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At least 703 records · Page 39Linked to original sources
PubMed2003

Multilevel selection in models of prebiotic evolution: compartments and spatial self-organization.

In this paper we explore the impact of new levels of selection in models of early evolution. We contrast two types of higher levels of selection. On the one hand we look at spatially explicit models of replicators in which, by a process of self-organization, new levels of selection arise as large scale spatial patterns with a dynamics of their own. Alternatively externally imposed levels of selection above the basic replicators are created by enclosing the replicators in vesicles. In this paper we first review some results on the impact of emerging higher levels of selection on the evolutionary persistence of interacting co-evolving replicator systems. Moreover, we present a vesicle model, which can potentially integrate emerging and imposed levels of selection. We use the models to examine the classical problem information integration in early evolution.

Biological Evolution↗
PubMed2006

Interleukin-7 induces HIV replication in primary naive T cells through a nuclear factor of activated T cell (NFAT)-dependent pathway.

Interleukin (IL)-7 plays several roles critical to T cell maturation, survival, and homeostasis. Because of these functions, IL-7 is under investigation as an immune-modulator for therapeutic use in lymphopenic clinical conditions, including HIV. We reported that naive T cells, typically not permissive to HIV, can be productively infected when pre-treated with IL-7. We evaluated the mechanism by which IL-7-mediates this effect. IL-7 potently up-regulated the transcriptional factor NFAT, but had no effect on NFkappaB. Blocking NFAT activity using a number of reagents, such as Cyclosporin A, FK-506, or the NFAT-specific inhibitor known as VIVIT peptide, all markedly reduced IL-7-mediated induction of HIV replication in naive T cells. Additional neutralization of cytokines present in IL-7-treated cultures and/or those that have NFAT-binding sequences within their promotors indicated that IL-10, IL-4, and most significantly IFNgamma, all contribute to IL-7-induction of HIV productive replication in naive T cells. These data clarify the mechanism by which IL-7 can overcome the block to HIV productive infection in naive T cells, despite their quiescent cell status. These findings are relevant to the treatment of HIV disease and understanding HIV pathogenesis in the naive CD4+ T cell compartment, especially in light of the vigorous pursuit of IL-7 as an in vivo immune modulator.

Antigens, CD↗
PubMed2006

Rotavirus NSP4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms.

Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.

Calcium↗
PubMed1990

Epithelial cell kinetics in the remaining colorectal mucosa after surgery for cancer of the large bowel.

We used microautoradiography in order to evaluate cell replication of the remaining colorectal mucosa in 20 patients previously operated on for cancer of the large bowel. The results were compared to those of 24 controls without neoplasms or other relevant colorectal disease. Samples of colorectal mucosa were taken during endoscopy. At histological examination each labeled intestinal hemicrypt was divided into 5 longitudinal compartments, from the base to the surface, and S-phase cells in each compartment were counted. Total labeling index (LI, ratio of labeled to total cells x 100) and labeling index per crypt compartment were similar in surgical patients and in controls. In contrast, both total LI and labeling index in the upper portions of the crypt (compartments 3, 4, and 5) were significantly higher in the 9 patients who showed recurrence of polyps than in those (n = 11) without recurrence. The LI in compartments 4 and 5 (the "high crypt region") was 4.37 +/- 0.95 (SEM) in patients with recurrence versus 0.88 +/- 0.21 (P less than 0.001) in patients with negative endoscopy finding and 1.47 +/- 0.22 in controls. Moreover, the fifth compartment was labeled in 8 of 9 individuals in whom polyps recurred but in only 2 of 11 patients without recurrence and 3 of 24 controls. In conclusion, after resection for large bowel cancer colonic epithelial cell proliferation tends to become more quiescent and similar to that of controls. However, in the subgroup of patients in whom polyps reappear, the colorectal mucosa maintains a hyperproliferative state with an expansion of the replicative zone to the most superficial portions of the crypt. These findings support the sequence adenoma-carcinoma and suggest that the evaluation of cell proliferation might be useful in the identification of subjects at increased risk for multiple tumors of the large bowel.

Cell Division↗
PubMed2006

Epstein-Barr virus BNRF1 protein allows efficient transfer from the endosomal compartment to the nucleus of primary B lymphocytes.

