An improved and simple micro-method of sphingomyelinase assay in leukocytes and urine. A simple one-vial-method was developed for the quantitative determination of sphingomyelinase activity in human leukocytes and urine, using [14C-methyl] sphingomyelin. The measured activities of healthy control persons show a higher scatter in (n=50) urine (1.2 +/- 0.5 nmol/h . ml urine) than in (n=9) leukocytes (2.15 +/- 0.35 nmol/h . mg protein). Long term tests showed that the enzyme activities in urine can best be correlated to the 24-h-creatinine excretion. A distinct loss of enzyme activity was found in dialyzed urine starting at about the third day; this did not occur in undialyzed urine. The method also shows good reproducibility in micro-tests. It is therefore suitable for screening tests (urine of persons suffering from Niemann Pick disease) and for the prenatal diagnosis of sphingomyelinosis. For one out of two children with symptoms of sphingomyelinosis (hepatosplenomegaly, mental retardation, and neurological deterioration) the diagnosis was confirmed by morphological examination of tissues obtained by biopsy. In both cases leukocytes and urine revealed normal sphingomyelinase activity. These biochemical results in conjunction with the clinical and morphological picture were indicative of type C Niemann-Pick disease.


The spatiotemporal transfer function of the Limulus lateral eye. The dynamics of the Limulus retina may be well described by the spatiotemporal transfer function, which measures the response of the eye to moving sinusoidal gratings. We consider a model for this system, which incorporates an excitatory generator potential, and self- and lateral inhibitory processes. Procedures are described which allow estimation of parameters for the model consistent with the empirical transfer function data. Transfer functions calculated from the model show good agreement with laboratory measurements, and may be used to predict accurately the response of the eye to arbitrary moving stimuli. The model allows convenient interpretation of the transfer function measurements in terms of physiological processes which underly the response of the Limulus retina.


The fine structure of cells infected with temperature-sensitive mutants of herpes simplex virus type 2. The fine structure of cells infected with the HG 52 strain of herpes simplex virus type 2 and 13 temperature-sensitive mutants derived from it was investigated. In cells infected with the wild-type virus, development of virions appeared to be similar to that described in previous reports. However there were two exceptions to this: (1) capsid envelopment apparently occurred de novo in the nucleus; (2) densely staining vacuolar accumulations were seen, frequently surrounding virus capsids. The 13 temperature-sensitive mutants of the virus were divided into three classes according to the type of capsid, if any, produced in cells infected and maintained at the non-permissive temperature. Class I mutants produced no capsids, Class II mutants produced empty and partial-cored capsids and Class III mutants produced empty, partial- and dense-cored capsids. Cellular alterations were also determined. Membranous tubular structures, previously unreported for herpes simplex virus, were observed in cells infected with Class III mutants and very occasionally with wild-type virus at the non-permissive temperature. Cytoplasmic particles were also found, but could not be correlated with any particular class of mutant.


Simultaneous production of mouse endogenous virus and Rous sarcoma virus by Schmidt-Ruppin virus infected mouse cells. Schmidt-Ruppin Rous sarcoma virus infected chick cells injected into newborn C3H/f mice gave rise to tumours at the site of inoculation. These tumours were transplantable in adult C3H/f mice and were able to induce tumours in the wing of adult Leghorn chickens. Tumour cells from the 18th passage in mice were used to establish a cell line in tissue culture (C3HSR). These cells released C-type virus particles that produced foci and were able to propagate in chick cells. Cloning of the C3HSR cells demonstrated that the same cell expressed both avian and murine antigens. Mouse cells infected with virus released by C3HSR cells produced murine leukaemia virus-like particles as revealed by the reverse XC syncytial test and by immunofluorescence tests.


The effect of trypsin on the growth of rotavirus. It has been found that 1000-fold more bovine rotavirus is obtained when trypsin is incorporated in the maintenance medium and allowed to remain throughout the growth cycle. This holds true for primary calf kidney (CK) cells and also for several continuous and semi-continuous cell lines. In the presence of trypsin it has been possible to pass the virus serially on continuous cell lines seven times. Concentrations of 1 to 10 microgram/ml of trypsin are found to be effective. Preliminary results suggest that the same technique will be effective for the in vitro propagation of human rotavirus.


The use of guanidine-HCl for the isolation of both RNA and protein from RNA tumour viruses. The RNA components of two C-type RNA viruses, avian myeloblastosis virus and Friend leukaemia virus, have been isolated by treatment of the viruses with 6 M-guanidine-HCl and precipitation with ethanol. The virus proteins were recovered by lyophilization of the guanidine-HCl-ethanol supernatant after thorough dialysis against 0.5 mM-dithiothreitol. This simple method yielded RNA of similar quality to the phenol and sodium dodecyl sulphate (SDS) extraction methods, and the same amount of 60-70S RNA, although a fraction of the smaller (4S) species remained in the protein fraction. The sedimentation patterns of heat-denatured RNA extracted by either method were similar. Electrophoretic analyses of the extracted proteins in polyacrylamide gel gradients containing SDS gave patterns that were very similar to those obtained by direct analysis of SDS disrupted viruses.


Infection of mouse liver by human adenovirus type 5. CBA mice, inoculated intravenously with large doses of adenovirus type 5, showed raised levels of serum aspartate aminotransferase (SAAT; EC 2.6.I.I) and died within a few days from histologically demonstrable hepatic necrosis. After inoculation of I LD50, virus was rapidly taken up by the tissues where infectivity then declined greatly. Organ titres then increased about 100-fold by 48 h p.i. but, in the liver, which showed intranuclear inclusion bodies, and by electron microscopy, scattered intranuclear and intracytoplasmic adenovirions, the increase was 10000- to 100000-fold. P antigen was detected by single radial diffusion in liver extracts, and by immunofluorescence in 80% of liver cells at 36 h p.i. Hexon, penton base and fibre antigens appeared later and in fewer cells. The maximum amount of hexon, of demonstrable type 5 specificity, was shown by radioimmunoassay to be equivalent to up to 5 x 1011 whole adenovirions/g liver. It is concluded that human adenovirus type 5 undergoes an abortive but lytic infection in most liver cells but that replication may proceed to completion in a few.


Defective herpes simplex virus DNA: circular and circular-linear molecules resembling rolling circles. The formation of defective herpes simplex virus (HSV) in BSC-1 cells and the synthesis of defective virus DNA was studied. The fourth consecutive passage of undiluted virus yielded defective DNA that was 0.008 g/ml more dense than wild type (w.t.) virus DNA. The amount of defective DNA increased at passage 6 concomitantly with the decrease in infectious virus progeny. The synthesis of defective DNA was always accompanied by w.t. virus DNA synthesis. Defective DNA from both infected nuclei and defective virions had a mol. wt. of 100 X 10(6) and was linear as determined by electron microscopy. Electron microscopy of defective virus DNA at passage 6 revealed circular molecules varying in size in addition to linear DNA molecules with the length of intact virion DNA. The circular DNA molecules had contour lengths of 10, 5, 2.5 and less than 2.5 micron. The smallest circular DNA molecules had a contour length of 0.3 micron, possibly one virus gene. In addition, circular-linear DNA molecules were observed in which both the circular and the linear components varied in length. Most of these DNA molecules had circular components of either 2.5 or 5.0 micron, and linear components varying in length from less than 1 to 50 micron. Based on the present study, it is proposed that the S component of w.t. virus DNA is fragmented into small circular molecules that serve as templates for DNA synthesis, possibly by the rolling circle mechanism.


Photochemical inactivation of DNA and RNA viruses by psoralen derivatives. Western equine encephalitis virus, and RNA virus, and herpes simplex virus type I, a DNA virus, were efficiently inactivated in less than I min by exposure to long-wave ultraviolet light (320 to 380 nm) in the presence of several psoralen derivatives. The psoralen photochemical reaction was chosen for study due to its known specificity for nucleic acids. Neither the light nor any of the drugs alone caused appreciable inactivation. The inactivation kinetics and dependence on light intensity and on different derivatives of psoralen were studied. The high solubility of a new aminomethyl psoralen derivative was found to be advantageous in the photochemical inactivation of the RNA virus, but was not in the case of the more easily inactivated DNA virus. Within its limited solubility range trimethylpsoralen was superior to its aminomethyl derivative on a molar basis for the inactivation of both types of viruses under most of the conditions studied.


Attachment of herpes simplex virus to neurons and glial cells. Cells of brain tissue of rabbits, rats and mice were dissociated and glial cells, neuronal perikarya and synaptosomes were separated by centrifugation on discontinuous Ficol gradients. HSV was shown to attach well to rat and rabbit glial cells and synaptosomes but not to neuronal perikarya. Of intracerebrally infected mice the fractions with glial cells contained the infective virus. The interactions between HSV and neuronal cells and the implication of the observations on the HSV infection of the nervous system are discussed.


Potentiation of the neurtalization of feline leukaemia virus in hypotonic medium. The neutralization of feline leukaemia virus could be enhanced by performing the reaction in hypotonic medium. Absorption of antibody by virus specific antigens was also made more sensitive under these conditions.


Replication of herpesviruses in human cells transformed by cytomegalovirus. The replication of human cytomegalovirus (CMV) and herpes simplex virus (HSV) was studied in three human embryo cell lines (CMV-Mj-HEL-I, CMV-Mj-HEL-2, and CMV-Mj-HEL-2,T-I) transformed in vitro by human CMV. Growth studies revealed that these cells were completely resistant to infection by CMV strains ADI69 and Mj and partially resistant to HSV types I and 2. Neither virus DNA nor virus proteins were synthesized in the transformed cells infected with CMV AD169. The HSV production in CMV-transformed human embryo lung (HEL) cells was delayed when compared to the virus production in normal HEL cells and spread of HSV c.p.e. was slower in the transformed cells. The treatment of normal HEL cells with a crude extract of CMV-transformed HEL cells also resulted in inhibition of the spread of c.p.e. of HSV types I and 2. The inhibitory effect was not due to interferon since vesicular stomatitis virus replication was not affected and several experiments showed that it was not due to mycoplasma. The presence of virus inhibitor molecules in CMV-transformed cells absent in normal HEL cells is postulated.


Sensitivity of the transforming and replicative functions of Epstein--Barr virus to inhibition by phosphonoacetate. Disodium, phosphonoacetate (PA), at concentrations of 50 to 200 microgram/ml, which still allowed continued growth of the EB virus-transformed B95-8 cell line on a routine culture regimen, was able to inhibit the production of virus capsid antigen and of virus particles by these cells down to very low but finite levels which persisted despite prolonged treatment. Further experiments measured the effects of these same drug concentrations on the EB virus-induced in vitro transformation of foetal cord blood lymphocytes and on the colony forming ability of already established EB virus-transformed foetal cell lines; in both types of culture, doses of PA up to and including 50 microgram/ml did not affect cell growth within the 8-week observation period, whereas doses of 100 microgram/ml and above were increasingly inhibitory. The cell lines established by EB virus-induced transformation in the continual presence of PA at 50 to 150 microgram/ml contained multiple copies of the virus genome per cell just as did the corresponding cell lines established in control medium. The results argue against the existence of any PA-sensitive event unique to the EB virus-induced transformation process.


