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At least 469 records · Page 26Linked to original sources
PubMed2003

Identification of Ets-1 as an important transcriptional activator of CTP:phosphocholine cytidylyltransferase alpha in COS-7 cells and co-activation with transcriptional enhancer factor-4.

Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT). Transcriptional enhancer factor-4 (TEF-4) enhances the transcription of CTalpha in COS-7 cells by interactions with the basal transcription machinery (Sugimoto, H., Bakovic, M., Yamashita, S., and Vance, D.E. (2001) J. Biol. Chem. 276,12338-12344). To identify the most important transcription factor involved in basal CTalpha transcription, we made CTalpha promoter-deletion and -mutated constructs linked to a luciferase reporter and transfected them into COS-7 cells. The results indicate that an important site regulating basal CTalpha transcription is -53/-47 (GACTTCC), which is a putative consensus-binding site of Ets transcription factors (GGAA) in the opposite orientation. Gel shift analyses indicated the existence of a binding protein for -53/-47 (GACTTCC) in nuclear extracts of COS-7 cells. When anti-Ets-1 antibody was incubated with the probe in gel shift analyses, the intensity of the binding protein was decreased. The binding of endogenous Ets-1 to the promoter probe was increased when TEF-4 was expressed; however, the amount of Ets-1 detected by immunoblotting was unchanged. When cells were transfected with Ets-1 cDNA, the luciferase activity of CTalpha promoter constructs was greatly enhanced. Co-transfection experiments with Ets-1 and TEF-4 showed enhanced expression of reporter constructs as well as CTalpha mRNA. These results suggest that Ets-1 is an important transcriptional activator of the CTalpha gene and that Ets-1 activity is enhanced by TEF-4.

Animals↗
PubMed2003

Dynamic properties of APC-decorated microtubules in living cells.

The adenomatous polyposis coli (APC) tumour suppressor protein is a component of the Wnt signalling pathway in which it plays a major role in controlling nuclear accumulation of beta-catenin and hence in the modulation of beta-catenin-regulated gene transcription. APC also associates with microtubules at the ends of cytoplasmic extensions in epithelial cells, a distribution that can be reproduced in COS cells ectopically expressing APC. To examine the effect of APC on microtubule properties, we monitored directly the behaviour of APC and of APC-decorated microtubules by time-lapse imaging of cytoplasmic extensions in live COS cells expressing APC tagged with a green fluorescent protein. On the proximal part of microtubules, APC was visualised as particulate material moving unidirectionally towards the plus end of microtubules. The distal parts of microtubules were uniformly decorated by APC and were animated by a motile behaviour in the form of aperiodic bending. This behaviour is likely to be the consequence of compression forces acting on microtubules encountering obstacles while elongating. The majority of APC-decorated microtubules in transfected COS cells was sensitive to depolymerisation by nocodazole, but they contained detyrosinated and acetylated alpha-tubulin, suggesting a reduction in the rate of subunit exchange at their growing end. Taken together, these results demonstrate that microtubule domains uniformly decorated by APC display dynamic and motile properties that may be significant for the postulated role of APC in targeting microtubules to specialised membrane sites.

Adenomatous Polyposis Coli Protein↗
PubMed2000

[Cloning of human TRAIL cDNA and its expression in COS-7 cells].

The human TRAIL cDNA was amplified with the total RNA from the human acute promyelocytic leukemia cell line HL-60 by means of RT-PCR, and was cloned into the pGEM-T vector. The DNA sequence analysis showed that it was consistent with the published sequence. Then, the insert of human TRAIL cDNA was subcloned into the mammalian expression vector pcDNA3. The hybrid plasmid pcDNA3-hTRAIL was transformed into COS-7 cells, and transiently expressed in the COS-7 cells. The activity of the expressed product could induce apoptosis in U937 cell line.

Animals↗
PubMed2000

Selective inhibition of monoamine neurotransmitter transporters by synthetic local anesthetics.

Synthetic local anesthetics (LAs) have been found to have cocaine-like characteristics with some psychotomimetic action, possibly through monoaminergic neurotransmission. To gain insight into the relation between LA action and monoamine transporters, we investigated the effect of synthetic LAs on neurotransmitter transporters, including monoamine transporters. We used cloned transporter cDNAs and examined transient functional expression in COS cells and stable expression in HeLa cells. Among the LAs tested, procaine and other ester-type LAs inhibited [3H]DA uptake and binding of [3H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (CFT), a cocaine analogue, in COS cells expressing rat dopamine transporter (DAT). The inhibition was concentration-dependent. The inhibitory effect on [3H]DA uptake was reversible and not dependent on pH, as observed in HeLa cells stably expressing DAT. Procaine also inhibited uptake of norepinephrine (NE) and serotonin (5-HT) by the norepinephrine transporter (NET) or serotonin transporter (SERT) expressed in COS cells. On the other hand, procaine and other LAs had little or no effect on [3H]GABA and [3H]glutamate uptake in COS cells expressing mouse GABA or rat glutamate/aspartate transporter. IC50 values for [3H]DA uptake inhibition correlated well with those for [3H]CFT binding inhibition, but not with intrinsic anesthetic potency. Kinetic analysis of monoamine uptake inhibition by procaine in COS cells expressing rat DAT, NET or SERT revealed a competitive action similar to that of cocaine. These results demonstrate that certain LAs selectively inhibit monoamine transporters. This might contribute to the cocaine-like psychotomimetic action of certain LAs.

