Comparative activity of immunofluorescent antibody and complement-fixing antibody in cytomegalovirus infection. Three different tests for detection of antibodies to human cytomegalovirus (CMV), complement fixing with antigen prepared by freeze-thaw disruption (CF-FT) or with antigen prepared by extraction with alkaline glycine buffer (CF-GE) and immunofluorescent staining (FA), were compared in renal transplant recipients and their healthy donors, FA and CF-GE tests yielded positive results at an identical and significantly higher frequency than CF-FT in both donors and recipients. CF-GE and FA performed on donors and recipients predicted all virus shedding post-transplant, whereas CF-FT did not. In the individuals who developed primary infection concurrent with the transplanted kidney, FA developed earlier than other antibodies in about one-half and at the same time in the remainder. In addition, the FA test could be completed more quickly and all sera could be interpreted, which made the FA test more useful than the CF-GE, but both of these tests were clearly superior to CF-FT.


Application of freeze-dried, one-day-old chick erythrocytes to viral hemagglutination and hemagglutination-inhibition tests. Erythrocytes collected from 1-day-old chicks were stabilized by fixation with formaldehyde and by freeze-drying after treatment with carbon monoxide. Suspensions of freeze-dried erythrocytes in distilled water or physiological saline had a homogeneous bright reddish-purple color. Freeze-dried erythrocytes were compared with fresh erythrocytes for hemagglutination and hemagglutination-inhibition tests for various viruses including rubella, Japanese encephalitis, influenza, mumps, Newcastle disease, and Sendai viruses. After storage at 4 degrees C for 1 year or more, freeze-dried erythrocytes maintained their original appearance and sensitivity to hemagglutination antigens.


Replication of murine paramyxoviruses in hamster tracheal organ culture and comparison with standard tissue culture methods. Replication of Sendai virus, pneumonia virus of mice, and SV5 was investigated in tracheal organ cultures from 2- to 4-day-old and 2- and 4-week-old hamsters, and viral infectivity in tracheal explants was compared with that in tissue culture monolayers. Explants from 2- to 4-day-old hamsters produced higher titers of the three paramyxoviruses, as detected by hemadsorption with guinea pig and murine erythrocytes in primary rhesus monkey kidney cells. Tracheal cultures from 2- and 4-week-old hamsters yielded 1.5 and 2.5 log10 lower infectivity titers. Infected explants exhibited cytopathological changes that correlated well with cessation of ciliary activity. Viral titers in BHK-21, Vero, and BS-C-1 monolayer cells, the systems commonly used for isolation and propagation of murine paramyxoviruses, were lower than those in 2- to 4-day-old hamster tracheal explants. These observations suggest that hamster trachea organ culture could have practical application as an aid for primary isolation of paramyxoviruses from clinical specimens from rodents with respiratory ailments.


In vitro action of bombesin and bombesin-like peptides on amylase secretion, calcium efflux, and adenylate cyclase activity in the rat pancreas: a comparison with other secretagogues. Bombesin (a tetradecapeptide), the C-terminal nonapeptide of bombesin (bombesin-NP), and litorin (a parent nonapeptide), each stimulated amylase secretion from rat pancreatic fragments. These responses were not affected by atropine. The concentrations that produced half-maximal stumulation of secretion were 0.25 nM for bombesin, 0.30 nM for bombesin-NP, and 0.07 nM for litorin, as compared to 0.12 nM for caerulein and 0.80 muM for the cholinergic agent carbamylcholine. When used at maximal concentrations, bombesin, bombesin-NP, and litorin showed no action on cyclic AMP levels in the presence of 5 mM theophylline. By contrast, caerulein and secretin increased cyclic AMP levels by 27 and 208%, respectively. Bombesin, bombesin-NP, and litorin did not activate adenylate cyclase in a purified pancreatic plasma membrane preparation, whereas caerulein and secretin increased this activity 20 and 16-times, respectively...


Inhibition of lymphoproliferation by hyperlipoproteinemic plasma. Plasma for patients with primary type IV or V hyperlipoproteinemia inhibited [3H]thymidine incorporation by cultured mononuclear leukocytes. This previously unreported abnormality affected mononuclear leukocytes from patients with type IV or V hyperlipoproteinemia and from normal subjects. Patient cells incorporated [3H]thymidine normally when washed and incubated in medium containing normal plasma. Both spontaneous incorporation and stimulated incorporation in response to various mitogens and antigens were inhibited. The inhibitory effect was identified with the chylomicron and very low density lipoprotein fractions isolated from plasma and was concentration-dependent. Lectin used to stimulate cultured cells and [3H]thymidine used to measure responses were not bound to the lipoproteins in appreciable amounts. [3H]-Thymidine incorporation correlated well with morphologic evidence of lymphoproliferation. The mechanism of the inhibitory effect of type IV or V hyperlipoproteinemic plasma upon the response tested was not identified by may be related to interaction between lipoproteins and the cell membranes. We suggest that these lipoproteins may also interfere with the function of other cells.


Selective inhibition of osmotic water flow by general anesthetics to toad urinary bladder. Vasopressin increases the permeability of the total urinary bladder, an analogue of the mammalian renal collecting duct, to water and small solutes, especially the amide urea. We have observed that three general anesthetic agents of clinical importance, the gases methoxyflurane and halothane and the ultrashortacting barbiturate methohexital, reversibly inhibit vasopressin-stimulated water flow, but do not depress permeability to urea, or the the lipophilic solute diphenylhydantoin. In contrast to their effects in vasopressin-treated bladders, the anesthetics do not inhibit cyclic AMP-stimulated water flow, consistent with an effect on vasopressin-responsive adenylate cyclase. The selectivity of the anesthetic-induced depression of water flow suggests that separate adenylate cyclases and cyclic AMP pools may exist for control of water and urea permeabilities in to toad bladder. Furthermore, theophylline's usual stimulatory effect on water flow, but not its effect on urea permeability, was entirely abolished in methoxyflurane-treated bladders, suggesting that separate phosphodiesterases that control water and urea permeabilities are present as well. We conclude that the majority of water and urea transport takes place via separate pathways across the rate-limiting luminal membrane of the bladder cell, and that separate vasopressin-responsive cellular pools of cyclic AMP appear to control permeability to water and to urea.


Viral infections.

Viral infections. Although rubella is the only virus which can be regarded in the strict sense of the term a teratogen, there is no convincing evidence that other viruses can cause fetal damage of varying severity. The risk to the fetus appears to depend on the nature of the infectious agent, the maternal immune status and the gestational age when infection takes place. The possibility that subclinical maternal infections may cause damage must not be overlooked. As some of the viruses referred to can cause damage after the period of organogenesis, the use of the term 'teratogenic efect' in relation to viral infections is considered to be inappropriate.


Relations between behavioral arousal and plasma cortisol levels in monkeys performing repeated free-operant avoidance sessions. The commonly believed hypothesis that increases in adrenocorticoid levels sensitively reflect behavioral arousal was tested by subjecting monkeys to repeated sessions of free-operant avoidance; these sessions produced varying degrees of behavioral arousal over time, which were quantified by a behavioral scoring technique. Cortisol was reliably elevated only in the most aroused subjects early in the first avoidance session. Although subjects were still aroused later in the session, cortisol had returned to basal levels. During subsequent avoidance sessions, cortisol returned to basal levels and did not increase significantly even when arousal was further manipulated by the superimposition of unavoidable shock during the final avoidance session. The addition of unavoidable shock was associated with a significant correlation between arousal, as reflected by increases in response rate, and magnitude of change, usually decreases, in mean cortisol levels. The frequent occurrence of dissociations between cortisol levels and behavioral arousal, as reflected by behavioral score, operant rate, and shock frequency, indicates that cortisol levels are of little use as a neuroendocrine index of arousal.


Hypnotic efficacy of triazolam: sleep laboratory evaluation of intermediate-term effectiveness. Triazolam (U-33030), an investigational hypnotic drug, was evaluated in seven insomniac subjects in the sleep laboratory. The protocol consisted of 22 consecutive nights: four placebo nights for adaptation and baseline, two weeks of drug administration (0.5 mg triazolam) for short- and intermediate-term drug effectiveness, and four placebo nights for withdrawal effects. With short-term drug use, both sleep induction and sleep maintenance improved, with total wake time decreasing markedly--a 45 per cent decrease from baseline. At the end of two weeks of drug use, none of the efficacy parameters was significantly decreased from baseline; there was only a 17 per cent decrease in total wake time. Following drug withdrawal, sleep difficulty significantly increased above baseline levels. Two of the subjects experienced episodes of amnesia during the drug administration period. The per cent of REM sleep decreased significantly during both short and intermediate drug conditions. Following drug withdrawal, the per cent of REM sleep was similar to baseline. Slow-wave (stages 3 and 4) sleep was significantly decreased for both drug conditions; and following drug withdrawal, it returned completely to the baseline leve. These data indicate that triazolam is effective for short-term use, loses most of its effectiveness with intermediate-term use, and its withdrawal is followed by a significant sorsening of sleep. These findings are discussed in relation to the potential labeling and promotion of triazolam. Finally, the findings of amnesia associated with triazolam administration need to be more thoroughly evaluated.


Absorption and biotransfomation of cholecalciferol in drug-induced osteomalacia. The gastrointestinal absorption of vitamin D3 and its biotransformation to 25-hydroxycholecalciferol was studied in patients with drug-induced osteomalacia. The mean coefficient of absorption was virtually identical in the drug-treated group (82.8%) and the control (83.8%). The biotransformation of vitamin D3 to 25-hydroxycholecalciferol was significantly accelerated (P less than 0.03) in the drug-treated group compared to the control group. These data suggest that vitamin D absorption is not significantly altered but that biotransformation of vitamin D3 to 25-hydroxycholecalciferol is accelerated in drug-induced osteomalacia.


Kinetics of irreversible activation of adenylate cyclase of fat cell membranes by phosphonium and phosphoramidate analogs of gtp1. The ability of guanylylimidodiphosphate (GMP=P(NH)P) and guanylylmethylenediphosphonate (GMP-P(CH2)P) to activate adenylate cyclase activity has been studied by incubating these analogs with fat cell membranes followed by thorough washing of the membranes before assay of enzyme activity. GMP-P(NH)P is hydrolyzed by membrane preparations from several tissues. A pyruvate kinase regenerating system maintains the concentration of GMP-P(NH)P and thereby augments the ability of suboptimal concentrations of GMP-P(NH)P to activate adenylate cyclase. GTP inhibits activation of fat cell membrane adenylate cyclase by GMP-P(NH)P but this inhibition is overcome by time. This is consistent with the virtually irreversible nature of the GMP-P(NH)P activation, and with the inability of GTP to reverse the stimulated state of the enzyme. Although the initial rate of enzyme activation is highly dependent on the concentration of GMP-P(NH)P, with increasing times of incubation nearly the same maximal extent of activation is seen over a wide range of concentrations. Thus, it is not possible to estimate true affinity constants (at equilibrium) for GMP-P(NH)P, as anticipated from the virtually irreversible character of the activation process.


