Oncogenic transformation of murine lymphoid cells by in vitro infection with Abelson leukemia virus. Spleen cell cultures stimulated to DNA synthesis by antigen or mitogen were infected with Abelson virus, a C-type RNA virus inducing nonthymic lymphomas in mice. After 3 days the cells were transferred to mice and caused 100 percent incidence of lymphomas in as few as 29 days. That a number of the tumors were of donor origin, as shown by female karyotypes in recipient male mice, indicated that cells infected by virus in vitro were transformed. The process depended upon both virus and stimulation of lymphocytes in culture. Lymphoid tumors did not develop in mice receiving cells from virus-infected cultures not exposed to antigen or mitogen.


Effect of protease inhibitors on focus formation by murine sarcoma virus. The effect of six protease inhibitors, isolated from various species of actinomycetes, on focus formation by murine sarcoma virus was examined. Pepstatin was the only inhibitor. The treatment of cells with pepstatin at various times possibly retards the early stage of infection with murine sarcoma virus.


Detection of oncornavirus antigenic activity in human urothelial tissues. Our studies indicate that certain urologic tissues demonstrate evidence of proteins which compete with the interspecies antigens of C-type ribonucleic acid viruses for binding sites of the feline and murine oncornavirus antiglobulins. This fact indicates either the association of viral antigens within these tissues or the presence of a protein so similar to the interspecies antigen that it cross reacts. Studies are now underway to localize reacting antigens within these urologic cells, to correlate the immunologic properties with enzymatic properties known to be specific for oncornaviruses and to assay the activity of patient sera with isolated interspecies antigens. Sera from patients with urologic malignancies will be tested to determine whether the sera can effectively absorb reactivity from tissue extracts or compete with reagent rabbit antisera raised against the interspecies component of the viruses. Should either activity be detected in patient sera, clinical screening will be undertaken to determine if this approach is applicable to early detection of urologic malignancies.


Androblastoma and germ cell tumor of the same testis: a case report. The rare case of a testicular androblastoma associated with a complex germ cell tumor of the same testis is reported. Gynecomastia preceded the clinical appearace of testicular swelling by 3 months, suggesting that the 2 tumors developed contemporaneously and that, in theory, the Sertoli cell tumor is probably dependent on the germ cell tumor.


[Comparative studies of the therapeutic effect of colistin methanesulfonate administered intramuscularly and intravenously on the acute bacterial infection in mice (author's transl)]. The therapeutic potencies of colistin methanesulfonate (CLM) was assessed quantitatively in acute infection of mice with clinically isolated strains of Escherichia coli and Pseudomonas aeruginosa, and effect of different routes of administration was compared. There was no detectable difference in the therapeutic effect of CLM when intramuscular (im) or intravenous (iv) administration was initiated one hour after the infection. On the other hand, a significant difference in ED50 given by im and iv administrations was observed, indicating the superiority of iv administration, when the treatment started 4 to approximately hours after the infection. No difference in the therapeutic effect of polymyxin B (PMB) and tetracycline (TC) administered via either im or iv route was found even in the delayed administration. In contrast to PMB and TC, lower toxicity of CLM was determined when it was administered iv rather than im.


Replication of Herpesvirus saimiri in cultured lymphocytes of infected owl monkeys (Aotus trivirgatus). An electron microscopic and immunofluorescent study. The lymphocytes of five owl monkeys infected with Herpesvirus saimiri (HVS) and three control monkeys were studied. Lymphocytes were separated on Ficoll-Hypaque gradients, incubated in suspension cultures, and prepared for electron microscopic and immunofluorescent study at 24, 48, and 72 hours after the beginning of culture (AC). Buffy coats of whole blood immediately after bleeding were also prepared for study. At the time of the study, four of the five infected monkeys had died of malignant lymphoma and lymphocytic leukemia. HVS virions were demonstrated by electron microscopy and HVS antigens by immunofluorescence in 1 to 20 per cent of the lmyphocytes from infected monkeys in two of five cultures at 24 hours AC, four of five at 48 hours AC, and all five at 72 hours AC. There was quite good agreement between the electron microscopic and the immunofluorescent data. None of the control monkey cultures and none of the buffy coat preparations contained HVS virions or antigens. By electron microscopy the great majority of the virus particles were nucleocapsids within the nuclei of lymphocytes. Very seldom were enveloped virions observed. There was some evidence to suggest that the higher the percentage of lymphocytes containing HVS the poorer the prognosis for the monkey. HVS was isolated from all five infected monkeys by cocultivation of lymphocytes with Vero cells. Control lymphocyte cocultivations were negative.


Restoration of myocardial bioenergetic metabolism in swine after periods of ischemic ventricular fibrillation. Myocardial mitochondrial function and high energy phosphate levels were measured in normal swine, in swine after either 5 or 10 minutes of ischemic ventricular fibrillation (IVF) while on cardiopulmonary bypass, and in swine defibrillated after either 5 or 10 minutes of IVE. The damage to myocardial mitochondria induced by IVF, such as partial uncoupling, decreased oxygen uptake, and loss of cytochrome oxidase activity, was completely reversed almost instantly by coronary artery perfusion and the restoration of sinus rhythm. After either 5 or 10 minutes of IVF followed by coronary artery reperfusion and defibrillation, myocardial creatine phosphate (CP), adenosine monophosphate (AMP) and adenosine diphosphate (ADP) return to normal levels very rapidly. However, adenosine triphosphate (ATP) levels remain significantly lower than control levels. If the bioenergetic mechanisms of swine and human myocardium are similar, it appears that IVF at least for a 10 minute period produces no damage to myocardial mitochondria that is not corrected by perfusion of the coronary arteries and re-establishment of sinus rhythm. Furthermore, sinus rhythm can be re-established and maintained despite signficantly lower levels of myocardial ATP.


Primary adenoid cystic carcinoma of the esophagus. Report of a case and review of the literature. A case of primary adenoid cystic carcinoma of the esophagus is reported and a total of 15 cases was reviewed. There were 9 men, although earlier reports indicated the disease was more common in women. The lesion was located in the middle third of the esophagus in 10 cases. The possibility of tumor development from embryonal rests of the tracheobronchial tree in the esophagus is discussed. The prognosis of adenoid cystic carcinomas is not well known, but the treatment and prognosis appear similar to those of epidermoid carcinomas of the esophagus.


Gluconeogenesis in isolated hepatic parenchymal cells. VII. Effects of monobutyryl cyclic adenosine monophosphate on gluconeogenic intermediates, phosphofructokinase, and fructose diphosphatase. Isolated parenchymal hepatocytes prepared from fasted (24 hours) rats convert added dihydroxyacetone and xylitol to glucose. Monobutyryl cyclic adenosine-3',5'-mono-phosphate (mb-cAMP) stimulated the rate from dihydroxyacetone (plus 32%; N equals 28) but not from xylitol (plus 4%; N equals 27). Iodoacetate (0.15 to 0.20 mM) was an effective inhibitor of gluconeogenesis from lactate-pyruvate mixtures (72% inhibition); quinolinate (2.5 to 3.0 mM) was relatively ineffective (26% inhibition). Measurements of glucogenic intermediates formed from added dihydroxyacetone in hepatocytes (20% cell suspension) preincubated with iodoacetate provided evidence that phosphoglyceraldehyde dehydrogenase was inhibited. Inhibition of additional glycolytic-gluconeogenic enzyme(s) involved in dihydroxyacetone metabolism was not disclosed by the metabolite concentration data. Because iodoacetate partially inhibited gluconeogenesis from dihydroxyacetone in 5% cell suspensions but did not prevent stimulation of gluconeogenesis by mb-cAMP (plus 31%), additional inhibition, probably nonspecific, occurred. With dihydroxyacetone as substrate, mb-cAMP had only a slight effect on the concentration of fructose diphosphate (decrease) relative to control experiments (no mb-cAMP), but preinculation of the cells with iodoacetate made the mb-cAMP-induced decrease much greater. mb-cAMP caused a 47% decrease (SE, 8;N equals 9) in the assayable activity of phosphofructokinase in liver cells incubated with dihydroxyacetone but did not alter fructose diphosphatase activity significantly. Cyclic GMP and cIMP were shown to stimulate gluconeogenesis from dihydroxyacetone; both nucleotides also cause a decrease in the assayable activity of phosphofructokinase. With xylitol as substrate, mb-cAMP did not cause stimulation of gluconeogenesis, decrease in fructose diphosphate concentration, or decrease in phosphofructokinase activity. Our results indicate that phosphofructokinase might be an important controlling enzyme in gluconeogenesis and subject to regulation by cAMP.


Management of parotid gland tumors in infants and children. Seventy-four pediatric patients with parotid gland tumors were seen during a 52-year period. Seventy percent of the lesions were benign and 30% were malignant. When the primary treatment is either subtotal or total parotidectomy, the recurrence rate was 2.3%.  The facial nerve should be dissected free of parotid parenchyma and preserved, except if the lesion was malignant--a situation in which the nerve might have to be sacrificed totally or in part to ensure adequate tumor removal. No patient died of a benign or malignant lesion in this series, and the morbidity after surgical removal of primary tumors of the parotid gland in children and infants was low.


Lipids of cultured hepatoma cells. VI. Glycerolipid and monoenoic fatty acid biosynthesis in minimal deviation hepatoma 7288C-1. 1-14C-Acetic, 1-14C-palmitic, or 1-14C-stearic acid was incubated with minimal deviation hepatoma 7288C cells grown in culture to assess: de novo fatty acid synthesis, oxidation, desaturation, and elongation of saturated fatty acids, as well as the ability of media fatty acids to serve as precursors of cellular glycerolipids. Distribution of radioactivity in the individual lipid classes and the various fatty acids of triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine was determined. The radioactivity among the monoenoic acid isomers derived from triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine was analyzed by reductive ozonolysis. Only small amounts of the labeled substrates were oxidized to carbon dioxide. Except for labeled stearic acid, which also was incorporated heavily into phosphatidyl inositol and phosphatidyl serine, most radioactivity was recovered in triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine. Synthesis of cholesterol and long chain fatty acids from labeled acetic acid demonstrated that these cells can perform de novo synthesis of fatty acids and cholesterol. Both labeled palmitic and stearic acids were desaturated to the corresponding delta9 monoenes, and palmitic and palmitoleic acids were elongated. The nexadecenoic acid fraction isolated from triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine, when acetic or palmitic acid was the labeled substrate, showed that greater than 70 percent of the labeled acids were the delta9 isomer. Radioactivity of the octadecenoic acid fraction derived from labeled acetic or palmitic acids was nearly equally divided between the delta9 isomer, oleic acid, and the delta11 isomer, vaccenic acid. Desaturation of labeled stearic acid produced only oleic acid. These data demonstrate that the biosynthesis of vaccenic acid in these cultured neoplastic cells proceeds via the elongation of palmitoleic acid. The relatively high level of vaccenic acid synthesis in these cells suggests that the reported elevation of "oleic acid" in many neoplasms may result from increased concentration of vaccenic acid.