Anesthetics, Local↗
PubMed1991

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.

Amino Acid Sequence↗
PubMed1992

Functional expression of bovine 17 alpha-hydroxylase in COS 1 cells is dependent upon the presence of an amino-terminal signal anchor sequence.

Microsomal forms of eukaryotic cytochrome P450 proteins are integral membrane proteins of the endoplasmic reticulum (ER) membrane which are targeted to the ER via the signal recognition particle pathway. A hydrophobic amino terminus serves as a combined signal sequence and major membrane anchor (signal-anchor sequence) for the microsomal P450s. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the ER of COS 1 cells in order to evaluate the role of membrane insertion of the amino-terminal signal-anchor of microsomal P450s as a functional determinant for these enzymes. Previously, we have shown that deletion of the hydrophobic amino terminus from P45017 alpha reduced membrane targeting and insertion by 5-fold compared with the wild-type protein, abolished enzymatic activity, and resulted in an aberrant CO difference spectrum. In the present study we have replaced the amino terminus of P45017 alpha with two heterologous signal-anchor sequences, one that is similar and one that is very different from the P45017 alpha sequence. The chimeric proteins were expressed in COS 1 cells. Immunoblot analysis of isolated microsomal membranes show that the heterologous signal-anchor sequences functioned to target the P45017 alpha protein to the ER. Enzymatic assays in intact COS 1 cells indicate that both the chimeric proteins are efficient 17 alpha-hydroxylase enzymes. The amino terminus of P45017 alpha was also replaced with a sequence that is not a signal-anchor, and the expressed protein was neither targeted to the ER nor was functional in COS 1 cells. In conclusion, both the structure and catalytic activity of P45017 alpha in COS 1 cells is dependent upon an amino-terminal sequence that functions as a signal-anchor sequence and not upon the precise sequence of the amino terminus.

Amino Acid Sequence↗
PubMed1996

Uncleaved env gp160 of human immunodeficiency virus type 1 is degraded within the Golgi apparatus but not lysosomes in COS-1 cells.

The fate of newly synthesized human immunodeficiency virus type 1 env gp160 was examined in COS-1 cells. The results of morphological chase experiments involving cycloheximide demonstrated that gp160 was retained in the Golgi apparatus for longer than the half-life of the molecule. The degradation of gp160 was insensitive to both bafilomycin A1 and leupeptin (< 0.2 mM), which block lysosomal proteolysis. However, degradation was effectively suppressed by leupeptin at higher concentrations, maximally at 1.7 mM. Furthermore, undegraded gp160 was accumulated in the Golgi apparatus, but was not detected in lysosomes. These results indicate that in COS-1 cells gp160 is not degraded in lysosomes, but rather that degradation takes place in the Golgi apparatus.

Animals↗
PubMed1997

Two Eph receptor tyrosine kinase ligands control axon growth and may be involved in the creation of the retinotectal map in the zebrafish.

The isolation and characterisation of two zebrafish Eph receptor ligand cDNAs which we have called zfEphL3 and zfEphL4 is described. These genes are expressed in the presumptive midbrain of developing embryos from 6 somites. By 24 hours L3 is expressed throughout the midbrain including the region of the presumptive tectum whereas L4 is strongly expressed in the midbrain caudal to the presumptive tectum. At later stages of development L3 is expressed in a graded fashion throughout the tectum and L4 is maintained at its posterior margin. Growth cone collapse and pathway selection assays demonstrate that both these proteins have a collapse activity for retinal ganglion cells. When faced with a choice of substrate on which to grow, temporal axons from chick retinal ganglion cells selectively avoided membranes from Cos cells transfected with L3, whereas nasal axons did not. Both temporal and nasal axons avoided membranes from Cos cells transfected with L4. The expression patterns together with the functional data suggest that although both ligands may be able to guide retinal ganglion cells axons in vitro, they have different roles in the guidance of retinotectal projections in vivo. The expression of L3 is consistent with a role in the guidance of retinal ganglion cells to their targets on the tectum whereas that of L4 suggests a role in delineating the posterior boundary of the optic tectum.