Complement-mediated cytolysis of HSV-1 and HSV-2 infected cells: plasma membrane antigens reactive with type-specific and cross-reactive antibody. Surface antigens of BHK-21 cells infected with HSV-1 or HSV-2 were radioiodinated (125I) with lactoperoxidase, immune precipitated and analysed by polyacrylamide gel electrophoresis (PAGE). Experiments using antiserum to HSV-1 or HSV-2, absorbed with appropriate hemotypic or heterotypic antigens, revealed that both type-specific (homotypic) and cross-reactive antibody combined with surface glycoproteins to form a single large radioactive peak. This peak, which constituted the major glycoprotein region (region a) observed in electropherograms, represented a range in mol. wt. from 115000 to 130000. Sensitization of cells to complement lysis, neutralization of infectious virus and immune precipitation of surface glycoproteins (region a) were found to be generally correlated properties of all the antibody preparations analysed, including antibody prepared specifically against region a antigens. These findings suggest a major immunological role for the surface glycoproteins migrating in PAGE region a.


Improved detection of virus-specific IgM antibodies. Elimination of non-specific IgM binding. A non-specific reaction between human IgM and cytoplasmic structures of virus infected cells can often be observed if MgM antibodies to virus antigens are detected by indirect immunofluorescence or by immuno enzyme assays. Formaldehyde selectively inactivates the cytoplasmic receptors for human IgM without affecting the virus structural proteins. Alternatively, receptor-free antigens can be obtained by isolation of nuclei from virus infected cells. Due to reduced background, a more specific and more sensitive detection of IgM antibodies to Epstein-Barr virus, cytomegalovirus or central European encephalitis virus is possible.


Metabolism of myoinositol in avian and mammalian cells infected with naked and enveloped DNA and RNA viruses. The uptake of 3H-inositol into the pool of free inositol and its incorporation into the lipid phosphatidylinositol have been studied in various avian and mammalian cells infected by different viruses. In all the virus-cell systems investigated, virus infection results in a drastically reduced amount of free 3H-inositol about 3 to 5 h post-infection, demonstrable in the infected cells as compared to the mock-infected controls. In contrast, the incorporation of 3H-inositol into lipid can be enhanced, reduced, or not influenced at all, depending on the virus-cell system under observation.


Development of early nuclear antigen in cytomegalovirus infected cells in the presence of RNA and protein synthesis inhibitors. Human cytomegaloviruses (HCMV) induce nuclear antigens as early as I h after infection as detected by immunofluorescence. Early antigen development occurs in the presence of DNA, RNA, and protein synthesis inhibitors and is dependent on intact viral DNA in the inoculum.


Properties of the viruses selected during persistent infection of L cells with VSV. Two virus clones, VSV-mp and VSV-sp, were isolated from L cells persistently infected with VSV (New Jersey serotype). Both clones were more temperature sensitive than the parent virus, VSV-o, and grew more slowly, gave smaller plaques, less c.p.e. and lower virus yields in L cells. Unlike the parent virus, both persistent viruses induced interferon production in L cells. Stable carrier cultures could be obtained from L cells infected with VSV-sp at low multiplicities without pretreatment with interferon. This may be related to the fact that VSV-sp is more sensitive to interferon than either VSV-mp or VSV-o.


Studies of Epstein-Barr virus (EBV)-associated nuclear antigen: solubilization from Raji cell chromatin with 5 M-urea-2 M-nacl and fractionation on hydroxyapatite. EBNA-containing chromatin from Raji cells was solubilized by treatment with high concentrations of urea and salt and fractionated by hydroxyapatite chromatography. Fractions eluting at different phosphate concentrations were analysed for the presence of EBNA by means of an anti-EBNA-specific 125I-labelled IgG absorption assay. The antigen was found in fractions containing non-histone chromatin proteins.


Persistence of both human cytomegalovirus and Epstein-Barr virus genomes in two human lymphoblastoid cell lines. By DNA-DNA reassociation kinetic analysis, less than one genome equivalent per cell of human CMV-DNA was found in two lymphoblastoid cell lines, one derived from the peripheral blood of a congenitally infected male infant at the age of 21 months (D4 cell line), the other obtained by co-cultivation of lethally X-irradiated cells from the 9-month lymphoblastoid cell line previously described by Joncas et al. (1975) with cord blood leukocytes of a female newborn (M1 cell line). Human CMV antigens could not be detected and virus could not be rescued from these cells by co-cultivation with fully permissive human fibroblasts. It may be that the CMV-DNA is defective. Epstein-Barr virus DNA as well as EBNA and EBV-EA antigens were present in these cell lines. Both lines express surface markers characteristic of thymus-independent, B lymphocytes. The CMV-DNA of the CMV-DU strain, isolated from this infant's urine five times successively from the age of 1 day to 30 months, appears to be closely related to the DNA of the AD-169 strain by reciprocal hybridization and by electrophoretic pattern analysis of the restriction enzyme cleavage products. Experimental attempts to transform cord blood leukocytes with this urine strain of CMV before or after u.v. irradiation have so far failed.


Herpes simplex virus type-1 and human lymphocytes: virus expression and the response to infection of adult and foetal cells. The growth of Herpes simplex virus type I (HSV-I) in human lymphocytes of adult and foetal origin was studied. Virus DNA synthesis, antigen and particle production and the yield of infectious progeny were determined in cultured lymphocytes with or without exposure to stimulating concentrations of the mitogens phytohaemagglutinin and pokeweed mitogen. Separated sub-populations of cells were examined and the conclusion reached that only the stimulated T-lymphoblast was permissive for full virus expression. Stimulation of cell DNA synthesis in response to infection was observed in cultures of adult and foetal lymphocytes under conditions which were nonpermissive for virus growth. Morphological change and prolonged culture survival were a feature of foetal lymphocytes exposed to u.v. irradiated HSV-I.


Herpes simplex virus necleic acid synthesis following infection of non-permissive XC cells. DNA hybridization kinetic analysis of cellular DNA following high multiplicity infection of non-permissive XC cells by herpes simplex virus type I showed that HSV DNA penetrates to the nucleus of the cell but that the number of virus DNA copies present in each cell quickly begins to decline. There did not appear to be any net virus DNA synthesis and the loss of virus DNA copies continued until there was approximately one per haploid genome equivalent. HSV-2 likewise did not show any detectable virus DNA replication. The residual virus information was stable for more than 48 h. CsCl density gradient analysis of the infected cell DNA suggested an association between the HSV DNA and that of the cells. Network analysis also supported the suggestion that a stable association between the virus DNA and host DNA begins shortly after infection. Cell division resulted in the segregation of the virus DNA but not its loss from the cell population. Virus-specific RNA synthesis was easily detectable and 40 to 50% of a labelled DNA probe was converted to an RNA:DNA hybrid.


OK 10 virus, an avian retrovirus resembling the acute leukaemia viruses. The OK 10 virus complex was isolated from a liver tumour of a chicken which, as an embryo, had been inoculated intravenously with a field isolate of an avian leukosis virus. The OK 10 virus complex contains at least two viruses: the interference assay and serum neutralization test indicate that the helper virus belongs to subgroup A. One of the viruses, OK 10 V, induces distinct foci in chick embryo cells under agar overlay and cells from the foci form colonies in soft agar. These properties allow in vitro assay of the virus. Injection of virus or infected cells into chicks induces acute leukaemia but no local tumours. Another virus, OK 10 AV (associated virus), comprises about 99% of the OK 10 complex. The virus does not induce foci in chick embryo cells. In chickens it causes leukosis 17 months after injection. Electron micrographs of OK 10 virus stocks show typical C type virus particles. These particles have a density of 1.16 g/ml and contain 70S RNA which, after heat denaturation, releases type b RNA subunits. The OK 10 virus complex apparently represents a strain of acute leukaemia viruses.


A plaque assay for Mount Elgon bat virus based on intrinsic interference. A plaque count infectivity assay was developed in which chick cells infected with the Mount Elgon bat virus were completely resistant to superinfection with large doses of Sendai virus. Several variables markedly affected the assay sensitivity. The defined plaque assay was simple, highly reproducible and sensitive. It allowed determination of virus neutralizing antibody in a simple and reproducible test. Both actinomycin D and 5-iododeoxyuridine were without effect on plaque formation.


Reversion of temperature sensitive transformation mutants of Rous sarcoma virus and its effect on the expression of tumour specific surface antigen. Rous sarcoma virus (RSV) mutants, which bear temperature sensitive (ts) defects in both the maintenance of cell transformation and the expression of tumour specific cell surface antigen(s) (TSSA), have yielded a number of revertants. In seven revertants studied, the acquisition of wild type transforming capacities is always accompanied by a wild type TSSA expression. This strongly indicates that transformation and TSSA expression in RSV are affected by the same mutation.


Noradrenaline receptor sensitivity after chronic ethanol administration. The effects of the central noradrenaline receptor stimulating agent clonidine on blood pressure, heart rate, and flexor reflex activity were studied in intact rats and in rats chronically treated with ethanol. The clonidine-induced changes were similar in ethanol-treated animals and in animals never subjected to ethanol. The results suggest that the sensitivity of central noradrenaline receptors involved in the mediation of the circulatory changes and of the flexor reflex activity after clonidine is not altered by chronic administration of ethanol.


Physiological properties of afferents and synaptic reorganization in the rat cerebellum degranulated by postnatal X-irradiation. Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x-irradiation applied during the first two weeks of postnatal life. Electrical stimulation of the brain stem and peripheral limbs was employed to investigate the properties of afferent cerebellar pathways and the nature of the reorganized neuronal synaptic circuitry in the degranulated cerebellum of the adult. Direct contacts of mossy fibers on Purkinje cells were indicated by short latency, single spike responses: 1.9 msec from the lateral reticular nucleus of brain stem and 5.4 msec from ipsilpateral forelimb. These were shorter than in normal rats by 0.9 and 2.1 msec, respectively. The topography of projections from peripheral stimulation was approximately normal. Mossy fiber responses followed stimulation at up to 20/sec, whereas climbing fiber pathways fatigued at 10/sec. The latency of climbing fiber input to peripheral limb stimulation in x-irradiated cerebellum was 23 +/- 8 (SD) msec. In x-irradiated rats, the climbing fiber pathways evoked highly variable extracellular burst responses and intracellular EPSPs of different, discrete sizes. These variable responses suggest that multiple climbing fibers contact single Purkinje cells. We conclude that each type of afferent retains identifying characteristics of transmission. However, rules for synaptic specification appear to break down so that: (1) abnormal classes of neurons develop synaptic connections, i.e., mossy fibers to Purkinje cells; (2) incorrect numbers of neurons share postsynaptic targets, i.e., more than one climbing fiber to a Purkinje cell; and (3) inhibitory synaptic actions may be carried out in the absence of the major inhibitory interneurons, i.e., Purkinje cell collaterals may be effective in lieu of basket and stellate cells.


