Neural factors in experimental degenerative arteriopathy. Intermittent electrical stimulation of the lateral hypothalamus of rats performed for 30 min to 6 hr, results in hyperlipidemia and endothelial cell damage of the aorta and coronary arteries. Hyperlipidemia is related to transient biliary obstruction elicited by hypothalamic stimulation and is characterized by elevation of the cholesterol, phospholipid, and triglyceride fractions. Endothelial cell damage is observed ultrastructurally as plasma membrane degeneration with detachment and the formation of large spaces ("vacuoles"). Thus, neural factors may be implicated in inducing conditions associated with early atherogenesis. Stimulation carried out for longer time intervals would be expected to produce more advanced lesions. However, the role of neural transmission per se (i.e., without hyperlipidemia) in producing arteriopathy is not clearly defined from these experiments. In rats, the lesser splanchnic nerve forms the major innervation of the abdominal aorta. In animals fed normal diets, chronic intermittent stimulation of this nerve (up to 3 weeks) resulted in advanced arteriosclerotic changes with intimal fibrosis and calcification. On histologic examination, lipid deposits appeared to be absent from these lesions. Animals stimulated for shorter periods of time exhibited earlier changes associated with atherogenesis, such as endothelial damage, elastic reduplication, and adherent microthrombi. Thus, direct neural transmission, especially if excessive, plays a role in producing arteriopathy. Hyperlipidemia, if persistent, could modify these lesions so that they would accumulate plasma lipids. Experiments to test this hypothesis are currently in progress.


The distribution of dietary plant sterols in serum lipoproteins and liver subcellular fractions of rats. Rats were fed plant sterols containing campesterol and beta-sitosterol in the differerent proportions, and their distribution in serum lipoproteins and in liver subcellular fractions was determined. In serum lipoproteins, the percentage as well as the concentration of plant sterols increased with the increase in the density of lipoproteins. Thus, high density lipoprotein (HDL) contained the highest and very low density lipoprotein (VLDL), the lowest. Also, there were distinct differences in the ratio of campesterol to sitosterol among lipoproteins, it was the highest in VLDL and lowest in HDL. Quantitatively, more than 75% of campesterol and 80% of sitosterol were carried in HDL; the values were significantly different from those of cholesterol (ca. 70%) in relation to total cholesterol. The distribution of plant sterols in liver subcellular fractions was virtually the same with that of cholesterol. Both nuclei and microsomes contained approximately 40% of total plant sterols.


Effect of phentolamine on the somatostatin-induced inhibition of glucagon and insulin secretion. Synthetic cyclic somatostatin was infused into either the cranial pancreaticoduodenal artery or the femoral vein of anesthetized dogs with or without previous administration of phentolamine. Somatostatin infused into the pancreatic artery at a dose of 50 ng/kg/min for 10 min caused significant decreases in blood flow and plasma basal concentrations of both glucagon and insulin in the cranial pancreaticoduodenal vein, resulting in a profound decline of bihormonal output during the infusion. Arterial plasma glucose was not reduced during the administration of somatostatin in the pancreatic artery. These somatostatin-induced decreases failed to be eliminated by a 0.2 mg/kg injection of phentolamine into the femoral vein followed by a 9-min infusion of this alpha-adrenergic blocker (0.02 mg/kg/min) into the pancreatic artery immediately prior to the somatostatin administration. An inhibition of glucagon and insulin output and a fall of plasma glucose caused by somatostatin (1.7 microgram/min) infused into the femoral vein for 30 min also were not abolished by a prolonged and simultaneous infusion of phentolamine (0.2 mg/min) into the femoral vein over a period of 2 hr. These results indicate that alpha-adrenergic receptor mechanisms do not play a major role in the inhibition of islet glucagon and insulin secretion by somatostatin.


Establishment of a persistent measles virus infection in HEp-2 cells. A productive measles virus persistent infection has been established in HEp-2 cells. Greater than 90% of the persistently infected HEp-2 cells (H2MV) exhibited measles specific immunofluorescence and haemadsorption. Although most of the H2MV cells contained measles specific antigens, only a small percentage (less than 1%) actually produced infectious measles virus as determined by infectious centre assays. The measles virus produced by H2MV cells exhibited properties different from the initiating parent Edmonston strain virus, being reduced in virulence and also temperature sensitive for replication at 39 degrees C. The role of these altered virus properties in the establishment of persistence is considered.


Common antigen between coxsackievirus A 16 and enterovirus 71. Cross immunofluorescence revealed that coxsackievirus A 16 (CA 16) shared a common antigen with enterovirus 71 (E 71). The cross reactivity of these two serotypes was also examined by complement fixation test with purified virus preparations fractionated by sucrose density gradient centrifugation and two peaks of antigenicity were detected, one being type-specific and the other cross-reacting. The common antigen was heat-stable and attributable to empty capsids. Immuno-diffusion also revealed the common antigen. Infants without antibody to E 71 developed complement fixing and precipitin antibody to E 71 after recovery from hand, foot and mouth disease caused by CA 16.


Protective effect of an oral infection with herpes simplex virus type 1 against subsequent genital infection with herpes simplex virus type 2. The problem of whether oral Herpes simplex virus type 1 (HSV-1) infection provides protection against subsequent genital infection by Herpes simplex virus type 2 (HSV-2) was investigated. Mice were used as models. Following conditions in man, both the oral and genital infections applied were noninjurious. Mice infected orally with HSV-1 were weakly protected against virus 'take' following vaginal challenge with HSV-2. Genital 'takes' were found in 67% of the immunized mice, as compared with 83% of the controls (protection rate 20%, P = 0.002). The course of genital infection in the immunized mice, however, was relatively mild: Lethality decreased from 97% in the controls to 35% in the immunized mice (protection rate 63%, P less than 0.001). Furthermore, local and neurologic symptoms occurred less frequently. Attempts to isolate the virus from homogenized brain and spinal cord of immunized mice that died after genital challenge with HSV-2 failed in most cases. Also virus could not be recovered from the liver of infected mice, irrespective of the experimental group.


BK virus: I. Seroepidemiologic studies and serologic response to viral infection. Sera of 656 patients in seven age groups were tested by hemagglutination=inhibition (HI) and complement=fixation (CF) tests for antibodies to BK virus. Both tests revealed that BK virus antibodies are common in the population of southern Germany, and that primary infection is apparently acquired during childhood. The highest rate of positive sera, 71% in the HI test and 56% in the CF test, was found in the 15-30 year old group. The antibody titers correlated in both serologic tests, but HI tests were more sensitive and reliable than CF tests. An indirect immunofluorescence assay for the detection of BK virus-specific IgM antibodies was also developed. Studying umbilical cord blood sera from 846 healthy donors, BK virus IgM antibodies were detected in 77 sera (9.1%).


BK virus: II. Serologic studies in children with congenital disease and patients with malignant tumors and immunodeficiencies. Sera of 451 children with congenital diseases and 185 tumor patients were tested for BK virus-specific antibodies by hemagglutination inhibition and IgM-immunofluorescence tests. Compared to age-matched control groups, higher percentages and significantly elevated geometric mean titers of HI antibodies were found in all patient groups tested. Of children under six months of age with congenital diseases such as dysplasia, cerebral defects, and hyperbilirubinemia and hepatosplenomegaly, 4.2% (17/402) had BK virus-specific IgM antibodies. No positive sera were found in 68 control sera. Of tumor patients 5--15 years of age, 8.6% (16/185) had IgM antibodies to BK virus. In the control group, 30% (3/99) had them. Serial serum samples from 76 tumor patients treated with cytostatic drugs showed seroconversion in three cases. No relationship between certain clinical features and BK virus infection was noted. Isolation of BK virus was successful from urines of two infants with connatal defects, six patients suffering from malignant tumors, and four patients with inherited immunodeficiencies.


Localization of papillomavirus and virus-specific antigens in the skin of tumor-bearing Mastomys natalensis (GRA Giessen). In skin neoplasms (so-called keratoakanthomas, papillomas, and squamous cell carcinomas) of Mastomys natalensis (GRA Giessen) virus particles and viral antigens were demonstrated in distinct epithelial layers. Nuclei of the upper parts of the stratum granulosum and disintegrated cell parts of the stratum corneum contain viral structures. The localization of virus particles and viral antigens in any skin neoplasms of M. natalensis (GRA Giessen) corresponded to the sites where papillomavirus-specific material had been demonstrated in papillomas of other species.


Physiological consequences of mitochondrial antibiotic-resistant mutations in Paramecium: growth-rates, cytochromic defects and cyanide-insensitive respiration of mutant and erythromycin-treated wild-type strains. A set of mitochondrial antibiotic-resistant mutants of Paramecium have been analyzed with respect to their growth-rates, cytochromic content and respiratory properties. The mutants could be arranged in a continuous series ranging from strains equivalent to wild-type to severely affected ones; affected strains display longer generation times, reduced amount of cytochrome oxidase and very high levels of cyanideinsensitive respiration. Perfect phenocopies of the mutants were obtained by treating wild-type cells with low concentrations of erythromycin suggesting that the mutations exert their pleiotropic effect by perturbating mitochondria protein synthesis in agreement with the idea that these mutations affect the mitochondrial ribosomes. In the mitochondria of some of the mutants, electrons can be channelled with equal efficiency into the "classical" cyanide-sensitive pathway and the alternate cyanide insensitive (and SHAM-sensitive) one, providing direct demonstration of the branching of these two respiratory pathways. In the absence of any added inhibitor, however, electrons tend to be channelled in the cyanide-sensitive pathway. All the physiological data fit perfectly the genetic data concerning the "stability" of the various mutations in "mixed mitochondrial populations", i.e., markers that were known to be strongly counter-selected with respect to wild-type in such populations correspond to severely affected strains, while markers that were known to be "stable" correspond to "healthy" strains. A more quantitative analysis of the data shows that that there is little or no "complementation" between wild-type and mutated mitochondria in mixed cells indicating a high extent of functional autonomy of mitochondria in Paramecium.


Viomycin favours the formation of 70S ribosome couples. The peptide antibiotic viomycin at a concentration of 10 muM inhibits E. coli ribosomes to the extent of about 70% as measured in the poly (U) system, and to about 85% in a natural mRNA (R17) system. Ribosomes from M. smegmatis show no activity at all at this concentration of the antibiotic. Experiments on the Mg+2 dependent dissociation and association of the ribosomal subunits revealed that viomycin stabilizes the 70S couples and promotes association of ribosomal subunits. This response is related to the drug action as indicated by the observation that viomycin resistant strains are not affected by viomycin with respect to dissociation and 70S couple information. A model for the inhibitory action of the drug is proposed.


