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PubMed2005

Insights into a CRM1-mediated RNA-nuclear export pathway in Trypanosoma cruzi.

Nuclear export and import of proteins and RNAs is a regulated process that permits the control of protein expression during cell development and differentiation. In all eukaryotic organisms transport of proteins to specific cellular compartments requires specific signaling sequences. Proteins that shuttle between nucleus and cytoplasm bear nuclear localization signals (NLS) and/or nuclear export signals (NES) and some of them can carry mRNAs, as part of shuttling ribonucleoprotein complexes. In this work we describe in the protozoan parasite Trypanosoma cruzi, a CRM1/exportin1 nuclear export factor named TcCRM1. This protein contains the conserved central region (CCR) that interacts with NES sequences present within cargo molecules, and the Cys residue involved in covalent binding to the Streptomyces metabolite leptomycin B (LMB). By subcellular fractionation we show that TcCRM1, a protein of about 117 kDa, has nuclear localization. We also demonstrate that LMB inhibits the replication of T. cruzi in a dose-dependent manner. In situ hybridization experiments performed with a Texas red-coupled oligo(dT) probe revealed that LMB produced a partial short-term accumulation of a poly(A)+RNA subset in the nucleus. Some mRNAs such as HSP70, TcUBP2/1 and TcPABP1 are reduced or disappeared from the cytoplasm of LMB treated cells. In sharp contrast with metazoans, no effect was observed on two U snRNAs subcellular localization, implying that a different export route might exist for these RNAs in trypanosomes.

Active Transport, Cell Nucleus↗
PubMed2002

The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host.

Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.

Amino Acid Sequence↗
PubMed2003

Increased expression of p21(waf) cyclin-dependent kinase inhibitor in asthmatic bronchial epithelium.

Because the asthmatic bronchial epithelium is characterized by widespread damage, we postulated that this is associated with expression of cell cycle inhibitors that control proliferation. Using bronchial biopsies, the epithelium was the major site of expression of the cyclin-dependent kinase inhibitor, p21(waf). Immunostaining usually occurred in the cytoplasm of columnar cells; however, in severe asthma, nuclear staining was also evident in the proliferative, basal cell compartment. p21(waf) expression was significantly higher in asthmatic versus nonasthmatic epithelium and was unaffected by corticosteroid treatment; proliferating cell nuclear antigen was not significantly different in any group. p21(waf), but not p27(kip1), mRNA and protein were induced by treatment of bronchial epithelial cells in vitro with transforming growth factor (TGF)-beta or H2O2, but not by dexamethasone, which induced p57(kip2). TGF-beta and dexamethasone inhibited epidermal growth factor (EGF)-induced DNA synthesis, whereas low concentrations of H2O2 synergized with EGF; at higher doses, growth inhibition and induction of apoptosis occurred. TGF-beta caused p21(waf) to become nuclear, suggesting interaction with the replicative machinery; however, in oxidant-stressed cells, p21(waf) was predominantly cytoplasmic, where it has been linked to cell survival. We conclude that p21(waf) overexpression in asthma influences cell proliferation and survival. This may cause abnormal repair responses that contribute to airway inflammation and remodeling.

Asthma↗
PubMed2003

Th1 cell-mediated resistance to cutaneous infection with Leishmania major is independent of P- and E-selectins.

Studies in several models of inflammation have underscored the importance of P- and E-selectins in the migration of T cells to inflamed tissues. However, the role of the endothelial selectins in infection-induced cutaneous inflammation and host-protective immunity has not been investigated. In this study, we demonstrate that CD4(+) T cells recruited to the cutaneous compartment during infection with Leishmania major express P- and E-selectin ligands. Furthermore, expression of P- and E-selectin ligands correlates with activated Leishmania-specific Th1 cells and is dependent upon IL-12. To investigate the functional role of the endothelial selectins during leishmaniasis, we infected mice either singly or doubly deficient in the expression of P- and E- selectins. Mice lacking both P- and E-selectins developed significantly less inflammation at the site of a primary and secondary infection, and exhibited an impaired delayed-type hypersensitivity response. Surprisingly, the absence of the endothelial selectins had no effect on the control of parasite replication or immunity to reinfection. Thus, these data demonstrate that although the endothelial selectins contribute to the inflammatory response, they are not required for protective immunity to L. major. Moreover, these data suggest that by blocking P- and E-selectins, the immune pathology associated with cutaneous leishmaniasis might be ameliorated without compromising immunity to infection.