A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems. Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P0 protein and a basic protein of slightly larger mol wt. than the P1 protein of rat myelin.


Herpes simplex virus types 1 and 2 in organotypic cultures of mouse central and peripheral nervous system. I. Light microscopic observations of myelin degeneration. Mature mouse spinal cord-ganglion cultures, which contain both peripheral and central nervous system as one unit, were infected with herpes simplex virus type 1 (HSV 1) or type 2 (HSV 2) and observed by bright field microscopy for up to 72 hours. There was degeneration of both central and peripheral myelin in cultures infected with either virus, but the pattern of peripheral myelin degeneration associated with HSV 1-infected cultures was differrnt from that in HSV 2-infected cultures. Type 1 was charcterized by focal dilatations; type 2 by "sausage-shaped" swellings, and the cytopathic effect of HSV 2 both began (6 hours p.i.) and was completed (36 hours p.i.) earlier than in cultures infected with HSV 1 (12 hours and 48 hours p.i. respectively). In central nervous tissue, the apperance of degenerating myelin after infection with HSV 1 was indistinguishable from  that in HSV 2-infected cultures, but the rate of myelin loss was greater in cultures infected with the type 2 virus. Evidence is presented which suggests that, at least in the peripheral nervous system,myelin degeneration did not appear to be dependent on neuronal or axonal dysfunction or death, but was a direct result of virus infection.


Morphogenesis of the Hirano body in neurons of the squirrel monkey dorsal horn. Electron microscopic investigation of laminae I-III in the lumbosacral region of the squirrel monkey spinal cord has disclosed possible stages in the morphogenesis of the Hirano body. These bodies occur occasionally in nerve cell bodies and dendrites. They are round to oval in shape, measure up to 2.2 micrometer in diameter and are composed of circular layers of 10 nm filaments. In several instances in the present study, Hirano bodies were observed in close association with the Golgi apparatus. Here Golgi-related vesicles were attached to the external surface of the Hirano bodies. In one instance a cluster of developing Hirano bodies of various sizes was observed. The smaller bodies were located nearest the Golgi apparatus while the larger were further away. In this case several short filaments, which may be precursors of the longer filaments which make up the layers of the inclusion, were observed in the cytoplasm between the Golgi cisternae and the smaller Hirano bodies. These observations suggest that the Golgi apparatus plays a major role in the production of the Hirano body. The significance of the occurrence of these inclusions is discussed.


Schwann cell abnormalities in 2,5-hexanedione neuropathy. Distinctive cytoplasmic alterations of Schwann cells were observed by electron microscopy in rats and mice with peripheral neuropathy induced by chronic exposure to 2,5-hexanedione. Pronounced enlargement of Schwann cells was due to accumulation of 100 angstrom cytoplasmic filaments and endoplasmic reticulum and was most often observed after 12--15 weeks exposure to 2,5-hexanedione. Examination of teased nerve fibres revealed segmental demyelination and remyelination involving axons of normal diameter as well as giant axons. The filament disorder induced by 2,5-hexanedione administration is not limited solely to axoplasmic contents. Possible mechanisms of demyelination are discussed and the changes are compared to those observed in human neuropathy for which 2,5-hexanedione appears to be the closest experimental model.


Inhibition of Na+-K+-activated ATPase activity following experimental spinal cord trauma. The specific activity of the membrane-bound enzyme, Na+-K+-activated adenosine triphosphatase (ATPase), has been shown to be decreased following experimental impact injury (400 gm-cm) to the spinal cord in dogs. The prompt and significant (p less than 0.01) fall in activity was evident as early as 5 minutes after injury, and remained at 56% to 67% of control for the 1-hour period studied. This decrease was most prominent in the central core of the traumatized segments of spinal cord. Central core samples, excised immediately adjacent to the trauma site, gave values for the Na+-K+-activated enzyme intermediate to those of the trauma and control sites. For these same samples, the activity of the Mg+2-dependent ATPase did not change appreciably. No alterations were observed in the tissue surrounding the zone of maximum injury at these early time periods. The relationship of membrane-bound enzyme alterations to blood flow, clotting mechanisms, and abnormal free radical reactions are briefly discussed.


Delayed cure of Cushing's disease after transsphenoidal surgery of pituitary microadenomas. Report of two cases. Transsphenoidal microdissection has been proposed as a preferred means of treating Cushing's disease. This procedure allows the surgeon to remove a pituitary microadenoma and at the same time to preserve normal tissue. Two cases described here were treated by this method. An interesting and important observation was that neither patient appeared to be cured for 2 to 6 weeks after surgery, as assessed by dexamethasone suppression. Later, normal suppressibility occurred and the course of each patient was compatible with cure. Patients treated by this method should not be automatically retreated because of adrenocorticotropic hormone (ACTH) non-suppressibility in the early postoperative period.


Inhibitory effect of cadmium on vitamin D-stimulated calcium transport in rat duodenum in vitro. In vitro effect of cadmium on vitamin D-stimulated calcium transport in the rat was studied using the everted gut sac technique. Calcium transport was significantly inhibited by the addition of cadmium to the mucosal incubation medium. Furthermore, the kinetic analysis of inhibitory effect of cadmium on calcium transport revealed that the type of inhibition was competitive. The apparent "Km" and "Vmax" value for calcium was 1.0 mM, and 2.86 mumoles ml-1.hr-1, respectively. An apparent "Ki" for cadmium was 0.02 mM.


Prevalence of growth hormone deficiency in children with cleft lip or palate. Two hundred children 7 to 14 years of age with isolated cleft defects of the lip, palate, or both were surveyed for stature. Twelve percent were less than the third percentile in height and were designated "short." All of the short children received an endocrine evaluation. Endogenous growth hormone was examined after two days' pretreatment with stilbesteral. Four of the 25 short children with CLP had total, and four had partial, GH deficiency. Three of the GH-deficient patients were also deficient in ACTH or TSH. In contrast, in a series of 75 short (less than third percentile) children 7 to 14 years of age without cleft defect or other apparent congenital abnormality, only two had total and two had partial GH deficiency. The data suggest that children with isolated CLP have short stature about four times more often, and GH-deficiency about 40 times more often, than children without CLP. The increased prevalence of GH-deficiency may stem from the embryologic relation of adenohypophysis and oral ectoderm.


Prospective study of enteropathogens in children with diarrhea in Houston and Mexico. During a 22-month period, 595 children with diarrhea and 210 age-matched controls attending clinics in Houston (367 children) and Mexico (438) were prospectively evaluated for enteric pathogens. Enteropathogens associated with disease were Shigella (18%), rotavirus (14%), Salmonella (9%), toxigenic Escherichia coli (6%), and others (12%), including 14 Proteus isolates that caused rounding of adrenal cells. Enteropathogens were isolated from a greater (P less than 0.001) number of children with diarrhea (59%) than from asymptomatic controls (6%). Paired sera tested for antibody to heat-labile toxin of E. coli rarely demonstrated a fourfold rise during episodes of diarrhea. This study demonstrates: (1) more striking illness in children from Mexico; (2) more common occurrence of Shigella in Houston, and of rotavirus and Salmonella in Mexico; (3) lack of seasonal occurrence of rotavirus isolation in either population and a summertime occurrence of Shigella in Houston; (4) lack of toxigenic E. coli isolation in endemic diarrhea of either population; and (5) a significant (P less than 0.001) age-related acquisition of E. coli LT antibodies.


Extrarenal Wilms tumor.

Extrarenal Wilms tumor. The previous reports of extrarenal Wilms tumor are reviewed and two additional patients presented. The clinical and pathologic features are discussed. The diagnosis of extrarenal Wilms tumor does not imply a bad prognosis. Treatment should utilize multidrug chemotherapy similar to the guidelines set by the National Wilms Tumor Study.


Preparation and immunological cross-reactions of penicilloic and penilloic acids. Methods are described for the preparation of pure crystalline samples of the penicilloic and penilloic acids of penicillin G, carbenicillin, cloxacillin, floxacillin, methicillin, penicillin V, and ticarcillin and the penicilloic acids of amoxicillin, ampicillin, phenethicillin, and propicillin. The interaction between the compounds and rabbit antibenzylpenicilloyl antibodies was evaluated by hemagglutination inhibition measurements. A significant correlation was found in this system between the reactivity of penicilloic acids and the corresponding penilloic acids; on average, the penicilloic acids were more reactive on a molar basis by a factor of 11. The results are discussed in terms of the general immunochemistry and side-chain structure of the parent penicillins.


Steady-state urinary excretion method for determining bioequivalence of conjugated estrogen products. The steady-state excretion of conjugated estrogens in the urine of postmenopausal women dosed with conjugated estrogens tablets was studied using a modification of a previously published method. The procedure was used to quantitate the estrogens both before and during conjugated estrogens replacement therapy. The method, which is relatively specific, involves enzyme hydrolysis of urine samples a number of classical extraction and purification steps, and analysis of the silylated estrogens on a 2.7-m, 1.7% diethylene glycol succinate column using flame-ionization detection. The results indicate that steady-state urinary estrogen excretion levels were obtained within 17 days of dosing. Furthermore, the urinary estrogen excretion profile was significantly different from the composition of the estrogens in the dosage form.


Reaction of sodium hydroxymethanesulfonate with substituted anilines. The reactions of substituted anilines with sodium hydroxymethanesulfonate to form the anilinomethanesulfonates were studied in 50% ethanol--water at 0--50 degrees. The Arrhenius rate constants were 5.4 X 10(10) exp(--16,400/RT) M-1 min-1 for aniline, 4.8 X 10(11) exp(--17,100RT) M-1 min-1 for p-anisidine, 7.1 X 10(9) exp(--14,500/RT) M-1 min-1 for p-toluidine, 1.5 X 10(13) exp(--21,100/RT) M-1 min-1 for p-chloroaniline, and 1.1 X 10(12) exp(--19,800/RT) M-l min-1 for p-bromoaniline. Some equilibrium constants and hydrolysis rate constants of the products also were calculated. Hydrolysis rate constants were temperature independent. These reactions had a p value of --3.40 in the Hammett equation. The solvent concentrations used proved to be very convenient for obtaining high yields of the aminomethanesulfonates.