Amino Acid Sequence↗
PubMed1987

Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide.

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.

1-Deoxynojirimycin↗
PubMed1993

The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils, and monocytes.

CD22 is a B lineage-restricted member of the Ig superfamily that serves as an adhesion receptor expressed by mature B lymphocytes. In this study, the ability of different cell types to attach to COS cells transiently transfected with a full-length CD22 cDNA (COS-CD22) was examined to determine the cellular distribution of the ligand for CD22. T and B lymphocytes, monocytes, erythrocytes, and neutrophils formed specific rosettes with COS-CD22 cells at 4 degrees C. A panel of 33 new mAb directed against CD22 were developed to examine the regions of CD22 that mediate adhesion. Four of these mAb, HB22-7, -22, -23, and -33 (at 1 to 5 micrograms/ml) specifically blocked adhesion (75 to 95%) of all cell types to COS-CD22 cells. Each of these mAb cross-blocked each other's binding, suggesting that ligand binding occurs through a single region of CD22. These mAb also identify a region of CD22 distinct from those defined by previously described CD22 mAb. CD22-mediated adhesion of cell lines to COS-CD22 cells was independent of CD45RO and CDw75 expression, and it was not inhibited by mAb against known integrins. Although alpha-2,6-linked sialic acid expressed on the surface of COS cells did not serve as a ligand for CD22, the CD22 ligand may contain a critical sialic acid determinant, as neuraminidase treatment of all target cells eliminated CD22-mediated adhesion. CD22-mediated adhesion was Ca2+/Mg2+ independent, again suggesting that integrins were not involved. An inhibitory substance for CD22-mediated adhesion was found to be present in FCS and some ascites fluid. Analysis of CD22 mRNA and protein revealed that although multiple mRNA splice variants of CD22 mRNA can be detected, only a single protein isoform was detected on the cell surface. Therefore, although the identity of the CD22 ligands remains incompletely characterized, it is possible that a single major ligand is expressed by RBC and leukocytes, which binds to a single region of CD22.

Animals↗
PubMed1988

Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells.

Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis.

Animals↗
PubMed1994

Functional analysis of myosin missense mutations in familial hypertrophic cardiomyopathy.

To analyze potential functional consequences of myosin heavy chain (MHC) mutations identified in patients with familial hypertrophic cardiomyopathy (FHC), we have assessed the stability of the mutant MHCs and their ability to form thick filaments. Constructs encoding wild-type rat alpha MHC and seven corresponding FHC missense mutants were transfected into COS cells. Immunoblot analysis suggested that FHC mutations do not grossly alter protein stability. Wild-type alpha MHC transfected into COS cells forms structures previously shown to be arrays of thick filaments, which also resemble myosin structures observed early in differentiation of muscle cells. Surprisingly, up to 29% of COS cells transfected with the FHC mutants failed to form filamentous structures. To assess whether this phenotype was specific for the FHC mutants and not generalizable to any myosin mutation, COS cells were transfected with a construct encoding an MHC with a 168-amino acid deletion of the hinge/rod region. This deletion construct formed filamentous structures with the same frequency as wild-type MHC. Biochemical analysis of one FHC mutant (Arg-249-->Gln) demonstrates that the structures formed by the mutant are solubilized at a lower ionic strength than those formed by wild-type MHC. We conclude that although the FHC mutant MHC is not labile, its assembly properties may be impaired.

Animals↗
PubMed1999

Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells.

The interactions of CYP11B1 (cytochrome P-45011beta), CYP11B2 (cytochrome P-450aldo) and CYP11A1 (cytochrome P-450scc) were investigated by cotransfection of their cDNA into COS-1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS-1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11beta-hydroxylation activity of CYP11B1 and in a decrease of the 18-hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.

Adrenodoxin↗
PubMed2000

The propeptide domain of membrane type 1-matrix metalloproteinase acts as an intramolecular chaperone when expressed in trans with the mature sequence in COS-1 cells.

It has been assumed that cleavage of the N-terminal propeptide domain of membrane type-1 matrix metalloproteinase (MT1-MMP) is required for enzyme function. We recently demonstrated that the propeptide domain of MT1-MMP is not cleaved and actually is required for function of the membrane-bound enzyme in transfected COS-1 cells (Cao, J., Drews, M., Lee, H. M., Conner, C., Bahou, W. F., and Zucker, S. (1998) J. Biol. Chem. 273, 34745-34752). In this report, we have inserted the cDNA encoding the signal and propeptide sequences of MT1-MMP (MT(1-109)) and the cDNA encoding propeptide-deleted mature MT1-MMP (MT delta pro) in expression vectors that were then transfected into matrix metalloproteinase-deficient COS-1 cells. Co-expression of both the mature sequence and the prosequence of MT1-MMP as independent polypeptides (in trans) in COS-1 cells resulted in reconstitution of MT1-MMP function in terms of facilitating (125)I-labeled tissue inhibitor of metalloproteinase 2 binding to transfected cells and subsequent activation of progelatinase A. Transfection of cells with either cDNA alone resulted in non-functional cells. These results are consistent with the propeptide sequence of MT1-MMP functioning as an intramolecular chaperone involved in protein folding and trafficking to the cell surface.

