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At least 883 records · Page 49Linked to original sources
PubMed2002

The hepatitis C virus RNA-dependent RNA polymerase membrane insertion sequence is a transmembrane segment.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) belongs to a class of membrane proteins termed tail-anchored proteins. Here, we show that the HCV RdRp C-terminal membrane insertion sequence traverses the phospholipid bilayer as a transmembrane segment. Moreover, the HCV RdRp was found to be retained in the endoplasmic reticulum (ER) or an ER-derived modified compartment both following transient transfection and in the context of a subgenomic replicon. An absolutely conserved GVG motif was not essential for membrane insertion but possibly provides a docking site for transmembrane protein-protein interactions. These findings have important implications for the functional architecture of the HCV replication complex.

Cell Membrane↗
PubMed1990

[Use of hepatocytes in primary cultures to predict potential hepatocarcinogenicity of compounds]

Short-term tests for genotoxic activity will detect a variety of potential carcinogens. However, experience over the last few years has shown that many chemical carcinogens are not detected in these tests. A large proportion of these non-mutagenic (non-genotoxic) carcinogens induce tumors in the rodent liver after life-long feeding. One class of such carcinogens are the hypolipidemic drugs many of which induce a characteristic hepatomegaly upon subchronic treatment. Typically this liver enlargement is accompanied by increased mitotic activity and a proliferation of the hepatic peroxisome compartment. Results also suggest a correlation between the degree and the nature of the hepatomegalic response to a compound, and its hepatocarcinogenic potency, but it remains unclear whether the growth stimulation and/or the peroxisome proliferation is correlated with the carcinogenicity. Both, the induction of peroxisomal enzyme and of replicative DMA synthesis may be measured in primary cultures of adult rat hepatocytes following in vitro exposition. Moreover, the ability of the compounds tested to induce in vitro replicative DNA synthesis in primary cultures of adult rat hepatocytes correlated well with the potency of the hepatomegalic response induced in vivo in treated rodents. Consequently, studies using such cultures might be useful to characterize and possibly predict in vitro the hepatocarcinogenic potency of some new compounds. The early recognition of the possible carcinogenic property of a new substance might accordingly contribute to the reduction of a number of life-long studies.

Journal Article↗
PubMed1985

Effect of amantadine on rhabdovirus infection.

Enveloped viruses enter host cells by fusion or viropexis. The latter mechanism is the prevalent entry pathway of rhabdoviruses into susceptible cells. Amantadine, a lysosomotropic agent, inhibits the multiplication of various groups of viruses. The effect of this drug was investigated on vesicular stomatitis virus and rabies fixed virus strain replication in fibroblasts. Amantadine was added to cells before, during and after infection to detect the phase of viral replication affected by the drug. Cells were inoculated with viruses at 4 degrees C and the incubation temperature was progressively raised to 37 degrees C in order to observe the effect of amantadine on attachment and early stages of viral replication. Experimental results indicated that the compound inhibited rhabdovirus infection in CER cells. Viral attachment and penetration did not appear to be affected by the drug, while later steps were inhibited, probably at the level of uncoating when the virus is released from the lysosomes into the intracytoplasmic compartment.

Amantadine↗
PubMed2006

Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus.

Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported "membranous web." However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle.

Capsid Proteins↗
PubMed1992

Nickel nitrate: a new junction permeability tracer for the study of the blood-testis barrier.