Epstein-Barr virus (EBV) is a tumor virus with marked B lymphotropism. After crossing the B-cell membrane, the virus enters cytoplasmic vesicles, where decapsidation takes place to allow transfer of the viral DNA to the cell nucleus. BNRF1 has been characterized as the EBV major tegument protein, but its precise function is unknown. We have constructed a viral mutant that lacks the BNRF1 gene and report here its in vitro phenotype. A recombinant virus devoid of BNRF1 (DeltaBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the DeltaBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented DeltaBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or DeltaBNRF1 virus was blocked by leupeptin addition, showing that both viruses reach the endosome/lysosome compartment. These data were confirmed by direct observation of the mutant virus in endosomes of infected B cells by electron microscopy. However, we observed a 20-fold reduction in the number of B cells expressing the nuclear protein EBNA2 after infection with a DeltaBNRF1 virus compared to wild-type infection. Likewise, DeltaBNRF1 viruses transformed primary B cells much less efficiently than EBV-wt or BNRF1 trans-complemented viruses. We conclude from these findings that BNRF1 plays an important role in viral transport from the endosomes to the nucleus.

Active Transport, Cell Nucleus↗
PubMed1995

In-vitro models of stroma-dependent lymphopoiesis.

The differentiation of hematopoietic stem cells into lymphocytes can be replicated ex vivo under the inductive influence of the stromal cells that frame the bone marrow and thymus. We summarize hereafter the development of culture systems where lymphopoiesis-supporting cell compartments are maintained in either their normal three-dimensional arrangement, in organotypic culture, or as culture dish-adherent monolayers and review the recent and current uses of those in-vitro models to investigate T- and B-cell differentiation in mouse and man.

Animals↗
PubMed2003

Different patterns of regulation of Tyr-phosphorylated STAT1 and STAT3 in human hepatoma Hep3B cells by the phosphatase inhibitor orthovanadate.

The cellular physiology of signal transducer and activator of transcription protein family (STAT) transcription factors includes activation by Tyr-phosphorylation (PY) in cytokine and growth factor receptor complexes at the level of plasma membrane rafts, subsequent cytoplasmic transit and nuclear import, and transcriptional regulation of target genes, followed by dephosphorylation and export back to the cytoplasm. The ubiquitous protein tyrosine phosphatase (PTP) called "T-cell protein tyrosine phosphatase" has been reported to mediate Tyr-dephosphorylation of both interferon-gamma (IFN-gamma)-induced PY-STAT1 and interkleukin-6 (IL-6)-induced PY-STAT3 in some cell lines. To test whether the same PTP regulated both PY-STAT1 and PY-STAT3 in human hepatocytes we used orthovanadate (VO(4); 0.01-1.0mM) as a PTP-inhibitory probe and evaluated the kinetics of PY-STAT3 and PY-STAT1 accumulation, nuclear trafficking, and dephosphorylation following cytokine (IL-6 or IFN-gamma) stimulation of Hep3B cells. As evaluated using DNA binding or Western blotting assays, in IL-6-treated hepatocytes VO(4) had a modest enhancing effect on peak levels of cytoplasmic and nuclear PY-STAT3 reached by 1h and on their subsequent decline. In contrast, in the same cells and at the same time, VO(4) caused a marked and continuing increase in cytoplasmic and nuclear levels of PY-STAT1 which, by 4h, were 5- to 10-fold higher than peak levels reached in VO(4)-free, IL-6-treated cells. Prolonged treatment of cells with VO(4) alone (for 4-8h) replicated this markedly selective enhancement of PY-STAT1 levels. Consistent with this selectivity, shorter term VO(4) treatment (1-2h) markedly increased PY-STAT1 levels in all cellular compartments of IFN-gamma-treated cells by >10-fold. The unexpected selectivity in the effects of VO(4) on PY-STAT1 compared to that on PY-STAT3 levels in Hep3B cells suggests that, at least in these hepatocytes, the regulation of PY-STAT1 and PY-STAT3 likely involves distinct protein tyrosine phosphatase mechanisms.

Biological Transport, Active↗
PubMed1996

Apoptosis in lymph nodes in acute African swine fever.

This paper reports apoptosis of lymph-node lymphocytes in swine experimentally inoculated with a virulent African swine fever (ASF) virus isolate (Malawi '83). Apoptosis was observed in both compartments of cortical tissue, but was more intense in diffuse lymphoid tissue (T area). Lymphopenia detected in peripheral blood was associated with T-lymphocyte depletion. No evidence of ASF virus replication was observed in lymphocytes in the lymph nodes studied. This finding, together with the high rate of virus replication recorded in macrophages in diffuse lymphoid tissue as compared with the low rate recorded for lymphoid follicles, suggests a mechanism for the induction of apoptosis related to virus replication in cells of the mononuclear phagocyte system.