Amino Acid Sequence↗
PubMed2004

Intracellular transport of human lysosomal alpha-mannosidase and alpha-mannosidosis-related mutants.

Human LAMAN (lysosomal a-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease a-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).

Animals↗
PubMed1992

Overexpression of the erythrocyte plasma membrane Ca2+ pump in COS-1 cells.

A full-length cDNA corresponding to the hPMCA4 plasma membrane Ca2+ pump was assembled and expressed in COS-1 cells. The original sequence of hPMCA4 gave a very low expression. The mutation of the initiation translation site of this sequence to the consensus A/G-X-X-AUG-G increased the production of the protein. The Ca2+ pump activity in transfected cells was 1.5-3.5-fold higher than in controls. The Ca(2+)-dependence and the calmodulin stimulation of hPMCA4 expressed in COS-1 cells were comparable with those of the erythrocyte Ca2+ pump. Immunohistochemistry experiments showed that most of the expressed protein remained in intracellular membranes. Possible explanations for this targeting of the pump are discussed.

Animals↗
PubMed2005

MARCO is the major binding receptor for unopsonized particles and bacteria on human alveolar macrophages.

Alveolar macrophages (AMs) avidly bind and ingest inhaled environmental particles and bacteria. To identify the particle binding receptor(s) on human AMs, we used functional screening of anti-human AM hybridomas and isolated a mAb, PLK-1, which inhibits AM binding of unopsonized particles (e.g., TiO2, latex beads; 63 +/- 5 and 67 +/- 4% inhibition, respectively, measured by flow cytometry; n = 11) and unopsonized bacteria ( approximately 84 and 41% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PLK-1, respectively). The PLK-1 Ag was identified as the human class A scavenger receptor (SR) MARCO (macrophage receptor with collagenous structure) by observing specific immunolabeling of COS cells transfected with human MARCO (but not SR-AI/II) cDNA and by immunoprecipitation by PLK-1 of a protein of appropriate molecular mass (approximately 70 kDa) from both normal human bronchoalveolar lavage cells (>90% AMs) and human MARCO-transfected COS cells. PLK-1 also specifically inhibited particle binding by COS cells, only after transfection with human MARCO cDNA. Immunostaining showed specific labeling of AMs within human lung tissue, bronchoalveolar lavage samples, as well as macrophages in other sites (e.g., lymph node and liver). Using COS transfectants with different truncated forms of MARCO, allowed epitope mapping for the PLK-1 Ab to MARCO domain V between amino acid residues 420 and 431. A panel of Abs to various SRs identified expression on AMs, but failed to inhibit TiO2 or S. aureus binding. The data support a dominant role for MARCO in the human AM defense against inhaled particles and pathogens.

Adult↗
PubMed1998

Extrahepatic and intrahepatic replication and expression of hepatitis C virus.

In order to determine the replication sites of hepatitis C virus, the in situ hybridization and immunohistochemical technique using digoxin-labeled 531 bp plus-strand and minus-strand HCVRNA probes were employed to detect HCVRNA in the liver tissues, bone marrow mononuclear cells and peripheral blood mononuclear cells (PBMCs) from the patients with chronic hepatitis C, and in HCV transfected COS cells. The results showed that both plus-strand and minus-strand HCVRNA were detected in 80% of liver tissues (4/5). Plus-strand HCVRNA could be detected in 90% of PBMCs and bone marrow mononuclear cells (18/20), minus-strand HCVRNA in 25% of PBMCs. In HCV transfected COS cells, plus-strand HCVRNA distributed evenly in 20% cellular nuclei and cytoplasms. No minus-strand HCVRNA was detected in the bone marrow mononuclear cells and HCV transfected COS cells. The positive signal appeared in more cells when the liver tissues, PBMCs and marrow mononuclear cells were hybridized by plus-strand probes than when hybridized by minus-strand probes. Our results suggested that the hepatocytic cytoplasms and PBMC cytoplasms were the replication sites of HCV, but the marrow mononuclear cells were not the replication sites of HCV although they were infected by HCV. HCV infection might be accounted for the pathogenesis of chronic hepatitis and relapse of hepatitis C after liver transplantation.

Animals↗