with an organic phosphate insecticide and antibacterial agents were added to the rations.


Evidence from auditory nerve and brainstem evoked responses for an organic brain lesion in children with autistic traits. In an attempt to resolve the question as to whether children with autistic traits have an organic nervous system lesion, auditory nerve and brainstem evoked responses were recorded in a group of 15 children with autistic traits. The most obvious results included a longer response latency of the auditory nerve and a longer brainstem transmission time, compared to normal children. Five of the autistic children were found to be profoundly deaf. These results strengthen the theory that an organic lesion of the nervous system can give rise to autistic traits.


Autistic symptoms in a child with congenital cytomegalovirus infection. A case of intrauterine cytomegalovirus infection with onset of autistic symptoms apparently after 6 months of age is reported. Physicians who find autistic symptoms in very young children might include cytomegalovirus in their differential to document the presence or absence of a correlation.


Changes in cyclic AMP levels during development in Myxococcus xanthus. Cyclic AMP levels doubled in Myxococcus xanthus under conditions in which cells aggregate and form fruiting bodies. In liquid medium, glycerol- or dimethyl sulfoxide-induced sporulating cultures exhibited a sharp but transient rise in cyclic AMP concentration after 45 min.


Arginine metabolism in Saccharomyces cerevisiae: subcellular localization of the enzymes. Subcellular localization of enzymes of arginine metabolism in Saccharomyces cerevisiae was studied by partial fractionation and stepwise homogenization of spheroplast lysates. These enzymes could clearly be divided into two groups. The first group comprised the five enzymes of the acetylated compound cycle, i.e., acetylglutamate synthase, acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, and acetylornithine-glutamate acetyltransferase. These enzymes were exclusively particulate. Comparison with citrate synthase and cytochrome oxidase, and results from isopycnic gradient analysis, suggested that these enzymes were associated with the mitochondria. By contrast, enzymatic activities going from ornithine to arginine, i.e., arginine pathway-specific carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, and argininosuccinate lyase, and the two first catabolic enzymes, arginase and ornithine aminotransferase, were in the "soluble" fraction of the cell.


Dimeric nature of apo-low density lipoprotein extracted with Triton X-100. Human low density lipoprotein (LDL) was dissolved in 0.3 to 2.0% Triton X-100 at pH 7.5 and apo-LDL (B protein) was extracted from LDL to form B protein-Triton complex. Sedimentation equilibrium study of this complex in a solvent nearly isopycnic to Triton X-100 showed that the molecular weight of the protein in the complex was 570,000. The complex eluted almost at the void volume of a Sepharose 6B column, as would be expected for a complex with a total molecular weight of roughly 900,000, on the assumption that 0.52 g of Triton was bound to 1 g of protein (Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656-3661). The sedimentation coefficient of the complex gave f/fmin = 2.2, indicating that the complex was either as asymmetric as a fibrinogen molecule or not compact. These results show that B protein exists in its complex with Triton X-100 as an elongated or a loosely expanded dimer based on the molecular weight of monomeric B protein of 270,000. B protein may also exist in LDL as a dimer.


Characterization of electron transport enzymes in the envelope of rat liver nuclei. Nuclei and microsomes were prepared from the livers of normal, phenobarbital (PB)-treated and beta-naphthoflavone (beta-NF)-treated rats, and the contents of several enzymes in both subcellular fractions were examined. In normal rats, the enzyme activities in the nuclear fraction were about one-third of those of microsomes on a phospholipid basis. The induction of some particular enzymes by the drugs was observed with nuclei as well as with microsomes. Cytochrome P-450 and NADPH-cytochrome c reductase were increased by PB treatment and cytochrome P-448 was induced by beta-NF treatment both in nuclei and in microsomes. The extents of inhibition of nuclear enzyme activities by the antibodies against corresponding microsomal enzymes were almost the same as those of the microsomal activities. It was concluded that a microsomal type electron transport system exists in rat liver nuclei, and that nuclear drug-oxidizing activities are inducible by PB or beta-NF as their microsomal counterparts are.


Role of phospholipid in adrenaline-induced lipolysis and cyclic AMP production. Lipid micelles consisting of a glyceride mixture (triolein, diolein, and monoolein) and lecithin bound adrenaline-14C more strongly than did micelles consisting of the glyceride mixture only. Lipid micelles consisting of the glyceride mixture and phosphatidic acid also bound adrenaline-14C effectivily, whereas lipid micelles consisting of the glyceride mixture and diglyceride, obtained from lecithin, did not bind the hormone strongly. Both phenoxybenzamine (an alpha- blocker) and propranolol (a beta-blocker) strongly inhibited the association between adrenaline-14C and lipid micelles consisting of the glyceride mixture and lecithin. Propranolol, inhibited adrenaline-induced lipolysis in both fat cells and fat globules, whereas, phenoxybenzamine, did not affect adrenaline-induced lipolysis. Both agents reduced adrenaline-induced adenyl-cyclase activation in fat cell ghosts. Phospholipid was also found to be related with adrenaline-mediated adenylcyclase activation.


beta-adrenergic receptor and adenylate cyclase in transverse tubules of skeletal muscle. Experiments were carried out to clarify the sites of action of beta-adrenergic agonists in skeletal muscle microsomes. Microsomes were fractionated into longitudinal reticulum, terminal cisternae, and isolated transverse tubules. Transverse tubules were selectively labeled and tracked with [3H]ouabain. beta-adrenergic receptor was identified by [3H]dihydroalprenolol binding. Assays of beta-adrenergic receptor, adenylate cyclase, and protein kinase-stimulated phosphorylation showed: 1) beta-adrenergic receptor was detected in transverse tubules with a receptor density of 0.61 pmol/mg of protein. No significant binding was detected in longitudinal reticulum or in terminal cisternae. 2) Isoproterenol-stimulated adenylate cyclase was present in microsomes but was similarly confined to the transverse tubular fraction. The activity of F- stimulated cyclase in transverse tubules was 2.3 nmol/mg of protein/min. 3) No phosphorylation of microsomes by cyclic AMP and protein kinase could be detected. We conclude that the action of epinephrine on skeletal muscle is mediated through receptors and adenylate cyclase in the external membrane.


The effect of tobacco smoke on the metabolism and function of rat alveolar macrophages. Alveolar macrophages harvested by bronchopulmonary lavage from rats exposed to tobacco smoke for 30 days ("smokers") showed alterations in oxidative metabolism, lactate production and phagocytosis of inert starch particles when compared with control macrophages. Phagocytosis of viable Staphylococcus aureus was unaffected by tobacco smoke. Glucose oxidation measured by conversion of glucose-1-14C to 14CO2 moderately affected while oxidation of glucose-6-14C to 14CO2 was not. Smokers routinely yielded fewer cells than controls, though these cells contained approximately 17% more protein than did controls. Opsonization of particles was not necessary for macrophages from either smoker or control animals to manifest a respiratory burst and increased superoxide and hydrogen peroxide release during phagocytosis. The glycolytic inhibitors, sodium fluoride and iodoacetamide, while effectively blocking glycolysis, did not inhibit phagocytosis by macrophages from either group. The results reported clearly distinguish alveolar macrophages from other phagocytic cells (peritoneal macrophages and polymorphonuclear leukocytes) and suggest a state of non-specific activation caused by exposure to tobacco smoke.


Serum-stimulated changes in calcium transport and distribution in mouse 3T3 cells and their modification by dibutyryl cyclic AMP. Serum stimulation of quiescent 3T3 cells returns the cells to a proliferative state. Changes in Ca content, transport and distribution during the transition through G1 and S phase have been investigated following serum stimulation of these cells. 45 Ca exchange data indicate at least two kinetically defined cellular compartments for Ca; a rapidly exchanging component presumably representing surface Ca which is removable by EGTA and a slowly exchanging component presumably representing cytoplasmically located Ca. Previous studies (Tupper and Zorgniotti, '77) indicate that the approach to quiescence in the 3T3 cells is characterized by a large increase in the surface Ca component. The present data demonstrate that this component is rapidly lost following serum stimulation. Furthermore, the serum induces an 8-fold increase in Ca influx into the cytoplasmic compartment and a reduction in the unidirectional efflux rate coefficient for Ca. The increased Ca uptake peaks at approximately six hours (mid G1) and is accompanied by a parallel increase in cellular Ca. Prior to entrance of the cells into S phase (10-12 hours), Ca uptake declines. This is followed by a slower decline in cytoplasmic Ca levels. Simultaneous addition to fresh serum plus 0.5 mM dibutryl cAMP inhibits the entrance of the cells into S phase. Under these conditions the loss of surface Ca is not blocked. However, the presence of 0.5 mM dibutyryl cAMP inhibits the increase in Ca uptake and, in turn, diminishes the increase in cellular Ca following serum stimulation. In contrast, a low level of dibutyryl cAMP (0.1 mM) enhances progression through G1 phase but also reduces both Ca uptake and Ca content of the cells. The data suggest that the serum induced changes in Ca content and transport are linked to intracellular cyclic nucleotide levels and progression through G1 phase and that extracellular cAMP elevating agents may enhance of inhibit these interactions in a concentration dependent manner.