A possible model for the structure of the Neurospora carbamoyl phosphate synthase-aspartate carbamoyl transferase complex enzyme. The pyrimidine-3 locus of Neurospora crassa specifies a multienzyme complex comprising pyrimidine-specific carbamoyl phosphate synthase (CPSpyr) and aspartate carbamoyl transferase (ACT). It appears to be divided into a translationally proximal CPS-specific region and a distal ACT-specific region. Levels of complementation for ACT activity between pairs of four pyr-3 CPS+ ACT- mutants showed a range from 12% to 68% of the wild-type level of the enzyme. This is interpreted as interallelic complementation, contradicting certain earlier suggestion of two dissimilar ACT subunits. Proteolysis of an extract from a heterokaryon formed from two of the above CPS+ ACT- alleles (alpha and beta) did not lead to loss of ACT activity, but led to the formation of a fragment with ACT activity with a similar molecular weight (92,000 daltons) to that produced in extracts of wild type strain. The pyr-3 polar mutant 43-174 which is enzymatically CPS+ ACT- and which fails to complement with any other CPS+ ACT- alleles, thus suggesting its location towards the proximal end of the ACT region, has CPS activity associated with a form of 180,000 daltons molecular weight. These findings are used to contruct a model for structure of the native enzyme complex.


Selection and characterization of nuclear mutations affecting mitochondria in Paramecium. A screening method, based upon resistance to a tetrazolium salt (TTC), is described which permitted the isolation in Paramecium of 28 mutants resistant to TTC. These mutants displayed various defects in mitochondrial functions (cytochromic content cyanide insensitive respiration). Some mutations seemed to affect directly the respiration chain while others seemed to cause indirect modifications, possibly altering mitochondrial protein synthesis. Genetic analysis of four mutants showed in all that the resistance to TTC was of nuclear origin.


Catabolite repression in Escherichia coli mutants lacking cyclic AMP. The regulation of catabolite repression of beta-galactosidase has been studied in Escherichia coli mutants deleted for the adenyl cyclase gene (cya delta), and thus unable to synthesize cyclic AMP. It has been found that, provided a second mutation occurs either in the crp gene coding for the catabolite gene activator protein (CAP) or in the Lactose region, these mutants exhibit catabolite repression. If the catabolite repression seen in the mutant strains corresponds to the mechanism operating in wild-type cells the results would suggest that the intracellular concentration of cyclic AMP cannot be the unique regulator of catabolite repression.


Catabolite modulator factor: physiological properties and in vivo effects. Catabolite modulator factor (CMF) specifically inhibits the expression of operons sensitive to catabolite repression. Systems known to be catabolite independent are not affected by CMF. The rate of metabolism of CMF depends on the extent of catabolite repression: it is slow under conditions of strong repression and high in catabolically derepressed cells. Cyclic AMP does not interfere with the rate of CMF metabolism. It has been found that a certain class of crp mutants are partially resistant to the repressive effect of CMF. Our results provide considerable support for the existence of an additional negative control in the regulation of catabolite repression.


Localization of glycosyl transferases in plasma membranes from Dictyostelium discoideum. Crude membranes from vegetative and aggregation competent cells of Dictyostelium discoideum Ax 2 were separated by a combination of differential and sucrose gradient centrifugation. A fraction mainly containing plasma membranes could be isolated. The high degree of purity was demonstrated by electron microscopy and by the presence of marker enzymes typical for the plasma membrane and the absence of enzymes characteristic for other subcellular compartments. Furthermore surface labelling with radioactive 1--fluoro--2,4--dinitrobenzene--14C and cAMP binding capacity were introduced as plasma membrane markers. In the pure plasma membrane fraction endogenous activities of D--mannosyl-, D--glucosyl- and N--Acetyl--D--glucosaminyl--transferases were present. The activities in plasma membranes of aggregation competent cells were up to thirty times higher than in membranes isolated from vegetative cells.


Cyclic AMP increase the Na+ permeability of the avian erythrocyte membrane by a process which does not involve protein phosphorylation. Preparations of avian erythrocyte plasma membranes have been made which are in the form of sealed vesicles. Using these preparations the permeability of the membranes to N+, K+, Mg2+ and Ca2+ was measured. Monobutyryl cyclic AMP and cyclic AMP increased the permeability to Na+ and Ca+ under conditions where no protein phosphorylation could occur. The only effect of phosphorylation of membrane proteins was to reduce Ca+ permeability. It is thus concluded that cyclic AMP increases Na+ permeability in the avian erythroycte by a direct effect which does not involve protein phosphorylation.


Decrease of collagen biosynthesis ability of rat kidney fibroblasts transformed with SV--40 virus. Rat kidney fibroblasts transformed with SV-40 produce "in vitro" a significantly lower amount of hydroxyproline-containing material which is collagenase sensitive as compared to normal cells. In contrast to normal fibroblast cultures, no collagenous material was found by histochemical methods in intercellular spaces of transformed cultures.


The effect of cyclic nucleotides and protein phosphorylation on the permeability of human erythrocyte ghosts to certain cations. Preparations of human erythrocyte membranes have been made which are in the form of sealed vesicles and which behave as osmometers on suspension in solutions of simple inorganic salts. Using these preparations the permeability of the membranes to Na+, K+, Mg2+ and Ca2+ was measured. Cyclic AMP (but not cyclic GMP) increased the permeability of the membranes to Ca2+ with a half maximal effect at a concentration of 25 microgram but did not affect the permeability to the other ions tested. Phosphorylation of proteins in the erthrocyte membrane lowered the permeability to Ca2+ without affecting the permeability to the other ions tested and there was a good correlation between the time course of protein phosphorylation and decrease in Ca2+ permeability. It is postulated that the system through which cyclic AMP causes an initial rapid rise in Ca2+ permeability followed by increased phosphorylation of membrane proteins and reduced Ca2+ permeability may have a widespread occurrence in biological systems and serve to control the concentration of Ca2+ in the cytoplasm.


EMS-induced reversion studies in the white locus of Drosophila melanogaster. 5 white-locus mutants of Drosophila melanogaster, respresenting 5 different sub-sites, were treated with EMS and tested for reversion to wild-type. 4 of them were genuine mutants and one was not. Moreover, the ability of the 4 mutants to revert to wild-type differed from one another which therefore reflects a qualitatively distinct alteration in the genetic material delimited by each mutant.


Inhibition of postreplication repair and the enhancement of induction of SV40 virus from transformed hamster kidney cells. The induction by ultraviolet light of simian virus 40 (SV40) from two SV40--transformed hamster kidney cell lines is enhanced by caffeine. In order to investigate the mechanism responsible for this enhancement, the effect of caffeine on postreplication repair of DNA damaged by UV light was studied utilizing alkaline sucrose-gradient sedimentation. Caffeine at concentrations of 0.5, 1.0 or 2.0 mM inhibited the filling of gaps during postreplication repair. In addition, caffeine was found to potentiate cell killing by mitomycin C, an alkylating agent, and to enhance SV40 induction by mitomycin C. We postulate that the persistence of gaps in DNA, caused by the presence of caffeine, results in the enhancement of SV40 virus induction.


Embryopathies due to spermatozoal impairment in Xenopus laevis. An in vitro test system, involving gametes of Xenopus laevis has been used to study the effect of antifertility agents upon spermatozoa as manifest in developing embryos. Exposure to either the isomeric forms of dimethylmyleran, methyl methanesulphonate or gamma-radiation led to development of a range of embryopathies, whereas treatment with steroidal drugs or the rodent epididymal chemosterilants alpha-chlorohydrin and trimethylphosphate was compatible with production of apparently normal offspring.


Mutagenicity of dimethylnitrosamine and ethyl methanesulfonate as determined by the host-mediated CHO/HGPRT assay. Host-mediated assays have been developed to allow determination of the mutagenic potential of promutagens and procarcinogens which require metabolic activation to exert their effects on indicator organisms. We report here the development of the host-(mouse)-mediated CHO/HGPRT system using the procarcinogen dimethylnitrosamine (DMN) as a model agent. Using a 2--h treatment time, we observed a linear dose-response relationship up to 250 mg of DMN per kg body weight. At 100 and 500 mg/kg DMN, mutation induction increased with time up to at least 6 h. DMN was not mutagenic when tested in vitro. Athymic (nude) mice, their phenotypically normal littermates, or BALB/c mice of both sexes were found to be suitable as hosts. A time- and dose-dependency of induced mutation frequency by a direct-acting agent, ethyl methanesulfonate (EMS), was observed in both the in vitro and the host-mediated assays.


Dose-rate effects of ethyl methanesulfonate on survival and mutation induction in cultured Chinese hamster cells. The dose-rate effects of ethyl methanesulfonate (EMS) on the survival and induction of mutations in Chinese hamster Don cells were investigated. The most effective time of exposure to EMS for reducing the surviving fraction of cells was 4 h, shorter and longer exposure times being less effective. The threshold or minimal concentration of EMS giving a surviving fraction of 0.5 was 0.05 mg/ml. The minimal effective time of exposure to EMS for cell death was 1 h. Corrected survival curves showed that longer exposure times at lower dose rates of EMS had less cytotoxic effect than shorter exposure times at higher dose rates. After exposure of Don cells to various doses of EMS for various times, the frequencies of mutations resistant to 6--thioguanine (6TG) were measured. An exposure time of 4 h produced a lower mutation frequency than shorter or longer exposure times that resulted in the same surviving fraction of cells. An exposure time of 20 h produced the highest induced mutation frequency. This system using cultured Chinese hamster cells should be useful as a sensitive procedure for detecting the mutagenic actions of chemicals.


Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures. Sodium selenite (Na2SeO3) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS)  or N-hydroxy-2-acetylaminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 X 10(-6) M Na2SeO3 resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na2SeO3 concentrations (7.90 X 10(-6) and 1.19 X 10(-5) M) resulted in a three-fold increase in the SCE frequency above background level (6--7 SCEs/cell). Exposure of lymphocytes to 1 X 10(-4) M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 +/- 0.75 while a similar exposure to 2.7 X 10(-5) M N-OH-AAF resulted in 13.61 +/- 0.43 SCEs/cell. Simultaneous addition of the high Na2SeO3 concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25--30% and 11--17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.


Cellular transformation and the 'morphologic phenotype' of transformed cells. Expression of the product of the transforming gene (src) of RNA tumour viruses promotes growth and usually alters the adhesion, appearance and surface properties of cultured fibroblasts. The latter group of properties termed the 'morphologic phenotype' of transformed cells is largely due to diminished cell-to-substratum adhesion. The role of cyclic AMP, cell surface protein (CSP), and other factors in producing the 'morphologic phenotype' are discussed. The effects of src expression bear a striking resemblance to the action of peptide hormones such as insulin on appropriate target cells.