Hepatoma, host liver, and normal rat liver neutral lipids as affected by diet. Groups of normal and hepatoma (7288CTC) bearing rats were maintained on normal chow and fat-free diets for 4 weeks. Normal liver, host liver, and hepatoma neutral lipids were examined in detail and compared. Water content, unaffected by diet was: hepatoma, 82 percent; host liver, 71 percent; and normal liver, 67 percent. The fat-free diet had no effect upon the hepatoma neutral lipids but elevated the triglyceride level in normal and host liver, shifted the triglyceride carbon number distribution to lower mol wt, and elevated the percentage of monoenoic acids in triglycerides and cholesteryl esters. Host triglyceride concentrations were ca. half, and cholesterol levels were reduced moderately relative to normal liver values. Hepatoma cholesterol levels were higher and triglyceride concentrations lower than normal and host liver values. Hepatoma triglycerides differed dramatically from liver and were characterized by increased concentrations of high mol wt species and a fivefold increase in the percentage of C-20 and C-22 fatty acids. The percentage of C-20 and C-22 fatty acids in hepatoma cholesteryl esters also increased ca. fivefold relative to liver. The data indicate that the systems that regulate triglyceride and cholesteryl ester fatty acid composition in liver do not control the compositions of these lipid classes in this hepatoma. The unchanged high level of essential fatty acids in the hepatoma lipids from the fat-free fed animals demonstrates the hepatoma's ability to absorb and conserve specific fatty acids.


Effects of diet and type IIa hyperlipoproteinemia upon structure of triacylglycerols and phosphatidyl cholines from human plasma lipoproteins. Four normal and two individuals with Type IIa hyperlipoproteinemia were placed on the National Heart and Lung Institute Type IIa diet (low cholesterol, smaller than 300 mg/day, high polyunsaturated, low saturated fat diet) for 1 week and on a normal diet the following week. Plasma samples were obtained and the triacylglycerols, phospholipids, and cholesterol contents of plasma and of very low density lipoproteins, low density lipoproteins, and high density lipoproteins determined. Triacyglycerol fatty acid composition was determined and stereospecific analyses of triacglycerols and phosphatidyl cholines performed. Structural determinations were limited to one normal and one Type IIa individual. In normal and Type IIa individuals, chylomicrons contained twice the amount of 18:0 as did the very low density lipoproteins, low density lipoproteins, or high density lipoproteins. The structure of the triacyglycerols from the very low density lipoproteins and low density lipoproteins was asymmetric with at least 50M% 16:0 in the sn-1 position and mostly 18:1 in positions sn-2 and 3. There was a marked difference in the distribution of 18:2 in low density lipoproteins of the normal and Type IIa individuals. The control contained equal amounts of 18:2 in the sn-1 and sn-3 positions, whereas IIa low density lipoprotein was asymmetric with 26% of the 18:2 in position sn-1 and 3% in the sn-3 position. Very low density lipoprotein was asymmetric with regard to 18:2 in control and IIa samples with an average of 5% of the 18:2 in position sn-1 and 40% in position sn-3. The phosphatidyl cholines contained predominantly 16:0 and 18:0 in position sn-1, whereas the acids in position sn-2 were unsaturated with very little difference between lipoprotein classes. Neither the short dietary periods nor source of plasma affected the structure of the phosphatidyl cholines.


Reaction of anti-phosphatidyl inositol antisera with neural membranes. Ca. 15% of the phosphatidyl inositol in myelin and microsomal membranes from rat brain was detectable by antiphosphatidyl inositol antibody. Antibody-detectable phosphatidyl inositol in myelin and microsomes readily increased when the membranes were incubated at 45 C with the antiserum. Synaptic membranes also had only a limited capacity to adsorb antibody. Quantitative binding studies with synaptic membranes and mitochondria were limited, because these membranes contain cardiolipin, which cross reacts with phosphatidyl inositol antisera. Moreover, highly purified synaptic and mitochondrial membranes contain appreciable amounts of other membrane fractions.


Site of free-fatty-acid inhibition of lipolysis by human adipocytes. When human adipocytes were incubated in albumin-free buffer, isoproterenol failed to stimulate an increase in either cyclic AMP or glycerol release. Cells incubated for 1/2 hr with 4% albumin and isoproterenol had a striking increase in cyclic AMP; this effect was markedly reduced when FFA concentration was increased by the addition of sodium oleate. When incubation was prolonged to 4 hr, the cyclic AMP concentration of stimulated cells fell towards the basal level. This decline in the level of cyclic AMP was prevented by frequent change in buffer. The ability of epinephrine and sodium fluoride to stimulate the adenylyl cyclase of human adipocyte membranes was not affected by the addition of sodium oleate. However, when intact cells were preincubated for 1 hr with added sodium oleate, the responsiveness to epinephrine of membranes derived from the cells was reduced. No such alternation in responsiveness to sodium fluoride occurred. These results indicate that the inhibitory effect of FFA on lipolysis is associated with a reduced production of cyclic AMP; the latter effect may be the consequence of FFA inhibition of adenylyl cyclase.


Biphasic effect of the alpha-adrenolytic phentolamine on hormone-stimulated formation of cyclic adenosine-3',5'-monophosphate in isolated fat cells of rats. Isolated fat cells from rat epididymal adipose tissue were incubated with various lipolytic hormones in the absence and presence of the alpha-adrenergic blocking agent phentolamine. Lipolysis, stimulated by noradrenaline, isoproterenol, or ACTH, was inhibited dose-dependently by phentolamine. At concentrations of phentolamine where lipolysis was already inhibited, phentolamine had a biphasic effect on hormone-stimulated formation of cAMP. Low concentrations of phentolamine enhanced cAMP formation, while high concentrations inhibited cAMP. The additional increase of cAMP formation by phentolamine was only seen with maximally effective concentrations of noradrenaline, isoproterenol, and ACTH. Half-maximally effective concentrations were invariably inhibited by phentolamine. The activity of noradrenaline-stimulated adenylate cyclase of fat-cell plasma membranes was inhibited by phentolamine, whereas cAMP phosphodiesterase activity was unaffected.


The glucagon receptor and adenylate cyclase. Acidic phospholipids play a critical role in the hormone activation of adenylate cyclase. Solubilized myocardial adenylate cyclase is unresponsive to glucagon and the catecholamines, two of the hormones which activate the membrane-bound enzyme. Phosphatidylserine, purified from bovine brain restored glucagon responsiveness of the solubilized adenylate cyclase. Monophosphatidylinositol, also purified from bovine brain, restored catecholamine responsiveness. Solubilized preparations of myocardial adenylate cyclase bind 125-I-glucagon either in the presence of added phosphatidylserine, thereby providing a clear separation of the processes of activation and binding. Solubilized myocardial adenylate cyclase has a molecular weight of about 160,000. Sephadex G-100 chromatography of the solubilized enzyme following the binding of 125-I-glucagon to its myocardial receptor reveals two distinct peaks; one, having catalytic activity and a molecular weight greater than 100,000 and two, the binding material having no catalytic activity and a molecular weight of 24,000-28,000. These data are consistent with the presence of a dissociable glucagon receptor site. The role of this dissociation in the activation-inactivation of the enzyme remains to be explored. It is postulated that phospholipids induce the required configurational change in the catalytic site following the binding of hormone to its receptor, and by this means couples the receptor to the catalytic site. This model may be applicable to certain clinical situations. Cardiac adenylate cyclase is unresponsive to glucagon in chronic congestive heart failure. The defect may reside either in the binding of glucagon to its receptor site or in the metabolism of a specific acidic phospholipid such as phosphatidylserine.


The role of cyclic GMP in the regulation of cyclic AMP hydrolysis. A rat-heart cyclic nucleotide phosphodiesterase has been chromatographically separated from related enzymes and its kinetic properties have been studied. The enzyme can hydrolyze both cyclic AMP and cyclic GMP and has about the same maximum velocity and apparent KM (greater  than 10-5 M) for the two nucleotides. Kinetic plots indicate positive cooperative behavior for both substrates. Cyclic GMP at low concentrations is a potent activator of cyclic AMP hydrolysis and this activation, as well as the cooperativity, can be abolished by treatment with solvents or sulfydryl reagents under conditions which do not destroy the catalytic function. A kinetic model for this enzyme is porposed and the physiologic role is discussed.


Divergent actions of protein kinase modulator in regulating mammalian cyclic GMP-dependent and cyclic AMP-dependent protein kinases. Protein kinase modulator can either augment or depress phosphorylation of substrate proteins catalyzed by cyclic GMP-dependent and cyclic AMP-dependent protein kinases prepared from mammalian and arthropod tissues. Alteration by the modulator of the phosphorylating activity of the protein kinases is considered to be due to modification of protein substrate specificity subsequent to interaction of the modulator with the catalytic subunits of the enzymes. It is likely that the physiologic role of the modulator is to monitor the opposing effects of cyclic GMP and cyclic AMP by regulating the activity of respective protein kinases. Thus the modulator may function as a biologic "fine tuner" providing on additional mechanism by which the signals imparted to cells by physiologic stimuli can be correctly expressed.


Widespread occurrence of a specific protein in vertebrate tissues and regulation by cyclic AMP of its endogenous phosphorylation and dephosphorylation. A protein whose endogenous phosphorylation and dephosphorylation are affected by cAMP has been found in the soluble and particulate fractions of all vertebrate tissues studied. This phosphoprotein, which contained a substantial proportion of the radioactive phosphate observed on SDS-polyacrylamide gels, was estimated to have an apparent molecular weight of 49,000. In the presence of Zn++, cAMP inhibited the endogenous phosphorylation of this protein (protein 49) in the cytosol and microsomal fractions. In the presence of Mg++, cAMP stimulated the phosphorylation of protein 49 in the cytosol fractions, but had only slight effects in the microsomal fractions. The dephosphorylation of protein 49 by an endogenous protein phosphatase was markedly stimulated by cAMP in the cytosol and microsomal fractions of all tissues studied. The binding of 8-azido-cAMP (a photoaffinity analog of cAMP, which reacts specifically with cAMP-binding sites) to subcellular fractions was also studied. This binding was principally to a protein of molecular weight 49,000. These and other data suggest that a cAMP-binding protein with a molecular weight of 49,000 capable of undergoing cAMP-dependent phosphorylation and dephosphorylation, occurs in a variety of tissues.