Immunohistochemical localization of cyclic nucleotides during testicular development. The potential role of cyclic nucleotides in rat testicular development was evaluated by measuring tissue levels of cyclic AMP and cyclic GMP during a time of rapid testicular growth. The levels of the nucleotides were correlated with their distribution in various cell types as well as intracellularly by an immunocytochemical technique. The concentration of cyclic AMP was high during the infantile phase, fell steadily to a nadir prior to puberty and then increased through sexual maturation. Cyclic GMP levels were also elevated during the infantile phase and decreased steadily through sexual maturation. Early in development the distribution patterns for the two cyclic nucleotides were quite similar with fluorescence in almost all cell types. With maturation, cyclic AMP was localized within tubules, primarily in Sertoli cells and spermatogenia, and was observed in cells in the intertubular area. In the mature testis, cyclic GMP fluorescence was associated with cells representative of most stages of spermatogenesis. Cyclic GMP was localized in nuclear elements of many cell types: in nucleoli of Sertoli cells, spermatogonia and of cells in the intertubular area. Staining was also observed along pachytene chromosomes and in a reticular nuclear pattern in a number of cell types. Cyclic AMP was seen in nuclear elements of certain cells, particularly early in testicular development, but the staining pattern was more indistinct than that of cyclic GMP. The distribution patterns of nucleotides at several stages of testicular development suggest that these compounds may be associated with cellular events related to hormone-dependent maturation of the testis.


Induction of ornithine decarboxylase in Reuber H35 rat hepatoma cells. Stimuli known to induce tyrosine aminotransferase in H35 cells were tested relative to their ability to induce ornithine decarboxylase, the initial enzyme in the polyamine biosynthetic pathway. Dibutyryl cyclic AMP (0.5 mM), parachlorophenylthio-cyclic AMP (0.1 mM) and dexamethasone (1 muM) stimulated the activity of ornithine decarboxylase 7- to 8-fold by 5 hr of induction. There was a delay of 1 hr before any increase in enzyme activity was detectable. Insulin administered alone failed to significantly change ornithine decarboxylase activity. The ability of dibutyryl cyclic AMP to elevate ornithine decarboxylase activity was found to be concentration-dependent, and a dose-response relationship very similar to that for the induction of tyrosine aminotransferase by dibutyryl cyclic AMP was observed in these cells. The ability of various 8-substituted cyclic AMP analogues to increase the activity of ornithine decarboxylase was correlated with their ability to activate purified protein kinase.


Lithium-angiotensin interaction in rat neurohypophysis. The effects of various concentrations of lithium upon basal and angiotensin-stimulated cyclic AMP accumulation were studied in isolated rat neurohypophysis. High doses of lithium diminished significantly the spontaneous accumulation of the nucleotide. This effect was not influenced by pre-incubation. Angiotensin-stimulated accumulation was inhibited by lithium at a dose which did not interfere with the basal accumulation of cyclic AMP. The possible physiological significance and mechanism of action are discussed.


Regulation of adenosine 3':5'-monophosphate content of human astrocytoma cells: desensitization to catecholamines and prostaglandins. Human astrocytoma cells (1321N1) exhibit adenylate cyclase activities coupled to independent receptors for catecholamines and prostaglandins of the E-series. Exposure of the cells to either norepinephrine or prostaglandin E1 (PGE1) results in an initial rapid accumulation of cyclic AMP but also results in a progressive loss of responsiveness of the cells to agonists. Initially, the desensitization is in large part agonist-specific. However, with continued exposure to high concentrations of norepinephrine, partial loss of responsiveness to PGE1 occurs, and vice versa. The mechanism underlying this phenomenon does not appear to involve inactivation of the effectors, formation of an inhibitory substance in the culture medium or an increase in the rate of excretion of cyclic AMP from the cell. Blockade of protein synthesis (85%) by 5 mug/ml cycloheximide did not change the rate or extent of desensitization. When desensitized cells were incubated in the presence of the effectors, responsiveness was essentially completely recovered with a t1/2 of 5-7 hr. Cycloheximide recovery reduced. Norepinephrine-induced desensitization to either norepinephrine or PGE1 was blocked by sotalol, a beta receptor blocking agent. Incubation of the cells with dibutyryl cyclic AMP caused desensitization to both norepinephrine and PGE). The results suggest that catecholamine-induced desensitization occurs as a result of interaction of the agonist with the same receptor that is linked to activation of adenylate cyclase. Cyclic AMP appears to mediate at least the non-specific aspect of agonist-induced desensitization.


Survival of foot-and-mouth disease virus in cheese. Persistence of foot-and-mouth disease virus during the manufacture of Cheddar, Mozzarella, Camembert cheese prepared from milk of cows experimentally infected with the virus was studied. Cheese samples were made on a laboratory scale with commercial lactic acid starter cultures and the microbial protease MARZYME as a coagulant. Milk was heated at different temperatures for different intervals before it was made into cheese. Food-and-mouth disease virus survived the acidic conditions of Cheddar and Camembert cheese processing but not that of Mozzarella. Foot-and-mouth disease virus survived processing but not curing for 30 days in Cheddar cheese preparaed from heated milk. However, the virus survived curing for 60 days but not for 120 days in cheese (pH 5) prepared from unheated milk. Foot-and-mouth disease virus survived in Camembert cheese (pH 5) for 21 days at 2 C but not for 35 days.


Regulation of adenosine 3':5'-monophosphate content of human astrocytoma cells: mechanism of agonist-specific desensitization. The mechanism underlying agonist-induced loss of responsiveness to catecholamines and prostaglandins has been investigated in human astrocytoma cells. Pulse-labeling of the cells with [3H] adenine during the time course of exposure to either norepinephrine or prostaglandin E1 (PGE1) demonstrated a reduction of the rate of incorporation of label into cyclic AMP within 5 min after exposure of the cells to either agonist. The loss of responsiveness observed by this technique was essentially agonist-specific during the first 30 min of exposure of the cells to either norepinephrine or PGE1. The rate constant for degradation of cyclic AMP throughout a 60 min exposure to either norepinephrine or PGE1 did not change suggesting that loss of responsiveness is not related to increased phosphodiesterase activity. The results are discussed in terms of a standard theoretical model for the regulation of the steady state level of an intermediate in a reaction sequence in which the rate of synthesis of the intermediate follows zero order kinetics and the rate of degradation follows first order kinetics. The hypothesis is put forth that agonist-induced desensitization is caused by an agonist-specific reduction in the rate of synthesis of cyclic AMP that follows rapidly after the initial stimulation of adenylate cyclase activity.


Catecholamine-mediated elevation of cyclic GMP in the rat C-6 glioma cell line. Catecholamines cause an elevation of both cyclic GMP and cyclic AMP levels in the rat C-6 glioma cell line. The response is mediated by a beta-receptor, with a Ka for stimulation of cyclic GMP of 2.6 X 10(-7)M. Maximum levels of cyclic GMP are reached by 5 min. whereas cyclic AMP levels are maximal by 10 min. Removal of calcium decreases the cyclic GMP elevation by 60%. Refractoriness to a second treatment with catecholamine develops for both responses. Catecholamine sensitivity of the cyclic GMP-generating system appears in the cells only as they start to contact and enter the stationary growth phase. In contrast to the effects of catecholamines, cholinergic agonists have no effect on either cyclic GMP or cyclic AMP levels.


Preoperative management of glucocorticoid-dependent pedodontic patients. Patients with adrenal insufficiency, allergic disorders, blood dyscrasias, collagen, malignant, and gastrointestinal diseases, and renal disorders may be receiving long-term courses of glucocorticoids; mismanagement could result in Addisonian crisis. If dental procedures for these patients involve moderate to severe stress, increases in the dosages of glucocorticoids shoudl be instituted before dental treatment begins.


Studies on the stimulation of cAMP metabolism by heparin solutions containing benzyl alcohol. Heparin solutions containing benzyl alcohol as a preservative increase intracellular cAMP concentrations in human lymphocytes by as much as 5 fold. Similar changes but of a lesser magnitude were also observed in human polymorphonuclear leukocytes and platelets and rabbit alveolar macrophages. Further analysis demonstrated that benzyl alcohol used as a preservative was responsible for this finding and that heparin per se had no effect on cAMP metabolism. The cAMP response to benzyl alcohol was a time- and dose-dependent process. In human peripheral blood lymphocytes, cAMP levels increased in a linear fashion over a benzyl alcohol concentration range from 0.015-0.30% (v/v). The increased cAMP concentrations in lymphocytes as measured by radioimmunoassay were not due to formation of a cross-reacting adenosine-alcohol adduct since the immunoreactive material produced was destroyed by phosphodiesterase and co-migrated with 3H-cAMP in thin layer chromatographic studies. Concentrations of benzyl alcohol which affect cAMP metabolism are commonly present in biologic studies employing heparin solutions. In view of these findings certain previously described effects of heparin may need to be reinterpreted and in the future heparin solutions containing benzyl alcohol should not be used when studying biologic processes known to be influenced by alterations in cAMP metabolism.


Stimulation of myocardial contractility by a new cyclic nucleotide analog, 8-(benzylthio)-N6-n-butyladenosine cyclic 3',5'-phosphate (SQ 80122). The cyclic nucleotide analog, 8-(benzylthio)-N6-n-butyladenosine cyclic 3',5'-phosphate (SQ 80122) increases contractile force of excised cat right ventricular papillary muscle and stimulates myocardial contractility in the anesthetized dog. The inotropic action of this compound is independent of catecholamines and stimulation of beta-adrenergic receptors in the heart.


Hypersensitivity to histamine and systemic anaphylaxis in mice with pharmacologic beta adrenergic blockade: protection by nucleotides. The effects of exogenous nucleotides on the histamine hypersensitivity of pharmacologically beta-blocked mice were investigated. Female HLA-SW (ICR) mice, 27-29 gm, were injected intraperitoneally with 20 to 100 mug of propranolol 45 min before intraperitoneal challenge with 1 mg histamine. These animals had a mortality which averaged approximately 80%. At various time intervals before histamine, doses of from 0.5 to 12 mumoles of nucleotides were administered intravenously. Noncyclic nucleotides, adenosine, adenosine 5'-monophosphate (AMP), and guanosine 5'-monophosphate (GMP) showed clear, dose-response protection against histamine death of propranolol-treated mice when they were given 45 to 90 min before histamine. Cyclic AMP showed significant protection only when it was given at a dose of 8 mumoles 45 to 90 min before histamine, and lower or higher doses gave equivocal or no protection. Cyclic GMP WAS Not protective at any dose tested. Propranolol treatment also produced enhanced sensitivity to passive systemic anaphylaxis. Mice were passively sensitized by intraperitoneal injection of mouse anti-egg albumin antibody 6 hr before intravenous challenge with 0.5 mg egg albumin. The mortality from anaphylaxis in the group treated with 20 mug propranolol 45 min before antigen challenge increased to 83%, while that of the group not given propranolol was only 10%. Nucleotides were given intravenously 45 min before antigen challenge. The nucleotides that protected mice from death due to histamine challenge also protected them from death due to systemic anaphylaxis. These protective nucleotides were the same nucleotides that had been reported previously to be protective against Bordetella pertussis-induced hypersensitivity to histamine and anaphylaxis.