Animals↗
PubMed2005

Functional and phenotypic characterization of CD57+CD4+ T cells and their association with HIV-1-induced T cell dysfunction.

HIV-1 replication is associated with reduced or absent HIV-1-specific CD4+ T cell proliferation and skewing of HIV-1-specific CD4+ T cells toward an IFN-gamma-producing, CCR7- phenotype. The CCR7- T cell population is heterogeneous and can be subdivided based on the expression of CD57. Although CD57 expression on CD8+ T cells is associated with proliferation incompetence and replicative senescence, less is known about the function of CD57-expressing CD4+ T cells. In this study, the frequency, phenotype, and function of CD57+CD4+ T cells were evaluated in 25 HIV-1-infected subjects and 10 seronegative controls. CD57+CD4+ T cells were found to be proliferation incompetent, even after strong mitogen stimulation. Percentages of CD4+ T cells that expressed CD57 were significantly higher in untreated HIV-1-infected subjects than in HIV-1-seronegative donors, and CD57 expression did not normalize in subjects receiving at least 6 mo of effective antiretroviral therapy. CD57 was predominately expressed on the CCR7- fraction of the CD4+ T cell compartment and accounted for the majority of cells in the CCR7-CD45RA+ population from untreated HIV-1-infected subjects. HIV-1-specific CD4+ T cells producing only IFN-gamma had the highest expression of CD57, whereas few cells producing IL-2 alone expressed CD57. These findings further define a novel population of proliferation-incompetent CD4+ T cells that are generated in the presence of chronic Ag exposure. A better understanding of the generation and persistence of CD57+ T cells in HIV-1 infection could provide important insights into the immunopathogenesis of this disease.

CD4-Positive T-Lymphocytes↗
PubMed2004

The post-translational modifications of proliferating cell nuclear antigen: acetylation, not phosphorylation, plays an important role in the regulation of its function.

The diverse function of proliferating cell nuclear antigen (PCNA) is thought to be due, in large part, to post-translational modifications. Here we show by high resolution two-dimensional PAGE analysis that there are three distinct PCNA isoforms that differ in their acetylation status. The moderately acetylated main (M) form was found in all of the subcellular compartments of cycling cells, whereas the highly acetylated acidic form was primarily found in the nucleoplasm, nuclear matrix, and chromatin. Interestingly, the deacetylated basic form was most pronounced in the nucleoplasm of cycling cells. The cells in G(0) and the cytoplasm of cycling cells contained primarily the M form only. Because p300 and histone deacetylase (HDAC1) were co-immunoprecipitated with PCNA, they are likely responsible for the acetylation and deacetylation of PCNA, respectively. We also found that deacetylation reduced the ability of PCNA to bind to DNA polymerases beta and delta. Taken together, our data support a model where the acidic and M forms participate in DNA replication, whereas the basic form is associated with the termination of DNA replication.

Acetylation↗
PubMed2006

Density-dependent decay in HIV-1 dynamics.

The decay of HIV-1-infected cell populations after treatment with antiretroviral therapy has been measured using simple exponential decay models. These models are unlikely to be realistic over periods longer than a few months, however, because the population dynamics of HIV are complex. We considered an alternate model developed by Perelson and colleagues that extends the standard model for biphasic viral load decline and allows for nonlinear log decay of infected cell populations. Using data from 6 children on highly active antiretroviral therapy (HAART) and a single parameter in the new model, the assumption of log linear decay of infected cell populations is tested. Our analysis indicates that the short-lived and long-lived infected cell populations do not decay according to a simple exponential model. Furthermore, the resulting estimates of time to eradication of infected cell compartments are dramatically longer than those previously reported (eg, decades vs. years for long-lived infected cell populations and years vs. weeks for short-lived infected cell populations). Furthermore, estimates of the second-phase decay rates are significantly different than 0 for most children when obtained using the Perelson biphasic decay model. In contrast, this rate is not significantly different than 0 when the density-dependent decay model is used for parameter estimation and inference. Thus, the density-dependent decay model but not the simple exponential decay model is consistent with recent data showing that even under consistent HAART-mediated suppression of viral replication, decay rates of infected cell reservoirs decay little over several years. This suggests that conclusions about long-term viral dynamics of HIV infection based on simple exponential decay models should be carefully re-evaluated.

