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T Albrecht

Publications and source records attributed to T Albrecht.

At least 109 records · Page 6Linked to original sources
PubMed1990

Human cytomegalovirus stimulates arachidonic acid metabolism through pathways that are affected by inhibitors of phospholipase A2 and protein kinase C.

Inhibitors of phospholipase A2, tetracaine and quinacrine, inhibitors of protein kinases, H-7 and H-8, and a diacylglycerol lipase inhibitor reduced the level of CMV-induced [3H]AA release. A combination of H-7 and quinacrine inhibited stimulation of [3H]AA by about 80%. LU cells chronically treated with TPA and infected with CMV, had a reduced level of CMV-induced [3H]AA release and in the presence of quinacrine it was completely inhibited. These results suggest that CMV-induced stimulation of AA metabolism is mediated by pathways which are associated with activation of PLA2 and protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Human cytomegalovirus. Stimulation of [3H] release from [3H]-arachidonic acid prelabelled cells.

Exposure of human lung fibroblasts to human cytomegalovirus (HCMV) stimulated a rapid increase in the release of [3H] from cells prelabelled with radiolabelled arachidonic acid ([3H]AA). Maximum stimulation of [3H] release was observed at 20 min postinfection and was quantitatively similar to that induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA: 10 nM) or fetal calf serum (5%). The level of [3H] release was dependent on the multiplicity of infection, and appeared to be mediated by a component(s) of the virion, since the findings from three series of experiments suggested that neither infectious virus, nor HCMV-specific macromolecular synthesis was required for stimulation of [3H] release. (1) Inactivation of HCMV infectivity with ultra-violet (UV) light (approximately 254 nm, 4.80 x 10(4) ergs/mm2) did not diminish the stimulation of [3H] release. (2) Significant reduction in the level of [3H] release was not observed when infected cells were maintained in the presence of a protein synthesis inhibitor, cycloheximide (50 micrograms/ml), or an inhibitor of mRNA synthesis, 3'-deoxyadenosine (cordycepin, 50 micrograms/ml). (3) No correlation was established between the expression of HCMV immediate early (IE) antigens and the induction of [3H] release, since there was little, if any, synthesis of HCMV IE antigen detectable by anticomplement immunofluorescence through the first 30 min postinfection. These findings suggesting that the HCMV particle rapidly stimulates AA metabolism are consistent with the view that the interaction of a HCMV virion component(s) with the cell surface may initiate membrane-associated events similar to those induced by growth factors.

Antigens, Viral↗
PubMed1990

Taxol inhibits stimulation of cell DNA synthesis by human cytomegalovirus.

The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 micrograms/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 micrograms/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle.

Alkaloids↗
PubMed1990

Compensation of respiratory alkalosis induced after acclimation to simulated altitude.

Conscious intact rats previously acclimated for 3 wk to barometric pressure of 370-380 Torr (3WHx) were made alkalotic for 3 h by a decrease in inspired O2 fraction from 0.10 to 0.075 at ambient barometric pressure (730-740 Torr). Controls were normoxic littermates (Nx) in which inspired O2 fraction was lowered from approximately 0.21 to 0.10 for 3 h. Arterial PCO2 decreased progressively and similarly in both groups (65-70% of control at 15 min). Initially, arterial pH increased less in 3WHx (0.09 +/- 0.004 vs. 0.15 +/- 0.008). As hypocapnia continued, delta[HCO3-]/delta pH (mmol.l-1.pH) became more negative in Nx, from -15.2 +/- 2.5 at 15 min to -37.0 +/- 2.9 at 3 h, indicating nonrespiratory compensation of alkalosis. In 3WHx, delta[HCO3-]/delta pH did not change during alkalosis. Cumulative renal excretion of base (mueq/100 g) during alkalosis increased by 73.2 +/- 11.1 in Nx and 25.4 +/- 7.3 in 3WHx. This difference was mainly due to a larger increase in HCO3- excretion in Nx. The data suggest that the smaller compensation of hypocapnic alkalosis in 3WHx is partly due to the smaller increase in renal base excretion. Because base availability limits renal base excretion, the smaller renal response of 3WHx may be secondary to the low plasma HCO3- concentration that accompanies altitude acclimation.