With the purpose of evaluating a new intercellular tracer, nickel-K ferrocyanide, we compared results yielded by lanthanum with information provided by nickel. This was done in the seminiferous epithelium of Holtzman rats of several postnatal ages and in a wild local seasonal breeder Galea musteloides. Tissues were studied with transmission electron microscopy and freeze-fracture replications. Nickel tracing proved to delineate cell contours more intensely and less interruptedly than lanthanum. With regard to seasonal variations in adult galea, the limits of the barrier were similar to those described in other mammals: spermatogonia, preleptotene, and leptotene spermatocytes were surrounded by the tracer in the basal compartment. The zygotenepachytenes were contained in the lumenal compartment and tracers were stopped at the inter-Sertoli cell tight junctions. During the inactive spermatogenic phase in winter, the seminiferous epithelium contained Sertoli cells and occasional germ cells, never beyond the spermatocyte stage. The tracer filled intercellular spaces, indicating that the barrier was incompetent. Some resting germ cells showed nuclear hyperchromasia, karyolysis, organelle loss, cell shrinkage, and cell fusion leading to a multinucleated cells. The inter-Sertoli tight junctions were scanty and had randomly oriented and discontinuous junctional strands. Moreover, inter-Sertoli cell gap junctions proliferated. During the active spermatogenic phase in summer, junctions were numerous. Their junctional strands were parallel to each other, and continuous.

Animals↗
PubMedDate not supplied

Telomeres, telomerase, and hematopoietic stem cell biology.

Telomeres are composed of the tandem DNA repeats and associated proteins that cap the end of linear chromosomes. They provide stability to the chromosome and protect against DNA loss associated with cellular replication. Telomeres are maintained by the reverse transcriptase, telomerase. The regulation of telomere length and telomerase activity is a complex and dynamic process that is tightly linked to cell cycle regulation. Hematopoietic stem cells have an impressive but finite proliferative potential and demonstrate telomeric shortening during replicative aging despite expression of low levels of telomerase. Recently, the important role of telomeres in human illness has been highlighted by studies of the rare genetic disorder dyskeratosis congenita. Here we review the role of telomeres and telomerase in the function and regulation of the hematopoietic stem cell compartment and their importance in hematologic disease.

Cell Lineage↗
PubMed2004

Kinetics of hepatitis B viral load during 48 weeks of treatment with 600 mg vs 100 mg of lamivudine daily.

Oral therapy for chronic hepatitis B remains suboptimal. Mathematical modelling of viral decay kinetics to rapidly assess potential antiviral regimens has proved valuable for human immunodeficiency virus and cytomegalovirus. We defined the kinetics of viral replication in 10 chronic hepatitis B patients randomized to lamivudine 100 mg vs 600 mg for 48 weeks. Viral decay kinetics conformed to a biphasic pattern in nine of 10 subjects. Persons receiving 600 mg daily of lamivudine exhibited a 1.6-fold faster decay rate in the infected cell compartment (0.028/day vs 0.017/day, P = 0.06) and a greater overall change in serum viral load when compared with those receiving 100 mg (4.06 vs 1.52 log(10) copies/mL, P = 0.08). More potent therapy appeared to result in more rapid decrease in the infected cell population. Studies using mathematical modelling of viral decay may be a useful method to evaluate single or combination therapy for HBV infection in vivo.

Adult↗
PubMed2005

Genetic composition of human immunodeficiency virus type 1 in cerebrospinal fluid and blood without treatment and during failing antiretroviral therapy.

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) is a significant cause of morbidity. The requirements for HIV adaptation to the CNS for neuropathogenesis and the value of CSF virus as a surrogate for virus activity in brain parenchyma are not well established. We studied 18 HIV-infected subjects, most with advanced immunodeficiency and some neurocognitive impairment but none with evidence of opportunistic infection or malignancy of the CNS. Clonal sequences of C2-V3 env and population sequences of pol from HIV RNA in cerebrospinal fluid (CSF) and plasma were correlated with clinical and virologic variables. Most (14 of 18) subjects had partitioning of C2-V3 sequences according to compartment, and 9 of 13 subjects with drug resistance exhibited discordant resistance patterns between the two compartments. Regression analyses identified three to seven positions in C2-V3 that discriminated CSF from plasma HIV. The presence of compartmental differences at one or more of the identified positions in C2-V3 was highly associated with the presence of discordant resistance (P = 0.007), reflecting the autonomous replication of HIV and the independent evolution of drug resistance in the CNS. Discordance of resistance was associated with severity of neurocognitive deficits (P = 0.07), while low nadir CD4 counts were linked both to the severity of neurocognitive deficits and to discordant resistance patterns (P = 0.05 and 0.09, respectively). These observations support the study of CSF HIV as an accessible surrogate for HIV virions in the brain, confirm the high frequency of discordant resistance in subjects with advanced disease in the absence of opportunistic infection or malignancy of the CNS, and begin to identify genetic patterns in HIV env associated with adaptation to the CNS.

