Immunosuppressive activity of antibody directed against endogenous C-type virus interferes with early events of the immune response. We have analyzed the effects of an antiserum prepared against BALB/c endogenous xenotropic C-type virus on the humoral immune response of mice. Both in vivo and in vitro, this serum suppresses the response to sheep red blood cells, an effect that can be absorbed out by purified BALB/c xenotropic C-type virus or Friend leukemia virus, but not by Rous sarcoma virus. The serum produces its maximum effect when administered together with or before the antigen, but not 24 hr later. This suggests that it acts on an early event of the immune response. Evidence is presented to show that the critical viral antigen is expressed before the spleen cells are experimentally stimulated by antigen. The same immunosuppressive effect was observed in a variety of mouse strains, including the high-leukemia incidence AKR strain and virus-free 129/J mice, indicating that it is independent of the expression of endogenous virus. The finding that a viral antigen is involved in the transition from a resting to a dividing lymphocyte is discussed with respect to viral involvement in leukemia.


Spin membrane immunoassay: simplicity and specificity. The many disadvantages of radioimmunoassay (RIA) have stimulated attempts to develop non-radioactive assays. These include spin immunoassay (SIA), which is simple, specific, and requires no separation procedures but is much less sensitive than RIA. The membrane immunoassay (MIA) described here is more sensitive than the SIA. Serum is prepared as for RIA. The MIA employs liposomes sensitized with epsilon-dinitrophenylated aminocaproyl phosphatidylethanolamine. It records liposome lysis induced by specific anti-Dnp antibodies, and complement which is monitored by the release of trapped spin labels (N-(2,2,6,6-tetramethylpiperidinyl-1-oxyl)-choline chloride). The sensitized liposomes are stable and give reproducible results for up to 4 weeks. The system's sensitivity is limited by the antibody's affinity (Ka approximately 10(8) M-1) rather than the sensitivity of the electron spin resonance spectrometer (approximately 1 X 10(-7) M). The inhibition of liposome lysis is hapten specific: (epsilon-Dnp-aminocaproic acid,epsilon-Dnp-lysine) greater than alpha-Dnp-glycine; o-nitroaniline and epsilon-dansyl-lysine are ineffective. Inhibition is quantitative without augmentation.


Immunoabsorbents prepared from cell membrane antigens: efficiency of incorporation and shedding of protein during use. Immunoabsorbents for the removal of antibodies against cell membrane antigens have been prepared by a variety of techniques in common use. Cells were surface-labelled by lactoperoxidase-catalysed radioiodination, and the different immunoabsorbent preparations were compared in terms of efficiency of incorporation and shedding of membrane material during use. Whole cells, either unfixed or fixed with glutaraldehyde, shed substantial amounts of material during absorption. Detergent extracts of surface-labelled cells were coupled covalently to agarose beads by cyanogen bromide activation, periodate oxidation, or epoxy activation of the gel. Although these immunoadsorbents were much more stable than whole cells, shedding could not be eliminated entirely. Minimal shedding was achieved using freshly washed epoxy-based immunoadsorbents. The possible consequences of shed protein in immunoabsorbed sera are emphasised.


Partial purification and characterization of the inherited Hl 1 and R 67 antigens of rabbit serum high density lipoprotein. Purification and characterization of the Hl 1 antigen revealed a polypeptide with molecular weight of 20,000 by gel filtration. The peptide contained 20% neutral sugar and 4.5% neuraminic acid. Amino acid analysis as well as the N-terminal sequence for thirteen amino acid residues and three C-terminal amino acids were determined. Difference index analysis gave an indication that the Hl 1 polypeptide is unique. Purification of R 67 antigen did not give a pure polypeptide in sufficient amount to do chemical analysis. A crude fraction of R 67 antigen had three bands on SDS-PAGE, all with R 67 antigenic activity. The crude fraction contained 10% neutral sugar and 3.5% neuraminic acid. No N- or C-terminal amino acids could be determined for the crude fraction of R 67 antigen.


Selective impairment of lymphocyte reactivity to varicella-zoster virus antigen among untreated patients with lymphoma. Herpes zoster is a frequent complication of lymphoreticular malignancy. In this study two assays of in vitro cellular immune response to varicella-zoster virus (VZV) antigen, lymphocyte transformation and interferon production, were performed in normal subjects with recent and remote VZV infection. The responses of patients with lymphoma were measured before treatment and during long-term remission and then compared with those of normal subjects. Despite levels of antibody to VZV that were equivalent to those in normal subjects, 44% of the untreated lymphoma patients showed a lower transformation response to VZV antigen than the normal patients. Production of interferon in response to VZV antigen was absent in 32% of the untreated patients. In contrast, lymphocyte responses in untreated patients to herpes simplex virus antigen were within the range observed in a normal population. Interferon production by lymphocytes in response to cytomegalovirus antigen was also lower among untreated lymphoma patients than among normal patients, but lymphocyte transformation was not. Twenty-two percent of lymphoma patients in long-term remission continued to have diminished cellular immune responses to VZV antigen. Observations in these patient populations and in normal subjects with acute herpes zoster suggest that deficiencies in in vitro lymphocyte responses may correlate with increased susceptibility to clinical infection with VZV.


Cell-mediated immunity of cytomegalovirus infection in normal subjects and cardiac transplant patients. Two assays of cell-mediated immunity, lymphocyte transformation and interferon production, were adapted to test for specific immunity to cytomegalovirus (CMV). Normal individuals seropositive for CMV had a mean transformation index of 7.9 in response to antigen of the Davis strain of CMV, whereas all of 14 seronegative normal individuals had transformation indexes of less than or equal to 3.0. Interferon production in seropositive and seronegative individuals was not statistically different. One to two months after CMV mononucleosis (after the termination of viruria), normal individuals had increased transformation indexes. Recipients of cardiac transplants within six months after transplant had normal levels of antibody to CMV; lymphocyte transformation and interferon production in these subjects were markedly decreased and returned to normal by three years and between one and three years after transplant, respectively. A syndrome of unexplained fever, hepatitis, pneumonitis, leukopenia, and atypical lymphocytes was common in a group of recipients with primary CMV infection. Shedding of virus was frequent in these symptomatic patients and in patients with repeat infection during the first three years after transplant. These assays appear to identify periods of immune deficits correlating with increased incidence of infection with CMV.


Productive infection with cytomegalovirus and herpes simplex virus in renal transplant recipients: role of source of kidney. Forty patients were prospectively studied for infection with cytomegalovirus (CMV) and herpes simplex virus (HSV) after renal transplantation. Although 85% had antibody to CMV and 95% had antibody to HSV prior to transplantation, excretion of CMV was found in 92.5%, usually as viruria, and HSV was recovered from 70%, most often from the oropharynx. Shedding of HSV reached a peak within the first month after transplantation when immunosuppression was most intense and then rapidly declined. In contrast, excretion of CMV was found in a greater proportion of patients during each interval up to about six months after transplantation and subsequently persisted in the majority of patients. Both serologic responses and viral isolation rates indicated more rapid activation of CMV but not of HSV in recipients of kidneys from cadavers than in patients who received kidneys from living related donors. At 60 days after transplantation, 76% of the recipients of kidneys from cadavers and only 33% of the recipients of kidneys from living, related donors had shed CMV (P = 0.003). These differences could not be accounted for by immunosuppressive treatment. Excretion of HSV was clearly associated with the time of most intense immunosuppression, but the major factor in initiation and maintenance of productive infection with CMV appeared to be the host vs. graft reaction.


Perinatal cytomegalovirus infection: influence of placentally transferred maternal antibody. The kinetics of the response to passively transferred maternal neutralizing antibody were studied for determination of whether this antibody offered protection against primary cytomegalovirus (CMV) infection in the offspring. Antibody response was determined in 17 infants infected in the immediate perinatal period and in 18 appropriate control subjects. Levels of neutralizing antibody in serum obtained at birth and at one month of age were similar in infected and in exposed, uninfected neonates. In addition, the quantity of neutralizing antibody did not influence the time of onset of viruria. Clearly, passive humoral immunity failed to prevent naturally acquired primary CMV infection in a significant number of young infants exposed to virus during and shortly after delivery.


Salivary neutralizing activity against herpes simplex virus type 1. Samples of saliva collected from 40 patients, of whom 16 had a previous clinical history of herpes simplex virus type 1 (HSV-1) infection, were concentrated and sterilized. Salivary neutralizing activity, as measured by a plaque-reduction method, was found in 62% of those patients with a history of oral lesions. Studies with antisera to human immunoglobulin indicated that the IgG class of immunoglobulin was a major HSV-1-neutralizing component of both saliva and serum.


Splenic necrosis in mice infected with cytomegalovirus. Necrosis of the spleen is seen in mice of certain strains after injection of large doses of murine cytomegalovirus. Necrosis begins in perifollicular areas containing infected cells which do not appear to be macrophages, and in severe cases the entire spleen is involved. Necrosis is first visible three days after infection and is not prevented by treatment of mice with corticosteroids, cyclophosphamide, or theta antiserum. There is a striking loss of nucleated cells from the spleen at two to four days after infection, in the absence of splenic necrosis. The pathogenesis of splenic necrosis, which is seen also in certain other viral infections, remains unexplained.


A practical method for preparation of varicella-zoster immune globulin. Outdated blood from blood banks in Massachusetts was screened for complement-fixing antibody to varicella-zoster virus (VZV). Approximately 15% of the plasma units had a titer greater than or equal to 16, and one-half of these had a titer of greater than or equal to 32. Plasma units with titers of CF antibody to VZV of greater than or equal to 16 were pooled. This pool had a CF antibody titer of 32 and a titer of fluorescent antibody of 64. Varicella-zoster immune globulin was prepared from the selected plasma pool by alcohol fractionation and analyzed for VZV-specific antibody. Varicella-zoster immune globulin had a fluorescent antibody titer of 2,048 and a neutralizing antibody titer of 1,024. This antibody concentration is equivalent to that in preparations of zoster immune globulin made from plasma of donors recovering from recent VZV infection.