Binding of (3H)dihydroalprenolol to beta adrenoceptors of cells isolated from adult rat heart. Myocardial cells isolated from adult rat heart bind (3H)dihydroalprenolol. Sixty-one percent of this binding appeared to be at the beta adrenoceptors since it was inhibited by saturating quantities of the beta antagonist propranolol or by the beta agonist isoprenaline. The binding is stereoselective as the l-isomer of isoprenaline caused greater inhibition than the d-isomer. The binding of (3H)dihydroalprenolol to beta adrenoceptors was saturable; half maximum binding occurred at about 8 nM and full saturation at 30--40nM.


A simple method for sampling murine pulmonary and cardiac tissues for analysis of cyclic nucleotide levels. A simple method for the rapid removal and freezing of mouse cardiac and pulmonary tissues is described. Samples thus obtained were judged to be suitable for valid estimation of in vivo levels of cyclic AMP and cyclic GMP based on the following findings: (a) the samples could be obtained and frozen in the very short time period of a few seconds; (b) no indication of adverse effects of the collection procedure was found upon examination of chemical indicators of energy metabolism; (c) the apparent rates of change of cyclic AMP and cyclic GMP levels during the first seconds after tissue isolation could produce small, but acceptable errors; and (d) dose-dependent elevations of pulmonary cAMP levels consistent with known effects in vitro were found after in vivo administration of isoproternol.


Effects of Mg2+, Mn2+ and Ca2+ on adenylcyclase activity. Evidence for a metallic site. In membrane preparations from immature erythrocytes from rats the effects of the divalent cations Mg2+, Mn2+ and Ca2+ on basal activity of adenylcyclase as well as on enzyme activity stimulated by isoprenaline (Ipn) or guanylyl-imidodiphosphate [Gpp(NH)p] were investigated.--Mn2+ is a ten-fold stronger activator of the enzyme than Mg2+ irrespective of the stimulant used. At suboptimal concentrations of the cations at all concentrations of Gpp(NH)p used (10(-6) to 10(-3) M) reaction velocities increase progressively over an incubation period of 40 min. Optimal cation concentrations, however, i.e. 3 x 10(-3) M Mn2+ and 3 x 10(-2)M Mg2+ elicit a constant and at 10(-4) M Gpp(NH)p maximal reaction velocity. In contrast, the Ipn-stimulated  cAMP synthesis proceeds linearly at all Ipn concentrations used; a change of cation concentrations elicits only a change in reaction velocity, which is maximal at 10(-3) M Mn2+ and 10(-2) M Mg2+ respectively.--Ca2+ inhibits adenylcyclase activity in a non-competitive manner, irrespective of the stimulant and ion concentration used. The Mg2+-activated enzyme, however, is more susceptible to the inhibiting effect of Ca2+ than the Mn2+-activated enzyme.--It is concluded that Mn2+ and Mg2+ are allosteric effectors of the enzyme adenylcyclase, acting at a Me2+-site of the catalytic unit of adenylcyclase.


Further evidence for a change in central alpha-adrenergic receptor sensitivity after withdrawal from long-term haloperidol treatment. Phenoxybenzamine, FLA-63 and alpha-MT produced less locomotor depression in mice withdrawn for 4 days from a 21 day treatment with haloperidol than that produced in vehicle-treated animals. There were no differences between the two groups when challenged with yohimbine or phentolamine. The data support the hypothesis that central alpha-adrenergic receptors had become supersensitive and suggest that the sensitivity changes are restricted to post-synaptic receptors.


Structural and steric requirements for beta-phenethylamines as agonists of the noradrenergic cyclic AMP generating system in the rat limbic forebrain. The present studies were undertaken to assess the structural and steric requirements for beta-phenethylamines as agonists of the noradrenergic cyclic AMP generating system in slices of the rat limbic forebrain. Significant agonist activity of beta-phenethylamines requires a beta-3,4-dihydroxyphenethylamine with a beta-hydroxyl group in the R configuration. Thus, dopamine did not stimulate the system at concentrations up to 10(-3) M. Moreover, beta-hydroxyphenethylamines without a 3,4-catechol group (octopamine, phenylephrine, p-hydroxynorephedrine, metaraminol and methoxamine) - though exerting alpha-agonist activity in peripheral tissues - lack agonist activity in this particular cyclic AMP generating system. The effects of (R)-norepinephrine and (R)-isoproterenol at maximal concentrations were not additive. The results lend further support to the view that the cyclic AMP generating system in slices of the limbic forebrain is part of a norepinephrine receptor coupled adenylate cyclase system with a subpopulation of receptors that are beta in nature.


Differential effects of benzamides and thioxanthenes on dopamine-elicited accumulation of cyclic AMP in isolated rabbit retina. Benzamides or thioxanthenes were tested as potential antagonists of the cyclic AMP accumulation induced by 10(-4) M dopamine in intact rabbit retinae in vitro in the presence of 5 to 7 mM theophylline. The neuroleptic sulpiride (10(-4) M) was found to be totally inactive whereas a substituted benzamide (clebopride) had small but significant antagonist effect at the same concentration. Among thioxanthenes, isomers of cisconfiguration, which are potent neuroleptics, completely inhibit the cyclic AMP accumulation induced by dopamine, in contrast to trans-isomers which had no inhibitory effects. These data would confirm that retina in vitro is another suitable model for screening antipsychotic activity of classical neuroleptics and that the mechanism of action of sulpiride still needs further investigation.


Alpha-receptor-mediated modulation of 3H-noradrenaline release from rat brain cortex synaptosomes. The effects of oxymetazoline and noradrenaline (in the presence of desipramine) on the release of 3H-noradrenaline from rat brain cortex synaptosomes were studied using a superfusion technique. Both drugs (at 1 micrometer concentrations) were found to reduce the depolarization-induced (15 mM K+) release of 3H-noradrenaline. The release-modulating effect of noradrenaline was antagonized by phentolamine and yohimbine. The data provide direct evidence for the hypothesis that alpha-receptors modulating the release of noradrenaline are localized on varicosities of central noradrenergic neurones.


[Transformation of long reticulofugal impulse trains by interneurons monosynaptically linked to the reticulospinal tract]. Transmission of the reticulofugal activity via the interneurons localized in the ventromedial grey matter of the lumbar region and monosynaptically activated from the reticulospinal fibres was studied in anaesthetized immobilized cats. Interneurons usually generated stationary discharges when reticulospinal pathways were rhythmically stimulated with relatively low frequencies (up to 80-100/s); with further increase of stimulation frequency the discharges became non-stationary (initial high-frequency phase was followed by partial or complete discharge suppression). Maximal firing rate at the initial phase could not exceed 180-230 imp./s. The "transfer function" (the ratio of response frequency to stimulation frequency) reached 0.7-0.8 at low stimulation frequencies and decreased substantially at higher ones. Using mathematical modelling of the excitatory and post-activation inhibitory actions in these neurons, the parameters have been estimated which determine the transfer properties, of the interneuronal population relaying reticulofugal activity.


[Responses of accessory nerve nucleus motor neurons to stimulation by different sensory inputs]. Monosynaptic EPSPs in the cat accessory motoneurons were evoked by the stimulation of the cervical dorsal roots (C1 and C2). Responses of these units to the accessory nerve muscle rami stimulation were unstable and polysynaptic. It is supposed that the accessory muscles in the cat (m. sternocleidomastoideus and m. trapezius) receive proprioceptive innervation via the dorsal rami of the upper crevical nerves, and the function of accessory nerve is predominantly motor.


[Herpetic meningo-encephalitis : emergency E.E.G. diagnosis and neurosurgical treatment (author's transl)]. Reporting two cases of true herpetic encephalitis, the authors insist on the great diagnostic value of some nearly constant, but transitory, aspects of localised periodic waves in the E.E.G. An emergency cerebral (usually temporal) resection is then advised. Thus focal infection is reduced and intra-cranial hypertension is prevented. Virologic and electron microscopic studies of the cortical specimen establish with certainty the diagnosis of herpetic encephalitis.


Noradrenergic and dopaminergic regulation of GH and prolactin in baboons. Aminergic regulation of growth hormone (GH) and prolactin (Prl) was studied in male adolescent baboons by i.v. infusion of dopamine (DA), norepinephrine (NE), thyrotropin-releasing hormone (TRH), phentolamine, haloperidol, and inhibitors of DA-beta-hydroxylase and peripheral decarboxylase. 20-min infusion of DA (40 microgram/kg.min) and 60-min infusion of NE (0.4 microgram/kg.min) stimulated GH release. The DA-induced GH release was suppressed by concomitant infusion of FLA 63 (inhibitor of NE synthesis from DA) and by phentolamine, indicating alpha-adrenergic mediation of GH release. Microinfection of DA (0.8 microgram/kg) into the medial basal hypothalamus (MBH) lowered basal GH. Prl was released by i.v. TRH, and this effect was suppressed by i.v. DA but not by i.v. NE. Blockade of peripheral decarboxylase by carbidopa elicited a marked and sustained rise in Prl which was inhibited by i.v. DA. Microinjection of NE (0.8 microgram/kg) into the MBH released Prl. These data indicate that in the MBH, alpha-adrenergic mechanisms release GH and Prl, and that dopaminergic mechanisms suppres GH.


Luteinizing hormone secretion and gonadotropin releasing hormone binding in heifer and steer anterior pituitaries. Luteinizing hormone (LH) release and tissue content were studied in explanted bovine pituitaries from heifer and steer during superfusion in vitro. Compared to steer, heifer pituitaries contained significantly more LH and released more LH into the incubation medium. Quantitative response in LH release to gonadotropin releasing hormone (GnRH) stimulation was about equal in heifer and steer, but relative increase was more pronounced in steer. Specific 125I-GnRH binding to dispersed anterior pituitary (AP) cells and isolated plasma membranes was also analyzed. Concentration dependent binding curve of 125I-GnRH to dispersed cells exhibited sigmoid characteristics suggesting positive cooperativity. There was a sex dependent difference, the binding curve for heifer being shifted to the left and reaching saturation at lower GnRH concentrations than that for steer. "Multisigmoid" concentration dependent GnRH binding curves were obtained using isolated plasma membranes, revealing the presence of multiple binding sites. One of the binding sites, present in heifer, was absent in the steer derived materials, but could be induced by pretreatment of the steer plasma membranes with 17-beta estradiol as low as 50 pg/ml. It is concluded that the differences in GnRH binding may be responsible, at least in part, for the observed differences in LH secretion pattern between heifer and steer.


[Unusual clinical course in a case of carcinomatosis of cerebrospinal meninges]. The authors report the clinical course of a case of carcinomatosis of the cerebrospinal meninges with frequent sudden rises of intracranial pressure and with lethal outcome. The primary tumour was a mucinous carcinoma of the ovary. In treatment Pudenz valve was used successfully for connecting the subarachnoid space of the spinal cord with the peritoneal cavity. This shunting of the cerebrospinal fluid caused no dissemination of the neoplasm within the peritoneal cavity.