AIDS Dementia Complex↗
PubMed2002

Aberrant expression of minichromosome maintenance proteins 2 and 5, and Ki-67 in dysplastic squamous oesophageal epithelium and Barrett's mucosa.

BACKGROUND: Minichromosome maintenance (Mcm) proteins are essential for eukaryotic DNA replication, and their expression implies potential for cell proliferation. Expression is dysregulated in dysplastic states but data for oesophageal squamous mucosa and Barrett's mucosa have not been published. AIM: To test the hypothesis that Mcm proteins are downregulated together with the proliferation marker Ki-67 in differentiating epithelial compartments of non-dysplastic squamous and Barrett's epithelium, and that this process does not occur in dysplastic mucosae. METHODS AND CASES: Forty five patients with Barrett's oesophagus included 20 with glandular dysplasia (10 low grade, eight high grade, two both, and four with invasive adenocarcinoma). Twenty five other patients included 12 with oesophageal squamous dysplasia (three low grade, six high grade, three both, and four with invasive squamous carcinoma). Formalin fixed paraffin embedded tissue sections from biopsy series and resections were immunostained using antibodies to Mcm2, Mcm5, and Ki-67. Percentage of nuclei positive for Mcm2, Mcm5, and Ki-67 was estimated and scored from 0 to 6 as: 0, none +; 1, <10%+; 2, 10-30%+; 3, 30-70%+; 4, 70-90%+; 5, >90%+; 6, all+. Four separate epithelial strata were scored: in squamous epithelium the basal layer and thirds to the surface, in Barrett's mucosa the luminal surface, upper and lower crypt, and deep glands. RESULTS: In non-dysplastic squamous epithelium and Barrett's mucosa, high level expression of Mcm2, Mcm5, and Ki-67 proteins was largely confined to the proliferative compartments and downregulated in differentiated compartments. Expression persisted up to the mucosal surface in dysplastic squamous epithelium and Barrett's mucosa. CONCLUSIONS: Persistent expression of Mcm2, Mcm5, and Ki-67 proteins in luminal compartments of dysplastic oesophageal squamous epithelium and dysplastic Barrett's mucosa may be diagnostic markers and imply disruption of cell cycle control and differentiation in these dysplastic epithelia.

Barrett Esophagus↗
PubMed2003

Integration of apoptosis and telomere erosion in virus-specific CD8+ T cells from blood and tonsils during primary infection.

Human-virus-specific CD8+ T cells that are found during primary infection have been studied almost exclusively in the peripheral blood, and it is unclear whether these cells are regulated in the same way as those in secondary lymphoid tissue. We investigated, therefore, the control of apoptosis and telomere erosion of Epstein-Barr virus (EBV)-specific CD8+ T cells found in the blood and tonsils of the same patients during acute infectious mononucleosis (AIM). Although the clonal composition of CD8+ T cells as determined by heteroduplex analysis was similar in both compartments, there was greater CD28 expression in the tonsil population, indicating that they were less differentiated. EBV-specific CD8+ T cells in both tissue types were extremely susceptible to apoptosis related to low Bcl-2 expression and were dependent on exogenous cytokines such as interleukin-2 (IL-2), IL-15, and interferon-alpha/beta (IFN-alpha/beta) for survival. In both compartments, however, these cells maintained their telomere lengths through telomerase induction. Thus, apoptosis-prone EBV-specific CD8+ T cells found during acute infection have to be rescued from death to persist as a memory population. However, signals that induce telomerase ensure that the rescued cells retain their replicative capacity. Significantly, these processes operate identically in cells found in blood and secondary lymphoid tissue.