African Swine Fever↗
PubMed1982

A randomized-blinded evaluation of analytical error associated with the enzyme immunoassay (EMIT) of gentamicin.

Analytical error is a potential source of inaccuracy and imprecision in the pharmacokinetic dosing of the aminoglycosides. The present study was undertaken to determine the analytical error (precision and accuracy) associated with the EMIT assay for gentamicin as it is routinely used in a clinical pharmacokinetics laboratory, and to assess what impact this error would have on estimates of pharmacokinetic model parameters. Serum samples spiked with known amounts of gentamicin were assayed over a 6-week period along with routine clinical samples by a medical technologist who was blinded with regard to the expected concentration or number of replicates of each sample. Under these conditions, the assay method had a coefficient of variation (CV) of 4.8%. When this analytical error was incorporated into a first-order, one-compartment pharmacokinetic model, the CV was 0.8% for the y intercept and 1.2% for the slope. When all measured concentrations were compared with actual values, the slope and intercept were not significantly different from the line of reference. As indicated by these data, the EMIT assay for gentamicin as it is routinely used in a clinical laboratory is both precise and accurate, indicating that analytical error is not the major source of inaccuracy in pharmacokinetic dosing of gentamicin.

Gentamicins↗
PubMed1994

Invasion and intracellular survival of Bordetella bronchiseptica in mouse dendritic cells.

We have studied the interaction between the respiratory pathogen Bordetella bronchiseptica and murine spleen dendritic cells, important antigen-presenting cells that are found in the airway epithelium. Wild-type B. bronchiseptica 5376 attached very efficiently to dendritic cells, whereas the bvg mutant ATCC 10580, wild-type strain BB7865, and its spontaneous delta bvgS mutant BB7866 bound less efficiently. However, all tested B. bronchiseptica strains were able to invade dendritic cells and survive intracellularly for at least 72 h. These results suggest that bvg-independent or bvg-downregulated products are involved in the uptake and intracellular survival. Transmission electron microscopic analysis revealed that bacteria grew and replicated intracellularly and were present in typical phagosomes, which fused with lysosomes during the initial infection period. However, in later infection stages some bacteria seemed to escape into an unfused endocytic compartment, where individual bacteria were tightly surrounded by a membrane. The in vitro interaction of B. bronchiseptica with dendritic cells reported here may be relevant to natural infections caused by this organism that lead to chronicity or an altered immune response.

Animals↗
PubMed2004

Intact purine biosynthesis pathways are required for wild-type virulence of Brucella abortus 2308 in the BALB/c mouse model.

Brucella abortus 2308 derivatives with mini-Tn5 insertions in purE, purL, and purD display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn5 insertions in pheA, trpB, and dagA display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and replication of the brucellae in host macrophages. In contrast to previous reports, however, these results indicate that exogenous tryptophan and phenylalanine are available for use by the brucellae in the phagosomal compartment.

Animals↗
PubMed1999

The YXXL sequences of a transmembrane protein of bovine leukemia virus are required for viral entry and incorporation of viral envelope protein into virions.

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.

Amino Acid Sequence↗
PubMed2005

Human papillomavirus type 16 E1circumflexE4 contributes to multiple facets of the papillomavirus life cycle.

The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation program of the host's stratified epithelia that it infects. E1(circumflex)E4 is a viral protein that has been ascribed multiple biochemical properties of potential biological relevance to the viral life cycle. To identify the role(s) of the viral E1(circumflex)E4 protein in the HPV life cycle, we characterized the properties of HPV type 16 (HPV16) genomes harboring mutations in the E4 gene in NIKS cells, a spontaneously immortalized keratinocyte cell line that when grown in organotypic raft cultures supports the HPV life cycle. We learned that E1(circumflex)E4 contributes to the replication of the viral plasmid genome as a nuclear plasmid in basal cells, in which we also found E1(circumflex)E4 protein to be expressed at low levels. In the suprabasal compartment of organotypic raft cultures harboring E1(circumflex)E4 mutant HPV16 genomes there were alterations in the frequency of suprabasal cells supporting DNA synthesis, the levels of viral DNA amplification, and the degree to which the virus perturbs differentiation. Interestingly, the comparison of the phenotypes of various mutations in E4 indicated that the E1(circumflex)E4 protein-encoding requirements for these various processes differed. These data support the hypothesis that E1(circumflex)E4 is a multifunctional protein and that the different properties of E1(circumflex)E4 contribute to different processes in both the early and late stages of the virus life cycle.

