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PubMedDate not supplied

State-dependent effects of Na channel noise on neuronal burst generation.

We explore the effects of stochastic sodium (Na) channel activation on the variability and dynamics of spiking and bursting in a model neuron. The complete model segregates Hodgin-Huxley-type currents into two compartments, and undergoes applied current-dependent bifurcations between regimes of periodic bursting, chaotic bursting, and tonic spiking. Noise is added to simulate variable, finite sizes of the population of Na channels in the fast spiking compartment. During tonic firing, Na channel noise causes variability in interspike intervals (ISIs). The variance, as well as the sensitivity to noise, depend on the model's biophysical complexity. They are smallest in an isolated spiking compartment; increase significantly upon coupling to a passive compartment; and increase again when the second compartment also includes slow-acting currents. In this full model, sufficient noise can convert tonic firing into bursting. During bursting, the actions of Na channel noise are state-dependent. The higher the noise level, the greater the jitter in spike timing within bursts. The noise makes the burst durations of periodic regimes variable, while decreasing burst length duration and variance in a chaotic regime. Na channel noise blurs the sharp transitions of spike time and burst length seen at the bifurcations of the noise-free model. Close to such a bifurcation, the burst behaviors of previously periodic and chaotic regimes become essentially indistinguishable. We discuss biophysical mechanisms, dynamical interpretations and physiological implications. We suggest that noise associated with finite populations of Na channels could evoke very different effects on the intrinsic variability of spiking and bursting discharges, depending on a biological neuron's complexity and applied current-dependent state. We find that simulated channel noise in the model neuron qualitatively replicates the observed variability in burst length and interburst interval in an isolated biological bursting neuron.

Action Potentials↗
PubMed2004

Human cytomegalovirus load in plasma and bronchoalveolar lavage fluid: a longitudinal study of lung transplant recipients.

BACKGROUND: In lung transplant recipients (LTRs), the measurement of human cytomegalovirus (HCMV) load dynamics in the local environment of the lung allograft may offer distinct advantages over their assessment in the peripheral blood compartment. METHODS: We compared HCMV load in paired bronchoalveolar lavage (BAL) and plasma samples in a prospective cohort of LTRs. RESULTS: In all, 182 paired samples were collected from 41 LTRs. Qualitative findings were concordant for 79.7% of the paired samples (virus load detected or absent in 8.1% and 71.5% of paired samples, respectively). In 35 paired samples (20.3%), HCMV was detected in the BAL but not the plasma sample; the converse was not found for any of the paired samples. HCMV inclusions were histologically detected in the lungs of 8 patients on 9 occasions. HCMV was detected in both BAL and plasma samples on 7 of the 9 occasions and in only the BAL sample on 2 occasions. Application of a threshold virus load in BAL samples, to predict histologically proven HCMV infection, was associated with improved diagnostic sensitivity and specificity. CONCLUSIONS: Quantification of HCMV in the lung allograft correlates with histological detection and, in addition, offers the potential to monitor subclinical viral replication in the lung allograft.

Adolescent↗
PubMed2001

Yellow fever virus encephalitis: properties of the brain-associated T-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for CD4+ lymphocytes.