The pharmacological properties of some crustacean neuronal acetylcholine, gamma-aminobutyric acid, and L-glutamate responses. 1. A study was performed of the L-glutamate, gamma-aminobutyric acid (GABA), and acetylcholine (ACh) responses of cells in the stomatogastric ganglion of the crab, Cancer pagurus. 2. Ionophoretic or pressure application of L-glutamate revealed three classes of responses: a K+-dependent inhibition which reversed at 15-20 mV more negative than the resting potential; a Cl- dependent inhibitory response which was at equilibrium at the resting potential; and a depolarizing response. 3. Ionophoretic or pressure applications of GABA likewise produced three kinds of responses: an increase in K+ conductance, an increase in Cl- conductance, and a depolarizing response. 4. Picrotoxin (10(-6)-10(-5) M) was effective in blocking both the glutamate inhibitory responses. 10(-4) M-picrotoxin, which was necessary to produce a 50% block of the GABA-K+-dependent response, had no effect on the GABA-Cl- response. 5. beta-Guanidinopropionic acid (beta-GP) was found to be an agonist for the GABA-K+ response, but was ineffective in mimicking or blocking the GABA-Cl- response. 6. ACh applications produced large depolarizing responses with a pharmacological profile similar to that of the nicotinic ganglionic response in vertebrates. 7. The muscarinic agonist, acetyl-beta-methyl choline (MeCh), produced depolarizations which decreased in amplitude as the membrane was hyperpolarized from -40 to -100 mV. Pilocarpine and oxotremorine produced changes in the endogenous activity of ganglionic neurones. 8. Implications of these results for the identification of synaptic transmitters in the somatogastric ganglion are discussed.


Transmission along and between rods in the tiger salamander retina. 1. The electrical pathways that couple the rods and that link the outer segments of the rods to the coupled network, were evaluated. Two separate micro-electrodes were inserted into the inner or outer segments of the same or neighbouring rods under visual control. Current was passed through one electrode, and the resulting potential recorded with the other. 2. The input resistance, measured at the inner or outer segment in a rod in the network, is strongly outward rectifying. It is typically near 40 Momega when the membrane is hyperpolarized 10 mV or more by extrinsic current, less than 10 Momega when the membrane is depolarized by 5 mV or more, and near 30 Momega at the no-current level. 3. When current is injected into the outer segment, the response in the inner segment is nearly identical with that at the outer segment, suggesting that the resistance coupling the segments is not high relative to the input resistance of the rod in the network. 4. Under voltage clamp the light response current for a rod in the network is of constant magnitude for potential levels between -80 and -20 mV. This suggests that there is little or no measurable light elicited conductance change associated with the response, possibly a consequence of coupling between rods. 5. The rod response increases with increasing diameter of a concentric test flash up to about 200 micron, or about 16 rod diameters. 6. When current is injected into one rod, the response in its immediate neighbours is between a quarter and one tenth that recorded in the injected rod for all potential levels in the injected rod. 7. The membrane time constant, measured in a rod in the network, is proportional to the voltage-dependent input resistance at 0.16 msec/Momega. With assumptions about the geometry of the rod network this represents a membrane capacitance of 1.5 muF/cm2. 8. The data can be approximated by a network model of square array. The model predicts that: the outer segment contributes less than half the current for the total rod response, the membrane resistance of an individual rod is greater than twice the measured input resistance for the rod in the network, near 60 Momega, and the coupling resistance for each arm of the network is about 4 times the individual rod resistance, near 240 Momega.


Regulation by patterned preganglionic neural activity of transmitter stores in a sympathetic ganglion. 1. The effect of patterned preganglionic stimulation on acetylcholine (ACh) stores was studied in the superior cervical ganglion of the cat. 2. Patterns of stimulation based on 0.5 and 1.0 s trains at frequencies from 10 to 80/s and intervals between trains of 2-180 s increased ACh stores, over periods of 1-3 h stimulation, to the same extent (30-70%) as found earlier to result from continuous stimulation. 3. If the mean frequency of the overall pattern was greater than 5/s the increase in stores developed after termination of the stimulation. If the mean frequency was less than 5/s the increase developed progressively during stimulation and it was then maintained without accommodation over 7 h of stimulation. 4. The magnitude of the increase in ACh stores was linearly related to the frequency within the trains from 10 to 40/s, decreasing with further increase in frequency, and absent below 6-7/s. 5. The ACh stores were increased by patterns of stimulation that mimicked discharging of preganglionic neurones driven by the central respiratory drive under hypercapnic or asphyxial stress; and were increased similarly in animals respired with hypercapnic or partially asphyxial gas mixtures when the preganglionic innervation was intact, but not when it was severed. 6. The variance between ACh stores of ganglia from different animals was reduced in animals held in a controlled environment for 1-2 weeks before experimentation. 7. It is concluded that patterning of preganglionic inputs into high frequency trains is a code for the ongoing modulation of transmitter stores in a sympathetic ganglion. 8. The findings are discussed in relation to current knowledge of ganglionic transmission and to after-hyperpolarization in mammalian unmyelinated nerve fibres.


Target tissues for relaxin identified in vitro with 125I-labelled porcine relaxin. Various tissues from the mouse, rat and guinea-pig were used to examine the binding of a biologically active, esterified and 125I-labelled porcine relaxin. Binding to mouse symphysial homogenates was time- and temperature-dependent. Other peptide hormones did not complete with relaxin for binding. Mouse uterine tissue displayed similar binding characteristics. Fractionated mammary tissue from 15- and 20-day-pregnant rats exhibited significant relaxin binding activity, as did homogenates of the guinea-pig public symphysis and cervix. Under the conditions used, no relaxin receptors were noted in the liver, spleen or heart from any of the species investigated.


Binding of 125I-labelled FSH in the mouse testis in vitro. In-vivo testicular binding of highly purified pituitary FSH, labelled by a method which did not significantly affect biological potency, was hormone-specific, tissue-specific and dose-dependent. Hypophysectomy of mice was followed by a progressive increase in the amount of 125I-labelled FSH per unit weight of testis but not in the total amount of hormone taken up by the testis. Maximum binding occurred at 4 h in a membrane-containing fraction prepared by high-speed ultracentrifugation of testicular homogenate. This is considerably later than has been reported for testicular tissue incubated in vitro at 37 degrees C.


Synthesis and antiviral acticity of some phosphates of the broad-spectrum antiviral nucleoside, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin). 1-beta-D-Ribofuranosyl-1,2,4-triazole-3-carboxamide 5'-phosphate (2) was prepared and converted into the following derivatives: the 5'-phosphoramidate 3, the 5'-diphosphate 4, the 5'-triphosphate 5, and the cyclic 3',5'-phosphate 6. The cyclic 2',3'-phosphate 7 was prepared from the parent nucleoside, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (1), and was opened to the 2'(3')-phosphate 8. These compounds were found to exhibit significant antiviral activity against several viruses in cell culture. Ribavirin 5'-phosphate (2) was shown to be effective when tested against lethal infections in mice caused by influenza A2, influenza B, and murine hepatitis viruses.


Synthesis and biological activity of new 2-nitroimidazole derivatives. In an earlier paper we described the synthesis and the antitrichomonas activity of 2-nitro-alpha,alpha,1-trimethyl-1H-imidazole-5-methanol (2). Starting from this compound, several derivatives have been synthesized. Among these, the phenyl carbonate 8 has been shown to possess activity equal to that of 2 and to be less toxic. This compound therefore is interesting in comparison with some antitrichomonas agents currently in use clinically. Before undertaking an in-depth investigation, compound 8 was subjected to some studies to see whether it has any effects on the central nervous system (CNS). Preliminary results show that, at therapeutic doses, it might induce unwanted CNS effects to a lesser degree than metronidazole.


The role of cyclic AMP in parathyroid hormone action in the toad bladder. Parathyroid hormone (PTH) inhibited active transport of inorganic phosphate and stimulated an increase in cyclic AMP concentration in the urinary bladder of the toad, Bufo marinus. Active transport of phosphate in the toad bladder was also inhibited by an analog of cyclic AMP (dibutyryl cyclic AMP) and by other drugs (pitressin and theophylline) which increase toad bladder intracellular cyclic AMP concentration. These data support the concept that cyclic AMP may be the mediator of PTH-induced phosphate transport inhibition in the toad bladder.


Demonstration of Marek's disease tumor-associated surface antigen in chickens infected with nononcogenic Marek's disease virus and herpesvirus of turkeys. The presence of Marek's disease tumor-associated surface antigen (MATSA) was demonstrated on spleen cells from P-line chickens inoculated 5--6 days earlier with herpesvirus of turkeys and SB-1 (a nononcogenic Marek's disease virus). Antisera against MATSA expressed on five Marek's disease lymphoblastoid cell lines were able to recognize the MATSA present on SB-1-infected spleen cells. No viral membrane antigens and only a low incidence of viral internal antigens could be demonstrated.


Ependymomas, malignant tumors of pancreatic islets, and osteosarcomas induced in hamsters by BK virus, a human papovavirus. BK virus (BKV), a human papovavirus, was inoculated iv into 3-week-old Syrian golden hamsters. Between 2 1/2 and 9 months after inoculation, 82% of the animals developed tumors. The induced neoplasms were ependymoma, carcinoma of the pancreatic islets, osteosarcoma, adenocarcinoma, angiosarcoma, angioma, lymphoma, and seminoma. Hypersecretion of insulin, glucagon, C-peptide, and calcitonin was detected in tumors of pancreatic islets. BKV etiology of tumors was supported by the following evidence: 1) No tumors with BKV-specific markers appeared in animals given injections of buffer, animals inoculated with BKV neutralized by anti-BKV-specific serum, or uninoculated controls; 2) BKV tumor (T) antigen was detected by immunofluorescence and complement fixation tests in tumors of animals inoculated with infectious BKV and in transplanted tumors; 3) antibodies to BKV T-antigen were detected in sera of animals bearing primary or transplanted tumors; 4) BKV could be activated by Sendai virus-mediated fusion of neoplastic cells with susceptible Vero cells; and 5) no endogenous hamster oncornaviruses were found in tumors.


Preparation of plasma membranes from line 10 and line 1 guinea pig hepatomas. Gentle homogenization followed by differential and density gradient centrifugation was used to purify line 10 and line 1 guinea pig hepatoma plasma membranes in the form of ghosts. Yields of 15--25% allowed enough membranes to be obtained from a single ascites tumor-bearing animal for immunologic and biochemical studies. Although the plasma membrane marker enzyme (Na+ + k+)atpase was present in normal concentrations in both line 10 and line 1 hepatomas, 5'-nucleotidase was reduced over 100-fold in both tumors and phosphodiesterase I was increased 210-fold in the line 10 hepatomas.