Cell Line↗
PubMed1998

Ultrastructural pathology of the bone marrow in pigs inoculated with a moderately virulent strain (DR'78) of African swine fever virus.

Interpretation of changes in bone marrow during infectious processes is quite complex. This paper reports bone marrow lesions observed in pigs inoculated with a moderately virulent ASF virus strain and studies their relationship to the pathogenesis of the disease. In this work, we have carried out the structural and ultrastructural study of the bone marrow of 14 Large White x Landrace pigs that were inoculated by the intramuscular route with 10(5) 50% hemodsorbing doses (HAD50) of the Dominican Republic'78 ASF virus strain. The inoculated pigs were killed at 3, 5, 7, 9, 11, 13, 15 and 17 days postinjection. Analysis of cells and structures belonging to the two main bone-marrow compartments, the hematopoietic cells and the hematopoietic micro-environment, showed that after inoculation with a moderately virulent strain, the most significant changes occurred in macrophages and megakaryocytes, consisting in virus replication in these cell populations and apoptosis of megakaryocytes, related with the sudden and transitory thrombocytopenia detected in the subacute ASF.

African Swine Fever↗
PubMed2003

HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T cell immunity in HIV-1-uninfected individuals.

A vaccine regimen that can rapidly control HIV-1 replication at the site of exposure following sexual contact is likely to be the most effective in preventing HIV-1 infection. As part of a larger, phase II clinical trial, we evaluated the ability of a recombinant canarypox HIV-1 vaccine to induce CTL that can be detected in both the systemic and mucosal compartments following i.m. immunization in 12 low- and high-risk HIV-1 seronegative volunteers. In the 7 volunteers receiving four immunizations with live recombinant canarypox ALVAC-HIV vaccine with or without rgp120/SF-2, HIV-1-specific CTL were detected in the blood of 5 (71%) and in the rectum of 4 (57%). CTL responses were observed in both risk strata. In contrast, 5 volunteers receiving placebo had undetectable responses in both compartments. Vaccine-induced, HIV-1-specific effector activities included IFN-gamma secretion and class I MHC-restricted CD8(+) CTL. Rectal and systemic CD8(+) CTL clones established in 1 vaccine recipient revealed similar Env-specific responses and MHC restriction. These findings indicate that parenteral vaccination can induce HIV-1-specific CTL that localize to sites of HIV-1 acquisition, where their presence may be critical in the control of initial viral replication and eventual dissemination. Determination of the optimal strategy to induce mucosal T cells requires future clinical studies.

AIDS Vaccines↗
PubMedDate not supplied

Reservoirs of HIV-1 in vivo: implications for antiretroviral therapy.

The eradication of HIV-1 from infected individuals remains the ultimate goal of all anti-HIV therapeutics. Although highly active antiretroviral therapy (HAART) has led to a profound decrease in morbidity and mortality in infected people by suppressing HIV replication, the virus continues to evolve slowly during therapy even when patients achieve below detectable levels of HIV in plasma. HIV-1 persists in latently infected memory CD4+ T cells and there is minimal decay of HIV in this compartment despite prolonged HAART. Various other reservoirs and sanctuary sites harboring HIV are also established in vivo during antiretroviral therapy. Collectively these sites represent a major impediment to the eradication of HIV-1. This review presents a detailed overview of various reservoir sites in vivo, and discusses their impact on the success and failure of HAART for HIV patients. In addition, it addresses the effect of sub-optimal drug concentrations on reservoir establishment and outlines future therapeutic strategies to counteract these reservoirs and sanctuaries.

Antiretroviral Therapy, Highly Active↗
PubMed1999

Analysis of hepatitis G virus/GB virus C quasispecies and replication sites in human subjects.

Although the hepatitis G virus is unlikely to be a primary hepatotropic virus, its replication sites remain unclear. Using highly strand-specific Tth-based reverse transcriptase PCR we searched for the presence of the viral RNA negative strand in various autopsy tissues in two patients who died of end-stage liver disease. In addition, amplified viral sequences were compared in the 5' untranslated and the putative capsid regions by the single-strand conformation polymorphism (SSCP). Negative strand HGV RNA was detected in bone marrow and spleen from both patients and in lymph node tissue from one. All amplified sequences from a given patient were identical when compared by SSCP and direct sequencing. This lack of difference in the composition of quasispecies recovered from various tissues suggests the presence of a single, common viral compartment in the infected host.

5' Untranslated Regions↗
PubMed2003

Replication complex of human parechovirus 1.

The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.

Amino Acid Sequence↗