Addition of fluoride to pit and fissure sealants--a feasibility study. The data obtained in this in vitro study indicate that contact with pit and fissure sealants to which NaF has been added in amounts ranging from 2 to 5% substantially increases the fluoride content of the enamel and reduces its solubility in acid. The properties of the materials do not seem to be impaired by the addition of fluoride in these amounts. It thus appears that this approach to providing a backup anticariogenic mechanism may, indeed, be feasible. However, further investigation must be done to confirm the anticariogenic effect and to establish the most efficacious means of fluoride incorporation in the materials.


Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate. Batches of rabbit anti-human immunoglobulin G antibodies were labeled either with horseradish peroxidase, using the two-step glutaraldehyde method or the periodate method, or with fluorescein isothiocyanate (FITC). The peroxidase conjugates were isolated by chromatography using two different gel types. The five types of conjugates thus obtained were standardized to the same amount of rabbit immunoglobulin G. The antibody activity, as estimated by means of single radial immunodiffusion and passive hemagglutination, and the enzyme activity, determined with orthodianisidine, were compared. The ultimate dilutions and absolute amounts of the five conjugates giving positive reactions were determined in direct and indirect immunohistochemical tests, using both cryostat sections of skin and the agarose bead model system. It appeared that during the peroxidase conjugation procedures there was a considerable loss of abtibody and enzyme activity, whereas in the FITC conjugation procedure the antibody activity remained intact. Neverthe less, peroxidase conjugates prepared with glutaraldehyde still gave positive staining reactions in equal or somewhat higher dilutions than the fluorescin conjugate did. The peroxidase conjugates prepared with periodate could not be diluted to the same extent. For the detection of antibodies by indirect immunohistochemical methods, the peroxidase conjugate, prepared with glutaraldehyde, was comparable to the FITC conjugate. The peroxidase conjugate, prepared with periodate, was less effective.


Application of the fluorescent antibodies method for rapid diagnosis caused of diseases by pox viruses. The results of application of the fluorescent antibodies method (MFA) with the diagnostic purpose of detecting pox antigen in specimens from smallpox patients are presented, The method was found highly effective for the examination of material taken directly from the patients (smears) in the early stages of disease (maculo-papular and vesicular). The results of examination of smears from pustular fluid and scabs were not reliable in 44% of cases due to the presence of nonspecific fluorescence of tissue fragments and leukocytes. The technique of preliminary accumulation of the virus in a sensitive cell culture made the MFA suitable for the examination of material taken at any stage of disease. Results of the assessment of sensitivity of MFA in comparison with other methods of rapid diagnosis of smallpox are presented.


Application of the fluorescent antibodies method for rapid diagnosis caused of diseases by pox viruses. The results of application of the fluorescent antibodies method (MFA) with the diagnostic purpose of detecting pox antigen in specimens from smallpox patients are presented. The method was found highly effective for the examination of material taken directly from the patients (smears) in the early stages of disease (maculo-papular and vesicular). The results of examination of smears from pustular fluid and scabs were not reliable in 44% of cases due to the presence of nonspecific fluorescence of tissue fragments and leukocytes. The technique of preliminary accumulation of the virus in a sensitive cell culture made the MFA suitable for the examination of material taken at any stage of disease. Results of the assessment of sensitivity of MFA in comparison with other methods of rapid diagnosis of smallpox are presented.


Surveillance of pertussis in the CSSR. IV. Immunological surveys of antibodies to pertussis and parapertussis in the Bohemian regions and in Slovakia in 1958 - 1971. A comparison of the results of repeated immunological surveys with the notification of morbidity and mortality to pertussis and parapertussis revealed direct dependence and simultaneously confirmed the effectiveness of Czechoslovak vaccine. Immunological surveys should be continued in spite of the fact that morbidity of pertussis is low at present, because immunological surveys may lead to timely detection of shortcomings in the quality of vaccination and vaccine. The results after 14 years of systematic vaccination and revaccination and consequent follow-up to the state of immunity in the population and of other factors in the pertussis surveillance programme rank among the other achievements of epidemiology and hygiene in Czechoslovak health services. Contemporary immunological surveys show that the state of immunity in Czechoslovak child population is such as to exclude the occurrence of pertussis in epidemics in the nearest future. The situation is different in parapertussis where epidemic incidence is still possible and where epidemics regularly occur in children of prae-school age.


Melanocyte-stimulating hormone receptors on cultured guinea-pig melanocytes. Guinea-pig melanocytes in mixed epidermal cell cultures bind melanocyte-stimulating hormone in a distinct focal surface area in their perinuclear field and thus follow the same pattern previously described for Cloudman melanoma cells. The labeling index ranged from 18 to 34%. Pretreatment of cultures with trypsin leads to destruction of melanocyte-stimulating hormone receptors whereas neuraminidase has no such effect.


Susceptibility of "enterobacteria" to aminoglycoside antibiotics: comparisons with tetracyclines, polymyxins, chloramphenicol, and spectinomycin. Strains of enterobacteria, including common, aerobic, pathogenic gram-negative bacilli, and enterococci were tested for susceptibility to 11 aminoglycoside antibiotics by a twofold agar-dilution method with an inocula replicator. For comparison, similar tests were done with seven tetracycline analogues, two polymyxins, chloramphenicol, and spectinomycin. Tobramycin compared favorably with the more active aminoglycosides, with some exceptions related to individual strains of species. The tetracyclines and chloramphenicol were generally less active than the more active aminoglycosides. The polymyxins were as active or more active against most species of gram-negative rods but were essentially inactive against Proteus, Providencia, and many strains of Enterobacter.


A possible role for cyclic nucleotides in the regulation of erythrocyte shape and permeability. Erythrocytes treated with vinblastine or colchicine become spheroidal, more permeable to sodium, and of diminished deformability; upon reinjection they are rapidly sequestered and destroyed in the spleen. These deleterious effects are competitively inhibited by the cyclic nucleotides, guanosine 3',5'-monophosphate or adenosine 3',5'-monophosphate. The data suggest that cyclic nucleotides may be involved in normal erythrocyte shape and survival by interacting with membrane components that also react with vinblastine and colchicine. Similar interactions have been noted previously in diverse proliferating cells as well.


Cyclic AMP and bicarbonate responses of the dog pancreas to vasoactive intestinal peptide (VIP) and secretion. The effects of secretin (3 CU per kilogram) and vasoactive intestinal peptide (VIP; 8 mug per kilogram) on bicarbonate and cyclic AMP secretions in pancreatic juice (with pancreatic duct perfusion) and on pancreatic tissue cyclic AMP were investigated as a function of time in 13 anesthetized dogs. The peptides were given by rapid intravenous injection. Even 30 sec. after peptide administration, tissue cyclic AMP levels were elevated, reaching peak values within the first minute and a second peak at about 3 min. Bicarbonate and cyclic AMP secretions in pancreatic juice started with a lag of 1 min. after peptide injection. Following the injection of VIP, peak pancreatic response developed within the first 5 min. and the pancreatic response actually disappeared after 15 min., whereas secretion evoked by secretin was sustained for at least 30 min. The mean +/- S.D. observed maximal bicarbonate response to VIP (100 +/- 49 muEq./5 min.) was about one sixth of the maximum output following secretin (592 +/- 181 muEq./5 min.). Increases in pancreatic tissue and juice cyclic AMP caused by VIP were significant (p less than 0.05) at 1 and 4 min.; however, they were but moderate if compared with the rise achieved by secretin. The results presented confirm previous reports that VIP is a secretin-like partial agonist of pancreatic bicarbonate secretion and are compatible with the hypothesis that both secretin and VIP elicit canine pancreatic bicarbonate secretion via the second messenger system of cyclic AMP.


The transport of esterified cholesterol in plasma high density lipoproteins of human subjects: a mathematical model. The pattern of labeling of free and esterified cholesterol in the plasma high-density lipoproteins after an intravenous injection of [3H]-mevalonic acid has been examined in six human subjects with a variety of plasma lipoprotein phenotypes. Attempts have been made to fit these data to theoretical models of high-density lipoprotein esterified cholesterol transport constructed on the basis of previous experimental observation. The first model, which assumed that the high-density lipoprotein esterified cholesterol transport constructed on the basis of previous experimental observation. The first model, which assumed that the high-density lipoprotein esterified cholesterol comprised a single homogeneous pool, was incompatible with the observed data. The second model assumed a two-pool model in which all of the plasma esterified cholesterol was produced in a small, rapidly turning over subfraction of the high-density lipoproteins. From this pool it was assumed that esterified cholesterol was transferred to other lipoprotein fractions, including transfer to a larger and much more slowly turning over subfraction of the high density lipoproteins. The second model was quite compatible with the observed data. Furthermore, the calculated net transport of esterified cholesterol through the fast turning over pool was in a range similar to that reported elsewhere for whole plasma. It has been concluded that the two-pool model not only fits the experimental data, but also accords well with previously documented observations of plasma esterified cholesterol transport.


Low density and high density lipoprotein turnover following portacaval shunt in swine. Turnover of 125I-low density lipoprotein (LDL) and of 131I-high density lipoprotein (HDL) was determined before and after end-to-side portacaval shunt in eight swine. LDL (d 1.019-1.063) and HDL (d.1.09-1.21) were isolated by ultracentrifugation and iodinated by the iodine monochloride technique. Immediately postoperatively there was no consistent change in the fractional catabolic rate (FCR) of LDL compared to preoperative control values, while in all animals FCR of HDL was significantly increased (by as much as 300%). After recovery from surgery, neither LDL nor HDL catabolic rates were significantly elevated above control values in four swine. However, plasma levels of LDL and HDL protein, and of LDL and HDL cholesterol were significantly reduced 10-12 weeks after the portacaval shunt. The reduced levels of LDL and HDL associated with normal fractional clearance rates imply a reduction in synthesis of LDL and HDL following portal diversion.