Multiple mononeuropathy in lymphomatoid granulomatosis: similarity to leprosy. Multiple mononeuropathy and anesthetic granulomatous skin lesions were the first manifestations of lymphomatoid granulomatosis in a young woman. The skin lesions resembled those of leprosy. Lymphomatoid granulomatosis is related to midline granulomas and other idiopathic granulomatous angiitic lesions.


[Specific cytoplasmic receptors for steroid homones in benign and malignant breast diseases, in normal glandular tissue and pectoral muscle]. An improvement to the DCC (destran-coated charcoal) method for determining receptors for 17-beta-oestradiol, 5-alpha-dihydrotestosterone and progesterone in benign and malignant breast conditions, normal glandular tissue and muscular tissue is reported. Preliminary results on 16 malignant cancers and 4 fibrocystic mastopathies are discussed together with a critical review of the technique employed and the results obtained in hormone competition.


Estrogen binding in mammary tissue of C3H mice with or without the mouse mammary tumor virus. Substrains of C3H mice, with and without the milk-transmitted form of the mouse mammary tumor virus (MMTV)1, were compared to determine if the presence of MMTV altered estrogen action. Uterine weight response to oral diethylstilbestrol was the same for both substrains, (MMTV+ and MMTV-). The concentration of high-affinity estrogen binding sites ("receptors") in lactating mammary tissue did not differ significantly for MMTV+ versus MMTV- mice; the affinity of the estradiol-receptor interaction appeared higher, however, in cytosol from MMTV- mice. Sucrose density gradient analysis revealed no significant differences in sedimentation properties of cytosol receptor from the two substrains.


Immunotherapy with tumor cells and BCG in the guinea pig, studied by immunological in vivo and in vitro experiments. The effect of intradermal immunisation with viable line 1 hepatoma cells and BCG was studied in syngeneic strain 2 guinea pigs. Immunisation with 1.5 X 10(6) hepatoma cells admixed to 200 microgram BCG induced tumor regression in 7/8 animals. Higher or lower doses were less effective and sometimes led to enhanced tumor growth. It appeared that regression or progression of tumors is established as early as 3 weeks after tumor inoculation. Aspects of humoral and cell-mediated tumor immunity in immunised and/or tumor-bearing animals were studied in vitro by the use of mixed cell cultures and of cytotoxicity tests. Immunised tumor-bearing animals reacted more vigorously than those from non-immunised animals when the tumor was small. With increasing tumor volume the specific tumor immunity disappeared in both immunised and non-immunised animals. Sera obtained from immunised or regressor animals were cytotoxic and partially blocked the mixed cell interaction. Sera obtained from progressor animals were neither cytotoxic and nor did they block the mixed cell interaction. Cytotoxic activities resided within immunoglobulin fractions enriched in IgG1 or IgG2, whereas blocking activity was mainly associated with IgG2. The effector cell function was not inhibited by the immunoglobulin fractions tested. In the discussion it is suggested that the ratio of IgG1 and IgG2 antibodies elicited during immune treatment may critically influence the success or failure of immunotherapy.


[Expression of paternal genes controlling cytochrome oxidase activity in hybrid fish]. The heat resistance of the oxygen consumption by the mitochondria, temperature dependence of the Michaelis' constant (CM) and heat resistance of cytochrome oxidase were studied in the embryos and larvae of fish hybrids (Misgurnus X Brachydanio). The oxygen consumption by the mitochondria from the larvae of Misgurnus ceased (following the 10 min heating) at 50 degrees, from Brachydanio at 54 degrees and from the hybrids at 52 degress suggesting control of the respiratory function. CM of cytochrome oxidase has the same minimum in the larvae of Misgurnus and Brachydanio, therefore this criterion was not used to study the genetic control in their hybrids. The heat resistance of cytochrome oxidase (T50) differed in Misgurnus and Brachydanio and was of intermediate value in their hybrids. At the early stages of hybrid development T50 was of maternal type (Misgurnus) but beginning from the mid-gastrula stage T50 increased and attained the maximum prior to the hatching. Chloramphenicol did not affect the increase of T50 in hybrids, but actinomycin decreased it almost down to the level characteristic of Misgurnus. The data obtained suggest that the genetic control of cytochrome oxidase activity begins earlier than that of other studied enzymes.


Biochemical effects of formocresol on bovine pulp tissue. Calf pulp was treated with full-strength formocresol, diluted formocresol, or saline for 4 hours. After washing and homogenization, the water extractable supernates were analyzed for total amino acid, carbohydrate, and hydroxyproline content. Additional samples were tested against trypsin, pepsin, collagenase, and hyaluronidase. Other tissue samples treated with 1/5, 1/10, and 1/25 dilutions of formocresol were subjected to trypsin and collagenase. The control tissue gave 50 per cent more extractable material, which contained over 300 per cent more total amino acids and hydroxyproline but only slightly more carbohydrate than the treated tissue. Formocresol treatment produced an 80 to 90 per cent reduction in reactivity to trypsin, pepsin, and collagenase but little change from hyaluronidase action. The increase in reactivity of the tissue to enzyme hydrolysis paralleled the increase in dilution of formocresol. These results indicate a profound effect on the protein fraction of pulp exposed to full-strength formocresol.


[Perinatal acquired cytomegalovirusinfection with following polyneuritis as a rare complication (author's transl)]. Case report of a 3 week old baby with polyneuritis caused by perinatal acquired cytomegalovirusinfection. Neurological complications are in the acquired form of cytomegalovirusinfection. In acute paralyses of unknown origin CMV-infection should also be included in diagnostic considerations in childhood.


Assessment of third trimester choline phosphotransferase activity in uncomplicated human pregnancies. CDP--choline:1,2-diglyceride choline phosphotransferase (CPT; EC 2.7.8.2) undergoes a marked surge in activity in human amniotic fluid when assayed from 30 weeks of gestation to term. The activity of this enxyme, plotted against gestational age, follows a highly significant regression correlation from which an equation can be obtained for the prediction of gestational age.


Infections and other maternal factors as risk indicators for congenital malformations: a case-control study with paired serum samples. An obstetric population of 48,000 individuals was prospectively followed up for evidence of possible teratogenic factors that might be associated with congenital malformations. Serum samples from 274 mothers of defective children and from paired controls were collected during early pregnancy and approximately one month after delivery and tested for antibodies against ten different viruses, Mycoplasma pneumoniae, and Toxoplasma. These data were supplemented with clinical information on infections, other diseases, drug intake, and other potentially teratogenic factors during pregnancy. Mothers of defective children had more seroconversions (fourfold or greater increase in titer) than the controls, 123 vs. 86. This difference was mainly due to an increase in herpes simplex viruses 1 and 2, cytomegalovirus, varicella-zoster virus, and Toxoplasma titers. In addition, the number of reported diseases during the pregnancy, the intake of drugs (especially analgesics and hormones), and the number of earlier abortions were greater in the mothers of the defective children than in the controls.


Daytime state and night-time sleep; a sleep study after a marathon group experience. The nocturnal sleep of 9 subjects was recorded under two conditions following a "normal" weekend and following a weekend in which the subjects participated in a psychodynamic marathon group activity. Differences between the two experimental conditions were found for sleep latency and on one measurement of D-latency. Both latencies were reduced after the marathon group. No differences were found on the remaining 12 measurements. These findings tend to reaffirm the stability of sleep patterns under various pre-sleep conditions.


Dissociation between antidiuretic response and renal medullary cyclic AMP levels in the rat. In vivo experiments were performed in male Wistar rats to elucidate the probable relation between renal concentrating ability and medullary cyclic AMP content as influenced by changes of hydration and by administration of antidiuretic hormone (ADH). Cyclic AMP levels were 37% lower in water diuretic than in control animals (P less than 0.01), but were not significantly changed during prolonged antidiuresis induced by dehydration or ADH administration. Nor could any change of cyclic AMP levels be demonstrated between 2 and 20 min after ADH injection. Significant increases of medullary cyclic AMP content occurred following stress, anesthesia, and administration of isoproterenol and 3-isobutyl-1-methylxanthin. The results suggest that the level of cyclic AMP in the renal medulla may not be an important determinant of the antidiuretic response produced by ADH in rats.


["Small cell" carcinoma of the lung. Results obtained using combination chemotherapy (adriamycin, vincristine, cyclophosphamide) in 38 patients (author's transl)]. A cyclic and sequential combination of adriamycin, vincristine (or VM 26) and cyclophosphamide was used, with a follow up of 4 to 33 months in 38 patients with a "micro-cellular" carcinoma of the lung. Radiotherapy was used in association in a variable manner. Chemotherapy alone resulted in marked regression of the lesions (regression is greater than 50%) in approximately 65 per cent of cases and a level of complete regression which varied from 31 to 45 per cent according to the degree of diffusion of the lesions at the time of admission. In this study, survival in apparently localised forms was 70 per cent after one year and 55 per cent at eighteen months, whilst it was 34 per cent at one year and nil at eighteen months in patients with evidence of metastatic disease at the time of entry. These encouraging results indicate a hope for improvement, in certain patients reacting favourably to this protocol, in the still grave prognosis of "small cell" carcinoma of the lung.


A comparative study of the cardiac troponin inhibitory factor (TNI) from mammalians. Troponin inhibitory factor, TNI, was prepared by affinity chromatography from different mammalian hearts. (i) Structure. These different TNI have the same M.W. (28000), which is higher than that found in rabbit skeletal muscle (23000). Nevertheless they differ with respect of their charge as shown by alkaline urea polyacrylamide gel electrophoresis using cardiac TNI which has previously been bound to an excess of skeletal troponin Ca2+-binding factor. These changes do not correlate with the PO4 content of TNI. They are associated with structural differences demonstrated by peptide mapping of the unfolded molecule after papain treatment. The structure of cardiac TNI from rat and rabbit differs clearly from that of crow and pig. (ii) Biological activity. These different TNI have the same inhibitory effect on skeletal actomyosin. ATPase, the same content of PO4 and the same ability to be phosphorylated in-vitro by a bovine heart c-AMP-dependent protein kinase.


[Epidemiology of primary bronchus cancer from which the African Negro suffers (author's transl)]. The study of 86 cases collected along 5 years regarding the primary bronchus cancer from which the African Negro in Abidjan suffers, points out the recent increase of this pathology, the predominating epidermolid histological type, the fact that men reaching a critical age of 45 years old are mostly concerned and especially those living in the rural areas. Because of the absence of industrial pollution and occupational exposure in Ivory Coast, the very important relation between bronchus carcinoma and smoking cigarettes is suggested.