Determinants of measles virus (hamster neurotropic strain) replication in mouse brain. In newborn mice the hamster neurotropic strain of measles virus produces a severe meningoencephalitis with readily recoverable virus, while in weanling mice a fatal encephalopathy is produced with scant histopathology and no viral infectivity in brain homogenates. In this study various host factors that may change with maturation and determine the restriction of viral expression were investigated, including immune response, interferon production, host temperature, and the possible role of proteases. None of these factors appeared to be responsible for host restriction of viral expression. Recovery of virus from brains of weanling mice was not significantly enhanced by cocultivation with Vero cells, complementation with temperature-sensitive mutants, or phenotypic mixing with the Edmonston strain of measles virus. These data, combined with our previous observations of production of viral proteins including nucleocapsid proteins without development of recognizable nucleocapsids in neurons of weanling mice, suggest that with maturation the neural cells fail to replicate sufficient amounts of encapsidated viral ribonucleic acid.


Synergistic infection with murine cytomegalovirus and Pseudomonas aeruginosa in mice. A previous report from this laboratory demonstrated that mice infected intraperitoneally with a 0--20% lethal inoculum of murine cytomegalovirus (CMV) exhibited markedly enhanced mortality rates (90%--100%) within 48 hr after an intravenous injection of a 0--20% lethal inoculum of Pseudomonas aeruginosa. The current study demonstrated that mice infected with murine CMV alone had high titers of virus in multiple organs over a 20-day period, whereas mice injected with P. aeruginosa alone had a self-limited infection confined to the kidney. In the combined murine CMV-P. aeruginosa infection, titers of virus in tissues were changed very little. In contrast, P. aeruginosa was recovered in high concentrations from multiple organs, a finding which demonstrated a progressive systemic infection closely resembling that produced by a 100% lethal inoculum of P. aeruginosa alone in normal mice. An increased mortality rate due to P. aeruginosa in murine CMV-infected mice was dependent on inoculation of live bacteria and could not be explained by enhanced susceptibility to endotoxin. These results indicate that murine CMF markedly enhanced the suceptibility of mice to infection with P. aeruginosa and suggest that the virus altered mechanisms of host resistance important in recovery from bacterial infections.


Granular cell myoblastoma of the larynx. Two cases of granular cell myoblastoma of the larynx are presented. In the first case the tumour was excised by laryngofissure, and in the second case by microlaryngoscopy. Based on this experience a review of this interesting tumour is made, and emphasis is given to the fact that this neoplasm may be mistaken for squamous cell carcinoma.


Effects of dietary fat composition on the Ehrlich ascites tumor fluid lipoproteins. Mice bearing the Ehrlich ascites tumor were fed diets rich in either coconut oil or sunflower oil. From 20 to 40% less lipid was present in the ascites tumor fluid when the mice were fed the sunflower oil diet. This was associated with a reduction in the amount of very low density lipoproteins (VLDL) and high density lipoproteins (HDL), the main lipoprotein fractions present in the ascites tumor fluid. The VLDL from the mice fed sunflower oil contained more cholesteryl esters and a lower free to esterified cholesterol ratio than those from the mice fed coconut oil. Very little change occurred in the composition of the HDL. All of the lipids contained in both lipoprotein fractions exhibited appreciable differences in fatty acid composition. Much more monoenoic and less polyenoic fatty acid were present in the lipids from the mice fed the coconut oil diet, but no appreciable change in saturated fatty acid content occurred. Similar changes in fatty acid composition were observed in the blood plasma of the tumor-bearing mice. There was no qualitative difference in the apolipoprotein patterns of either the ascites fluid VLDL or HDL. Pyrene fluorescence studies indicated that the fluidity of the VLDL was increased when the mice were fed the sunflower oil diets. No difference in HDL fluidity, however, was observed by this technique. These results indicate that the amount, composition, and physical properties of certain of the lipoproteins contained in the ascites tumor fluid can be modified by changing the composition of the dietary fat fed to mice bearing the Ehrlich ascites tumor.


Pulmonary surfactant synthesis. A highly active microsomal phosphatidate phosphohydrolase in the lung. Lung cell-free homogenate, which contains about twice the units of phosphatidate phosphohydrolase per mg of protein compared to liver, was fractionated by differential centrifugation and the fractions were assayed for phosphatidate phosphohydrolase and marker enzymes of endoplasmic reticulum, mitochondria, and lysosomes. Over 60% of the lung phosphatidate phosphohydrolase was associated with the endoplasmic reticulum, compared to 50% of the total liver enzyme. Thus a major portion  of the more active lung enzyme is potentially involved in lipid biosynthesis by the endoplasmic reticulum. Less than 0.2% of the total lung enzyme was found in a lamellar body fraction, consistent with previous findings. The lung microsomal phosphohydrolase was specific for lipid substrates, showing equal activity towards phosphatidic acid or lysophosphatidic acid and relatively low activities towards glycerophosphates. It had a neutral pH optimum, similar to the liver enzyme, but differed somewhat in its relative activity at extremes of pH. Stability at 65 degrees C was greater for the lung enzyme. Fluroide inhibited lung (or liver) microsomal phosphatidate phosphohydrolase, while tartrate, MgCl2, or EDTA had no effect. The presence of a high activity of phosphatidate phosphohydrolase in lung endoplasmic reticulum is consistent with the rapid synthesis of pulmonary surfactant phosphatidylcholine.


Proteolytic digestion in the elucidation of the structure of low density lipoprotein. The apoprotein (apoB) of low density lipoprotein (LDL) is reported to be a large polypeptide, and it is proposed that there are two similar-sized subunit proteins in LDL (Smith, Dawson, and Tanford. 1972. J. Biol. Chem. 247: 3376-3381.). When apoB is isolated under conditions that minimize artifactual proteolysis, only a single, large molecular weight protein appears on polyacrylamide gel electrophoresis in SDS. To investigate the organization of apoB as it exists within native LDL, limited proteolysis with trypsin has been used as a structural probe. Tryptic digestion for 1 hr at pH 7.6 with enzyme-to-protein ratios of 1:100 and 1:5 results in the liberation of approximately 10% and 30% of apoB as smaller, water-soluble peptides. These peptides may be separated from the partially digested but still intact tryptic core (T-core) of the lipoprotein by chromatography on Sephadex G-75. Repeatedly, the 1:5 T-core of native LDL is found to contain a family of polypeptides of 14,000-100,000 molecular weight. Although they have lost significant quantities of apoprotein, these T-cores sustain an appearance of homogeneity, as studied by analytical ultracentrifugation. Their measured molecular weights do not differ appreciably from those of the native LDL, and the carbohydrate content of the 1:5 tryptic T-core of LDL is similar to that of the native LDL. In normolipemic individuals, LDL generally exists in a monodisperse state, but, in different individuals, monodisperse LDL may range in molecular weight from 2.4 to 3.9 x 10(6). Limited tryptic digestions were used to probe the organization of apoB in these different molecular weight LDL. As assayed by SDS-acrylamide gel electrophoresis of the larger polypeptides and fingerprinting of the smaller released peptides, those regions of LDL exposed to trypsin digestion are identical in monodisperse LDL of 2.5 and 3.4 x 10(6) molecular weight. Thus, the different quantities of lipid bound in these various LDL must interact with apoB so that the same regions of the apoprotein are exposed to the action of trypsin in these different molecular weight lipoproteins.


A convenient synthesis of 3-keto bile acids by selective oxidation of bile acids with silver carbonate-Celite. A number of 3-keto bile acids were synthesized by the selective oxidation of bile acid methyl esters with silver carbonate-Celite in refluxing toluene. The pure 3-keto bile acids were isolated simply by filtering the reaction mixture and concentrating the filtrate. The relation of the bile acid structure to the oxidation rate is also discussed.


A spectrophotometric method for the assay of cytidine 5'-diphospho-1,2-diacyl-sn-glycerol-dependent enzymes of phospholipid metabolism. Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride) hydrolase, CDP-diglyceride:L-serine O-phosphatidyltransferase, and CDP-diglyceride:sn-glycero-3-phosphate phosphatidyltransferase all release CMP from their liponucleotide substrate, CDP-diglyceride. We have developed a spectrophotometric assay for these enzymes using CMP kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of CMP with the oxidation of NADH. The assay for each of the phospholipid-dependent enzymes was found to be linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzymatic activity, determination of initial rates of reaction, and detailed kinetic analysis of these enzymes. Since several enzymes and substrates are used in the coupled assay system, the method is limited to analysis of partially purified preparations lacking competing activities.


Calcium and cyclic nucleotide regulation in incubated mouse retinas. When retinas from dark-adapted C57BL/6 mice were incubated in the dark for 5 min at 37 degrees C in Earle's medium, they contained 80-120 pmol/mg protein of cGMP and about 13 pmol/mg protein of cAMP. When the incubation in darkness was in calcium-deficient Earle's medium with 3 mM EGTA, a 10-20 fold increase occurred in the cGMP level, peaking at 2-3 min, but no change occurred in cAMP. This elevated level fell in 3 min to normal dark levels on return to normal Earle's medium, but was still about three times that of control levels after 15 min in EGTA-containing solution. Bright light after 2 min of dark incubation of dark-adapted retinas resulted in a 40-50% fall in cGMP, and bright light sharply reduced the elevated dark cGMP level of retinas in calcium-deficient media with 3 mM EDTA. However, no depression of normal dark levels of cGMP has thus far been obtained by increasing external calcium levels, even in the presence of the ionophore A23187. All the above phenomena involving dark cGMP levels and calcium are similar in Earle's medium with 100 mM of K+ substituted for Na+. Congenic rodless (rd/rd) mouse retinas have less than 5% of control cGMP and show only traces of calcium sensitivity. Thus, the above phenomena in controls are likely to be largely occurring in rods. The data suggest a dependency of the dark cGMP level on the calcium level, but that the light-induced fall in cGMP may largely be calcium insensitive.


The effect of heat-inactivated murine cytomegalovirus on host DNA synthesis of different cells. Heat-inactivated murine cytomegalovirus (MCMV) stimulates cellular DNA synthesis in WME, NMG, 3T3, WgIA, chick and NIK-8 cells, but active or u.v.-irradiated MCMV does not. The stimulation of DNA synthesis in NIL-8 and chick cells was studied in detail. We found that both the nuclear and the mitochondrial DNA synthesis were stimulated in these cells. There was no virus DNA synthesis during the period we studied (48 h). The stimulation of nuclear DNA synthesis was about threefold in NIL-8 and 2.5-fold in chick cells as measured by the rate of incorporation of 3H-thymidine (3H-dThd) in the CsCl fractions which banded at the density of cell DNA. The stimulation was about 9.5-fold in NIL-8 and 1.7-fold in chick cells as detected by autoradiography. There was a 3-fold and a 2.2-fold increase in the degree of incorporation of 3H-dThd into mitochondrial DNA of NIL-8 and chick cells, respectively. The amount of mitochondrial DNA obtained in infected cells of both kinds was about twice that in control cells. The synthesis of mitochondrial DNA was also stimulated by a factor of 2 in the thymidine kinaseless 3T3 cells which incorporate exogenous thymidine exclusively into mitochondrial DNA. There were no MCMV specific antigens detectable by immunofluorescence 5 h after infection, but diffuse nuclear fluorescence could be demonstrated 24 h after infection. Our results indicate that the heat-inactivated virus penetrates the cells, stimulates host DNA synthesis and induces synthesis of early MCMV antigens.


