Modulation of transmission at an inhibitory synapse in the central nervous system of the leech. The synaptic interactions among a group of cells in the leech C.N.S. that regulate the animal's heartbeat exhibit several remarkable features (Thompson & Stent, 1976 a, b, c). We have examined in detail the properties of the inhibitory synapse between two of these cells, the heart interneurone (HN cell) and the heart excitor motoneurone (HE cell). 1. Impulses in the presynaptic HN cell gave rise to monosynaptic i.p.s.p.s in the HE cell that were blocked by high concentrations of Mg and were reversed when the membrane potential of the post-synaptic motoneurone was hyperpolarized beyond--75 m V or when Cl was injected into the cell body. These i.p.s.p.s were chemically mediated, and involved an increase in chloride conductance. 2. In contrast to chemical synapses between sensory and motor cells in the leech C.N.S., little facilitation or depression of transmission occurred when the HN cell was stimulated at frequencies of 0.1--50 Hz. 3. Steady subthreshold depolarization of the presynaptic HN interneurone evoked a maintained hyperpolarization of the post-synaptic HE cell, indicating that currents injected into the HN cell body could spread to the terminals and cause continuous release of transmitter. 4. The size of the i.p.s.p. evoked in the HE motoneurone by an action potential in the HN interneurone varied with the resting membrane potential of the presynaptic cell. An impulse superimposed on a prolonged, subthreshold, depolarizing pulse produced a larger i.p.s.p.; conversely, prolonged hyperpolarization of the HN interneurone reduced the i.p.s.p. amplitude recorded in the HE cell. This effect was most obvious when the natural, rhythmical bursts of activity in the HN interneurone were interrupted by bathing the preparation in leech Ringer fluid containing elevated concentrations of Mg. Under these conditions a 10 mV depolarization of the HN cell increased the size of the i.p.s.p. in the HE cell approximately sixfold. Significant changes in i.p.s.p. amplitude occurred without any noticeable change in the amplitude and duration of the presynaptic action potential. With large presynaptic depolarizations, which produced the biggest i.p.s.p.s, there was some reduction in the amplitude and increase in the duration of the action potential. 5. Following a step depolarization of the presynaptic cell, the size of successive i.p.s.p.s increased with a time constant of about 1 sec. Upon repolarization the i.p.s.p.s decreased in amplitude to the original level. 6. stimulation of one HN cell also gives rise to an i.p.s.p. in its contralateral homologue (Thompson & Stent, 1976c). Trains of i.p.s.p.s produced in this way hyperpolarized at HN cell to such an extent that the size of the synaptic potential it evoked in an HE cell was reduced. 7. Thus, an HN interneurone inhibitis transmission between the contralateral HN and HE cells presynapitcally in addition to inhibiting directly the ipsilateral HE motoneurone.


Quantal analysis of transmitter release at an inhibitory synapse in the central nervous system of the leech. The quantal nature of transmitter release has been analysed at central inhibitory synapses in the leech nervous system between an interneurone (HN) and a motoneurone (HE) that regulate the heartbeat. 1. Ganglia were bathed in leech Ringer fluid containing 20 mM-Mg and 1.8 mM-Ca and the membrane of the presynaptic HN interneurone was hyperpolarized by current injection. Under these conditions successive inhibitory potentials in the HE motoneurone, evoked by impulses in the HN interneurone, showed striking fluctuations in amplitude. 2. Assuming a Poisson distribution of the i.p.s.p.s and estimating the number of failures from the amplitude histograms of the observed responses, the mean size of the quantal unit was estimated as 0.25 +/- 0.015 mV (S.E. of mean, n = 26). When m, the mean number of quanta released per trial, was varied by changing the membrane potential of the presynaptic HN cell (Nicholls & Wallace, 1978), the experimentally observed amplitude distributions could be predicted by the Poisson theory. 3. An independent estimate of the unit size was obtained by noise analysis. A long subthreshold depolarizing pulse applied to the presynaptic HN interneurone evoked a sustained hyperpolarization of the HE motoneurone, apparently caused by an increase in the rate of on-going release of quanta by the HN cell terminals. From the mean change in membrane potential and the increase in variance, the size of the unit was calculated as 0.21 +/- 0.039 mV (S.E. of mean, n = 11). For ten pairs of cells an estimate of unit amplitude was made both from the Poisson analysis and the analysis of variance, again with good agreement. For these cells the estimated unit sizes were 0.24 +/- 0.023 mV (S.E. of mean, n = 10) from the failures and 0.21 +/- 0.043 m V (S.E. of mean, n = 10) from the noise. 4. A similar analysis was made of the inhibitory synaptic potentials evoked in one HN interneurone by stimulation of its contralateral homologue. Transmission again appeared to be qualtal; the mean unit amplitude from Poisson analysis was 0.31 +/- 0.022 mV (S.E. of mean, n = 19) and from the noise 0.29 +/- 0.027 mV (S.E. of mean, n = 3). 5. We conclude that transmitter is released from the terminals of the HN interneurone in quantal units that evoke miniature i.p.s.p.s of about 0.25 mV in the post-synaptic cells. Furthermore, modulation of transmission proudced by variation in the presynaptic resting potential and during presynaptic inhibition results from changes in the mean number of quanta released by each impulse.


An alpha-adrenergic receptor mechanism controlling potassium permeability in the rat lacrimal gland acinar cell. 1. Rat lacrimal gland slices, incubated in a balanced, buffered salt solution, were found to be physiologically stable for up to 2 hr with respect to O2 consumption, extracellular space, and water and ion content. 2. The release of 86Rb serves as a good substitute for 42K in monitoring the movement of K through the cell membrane. 3. Adrenaline appears to increase membrane permeability to K as evidenced by an increase in the rate of 86Rb efflux. 4. This response to adrenaline was blocked by phentolamine but not by propranolol and was mimicked by phenylephrine but not by isoprenaline. 5. The magnitude of the 86Rb release indicates that it is being released, at least in part, from the lacrimal gland acinar cell. 6. It is concluded that the lacrimal gland acinar cell has an alpha-adrenergic receptor, activation of which leads to an increase in membrane permeability to K.


The role of calcium in the receptor mediated control of potassium permeability in the rat lacrimal gland. 1. In the presence of extracellular Ca, adrenaline stimulated a large increase in the rate of K (86Rb) release from rat lacrimal slices, followed by a lower, more sustained rate. 2. In the absence of extracellular Ca, adrenaline elicited only a transient release of 86Rb. 3. The artificial introduction of Ca into the cytosol by the ionophore A-23187 could also initiate the release of 86Rb. 4. In a zero-Ca medium, if either adrenaline or carbachol produced a transient release of 86Rb, the tissue could not respond to the other agonist with a transient release unless Ca was momentarily reintroduced to the medium. 5. If Ca was present in a limiting concentration, the Ca-dependent rate of 86Rb release elicited from a lacrimal slice exposed simultaneously to carbachol and adrenaline was not significantly different from the release seen with carbachol alone. 6. It is concluded that the agonist-induced release of K from the lacrimal gland consists of both a Ca-independent phase which is initiated by the release of a limited pool of Ca, and a Ca-dependent phase which is mediated by the influx of extracellular Ca. 7. It is also concluded that both alpha-adrenergic and muscarinic receptor occupation activate a common, post-receptor mechanism which may be responsible for both phases of K release.


Role of calcium in the fade of the potassium release response in the rat parotid gland. 1. The 86Rb release response in the parotid due to alpha-adrenergic (epinephrine), muscarinic (carbachol) or peptide (Substance P) receptor activation exhibited 'fade': a return of efflux to control levels despite the continuing presence of agonist. 2. The time course of fade of the response to all three agonists was independent of the concentration of the agonist. 3. After fade was fully developed to one agonist, the response to an agonist acting on a different receptor was either absent or greatly diminished. 4. Removal of carbachol from muscarinic receptors with atropine 10 min prior to Substance P partially restored the ability of Substance P to produce a response. 5. Fade of the response with all three agonists was greatly retarded by the omission of Ca. 6. release of alpha-amylase did not appear to fade following exposure to carbachol or Substance P. 7. It is concluded that the K+ release response may be inactivated with time due to diminution in responsiveness of the K+ channel to an increase in internal Ca2+.


A voltage-clamp study of the permeability change induced by quanta of transmitter at the mouse end-plate. 1. Miniature end-plate currents (m.e.p.c.s) were recorded from mouse diaphragm using a point voltage-clamp. The relation between m.e.p.c. amplitude and membrane potential was determined in bathing solutions of varied composition. 2. In solution containing normal sodium the relation between m.e.p.c. height and membrane potential (Im.e.p.c./Vm relation) was always linear, at least in the range +30 to -100 mV; the reversal potential (Vr) at which Im.e.p.c. was zero was close to 0. The slope of the Im.e.p.c./Vm line varied little between junctions (coefficient of variation about 20%) and was about 50 nS, or 1nA per 20 mV. The Im.e.p.c./Vm relation was not altered by withdrawal of Ca2+, addition of ethanol, or substitution of NO-3 or SO2-(4) for Cl-. 3. Alteration of K+ concentration in the bathing medium, in the range 10 to 1 mM, had no apparent effect on the Im.e.p.c./Vm relation. 4. Reduction of Na+ concentration, with isosmotic substitution of sucrose, caused rapid alteration of the Im.e.p.c./Vm relation, which became rectifying, with a slope at negative Vm less than at positive Vm. Vr was shifted in the negative direction. Quantitatively these changes were close to those predicted by the Goldman-Hodgkin-Katz formulation for permeation of monovalent ions through a membrane with constant field. 5. In solution with low Na+ (2 mM) and partial substitution of K+ for Na+, the Im.e.p.c./Vm relation was indistinguishable from that in solutions with Na" as the predominant extracellular cation. With complete substitution of K+ for Na+ the Im.e.p.c./Vm relation was a little less steep (at negative Vm) than in Na+ solution and Vr was shifted slightly in the negative direction. 6. With substitution of NH+4 for Na+, the Im.e.p.c./Vm relation was little changed (about 10% steeper at negative Vm). With substitution of Li+ for Na+, the Im.e.p.c./Vm relation remained linear, but was made less steep, at positive as well as negative Vm, and Vr was shifted slightly in the positive direction. 7. These results indicate that the permeability change associated with generation of the m.e.p.c. (i.e., evoked by a quantum of transmitter) corresponds to the opening of a single species of membrane channel that allows the free movement of K+, Na+, NH+4, AND Li+ ions along their electrochemical gradients. The channel discriminates little between these ions. The apparent order of permeability is Li+ greater than NH+4 greater than Na+ greater than or equal to K+. The apparent permeability per channel corresponds to that expected for channels of about 6.4 A diameter, 100 A length, and ionic mobility the same as in dilute solution.


