Release of glycopeptides and mucopolysaccharides from ascites hepatoma cells by tryptic treatment. Two types of ascites hepatoma cells, AH 66 and AH 130 FN, were treated with trypsin to observe the release of complex carbohydrates constituting the plasma membranes. From AH 66 cells, mucopolysaccharide (heparan sulfate) was preferentially released. From AH 130 FN cells, N-glycosidic glycopeptides were preferentially released whereas no mucopolysaccharide (chondroitin sulfate A) was released.


The DNA untwisting enzyme from Saccharomyces cerevisiae. Partial purification and characterization. The DNA untwisting enzyme has been partially purified from Saccharomyces cerevisiae. The enzyme exhibits a pH optimum of 7.3 to 7.6 in phosphate buffer, appears to require 0.15 M KCl for activity as determined by a DNA filter-binding assay, and is inhibited by N-ethylmaleimide. Like the untwisting enzymes from other eucaryotic cells, it can remove both positive and negative superhelical turns. A DNA molecule containing a single strand break was shown to be an intermediate in the untwisting reaction. Thermal stabilities of the enzyme from selected conditional lethal mutants defective in DNA synthesis have been examined and were found to be indistinguishable from the wild type enzyme.


Purification and characterization of a Ca2+-binding protein in Lumbricus terrestris. A Ca2+-binding protein which is capable of activating mammalian Ca2+-activatable cyclic nucleotide phosphodiesterase has been purified from Lumbricus terrestris and characterized. This protein and the Ca2+-dependent protein modulator from bovine tissues have many similar properties. Both proteins have molecular weights of approximately 18,000, isoelectric points of about pH 4, similar and characteristic ultraviolet spectra, and similar amino acid compositions. Both proteins bind calcium ions with high affinity. However, the protein from Lumbricus terrestris binds 2 mol of calcium ions with equal affinity, Kdiss = 6 X 10(-6) M, whereas the Ca2+-dependent protein modulator from bovine tissues binds 4 mol of calcium ions with differing affinities. Although the Ca2+-binding protein of Lumbricus terrestris activates the Ca2+-activatable cyclic nucleotide phosphodiesterase from mammalian tissues, we have failed to detect the existence of a Ca2+-activatable phosphodiesterase activity in Lumbricus terrestris. The activation of phosphodiesterase by the Ca2+-binding protein from Lumbricus terrestris is inhibited by the recently discovered bovine brain modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184). Since the modulator binding protein has been shown to associate with the mammalian protein modulator to result in phosphodiesterase inhibition, it can be concluded that the Lumbricus terrestris Ca2+-binding protein also associates with the bovine brain modulator binding protein. Attempts to demonstrate the existence of a similar modulator binding protein in Lumbricus terrestris have been unsuccessful.


Sarcoplasmic reticulum ATPase. Spin labeling detection of ligand-induced changes in the relative reactivities of certain sulfhydryl groups. In sarcoplasmic reticulum fragments, chemical reactivity of calcium ATPase -SH groups toward N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-iodoacetamide (ISL) was estimated by measuring the steady reduction in free label spectrum intensity during the labeling reaction. A few -SH groups reacted easily with ISL and activity was not inhibited. The reaction rate was highly sensitive to pH and temperature. Calcium chelation in the presence of magnesium accelerated the reaction slightly, and nucleotides accelerated if severalfold in the presence of calcium. The resulting spectra were also studied for the bound labels, after extensive washing of the nonreacted label. Compared to the spectrum obtained after labeling in the control calcium medium, the "weakly immobilized signal" of the spectrum of vesicles labeled in a chelated calcium medium was enhanced. On the other hand, the "strongly immobilized signal" was enhanced when vesicles were labeled in a medium containing calcium and nucleotides. This was taken as evidence that different -SH groups are selectively alkylated, according to the labeling medium. The present study confirms the calcium-induced modifications in the -SH environment reported previously and suggests new ways of searching for possible conformational events during the transport cycle in the membrane.


A copper-binding immunoglobulin from a myeloma patient. Purification, identification, and physical characterization. A copper.protein complex present in the serum of a hypercupremic myeloma patient has been purified to homogeneity using gel filtration, DEAE-cellulose chromatography, and concanavalin A/Sepharose affinity chromatography. Immunoelectrophoresis and hemagglutination inhibition tests showed the copper-bound protein to be an IgG1-type immunoglobulin with lambda light chains. The immunoglobulin is of normal molecular weight (150,000) with normal size light and heavy chains (28,000 and 56,000, respectively). The carbohydrate portion of the molecule appears to be abnormal in that it interacts with concanavalin A, whereas most immunoglobulins of the gammaG-type do not. The copper in the native copper.IgG complex is in an EPR-indeterminable valence state. Copper was efficiently removed from the copper.IgG complex by dialysis against 0.1 M potassium cyanide. The apo-IgG was separated from the copper.cyanide complex by gel filtration. The copper complex was reconstituted by equilibrating the apo-IgG with 7.7 muM cupric ions.


Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus. Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.


Identification of a high molecular weight presumptive precursor to albumin mRNA in the nucleus of rat liver and hepatoma cell line H4AZC2. Nuclear RNAs prepared from rat liver and rat hepatoma cell line H4AZC2 have been fractionated and examined for albumin mRNA sequences by annealing to specific albumin [3H]cDNA. In both instances, sucrose gradient analysis revealed nuclear RNA molecules containing albumin RNA sequences which sedimented at 26 S (26 S albumin RNA). In contrast, cytoplasmic albumin messenger RNA sediments exclusively at 17 S. 26 S albumin RNA is resistant to both heat denaturation (65 degrees C X 5 min) and denaturation in 85% formamide (v/v), and 75% of these molecules are polyadenylated. These results provide evidence for the existence of an intact, high molecular weight, polyadenylated nuclear RNA which contains albumin mRNA sequences.


The reduced nicotinamide adenine nucleotide-activated phosphoenolpyruvate carboxylase from Pseudomonas MA. Further studies on regulatory properties. Phosphoenolpyruvate carboxylase from Pseudomonas MA grown on methylamine as a sole carbon source is an allosteric enzyme activated by NADH. Activation is accompanied by a change in the sedimentation value of the enzyme from 12 S to 9 S. In this paper ADP is shown to be an inhibitor of the enzyme. ADP has its most potent effect on the NADH-activated enzyme. Kinetics of ADP inhibition in the presence of NADH and of NADH activation in the presence of ADP are allosteric. The presence of ADP prevents the decrease in sedimentation value in the presence of NADH. Cross-linking studies indicate that the 12 S form of the enzyme is a tetramer of identically sized subunits and that the 9 S form corresponds to a dimer. The cross-linked enzyme is active and is activated by NADH and inhibited by ADP. It is proposed that NADH and ADP are a regulatory pair for this enzyme and reflect the energy status of the organism, allowing the carboxylase to control the flow of carbon into anabolic or catabolic pathways.


Regulation of adenosine 3':5'-monophosphate content of rous sarcoma virus-transformed human astrocytoma cells. Effects of cholera toxin on the responsiveness to catecholamines and prostaglandins. Human astrocytoma cells (EH118MG) respond to catecholamines and prostaglandins with a marked increase in the rate of formation of cyclic AMP. Treatment of EH118MG cells with cholera toxin (10 to 100 ng/ml) for 45 to 60 min caused an increase in cellular cyclic AMP content (5- to 10-fold over basal). Cholera toxin also decreased the K0.5 for isoproterenol 10- to 50-fold and decreased the K0.5 for prostaglandin E1 (PGE1)30- to 100-fold, while increasing the maximal response to PGE1 by 1.5- to 3-fold. Treatment with cholera toxin did not change the K1 values for beta-adrenergic receptor antagonists such as propranolol, alprenolol, and sotalol. Direct binding studies using [125I]iodohydroxybenzylpindolol indicated no significant changes in the number of beta-receptors or in the kinetics of the interaction of the radioligand with receptors after treatment of cells with the toxin. Competition binding studies with propranolol and sotalol revealed no toxin-induced change in Kd values for these antagonists. Treatment with cholera toxin caused only small decreases (2- to 3-fold) in the Kd values for binding of isoproterenol and norepinephrine. It is concluded that cholera toxin has little direct effect on the binding of agonists or antagonists to beta-receptors, but instead increases the efficiency of coupling of receptor and catalytic moieties of adenylate cyclase.


Rabbit mammary prolactin receptors. Demonstration of a late puerperal increase in affinity. Crude receptor preparations of rabbit mammary gland were made by differential centrifugation and reacted with lactoperoxidase-iodinated ovine prolactin (oPRL) in order to determine their binding characteristics. Receptors prepared from the mammary glands of animals less than 4 days postpartum bound oPRL with high affinity (Ka = 3.50 X 10(9) M-1), in good agreement with previous results of other investigators. The binding capacity of these preparations was 107 +/- 16.3 fmol/mg of protein. In contrast, receptors prepared from the mammary glands of late lactating rabbits (Days 25 to 30 of lactation) showed a 2.5-fold increase in binding affinity (Ka = 8.63 X 10(9) M-1, p less than 0.001) without a significant increase in binding capacity (135 +/- 21.4 fmol/mg, p greater than 0.2). Kinetic experiments revealed that the rates of association of hormone and receptor were identical in early and late receptor preparations, and that the 2.5-fold decrease the dissociation rate observed in the late preparations was fully explanatory of the differences in equilibrium binding. The mechanism of this affinity increase is not known. Such a change in binding characteristics, which would tend to enhance tissue responsiveness, may underlie the well characterized maintenance of full lactation in women despite falling concentrations of prolactin.


Nucleotide sequences of DNA encoding the 3' ends of SV40 mRNA. I. The sequence of the DNA fragment Hi-DII,III-G. The 3' ends of the mRNA coding for the early and late proteins of SV40 DNA overlap. We have analyzed the restriction endonuclease fragment of SV40 DNA complementary to the 3' untranslated ends of the mRNA and the codons for the COOH-terminal amino acids of early and late protein. The sequence of this DNA fragment is presented.


Nucleotide sequence of DNA template for the 3' ends of SV40 mRNA. II. The sequence of the DNA fragment EcorII-F and a part of EcorII-H. The nucleotide sequence for two-thirds of restriction endonuclease fragment EcoRII-F and part of RII-H of SV40 DNA is presented. This segment of SV40 DNA is complementary to the sequence near the 3' end of early mRNA. This sequence could be translated in one reading frame to form a large protein. However, in a second translational frame there are four AUG codons followed by 91 sense triplets, followed by a termination codon. These results provide the sequence for the entire 3' untranslated ends of SV40 early and late mRNAs and for the DNA beyond the 3' ends of the mRNAs. The ends of early and late mRNA are transcribed from the opposite strands of the same segment of DNA. At or beyond the 3' ends of both early and late mRNA are sequences whose transcripts would include uridylic acid-rich products.