Relationship of endogenous murine xenotropic type C virus production to spontaneous transformation of cultured cells. Spontaneous production of endogenous xenotropic viruses by clones of New Zealand Black (NZB) embryo cells occurs at a constant level even after several cell transfers. Foci of cell alteration occur spontaneously in some of the clonal lines after 6 to 10 passages. The amount of virus generated does not correlate with spontaneous transformation.. of these NZB cells nor of cells from NIH Swiss or BALB/c mice. The data demonstrate an intracellular regulation of xenotropic virus expression which remains stable over several cell generations and differs from that controlling cell transformation


On the association of virus proteins with the nuclei of cells infected with herpes simplex virus. In cells infected with herpes simplex virus, HSV-I, newly synthesized polypeptides accumulated in the nucleus at different rates, which did not change during the first 6 h after infection. Canavanine, an arginine analogue, prevented the nuclear accumulation of ICP (infected cell polypeptides) 5 and 8 and azetidine, a proline analogue, prevented that of ICP 5 and 7. The transfer of polypeptides to the nucleus was inhibited at 4 degrees C but not by dinitrophenol. Some of the nuclear polypeptides could be released by washing isolated nuclei with hypertonic salt solutions. ICP 17 was particularly sensitive to high salt treatment while ICP 5 and II were resistent. ICP 4b, a modified form of the alpha polypeptide ICP 4, was released by EDTA, and the detergent NP40 removed ICP II. Treatment of nuclei with DNase selectively reduced the amount of bound alpha polypeptides ICP 4c (the second modified form of ICP 4), 0 and 27 as well as ICP 8 and 25. Nuclei isolated from infected or uninfected cells and incubated in labelled cytoplasmic extracts took up primarily ICP 8 and 32. Alpha polypeptides were taken up to a lesser extent and ICP 6 and 10 were excluded. It is concluded that affinities for various constituents of host cell nuclei are likely to determine the nuclear accumulation of specific virus polypeptides.


Ribonucleoprotein-like structures from coronavirus particles. The structure of the ribonucleoprotein (RNP) complex of three coronaviruses was investigated. A single-stranded helix of diam. 14 to 16 nm and up to 320 nm in length was released from disrupted particles of human coronavirus strain 229E and mouse hepatitis virus strain 3 after incubation in mild conditions. The helical complexes appeared to be composed of globular subunits with long axes of 5 to 7 nm surrounding a hollow core of diam. 3 to 4 nm. The complexes were shown to be sensitive to both pancreatic RNase and to pronase. No undegraded internal component was obtained from disrupted avian infectious bronchitis virus particles. We conclude that these structures are RNP complexes. The similarity between these RNPs and those of other large lipid containing RNA viruses is discussed.


Evidence against the role of K+ in the shut-off of protein synthesis by vesicular stomatitis virus. Intracellular K+ ion concentration and transport were measured in L cells infected with wild type VSV, which rapidly inhibits total protein synthesis in infected cells, and with a mutant, RI, defective in this function. No alterations in intracellular K+ ion concentration or transport were observed until late in infection and the late changes seen were similar for both viruses. Thus the inhibition of total protein synthesis by VSV cannot be ascribed to virus induced changes in host K+ ion concentration.


Studies on the replication of KRV single-stranded linear DNA. The autonomous parvovirus, Kilham rat virus (KRV), is composed of three structural proteins (Salzman & White, 1970) and a single molecule of DNA. The DNA molecule is linear and single-stranded (ss). It is believed to have both 3' and 5'-terminal palindromic sequences (Salzman, 1977). Replication of the ss DNA may involve double-stranded (ds) DNA intermediates of one unit length as found in the virion or multiple length concatamers (Salzman & White, 1973; Gunther & May, 1976; Lavelle & Li, 1977). We have attempted to determine when the synthesis of KRV ds DNA can be determined in synchronized infected cells and the structure of the ds DNA molecules.


Determination of acetylcholine receptor antibody in myasthenia gravis: clinical usefulness and pathogenetic implications. Antibodies to cholinergic receptor structures were found in 75% of 76 Finnish and 93% of 175 Swedish patients with myasthenia gravis. The amount of antibodies showed a positive correlation to the severity of the disease, and was reduced during immunosuppressive treatment, and by thymectomy. Thymoma patients had high values. The antibody was also found in the cerebrospinal fluid. Two healthy newborn babies of myasthenic mothers had antibodies during the first weeks of life, in spite of no clinical symptoms. The occurrence of IgM antibodies before IgM antibodies in two patients during the early stages of myasthenia gravis suggests that the antibody is not a primary cause of the disease.


Central nervous system susceptibility to herpes simplex infection. Four days after inoculation of herpes simplex virus (HSV) on the rabbit cornea, distinctive and reproducible lesions appear in the trigeminal root entry zone. These viral lesions, situated in the central nervous system (CNS) portion of the root, consist of severe myelin destruction accompanied by mononuclear cell infiltration and partial sparing of axons. Immunofluorescent study demonstrated abundant viral antigen, and by electron microscopy viral nucleocapsids were found to be numerous within astrocytes and were rarely found in other cell types. In contrast, the adjacent peripheral nervous system (PNS) tissue appears unaffected by the presence of virus. The mechanism for this marked difference in response of the central nervous system and the peripheral nervous system may depend upon the susceptibility of astrocytes to viral infection and replication. The selective nature of the lesion provides an easily reproducible model for further investigation of the response of nervous system tissue to HSV.


A second sensory--motor--interneuron with neurosecretory granules in Hydra. Using serial-sectioning techniques for conventional transmission and high-voltage electron microscopy, we characterized the ultrastructural features and synaptic contacts of the sensory cell in tentacles of Hydra. The sensory cell has an apical specialization characterized by a recessed cilium surrounded by three rodlike stereocilia. This ciliary--stereociliary complex constitutes the receptive or dendritic pole of the sensory cell. The dense filamentous cores of the stereocilia project proximally into a narrow circumciliary cytoplasmic region connected by septate junctions to marginal processes of an enveloping epitheliomuscular cell. The central cilium has a characteristic marginal flare midway along its length and a dense filamentous substructure at its base. Pairs of branched, striated rootlets extend from the axial centriole into a mitochondria-rich region of the cell. Pigment-like granules are present in the cytoplasm around the circumciliary space. The perikaryon is characterized by an elongate nucleus surrounded by a narrow rim of cytoplasm containing prominent Golgi complexes, assorted vacuoles and dense-cored vesicles, free ribosomes, short segments of rough endoplasmic reticulum, microtubules, glycogen particles, and lipid droplets. Generally, one or two thin, naked axons extend laterally from the perikaryon into the nerve net region above the myonemes of the large epitheliomuscular cells. Within the axons are found occasional aggregates of dense-cored vesicles and en passant synapses characterized by the presence of clear or dense-cored vesicles in contact with paramembranous densities and associated intracleft cross filaments. Using these ultrastructural criteria, we demonstrated for the first time that the granule-containing sensory cells have synaptic contacts with other neurons, nematocytes, and epitheliomuscular cells hence, we considered these cells to be sensory--motor--interneurons with neurosecretory granules. We hypothesize that this unique, apparently multifunctional neuron may be a modern representative of a primitive stem cell that give rise evolutionarily to the sensory cells, motor neurons, interneurons, and neurosecretory cells of higher animals.


Control of pedal and parapodial movements in Aplysia. II. Cerebral ganglion neurons. 1. Intracellular stimulation of individual neurons in the two symmetrical A neuron clusters of the cerebral ganglion evoked contractions of both the foot and parapodia. Electrical stimulation of pedal and parapodial nerves caused antidromic action potentials in A neurons. Units recorded in the nerves followed the driven somatic spike 1:1. This suggests that the A neurons are presumptive pedal and parapodial motor neurons.2. Individual A neurons evoked both bilteral and unilateral contractions of the parapodia or split foot. Contractions in the parapodia were independent of those in the foot. An individual A neuron caused contractions in either the foot or the parapodia, but not both. Sequential transection of parapodial nerves had only a slight effect until a key nerve was cut. The contractions produced by a single A neuron on one side were then abolished. These data suggest that the motor fields of the A neurons are well defined within the foot or the parapodia. 3. Parapodial contractions produced by individual A neurons are not dependent on the excitation of follower motor neurons. Blocking synaptic transmission by the addition of CoCl2 did not eliminate the contractions produced by driving individual A neurons. This is consistent with the A neurons being motor neurons. 4. Intracellular stimulation of individual neurons in the symmetrical B neuron clusters of the cerebral ganglion also evoked pedal and parapodial contractions. Electrical stimulation of the pedal and parapodial nerves elicited antidromic spikes in these neurons. Individual B neurons caused contractions in both the foot and parapodia. This suggests that the B neurons are motor neurons with very large motor fields. 5. Filling the pedal and parapodial nerves with cobalt primarily filled the cell bodies of neurons located in the pedal and pleural ganglia. The somata of A and B neurons were also occasionally filled. This is consistent with the electrophisiological results. 6. Other neurons also evoked parapodial contractions. Intracellular stimulation of neurons in the pedal and pleural ganglia caused parapodial contractions in intact animals. Some of these neurons were excited by stretching the parapodia or touching the tentacles. 7. The B neurons are strongly excited by tactile stimulation of the tentacles. Since they can cause pedal and parapodial contractions they may mediate reflex contractions elicited by tentacular stimulation. Stretching the parapodia only occasionally caused the A neurons to fire. This makes it unlikely that they make a major contribution to pedal and parapodial proprioceptive reflexes. These reflexes are probably controlled by neurons in the pedal and pleural ganglia.