Multiple innervation of normal and re-innervated parasympathetic neurones in the frog cardiac ganglion. 1. Multiple innervation of parasympathetic neurones was examined in normal and re-innervated frog cardiac ganglia. The number of synaptic inputs impinging upon individual ganglion cells was determined by recording intracellularly and stimulating the vagosympathetic nerves. 2. In unoperated cardiac ganglia most neurones (93%) received a large, suprathreshold synaptic input. Some ganglion cells received additional, small synaptic inputs. Roughly equal numbers of cells encountered were singly and doubly innervated, and only 8% received more than two inputs. 3. Re-innervation of cardiac ganglion cells began three weeks after bilateral crush of the vagosympathetic nerves. By 7 weeks more than 90% of the ganglion cells were re-innervated. At this stage the pattern of multiple innervation was significantly different than normal: doubly innervated neurones outnumbered singly innervated ones, and 31% of the cells encountered received more than two inputs. This pattern was stable for at least a year. 4. These results indicate that polyneuronal innervation of cardiac ganglion cells is more widespread after re-innervation than it is normally and, furthermore, that synapse elimination does not occur during re-innervation of these cells.


A follow-up of self-poisoned patients.

A follow-up of self-poisoned patients. Over half the patients who were admitted to hospital after self-poisoning had seen their general practitioner in the previous month, and in half of those discharged from hospital no definite after-care arrangements were made. Opportunities for prevention of occurrences are being missed. Thorough follow-up of 135 such patients identified many non-medical problems, and over 80 per cent were helped by appropriate referral. Although only five of these patients made a further attempt during the period of review, the method of selection of patients does not allow definite conclusions to be made.


[Virus diarrhea in calves in South Africa]. The importance of diarrhoea in calves is briefly discussed. The role of viruses as primary invaders of the intestinal epithelium is stressed. The pathogenesis and problems encountered in diagnosing virus associated diarrhoea as well as the morphology of rota and coranavirus are discussed. Possible methods of prevention and treatment are briefly mentioned.


Cytogenetic diagnosis of cancer: abnormalities of chromosomes and polyploid levels in the bone marrow of patients with small cell anaplastic carcinoma of the lung. The chromosomes of metastatic cells and polyploid levels in the bone marrow of 26 patients with small cell anaplastic carcinoma were studied by direct bone marrow preparation and trypsin-Giemsa banding. Eighteen of these patients had received no tumor therapy and 8 had had chemotherapy and/or radiation therapy; 18 patients, including 5 who had received therapy, had karyotypic abnormalities with or without elevation of the polyploid level. Modal numbers and chromosome abnormalities were highly variable in treated and untreated patients. Modes ranged from hypodiploid to polyploid, but polyploid modes were the most frequently observed abnormal modes. Polyploid modes were not seen, however, in post-therapy patients with the exception of one who had received radiation therapy to the mediastinum for only 4 days prior to withdrawal of the specimen for chromosome analysis. Ten patients had elevated polyploid levels that ranged from 4.24 to 44.8% and always occurred in conjunction with karyotypic abnormalities. Both aneusomy (abnormal number) of normal chromosomes and structural aberrations (markers) occurred frequently. Some markers were consistent within an individual, but other variable aberrations were also typically present. Very few markers were common to 2 or more patients. The no. 1 chromosome participated in marker formation in 14 of the 18 patients with karyotypic abnormalities. Of the 26 patients, 5 were negative for metastasis to the marrow by pathologic examination but positive by cytogenetic diagnosis, whereas none were positive by pathologic examination and negative by cytogenetic diagnosis; this demonstrated that cytogenetics may be used as a rapid adjunct diagnostic procedure for the detection of metastasis in the marrow.


Nucleases and adenosine 3',5'-cyclic monophosphate phosphodiesterase activities in murine sarcoma virus (Moloney)-infected mice. To provide information on the role of nucleases in oncogenic virus infection, the activities of 3'-nucleotide phosphodiesterase (3'-NPDase), 5'-nucleotide phosphodiesterase (5'-NPDase), acid deoxyribonuclease (DNase II), and 3',5'-cyclic AMP phosphodiesterase (cAMPDase) in spleen extracts of murine sarcoma virus-infected C57BL/6 inbred mice were studied. At the peak of tumor growth and of the cell-mediated cytotoxic response (CMC) against tumor-associated antigens, 3'-NPDase, 5'-NPDase, and DNase II all showed depressed activities in the spleen, whereas the activity of cAMPDase in the spleen increased at the peak of CMC and remained elevated thereafter. Serum enzyme activities of the infected mice were also determined, and only 3'-NPD-ase in serum correlated well with CMC. Inasmuch as the correlation of the tumor growth with CMC was established in this system, further study on tumors with variance between CMC and growth is necessary to determine if serum 3'-NPDase is a useful biochemical marker for CMC in vivo.


Natural transmission of gibbon leukemia virus. Gibbon leukemia virus can infect prenatal gibbons through in utero infection or postnatal gibbons through contact transmission. The transmission of infectious virus was from viremic gibbons and not from uninfected or antibody-positive animals. The two modes of transmission could be distinguished by the amount of proviral DNA integrated into the muscle tissue of viremic gibbons. Muscle of gibbons infected postnatally had little or no proviral DNA, whereas gibbons infected prenatally had a large quantity of proviral DNA.


Genetic analyses of differences in incidence of mammary tumors and reticulum cell neoplasms with the use of recombinant inbred lines of mice. The influence of genes, in addition to genes in the H-2 complex, that effect the genesis of mammary tumors was studied. The recombinant inbred (RI) CXB lines were chosen for this investigation, because they are well suited for the study of the genetics of a trait for which the genotype affects probability of phenotype expression and which therefore is measured as incidence. Females of seven RI lines (CXBD, CXBE, CXBG, CXBH, CXBI, CXBJ, and CXBK) and their progenitor strains C57BL/6By (B6) and BALB/cBy (BALB/c) were given ip injections of MuMTV at 3 months of age and were force bred. They were observed for mammary tumors. The B6 strain was least susceptible, and mammary tumors appeared late in life. The BALB/c strain was most susceptible, and the tumors appeared early in life. The course of tumor development in the RI lines fell between these extremes. The RI strain distribution pattern of mammary tumor incidence indicated that at least one and probably several loci in addition to those at H-2 determined the difference between the BALB/c and B6 strains. Effects of the other gene(s) appeared to be even more important than those of H-2. The locations of those loci were not made clear by this study. The spontaneous incidence of reticulum cell neoplasms was also recorded. The most frequently formed neoplasm of the reticular system was a Hodgkin's-like lesion. The data suggested an influence of the H-2 complex.


Antibody-dependent lysis of tumor cells in vivo. II. Elimination of chromium-51 as a measurement of cytolysis. The effect of antibody on tumor cells was studied in vivo by measurement of the rate of elimination of 51Cr in A/J mice inoculated with labeled Ehrlich tumor cells. Mice receiving ip injections of tumor cells and normal serum eliminated 51Cr rapidly during the first 24 hours and at a much slower rate thereafter. This biphasic pattern of elimination was due to the fact that 51Cr predominantly labels small (less than 13,000 mol wt) intracellular molecules, which the mouse rapidly eliminates, whereas larger labeled molecules are eliminated slowly. Antibody to tumor cells significantly accelerated the elimination of 51Cr at concentrations that regularly suppressed tumor growth. Antibody also induced a faster elimination rate in mice treated with cobra venom factor but not in mice treated with silica or inoculated sc with the tumor cells. Unlabeled tumor cells inhibited antibody-induced 51Cr clearance in normal mice but not in proteose peptone-treated mice. These results suggested that peritoneal cells are required in the induction of antibody-dependent cytolysis in vivo. In addition, actively or passively alloimmunized mice exhibited a similar accelerated 51Cr elimination rate when inoculated with the appropriate labeled target cells.


Particle-enhanced membrane uptake of a polynuclear aromatic hydrocarbon: a possible role in cocarcinogenesis. One possible mechanism by which cocarcinogenesis occurs was investigated. The effect of a particulate, silica, upon the rate of membrane uptake of a polynuclear aromatic hydrocarbon, benz[a]anthracene (BA), was studied. The fluorescence emission spectrum and quantum yield of BA, when adsorbed to silica, underwent spectral shifts upon addition of phospholipid vesicles. These spectral changes appeared to result from the transfer of BA from the silica surface into the lipid bilayer. We used these spectral changes to measure the rates of membrane uptake of BA from the silica-adsorbed state and from the crystalline and microcrystalline states of BA. Our studies demonstrated that adsorption of BA to silica resulted in an increased rate of uptake of BA into dipalmitoyl-L-alpha-phosphatidylcholine vesicles compared to the uptake rate into these vesicles from crystalling states. Such particle-enhanced membrane uptake of chemical carcinogens may be of importance in explaining the enhanced carcinogenicity of the polynuclear aromatic hydrocarbons in the presence of particulate matter.