The effect of radiotherapy in the treatment of adenoid cystic carcinoma of the head and neck arising in minor salivary glands. Eighteen cases of adenoid cystic carcinoma of the minor salivary glands are reviewed. Noteworthy in the history is the report of pain at the site of the lesion which radiates elsewhere, or of numbers or tingling in its area. Radiation therapy is as able to control the primary as local surgery. Involvement of a much wider field than is required to treat the primary may control the perineural spread common to this tumor and avoid the massive procedures necessary to cure it by surgical means. Metastases to the lung, bone, and brain by venous spread can probably be avoided only by early diagnosis.


Herpesvirus infection modifies adenovirus RNA metabolism in adenovirus type 5-transformed cells. The effect of herpes simplex virus (HSV) infection of mRNA metabolism was examined in a system where the fate of specific RNA sequence can be assayed. Adenovirus type 5-transformed rat embryo cell line 107 synthesizes adenovirus-specific RNA (ad-RNA), which functions in the cytoplasm as mRNA. We have utilized ad-RNA as a model for mRNA metabolism, and in a preliminiary study we characterized ad-RNA in the nucleus and cytoplasm by hybridization to filter-bound adenovirus DNA. The results indicated the as-RNA accumulates in the nucleus and that cytoplasmic polyadenylic acid [poly(A)]-containing ad-RNA turns over with a half-life of a few hours. Pulse-chase experiments confirmed these observations and a half-life of about h was determined for the poly(A)-containing cytoplasmic ad-RNA. A second class of ad-RNA remains in the nucleus, where it turns over with a longer hlaf-life (about 24 h). The infection of 107 cells by HSV was restricted at 37 degree C, giving a burst size of 5 PFU per cell and allowing continued host DNA synthesis. Protein synthesis was inhibited greater than 50% by 7 h after infection, and total RNA synthesis was 50% inhibited by 4 h after infection. During the first 8 h after infection, HSV has little effect on the rate of synthesis of ad-RNA as determined by hybridization of nuclear RNA samples, but,during the same period, HSV inhibits the accumulation of poly(A)-containing ad-RNA in the cytoplasm. The degree of this inhibition increases steadily throughout this period and reaches 60% by 6.5 to 8 h after infection. Nosignificant effect was seen on the accumulation of total cellular poly(A)-containing RNA. It was concluded from these experiments that HSV infection alters the metabolism of ad-RNA so as to prevent the normal appearance of the poly(A)-containing mRNA in the cytoplasm. The result for ad-RNA may not represent the behavior of total cellular poly(A)-containing RNA under conditions where infection is restricted.


Photochemical addition of the cross-linking reagent 4,5', 8-trimethylpsoralen (trioxaslen) to intracellular and viral simian virus 40 DNA-histone complexes. We demonstrated here that 4,5', 8-trimethylpsoralen (trioxsalen) is a valuable probe for the structure of SV40 DNA-histone complexes. Trioxsalen readily penetrated intact cells and, in the presence of 340- to 380-nm light, covalently cross-linked DNA preferentially at the sites available for micrococcal nuclease digestion. Histograms of the lengths of the regions of SV40 DNA protected from cross-linking, as visualized by electron microscopy, indicated a repeating pattern of base pairs in DNA from both infected cells and virus particles. The ability of the trioxsalen probe to act in vivo and to map the location of protected  regions may provide a powerful tool for analyzing the role of nucleosomes in the structure of the virus particle and in intracellular complexes such as transcription templates and replication intermediates.


Oncogenicity of AKR endogenous leukemia viruses. Four biologically distinct groups of endogenous murine leukemia virus (MuLV) have been isolated from AKR mice. These viruses included (i) ecotopic XC+ MuLV that occur in high titer in normal tissues and serum of AKR mice throughout their life span, (ii) ecotropic XC- MuLV that are produced in high titers by leukemia cells, (iii) xenotropic MuLV that are readily demonstrable only in aged mice, and (iv) polytropic MuLV thatarise in the thymuses of aged mice as a consequence of genetic recombination between ecotropic and xenotropic MuLV. Virus of each of  these biological classes were assayed in AKR mice for their ability to accelerate the occurrence of spontaneous leukemia. Certain isolates of ecotropic XC- MuLV and polytropic MuLV were found to have high oncogenic activity. These viruses induced 100% leukemias within 90 days of inoculation. In contrast, ecotropic XC+ MuLV that were obtained from AKR embryo fibroblasts and xenotropic MuLV that were obtained from the lymphoid tissues of aged AKR mice did not demonstrate oncogenic activity. These findings demonstrate fundamental differences between XC- and XC+ ecotropic MuLV that are found in leukemic and normal tissues, respectively. Furthermore, these findings point to the role of ecotropic XC- and polytropic MuLV in the spontaneous leukemogenesis of AKR mice.


Isolation and preliminary characterization of temperature-sensitive mutants of encephalomyocarditis virus. Thirty temperature-sensitive mutants of encephalomyocarditis virus have been isolated and partially characterized. Fifteen of these mutants are phenotypically RNA+ thirteen are RNA-, and two are RNA +/-. Six RNA + mutants, one RNA- mutants, and one RNA +/- mutant have virions which are more thermosensitive at 56 degree C than the wild-type virions. Hela cells infected at the nonpermissive temperature with any of the RNA+ mutants produced neither infective nor noninfective viral particles. The cleavage of the precursor polypeptides in cells infected with 11 of the RNA+ mutants was defective at the nonpermissive temperature. This defect in cleavage occurred only in those precursor polypeptides leading to capsid proteins.


Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid and dithiothreitol. Treatment of polyoma virions with ethyleneglycol-bil-N,N'-tetraacetic acid (EGTA) and dithiothreitol (DTT) at pH 8.5 resulted in the dissociation of the virions into a DNA-protein complex and individual structural capsomere subunits. The sedimentation value of the DNA-protein complex in sucrose gradients was approximately 48S, and it had a density of 1.45 g/cm3 in equilibrium CsCl gradients. Alkaline sucrose analysis of the DNA within this DNA-protein complex demonstrated that approximately 75% of the DNA is component 1. The proteins associated with the DNA were dissociated by treatment with either NaCl or the anionic detergent Sarkosyl. VP1 and the histone proteins VP 4--7 were the major proteins associated with the DNA. Treatment of the DNA-protein complex with alkaline pH resulted in the specific removal of FP1. Electron microscopy of the 48S DNA-protein complex demonstrated that it is a very tightly coiled structure that is slightly larger than the intact virion. Treatment of the complex with either NaCl or with pH 10.5 buffer resulted in the loss of protein and subsequent loosening of the DNA-protein complex such that the DNA could be visualized. The capsomere subunits released as a result of the EGTA-DTT treatment sedimented as 18S, 12S, and 5S subunits in sucrose gradients. Electrophoretic analysis of the isolated capsomeres demonstrated that VP1, VP2, and VP3 were present in each species, although the ratios of the proteins varied. In addition to the structural proteins, histones VP 4--7 were found to be predominantly associated with the 5S capsomere subunit.


Measurements of the molecular size of the simian virus 40 large T antigen. A measure of the molecular weight of the large simian virus 40 T antigen was sought by SDS-polyacrylamide gel electrophoresis, random-coil chromatography, and sedimentation-velocity analysis in a density gradient. Large T antigen obtained from a simian virus 40-transformed human cell line either by immunoprecipitation or by standard preparatory methods migrated like a 94,000-molecular-weight (approximately  94K) polypeptide in SDS-gels but was found to have an approximate was observed with T antigen obtained from lytically infected monkey cells. In view of the strong theoretical basis for the guanidine method and the agreement with the sedimentation data, these findings suggest that the molecular weight of this protein is approximately 75 to 80K as opposed to 94 to 100K and, therefore, that considerably less than the entire early region of simian virus 40 is required to encode it. This size estimate is in keeping with earlier results which revealed a normal-size T antigen in cells infected with viable deletion mutants lacking as much as 10% of the early region.


Purification of virus-specific RNA from chicken cells infected with avian sarcoma virus: identification of genome-length and subgenome-leghth viral RNAs. Avian sarcoma virus (ASV)-specific RNA was purified from ASV-infected cells by using hybridization techniques which employ polydeoxycytidylic acid-elongated DNA complementary to ASV RNA as well as chromatography on polyinosinic acid-Sephadex columns. The purity and nucleotide sequence composition of purified, virus-specific RNA were established by rehybridization experiments and analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. Polyadenylic acid-containing RNA purified from ASV-infected cells contained approximately 1 to 4% virus-specific RNA, compared with 0.06 to 0.15% observed in uninfected cells. Sucrose gradient analysis of virus-specific RNA isolated from ASV-infected cells revealed two major classes of polyadenylated viral RNA with sedimentation values of 36S and 26-28S. Cells infected with transformation-defective ASV (virus containing a deletion of the sarcoma gene) contained 34S and 20-22S viral RNA species. Double-label experiments employing infected cells labeled initially for 48 h with [3H]uridine and then for either 30, 60, or 240 min with [32P]phosphate showed that the intracellular accumulation of genome-length RNA (36S) was significantly faster than that of the 26-28S viral RNA species.


Anatomy of bovine mammillitis DNA II. Size and arrangements of the deoxynucleotide sequences. We previously reported that bovine mammillitis virus (BMV) DNA consists of two covalently linked components designated L and S and estimated to be 71.5 x 10(6) and 15.7 x 10(6) in molecular weight, respectively; the components invert relative to each other, giving rise to four equimolar populations differing soley in the relative orientation of the two components. We now report that (i) BMV DNA has a contour length corresponding to a molecular weight of 89 x 10(6). (ii) Component L consists of a unique sequence (Ul) bracketed by sequences ab and its inverted repeat b'a', estimated to be of molecular weights 66.1 x 10(6), 2.7 x 10(6),  and 2.7 x 10(6), respectively. (iii) Component S consists of a unique sequence (Us) bracketed be sequence ca and its inverted repeat a'c', estimated to be of molecular weights 8.3 x 10(6), 3.7 x 10(6), and 3.7 x 10(6), respectively. (iv) The a sequences present at the termini of a complete linear molecule (abUlb'a'a'c'Usca) are arranged in tandem so that the DNA can circularize after limited digestion with arranged in tandem so that the DNA can circularize after limited digestion with lambda 5'-exonuclease. The size of the a sequences was estimated to be 0.7 x 10(6) in molecular weight. (v) At least portions of the a sequences are repeated in an inverted orientation immediately adjacent to or near the a sequence. Thus, BMV DNA mimics herpes simplex virus type 1 DNA with respect to the arrangement but not size of deoxynucleotide sequences. The evolutionary relationship of BMV DNA relative to other herpesvirus DNAs is discussed.