Effect of essential fatty acid deficiency on the lipid composition of the Yoshida ascites hepatoma (AH 130) and of the liver and blood plasma from host and normal rats. In order to study the response of a poorly differentiated tumor to nutritional manipulation, the Yoshida ascites hepatoma (AH 130) was grown in rats fed an essential fatty acid (EFA)-deficient diet and in rats fed a control diet. Hepatomas, livers, and blood plasma from host rats and normal rats were studied as to the effects of EFA deficiency on the lipid composition. Normal rats fed an EFA-deficient diet showed an increased concentration of triglycerides and cholesteryl esters in the liver and a reduced level of total phospholipids in plasma. Host rats fed the EFA-deficient diet showed a lower concentration of triglycerides in the liver when compared with the host rats fed a control diet. In addition, EFA-deficient host rats had reduced levels of plasma free fatty acids and triglycerides. These latter were markedly high in host rats under normal dietetic conditions. As compared to the livers of either host rats or normal rats fed the control diet, the Yoshida hepatoma cells had a lower content of total phospholipids and free fatty acids as well as a higher level of free cholesterol; they also showed a typical fatty acid pattern in their phospholipids. The main characteristics of this pattern were a high content of oleic and palmitoleic acids and a low level of C20 and C22 polyunsaturated fatty acids. Exposure of Yoshida hepatoma cells to an EFA-deficient environment resulted in a decrease in the concentration of total phospholipids and free fatty acids and in changes in the fatty acid composition similar to those observed in the livers of normal and host rats. These changes suggest that, under the experimental conditions used, the Yoshida hepatoma cells are responsive to EFA deficiency.


Studies on the transport of vitamin D and of 25-hydroxyvitamin D in human plasma. A study was conducted to investigate whether human plasma contains one or more than one protein for the transport of vitamin D and of 25-hydroxyvitamin D (25-OH-D). Serum was labeled in vivo with a mixture of radioactive vitamin D3 (derived from orally administered tracer vitamin D3) and of endogenously synthesized labeled 25-OH-D3. Samples of such serum were subjected to several different protein fractionation procedures. Only a single peak of protein-bound radioactivity was observed after each of these procedures. The fraction comprising the ascending and the descending limbs of the single peak of protein-bound radioactivity (after each procedure) were separately pooled. In each instance the ratio of radioactive 25-OH-D3 to radioactive vitamin D3 was found to be almost identical in both the ascending and the descending limbs. Taken together, these findings provide strong evidence that human serum contains only a single binding protein responsible for the normal transport of both vitamin D and 25-OH-D. Plasma labeled in vitro with added 3H-labeled 25-OH-D3 was subjected to gel filtration on Sephadex G-200 and to chromatography on columns of DEAE-cellulose and of SP-Sephadex. After each of these procedures a single peak of protein-bound radioactivity was observed, with elution profiles of protein and of radioactivity that were identical with those observed with in vivo labeled serum. These data indicate that tracer 25-OH-D3 added to plasma in vitro binds to the same plasma protein normally responsible for the transport of vitamin D and of 25-OH-D.


Effect of an anti-lipoprotein lipase serum on plasma triglyceride removal. Anti-lipoprotein lipase sera injected intravenously in roosters blocked quantitatively the catabolism of very low density lipoprotein (VLDL) triglyceride. Antibodies were produced in rabbits immunized with highly purified lipoprotein lipase (LPL, glycerol ester hydrolase, E C 3.1.1.3) prepared from chicken adipose tissue. Following anti-LPL serum injection there was a linear increase in plasma triglyceride concentration. The rate of entry of triglyceride in plasma was estimated from the rate of triglyceride accumulation in the plasma of animals injected with anti-LPL serum, or from the disappearance curve of biologically labelled VLDL. In instances where both measurements were conducted in the same animals there was very close agreement between the two procedures. Inhibition of VLDL triglyceride catabolism of anti-LPL serum provided a way to characterize newly secreted VLDL that exhibited a broad spectrum of particle sizes with a median of 625 A degrees. They contained 76.2 +/- 1.2% triglyceride and had a high ratio of free to ester cholesterol (2.46 +/- 0.45). In control VLDL samples there was 46.1% triglyceride, and the ratio of free to ester cholesterol was 1.19. The complete inhibition of triglyceride removal by an antiserum prepared against adipose tissue LPL demonstrates that the NaCl-inhibited, serum-activated lipase prepared by affinity chromatography on heparin-Sepharose and concanavalin A-Sepharose columns is the enzyme responsible in vivo for the catabolism of VLDL triglyceride. Further, the kinetics of triglyceride accumulation in the plasma provide evidence that the site of degradation of VLDL triglyceride is within the plasma compartment.


Incorporation of 14C-labelled amino acids into corticotrophin-like intermediate lobe peptide and alpha-melanocyte-stimulating hormone by the rat pituitary neurointermediate lobe in vitro, and the identification of four new pars intermedia peptides. At least seven radioactive peptides, which fractionated on Biogel P6, were found in rat neurointermediate lobes after incubation for 6 h with [14C]proline. Only three of these could be tentatively identified; one as alpha-melanocyte-stimulating hormone (alpha-MSH) and two as forms of corticotrophin-like intermediate lobe peptide (CLIP). One other cross-reacted partially with a beta-malanocyte-stimulating hormone (beta-MSH) antiserum, was acidically charged and eluted on Biogel P6 in roughly the same position as ACTH. The other three peptides showed no resemblance to alpha-MSH, CLIP, beta-MSH or ACTH.


Effects of corticosterone on adrenocorticotrophin-induced mitochondrial differentiation with special reference to 11 beta- and 18-hydroxylation. The effects of corticosterone in concentrations found in adrenal venous plasma on ACTH-induced changes in cultured cortical cells derived from foetal rat adrenals were studied. Corticosterone at a concentration of 5-7 X 10(-5) mol/l completely inhibited mitochondrial differentiation to fasciculte-like morphology. The same cultures revealed significant inhibition of 11beta- and 18-hydroxylation compared with cultures treated with ACTH only. This was shown by the reduced formation of corticosterone and 18-OH-deoxycorticosterone (48%, P less than 0-001) and simultaneous enhancement of deoxycorticosterone formation (33%, P less than 0-05) from added [4-14C]progesterone. Similar inhibition was observed when dibutyryl cyclic AMP replaced ACTH as an inducer of differentiation. Lower concentrations of corticosterone (1-2 X 10(-5) and 2-4 X 10(-5) mol/l) inhibited ACTH-stimulated formation of corticosterone and 18-OH-deoxycorticosterone from endogenous precursors. The results demonstrate that corticosterone regulates the stage of differentiation in cultured adrenocortical cells. The possible role of corticosterone in the regulation of growth and steroidogenic capacity of the adrenal cortex is discussed.


Effects of follicle-stimulating hormone and luteinizing hormone on ovarian cyclic AMP and prostaglandin E in vivo in rats treated with indomethacin. The role of prostaglandins in both FSH- and LH-mediated increases in ovarian cyclic AMP was examined in rats in vivo. Dose-related increases in ovarian cyclic AMP levels were observed 10 min after intravenous injection of FSH or LH. No changes in ovarian prostaglandin E (PGE) content were detected after injection of either gonadotrophin at doses which substantially enhanced cyclic AMP accumulation. Prior treatment of rats with indomethacin produced a greater than 50% decrease in ovarian PGE levels, but did not prevent the FSH- or LH-induced rise in ovarian cyclic AMP concentration. These in vivo experiments do not support the concept that PGE plays a role as an obligatory mediator for the gonadotrophin-induced rise in ovarian cyclic AMP levels.


The development of a radioimmuno-assay for carcino-embryonic antigen with some applications. Clinical evaluation of carcino-embryonic antigen, I. This paper, the first in a series devoted to the study of the clinical usefulness of estimations of carcino-embryonic antigen (CEA) in serum and urine, describes in detail a direct radioimmunoassay for CEA in serum and urine (a modified Egan technique, Egan, M.L. et al. (1972), Immunochemistry, 9, 289-299). A comparative study of the behaviour of CEA batches and anti-CEA antisera from different laboratories in the radioimmunoassay is presented. The incidence of increased serum CEA levels in healthy smokers was found to be related to smoking habits. Assays of CEA in serum obtained by the direct technique and the Hansen perchloric acid--zirconyl gel technique (Hansen, H.J. et al. (1971), Clin. Res. 19, 143-147) showed comparable results. Various problems affecting the assay of CEA in serum and urine are critically discussed, e.g. criteria for absorbing-out of anti-CEA antisera, identification of CEA, factors causing false CEA values, use of reference materials, acceptability of a strict cut-off level to indicate increased CEA levels, and factors governing the choice of antisera. In this connection we present a new approach which maintains a reliable and consistent cut-off level in follow-up studies. Evidence is presented that urinary CEA is heterogeneous and perchloric acid-unstable.


The application of differential thermal analysis and thermogravimetric analysis to dating bone remains. Thirty-four samples of bone remains of known time since death were studied by using DTA, TGA, and derivative-TGA techniques. The DTA patterns enable us to distinguish recent from old (more than 100 years) bones. The TGA curve is also significant for an extreme series. Both DTA and TGA curves show a correlation that allows us to obtain patterns with high significance for the extreme series. They also make evident the decomposition grade that bone organic material undergoes during the postmortem putrefactive process.


Annealing and hybridization properties of herpes simplex virus type 1 DNA. The hybridization properties of the herpes simplex virus type 1 (HSV) genome have been analysed. The DNA has a kinetic complexity of 1 X 10(-8). E. cole RNA polymerase was found to initiate synthesis at about 70 sites on the HSV DNA. The in vitro RNA product from this reaction was complementary to about 80% of the HSV genome. The RNA-DNA hybridization rate constant (Kh) was determined using conditions of both RNA excess and DNA excess. Using this rate constant one can analyse the content of HSV sequences in any RNA population.


Genesis and maintenance of a persistent infection by canine distemper virus. Vero cells were persistently infected with canine distemper virus by continuous undiluted passage of virus harvests. The cells were refractory to superinfection by both measles virus and canine distemper virus. These persistently infected cells produced and released into the medium a labile component which had a potent and selective inhibitory effect on the replication of canine distemper and measles virus. The inhibitory agent was not inactivated by u.v.-irradiation or sedimented by ultracentrifugation. Antisera against canine distemper virus or SSPE sera were able to block this inhibitory effect. We propose that these persistently infected cells produce an excess of a virus-induced regulatory protein.


Further studies in genetic resistance of fowl to RSV(RAV O): evidence for interaction between independently segregating tumour virus b and tumour virus e genes. The segregation of resistant and susceptible phenotypes in response to infection by RSV(RAV 2), RSV(RAV 50) and RSV(RAV 0), of avian RNA tumour virus subgroups B, D and E, respectively, was analysed in several test-crosses using chickens from the RPRL line 7-2, HPRS-synthetic line E and the Reaseheath line C. The results were fully consistent with out view reported previously that the genes at the tve and tvb loci segregate independently and recombine under the Mendelian second law of independent assortment. The dominant susceptibility es gene is expressed phenotypically when associated with the dominant susceptibility bs gene, but its expression is suppressed when associated with two doses of the recessive br gene. Genetic causes such as lack of penetration, recessive epistasis, and/or complementary intereaction between the tvb and tve genes have been discussed to account for the modified phenotypic expression of brbreses cells, i.e. resistance to subgroup E virus. Also, as reported previously, it was observed in this study that the tvb genes control the cellular response to subgroup D virus.