Glucagon binding and adenylate cyclase activity in liver membranes from untreated and insulin-treated diabetic rats. To investigate the role of hepatic glucagon receptors in the hypersensitivity to glucagon observed in insulin-deprived diabetics, liver plasma membranes were prepared from control rats and from streptozotocin-induced diabetic rats some of whom were treated with high-dose and low-dose insulin. The untreated diabetic animals exhibited hyperglycemia, weight loss, hypoinsulinemia, and hyperglucagonemia. High-dose insulin treatment (2 U Protamine-zinc-insulin/100 g per day) resulted in normoglycemia, normal weight gain, mild hyperinsulinemia, and return of glucagon levels toward base line. The low-dose (1 U protamine-zinc-insulin/100 g per day) insulin-treated diabetic group demonstrated chemical changes intermediate between the untreated and the high-dose insulin-treated animals. In liver plasma membranes from the untreated diabetic rats, specific binding of (125)I-glucagon was increased by 95%. Analysis of binding data suggested that the changes in glucagon binding were a consequence of alterations in binding capacity rather than changes in binding affinity. Furthermore, in the untreated diabetic rats, both basal and glucagon (2 muM)-stimulated adenylate cyclase activity were twofold higher than in controls. In the high-dose insulin-treated diabetic rats, glucagon binding and basal and glucagon-stimulated adenylate cyclase activity were normalized to control values, whereas low-dose insulin treatment resulted in changes intermediate between control and untreated diabetic rats. In contrast to glucagon-stimulated adenylate cyclase activity, fluoride-stimulated adenylate cyclase activity was similar in all groups of rats. Liver plasma membranes from untreated and insulin-treated diabetic animals degraded (125)I-glucagon to the same extent as control rats. The specific binding of (125)I-insulin in the untreated diabetic animals was 40% higher than in control rats. In low-dose insulin-treated diabetic rats, insulin binding was not significantly different from that of control rats, whereas in the high-dose insulin-treated group in whom plasma insulin was 70% above control levels, insulin binding was 30% lower than in control rats. These findings suggest that alterations in glucagon receptors may contribute to the augmented glycemic and ketonemic response to glucagon observed in insulin-deprived diabetics.


Genetics of the low density lipoprotein receptor. Diminished receptor activity in lymphocytes from heterozygotes with familial hypercholesterolemia. Using circulating mononuclear cells as a readily available tissue and using the rate of high affinity degradation of 125-I-labeled low density lipoprotein (LDL) as an index of cell surface LDL receptor activity, we have measured receptor activity in cells from 53 individuals. This group includes 32 healthy subjects, 15 subjects with the heterozygous form of familial hypercholesterolemia, and 6 subjects with hyperlipidemic disorders other than familial hypercholesterolemia. 7 of the healthy subjects and 10 of the heterozygotes were members of a single large kindred with five-generation transmission of the mutant familial hypercholesterolemia gene. LDL receptor activity was assayed in blood mononuclear cells under two sets of conditions. First, 125I-LDL degradation was measured in purified lymphocytes that had been incubated for 3 days in the absence of lipoproteins so as to induce a high level of LDL receptor activity. Phase-contrast autoradiograms of cells incubated with 125I-LDL and electron micrographs of cells incubated with ferritin-labeled LDL confirmed the existence of LDL receptors on lymphocytes. Second, 125I-LDL degradation was measured in mixed mononuclear cells (85-90% lymphocytes and 5-15% monocytes) immediately after their isolation from the bloodstream. This assay represented an attempt to assess the number of receptors actually expressed on the cells when they were in the circulation. Under both sets of conditions, cells from the familial hypercholesterolemia heterozygotes expressed an average of about one-half the normal number of LDL receptors. The current findings are consistent with the conclusion that heterozygotes with familial hypercholesterolemia possess only one functional allele at the LDL receptor locus and that the consequent deficiency of LDL receptors produces the clinical syndrome of heterozygous familial hypercholesterolemia.


Catabolism of very low density lipoprotein B apoprotein in man. The turnover and the catabolic fate of the B apoprotein of very low density lipoprotein (VLDL-B) was studied in 15 normal and hyperlipidemic subjects using reinjected autologous VLDL labeled with radioiodine. The specific radioactivity-time curve of the B apoprotein in total VLDL (S(f)20-400) was multiexponential but conformed to a two-pool model during the first 48 h of catabolism. The flux was highest in several hypertriglyceridemic subjects. The mass of pool A exceeded the intravascular content of VLDL-B by 30% on average, indicating extravascular metabolism of VLDL. The two-pool model might reflect the input of several populations of particles or heterogeneity of catabolic processes or pools. The flux of B apoprotein was also measured in several subclasses of VLDL, in smaller intermediate density lipoproteins, and in low density lipoproteins (LDL). In three subjects the flux was similar in S(f) 60-400 and in S(f) 12-60 lipoproteins, suggesting that VLDL was catabolized at least to a particle in the density range S(f) 12-60. Subsequent catabolism appeared to proceed by two pathways: in normotriglyceridemic subjects, B apoprotein flux in the S(f) 20-400 and in S(f) 12-20 lipoproteins was similar, whereas in hypertriglyceridemic subjects flux through S(f) 12-20 accounted for only part of the VLDL-B flux. The flux of low density lipoprotein B apoprotein (LDL-B), which is believed to be derived from VLDL catabolism, was calculated from the area between the specific activity time curves of VLDL-B and LDL-B. In subjects with normal plasma triglyceride concentration, LDL-B flux was from 91% to 113% of that of VLDL-B; but in three hypertriglyceridemic subjects showing high rates of VLDL-B transport, LDL-B flux was only one-third that of VLDL-B. This suggests that when VLDL-B flux is high, VLDL is substantially catabolized by a route other than through LDL and possibly leaves the circulation as a particle in the S(f) 20-60 density range.


Free-sporing Cl. welchii in ordinary laboratory media and conditions. A strain of Clostridium welchii produced spores in ordinary blood agar plates. Investigations confirmed that it was the character of this particular strain and that the laboratory media were not inducing sporulation. During a period of 12 months a total of 100 strains of Cl. welchii were studied. None of them produced spores in ordinary laboratory media and conditions when examined microscopically.


Human polyomavirus (BK) infection and ureteric stenosis in renal allograft recipients. Human polyomavirus (BK) was detected in two renal allograft recipients as a result of routine examination of Papanicolaou-stained smears of urinary sediment in the light microscope. Infection with this recently identified virus was confirmed by virus isolation and electron microscopy. The cytological, histological, and ultrastructural changes due to the virus are described, and virus excretion is correlated with the clinical progress of the patients and the pathological findings. The transplant ureters in both patients were found to be ulcerated and stenosed, and virus-infected cells were observed in the ureteric epithelium. We suggest that the administration of high-dose steroids in transplantation may permit active infection with human polyomavirus to occur in ureteric epithelium which has been damaged by ischaemia or inflammation.


Chemical and kinetic study of the lipoproteins in abetalipoproteinaemic plasma. Abetalipoproteinaemic plasma lipoproteins were fractionated by molecular sieve chromatography into two classes on the basis of size. Each class had the same chemical and immunochemical composition and seemed to be interconvertible in vitro, presumably as a result of aggregation/disaggregation. The low levels of circulating apolipoprotein A-I found in abetalipoproteinaemic subjects have been shown by kinetic analysis to result from reduced synthesis of the apoprotein and not from increased catabolism or redistribution between vascular and extravascular compartments.


Multiple effects of N, N' dicyclohexyl carbodiimide on the beta-adrenergic receptor--adenylate cyclase system in frog erythrocytes. Treatment of frog erythrocytes with N,N' dicyclohexylcarbodiimide (DCCD) leads to a loss of catecholamine stimulated adenylate cyclase activity without any decrease in fluoride or PGE1 stimulated cyclase. However, the concentrations of the reagent which inhibit catecholamine sensitive adenylate cyclase activity are 10 fold lower than those which inhibit specific [3H]dihydroalprenolol ([3H]DHA) beta-adrenergic receptor binding. By contrast binding of the readiolabeled beta-adrenergic agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) is considerably more sensitive than antagonist binding to the effects of DCCD. The data suggest that low concentrations of the reagent may modify the effector portion of the beta-adrenergic receptor leading to functional uncoupling of the beta-receptor adenylate cyclase system. At higher concentrations of the reagent the  ligand bidning site of the beta-receptor appears also to be altered.


On the role of the cAMP and cGMP-dependent protein kinases in cell function. Cyclic AMP and cGMP-dependent protein kinases have many similarities in physical and kinetic properties. Thus, the two enzymes appear to be homologous proteins, even though the subunit compositions of the two enzymes differ. Of several possible evolutionary schemes, two likely ones are proposed: (i) the ancestral protein kinase was composed of a single type of subunit which evolved into separate regulatory and catalytic subunits by gene splitting; or (ii) the ancestral protein kinase was composed of separate regulatory and catalytic subunits which evolved into a single type of subunit by gene fusion. The evolutionary parallelism poses interesting questions on the functional relatwonships between the two enzymes. Although, there is overlapping substrate specificity between the two kinases, the cAMP kinase is generally a more efficient and versatile catalyst than the cGMP kinase. This difference in catalytic versatility could have offered an evolutionary advantage to the cAMP kinase, and could explain the more widespread distribution of this enzyme in mammalian tissues. The cGMP kinase is proposed to be a more specific enzyme than the cAMP kinase. This implies a less diverse role for cGMP in the regulation of cell function.


8-P-Chlorophenylthio-cyclic AMP: a potent partial simulator of antidiuretic hormone action. In the toad urinary bladder 8-p-chlorophenylthio-cyclic AMP mimics the stimulatory effects of antidiuretic hormone on osmotic water permeability, 3H2O diffusion, and transepithelial sodium transport; but unlike the hormone does not cause an increase in urea permeability. Trheshold activation for the hydroosmotic response is observed at 1 micrometer and full activation at 100 micrometer. These results suggest that cyclic AMP may not mediate all the physiological effects of antidiuretic hormone and that this highly potent cyclic AMP analog may be useful in elucidating the precise role of cyclic AMP in other biomediate hormone action.