Cytogenetic studies of a simian virus 40 temperature-sensitive mutant-transformed Chinese hamster cell clone at the permissive and nonpermissive temperature. A clone of Chinese hamster embryo cells transformed by tsA58, the temperature-sensitive mutant of simian virus 40, was analyzed for chromosome abnormalities at the permissive temperature (37 degrees C) and nonpermissive temperature (40.5 degrees C). Trypsin-Giemsa-banded metaphases were analyzed 1 week after the temperature shift. The metaphases from cells at both temperatures were pseudodiploid, with numerous chromosome changes primarily affecting chromosomes no. 1 and 2. Other chromosomes (no. 6, 11, and the X) were also frequently involved. A marker chromosome, LM, was present in 35% of the cells at 40.5 degrees C.


Isolation of two human tumor epithelial cell lines from solid breast carcinomas. Most of the available human breast tumor cell lines have been derived from pleural effusions. The two cell lines herein described, BT-474 and BT-483, were derived from solid, invasive ductal breast carcinomas. Both are epithelial and neoplastic as judged by their general morphology, their fine structure, and their ability to produce growing nodules in nude mice and colonies in soft agar and methocel. BT-474 and BT-483 are human as expressed by chromosome morphology and aneuploid with a modal number of 55 and 72 chromosomes, respectively. Trypsin-Giemsa banding did not reveal the presence of obvious HeLa markers, and the glucose 6-phosphate dehydrogenase electrophoretic migration pattern was of the B-type. Furthermore, the migration of lactic dehydrogenase, malic dehydrogenase, and 6-phosphogluconate dehydrogenase isoenzymes was consistent with a human pattern and different from that of the mouse, rat, or hamster. Quarterly tests to detect the presence of aerobic and anaerobic mycoplasmas were repeatedly negative. A culture medium containing insulin, increased amounts of amino acids, vitamins, and glucose facilitated the isolation of the tumor cells. Cell replication was maintained with 10% fetal calf serum absorbed with activated charcoal and dextran. No production of alpha-lactalbumin was detected by radioimmunoassays, but high levels of progesterone receptors were found in both cell lines.


Colony formation by simian virus 40-transformed human parapharyngeal cells cultured in semisolid agar. A simian virus 40 (SV40)-transformed line of human parapharyngeal cells (SV-TGo) was cultured in semisolid agar to determine its ability to grow in the absence of an anchoring substratum and to evaluate any phenotypic changes that might have resulted during the isolation of sublines specifically selected for anchorage independence. After 2--3 weeks and 14--15 population doublings in culture, SV-TGo plated with over 1,000% higher efficiency than negative controls (F2408 cells). Sublines, 0.3--2.0 mm in diameter, were isolated and transferred to Leighton tubes in which they underwent an additional 0--7 divisions before senescence after 39--44 total population doublings. Subline phenotype was identical to the original parental phenotype, including epithelioid morphology, organized pattern of growth, extreme sensitivity to density-dependent inhibition of growth, and continuous production of infectious SV40 as detected by the combined tests of cocultivation and direct isolation. Limited division potential was within the range observed for the parental line. The ability to grow in agar without identifiable phenotypic changes was therefore confirmed for this line of SV40-transformed human epithelioid cells.


Suppression of the hypothalamic-pituitary-adrenal axis after oral hydrocortisone succinate ingestion in rats. Groups of Holtzman female rats were fed 10 mg/day of hydrocortisone succinate orally to study the responsiveness of the hypothalamic-pituitary-adrenal axis to acute stress. Pituitary ACTH content, plasma ACTH, adrenal venous corticosterone, and adrenal weights were studied simultaneously in experimental and control rats before, during, and up to two weeks after oral hydrocortisone administration. There was a significant decrease in pituitary ACTH content (p=<0.001), suppression of plasma ACTH and corticosterone in response to acute stress (p=<0.001), and adrenal atrophy during and following oral hydrocortisone administration. After discontinuing the hydrocortisone it required three to five days for the rats to respond adequately to acute stress. However, it was seven to ten days post-hydrocortisone before plasma ACTH and corticosterone responses to acute stress had returned to basal values, but decreased pituitary ACTH content and partial adrenal atrophy continued throughout the ten-day post-hydrocortisone study interval. Recovering from the suppressive effects of oral hydrocortisone was more rapid than following parenteral hydrocortisone. However, oral hydrocortisone causes identical but less sustained suppression of the hypothalamic-pituitary-adrenal axis as observed in animals treated with parenteral glucocorticoid preparations.


Endocrine function in a patient with asymmetrical acral hypertrophy and giantism: a possible variant of the Kippel-Trenaunay syndrome. Endocrine function was evaluated in a 38-year-old man who had patchy asymmetrical acral hypertrophy and giantism. The history and clinical manifestations were consistent with previously described cases of the Klippel-Trenaunay syndrome. Pituitary and peripheral hormone concentrations were generally elevated, but his endocrine status appeared normal by clinical evaluation. Of particular interest were elevated growth hormone and somatomedin-A concentrations and responses to provocative tests. These findings suggest that this patient had abnormal cell receptor pathophysiology as the cause of the asymmetrical acral hypertrophy and giantism, which often occurred in the same anatomical site.


env Gene of chicken RNA tumor viruses: extent of conservation in cellular and viral genomes. The env gene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode env; our work exploited the availability of radioactive DNA (cDNA(gp)) complementary to most or all of env. env sequences were detectable in the DNAs of chickens which synthesized an env gene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no env gene product (chick helper factor negative). env sequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral env only in chicken DNA contrasts sharply with the demonstration that the transforming gene (src) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London) 260: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNA(gp) and chicken DNA and also between cDNA(gp) and RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNA(gp) did not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the env genes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin.


Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus. Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.


Collaborative complementation study of temperature-sensitive mutants of herpes simplex virus types 1 and 2. Twenty-three complementation groups of herpes simplex virus type 1 (HSV-1) and 20 of HSV-2 were identified by qualitative and quantitative complementation analysis from among 43 temperature-sensitive (ts) mutants of HSV-1 and 29 ts mutants of HSV-2 which had been isolated independently in 10 laboratories.


Timing of some of the molecular events required for cell fusion induced by herpes simplex virus type 1. The timing of some of the molecular events that are required for cell fusion was investigated. Cell fusion was produced by a mutant of herpes simplex virus type 1 that causes extensive cell fusion during infection. The timing of molecular events required for fusion was established by the use of blocking agents. Phosphonoacetic acid blocks viral DNA synthesis; actinomycin D blocks RNA synthesis; cycloheximide blocks protein synthesis; 2-deoxyglucose blocks glycosylation of glycoproteins; high temperature, NH(4)Cl, and adamantanone block unknown steps required for cell fusion. For cells infected at a low multiplicity of infection, phosphonoacetic acid decreased the rate but not the final amount of fusion, but at a multiplicity of infection of 10 it had no effect on the rate of cell fusion. RNA synthesis was required for fusion until 4 h after infection, protein synthesis until 5.5 h after infection, and glycosylation until 7 h after infection. The temperature-dependent step occurred before 6 h after infection, whereas NH(4)Cl and adamantanone acted at steps that occurred until 8 h after infection. Cycloheximide, temperature, NH(4)Cl, and adamantanone acted reversibly; actinomycin D and 2-deoxyglucose acted irreversibly. The same order of action of the inhibitors was also determined by using pairs of inhibitors sequentially. These experiments also indicated that the fusion factor was not an alpha-polypeptide. Virus growth and cell fusion were both found to be highly dependent on temperature in the range of 30 to 40 degrees C. Wild-type infections are apparently characterized by the presence of a fusion factor and a fusion inhibitor. The fusion-blocking agents were added to wild-type-infected cells under a variety of conditions in an attempt to selectively block the production of the fusion inhibitor molecule and thereby cause extensive cell fusion. However, fusion was not observed in any of these experiments.


Involvement of mouse mammary tumor virus in spontaneous and hormone-induced mammary tumors in low-mammary-tumor mouse strains. The involvement of the mouse mammary tumor virus (MTV) in spontaneous and hormone-induced mammary tumors in low-mammary-tumor mouse strains was studied by comparing the amounts of MTV RNA and MTV DNA sequences in mammary tumors and other tissues of mice with an without hormonal treatments. The following results were obtained. (i) Mammary tumors which appeared in C3H mice as a result of an infection with MTV contained more MTV DNA compared with noninfected organs; these mammary tumors also contained more MTV RNA than was present in lactating mammary gland cells. (ii) Hormonal stimulation by administration of excessive amounts of prolactin via hypophyseal isografts in C3Hf and O20 mice resulted in an increased expression of MTV RNA in the mammary glands. This elevated level of MTV RNA expression was, however, not maintained in the hormone-induced mammary tumors. (iii) Spontaneous mammary tumors in BALB/c mice contained similar levels of MTV DNA and MTV RNA sequences as were found in other cells of these animals.


Rebinding of transcriptase components (L and NS proteins) to the nucleocapsid template of vesicular stomatitis virus. The L and NS proteins of vesicular stomatitis virions (New Jersey serotype) were solubilized with Triton X-100 and high-salt buffer and recombined with purified nucleocapsids under conditions similar to those used to reconstitute transcriptase activity in vitro. The nucleocapsid-bound L and NS proteins were separated from unbound proteins on a glycerol gradient. The rebinding of L and NS proteins mimics the in vivo binding in that at saturation the ratio of L and NS molecules to N molecules is approximately the same as observed in the intact virion. L and NS proteins were separated and added back independently and in combination to the template. The purified NS protein bound to the template in the absence of L protein. However, the L protein binding appeared to depend on the presence of NS protein. The presence of Mg2+ and nucleotides, which is required for transcription, was not necessary for the rebinding of L and NS proteins.