Epstein-Barr nuclear antigen (EBNA) carrying lymphocytes in human palatine tonsils. The presence of Epstein-Barr virus (EBV) antigens in human palatine tonsilderived lymphocytes (TDL) was investigated using the indirect fluorescent antibody (FA) technique. The TDL were screened for the presence of EBV early antigen (EA), virus capsid antigen (VCA), and EBV nuclear antigen (EBNA). In 76% of the patients diagnosed as recurrent exudative tonsillitis, and in 33% diagnosed as recurrent tonsillitis and/or serous otitis media, EBNA was demonstrated in the purified TDLs. No EA- or VCA-producing cells were found in either the glass adsorbed or TDL cell preparations from all of the patients. These data suggest that in our patient sample, the tonsils may serve as a reservoir for EBV carrying lymphocytes and a basis for recurrent disease.


Morphological observations on the replication of herpesvirus saimiri in monkey kidney cell cultures. Owl and African green monkey kidney cell cultures have been infected with 1 p.f.u./cell of herpesvirus saimiri and sample cultures have been taken for examination by electron microscopy at 3 to 6 hourly intervals over a period of 7 days; the experiments were repeated several times. The peculiarly slow replication cycle of Herpesvirus saimiri has enabled distinct cytoplasmic and nuclear phases in virus maturation to be clearly distinguished; the overall fine structural features were similar in both cell types. Immature particles were first detected in the nucleus and cytoplasm 63 h after infection. Thereafter, abundant cytoplasmic immature particles matured by budding through cytoplasmic membranes until about 100 h, whereas nuclear immature particles budded through the inner nuclear membrane or intranuclear invaginations of it later, from about 100 h until cytolysis was complete at 160 h. Morphological differences were also observed between particles budding at cytoplasmic membranes and the nuclear envelope. At the former site the membrane overlying the bud showed an electron opaque thickening which imparted to the mature particle an asymmetrical appearance. Such thickenings of the envelope were not observed in mature particles of nuclear origin. Unusual tubular and laminated nuclear structures were seen towards the end of the replicative cycle corresponding with the phase of nuclear virus maturation by budding; the morphology of the latter structures is described.


Virus specified enzyme activity and RNA species in herpes simplex virus type 1 transformed mouse cells. LMTK-cells infected with u.v.-irradiated herpes simplex virus type 1 have been selected for the presence of the enzyme thymidine kinase. These cells have an altered morphology compared to the control cells and contain herpes-specific antigens in their cytoplasm. The thymidine kinase activity present in these cells has been shown, on the basis of a number of biochemical properties, to be identical to the herpes virus specified deoxypyrimidine kinase found during lytic infection of this virus. In addition it has been possible to detect herpes simplex-specific RNA sequences in the transformed cells and this occurs in both the polyadenylated and non-polyadenylated cytoplasmic and nuclear fractions.


Individual EPSPs produced by single triceps surae Ia afferent fibers in homonymous and heteronymous motoneurons. 1. The individual EPSPs evoked by the action of single Ia fibers from cat triceps surae (MG, LG, SOL) were recorded in homonymous and heteronymous motoneurons innervating these same three muscles. 2. In general, Ia fibers projected to a greater percentage of homonymous than heteronymous motoneurons. One class of Ia afferent evoked EPSPs in virtually all homonymous motoneurons; the other had a substantially lower projection frequency. Possible difficulties introduced by the limited resolution of the averaging technique are discussed. 3. Individual EPSPs were larger on the average if evoked a) in SOL rather than in MG or LG motoneurons, b) by LG rather than by MG or SOL afferent fibers, or c) in homonymous rather than in heteronymous motoneurons. The mean EPSP was larger in homonymous than in heteronymous motoneurons because the largest EPSPs (greater than 150 muV) were found mainly in homonymous motoneurons. 4. Rise times of EPSPs were only slightly shorter in homonymous than in heteronymous motoneurons, suggesting that other factors besides relative location of Ia terminals account for the observed EPSP amplitude differences. Rise times in SOL motoneurons were longer than those in MG or LG. 5. LG afferent fibers tended to produce larger EPSPs in rostral than in caudal LG motoneurons, and MG afferents produced larger EPSPs in caudal than in rostral MG motoneurons. These spatial effects were in accord with the more rostral entry of LG than MG Ia afferents into the spinal cord. The differential projection of SOL afferents to MG and SOL motoneurons which overlap spatially in the spinal cord suggests a species specificity in addition to a location specificity.


Interaction of electrosensory and electromotor signals in lateral line lobe of a mormyrid fish. A signal associated with the neural command to discharge the electric organ is recorded in cells of the lateral line lobe. Responses of cells activated by medium receptor inputs are facilitated or less frequently inhibited during this command-associated signal. Only responses to disynaptic inputs are affected, the monosynaptic response is not altered. The periods of facilitation and inhibition occur at times at which electroreceptor activity evoked by organ discharge reaches the lateral line lobe. Presumably the command-associated signal is important in electrolocation. Cells responding to large receptor inputs are inhibited by the command-associated signal. Activity evoked by large receptors is transmitted in a mesencephalic fiber tract. The tract response is also inhibited by the command-associated signal. Since each organ discharge would excite all the large receptors at short latency, there would be little information contained in their responses. Inhibiting discharge-evoked activity may allow the system to return to maximum sensitivity most rapidly.


Pathologenesis of hematogenous bacterial meningitis in rabbits. The authors present data obtained from a series of 27 rabbits studied following intracarotid injection of saline, brain-heart infusion broth, aerobic, or anaerobic bacteria. These data support the hypothesis that injections of cultures of both aerobic and anaerobic organisms by way of the carotid artery disrupts the blood-brain barrier over the cerebral convexities within 15 minutes; however, the inflammatory response and bacterial proliferation occur much more rapidly in the ventricular system. Within 3 hours,the barrier over the convexities is intact, but leukocytes and organisms may be seen in the long cilia of the aqeuductal region. A relative failure of leucotaxis over the convexities of the brain is the most likely explanation of these preliminary findings in this experimental model.


Evaluation of radiopharmaceuticals sequestered by acutely damaged myocardium. Eighteen radiopharmaceuticals were screened in a small-animal model as potential infarct-localizing agents. Twelve of the 18 compounds were labeled with 99mTc, four with 203Hg, one with 131I, and one with either 203Hg or 125I. The model used heat-induced myocardial lesions in the rat. The absolute concentration within the lesion and also the activity ratios of injured myocardium to normal heart tissue, blood, and muscle were determined for all compounds. Among the 99mTc-labeled agents, two bone-seekers [pyrophosphate (PPi) and 1-hydroxyethylidine-1,1-diphosphonate (HEDP)] showed the most promise; these were followed by 99mTc-tetracycline analogs and 99mTc-glucoheptonaate. The tracer showing the most favorable concentration in the lesion and the best target-to-nontarget ratios was an iodinated derivatives of hydroxymercurifluorescein labeled with either 125I or 203Hg. Consideration of the structure of these compounds suggests that the presence of mercury or of a polycyclic aromatic structure, such as that found is tetracycline and fluorescein, was associated with localization in damaged myocardial tissue. Mercury bound to such an aromatic moiety may produce an additive or even a synergistic effect.


alpha-Aminoisobutyric acid transport in liver slices from rats fed low protein meals. The effect of low protein intake on alpha-aminioisobutyric acid (AIB) transport has been examined in liver slices from meal-fed rats. Treatments (force-feeding casein or gelatin hydrolysates, glycine, or potassium chloride; injecting glucagon, or preincubating liver slices with cyclic AMP) which stimulated transport in control rat fed 3-hour meals containing 18% casein were less effective in rats fed 6% casein meals for 8 days. Responses of protein-depleted rats to glucagon or cyclic AMP had become essentially normal after they had consumed 18% casein meals for 2 days, whereas between 6 to 10 days were required for complete recovery of the response to casein hydrolysate. Stimulation of AIB transport by casein hydrolysate was also normal in depleted rats after they had consumed 40% casein meals for 2 days. Basal, non-stimulated transport of AIB was not significantly depressed in rats fed the low protein diet. Casein hydrolysate-induced increases in hepatic cyclic AMP concentrations were smaller in rats fed low protein meals. The results show that, although various stimuli of hepatic AIB transport become less effective in the protein-deprived rat, relatively rapid recovery of the response can occur upon refeeding adequate or high levels of protein.


The redox state and the concentration of ketone bodies in tissues of rats fed carbohydrate free diets. The effect of carbohydrate free-low protein diets on the redox potential and ketone body concentrations in liver, muscle, and blood was investigated. Two carbohydrate free diets were fed: 1) A diet in which all the non-protein energy was provided by fatty acids (FA); 2) A similar diet in which the fatty acids were substituted by neutral fat (NF). A carbohydrate rich diet (HC) was fed for comparison. The redox potentials in cytoplasma and mitochondria were calculated from the relative concentrations of the [lactate]:[pyruvate] and [beta-hydroxy-butyrate]:[acetoacetate] couples respectively. In fed rats the cytoplasmic redox potential in liver was much higher in FA than in NF rats, but in fasted NF rats it increased markedly and equaled that of FA rats. In liver mitochondria the redox potential was lower in fed FA than in fed NF rats, while after a 24 hour fast it increased in FA rats and decreased in NF rats. Total ketone body concentrations were high in fed NF rats and increased further in fasted rats, however, in FA rats the high concentrations in the fed state decreased after the short fast. The changes of total ketone-bodies in blood and muscle followed those in liver in both fed and fasted states.


Gas-liquid chromatographic determination of vitamin D in tuna liver and vitamin D resin oils. Gas-liquid chromatographic (GLC) determination of vitamin D3 in tuna liver and vitamin D3 resin oils was investigated and a routine method slightly modified from the previously reported methods (1, 2) was established. Since both tuna liver and vitamin D3 resin oils contained large amounts of sterols, digitonin-Celite column chromatography according to SHEPPARD et al.(4) was used to remove the sterol influence from the unsaponifiable matters of the oils. After collecting the eluate and evaporating the solvent, the residue was subjected to thin-layer chromatography (TLC) using Kieselgel GF254 as an adsorbent and a mixture of n-hexane-ethyl acetate (4: 1) as a developing solvent. The scraped zones corresponding to vitamin D3 and pre-D3 were trimethylsilylated and then applied to the GLC using 1.5% OV-17 packed on Shimalite W (80-100 mesh) as a stationary phase. Trimethylsilylation of the gas chromatograms was an essential procedure, because the peaks of unknown substances in tuna liver oils and lumisterol in vitamin D3 resin oils could not be separated from the peak of pyro-D3 without trimethylsilylation. When the proposed method was applied to the samples, satisfactory results were obtained.