Activation of cyclic AMP-dependent protein kinase(s) by growth hormone in the liver and adrenal gland of the rat. A single dose of growth hormone (10 mg/kg, i.p.) was injected into male weanling rats (50--60 g), and the temporal changes in cyclic AMP concentration, protein kinase activation, and ornithine decarboxylase activation were measured in the liver and adrenal gland. The level of cyclic AMP did not change significantly from control values in either liver or adrenal following growth hormone administration. Cyclic AMP-dependent protein kinase(s); however, was markedly activated in liver and adrenal within 30 min. Protein kinase remained activated for more than 4 hr in the liver, while activation of protein kinase in the adrenal returned to control value within 2 hr. Ornithine decarboxylase activity was elevated 20-fold in liver within 4 hr of injection and was increased 7- to 8-fold in be adrenal within l hr. These observations are discussed with regard to the generality of the role of cyclic AMP as the second messenger for target-specifici trophic hormone action and the significance of protein kinase activiation as an index of the cyclic nucleotide involvement in the growth response.


Beta-adrenergic receptors and myogenesis. The rat myogenic cell line, L8, contains a beta-adrenergic catecholamine-sensitive adenylate cyclase. Prior to cell fusion, and continuing thereafter, beta-adrenergic sites, as determined by the stereospecific binding of (125I)-hydroxybenqylpindolol, I1(125I)IHYP] increases from 470 to 2000 sites/cell. There is also an increase in adenylate cyclase (2-5 fold) and endogenous cAMP (5-30 fold) following stimulation by catecholamine. The dissociation constant (KD) of (125I)IHYP for unfused and fused cell-homogenates, as determined by estimation with Scatchard analysis, by direct determination at receptor concentrations well below the KD, or by association (4.6 X 10(8) M-1 min-1); and dissociation (0.028 min-1) kinetics; ranged from about 40 to 70 pM. The acquisition of beta-receptors prior to fusion in L8 cells may implicate this system in the regulation of myogenesis.


Effect of adrenocorticotropic hormone on plasma glucocorticoids and antidiuretic hormone of cattle exposed to 20 and 33 C. Four open, dry Holstein cows in the University of Missouri Climatic Laboratory were used to study the effect of adrenocorticotropic hormone administration on glucocorticoids and antidiuretic hormone of plasma in two reversal trials at thermoneutral (20 C, 50% relative humidity) and heat (33 C, 50% relative humidity) conditions. Average glucocorticoids (saline versus adrenocorticotropic hormone) at thermoneutral were 8.3 and 41.2 ng/ml while values for heat were 6.2 and 40.4 ng/ml. The effect of heat on glucocorticoids of plasma was not significant. Injection of adrenocorticotropic hormone under thermoneutrality and heat caused a marked increase in antidiuretic hormone within 5 min which lasted to 120 min. At thermoneutral average antidiuretic hormone levels (saline versus adrenocorticotropic hormone) were .5 and 1.9 pg/ml while values for heat were 1.8 and 3.6 pg/ml. Antidiuretic hormone concentrations under heat were higher than thermoneutral in cows injected with saline and andrenocorticotropic hormone. The fast response of antidiuretic hormone of plasma to exogenous adrenocorticotropic hormone suggests that adrenocorticotropic hormone might act directly at higher levels of the central nervous system causing release of antidiuretic hormone.


Changes in blood and urine composition from feeding polybrominated biphenyls to pregnant Holstein heifers. The toxicity of a commercial preparation of polybrominated biphenyl was determined in 24 pregnant Holstein heifers that were alloted randomly to one of four experimental groups given 0 (I), .25 (II), 250 (III), and 25,000 (IV) mg per day of fireMaster BP-6 for 60 days or until the animals became moribund. Clinicopathologic determinations were on day -1 prior to dosing, days 15, 30 and 60 during dosing, and following dosing on days 80, 110, 150, and 190 from start of dosing. In addition, samples were collected from moribund heifers of Group IV immediately prior to necropsy. Toxicity was not evident in heifers in Groups I, II, or III. Toxicity was induced in heifers in Group IV. Glutamic-oxaloacetic transaminase of serum was increased and calcium decreased as early as day 15 whereas significant increases in lactate dehydrogenase, urea nitrogen, and bilirubin, and decreases in albumin were not observed until day 30 in heifers of Group IV. Analysis of urine from moribund heifers of Group IV revealed moderate proteinuria and decreased specific gravity. Evaluation of clinicopathologic data has suggested that the polybrominated biphenyls fed were renal toxins with no clear evidence of hepatotoxicity.


Activation of human B lymphocytes VII. the regulatory effect of cyclic adenosine monophosphate on human B cell activation. Cyclic adenosine monophosphate (cAMP) has been demonstrated to play an integral role in the regulation of B cell activation. By employing a plaque-forming cell (PFC) assay for polyclonal activation of human B lymphocytes, it was demonstrated that dibutyryl cyclic adenosine monophosphate (DB-cAMP) markedly increased the PFC response of pokeweed mitogen (PWM)--stimulated lymphocytes. Inducers of intracellular cAMP effected a comparable enhancement. Co-cultures of fresh lymphocytes with autologous T cells which had been pre-incubated with DB-cAMP produced an enhancement of B cell activation by a selective effect on the T cells. The mechanism of action of this enhancement of the B cell response is most likely a relative increase in helper T cell function resulting from a selective inhibition of suppressor T cells.


LPS-or 8Br-cyclic AMP-induced production of T cell-activating factor(s) in macrophage tumor cell line J774.1. The macrophage tumor cell line J774.1 replaced the function of normal macrophages in the induction of polyclonal killer T cells with 2-mercaptoethanol. J774.1 does not normally release soluble factor(s) which we have shown to be responsible for the differentiation of T cells to killer T cells. However, stimulation of J774.1 with LPS induced soluble factor(s) for T cell activation. An optimum concentration of LPS for the production of soluble factor(s) was 1 to 10 microgram/ml, which completely inhibited growth of the tumor cells. The production of soluble factor(s) was observed within 6 hr after LPS stimulation and reached its maximum level at 24 hr. Incubation of the cell line with 8Br-cyclic AMP and theophylline induced soluble factor(s), suggesting that LPS stimulation induced an increase in intracellular cyclic AMP which leads to the synthesis of soluble factor(s).


The central nervous system-associated immune response to parainfluenza type I virus in mice. The immune response in the cerebrospinal fluid (CSF) and serum of BALB/c mice was compared after intracerebral (i.c.) inoculation with inactivated parainfluenza type 1 virus. The antiviral antibody response in CSF peaked approximately 11 days after primary i.c. inoculation coinciding with or even slightly preceding the response in the serum. Prior extracerebral priming of the mice by the intranasal or i.v. route did not alter the kinetics of the response in CSF. However, the antibody response in CSF after i.c. inoculation was accelerated if the mice were primed previously by the i.c. route. In all instances, CSF and serum differed markedly with regard to the isotype composition, which was characterized by a 20- to 80-fold increase in IgA over IgG1 and IgG2 in CSF. Taken together, the results prove that part of the antiviral antibodies in CSF are locally produced. In addition, the results indicate that after primary i.c. inoculation with virus, the CNS acquired immunocompetence with regard to the secondary anti-parainfluenza response.


The distribution of the inherited Hl 1 and R 67 antigens on rabbit serum high density lipoprotein (HDL) particles. Qualitative and quantitative analysis of the distribution of Hl 1 antigen and R 67 antigen on high density lipoprotein (HDL) particles were conducted on six rabbit sera. The Hl 1 antigen showed non-identity with A-I-containing HDL particles, and antiserum to Hl 1 did not remove A-I-containing HDL particles. The R 67 antigen showed partial identity with A-I-containing HDL particles. Antiserum to R 67 antigen removed 30--40% of A-I-containing HDL particles from the sera of animals which were heterozygous for R 67, and 65% of the A-I-containing HDL particles from the serum of one R67 homozygous animal.


Purification and partial characterization of the apoA-I of rabbit high density lipoprotein. Purification of apoA-I from rabbit high density lipoproteins (HDL) gave one single band in sodium dodecylsulphate-polyacrylamide disc electrophoresis and reacted only with antiserum to apoA-I. The molecular weight was about 25000. The amino acid composition of rabbit ,poA-I gave a difference index of 7.4 compared to human apoA-I and of 6.5 compared to dog apoA-I. Of the three carboxy terminal amino acid residues the rabbit protein has one in common with the dog. The amino acid sequence of twenty-nine amino terminal residues showed 62% homology with the human protein; a minimum of thirteen base changes were required in the DNA sequences that encoded these two proteins. When the same sequence was compared with dog apoA-I, it was found that ten base changes would be sufficient to account for the differences between the proteins.