Poliovirus single-stranded RNA and double-stranded RNA: differential infectivity in enucleate cells. The ability of poliovirus virion RNA and double-stranded RNA (replicative form) to replicate in enucleate mouse L cells was investigated. Virion RNA replicated successfully in the absence of the cell nucleus, whereas replicative form infection did not produce any detectable progeny in enucleate cells. The results provide direct evidence of a nuclear requirement early in the infection initiated by replicative form RNA.


Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines. The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.


Intracellular restriction of ecotropic murine leukemia virus in rat NRK cells and its abolishment by adaptation. Ecotropic murine leukemia viruses, both N-tropic FN-2 (purified helper component of Friend leukemia virus) and B-tropic WNB-2 (purified WN1802B BALB/c-derived endogenous virus), were partially restricted in rat NRK cells. In NRK cells, they produced obscure small plaques at reduced efficiencies relative to their plaque-producing efficiencies in mouse SC-1 cells (10-fold for FN-2 and 100-fold for WNB-2). After three or four passages in NRK cells, the plaquing efficiencies of the viruses in NRK cells increased to levels close to their efficiencies in mouse cells, and the plaques in NRK cells became larger and clearer. The adaptation was more complete with FN-2 than with WNB-2. The adaptation was not due to simple selection of a virus in the FN-2 stock, but was host induced, as the viruses had been submitted to successive limiting dilutions in SC-1 cells before propagation in NRK cells. Possible commitment of xenotropic virus in the adaptation was excluded. The change was stable, even if the adapted viruses were propagated back into SC-1 cells. The NRK-adapted viruses were restricted in other rat cell lines of different origins, and the virus adapted in another rat cell line, RFL, was still restricted in NRK cells. The adaptation was mainly brought about by increased viral growth within the rat cells and not by an increased efficiency of viral penetration into the rat cells. This inversely suggests that the restriction of the ecotropic murine leukemia viruses in NRK cells was a mainly intracellular event. The mobilities of gp69/71 and p30 in sodium dodecyl sulfatepolyacrylamide gel electrophoresis remained unchanged after adaptation of FN-2 in NRK cells.


Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected wtih herpes simplex virus type 1. We describe experiments which demonstrate that shortly after infection of Friend erythroleukemia cells with herpes simplex virus (HSV), polyribosomes dissociate and cellular mRNA degrades. Analysis of infected cell extracts on sucrose density gradients demonstrates that the majority of the polyribosomes have dissociated to monoribosomes at 2 h postinfection. Physical measurements of infected-cell RNAs support this conclusion and demonstrate that the polyadenylated RNAs decrease in size. The degradation of mRNA is apparently a stochastic process as judged by the failure to detect a shift in the Crt1/2 when polyadenylated RNA extracted from infected cells at different times is hybridized to globin complementary DNA. In experiments designed to determine whether dissociation of polyribosomes is sufficient to cause degradation of globin mRNA, the amount of globin mRNA in uninfected cells did not change when cells were treated with NaF or pactamycin at concentrations sufficient to dissociate all polyribosomes. In cells infected with UV-irradiated virus polyribosomes dissociate but globin mRNA does not degrade, suggesting that it is possible to separate dissociation from degradation.


Comparison of initiation rates of encephalomyocarditis virus and host protein synthesis in infected cells. The relative initiation rates for encephalomyocarditis virus mRNA and host mRNA's in infected cells were measured using two independent techniques. In both cases the results showed that viral mRNA initiates at a much higher rate than host mRNA'S. This difference was observed midway in the infectious cycle, well before virus-induced cytopathic effects (leakage of low-molecular-weight metabolites, failure to exclude trypan blue) were apparent. These results confirm that encephalomyocarditis viral mRNA is a more efficient initiator than host mRNA's in vivo, as has previously been demonstrated in in vitro experiments.


Specificity of interferon action in protein synthesis. Inhibitors of elongation steps in protein synthesis such as cycloheximide and anisomycin mimic interferon treatment in that they specifically inhibit the synthesis of certain viral proteins. These specific effects are seen only at very low concentrations of the antibiotics, under conditions where host cellular protein synthesis, as well as cell viability, are not severely reduced. A qualitatively as well as quantitatively close correlation between the effects of the two types of agents has been established for encephalomyocarditis virus, vesicular stomatitis virus and murine leukemia virus protein synthesis. It is concluded that one of the primary mechanisms of interferon action may be a nonspecific retardation of one or more elongation steps, and that this may be sufficient to account for its effects on the replication of certain viruses such as encephalomyocarditis and vesicular stomatitis viruses.


Characterization of the env gene in avian oncoviruses by heteroduplex mapping. The genome of ring-necked pheasant virus, an avian oncovirus, is largely homologous to the genomes of chicken oncoviruses except for a specific nonhomology in env, the gene coding for the surface glycoprotein of the virion (J. Tal, D. J. Fujita, S. Kawai, H. E. Varmus, and J. M. Bishop, J. Virol. 21:497--505, 1977). We have used this nonhomology between ring-necked pheasant virus and chicken oncoviruses in electron microscopic studies of heteroduplex molecules. The env-specific region of nonhomology is 1.5 to 1.7 kilobases in length. Its 3' boundary is located 0.6 to 0.7 kilobases from the 3' end of the genome in transformation-defective viruses and 2.5 kilobases from the 3' end in nondefective avian sarcoma viruses. Comparison of several strains of avian oncoviruses shows that the 3' half of this env region is conserved, while the 5' half is more diverged. A small area at the very 3' end of env also shows divergence between different avian oncoviruses. We found no evidence for the presence of a previously unrecognized gene between env and src. An electrophoretic comparison of the glycoproteins from various avian oncoviruses shows that those of ring-necked pheasant virus and Chinese quail virus differ in molecular weight from the glycoproteins of the chicken oncoviruses.


Strain-specific markers for the major structural proteins of highly oncogenic murine mammary tumor viruses by tryptic peptide analyses. Tryptic peptide analyses were performed on the major structural 52,000- and 36,000-dalton glycoproteins (gp52 and gp36-38) and the nonglycosylated 28,000-, 14,000-, and 10,000-dalton proteins (p28, p14, and p10) of the highly oncogenic murine mammary tumor viruses (MMTVs) of C3H, RIII, and GR mice, i.e., MMTV(C3H), MMTV(RIII), and MMTV(GR), respectively. Each virus was grown in both murine and feline cells to ensure the virus-coded nature of each peptide analyzed. The gp36-38 peptide maps of all three MMTVs were indistinguishable, as were the p14 maps of the different MMTVs. Both the p28 and the gp52 of MMTV(C3H), however, could be clearly distinguished from the corresponding proteins of MMTV(RIII) and MMTV(GR), regardless of whether the viruses were grown in feline or murine cells. The p1o of MMTV(RIII) was clearly different from that of MMTV(C3H) and MMTV(GR). Therefore, tryptic peptide analysis of three proteins, gp52, p28, and p10, can serve to distinguish these three viruses from one another. These studies further characterize the heterogeneity in polypeptides among MMTVs.


Cleavage map of BK virus DNA with restriction endonucleases MboI and HaeIII. Specific cleavage of BK virus (MM) DNA with restriction endonuclease MboI gives rise to 10 fragments. Two techniques were used to determine the location of these fragments on the viral genome with respect to the three known sites for HindIII cleavage. In the first method, reciprocal digestion, individual MboI fragments were digested with HindIII and individual HindIII fragments were digested with MboI. In the second method, single-end 32P-labeled HindIII subfragments were partially digested with MboI, and then the sizes of the radioactive partial products were used to deduce the nearest neighboring fragment. Information from these two methods is more than adequate to map all the MboI enzyme sites. Cleavage of BK virus (MM) DNA with restriction enzyme HaeIII produces 21 fragments. With the aid of the same two methods, these fragments have also been ordered with respect to the known map locations of the HindIII and MboI sites.


Identification of a herpesvirus isolated from cytomegalovirus-transformed human cells. Human cells transformed by cytomegalovirus and transplanted to athymic nude mice yielded a cytopathic virus, Hershey Medical Center virus, following prolonged in vitro passage of the tumor cells. The virus is a double-enveloped herpesvirus, is sensitive to ether, and is inhibited by iododeoxyuridine. No significant antigenic relationship to herpes simplex virus was detected using herpes simplex virus-immune sera in neutralization and immunofluorescence tests, but indirect immunofluorescence tests revealed cytomegalovirus-related antigenicity. Further immunological tests revealed that Hershey Medical Center virus is antigenically indistinguishable from infectious bovine rhinotracheitis virus. Thus, it appears that Hershey Medical Center virus is infectious bovine rhinotracheitis virus, which presumably appeared in the cell culture as a contaminant from fetal calf serum.


Viral gene expression in murine sarcoma virus(murine leukemia virus)-infected cells. NIH 3T3 cells infected with Moloney murine sarcoma virus (murine leukemia virus) produce virions which contain about 99% murine sarcoma virus RNA and 1% murine leukemia virus RNA. This report describes experiments which measured intracellular concentrations of proviral DNA and RNA transcripts for each of the viruses. We found that three to four copies of proviral DNA from each virus were integrated into the cellular DNA. Measurements of RNA specific for each of the genomes by hybridization to specific cDNA reagents revealed a 10- to 15-fold difference in concentration in both nuclear and polysomal RNA fractions, with murine sarcoma virus RNA predominating in both cases. Unless there are major differences in stability between the two viral RNAs, our results suggest that transcriptional control is responsible for much of the difference in final levels of virus synthesis.