Evidence for nonsynaptic neuronal interaction. 1. Evidence is presented that synaptic interactions within and between the statocyst and visual pathways of Hermissenda are eliminated after 0.5-4 min exposure to 20-40 mM Co2+. 2. Synaptic blockade was also produced by perfusion with low Ca2+ (5mM) plus 10-20 mM Co2+. 3. Depolarization of hair cells by impulses of type A photoreceptors remains after the same exposure to Co2+, or low Ca2+ plus Co2+. 4. The increased resistance previously observed during this depolarization of hair cells cannot be observed after exposure to Co2+. 5. The depolarization which remains after exposure to Co2+ did not change with different levels of membrane potential from -20 mV below to +10 mV above the resting level. 6. The time course of potassium accumulation, monitored by the amplitude of the type A impulse afterpotential, closely followed the time course of hair cell depolarization and also of changes in the amplitude of the hair cell afterpotential. 7. The depolarization of hair cells by type A impulses decreased with increased extracellular potassium, but was only slightly reduced by lowered extracellular potassium. 8. The amount of potassium accumulation following a type A impulse train could be estimated from the effects of changes in extracellular potassium in the perfusate on the type A impulse afterpotential. From this extimated increase of extracellular potassium it was possible to predict with some accuracy, the observed hair cell depolarization. 9. Although type A cells are not electrically coupled to ipsilateral hair cells, firing of these hair cells slightly depolarized the type A photoreceptor which excites them. 10. Strophanthidin (10-4 M) did not block the depolarization of hair cells by type A impulses. 11. The data are evidence for nonsynaptic excitation of hair cells by type A photoreceptor impulses. The data are also consistent with the interpretation that the excitation arises from potassium accumulation around the type A and hair cell axonal membranes.


Neuronal transmission through hippocampal pathways dependent on behavior. 1. In chronically prepared, freely moving rats, electrical stimulation was applied to the angular bundle, and responses were recorded extracellularly at a variety of sites in the ipsilateral hippocampal formation. At each recording site the stimulus-response relationship was tested during four different behavioral states. These were slow-wave sleep (SWS), REM sleep ( REM), and also two waking behaviors consisting of the still, alert condition (labeled SAL), and voluntary movement (AW theta). 2. Two varieties of evoked responses were recorded: those due to the synchronous firing of neuronal action potentials (EAPs) and those produced by excitatory synaptic activity (ESPs). The overall pattern of monosynaptic, di-, and trisynaptic responses found was similar in the rat to that found by Andersen et al. (3-5) in cat and rabbit. 3. When the trisynaptic EAP was recorded in CA1, the threshold was similar during all four behavioral states. However, suprathreshold stimuli evoked a greater response during SWS than during the other three states. The trisynaptic ESP was also greater during SWS. 4. Disynaptically, EAPs were recorded in CA3. These were greater in magnitude during SWS than during SAL, but were intermediate in mean amplitude during AWtheta and REM. Response variability was much greater during AWtheta and REM. 5. The monosynaptic EAP recorded in the upper blade of the dentate gyrus (DG) exhibited the same behaviorally correlated properties found disynaptically in CA3. 6. The monosynaptic ESP recorded in the DG, in contrast to the EAP, was greater in magnitude during SAL than during SWS. 7. The primary afferent volley was also recorded at high gain in the DG. The amplitude of this was found to be dependent solely on stimulus intensity and not on behavioral state. 8. The results are interpreted as suggesting that the granule cell membranes in the DG are relatively hyperpolarized during SAL compared with SWS as the result of either tonic excitatory bombardment occurring during SWS or tonic inhibitory bombardment during SAL.


Death following external carotid artery embolization for a functioning glomus jugulare chemodectoma. Case report. A 24-year-old man with left sixth through twelfth cranial nerve palsies and severe, fluctuating, systemic arterial hypertension was found to have a large, vascular, catecholamine-forming glomus jugulare chemodectoma. After his electrolyte imbalance was corrected, left external carotid embolization (using Gelfoam) was carried out. The systemic blood pressure stabilized at around 160/100 mm Hg over the next 12 hours. Ten hours later the hitherto conscious patient developed acute arterial hypotension, lapsed into coma, and died. Autopsy showed tumor infarction, swelling, cerebellar tonsillar herniation, and medullary compression. An unusual complication of glomus tumor embolization is highlighted. The roles of preliminary decompressive surgery and urgent resuscitation by vasopressors are discussed.


Transsphenoidal microsurgery for pituitary tumors associated with hyperprolactinemia. The results of transsphenoidal microsurgery in treating 37 patients (30 women and seven men) with pituitary tumors associated with hyperprolactinemia are presented. Immediate (10-day) postoperative fasting prolactin levels were normal (less than 25 ng/ml) in 19 of 26 patients whose preoperative prolactin level was less than 200 ng/ml, and in only three of 11 patients in whom preoperative prolactin was greater than 200 ng/ml. Twelve of 13 patients with normal preoperative pituitary-target organ function maintained normal axes postoperatively. Thirteen other patients had preoperative deficiencies in one or more pituitary-target organ axes. Postoperatively, in these latter 13 patients, a pituitary-target organ axis that was deficient preoperatively returned to normal in six cases; there was no change in five, and there was impairment in another axis in four instances. Although gross total tumor removal was believed to be complete in 35 of 37 patients, serial postoperative prolactin determinations in four of these 35 patients indicate tumor regrowth. The authors conclude that transsphenoidal microsurgery is currently the operative procedure of choice for the majority of pituitary tumors associated with hyperprolactinemia.


Fast-neutron irradiation of glioblastoma multiforme. Neuropathological analysis. Various modes of therapy, alone or in combination, have had little effect in improving the survival of patients with glioblastoma multiforme. Recently, in a pilot study, 34 patients with glioblastoma were treated by fast-neutron-beam irradiation of the whole brain. Following treatment, the patients became steroid-dependent and pursued a gradual downhill course with increasing obtundation. Although there was no improvement in the length or quality of survival of these patients, neuropathological studies in the 13 patients who came to autopsy showed the following: 1) extensive coagulative necrosis of much of the tumor mass; 2) dense infiltration by collagenous connective tissue; 3) minimal phagocytic reaction; 4) marked reduction in the amount of viable tumor; 5) abnormal astrocytic proliferation, which may represent either astrocytoma or a radiation-induced bizarre gliosis, and 6) areas of gliosis and white matter degeneration in the brain stem, remote form the tumor site. These observations suggest that continued efforts to further refine this mode of therapy for glioblastoma are warranted.


The distribution of nuclear DNA from human brain-tumor cells. Flow cytometry (FCM) is a technique that measures the quantity of DNA contained in individual nuclei and records a frequency distribution of the DNA content per nucleus in the sampled cell population. Nuclei from a variety of human brain-tumor types were isolated by means of tissue grinding, purified by centrifugation through 40% sucrose (15 minutes at 4000 rpm), fixed with 10% formalin, stained with acriflavin-Feulgen, and analyzed by FCM. Profiles of DNA distribution in histologically benign tumors, such as meningiomas, pituitary adenomas, neuroblastomas, and low-grade astrocytomas, revealed a large diploid population (2C) with a few nuclei in DNA synthesis, as well as a small premitotic population (G2 cells) that contains a 4C DNA complement. In contrast, malignant gliomas, including glioblastomas, consist of more cells in DNA synthesis; these tumor cells show a highly variable distribution of ploidy consisting not only of diploid, and/or aneuploid, but also of triploid, tetraploid, and possibly octaploid populations. Also, a large variability between different regions of each tumor was always observed. In contrast, metastatic brain tumors, despite the fact that they contain a considerable number of cells undergoing DNA synthesis, demonstrate little variability within each individual tumor. The ability to rapidly characterize the cell populations of human brain tumors with FCM may enhance the effectiveness of their clinical management.


Metabolism of fatty acids and the levels of ketone bodies in the livers of pyridoxine-deficient rats. Lipid metabolism was examined in rats fed a high-protein pyridoxine-deficient diet, and their livers were found to contain large amounts of lipids, mainly in the forms of triglycerides and cholesteryl ester. The contents of ketone bodies in the livers of pyridoxine-deficient and the control rats were similar. Their NAD+/NADH ratios, calculated from the amounts of ketone bodies, were also similar in pyridoxine-deficient and control groups when the animals were fed, but the ratio in pyridoxine-deficient rats was lower than that of control rats when the animals were starved. After injection of 14C-linoleic acid, the amounts of expired 14CO2 in pyridoxine-deficient and control rats were similar. The pattern of incorporations of 14C-linoleic acid into various lipid components of the livers were examined; incorporation into the phospho-lipid fraction was similar in control and deficient rats, but the incorporation into the triglyceride fraction was slower, and the incorporation into cholesterol was faster in deficient animals than in controls.


In vivo and in vitro conversion of 7-dehydrocholesterol into vitamin D3 in rat skin by ultraviolet ray's irradiation. In order to confirm the photochemical conversion of 7-dehydrocholesterol (7-DHC) into vitamin D3 in rat skin, the following in vitro and in vivo experiments were carried out. In the first (in vitro) experiment, the skin stripped off from a sacrificed normal rat was irradiated with an ultraviolet (UV) lamp for a constant period. In the second (in vivo) experiment, the normal rat, irradiated under the same condition mentioned above, was sacrificed and then the skin was stripped off. Lipids were individually extracted with chloroform-methanol (1:1) from the skin obtained in the two experiments and the solvent was evaporated. The resulting residue was saponified and the unsaponifiable matter extracted with benzene was purified by application to hydroxyalkoxy-propyl (HAP) Sephadex column chromatography. The resulting purified vitamin D3 fraction was applied to high-performance liquid chromatography (HPLC) in order to estimate vitamin D3. No peak, aside from that of alpha-naphthol as an internal standard, was observed in the HPLC chromatogram on the skin obtained from the non-irradiated rat, whereas the peak corresponding to vitamin D3 was observed in each HPLC chromatogram on both the irradiated skin (in vitro experiment) and the skin obtained from the irradiated rat (in vivo experiment). The peaks, confirmed to be due to vitamin D3 by the results of co-chromatography, were increased according to the increase of irradiation energy and there were little differences between the corresponding estimated values of vitamin D3 in the two experiments. These results prompted the conclusion that 7-DHC in rat skin was photochemically converted into vitamin D3 by UV irradiation and that the in vivo conversion mechanism might be the same as the in vitro one.