Cytomegalovirus infection in malignant blood diseases:clinical and laboratory data in 29 patients. Twenty-nine patients treated for malignant blood diseases developped CMV infection. Their clinical and laboratory features were studied. The results indicated that this infection apparently did not influence the prognisis of the underlying disease. The main hematological feature was pancytopenia. The data of viremia suggested active infection. A marked increase of CMV CF antibodies were observed in 27/29 patients, and the peak titers of 42% of our cases were greater than 1:1024. Homogenous Ig were detected in 7/29 patient's serum.


Binding of phosphorylated histone H1 to DNA. A chromatin associated protein kinase was used to add 3 moles of phosphate to seryl side chains of 1 mole of histone H1. The DNA binding properties of this in vitro phosphorylated H1 were compared with those of unmodified H1. Considerably more radioactive superhelical DNA was retained on nitrocellulose filters at 20mM-40mM NaCl by phosphorylated H1 than by unmodified H1. However, zone velocity sedimentation analysis of histone-DNA complexes indicated that similar amounts of phosphorylated and unmodified H1 are bound to DNA. It is therefore concluded that phosphorylated H1 binds distributively to many or all DNA molecules available (depending on the histone/DNA ratio) while unmodified H1 binds cooperatively to a fraction of the DNA molecules in the reaction mixture.


Characterization of the mRNA for herpes simplex virus thymidine kinase by cell-free synthesis of active enzyme. The cytoplasmic mRNA which codes for the herpes simplex virus specific thymidine kinase (TK) is polyadenylated and is 14.5s in size. This corresponds to an RNA of 1400 nucleotides. The TK polypeptide is about 42,000 daltons, which requires 1100 nucleotide. We conclude that the cytoplasmic mRNA is monocistronic. The reticulocyte lysate cell-free translation system synthesizes enzymatically active HSV-TK which can be assayed with high specificity and sensitivity by use of 125I-iododeoxycytidine as a substrate.


Localization of the 5' terminus of late SV40 mRNA. A cap-containing oligonucleotide has been isolated from a T1 ribonuclease hydrolysate of total Simian Virus 40-specific late RNA and its structure has been determined as 7mG(5')ppp(5') mAmpU(m)pUp(Up,Cp)ApGp. This oligonucleotide constitutes the major 5' terminus of both late mRNA species, 16S and 19S. Assuming that viral mRNA "caps" are derived from a 5'-terminal triphosphate or diphosphate, these results mean that late transcription is (mainly) initiated at nucleotide L 308 (numbering system of Simian Virus 40 DNA, cf. Fiers et al. (1978) Nature, 273, 113-120). The cap is followed by a contiguous leader sequence which is at least 194 nucleotides long.


A rapid method for the measurement of the unwinding angle of intercalating agents and the superhelix density of circular DNAs. By incubating covalently-closed circular DNA in the presence of calf thymus topoisomerase and unwinding ligands (intercalating drugs and proteins) DNAs of different superhelix density can be produced. These changes in superhelix density can be monitored by an ethidium fluorescence assay, since the level of ethidium binding varies with the superhelix density of a DNA. Thus the equivalence point, in a titration between an unwinding ligand and a supercoiled DNA, can be found. This forms the basis for an extremely rapid method for measuring unwinding angles and superhelix densities. Results are presented which agree well with those reported by previous authors using different techniques. The present method compares very favourably with others when evaluated in terms of rapidity, cost of materials, cost of equipment, accuracy and also applicability.


Relation of cell type and cell density in tissue culture to the isoaccepting spectra of the nucleoside Q containing tRNAs: tRNATyr, tRNAHis, tRNAAsn and tRNAAsp. An examination, using reversed-phase chromatography and cyanogen bromide treatment, of tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp from SV40-transformed mouse fibroblasts grown to different cell densities, untransformed cells grown to confluence, and mouse liver indicates that: (1) The tissue cultured mouse fibroblasts examined here are hypomodified with respect to nucleoside Q, while liver tRNA is almost completely modified with respect to Q. (2) Cell density and/or proliferative state do not present as major variables in controlling the expression of Q in the present system. (3) SV40 virus transformation is not a major variable controlling the expression of Q in the present system. The present results support previous use of cyanogen bromide effected shifts in chromatographic elution as an assay for nucleoside Q.


Okazaki pieces grow opposite to the replication fork direction during simian virus 40 DNA replication. Simian virus 40 replicating DNA was pulse labeled with alpha-32P-dATP using an acellular DNA replication system. Nascent DNA chains of less than 200 nucleotides (Okazaki pieces) were then isolated from the denatured replicating DNA by electrosieving through a polyacrylamide gel column. The purified Okazaki pieces were hybridized to separated strands of Bg1(1)+Hpa1 simian virus 40 DNA restriction fragments immobilized on nitrocellulose filters. Only strands with polarity of the DNA replication fork direction hybridized with Okazaki pieces. Hence, Okazaki pieces in simian virus 40 are synthesized against the DNA replication fork direction.


Alterations in chromatin of cells infected with RNA tumor viruses. Chromatins were isolated from murine leukemia or sarcoma virus infected lymphocyte-like TB cells and compared by immunological and biochemical methods. Chromatin from virus infected cells did not fix complement as well as uninfected cell chromatin suggesting that conformational changes had occurred in chromatin from virus infected cells. This alteration was detected within 24 hours after infection. Infected cell chromatins, examined by electrophoretic methods after radiolabeling displayed alterations in nonhistone proteins, whereas the histones appeared unaltered. The non-histones were synthesized in greater amounts in infected compared to normal cells, particularly a 60,000 D protein, while the amount of histone did not vary. The above changes should not have been due to cell growth or cycle variations, for the cells had similar growth rates and were harvested from the same stage of cell confluency during exponential growth to ensure uniformity of culture conditions.


The nursing care of lung cancer patients: emphasizing chemotherapy. The experience of coping with lung cancer--from diagnosis to treatment to inevitable death--is indescribably difficult for the cancer patient. The nurse, with her basic knowledge of the disease process, diagnosis, prognosis, and treatment, and by employing her sensitivity to the patient's emotional needs, can ease the pain of this experience. The nurse must use her technical knowledge and understanding to provide the patient with the knowledge he needs to participate in his own care as well as the emotional strength to deal with the physiologic strains of the disease.


[Mediastinal surgery in children].

[Mediastinal surgery in children]. From 96 surgical interventions in the child, the authors studied the repartition of tumours according to their histological nature, their benignity or malignancy. For each kind of tumour they gave its frequency and clinical, radiological and biological data. Above all they studied the distant results of these interventions with an important set back. The results are excellent for benign tumours and promising for malignant ones and enable the deduction of therapeutical indications.


[One case of severe tracheobronchopathy with immune depression : herpetic disease?]. The authors report a case of severe obliterating laryngo-tracheo-bronchitis without any evident etiology in a young patient with depressed immunity. A herpetic virus was isolated by distal bronchial brushing and by larynx biopsy. The level of specific antibodies rose significantly. These facts together with the clinical history and unsuccessful microbiological searches, suggest the herpetic nature of this affection, albeit exceptional. At this opportunity, a review of the literature is presented showing the imprecision of diagnostic criteria in this field of pathology.


Effect of dietary aflatoxin on vitamin D3 metabolism in chicks. Male broiler chicks were fed diets containing either O (control) or 2.5 p.p.m. aflatoxin (toxin) for four weeks. A group of eight birds fed each diet was infected intravenously with radioactive vitamin D3 (D3) and a second group with radioactive 25-hydroxy vitamin D3 (25-OH D3). Plasma was obtained 24 hr. after dosing with D3 and 6 hr. after dosing with 25-OH D3. The vitamin D metabolites were extracted from the plasma an chromatographed on Sephadex LH-20 for separation. The four peaks of radioactivity separated corresponded to D3, 25-OH D3, 24,25-dihydroxy vitamin D3 (24,25-(OH)2D3) and 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3). Percentages of radiation in each peak were (1) controls given D3--6.55, 64.30, 5.94 and 4.04; (2) toxin given D3--10.05, 56.96, 8.95 and 4.68; (3) control given 25-OHD3-2.16, 85.80, 4.11 and 2.04; and (4) toxin given 25-OH D3--1.53, 79.84, 5.56 and 2.14. The only significant differences between the control and toxin groups were in D3 and 24,25-(OH)2D3 in chicks given D3. Even these changes were small and the data would suggest that feeding 2.5 p.p.m. aflatoxin for four weeks does not greatly alter vitamin D metabolism.


Vaccination immunity to selected diseases in chickens fed the androgen analog mibolerone. Chickens fed the androgen analog mibolerone during the first 7 weeks of life regress their bursa of Fabricius but can be properly immunized by vaccination against avian pathogens of major economic importance such as Newcastle disease virus, infectious laryngotracheitis virus, avian encephalomyelitis virus, infectious bronchitis virus, fowl pox virus, Marek's disease virus, and Pasteurella multocida, the pathogen causing fowl cholera. These findings on immunocompetence to infectious agents are important because we have previously shown that the administration of mibolerone prevents the development of lymphoid leukosis tumors.


Differentiation of progressive versus regressive Rous virus-induced avian sarcomas according to tumor and infiltrating lymphocyte fine structure. Electron microscope examination of progressing and regressing Rous virus-induced sarcomas in an inbred line of White Leghorns revealed that regressing tumors contained moderate to marked lymphocyte infiltration, frequent contact between lymphocytes and tumor cells, and extensive areas of necrosis. Lymphocytes infiltrating regressing tumors exhibited a polar accumulation of organelles at the point of contact between lymphocyte and target cell. On the other hand, progressing tumors contained low to moderate numbers of lymphocytes, infrequent lymphocyte-tumor cell interaction and less evidence of tumor cell degeneration. Lymphocytes from progressing tumors lacked the polar organization of organelles. This experimental system is offered as a means of studying the role of lymphocytes in tumor regression.


Lymphocytotoxicity of chickens bearing Rous sarcomas. A 51Cr uptake microcytotoxicity assay was used to determine the cytotoxicity of lymphocytes from chickens bearing Rous sarcomas. Differences in lymphocyte cytotoxicity among individual chickens were statistically significant. Lymphocytes from chickens regressing (or having regressed) their tumors were significantly more cytotoxic than lymphocytes from hosts with progressing tumors. Lymphocytes from chickens with progressing tumors tended to enhance tumor cell growth in vitro.


