Biomedical subjects

D Falke

Publications and source records attributed to D Falke.

At least 73 records · Page 4Linked to original sources

PubMed1985

Differences in humoral immunogenicity between herpes simplex virus types 1 and 2.

Infection of NMRI mice with increasing doses of six different strains of herpes simplex virus type 1 (HSV-1) induced increasing levels of neutralizing antibodies. In contrast, three strains of HSV-2, irrespective of the dose, induced only marginal antibody responses. Only strain HG 52 (HSV-2) at high doses of infection stimulated antibody formation. The virus content of some organs in 6- to 8-week-old mice appeared to be lower after HSV-2 than after HSV-1 infection. Application of immune-modulating drugs [silica or poly(I) X poly(C) coupled via L-lysine to CM-cellulose] resulted in little augmentation of antibody formation if compared to HSV-1 infection. Secondary infections with HSV-1 or HSV-2 after a primary dose of HSV-1 were followed by a marked booster response. In contrast, a primary infection with HSV-2 suppressed secondary responses of HSV-1 and HSV-2, thus indicating fundamental differences between the antibody-stimulating potency of HSV-1 and HSV-2 strains.

Animals↗

PubMed1985

Fusion from without induced by herpes simplex virus type 1.

Strains ANG and ANG path of herpes simplex virus type 1 (HSV1) produced fusion from without (FFWO) of cells in culture. FFWO required 45 min to become complete. In contrast, fusion from within (FFWI) was not detected until 3-4 h after infection, depending on the cell type. FFWO was temperature dependent: at 0 degrees no fusion could be observed, but increase of temperature increased the degree of fusion. The pH optimum for FFWO was 7.8-8.5. The FFWO activity of the virus was found to be slightly more heat stable at 46 degrees than was infectivity. FFWO was produced in Vero, CV-1 and BSC1 cells, but not in BHK clone 13 or in primary or secondary rabbit kidney cells. FFWO was linked to the presence of virus particles and perhaps to other sedimentable, infected-cell material but not to soluble factors. Actinomycin D, cycloheximide, and UV irradiation did not block this activity, indicating no direct activity of the HSV1 genome for FFWO.

Animals↗

PubMed1984

Inhibition of the herpes simplex virus-coded thymidine kinase-complex by 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) and 9-(2-hydroxyethoxymethyl)guanine-monophosphate (acyclo-GMP).

The thymidine kinase-complex isolated from herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) is associated with the following enzyme activities:ATP:dThd (dCyd) deoxypyrimidine kinase, ATP:dTMP thymidylate kinase, ADP:dThd- and AMP:dThd 5'-phosphotransferase. In kinetic experiments it is shown that ara-AMP inhibits AMP:dThd- and ADP:dThd phosphotransferase activity, while acyclo-GMP impairs ADP:dThd phosphotransferase reaction only; the inhibition was found to be non-competitive. The functional subunit ATP:dThd kinase was not affected by either compound.

Acyclovir↗

PubMed1984

Replication of HSV-1 in murine peritoneal macrophages: comparison of various virus strains with different properties.

The in vitro replication of eleven different strains of herpes simplex virus type 1 was studied in resident or thioglycollate-stimulated mouse macrophages. The strains of herpes simplex virus differed in the type of cytopathic effect, induction capacity for herpes simplex virus coded thymidine kinase and pathogenicity in the mouse. Herpes simplex virus replicated better in thioglycollate-stimulated macrophages than in resident macrophages. In vitro ageing of macrophages increased their replicative potency. Herpes simplex virus replicated better in macrophages from homozygous bg/bg C57/BL6J mice than in macrophages from their heterozygous littermates. Separation of macrophages on discontinuous Percoll-gradients revealed 4 fractions with identical potency for replication. The ability of herpesvirus to replicate in macrophages varied from strain to strain of virus i.e. Wal greater than Len, clone 4 of Len, greater than L3-2s, JES, Ang-, Ang + path, clone 2 of Len and greater than MDK clones. The ability to cause cytopathology also varied. Only strains Ang- and Ang + path showed limited or late cytopathology in macrophages. The cell-fusing property of herpes simplex virus appeared to be more closely correlated with lower replication rates than production cell rounding. Thymidine kinase- viruses replicated less well than thymidine kinase+ or thymidine kinase(+) strains. Strains of herpes simplex virus with high or low pathogenicity for mice replicated in macrophages to the same degree. The phagocytic activity of macrophages for IgM-coated sheep red blood cells was inhibited earlier by strains of herpes simplex virus of type 2 than by strains of herpes simplex virus of type 1.