Acid-Base Equilibrium↗
PubMed1990

Cell activation signals and the pathogenesis of human cytomegalovirus.

Cytomegalovirus (CMV) infection induces a series of cellular responses that resemble those observed in cells activated by growth factors or hormones including: hydrolysis of phosphatidylinositol-4,5-bisphosphate; Ca2+ influx and an increase in the cytosolic free [Ca2+]; an increase in Na+ entry; and, increases in cellular levels of cyclic AMP and cyclic GMP. The time courses for some of these responses appear to be markedly protracted relative to those observed for growth factors. The prolonged physiologic responses in CMV-infected cells appear to be related to modifications in the intracellular environment that are associated with the development of cytomegaly and with the phasing of CMV-directed macromolecular synthesis. For example, as the infected cell enlarges, the rate of CMV DNA synthesis increases by about 4-fold, late nuclear inclusions develop and progeny viruses are formed. When the CMV-induced activation signals are inhibited or their physiologic responses are blocked, then the yields of infectious CMV are substantially reduced. Furthermore, perturbation of the cell cycle resulting from induction of the cell activation process by CMV may be causally related to the induction of cellular damage by CMV, even in the absence of productive infection. Accordingly, the CMV-induced pathophysiologic cell activation responses represent potential targets for novel antiviral therapy.

Amiloride↗
PubMed1989

Interleukin 2 induced non MHC-restricted killing of herpes simplex type-1 (HSV-1) infected allogeneic and autologous lymphoblasts.

Peripheral blood lymphocytes (PBL) from herpes simplex type-1 (HSV-1) seropositive (sero+) and seronegative (sero-) individuals were assessed before and after in vitro culture with IL-2 for their ability to kill different tumor cells and to kill HSV-1 infected PHA lymphoblasts using a 4 hr 51Cr release assay. Fresh PBL from either sero+ or sero- individuals had little cytotoxic activity against HSV-1 infected autologous PHA lymphoblasts or against different NK-insensitive tumor targets. When PBL from either sero+ or sero- individuals were incubated 3-4 days in vitro with IL-2, significant levels of cytotoxic activity was observed against both allogeneic and autologous HSV-1 infected targets and against different NK-insensitive tumor cells. When comparing PBL from sero+ versus sero- individuals for IL-2 induced cytotoxic activity against HSV-1 infected autologous targets, PBL from sero+ individuals always had significantly higher levels of cytotoxic activity. Allogeneic HSV-1 infected targets were also killed by IL-2 activated PBL obtained from either sero+ or sero- individuals, but IL-2 activated PBL from sero+ individuals showed higher cytotoxic activity on HSV-1 infected autologous targets when compared to HSV-1 infected allogeneic targets. When IL-2 induced cytotoxic effector cells were treated with HSV-1 for 1 hr and recultured with IL-2, significant inhibition of cytotoxic activity against all target cells was observed. Greater than 50% inhibition of cytotoxic activity was observed when IL-2 induced cytotoxic effector cells were treated with HSV-1 and cultured for 6 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

[Secondary messenger level of inositol triphosphate in fibroblasts after treatment with papaverine].

Ten per cent foetal bovine serum had been added to human embryonic fibroblasts which had previously been incubated in serum free cultures. The effect has been studied on quantity of synthetized DNA in next 30 hours and on inositol-1,4,5-phosphate contents of cells. On the effect of foetal bovine serum synthesis of DNA has increased four times and 1 minute after its addition inositol-1,4,5-phosphate level has doubled. Papaverine given together with FBS has suspended inductive effect of FBS on DNA synthesis and its increasing effect on level of inositol-1,4,5-phosphate.