In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome. Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase "large fragment" as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.


Phosphorylation of chromatin- and ribosome-associated proteins in cells transformed by adenovirus, murine sarcoma virus, and methylcholanthrene. Endogenous protein phosphorylation in chromatin and ribosomes of monkey, mouse, and rat cells transformed by DNA, RNA tumor viruses, and a chemical carcinogen revealed the association of a protein of approximately 90,000 daltons (90K), which is highly phosphorylated in vitro. Peptide map analysis showed that the 90K proteins associated with these organelles of various transformed cells are similar irrespective of the species of cells and transforming agents. This species of protein could not be detected, or was scarcely detected, by phosphorylation in chromatin and ribosomes of untransformed cells and in revertants of transformed cells. These results suggest that the alteration in the pattern of endogenous protein phosphorylation in these organelles is closely related to the transformed state of cells.


Murine leukemia virus (T-8)-transformed cells: identification of a precursor polyprotein containing gag gene-coded proteins (p15 and p12) and a nonstructural component. Mink cells nonproductively transformed by the T-8 strain of mink cell focus-inducing virus express two type C viral amino terminal gag gene-coded structural proteins, p15 and p12, in the form of a 90,000 to 110,000 molecular weight polyprotein that lacks detectable immunological reactivity with other known type C virus-coded translational products. The observation concurs with the previous demonstration of similar high-molecular-weight precursor polyproteins in cell lines nonproductively transformed by either of two other mammalian sarcoma viruses also limited in virus-coded structural protein expression to p15 and p12.


Growth control in simian virus 40-transformed rat cells: temperature-independent expression of the transformed phenotype in tsA transformants derived by agar selection. Fisher rat fibroblasts (FR 3T3), transformed with the tsA30 mutant of simian virus 40 and selected by colony formation in soft agar, maintained the transformed phenotype at high temperature, whereas most transformants isolated from foci were found to undergo a phenotypic reversion toward the normal state in their saturation density, ability to grow in soft agar, and rate of 2-deoxyglucose transport. The temperature-independent phenotype observed in agar-selected transformants was not due to a reversion of the viral mutation. These results, similar to those previously obtained with polyoma virus tsa mutants, further suggest that two distinct mechanisms may operate in both cases for maintaining the transformed phenotype. Immunofluorescence studies suggested a different regulation of T antigen synthesis in these two classes of transformants.


Biochemical properties of p15-associated protease in an avian RNA tumor virus. It was observed that the viral structural protein p15 from avian myeloblastosis virus emerges from ion-exchange column chromatography along with a proteolytic activity. p15 is apparently pure, as judged by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and isoelectric focusing. Increase and decrease in proteolytic activity coincided exactly with increasing and decreasing amounts of p15 during ion-exchange chromatography and during size fractionation by gell filtration. The proteolytic activity cleaved various substrates such as bovine serum albumin, ovalbumin, concanavalin A, and casein after denaturation by sodium dodecyl sulfate and heat. Highest enzyme activity was observed around pH 5.7. As judged from its cleavage pattern and its response to proteolytic inhibitors, the proteolytic activity appears papain-like, and the protease responsible for it may be classified as a thiol protease. If added to immunoprecipitated viral polyprotein precursor Pr76, p15 resulted in cleavage of Pr76,which could be inhibited by antibodies against p15.


Viral DNA synthesis in cells infected with temperature-sensitive mutants of herpes simplex virus type 1. Temperature-sensitive mutants of herpes simplex virus type 1 representing eight DNA-negative complementation groups were grouped into the following three categories based on the viral DNA synthesis patterns after shift-up from the permissive to the nonpermissive temperature and after shift-down from the nonpermissive to the permissive temperature in the presence and absence of inhibitors of RNA and protein synthesis. (i) Viral DNA synthesis was inhibited after shift-up in cells infected with tsB, tsH, and tsJ. After shift-down, tsB- and tsH-infected cells synthesized viral DNA in the absence of de novo RNA and protein synthesis whereas tsJ-infected cells synthesized no viral DNA in the absence of protein synthesis. The B, H, and J proteins appear to be continuously required for the synthesis of viral DNA. (ii) Viral DNA synthesis continued after shift-up in cells infected with tsD and tsK whereas no viral DNA was synthesized after shift-down in the absence of RNA and protein synthesis. Mutants tsD and tsK appear to be defective in early regulatory functions. (iii) Cells infected with tsL, tsS, and tsU synthesized viral DNA after shift-up and after shift-down in the absence of RNA and protein synthesis. The functions of the L, S, and U proteins cannot yet be determined.


Extraction and fingerprint analysis of simian virus 40 large and small T-antigens. A study of simian virus 40 (SV40) T-antigens isolated from productively infected CV1 cells using a variety of different extraction procedures showed that under some conditions the highest molecular weight form of T-Ag (large-T) isolated comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with large-T from SV40-transformed H65-90B cells. Other faster-migrating forms of large-T are probably generated during the extraction procedure by a protease which is active at low pH, and such forms are probably experimental artifacts. After extraction under conditions which minimize proteolytic degradation of large-T, a further form of T-antigen was isolated; this has an apparent molecular weight in the range 15,000 to 20,000 and is referred to as small-t. Fingerprint analysis of [35S]methionine-labeled SV40 proteins showed that small-t has 10 to 12 methionine peptides whereas large-T has 15 to 18 methionine peptides. All but two of the methionine tryptic peptides present in small-t are also present in large-T. The fingerprint data also showed that T-antigens have no peptides in common with SV40 VP1. Experiments using reagents which inhibit posttranslational cleavage of encephalomyocarditis virus polyproteins showed that these reagents do not affect the synthesis of small-t and suggest that it is not made by proteolytic cleavage of large-T in vivo. An alternative model, which proposes that large-T and small-t are synthesized independently, is discussed in terms of the fingerprint data and the number of methionine tryptic peptides predicted from the primary sequence of SV40 DNA.


Cell-free synthesis of simian virus 40 T-antigens. Polyacrylamide gel electrophoresis and tryptic peptide fingerprint analysis of the proteins made in a cell-free system derived from L-cells and immunoprecipitated with simian virus 40 (SV40) anti-T serum demonstrated that both SV40 large-T and small-T antigens are synthesized in vitro in response to mRNA isolated from productively infected CV1 CELLS. Sucrose density centrifugation in gradients containing 85% formamide showed that the mRNA's for both forms of T-antigen sediment at about 17.5S, with the mRNA for small-t sedimenting marginally, but reproducibly, ahead of the mRNA for large-T. Hybridization experiments using restriction endonuclease fragments Hae III-E and Hind II/III-B showed that all fractions active in the cell-free synthesis of both forms of T-antigen hybridized equally to both fragments. This suggests that the mRNA's for SV40 T-antigens are at least partly virus coded and that the bulk of the early SV40 mRNA contains sequence information from both ends of the early region. The data are consistent with the suggestion that the large-T mRNA is spliced. SV40 complementary RNA (the product of transcription of SV40 DNA using Escherichia coli RNA polymerase) was also translated in the L-cell system and gave two families of polypeptides which specifically immunoprecipitate with anti-T serum. One family (the small-t family) includes a polypeptide indistinguishable by gel electrophoresis and tryptic peptide fingerprinting from small-t isolated from cells. The other family (the 60K family) has a major component with molecular weight approximately 60,000 and includes other polypeptides with molecular weights ranging from approximately 14,000 to about 70,000. The 60K family has petides in common with large-T but not with small-T. Together, the peptides of the small-t and 60K families account for virtually all of the methionine peptides of SV40 large-T. We conclude from these results (i) that small-t is probably entirely, and large-T at least predominantly, virus coded; (ii) that the small-t and 60K families represent the translation products of two different portions of the early region of SV40 DNA (approximately 0.65 to 0.55 map units and 0.54 to 0.17 map units); and (iii) that although most, if not all, of the large-T and small-t peptides are present in the cell-free product, some feature of sequence arrangement of SV40 complementary RNA prevents the translation of full-length large-T and results instead in the synthesis of fragments. We suggest that the absence of a splice in the complementary RNA is responsible for this result.


Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA. In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.


Physical mapping of herpes simplex virus type 1 mutations by marker rescue. A generally applicable technique which permits the rescue of selected genetic markers from fragments of herpes simplex virus DNA is described. Baby hamster kidney cells infected at the nonpermissive temperature with intact DNA from temperature-sensitive mutants or with fragmented wild-type DNA produce no, or little, infectious progeny. Coinfection results in an increased yield of virus, demonstrating the rescue of genetic information from the DNA fragments. This progeny virus consists of both wild-type and temperature-sensitive virus, demonstrating that both recombination and complementation can occur in coinfected cells. Rescue experiments using isolated fragments produced with various restriction endonucleases have enabled us to locate five temperature-sensitive mutations on the herpes simplex virus type 1 physical map. An adaptation of the technique has allowed the physical mapping of a mutation which affects the herpes simplex virus type 1 pyrimidine deoxyribonucleoside kinase gene. Comparison of the genetic and physical maps for these mutants reveals several anomalies which are discussed.