Mixed tumor of skin.

Mixed tumor of skin. A 40-year-old white male consulted his dentist because of a mass in the upper lip to the right of the midline. The patient stated that the lesion had been present and slowly enlarging for at least the pat 10 years. A mass that appeared to be about the size of a hazel nut was apparent to direct visualization and it produced an elevation of the lip surface. On palpation, the mass was found to have a firm rubbery texture and to be freely movable. The history of slow growth of an apparently well delineated mass over a lengthy period of time, together with a lack of fixation to the surrounding tissues of the lip was considered indicative of a benign lesion. The mass was extirpated using an intraoral approach under local anesthesia.


Polydipsia, polyuria, and hypertension associated with renin-secreting Wilms tumor. A 16-month-old black male infant had unusual thirst, polyuria, hyponatremia, and hypertension. His polyuria was unresponsive to vasopressin therapy, and his high blood pressure was not effectively controlled by antihypertensive drugs. Radiographic examinations revealed an occult Wilms tumor in the right kidney. After removal of the tumor, the signs and symptoms were relieved. The tumor had a renin activity about 280 times that of the adjacent renal cortex, and many intracytoplasmic secretory granules were found on electron microscopy. The pathogenesis of these clinical manifestations appears to be mediated through the physiologic pathways of renin-angiotensin II and renin-aldosterone.


The biochemical basis for resistance to adenine arabinoside in a mutant of Toxoplasma gondii. We have previously reported the isolation and preliminary characterization of a mutant of Toxoplasma gondii that was resistant to adenine arabinoside. Fiftyfold higher concentrations of adenine arabinoside were required to inhibit the growth of the resistant parasite in human fibroblast cultures. To determine the enzymic basis for resistance, we measured the kinases and deaminases that act on adenosine or deoxyadenosine. All of these enzymic activities were found in uninfected human fibroblast cells. The mutant and wild type parasite proved to have similar activities of adenosine deaminase, deoxyadenosine deaminase, and deoxyadenosine kinase. However, the adenine arabinoside resistant mutant had less than 0.1% of the adenosine kinase activity observed in the wild type T. gondii. The mutant parasite is presumably resistant because without adenosine kinase to phosphorylate adenine arabinoside it cannot carry out the first step in the conversion of the analogue to adenine arabinoside triphosphate, the active form. A mutant of 3T6 (mouse) cells previously selected for a loss of adenosine kinase also proved to be resistant to adenine arabinoside.


Ultrastructure of erythrocytes parasitized by Haemobartonella felis. Experimental Haemobartonella felis infections were studied in 3 mature, intact cats by examining peripheral blood, lung, and spleen by electron microscopy. Coccoid, rod, or ring forms of the organism were found on or close to the erythrocytic membrane, and adjacent parasitized erythrocytes often were attached. Intracytoplasmic crystalloid inclusions occupying most of erythrocytic cytoplasm were seen in the 3 infected cats. The cat with the highest parasitemia had inclusions in about 10% of the erythrocytes. Less than 0.01% of the erythrocytes of a control cat contained inclusions. Parasitized erythrocytes, with and without inclusions, were seen in capillaries of the lung and spleen of infected cats. Macrophages in the lung and spleen of infected cats contained parasitized erythrocytes, either with or without inclusions. Some macrophages contained erythrocyte-free organisms in phagocytic vacuoles.


Effect of concomitant administration of magnesium trisilicate on GI absorption of dexamethasone in humans. The oral absorption of dexamethasone in humans was compared to its absorption when coadministered with magnesium trisilicate. The bioavailability of dexamethasone was estimated by measuring the suppressive effect of the drug on the daily excretion of endogenous steroids. Administration of 1 mg of dexamethasone to six healthy male volunteers significantly decreased the urinary excretion of 11-hydroxycorticosteroids. However, the coadministration of magnesium trisilicate with dexamethasone decreased its absorption, as indicated by the increased urinary excretion of 11-hydroxycorticosteroids. The decrease in absorption was attributed to drug adsorption on the antacid surface. These results confirm previous in vitro findings.


Synthesis and quantitative structure-activity relationships of antibacterial 1-(substituted benzhydryl)-4-(5-nitro-2-furfurylideneamino) piperazines. 1-Benzhydryl -4- (5-nitro-2-furfurylideneamino) piperazine and 11 substituted analogs were prepared and examined for in vitro antimicrobial activity. The compounds were active against Bacillus cereus 7, Bacillus megaterium 122, Bacillus subtilis 104, Clostridium perfringens 13, and the tetracycline-resistant Clostridium perfringens 37. Regression analyses on the antibacterial activity data based on the Hansch approach, using pi, pi2, and sigma parameters, yielded several statistically significant correlation equations. 1-Benzhydryl-4-(5-nitro-2-furfurylideneamino) piperazine stopped the protein and DNA syntheses in C. perfringens 13, as indicated by precipitable radioactivity. The compound, however, showed no effect on the cell wall synthesis in the bacteria.


New opiate-receptor model.

New opiate-receptor model. A new opiate-receptor model is proposed in which only one conformation of the receptor is needed for binding of both agonists and antagonists. There are two different spacially fixed amine-binding sites in this model: one agonist and one antagonist. The opiates undergo binding to their amine-binding sites via the lone electron pair on nitrogen. The role of the N-allyl or other such group in imparting antagonist properties is explained in terms of the steric requirements of this group. For this group to be accommodated without imposing severe steric interactions in the rest of the opiate molecule, the piperidine ring must assume a flexible (skew boat) conformation; in this conformation, the N-lone-pair electron lobe assumes the characteristic directionality of an antagonist toward its amine-binding site. If the N-lone-pair lobe is not rigorously maintained in this direction, the opiate molecule assumes both antagonist and agonist conformations and mixed antagonist-agonist activity is observed. The observed differences in the effect of sodium on the degree of binding of an agonist versus an antagonist can be explained in this model by the different effects of sodium on the two amine-binding sites. The antagonist activity of an N-methyl antagonist can be rationalized on the basis of the proposed model.


Comparison of the mechanism of isoproterenol-stimulated glycogenolysis in skeletal muscle of normal and phosphorylase kinase-deficient mice (I strain). Diaphragm extracts from mice with the phosphorylase kinase deficiency mutation (I strain) have only 3.7% of the phosphorylase kinase activity of muscle extracts of the control strain (C57BL). Nevertheless, previous studies have shown that isoproterenol-stimulated glycogenolysis in I strain diaphragm muscle at a rate 57% (average relative response at 10 isoproterenol concentrations) of that of C57BL (GROSS, S.R., MAYER, S.E. and LONGSHORE, M.A.: J. Pharmacol. Exp. Ther. 198: 523-538, 1976). The present studies were initiated to compare the mechanism of isoproterenol-stimulated glycogenolysis in I and C57BL diaphragms. Isoproterenol was found to stimulate phosphorylase b to a conversion with an EC50 of 8 nM in muscles from mice of either strain, and the maximum increase in phosphorylase alpha activity in I diaphragms was 23% of that in C57BL diaphragms. Moreover, the initial rate of increase in phosphorylase alpha activity in I diaphragms incubated with 40 nM isoproterenol was 24% of that in C57BL muscles. The isoproterenol-stimulated increases in cyclic AMP content in diaphragms of the two strains were the same. Incubation of I diaphragms with isoproterenol did not significantly increase the concentrations of AMP, IMP or inorganic phosphate, activators of phosphorylase beta activity, nor was there a decrease in ATP and glucose 6-phosphate content, allosteric inhibitors of phosphorylase beta activity. Thus, phosphorylase alpha formation is the principal, if not only, catalyst of isoproterenol-stimulated glycogenolysis in skeletal muscle of phosphorylase kinase-deficient mice, and no evidence was obtained indicating that allosteric regulation of phosphorylase beta activity is part of the mechanism.


Genetic control of opiate-induced locomotor activity in mice. Swiss-Webster stock male and female mice were tested for running activity after a 20 mg/kg dose of levorphanol. The best runners and worst runners were then bred. Within each litter, brother-sister pairs with the highest and lowest running activity after a test dose of levorphanol (20 mg/kg) were segregated and bred. This procedure was followed to the F4 generation. Running activity, analgesia and brain catecholamines were studied. The results showed that the "non-running" (NR) trait segregated at the F1 generation, while the "running" (R) trait diverged more slowly. However, by the third generation, the R mice differed significantly from the F0 stock mice. NR mice also did not show locomotor activity when tested with amphetamine. While the NR mice showed reduced running response to levorphanol they were significantly more sensitive than the F0 stock to the analgesic effect. Measurement of brain catecholamines showed no difference between R and NR mice in dopamine or serotonin, but did show a significant increase in norepinephrine in the NR strain.


A behavioral approach to physical rehabilitation: a case study. This report presents an application of a behavioral approach used in treatment of a 65-year-old multiple handicapped woman admitted to a 29-bed physical rehabilitation hospital unit. Measurement used was a combined multiple baseline and BABA design. Findings can be summarized as follows: 1) Inappropriate behavior decreased during treatment, i.e., call outs and appropriate behavior increased, time spent out of bed, adhering to her diet, and wearing of a back brace; 2) Treatment quickly generalized to other behaviors;3) Follow-up measures at 6 and 18 months showed continued improvement.


Analgesics. 1. Synthesis and analgesic properties of N-sec-alkyl- and N-tert-alkylnormorphines. A series of N-sec- and N-tert-alkylnormorphines was synthesized and evaluated for analgesic potency, antagonist activity, and opiate receptor binding. Computer-assisted conformational analysis profiles were utilized to assist in the selection of compounds for synthesis and correlation of receptor events with in vivo observations. N-tert-Alkylnormorphines 5a-c were devoid of agonist activity; however, some sec-alkyl analogues showed interesting mixed agonist-antagnoist actions. N-sec-Butyl- and N-(alpha-methylally)normorphine were separated into R and S isomers, which exhibited quantitative pharmacological differences. The N-sec-butyl S isomer 10a showed analgesia approximating morphine with nalorphine-like antagonist activity. Preliminary testing indicates only slight evidence for physical dependence with this compound.


Post-menopausal vaginitis.

Post-menopausal vaginitis. The organisms cultured from the vaginal swabs of 383 women over 50 years old with vaginal discharge were compared with those from 148 normal women of similar age attending a cytology clinic. The only significant difference was that yeasts were found in 20% and 6% of the two groups of women, respectively. It is therefore concluded that in post-menopausal women with vaginal discharge, the main microbiological requirement is for the diagnosis and treatment of yeast infection.