Cells, Cultured↗
PubMed1988

Induction of chromosome aberrations and mitotic arrest by cytomegalovirus in human cells.

Human cytomegalovirus (CMV) is potentially an effective but often overlooked genotoxic agent in humans. We report here evidence that indicates that infection by CMV can induce chromosome alterations and mitotic inhibition. The frequency of chromosome aberrations induced was dependent on the input multiplicity of infection (m.o.i.) for human lung fibroblasts (LU), but not for human peripheral blood lymphocytes (PBLs) when both cell types were infected at the GO phase of the cell cycle. The aberrations induced by CMV were mostly chromatid breaks and chromosome pulverizations that resembled prematurely condensed S-phase chromatin. Pulverized chromosomes were not observed in LU cells infected with virus stocks that had been rendered nonlytic by UV-irradiation at 24,000 ergs/mm2 or from infection of human lymphocytes. In LU cells infected with UV-irradiated CMV, the frequency of aberrations induced was inversely dependent on the extent of the exposure of the CMV stock to the UV-light. In permissive CMV infection of proliferating LU cells at 24 hr after subculture, a high percentage (greater than 40%) of the metaphase cells were arrested at their first metaphase and displayed severely condensed chromosomes when harvested 48 hr later. A significant increase (p less than 0.05) in the chromosome aberration frequency was also observed. Our study shows that CMV infection is genotoxic to host cells. The types and extent of damage are dependent on the viral genome expression and on the cell cycle stage of the cells at the time of infection. The possible mechanisms for induction of chromosome damage by CMV are discussed.

Cell Division↗
PubMed1988

Hydrolysis of inositol lipids: an early signal of human cytomegalovirus infection.

The production of inositol 1,4,5-triphosphate (IP 3) and 1,2-diacylglycerol (DG) as an early cellular response to human cytomegalovirus (HCMV) infection was investigated in human embryo lung (LU) cells. The results obtained show transient 85 and 30% increases in IP3 and DG, respectively, 20 min post-exposure to HCMV. Following heat inactivation or serum neutralization of HCMV stock or in the presence of papaverine we failed to detect increased levels of IP3 and DG relative to mock-infected LU cultures.

Antigens, Viral↗
PubMed1988

Cytomegalovirus: sodium entry and development of cytomegaly in human fibroblasts.

A possible relationship between net Na+ entry and the development of CMV-induced cytomegaly (cell enlargement) was investigated in human fibroblasts derived from skin-muscle and thyroid tissue. We found that inhibiting cellular Na+ uptake, either by pharmacological means (amiloride, an inhibitor of Na+/H+ exchange) or by replacement of extracellular Na+ (by N-methyl-D-glucamine or choline), inhibited the development of cytomegaly. Furthermore, we noted a temporal parallelism between the development of cytomegaly and enhancement of ouabain-sensitive (O-S) 86Rb+ uptake. O-S 86Rb+ uptake is a monitor for the activity of the sodium pump resident in the plasmalemma of the fibroblasts. The enhanced O-S 86Rb+ uptake reflects either an increased intracellular Na+ concentration or an increased number of sodium pump complexes per fibroblast. Amiloride inhibited the enhancement of O-S 86Rb+ uptake, as well as cytomegaly development. Addition of amiloride at selected times after infection suggested that the same phase of virus replication was sensitive to the inhibitory effect of this drug on the enhancement of O-S 86Rb+ uptake and on the development of cytomegaly. There was also a similar pattern of inhibition of O-S 86Rb+ uptake and cytomegaly with increasing concentrations of amiloride. Thus, there may be a relationship between CMV-induced Na+ entry through activation of the Na+/H+ exchanger and development of cytomegaly.

Amiloride↗
PubMed1987

Ca2+ responses in cytomegalovirus-infected fibroblasts of human origin.