Antiretroviral Therapy, Highly Active↗
PubMed1986

Variability in proteoglycan synthesis among six strains of normal human adult skin fibroblasts.

Several parameters of proteoglycan synthesis (incorporation of precursor isotope, distribution in culture compartments, sensitivity to chondroitinase ABC and Sepharose CL-2B elution profile) were investigated for six strains of normal human adult skin fibroblasts in culture in order to determine the range of variability in these parameters that could be expected in a normal population. When proteoglycan accumulation was measured in all cultures the uptake of Na2 35SO4 expressed per 10(6) cells had a coefficient of variation ranging from 17% to 50% in different experiments. Variability was always less when expressed per mg cell protein. The coefficient of variation was lowest when isotopic incorporation per mg cell protein was expressed as a percent of total incorporation of that isotope. Replicate tests indicated that the variability could not be explained by the assay methodologies alone. The protocol followed was designed to determine whether any consistent distinctions for individual cell strains could be identified. No significant difference in any parameter could be detected between the six cell strains by two-way analysis of variance although differences between experiments were significant in several cases and cultured bovine tendon fibroblasts were significantly different for every parameter tested.

Adult↗
PubMed1995

Molecular and cytotoxic effects of camptothecin, a topoisomerase I inhibitor, on trypanosomes and Leishmania.

Parasites pose a threat to the health and lives of many millions of human beings. Among the pathogenic protozoa, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani are hemoflagellates that cause particularly serious diseases (sleeping sickness, Chagas disease, and leishmaniasis, respectively). The drugs currently available to treat these infections are limited by marginal efficacy, severe toxicity, and spreading drug resistance. Camptothecin is an established antitumor drug and a well-characterized inhibitor of eukaryotic DNA topoisomerase I. When trypanosomes or leishmania are treated with camptothecin and then lysed with SDS, both nuclear and mitochondrial DNA are cleaved and covalently linked to protein. This is consistent with the existence of drug-sensitive topoisomerase I activity in both compartments. Camptothecin also inhibits the incorporation of [3H]thymidine in these parasites. These molecular effects are cytotoxic to cells in vitro, with EC50 values for T. brucei, T. cruzi, and L. donovani, of 1.5, 1.6, and 3.2 microM, respectively. For these parasites, camptothecin is an important lead for much-needed new chemotherapy, as well as a valuable tool for studying topoisomerase I activity.

Animals↗
PubMed1987

Simian virus 40 infection is not mediated by lysosomal activation.

The uptake of simian virus 40 (SV40) virions to the nucleus, the site of viral replication, proceeds via engulfment at the cytoplasmic membrane and transport in monopinocytotic vesicles through the cytoplasm to the nuclear membrane. In the case of Semliki Forest virus and poliovirus which undergo primary endocytosis in a similar manner, neutralization of the acid pH in these vesicles abolishes viral infectivity. We have examined the effects of the lysosomotropic agents chloroquine and ammonium chloride on the uptake of SV40 and find that neutralization of the acid pH in cellular organelles has no effect on the progress of SV40 infection. Although the initial endocytotic pathway appears similar for the viruses, the vesicular transport of SV40 to the nucleus proceeds, therefore, via an alternative endocytotic compartment which is not inhibited by increasing the endosomal pH.

Ammonium Chloride↗
PubMed1990

NRF-1: a trans-activator of nuclear-encoded respiratory genes in animal cells.

The assembly of the respiratory apparatus requires the coordinate expression of a large number of genes from both nuclear and mitochondrial genetic systems. In vertebrate organisms, the molecular mechanisms integrating the activities of these distinct genomic compartments in response to tissue demands for respiratory energy remain unknown. A potential inroad to this problem came with the discovery of nuclear respiratory factor 1 (NRF-1), a novel transcriptional activator defined by mutational and DNA binding analysis of the somatic cytochrome c promoter. Functional NRF-1 sites are now observed in several other recently isolated nuclear genes whose products function in the mitochondria. Among these are genes encoding subunits of the cytochrome c oxidase (subunit VIc) and reductase (ubiquinone-binding protein) complexes. In addition, a functional NRF-1 site resides in the MRP RNA gene encoding the RNA moiety of a ribonucleoprotein endonuclease involved in mitochondrial DNA replication. Synthetic oligomers of these sites competitively displace NRF-1 binding to the cytochrome c promoter. NRF-1-binding activities for each site also have the same thermal lability, copurify chromatographically, and make similar guanosine nucleotide contacts within each recognition sequence. Moreover, NRF-1 recognition in vitro correlates with the ability of each site to stimulate expression in vivo from a truncated cytochrome c promoter. The presence of NRF-1-binding sites in nuclear genes encoding structural components of the mammalian electron transport chain, as well as the mitochondrial DNA replication machinery, suggests a mechanism for coordination of nuclear and mitochondrial genetic systems through the concerted modulation of nuclear genes.