Viral encephalitis caused by neuroadapted yellow fever 17D virus (PYF) was studied in parental and gamma interferon (IFN-gamma)-deficient (IFN-gamma knockout [GKO]) C57BL/6 mice. The T-cell responses which enter the brain during acute fatal encephalitis of nonimmunized mice, as well as nonfatal encephalitis of immunized mice, were characterized for relative proportions of CD4+ and CD8+ cells, their proliferative responses, and antigen-specific expression of cytokines during stimulation in vitro. Unimmunized mice accumulated only low levels of T cells within the brain during fatal disease, whereas the brains of immunized mice contained higher levels of both T-cell subsets in response to challenge, with CD8+ cells increased relative to the CD4+ subset. The presence of T cells correlated with the time at which virus was cleared from the central nervous system in both parental and GKO mice. Lymphocytes isolated from the brains of challenged immunized mice failed to proliferate in vitro in response to T-cell mitogens or viral antigens; however, IFN-gamma, interleukin 4 (IL-4), and, to a lesser extent, IL-2 were detectable after stimulation. The levels of IFN-gamma, but not IL-2 or IL-4, were augmented in response to viral antigen, and this specificity was detectable in the CD4+ compartment. When tested for the ability to survive both immunization and challenge with PYF virus, GKO and CD8 knockout mice did not differ from parental mice (80 to 85% survival), although GKO mice exhibited a defect in virus clearance. In contrast, CD4 knockout and Igh-6 mice were unable to resist challenge. The data implicate antibody in conjunction with CD4+ lymphocytes bearing a Th1 phenotype as the critical factors involved in virus clearance in this model.

Animals↗
PubMed2003

The expression of the thyroid-stimulating hormone (TSH) receptor and the cAMP-dependent protein kinase RII beta regulatory subunit confers TSH-cAMP-dependent growth to mouse fibroblasts.

TSH activates its specific receptor in thyroid cells and induces cAMP, a robust stimulator of thyroid cell proliferation. Conversely, cAMP is a potent inhibitor of growth in mouse fibroblasts. To dissect the signals mediating cAMP-dependent growth, we have expressed in mouse fibroblasts the human thyrotropin receptor (TSHR) or a constitutively active mutant, under the control of the tetracyclin promoter. Both TSHR and cAMP levels were modulated by tetracyclin. In the presence of serum, activation of TSHR by TSH induced growth arrest. In the absence of serum, cells expressing TSHR stimulated with TSH, replicated their DNA, but underwent apoptosis. Co-expression of cAMP-dependent protein kinase (PKA) regulatory subunit type II (RIIbeta) inhibited apoptosis and stimulated the growth of cells only in the presence of TSH. Expression of RIIbeta-PKA, in the absence of TSHR, induced apoptosis, which was reversed by cAMP. Growth, stimulated by TSHR-RIIbeta-PKA in mouse fibroblasts, was also dependent on Rap1 activity, indicating cAMP-dependent growth in thyroid cells. As for the molecular mechanism underlying these effects, we found that in normal fibroblasts, TSH induced AKT and ERK1/2 only in cells expressing TSHR and RII. Similarly, activation of TSHR increased cAMP levels greatly, but was unable to stimulate CREB phosphorylation and transcription of cAMP-induced genes in the absence of RII. These data provide a simple explanation for the anti-proliferative and proliferative effects of cAMP in different cell types and indicate that RII-PKAII complements TSHR action by stably propagating robust cAMP signals in cell compartments.

Adenoviridae↗
PubMed1976

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland.

Mitochondrial biogenesis in the parenchymal cell of the mouse mammary gland appears to occur in two distinct phases: replication during cell proliferation, and maturation during cell differentiation. This study of the mitochondrial maturation phase in the mouse gland demonstrates a significant increase in organelle density on isopycnic sucrose gradient centrifugation during the transition from late pregnancy to day 8 of lactation. Differential fragility to high sucrose concentrations or changes in mitochondrial lipid composition do not satisfactorily explain the density increases. When organelle densities were assessed by centrifugation under iso-osmotic conditions with Ficoll gradients in 0.25 M sucrose, the mitochondria from pregnant glands were observed to be more dense than those from lactating glands. The two mitochondrial populations were also found to differ in their response to changes in sucrose concentration in the Ficoll gradients. When sucrose concentration was increased, the density of both pregnant and lactating gland mitochondria increased nonlinearly, the increase being greater with the lactating gland organelles. By use of mathematical models, the differing response was interpreted as a change in the density and osmotic activity of the mitochondrial internal compartment (inner membrane plus matrix space). We have proposed that the changes reflect a large expansion of the inner mitochondrial membrane and perhaps the matrix material during the transition into lactation in the differentiating parenchymal cell.