Animals↗

PubMed1984

Electron microscopic observations on primary hepatocyte cultures infected with herpes simplex virus types I and II.

The replication cycle of the Herpes simplex virus (HSV) strains I and II has so far been described mainly in established proliferative cell cultures. Most of the biochemical data and ultrastructural cell changes regarding the virus-cell interaction have been obtained from 'permissive' cells which allow almost unrestricted viral multiplication. It seems obvious, however, that the in vivo viral infections are not represented adequately by these experiments. In order to achieve a more realistic view of the ultrastructural events during HSV infection of adult tissue, cell cultures were prepared from adult mouse and rat livers and infected with several HSV strains. Established 'permissive' cell lines (BHK and RK cells), served as controls. Although the main principles of viral attachment, replication and release of viral particles, were similar in hepatocytes and proliferating cells, marked differences were observed regarding the better preservation of the nuclear structures, the lower replication rate of viruses, the hypertrophy of the endoplasmic reticulum, and the changes in the Golgi apparatus. Summarizing, it can be stated that hepatocytes infected by HSV in cell culture display the well known general features of adult cells infected by viruses in vivo.

Animals↗

PubMed1984

Enhancement of antibody formation against herpes simplex virus in mice by the T-cell mitogen bestatin.

The influence of the T-lymphocyte-stimulating dipeptide bestatin on the induction of neutralizing antibodies against herpes simplex virus type 1 in the mouse was investigated. Dose-response experiments revealed two active ranges from 1 ng/kg to 100 ng/kg and from 10 micrograms/kg to 10 mg/kg or more. Bestatin (10 mg/kg) enhanced antibody levels after primary infection, if injected between day 5 and day 8 after infection with a maximum effect at day 5. Following secondary infections, bestatin was most effective at day 1 after secondary infection. Moreover, the antibody-generating potency of a formalinized herpes simplex virus type 1 vaccine was elevated considerably. Bestatin and silica seemed to be effective systemically. Treatment of mice with silica before virus infection and additionally with bestatin at day 1 after infection resulted in an additive effect on antibody production. Comparable effects could be obtained when polyinosinic acid X polycytidylic acid or indomethacin was combined with bestatin at day 1. It was assumed that certain factors released by macrophages 'sensitize' the antibody-producing system for the enhancing activity of bestatin at day 1. Indeed, culture fluids of macrophages obtained from mice either pretreated with silica or infected by herpes simplex virus were active in enhancing antibody formation upon injection into mice at day 1 in combination with bestatin. Bestatin did not induce interferon activity. No influence of bestatin on the virus content of organs or on mortality was observed.

Animals↗

PubMed1984

Infection of murine hepatocyte cultures by herpes simplex virus (HSV) 1 and 2.

A study was undertaken of the interaction between liver cells and Herpes Simplex Virus (HSV) in vitro. Hepatocytes were obtained from HSV-resistant (C57/B16) and from HSV-susceptible (BALB/c, A/J, C3H) mouse strains and cultured according to standard methods. Each culture was infected with several strains of HSV-type 1 or of HSV-type 2, respectively. The multiplicity of infection was 5. The cytopathic effect was evaluated by light- and electron-microscopy. The number of infectious particles was determined using rabbit kidney or Vero cell cultures. All evaluations were made at different time intervals after infection. No difference concerning the replication rate of HSV-1 and 2 in isolated hepatocytes from resistant and susceptible mouse strains was detected. However, a marked difference was observed with respect to the production of infectious particles: HSV-1 exhibited a good multiplication rate, whereas no production of virus particles was observed with HSV-2. The data show that mice hepatocytes fail to bear the genotype for resistance to HSV infections.

Animals↗

PubMed1983

Identification and properties of the cell membrane bound leucine aminopeptidase interacting with the potential immunostimulant and chemotherapeutic agent bestatin.