Animals↗
PubMed1993

The potential for gene therapy in Duchenne muscular dystrophy and other genetic muscle diseases.

Dystrophin cDNAs have been introduced into skeletal muscle fibers of dystrophin-deficient mice (mdx) through direct DNA injection in plasmid expression vectors and by replication-defective recombinant adenovirus vectors. The introduced genes appear to protect those muscle fibers from necrosis in which they become expressed. By direct injection of dystrophin cDNA in plasmid expression vector, only 1-2% of adult mdx muscle fibers of the injected muscle expressed dystrophin. On the other hand, by recombinant adenovirus injection into very young mdx muscle, a better efficiency has been reported. We have discussed several putative and proven factors that may contribute to the thus far demonstrated relatively low efficiency of dystrophin gene transfer. These include poor uptake of gene constructs by muscle fibers, degradation of the injected DNA, and poor access of gene constructs to the nuclear compartment. Neutralization or elimination of these factors could improve the efficiency of gene transfer so that it might, in the future, qualify as an effective therapy for DMD and some other genetic diseases of muscle.

Animals↗
PubMed2004

Intracellular localization of the cyclin-dependent kinase inhibitor p21CDKN1A-GFP fusion protein during cell cycle arrest.

The cyclin-dependent kinase (CDK) inhibitor p21CDKN1A is known to induce cell cycle arrest by inhibiting CDK activity and by interfering with DNA replication through binding to proliferating cell nuclear antigen. Although the molecular mechanisms have been elucidated, the temporal dynamics, as well as the intracellular sites of the activity of p21 bound to cyclin/CDK complexes during cell cycle arrest, have not been fully investigated. In this study we have induced the expression of p21CDKN1A fused to green fluorescent protein (GFP) in HeLa cells, in order to visualize the intracellular localization of the inhibitor during the cell cycle arrest. We show that p21-GFP is preferentially expressed in association with cyclin E in cells arrested in G1 phase, and with cyclin A more than with cyclin B1 in cells arrested in the G2/M compartment. In addition, we show for the first time that p21-GFP colocalizes with cyclin E in the nucleolus of HeLa cells during the G1 phase arrest.

Cell Cycle↗
PubMed2005

Expression of exogenous or endogenous green fluorescent protein in adipose tissue-derived stromal cells during chondrogenic differentiation.

Pluripotent stem cells within the adipose stromal compartment, termed adipose-derived stromal cells (ASCs), have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. Imaging with expression of exogenous or endogenous green fluorescent protein (GFP) reporters facilitates the detailed research on ASCs' physiological behavior during differentiation in vivo. This study was aimed to confirm whether ASCs expressing GFP still could be induced to chondrogenesis, and to compare the expression of exogenous or endogenous GFP in ASCs during chondrogenic differentiation. ASCs were harvested from inguinal fat pads of normal nude mice or GFP transgenic mice. Monolayer cultures of ASCs from normal mice were passaged three times and then infected with replication-incompetent adenoviral vectors carrying GFP genes. Allowed to recover for 5 days, Ad/GFP infected ASCs were transferred to chondrogenic medium as well as the ASCs from transgenic mice cultured in vitro over the same passages. The level of GFP in transgenic ASCs maintained stable till 3 months after chondrogenic induction. Whereas, high level of GFP expression in Ad/GFP infected ASCs could last for only 8 weeks and then declined stepwise. Important cartilaginous molecules such as SOX9, collagen type I, collagen type II, aggrecan, collagen type X were assessed using immunocytochemistry, RT-PCR, and Western Blot. The results indicated that no matter the GFP was exogenous or endogenous, it did not influence the chondrogenic potential of ASCs in comparison with the normal controls. Moreover, chondrogenic lineages from ASCs also underwent phenotypic modulation called dedifferentiation as a result of long-term culture in monolayers similar to normal chondrocytes.