Factors influencing the effect of hormones on the accumulation of cyclic AMP in cultured human astrocytoma cells. The characteristics of the effects of catecholamines, prostaglandins, and adenosine on the adenosine 3',5'-monophosphate (cAMP) content of human astrocytoma cells are described. Catecholamines interact with a typical beta-adrenergic receptor, i.e., the order of potency of catecholamines is isoproterenol larger than or equal to epinephrine greater than norepinephrine greater than dopamine, and propranolol is an inhibitor but phentolamine is not. The prostaglandins interact with a receptor that recognized PGE-1, PGE-2, and PGA-1 but not PGF-2-alpha. The effects of PGE-1 are blocked by 7-oxa-13-prostynoic acid, indomethacin, and meclofenamic acid in a rapid, reversible manner. The cells contain another adenylate cyclase-linked receptor that recognizes adenosine and the adenine nucleotides but not guanosine, deoxyadenosine, or adenine. Theophylline and other methylxanthines are competitive inhibitors of the effect of adenosine. Each class of effector appears to stimulate adenylate cyclase by interacting with a structure-specific receptor. This follows from the observation that the effect of each class of agonists can be blocked selectively by the various inhibitors and is consistant with the observation that co-addition of different agonists results in additive effects on accumulation of cAMP. The magnitude of the effect of any of the classes of agonists can be influenced by a variety of factors, some of which may be related to the peculiarities of growth in culture: (1) The cells secrete cAMP into the medium, and the magnitude of this secretion for a given rise in intracellular cAMP is different for different agonists. (2) The exposure of the cells to catecholamines or prostaglandins leads to a loss of responsiveness to a subsequent challenge by the same agonist. The magnitude of the agonist-induced loss of responsiveness is dependent on the concentration of the agonist and the time of exposure. The process is at least partially agonist specific in that exposure of cells to isoproterenol can lead to greater than 90% loss in catecholamine responsiveness with less than 20% loss in responsiveness to prostaglandins. (3) The responsiveness of the cells also changes as a function of the age of the culture and as a function of cell density. (4) Finally, it can be demonstrated that cells maintained in culture for prolonged periods (months to years) may lose responsiveness to specific agonists while responsiveness to other agonists remains unchanges or actually increases. The advantages and disadvantages of the use of cells in culture for studies of the regulation of cAMP metabolism are discussed.


Studies on cyclic AMP metabolism in human epidermoid carcinoma (HEp-2) cells. Cultured human epidermoid carcinoma (HEp-2) cells were found to contain a highly responsive, catecholamine-sensitive adenyl cyclase activity in cellfree preparations. By contrast, cyclic AMP levels in intact HEp-2 cells were at best only marginally increased by catecholamines under a variety of conditions. The lack of an intact cell response could not be accounted for by escape of cyclic AMP to the medium, excessive phosphodiesterase activity, inactivation of the catecholamine, or by unusual kinetics of the system. However, in the presence of 1-methyl,3-iso-butylxanthine (MIX), a potent phosphodiesterase inhibitor, a moderate catecholamine response was observed in the intact cells. A significant elevation of cyclic AMP levels in the presence of MIX was observed at 0.3 muM epinephrine, and maximal levels occurred at 10 muM. Norepinephrine was much less effective than either epinephrine or isopropylnorepinephrine at 10 muM concentrations. In addition, intact cells slowly but steadily released cyclic AMP into the incubation medium over the course of 60-min incubations in the presence of MIX and epinephrine; maximum intracellular levels were reached by 5 min.


Phosphorylase kinase mediating the effects of cyclic AMP in muscle. In the classic view of the control of phosphorylase b to a conversion by catecholamines, cyclic AMP acts as the second messenger stimulating the activity of cyclic AMP-dependent protein kinase to covalently modify phosphorylase kinase. Phosphorylation of phosphorylase kinase converts this enzyme form with a nonactivated to an activated form with a markedly higher activity at pH 7. There is now considerable evidence that the activity of phospphorylase kinase is also regulated by changeds in the Ca-2+ concentration. The activity of both nonactivated and activated phosphorylase kinase is stimulated by Ca-2+ in the range of concentrations that have been reported to occur in the sacroplasm of contracting muscle, with the activated pphosphorylase kinase having a lower K-alpha for Ca-2+. Thus there are at leaset two mechanisms for the regulation of phosphorylase kinase activity in muscle. These mechanisms may act independently or in concert in controlling glycogenolysis stimulated by catecholamines, anoxia, or tetanic electrical stimulation...


Thyroid-stimulating hormone and cyclic adenosine 3',5'-monophosphate in the regulation of thyroid gland function. The initial step in TSH action reflects binding of the hormone to specific receptor sites on the plasma membrane. Such binding has been studied using plasma membranes, homogenates, isolated thyroid cells grown in culture, and thyroid slices. 3-H- and iodinated TSH preparations have been used; the latter have been prepared using both chloramine-T and lactoperoxidase. Some of the discrepancies reported in the literature might reflect the different thyroid and hormone preparations and the variable incubation conditions which have been used. In general, good correlation exists between binding of TSH and activation of adenylate cyclase in thyroid plasma membranes. Data is reviewed related to activation of protein kinase in intact thyroid cells by TSH. Although there is impressive evidence for cyclic AMP mediation of effects of TSH on the thyroid, some data that are inconsistent with this concept are considered, especially in relationship to 32-P incorporation into phospholipid. The role of cyclic GMP in thyroid function is discussed.


Cyclic nucleotides and gluconeogenesis by rat liver cells. Gluconeogenesis from lactate, pyruvate, fructose, alanine, and other substrates was accelerated by glucagon or epinephrine in hepatocytes isolated from rat liver. Glucagon and epinephrine also increased cyclic AMP accumulation by rat hepatocytes. Isoproterenol increased cyclic AMP but not gluconeogenesis, while phenylephrine accelerated gluconeogenesis. The activation of gluconeogenesis by epinephrine was unaffected by propranolol but blocked by dihydroergotamine. Dibutyryl cyclic AMP added to hepatocytes stimulated gluconeogenesis at concentrations as low as 1 muM. Exogenous cyclic GMP (0.1- muM) inhibited gluconeogenesis due to either glucagon or epinephrine without affecting basal gluconeogenesis. However, carbamylcholine did not affect gluconeogenesis by hepatocytes. Basal gluconeogenesis and the increases due to all agents were inhibited by removal of extracellular calcium or the presence of A-23187, D-600, or tetracaine. In contrast, added 0.1 muM cyclic  GMP, 2 mM NH-4-Cl, and 10 muM phenethylbiguanide inhibited glucagon- or epinephrine-stimulated gluconeogenesis without affecting basal values. Studies with hepatocytes indicate that the hormonal activation of gluconeogenesis is not limited to substrates entering prior to triose phosphate formation. Glucagon may act by increasing cyclic AMP which acts via unknown mechanisms to increase gluconeogenesis. In contrast, epinephrine acts via a cyclic AMP-independent mechamism which does not appear to involve cyclic GMP, Ca-2+ flux, of K+ flux.


The role of calcium and cyclic nucleotides in alpha-amylase release from slices of rat parotid: studies with the divalent cation ionophore A-23187. The divalent cation ionophore A-23187 caused a Ca-2+-dependent increase in alpha-amylase release from slices of rat parotid gland. The effect of A-23187 on alpha-amylase release was not caused by release of endogenous agonists since l-propranolol, phentolamine, and atropine had no effect. The magnitude of alpha-amylase release caused by A-23187 was small compared to the effect of isoproterenol. In this respect it more closely resembles the action of cholinergic and alpha-adrenergic agonists on alpha-amylase release. A-23187 inhibited the increase in the level of parotid adenosine 3,5'-monophosphate caused by isoprotrenol. The inhibitory effect required incubation of the slices with the ionophore before the addition of isoproterenol. The ionophore also caused a Ca-2+-dependent increase in the level of guanosine 3',5'-monophosphate (cyclic GMP). Theophylline enhanced the effect of A-23187 on the level of cyclic GMP. These results emphasize the role of Ca-2+ in the regulation of parotid cyclic nucleotide levels. Since the effects of the ionophore depended on the presence of Ca-2+, it is possible that some of the effects of agonists on parotid gland physiology are secondary to an action on intracellular Ca-2+ distribution.


Cyclic AMP and cyclic GMP: studies utilizing immunohistochemical techniques for the localization of the nucleotides in tissue. Antibodies to the cyclic nucleotides initially were utilized in radioimmunoassays for cyclic AMP and cyclic GMP which might be present in mammalian tissues. allowed measurement of the nucleotides on small amounts of tissue in physiologic studies. To gain further insight into the relative roles of cyclic AMP and cyclic GMP in cell function, these antibodies have been applied to immunohistochemical studies for the localization of the cyclic nucleotides in tissues and cells. This methodology is useful for determining in which cell type in a heterogeneous tissue increases in cyclic nucleotide concentrations occur. In addition, within individual cells, staining patterns for cyclic AMP and cyclic GMP are usually quite distinct. Cyclic GMP in canine thyroid is located to the follicular cell membrane while cyclic AMP is ubiquitously distributed in follicular cell cytoplasm. In both rat adrenal cortex and testis, there is prominent nuclear localization of cyclic GMP, suggesting a role for the nucleotide in growth regulation. These studies provide histologic evidence suggesting diverse roles for cyclic AMP and cyclic GMP in mammalian physiology. It is anticipated that this technique will also be useful in the ultrastructural localization of the cyclic nucleotides and for the identification of other cyclic nucleotides which might be present in mammalian tissues.


Effects of norepinephrine on cyclic nucleotide levels in the ductus deferens of the rat. The effect of 1-norepinephrine on cyclic nucleotide levels was studied in rat ductus deferens. Norepinephrine (0.01-0.3 mM) increased cyclic AMP and cyclic GMP levels in a concentration-dependent manner. Cyclic GMP was increased by about two-fold and cyclic AMP by 30%-40% by 0.1 mM norepinephrine after 3 min. Phenylephrine (0.1 mM) increased cyclic GMP levels about twofold without affecting cyclic AMP. Cyclic GMP was maximally increased by norepinephrine or phenylephrine after 3-10 min, but was not significantly changed after 20 sec, when contraction was fully developed. Atropine did not affect the norepinephrine-induced changes in cyclic nucleotide levels; phentolamine blocked the effect of norepinephrine on cyclic GMP, and propanolol blocked that on cyclic AMP. These findings indicate that in the ductus deferens alpha-adrenergic receptors are involved in the effect of catecholamines on cyclic GMP, and beta-adrenergic receptors are involved in the effect on cyclic AMP levels. The presence of Ca-2+ was required for the effect of norepinephrine on cyclic GMP, but not on cyclic AMP.