Vaccination against infectious bronchitis and the immunosuppressive effects of infectious bursal disease. An immunosuppressive effect was demonstrated in chickens which were infected with infectious bursal disease virus (IBDV) early in life and prior to or shortly after vaccination with infectious bronchitis virus (IBV). This effect was evident by an increased susceptibility to respiratory tract infection with IBV and reduced virus-serum neutralizing antibody levels. Chickens which were hatched from dams susceptible to infectious bursal disease (IBD) were less responsive to IBV immunization attempts, if exposed to IBDV, than were those individuals hatched from IBD immune dams. However, in some cases, chickens from IBD immune dams were also more susceptible to IBV challenge when they had been exposed to IBDV and when compared to birds unexposed to IBDV but vaccinated against IB. An effect of cyclophosphamide on the bursa of Fabricius also had an immunosuppressive action on IBV immunity which was similar to the results from IBDV exposure. The data engendered from these trials may explain the unsatisfactory immunity sometimes observed under field conditions when broilers and replacement pullets are vaccinated at an early age.


Monensin, Eimeria tenella infection, and effects on the bacterial populations in the ceca of gnotobiotic chickens. Bacteria-free chicks in separate plastic film isolators were inoculated orally with single species of bacteria. Within an isolator, half the birds were fed unmedicated feed and half received feed containing 100 ppm monensin. With clostridium perfringens as the established species of monoflora, bacterial counts from the duodenum were 10(4) times lower and counts from the ceca were three times lower in monensin-fed birds compared to unmedicated birds. Infection with Eimeria tenella stimulated an eight-fold increase in the numbers of C. perfringens in the ceca of unmedicated birds but no increase in monensin-fed birds. With Bacteroides sp. or Streptococcus faecalis as monoflora, there was no difference in the cecal or duodenal populations between medicated and unmedicated birds uninfected with coccidia. In contrast to C. perfringens, populations of Bacteroides sp. and S. faecalis in the ceca decreased five to 100-fold in both medicated and unmedicated chicks after infection with E. tenella. Duodenal populations of C. perfringens, Bacteroides sp., and S. faecalis were unaffected by the coccidial infection.


A comparative pathogenesis of two Mareks disease virus isolates. The pathogenesis of the JM and GA isolates of Marek's disease virus (MDV) infection was compared by studying the sequential tissue distribution of MDV associated antigens with gross and microscopic lesions in infected chickens. With both MDV isolates the initial sites of viral replication were the lymphoid organs, skin, and featers. Both the GA and JM isolates were viscerotropic as demonstrated by the presence of MDV antigens with gross and microscopic changes occurring in several visceral organs. The JM isolate was more neurotropic and had a greater affinity for the gonads than the GA isolates as evidenced by an early detection of MDV antigens and severe pathology in the peripheral nerves and gonads.


[Effect of amino acids on the beta-aspartokinase activity from Corynebacterium glutamicum of wild and mutant strains]. The effect of amino acids, their structural analogs and ammonium sulphate on the enzyme activity of beta-aspartokinase isolated from Corynebacterium glutamicum was studied. Two strains were used: one of the wild type and the other--a mutant strain whose growth remains uninhibited by a simultaneous addition of threonine and S-2-aminoethylcysteine, a lysine analog. Significant differences in the effect of amino acids and their analogs on the beta-aspartokinase activity of the parental and mutant strains were detected. These findings suggest that the lysine supersynthesis by the mutant strain of Corynebacterium glutamicum occurs due to genetically induced changes of allosteric properties of the mutant enzyme.


[Separation and purification of isoenzymes of Clostridium perfringens phospholipase C]. The procedure for separation and purification of isoenzymes of phospholipase C from Clostridium perfringens (PLC) was developed. The procedure included primary concentration of culture liquid proteins and isoenzyme separtion on DEAE-cellulose during negative sorption of the major isoenzyme. Further purification of the isoenzymes was achieved by (NH4)2SO4 fractionation by Sephadex gel-filtration and isoelectric focusing. During polyacrylamide gel electrophoresis and precipitation reaction in the agar with the antiperfringens hyperimmune serum alpha1-PLC preparations were homogenous. Their coefficient of sedimentation was 3.8 S and isoelectric point was 5.50. The minor isoenzyme alpha2-PLC had the same coefficient of sedimentation and the isoelectric point of 5.35.


[Effect of removal and stimulation of the superior cervical sympathetic ganglia on the activity of oxidation-reduction enzymes in the neurosecretory cells of the anterior hypothalamus in rabbits]. Experiments were conducted on rabbits. A study was made of the activity of the redox enzymes--glucose-6-phosphate dehydrogenase (G-6-PDH), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), NAD-and NADP-diaphorases, cytochromeoxidase (CCO), alpha-glycerophosphate dehydrogenase (alpha-GPDH) in the supraoptic and paraventricular nuclei of the hypothalamus and the posterior lobe of the hypophysis under conditions of stimulation and removal of the superior cervical sympathetic ganglia. There was revealed a correlation between the activity of the tissue respiration enzymes (SDH, MDH, NAD- and NADP-diaphorase, CCO) and the functional condition of the hypothalamo-neurohypophysial neurosecretory system. However, the enzymes of the pentose-phosphate (G-6-PDH) and glycerophosphate shunt (alpha-GPDH) and also of the anaerobic way of oxidation (LDH) reacted nonspecifically on the induced effects.


[Relationship between the hypophyseal-adrenal and the genital systems in silver-black foxes]. Interaction between the hypophysial-adrenal system (HAS) and the hypophysis-gonad system was studied. For this purpose the foxes were subjected to total gonadectomy 8 months before they were sacrificed. Functional activity of the HAS was judged by the hypophysis ACTH content, corticosteroid production by the adrenal cortex in vitro, and by the glucocorticoid concentration in the peripheral blood. Castration of male foxes foxes failed to influence the activity of various HAS links. Gonadectomy of females was accompanied by changes in the activity of individual HAS links in different direction--some reduction of ACTH in the hypophysis, a sharp and significant fall of the peripheral blood glucocorticoid level and a marked significant elevation of hydrococortisone production in the adrenal cortex in vitro.


Direct C-1 hydroxylation of vitamin D compounds: convenient preparation of 1alpha-hydroxyvitamin D3, 1alpha, 25-dihydroxyvitamin D3, and 1alpha-hydroxyvitamin D2. An efficient procedure for the direct C-1 hydroxylation of vitamin D compounds has been developed. The method involves conversion of vitamin D3 tosylates to 3,5-cyclovitamin D derivatives, allylic oxidation with selenium dioxide, and acid-catalyzed solvolysis to the 1 alpha-hydroxyvitamin D analogs. When applied to vitamin D3,25-hydroxyvitamin D3, and vitamin D2, this sequence give the corresponding 1alpha-hydroxylated derivatives in 10-15% yield.


Regulation of cholesterol biosynthesis in HeLa S3G cells by serum lipoproteins: dexamethasone-mediated interference with suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Depression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [mevalonate:NADP(+) oxidoreductase (CoA-acylating); EC 1.1.1.34] was elicited by the removal of serum from the growth medium of HeLa S3G cells with a concomitant expected increase in cellular sterol biosynthesis; if dexamethasone (9alpha-fluoro-11beta,17alpha,21-trihydroxy-16alpha-methyl-1, 4-pregnadiene-3,20-dione) was present in the serumless medium, there was an augmentation of HMG-CoA reductase activity but a suppression of sterol biosynthesis. When human serum, human low density lipoprotein, or calf serum was present in the medium, there was a reduction of both the enzyme activity and sterol biosynthesis, but the presence of dexamethasone resulted in an increase in HMG-CoA reductase activity as compared to the controls containing human serum, low density lipoprotein, or calf serum alone. In contrast, either low density lipoprotein or whole serum supplementation eliminated the differences in acetate incorporation into sterols between glucocorticoid-treated and untreated cells. Human high density lipoproteins had little effect on the enzyme activity and abolished the difference in sterol biosynthesis only at relatively high concentrations. Addition of low density lipoproteins to cells after preincubation in serumless medium elicited the same rate of decay of HMG-CoA reductase (t(1/2) 3.8-4.2 hr) regardless of the presence of glucocorticoids in the medium, but there was an exaggerated lag before the onset of suppression in the hormone-treated cells. If free cholesterol was present in the medium, the dexamethasone augmentation of HMG-CoA reductase was maintained, but the addition of either 7-ketocholesterol or 25-hydroxycholesterol abolished the difference between glucocorticoid-treated and control cells. These observations suggest that, under certain physiological conditions, HMG-CoA reductase activity no longer accurately reflects cellular sterol biosynthesis.


Mapping of linear and circular forms of mouse mammary tumor virus DNA with restriction endonucleases: evidence for a large specific deletion occurring at high frequency during circularization. Rat hepatoma cells infected with mouse mammary tumor virus contain multiple forms of unintegrated viral DNA when grown in the presence of glucocorticoids. Using the DNA transfer procedure of Southern, we have prepared restriction endonuclease fragment maps of these forms of viral DNA. The maps indicate that: (i) the major species of viral DNA is a linear molecule of 5.9 X 10(6) Mr located in the cytoplasm; (ii) the nuclei contain covalently closed circular viral DNA of two distinct sizes (5.1 X 10(6) and 5.9 X 10(6) Mr) in addition to linear molecules (5.9 X 10(6) Mr); (iii) the linear molecule has specific termini; (iv) there is extensive homology between regions at or near termini of the linear molecule; (v) the predominant form of circular DNA lacks 1.2 kilobase pairs present in both the larger circular molecule and the linear molecule; and (vi) the sequences deleted from the majority of the circular DNA molecules are located at the ends of the linear DNA that are joined during circularization.


Transduction of a bacterial gene into mammalian cells. The transduction of an Escherichia coli gene into mammalian cells is described. A supressor tRNA gene was linked to a simian virus 40 (SV40) vector in vitro and the recombinant was used to transfect rat embryo cells and monkey kidney cells. The hybrid SV40 genome, SV40-su+ III, retained genetic information required for autonomous replication and cellular transformation and had a 1300-base-pair DNA segment in the late gene region (between the restriction endonuclease sits Hpa II at 0.735 and EcoRI at 0/1.0 on the SV40 genetic map) replaced by an 870-base-pair bacterial DNA segment containing the suppressor tRNA gene, su+ III (tRNATyrsu+III). The structure and fate of the SV40-su+III chimera were determined by DNA reassociation kinetic analysis and restriction enzyme cleavage of the total cellular DNA from transformed rat embryo cells and persistently infected monkey cells. Hybridization with radiolabeled probes specific for vector (SV40) or su+III DNA sequences revealed primarily nonintegrated or free hybrid genomes. In cloned lines of both cell types, the bacterial DNA segment was recovered intact, as judged by the length of the segment excised by restriction endonucleases and its ability to hybridize to the radiolabeled bacterial DNA probe and not to the SV40 probe.


Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin. Highly fluorescent analogs of insulin and epidermal growth factor were prepared by the covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The alpha-lactalbumin was specifically linked to the lysine residue of insulin or to the alpha-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptor-hormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormone-receptor complexes can move laterally in the plane of the plasma membrane with a diffusion coefficient of (3-5) X 10(-10) cm2/sec.