Adipose Tissue↗
PubMed2003

Infectious HIV-1 assembles in late endosomes in primary macrophages.

Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.

Antigens, CD↗
PubMed1999

Acute activation of CD8+ T lymphocytes in interleukin-2-treated HIV-infected patients. ANRS-048 IL-2 Study Group. Agence Nationale de Recherches sur le SIDA.

CD8+ T lymphocytes play a key role in the control of HIV infection, through both cytotoxic and noncytotoxic mechanisms. To study in vivo effects of interleukin-2 (IL-2) treatment on this cell compartment, the level of activation of CD8+ T lymphocytes was evaluated before and just after 5-day administration of IL-2 in 16 HIV-infected patients. The serum level of soluble CD25 and of soluble CD8 significantly increased following IL-2 administration. The number of mRNA molecules coding for perforin and granzyme B, two enzymes that are contained in granules of cytotoxic cells, also significantly increased in peripheral blood mononuclear cells and in purified CD8+ cells (p < .001). Variations of plasma HIV viremia and perforin gene expression following IL-2 administration were inversely correlated (p = .023), suggesting that IL-2-induced activation of CD8+ T lymphocytes contributes to limit HIV replication in vivo. In contrast to perforin and granzyme B gene expression, IL-2 administration did not increase the expression of macrophage inhibitory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated-on-activation normal T-expressed and secreted (RANTES) genes. These findings indicate that CD8+ T lymphocytes in HIV-infected patients are acutely activated by IL-2 treatment, which may improve long-term control of HIV infection.

CD8 Antigens↗
PubMed2001

Water acquisition and partitioning in Drosophila melanogaster: effects of selection for desiccation-resistance.

We examined physiological features related to water balance in five replicate populations of Drosophila melanogaster that have undergone selection for enhanced resistance to desiccation (D populations) and in five replicate control (C) populations. Adult D flies contain 34 % more water than the control flies. We examined two hypotheses for increased water acquisition in the D flies: (i) that they accumulate more water early in development and (ii) that they have a reduced post-eclosion diuretic water loss. We found no evidence of differential water or dry mass acquisition between the C and D populations prior to adulthood. We also found no evidence of differential post-eclosion diuresis, i.e. both C and D groups showed insignificant changes in water volume in the 4 h period immediately after eclosion. In addition, we quantified water content in the intra- and extracellular compartments of the C and D populations and were able to identify the hemolymph as the primary storage site of the 'extra' water carried by the desiccation-resistant flies. We estimated that 68 % of the increased water volume observed in the D flies was contained in the hemolymph. Desiccation-resistance was strongly correlated with hemolymph volume and only weakly with intracellular water volume. Survival during desiccation was also strongly related to the carbohydrate content of the D flies. It has been presumed that the D flies accumulate carbohydrate primarily as intracellular glycogen, which would result in a significant increase in intracellular water volume. We found that carbohydrate content was weakly correlated with intracellular water volume and more strongly with hemolymph volume. The carbohydrate pool in the D flies may, therefore, be contained in the extracellular compartment as well as in cells. These results are suggestive of the importance of modifications in hemolymph volume and hemolymph solute concentrations in the evolution of enhanced desiccation-tolerance in populations of Drosophila melanogaster.

Animals↗
PubMed1998

Human immunodeficiency virus type 1 Vpr localization: nuclear transport of a viral protein modulated by a putative amphipathic helical structure and its relevance to biological activity.

Protein import into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly clear that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Human immunodeficiency virus type 1 (HIV-1) Vpr is one such protein which does not have an identifiable canonical NLS and yet efficiently localizes to the nuclear compartment. Here, we use confocal microscopy to demonstrate that mutations in the putative central hydrophobic helix of Vpr result in the retention of the protein in highly localized ring-like structures around the nuclear periphery with striking impairment in their ability to enter the nuclear interior. By characterizing other biological activities associated with this protein, such as its ability to incorporate into budding virions and its ability to arrest cells in G2, we show that this helical domain is specific for the nuclear translocation of the protein with very little effect on these other functions. Interestingly, however, perturbation of this helical motif also perturbs the protein's ability to augment viral replication in primary human macrophages indicating that the integrity of this secondary structure is essential for optimal infection in these non-dividing cells.

Amino Acid Sequence↗