Comparison of the genomic organization of Kirsten and Harvey sarcoma viruses. Current studies were undertaken to compare the genomes of Kirsten murine sarcoma virus (Ki-MuSV), Harvey murine sarcoma virus (Ha-MuSV), and the replication-defective endogenous rat virus to understand the function of these viral RNAs. Genome organization and sequence homology were studied by fingerprinting large RNase T1-resistant oligonucleotides and by cross-protecting homologous oligonucleotides against RNase A and T1 digestion with complementary DNA prepared from each of the other viral RNA. Ki-MuSV and Ha-MuSV were found to share an extensive series of rat-derived oligonucleotides begining ca. 1 kilobase (kb) from the 3' end and extending to within 1.5 kb of the 5'end of Ki-MuSV RNA. The total map distance covered in ca. 5.5 kb. The eight oligonucleotides covering the 1.5 kb at the 5' end of Ki-MuSV RNA were not found in Ha-MuSV RNA. Five out of these eight oligonucleotides, however, could be designated with certainty to be of rat virus origin. Since Ha-MuSV is 6.5 kb in size and Ki-MuSV is 8 kb in size, the major difference between them is the 1.5 kb from the replication-defective endogenous rat virus sequences at the 5' end of Ki-MuSV not present in Ha-MuSV. Consistent with the difference in the genome structure, these two sarcoma viral RNA'S yielded distinct major translation products in cell-free systems, I.E., A 50,000-dalton polypeptide (P50) from Ki-MuSV and a 22,000-dalton polypeptide (p22) from Ha-MuSV. These polypeptides  may provide the necessary protein makers for identifying in vivo virus-coded proteins.


Mapping early transcripts of herpes simplex virus type 1 by electron microscopy. RNA displacement loop patterns in intact herpes simplex virus DNA and herpes simplex virus DNA restriction fragments indicate that viral RNA associated with polyribosomes early after infection is transcribed from three major areas of the genome. One area of early transcription is in the short segment of the viral DNA and is roughly delineated by the inverted repeat sequences bounding this segment. The other two areas of early mRNA transcription map in the long segment. Each of three major areas of early mRNA transcription can be further resolved into several regions of freqent looping bordered by regions in which RNA displacement loops are rare. These regions range in size from about 1.5 kilobases to about 9 kilobases with a mean size of about 3.5 kilobases. Although the data do not allow precise assignment of individual early gene locations, it is seen, even at the lowest level of resolution, that the early genes are not completely contiguous but are distributed along the length of the herpes simplex type 1 viral genome.


Heterogeneity of Epstein-Barr virus. III. Comparison of a transforming and a nontransforming virus by partial denaturation mapping of their DNAs. The DNAs of a transforming and a nontransforming Epstein-Barr virus strain, B95-8 AND P3HR-1, were compared by partial denturation mapping. B95-8 viral DNA showed a homogeneous denaturation pattern. In contrast, P3HR-1 viral DNA was heterogeneous, containing at least two classes of molecules, classified into groups A and B and present in a ratio of about 2:1 to 3:1. No evidence could be obtained that molecules from both groups A and B contain identical sequences present in different orientations as described for herpes simplex viral DNA. The majority of sequences present in B95-8 and in P3HR-1 viral DNA group A could be correlated by assuming that different sequences, about 12,000 base pairs long, were inserted or deleted, respectively, at different position of both viral genomes.


New late gene, dar, involved in the replication of bacteriophage T4 DNA. II. Overproduction of DNA binding protein (gene 32 protein) and further characterization. We have previously shown that the arrested DNA synthesis of mutant defective in T4 phage gene 59 can be reversed by a mutation in dar. In this paper, we have examined the effect of the dar mutation on the kinetics of gene 32 protein (DNA binding protein) synthesis, DNA packaging, progeny formation, and several other porcesses. Several lines of evidence are presented showing that the regulation of synthesis of gene 32 protein is abnormal in dar 1-infected cells. In these cells, gene 32 protein, an early protein, is also expressed late in the infectious cycle. Our data also indicate that the packaging og DNA into T4 phage heads is delayed in dar mutant-infected cells, and this in turn results in a 6- to 8-min delay in intracellular progeny formation, although the synthesis of late proteins appears to be normal, as shown by gel electrophoresis. We have also studied the phenotypes of the double mutant dar-amC5 (gene 59). The increased sensitivity to hydroxyurea caused by a mutation in the dar gene can be alleviated by a  second mutation in gene 59, but an increased sensitivity to UV irradiation caused by a mutation in gene 59 cannot be alleviated by a second mutation in the dar gene. Therefore, the double mutant still exhibits abnormalities in the repair of UV lesions.


Regulation of murine cytomegalovirus gene expression. I. Transcription during productive infection. Murine cytomegalovirus RNA synthesis in productively infected mouse embryo cultures was measured by reassociation kinetics with iodinated viral DNA. The data were analyzed by a computer program and indicated the following: before DNA replication approximately 25% of the genome was transcribed into asymmetric transcripts, of which slightly fewer than half of the sequences were recovered from the cytoplasm. After viral DNA replication, approximately 38% of the genome was transcribed (5% as symmetric transcripts), and again less than half of the sequences appeared in the cytoplasm. Both early and late RNA comprised two abundance classes differing about 8- to 10-fold in concentration. Early RNA was a subset of late RNA. The RNE sequences synthesized in late-infected cells in the presence of cytosine arabinoside or cycloheximide were similar to early RNA. Thus, murine cytomegalovirus displays temporal, quantitative, and post-transcriptional controls over gene expression, but the pattern differs considerably from herpes simplex virus.


Evidence for a herpes simplex virus-specific factor controlling the transcription of deoxypyrimidine kinase. A cistron-specific, enzyme-forming-capacity method was used to study the control of herpes simplex virus (HSV)-specific deoxypyrimidine kinase (dPyK) mRNA synthesis. A virus-specific factor was formed by a primary infecting virus, and this factor effected the transcription of dPyK mRNA of a superinfecting virus in the presence of cycloheximide, suggesting that the factor acted in "trans" and was a diffusible one. After the addition of actinomycin D to prevent further transcription and upon removal of cycloheximide, the dPyK mRNA was allowed to express into dPyK activity. A factor from HSV-1 could effect the transcription of dPyK mRNA of both HSV-1 and HSV-2. Amino acid analogs, canavanine or ethionine, inhibited the action of this factor, suggesting that a protein was involved. This protein factor was shown to belong to the alpha (or immediate-early) group of HSV-Specific polypeptides in preductively infected cells.


Interference with simian virus 40 DNA replication by adenovirus type 2 during mixed infection of monkey cells. Infection of monkey cells with human adenovirus (Ad) is abortive, but the infection can be enhanced by coinfecting with simian virus 40 (SV40). However, in the coinfected monkey cells, Ad interferes strongly with SV40 DNA biosynthesis. This interference was found to be a reproducible, delicately controlled phenomenon that was proportional to the multiplicity of infection of Ad and dependent on the active expression of the Ad genome. Newly synthesized SV40 DNA was not broken down in cells after delayed superinfection with Ad, and several early events of SV40 infection such as adsorption, penetration, uncoating, induction of cellular DNA synthesis, and enhancement of Ad infection were not markedly influenced by Ad-mediated interference. It is unlikely that interference is simply due to competition between SV40 and Ad for metabolites, enzymes, or replication sites. The interference effect could be partially neutralized by an increase in the multiplicity of coinfecting SV40 or by an increase in the time interval between SV40 infection and Ad coinfection. Interference was shown to be due to the activity of an Ad early gene product. However, the detailed mechanism of this Ad interference is still unclear.


Transfer of Fv-1 locus-specific resistance to murine N-tropic and B-tropic retroviruses by cytoplasmic RNA. A standardized bioassay for transfer of Fv-1 gene-specific resistance to N-tropic and B-tropic murine retroviruses was developed using X plaque reduction in SC-1 (Fv-1-) cells inoculated with virus. Testing of subcellular fractions of restrictive cells showed that the resistance transfer activity was present in the cytoplasmic (microsomal and cytosol) fractions. The activity of the cytoplasmic extract was destroyed by treatment with ribonuclease, but not with deoxyribonuclease or proteases. RNA prepared by phenol-chloroform extraction of mouse tissues, including embryos and livers of weanling mice, transferred Fv-1 locus-specific resistance into DEAE-dextran-treated SC-1 cells. The activity of isolated RNA preparations against virus of the appropriate host-range type has been demonstrated to correspond to the Fv-1 genotypes of the cell sources. The specific transfer of resistance with cellular RNA was effective within a 5- to 6-h period from 2 h before to 4 to 5 after virus infection. Sucrose gradient centrifugation of the RNA showed that the activity sedimented as a broad peak, with an apparent maximum in the 22S region. Affinity chromatography of whole-cell RNA on polyuridylic acid-Sepharose tended to separate more activity into the polyadenylic acid RNA fraction than the non-polyadenylic acid RNA fraction. Except for the reciprocal inhibitory activity for the two host-range virus types, the RNAs of Fv-1n and Fv-1b specificities showed similar properties in all aspects studied.


Presence of free viral DNA in simian virus 40-transformed nonproducer cells. Extracts from several simian virus 40 (SV40)-transformed nonproducer cells were prepared by the hot-phenol procedure normally used to extract cellular RNA. These extracts contained SV40 infectious units. Part of the infectious units were identified as SV40 form I DNA molecules. The results of reconstruction experiments suggest that SV40 form I DNA is extractable by the hot-phenol procedure because of its fast renaturation rate. The significance of the presence of free viral DNA in nonproducer transformed cells is discussed.


Interaction of vesicular stomatitis virus with lipid vesicles: depletion of cholesterol and effect on virion membrane fluidity and infectivity. Interaction with excess unilamellar phosphatidylcholine (PC) vesicles resulted in depletion of as much as 90% of the cholesterol from the membrane of intact vesicular stomatitis (VS) virus. The cholesterol depletion was not significantly influenced by the proteolytic removal of virion glycoprotein spikes, but it was temperature dependent. Cholesterol depletion caused substantial reduction in anisotropy of the VS virion membrane as measured by fluorescence depolarization of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene; residual adsorbed vesicles represent a significant factor in this apparent increase in virion membrane fluidity. Interaction with PC vesicles resulted in a substantial loss of VS viral infectivity as measured by plating efficiency on L-cell monolayers. Reduction in infectivity appeared to be related to temperature-dependent depletion of virion cholesterol by PC vesicles. Interaction of VS virions with cholesterol-containing PC vesicles resulted in significantly less decline in infectivity, but attempts to restore cholesterol and infectivity to depleted VS virions were unsuccessful. Depletion of virion cholesterol apparently results through collision with PC vesicles rather than movement of cholesterol monomers or micelles through the aqueous phase, because PC vesicle-virion interaction in the presence of cholesterol oxidase did not result in substantial oxidation of translocated cholesterol.