Cytomegalovirus (CMV) infection of fibroblasts of human origin is associated with a cascade of cellular responses (rounding, "contraction," "relaxation," and enlargement). Since in other systems these cellular responses are regulated by intracellular free Ca2+ activity ([Ca2+]i), we measured intracellular Ca2+ responses to CMV infection. At relatively high multiplicities of infection (m.o.i), an influx of Ca2+ was observed within the first hour after CMV infection (p.i.) at which time it was at its maximum rate. Both the time of occurrence and the magnitude of this Ca2+ influx were dependent on the calculated input m.o.i. In CMV-infected cells, [Ca2+]i rose gradually from 80 nM at 0 hr to 174 nM at 48 hr p.i. (about 2.7 times the [Ca2+]i found in mock-infected cells at this time). At 8 and 12 hr p.i. CMV-infected cells consistently contained a somewhat greater level of 45Ca2+ than mock-infected cells, despite the fact that there was only a small increase in [Ca2+]i between CMV and mock-infected cells in the same period. This observation suggests that there may be significant amounts of Ca2+ taken up into intracellular stores. This Ca2+ in intracellular stores may, at later times after infection, contribute to the increase in [Ca2+]i observed from 12 to 48 hr p.i. Ca2+ influx blockers, such as nifedipine and verapamil, inhibited the rise in [Ca2+]i. The increase in [Ca2+]i in response to CMV infection was shown to be dependent on the m.o.i., require infectious virus, and occur under conditions consistent with the expression of immediate-early CMV genes. The capability of inducing such Ca2+ responses was conserved among three laboratory strains of CMV. The CMV-induced Ca2+ responses may be related not only to the development of CMV cytopathology, but also to the replication of CMV, since in other studies cyclic nucleotide modulators and Ca2+ influx blockers were found to inhibit the replication of CMV.

Calcium↗
PubMed1987

Inhibition of cytomegalovirus replication by smooth-muscle relaxing agents.

The effect of the smooth-muscle relaxing agents, papaverine, sodium nitroprusside, and verapamil, on the replication of human cytomegalovirus (CMV) was investigated. At a concentration of 100 microM, infectious yields of CMV were reduced by 1.23 to 5.72 log10 by these drugs (papaverine, 5.72 log10; nitroprusside, 1.85 log10; verapamil, 1.23 log10). The ED50 for papaverine was found to be somewhat less than 1 microM, a concentration which appears to be within the range achieved clinically. Papaverine did not irreversibly modify treated cells to a virus-resistant state since treatment of cells with papaverine from 24 hr before until immediately prior to CMV infection did not significantly reduce CMV yields. Replication of CMV was most sensitive to inhibition when papaverine was added at or before 6 hr after CMV infection. Addition of papaverine at later times resulted in a substantial reduction of the inhibitory effect on virus yields, suggesting that the phase of CMV replication sensitive to papaverine inhibition occurred early in the replication cycle. These results, particularly in light of the potency of papaverine, indicate that some smooth-muscle relaxing agents have significant antiviral activity toward the replication of CMV.

Cytomegalovirus↗
PubMed1986

A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells.

To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins.

Animals↗
PubMed1986

Changes in the extent of microtubule assembly can regulate initiation of DNA synthesis.

We have shown that MT depolymerization by colchicine and other drugs is sufficient to initiate DNA synthesis in serum-free cultures of embryonic fibroblasts and that stabilization of MTs with taxol inhibits this initiation. Growth factors and oncogenic DNA viruses also initiate DNA synthesis by a taxol-sensitive mechanism that appears to require MT depolymerization or rearrangements. Because we have shown that microtubule heterogeneity exists within single fibroblastic cells, we have carried out a series of experiments to determine the extent of microtubule disruption necessary to initiate DNA synthesis. We have compared the effects of various concentrations of colchicine and taxol on initiation of DNA synthesis with their effects on cytoplasmic MT complexes as visualized by indirect immunofluorescence microscopy and quantitated by direct binding of radiolabeled monoclonal antibody to cytoskeletons. The opposing effects of these drugs on MTs shows that there is a correlation between the extent of MT depolymerization and initiation of DNA synthesis. Initiation of DNA synthesis by colchicine in the presence of taxol is half-maximal when taxol and colchicine are added to cultures at a ratio of about 13 to 1. At this drug ratio, taxol stabilizes MTs near the nucleus, but MTs near the cell periphery are depolymerized. Maximal inhibition of DNA synthesis by taxol occurs only at taxol to colchicine ratios where MTs extend throughout the cytoplasm to the cell periphery. Thus, depolymerization of a small fraction of total MTs, particularly those near the periphery, may be sufficient to initiate proliferative events.