A deoxyribonucleic acid unwinding protein isolated from regenerating rat liver. Physical and functional properties. A DNA-unwinding protein has been purified from regenerating rat liver cytosol to apparent homogeneity. The protein is present in about 10(6) copies per cell. It is a tetramer, composed of 25,000-dalton subunits which does not exhibit enzymatic activity for ATPase, DNA polymerase, or DNase. The protein is able to unwind the double helix of poly[d(A-T)], depressing the melting point of this synthetic polymer by about 40 degrees. It also binds to supercoiled SV40 DNA, probably by melting A-T-rich regions in the genome. The fully saturated complex of protein and SV40 DNA sediments at 30 S. Homologous DNA polymerases-alpha and -beta are stimulated by the protein at a different level depending on the templates used. This result argues in favor of the intervention of the unwinding protein in replication processes.


Cell contact-induced inhibition of division in Dictyostelium. The developmental stage at which Dictyostelium discoideum strain v12/M2 cells was inhibited in resuming cell division was monitored by a Coulter counter. Neither vegetative nor pre-aggregating v12/M2 amoebae used as inocula in liquid growth cultures exhibited a delay in recommencing cell division. Inocula prepared from aggregates were delayed for 3 h before the onset of division. Although cyclic AMP had no effect upon vegetative or preaggregating stages the aggregating amoebae were inhibited for 9 h before resuming division. Following the onset of division by both controls and cyclic AMP-treated cells the normal growth rate of vegetative amoebae (3.2 h/generation) was attained. Aggregateless mutant vegetative amoebae and those of comparable ages to vas/M2 aggregates were not inhibited in the rate of cell division by cyclic AMP. Cyclic AMP sustains the delay in cell division only among amoebae derived from aggregates. Cyclic AMP induces structural components in the plasma membranes which may be necessary in cell adhesion and interactions. Consequently interactions between the membranes of apposing cells may transmit stimuli intracellularly to inhibit cell division.


Eradication of Aleutian disease of mink by eliminating positive counterimmunoelectrophoresis test reactors. The counterimmunoelectrophorsis test was applied on three Aleutian disease virus-infected mink ranches for the detection of specific Aleutian disease virus antibody. All mink on the ranches were tested during the pelting season and before the breeding season for 4 consecutive years. Aleutian disease has been eliminated from the three commercial mink ranches by culling out all mink that were positive for Aleutian disease virus antibody.


Detection of alcohols and volatile fatty acids by head-space gas chromatography in identification of anaerobic bacteria. A head-space gas chromatographic technique for the analysis of volatile bacterial metabolites is described. Bacteroides fragilis, Clostridium perfringens, and Propionibacterium acnes, cultured in a glucose-containing peptone yeast extract medium, were studied. The head-space technique was compared with the injection of the complete liquid culture medium, and solvent extracts thereof, into the gas chromatograph. Volatile fatty acids could be detected by all three methods, whereas alcohols produced by C. perfringens and P. acnes were detectable only in the head-space chromatograms. Both FFAP and Porapak Q were used as gas chromatography stationary phase. Porapak Q was found more suitable than FFAP for the separation of alcohols. The head-space technique requires a minimum of preparation before the analysis and is well suited to automation.


Production of human lymphoblastoid interferon by Namalva cells. Optimum conditions for growth and interferon production by a human lymphoblastoid cell line, Namalva, have been studied. Adaptation to large-scale production is possible utilizing either Sendai virus or Newcastle disease virus. Priming of cultures before induction is unnecessary. The interferon produced has properties similar to human leukocyte interferon. The production of lymphoblastoid interferon per cell is increased two- to fourfold after dilution with serum-free medium of a saturation-density culture of Namalva induced with Newcastle disease virus. Maximum interferon yields were obtained 27 h after the addition of virus, using cultures diluted to 4 X 10(5) to 9 X 10(5) cells per ml. The presence of glutamine in the dilution medium was required for maximum interferon production. Newcastle disease virus appeared to inhibit the rates of RNA and protein synthesis more effectively in the diluted cultures.


Quantitation of antibodies to varicella-zoster virus by immune adherence hemagglutination. Immune adherence hemagglutination was compared with the complement fixation test as a means of measuring antibodies to varicella-zoster virus. Analysis of acute- and convalescent-phase sera from patients infected with varicella-zoster or with herpes simplex virus showed the immune adherence hemagglutination test to be more sensitive than the complement fixation test, and greater cross-reactivity between the two viruses appeared to be associated with the increased sensitivity. The two assay methods were used to measure antibodies to varicella-zoster virus in 265 sera obtained from patients of different ages as well as sera from 26 patients with leukemia. There were 35 cases where antibodies were detected by immune adherence hemagglutination but not by complement fixation, whereas in five cases the converse was found. Our findings support the contention that immune adherence hemagglutination is the method of choice for detecting antibodies to varicella-zoster virus.


Viral antibodies in cerebrospinal fluid of multiple sclerosis and control patients: comparison between radioimmunoassay and conventional techniques. Cerebrospinal fluid antibodies to measles, rubella, vaccinia, herpes simplex, and varicella-zoster viruses in four patient study groups (clinically definite multiple sclerosis [MS], early probable MS, optic neuritis, and control patients with other neurological diseases) were assayed by radioimmunoassay, complement fixation, hemagglutination-inhibition, or complement-enhanced plaque reduction methods. Antibodies were more frequently found and at higher dilutions by radioimmunoassay than by other techniques. Measles virus antibody, the most frequently found antibody, was present in the cerebrospinal fluid of 72% of MS patients and 5% of control patients. The differences between the numbers of MS patients and control patients with antibodies to other viruses were not as marked. Thus, 58% of MS patients versus 21% of control patients had antibody to rubella virus, 20 versus 3% had antibody to vaccinia virus, 50 versus 33% had antibody to herpes simplex virus, and 25 versus 8% had antibody to varicella virus. Sixty-seven percent of MS patients and 26% of control patients had antibodies to two or more viruses in their cerebrospinal fluid.


Serotyping herpes simplex virus isolated by enzyme-linked immunosorbent assays. An enzyme-linked immunosorbent assay (ELISA) using a double-antibody sandwich tecnhique has been developed to serotype isolates of herpes simplex virus from clinical sources. The results obtained using this procedure were in agreement with those obtained with a standard neutralization test in typing stock cultures and 32 clinical isolates of herpes simplex virus. Clear differentiation between the two viral serotypes was obtained using rabbit immunoglobulin cross-absorbed with heterologous virus antigen. The ELISA procedure described appears to be a convenient and accurate substitute for the neutralization test in typing herpes simplex viruses. ELISA techniques require relatively small amounts of antigen and antibody and can be performed with very simple equipment.


Rapid identification of Neisseria gonorrhoeae and Neisseria meningitidis by using enzymatic profiles. The enzymatic profiles of Neisseria gonorrhoeae, N. meningitidis, and related species were determined, using a total of 48 chromogenic substrates. Enzyme classes assayed for included glycosidases, aminopeptidases, phosphoamidases, proteases, lipases, esterases, and aryl sulfatase. A final test selection of 10 substrates, based upon their differential and reproducible characteristics, allowed the separation of N. gonorrhoeae and N. meningitidis from each other and from all species tested within 4 h after primary isolation on modified Thayer-Martin medium. The need for subculturing suspect colonies from modified Thayer-Martin medium to chocolate medium with a subsequent loss of 18 to 24 h of identification is eliminated.


Incidence in South-west Scotland of hepatitis B surface antigen in the liver of patients with hepatocellular carcinoma. A retrospective examination in South-west Scotland of formalin-fixed paraffin-embedded liver tissue by an immunoperoxidase technique revealed hepatitis B surface antigen (HBsAg) in eight out of 81 cases (10%) of primary hepatocellular carcinoma (PHC) and in four out of 82 cases (5%) of cirrhosis. No positive staining was found in 112 controls without overt liver disease matched for age and sex. Unlike most previous studies showing an association between HBsAg and PHC, the present investigation was carried out in an area where HBs antigenaemia is infrequent and PHC is an uncommon tumour. While possibly hepatitis infection is an important cause of PHC, the association between HBsAg and PHC could be due merely to activation by the tumour of latent virus B in a previously infected person.


'Pseudocirrhosis' in hereditary haemorrhagic telangiectasia. Telangiectasia-associated hepatic fibrosis (TAHF) in a 68-year-old woman with hereditary haemorrhagic telangiectasia (HHT) is described. The patient died of oat-cell carcinoma of the lung. In addition to the structural alterations which have been described previously in HHT, the liver exhibited focal midlobular hepatocytic necrosis and tumour metastases. The possibility that treatment of HHT was causally related to some of the hepatic abnormalities found in our patient and the differentiation of TAHF from true cirrhosis are discussed.


ACTH and ACTH4-10 modification of neophobia and taste aversion responses in the rat. A series of experiments assessed the effects of ACTH and the ACTH analogue ACTH4-10 on drinking in conditioned taste aversion and neophobic situations. Both substances delayed the extinction of a conditioned taste aversion established by a single pairing of lithium chloride with milk (Experiment 1). However, in this situation, the ACTH parent peptide was more potent behaviorally. Administration of ACTH suppressed milk consumption in animals with no toxicosis experience (Experiment 2). This effect was apparently not due to the conditioning of a taste aversion (Experiment 3) with ACTH serving as a weak aversive unconditioned stimulus. Administration of exogenous ACTH (Experiment 4) or ACTH4-10 (Experiment 5) did not enhance neophobia; however, repeated injections of ACTH suppressed drinking. This ACTH suppression was related to the familiarity/novelty of the subtance being consumed. The neophobic response to milk eas no accompanied by pituitary-adrenal activation (Experiment 6). Both neophobic and conditioned taste aversion situation appear to be useful for assessing peptide effects on consummatory behavior.


Multiple glomus tumors.