Acute Disease↗
PubMed1995

Abortive infection of Vero cells by an influenza A virus (FPV).

We have discovered a new type of abortive replication in Vero cells infected with fowl plague virus. In these cells there is an enhanced splicing of the colinear mRNAs of segment 7 and presumably also of segment 8, leading to an extreme overproduction of M2 and NS2 proteins. The cleavage of the hemagglutinin (HA) into HA1 and HA2 and the processing of its carbohydrate side chains are markedly retarded and incomplete. Although some of the HA is incorporated into the plasma membrane, leading to a positive hemadsorption, most of it accumulates in a discrete compartment close to the nuclear membrane, representing presumably the reticuloendothel and/or the Golgi network. Neuraminidase activity in Vero cells is extremely low. The nucleoprotein is normally released from nuclei late in infection. Very little infectious virus is released, and its spread is highly impeded.

Animals↗
PubMed2004

Simian immunodeficiency virus promoter exchange results in a highly attenuated strain that protects against uncloned challenge virus.

Among the many simian immunodeficiency virus (SIV) immunogens, only live attenuated viral vaccines have afforded strong protection to a natural pathogenic isolate. Since the promoter is crucial to the tempo of viral replication in general, it was reasoned that promoter exchange might confer a novel means of attenuating SIV. The core enhancer and promoter sequences of the SIV macaque 239nefstop strain (NF-kappaB/Sp1 region from -114 bp to mRNA start) have been exchanged for those of the human cytomegalovirus immediate-early promoter (CMV-IE; from -525 bp to mRNA start). During culture of the resulting virus, referred to as SIVmegalo, on CEMx174 or rhesus macaque peripheral blood mononuclear cells, deletions arose in distal regions of the CMV-IE sequences that stabilized after 1 or 2 months of culture. However, when the undeleted form of SIVmegalo was inoculated into rhesus macaques, animals showed highly controlled viremia during primary and persistent infection. Compared to parental virus infection in macaques, primary viremia was reduced by >1,000-fold to undetectable levels, with little sign of an increase of cycling cells in lymph nodes, CD4(+) depletion, or altered T-cell activation markers in peripheral blood. Moreover, in contrast to wild-type infection in most infected animals, the nef stop mutation did not revert to the wild-type codon, indicating yet again that replication was dramatically curtailed. Despite such drastic attenuation, antibody titers and enzyme-linked immunospot reactivity to SIV peptides, although slower to appear, were comparable to those seen in a parental virus infection. When animals were challenged intravenously at 4 or 6 months with the uncloned pathogenic SIVmac251 strain, viremia was curtailed by approximately 1,000-fold at peak height without any sign of hyperactivation in CD4(+)- or CD8(+)-T-cell compartment or increase in lymph node cell cycling. To date, there has been a general inverse correlation between attenuation and protection; however, these findings show that promoter exchange constitutes a novel means to highly attenuate SIV while retaining the capacity to protect against challenge virus.

AIDS Vaccines↗
PubMed1992

Infectious entry pathway for canine parvovirus.

We have investigated whether canine parvovirus (CPV) infection involves a low pH-dependent entry pathway. The effects of two lysosomotropic bases, NH4Cl and chloroquine, on CPV entry were studied by immunofluorescence and ultrastructural and biochemical methods. In the presence of these reagents, input virions appear to accumulate in large vacuoles. Ultrastructural studies indicated that uptake of virions takes place predominantly in small uncoated vesicles that appear to fuse with larger vesicles. In the presence of NH4Cl, virions accumulate in the latter structures and their uncoating appears to be prevented. Viral DNA as well as antigen synthesis were found to be significantly inhibited in the presence of these reagents. In addition, inhibition of viral DNA and antigen synthesis appeared to be most extensive when NH4Cl was present from 30 min preinfection, whereas no significant inhibition was observed when the cells were treated after 2 hr postinfection. Thus, the results indicate that CPV requires exposure to low pH in an endosomal compartment to initiate a productive infection.