Animals↗
PubMed2004

Cdt1 and geminin are down-regulated upon cell cycle exit and are over-expressed in cancer-derived cell lines.

Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 was shown to predispose cells for malignant transformation. We show here that Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3-6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. Our data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. We show that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and that the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. Our data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells.

Animals↗
PubMed2002

Cervicovaginal lamina propria lymphocytes: phenotypic characterization and their importance in cytotoxic T-lymphocyte responses to simian immunodeficiency virus SIVmac251.

Most human immunodeficiency virus (HIV) type 1 infections occur by the mucosal route. Thus, it is important to assess the immune responses to HIV in the vaginal, cervical, and rectal compartments. Here we quantitated the virus-specific CD8+ T-cell response and characterized the phenotype of lymphocytes in the genital tracts of naive macaques, macaques acutely or chronically infected with simian immunodeficiency virus SIVmac251, and macaques chronically infected with chimeric simian/human immunodeficiency virus SHIV(KU2.) Vaginal biopsy samples or samples obtained at the time of euthanasia were used in this analysis. The percentage of Gag-specific, tetramer-positive T cells was as high as 13 to 14% of the CD3+ CD8+ T-cell population in the vaginal and cervical laminae propriae of both SIVmac251 and SHIV(KU2) chronically infected macaques. In most cases, the frequency of this response in the cervicovaginal compartment far exceeded the frequency in the blood or the draining iliac lymph node. Vaginal laminae propriae of naive macaques contained 55 to 65% CD3+ CD8+ cells and 28 to 34% CD3+ CD4+ cells, while the majority of intraepithelial cells were CD8+ T cells (75 to 85%). For the same cells, the surface expression of CD62L was low whereas that of alphaEbeta7 was high. No difference in the expression of CD45RA on CD8+ T cells was observed in the chronic stage of SIVmac251 infection. Although no decrease in the percentage of CD4+ cells in the genital tract was observed within the first 12 days of infection, by 6 weeks from SIVmac251 infection and thereafter the percentage of CD4+ T cells was decreased in the laminae propriae of the vagina and cervix. Expression of CD45RA did not differ in naive and acutely SIVmac251 infected macaques. Information on the quality and quantity of local immune responses may help in the design of vaccine strategies aimed at containing viral replication at the site of viral encounter.

Animals↗
PubMed2006

Expansion and diversification of virus-specific T cells following immunization of human immunodeficiency virus type 1 (HIV-1)-infected individuals with a recombinant modified vaccinia virus Ankara/HIV-1 Gag vaccine.

Affordable therapeutic strategies that induce sustained control of human immunodeficiency virus type 1 (HIV-1) replication and are tailored to the developing world are urgently needed. Since CD8(+) and CD4(+) T cells are crucial to HIV-1 control, stimulation of potent cellular responses by therapeutic vaccination might be exploited to reduce antiretroviral drug exposure. However, therapeutic vaccines tested to date have shown modest immunogenicity. In this study, we performed a comprehensive analysis of the changes in virus-specific CD8(+) and CD4(+) T-cell responses occurring after vaccination of 16 HIV-1-infected individuals with a recombinant modified vaccinia virus Ankara-vectored vaccine expressing the consensus HIV-1 clade A Gag p24/p17 sequences and multiple CD8(+) T-cell epitopes during highly active antiretroviral therapy. We observed significant amplification and broadening of CD8(+) and CD4(+) gamma interferon responses to vaccine-derived epitopes in the vaccinees, without rebound viremia, but not in two unvaccinated controls followed simultaneously. Vaccine-driven CD8(+) T-cell expansions were also detected by tetramer reactivity, predominantly in the CD45RA(-) CCR7(+) or CD45RA(-) CCR7(-) compartments, and persisted for at least 1 year. Expansion was associated with a marked but transient up-regulation of CD38 and perforin within days of vaccination. Gag-specific CD8(+) and CD4(+) T-cell proliferation also increased postvaccination. These data suggest that immunization with MVA.HIVA is a feasible strategy to enhance potentially protective T-cell responses in individuals with chronic HIV-1 infection.