Multiple glomus tumors. A man had widespread, slowly evolving vascular lesions since infancy suggestive of the Blue Rubber Bleb Nevus Syndrome. His son had two painless lesions typical of Multiple Glomus Tumors. Many of the man's nodular lesions were painful. Post-excision recurrences were noted. Histologic studies of asymptomatic tumors from both cases showed irregular, dilated, vascular channels surrounded by narrow mantles of glomus cells, whereas a painful tumor had large foci of glomus cells with wider mantles around the flattened channels. Electron microscopy showed the glomus cells to be modified smooth muscle cells. The anatomy and pathology of glomus tumors are reviewed. Differentiation from other syndromes of multiple hemangiomata, particularly the Blue Rubber Bleb Nevus Syndrome is stressed. It is suggested that Multiple Glomus Tumors may be derived from simple cutaneous vessels and not the Sucquet-Hoyer canal of the normal cutaneous glomus body described by Masson in 1924.


The regulatory GTPase cycle of turkey erythrocyte adenylate cyclase. It has recently been suggested that adenylate cyclase activity is controlled by a regulatory cycle consisting of two reactions: a hormone induced formation of the active adenylate cyclase-GTP complex, and a subsequent turn-off reaction in which hydrolysis of the bound nucleotide reverts the system to the inactive state. To test this model each of the two reactions was measured separately and their rate constants were used to estimate the steady state adenylate cyclase and GTPase activities. The first order rate constants were kon = 3 min-1 for the activation reaction and koff = 15 min-1 for the turn-off reaction. Substitution of these rate constants in the steady state equation of the regulatory cycle gave values of hormone stimulated adenylate cyclase and GTPase activities similar to those determined by direct measurements. Treatment of the adenylate cyclase with cholera toxin caused a decrease of 96% in the rate constant of the turn-off reaction. In this case too the activities calculated from the steady state equation were in good agreement with those determined directly.


Dissociation of cyclic GMP from the negative inotropic action of carbachol in guinea pig atria. The relationship between the negative inotropic action of carbachol and its ability to elevate cyclic GMP was determined in isolated paced guinea pig atria. A clear dissociation was observed between that concentration of carbachol which depressed contractility and that which elevated tissue cyclic GMP content. Doses as low as 0.03 micronM caused a negative inotropic effect while cyclic GMP was not elevated until concentrations nearly 100-fold higher were used. Thus a correlation between tissue cyclic GMP content and the negative inotropic action of carbachol was not found to exist in guinea pig atria.


Purification of a protein inhibitor of adenosine 3':5'-monophosphate-dependent protein kinase from bovine myocardium by a non-denaturing procedure. The activities of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase may be partially controlled by a ubiquitous acidic heat-stable protein which inhibits the phosphotransferase reaction by interaction with the catalytic subunit of protein kinase (Walsh, D.A. et al. (1971), J. Biol. Chem. 246, 1977-1985). Since reported purification of this inhibitor involved subjecting tissue extracts to denaturing conditions, its existence under physiological conditions remained uncertain. A protein inhibitor, molecular weight 22,500, has been isolated from bovine myocardium by methods that do not include exposure to extreme heat or acid precipitation. The activity of this acidic protein is destroyed by exposure to trypsin and is unaffected by treatment with neuraminidase, RNAse or DNAse.


Inhibition of adenosine 3':5'-monophosphate-dependent protein kinase: comparison of a protein inhibitor with polyanions and substrate analogs. The mechanism of inhibition of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase was studied using a protein inhibitor isolated by a non-denaturing procedure from bovine heart. This protein inhibitor interacts with the catalytic subunit of protein kinase and binds to some substrates of the kinase. Protein kinase activity can also be inhibited by polyanions which, like the protein inhibitor, bind to basic substrates but do not bind to the catalytic subunit of protein kinase. Peptides such as L-lysyl-L-tyrosyl-L-threonine that resemble the phosphate accepting site of protein kinase substrates competitively inhibit phosphorylation of histone. Protein kinase activity can thus be inhibited in vitro by interaction of the protein inhibitor with substrates, and/or the catalytic subunit of the kinase, by competition of substrate analogs with "natural" substrates and by direct interaction of polyanions with basic protein substrates for the phosphotransferase reaction.


Adrenergic desensitization in leukocytes of normal and asthmatic subjects. Cyclic AMP was measured in leukocytes of normal and asthmatic subjects before and after one week of treatment with equal amounts of ephedrine. During the control and placebo periods, the measurements of cyclic AMP in leukocytes of asthmatic subjects were similar to those of normal individuals. After one week of treatment with ephedrine, both groups exhibited suppression of the leukocyte cyclic AMP response to adrenergic stimulation in vitro: however, the suppression of response was significantly greater in asthmatic subjects (p less than .u1). Subcutaneous administration of epinephrine was followed by further suppression of the leukocyte cyclic AMP response to in vitro stimulation which was similar in both groups during all treatment periods. The results indicate that in vivo exposure to adrenergic medications is followed by desensitization of the leukocyte responses to subsequent adrenergic stimulation in vitro. After administration of small doses of medication, the severity and/or duration of desensitization is significantly greater in asthmatic leukocytes.


Dental and skeletal ages in hypopituitary patients. Skeletal and dental ages were radiographically determined in 14 cases of confirmed growth hormone deficiency. The cases were in the 6.5- to 12-year-age group, and were receiving hormone therapy. Delay in skeletal age ranged from 24.8 to 32.3%, the average being 27.9%. There was very much less delay in dental age, ranging from only 4.3 to 19.7%; the average delay being approximately 9%. Most cases thus fell within normal limits. Seven patients had dental ages assessed before therapy and their dental age delays were comparable to those of the group as a whole. The four patients in the 12-year-old group showed a greater dental age delay then did the others. This could have been simply due to individual variation, or this delay could possibly support the view that dental development does become androgen dependent at puberty. None of the radiographs examined revealed any defects in dental development as has been reported elsewhere. While the small normal patient followed the general trend, the three low birth weight patients did not and it would not be instructive to measure the dental ages of larger numbers of these cases.


Myocardial infarct imaging agents. III. Synthesis and evaluation of [203Hg] hydroxymercuriphthaleins. Four Hg-203-mercurated phthaleins were prepared, purified, and compared with [203Hg] mercuric nitrate, [3H] phenolphthalein [203Hg] hydroxymercurifluorescein and Tc-99m-pyrophosphate in a rat model of myocardial necrosis to determine their specificities for damaged myocardium. The ratios of damaged myocardium to normal myocardium, and to blood, for the [203Hg] hydroxymercuriphthaleins (20.7-34.1 and 12.1-20.1, respectively) were somewhat lower than those reported previously for [203Hg] hydroxymercurifluorescein, but were higher than those found with [203Hg] mercuric nitrate, [3H] phenolphthalein, and Tc-99m pyrophosphate. Both the hydroxymercuri- functional group and the phthalein moiety are required for selectivity. The removal of the oxygen bridge present in fluorescein, and the replacement of carboxylic acid by sulfonic acid, had no significant effects on the sequestration process in damaged tissue.


Metastasis of adenoid cystic carcinoma of the mandible to the Gasserian ganglion. A case of adenoid cystic carcinoma of the mandible in a 47-year-old woman metastasized to the Gasserian ganglion probably through the perineural space of the inferior alveolar nerve. The patient was treated by irradiation after excision of the tumor from the ganglion. She is currently an outpatient in the oral and maxillofacial surgery department; two years and seven months after the first examination, there is no sign of recurrence or metastasis of adenoid cystic carcinoma.


Electroencephalographic sleep recordings and depression in the elderly. Eighteen patients over the age of 60 who were experiencing a major depressive episode were studied on a Clinical Research Unit after they had been drug-free for at least two weeks. All-night electroencephalographic (EEG) recordings revealed considerable fragmentation of sleep, a mean sleep efficiency of 58 percent, and very little delta sleep. The findings of reduced sleep time, shortened REM latency, and high REM density were similar to those in depressed patients under the age of 60. These preliminary findings support the application of EEG sleep recordings as a tool for the differential diagnosis of depression in the elderly.


Adenylate kinase activity in various organs and tissues of mice with the obese-hyperglycemic syndrome (gene symbol Ob/Ob). The activities of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in lyophilized cryostat sections of various organs from mice with the obese-hyperglycemic syndrome (gene symbol ob/ob) were measured fluorometrically. Skeletal and heart muscle tissues had the highest enzyme activities, i.e. 258 and 155 mmoles substrate converted per kg dry weight and per hr (MKH), respectively. Pancreatic islet specimens, which were composed mainly of B-cells, possessed the highest activitty of all parenchymatous cells studied, with 18 MKH as compared to 16 MKH for the cortical tubules of the kidney and 7 and 6 for the exocrine pancreas and liver cells, respectively. The granular layer of the cerebellum had enzyme activity of 31 MKH. The adenylate kinase activity in the B-cells was apparently not influenced by hyperglycemia since it was similar both in obese-hyperglycemic mice of different ages and in lean mice, although the blood sugar concentrations in such animals are much different.


In situ embedding method of cells grown in beem capsules for immunoelectron microscopic studies of oncornaviruses. A technique of in situ embedding of cells grown in BEEM capsules has been devised for immunoelectron microscopic studies of oncornaviruses. As compared to other immunoelectron microscopic procedures, this technique is less time and reagent-consuming. The quality and specificity of this method were tested on well-characterized mouse mammary tumor virus (type B virus) and murine sarcoma virus (type C virus particles). This method gave good results in labeling of the virus particles with ferritin or peroxidase in the cells of mouse tissue cultures. In an application of this method, peroxidase labeling of type B virus particles was obtained in frozen sections of normal prostatic tissues of C3H/Dm and A/Dm strain mice treated with rabbit antiserum to mouse mammary tumor virus from A/Dm strain mouse milk. These results indicate that this method is useful and reliable for immunoelectron microscopy studies of oncornaviruses in tissue culture cells and also in frozen sections of tissues.


Passive protection of mice with the 19 S and 7 S fractions of anti-pertussis sera in experiment. Protective activity of anti-persussis rabbit and mouse sera and 19 S and 7 S fractions obtained from these sera was investigated in the test of passive protection of mice on a model of pertussis meningoencephalitis. The method of simultaneous intracerebral administration of the serum or fraction with live culture of a virulent B. pertussis strain was used. Hyperimmune rabbit serum containing mercaptoethanol-resistant agglutinins in a high titre was found to have the most pronounced protective effect. Serum of mice, collected 14 days after single immunization of the animals, did not show any protective properties. A small amount of protective activity was observed in the serum collected on the 30th day after a single administration of the vaccine. A sharp increase in the protective activity of the serum was observed after double immunization of mice. Correlation was found between the increase in the titre of agglutinins (in particular of 7 S antibodies) and the protective activity of the serum. Protective properties of 19 S and 7 S fractions isolated from immune rabbit and mouse sera by the method of gel filtration were investigated. Both fractions were found to possess protective properties, but fraction 7 S was more active than fraction 19 S.