Ammonium Chloride↗
PubMed2000

Nucleotide and amino acid complexity of hepatitis C virus quasispecies in serum and liver.

The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral replication. Several studies have tried to unravel, through the parameters that characterize HCV circulating quasispecies, prognostic markers of the disease. In a previous work we demonstrated that the parameters of circulating viral quasispecies do not always reflect those of the intrahepatic virus. Here, we have analyzed paired serum and liver quasispecies from 39 genotype 1b-infected patients with different degrees of liver damage, ranging from minimal changes to cirrhosis. Viral level was quantified by real-time reverse transcription-PCR, and viral heterogeneity was characterized through the cloning and sequencing of 540 HCV variants of a genomic fragment encompassing the E2-NS2 junction. Although in 95% of patients, serum and liver consensus HCV amino acid sequences were identical, quasispecies complexity varied considerably between the viruses isolated from each compartment. Patients with HCV quasispecies in serum more complex (26%) than, less complex (28%) than, or similarly complex (41%) to those in liver were found. Among the last, a significant correlation between fibrosis and all the parameters that measure the viral amino acid complexity was found. Correlation between fibrosis and serum viral load was found as well (R = 0.7). With regard to the origin of the differences in quasispecies complexity between serum and liver populations, sequence analysis argued against extrahepatic replication as a quantitatively important contributing factor and supported the idea of a differential effect or different selective forces on the virus depending on whether it is circulating in serum or replicating in the liver.

Adult↗
PubMed2006

Efferent projections of rat rostroventrolateral medulla C1 catecholamine neurons: Implications for the central control of cardiovascular regulation.

A replication-defective lentivirus vector that expresses enhanced green fluorescent protein (EGFP) under the control of a synthetic dopamine-beta-hydroxylase (DbetaH) promoter was used to define efferent projections of C1 catecholamine neurons in rat rostral ventrolateral medulla (RVLM). EGFP expression was restricted to C1 neurons and filled their somatodendritic compartments and efferent axons 7-28 days after vector injection. This included the descending projections to thoracic spinal cord and a network in brainstem, midbrain, and diencephalon. In caudal brainstem, restricted terminal fields were present in the dorsal motor vagal complex, A1, raphe pallidus and obscurus, and marginal layer of ventrolateral medulla. Innervation of raphe nuclei was most dense at the level of RVLM, but rostral levels of pallidus were devoid of innervation. A sparse commissural projection to contralateral RVLM was observed, and pericellular arbors were present in the dorsal reticular formation among the projection pathway of catecholamine axons. Rostral brainstem contained a dense innervation of locus coeruleus and the nucleus subcoeruleus. A restricted innervation of the ventrolateral column of the periaqueductal gray distinguished the midbrain. Forebrain labeling was restricted to the diencephalon, where distinctive terminal fields were observed in the paraventricular thalamic nucleus; the lateral hypothalamic area; and the paraventricular, dorsomedial, supraoptic, and median preoptic nuclei of hypothalamus. Projection fibers also coursed through the tuberal hypothalamus into the median eminence. Collectively, these data demonstrate that RVLM C1 neurons modulate the activity of other central cell groups known to participate in the regulation of cardiovascular and autonomic function.

Animals↗
PubMed1976

Notions about human stem cells.

From an analysis of the known red cell and granulocytic cell turnover rates in the peripheral blood, absolute bone marrow cellularity of man, mitotic index of human marrow, tritiated thymidine labeling index of human marrow, the proportional distribution of the different cell types in the bone marrow of man and the measured DNA synthesis time of differentiated cell lines in human bone marrow, the flux of stem cells into the differentiated compartment has been calculated. By assuming that the same fraction of committed stem cells of man and mouse are in DNA synthesis, the number of cells in DNA synthesis and the size of the stem cell compartments have been calculated. The calculation gives an abundance of stem cells in human marrow that is much larger than that estimated by in vitro culture techniques. On the basis of the calculations, it is hypothesized that the pool of cells preceding the cytologically differentiated pools may consist of self replicating committed stem cells and differentiated cells undergoing amplification that have not developed cytologic markers characteristics of differentiated cells.