Shedding of the glycoprotein from vesicular stomatitis virus-infected cells. In a culture of Chinese hamster ovary cells infected with vesicular stomatitis virus, there is specific shedding of viral antigens into the medium. This shedding appears to be unrelated to progeny formation or to cell lysis. Although all five of the virus-specific proteins are detected in the extracellular soluble fraction, the major antigen is the Gs protein. This protein has a molecular weight of 54,000. Indirect analysis of the content of sialic acid as well as peptide analysis of the Gs and G proteins of vesicular stomatitis virus suggest that the Gs protein is derived from the G protein by proteolysis. Both proteins are hydrophobic when analyzed by charge-shift electrophoresis. The presence of phenylmethylsulfonyl fluoride in the culture medium or the removal of serum from the culture medium partially reduces the shedding of Gs protein. Increased shedding of the Gs protein is seen when there is an unstable M or matrix protein synthesized by a temperature-sensitive mutant, tsG31. These results indicate that the G protein is cleaved at the cell surface, thus releasing Gs protein into the medium. Furthermore, the stability of G protein at the cell surface appears to be dependent on its association with the M protein.


Identification of a protein linked to nascent poliovirus RNA and to the polyuridylic acid of negative-strand RNA. A protein similar to that previously demonstrated on poliovirus RNA and replicative intermediate RNA (VPg) was found on all sizes of nascent viral RNA molecules and on the polyuridylic acid isolated from negative-strand RNA. 32P-labeled nascent chains were released from their template RNA and fractionated by exclusion chromatography on agarose. Fingerprint analysis using two-dimensional polyacrylamide gels of RNase T1 oligonucleotides derived from nascent chains of different lengths showed that a size fractionation of nascent chains was achieved. VPg was recovered from nascent chains varying in length from 7,500 nucleotides (full-sized RNA) to about 500 nucleotides. No other type of 5' terminus could be demonstrated on nascent RNA, and the yield of VPg was consistent with one molecule of the protein on each nascent chain. These results are consistent with the concept that the protein is added to the 5' end of the growing RNA chains at a very early stage, possibly as a primer of RNA synthesis. Analysis of the polyuridylic acid tract isolated from the replicative intermediate and double-stranded RNAs indicated that a protein of the same size as that found on the nascent chains and virion RNA is also linked to the negative-strand RNAs. It is likely that a similar mechanism is responsible for initiation of synthesis of both plus- and minus-strand RNAs.


Structure of the joint region and the termini of the DNA of herpes simplex virus type 1. Analysis of restriction endonuclease cleavage sites within the inverted, repeated sequences in the joint region of the DNA of herpes simplex virus type 1 strain KOS revealed the presence of two types of sequence heterogeneity. The first was an insertion of 280 base pairs or multiples of 280 base pairs which was found in approximately half of all DNA molecules from every plaque-purified stock of virus. These insertions seemed to be tandem duplications of sequences which were present at the joint and correspond closely to the inverted terminal redunancy. The second type of heterogeneity was due to variable insertions and deletions which were present in some, but not all, plaque-purified virus stocks. Comparison of restriction fragments from the joint region with fragments from the termini indicated that in the simplest observed molecules of herpes simplex virus type 1 DNA, only one copy of the inverted terminal redundancy was present at the joint. A map of restriction endonuclease cleavage sites in the joint region is presented.


DNA of Epstein-Barr virus. III. Identification of restriction enzyme fragments that contain DNA sequences which differ among strains of Epstein-Barr virus. Previous kinetic and absorption hybridization experiments had demonstrated that the DNA of the B95-8 strain of Epstein-Barr virus was missing approximately 10% of the DNA sequences present in the DNA of the HR-1 strain (R.F. Pritchett, S.D. Hayward, and E. Kieff, J. Virol. 15:556-569, 1975; B. Sugder, W.C. Summers, and G. Klein, J. Virol. 18:765-775, 1976). The HR-1 strain differs from other laboratory strains, including the B95-8 and W91 strains, and from virus present in throat washings from patients with infectious mononucleosis in its inability to transform lymphocytes into lymphoblasts capable of long-term growth in culture (P. Gerber, Lancet i:1001, 1973; J. Menezes, W. Leibold, and G. Klein, Exp. Cell. Res. 92:478-484, 1975; G. Miller, D. Coope, J. Niederman, and J. Pagano, J. Virol. 18:1071-1080, 1976; G. Miller, J. Robinson, L. Heston, and M. Lipman, Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974). In the experiments reported here, the restriction enzyme fragments of Epstein-Barr virus DNA which contain sequences which differ among the HR-1, B95-8, and W91 strains have been identified. The DNA of the HR-1, B95-8, and W91 strains each differed in complexity. The sequences previously shown to be missing in the B95-8 strain were contained in the EcoRI-C and -D and Hsu I-E and -N fragments of the HR-1 strain and in the EcoRI-C and Hsu I-D and -E fragments of the W91 strain. The HR-1 strain was missing DNA contained in EcoRI fragments A and J through K and Hsu I fragment B of the B95-8 strain and in the EcoRI-A and Hsu I-B fragments of the W91 strain. The relationship of these data to the linkage map of restriction enzyme fragments of the DNA of the B95-8 and W91 strains (E. Kieff, N. Raab-Traub, D. Given, W. King, A.T. Powell, R. Pritchett, and T. Dambaugh, In F. Rapp and G. de-The, ed., Oncogenesis and Herpesviruses III, in press; D. Given and E. Kieff, submitted for publication) and the possible significance of the data are discussed.


Polyoma virus has three late mRNA's: one for each virion protein. Polyoma virus mRNA, isolated from the cytoplasm of 3T6 cells late after infection and purified by hybridization to HpaII fragment 3 of polyoma virus DNA, was separated on 50% formamide-containing sucrose density gradients, and the fractionated RNA was recovered and translated in vitro. Analysis of the cell-free products showed that the minor virion protein VP3 was synthesized from an mRNA sedimenting at approximately 18S betweeen the 19S VP2 mRN and the 16S VP1 mRNA. Other experiments showed that the VP2 and VP3 can be labeled with formyl methionine from initiator tRNA. We conclude that there are three late polyoma virus mRNA's, each directing the synthesis of only one viral capsid protein.


Chromatographic separation of the polyoma virus proteins and renaturation of the isolated VP1 major capsid protein. Treatment of purified polyoma virions with 6 M guanidine-hydrochloride and 0.01 M beta-mercaptoethanol resulted in the immediate loss of both hemagglutinating and plaque-forming ability. Gel filtration through Sepharose CL-6B beads allowed separation of the dimer, VP1, VP2, VP3, and histone proteins VP4-7 in highly purified form. Renaturation of the purified VP1 protein resulted in the formation of subunits that were morphologically, biophysically, and immunologically similar to native virion capsomeres.


Proteolysis of noncapsid protein 2 of type 3 poliovirus at the restrictive temperature: breakdown of noncapsid protein 2 correlates with loss of RNA synthesis. Inhibition of RNA synthesis of the LH strain of type 3 poliovirus at the restrictive temperature occurs concurrently with the breakdown of noncapsid virus-specific peptide 2. This finding, along with other reported data (B.D. Korant, In Proteases and Biological Control, p. 621-644, 1975), suggests that noncapsid virus-specific peptide 2 is a component of the viral replicase.


Skin papillomas in an impala (Aepyceros melampus) and a giraffe (Giraffa camelopardalis). Viral particles, typical of the papovavirus family, were demonstrated by electronmicroscopy in small papillomas found on the feet of an impala (Aepyceros melampus) and on the face of a giraffe (Giraffa camelopardalis) in Kenya. Histologically the tissues proved to be typical papillomas. The viral particles measured 38 nm and 40 nm in diameter in all tissue sections from the impala and giraffe respectively.


Natural pox infection in a common murre (Uria aalge). Natural pox infection occurred in a free-living, immature common murre (Uria aalge) in northern California. Cutaneous and diphtheritic lesions were present. Death of the bird was attributed to respiratory insufficiency and starvation resulting from impairment of breathing and feeding, respectively.


Rotavirus (reovirus-like) infection of neonatal ruminants in a zoo nursery. An outbreak of a pneumoenteric disease occurred in neonates in a zoo nursery. Four of seven affected animals died. Rotaviruses were observed in the feces of an affected 4-day old impala (Aepyceros melampus), a Thomson's gazelle (Gazella thomsonii) and an addax (Addax nasomaculatus). Encapsulated Escherichia coli also were isolated from the feces. The recovered rotaviruses was antigenically related to bovine rotavirus. A bovine rotavirus vaccine was given orally and no adverse effects were noted.


Neutralising antibodies to parainfluenza 3 virus in African wildlife, with special reference to the Cape buffalo (Syncerus caffer). As part of a study to assess the prevalence of common viral agents in African wildlife, nearly 3,300 sera from 44 different wild species, from eight African countries, have been examined for neutralising antibodies to parainfluenza 3 (PI3) virus. Antibody was demonstrated in 20 of the 44 species examined, including seven species not previously reported as sero-positive. Sera were collected between 1963 and 1977 and results indicated that infection has been widespread for a considerable time. The high prevalence of antibody, and the range of titres, to PI3 virus found in free-living populations of buffalo suggest that this species is particularly important as a reservoir of infection in the wild.


Parainfluenza virus type 3: isolation from CSF of a patient with Guillain-Barré syndrome. We report the isolation of parainfluenza virus type 3 from the CSF of a 19-year-old man with Guillain-Barré syndrome. Although parainfluenza viruses are usually associated with respiratory tract illnesses, some strains show a neurotropism not previously appreciated. Thus, parainfluenza viruses may be responsible for a portion of cases of Guillain-Barré syndrome.


Effects of several monoamine-related compounds on the reserpine-induced spikes recorded from the medial nucleus trapezoides in rabbits. Effects of several monoamine-related compounds on the reserpine-induced spikes recoreded from the medial nucleus Trapezoides (reserpine-induced Tr spikes) in rabbits were investigated. 5HTP (30--50 mg/kg, i.p.) showed marked suppression of reserpine-induced Tr spikes. Parachlorophenylalanine (PCPA) alone induced spikes similar to reserpine-induced Tr spikes after repeated doses (250 mg/kg twice daily for 3 successive days). A marked enhancement of the generation of reserpine-induced Tr spikes was elicited, when reserpine was given to rabbits pretreated with PCPA. L-DOPA (30--50 mg/kg, i.p.) showed a slight but significant suppressive action 20 to 40 min after injection. alpha-methyl-p-tyrosine (alpha-MT) (200 mg/kg, i.p.) produced no significant change within 6 hr, but did show a marked facilitatory effect on the generation of the spikes when reserpine was given to rabbits pretreated with alpha-MT. alpha-methyl-metatyrosine (alpha-MMT) (100 mg/kg, i.v.) caused a long-lasting, marked suppression. These findings suggest that both catecholamines and 5HT have a suppressive action on the generation of reserpine-induced Tr spikes.