AIDS Vaccines↗
PubMed2006

Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines.

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.

Calcium↗
PubMed1996

The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum.

Legionella pneumophila is an intracellular parasite of protozoa and human phagocytes. To examine adaptation of this bacterium to parasitize protozoa, the sequence of events of the intracellular infection of the amoeba Hartmannella vermiformis was examined. The previously described uptake phenomenon of coiling phagocytosis by human monocytes was not detected. A 1 h postinfection with wild-type strain AA100, mitochondria were observed within the vicinity of the phagosome. At 2.5 h postinfection, numerous vesicles surrounded the phagosomes and mitochondria were in close proximity to the phagosome. At 5 h postinfection, the bacterium was surrounded by a ribosome-studded multilayer membrane. Bacterial multiplication was evident by 8 h postinfection, and the phagosome was surrounded by a ribosome-studded multilayer membrane until 15 h postinfection. The recruitment of organelles and formation of the ribosome-studded phagosome was defective in an isogenic attenuated mutant of L. pneumophila (strain AA101A) that failed to replicate within amoebae. At 20 h postinfection with wild-type strain AA100, numerous bacteria were present in the phagosome and ribosome were not detected around the phagosome. These data showed that, at the ultrastructural level, the intracellular infection of protozoa by L. pneumophila is highly similar to that of infection of macrophages. Immunocytochemical studies provided evidence that at 5 h postinfection the phagosome containing L. pneumophila acquired an abundant amount of the endoplasmic reticulum-specific protein (BiP). Similar to phagosomes containing heat-killed wild-type L. pneumophila, the BiP protein was not detectable in phagosomes containing the mutant strain AA101A. In addition to the absence of ribosomes and mitochondria, the BiP protein was not detected in the phagosomes at 20 h postinfection with wild-type L. pneumophila. The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is dependent on its capacity to reside and multiply within a phagosome surrounded by the rough endoplasmic reticulum. This compartment may constitute a rich source of nutrients for the bacteria and is probably recognized as cellular compartment. The remarkable similarity of the intracellular infections of macrophages and protozoa by L. pneumophila strongly supports the hypothesis that adaptation of the bacterium to the intracellular environment of protozoa may be the mechanism for its ability to adapt to the intracellular environment of human alveolar macrophages and causes pneumonia.

Adaptation, Physiological↗
PubMed1987

Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enterocolitica to enter and replicate within HEp-2 cells.

Salmonella typhimurium, enteroinvasive Escherichia coli, Yersinia pseudotuberculosis, and Yersinia enterocolitica possess the ability to enter intestinal epithelial cells. We used a quantitative tissue culture model employing HEp-2 cells to compare the abilities of these bacteria to enter epithelial cells. S. typhimurium and Yersinia species were highly infective for HEp-2 cells but were unable to replicate extensively intracellularly. Enteroinvasive E. coli exhibited low infectivity but replicated extensively intracellularly. The growth of enteroinvasive E. coli led to destruction of the HEp-2 monolayer, whereas Yersinia spp. and S. typhimurium were maintained intracellularly for prolonged periods without damage to the monolayer. The ability of enteroinvasive E. coli to enter HEp-2 cells required prior growth at 37 degrees C; neither S. typhimurium nor Yersinia spp. exhibited this temperature dependence for cell entry. An E. coli K-12 derivative containing a 230-kilobase plasmid from enteroinvasive E. coli was constructed. This derivative shared all the invasive characteristics of the parental enteroinvasive strain, suggesting that determinants required for cell entry and intracellular multiplication were at least partially plasmid encoded. An HB101 derivative containing a cloned invasion determinant from Y. pseudotuberculosis was constructed in our laboratory. HEp-2 monolayers were coinfected with these two K-12 derivatives to compare invasion determinants from enteroinvasive E. coli with those of Y. pseudotuberculosis in a common genetic background. Results from these experiments suggest that these organisms reside within separate intracellular compartments.