Prolyl hydroxylase half reaction: peptidyl prolyl-independent decarboxylation of alpha-ketoglutarate. Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) is a mixed-function oxygenase that hydroxylates peptidyl proline with the simultaneous and stoichiometric decarboxylation of alpha-ketoglutarate to succinate and CO2. It has been found that highly purified preparations of the enzyme can decarboxylate alpha-ketoglutarate in the absence of a peptidyl proline substrate. The uncoupled decarboxylation proceeds at only a fraction of the rate of the whole reaction and for study requires substrate quantities of the pure enzyme, as well as oxygen, ferrous ion, and ascorbate. No hydroxyproline is formed under these conditions. Immobilized antiserum to prolyl hydroxylase was found to remove both activities from enzyme preparations. However, addition of free antiserum during incubation inhibits only the complete reaction. Poly(L-proline), a specific inhibitor of prolyl hydroxylation, enhances the uncoupled decarboxylation of alpha-ketoglutarate without itself being hydroxylated. All of these findings prove that alpha-ketoglutarate can serve as substrate in the absence of peptidyl proline and is most likely the initial site of attack by oxygen. In the coupled reaction an oxidized form of the keto acid, perhaps a peroxy acid, then attacks prolyl residues in the unhydroxylated substrate.


BK virus DNA: cleavage map and sequence analysis. A detailed physical map of the BK virus (MM strain) genome has been constructed with respect to the cleavage sites of 11 different restriction enzymes. The enzymes cut BKV(MM) DNA at 61 specific sites whose locations have been determined. Preliminary nucleotide sequence was carried out in the region from 0.70-0.75 map positions on BKV(MM) DNA. An 80% homology was found at 0.714-0.744 map positions on BKV(MM) DNA with 0.722-0.752 map positions on simian virus 40 DNA. This region of simian virus 40 DNA codes for the synthesis of the leader sequence of late mRNA.


Nucleotide sequence of the simian virus 40 small-t gene. The nucleotide sequence of the segment of simian virus 40 DNA between standard map positions 0.53 and 0.65, i.e., approximately half of the restriction fragment Hind A, is reported. This segment is located near the beginning of the early region and is transcribed counterclockwise. There is a potential initiating ATG signal at 13 nucleotides from the Hind C-Hind A junction in the strand with the same polarity as the early mRNA. From this signal on, an open reading frame is present which would allow the synthesis of a polypeptide of 174 amino acids until a TAA termination codon is reached at nucleotide 602 (map position 0.547). This polypeptide, revealed by the DNA sequence, corresponds almost certainly to small-t antigen. Correlation of the deduced amino acid sequence with the NH(2)-terminal sequences of small-t and large-T (tumor) antigens of simian virus 40, as established by Paucha et al. [Paucha, E., Mellor, A., Harvey, R., Smith, A. E., Hewick, R. M. & Waterfield, M. D. (1978) [Proc. Natl. Acad. Sci. USA 75, 2165-2169], strongly argues that both proteins are indeed initiated at the ATG triplet. Because the DNA region between 0.547 and 0.534 is blocked for translation in all three reading frames by multiple termination condons, we conclude that the large-T antigen must be coded for by two noncontiguous DNA segments: the segment from 0.65 to around 0.60, which small-t and large-T antigens share, and another segment starting at some point after position 0.534 and continuing counterclockwise until it terminates at map position 0.174. Small-t antigen is methionine-rich and has a remarkably high number of cysteine residues clustered mainly in its COOH-terminal half. It is rich in both basic and acidic residues, the former being slightly in excess.


Large and small tumor antigens from simian virus 40 have identical amino termini mapping at 0.65 map units. Large and small tumor (T)antigens of simian virus 40 were synthesized in vitro with L-cell extracts that had been treated by the method of Palmiter to prevent amino-terminal acetylation of nascent proteins. Partial amino-terminal amino acid sequences of both forms of T-antigen were determined and found to be identical. Methionine residues were located at positions 1 and 14, a lysine residue at position 3, and leucine residues at positions 5, 11, 13,16, 17, and 19. These amino acid sequence data match perfectly the amino acid sequence predicted from a sequence of nucleotides in the E strand of simian virus 40 DNA which begins near the junction between HindII/III fragments A and C at about 0.65 map units. This strongly suggests that the sequence coding for the amino terminus of both proteins is located at this position. Furthermore, the data are consistent with a model for the synthesis of both forms of T-antigen that predicts that (i) small T-antigen is coded for by a sequence of nucleotides from the 5' end of the early region and (ii) large T-antigen is coded for by nucleotide sequences from two noncontiguous regions of simian virus 40 DNA.


Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome. DNA from simian virus 40 (SV40) was prepared for local mutagenesis by nicking the molecule at a specific site with a restriction endonuclease that recognizes one site in SV40 DNA and then extending the nick enzymatically to expose a short, single-stranded segment of DNA. The "gapped" DNA was treated with a single-strand-specific mutagen, sodium bisulfite, which converts cytosine to uracil. After mutagenesis, the gap was repaired with DNA polymerase, generating molecules resistant to the restriction enzyme used to make the initial nick. From cells infected with DNA thus modified, SV40 mutants were isolated that had enzyme-resistant genomes. In some cases, precise positions of G.C to A.T transitions could be inferred from the patterns of susceptibility of mutant DNA to other restriction endonucleases whose recognition sequences were altered by the mutagenesis procedure. One of the restriction endonuclease sites mutagenized (Bgl I) maps at the origin of SV40 DNA replication and near sequences corresponding to the 5' ends of viral mRNAs. Many of the resulting Bgl I-resistant mutants yielded small plaques, suggesting partial defectiveness in DNA replication or transcription.


Binding and kinetic data for rabbit liver fructose-1,6-bisphosphatase with Zn2+ as cofactor. Atomic absorption determinations of zinc content were employed to demonstrate the technique to obtain zinc-free rabbit liver fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11). Reactivation of the apoenzyme by Zn(2+) is rapid (within 1 min) and restores up to 96% of the initial specific activity. Gel filtration measurements showed that the enzyme contains four binding sites for zinc per molecule, one per subunit. The dissociation constants for the initial two binding sites are less than 0.1 muM. In the presence of a substrate analog, (alpha + beta) methyl D-fructofuranoside 1,6-bisphosphate, at a level where two analog molecules are bound per phosphatase molecule, a total of eight Zn(2+) ions bind at 8 muM Zn(2+), revealing the presence of additional binding sites, including the catalytic one. The activity in the presence of Zn(2+) is maximal at ca. 8 muM Zn(2+), which corresponds to saturation of the four subunit sites plus the catalytic sites in the presence of substrate. At metal ion concentrations less than 10 muM, the order of activation is Zn(2+) > Mn(2+) > Mg(2+). In kinetic assays with two metal cofactors the effect of Zn(2+) at concentrations less than 10 muM on either the Mg(2+) or the Mn(2+) assays is inhibitory owing to the apparent formation of mixed (two different elements) metal ion-enzyme complexes possessing a catalytic activity that is measureable but lower than anticipated if the catalysis by the various metal ions is simply additive. Hence the activation by EDTA of the Mg(2+) and Mn(2+) assays is explicable in terms of Zn(2+) removal, thus eliminating mixed metal species. Collectively these observations suggest that fructose-1,6-bisphosphatase may function in vivo as a Zn(2+) metalloprotein.


Adenosine 5'-O-([gamma-18O]gamma-thio)triphosphate chiral at the gamma-phosphorus: stereochemical consequences of reactions catalyzed by pyruvate kinase, glycerol kinase, and hexokinase. The 2-[18O]phosphorothioate of D-glycerate, chiral at phosphorus, was prepared. The chiral phosphoryl group was transferred enzymically to ADP [by using enolase and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase; EC 2.7.1.40)] resulting in the synthesis of adenosine 5'-O-([gamma-18O],gamma-thio)triphosphate. This labeled ATP was used as a thiophosphoryl group donor in the reactions catalyzed by glycerol kinase (ATP:glycerol 3-phosphotransferase; EC 2.7.1.30) and by hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1). The product from the latter (glucose 6-phosphorothioate) was converted enzymically into glycerol phosphorothioate. Determination of the relative configurations and diastereoisomeric purities of the samples of glycerol phosphorothioate demonstrates that all three phosphokinases (pyruvate kinase, glycerol kinase, and hexokinase) transfer the thiophosphoryl group with complete stereospecificity, and further shows that these reactions follow an identical stereochemical course.


Recognition site of Escherichia coli B restriction enzyme on phi XsB1 and simian virus 40 DNAs: an interrupted sequence. Methyl groups placed on varphiXsB1 replicative form DNA by the Escherichia coli B modification enzyme are located in the overlap between fragments Mbo II-3 and Alu I-2, a 61-base-pair DNA segment. Mutations that led to loss of susceptibility to restriction by E. coli B occurred within this segment at three positions spanning 14 nucleotides. A sequence difference between varphiXsB1 and varphiXam3cs70, a varphiX174 strain not restricted by E. coli B, occurs at one of these positions. The site on simian virus 40 DNA methylated by the modification enzyme is located in the 115-base-pair overlap between fragments Hae III-I and Alu I-G. The sequences of these segments of varphiXsB1 and simian virus 40 DNA and two regions of phage f1 DNA recognized by the E. coli B restriction enzyme [Ravetch, J. V., Horiuchi, K. & Zinder, N. D. (1978) Proc. Natl. Acad. Sci. USA 75, 2266-2270] contain a homology of nine bases in the configuration:5'-T-G-A... 8N... T-G-C-T... 9N... T-N-N-T-3'. The sequence 5'-T-G-A... 8N... T-G-C-T-3' may constitute the restriction enzyme recognition site since it does not occur in varphiXam3cs70 DNA and occurs only once in simian virus 40 DNA, and since all observed mutations leading to loss of the site occur at one of the bases specified by this sequence. Analysis of the sequence of varphiXam3cs70 showed that if no other residues are recognized, all seven of these bases are essential for recognition and the interval between the two groups of specified bases must be precisely eight.


Identification of the Mtv-2 gene responsible for the early appearance of mammary tumors in the GR mouse by nucleic acid hybridization. In the mouse strain GR, the Mtv-2 gene controls the expression of large amounts of mammary tumor virus (MTV) antigens in the milk at first lactation. It also controls the early appearance of mammary tumors. We have investigated the number of MTV proviral sequences associated with this Mtv-2 gene by nucleic acid hybridization between MTV [(3)H]cDNA and DNA from GR, B10, and GR-Mtv-2(-) mice. B10 and GR-Mtv-2(-) mice lack Mtv-2 gene expression. The molecular hybridizations revealed that the DNA of GR mice contains 12 copies of MTV proviral sequences, whereas only 4 copies are present in the DNA of B10 and GR-Mtv-2(-) mice. We therefore conclude that the Mtv-2 gene in the GR mouse strain is associated with eight additional MTV proviral sequences. The four Mtv proviral sequences in the GR-Mtv-2(-) DNA might represent another Mtv gene in the GR mouse. Different amounts of MTV RNA are detected in mammary glands at first lactation of B10 and GR-Mtv-2(-) mice, even though both contain four copies of MTV proviral sequences. This indicates a difference between these two mouse strains either in the regulation of expression of these MTV proviral sequences or in the location of these sequences in the murine genome.