Modulation of human neutrophil chemotactic responses by cyclic 3',5'-guanosine monophosphate and cyclic 3',5'-adenosine monophosphate. Cyclic 3',5'-guanosine monophosphate (cGMP) and cyclic 3',5'-adenosine monophosphate (cAMP) and compounds known to effect the intracellular concentrations of these nucleotides were examined for their ability to effect human neutrophil (PMN) responsiveness to chemotactic stimulation. Incubation of neutrophils with agents recognized to promote increases in intracellular cAMP in a variety of tissues (i.e., epinephrine, norepinephrine, isoproterenol, histamine, cholera toxin, and prostaglandin E-1 and E-2) or with cAMP inhibited the leukotactic response to a bacterial chemotactic factor. In contrast, cGMP and compounds which have been shown to promote increases in intracellular cGMP concentration (i.e., acetylcholine, carbamylcholine, phorbol myristate acetate, and prostaglindin F-2-alpha) markedly enhanced the neutrophil chemotactic response. The inhibitory or stimulatory influences on chemotactic responsiveness promoted by several of the agents could be shown to be blocked by a specific pharmacologic antagonist of the particular compound tested. These data support the hypothesis that cGMP and cAMP can provide opposing regulatory influences on certain cellular functions; in this case, directed motility of leukocytes.


The mechanism of the potentiating effect of glucocorticoids on catecholamine-induced lipolysis. Preincubation of isolated epididymal fat cells with dexamethasone or treating rats with cortisol enhances the epinephrine-stimulated lipolysis of the cells as well as cAMP-dependent protein kinase activity in homogenates of these fat cell suspensions. During maximal inhibition of phosphodiesterase activity by theophylline or dibutyryl cAMP, this potentiating effect of glucocorticoids on the fat cells was also present. There was no lowering of the total phosphodiesterase activity in homogenates of fat cell suspensions of rats that were treated with cortisol, but there appeared to be a lower activity of the low KM phosphodiesterase activity. It is concluded that induction of protein kinase by glucocorticoid hormone is responsible for its special type of stimulative action on lipolysis.


Human plasma triglyceride labeling after high sucrose feeding. II. Study on triglyceride kinetics and postheparin lipolytic activity. Kinetic studies of the very-low-density lipoprotein triglycerides (VLDL-TG) turnover by endogenous labeling with glycerol-2-3-H were performed in 13 patients in the postabsorptive state, first after 10-14 days on a low-sucrose high-starch diet, then again after 10-14 days of isocaloric high-sucrose low-starch diet (HSD). After HSD, a significant decrease in the fractional turnover rates of VLDL-TG was observed, as well as a modest but significant increase in its pool size, but the net turnover rates remained unchanged. Using Michaelis-Menten formulation, we have further calculated the Vmax and Km's of the removal system for VLDL-TG and found that the Vmax and Km's do not differ significantly between the two dietary periods. These results suggest that the removal mechanism for VLDL-TG has not changed after 10-14 days on the HSD, at least when the patients are studied in the postabsorptive state. Measurements of postheparin lipolytic acitivty under fed condition in 17 patients (including the 13 patients above) have shown a decrease after HSD. However, a defect in the removal of plasma-TG related to decreased activity of tissue-lipoprotein lipase in the fed state has not been conclusively uncovered by the kinetic studies performed in the postabsorptive state, and cannot contribute significantly to the expansion of VLDL-TG pool.


Clinical testing of the hypothalamic-pituitary-adrenocortical system in states of hypo- and hypercortisolism. Cortisol production is appropriately maintained by a complex control system which involves primarily the hypothalamus, the pituitary, and the adrenal cortices. Very small quantities of ACTH stimulate cortisol production, and maximum stimulation occurs with serum concentrations of only 3 mU/100 ML. Under normal circumstances, cortisol is secreted in bursts about ten times each day and circulates predominately bound to a specific binding protein which is rather completely saturated. Radioimmunoassay of plasma cortisol is now generally available and is the assay method of choice, but because of the episodic nature of its secretion, random values of plasma cortisol must be interpreted with great reservation, and even the comparison of morning and evening values in assessing circadian rhythmicity is not often helpful. Urinary free cortisol determinations provide excellent discrimination between normal function and all forms of hypercortisolism. Although the response of the adrenal cortices to ACTH may be evaluated in a number of different ways, the simplest but most definitive procedure involves continuous intravenous administration over a 48-hr period. Of the various tests which indirectly assess the potential for ACTH secretion, the use of metyrapone is most helpful. In the test of greatest utility, plasma cortisol and 11-desoxycortisol are determined the morning after a single midnight oral dose of 30 mg/kg. The detection of all forms of pathologic hypercortisolism is still best accomplished by the oral administration of dexamethasone. Plasma cortisol can be determined the morning after a single midnight dose of 1 mg, or urinary 17-hydroxycorticosteroids can be determined after 2 days in which 0.5 mg is given at 6-hr intervals. Patients with hypercortisolism of hypothalamic-pituitary origin usually evidence appropriate suppression of urinary steroids if the dose is increased to 2.0 mg every 6 hr for another 48 hr. In patients who do not suppress on this or even higher doses of dexamethasone, the distinction between those with adrenal tumor and those with the ectopic ACTH syndrome can be accomplished most definitively by the assay of plasma ACTH where this determination is available.


Adenosine 3',5'-cyclic monophosphate in rabbit cerebrospinal fluid during fever induced by E. coli-endotoxin. In rabbit cerebrospinal fluid during fever induced by E. coli-endotoxin adenosine 3',5'-cyclic monophosphate concentrations are about 2-fold higher in comparison to normal values. In addition to prostaglandins of the E series adenosine 3',5'-cyclic monophosphate might act as another mediator in the genesis of fever during infectious diseases.


Attempts to detect virus-specific DNA sequences in human tumors. III. Epstein-Barr viral DNA in non-lymphoid nasopharyngeal carcinoma cells. Fourteen tumors of the nasopharyngeal region were analyzed for the presence of Epstein-Barr virus-specific DNA by DNA-cRNA hybridization. These data were compared to the histology of the respective tumors and the seroreactivity of the tumor-bearing patient against EBV-related antigens. With one exception, all tumor pieces containing nasopharyngeal carcinoma cells hybridized significantly with EBV-cRNA. Tumors of predominantly epithelial morphology annealed in the highest range. In situ-hybridization of freeze sections from a tumor containing almost equal amounts of tumor cells and lymphocytes revealed hybridizing DNA within nuclei of non-lymphoid cells. Although these data do not exclude the presence of EBV-DNA within lymphoid cells, they clearly demonstrate that in nasopharyngeal carcinomas the vast majority of EBV-specific DNA rests within non-lymphoid cells.


In vitro cultivation of cells from the chorioallantoic membrane of chick embryos. By treatment of chorioallantoic membranes from embryonated eggs with collagenase and hyaluronidase before the conventional application of trypsin cells could be grown in culture which supported growth of a large variety of myxoviruses, herpesviruses, avian reoviruses and the infectious bronchitis virus of chickens. The cultures could be used for sensitive plaque assays and neutralization tests.


Investigation of electron transport in the chloroplasts and their fragments by the ESR method. II. Light-induced interaction of water-soluble nitroxide radical with chloroplasts and chlorophyll containing protein-lipid micelles. A light-induced reduction of the water-soluble nitroxide radical by chlorophyll in lipid and protein--lipid micelles was demonstrated. In contrast to model systems, in whole chloroplasts the NR is photoreduced by the electrons of the noncyclic electron transport chain. The initiation of cyclic electron transport in light particles, containing only photosystem I, does not lead to photoreduction of NR.  When exogenous protein -- human serum albumin -- is added to the light particles, the nitroxide radicals are intensively reduced. The specific role of protein in electron transport from P700 to the exogenous acceptor is discussed.


Protein biosynthesis in a homologous, cell-free system in the presence of chick embryo RNA isolated from free and membrane-bound polyribosomes. Protein synthesis has been studied in a cell-free system from chick embryo, in the presence of homologous RNA isolated from free and endoplasmic reticulum-bound polyribosomes. The two RNA fractions showed equal activities in total protein synthesis. However, while the RNA from bound polyribosomes mainly supported synthesis of high molecular weight, TCA-insoluble polypeptides, the RNA from free polyribosomes was more active in the synthesis of low molecular weight, TCA-soluble polypeptides. Optimal conditions for translation of the two RNA's under study were different when studied in a cell-free system with reduced content of endogenous matrix. Collagen synthesized in the system was identified by collagenase digestion. Collagen synthesis was demonstrated only in the presence of RNA from endoplasmic reticulum-bound polyribosomes, and represented 16-19% of total protein synthesis.


Effects of DNA-polymerase-defective and recombination-deficient mutations on the ultraviolet sensitivity of Bacillus subtilis spores. The DNA of UV-irradiated Bacillus subtilis spores, which contains 5-thyminyl-5,6-dihydrothymine (TDHT) as the major thymine photoproduct, is known to be repaired during germination by two complementary mechanisms: (I) the well-known excision repair, and (2) a special process, "spore repair", which destroys TDHT in situ without rendering it acid-soluble. In the absence of both mechanisms TDHT is not removed, and spores are highly UV-sensitive. When either of two mutations (pol-59 and pol-151) giving defective DNA polymerase, or one (rec-A1) giving a recombination deficiency are introduced into strains defective in one of these known TDHT removal processes, the chemically measured elimination of TDHT from spore DNA is unaltered, but spore UV-sensitivity is increased. The pol mutations produce their greatest sensitivity increase in spores of strains already deficient for the in situ destruction of TDHT, while the rec mutation gives its maximum sensitivity increase to spores of strains lacking excision. These facts argue that the pol mutations interfere mostly with excision repair (presumably its later resynthesis step), shile the rec mutation impairs "spore repair" in some step occurring subsequent to the TDHT destruction in situ. With either of these impairments of the later repair steps, DNA of UV-irradiated and germinated spores is considerably degraded, unless germination is carried out in the presence of chloramphenicol.


Genetic analysis of radiation sensitive and chemical-mutagen sensitive mutants of Pseudomonas aeruginosa. Thirty-five mutants of Pseudomonas aeruginosa sensitive to methyl methanesulfonate (MMS) have been genetically characterized. They constitute ten separable groups as defined by transduction and conjugation. Three of the groups have been shown to be cotransducible with auxotrophic markers.


Treatment of carbamyl phosphate synthetase deficiency with keto analogues of essential amino acids. Congenital carbamyl phosphate synthetase deficiency was diagnosed by liver biopsy in a 13-year-old girl, alpha-Keto analogues of essential amino acids have been shown to spare nitrogen by reducing urea formation; hence, they were given to this patient in the hope of reducing hyperammonemia and improving protein tolerance. After intravenous infusion of the keto analogues of valine, leucine, isoleucine, methionine and phenylalanine, the corresponding plasma amino acids, including alloisoleucine and tyrosine, rose sharply. Twenty-four hours later, fasting plasma ammonia had fallen from the preinfusion value of 0.050 to 0.028 mM. Protein intake was kept at 0.5 g per kilogram for two weeks. Addition of keto acids by mouth reduced plasma ammonia and alanine to normal or near normal levels. Seizures and episodes of vomiting and lethargy decreased in frequency. Urinary nitrogen decreased, suggesting that nitrogen balance improved. These data indicate that keto acids may be useful in the treatment of congenital hyperammonemia.