Cells, Cultured↗
PubMed1998

Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts.

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.

Animals↗
PubMed1993

Lipids alter the level and distribution of mouse mammary tumor virus gp52 in vitro.

Linoleic acid, cholesterol, dexamethasone and progesterone were tested by immunocytochemistry and immunoprecipitation for their single and combined effects in vitro on mouse mammary tumor virus (MMTV) gp52 distribution among three compartments: cell-associated antigen, extracellular virus particles and extracellular shed antigen unassociated with virus particles. Results indicated that all additives significantly increased total MMTV gp52 levels and altered the distribution. Linoleic acid and dexamethasone induced the greatest relative proportion of extracellular gp52, whereas cholesterol and progesterone induced the greatest proportion of cell-associated gp52. The implications of these findings for the immune response to mammary tumors is discussed.

Analysis of Variance↗
PubMed1990

Putative site for the acquisition of human herpesvirus 6 virion tegument.

The virion of human herpesvirus 6 (HHV-6) contains a very distinct tegument layer, occupying the space between the nucleocapsid and the virion envelope. Ultrastructural analyses of thymocytes infected with HHV-6 revealed the presence of intranuclear spherical compartments, approximately 1.5 microns in diameter, in which tegumentation seems to take place. These compartments, termed tegusomes, were bounded by two membranes and contained ribosomes, consistent with their derivation by cytoplasmic invagination into the nucleus. Capsids located within the nucleus outside the tegusomes were all naked, while those located in the cytoplasm were uniformly tegumented. In contrast, capsids present inside the tegusomes contains teguments of variable thicknesses. In addition, nucleocapsids were documented in the process of budding into the tegusomes. We thus suggest that the tegusomes represent a cellular site in which HHV-6 virions acquire their tegument.

Cell Nucleus↗
PubMed2005

Mechanism for removal of tumor necrosis factor receptor 1 from the cell surface by the adenovirus RIDalpha/beta complex.

Proteins encoded in adenovirus early region 3 have important immunoregulatory properties. We have recently shown that the E3-10.4K/14.5K (RIDalpha/beta) complex downregulates tumor necrosis factor receptor 1 (TNFR1) expression at the plasma membrane. To study the role of the RIDbeta tyrosine sorting motif in the removal of surface TNFR1, tyrosine 122 on RIDbeta was mutated to alanine or phenylalanine. Both RIDbeta mutations not only abolished the downregulation of surface TNFR1 but paradoxically increased surface TNFR1 levels. RID also downregulates other death receptors, such as FAS; however, surface FAS expression was not increased by RIDbeta mutants, suggesting that regulation of TNFR1 and that of FAS by RID are mechanistically different. In the mixing experiments, the wild-type (WT) RID-mediated TNFR1 downregulation was partially inhibited in the presence of RIDbeta mutants, indicating that the mutants compete for TNFR1 access. Indeed, an association between RIDbeta and TNFR1 was shown by coimmunoprecipitation. In contrast, the mutants did not affect the WT RID-induced downregulation of FAS. These differential effects support a model in which RID associates with TNFR1 on the plasma membrane, whereas RID probably associates with FAS in a cytoplasmic compartment. By using small interfering RNA against the mu2 subunit of adaptor protein 2, dominant negative dynamin construct K44A, and the lysosomotropic agents bafilomycin A1 and ammonium chloride, we also demonstrated that surface TNFR1 was internalized by RID by a clathrin-dependent process involving mu2 and dynamin, followed by degradation of TNFR1 via an endosomal/lysosomal pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Low level expression of the HIV-1 Nef protein in transiently transfected COS-1 cells in culture.