Cyclic nucleotide-phosphodiesterase in the uninvolved and involved skin of psoriasis. In the present study we have compared cyclic nucleotide-phosphodiesterase activities and affinity of phosphodiesterase for substrates (Km) in enzyme preparations obtained from the involved and uninvolved skin of psoriatic patients. With crude skin homogenates we consistently obtained two Km values (high and low) for both the involved and uninvolved, and both Km values were nearly identical between the involved and uninvolved. The same conclusion is also drawn from the Km determinations with partially purified preparations. Cyclic AMP-phosphodiesterase activities with crude homogenates showed no statistically significant differences between the involved and the uninvolved skin. However, when cyclic AMP- and cyclic GMP-phosphodiesterase activities were compared with a highly sensitive assay method in "pure" epidermal samples, which were microdissected free from stratum corneum, dermis and skin appendages, the involved skin contained 40% more activity of the low Km enzyme and 100% more of the high Km enzyme of both cyclic AMP- and cyclic GMP-phosphodiesterase. It is suggested that this may be due to a higher proportion of germinative cells in the lesional epidermis.


Cyclic AMP accumulation in psoriatic skin: differential responses to histamine, AMP, and einephrine by the uninvolved and involved epidermis. Using the uninvolved and involved skin from psoriatic patients, we investigated the effects of histamine and AMP (or adenosine) in vitro on the intracellular cyclic AMP levels. Both agents activated adenylate cyclase of the uninvolved and involved resulting in the accumulation of cyclic AMP. Without a cyclic nucleotide phosphodiesterase (PDE) inhibitor, these responses were biphasic and the maximal accumulation was observed in 5 min. With the PDE inhibitor both responses were markedly potentiated and high levels of cyclic AMP were observed for more than 20 min. The response to histamine by the involved skin was much greater than that by the uninvolved. The degree of the response to adenosine was approximately equal. In accordance with our previous work, the response to epinephrine by the involved skin was much less than that by the uninvolved. Thus adenylate cyclases of involved skin from psoriatic patients exhibit a markedly diminished response to epinephrine while at the same time exhibiting a markedly enhanced response to histamine. This precludes the possibility that the unresponsiveness to epinephrine can be due to a generalized inability of the epidermal psoriatic plaque cell to make a functioning cell membrane.


An immunoelectron microscopic localization of wart associated antigens present in human papilloma virus (HPV) infected cells. Wart associated antigens were observed by the indirect immunoperoxidase technique in light and electron microscopy. Positive reaction products could be found in the nuclei of wart cells, in which virus particles were labeled with a specific immune rabbit serum and with human sera from patients with warts. Tissue antigens, differing from the viral labeled inclusions, were detected within the cytoplasm of some infected cells, by means of IgM and IgG antibodies from patients with warts. In these particular cells the positive reaction occurred in irregular patterns giving microgranular precipitates, which suggests that the antigen was not uniform. No staining was observed on the cell surface. This study has shown direct immunomorphological evidence in vivo of a specific immune reaction in man directed against whole virus and wart specific cellular antigens.


Biochemical characterization of pituitary-dependent hyperadrenocorticism in the dog. The biochemical characterization of 22 cases of pituitary-dependent hyperadrenocorticism in the dog, is reported. The principal characteristics of the disease include excessive and non-rhythmic production of cortisol, decreased sensitivity of the hypothalamic-pituitary system to the suppressive effects of dexamethasone, decreased responsiveness of the pituitary-adrenocortical system to the stimulus of insulin-induced hypoglycaemia and increased responsiveness of the system to stimulation with lysine-vasopressin. From these observations it is concluded that pituitary-dependent hyperadrenocorticism in the dog is a valid model for study of the pathogenesis of the disease in man. For the diagnosis of hyperadrenocorticism itself, the measurement of the concentration of corticosteroids in a single sample of plasma obtained 8 h after intravenous injection of 0.01 mg dexamethasone/kg was sufficient. The level of 11-hydroxycorticosteroids was less than 140 nmol/1 plasma in normal dogs, whereas higher values were found in dogs with hyperadrenocorticism. For purposes of differential diagnosis, measurement of the level of corticosteroids in the plasma both before and 4 h after intravenous injection of 0.05 mg dexamethasone/kg is adequage: suppression is obtained only in cases of pituitary-dependent hyperadrenocorticism.


Possible role of gamma-aminobutyric acid synthesis in the mechanism of dexamethasone feedback action. Specific inhibition of glutamic acid decarboxylase (GAD, EC 4.1.1.15; the main enzyme involved in the synthesis of gamma-aminobutyric acid) by mercaptopropionic acid interferes with the effect of dexamethasone on both the resting and stress-induced secretion of ACTH. It is postulated that dexamethasone may, at least in part, inhibit the secretion of ACTH via the induction of GAD, thereby raising the level of gamma-aminobutyric acid in the central nervous system.


Properties of rat adrenal zona reticularis cells: preparation by gravitational sedimentation. An enriched fraction of zona reticularis cells was obtained by unit gravity sedimentation of decapsulated adrenal glands from female rats. From light microscopic and ultrastructural studies of the whole gland and the isolated cell fractions, the zona reticularis cells of the adrenal gland can be classified mainly on the bases of size, position and mitochondrial morphology. This cell population consists of two types of cell, the 'true' zoma reticularis cells (Type I, modal diameter 9 micrometer), which usually constitute 90% of the isolated reticularis fraction and 80% of the intact reticularis tissue, and cells (Type II, modal diameter 13 micrometer) with fasciculata-like properties (rich in lipid and spherical mitochondria with vesicular cristae). Staining of the cell preparation for 3beta-hydroxysteroid dehydrogenase activity also demonstrates the existence of two types of cell in the zona reticularis. The zona reticularis cell fraction, like the zona fasciculata cell fraction, was capable of producing the subsequent steroids from radioactive pregnenolone: corticosterone, deoxycorticosterone, 18-hydroxydeoxycorticosterone, 11-dehydrocorticosterone, progesterone and androstenedione. However, the pattern of steroid production differed markedly between the zona reticularis and zona fasciculata cells, particularly with respect to the production of deoxycorticosterone and corticosterone (and its correlated steroids, 11-dehydrocorticosterone and 18-hydroxydeoxycorticosterone). When R (the ratio of deoxycorticosterone : corticosterone plus 11-dehydrocorticosterone) for the purest preparation of reticularis cells was compared with R for the corresponding preparation of fasciculata cells, the normalized ratio was found to be 6.4, 16.4 and 20.1 in three experiments. The pattern of production of androstenedione per cell was similar in the reticularis and fasciculata cell fractions. The exact mechanism for the altered pattern of steroid metabolism remains to be elucidated. However, these results establish that the corticosteroids produced by the cells of the zona reticularis may be quantitatively, if not qualitatively, different from those produced by the zona fasciculata cells.


The effect of nalidixic acid group compounds on reduction of cytochrome c from horse heart and Candida krusei. Reduction of cytochrome c from both horse heart and Candida krusei by FeSO4 has been demonstrated. This reaction was stimulated by nalidixic acid and structurally related compounds, and the effect was more pronounced for the yeast cytochrome. Divalent metal ions other than Fe2+ lessened or abolished the stimulation by these compounds. Fe2+ and other metal ions altered the spectra of nalidixic acid and related compounds indicating the formation of metal chelate complexes. 1,10-Phenanthroline inhibited reduction of cytochrome c b Fe2+. Other divalent metal ions relieved the inhibition, probably by forming chelates with 1,10-phenanthroline. The results suggest that metal ion chelation may be involved in the molecular mode of action of nalidixic acid and related drugs. The relevance of this artificial electron transfer system to bacterial electron transfer in vivo is discussed.


Feedback synaptic interaction in the dragonfly ocellar retina. The intracellular response of the ocellar nerve dendrite, the second order neuron in the retina of the dragonfly ocellus, has been modified by application of various drugs and a model developed to explain certain features of that response. Curare blocked the response completely. Both picrotoxin and bicuculline eliminated the "off" overshoot. Bicuculline also decreased the size of response and the sensitivity. gamma-Aminobutyric acid (GABA), however, increased the size of response. The evidence indicates the possibility that the receptor transmitter is acetylcholine and is inhibitory to the ocellar nerve dendrite whereas the feedback transmitter from the ocellar nerve dendrite may be GABA and is facilitory to receptor transmitter release. The model of synaptic feedback interaction developed to be consistent with these results has certain important features. It suggests that the feedback transmitter is released in the dark to increase input sensitivity from receptors in response to dim light. This implies that the dark potential of the ocellar nerve dendrite may be determined by a dynamic equilibrium established by synaptic interaction between it and the receptor terminals. Such a system is also well suited to signalling phasic information about changes in level of illumination over a wide range of intensities, a characteristic which appears to be a significant feature of the dragonfly median ocellar response.


Structures of polyoma virus: on the histone component and virion core. We have observed that purified polyoma virus is able to take up an amount of calf thymus histone equivalent to 10 to 50% of its normal histone content under conditions allowing the binding of considerably lesser amounts of several other proteins. Some of the bound histone could not be released by procedures procedures routinely used for virus purification. We have also found that some of the histone present in purified polyoma virus could be selectively released without major breakdown of virus particles. Possible models for virus structure are discussed in the light of the present and other recent data.


The fate of parental herpesvirus DNA after infection with normal and photoinactivated virions. The fate of the 3H-dT-labelled parental DNA of normal and photoinactivated herpes simplex virus type 1 (KOS strain) was followed for 8 h after infection. The sedimentation coefficients of parental virus DNA from cells infected with normal virons increased and became associated with the replicative intermediates of HSV-1 DNA. When cultured in 5-bromodeoxyuridine-containing medium, the buoyant density of the normal parental virus DNA shifted to the hybrid region (containing heavy and light molecules) of the caesium chloride density gradient. The parental virus DNA of photoinactivated virions, however, degraded quickly and did not participate in progeny virus DNA replication.