Etiology of nongonococcal urethritis.

Etiology of nongonococcal urethritis. Chlamydia trachomatis was isolated from the urethra from 48 (42 per cent) of 113 men with non-gonococcal urethritis (NGU), four (7 per cent) of 58 without overt urethritis, and 13 (19 per cent) of 69 with gonorrhea. Postgonococcal urethritis (PGU) developed in 11 of 11 men who had C. trum antibody to C. trachomatisis developed. The immunotype specificity of chlamydial antibody corresponded to the immunotype isolated. Among culture-negative patients. chlamydial antibody prevalence correlated with the number of past sex partners and with previous NGU. Herpesvirus hominis, cytomegalovirus, T-mycoplasma, Mycoplasma hominis, other bacteria, and Trichomonas vaginalis were not implicated in NGU or PGU. Thus, the cause of chlamydia-negative NGU and PGU remains obscure. Endocervical chlamydia were found in sex partners of 15 of 22 NGU patients with and two of 24 without urethral chlamydial infection (p smaller than 0.001). Tetracycline treatment of both sex partners appears advisable.


Acquired agammaglobulinemia after a life-threatening illness with clinical and laboratory features of infectious mononucleosis in three related male children. Three males in one family (two siblings and one maternal cousin) had an illness with cervical adenopathy, hepatosplenomegaly, and a fulminant febrile course. In the two survivors agammaglobulinemia developed. One of them became ill at the age of six months and had an Epstein-Barr-virus antibody titer of 1:10 during illness and convalescence. The white-cell count was 120,000 with 90 per cent lymphocytes, most being atypical and forming increased numbers of sheep erythrocyte rosettes. IgM was elevated, IgA normal and IgG decreased. Subsequently, all immunoglobulins were absent, and the Epstein-Barr-virus antibody titer became negative. Peripheral B-cell number remained normal, but abnormal lymph-node architecture associated with failure to respond to antigenic challenge indicated B-cell dysfunction. The pathogenesis of this entity may involve an abnormal T-cell response to transformation of B cells by Epstein-Barr virus, leading to B-cell dysfunction and agammaglobulinemia.


Evidence for normal thyroidal adenyl cyclase, cyclic AMP-binding and protein-kinase activities in Graves' disease. The adenyl cyclase, cAMP-binding and protein-kinase activities have been studied in thyroid glands from patients with Graves' disease in comparison with normal thyroid glands. The basal and TSH-stimulated adenyl cyclase activities were tested in crude plasma membrane preparations. The characteristics of the intracellular binding of cAMP, i.e., the maximal binding capacity (MBC) for cAMP and affinity constant (Ka) of the binding, and the basal and cAMP-stimulated protein-kinase activities, were estimated in both the soluble and particulate fractions of thyroid tissue. All of these parameters studied were essentially normal in Graves' disease. It is concluded that hyperthyroidism in Graves' disease is probably not a result of qualitative or quantitative abnormalities in the adenyl cyclase-cAMP protein-kinase system.


A sensitive bioassay for the determination of human plasma ACTH levels. A sensitive bioassay for the measurement of plasma ACTH is presented. The use of silicic acid adsorption of plasma, with a subsequent acid wash and aqueous acetone desorption, was successful in removing those substances which had interfered with the steroidogenic response of dispersed adrenal cells when unextracted plasma was employed. This extraction procedure extracted 72-76% of ACTH present in plasma. Two pg ACTH1-39 could be consistently detected. Alpha-hACTH1-39 and alpha-pACTH1-39 exhibited equal potencies. Beta-MSH was ineffective at dosage levels up to 2 x 10(8) pg. One x 10(8) pg of ACTH1-10, ACTH4-10, or alpha-MSH had a steroidogenic effect equivalent to that of 40 pg ACTH1-39. ACTH 17-39 and ACTH 11-24 were incapable of stimulating steroid production at doses of 1 x 10(8) pg. Excesses of the latter, but not of the former appeared to be able to antagonize the steroidogenic effect of ACTH1-39. Plasma from normal subjects, bioassayed by this extraction procedure, contained 12-186 pg/ml ACTH at 0400-0800: 14-93 pg/ml ACTH at 1000-1300, and less than 10-34 pg/ml ACTH at 1600-2200. Hypoglycemia and vasopressin administration were followed by increases in plasma ACTH concentratrations. Plasma ACTH concentrations in untreated patients with Cushing's disease (sampled over the period 0900-1300) ranged from 65-220 pg/ml. Three patients with Addison's disease (untreated or 12 h following replacement steroid withdrawal) had ACTH concentrations of 223, 370 and 1226 pg/ml. Markedly elevated ACTH concentrations were observed in a patient with Nelson's syndrome (391 and 835 pg/ml). Bioassayable ACTH was not detected in 2 patients with panhypopituitarism.


5'-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation. Unmethylated reovirus and VSV mRNAs are specifically methylated to form 5'-terminal structures of the type, m-7-G(5')ppp(5')N by protein synthesising extracts prepared from wheat germ and mouse L cells. Reticulocyte mRNA also contains 5'-terminal m-7-G. MRNAs having 5'-terminal m-7-G stimulate protein synthesis in vitro. Removal of m-7-G by beta-elimination abolishes translation of the mRNAs.


Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides. The binding of concanavqlin A to T but not B mouse spleen lymphocytes increases Ca-2+ uptake in these cells which is measurable by 45 s and complete by 1 min. Dibutyrl cyclic AMP, but not sodium azide inhibits induced Ca-2+ uptake, wheras dibutyryl cyclic GMP enhances it. B cell mitogens do not cause a similar Ca-2+ uptake in mouse B lymphocytes. The induction of increased Ca-2+ uptake by T cells is discussed in terms of gated membrane channels for Ca-2+.


Role of the renin-angiotensin-aldosterone system in the regulation of plasma potassium in chronic renal disease. The relationship between plasma potassium concentration and the renin-angiotensin-aldosterone system was evaluated in ten patients with chronic renal failure (creatinine clearance 10-56 ml/min).  Under basal conditions and following various stimulation maneuvers, normokalemic patients demonstrated normal plasma renin and aldosterone levels.  Five of six patients with hyperkalemia had diminished function of the renin-angiotensin-aldosterone system; their ability to conserve sodium during salt depletion was less than that of normokalemic patients.  The data suggest that the maintenance of plasma potassium levels in these patients is dependent of the presence of a normally functioning renin-angiotensin-aldosterone system; aldosterone activity may be an important determinant of sodium conservation in patients with renal failure.


Mononeuropathy multiplex as a complication of amphetamine angiitis. A patient who abused multiple drugs developed a rapidly progressive mononeuropathy multiplex, which appeared to respond to corticosteroid therapy with partial resolution. Intravenous methamphetamine had been used almost exclusively from the fourth month prior to the onset of symptoms. Biopsy material revealed a necrotizing angiitis involving medium and small sized arteries, capillaries, and venules, typical of a hypersensitivity-type angiopathy, rather than the previously reported polyarteritis nodosa-type lesions secondary to illicit drugs. The apparent response to corticosteroids suggests that these agents might be useful in the treatment of some complications of drug abuse.


A cytochemical study of some enzyme activities in biliverdin-treated cell cultures. The authors studied the modifications of the activities of some enzymes in cell cultures submitted to the action of biliverdin. This biliary pigment rapidly induces a remarkable increase in alkaline phosphatase and ATP-ase activities and subsequently, an activation of acid phosphatase and beta-glucuronidase. On the contrary, 5'-nucleotidase and glucose-6-phosphatase activities remain unchanged. These results are discussed and compared with those obtained in our and other laboratories by using unconjugated bilirubin on different biological substrates.


[Immunochemical study of liver metastases of colonic tumors]. This work demonstrated that anti-CEA and anti-NCA antibodies can be obtained from liver metastases of colonic carcinoma. The various antigens that have been identifiedin colonic tumor are found in liver metastases. Furthermore, the healthy parts of a metastatic liver contain not only CEA but also NCA.


[Viral contamination in laboratories and hospital units]. Viral contamination is at least as important in hospital laboratories and wards as contamination by bacteria or microscopic fungi, but it is much more insidious and sometimes unrecognized. There are two main types: The first has a purely technical effect and only interest the virologist. This is contamination of reagents, reference strains, cell cultures, etc.., by foreign viral agents. It may be the cause of errors on diagnosis or regrettable errors of interpretation of certain experimental data. It is most difficult to detect, if not to avoid. The second is much more worrying as it is liable to cause disease in man, which may induce severe, and even fatal infections in patients or in the medical, para-medical and technical personnel. This is the case with type B hepatitis virus which tends to invade surgical units using extra-corporeal circulation, hemodialysis units and transplantation units, blood transfusion centres, dental units and even causes victims in routine laboratories. However, type B hepatitis is not the only virus which may lead to severe infections; other viruses include: poxvirus, cytomegalovirus, arbovirus, etc. Finally, other often severe accidents may occur in research laboratories and in the pharmaceutical industry, owing to manipulation of dangerous viruses or by contact with experimental animals, e.g. rodents, or monkeys, which contain the virus in a latent state, e.g. lymphocytic choriomeningitis, Sabin virus, Marburg virus, type A hepatitis virus, etc. With regard to such accidents, we are almost completely powerless from the therapeutic point of view and, even poorly equipped, from the point of view of prophylaxis.


[Prolactin and breast cancers].

[Prolactin and breast cancers]. Prolactin has a cocarcinogenic activity in the rat and mouse as its administration or stimulation of endogenous secretion increases the incidence and reduces the latent period of breast tumours, whether spontaneous or induced by chemical carcinogens. Prolonged hyperprolactinemia obtained by neutralizing the inhibitory influence of the hypothalamus on its secretion and liberation, favours the development of breast carcinoma and inversely. (Estrogens and prolactin exert a synergistic effect, both centrally and peripherally, on the acinar cells of the breast. Prolactin dependency of these mammary carcinomas is due to the persistence of specific cell receptors. In human pathology, it has not yet been possible to demonstrate a comparable effect of prolactin. Radioimmunoassay of plasma prolactin and specific receptors, together with epidemiological enquiries, should rapidly confirm or refute the cocarcinogenic effect of prolactin.


Effects of therapeutic irradiation delivered in early childhood upon subsequent lung function. To determine the long-term effects of therapeutic pulmonary irradiation and treatment with actinomycin D during a period of lung growth, 12 patients treated for Wilms' tumor metastatic to the lung and 8 patients treated for Wilms' tumor with no evidence of pulmonary metastases were studied 7 to 14 years after their initial tumor therapy. All patients had received irradiation to the tumor bed and treatment with actinomycin D. Group 1 had received a single course of bilateral pulmonary irradiation; group 2 had received additional pulmonary irradiation and/or thoracic surgery; group 3 had received no therapeutic irradiation directed primarily to the chest. Total lung capacity (TLC) averaged 71% of predicted value in group 1, 58% in group 2, and 94% in group 3. Diffusing capacity in groups 1 and 2 was reduced to the same extent as lung volume. Quasi-static pressure-volume relationships, studied in three of six patients in group 1, were within the normal range when lung volume was expressed as percentage of observed TLC. Airway resistance, evaluated by spirometry, maximum expiratory flow-volume curves, and resistance of the total respiratory system, was normal or reduced. The data support the hypothesis that therapeutic irradiation during a period of lung growth primarily affects the lung parenchyma and produces a decrease in subsequent size of both the lung and chest wall. No effect of actinomycin D alone upon the lung could be demonstrated.