Arboviruses in birds captured in Slovakia. Virus neutralizing (VN) antibodies against Sindbis, tick-borne encephalitis (TBE), West Nile (WN), Tahyna and Calovo viruses were found in birds captured in Slovakia. In parallel, Sindbis, TBE and WN viruses were isolated from the blood, brain and liver of migrating birds.


Spontaneous regression of Friend virus-induced erythroleukemia. III. The role of macrophages in regression. The spontaneous regression of erythroleukemia induced by the RFV strain of Friend virus is a macrophage-dependent process. Functional suppression or elimination of the macrophage population in leukemic mice with silica, carrageenan, anti-macrophage serum, or trypan blue inhibited regression. Prior protection of the macrophages with PVNO allowed regression in silica or carrageenan-treated mice. Macrophage phagocytic activity was inhibited in about half the RFV-induced leukemic mice at 25 to 30 days post virus inoculation. Those animals with normal macrophages regressed, whereas whereas those with inhibited macrophages did not. Progressor mice could be induced to regress by inoculation with normal syngeneic macrophages; other cell types were ineffective. The inhibition of macrophage function in leukemic mice was the result of infection of the macrophages by virus. Removal of the infected cells by cytolysis with anti-virus antiserum and C restored the phagocytic activity of the population. Inhibited macrophages were less capable of responding to immobilized antigen-antibody complexes than normal macrophages, suggesting that the loss of function was due to a change in their Fc receptor.


Replication of herpes simplex virus and cytomegalovirus in human leukocytes. Human peripheral blood leukocytes, lymphocyte subpopulations, and hemic cell lines were examined for their ability to supprot HSV and CMV replication. Mitogen-stimulated mononuclear leukocytes, B lymphocytes, and T lymphcytes supported the replication of HSV to high titers over 3 to 5 days of infection. HSV replicated in unstimulated mononuclear leukocyte cultures of one of five donors, and to a limited degree in untreated B lymphocytes of three of five donors; HSV replication was not detected in unstimulated T lymphocytes (five donors). There was no evidence of enhanced uptake of 3H-thymidine in the untreated donor cells that replicated HSV. CMV replication was not detected during 9 to 10 days of infection in untreated or mitogen-treated mononuclear leukocytes and lymphocyte subpopulations from the same adult donors or in neonatal cord blood leukocytes. The ability of the cells to support HSV or CMV replication did not correlate with the presence of specific antiviral antibodies in the donor serum. HSV replication in B, T, and myeloid cell lines to high titers over 5 days of infection, whereas CMV failed to replicate in any of the hemic cell lines. A persistent HSV infection has been established in a T cell line (CEM) with high titers of infectious virus being produced concurrently with growth of the cells over the first 11 weeks of infection.


Specific lethality of silica for human peripheral blood mononuclear phagocytes, in vitro. Human peripheral blood mononuclear cells, cultured in the presence of 100 microgram/ml protein-coated silica particles, were studied to determine changes in number and function of monocytes, immunoglobulin bearing (B), sheep red blood cell rosetting (T) lymphocytes and the effector cells of antibody dependent cell-mediated cytotoxicity (ADCC). After 24-48 h, phagocytic cells were effectively eliminated from culture but there was no significant reduction in number or function of T or B lymphocytes or in ADCC to cell line targets. ADCC to erythrocyte targets was inhibited but not completely blocked. It is concluded that silica is a specific toxin for human peripheral blood mononuclear phagocytes and may be useful in in vitro immunological studies as a means of eliminating or determining the role of these cells without resort to separation methods which result in losses of cells other than monocytes.


A method using toluene-2,4-diisocyanate and glutaraldehyde to stabilize and conjugate antigens to erythrocytes for use in passive hemagglutination tests. Erythrocytes were stabilized and activated for conjugation by simultaneous treatment with toluene-2,4-diisocyanate and glutaraldehyde. Following conjugation with an antigen, these cells were suitable for use in passive hemagglutination tests and could be stored in either hypo- or hyper-tonic solutions at 4 degrees C for at least 3 months without hemolysis or loss of agglutinating properties. Furthermore, these cells can be lyophilized and reconstituted with minimal loss of conjugation ability or of agglutination titer.


Preparation of sera for typing of adenovirus infections by immunofluorescence. The conditions for preparation of adenovirus antisera with group, subgroup and type-specific reactivity to be used in the immunofluorescence technique have been evaluated. Group-specific antibodies were prepared by passing an antiserum against virions or hexons of one serotype through an immunosorbent column containing soluble viral components of an adenovirus type belonging to a different subgroup. The group-specific antibodies were recovered from the solid phase by elution at pH 2.8. Reagents specific for subgroup I and II were obtained by passing anti-dodecon sera through an immunosorbent column containing soluble viral components from heterologous subgroups. In this case antibodies not attaching to the absorbent were recovered. Sera against fibers of subgroup III members could be used as subgroupspecific sera without absorption. Type-specific antibodies were prepared by removal of antibodies of other specificities by passing anti-virion or anti-hexon sera through immunosorbent columns containing soluble viral components of an adenovirus type belonging to the same subgroup. Reagents specific for seven adenovirus types representing Rosen's three subgroups were prepared according to the outlined procedures.


The microbiological flora of penile ulcerations. The penile ulcerations of 100 consecutive men were tested for microorganisms. A polymicrobial flora was identified in the ulcers of 97 men. The microorganisms recovered from these ulcers included combinations of anaerobic and aerobic bacteria (including Mycoplasma), herpes simplex virus, yeasts, and filamentous fungi. Fifty-three study entrants had microorganisms, identified by culture or serologic tests, that were considered primary in ulcer pathogenesis. Herpes simplex virus was the most prevalent and Treponema pallidum was the next most prevalent pathogen identified. Of our patients, 5% had two recognized pathogens confirmed by laboratory tests, and only one of these was suspected at clinical examination. In addition, the study suggests that microorganisms other than Haemophilus ducreyi can produce ulcers with a morphology mimicking chancroid.


Effect of a polynucleotide interferon inducer of fungal origin on experimental rhinovirus infection in humans. A double-stranded RNA of fungal origin (BRL 5907) was given intranasally to volunteers. Apart from mild local irritancy of the higher dosage, the compound was well tolerated. A double-blind placebo-controlled trial of a three-day course (5 mg per day) of BRL 5907 against challenge with rhinovirus type 4 showed that treatment was associated with a delay in onset of symptoms and a reduction in shedding of virus, but the differences were not statistically significant. Low titers of interferon were found in nasal washings.


Central nervous system immunity in mice infected with theiler's virus. I. Local neutralizing antibody response. Experimental Theiler's mouse encephalomyelitis virus (TMEV) infection in mice is atypical of most other picornavirus infections because virus persists in the host. It was shown previously that low levels of infectious virus are readily detectable in the central nervous system (CNS) despite the presence of substantial titers of serum neutralizing antibody. In this study antibody assays were performed on CNS tissue homogenates, and neutralizing antibody was regularly found in the CNS of TMEV-infected mice. That neutralization by infected CNS extracts was due to antibody was demonstrated by the specificity of neutralization for TMEV and by elimination or marked reduction of neutralization by in vitro treatment with goat antiserum to mouse IgG. In addition, immunofluorescent staining consistently revealed IgG- but not IgM-containing cells in perivascular cuffs and parenchymal lesions of the brains of infected animals. Evidence of local antibody formation in the CNS was found in the actual reversal of the serum-CNS antibody ratio in about one-third of infected mice after three weeks. In contrast, normal mice had a mean serum-CNS antibody ratio of approximately 100:1 after passive transfer of antibody. Possible reasons for the fact that TMEV is not neutralized by antibody and chronic infection is not aborted include the formation of complexes of infectious virus and antibody in the CNS and the production of antibodies with low affinity for TMEV.


Persistent infection of a human lymphocyte cell line (Molt-4) with the kilham rat virus. Inoculation of a human thymus-derived lymphocyte cell line (Molt-4) with the Kilham rat virus resulted in persistently infected cultures, that released infectious virus for periods up to seven months. At any time following infection, a small percentage of the cells were positive for rat virus antigens as determined by immunofluorescene assay.


Effects of adenosine and guanosine cyclic phosphates and their corresponding nucleotides and nucleosides on vitamin A-induced epidermal tumor promotion and growth in hamster cheek pouch. Adenosine and guanosine cyclic monophosphates (cAMP and cGMP) exerted opposite effects at similar concentration and similar effects at markedly different concentrations on the yields of tumors promoted by vitamin A in hamster cheek pouch. Increasing the concentration of cAMP between 10(-5) M and 10(-3) M was associated with increases in tumor yield and diameter which partially or completely overcame the net tumor promotion inhibitory effects which were sometimes seen at 10(-5) M cAMP. In contrast, increasing the concentration of cGMP between 10(-5) M and 10(-3) M caused the net effects to change from increases to decreases in tumor yield without much change in diameter. The relative magnitudes of the tumor yield increasing and decreasing effects of the cyclic nucleotides varied substantially between experiments at constant concentration of cyclic nucleotide. Adenosine-5'-monophosphate (AMP) showed simultaneous tumor promotion inhibitory and growth stimulatory effects, the former effect being similar to that of cAMP but tumor yield decreased rather than increased as concentration of AMP was increased whereas the opposite occured with cAMP. Guanosine-5'-monophosphate (GMP) showed the tumor yield increasing effect of low concentraitons of cGMP but not the yield decreasing effect of high concentrations of cGMP. Adenosine (10(-3) M) showed a significant tumor yield increasing effect similar to that of 10(-3) M cAMP. Guanosine showed no significant effects at either 10(-3) M or 10(-5) M concentrations.