Trauma to the skin causes recurrence of herpes simplex in the mouse. Mild trauma was induced in the skin of mice latently infected with herpes simplex virus type I by stripping the originally infected ear with cellophane tape. Recurrent herpes simplex developed at this site 2 to 5 days later. It was detected clinically by the development of erythema and vesicles and by the appearance of virus in the skin. On any one occasion about 30% of mice showed reactivated disease and increasing the severity of trauma did not increase this proportion. However the majority of animals developed reactivated disease on some occasions when stripping was repeated at monthly intervals. The results are discussed in relation to the skin trigger theory of reactivation of herpes simplex.


Infection by herpes simplex virus and cells of nervous system origin: characterization of a non-permissive interaction. Replication of herpes simplex virus type I (HSV-I) was studied in various cell lines of rat nervous system origin. Infection of neonatal rat glial primary cells with HSV-I, strain KOS, produced normal yields of progeny virus. Glioma lines B9 and B15 were permissive, the neuronal line B50 was partially restricted (10 to 100-fold reduction) and the neuronal line B103 was non-permissive (greater than 1000-fold reduction) for HSV-I (KOS) replication. Synthesis of virus DNA in infected B103 cells was not detected. However, at least some virus macromolecular synthesis was induced, including production of thymidine kinase, DNA polymerase and virus structural proteins.


Cyclic excretion of hepatitis A virus in experimentally infected chimpanzees: biophysical characterization of the associated HAV particles. Experimental infection of two chimpanzees with the Phoenix Antigen strain of HAV resulted in the cyclic excretion of virus particles on days 9-11, 14-15, and 20-21 postinoculation. Isopycnic banding in CsCl of stool suspensions prepared from 9-11; 14-15; and 17, 19, 21 dav stool pools revealed multiple buoyant densities for the associated HAV particles. Hollow HAV particles found in the 9-11 day pool banded primarily at a buoyant density of 1.30 g/cm3. HAV in the 14-15 day stool banded bimodally in a CsCl gradient, with antigen peaks at buoyant densities of 1.29 and 1.33 g/cm3. HAV in the days 17, 19, 21 stool pool also banded bimodally in a CsCl gradient; however, the antigen peaks occurred at buoyant densities of 1.33 and 1.40 g/cm3.


The virion and soluble antigen proteins of variola, monkeypox, and vaccinia viruses. The structural proteins in purified preparations of variola, monkeypox, and vaccinia viruses were separated and compared by using a high resolution SDS-polyacrylamide gel electrophoresis system. About 30 proteins were resolved for each virus by autoradiography of longitudinally-sliced gel rods. Although the autoradioelectropherograms of each virus were similar, it was possible to differentiate them by their unique protein pattern in the 30,000 to 40,000 molecular weight region of the gels. A single virion glycoprotein (mol. wt. = 38 X 10(3)) and a virion phosphoprotein (mol. wt. = 12 X 10(3)) were associated with each of the virus preparations. Cross-absorbed monospecific immune sera against variola, monkeypox, and vaccinia virus-infected cells were used in immunodiffusion tests to precipitate radiolabeled, homologous, soluble antigen proteins. The predominant antigen protein associated with each immunospecific precipitate had a molecular weight of approximately 73,000.


Residual effects of ethanol and chlordiazepoxide treatments for alcohol withdrawal. Eighteen male alcoholics were randomly assigned to one of two alcohol detoxification treatments. One group received a low dose ethanol treatment while the other group received a chlordiazepoxide treatment. This study compares recovery of sleep EEG and clinical symptomatology following these two detoxification treatments. Sleep EEG and clinical measures were obtained for the final medication day and during a 6-day postmedication "recovery" period. The chlordiazepoxide treatment produced suppression of rapid eye movement (REM) sleep lasting for about 4 days and virtually eliminated delta sleep (stages III and IV) during the recovery period. The low dose ethanol treatment regimen produced less disruption of REM and delta sleep during the recovery period. These findings suggest that under some circumstances an ethanol treatment regimen may prove more beneficial to the healthy alcoholic patient than current regimens which employ other psychoactive medication. In particular, the long lasting suppression of delta sleep during the recovery period in subjects treated with chlordiazepoxide suggests a vulnerability of the slow wave sleep mechanisms during early alcohol abstinence and raises the possibility that this regimen prolongs functional tolerance to alcohol effects. Continued clinical evaluation of low dose ethanol detoxification treatment is suggested.


Vertebral and epidural hemangioma with paraplegia in Klippel-Trenaunay-Weber syndrome. Case report. A patient with a rare case of Klippel-Trenaunay-Weber syndrome presented with paraplegia due to compression by a vertebral and epidural cavernous hemangioma. The metameric distribution of the large cutaneous vascular nevus provided the clinical clue to the nature of the spinal lesion. The association of the two lesions is explained in the basis of developmental anomaly.


Ga-67 citrate imaging in tumors of the genito-urinary tract: report of cooperative study. Whole-body imaging with Ga-67 citrate in 127 tumors of the genito-urinary tract has been evaluated by a cooperative group using a uniform protocol. Primary sites of tumor were not detectable by imaging, except for one bladder and one kidney tumor. Proven and apparent metastases yielded positive scans, however, in 51% of prostatic, 50% of bladder, 72% of kidney, and 53% of testicular neoplasms. In bladder and kidneys metastases, if bone sites are excluded, detection of soft tissue metastases was 61% and 75%, respectively. In embryonal-cell carcinoma of the testicle, 74% of metastatic foci were detected.


Factors influencing paper chromatographic analysis of technetium-99m phosphorus compounds: concise communication. A single-paper chromatographic system has been developed, capable of resolving Tc-99m dioxide, [99mTc] pertechnetate and Tc-99m phosphorus compounds. The best separations are obtained with CM82 paper developed in 0.5 m naCl, and 3MM or ashless No. 40 paper developed in 1 M sodium acetate buffer. In these systems, 99TcO2 remains at the origin, while 99mTcO4--and Tc-99m phosphorus compounds move with Rf values of 0.56--0.75 and 0.80--1.0, respectively.


Stimulation of lead absorption by vitamin D administration. Two methods for the determination of lead absorption have been developed using the rat as the test animal. One is an everted intestinal sac method in which the time course of 210Pb transport across intestinal wall can be followed in vitro; and the other, 210Pb absorption from the intestine following gastric intubation can be followed in vivo. By using these techniques it can be clearly demonstrated that vitamin D markedly enhances lead absorption. Both techniques provide evidence that the distal small intestine is the major site of lead absorption and the site of greatest vitamin D stimulation. Lead acetate is apparently absorbed to the extent of 45% as it passes through the small intestine at a concentration of 0.01 mM.


Adenoid cystic carcinoma of the parotid gland: report of case. A case of adenoid cystic carcinoma of the parotid gland is reported, in which the initial symptom was facial paresthesia. Pathologic features, treatment, and prognosis of the tumor have been discussed.


Malignant mixed tumor of the soft palate. Malignant mixed tumor (carcinoma within a pleomorphic adenoma) is an unusual tumor that rarely occurs in the minor salivary glands of the soft palate. We have reported a cases and a review of the terminology, diagnosis, and treatment.


Antibody response to live virus vaccines in congenital and neonatal cytomegalovirus infections. When 42 infants with congenitally or neonatally acquired cytomegalovirus infections were one year of age, they were given live attenuated measles-mumps-rubella combined vaccine to determine what effect, if any, the existence of chronic CMV infection has on the antibody response to measles and rubella antigens. When the infected infants were compared with 60 healthy non-CMV-infected control infants, no significant difference in the seroconversion rates or ultimate antibody levels was observed with either measles or rubella antigens. Persistence of antibody, when measured three years after vaccination, was likewise similar in each group; levels had waned only slightly. These results demonstrate the intactness of humoral immunity in children with pre- and perinatal CMV infection.


Sexual precocity in association with septo-optic dysplasia and hypothalamic hypopituitarism. Sexual precocity in association with abnormalities of the central nervous system is well known, but its occurrence with hypothalamic hypopituitarism is most unusual. We report five females with septo-optic dysplasia, blindness, and multiple pituitary tropic hormone deficiencies: all were growth hormone and adrenocorticotropic hormone deficient; two had diabetes insipidus; one had sexual precocity, and one had early pubertal maturation, whereas three were prepubertal and responded to administration of synthetic gonadotropin-releasing hormone. These children retained ability to secrete gonadotropins despite the presence of anterior hypothalamic disease. Experimental data from primates plus our observations on these patients raise questions about the role of the anterior hypothalamus in gonadotropin secretion in man.


Synthesis of as-triazines as potential antiviral agents. Four acenaphtho[1,2-e]-as-triazines and 11 5,6-diaryl-as-triazines, all substituted with an aliphatic or aromatic amino function in the 3-position, were synthesized. Two acenaphthotriazines were active against vesicular stomatitis virus in tissue culture.