Inotropic effect of cyclic AMP in calf ventricular muscle studied by a cut end method. 1. Cyclic AMP was introduced into ventricular muscle by a cut-end method. Trabecular bundles were pulled through a partition which divided the preparation into a loading region and a test region. The loading region was exposed to Ca-free solution, cut transversely near the partition, and then briefly exposed to cyclic AMP. The test region was continually superfused with Tyrode soltuion. 2. In preliminary experiments, cell-to-cell movements were studied in long bundles by including [3H]cyclic AMP in the loading procedure and allowing redistribution to occur. After suitable test periods, the bundles were removed, frozen, and sliced into segments. Segment radioactivity was plotted against distance and fitted by a theoretical diffusion curve. 3. The results showed longitudinal redistribution of label over many cell lengths with an average effective diffusivity of 8 X 10(-7) cm2/sec. This value did not appear sensitive to the length of the test period or to the presence of a phosphodiesterase inhibitor. 4. The metabolic fate of cyclic AMP introduced by the cut-end method was determined by chromatographic separation of [3H]cyclic AMP and its break-down products. Most of the cyclic AMP was metabolized, but the results suggest that cell-to-cell movements of cyclic AMP contribute to the overall redistribution of label. 5. The cut-end method was used to study the influence of cyclic AMP on the contractile activity in the test region. Introduction of cyclic AMP evoked a delayed increase in twitch tension, about 25% above control. The inotropic effect peaked about 50 min after the end of the loading procedure, a delay which seemed compatible with slow longitudinal diffusion into the test region. 6. In control experiments, the cut-end procedure was repeated with 5'AMP (the immediate break-down product of cyclic AMP) instead of cyclic AMP. No delayed increase in twitch tension was observed. 7. Introduction of dibutyryl cyclic AMP increased twitch amplitude by 130%, with a delayed time course similar to that found for cyclic AMP. 8. The results using the cut-end procedure provide new evidence that cyclic AMP helps mediate adrenergic effects on the strength of contraction.


Physiological and morphological effects of post-ganglionic axotomy on presynaptic nerve terminals. 1. Electrophysiological and electron microscope studies were done on cells in the ciliary ganglion of chickens which had been axotomized on the day of hatching. 2. By the third day after post-ganglionic axotomy both electrical and chemical transmission through the ganglion were severely depressed; by the fifth day ganglionic transmission had disappeared. 3. Action potential initiation and conduction in axotomized cells and in their associated presynaptic nerve terminals were unimpaired 3-4 days after axotomy. 4. Depression of ganglionic transmission in 3-4 day axotomized preparations was due to a reduction in amplitude of both the excitatory post-synaptic potential (e.p.s.p.) and the electrical coupling potential in individual ganglion cells. 5. In addition to being reduced in amplitude, e.p.s.p.s in axotomized cells were more subject to fatigue during low frequency (1/sec) stimulation. 6. The reduction in e.p.s.p. amplitude was due to a reduction in both the mean quantal content of the e.p.s.p.s and the calculated depolarization produced by an individual quantum of transmitter. On the average the e.p.s.p. was reduced by a factor of about 4, the mean quantum content to about two thirds normal and the quantal size to about a third normal, compared with responses in unaxotomized cells of the same age. 7. Ultrastructural studies revealed a progressive maturation of pre-synaptic terminals in normal ganglia between 0 and 9 days after hatching. Over this period the content of synaptic vesicles and mitochondria in the terminals increased and the background matrix became more dense. 8. After axotomy these signs of maturation was abolished or reversed, particularly from the third day onward. In addition there was an increase in the number of cell sections in which no synaptic terminals were observed. 9. It was concluded that loss of synaptic transmission was due to at least three factors: a reduction in release of transmitter from presynaptic terminals, a reduction in quantal size, probably due to a loss of post-synaptic sensitivity, and a partial loss of presynaptic contact.


Reduction in acetylcholine sensitivity of axotomized ciliary ganglion cells. 1. Isolated cell clusters from ciliary ganglia of 3- to 4-day-old chickens were used to examine the electrical characteristics and sensitivity to iontophoretically applied acetylcholine (ACh) of normal cells and cells that had been axotomized on the day of hatching. 2. Resting potentials, input resistances and capacitances were the same in axotomized cells as in normal cells. These averaged about 70 mV, 165 Momega and 35 pF respectively. 3. Sensitivity to iontophoretically applied ACh was lower in axotomized cells than in normal cells by a factor of about 8. The rise times of the ACh potentials were the same in the two groups; indicating that the reduced sensitivity was not due to a diffusion barrier. 4. The slopes of the dose-reponse curves, plotted on a double-logarithmic scale, suggested that the co-operative action of two ACh molecules was involved in activating a post-synaptic conductance channel. This relation was unaltered by axotomy. 5. The estimated reversal potential for the action of ACh was unchanged after axotomy. 6. Cells in isolated clusters were similar to those in intact ganlia with respect to threshold depolarization, amplitude and time course of action potentials and ability to generate repetitive action potentials. There were no differences in these characteristics between normal and axotomized cells. 7. Cells in the isolated clusters had input resistances which were larger by a factor of 2-3 and capacitances that were smaller by a factor of about 2 than those of cells in intact ganglia. It is suggested that these differences were due to loss of initial segments of axons from the isolated cells. 8. Normal cells in the isolated clusters displayed spontaneous miniature synaptic potentials, indicating that synaptic integrity was maintained during the isolation procedure. As in intact ganglia, no spontaneous activity was observed in axotomized cells.


Current clamp and voltage clamp study of the inhibitory action of DNP on membrane electrical properties of frog auricular heart muscle. Current clamp studies showed that after 10 minutes under DNP 10(-4) M the membrane potential does not change significantly while an important shortening of the action potential duration and a diminished amplitude are observed. Voltage clamp studies have been performed on the slow inward and delayed outward currents. DNP 10(-4) M induced a marked decrease of the slow inward current related to the reduction in both conductance and driving force, and a decrease in the amplitude of the delayed current. The decrease of the slow inward current seems to be mainly responsible for the suppression of the plateau of the action potential during metabolic inhibition.


Effects of botulinum toxin on neuromuscular transmission in the rat. 1. Botulinum toxin (BoTx) type A partially blocks spontaneous transmitter release from nerve terminals in the rat. Minature end-plate potentials (m.e.p.p.s) are present at all end-plates, initially with a low frequency but increasing with time after posoning. Their amplitude distribution is at first skew with a predominace of very small m.e.p.p.s but, after a few days, larger than normal m.e.p.p.s appear. 2. Tetanic nerve stimulation, Black Widow Spider Venom, the Caionophore A 23187 or mechanical damage to nerve terminals increases the frequency of m.e.p.p.s and alters the amplitude distribution of m.e.p.p.s towards a normal Gaussian one; the m.e.p.p. size approaches that seen at normal end-plates. This was seen at any time after poisoning. 3. Nerve stimulation gives rise to end-plate potentials (e.p.p.s) of low amplitude and high failure rate. Statistical analysis indicates that evoked release is quantal in nature and follows Poisson statistics, quantum size being initially very small, but after a few days approaching normal size. Short-term tetanic nerve stimulation reversibly increases the quantum content of e.p.p.s and during early stages of paralysis long-term (2 hr) stimulation causes an apparently permanent increase in quantum size. 4. Raising the extracellular Ca concentration from 2 to 16 mM increases the frequency of m.e.p.p.s in normal muscle but not in BoTx poisoned ones. K-free medium or ouabain, which are believed to raise the intracellular Ca concentration in nerve terminals, similarly increases m.e.p.p. frequency in normal but not in poisoned muscles. When the Ca-ionophore A 23187 is used together with high extracellular Ca (greater than 4 mM) massive release of transmitter occurs from poisoned terminals. 5. The extracellular Ca concentration which causes  a certain level of transmitter release in reponse to nerve impulses is considerably higher at BoTx poisoned end-plates than at normal ones. The slope value for Ca dependence of transmitter release is about 1-5 compared with about 3 at normal end-plates. 6. Tetraethylammonium (TEA) greatly increases the amount of transmitter released by nerve impulses and restores neuromuscular transmission during all stages of poisoning, although it has not effect on spontaneous transmitter release. In the presence of TEA the power relation between Ca concentration and quantum content at the BoTx poisoned end-plate is similar to that seen at normal end-plates. 7. It is suggested that in BoTx poisoning the mechanism for transmitter release has a reduced sensitivity to Ca, and the level for activation by intracellular Ca is elevated. Once the intracellular concentration of Ca is raised to this level, by tetanic nerve stimulation, mechanical injury to nerve terminals, the Ca-ionophore or the prolongation of the nerve action potential with TEA, augmented transmitter release occurs, similar to that which occurs in normal nerve terminals at a lower level of Ca.


Behavior of the contractile vacuole of Tetrahymena pyriformis W: a redescription with comments on the terminology. The behavior of the contractile vacuole of Tetrahymena pyriformis W has been recorded and analyzed quantitatively by cinephotography. The vacuole fills in a stepwise fashion by the confluence of ampullae which appear regularly at the beginning of systole and whose membranes are continuous with that of the contractile vacuole throughout the cycle. The vacuole may subsequently fill slowly by a means not discernible by light microscopy. The vacuole rounds up at the beginning of systole and shortly thereafter the ampullae reappear around the perimeter of the vacuole. They are expanded by fluid forced into them from the vacuole. Round-up and the mode of growth of the ampullae indicate that the contractile vacuole is truly contractile. Expulsion occurs soon after the appearance of the ampullae and terminates the cycle. Contraction is initiated at regular intervals by a timing mechanism which is independent of the size of the vacuole. Suitable terminology to describe the structure and behavior of the contractile vacuole is discussed.


Changes in rat ovaries of specific binding for LH, FSH and prolactin during the oestrous cycle and pregnancy. In cyclic rats, the highest ovarian specific binding for LH was 6-0+/- 2-2% inpro-oestrus. During pregnancy, the specific binding of 125I-labelled bovine LH by rat ovaries increased gradually and reached a maximum of 24-1+/-4-9% between Days 14 and 18 of gestation; a slight decrease in binding was observed at Day 20 of pregnancy. Ovarian specific binding for FSH was also highest in pro-oestrus (8-9+/-2-1%), decreasing to about 50% in oestrus and metoestrus, but staying relatively constant during pregnancy. For prolactin, the specific binding in rat ovaries was highest (7-1+/-1-6%) in pro-oestrus, quite high in metoestrus and dioestrus and low in oestrus. Specific binding increased gradually only after Day 14 of pregnancy. Serum concentrations of rat LH, FSH and prolactin at different stages of the oestrous cycle and during pregnancy were determined by radioimmunoassays, and no obvious correlation was observed between levels of circulating hormones and the specific binding of these hormones in ovarian tissues. Affinity constants (Ka) for the hormones were very similar between ovaries from pro-oestrous rats and late-pregnant rats, being 0-31 X 10(9) M-1 for LH, 0-65 X 10(10)M-1 for FSH, and 1-14 X 10(10)M-1 for prolactin. Increases in specific binding for different hormones were due to increases of total binding sites in the ovary under different physiological states.