Nef (negative factor) is a membrane-associated cytoplasmic accessory gene product of HIV-1 that induces increased viral load and pathogenesis in the host. In the present study, we have utilized a transient co-transfection assay with the HIV-1lai nef gene and the gene for green fluorescent protein (GFP) in the monkey kidney cell line, COS-1, to analyze Nef expression. We have observed significantly lower level expression of the Nef protein relative to expression of the GFP reporter protein, even when identical vector constructs and similar plasmid DNA concentrations were used. Moreover, Northern blot analysis have indicated that comparable levels of both the Nef and GFP transcripts were transcribed from the co-transfected nef and gfp plasmid constructs, respectively. Western blot analysis have also indicated that the intracellular accumulation of the Nef protein was not visibly affected by proteolytic inhibitors of two major subcellular compartments, lysosomes and proteosomes. In addition, the Nef protein does not appear to be secreted into the culture medium up to 24 hr post-transfection. Our results indicate the possible selective translational expression or post-translational down-regulation of the Nef protein in cultured COS-1 cells that were transiently co-transfected with the nef and gfp genes. However, the mechanism(s) underlying this phenomenon has not been determined.

Animals↗
PubMed2003

Short-term effect of zidovudine on plasma and genital human immunodeficiency virus type 1 and viral turnover in these compartments.

The effect of zidovudine on plasma and genital human immunodeficiency virus type 1 (HIV-1) was determined in 42 antiretroviral-naive HIV-1-seropositive women in Nairobi. After 7 days of zidovudine treatment, HIV-1 RNA levels decreased by 0.5 to 1.1 log(10) in plasma and genital secretions. HIV-1 RNA half-life following zidovudine treatment was 4.7, 1.3, and 0.9 days in plasma, cervix, and vagina, respectively, and significantly shorter in genital secretions than in plasma (P < 0.001). Defining the short-term effect of zidovudine on plasma and genital HIV-1 is important for improving perinatal HIV-1 interventions.

Cervix Uteri↗
PubMed1991

Cell-cycle specific cytotoxicity mediated by stizophyllin (2 alpha,3 beta,12 beta-trihydroxypregna-4,7,16-trien-20-one), a novel electrophilic pregnane isolated from Stizophyllum riparium.

A structurally-novel pregnane derivative, 2 alpha,3 beta,12 beta-trihydroxypregna-4,7,16-trien-20-one (stizophyllin), was isolated from an extract of Stizophyllum riparium (H.B.K.) Sandw. on the basis of bioactivity-guided fractionation and confirmed to mediate a potent cytotoxic response with cultured P-388 cells. We presently report a detailed isolation procedure and the results of studies designed to examine its mechanism of action. By means of a Michael-type addition, stizophyllin formed adducts with nucleophilic substances such as L-cysteine and beta-mercaptoethanol. The adduct with beta-mercaptoethanol was isolated, structurally characterized, and found to be 20-fold less cytotoxic than stizophyllin. Stizophyllin interacted with DNA, but no mutagenicity was observed with Salmonella typhimurium strain TM677 or cultured Chinese hamster ovary cells, and no in vitro reaction occurred with guanosine. Relative to control cell cultures, the total biosynthesis of DNA, RNA or protein was reduced when P-388 cells were treated with stizophyllin. However, the cell number did not increase in the presence of inhibitory stizophyllin concentrations (e.g., 4 micrograms/ml), and the DNA content (per cell) actually doubled after approximately 48 h. Consistent with this, stizophyllin blocked the cells in the G2 + M compartment of the cycle, and the block appeared to be specific for the G2 phase. Accordingly, stizophyllin did not modulate in vitro tubulin polymerization reactions nor did it affect the morphology of dibutyryl cAMP-treated astrocytoma cells in culture. These data suggest that the cytotoxic activity of stizophyllin is mediated by covalent reaction with a cellular component (such as a sulfhydryl-containing protein) by means of a Michael-type addition. Based on the cell-cycle specificity of the response, it appears that this interaction prevents the cells from progressing through mitosis.

Animals↗