Neostigmine-induced alterations at the mammalian neuromuscular junction. I. Muscle contraction and electrophysiology. The effects of single and repetitive injections of neostigmine on neuromuscular physiology were examined in rat extensor digitorum longus muscles. The characteristic facilitation of neuromuscular transmission associated with acute anticholinesterase treatment was accompanied by significant pre- and postsynaptic alterations in neuromuscular transmission. Three days of neostigmine treatment caused a decrease in indirectly and directly elicited muscle contraction. Miniature end-plate potential amplitude and frequency, end-plate potential amplitude, junctional acetylcholine sensitivity and quantal content of nerve-evoked end-plate potentials were also decreased by this treatment. By 22 to 25 days of continued treatment, the decreased rate of transmitter release had returned almost to normal, whereas the alterations of the postsynaptic membrane persisted for as long as 106 days. Alterations were also found in the muscle action potential and in certain passive electrical properties of the extrajunctional muscle membrane. In addition, many of the physiological changes were correlated directly with the morphological changes observed in rats treated similarly. We conclude that neostigmine treatment in rats in therapeutic doses has deleterious effects on neuromuscular physiology and neuromuscular ultrastructure. Although the pattern of these changes is not identical with that seen in rabbit and human myasthenia gravis, the neostigmine treatment used in patients with myasthenia gravis may contribute in part to the neuromuscular alterations observed in this disease.


Pathosis of the hard and soft palate.

Pathosis of the hard and soft palate. In this article a few of the more recently emphasized and pertinent conditions that may affect the hard and soft palate have been discussed. The purpose of the article is not to present in all-inclusive classification of lesions occurring the palate. Disease of the palate may be local in nature or may reflect a systemic condition. Dentists must be conscious of the palate as the site of many possible pathoses. The prosthodontist in particular must observe and carefully evaluate the palate and insure its good health before he can prescribe a prosthesis to cover it. Dentistry provides a health service which affects the entire human organism and not just the oral cavity. Therefore an understanding of the varied nature of pathosis as it relates to the oral cavity, and especially the palate, is essential. Continuous surveillance of the palatal regions insures that the dentist's obligation to detect oral abnormalities in patients is in large measure fulfilled.


Presence of leucocyte inclusions in spleen and bone marrow of patients with Felty's syndrome. The spleens from two patients with Felty's syndrome and the bone marrow from one patient with Felty's syndrome were examined for the presence of intracytoplasmic inclusions in neutrophils when stained with antibodies to IgG, IgM, IgA, and betalc. Four normal spleens from trauma patients were similarly examined. In both spleens and the bone marrow from the Felty patients, large inclusions were noted. In none of the four normal spleens were large inclusions present. These studies suggest that phagocytosis of immune complexes by neutrophils in Felty patients may induce sequestration of these cells in the spleen and bone marrow.


Characterization of the inhibitory effects of retinoids on the in vitro growth of two malignant murine melanomas. The in vitro proliferation of murine melanoma cell lines S91 and B16 was inhibited by retinoic acid and retinyl acetate. The inhibitory effects were dependent on retinoid concentration and increased from 55 and 30% at 10(-9) M retinoic acid to 85 and 82% at 10(-5) M retinoic acid for S91 and B16 melanoma cells, respectively. S91 melanoma cells were more sensitive than B16 melanoma cells to inhibition by either retinoid, and both cell lines were more sensitive to retinoic acid than to retinyl acetate. When exposed to 10(-5) M retinoic acid, the two cell types grew at the same rate as did control cells for 48 hours, whereupon the growth rates of retinoid-treated cells decreased. After 6 days, the number of cells in control cultures increased 140 times (S91 melanoma cells) and 265 times (B16 melanoma cells), whereas retinoic acid-treated cells increased only 14 times (S91 melanoma cells) and 40 times (B16 melanoma cells). The degree of growth inhibition by retinoic acid was not dependent on initial cell density. Cortisone and hydrocortisone failed to prevent or reduce the inhibitory effect of retinoic acid; the release of lysosomal acid phosphatase was not increased and the intracellular level of 3',5'-cyclic AMP in cells grown for 5 days in the presence of 10(-5) M retinoic acid was not elevated. Viability of S91 and B16 cells after 8 days' exposure to 10(-5) M retinoic acid was similar to that in control cultures. The reduced growth rate of retinoic acid-treated cells reversed to the control rate 48-72 hours after removal of retinoic acid from the growth medium.


Enhancement of the tumorigenicity of preneoplastic mammary nodule lines by enzymatic dissociation. Preneoplastic mammary nodule lines D1, D2, and C4 were enzymatically dissociated with collagenase, hyaluronidase, and pronase or with only collagenase and hyaluronidase to produce high yields of viable single cells; 10(5) cells were injected into the cleared mammary fat pads of syngeneic BALB/cCrgl mice. In 11 experiments involving three different preneoplastic nodule lines with different tumor potentials, all dissociated nodule cell lines showed a marked increase in tumorigenicity as compared to the same tissues transplanted as 1-mm3 pieces. The results could not be explained on the basis of differences between the amounts of cells transplanted in the two procedures. In a second series of experiments, normal mammary cells from virgin, pregnant, or lactating mice were mixed in different ratios with 10(5) nodule cells and injected into the mammary fat pads. The presence of normal cells reversed the marked increase in the tumorigenicity of enzymatically dissociated nodule cells to a level equal to or less than the tumorigenicity of control transplants (1-mm3 pieces). The growth of 10(5) mammary tumor cells was not inhibited when tumor cells were mixed with 3 x 10(5) normal cells and transplanted into the mammary fat pads. These results showed that enzymatic dissociation can lead to an increase in tumor potential of preneoplastic mammary nodule lines, and they supported the hypothesis that nodule cells, but not neoplastic cells, are sensitive to the growth-inhibitory effects of normal mammary cells.


Carcinogenicity and acute toxicity of dimethylnitrosamine in rainbow trout (Salmo gairdneri). Four-week-old rainbow trout (Salmo gairdneri) were fed diets containing 0, 3, 50, 200, 400, and 800 ppm dimethylnitrosamine (DMN) for 52 weeks. At the end of 52 weeks, the fish were fed a control diet without DMN for an additional 26 weeks. Samples were taken at 26, 52, and 78 weeks to determine tumor incidence. A dose-related carcinogenic response was established from these results, and an equation was derived to relate the level of the carcinogen to the hepatocellular carcinoma incidence. From a published dose-response study that used outbred Porton rats, a second equation was derived for comparison. Rats and trout were approximately equal in their sensitivity to DMN carcinogenesis. The median lethal dose after ip injection of DMN was 1,770 mg/kg body weight in rainbow trout. Relative to the range of 15-50 mg/kg body weight reported for several mammalian species, trout were resistant to the acute toxicity of DMN.


Aflatoxin B1 induction of hepatocellular carcinoma in the embryos of rainbow trout (Salmo gairdneri). Liver cancer in rainbow trout was induced by exposure of fertile eggs to an aqueous, 0.5 ppm (microgram/ml) solution of aflatoxin B1 (AFB1) for 1 hour. Single treatments, given on alternate days during the embryonic period, produced a low cancer incidence (less than 20%) prior to formation of the embryonic liver on day 14, but a steadily increasing incidence from day 15 (31.7%) until day 23 (58.3%), in fish examined 1 year later. Treatment of trout eggs with [14C]AFB1 was used to quantitate the amount of AFB1 absorbed by the eggs. Twenty-one-day-old rainbow trout eggs absorbed approximately 30 ng of [14C]AFB1 during a 1-hour exposure to 0.5 ppm aqueous [14C]AFB1. After 1 day 85-90% of the [14C]AFB1 was either metabolized and excreted or leached from the egg. The residual [14C]AFB1 remained constant until hatching when an additional 50% was lost. Comparison of the amount of AFB1 absorbed by eggs with the amount of AFB1 consumed per fish during a 1-year feeding trial at 4 ppb in the diet indicates that the trout embryo is even more sensitive than juvenile trout to the carcinogenic properties of AFB1.


Cyclic adenosine 3',5'-monophosphate-binding protein: role in ovariectomy-induced regression of a hormone-dependent mammary tumor in Sprague-Dawley female rats. Cyclic AMP- and estrogen-binding activities were present in 7,12-dimethylbenz[a]anthracene-induced mammary carcinomas in Sprague-Dawley female rats. Soon after ovariectomy of the host, cyclic AMP binding markedly increased and estrogen binding decreased in regressing tumors. These changes were reversed when tumor growth was resumed following the injection of estradiol-17beta. The data suggested the possible involvement of cyclic AMP binding in the growth control of a hormone-dependent mammary tumor.


State of Epstein-Barr virus DNA in an American Burkitt's lymphoma line. A human lymphoma cell line, positive for the Epstein-Barr virus (EBV)-associated nuclear antigen, was recently established from a North American Burkitt's lymphoma. This cell line, SU-AmB-2, contained EBV DNA both in the form of circular, nonintegrated DNA molecules of viral genome length, present in multiple copies per cell, and as integrated sequences. Having DNA present in both of these forms, it resembled cell lines established from African Burkitt's lymphomas. In studies on EBV strain differences, the episomal viral DNA in Burkitt's lymphoma cells may now be compared with viral DNA in nonmalignant cells with the use of cell lines from Burkitt's lymphoma patients of similar geographic origin.


Leukemogenic activity of thymotropic, ecotropic, and xenotropic radiation leukemia virus isolates. Thymotropic, ecotropic, and xenotropic oncoviruses were isolated from the C57BL/6 mouse radiation leukemia system and were propagated in culture. The purified viruses were inoculated singly and in various combinations into groups of mice, and leukemia incidence was determined. Only the thymotropic virus was leukemogenic in vivo.