Effect of human chorionic gonadotrophin and ovine luteinizing hormone on rat ovarian macromolecular metabolism. Administration of human chorionic gonadotrophin (HCG) or ovine LH to immature rats primed with pregnant mare serum gonadotrophin (PMSG) stimulated the rate of synthesis of polyadenylic acid (poly A)-rich RNA in the ovaries. The rate of total RNA synthesis was not affected significantly by hormone treatment, whereas protein synthesis was enhanced. The increase in the rate of synthesis of poly(A)-rich RNA in the ovaries could be inferred as induction of messenger RNA synthesis after the hormone treatment. The poly(A)-rich nature of the isolated RNA was established by oligo(dT)-cellulose chromatography, binding the Millipore filter disks and hydridization with [3H]polyuridylic acid. The level of cyclic AMP in the ovaries of such rats was also raised after administration of LH, the increase coincided with the increase in the rate of synthesis of poly(A)-rich RNA. The implications of these results are discussed in the light of the biochemical basis of luteinization and the action of LH.


Regulatory substances produced by lymphocytes. VI. Cell cycle specificity of inhibitor of DNA synthesis action in L cells. IDS inhibits DNA synthesis and mitosis of L cells only when present during the late G1 phase of the cell cycle, as shown with L cells synchronized by a variety of methods. This corresponds well with earlier findings that IDS inhibits DNA synthesis in mitogen-stimulated lymphocytes when present between 16 and 24 h after adding mitogen. In both cell types, the inhibition produced by IDS appears to be totally the result of elevation of cAMP level. Thus, inhibitors of cAMP phosphodiesterase work synergistically with IDS, and activators of cAMP phosphodiesterase overcome the inhibition by IDS. This paper shows that IDS raises cAMP levels in L cells only within a narrow interval of the cell cycle, around 6-8 h after mitosis. This cell cycle specificity, which may be related to appearance of receptors for IDS only at discrete times, may be important in limiting IDS action to suppression, as elevated cAMP levels have a variety of other effects during other phases of the cell cycle.


Bvr-1, a restriction locus of a type C RNA virus in the feline cellular genome: pleiotropic restriction of endogenous BALB virus in cat X mouse somatic cell hybrids. Bvr-1 is a dominant X-linked feline gene which restricts the replication of B-tropic murineleukemia virus (B-MuLV) in somatic cell hybrids between murine BALB/c-RAG cells and FL-74 feline cells. Since the hybrids were originally derived by the hypoxanthine aminopterin thymidine selection scheme, counter selection experiments on 6-thioguanine result in preferential survival of hybrid cells which have spontaneously lost the feline X-chromosome on which is located the structural gene for hypoxanthine guanine phosphoribosyl transferase (IMP: pyrophosphate phosphoribosyl transferase, E.C. 2.4.2.8) and Bvr-1. Back selected Bvr-1- cells express high parental levels of B-MuLV. Bvr-1 effectively restricts the IdU-mediated induction of the endogenous xenotropic BALB virus (BALB: virus 2) but not the endogenous N-tropic virus (BALB: virus 1). Pleiotropic restriction of B-MuLV and X-MuLV, but not N-MuLV suggests that the viral targets of Bvr-1 (either viral components or functions in viral assembly) of the B-tropic and X-tropic endogenous BALB viruses are similar to each other but distinct from the target in the N-tropic virus. Very low levels of B-MuLV are detected in restricted cells, but this residual virus is not infectious in either NIH-3T3 or BALB-3T3 mouse cells which are genotypically Fv-1N/Fv-1N and Fv-1B/Fv-1B, respectively. Passage of residual virus through host cells without Fv-1 related restriction (SC-1) results in production of infectious B-MuLV indistinguishable from that produced by RAG parent cells.


Plasma membrane localization and metabolism of alkaline phosphodiesterase I in mouse peritoneal macrophages. Alkaline phosphodiesterase I activity is demonstrable in lysates of mouse resident peritoneal macrophages (1.43 mU/mg), endotoxin-stimulated macrophages (1.36 mU/mg), and thioglycollate-stimulated macrophages (3.91 mU/mg), as well as in the lysates of several mouse cell lines. The enzyme showed little variation in culture, although serum deprivation caused a 50% decrease in enzyme activity. In each of the three macrophage types about 80% of the enzyme is inactivated by the diazonium salt of sulfanilic acid, indicating that this enzyme is a component of the plasma membrane. In thioglycollate-stimulated cells about the same fraction of enzyme can be inactivated with papain corroborating this assignment. The enzyme is inactivated with a half-time of 14.1 h in resident cells, but this is decreased to 8.2 h in endotoxin cells, and to 5.7 h in thioglycollate cells. These results are consistent with the hypothesis that the endogenous pinocytic rate is a major determinant of plasma membrane turnover. In addition, the different synthetic rates measured in resident and inflammatory cells support the concept that macrophage activation is a differentiative process leading to a qualitatively new cell type.


Human granulocyte generation of hydroxyl radical. Human granulocytes were capable of oxidizing 2-keto-4 thiomethylbutyric acid to ethylene during phagocytosis or membrane perturbation. The reaction required hydrogen peroxide and superoxide and in addition was inhibited by various hydroxyl radical (OH) scavengers. These observations represent direct evidence for the generation of OH by human granulocytes. Further, inhibition of ethylene generation by azide and cyanide suggests that OH generation in granulocytes may be linked to myeloperoxidase.


Inborn resistance of mice to myxoviruses: macrophages express phenotype in vitro. A strain of avian influenza A virus was adapted to grow in mouse peritoneal macrophages in vitro. The adapted strain, called M-TUR, induced a marked cytopathic effect in macrophages from susceptible mice. Mice homozygous (A2G) or heterozygous (F1 hybrids between A2G and several susceptible strains) for the gene Mx, shown previously to induce a high level of resistance towards lethal challenge by a number of myxoviruses in vivo, yielded peritoneal macrophages which were not affected by M-TUR. Peritoneal macrophages could be classified as resistant or susceptible to M-TUR without sacrificing the cell donor. Backcrosses were arranged between (A2G X A/J)F1 and A/J mice. 64 backcross animals could be tested individually both for resistance of their macrophages in vitro after challenge with M-TUR, and for resistance of the whole animal in vivo after challenge with NWS (a neurotropic variant of human influenza A virus). Macrophages from 36 backcross mice were classified as susceptible, and all of these mice died after challenge. Macrophages from 28 mice were classified as resistant, and 26 mice survived challenge. We conclude that resistance of macrophages and resistance of the whole animal are two facets of the same phenomenon.


Properties of a transforming virus, KiMSV(RHHV), isolated from a co-culture of rat HTC-H1 cells with K-NRK cells. Some morphological, biological, immunological and biochemical characterizations of a virus, rat helper virus pseudotype Kirsten sarcoma virus, KiMSV(RHHV), have been presented here. KiMSV(RHHV) has a type C virus ultrastructure. It is strictly rat tropic and is able to transform rat cells in vitro. The possibility of its being a xenotropic mouse virus has been carefully ruled out by exhaustive analyses of host range and immunological studies. Antigenically KiMSV(RHHV) demonstrates cross reactivity with an antiserum specific against rat leukaemia virus, no cross reactivity with antiserum against Moloney leukaemia virus, and only minor cross reactivity with antiserum against cat leukaemia virus. Analysis of virus proteins and glycoproteins by equilibrium acrylamide gradient gel electrophoresis showed that the virus complex possesses both a gp70 fraction and a p30 fraction. KiMSV(RHHV) sediments isopycnically in a linear sucrose gradient at 1.145 to 1.155 g/ml and possesses RNA and reverse transcriptase activity.


The isolation of defective variants of simian virus 40 whose genomes contain sequences derived from adenovirus 2 DNA. A new set of hybrid viruses has been isolated whose closed circular genomes 5 to 6 kB in size, contain DNA sequences derived in part from adenoviruses 2 and in part from SV40. The structure of these genomes is complex, but in the simplest case, analyses by restriction endonuclease digestion and hybridization indicate that the adenovirus 2 DNA is present as a continuous block, of maximum size 2.8 kB. Different hybrids contain sequences derived from different segments of the adenovirus 2 genome.


Tryptic peptide analysis of the structural proteins of vesicular stomatitis virus. The individual structural polypeptides of vesicular stomatitis virus have been examined by tryptic peptide analysis of 35S-methionine preparations labelled in vivo and 125I-preparations labelled in vitro. Isolates of the two classical serotypes of the virus (Indiana and New Jersey) and of a sub-type of the Indiana serotype, Brazil virus, were compared. The study showed that the major internal proteins of all three viruses gave similar maps, whereas the surface glycoproteins gave distinct maps that had very few spots in common. The map of the glycoprotein of Brazil virus, which has been shown previously to be more closely related serologically to Indiana virus than to New Jersey virus, did not show any greater similarity to the Indiana virus than to the New Jersey virus glycoprotein. On the other hand, peptide maps of the nucleoprotein and matrix protein showed Indiana and Brazil viruses to be more closely related to each other than to New Jersey virus.


Stable transformation of mouse, rabbit and monkey cells and abortive transformation of human cells by BK virus, a human papovavirus. Semi-permissive mouse, rabbit and monkey cells were stably transformed by BK virus (BKV). The specificity of transformation was demonstrated by the presence of BKV tumour (T) antigen in nuclei of transformed cells and by virus rescue with Sendai virus-mediated fusion or transfection. Two out of seven BKV-transformed cell lines were oncogenic. Permissive human cells were only abortively transformed by BKV, since morphologically modified cells persisted in culture for a few passages and eventually died.


Observations on the growth and plaque assay of BK virus in cultured human and monkey cells. Although human embryo kidney (HEK), muscle (HEM) and lung (HEL) cells are capable of supporting the replication of BK virus (BKV) through passage levels 9, 12 and 12 respectively, only third, fourth and fifth passage level HEK cells were found to be satisfactory for the plaque assay of the virus. BSC-I and VERO cells can also be used for the plaque assay of BKV. However, in HEK cells plaques can be visualized in 20 days (compared to 28 days in BSC-I cells), and since in VERO cells the plaques are poorly defined and the titre about I log10 lower than in either HEK or BSC-I cells, HEK cells were the ones chosen for use.


Experimental lipid storage myopathy. A quantitative ultrastructural and biochemical study. A lipid storage myopathy was induced in rats treated with daily doses of 2.5 g/kg brominated vegetable oil. As in human lipid storage myopathies, type I fibers were selectively severely affected, the lipid deposits were surrounded by mitochondria and the mitochondrial fraction of the affected fibers was increased. The oxidation of [U-14C]palmitate, [1(-14)C]octanoate and beta-[3(-14)C]hydroxybutyrate was significantly depressed but [1(-14)C]palmitate, as well as labeled pyruvate and succinate were oxidized at normal rates. The activities of long-chain, medium-chain and short-chain carnitine acyltransferases and the muscle carnitine levels were normal. The lipid storage is attributed to impaired beta oxidation of medium-chain and short-chain fatty acyl residues. An approach to the investigation of those human lipid storage myopathies not due to defects in the carnitine system is suggested.