Bone Marrow↗
PubMed2005

Microcosm evaluation of the fate, toxicity, and risk to aquatic macrophytes from perfluorooctanoic acid (PFOA).

Perfluorooctanoic acid (PFOA) is an anthropogenic contaminant detected in various environmental and biological matrices. This compound is a fluorinated surfactant, belonging to a class of molecules known for persistence and their global distribution, but for which little ecotoxicological data are currently available, especially under field conditions. The environmental fate and toxicity of PFOA to the aquatic macrophytes Myriophyllum sibiricum and M. spicatum were investigated using 12,000 L outdoor microcosms. Replicate microcosms (n = 3) were treated with 0.3, 1, 30, and 100 mg/L PFOA as the sodium salt, plus controls, and assessed at regular intervals over 35 days. PFOA showed no significant dissipation from the water column, except at the greatest concentration, where partitioning from the water column into other compartments is suspected. The two species of Myriophyllum were similar in their sensitivity to PFOA under these simulated field conditions. Toxicity after 14 to 35 days of exposure in the evaluated endpoints for M. spicatum was > or = 5.7 mg/L PFOA for EC10s and > or = 31.8 mg/L PFOA for EC50s and in M. sibiricum was > or = 8.4 mg/L PFOA for EC10s and > or = 35.8 mg/L PFOA for EC50s. The no observed effects concentrations (NOECs) for Myriophyllum spp. were consistently > or = 23.9 mg/L PFOA. A risk assessment for these plant species estimated a negligible probability of toxicity being observed from PFOA exposure at current environmental concentrations.

Caprylates↗
PubMed1999

Mutant cells selected during persistent reovirus infection do not express mature cathepsin L and do not support reovirus disassembly.

Persistent reovirus infections of murine L929 cells select cellular mutations that inhibit viral disassembly within the endocytic pathway. Mutant cells support reovirus growth when infection is initiated with infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly formed following proteolysis of viral outer-capsid proteins. However, mutant cells do not support growth of virions, indicating that these cells have a defect in virion-to-ISVP processing. To better understand mechanisms by which viruses use the endocytic pathway to enter cells, we defined steps in reovirus replication blocked in mutant cells selected during persistent infection. Subcellular localization of reovirus after adsorption to parental and mutant cells was assessed using confocal microscopy and virions conjugated to a fluorescent probe. Parental and mutant cells did not differ in the capacity to internalize virions or distribute them to perinuclear compartments. Using pH-sensitive probes, the intravesicular pH was determined and found to be equivalent in parental and mutant cells. In both cell types, virions localized to acidified intracellular organelles. The capacity of parental and mutant cells to support proteolysis of reovirus virions was assessed by monitoring the appearance of disassembly intermediates following adsorption of radiolabeled viral particles. Within 2 h after adsorption to parental cells, proteolysis of viral outer-capsid proteins was observed, consistent with formation of ISVPs. However, in mutant cells, no proteolysis of viral proteins was detected up to 8 h postadsorption. Since treatment of cells with E64, an inhibitor of cysteine-containing proteases, blocks reovirus disassembly, we used immunoblot analysis to assess the expression of cathepsin L, a lysosomal cysteine protease. In contrast to parental cells, mutant cells did not express the mature, proteolytically active form of the enzyme. The defect in cathepsin L maturation was not associated with mutations in procathepsin L mRNA, was not complemented by procathepsin L overexpression, and did not affect the maturation of cathepsin B, another lysosomal cysteine protease. These findings indicate that persistent reovirus infections select cellular mutations that affect the maturation of cathepsin L and suggest that alterations in the expression of lysosomal proteases can modulate viral cytopathicity.

Animals↗