The effect of maternal cytomegalovirus infection on preimplantation development in the mouse. Mice were inoculated with murine cytomegalovirus at 14 and 7 days before and 1 and 4 days after mating. The effects of maternal infection on early pregnancy were investigated. Inoculation 7 days before and 1 day after mating, i.e. around ovulation and implantation, significantly reduced pregnancy rate. Embryos in these females were developmentally retarded, perhaps because of the inflammatory effect of the infection on the genital tract. Retarded embryos developed normally when cultured.


Synthesis and antiviral and antimicrobial activity of certain 1-beta-D-ribofuranosyl-4,5-disubstituted imidazoles. Starting with AICA ribonucleoside the following nucleosides were prepared. Methyl 5-amino-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carboxylate (5) was converted into methyl 5-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carboxylate (6) via diazotization in the presence of cuprous chloride. Similarly, 5-amino-1-(2,3,5-tri-O-acetyl-beta-D-ribofuanosyl)imidazole-4-carbonitrile (9) was converted into 5-chloro-, 5-bromo-, and 5-iodo-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carbonitrile derivatives. These 5-halogenated imidazole nucleosides were treated with several nucleophiles such as ammonia, hydroxylamine, and hydrogen sulfide to provide, respectively, 5-haloimidazole-4-carboxamide, 5-haloimidazole-4-carboxamidoxime, and 5-haloimidazole-4-thiocarboxamide ribonucleosides. 5-Chloro- or 5-bromo-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carbonitrile was treated with potassium hydrosulfide to yield 5-mercapto-1-beta-D-ribofuranosylimidazole-4-thiocarboxamide (16). The catalytic reduction of 5-chloro- or 5-bromo-1-beta-D-ribofuranosylimidazole-4-carboxamidoxime provided 1-beta-D-ribofuranosylimidazole-4-carboxamidines as their hydrochloride and hydrobromide salts, respectively. These nucleosides were tested for in vitro antiviral, antifungal, and antibacterial activity. The 5-halo analogues of 1-beta-D-ribofuranosylimidazole-4-carboxamide showed significant antiviral activity whereas compound 16 was found inhibitory to fungi.


Synthesis and anti-DNA -virus activity of the 5'-monophosphate and the cyclic 3',5'-monophosphate of 9-(beta-D-xylofuranosyl) guanine. 9-(beta-TD-xylofuranosyl)guanine (xylo-G) was converted chemically to the 9-(beta-D-xylofuranosyl)guanine 5'-monophosphate (xylo-GMP) and 9-(beta-D-xylofuranosyl)guanine cyclic 3',5'-monophosphate (c-xylo-GMP). These compounds were tested against a variety of DNA viruses in tissue culture in parallel with 9-(beta-D-arabinofuranosyl)adenine (ara-A). This evaluation revealed that xylo-G, xylo-GMP, and c-xylo-GMP were all moderately active but less effective than ara-A. When the four compounds were administered intracerebrally as a treatment for herpes virus, type 1 induced encephalitis in mice, c-xylo-GMP exhibited superior activity to that shown by the other three. When administered intraperitoneally, c-xylo-GMP was found to have a therapeutic index of about 4, which is less than that for ara-A (approximately 30) in the same system.


Centrally acting emetics. 10. Rigid dopamine congeners derived from octahydrobenzo[f]quinoline. In a study of conformational requirements for certain dopaminergic agonist molecules, a series of conformationally predictable dopamine congeners related to cis- and trans-octahydrobenzo[f]quinoline was prepared. The complexity and equivocal character of the reduction of variously substituted 4-methyl-1,2,3,4,5,6-hexahydrobenzo[f]quinolines were demonstrated and studied. It was shown that several literature methods for reduction of these systems were in error regarding the stereochemical nature of the product(s). It has been concluded that geometrically specific and predictable reductions of these hexahydrobenzo[f]quinolines seem unlikely to attain, and a plausible rationalization for this conclusion has been proposed. Pharmacologic data on the compounds prepared are consistent with our earlier proposals of a biologically significant conformation of dopamine for emesis, the pecking syndrome in pigeons, and other physiological effects.


Synthesis and biological properties of some spin-labeled 9-aminoacridines. Five spin-labeled 9-aminoacridines, each bearing either a 4-(2,2,6,6-tetramethyl-1-piperidinyloxy) or a 3-(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety in the 9 position, have been synthesized and assayed for biological activity in three different test systems. Sedimentation velocity measurements indicated that the labels caused unwinding of calf thymus DNA. Those acridines which contained both 6-chloro and 2-methoxy substituents were less toxic to leukemia L1210 in static culture than the corresponding unsubstituted analogues. While the unsubstituted aminoacridines were quite good inhibitors of Escherichia coli DNA-primed RNA polymerase, the 6-chloro-2-methoxy-substituted compounds stimulated this enzyme system. In the presence of E.coliDNA, the ESR spectrum of 4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2,2,6,6-tetramethyl-1-piperidinyloxyl (12) became broad and highly asymmteric with a maximal hyperfine splitting of 57.5 G. This observation suggests that when 12 intercalates into DNA the piperidinyl moiety that bears the nitroxide group becomes highly immobilized. These results suggest that the spin-labeled 9-aminoacridines will be useful probes for nucleic acids.


Simultaneous measurements of optical and electrical properties of artificial membranes composed of mitochondrial lipids and their interaction with cytochrome c. A newly constructed cell, which allows simultaneous measurements of optical and electrical properties, was used to study bimolecular black membranes composed of beef heart mitochondrial lipids and their interaction with cytochrome c. The results show that these highly charged membranes are stable only in relatively limited ranges of boundary conditions. In 0.1 n KCl their maximum direct current (dc) resistance is 7 X 10(8) Ohm cm2 +/- 10%; the series capacity at 1 kHz is 0.43 muF/cm2 +/- 3%; the entire thickness, determined by optical reflectivity, is 5.8 +/- 0.2 nm. The interaction between oxidized cytochrome c and these lipid membranes is primarily of electrostatic nature, and dependent on the presence of highly charged phospholipids, such as diphosphatidyl glycerol (cardiolipin) and phosphatidyl ethanolamine. The attachment of cytochrome c maximally causes a 2.5-fold increase in reflectivity, without any noticeable change in the capacity. This leads to a subsequent instability of the membrane (i.e., rupture) preceded by a rapid increase of the dc conductivity. This behavior is far less pronounced with reduced cytochrome c.


Localization and histochemical correlation of 73As by whole-body autoradiography in mice. Whole-body sagittal sections of mice administered [73As] H3AsO4 intravenously were processed for autoradiography and histochemistry for glucose-6-phosphatase or alkaline phosphatase. The intense localization of 73As in bone, kidney cortex, intestinal mucosa, and hair follicles corresponded exactly with the histochemical activity of glucose-6-phosphatase and alkaline phosphatase in sections containing no arsenic. The activity of glucose-6-phosphatase was inhibited in all of these sites in sections containing arsenic.


Hemifacial spasm: importance of a complete investigation. The authors report the experience of the Clinique d'O.R.L. de l'Université de Bordeaux II in the management of hemifacial spasm. The recent diagnostic and therapeutic progress in otoneurology has revealed an organic etiology for many cases of hemifacial spasm. The recent diagnostic and therapeutic progress in otoneurology has revealed an organic etiology for many cases of hemifacial spasm. From their experience, they propose a complete investigation for all cases of hemifacial spasm. The disorder is labelled as being idiopathic only if the complete investigation is negative.


Induction of murine p30 by superinfecting herpesviruses. The interaction of endogenous type C viruses with superinfecting herpes simplex virus type 2 (HSV-2) was investigated in two murine cell lines. Replication of HSV-2 was suboptimal in random-bred Swiss/3T3A cells and, in initial experiments, infection with a low virus-to-cell ratio resulted in carrier cultures with enhanced murine leukemia virus (MuLV) p30 expression. Immunofluorescence tests with Swiss/3T3A cells productively infected with HSV-2 also showed HSV-associated cytoplasmic antigens and enhanced MuLV p30 expression when compared with uninfected controls. Inactivation of HSV-2 with UV light did not abolish this reaction, although the number of cells expressing p30 was reduced. HSV-2 replicated more efficiently in a line of NIH Swiss cells (N c1 A c1 10). These cells are not readily inducible for type C expression by conventional methods; however, untreated and UV-inactivated HSV-2 induced both HSV-2-associated antigens and MuLV p30 in these cells. Although the Birch strain of human cytomegalovirus induced MuLV p30, neither mouse cytomegalovirus nor vesicular stomatitis virus induced MuLV p30 in either cell line.


Antigens and DNA of a chimpanzee agent related to Epstein-Barr virus. Biological and biochemical studies of the herpesvirus of chimpanzees previously demonstrated to be antigenically related to human Epstein-Barr virus (EBV) indicated that the agent is similar to EBV in that: (i) leukocyte culture of chimpanzees whose sera contained antibody against EBV capsid antigen could yield long-term lymphoblastoid cell lines (Ch-LCL) with B-cell characteristics; (ii) the DNA of Ch-LCL contained sequences homologous to approximately 35 to 45% of human EBV; (iii) Ch-LCL contained an intranuclear antigen, Ch-NA, that could be identified with some chimpanzee or orangutan serum in anticomplimentary immunofluorescence assays; and (iv) treatment of Ch-LCL with iododeoxyuridine resulted in expression of new antigenic activity that reacted with EA+ but not EA- human sera. Two lines of evidence indicate that the chimpanzee agent, although related to human EBV, is a distinct agent: (i) Ch-NA was antigenically distinct from EBV-rebv infection although it cross-reacts of a limited extent with a minor component of EBNA; and (ii) Ch-LCL are missing 55 to 65% of the DNA sequences of human EBV.


Structure of a defective simian virus 40 genome bearing an operator from bacteriophage lambda. We examined further the physical structure of the simian virus 40 (SV40) and bacteriophage lambda DNA sequences in an SV40-lambda hybrid that had been propagated in monkey kidney cells. The SV40 vector portion of the hybrid, which was a small fragment isolated from a reiteration mutant of SV40, contained the site for initiation of SV40 DNA replication. Electron microscope heteroduplex and restriction endonuclease analyses revealed a tandem duplication of the SV40 vector segment linked to a 2,300-base pair portion (lambda map units 71 to 76) of the lambda immunity region. The defective hybrid genome thus harbors two origins for SV40 DNA replication in addition to the leftward operator and the N gene of lambda.