Ultraviolet reactivation of herpes simplex virus is mutagenic and inducible in mammlian cells. The survival of UV-irradiated herpes simplex virus on UV-irradiated Vero cells was increased over that on unirradiated cells. A time period between irradiation of the host cells and infection with virus was needed to achieve maximum reactivation. In parallel experiments in which the frequencies of occurrence of the forward mutation in the thymidine kinase gene of the virus were measured, growth of herpes simplex virus on UV-irradiated cells yielded progeny virus that had higher frequencies of TK- mutants than did progeny from infections of control cells. The time course of development of this mutagenic effect was the same as that for the development of the UV-reactivation capacity. Furthermore, development of the UV reactivation could be blocked by inhibition of protein synthesis. These results suggest that an "error prone" inducible UV-reactivation phenomenon exists in mammalian cells.


Homologous genes for enolase, phosphogluconate dehydrogenase, phosphoglucomutase, and adenylate kinase are syntenic on mouse chromosome 4 and human chromosome 1p. It is possible to generate interspecific somatic cell hybrids that preferentially segregate mouse chromosomes, thus making possible mapping of mouse genes. Therefore, comparison of the linkage relationships of homologous genes in man and mouse is now possible. Chinese hamster x mouse somatic cell hybrids segregating mouse chromosomes were tested for the expression of mouse enolase (ENO-1; EC 4.2.1.11, McKusick no. 17245), 6-phosphogluconate dehydrogenase [PGD; EC 1.1.1.44, McKusick no. 17220], phosphoglucomutase-2 (PGM-2; EC 2.7.5.1, McKusick no. 17190), and adenylate kinase-2 (AK-2; EC 2.7.4.3, McKusick no. 10302). In man, genes coding for the homologous forms of these enzymes have been assigned to the short arm of human chromosome 1. Analysis of 41 primary, independent, hybrid clones indicated that, in the mouse, ENO-1 and AK-2 are syntenic with PGD and PGM-2 and therefore can be assigned to mouse chromosome 4. In contrast, they were asyntenic with 21 other enzymes including mouse dipeptidase-1 (DIP-1, human PEP-C; EC 3.4.11.(*), McKusick no. 17000) assigned to human chromosome arm 1q and mouse chromosome 1. Karyologic analysis confirmed this assignment. These data demonstrate that a large autosomal region (21 map units in the mouse and 51 map units in the human male) has been conserved in the evolution of mouse chromosome 4 and the short arm of human chromosome 1. Identification of such conserved regions will contribute to our understanding of the evolution of the mammalian genome and could suggest gene location by homology mapping.


Restricted classes of immunoglobulin produced by a lymphoid cell line from a patient with agammaglobulinemia. Restricted expression of immunoglobulin by a long-term B lymphoid cell line derived from a patient with X-linked agammaglobulinemia is reproted. The patient had peripheral blood B lymphocytes with surface IgD and IgM. Culture of the B cells in vitro with mitogens did not stimulate immunoglobulin secretion, although pokeweed mitogen stimulation resulted in the development of cytoplasmic IgD and IgM. The lymphoid cell line established from these B lymphocytes primarily bore surface IgD, with a small population of cells also bearing surface IgM. These cells also had cytoplasmic immunoglobulin, primarily IgD. The cell line did not have a cytoplasmic pool of immunoglobulin for export and did not secrete immunoglobulin. This B lymphoid cell line is an in vitro analogue of the arrest in differentiation of the patient's B lymphocytes. The IgD-bearing phenotype may represent a normal transitional stage in the differentiation of B lymphocytes.


Type C RNA virus-specific antibody in human systemic lupus erythematosus demonstrated by enzymoimmunoassay. Postmortem study of proliferative glomerulonephritis associated with human systemic lumpus has previously shown that an antigen related to mammalian type C RNA viral core (p30) proteins is deposited in the renal glomerular lesions with human immunoglobulins in an immune-complex pattern. In the present work, human immunoglobulins were sequentially eluted from the lupus glomerular immune deposits and were assayed by a sensitive enzymoimmunoassay developed for the measurement of anti-p30 antibody activity against purified viral p30 proteins of mammalian type C viruses. Human immunoglobulins showing specific anti-p30 antibody activity, particularly against p30 antigen of feline endogenous virus RD-114 and to a smaller extent against p30 antigen of murine type C virus, were eluted by acid buffer from the glomerular immune deposits in two patients with lupus proliferative glomerulonephritis who have deposits of viral p30-related antigen in the same tissue lesions. This study adds support for the hypothesis that expression of type C viral antigen may be involved in the multifactorial pathogenesis of proliferative glomerulonephritis associated with human systemic lupus.


Generation of oncogenic type C viruses: rapidly leukemogenic viruses derived from C3H mouse cells in vivo and in vitro. A type C virus was isolated from C3H/10T1/2 mouse cells in culture after activation with iododeoxyuridine. This virus was poorly infectious for mouse cells and did not cause tumors upon inoculation into newborn NIH Swiss mice. Variants with increased infectivity for mouse cells were then derived both in vivo and in vitro by selecting for variants able to grow to high titers. The highly infectious variants were found to induce mouse fibroblasts to grow in soft agar. When the viruses were inoculated into newborn NIH Swiss mice, 100% of the animals died of leukemia within 4 months. Solid tumors developed at the injection site. Both mouse-tropic and dualtropic viruses were isolated from the leukemic mice and plaque purified. The first group of viruses produced large syncytial plaques on rat XC cells and did not grow in mink cells. The viruses of the other group replicated well in both mouse and mink cells, producing morphologic changes similar to transformation but not XC syncytia; they, therefore, are members of the newly described MCF class of mouse type C viruses. Isolates from either group were highly leukemogenic on retesting, the mean latent period being 67 days for a mouse-tropic virus and 105 days for one of the dual-tropic viruses. The results led to the conclusion that the better a mouse type C virus grows in cell culture the more effective it is as a leukemogen. Further, it is possible to start with a weakly infectious, nonleukemogenic virus and to convert it to a rapidly replicating, highly leukemogenic virus by passage either in cell culture or in the animal. The availability of a defined series of viruses from a low-leukemia mouse strain that differ greatly in their biologic properties should facilitate studies of the molecular basis for the acquisition of type C virus oncogenicity.


New region of the simian virus 40 genome required for efficient viral transformation. Viable mutants of simian virus 40 with deletions in three regions of the virus genome (map coordinates 0.21-0.17, 0.59-0.54, and 0.67-0.74) have been tested for their ability to transform rat fibroblasts to anchorage independence. Only those mutants whose deletions occur between 0.59 and 0.55 in the proximal part of the early region are defective in transforming ability. The most severely defective of these transform with less than one-hundredth the efficiency of wild type. They retain their defect when tested in Chinese hamster lung cells and when infection is initiated with viral DNA instead of intact virions. Complementation for transformation can be observed between these transformation-defective deletions and a simian virus 40 temperature-sensitive A mutant.


Identification of an Abelson murine leukemia virus-encoded protein present in transformed fibroblast and lymphoid cells. Extracts from lymphoid and fibroblast cell lines transformed by Abelson murine leukemia virus (A-MuLV) contain a protein of molecular weight 120,000 (P120). Immunoprecipitation with specific sera shows that P120 contains regions homologous to the 5'-terminal segment of the MULV gag gene complex--p15, p12, and at least part of p30--but lacks detectable determinants of p10, reverse transcriptase, and the envelope glycoprotein. P120 is phosphorylated and has an intracellular half-life of 3--6 hr. In vitro translation of virion RNA from A-MuLV, with Moloney MuLV as helper, yields a product of molecular weight 120,000 with serological reactivity similar to that of the cellular P120. Translation of the RNA from the helper gave no P120. P120 is expressed in all lymphoid and fibroblastic cell lines we have tested that were transformed by A-MuLV but is not detectable in a lymphoid line in which the A-MuLV genome was established by infection but was not responsible for the transformation. Expression of P120 is selectively retained in clones of A-MuLV-transformed lymphocytes that convert to a nonproducer state after loss of expression of helper MuLV intracellular precursors. These results suggest that the P120 product of the A-MuLV genome may be responsible for maintenance of the transformed phenotype of lymphoid and fibroblast cells transformed by the virus.


Quantitative visualization of gamma-aminobutyric acid receptors in hippocampus and area dentata demonstrated by [3H]muscimol autoradiography. Muscimol, a potent gamma-aminobutyric acid (GABA) agonist, was used in a radioactively labeled form for the quantitative localization of GABA receptors in the rat's hippocampus (CA(1) to CA(4)) and area dentata. [(3)H]Muscimol was injected directly in vivo or used in the incubation medium of tissue slices, and the tissues were then fixed and prepared for autoradiography. [(3)H]Muscimol-bound GABA receptors are weakly though evenly distributed over the fimbria of the fornix. There was a laminar distribution in CA(1) to CA(4) and the area dentata, with an increasing density of the GABA receptors in that order. The lowest density was found in the alveus of CA(1) and CA(2) and the highest in the stratum granulosum of the area dentata. The greatest density was found in the neuropil between granule cells, in which are found dendrites and the basket-like plexuses of the inhibitory GABA-containing local circuit neurons. The molecular layers of the area dentata, CA(1), and CA(2) also have a high density of GABA receptors, indicating a probable distribution over the dendrites of granule and pyramidal cells. The laminar distribution of GABA receptors in the hippocampus and area dentata is similar to the distributions of the GABA-synthesizing enzyme, glutamate decarboxylase, and of GABA previously published. Neurons with label of various density are found in the polymorphic cell layer of the area dentata, in the stratum radiatum of CA(3), and in CA(4). These are possibly the GABA-containing basket local circuit neurons.


ACTH induced sodium appetite in the rat. Subcutaneous injections of long-acting synthetic ACTH (5 U/day) caused a large increase in the intakes of both 0.5 M NaCl and water in rats. By the fifth day of treatment the rats were turning over an amount of sodium approximating their own total body sodium. The mineral appetite was specific for NaCl. Intakes of KCl, MgCl2 and CaCl2 were unchanged. ACTH was ineffective in adrenalectomized rats suggesting that the appetite was dependent on adrenal hormones.