Integrated viral DNA sequences in Epstein-Barr virus-converted human lymphoma lines. Most human lymphoid cell lines contain multiple copies of circular, nonintegrated Epstein-Barr virus (EBV) DNA molecules as well as viral DNA sequences with properties of integrated DNA. The physical state of the EBV DNA in a human lymphoma line that only contains one virus genome equivalent per cell has now been studied by three different methods, neutral CsCl density gradient centrifugation, actinomycin D-CsCl gradient centrifugation, and Hirt fractionation. This cell line, AW-Ramos, has been obtained by EBV infection in vitro of the apparently EBV-negative Ramos lymphoma line. The results indicate that the EBV DNA in AW-Ramos is present exclusively in a linearly integrated form. Similar data were obtained with two other EBV-converted sublines of Ramos cells.


Polyoma virus-specific RNA synthesis in an inducible line of polyoma virus-transformed rat cells. Viral RNA present in the inducible LPT clone 1A of polyoma virus-transformed rat cells was characterized before and after mitomycin C induction by hybridization with 32P-labeled separated E and L strands of polyoma viral DNA restriction endonuclease fragments. In clone 1A cells maintained under normal growth conditions, the cytoplasm contained a transcript of the E-strand DNA from the "early" region similar to that previously identified in lytically infected cells, as well as minor quantities of RNA complementary to less than one-half of the L- and the E-strand DNA from the "late" region. Nuclei of normally growing cells contained the same species found in the cytoplasm, as well as an additional abundant RNA complementary to one-half of the L-strand DNA of the late region. No significant changes occurred in the cytoplasmic viral RNA after mitomycin C treatment before the onset of viral DNA replication, but the concentration of the nuclear L-strand DNA transcript diminished. After the onset of viral DNA replication after mitomycin C treatment, transcripts of virtually the entire L-strand DNA were found in the nuclei, and a 10-fold increase was observed in the abundance of RNA transcribed from the E strand of the early region. In the cytoplasm, the abundance of the early RNA increased about 25-fold and late RNA complementary to the L-strand DNA of the late region was found in a similar quantity. The synthesis of both the early and the late RNA species was inhibited if viral DNA replication was blocked with 5-fluorodeoxyuridine. We conclude that the induction of viral DNA replication in LPT cells is not determined at the level of mRNA synthesis.


Analysis of cytoplasmic RNA and polyribosmomes from feline leukemia virus-infected cells. Cytoplasmic virus-specific RNA and polyribosomes from a chronically infected feline thymus tumor cell line, F-422, were analyzed by using in vitro-synthesized feline leukemia virus (Rickard strain) (R-FeLV) complementary DNA (cDNA) probe. By hybridization kinetics analysis, cytoplasmic, polyribosomat, and nuclear RNAs were found to be 2.1, 2.6, and 0.7% virus specific, respectively. Size classes within subcellular fractions were determined by sucrose gradient centrifugation in the presence of dimethyl sulfoxide followed by hybridization. The cytoplasmic fraction contained a 28S size class, which corresponds to the size of virion subunit RNA, and 36S, 23S, and 15 to 18S RNA species. The virus-specific 36S, 23S, and 15 to 18S species but not the 28S RNA were present in both the total and polyadenylic acid-containing polyribosomal RNA. Anti-FeLV gamma globulin bound to rapidly sedimenting polyribosomes, with the peak binding at 400S. The specificity of the binding for nascent virus-specific protein was determined in control experiments that involved mixing polyribosomes with soluble virion proteins, absorption of specific gamma globulin with soluble virion proteins, and puromycin-induced nascent protein release. The R-FeLV cDNA probe hybridized to RNA in two polyribosomal regions (approximately 400 to 450S and 250S) within the polyribosomal gradients before but not after EDTA treatment. The 400 to 450S polyribosomes contained three major peaks of virus-specific RNA at 36S, 23S, and 15 to 18S, whereas the 250S polyribosomes contained predominantly 36S and 15 to 18S RNA. Further experiments suggest that an approximately 36S minor subunit is present in virion RNA.


Inhibition of RNA synthesis in mouse myeloma cells infected with vesicular stomatitis virus. Infection of mouse myeloma cells (MPC-11) with vesicular stomatitis (VS) virus resulted in rapid and marked reduction in cellular RNA synthesis considerably before cell viability was compromised. Mouse myeloma cells responded maximally to viral infection at a multiplicity of 1 and were considerably more se;sitive to shut-off of RNA synthesis than were mouse L cells or BHK-21 cells. This inhibition of cellular RNA synthesis was shown not to be caused by differential membrane permeability of infected and uninfected MPC-11 cells to [3H]uridine, nor was it due to greater degradation of previously synthesized RNA. VS viral infection appeared not to impede transport of newly synthesized nuclear RNA to the cytoplasm; moreover, infected cells accumulated polyadenylated mRNA at the same rate as did uninfected cells. Polyacrylamide gel electrophoresis of newly synthesized nuclear RNA demonstrated that the polydisperse nature and size distribution were not affected by VS viral infection. Isolated nuclei of infected MPC-11 cells also inhibited greatly impaired capacity to synthesize RNA despite the absence of cytoplasmic factors. Infected-cell cytosol did not inhibit transcription by uninfected-cell nuclei, nor did uninfected-cell cytosol reverse viral inhibition of nuclear transcription. Studies with alpha-amanitin revealed that VS viral infection inhibited the activity of polymerases I, II, and III, but only polymerase II was affected progressively throughout infection and to a much greater extent. These data suggest that, even at low multiplicities of infection, VS virus rapidly shuts off cellular RNA synthesis at the level of nuclear transcription.


Characterization of the 5'-terminal capped structures of late simian virus 40-specific mRNA. 32P-labeled, late simian virus 40-specific RNA was isoalted from infected CV1 cells and completely degraded with RNase T2 and bacterial alkaline phosphatase. The RNase-resistant material was fractionated two dimensionally and further characterized with Penicillium nuclease and nucleotide pyrophosphatase. Two major 5' termini were identified in late simian virus 40 RNA, namely, 7-methyl Gppp 2',6-dimethyl ApUp and 7-methyl Gppp 2',6-dimethyl Ap 2'-methyl, UpUp. Both 5' termini are present in unfractionated viral RNA as well as in the separated 16S and 19S species. As both caps differ only in secondary modification, it is possible that they are derived from the same site on the DNA. The relatively higher cap II content of the 16S mRNA may be related to its slower rate of turnover.


Intracellular precursors to the major glycoprotein of avian oncoviruses in chicken embryo fibroblasts. A 96,000-dalton glycoprotein, p(96), was present in cell extracts obtained from gs-chf- chicken embryo fibroblasts infected with the avian RNA tumor viruses Rous-associated virus-2 subgroup B (RAV-2) and the Schmidt-Ruppin strain of Rous sarcoma virus subgroup A (SR-RSV-A), as well as from uninfected gsLchf+ (HE) cell extracts. It was not found in cell extracts from uninfected gs-chf- or gs+chf+ (HH) cells, nor from gs-chf- cells infected with envelope-deficient Bryan high-titer Rous sarcoma virus. Immunoprecipitation, kinetic, and biochemical data indicate the this polyprotein contains information that gives rise to the major virion glycoprotein gp85. A second polyprotein of 80,000 daltons, p/80), is also present in the RAV-2- and SR-RSV-A-infected gs-chf- cells. This second polyprotein contains less carbohydrate than p(96), and kinetic and biochemical data indicate that p(80) may be an immature form of p(96).


Solubilization and characterization of Herpesvirus saimiri-induced membrane antigens. Treatment of Herpesvirus saimiri (HVS)-infected owl monkey cells by limited papain digestion removed the HVS-induced membrane antigen (MA) as determined by membrane immunofluorescence and antibody-dependent lymphocyte cytotoxicity (ADLC). Soluble antigenically active HVSMA was detected by inhibition of ADLC and by the decreased binding of 125I-labeled staphylococcus protein A to HVS-infected cells after absorption of an anti-MA-positive serum with papain extracts. Approximately 38% of the inhibitory activity of the papain extracts was sedimentable at 100,000 X g, indicating that the released MA was heterogeneous in size. Preliminary investigations by gel filtration chromatography identified a major peak of MA with a molecular weight between 20,000 and 50,000.


Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus. The function of the A gene of simian virus 40 (SV40) in transformation of BALB/c-3T3 cells was investigated by infecting at the permissive temperature with wild-type SV40 and with six tsA mutants whose mutation sites map at different positions in the early region of the SV40 genome. Cloned transformants were then characterized as to the temperature sensitivity of the transformed phenotype. Of 16 tsA transformants, 15 were temperature sensitive for the ability to overgrow a monolayer of normal cells, whereas three of three wild-type transformants were not. This pattern of temperature sensitivity of the transformed phenotype was also observed when selected clones were assessed for the ability to grow in soft agar and in medium containing low concentration of serum. The temperature resistance of the one exceptional tsA transformant could be attributed neither to the location of the mutation site in the transforming virus nor to transformation by a revertant virus. This temperature-resistant tsA transformant, however, was demonstrated to contain a higher intracellular concentration of SV40 T antigen than a temperature-sensitive line transformed by the same tsA mutant. A tsA transformant displaying the untransformed phenotype at the nonpermissive temperature was found to be susceptible to retransformation by wild-type virus at this temperature, demonstrating that the temperature sensitivity of the tsA transformants is due to the viral mutation and not to a cellular defect. These results indicate that continuous expression of the product of the SV40 A gene is required to maintain the transformed phenotype in BALB/c-3T3 cells.


T antigen of BK papovavirus in infected and transformed cells. BK virus T antigen from BKV-transformed rat and hamster cells and from productively infected monkey cells has been examined by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Forms of the antigen that migrate as proteins of 86,000 and 92,000 daltons have been identified. Both forms can be labeled by 32P.