Microviscosity of mucosal cellular membranes in toad urinary bladder: relation to antidiuretic hormone action on water permeability. The microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad, Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23 degrees C, 5 mM dibutyryl cyclic 3',5'-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P less than 0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.


Evidence for involvement of microtubules in the action of vasopressin in toad urinary bladder. I. Functional studies on the effects of antimitotic agents on the response to vasopressin. The antimitotic agents colchicine, podophyllotoxin, and vinblastine inhibit the action of vasopressin and cyclic AMP on osmotic water movement in the toad urinary bladder. The alkaloids have no effect on either basal or vasopressin-stimulated sodium transport or urea flux across the tissue. Inhibition of vasopressin-induced water movement is half-maximal at the following alkaloid concentrations: colchicine, 1.8 X 10(-6) M; podophyllotoxin, 5 X 10(-7)M; and vinblastine, 1 X 10(-7)M. The characteristics of the specificity, time-dependence and temperature-dependence of the inhibitory effect of colchicine are similar to the characteristics of the interaction of this drug with tubulin in vitro, and they differ from those of its effect on nucleoside transport. Inhibition of the vasopressin response by colchicine, podophyllotoxin, and vinblastine is not readily reversed. The findings support the view that the inhibition of vasopressin-induced water movement by the antimitotic agents is due to the interaction of these agents with tubulin and consequent interference with microtubule integrity and function. Taken together with the results of biochemical and morphological studies, the findings provide evidence that cytoplasmic microtubules play a critical role in the action of vasopressin on transcellular water movement in the toad bladder.


Formation of P1, P2-dinucleoside 5'-pyrophosphates under potentially prebiological conditions. Organic pyrophosphates such as UppA and NAD are formed when a solution containing a nucleotide, a nucleoside 5'-polyphosphate, Mg2+ and imidazole are allowed to dry out. We suggest that this synthesis may have occured concurrently with oligonucleotide formation.


Evolutionary change in invertebrate cytochrome C. Recently published amino acid sequences are compared to those of other cytochromes c. Molecular phylogenies constructed by using an ancestral sequence method are compared to the classical biological view of invertebrate evolution. Problems associated with the analysis of sequences of different chain lengths and of high variability are discussed, and the logistics of increasing the representation of key invertebrate phyla is assessed.


Parallel evolution of pairs of dehydrogenase isoenzymes. Lactate dehydrogenase and glycerol 3-phosphate dehydrogenase are metabolically coupled by the anaerobic dismutation of glyceraldehyde 3-phosphate and by the NAD redox state. This causes the concentrations of lactate and glycerol 3-phosphate to accumulate proportionally during anaerobic muscle contraction; these concentrations are high relative to those in aerobic tissues such as liver. We show that the isoenzymes of lactate dehydrogenase and glycerol 3-phosphate dehydrogenase from chicken breast muscle have Km values for lactate and glycerol 3-phosphate, respectively, that are 10-fold higher than the Km values measured for the lactate dehydrogenase and glycerol 3-phosphate dehydrogenase isoenzymes from chicken liver. The association of proportionally higher Km values with the potential for proportionally higher accumulation of substrates suggests that the isoenzymes of lactate dehydrogenase and glycerol 3-phosphate dehydrogenase from chicken muscle have evolved in parallel as a coupled metabolic unit distinct from the coupled isoenzymes in liver. The parallelism observed for the reduced substrates extends to the oxidized substrates, and to the coenzymes, NAD+ and NADH.


Mechanism of steroid binding to serum proteins. Association and dissociation rate constants of steroid complexes with progesterone-binding globulin (PBG) and with corticosteroid-binding globulin have been determined, utilizing the fluorescence quenching phenomenon observed on steroid binding to protein. Stopped-flow techniques were used in most cases. The dissociation rates of the complexes with steroid-binding proteins of serum are much greater than those of steroid-receptor complexes, in accordance with the biological functions of these two types of proteins. Association of steroids with PBG is accompanied by conformational changes in both components of the complexes. Chemical modification of tryptophan, lysine, and tyrosine in PBG results in inactivation of the binding site; complex formation with progesterone protects against this inactivation. A comparison of the affinity constants of PBG complexes with steroids of different structures leads to a conceptual image of the binding site and to localization of the various forces of interaction over the binding site area.


Alkaline DNase activity in cells infected with a temperature-sensitive mutant of herpes simplex virus type 2. BHK cells infected with the temperature-sensitive mutant ts13 of herpes simplex virus type 2 at a nonpermissive temperature lack the alkaline nuclease activity, which is induced by the mutant at a permissive temperature and by wild-type virus at either temperature. For ts13, enzyme activity could be induced by a temperature shift to permissive conditions, but not in the presence of cycloheximide. After a shift from permissive to nonpermissive conditions in the presence of cycloheximide, the activity was stable in wild-type, but not in mutant-infected, cells. After extensive purification, the wild-type nuclease was fourfold more heat stable in the presence of substrate than was the mutant enzyme. Mixtures of both purified enzymes showed the predicted intermediate stabilities. The results strongly suggest that the enzyme is virus coded and that the mutant possesses a lesion in the structural gene of the enzyme.


Nonsteroidal antiestrogens: their biological effects and potential mechanisms of action. The uterotropic and antiuterotrapic effects of a variety of structural derivatives of the nonsteroidal antiestrogen tamoxifen have been determined in the rat and the mouse. One derivative, monohydroxytamoxifen, was found to be a potent antiestrogen in the rat, with a high affinity for the estrogen receptor. Various techniques of sucrose density gradient analysis were used to demonstrate that estradiol and tamoxifen bind to the rat uterine cytoplasmic estrogen receptor. Estrogens and antiestrogens provoke the translocation of estrogen receptors to the nucleus and deplete the cytoplasmic estrogen receptor pool for short or long periods depending on the dose administered. Estradiol stimulates endometrial hyperplasia with an increase in total uterine DNA content, whereas tamoxifen stimulates endometrial hypertrophy with only a slight increase in uterine DNA content. It is concluded that the molecular shape of the ligand that binds to the estrogen receptor determines antiestrogenic activity.


Presence of 5'-terminal cap structures in virus-specific RNA from feline leukemia virus-infected cells. The F-422 line of feline thymus tumor cells, chronically infected with the Rickard strain of feline leukemia virus (R-FeLV), was labeled with 32P, and the total cytoplasmic RNA was isolated. The RNA was centrifuged through sucrose gradients, and R-FeLV virus-specific RNA (vRNA) was located by hybridization of portions of the gradient fractions to R-FeLV complementary DNA. vRNA classes with average sedimentation coefficients of approximately 36S, 28S, 23S, and 15S were identified. Each class of RNA was recovered by hybridized with mercurated R-FeLV complementary DNA, and the hybrids were chromatographed on columns of sulfhydryl-Sepharose to separate them from unhybridized cellular RNA. Although insufficient amount of 36S and 28S vRNA were obtained for further analysis, the 23S and 15S VRNA classes were analyzed to determine the nature of their 5' termini. Each of these vRNA classes was found to contain stoichiometric amounts of cap structures per unit length of RNA, consistent with the presence of one cap per molecule. The structure of the 23S vRNA cap was found to be m7G5'ppp5'GmpAp, whereas that of the 15S vRNA cap was m7G5'ppp5'GmpGp.


Hormonal regulation of cultured rabbit endometrial cells. A technique for culturing primary explants of rabbit endometrial cells in chemically defined medium has been developed. Diethylstilbestrol and natural estrogens were found to increase the rate of DNA initiation while progesterone had the opposite effect. Ultrastructural studies revealed that with estrogens, the cultures had the appearance of rapidly dividing cells having large euchromatic nuclei and prominent nucleoli, with aboundant free ribosomes in the cytoplasm. On the other hand, progesterone induced the formation of large multinucleated cells and also converted the cells to a more secretory type. When the cultures appear secretory, a protein called blastokinin (uteroglobin) is believed secreted. We have found that cultured cells synthesize blastokinin and progesterone is necessary for its continuous synthesis. The cultures were found to have receptors for the ovarian hormones and these receptors exhibited saturation kinetics. The dissociation constants for the receptors were also determined. A hypothetical model for the hormonal regulation of cell proliferation and differentiation is proposed.


RNA of mouse hepatitis virus.

RNA of mouse hepatitis virus. The RNA of mouse hepatitis virus, a coronavirus, was isolated from the virus released early in the infection and analyzed by sucrose gradient sedimentation and electrophoresis. It was found to consist of a piece of single-stranded RNA of about 60S. Its molecular weight was estimated to be 5.4 X 10(6) by electrophoresis in methylmercury-agarose gels. At least one third of the RNA contained polyadenylated sequences. It is, therefore, probably positive stranded. The virus harvested late in the infection contained, in addition to 60S, some 30 to 50S RNA that are possibly degradation products of the 60S RNA. No difference in the electrophoretic behavior could be detected between the RNA isolated from a pathogenic (JHM) and a nonpathogenic (A59) strain.


Mouse mammary tumor virus genes: regulation of expression by glucocorticoids and structural analysis with restriction endonucleases. The production of mouse mammary tumor virus (MTV) is stimulated by treatment of mammary carcinoma cells with glucocorticoid hormones (e.g., dexamethasone). The reaction appears to be a primary and receptor-mediated phenomenon in which the rate of synthesis of MTV RNA is selectively increased; the maximally induced rate is reached within 15 min after addition of the hormone. Production of MTV RNA is also stimulated by dexamethasone in some but not all clonal isolates of nonmurine cells infected by the virus. Using restriction endonucleases, we have characterized both integrated and unintegrated viral DNA from clones of infected rat hepatoma (HTC) cells. We suggest that the site(s) in the rat cell genome into which MTV DNA integrates may be a determining factor for MTV RNA synthesis.


Purification and characterization of varicella-zoster virus-induced DNA polymerase. Infection of WI-38 human fibroblasts with varicella-zoster virus led to the stimulation of host cell DNA polymerase synthesis and induction of a new virus-specific DNA polymerase. This virus-induced DNA polymerase was partially purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. This virus-induced enzyme could be distinguished from host cell enzyme by its chromatographic behavior, template specificity, and its requirement of salt for maximal activity. The enzyme could efficiently use poly(dC).oligo(dG)12-18 as well as poly(dA).oligo(dT)12-18 as template-primers. It required Mg2+ for maximal polymerization activity and was sensitive to phosphonoacetic acid, to which host alpha- and beta-DNA polymerase were relatively resistant. In addition, this induced DNA polymerase activity was enhanced by adding 60 mM (NH4)2SO4 to the reaction mixture.