Bestatin was found to be a competitive inhibitor (with respect to the Leu-NA substrate) not only of the isolated microsomal and cytosolic leucine aminopeptidases (Leu-APm and Leu-APc) but also of the aminopeptidases (APs) present in membrane preparations (from mouse liver) and on the cell surface of L5178Y cells. Kinetic parameters indicate that cellular AP is identical to Leu-APm. To rule out the possibility that AP-B is involved in the inhibition reactions, comparable studies with amastatin were performed. Electrophoretical studies revealed the solubilized cell membrane bound AP to co-migrate with Leu-APm in polyacrylamide gels. The activity of the separated membrane AP was inhibited by bestatin in situ. The cell membrane bound AP activity was found to be lowest in lymphocytes, higher in tumor cells and highest in bone marrow cells and macrophages. Using synchronized L5178Y cells, the AP activity changes during the cell division cycle; the lowest activity was determined during the G1-phase and 35% higher values were measured during the S/G2-phase. The fluctuation of the cell surface associated AP activity parallels with changes in the number of binding sites for bestatin.

Animals↗

PubMed1983

Kinetics and genetics of herpes simplex virus-induced antibody formation in mice.

The kinetics of antibody synthesis was investigated after intraperitoneal, subcutaneous, and footpad infection of various strains of mice with herpes simplex virus. Immunoglobulin M antibodies appeared 5 days after and immunoglobulin G antibodies appeared 10 to 12 days after intraperitoneal infection with herpes simplex virus type 1. The major histocompatibility complex and the background genome of inbred mice were not found to have a systematical influence on antibody synthesis. Female mice, however, consistently produced more antibodies than did male if the infection was done intraperitoneally, but not if it was done subcutaneously or into footpads. Castration considerably increased the amount of antibodies produced by male mice. The difference in antibody formation between females and males could be abolished by injection of silica; moreover, antibody titers were enhanced by this treatment. This has also been found by immunization with a Formalin-inactivated herpes simplex virus vaccine. The effect of silica in enhancing antibody formation could be observed up to 12 days after infection. Infectious virus could be detected up to 2 days after infection, and herpes simplex virus type 1 antibody-stimulating antigens could be detected up to 4 days in ultrasonicates of macrophages. The assumption is made that androgen-sensitive cell populations, including macrophages and their soluble products, are involved in antibody-depressing mechanisms.

Animals↗

PubMed1983

Adenosine- and ADP-phosphorylating capacity of herpes simplex virus-induced thymidine kinase enzyme complex.

The herpes simplex virus(HSV)-coded thymidine kinase (TK) enzyme complex was isolated from HSV type 1 strain Lennette(TK+)-infected CLID (TK-) cells and was enriched by streptomycin sulfate and ammonium sulfate fractionation. This enzyme preparation was tested for dTMP:Ado (adenosine) and for dTMP:ADP phosphotransferase activities. The presence of dTMP:Ado phosphotransferase activity was proven by time-course studies with cells infected for 0-18 h, by biophysical studies in polyacrylamide gels, by affinity chromatography studies using AMP- and dTMP-Sepharose, and by immuno-neutralization experiments. The presence of dTMP:ADP phosphotransferase activity was demonstrated by kinetic experiments. These results were taken as evidence that the two functional subunits of the HSV-TK enzyme complex, AMP:dThd (deoxythymidine) phosphotransferase and ATP: dThd kinase, catalyze highly reversible enzyme reactions. New data are presented indicating that the ATP:dThd kinase is a nonspecific enzyme with respect to the nucleoside acceptor.

Adenosine↗

PubMed1982

Adenosine diphosphate: thymidine 5'-phosphotransferase, a new enzyme activity, associated with the Herpes simplex virus-induced deoxypyrimidine kinase.

The deoxypyrimidine kinase induced in mouse fibroblasts, strain CLID (TK-) infected with either herpes simplex virus (HSV) type 1 or type 2, possesses besides deoxypyrimidine kinase (ATP:dThd/dCyd phosphotransferase) two further enzyme activities: an AMP:dThd phosphotransferase and an ADP:dThd phosphotransferase. The latter enzyme activity, described in this report, was found to be inhibited by antiserum against the HSV deoxypyrimidine kinase and to be absent after infection with TK- mutant MDK 10 (B 2006). The ADP:dThd phosphotransferase, which had been purified approx. 340-fold, differs by a series of physicochemical properties from the viral AMP:dThd- and ATP:dThd phosphotransferase.

Animals↗

PubMed1982

Alkaline phosphodiesterase I and alkaline phosphatase I in plasma membranes of herpes simplex virus type 1 transformed hamster cells.