Clinical comparison of technetium-99m diphosphonate and pyrophosphate in bone scintigraphy: concise communication. Thirty patients had bone scintigraphy with both Tc-99m pyrophosphate (Tc-PPi) and Tc-99m diphosphonate (Tc-HEDP). The images were given a composite rating for quality and the basis of three sets of criteria, and were also compared for the number of lesions detected by each agent. The two agents provided no difference in scan quality. Nevertheless, in ten of the 30 patients, at least two of the three readers detected with Tc-HEDP lesions that were not seen with Tc-PPi, and in two such cases all three readers considered the Tc-PPi scan normal. In another of these ten, two of three readers felt the Tc-PPi image was norm, whereas all three detected the lesion with Tc-HEDP. The reverse never occurred (P less than 0.01).


Radiometric detection of herpes simplex viruses. Two radiometric techniques were investigated as possible means of detecting viruses in clinical specimens. The effect of herpes simplex virus on DNA synthesis by monolayers of human embryonic lung fibroblasts was monitored by the incorporation of [3H] thymidine or [125I] iododeoxyuridine ([125I] IdU). Radiometric results were compared with those obtained from visual examination for cytopathic effects in the same cell line. Cells infected with herpes simplex virus type-2 (HSV-2, 10(6.8) virions) showed a marked increase in [3H] thymidine incorporation 2--6 hr after infection. Types 1 and 2 herpes simplex virus yielded similar results, uith increased incorporation of tracer being observed 72 hr after infection with 10 virions. The [3H] thymidine technique was used successfully to assay mouse brains infected with HSV-1. Increased [125I] IdU incorporation was observed 6 hr after infection with 10(5)--10(6.8) HSV-1 virions, 24 hr after 10(4) virions, 48 hr after 10(3) virions, and 72 hr after 10--100 virions. The increased [125I] IdU incorporation was completely inhibited by preneutralization with immune serum. These radiometric techniques for detection of viral effect on cellular metabolism are simple, objective, and quantitative.


Effect of cytomegalovirus infection on metabolism of WI-38 cell cultures: concise communication. The effect of cytomegalovirus on the metabolism in monolayers of human embryonic lung fibroblasts (WI-38 cells) was studied. Effects of viral infection were examined by comparing [3H] thymidine incorporation in infected cells with that in uninfected cells. The time for detecting changes in cellular metabolism using the radiometric method was compared with that for observing cytopathic effects in infected cells. Compared with uninfected cells, cells infected with 10(4) TCID50 of virus showed nearly 400% increased in [3H] thymidine uptake 48 hr after infection. The radiometric method was able to detect 10(3) TCID50 of virus, with about 250% stimulation, 24--48 hr before visible signs of cytopathic effects. Our results suggest that with further development, radiometric measurement of metabolism in cytomegalovirus-infected cell cultures might provide a means of detecting viral presence in clinical assays. The radiometric method has the advantage of objectivity and potential for automation.


Side effects of diphenylhydantoin: a review. For almost 40 years, diphenylhydantoin has been the preferred drug in the treatment of seizure disorders. Soon after the drug was introduced into clinical practice, gingival enlargement was noticed as a side effect and, despite much research effort, the etiology of this condition remains unknown. More recently, diphenylhydantoin-induced osteomalacia and the teratogenic effects of the drug have been reported. Recent advances in our knowledge of these conditions will be discussed and future directions for research will be outlined.


A method for detecting therapeutic activity against Schistosoma mansoni in mice. A simple and rapid assay, suitable for routine screening against Schistosoma mansoni in mice, can be achieved by using a reduction in the severity of hepatic lesions as the chief criterion of efficacy. Previous attempts to use this criterion were largely hampered by the use of inappropriate time schedules. Provided the timing of treatment and necropsy is restricted to a certain schedule, a mere glance at the opened abdomen of an infected mouse is sufficient to determine whether schistosome reproduction has been suppressed (by chemosterilization or by broader anthelmintic effects). The essence of the necessary schedule is treatment beginning at 4 weeks after infection and prolonged (continuously or intermittently) for 2 weeks, followed by necropsy at 8 weeks after infection. Using the methods described, two persons can easily examine mice for therapeutic response at the rate of 300 per hour. The assay has been shown to detect both schistosomaticidal and chemosterilizing compounds.


Release of endogenous serotonin from two identified serotonin-containing neurones and the physiological role of serotonin re-uptake. 1. The amounts of endogenous serotonin (5-HT) released into the medium by the cerebro-buccal ganglionic ring of Aplysia californica incubated in artificial sea water (ASW) were measured. The rate of spontaneous 5-HT release varied between 0.4 and 1.2 p-mole per hour, which is less than 1% of the total 5-HT present in this preparation.2. Direct stimulation of the ordinarily silent 5-HT-containing giant cerebral neurones resulted in a 80-100% increase of the 5-HT released, but only when the 5-HT uptake was blocked by chlorimipramine (1-10 muM).3. High K(+) media (50 mM) also caused a significant increase in the amount of 5-HT released from the preparation provided that chlorimipramine (1-10 muM) was present in the incubation fluid.4. Co(2+) ions (10-30 mM) added to the incubating medium blocked the spontaneous leak of endogenous 5-HT as well as the release, in the presence of chlorimipramine, evoked either by stimulation of the 5-HT-giant cerebral neurones or high K(+)-media.5. In the presence of chlorimipramine or desmethylimipramine, the duration and/or the amplitude of the excitatory or the inhibitory synaptic potentials evoked in the buccal neurones by the stimulation of the 5-HT giant cerebral neurones were markedly enhanced.6. These results strongly support the idea that 5-HT is the synaptic transmitter released at the excitatory and inhibitory junctions established by the 5-HT giant cerebral neurones in the ipsilateral buccal ganglia. In addition, they underline the role of amine re-uptake in the physiological inactivation of 5-HT as a transmitter.


Octanol reduces end-plate channel lifetime. 1. Post-synaptic effects of n-octanol at concentrations of 0.1-1 mM were examined in toad sartorius muscles by use of extracellular and voltage-clamp techniques.2. Octanol depressed the amplitude and duration of miniature end-plate currents and hence depressed neuromuscular transmission.3. The decay of miniature end-plate currents remained exponential in octanol solutions even when the time constant of decay (tau(D)) was decreased by 80-90%.4. The lifetime of end-plate channels, obtained by analysis of acetylcholine noise, was also decreased by octanol. The average lifetime measured from noise spectra agreed reasonably well with the time constant of decay of miniature end-plate currents, both in control solution and in octanol solutions.5. Octanol caused a reduction in the conductance of end-plate channels. Single channel conductance was on average about 25 pS in control solution and 20 pS in octanol.6. In most cells the normal voltage sensitivity of the decay of miniature end-plate currents was retained in octanol solutions. The lifetime of end-plate channels measured from acetylcholine noise also remained voltage-sensitive in octanol solutions. In some experiments in which channel lifetime was exceptionally reduced the voltage sensitivity was less than normal.7. In octanol solutions, tau(D) was still very sensitive to temperature changes in most cells although in some the temperature sensitivity of tau(D) was clearly reduced. Changes in tau(D) with temperature could generally be fitted by the Arrhenius equation suggesting that a single step reaction controlled the decay of currents both in control and in octanol solutions. In some cells in which tau(D) became less than 0.3 ms, the relationship between tau(D) and temperature became inconsistent with the Arrhenius equation.8. As the decay of end-plate currents in octanol solutions remains exponential, and the voltage and temperature sensitivity can be unchanged even when tau(D) is significantly reduced, it seems likely that octanol decreases tau(D) by increasing the rate of the reaction which normally controls the lifetime of end-plate channels.


Formation and elimination of foreign synapses on adult salamander muscle. 1. Synapses by flexor nerve were induced on denervated extensor muscle in adult salamander forelimbs. Excitatory potentials evoked by these ;foreign' synapses were at first small but increased to normal amplitude within several weeks, in the absence of correct nerve reinnervation.2. Upon return of the correct nerve the efficacy of foreign synaptic transmission began to decline. The time of initiation of this decline correlated well with the resumption of correct nerve transmission. The suppression of foreign transmission involved a reduction in mean quantal content of transmitter release.3. Suppression of foreign synapses was sufficiently thorough that most ceased transmitting entirely. Before reinnervation by the correct nerve 97% of the extensor muscle fibres received functional foreign synapses while 4-6 months after correct nerve return only 35% of the fibres retained foreign synapses, with weak transmission.4. Two lines of evidence indicate that suppressed foreign synapses are lost from the muscle: (a) a second correct nerve lesion 6-8 months after the initial denervation produced no significant increase in the proportion of fibres with foreign transmission and (b) four muscles which showed complete suppression of foreign transmission were bathed in medium containing horseradish peroxidase (h.r.p.) and the correct nerve was stimulated repetitively. Subsequent histochemical staining for h.r.p. and examination of synapses by electron microscopy revealed that 94% of the axon terminals had h.r.p. incorporated in vesicles. Thus at least that percentage of all identifiable synapses were from the correct nerve.5. This ability to eliminate incorrect synapses in favour of correct ones is speculated to be a general characteristic of embryonic nervous systems, which in adulthood is retained by salamanders but lost by most other animals.


The control of enzyme secretion from fly salivary glands. 1. Stimulation of fluid secretion from fly salivary glands by 5-hydroxytryptamine (5-HT) is known to involve calcium and cyclic AMP. Isolated salivary glands were used to investigate the role of these second messengers in the control of enzyme (sucrase) secretion.2. The protein component of secretion from isolated glands treated with 5-HT appears to be identical to that of saliva secreted by flies during feeding.3. Stimulation of fluid secretion by 5-HT follows a definite dose-response curve, but there is no consistent relationship between the rate of enzyme secretion and the stimulating concentration of 5-HT.4. Exogenous cyclic AMP causes secretion of enzymes as well as of fluid, thus mimicking the action of 5-HT. The phosphodiesterase inhibitor theophylline enhances the rate of 5-HT-stimulated enzyme secretion.5. Removal of calcium from the bathing medium enhances enzyme secretion in response to 5 or 10 nM-5-HT but has no effect on enzyme secretion stimulated by 100 nM-5-HT or by cyclic AMP.6. Addition of 0.1 mM-lanthanum to medium containing 2 mM-calcium mimics the effect of calcium-free solution on 5-HT-stimulated enzyme secretion.7. The ionophore A 23187 causes secretion of both fluid and enzyme. The secretory rate is initially high but soon declines and ceases after about 40 min.8. Enzyme secretion in response to 5-HT or to cyclic AMP is progressively inhibited as the concentration of potassium is increased from 10 to 80 mM. Secretion in response to A 23187 is initially inhibited by 80 mM-potassium but then partially recovers.9. The rate of enzyme secretion appears to be affected by the intracellular concentrations of both calcium and cyclic AMP. It is possible that the rate of enzyme secretion increases as the intracellular calcium concentration rises, until the optimal calcium concentration is reached when further increase in the level of calcium progressively inhibits secretion. The optimal calcium concentration for enzyme secretion is lower than that for fluid secretion, and 5-HT normally causes maximal fluid secretion and submaximal enzyme secretion.