Sudden infant death syndrome: hypothesis of causation. The airway of the young infant is anatomically vulnerable at the oropharyngeal level between the soft palate and the base of the skull.  Airway occlusion at this level might occur during the muscle relaxation which occurs during REM sleep, facilitated by a hypermobile mandible, by the hypotonia of infection, perhaps by an enlarged tongue with a strong backwards sucking action which might be the result of the artificial feeding of the infant.  It is possible that "cot deaths" (SIDS) may be precipitated by such oropharyngeal airway occlusion, cardiac arrest following variable periods of partial or complete oxygen deprivation.


Antiviral chemotherapy and neonatal herpes simplex virus infecition: a pilot study--experience with adenine arabinoside (ARA-A). Among 13 neonates with herpes simplex virus (HSV) infection, eight had disseminated disease, one localized CNS disease, and in four the infection was confined to the skin and eyes. Ara-A, a purine nucleoside with anti-viral activity against DNA-VIRUSES, WAS GIVEN (10 TO 20 MG/MG/DAY) BY A CONTINUOUS 12-HOUR INTRAVENOUS DRIP FOR 10 TO 15 DAYS. In all, ara-A administration was begun within three to eight days after the appearance of skin vesicles which represented the hallmark of the disease. Both diagnosis and ara-A treatment were much delayed in one infant without skin vesicles and four infants whose skin vesicles appeared late, long after the occurrence of CNS damage. In this group of infants with disseminated disease, four died and one infant was left with severe neurological deficits. Eight infants (four with disseminated and four with localized skin disease) with skin vesicles as the earliest sign of infection received ara-A early, within three days after the onset of neurologic signs. All survived with no neurologic deficit at 6 months to 1 year of age. There was no apparent toxicity of ara-A to the bonemarrow, liver, or kidney.


The absorption of iron as supplements in infant cereal and infant formulas. The absorption of iron was measured from isotopically tagged salts used in supplementing infant cereals and as the iron supplement in cow's milk and soy-based formulas.  Iron as sodium iron pryophosphate and ferric orthophosphate were poorly absorbed from infant cereal (mean, smaller than 1.0%) and thus are not dependable sources of iron to meet the nutritional needs of infants.  Reduced iron of very small particle size and ferrous sulfate when added to cereal was absorbed to a greater extent (mean, 4.0% and 2.7% respectively).  For technical reasons, these two forms of iron had not been added to commercial cereal products because of discoloration, distribution problems of the iron in the product, and shortened shelf life.  Therefore, at the present time, iron supplementation of infant cereals with sodium iron pyrophosphate, ferric orthophosphate, and reduced iron of large particle size does not provide a predictable and available source of iron to meet the needs of infants.  Supplemental iron as ferrous sulfate in milk- and soy-based formulas gave a mean absorption of 3.4% and 5.4%.  The iron supplements in these formulas can essentially meet the needs for dietary iron of healthy infants.


[Chemotherapeutic combinations of mutually potentiating drugs. 3-Application to primary tumors of the central nervous system]. 17 patients suffering from primary tumours of the central nervous system recurring after surgery and/or radiotherapy, received chemotherapy consisting of the administration of VM 26 followed by CCNU. Each cycle of treatment lasted four days and was restarted only after an average interval of 30 days to allow for haematological recovery. All of the patients in the trial have been under treatment for eight months. Five (28 p.cent) are at present in complete remission with no residual clinical, isotopic or radiological signs. In six others there was tumour regression and in four neurological stabilisation. In all those cases where objective tumour regression was obtained functional improvement was noted.


[Peculiarities of lymphocytes during dermatosis. Ultrastructural study of 100 cases]. Peripheral blood lymphocytes from one hundred patients with skin disorders have been examined under the electron microscope. The bloods of 20 donors were used as controls. One observed: Branched Tubular Structures (so-called Lupus type inclusions) in 8 cases (4 systemic Lupus Erythematosus out of 15; I mixed connective tissue disorder; 3 cutaneous lymphomas). As regards Lupus, these findings are in agreement with those already reported in the literature. However, the presence of such inclusions in Lymphomas calls attention once again on the fact that the lymphocyte appears like a possible common denominator to both autoimmune diseases and lymphomas; Intented or cerebriform nuclei in 12 cases, all lymphomas, including 8 cases of Sezary Syndrome out of 8 and 4 Mycosis Fungoïdes out of 12; Lamellar type inclusions, similar to those described by Hovig, in 13 cases (connective tissue disease 1, lymphomas 2, psoriasis 4, miscellaneous 4, healthy controls 2). Their meaning is unclear; they may be artifacts.


[Is progesterone insufficiency carcihogenic?]. Estradiol is a natural estrogen which favors cellular hyperplasia at the level of its target organs. As for progesterone, its activity may be synergic or antagonist of estradiol. In the first case, progesterone has generally a secretory activity upon the target cells "prepared" by estrogens, in particular the cells of the female genital tract (uterus, breast). In the second case, progesterone may be an antiestrogen not only at the level of genital cells but also at the level of other receptors such as vascular system, etc. If an unbalance occurs in the quantitative and chronological ratio between estradiol and progesterone production (which determines the number of target cells and their secretory activity), a cellular dysplasia may be observed. This unbalance is generally characterized by an insufficiency in progesterone secretion which may be physiological  (puberty, menopause) or pathological. Such a progesterone insufficiency may play a role in the pathogenesis of genital cancers (endometrial and possibly mammary). Experimental data which give some support to such an hypothesis are reported. They emphasize the importance of treatment of the progesterone insufficiency of the premenopausal period in women with the aim of preventing cellular dysplasia of the female genital tract.


[Relation between endocrinology and psychiatry. Some recent developments]. Relations between Endocrinology and Psychiatry are developing in two main directions: Behavioural effects of hormones. After the effects of peripheral hormones, recent works are concerned with A.C.T.H. and Antidiuretic Hormone (which interfere with memory process) and with Hypothalamic Peptides (which might have psychotropic properties, independant of their endocrine regulation function). -Correlations between psychiatric syndroms and endocrine disorders. In some mentally depressed patients, disorders of the hypothalamic control of Coricotrophin, Growth Hormone and Thyreo-stimulating Hormone, have been observed. As many psychotropic drugs can influence the secretion of Prolactin, a systematic survey of this hormone in various mental illnesses, is suggested.


Determination of the 3' terminal nucleotide of DNA fragments. A new method for determining the 3'-terminal nucleotide of a DNA strand is presented. Use is made of the fact that one (and only one) 2',3'-dideoxyribonucleotide can be added to the 3'-end of a DNA fragment with calf thymus terminal transferase. Addition of more than one nucleotide analog per strand is impossible due to the absence of a 3'-terminal hydroxyl group. If the terminatind dideoxyribonucleotide contains an (alpha 32p) label, the resulting 3'-blocked strand can be digested by "nearest neighbor" techniques and the original 3'-endgroup determined. Picomole quantities of DNA strands can be labeled and the 3'-end determined.


Antiviral activity of polynucleotides: copolymers of inosinic acid and N2-dimethylguanylic of 2-methylthioinosinic acid. Complexes of poly(C) with copolymers of inosinic acid containing various amounts of mismatched bases (see journal for formula) have been examined for direct resistance to virus infection, interferon induction and toxicity in two different cell cultures (primary rabbit kidney cells and mouse L-929 cells). Complexes in which 20% of the hypoxanthine bases were replaced by (see journal for formula) or ms-2I were partially active whereas complexes in which 40% or more of the hypoxanthine bases were replaced by the odd bases were entirely inactive. The decrease in biological activity observed upon intrusion of (see journal for formula) or ms-2I in the poly(I) strand of poly(I) with poly(C) closely paralleled the amount of odd bases introduced irrespective of the system employed to assess the biological activity (resistance to virus infection, interferon induction or toxicity).


The public health nurse in mental health follow-up care. To discover if psychiatric patients who received follow-up care in the home by public health nurses could assume a functional role in the family. and in the community to a greater extent than similar patients who did not receive home care, a sample of all chronic undifferentiated and paranoid schizophrenics who had been released from a Georgia mental hospital within a one-year period and who were receiving follow-up care in the home by the public health nurse was selected from County A and matched according to diagnosis, time released from the hospital, age, sex, race, income, education, and marital status as closely as possible with a sample of patients from County B who were not receiving such follow-up care. Eight pairs of patients were interviewed and rated according to the Psychiatric Status Schedule; six pairs of patients were rated on a questionnaire by a significant member of the family as to the amount of burden they imposed on the family. The rate of patients readmitted to the hospital was also calculated for each sample group and compared. No statistically significant differences were found in the readmission rate, functioning level in the family and the community, or burden on the family


[The functional state of the hypothalamo-hypophyseal system in tumors of the pituitary and diencephalic area]. A study was made of 38 persons, including 25--with acromegaly, 5--with chromophobic adenomas of the hypophysis, 3--with diencephalic tumours and 5 healthy individuals.  STH, TTH and ACTH were determined in the blood plasma with the aid of radioimmunological methods under basic conditions and L-DOPA loading (10 mg per 1 kg of weight) with insulin (0.10 U/kg).  There was an increase in the plasma content of STH, TTH and ACTH with a maximum 60 min after the administration of L-DOPA.  In the patients with acromegaly STH increased considerably, and in cases with chromophobic adenomas and diencephalic tumours--less.  ACTH level was also elevated.  STH and ACTH level rose after insulin hypoglycemia.  Possible mechanisms of a stimulating action of L-DOPA and insulin are discussed.


Induction of erythroid differentiation in murine virus infected eythroleukemia cells by highly polar compounds. Murine-virus-infected erythroleukemia cells cultured in a medium with dimethylsulfoxide or N,N-dimethylformamide are induced to differentiate to erythroid cells. A number of highly polar compounds have a similar effect in inducing erythroid differentiation of the virus-infected cells, as assayed by the appearance of hemoglobin. These compounds are 1-methyl-1-2-piperidone, N,N-dimethylacetamide, N-methylpyrrolidinone, N-methylacetamide, 2-pyrrolidinone, propionamide, pyridine-N-oxide, piperidone, N-methylformamide, acetamide, and triethylene glycol. It has been previously reported that dimethylsulfoxide must be present during DNA synthesis and, possibly, shortly therafter, to induce differentiation. These findings are consistent with the hypothesis that dimethylsulfoxide and related polar compounds act by changing the conformation of DNA or a DNA-protein complex, causing an alteration in transcription that leads to the expression of the program of erythroid differentiation.