Plasma membrane extracts from Herpes simplex virus type 1 transformed hamster embryo fibroblasts were chromatographed on Lens culinaris lectin coupled to Sepharose (LcH-Sepharose) and analysed by dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie blue-staining revealed two major protein bands with apparent molecular weights of 125 000 and of about 75 000-90 000. In plasma membranes isolated from these tumor cells prior labeled with [3H]fucose or [3H]glucosamine these bands contained the highest amounts of incorporated radioactivity. Separation by LcH-Sepharose-affinity chromatography as well as metabolic labeling clearly demonstrates their glycoprotein character. The 125 000 protein coincides with alkaline phosphodiesterase I activity with a Km of 6 . 10(-4) M for TMP p-nitrophenyl ester and is competitively inhibited by UDP-N-acetylglucosamine. This enzymatic activity is also present in normal hamster embryo fibroblasts. Gel electrophoresis of the Lens culinaris lectin-binding glycoproteins from plasma membranes of normal hamster embryo fibroblasts additionally revealed a strong alkaline phosphatase activity represented by an apparent molecular weight of 150 000, while HSV1 hamster tumor cells contain only a very weak activity of this enzyme activity. HSV-lytically infected cells, however, have unchanged levels of alkaline phosphatase activity, whereas alkaline phosphodiesterase activity increases slightly.

Alkaline Phosphatase↗

PubMed1982

Analysis of the TK enzyme complex induced by HSV types 1 and 2 by means of isoelectric focusing and polyacrylamide gel electrophoresis.

Recently we have described that the Herpes simplex virus (HSV)-induced thymidine kinase (TK) induces AMP- and ADP-dThd-5'-phosphotransferase activities. We now demonstrate the heterogeneity of the described activities in isoelectric focusing experiments and polyacrylamide gel electrophoresis. A TK--mutant of HSV type 1 fails to induce these activities. The activities of the type 1 enzyme complex was neutralized by an anti-HSV-serum. The TK-enzyme complex expressed in LTK--cells transformed to a TK+-phenotype by sheared HSV-1 DNA was compared with the wild type TK complex in isoelectric focusing experiments. Additionally we demonstrate that the HSV type 1 enzyme complex has thymidylate kinase activity, while the type 2 TK complex did not exhibit thymidylate kinase activity. Feedback regulation mechanisms by dTMP, dTDP and dTTP were investigated using partially purified enzyme preparations of HSV types 1 and 2 infected TK--cells.

Electrophoresis, Polyacrylamide Gel↗

PubMed1982

Properties and specificity of binding sites for the immunomodulator bestatin on the surface of mammalian cells.

The binding of the immunomodulator bestatin, an inhibitor of cell surface bound leucine aminopeptidase and aminopeptidase B, to mammalian cells of varying origin has been studied. The specific binding of [3H] bestatin was a rapid and saturable process exhibiting one affinity, characterized by an association constant of 0.8 x 10(5) M-1, as determined in the L5178y mouse lymphoma system. Optimal binding was observed at 37 degrees C. L-leucine and L-leucine-beta-naphthylamide prevented the binding, suggesting that the complex was formed between leucine aminopeptidase and bestatin. The protein nature of the bestatin-"receptor" was suggested by its susceptibility to trypsin. Under the conditions used here intracellular translocation of bestatin appeared to be negligible. A maximum of about 2.2 x 10(6) bestatin molecules could bind to L5178y mouse lymphoma cells. Under identical conditions by far the highest amount of bestatin was bound to macrophages from mice. Lower levels were measured with T-lymphocytes; very low binding capacity was observed with B-lymphocytes. Experiments with synchronized L5178y cells revealed a cell cycle dependent change of binding capacity for bestatin; the highest level was observed during the transition from S-to G2 phase and the lowest during G1- and early S phase. These data lend further support to the assumption that the immuno-potentiating activity of bestatin is due to a stimulation of T-lymphocyte proliferation probably mediated through the activation of macrophages.

Adjuvants, Immunologic↗

PubMed1981

DNA-replication complex from cells infected with herpes virus.