Rescue of endogenous 30S retroviral sequences from mouse cells by baboon type C virus. Mus musculus SC-1 cells were infected with M7 baboon type C virus. The progeny of this infection included viral pseudotypes that contained M7 helper virus and endogenous 30S retrovirus-associated sequences derived from SC-1 cells (RAS). The RAS sequences are unrelated by nucleic acid hybridization criteria to previously described types of murine retroviruses and do not code for known murine viral structural proteins. The RAS genome is present in multiple copies in the DNA of laboratory (M. musculus) and Asian (M. caroli and M. cervicolor) mice, is expressed in the RNA of uninfected mouse cells, and can be efficiently rescued by type C, but not type B, viruses. RAS is closely related to 30S virus-associated RNA in NIH/3T3 and BALB/c JLSV-9 cells and may be analogous to the defective 30S RNA sequences found in rats.


Rescue of infectious virus from permissive monkey cells containing simian virus 40 DNA fragments. Permissive TC7 cells were separately transfected with simian virus 40 (SV40) EcoRI/Hap II A (74% genome) DNA fragments and EcoRI/Hap II B (26% genome) DNA fragments in the presence of DEAE-dextran. Fusion of the progeny of recipient cells receiving the A fragment, TC7 (SV40/74) cells, with TC7 (SV40/26) cells, which had received the B fragment, resulted in SV40 rescue. TC7 (SV40/74 + 26) cells, which had simultaneously received both complementary subgenomes, either spontaneously produced SV40 upon subculture or yielded virus upon treatment with iododeoxyuridine. In addition, fusion of rat cells containing the EcoRI/Hap II A fragment with TC7 (SV40/26) cells resulted in SV40 rescue. Cytopathology, V-antigen production, neutralization, and electron microscopy were parameters used to verify that the rescued virus was SV40. No infectious virus was produced when the combinations of cells fused did not total a complete SV40 genome equivalent.


Synthesis of viral and host DNA in isolated chromatin from herpes simplex virus-infected HeLa cells. DNA synthesis in chromatin isolated from herpes simplex virus type 1-infected HeLa cells (HSV chromatin) was examined in vitro. The HSV chromatin was found to carry out an initial limited synthesis of DNA in vitro, 50 to 64 pmol of dTMP incorporated in 10(6) nuclei per 10 min, which is comparable to that found in nuclei isolated from HSV-infected cells. DNA synthesis in vitro proceeded for only 30 min, and both HSV DNA and host DNA were synthesized in significant amounts. The HSV and host DNA synthesis in isolated chromatin were inhibited to the same extent by anti-HSV antiserum or by phosphonoacetic acid. The results indicate that the HSV-induced DNA polymerase is most likely involved in the synthesis of host and HSV DNA in isolated chromatin, even though this chromatin contains small amounts of the host gamma-polymerase in addition to the HSV-induced DNA polymerase. The HSV chromatin contains no detectable levels of DNA polymerases alpha and beta, even though infected cells have normal, or increased, levels of these enzymes.


Mechanism of poliovirus inactivation by ammonia. Poliovirus inactivation by ammonia causes a slight reduction in the sedimentation coefficients of viral particles, but has no detectable effect on either the electrophoretic pattern of viral capsid proteins or the isoelectric points of inactivated particles. These virions still attach to cells, but are unable to repress host translation or stimulate the synthesis of detectable amounts of viral RNA. Although ammonia has no detectable effect on naked poliovirus RNA, it causes cleavage of this RNA when still within viral particles. Therefore, the RNA genome appears to be the only component of poliovirus significantly affected by ammonia.


Recently replicated simian virus 40 DNA is a preferential template for transcription and replication. The template activities for the processes of replication and transcription were compared for recently replicated ("new") and uniformly labeled ("old") simian virus 40 (SV40) DNA in infected monkey cells (line TC7). New SV40 DNA (pulse-labeled for 1 h) served as a template for a second round of replication with a relatively high probability (8% of the DNA replicated per h) for a period of 5 h, after which time its template activity rapidly decreased by severalfold. Old SV40 DNA (labeled for 24 h) functioned as a template for replication with a constant probability (1.8% of the DNA replicated per h) for at least 12 h. The proportion of RNA polymerase with nonreplicated and with recently replicated (bromodeoxyuridine-substituted) viral DNA was determined by an assay that used the Triton-soluble SV40 transcription complex. The proportion of RNA polymerase associated with nonreplicated SV40 DNA decreased very slowly (to 50% in 6 h), strongly suggesting that replicating viral genomes are not required as templates for the initiation of late transcription. This hypothesis was supported by the finding that the RNA synthesized in vitro was associated with covalently closed circular SV40 DNA. Furthermore, after 9 h in bromodeoxyuridine, the recently replicated viral DNA had nearly three times more RNA polymerase per unit of DNA than did the nonreplicated DNA. We thus conclude that recently replicated SV40 DNA is utilized preferentially as a template for transcription and for replication.


More precise location of the polycytidylic acid tract in foot and mouth disease virus RNA. The polycytidylic acid [poly(C)] tract in foot and mouth disease virus RNA has been located about 400 nucleotides from the 5' end of the RNA by analysis of the products from the digestion of the RNA with RNase H in the presence of oligodeoxyguanylic acid [oligo(dG)]. This treatment produces a small fragment (S) containing the small protein covalently linked to the RNA and a large fragment (L) that migrates faster than untreated RNA on low-percentage polyacrylamide gels, lacks the poly(C) tract as shown by RNase T1 digestion and oligo(dG)-cellulose binding, and is no longer infective. Polyacrylamide gel electrophoresis of fragment S suggests that it is about 400 nucleotides long, in agreement with the size estimated from the proportion of radioactivity in the fragment. Analysis of the RNase T1 digestion products of S shows that it contains only those oligonucleotides mapping close to the poly(C) tract that is situated near the 5' end of the virus RNA.


Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei. Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.


Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 X HSV-2 recombinants. In an earlier paper (Morse et al., J. Virol 24:231--248, 1977) we reported on the provenance of the DNA sequences in 26 herpes simplex virus type 1 (HSV-1) X HSV-2 recombinants as determined from analyses of their DNAs with at least five restriction endonucleases. This report deals with the polypeptides specified by the recombinants and by their HSV-1 and HSV-2 parents. We have identified (i) the corresponding HSV-1 and HSV-2 polypeptides with molecular weights ranging from 20,000 to more than 200,000, (ii) the polypeptides that undergo rapid post-translational processing, and (iii) polypeptides that vary intratypically in apparent molecular weight. By comparing the segregation patterns of the polypeptides with those of the DNA sequence of the recombinants, we have mapped the templates specifying 26 polypeptides and several viral functions on the physical map of HSV DNA. The data show the following: (i) alpha polypeptides map at the termini of the L and S components of the HSV DNA. Although alpha ICP 27 maps entirely within the reiterated region of the L component, the template for alpha ICP 4 may lie only in part within the reiterated sequences of the S component. Of note is the finding that cells infected with a recombinant that contains both HSV-1 and HSV-2 DNA sequences in the S component produced alpha ICP 4 of both HSV-1 and HSV-2. (ii) Templates specifying beta and gamma polypeptides map in the L component and appear to be randomly distributed. (iii) Thymidine kinase and resistance to phosphonoacetic acid mapped in the L component. In addition, we have taken advantage of the rapid inhibition of host protein synthesis characteristic of HSV-2 infections and syncytial plaque morphology to also map the template(s) responsible for these functions in the L component. The implications of the template arrangement in HSV DNA are discussed.


Biosynthesis, immunological specificity, and intracellular distribution of the simian virus 40-specific protein induced by the nondefective hybrid Ad2+ND1. Ad2(+)ND(1), a nondefective hybrid virus containing a segment of the early region of simian virus 40 (SV40) DNA covalently inserted into the human adenovirus 2 genome, enhances the growth of human adenoviruses in simian cells and induces the SV40 U antigen. This hybrid previously has been shown to code for a 28,000 (28K) molecular weight protein not present in wild-type adenovirus 2-infected cells. By radioimmunoprecipitation using sera from hamsters bearing SV40-specific tumors, we have established that the Ad2(+)ND(1)-induced 28K protein is SV40-specific. This Ad2(+)ND(1)-induced protein is synthesized as a 30K molecular weight precursor, which is detectable only when infected cells are pulse-labeled in the presence of the protease inhibitor tosylamino phenylethyl chloromethyl ketone. Upon fractionation of labeled cell extracts, about 80% of the 28K protein is found in the plasma membrane fraction, whereas the remaining 20% is associated with the outer nuclear membrane. This protein is not detectable either in the nucleus or in the cytoplasm. Blockage of proteolytic cleavage by tosylamino phenylethyl chloromethyl ketone did not alter the topographic distribution of this SV40-specific protein, although the amount of the precursor protein in the outer nuclear membrane increased fourfold while that in the plasma membrane was proportionately decreased. This result suggests that the 28K protein is transferred from the outer nuclear membrane to the plasma membrane after posttranslational cleavage of the 30K precursor polypeptide. These data offer further support to the proposal that the 28K protein contains the determinants for SV40 U antigen and is responsible for SV40 enhancement of adenovirus growth in simian cells.


Intramembrane changes occurring during maturation of herpes simplex virus type 1: freeze-fracture study. During the maturation of two strains of herpes simplex virus type 1 (VR3 and Patton), intramembrane changes were detected with the freeze-fracture technique in the viral envelope and the infected cell plasma membrane, and these changes were compared with data obtained from thin sections. Regardless of the strain, the inner leaflet of the viral envelope of extracellular virions was characterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and plasma membrane. Addition of IMP, which probably represent virus-coded proteins, was detected in the viral envelope only after budding from the nuclear membrane, whereas it occurred during envelopment of capsids at cytoplasmic vacuoles. Fused membranes also showed one of their fracture faces covered with a high density of IMP similar to that of the mature virion envelope. The internal side of the membrane leaflet bearing these numerous particles was always characterized by the presence of an electron-dense material in thin sections. In addition, the plasma membrane of fibroblasts and Vero cells showed strain-specific changes: patches of closely packed IMP were observed with the VR3 strain, whereas ridges almost devoid of IMP characterized the plasmalemma of cells infected with the Patton strain. These intramembrane changes, however, were not observed as early as herpes membrane antigens. Thus, application of the freeze-fracture technique to herpes simplex virus type 1-infected cells revealed striking structural differences between viral and uninfected cell membranes. These differences are probably related to insertion and clustering of virus-coded proteins in the hydrophobic part of the membrane bilayer.