[A comparative analysis of the behavior of two labeled ACTH preparations in the body]. Experiments were conducted on male Wistar rats.  A study was made of the intensity of incorporation into the liver, kidneys, the thyroid gland, the adrenal glands and the rate of elimination from the blood plasma of two ACTH preparations iodated by Greenwood's method: International Standard for Corticotropin, Mill Hill, London; Hum ACTH--Hid-28, Richter.  It appeared that the preparations of natural swine -125-I-ACTH and of synthetic -131-I-ACTH were identical chromatographically and by saturation with iodine-125 and iodine-131, respectively, but differed by their behaviour in the organism.  T1/2 for -125-I-ACTH (swine) was 4.7 min; the hormone was not incorporated into hepatic parenchyma, and only very weakly--into the kidneys, but it was intensively accumulated by the adrenal galnds.  Radioactivity peak was recorded in the adrenal cortex 6 minutes after the administration of the labeled hormone.  -131-I-ACTH (synthetic) disappeared from the blood with a falf-period of 11.2 min, was incorporated into the liver and particularly into the kidneys, and was not accumulated in the adrenal gland.


Host induced alteration of avian sarcoma virus B-77 genome. The genome of an avian oncornavirus was altered after infection of a heterologous host. This was studied with avian sarcoma virus B-77 in duck embryonic fibroblasts (DEF) and chicken embryonic fibroblasts (CEF). To detect alteration of the viral genome, we hybridized 35S B-77 RNA with normal duck DNA by either one of two techniques:when viral RNA was in excess and when DNA was in excess. The RNA of B-77 passaged only in gs minus chf minus CEF does not have homology with duck DNA. However, after several passages of B-77 through DEF the viral genome acquired duck specific RNA sequences. After 4 and 10 passages, B-77 RNA acquired 2.2 and 6.6%, respectively, complementarity to normal duck DNA. The duck specific RNA sequences were found to be covalently linked to the B-77 RNA genome. Also, the host specific sequences acquired by the virus appear to be from a region of the duck DNA which is repeated four to six times per cell. After 5 back passages in CEF some of the duck specific RNA sequences in the viral genome were lost.


[The effect of estrogens on the concentration of estradiol receptors in rat uterus, kidneys and liver]. The concentration of receptors to E2 decreased in the uterine cytoplasm of rat uterus and increased in the nuclei during sexual maturation.  With ageing this index in the cytoplasm and the nuclei of the uterus fell, and failed to alter during pregnancy.  After ovariectomy the concentration of the receptors increased in the uterine cytoplasm, but no marked changes in the receptors occurred in the nuclei.  No significant changes were revealed in the receptors to E2 in sexual maturation, pregnancy and ovariectomy in the cytoplasm and the nuclei of the kidneys and the liver.  Administration to rats of E2 produced a reduction of the concentration of receptors to E2 in the cytoplasm of the uterus, kidneys and the liver and an increase in their content in the nuclei of the uterus and the liver.


[Stimulation of the protein synthesizing capacity of thyroid gland polyribosomes in vitro under the influence of cyclic adenosine-3', 5'-monophosphate]. It was shown that cAMP increased the capacity of polyribosomes of the thyroid glands to the labeled phenylalanine incorporation into the protein by 51 per cent.  Preincubation of microsomes or ribosomes with cAMP in the cell-free protein-synthesizing system also increased the incorporation of the labeled amino acids into the protein by 54 and 49 per cent, respectively.  The optimal effect occurred with the concentration of cAMP of 0.2 mg/ml. cAMP in a concentration of 0.8 mg/ml inhibited the incorporation of the labeled amino acids by 32 per cent.  This can possibly be attributed to the forming products of cAMP degradation.  Modification of the protein-synthesizing system (ATP--1 mM. GTP--1.6 mM) led to the fact of an increase in the level of the labeled amino acid incorporation into the protein by 54 per cent; the simulating effect of cAMP persisted.  In the absence of ATP in the incubation medium addition of cAMP failed to stimulate the protein synthesis.  Thus, cAMP stimulated protein synthesis in the thyroid gland in vitro, with utilization of ribonucleoproteid particles.


Interaction of a spin-labeled long chain acylcholine with the cholinergic receptor protein in its membrane environment. The choline ester of a spin-labeled fatty acid, 8-doxylpalmitocylcholine, CH3--(CH2)7--CR-(CH2)6-- + COO--(CH2)2--N(CH3)3, where R is the paramagnetic 4',4'-dimethyloxazolidine-N-oxyl (doxyl) ring has been synthesized. 8-Doxylpalmitoylcholine blocks reversibly the depolarization of Electrophorus electroplaque elicited by the bath application of carbamylcholine. It slows down the initial rate of binding of the alpha-[3-H]toxin from Naja nigricollis to receptor-rich membranes fragments from Torpedo, and it displaces [3-H]acetylcholine bound to the cholinergic receptor site present in these fragments. Electron spin resonance spectra of 8-doxylpalmitoylcholine in the presence of the receptor-rich membrane fragments show complete immobilization of the spin label. Various cholinergic agents tested, including N. nigricollis alpha-toxin, reverse this immobilization, probably by displacing the 8-doxylpalmitoylcholine from its complex with the cholinergic receptor protein to the lipid phase of the membrane.


Cyclic AMP and cyclic GMP concentrations in serum- and density-restricted fibroblast cultures. Mouse fibroblasts transformed by simian virus 40 (SV3T3 cells) are characterized by cyclic AMP and cyclic GMP levels, respectively, about half and twice those found in growing untransformed 3T3 cells. Density-dependent inhibition of growth is correlated with reduced cyclic GMP concentrations in 3T3 and four different density-restricted revertant lines derived from SV3T3. The levels of cyclic AMP are not increased at confluence. Upon serum restriction, serum-dependent cell lines show a greater increase in intracellular cAMP than serum-insensitive lines. Cyclic GMP levels are greatly reduced, even in serum-insensitive density revertants, but not in SV3T3. Serum readdition to all serum-dependent lines is followed by a rapid decrease in cyclic AMP and increase in cyclic GMP concentrations. The magnitude of these responses is decreased in SV3T3 and density revertants.


Plasminogen-independent fibrinolysis by proteases produced by transformed chick embryo fibroblasts. The fibrinolytic activity of proteases secreted by chick embryo fibroblasts infected with Rous sarcoma virus was studied by use of a procedure in which a fibrin clot was formed with highly purified fibrinogen and thrombin above the cell layer. This procedure results in the formation of fibrin that is apparently a more suitable substrate for studies on fibrinolysis than is fibrin prepared by other methods. Since neither plasminogen nor serum were included in the assay system in the present studies, the fibrinolytic activity observed cannot be ascribed to the conversion of the plasminogen in serum to plasmin by a plasminogen activator produced by transformed cells. Our procedure, therefore, measures proteolytic activities other than those reported by previous investigators. Maintenance of some of the transformed phenotypes of Rous sarcoma virus transformed chick embryo fibroblasts such as morpholigical change and increased rate of glucose uptake apparently does not depend on the presence of plasminogen in the culture medium.


Affinity chromatography of viral DNA polymerases on pyran-sepharose. Pyran covalently linked to cyanogen bromide-activated Sepharose has been shown to be an effective affinity matrix for several viral DNA polymerases. Differential salt elution of viral compared with cellular polymerases, as well as substrate elution, suggests the affinity nature for the matrix. Unlike some other affinity systems described, pyran-Sepharose is totally resistant to nuclease digestion and is stable at 4 degrees for several months. DNA polymerases isolated from several viruses by detergent treatment were recovered in good yield. Analysis of iodinated proteins by sodium dodecyl sulfate-gel electrophoresis revealed that the DNA polymerase of avian myeloblastosis virus found in crude preparations of the virus could be purified nearly to homogeneity by a single passage through the column. These results suggest that pyran-Sepharose is an effective affinity column that is potentially adaptable as part of a general purification procedure for viral DNA polymerases.


Stimulation of DNA polymerase by factors isolated from Novikoff hepatoma. Extracts of Novikoff hepatoma cells contain factors capable of stimulating in vitro DNA synthesis several fold. The activity can be resolved into three separate protein peaks on DEAE-Sephadex. Two of these, factors II and III, have been purified and partially characterized. Both factors increase the initial rate of DNA synthesis and allow synthesis to proceed much longer. If either factor is added after synthesis by the DNA polymerase has reached a plateau, resumption of synthesis occurs. The factors appear to have different modes of action or sites of action since they show an additive effect even when a single one is used at saturating conditions. These factors are present in normal rat liver but at a concentration less than 5% of that found in the tumor cells. When tested with several highly purified DNA polymerases (DNA nucleotidyltransferase, EC 2.7.7.7), the factors show a much greater stimulation of homologous, non-mitochondrial enzymes (rat liver nuclear-, rat liver cytoplasmic-, or Novikoff-DNA polymerases) when compared with rat liver or calf liver mitochondrial-, Escherichia coli I-, or sea urchin nuclear-DNA polymerases. The mechanism of action of these factors is not known at present. No enzymatic activity has been associated with factor III. Highly purified, but not homogeneous, preparations of factor II contain low levels of endonuclease; it has not been established whether endonuclease is a contaminant or is responsible for the stimulating activity.


Methylation-dependent translation of viral messenger RNAs in vitro. Methylated reovirus and vesicular stomatitis virus mRNAs, synthesized in vitro in the presence of S-adenosylmethionine by the virion-associated polymerases (RNA nucleotidyltransferases, EC 2.7.7.6), stimulate protein synthesis by wehat germ extracts to a greater extent than unmethylated mRNAs. Addition of S-adenosylmethionine to a cell-free extract programmed with unmethylated mRNA stimulates protein synthesis and results in methylation of the mRNA. An inhibitor of mRNA methylation. S-adenosylhomocysteine, blocks translation of unmethylated, but not of methylated, mRNAs. Aurintricarboxylic acid, which inhibits polypepetide chain initiation, also prevents mRNA methylation by wheat germ extracts. In contrast, sparsomycin, which inhibits polypeptide chain elongation, does not reduce mRNA methylation. The results indicate that methylation of viral mRNA is required for translation in vitro and suggest that mRNA methylation occurs at the initiation step of protein synthesis.


Physical properties of New Jersey serotype of vesicular stomatitis virus and its defective particles. The wild-type New Jersey serotype of vesicular stomatitis virus generated two types of defective interfering T-particles. The physical properties of these particles and the wild-type virion were determined by laser light scattering spectroscopy, sedimentation measurements, and electron microscopy.