Alkaloids↗
PubMed1986

Human cytomegalovirus: development and progression of nuclear inclusions by primary clinical isolates and laboratory-adapted strains.

The morphogenesis of cytomegalovirus (CMV) nuclear inclusions (NIs) was investigated using unadapted clinical isolates and adapted laboratory strains. Both adapted and unadapted strains of CMVs induced NIs whose morphologic appearance was similar in human fibroblastic cells. Early NIs appeared as ring-like structures composed of dense granular and fibrillar material, while late NIs appeared to consist of multiple electron-lucent areas containing coarse granules and bounded by electron-dense fibrillar material (cellulae). Capsids and nucleocapsids were associated primarily with the electron-dense fibrillar material; however, developing nucleocapsids were most often observed at the interface of the electron-dense and -lucent areas. Although there was some variation in the rate of development and maturation of the NIs with the intensity of infection, all CMVs examined produced late NIs with similar organizational patterns consisting of cellulae. Substitution of human fibroblastic cells derived from various tissues as cellular substrate did not appreciably affect the results. Thus, the unique organization of the CMV late NI, consisting of multiple cellulae, appears to be an intrinsic feature of CMV replication since it seems to be independent of the extent of laboratory adaptation, the virus strain, the intensity of infection, or the cell type.

Cell Nucleus↗
PubMed1986

Defective NK cell activity following thermal injury.

Peripheral blood mononuclear lymphocytes (PBL) from thermal injury patients were examined for their ability to mediate natural killer (NK) cell activity against K562 tumour cells and against herpes simplex virus type 1 (HSV-1) infected Raji tumour cells. Using fluorescein isothiocyanate-conjugated monoclonal antibodies, the number of T3, T4, T8, Leu11, and Leu7 positive cells in PBL obtained from patients and normal controls was determined. Thermal injury patients had decreased levels of T3+ cells and a T4:T8 ratio which was significantly lower than that found in normal control individuals. Although patients had normal percentages of Leu7+ and Leu11+ cells, they had depressed NK cell activity against both K562 tumour cells and HSV-1 infected Raji cells. NK cell activity against K562 tumour cells was severely depressed during the first 20 days after injury. This defective NK cell activity did not appear to be due to a defect in PBL binding to the K562 tumour cells. In patients, the level of NK cell activity against HSV-1 infected cells did not correlate with the level of NK cell activity against K562 tumour cells. This finding further supports previous reports showing that NK cells which kill K562 tumour cells are different from the NK cell population which kills HSV-1 infected cells. Pretreatment of PBL obtained from patients with IL-2 or IFN-alpha, in some cases greatly enhanced NK cell killing of K562 tumour cells. However, IL-2 or IFN-alpha did not enhance NK cell activity in patients who had severely depressed levels of NK cell activity. Interestingly, in some patients, differential responsiveness to IL-2 and IFN-alpha was observed. In some patients, NK cell activity was enhanced by IL-2 but not by IFN-alpha. These results, while only suggestive, may indicate that different populations of NK cells respond preferentially to IL-2 and that IFN-alpha and/or IL-2 enhance NK cell activity in PBL obtained from some, but not all, thermal injury patients. Finally, this study clearly shows that thermal injury patients have defective NK cell activity not only against K562 tumour cells but also against virus-infected cells.

Adult↗