Structural components of mouse mammary tumor virus. II. Isolation and purification of virion polypeptides. Mouse mammary tumor virus (MMTV) glycoproteins and nonglycosylated polypeptides were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Primary amino groups were labeled with fluorescamine to enable visualization of MMTV polypeptides in the gels. Protein bands were sliced from the gels and eluted with 90 to 95% recovery. Eight MMTV polypeptides, including three of the major viral components as well as five minor proteins, were routinely obtained. Double diffusion assays and immunoelectrophoresis confirmed the retention of antigenicity identical to that of untreated MMTV virions. Antisera obtained from MMTV-free BALB/c mice immunized with these purified proteins reacted with the polypeptide immunogen as well as with detergent-disrupted MMTV virions from mouse milk or cell culture. Double diffusion assays using the specific mouse antisera failed to detect any cross-reactivity among the isolated polypeptides. A hemagglutination-inhibition assay demonstrated that the ability of MMTV virions to inhibit the hemagglutinating properties of influenza virus resides in the glycosylated polypeptides gp52, gp37.7, and gp33.


Restriction enzyme sites on the avian RNA tumor virus genome. Full-size single-stranded DNA transcripts of the avian RNA tumor virus genome were isolated from the products of the endogenous reaction of detergent-disrupted avian sarcoma virus particles. These transcripts were converted with Escherichia coli DNA polymerase I and 32P-labeled nucleoside triphosphates into labeled double-stranded DNA. The latter DNA was used to map the sites of action of seven restriction enzymes (Pvu I, Hpa I, Kpn I, Xba I, EcoRI, HindIII, and Xho I) on the genome of three strains of avian sarcoma virus (Prague B, Prague C, and Bratislava 77).


Control of protein synthesis in extracts from poliovirus-infected cells. I. mRNA discrimination by crude initiation factors. By using cell-free systems prepared from uninfected and poliovirus-infected cells, we have been able to demonstrate that crude preparations of initiation factors from infected cells do not stimulate the initiation of translation by polyribosomes containing endogenous host cell mRNA. When tested with polysomes containing endogenous viral mRNA, however, they were able to stimulate initiation of translation nearly as well as uninfected cell initiation factors. The uninfected cell initiation factor preparations were able to stimulate initiation of translation of both cell and viral mRNA. The results indicate an mRNA-specific activity present in crude initiation factor preparations from infected cells. Furthermore, the ability of eIF2 from infected cells to form a ternary complex with GTP and formyl [35S]methionine-tRNAfmet, an mRNA-independent step in initiation, was found not to be deficient. Implications of these data for proposed mechanisms of poliovirus-induced host cell shutoff are discussed.


Identification of virus found in mouse lymphomas induced by HIX murine oncornavirus. Lymphomas induced by pure HIX murine leukemia virus in two mouse strains contained large amounts of virus with amphotropic properties. Analysis of tumor derived virus purified by limiting dilution techniques indicated that its interference and neutralization spectra were essentially identical to parental HIX virus and different from eco- and xenotropic viruses. Examination of virus progeny from many individual foci induced by virus derived directly from lymphomas indicated that each infectious unit contained only HIX virus. No evidence of ecotropic virus presence was observed.


Adenovirus-induced inhibition of cellular DNase. During the productive infection of KB cells by adenovirus type 5 (Ad5), there is a progressive decrease in the level of cellular DNase activity towards single-stranded DNA, in contrast to DNA polymerase which remains relatively constant throughout the infection. This decrease is prevented by the inhibition of protein synthesis by cycloheximide. The inhibition of DNase activity does not occur after infection by Ad5 ts125, a DNA-negative mutant which fails to induce the adenovirus-specific DNA binding protein. In contrast, infection by Ad5 ts36, a DNA-negative mutant which complements ts125, does result in decreased levels of DNase. A mechanism is discussed in which the DNA binding protein protects viral replicative intermediates from degradation by cellular DNase.


RNA polymerase associated with human rotaviruses in diarrhea stools. RNA polymerase activity was detected in six stools which were partially purified by high-speed centrifugation from infants with rotavirus gastroenteritis, but was not detected in five stools which were negative for rotavirus by counterimmunoelectrophoresis and radioimmunoassay. The polymerase activity was associated with the 1.38-g/ml rotavirus band after purification in a CsCl gradient.


Simultaneous serum and CSF antibodies in herpes simplex virus encephalitis. Serial concomitant paired sera (S) and CSF were taken from eight patients with biopsy-proved herpes simplex virus encephalitis (HSVE). These specimen pairs were compared with 28 others from patients with various neurologic conditions. Before and after reduction with 2-mercaptoethanol, a ratio of S/CSF antibody titers of less than or equal to 20 with either the passive hemagglutinating (PHA) or immune adherence hemagglutinating (IAHA) antibody tests occurred in every patient with HSVE. Diagnostic S/CSF ratios were noted in three patients before biopsy of the brain and in four patients by the tenth day of neurologic disease. Among control subjects, a ratio of S/CSF titers greater than 20 was observed in all but four patients. Each of the latter patients had neurologic diagnoses easily distinguishable from HSVE. The PHA or IAHA S/CSF ratio offers a rapid, reliable method for the diagnosis of HSVE (P less than .001).


Epidemiological studies on Japanese encephalitis in Kyoto City area, Japan. I. Evidence for decrease of vector mosquitoes. Mosquito collections by using light traps have been carried out at 10 to 11 stations in Kyoto City area at intervals of about 10 days every year. Mean percent indexes (MPI), being calculated from the data of mosquito collections, were used for comparison of the annual abundance of mosquitoes. It is no doubt that Culex tritaeniorhynchus summorosus has decreased recently and this decrease is correlated with the reduction of human patients of Japanese encephalitis. Wide use of two herbicides, CNP and nitrofen, for rice plant cultivation, may probably be one of the reasons for the decrease of the mosquitoes.


Epidemiological studies on Japanese encephalitis in Kyoto City area, Japan. II. Annual patterns of virus dissemination on virus recoveries from unfed Culex tritaeniorhynchus summorosus. Annual patterns of dissemination of Japanese encephalitis virus in vector mosquitoes have been investigated at the main collection station from 1965 through 1973 at some other stations from 1969 through 1971. The virus was recovered usually from Culex tritaeniorhynchus summorosus during a period of about a month from July to August every year till 1969, and from August to September after 1970, although at some of the stations the virus was recovered intermittently for longer or shorter terms. Higher infection rates were recorded with the mosquitoes caught at the stations near to pig sheds than at the stations far from pig sheds. The infection rates at the peak of virus recovery in high epidemic year (1965 to 1967) were higher, being over 2%, than those in lower or latent epidemic years (1968 to 1973). Human patients of Japanese encephalitis were found in 17 to 20 days after the appearance of the highest peak of the infection rate in mosquitoes.


Nature of catecholamine-like actions of ATP and other energy rich nucleotides on the bullfrog atrial muscle. The nature of the inotropic actions of exogeneously applied ATP and related compounds (AMP-PNP, GTP, GMP and guanosine) was studied in comparison with that of adrenaline on the bullfrog atrial muscle under voltage clamped and unclamped conditions with the double-gap method. ATP and GTP (10(-6-) - 10(-3) M) produced an immediate positive inotropic effect similar to that achieved with adrenaline. Dose-response curves of these drugs fitted the theoretical dose-response relations, but the curves of ATP and GTP were not significantly altered by propranolol. Under voltage clamped conditions, ATP, AMP-PNP (non-hydrolyzable analogue of ATP) and GTP augmentated the calcium inward current (ICa), ICa-dependent tension and the delayed outward current (Ix) as adrenaline. On the other hand, GMP and guanosine, which have a purine-ribose base but not a high energy phosphate bond, produced a negative inotropic effect, and depressed the Ix as adenosine. All these nucleotides and nucleosides inhibited the ICa-independent tonic tension. The facts that GTP and AMP-PNP showed the same effect as ATP indicated that neither the energy liberation from the phosphate bond nor the substrate for cyclic AMP formation is involved in their positive inotropic effects. It is proposed that the energy rich nucleotides modulate the adenylate cyclase activity, being mediated by some receptor located at the outer surface of the membrane other than beta-adrenergic receptor.


[Virilizing adrenal cortical tumors in childhood (author's transl)]. We report on four patients, aged seven months to seven years, with virilizing adrenal cortical tumors. Diagnosis was made by clinical and laboratory data. Tumor localisation was achieved preoperatively in three patients. In two patients both androgen and glucocorticoid excretion were elevated. In three patients histological examination showed a carcinoma, in one patient an adenoma was suggested. There were no metastases. In two patients surgical removal was followed by cytostatic therapy, in one of them additional irradiation treatment was carried out. This child died 10 1/2 years later from a metastasizing renal cell carcinoma which originated from the kidney being situated in the irradiated area.


[Decrease in the radiation effect in the lymphoid organs of animals adapting to altitude]. Adaptation to altitude hypoxia exerts a positive effect on the cell density of lymph organs of gamma-irradiated animals. It also reduces radiation degradation of DNA, its availability to DNase in the nuclear chromatin and disorders in the activity of DNA polymerases. The role of inhibition of radiation-induced degradation of chromatin DNA in the protective effect of altitude adaptation on the cell density of lymph organs is discussed.


Surgery of tumors of the subdiaphragmatic inferior vena cava. Report of two cases and review of the literature. Two cases of obstruction of the hepatic inferior vena cava (IVC) are presented. In Case 1, the obstruction was due to a calcified thrombus which presumably developed after lower limb trauma 19 months previously. Complete surgical correction was achieved with the aid of deep hypothermia and circulatory arrest. In Case 2, a right atrial tumor was mimicked by a propagating malignant hepatoma. Palliative removal of the tumor cleared the IVC passage. The etiology of tumors of the IVC and their surgical management are discussed.