Replication of nondefective parvoviruses: lack of a virion-associated DNA polymerase. We have examined four of the nondefective parvoviruses for an associated DNA polymerase. Virions were purified from neuraminidase-treated infected-cell lysates by isopycnic centrifugation in CsCl or from infected cell material by CaCl(2) precipitation and centrifugation through sucrose into CsCl. Preparations of bovine parvovirus or Kilham rat virus obtained by the former procedure contained DNA polymerase activity but were not free of contaminating cellular proteins. The latter method produced viral preparations free of contaminating cellular proteins, and no DNA polymerase activity was detected in light infectious particles of H-1, LuIII, bovine parvovirus, or Kilham rat virus. Examination of levels of each cellular DNA polymerase in these preparations from each step of both purification procedures revealed that DNA polymerase beta had a greater tendency to copurify with bovine parvovirus and Kilham rat virus than did DNA polymerases alpha or gamma. Disruption of infectious virions obtained by the second purification method with detergents and sonic treatment did not result in the detection of a DNA polymerase activity. The biological activity and purity of each of the four different viruses obtained by the latter procedure were determined by hemagglutination and infectivity assays, polyacrylamide gel electrophoresis, and electron microscopy. In each case, the virions banding at a density of 1.39 to 1.41 g/cm(2) in CsCl were infectious and contained only the virion structural proteins. DNA polymerase activity was not detected in any of these preparations, and we have concluded that a virion-associated DNA polymerase is not required for productive infection with the nondefective parvoviruses.


Characterization of the mRNA's for the polyoma virus capsid proteins VP1, VP2, and VP3. Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.


Synthesis of T4 DNA and bacteriophage in the absence of dCMP hydroxymethylase. Several lines of research have suggested that the dCMP hydroxymethylase (HMase) coded by bacteriophage T4 is an essential protein in a DNA replication complex, as well as a supplier of hydroxymethyl dCMP for phage DNA synthesis. We show that a mutant [HMase, dCTPase, endonuclease II, endonuclease IV] which lacked this enzyme made cytosine-containing DNA at about two-thirds of the normal rate. When coupled with an alc mutation which permitted synthesis of late proteins, a small burst of phage was produced whose DNA contained no hydroxymethylcytosine. This pentuple mutant made both early and late proteins with abnormal kinetics, whereas the HMase+ parent showed normal kinetics. However, intracellular phage DNA showed no gross abnormalities in alkaline sucrose gradients. We conclude that HMase is not required for DNA synthesis when hydroxymethyl dCMP is not needed as a substrate; however, its absence still impairs both replication and transcription.


Heteroduplex analysis of the sequence relations between the RNAs of mink cell focus-inducing and murine leukemia viruses. The sequence relationships betwen AKR ecotropic virus and an AKR-derived "mink cell focus-inducing" (MCF) isolate (AKR MCF 247), between Moloney murine leukemia virus (M-MLV) and an M-MLV MCF isolate (M-MLV83), and between AKR and M-MLV were studied by electron microscopic heteroduplex analysis. The MCF-specific sequences were found to map from 1.95 kilobases (kb) to 2.75 kb (+/- 0.15 kb) from the 3' end of the RNAs for both MCF isolates. The major sequence nonhomology regions between AKR and M-MLV lie between 0.9 and 3.5 kb from the 3' end. However, the AKR and M-MLV sequences immediately adjacent to the 1.95- and 2.75-kb junctions with MCF-specific sequences are relatively similar in AKR and M-MLV. Our results suggest that the env gene of MLVs maps from 1 kb to 3 kb from the 3' end of the genomic RNA and that the carboxyl end of the glycoprotein of each MCF strain is similar (or identical) to that of its ecotropic parent.


Biochemical transformation of deoxythymidine kinase-deficient mouse cells with UV-irradiated equine herpesvirus type 1. A line of 3T3 mouse cells lacking deoxythymidine kinase (dTK-) was stably transformed to the dTK+ phenotype after exposure to UV-irradiated equine herpesvirus type 1 (EHV-1). Biochemical transformants were isolated in a system selective for the dTK+ phenotype (Eagle minimal essential medium containing 10(-4) M hypoxanthine, 6 X 10(-7) M aminopterin, and 2 X 10(-5) M deoxythymidine). Transformation was accompanied by the acquisition of a dTK activity with immunological, electrophoretic, and biochemical characteristics identical to those of the dTK induced by EHV-1 during productive infection. The transformed cells have been maintained in selective culture medium for more than 50 passages and have retained the capacity to express EHV-1--specific antigens. Spontaneous release of infectious virus has not been detected in the transformed lines, and the the cells were not oncogenic for athymic nude mice. In contrast to normal dTk+ 3T3 cells, EHV-1 transformants were unable to grow in the presence of arabinosylthymine, a drug selectively phosphorylated by herpesvirus-coded dTK's. These results indicate that a portion of the EHV-1 genome is able to persist in the transformed cells for many generations and be expressed as an enzymatically active viral gene product.


Assembly of viral membranes: maturation of the vesicular stomatitis virus glycoprotein in the presence of tunicamycin. The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375--385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.


Two initiation sites for translation of poliovirus RNA in vitro: comparison of LSc and Mahoney strains. Previous studies in our laboratory provided evidence that the initiation of translation by the Mahoney strain of poliovirus type 1 RNA in vitro occurs at two unique sites. This study shows that the LSc strain of poliovirus type 1, a multistep, temperature-sensitive mutant of the Mahoney strain, also utilizes two sites for the initiation of translation in vitro. Incorporation of formyl-[35S]methionine into the amino terminus of newly synthesized polypeptides revealed the production of two labeled tryptic peptides which are identical in size and electrophoretic mobility with those produced by Mahoney virus. The polypeptides containing amino-terminal label showed similar patterns on sodium dodecyl sulfate-acrylamide gels, although one of the LSc polypeptides had a slightly faster mobility. The relative proportion of initiation at each site varied with the magnesium concentration for both viruses, but the LSc strain favored initiation at one site more so than did the Mahoney strain.


Identification of Sendai virus mRNA species. Cell-free translation of separated Sendai virus mRNA species identified the message for polypeptide M and suggested the identity of the message for polypeptide NP.


Simian virus 40 production after viral uncoating in the CV-1 cell nucleus. A normal yield of infectious simian virus 40 was produced by a CV-1 cell culture after inoculation of a few cells with an average of one particle per nucleus by microinjection.


Mechanism of transfection of chicken embryo fibroblasts by Rous sarcoma virus DNA. The mechanism of transfection by Rous sarcoma virus DNA was investigated by assaying DNA-mediated transformation under conditions which restricted secondary virus infection. Chicken embryo fibroblasts which were genetically resistant to virus infection as a result of the absence of receptors for virus penetration were also resistant to transformation by integrated or unintegrated Rous sarcoma virus DNA. In addition, DNA of replication-defective Bryan hightiter Rous sarcoma virus was noninfectious, and transformation by DNA of a temperature-sensitive DNA polymerase mutant was temperature sensitive. These results indicated that secondary virus infection was necessary for transformation by Rous sarcoma virus DNA. Since transformation was assayed by colony formation in soft agar, as well as by focus formation, the requirement for secondary virus infection was not an artifact of potential difficulty in detection of foci formed by division of single transformed cells. Therefore, it appeared that donor DNA did not stably transform recipient cells by direct integration. Instead, the results were consistent with the hypothesis that transfection of chicken embryo fibroblasts by Rous sarcoma virus DNA proceeded by transcription of donor DNA, formation of extracellular progeny virus, and secondary virus infection of sensitive cells.


Simian virus 40 DNA replication in isolated replicating viral chromosomes. Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.


Persistent infection of L cells with vesicular stomatitis virus: evolution of virus populations. A previous report (Youngner et al., J. Virol. 19:90-101, 1976) documented that noncytocidal persistent infection can be established with wild-type vesicular stomatitis virus (VSV) in mouse L cells at 37 degrees C and that a rapid selection of RNA(-), group I temperature-sensitive (ts) mutants consistently occurs in this system. To assess the selective advantage of the RNA(-)ts phenotype, evolution of the virus population was studied in persistent infections initiated in L cells by use of VSV ts 0 23 and ts 0 45, RNA(+) mutants belonging to complementation groups III and V. In L cells persistently infected with ts 0 23, the ts RNA(+) virus population was replaced gradually by viruses which had a ts RNA(-) phenotype. VSV ts 0 45 (V) has another marker in addition to reduced virus yield at 39.5 degrees C: a defective protein (G) which renders virion infectivity heat labile at 50 degrees C. Persistent infections initiated with this virus (ts, heat labile, RNA(+)) evolved into a virus population which was ts, heat resistant, and RNA(-). These findings suggest that the ts phenotype itself is not sufficient to stabilize the VSV population in persistently infected L cells and also indicate that the ts RNA(-) phenotype may have a unique selective advantage in this system. In addition to the selection of ts RNA(-) mutants, other mechanisms which also might operate in the maintenance of persistent VSV infections of L cells were explored. Whereas defective-interfering particles did not seem to mediate the carrier state, evidence was obtained that interferon may play a role in the regulation of persistent infections of L cells with VSV.


Nucleotide sequence relationship between intracisternal type A particles of Mus musculus and an endogenous retrovirus (M432) of Mus cervicolor. Intracisternal type A particles are retrovirus-like structures found in embryonic cells and many tumors of Mus musculus but having no clear relationship with other retroviruses of this mouse species. We have observed a partial nucleotide sequence homology between the high-molecular-weight (32S and 35S) RNA components of intracisternal A-particles from a neuroblastoma cell line and the 70S RNA fraction from M432, a type of retrovirus endogenous to the Asian mouse Mus cervicolor. M432 complementary DNA (cDNA) was hybridized to the extent of 30% by the A-particle RNAs. The hybrids showed a lower thermal stability (DeltaT(m), 7 degrees C) than those formed with homologous RNA. The reaction was commensurate with that found between M432 cDNA and divergent sequences in the M. musculus genome. The capacity to hybridize M432 cDNA was closely correlated with the concentration of A-particle sequences in the cytoplasmic RNA of several M. musculus cell types. The major RNA fraction of M432 virus showed a reciprocal partial reaction with the A-particle cDNA's; the virus, which was grown in NIH/3T3 (M. musculus) cells, also contained a small proportion of apparently authentic A-particle nucleotide sequences. A subset of A-particle sequences seemed to be almost totally lacking in the main M432 RNA. The A-particle cDNA's hybridized extensively with divergent sequences in M. cervicolor cellular DNA, indicating that this mouse species may contain not only the partially homologous M432 virogene, but also a more complete genetic equivalent of the intracisternal A-particle.