The effect of age on neuromuscular transmission. 1. Resting membrane potentials (RMPs), spontaneous miniature end-plate potentials (m.e.p.p.s), and evoked end-plate potentials (e.p.p.s) were recorded in phrenic nerve-hemidiaphragm preparations from rats at ages from 11 to 375 days. 2. The mean RMP increased from -64.1 +/- 1.2 mV (mean +/- S.E.) at age 11 days to -71.3 +/- 1.0 mV at age 30 days, after which there was no significant change with age. 3. The mean amplitude of m.e.p.p.s decreased from 1.088 +/- 0.070 mV at 11 days of age to 0.405 +/- 0.030 mV at 175 days of age, after which there was no significant change. 4. There was a rpaid, large increase in the frequency of m.e.p.p.s from 0.02/sec to 0.97/sec (geometric means) between 11 and 23 days of age, followed by a slower increase to 3.19/sec at 175 days of age. Subsequently there was a decrease by 2.58/sec at 375 days of age. 5. The mean of quantum content of plateau e.p.p.s elicited at a frequency of 10 Hz increased from 20.5 quanta/e.p.p. to 169.9 quanta/e.p.p. (geometric means) between 11 and 175 days of age and then decreased to 120.4 quanta/e.p.p. at 375 days of age. 6. The mean quantum content of the first e.p.p.s of trains of e.p.p.s increased from 44.2 quanta/e.p.p. to 468.8 quanta/e.p.p. (geometric means) between 11 and 175 days of age and then decreased to 358.1 quanta/e.p.p. at 375 days of age. 7. The calculated safety factor of neuromuscular transmission increased with age up to 110-175 days and subsequently decreased. 8. The change in all the above parameters occurred most rapidly in the first 6 weeks of life. The rapidity of these changes indicates that great care must be taken to ensure that control and experimental animals are adequately matched according to age, especially when rats weighing less than about 300 g are used.


Bisamidines of 2,6-diaminoanthraquinone as antiamebic agents. A series of bisamidines of 2,6-diaminoanthraquinone was synthesized and tested against cecal and hepatic forms of Entamoeba histolytica infections in rats and hamsters, respectively. A number of compounds were found to have good activity against infections in both species.


Chemical analysis of an angiofibroma from a patient with tuberous sclerosis. Amino-acid analyses on the acid hydrolysates of an angiofibroma and skin established that the former tissue contained less collagen than skin based on the reduced content of hydroxyproline, glycine, proline and alanine in the tumour. From lysosomal enzyme measurements it became evident that the specific activities of the hexosaminidases, beta-glucuronidase and beta-galactosidase were elevated. Analyses of the alcohol insoluble fraction following pronase digestion revealed that the tumour contained more acidic glycosaminoglycan (AG) than skin as assessed by uronic acid and hexosamine measurements. More outstanding, however, was the seven-fold increase in the total carbohydrate in the AG fraction of the tumour. The overall composition of this fraction was very similar to comparable material from foetal skin except that the tumour fraction contained increased sulphate concentration.


Transformation in vitro of glial hamster cells by Rous sarcoma virus. Cell lines from the brains of inbred CF hamster embryos were established in vitro. The morphology of the cells in the light and electron microscopes was that of glial cells, and the cells contained the nervous system-specific protein S-100. Infection with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup B, resulted in foci of transformation. The transformed cells were virogenic and upon intracerebral and sc inoculations into young hamsters, they developed into histologically typical gliomas.


Suppressor cells in mice with murine mammary tumor virus-induced mammary tumors. I. Inhibition of mitogen-induced lymphocyte stimulation. Suppressor cell activity was present in the glass-adherent fraction of spleen cells from C3H mice bearing murine mammary tumor virus-induced mammary tumors. These cells effectively suppressed the blastogenic response of syngeneic normal lymphocytes to concanavalin A (Con A). Suppression by the spleen cells from mammary tumor-bearing mice was not dependent on DNA synthesis. Removal of the suppressor cells from spleen cell suspensions of tumor-bearing mice was not dependent on DNA synthesis. Removal of the suppressor cells from spleen cell suspensions of tumor-bearing animals (TBA) by passage of the cells on glass wool columns increased the Con A response of the remaining cells by fourfold to eightfold. Characterization of the suppressor population indicated that the cells were also adherent to nylon wool but not to plastic and contained a significantly increased proportion of surface immunoglobulin-bearing and complement receptor-bearing lymphocytes. Depletion of macrophages and T-cells did not remove the suppressive activity from the spleens of the TBA. The results were consistent with the identification of the suppressor cell as a B-cell.


Isolation of a precipitating glycoprotein antigen from cell cultures persistently infected with bovine leukemia virus. A procedure was developed to isolate a glycoprotein with precipitating antigen activity from fluids from fetal lamb kidney cell cultures persistently infected with bovine leukemia virus (BLV). The antigen was precipitated by ammonium sulfate and subjected to affinity chromatography on concanavalin A Sepharose. The glycoprotein was eluted with alpha-methyl-D-mannoside and was further purified by gel filtration over Sephadex G-100. Antigen activity was determined by agar gel immunodiffusion (AGID) reactions with serum from cattle infected with the virus. The major portion of the AGID activity was eluted from the Sephadex G-100 in the 60,000-dalton elution region. In some experiments, identical AGID activity was also found in the 18,000-dalton elution region. The larger protein was discovered to have a molecular weight of 58,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its designation as a glycoprotein was confirmed by carbohydrate-positive staining. The isolated BLV glycoprotein antigen did not contain ovine or bovine proteins as indicated by gel immunodiffusion.


Immune function in healthy relatives of patients with malignant disease. Immune function studies of healthy members of families with multiple lymphoreticular neoplasms were compared with those of healthy members of families with multiple cancers and families with no known history of cancer. The lymphoma family group had a significantly elevated serum level of IgM and diminished responses to the Candida albicans skin test antigen and the T-cell mitogen concanavalin A. Subjects with Epstein-Barr virus-viral capsid antigen titers greater than or equal to 160 had lower responses to mitogens.


Epstein-Barr virus-associated and other antiviral antibodies during intense BCG administration to patients with Burkitt's lymphoma in remission. Patients with Burkitt's lymphoma in chemotherapy-induced remission received through dermal scarifications one or two doses per week of approximately 3 X 10(8) living BCG organisms (Pasteur Institute vaccine). This treatment was always followed by usually rapid increases by 1--4 log2 steps in the antibody titers to Epstein-Barr virus (EBV)-associated cell membrane antigens. Titer increases of less than 2.5 log2 steps within the first month after the start of BCG treatment correlated with a significantly elevated frequency of extradural relapse as compared to that seen in patients with larger titer rises. During this time, antibodies to EBV-associated viral capsid antigens and early antigens of D and R specificity, as well as antibodies against herpes simplex, varicella, cytomegalovirus, measles, and respiratory syncytial virus antigens, did not show any consistent or impressive changes.


Cell-surface antigens from human breast tumor cells. Biochemical and immunologic studies on breast cancer with the use of cells from a human ductal cell carcinoma, BOT-2, were initiated. Antigens were extracted from the cells by mild sonication and purified by gel filtration chromatography. Only one of the three peaks from gel filtration chromatography reacted with antiserum prepared against whole BOT-2 cells. Analysis by polyacrylamide gel electrophoresis of the BOT-2 cell extract revealed many protein bands, whereas analysis of the antibody-reactive peak after gel filtration chromatography revealed fewer protein bands. Immunologic tests to identify human serum antibodies against BOT-2 cells or cell extracts were performed by fixed cell immunofluorescence, living cell membrane immunofluorescence, and indirect hemagglutination. Depending on the test, the sera from women with diagnosed, untreated mammary cancer were positive in 45--80% of the cases, whereas the sera from women without apparent breast diseases (controls) were positive in only 5--10% of the cases. The results suggested that the antigens from the BOT-2 cells will be useful in understanding the processes involved in human mammary neoplasia.


Cell-mediated immune response to simian oncornavirus antigens in pregnant women. Tritiated thymidine incorporation into lymphocytes of 118 adult women was studied in the presence of mitomycin C-treated cells prepared from cell lines continuously producing Mason-Pfizer monkey virus (MPMV), baboon C-particle virus, or simian sarcoma virus (SSV) and in the presence of control cell lines documented for the absence of oncornaviruses. At the end of pregnancy, women who had 5-9 pregnancies showed a high frequency (53%) of specific positive responses to cells with MPMV antigens. The frequencies were 15% for pregnant women with smaller numbers of pregnancies and 3% for nonpregnant women with similar numbers of previous pregnancies as in the pregnancy group. None of these nonpregnant women had lymphocytes responding to stimulation by baboon C-virus antigens, but positive responses were obtained in 20 and 16% of the pregnant groups, respectively. No correlation was found between responses to MPMV or baboon C-virus antigens. Of 48 women (35 pregnant, 13 nonpregnant) who were tested for lymphocyte responses to SSV antigens, only 2 showed a positive response. The results indicated that two distinct antigens, related to MPMV and to baboon C-virus, may be expressed during pregnancy and may then induce a transient cell-mediated response.