[Deposits of immunoglobulins in the cells of chronic lymphocytic leukemia (author's transl)]. Cristalloid inclusions were demonstrated in the perinuclear space and the ergastoplasm of cells of chronic lymphocytic leukemia. These inclusions and the immunoglobulins of the cell membrane consisted of IgG. The leukemic cells were able to synthesize, but not to secrete, monoclonal immunoglobulins.


[Virilism due to an adrenal medullary tumor with ectopic ACTH syndrome (author's transl)]. The case of a 14-year-old girl with hirsutism and virilism due to the secretion of ectopic ACTH by an adrenal medullary tumor is described. At the age of 5 years changes in appearance had begun with masculinization. The effect of ACTH-like material, measured by radioimmunoassay in plasma and in tumor tissue, was compensated partially by the hypothalamo-pituitary-adrenal feedback mechanism. Increased concentrations of dehydroepiandrosterone, estrone and testosterone in plasma and of 17-ketosteroids and free cortisol in urine originated in the adrenals. After operation of the tumor menarche began spontaneously, hirsutism disappeared and testosterone plasma concentrations returned to normal. An adrenogenital syndrome was excluded.


Control of plasma aldosterone in a patient with Conn's syndrome before and during spironolactone medication. In a patient suffering from Conn's syndrome analysis of short-time fluctuations of plasma aldosterone, plasma cortisol and plasma renin activity were performed before and after a 9-months therapy period with spironolactone. Under the former conditions aldosterone was secreted episodically and a highly significant correlation was found between plasma aldosterone and plasma cortisol (r=0.817, p is less than 0.001) while plasma renin activity was undetectable (is less than 0.16 ng/ml/3hr). Following a 9-month therapy with spironolactone episodic secretion of aldosterone and the significant correlation between aldosterone and cortisol persisted (r=0.819, p less than 0.001) in the presence of an abnormally high plasma renin activity. First when the secretion of ACTH was suppressed by dexamethasone a weak correlation was found between renin activity and aldosterone (r=0.517, p is less than 0.05). Our results show that both before and after a 9-months therapy with spironolactone episodic aldosterone release of an aldosterone producing adrenal adenoma was mediated through ACTH and that endogeneous angiotensin II had no or only little influence.


[The enzymatic linkage of fibrinogenesis and fibrinolysis (author's transl)]. The coagulation cascade can be triggered by an extrinsic or intrinsic signal. The response is a highly complex activation sequence of several serine proteases. This physiological event of hemostasis comprises the activation of the kinin system as well as other plasma constituents. As an expression of the dynamic balance between fibrinogenesis and fibrinolysis plasminogen is activated either by proteases deriving from the blood clotting cascade or by its linkage to the kallikrein kinin system. Inhibitors of the fibrinolytic events have to be directed specifically against serine proteases or they may be used to interact with the substrate fibrin thus its degradation is delayed.


[Relationship between the plasma concentration of the high density lipoproteins (HDL) and the intravenous fat tolerance in normo-and hypertriglyceridaemics]. The relationship between the plasma concentration of high-density-lipoprotein(HDL)-cholesterol, very-low-density-lipoprotein(VLDL)-triglycerides, and low-density-lipoprotein(LDL)-cholesterol and the fractional removal rate (K2) of an intravenously administered fat emulsion (Intralipid) was investigated in 13 normo- and 34 hypertriglyceridaemics. A highly significant correlation (p less than 0,001) between the plasma concentration of HDL-cholesterol and the fractional removal rate of exogenous triglycerides was found for both groups. No significant relationship existed between the concentration of VLDL-triglycerides and of LDL-cholesterol and the removal rate of exogenous triglycerides. These observations suggest a major role of HDL in the removal of plasma triglycerides.


[Hodgkin's disease: effect of spenectomy on the immune status (author's transl)]. In 22 untreated Hodgkin's patients the following parameters were studied before and after splenectomy: the total WBC, lymphocytes, B- and T-cells and mitogenic stimulation of peripheral lymphocytes using autologous and control serum. The results were correlated to patient groups with favourable and unfavourable prognosis according to pathological stage, histology, spleen involvement and constitutional symptoms. 1. All Patients. Significant increase of the absolute number of peripheral lymphocytes and B-cells. Significant increase of the spontanous DNA synthesis in the presence of AB-control serum, but no change after mitogenic stimulation of peripheral lymphocytes. No change in the number of peripheral T-cells. 2. Patients with Favourable Prognosis. Significant increase of the absolute number of peripheral lymphocytes and B-cells. Significant increase of the spontanous DNA synthesis in the presence of AB-control-serum. Significant decrease of the T-cell function (PHA- and Con-A-stimulation in the presence of autologous serum). Significant decrease of the PHA-, Con-A- and PWM stimulation rate using control serum in the lymphocytic predominance and nodular sclerosis group. No change in the number of peripheral T-cells. 3. Patients with Unfavourable Prognosis. No change in the absolute number of B-cells, of the spontanous DNA-synthesis using autologous serum and of the PHA- and Con-A-stimulation. Significant increase of the EBV-induced blastogenesis in the mixed cellularity and lymphocytic depletion group.


Abnormal low density lipoproteins in children with familial hypercholesterolemia--effect of polyanion exchange resins. In 20 children and adolescents with familial Type II a hyperlipoproteinemia, serum lipids and lipoproteins were examined before and during treatment with polyanion exchange resins. The composition of LDL was compared to that of helthy siblings. The patients were given Colestyramine (0.6 g/kg body weight) and Colestipol (0.5 g/kg body weight) in a cross-over study for 8 weeks each, after they had been under dietary treatment for at least 12 months. In 6 children, drug treatment had to be stopped due to side-effects. The most common complaints were gastrointestinal discomfort and constipation. Cholesterol, triglycerides and phopholipids were measured in whole serum and cholesterol, triglycerides and Apolipoprotein-B in isolated lipoprotein fractions after ultracentrifugation. Apo-B was determined by radial immunodiffusion. The Apo-B: cholesterol ratio in whole serum and in the LDL fraction was identical in the patients and in the controls. The LDL triglyceride: Apo-B ratio, however, was about 50% lower in the patients. This abnormal LDL composition was not altered by therapy with polyanion exchange resins. HDL cholesterol levels were significantly lower in the patients than in healthy children, and remained low during therapy. The decrease of total and LDL cholesterol (25%) and Apo-B (20%) was similar under both Colestipol and Colestyramine. Triglycerides and phospholipids showed no significant changes in therapy.


Membrane assembly: synthesis and intracellular processing of the vesicular stomatitis viral glycoprotein. The glycoprotein (G) of vesicular stomatitis virus (VSV) is synthesized on membrane-bound polyribosomes. Approximately 30 min after its synthesis, it reaches the surface plasma membrane where it is incorporated into budding virus. The first part of this paper focuses on the 2 intracellular, membrane-bound, glycosylated forms of the glycoprotein which are intermediates in its biogenesis. All glycosylation and processing is completed in the smooth microsome fraction before the protein reaches the surface. Next, we turn to the mechanism by which G is synthesized on membrane-bound polyribosomes. All of the G mRNA is bound to membranes, and studies with puromycin suggest that this attachment of G mRNA is mediated by the nascent glycoprotein chain. After its synthesis G is a transmembrane protein with about 30 amino acids at the carboxyl terminus remaining on the cytoplasmic side of the endoplasmic reticulum. Since 95% of the glycoprotein, containing the carbohydrate residues, is resistant to attack by external proteases, it appears to be within the lumen of the endoplasmic reticulum or embedded within the lipid bilayer. Finally, we show that synthesis, glycosylation, and proper asymmetric insertion of G into the ER can be achieved in cell-free extracts. Both glycosylation of G and proper insertion into the ER membrane in this cell-free system require concomitant protein synthesis.


Small-angle x-ray scattering study of human serum low-density lipoproteins with differential reactivity for an arterial proteoglycan. The structure and thermal behavior of human serum low-density lipoproteins showing either a high or a low reactivity against a proteoglycan isolated from human arteries have been found to be different from each other. It is suggested that modifications in the lipoprotein surface structure induced by the physical state of the neutral lipids could modulate the affinity of the macromolecule for the arterial component.


Glycoprotein synthesis as a function of epithelial cell arrangement: biosynthesis and release of glycoproteins by human breast and prostate cells in organ culture. We demonstrate that a technique is available to investigate glycoprotein synthesis in organ cultures of human breast and prostate surgical specimens where the 3-dimensional epithelial cell arrangement remains intact. Malignant breast and prostate epithelium maintained their capacity to synthesize glycoproteins for at least 3 days as followed by the incorporation of [3H]glucosamine into macromolecules. Over 70% of incorporation was by malignant cells as judged by autoradiography. Labeled glycoproteins were released into glandular lumina and consequently into the culture fluid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed predominantly one group of macrmolecules released with an apparent molecular weight of 48,000 +/- 6,000 daltons. This glycoprotein was found in all of the breast specimens studied, which included 1 medullary, 1 infiltrating lobular, and 8 infiltrating duct carcinomas. The pattern was independent of the availability of estrogen receptors. A similar glycoprotein was also observed in the culture media from a Grade I and a Grade II well-differentiated infiltrating prostate carcinoma. Incorporation was below the level of detection in 4 of 6 cases of benign prostatic hyperplasia. A more complex pattern of labeled glycoproteins was found in the media of a Grade II and a Grade III poorly-differentiated prostate carcinoma. The established human mammary carcinoma cell line MCF-7 synthesized and released a similar 48,000 molecular weight glycoprotein but additional components with larger molecular weights were also released. An intriguing interpretation that 3-dimensional tissue integrity restricts some glycoprotein synthesis is discussed. Cells grown in 2-dimensional monolayers could escape from such a topographic restriction and express additional families of glycoproteins.


Viral infections.

Viral infections. In summary, viral infections of the skin that occur at adolescence include those typical of childhood and those associated with sexual maturity. Herpes simplex Type II, molluscum contagiosum, and venereal warts, diseases that have shown a marked increase, are in the latter group. Certain diseases, such as rubella, herpes simplex, varicella, and condylomata acuminata, which may lead to laryngeal papillomas, are particularly important because of their effects on the newborn infant. The epidemiological pattern of viral infections changes, and new viruses gain prominence while others fade in importance as causes of disease. Because of this, we can expect new viral entities and changes in earlier ones.


Evidence for increased degeneration of mitochondria in old rats. A brief note. An isotonic density gradient technique for separating mitochondria into two major components was applied to mitochondria isolated from young and old rats. Respiratory measuremtns indicate that the depression of state 3 respiration rates observed in the mitochondria from old rats when glutamate--malate is used as a substrate is due primarily to the faster sedimenting mitochondrial component.