Pathogenesis of essential hypertension with low renin: responses of plasma renin activity to various stimulation tests in essential hypertension. Plasma renin activity (PRA) was measured in 14 control subjects and 27 patients with essential hypertension (EH) (low renin group: 9, normal renin group: 11, and high renin group: 7) before and after the following stimulation tests. Test procedures: 1) Circadian rhythm (0600, 1600 and 2400h). 2) Adrenal stimulation test (ACTH: 12.5 I.U.). 3) Adrenal suppression test (Dexamethasone: 1.0 mg). 4) Metopirone test (1.5 g). 5) Angiotensin II infusion test (8 ng/kg/min). 6) Saline infusion test (1000 ml/hr). Patients with low PRA showed significantly lower levels of PRA than those of other two groups in circadian rhythm, after 2 hours of ACTH infusion and after angiotensin II infusion. Furthermore, these patients showed significantly higher responses of PRA than other two groups after furosemide test under dexamethasone and after metopirone test. In case of saline infusion test, patients with low and normal PRA did not show significantly decreased levels of PRA after the infusion, though all patients with high PRA and all control subjects showed significantly decreased levels of PRA. From the present studies, it might be concluded that patients with low PRA has an unknown mineralocorticoid excess which is ACTH dependent and 11 hydroxylated and some of hypertensive patients have an abnormality in their renin-angiotensin-aldosterone volume feed back loop as a factor for hypertension.


Hypertension induced by adrenocortical dysfunction--hypertension in 17 alpha-hydroxylase deficiency and metopirone-induced hypertension. Hypertension in 17 alpha-hydroxylase deficiency was studied by comparing it with hypertension in Cushing syndrome or that in primary aldosteronism. Furthermore, the role of endogenous increases of ACTH, deoxycorticosterone, and 18 alpha-hydroxy-deoxycorticosterone upon blood pressure was studied in rats  by administerating metopirone. Hypertension in 17 alpha-hydroxylase deficiency was considered to be more similar to that in primary aldosteronism from the studies on renin components, pressor responses to angiotensin II and norepinephrine, and renin responses to stimulations. Plasma catecholamines were slightly decreased in 17 alpha-hydroxylase deficiency. The hypertension was alleviated by the administeration of dexamethasone in 2 of 3 patients with 17 alpha-hydroxylase deficiency. However, in the remaining one who had an accelerated hypertension and normal renin, the hypertension was not alleviated by dexamethasone. In the animal studies, hypertension induced by metopirone was accelerated by salt loading of uni-lateral nephrectomy plus salt loading. In those rats, plasma ACTH, and deoxycorticosterone were markedly increased.


Effect of sodium ion on levels of cyclic adenosine 3',5'-monophosphate in guinea pig cerebral slices. Accumulation of cyclic AMP was studied in guinea pig cerebral slices when Na+ levels in the bathing medium were varied or agents which affect tissue Na+ content were added. When NaCl was gradually replaced with Tris-HCl or choline chloride, cyclic AMP formation was progressively enhanced. ;when Na+ was below 30 mM,  cyclic AMP formation reached the maximum (approximately 30 fold), but this increment was not blocked by tetrodotoxin. The stimulatory effect of high K+ was nearly linear over 120 mM and became much more prominent when Na+ also was not blocked by tetrodotoxin. Ouabain (10(-4) M), electrical pulses and glutamate (5 x 10(-3) M), each stimulated cyclic AMP formation about 17-, 7- and 5-fold, respectively. Tetrodotoxin (2 x 10(-6) M) completely blocked the effects of electrical pulses and partially blocked the effects of glutamate and oubain. It is suggested that the increase of cyclic AMP in cerebral cortical slices may be related to the decrease in Na+ gradient across the cell membrane.


[Mechanism of the change in adipocyte sensitivity to ACTH and adrenaline in spontaneous genetic hypertesion in rats]. As compared to fatty tissue cells of animals with normal pressure, those of SHR rats were found to be characterized by a higher lipolytic response and a larger increase in the cAMP content on exposure to the effect of ACTH. As compared to the controls, adrenalectomized SHR rats had an increased basal cAMP content and an increased lipolytic response of the adipocytes following adrenaline administration. In inhibition of phosphodiesterase in the fatty cells of adrenalectomized rats with normal pressure, the cAMP content grew by 20% as compared to that in SHR rats subjected to the operation. It is suggested that the changes in intracellular distribution of calcium, shown in this model of hypertension, may be the direct cause of the altered sensitivity of the "target" cells to the effect of hormones.


[Myoclonic-astatic seizures (Lennox syndrome) in the course of juvenile neuronal ceroid-lipofuscinosis (M. Batten-Spielmeyer-Vogt) (author's transl)]. EEG findings and the course of epileptic seizures in two patients with neuronal ceroid lipofuscinosis (Batten Spielmeyer Vogt syndrome) are presented. Both patients, during the course of disease, developed therapy resistant epileptic reactions with myoclonicastatic seizures. These seizures in connection with diffuse encephalopathy and EEG pattern with 2.5 to 3.5/sec slow-spike-wave meet the criteria of the Lennox syndrome. Pathogenetic questions regarding possible additional genetic predisposition for epileptic seizures are discussed. Since therapeutic effect of different medications is uncertain hormonal therapy may be considered.


Metabolic bone disease resembling osteosarcoma in a wooly monkey (Lagothrix lagotricha). A female pet wooly monkey with metabolic bone disease initially presented with a proliferating bony mass in the left humerus which had many features of osteosarcoma. At necropsy, parathyroid hyperplasia, osteoclastic resorption, proliferative osteoid deposition in the calvarium and cortex of long bones, and fibrous proliferation of the marrow indicated the presence of generalized osteodystrophia fibrosa. The dietary history of deficient vitamin D3 and protein and minimal exposure to sunlight supported this diagnosis, as did depressed levels of serum calcium and elevated levels of serum parathyroid hormone, alkaline phosphatase, and acid phosphatase.


Disseminated lymphoma in a rhesus monkey: a case report. A rhesus monkey was inoculated with rhesus cytomegalovirus. A leukocyte-associated herpes virus, unrelated to cytomegalovirus, was later isolated from the same monkey. Four years after the virus inoculation, the monkey developed a disseminated lymphoma.


Composition of bronchoalveolar lavage effluents from patients with pulmonary alveolar proteinosis. The lungs of patients with pulmonary alveolar proteinosis were lavaged with saline. The lavage effluents were examined for the presence of unusual components which might be considered characteristic of the disease. Soluble proteins were resolved by electrophoresis on polyacrylamide linear gradient (2.5 to 27 per cent acrylamide) gel slabs and compared with proteins found in the patients' sera and lavage effluents from nondiseased human lungs. Many proteins in lavage effluents from both diseased and nondiseased lungs appeared similar to those found in serum. Several proteins from diseased lungs were not present in serum nor lavage effluents from nondiseased lungs. A number of proteins present in lavage effluents from nondiseased lungs appeared to be absent from lungs with alveolar proteinosis. Particulate components of lavage effluents from diseased and normal lungs were examined and compared using the electron microscope. Many particulate components including degenerate and disintegrating cells, ultrastructurally abnormal alveolar macrophages, and myelin-like structures were present in lavage effluents from diseased lungs but absent from normal lungs. Much of the particulate material resembled cell debris. The insoluble accumulations present in the pulmonary alveoli and airways of patients with alveolar proteinosis do not come from serum leakage nor from hypersecretion of materials normally found in these regions of the lungs. The major insoluble components appear to arise from cellular disintegration and the extracellular formation of myelin-like structures.


Complications of leprosy.

Complications of leprosy. Leprosy is essentially a systemic disease and is a great "mimicker" of many other diseases. It affects apart from skin and peripheral nerves, haemopoietic, reticulo-endothelial and endocrine systems as well as eyes, bones and muscles.


Sleep during chronic ethanol administration and withdrawal in rats. After a priming dose, ethanol was administered at a rate relative to behavioral impairment for 4 days and its effects on sleep were monitored by EEG. EEG's during withdrawal indicated an initial sleep loss followed by a return of total sleep and a REM sleep rebound several days later.


Granular cell myoblastoma of the bronchus. Case report and literature review. A case of multiple granular cell myoblastomas of the tracheobronchial tree is presented. The patient is well 32 months following endobronchial removal. Only 46 cases of solitary and two cases of multiple lesions of the tracheobronchial tree have been described in the literature. These lesions occur with equal frequency in men and women; at a median age of 38 years; more frequently on the right than on the left; and most commonly with cough as the presenting symptom. Most patients have been treated with major surgical resections, but conservative endobronchial removal may be effective therapy.