Inhibition of deoxynucleotide-polymerizing enzyme activities of human leukemia lymphoblasts and simian sarcoma virus by tilorone and thirteen of its analogs. Tilorone, which is 2,7-bis[2-(diethylamino)ethoxy]-9H-fluoren-9-one dihydrochloride, and 13 of its analogs inhibited human cellular DNA polymerases alpha and beta assayed with activated DNA as template and also cellular DNA polymerase gamma and DNA polymerase from simian sarcoma virus assayed with poly(A) (dT)12-18 as template. Terminal deoxynucleotidyltransferase (TdT),  which has no template requirement, was not inhibited by any of the 14 compounds when d(A)12-18 or d(G)12-18 was used as initiator. Three compounds did not inhibit TdT assayed with activated DNA as initiator, but 11 compounds did, and these 11 compounds were generally less inhibitory to TdT than to the other DNA polymerases. The three compounds that did not inhibit TdT assayed with activated DNA but did inhibit the other DNA polymerases will be useful in the characterization of TdT activity. Modifications of the polycyclic ring structure of tilorone and the kinds of substituent groups attached to the ring structures influenced the degree of inhibition of all enzymes.


Investigation of human urogenital tract tumors of papovavirus etiology: brief communication. Cells cultured from human urogenital cancer and other cancers as well as cells from noncancerous tissues were examined by immunofluorescent staining with antibodies to T-antigens and capsid antigens of papovaviruses BK virus (BKV), JC virus, and simian virus 40(SV40), and to capsid antigens of herpes simplex virus types 1 and 2 and human cytomegalovirus (CMV). Cells from early passage cultures of 123 primary tissues and from 14 continuous lines derived from transitional or renal cell carcinoma were tested. None of the cell preparations was specifically stained with any of the antisera. A serologic comparison of patients with bladder cancer, patients with prostate cancer, and normal control groups of BKV hemagglutination-inhibiting and SV40-neutralizing antibodies showed no differences among the 3 groups. None of the sera in the 3 groups had SV40 or BKV T-antibodies. In tests of supernatants of 35 primary cultures for presence of virus, a single isolation, that of a cytomegalovirus, was made. The study revealed no evidence that infection with papovaviruses of the SV40-polyoma subgroup has any part in the production of bladder and prostate cancer.


Mammary tumors, hepatocellular carcinomas, and pancreatic islet changes in C3H-Avy Mice. Studies of tumor incidence and assorted lesions found in 187 C3H-Avy mice throughout their natural life-spans revealed the following: Hepatocellular carcinomas occurred in 54.3% of males, mammary carcinomas in 95% of females, pancreatic islet cell adenomas in 9.4% of males and in no females, and pancreatic islet cell hyperplasia in 41% of males and 23% of fefemales. Islet cell hyperplasia and adenomas appeared to consist predominantly of alpha and delta cells. Multiple tumors, or hyperplasia, or both, of a single site or of multiple sites occurred as frequently in males as they did in females--49.6% and 51.7% respectively. The most frequent neoplasms were hepatocellular carcinomas and islet cell tumors or hyperplasia in males (45.7%) and multiple mammary tumors in females (30%). Heretofore unreported tumors found in this strain of mouse were 12 islet cell adenomas, 2 spindle cell tumors of the meninges and olfactory lobes, a squamous cell carcinoma of the nasal turbinates, and a schwannoma of the spermatic cord.


Oncornavirus lytic activity in the serum of gibbon apes. Fresh blood serum from normal gibbon apes (Hylobates lar) contained heat-sensitive lytic activity for various mammalian oncornaviruses. Lytic activity quantitatively similar to that in gibbon serum was demonstrated in serum from three other primate species, including man; it was demonstrated to be low or absent in lower mammalian species with the exception of domestic cats, which had intermediate levels of serum lytic activity. Gibbons that acquired infectious gibbon ape leukemia virus, either naturally by exposure to a virus-shedding ape or experimentally by deliberate virus inoculation, had the same levels of serum lytic activity as did unexposed gibbons that had no detectable antibodies to gibbon ape leukemia virus. A leukemic-viremic gibbon had low or absent serum oncornavirus lytic activity. These results indicated that serum lytic activity does not necessarily protect against infection by oncornaviruses, although it may limit virus replication and/or dissemination.


Spontaneous and induced preleukemia cells in C57BL/6 mice:brief communication. A transplantation bioassay method was used to verify the presence of preleukemia cells in C57BL/6 mice shortly after leukemogenic treatment or in relation to age increase. Preleukemia cells were identified mainly among bone marrow cells of old C57BL/6 mice or within 10 to 30 days after leukemogenic treatment of young mice with radiation-induced leukemia virus variants, fractionated doses of irradiation, or 7,12-dimethylbenz[a]anthracene (DMBA), although the overt disease did not occur until many months later. Mice could carry preleukemia cells without necessarily developing overt leukemia. Since the leukemogenic agents used in the present studies induced T-leukemias, the role of the thymus in the induction of preleukemia cells was tested. Thymectomy affected viral transformation but did not diminish the number of preleukemia cells induced by DMBA or X-ray.


The clinical utility of ipsilateral stapedius reflex tests. Ipsilateral stapedius reflex testing has been evaluated in 166 patients. It proves of value in defining the state of an ear (1) opposite to a unilateral conductive loss; (2) the less hard-of-hearing ear in the presence of bilateral asymmetrical sensorineural hearing losses where the worse ear has sufficiently severe loss to prevent a contralateral reflex from being elicited; (3) in patients with absent contralateral acoustic reflexes at 500, 1000, and 2000 Hz; and (4) in suspected central lesions. The test is still bedevilled by artifacts which are produced in the ear rather than the machine; atypical results must be evaluated with extreme caution.


Effect of rhesus or vervet cytomegalovirus infection of DNA synthesis in untreated and 5-iodo-2'-deoxyuridine-arrested cells. Infection of permissive cells with either rhesus or vervet cytomegalovirus resulted in suppression of cellular DNA synthesis, only viral DNA was resolved by isopycnic centrifugation after 24 h postinoculation. Infection of 5-iodo-2'-deoxyuridine (IUdR)-arrested cells with either of the simian cytomegaloviruses, however, induced synthesis of cellular DNA of normal density; synthesis of cellular DNA substituted with IUdR as evidenced by resolution of a heavy DNA peak after isopycnic centrifugation was not observed. Stimulation of DNA synthesis in IUdR-arrested cells was not observed with virus inactivated with UV light.


Molecular mechanisms involved in the differential expression of gag gene products by clonal isolates of a primate sarcoma virus. Clonal isolates of an early passage stock of woolly monkey sarcoma virus (WSV) have been shown to code for different numbers of woolly monkey helper leukemia virus gag gene products. In the present report, the molecular mechanisms responsible for their differential expression of gag gene products have been analyzed. Three WSV RNA genomes were shown to possess sedimentation coefficients consistent with the differences demonstrated in their allotments of helper viral sequences. The WSV variant (WSV clone 9) that expressed no detectable proteins was shown to contain the largest amount of helper viral information. Moreover, there was no additive hybridization of the WLV complementary DNA probe by RNA of this WSV clone and that of a WSV clone coding for several gag gene products. These results suggest that the lack of expression of gag gene products by WSV clone 9 is not due to a major deletion of helper viral gag gene sequences. Similar levels of WLV-specific RNA were demonstrated in cells nonproductively transformed by each WSV clone, arguing that the ability to express gag gene proteins was not related to the magnitude of viral RNA transcription. Taken together, the results are most consistent with a mechanism by which small deletions or point mutations in the genomes of some WSV variants result in premature termination of translation or synthesis of immunologically nonreactive gag gene proteins. The present findings have implications concerning the effects of evolutionary selective pressures on helper viral genetic information in mammalian transforming viruses.


Human lymphoblastoid cells as hosts for parvoviruses H-1 and rat virus. A human T-cell line (Molt-4) was shown by viral hemagglutination and infectivity assays to support the replication of rat virus (RV) and H-1 virus. In addition, H-1 virus, but not RV, multiplied in two human B-cell lines, AV-1 and NC-37. The ability to bind radioactively labeled RV was demonstrated for each of the cell lines, but viral adsorption occurred to a greater degree with Molt-4 cells than with either AV-1 or NC-37 cells. After challenge with RV, virus-specific antigens were detected in cells of the B-cell lines by the indirect immunofluorescence technique. Infection of AV-1 or NC-37 cells by RV apparently results in an abortive cycle of virus replication. Differences among the three cell lines that might influence with H-1 virus or RV are discussed.


Quantitation and localization of Rous sarcoma virus-specific RNA in transformed and revertant field vole cells. Hybridization analysis of RNA from transformed clones of Rous sarcoma virus (RSV)-infected field vole cells and revertant subclones indicated the presence of similar amounts of viral-specific RNA in both cell types. Employing both a relatively uniform and representative complementary DNA probe and genomelength complementary DNA, we have demonstrated that the majority of RSV proviral DNA is transcribed into viral-specific RNA in both transformed and revertant clones. The viral-specific RNA is present in several size classes, the largest of which is genome-length 35S RNA. Studies on the intracellular distribution of viral-specific RNA indicate that both transformed and revertant cells contain viral RNA in their cytoplasm. Furthermore, the bulk of viral-specific nucleotide sequences are also associated with polyribosomes in both cell types. These data indicate that, contrary to previously reported studies with other retrovirus-revertant cell systems, the entire RSV provirus DNA is transcribed into viral RNA similarly in both transformed and revertant vole cells. Since we have previously demonstrated similarities in sarcoma-specific viral RNA in both revertant and transformed vole cells, these data collectively indicate that the loss of the transformed phenotype does not reflect changes in transcription of all or part of the RSV provirus, or the processing, transport, or polyribosome association of viral-specific RNA representing the entire RSV genome.


Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2. Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.


Cellular and cell-free synthesis of simian virus 40 T-antigens in permissive and transformed cells. mRNA extracted from a variety of simian virus 40 (SV40)-infected monkey cell lines directs the cell-free synthesis of viral T-antigen polypeptides with molecular weights estimated as 90,000 and 17,000. However, the size, abundance, and distribution of these T-antigens synthesized in vivo vary greatly over a range of permissive and transformed cell lines. To establish whether differences in the size of T-antigen polypeptides can be correlated with the transformed or lytic state, recently developed lines of SV40-transformed monkey cells that are permissive to lytic superinfection were analyzed for T-antigen. In these cells, regardless of the state of viral infection, the size and pattern of T-antigen are the same. However, species differences in the largest size of T-antigen are the same. However, species differences in the largest size of T-antigen do exist. In addition to the 90,000 T-antigen, mouse SV3T3 cells contain a 94,000 T-antigen polypeptide as well. Unlike the size variations in monkey cells, which are due to modification of T-antigen polypeptides, the 94,000 SV3T3 T-antigen results from an altered mRNA, since the cell-free products of SV3T3 mRNA also contains the 94,000 T-antigen polypeptide.