Physical map of the origin of defective DNA in herpes simplex virus type 1 DNA. The origin of defective DNA (dDNA) of the Patton strain of herpes simplex virus type 1 (HSV-1) was physically mapped with BamHI in the parental DNA. The dDNA obtained from virus passaged at high multiplicities of infection was resistant to cleavage with HindIII, whereas digestion with EcoRI yielded a cluster of fragments 5.4 to 5.7 megadaltons (Mdal) in size. Cleavage with BamHI gave a cluster of fragments 2.6 to 3.2 Mdal in size, plus two homogeneous, comigrating 1-Mdal fragments. One of the latter fragments contained the single EcoRI site approximately 65 base pairs from one end. Hybridization of in vitro labeled dDNA probe to EcoRI, HindIII, BamHI, and Hpa I digests of nondefective HSV-1 DNA demonstrated that, in addition to the S-region terminal repeat, only one end of the S region was involved in the generation of this class of dDNA. Thus, the dDNA probe did not hybridize to either the S region 3.0-Mdal HindIIIN fragment or a 3.0-Mdal BamHI fragment of the adjacent 8.7-Mdal HindIIIG fragment, but did hybridize to four BamHI fragments of HindIII G (approximately 5.7 Mdal). The cluster of 2.6- to 3.2-Mdal fragments obtained with BamHI digestion of dDNA appears to represent a novel junction between the termination of dDNA adjacent to the 3.0-Mdal BamHI fragment in HindIII G and the 2.0- to 2.3-Mdal BamHI fragment terminal in HSV-1 DNA.


High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures. Several 50 to 70S tumor viral RNAs have previously been shown by electron microscopy to be dimers, with the two monomer subunits joined near their 5' ends. Five additional naturally occurring type C RNA tumor viruses have now been examined: AKR, and endogenous murine ecotropic virus; NZB, an endogenous murine xenotropic virus; and ecotropic and an amphotropic virus isolated from a wild mouse; and the avian reticuloendotheliosis virus (REV). All five 50 to 70S RNAs have similar 5'-to-5' dimer structures. Therefore, the observations support the hypothesis that the dimer linkage is a structural feature common to all type C mammalian viruses. REV is the first example of an avian virus with a clear 5'-to 5' dimer linkage. All of the mammalian viral RNAs, but not REV, showed symmetrically placed loops in each subunit of the dimer. Possible molecular structures and biological functions of the dimer linkages and loops are discussed.


Characterization of DNA-protein complexes from simian adenovirus SA7. DNA-protein complexes prepared from purified simian adenovirus SA7 virions and from lytically infected monkey kidney cells exhibited similar properties when compared with respect to size by sucrose gradient centrifugation, to configuration by electron microscopy, and to susceptibility to a variety of treatments by electron microscopy and electrophoresis in agarose gels.


Retrovirus purification: method that conserves envelope glycoprotein and maximizes infectivity. A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques.


RNA tumor virus phosphoproteins: primary structural analysis and identification of phosphopeptides. Two-dimensional tryptic peptide mapping was used to compare the peptide sequences of the phosphoprotein (pp12) of cloned ecotropic and amphotropic wild mouse leukemia viruses, strains 1504 and 292. The maps of two ecotropic isolates were very similar to one another, as were the maps of two amphotropic isolates. There was also extensive similarity between the maps of this protein from ecotropic and amphotropic viruses, although characteristic peptide differences were readily recognized. These differences were consistent with the general type specificity of oncovirus phosphoproteins. The pp12 of the field isoalte of 292 virus contained five phosphopeptides, and the non-phosphorylated and variously phosphorylated species of this pp12 showed identical peptide maps, indicating differential phosphorylation of a single polypeptide.


Infectious primate type C virus proviral DNA in productively infected cells. Cell lines competent to infection by DNA from cultures chronically infected by type C viruses of the simian sarcoma virus and baboon endogenous virus groups were identified. Significant differences were observed in the relative susceptibility of some cell lines to infection by a given proviral DNA. Practical applications of transfection techniques for the separation of viruses from dually infected cultures and to free virus stocks from mycoplasmal contamination are described.


Transcription of BK virus DNA by Escherichia coli RNA polymerase: size and sequence analysis of RNA. Transcription of human papovarirus BK superhelical DNA by Escherichia coli RNA polymerase yielded symmetric RNA with an average chain length of 1,3000 nucleotides.  All regions of human papovavirus BK DNA were equally transcribed.  At least four initiation sites were available to the procaryotic enzyme.


Antiviral action of a rifamycin derivative: formation Rous sarcoma virus particles deficient in 60 to 70S RNA. Growth of Rous sarcoma virus-transformed cells in the presence of rifazone-8(2), a rifamycin derivative, results in the formation of noninfectious virus particles lacking 60 to 70S RNA.


Autonomic neuropathy and the genitourinary system. Autonomic and pudendal neuropathies are frequent and neglected diseases affecting the genitourinary system. In the initial stages they frequently are asymptomatic. Autonomic neuropathy is seen in association with peripheral neuropathy in a wide range of metabolic diseases, including diabetes mellitus. Diagnosis can only be made directly by electrophysiologic methods. Treatment is based upon an adequate laboratory study of genitourinary function.


Intraductal carcinoma. Long-term follow-up after treatment by biopsy alone. A follow-up period averaging 21.6 years was obtained for patients with low-grade papillary intraductal carcinoma initially treated only by biopsy between 1940 and 1950. Subsequent carcinoma was diagnosed in the same breast in seven of the ten patients after an average interval of 9.7 years. Six of the seven subsequent carcinomas were invasive. Two of the patients died of metastatic carcinoma and two were known to be alive with metastases when last contacted. Three patients were without carcinoma following mastectomy. When these results were combined with the few reports available in the literature, it appeared that at least 39% of patients with intraductal carcinoma treated by biopsy alone subsequently had clinically evident carcinoma, invariably in the same breat, with an average latent period of about ten years. This was undoubtedly a result of the multicentric nature of the disease in many patients.


Evaluation of zoster immune plasma. Treatment of cutaneous disseminated zoster in immunocompromised patients. Zoster immune plasma (ZIP) was evaluated for treatment of cutaneous disseminated zoster in immunocompromised hosts. Twenty patients were studied: 13 were enrolled in a double-blind protocol, five received ZIP under an open protocol, and two were observed without receiving a transfusion. In the double-blind study, eight patients actually received ZIP; five were given plasma lacking varicella-zoster virus antibodies (control plasma). The clinical course of zoster in the group given ZIP was the same as that of patients given control plasma or no transfusions. Because ZIP did not alter the clinical course of zoster and because zoster patients produced high-antibody titers without ZIP, we concluded that ZIP is not useful for treatment of cutaneous disseminated zoster and should be reserved for prevention or modification of varicella in exposed, susceptible immunocompromised patients.


[Content of testosterone and of different classes of lipoproteins in the blood plasma of men 40-59 years old]. The blood plasma of males aged from 40 to 59 (227 practically healthy individual and 48 persons suffering from ischemic heart disease) was tested for the content of testosterone, cholesterol, triglycerides, cholesterol of various classes of lipoproteins and the lipoprotein spectrum by electrophoresis in polyacrylamide gel. It was found that a change in the testosterone level in the blood plasma, even within the limits of physiological values, may affect the plasma lipoprotein spectrum: a decrease in the testosterone concentration is attended with an increase in the content of atherogenic (beta- and pre-beta-) pipoproteins and a decrease in the content of alpha-lipoproteins.


[Change in the reactions of isolated vascular segments to electric stimulation under the influence of inderal]. It is shown that under the effect of inderal the relaxation of isolated vascular segments in response to electric stimulation is replaced by their contraction. In the event of initial contractile reactions these are noticeably potentiated. The conditions of the experiment do not allow the effect observed to be explained by the block of specific beta-adrenergic receptors by inderal. Taking into account the data on the effect of inderal on the intracellular biochemical processes, the authors presume that one of the intimate mechanisms of changes in the vascular effects due to inderal are alterations in the content of intracellular calcium.


Influence of steroids on urea-cycle enzymes in chronic human liver disease. Activities of Krebs-Henseleit enzymes were determined in liver biopsies of normal persons and in patients suffering from alcoholic hepatitis and chronic active hepatitis. Prednisone was administered for five days in falling dosis (1.5 mg-0.5 mg/kg/body weight) to patients with alcoholic hepatitis and to controls. Patients with chronic active hepatitis received 15-20 mg prednisone daily for more than three months. In healthy persons prednisone did not influence the activities of Krebs-Henseleit enzymes. In patients with alcoholic hepatitis most of the urea-cycle enzymes are significantly decreased (p is less than 0.05) when compared to controls. After glucocorticoid administration enzyme activities remained unchanged. Activities of most of the urea-cycle enzymes are significantly (p is less than 0.05) decreased in untreated patients with chronic active hepatitis. In some of these patients, glucocorticoid administration was associated with a remission as proved by clinical, biochemical and histological data. Activities of the rate-limiting enzymes of the urea-cycle (ASAS, CPS) increased significantly in these patients. By contrast, alterations of enzyme activities could not be observed in patients who failed to respond favourably to steroid treatment.


Perhexiline induces generalized lipidosis in rats. Administration of perhexiline (Pexide) to rats causes generalized occurrence of lamellated and crystalloid cytoplasmic inclusions which resemble those described in patients with perhexiline-induced polyneuropathy. It is concluded that perhexiline being an amphiphilic cationic compound is a potent inducer of generalized lipidosis.