Coexistent bronchogenic carcinoma and active pulmonary tuberculosis. Sixty-four cases of coexistent bronchogenic carcinoma and active pulmonary tuberculosis were diagnosed between 1969 and 1976. The majority were male chronic cigarette smokers in their fifth and sixth decades. Human bacilli were isolated in 48 patients (88.9 percent) and atypical bacilli in six patients (11.1 percent). All of the atypical bacilli and 8.3 percent of the human bacilli were found to be resistant to the first line antituberculous drugs. All patients, except two who died following resection, were given a course of antituberculous drugs with 93.8 percent successful sputum conversion. Uncontrolled or disseminated tuberculous infection was not observed. Forty-five patients (70.3 percent) underwent pulmonary resection. Median survival time of those who had curative resection was 14.3 months with a 5 year survival rate of 13.2 percent. Median survival times of those treated by palliative resection plus anticancer chemotherapy and by anticancer chemotherapy alone were 8.3 months and 11.1 months, respectively. None of these patients survived more than 30 months. It appears that, clinically, each disease runs its own course with little effect on the other.


Prostaglandins; their biological and pharmacological role. The biological and pharmacological roles of prostaglandin system are reviewed. The most recent and important finding is the isolation and characterization of endoperoxides, thromboxanes and prostacyclin. In many respect they seem to be even more effective than the original prostaglandins and could explain many actions previously attributed to prostaglandins. Prostaglandins, endoperoxides, thromboxanes, and prostacyclin seem to have important physiological and pathophysiological roles, e.g. in the cardiovascular, gastro-intestinal and reproductive systems, the skin, and the central nervous system, as well as in inflammatory, immunological and metabolic reactions. Thus, prostaglandin-like substances, their analogues, prostaglandin antagonists, and the drugs that affect prostaglandin synthesis present good prospects for the treatment of many diseases affecting these systems. The mode of action of most anti-inflammatory and analgesic drugs can be explained by actions on the prostaglandin system. New potent and selective regulators of the prostaglandin system are being investigated and more indications are being found for using existing drugs. Although the field is generally promising, there are many contradictory findings. This calls for a cautious interpretation of the many interesting observations.


BK and Herpes simplex virus antibodies in renal cell carcinoma. Serum antibodies against BK virus (BKV) and seven other viruses were studied in 76 patients with renal cell carcinoma using either the haemagglutination inhibition or the complement fixation techneque. BKV and herpes simplex virus (HSV), but no other virus antibodies, showed a clear correlation with the clinical course of renal carcinomas. The patients without BKV antibodies or with marginal antibody titres (titre less than or equal to 10), had a significantly longer observed expectation of life (O.E. life, 44.1 months) than the O.E. life (26.7 months) of the patients with BKV antibodies of titre less than or equal to 20. Similarly, patients with low or undetectable HSV antibodies (titre less than or equal to 8) also had significantly longer O.E. life (54.0 months) than the rest of the patients (32.7 months). The results suggest that BKV and HSV have an as yet underfined relationship to renal cell carcinomas.


Small cell anaplastic (oat cell) carcinoma of the larynx: report of a case and review of the literature. The fifth reported patient with primary laryngeal oat cell carcinoma is described. Electron microscopy of tumor cells revealed typical neurosecretory-type granules similar to those described for pulmonary oat cell carcinoma. Analysis of clinical data from all five reported patients revealed that this neoplasm is a virulent one which spreads early and rapidly and kills quickly. Combination chemotherapy and/or radiotherapy, which has significantly prolonged life in patients with oat cell carcinoma of lung, may be indicated as an adjunct to surgery for this rare malignant tumor of the larynx.


[Ethanol and lipid metabolism (author's transl)]. Fatty liver and hyperlipoproteinemia are the main clinical manifestations of interrelationships between ethanol and fat metabolism. Elevation of VLDL is observed more, hyperchylomicronemia less frequently. In metabolic healthy volunteers reversible hypertriglyceridemia can be provoked as well. The pathogenesis of ethanol-induced hyperlipoproteinemia is based on metabolic alterations directly dependent on the oxidation of ethanol in the liver as well as on indirect effects: Besides decreased oxidation and increased de-novo-synthesis by the liver, the sources of fatty acids for the enhanced production of VLDL are the adipose tissue and alimentary fat. VLDL concentrations have been shown to correlate with risk of atherogenesis in the middle-aged. Enhancement of alpha/beta (HDL/LDL)-cholesterol which would indicate an antiatherogenetic effect have been observed in relation to ethanol intake. At the moment it is premature to conclude that ethanol-induced changes in plasma lipoproteins may favour or delay atherogenesis.


Binding and degradation of human 125I-HDL by rat adrenocortical cells. In order to learn more about the mechanism by which high density lipoprotein (HDL) cholesterol is taken up by the adrenal cortex, binding and degradation of human 125I-HDL by suspensions of intact rat adrenal cortical cells have been examined. Cellular accumulation of 125I-HDL was found to occur in two phases. Our results indicate that the initial phase of association results from reversible binding of 125I-HDL to a specific saturable set of membrane binding sites. Binding site affinity appears equal for both rat and human HDL while affinity for human LDL is approximately one order of magnitude less on the basis of apoprotein weight. In addition, isolated rat adrenal cortical cells were found to degrade human 125I-HDL at a rapid rate. Degradation, like binding, can be prevented by addition of excess unlabeled HDL suggesting that binding and degradation are linked. Thus, one mechanism that could account for adrenal uptake of HDL cholesterol is endocytosis, initiated by lipoprotein binding to the HDL specific membrane binding site.


Lipoprotein lipase activity in adipose tissue and skeletal muscle of runners: relation to serum lipoproteins. Physically well-trained people generally have lower VLDL-triglyceride and higher HDL-cholesterol levels than sedentary subjects. To examine the underlying mechanisms of this lipoprotein pattern, we measured the lipoprotein lipase (LPL) activity in needle biopsy specimens of adipose tissue and skeletal muscle of competitive runners and of body weight-matched, physically less-active controls. The active sportsmen were either sprinters, whose training program consisted mainly of athletics of short duration or long distance runners undergoing a strenuous endurance exercise program. In sprinters (all males) the serum lipid and lipoprotein concentrations did not differ significantly from those of controls and the mean LPL activities in muscle and adipose tissue were also similar in these two groups. The long distance runners (both sexes), on the other hand, had higher means levels of HDL-cholesterol than the respective controls. The LPL-activity of both adipose tissue (p less than 0.05) and skeletal muscle (p less than 0.01) was significantly higher in male long distance runners than in control males. Female runners had higher muscle LPL activity than controls (p less than 0.01) but in adipose tissue the difference in LPL activity was not significant. Rough estimates calculated for LPL activity present in whole body adipose tissue and skeletal muscle indicated that total LPL activity was 2.3 times higher in male long distance runners and 1.5 times higher in female long distance runners than in the respective controls. In combined groups of male runners and controls, there was a highly significant positive correlation between the serum HDL-cholesterol level and the LPL activity of adipose tissue expressed per tissue weight (r = +0.72, p less than 0.001) or per whole body fat (r = +0.62, p less than 0.001). The group means of HDL-cholesterol and adipose tissue LPL activity in the five cohorts studied (male sprinters, distance runners and controls and female distance runners and controls) were also positively correlated (r = +0.94). It is concluded that endurance training is associated with an adaptive increase of LPL activity not only in skeletal muscle but also in adipose tissue. These changes are not observed in sprinters who are trained by exercises of shorter duration. The high HDL-cholesterol levels of physically well-trained people are probably accounted for, at least partly, by the increased LPL activity and the concomitant rapid turnover or triglyceride-rich lipoproteins.


[Regulation of the biosynthesis of extracellular phosphohydrolases in Penicillium brevicompactum]. The effect of certain metabolites of Penicillium brevi-compactum on the biosynthesis of exocellular ribonucleases and phosphomonoesterase was studied. Their synthesis was found to be inhibited by RNA and AMP, as well as by high concentrations of these enzymes in the medium. The mechanism which regulates the biosynthesis of exocellular phosphohydrolases by both phosphate and the enzymes is discussed.


[Use of the electron paramagnetic resonance method for the comparative evaluation of the effectiveness of the protective media used in the lyophilization of actimomycetes]. The accumulation of free radicals correlates with an increase in the percentage of killed lyophilized spores of actinomycetes. Therefore, the EPR method in combination with the method of "accelerated storage" at 37 degrees C was used to evaluate rapidly (during one month) the effectiveness and to select certain protective media for stabilization of actinomycetes cultures. The EPR method can be applied for comparative evaluation of only those media which did not contain originally free radicals.


[Ethylmethane sulfonate induction of auxotrophic mutants of Rhizobium meliloti and their characteristics]. Ethylmethanesulfonate was used as a mutagen to induce auxotrophic mutants in Rhizobium meliloti L5-30. Survival of the culture depended on the period of time during which it was treated with the mutagen. Thirteen auxotrophic mutants were isolated as a result of screening 1404 clones, and their requirements in growtn factors were determined. Ethylmethanesulfonate induced mainly those mutants which required sulfur-containing amino acids. The activity of nitrogen fixation of the auxotrophic mutants was assayed by measuring the reduction of acetylene and the yield of lucerne. Mutants requiring methionine or cystein, cysteine or histidine, and adenine with thiamine were not effective whereas those requiring arginine, tryptophan, and thiamine were capable of nitrogen fixation. Among two methionine auxotrophs, one displayed the activity of nitrogen fixation and the other did not.


[Cytomorphology of the lipid inclusions of Caryophanon during its growth on an agarized medium]. The cytomorphology of lipid inclusions of Caryophanon latum and Caryophanon tenue was studied in the course of their growth on a solid medium. The paper presents a detailed scheme and microphotographs showing cytomorphological changes in the inclusions of the cultures under study. Staining of the preparations with the lipophilic dye Sudan Black B revealed two types of sudanophilic granules which varied in their location within trichomes, the time at which they appeared in bacteria, and their behaviour toward solvents. These lipid inclusions of Caryophanon are mainly represented by the polymer poly-beta-hydroxybutyric acid.