Suppression of glycoprotein formation of Semliki Forest, influenza, and avian sarcoma virus by tunicamycin. Tunicamycin, a new antibiotic, halts the formation of physical particles of Semliki forest and fowl plague virus, whereas avian oncornavirus particles which show a reduction in infectivity and do not contain detectable labeled glycoprotein are released in the presence of the drug. In Semliki forest virus-infected cells only the protein moieties of the glycoproteins could be labeled. In cells infected with fowl plague and avian sarcoma virus neither intact glycoproteins nor their protein moieties could be detected. By using a protease inhibitor (N-alpha-p-tosyl-L-lysin chloromethyl ketone, TLCK) it could be shown, however, that the carbohydrate-free hemagglutinin precursor of influenza virus is synthesized but is presumably degraded by intracellular proteases in the absence of TLCK as a consequence of the lack of carbohydrate.


Configurational variants of oncornavirus RNAs. Heating oncornavirus RNAs at temperatures insufficient for complete denaturation results in forms migrating between the native form (vRNA) and the completely denatured form (vRNA) after gel electrophoresis. Intermediate forms from Rous sarcoma virus or murine leukemia virus were isolated after heating of vRNA's at 58 degrees C and sedimenting in sucrose gradients, and at least four intermediates could be identified in each case. Melting of feline virus (RD-114) RNA produced one major intermediate which required a comparatively high temperature to denature, and a second intermediate occurring in conditions of low ionic strength. Although the subunit model for oncornavirus RNA is not excluded by these data, we propose that vRNA, vRNA', and intermediates may be configurational variants of the same molecule, and a monomer model for oncornavirus RNA is presented.


Physical and metabolic requirements for early interaction of poliovirus and human rhinovirus with HeLa cells. Attachment, ""tight binding'' and eclipse of radioactive poliovirus 2 (P2) and human rhinovirus 2 (HRV 2) were investigated. The activation energy for attachment of both HRV2 and P2 was about 13 kcal/mol. HRV2 differed from P2 in two respects: the Arrhenius plot for attachment of HRV2 showed a break at 15 to 19 degrees C when the cells were first treated several hours at 0 degrees C, and attachment of HRV2 was inhibited by treatment of cells with metabolic poisons able to reduce cellular ATP by more than 90%. Tight binding was determined by isolation of a specific P2-membrane complex or by loss of EDTA dissociability of HRV2. Tight binding of both viruses was slowed by 0.01 M iodoacetamide but not by 0.02 M F-; F- plus 0.002 M CN- slowed tight binding of HRV2 but not of P2. Eclipse, the irreversible alteration of parental virions, was detected by isolation of cell-associated subviral particles or by loss of cell-associated infectious virus. Eclipse of both viruses is slowed by iodoacetamide or F-. It seems likely that the early steps of infection with picornaviruses may be sensitive to alterations in the cell membrane produced by metabolic inhibitors or by treatment at low temperature.


Glycosylation sites of vesicular stomatitis virus glycoprotein. Detailed analysis on DEAE-Sephadex of the tryptic digestion products of the glycoprotein from vesicular stomatitis virus grown in HeLa suspension cultures revealed the presence of two major and several minor sugar-labeled species. The minor tryptic glycopeptides were converted to one of the two major glycopeptide species by treatment with neuraminidase. Thus, vesicular stomatitis virus glycoprotein contains only two oligosaccharide side chains that are heterogeneous in their sialic acid content.


Natural immunity in mice to structural polypeptides of endogenous type C RNA viruses. The immunological responses of inbred mice to structural components of one class of endogenous virus were investigated by means of radioimmunoassays utilizing highly purified viral proteins. Naturally occurring antiviral antibodies were demonstrated only in those strains possessing information for induction of a mouse cell-tropic endogenous virus. Moreover, these antibodies invariably appeared subsequent to the detection of spontaneous replication of this virus in the same animal. The immune responses elicited were much stronger against endogenous viral gp70 than p30, consistent with previous findings of tolerance in the mouse to the major structural antigen of its endogenous virus. However, the demonstration of an immune response to p30 under conditions of both natural and experimental immunization establishes that tolerance to this viral antigen can be overcome.


Evidence of ambiguous processing and selective degradation in the noncapsid proteins of rhinovirus 1A. Pulse-chase kinetics and extensive pactamycin mapping studies show that the translation of rhinovirus 1A proceeds in the order: initiate-P1-S-P2-terminate, where P1 is the precursor to the capsid proteins, S is a stable primary gene product, and P2 is the precursor to a family of noncapsid products. Initial examination of the molar stoichiometry of the families of rhinoviral proteins in infected cells suggested that both the P1 and P2 regions were translated more frequently than the S region. However, we show that this apparent asymmetry in translation is an artifact arising from two phenomena: (i) ambiguous cleavage sites which result in two alternative products from the S region, having apparent molecular weights of 47,000 and 38,000, and (ii) several fates for the P2 precursors, including degradation of 35 to 45% of the P2 family to small unidentifiable products. Another artifact, a time-dependent shift in the pactamycin mapping position of polypeptide r-39, was traced to a selective inhibition of the rate of cleavage of its precursor (peak 76). The processing rate of the capsid precursor (peak 92) was not retarded by pactamycin.


Cell-free translation of virion RNA from nondefective and transformation-defective Rous sarcoma viruses. Nondefective and transformation-defective virion subunit RNAs from two strains of Rous sarcoma virus (RSV) were translated in cell-free systems derived from Krebs IIA ascites cells, wheat germ, and L-cells. In each case the predominant viral-specific product was a polypeptide of molecular weight 76,000 that is related to the internal viral group-specific antigens, as judged by immunoprecipitation with monospecific antisera and tryptic peptide fingerprinting. No difference could be detected between the translation products of 35S RNA from nondefective and transformation-defective RSV virions, nor of 35S RNA from different strains of RSV. The 76,000-molecular-weight polypeptide synthesized in response to 35S RNA in vitro was labeled with formyl-methionine from initiator tRNA. Models for viral protein synthesis are discussed in the light of these results, and arguments positioning the group-specific antigen gene at the 5' end of the 35S RNA are presented.


Osteosclerotic myeloma.

Osteosclerotic myeloma. A 49-year-old man with an osteosclerotic lesion of a thoracic vertebra had peripheral neuropathy, lymphadenopathy, and elevated serum alkaline phosphatase and monoclonal immunoglobulin levels in the cerebrospinal fluid. These findings, not usually seen in ""classical'' myeloma, have been noted in different combinations in the osteosclerotic variety.


The mechanism of low-renin hypertension: aldosterone response to sodium restriction and upright posture, angiotensin II, ACTH and potassium in patients with hypertension. Plasma renin activity and aldosterone were measured simultaneously in 67 out-patients with essential hypertension. High aldosterone was more often in patients with high renin, and low levels of aldosterone were usual in those with low and normal renin. In order to study the mechanism by which aldosterone and renin acitvity are suppressed in low-renin hypertension, 25 patients (13 normal-renin hypertensives, 10 low-renin patients including 4 non-responders and two DOC excess hypertensives) were investigated as inpatients. Plasma renin activity, aldosterone and cortisol were determined by the following stimualtions with 3 days of sodium restriction and 2 hours of upright posture, angiotensin II infusion (at a dose which increased 20mmHg of diastolic blood pressure), ACTH administration (rapid i.m. injection of 0.25 mg of alpha 1-24  preparation) and potassium infusion (30 meq of potassium i.v.). Responses of aldosterone in normal-renin hypertensives to all stimulations were 3-5 fold increases from bases line values. Low-renin hypertensives except two of four non-responders showed the responses similar to those in normal-renin patients. The responses of two of the non-responders were similar to those in DOC excess hypertensives who showed reduced responses of aldosterone to some of these stimulations. Thus, it seems that low-renin hypertension is a clinical entity caused by a variety of mechanisms, and the mechanism by which low-renin hypertension is induced is not explained by one factor such as an unknown mineralocorticoid.


A scanning electron microscopic study of SV40 infected cells. The surface of the SV40-infected African green monkey kidney (AGMK) cells was studied morphologically by scanning electron microscopy. In 24 hr post infection (p.i.), the cell surface was covered with slightly elongated microvilli. The microvilli increased in number. In 96 hr.p.i. most of the cells showed SV40-specific cytopathic effects (CPE). Nuclear swellings and the elongation of microvilli were eminent. Microvilli were observed projecting with high densities especially on the nuclear portions of the cell surfaces. Features suggesting cytoplasmic vacuolization were also observed in some cells. Spherical particles viewed in some of the cells at the late stage of infection were considered SV40 virions. Their origin was also discussed.


Studies on the interaction between coxsackievirus A9 and HeLa cells. I. Plaque-forming ability of coxsackievirus A9 in HeLa cell cultures. Most of the coxsackievirus A9 (CA 9 virus) including the prototype strain formed plaques in HeLa cell monolayers under agar overlay, although they showed little or no cytopathogenicity under fluid medium. These viruses were isolated or passaged in primary cynomolgus monkey kidney (MK) cell cultures, and the infectivity of any strain in terms of plaque-forming units was much higher in MK cells than in HeLa cells, even after plaque purification of the virus in HeLa cell cultures. CA 9 virus contained in the original throat swabs as well as some clones obtained by plaque purification in MK cells failed to form plaques in HeLa cells, but virus preparations obtained after several undiluted passages through MK cells included plaque-formers in HeLa cells, suggesting that such plaque (HeLa)-forming viruses may have developed at a certain rate during multiplication of the original non-plaque (HeLa)-forming virus population in MK cells. Out of four lines of HeLa cells examined, two, including a clonal line S3, failed to support plaque formation by CA 9 virus.


Studies on the interaction between coxsackievirus A9 and HeLa cells. II. Mode of growth of coxsackievirus A9 in HeLa cell cultures and the effect of sulfated polysaccharide on plaque formation. For the purpose of clarifying the mechanism of plaque formation in HeLa cell cultures by coxsackievirus A9, which does not show definite CPE in fluid cultures, we investigated the growth pattern of the virus in HeLa cells, comparing plaque (HeLA)-forming and non-plaque (HeLa)-forming viruses. It was revealed that the yield of both viruses per cell was nearly the same, but non- plaque (HeLa)-forming virus was far less efficient in infecting HeLa cells. Dextran sulfate was effective in releasing more virus from cells, when HeLa cell cultures were infected with plaque (HeLa)-forming virus, but not in cultures infected with non-plaque (HeLa)-forming virus. From these experimental results, the mechanism by which plaques are formed in HeLa cell cultures by coxsackievirus A9 was discussed.