Lack of detectable change in cyclic AMP during the cardiac inotropic response to isoproterenol immobilized on glass beads. Changes in contractility and the levels of adenosine 3':5'-cyclic monophosphoric acid (cAMP) were assessed in isolated cat cardiac muscle in response to soluble isoproterenol and isoproterenol immobilized on glass beads. Drug-induced positive inotropic responses were compared to the maximum isometric force achieved with paired electrical stimulation, a potent physiological inotropic stimulus. Isoproterenol (1 muM) in solution increased the force of contraction 0.832 plus or minus 0.165 g at 60 sec in eight muscles tested, which at 60 and 120 sec averaged 65.5 plus or minus 6.5% and 82.9 plus or minus 8.8%, respectively, of the force with paired electrical stimulation. Isoproterenol immobilized on glass beads gave positive inotropic responses similar to those for the soluble form of the drug. Placement of only three isoproterenol-glass beads on the muscles increased the force of contraction 0.742 plus or minus 0.166 g at 60 sec (n equals 11), which at 60 and 120 sec averaged 45.1 plus or minus 7.0% and 58.6 plus or minus 6.4%, respectively, of the force with paired electrical stimulation. The magnitude of this response indicates that the increased force was developed by at least 60% of the cells in each muscle. Control levels of cAMP were 0.527 plus or minus 0.049 pmol/mg of tissue wet weight, n equals 11. cAMP levels 60 sec after 1 muM soluble isoproterenol was added were 1.212 plus or minus 0.085 pmol/mg; in contrast, the levels of cAMP in response to isoproterenol immobilized on glass beads at 60 sec were 0.490 plus or minus 0.060 pmol/mg, not significantly different from control levels. These data indicate that cAMP may not be involved in the propagation of the inotropic response that must have occurred in these cardiac muscles and raise questions as to the physiological significance of the large cAMP increases that occur in response to soluble drugs and hormones.


Epithelial cell death and cyclic AMP increase during palatal development. Cyclic AMP levels increase abruptly in the secondary palatal shelf (mesenchyme plus a two- to three-cell-layered epithelium) between day 15 and 16 of gestation in the rat embryo. The addition of dibutyryl cyclic AMP to the media in which immature palatal shelves (day 14) are cultured causes a precocious decrease in DNA synthesis, but increased glycoprotein synthesis and epithelial adhesiveness. These changes are most evident in the medial-edge epithelial cells and resemble the developmental events that occur normally on day 15 and 16 during formation of the secondary palate. The initiation of epithelial cell death and adhesiveness in the medial edge of the palatal shelf may be mediated through an increase in cyclic AMP.


Cyclic AMP, a nonessential regulator of the cell cycle. Flow-microfluorimetric analysis has been carried out on populations of exponentially growing S49 mouse lymphoma cells treated with dibutyryl cyclic AMP. The drug produces a specific concentration-dependent block in the G-1 phase of the cell cycle while other phases of the cycle are not perceptibly altered. The cell cycle of a line of mutant cells lacking the cyclic AMP-dependent protein kinase is not affected by the drug. Since these mutant cells have been shown to maintain a normal cell cycle, even in the presence of high levels of cyclic AMP, periodic fluctuations in the levels of the cyclic nucleotide cannot be required for or determine progression through the cell cycle.


Cyclic AMP and immune responses: changes in the splenic level of cyclic AMP during the response of mice to antigen. Intravenous injection of sheep erythrocytes into normal immunologically competent C57BL/6J mice results in significant and characteristic changes in the splenic level of 3':5'-cAMP with initiation of the immune response and proliferation of antibody-forming cells. The level increases 2- to 3-fold initially, peaks at 2 min, and returns to base level in an hour. Between 2 and 5 days there is a decrease, followed by a peak when the rate of proliferation of antibody-forming cells is maximal. Changes in splenic level of cAMP are thus transitory and biphasic, and they occur only in response to foreign substances that are immunogenic, such as heterologous erythrocytes, and not to antigenically inert carbon particles. They are also dependent upon the dose of immunogen. Moreover, the double-stranded hybrid of polyadenylate and polyuridylate, which acts synergistically with antigen in stimulating endogenous cAMP, is immuno-enhancing if given with sheep erythrocytes when the cAMP level is increasing, and immunosuppresive if given when cAMP is decreasing. These data provide direct evidence for a role of cAMP as a mediator in the activation and proliferation of immunocytes stimulated by antigen. With knowledge of the transitory and biphasic nature of the cAMP response induced by antigen, one can avoid indiscriminate use of drugs that modify the level of endogenous cAMP and instead employ them rationally in controlling the immune response, enhancing or suppressing it as desired.


Binding of ATP to the progesterone receptor. The possible interaction of progesterone--receptor complexes with nucleotides was tested by affinity chromatography. The cytosol progesterone receptor from hen oviduct was partially purified by ammonium sulfate precipitation before use. When progesterone was bound to the receptor, the resulting complex could be selectively adsorbed onto columns of ATP-Sepharose. This interaction was reversible and of an ionic nature since it could be disrupted by high-salt conditions. A competitive binding assay was used to test the specificity of receptor binding to several other nucleotides, including ADP, AMP, and cAMP. A clear specificity for binding ATP was evident from these studies. When ATP was added to receptor preparations, the nucleotide did not affect the sedimentation properties or hormone binding characteristics of the receptor. Although the function of ATP remains unknown, these studies indicate a role of this nucleotide in some aspect of hormone receptor activity.


Structure-function relationships and mode of replication of animal rhabdoviruses. Recently accumulated knowledge allows more precise comparison of the structural (and possibly evolutionary) relationships of several different animal rhabdoviruses: vesicular stomatitis virus, rabies virus, Kern Canyon virus, and spring viremia of carp virus. Each virus is composed primarily of a glycoprotein, an RNA-associated nucleoprotein, and one or two membrane proteins. Vesicular stomatitis virus group viruses contain lesser amounts of two additional distinct polypeptides, NS and L. The separate viruses undergo structural polypeptide phosphorylation in vivo according to characteristic patterns. In vesicular stomatitis virus the NS protein is selectively phosphorylated. In rabies group viruses and in spring viremia of carp virus, the nucleoprotein is the predominant phosphoprotein; in these viruses only the phosphorylated moiety is selectively cleaved off with trypsin. In Kern Canyon virus, only membrane protein and glycoprotein are weakly phosphorylated. Each virus possesses a virion-bound protein kinase. Vesicular stomatitis virus group viruses, Kern Canyon virus, and spring viremia of carp virus only contain virion-bound transcriptases of respectively decreasing levels of activity demonstrable in vitro. Vesicular stomatitis and Kern Canyon viruses replicate efficiently in enucleated cells; rabies virus does not. Based upon these observations, it is suggested that vesicular stomatitis virus may represent the most highly evolved of these rhabdoviruses, whereas spring viremia of carp and Kern Canyon viruses may represent "evolutionary links" between the vesicular stomatitis and rabies virus groups.


Melanoma cells resistant to inhibition of growth by melanocyte stimulating hormone. Melanocyte stimulating hormone (MSH) enhances melanization but inhibits proliferation of Cloudman S91 melanoma cells in culture. We have isolated variants of these cells that can grow in the presence of MSH. The conclusions we have reached from analyses of these cells are the following: (1) Basal tyrosinase activity (monophenol monooxygenase; monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1), i.e., the activity that is present in the absence of added MSH, is related through a common biochemical pathway to MSH-mediated control of growth. (2) MSH-inducible tyrosinase activity does not appear to be related to MSH control of growth. (3) The morphological changes that occur following the addition of MSH or cAMP are related to controls of growth and not to those of melanization.


Free radicals formed by exposure of pyrimidine solids to sodium atoms: an electron spin resonance study. Sodium atoms have been deposited on various pyrimidine powders under high vacuum at 77 degrees K and the electron spin resonance spectra of the resulting free radicals have been observed. Generally, the spectra show that the electron of the Na goes into a molecular orbital of the pyrimidine ring and the Na+ ions become attached to a carbonyl oxygen of the resulting pyrimidine anion. In 5-fluorouracil and 5-chlorouracil, however, the halogen is evidently abstracted by the Na to form NaF or NaC1 and the neutral uracil radical. Thymine shows evidence for H-addition radicals as well as the Na+-[thymine]-complex. The H source for the addition radicals may be an H-2-0 impurity in the sample, with which the Na atoms combine to release the H atoms. In addition to a resonance with g equals 2.00 from the pyrimidine anion radical, broad resonances with g greater than 2 were observed for 5-bromouracil, 5-chlorouracil and 5-iodouracil, as well as for alloxan and cytosine. These resonances, generally unstable at room temperature, are believed to arise from electrons trapped in interstitial sties or vacancies in the lattice.


Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids. We described previously a simple test on petri plates for detecting chemical carcinogens as mutagens, using an especially sensitive set of bacterial strains to detect mutagenic acitivty and a mammalian liver extract for carcinogen activity. We now extend the utility of the method by introducing two new bacterial strains which can detect with great sensitivity many carcinogens which we did not detect before or detected with less sensitivity. Among these carcinogens are aflatoxin B-1, sterigmatocystin, benzyl chloride, benzo[a]-pyrene, 7,12-dimethylbenzanthracene, 1'-acetoxysafrole, and the nitrofuran food additive furylfuramide (AF-2). The new strains TA100 and TA98 contain an R factor plasmid, pKM101, in our standard tester strains TA1535 and TA1538. The R factor increases mutagenesis with certain mutagens, but not others. We present evidence that the mutagens that become more effective work through an error-prone recombinational repair.


Biochemical method for mapping mutational alterations in DNA with S1 nuclease: the location of deletions and temperature-sensitive mutations in simian virus 40. S1 nuclease (EC 3.1.4.X), a single-strand-specific nuclease, can be used to accurately map the location of mutational alterations in simian virus 40 (SV40) DNA. Deletions of between 32 and 190 base pairs, which are at or below the limit of detectability by conventional electron microscopic analysis of heteroduplex DNAs, have been located in this way. To map a deletion, a mixture of unit length, linear DNA, prepared from the SV40 deletion mutant and its wild-type parent, are denatured and reannealed to form heteroduplexes. S1 nuclease can cut such heteroduplexes at the nonbase-paired region to produce fragments whose lengths correspond to the position of the deletion. Similarly, specific fragments are produced when S1 nuclease cleaves a heteroduplex formed from the DNAs of SV40 temperature-sensitive mutants and either their revertants or wild-type parents. Thus, the positions of the nonhomology between these DNAs can be determined.


Patterns of organization of actin and myosin in normal and transformed cultured cells. The patterns of distribution of intracellular actin and myosin were examined by specific immunofluorescence in a series of normal, simian-virus-40-transformed, and revertant cell lines of rat and mouse origin. A consistent correlation was found between sensitivity to anchorage-dependent growth control and the presence of large, thick sheaths of actin-containing material. The presence of these sheaths was temperature-dependent in a rat line transformed by a temperature-sensitive mutant in the complementation group A of the oncogenic virus simian virus 40.