Herpes simplex virus (HSV) DNA synthesis is initiated in an intact cell system by a 36-residue ribonucleotide stretch [W.E.G. Müller, R.K. Zahn, J. Arendes, and D. Falke (1979) Virology, 98, 200-210]. In the present study a nucleoplasmic fraction was isolated from rabbit kidney cells infected with HSV (type 1), which catalyzes DNA synthesis. By means of specific assays, containing single-stranded deoxyribopolymers, it was elucidated that the replication complex contains both an RNA-synthesizing and a DNA-synthesizing enzyme. These enzymes were characterized as host cell RNA polymerase II and HSV-induced DNA polymerase. The RNA polymerase II synthesizes an RNA initiator with an average chain length of 25 nucleotides, to which the newly synthesized DNA is covalently attached. The chemical nature of the RNA primer was proven by degradation experiments with endoribonuclease V. In the absence of any initiator in the reaction mixture the HSV-induced DNA polymerase is inactive. Kinetic experiments revealed that DNA synthesis in assays containing poly(dT), dNTPs and NTPs starts after a lag phase of 10 min during which the RNA initiator is synthesized. The HSV DNA NTPs starts after a lag phase of 10 min during which the RNA initiator is synthesized. The HSV DNA replication complex was further fractionated into HSV-induced DNA polymerase and the three cellular RNA polymerases. In the reconstructed system DNA synthesis, catalyzed by virus-induced DNA polymerase starts in a reaction assay, containing single-stranded DNA template, only in the presence of RNA polymerase II. The conclusion that the subnucleoplasmic complex, isolated from HSV-infected cells, is of biological significance for HSV DNA synthesis stems (a) from the the close correlations of the known biochemical events occurring during the initiation of HSV DNA synthesis in intact cells and in the nucleoplasmic system and (b) from control experiments with a subnucleoplasmic complex, isolated from uninfected cells, which is devoid of any potency to initiate DNA synthesis.

Animals↗

PubMed1981

Microtubules and microfilaments in HSV-Infected rabbit-kidney cells.

In rabbit kidney cells infected with strains of Herpes simplex virus producing either cell-rounding or polycaryocytosis. Vinblastine induced paracrystals. This could be shown by phase-contrast- and electron-microscopy. Infections were done under one-step-growth conditions or at low MOI. 90 per cent noninfected cells contained stress fibers as detected by Servablue R250-staining. Shortly after recruitment into polycaryocytes, stress fibres of normal length appearing in criss-cross arrangement can be seen in the periphery of these cells. Later they polymerize to very long fibers and finally they are partially destroyed. The time of destruction depends on the MOI employed. By using Actinomycin D and/or Cycloheximide as blocking agents, it could be shown that polymerization of microfilaments correlates in time with giant cell formation. In view of the fact that the virus synthesis is accompanied in parallel by a special rearrangement of microfilaments as well as polycaryocytosis, both these processes have to be considered as caused by early (and late ?) protein-synthesis (beta-/gamma-proteins) but not as induced by "very-early" proteins (alpha-proteins).

Animals↗

PubMed1981

Adenylic acid: deoxythymidine 5'-phosphotransferase: evidence for the existence of a novel herpes simplex virus-induced enzyme.

BHK (dPyK-) cells infected with herpes simplex virus type 1 (HSV-1) contain a virus-induced deoxythymidine (dThd)-phosphorylating enzyme. This enzyme uses AMP as phosphate donor and is called AMP :deoxythymidine 5'-phosphotransferase (or kinase). The enzyme was purified over 1300-fold and was found to be specific for an AMP substrate. It can thus be distinguished from virus-specific deoxypyrimidine kinase (dPyK). It is shown that the two substrates AMP and dThd participate in the reaction at a 1 : 1 molar ratio; the Km for AMP was 2 X 3 muM and for dThd it was 2 X 1 muM. The mol. wt. of the enzyme was estimated to be between 110 000 (by glycerol gradient centrifugation) and 90 000 (by gel filtration). For optimum activity, the phosphotransferase required an alkaline pH, and 37 degrees C; the activation energy of the reaction was 18 450 cal/mol. The appearance of the enzyme after infection parallels that of viral DNA synthesis-related functions.

Adenosine Monophosphate↗

PubMed1980

Infections of susceptible and resistant mouse strains with herpes simplex virus type 1 and 2.

The spread of HSV of type 1 and 2 was investigated after intraperitoneal, intraplantar and intracerebral infections of resistant (C57/bl) and susceptible (NMRI) mice. The virus spreads after i.p. infection to the spleen and the liver to the same extent in both strains of mice. However, virus is eliminated earlier in resistant mice. Intracerebral infections revealed a peculiar type of resistance of C57/bl mice especially for type 2 of HSV. HSV multiplies in the thymus at the early stage of infection and can be detected in this organ in sick mice of NMRI strain. HSV-1 and 2 can be detected in the spinal cord of C57/bl mice without sickness or death of these animals.

Animals↗