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At least 955 records · Page 53Linked to original sources
PubMed2005

Toxoplasma gondii Hsp90 is a potential drug target whose expression and subcellular localization are developmentally regulated.

Two replicative forms characterize the asexual cycle of the protozoan parasite Toxoplasma gondii: rapidly growing tachyzoites and slowly dividing encysted bradyzoites. The mechanisms that regulate the transition between these two stages are not clearly understood. However, stress inducers that also activate heat shock protein expression can trigger formation of bradyzoites in vitro. Here, we studied the association of the T.gondii Hsp90 with modulation of parasite differentiation and response to stress stimuli using RH DeltaUPRT parasites and the cystogenic strain ME49 and a clone derivative of that strain, PK. Our results show that Hsp90 transcript and protein levels increase under stress or bradyzoite differentiation conditions. Moreover, fluorescence microscopy studies revealed that Hsp90 is present in the cytosol of tachyzoites and both in the nucleus and cytosol of mature bradyzoites, suggesting a correlation between its subcellular organization and these two developmental stages. To further characterize the role for Hsp90 in bradyzoite differentiation, T.gondii tachyzoite mutants that are defective in differentiation showed the same staining pattern as tachyzoites under differentiation conditions. In addition, geldanamycin, a benzoquinone ansamycin antibiotic capable of binding and disrupting the function of Hsp90, blocked conversion both from the tachyzoite to bradyzoite and the bradyzoite to tachyzoite stage, suggesting an essential role for this protein in the regulation of stage interconversion. These results thus suggest Hsp90 may play a role in stage switch.

Amino Acid Motifs↗
PubMed2004

Structure and dynamics of the nucleocapsid-binding domain of the Sendai virus phosphoprotein in solution.

The RNA-dependent RNA polymerase of the Sendai virus (SeV) consists of the large protein (L) and the phosphoprotein (P). P plays a crucial role in the enzyme by positioning L (which carries the polymerase activity) onto the matrix for transcription and replication formed by the RNA and the nucleoprotein, the N-RNA. P has a modular structure with distinct functional domains: an N-terminal domain involved in binding to N degrees (N that is not yet bound to RNA) and a C-terminal domain that carries the oligomerisation domain, the N-RNA binding domain and the L binding domain and that, combined with L, is active in transcription. Structural data have previously been obtained on the N-terminal domain and on the oligomerisation domain of P, but not yet on its N-RNA binding domain (also-called the X protein). Here we present an NMR and a small angle neutron scattering study of the SeV X protein. We show that this molecule presents two subdomains linked by an 11-residue linker, with the N-subdomain lacking a well-defined conformation. The 3D structure of the C-subdomain consists of three alpha-helices revealing an asymmetric charge distribution that may be important for binding to RNA-bound nucleoprotein. The structure of the entire C-terminal domain of P is modelled from its constituent parts in combination with small angle scattering data on this domain.

Amino Acid Sequence↗
PubMed2006

Single-stranded DNA of Tomato leaf curl virus accumulates in the cytoplasm of phloem cells.

Geminiviruses have been reported to replicate in, and localize to, the nuclei of host plant cells. We have investigated the tissue and intracellular distribution of the monopartite Tomato leaf curl virus (TLCV) by in situ hybridization. Contrary to the current understanding of geminiviral localization, single-stranded (ss) DNA of TLCV accumulated in the cytoplasm. TLCV ssDNA was also found in the nucleus, as was lower levels of replicative form double-stranded (ds) DNA. Under the same conditions, Tomato golden mosaic virus (TGMV) ssDNA and dsDNA were found in nuclei. ssDNA of TLCV, TGMV, and Tomato yellow leaf curl Sardinia virus (TYLCSV) was detected in some xylem vessels under specific hybridization conditions. Tissue specificity of TLCV was partially released by co-infection with TGMV. Our observations suggest that the mechanism of TLCV movement may differ from that of bipartite begomoviruses.

Cell Nucleus↗
PubMed2006

The presence of human papillomavirus 16 in neural structures and vascular endothelial cells.

Human papillomavirus (HPV) is known as a strictly epitheliotropic pathogen. Our results raised the possibility that HPV 16 is present in neural cells and in the vascular endothelium. By in situ hybridization, we have detected HPV 16 E6 ORF sequence in small blood vessels and peripheral nerves adjacent to oral and cervical cancers. The same structures have clearly shown immunohistochemical reactivity for the E6 oncoprotein. These results were verified by PCR applied to E6 and L1 ORFs following microscopic laser dissection of the immunohistochemically positive nerves and vessels. These observations suggest that HPV 16 DNA and protein are present in neurons and endothelial cells in the vicinity of HPV-associated tumors. The HPV 16 genome presumably exists in a non-replicating form in the neurons and constitutively produces high levels of E6 and E7 proteins with an unknown neuropathological outcome.

Base Sequence↗
PubMed2001

Cutaneous lymphoma associated with Epstein-Barr virus infection in 2 patients treated with methotrexate.

Whether patients with rheumatoid arthritis (RA) have an increased risk of developing non-Hodgkin lymphoma is controversial, and opinions differ on the possible role of methotrexate in the occurrence of lymphomas in patients with RA. We report 1 T-cell lymphoma and 1 B-cell lymphoma restricted to the skin associated with Epstein-Barr virus infection that healed completely and spontaneously after discontinuation of methotrexate in a man with RA and a woman with dermatomyositis. Cutaneous infiltrating cells were infected by a replicative form of Epstein-Barr virus. After discontinuation of methotrexate, the cutaneous lesions disappeared completely in 15 days without recurrence. Discontinuation of methotrexate is necessary in patients with RA or dermatomyositis who have a lymphoproliferative disorder, and a follow-up period of several weeks should be observed before specific therapy is initiated.

Aged↗
PubMed2001

Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin.

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD-->GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity.

Animals↗
PubMed1998

Efficient cloning of cDNA from grapevine leafroll-associated virus 4 and demonstration of probe specificity by the viral antibody.

Using a random-PCR method, a cDNA clone (LR4) was constructed from the replicative form dsRNA of grapevine leafroll-associated virus 4 (GLRaV-4). Northern blot analysis showed hybridization of LR4 to dsRNA in an extract of a Thompson Seedless grapevine clone from which GLRaV-4 was isolated originally by Hu et al. (1990). The cDNA clone was sequenced and shown to be specific to GLRaV-4 by reverse-transcription-PCR using GLRaV-4 particles enriched by the virus antibody coupled to magnetic beads. Reverse-transcription-PCR was used successfully to screen different varieties of grapevines for the virus. Western blot analysis of GLRaV-4 extracts from different varieties of infected grapevines revealed two distinct species of capsid protein with estimated Mr of either 35500 or 38000 depending on the variety used. Both proteins reacted with polyclonal as well as monoclonal antibodies.

Antibodies, Viral↗
PubMed1986

Hepatitis B virus DNA in mononuclear blood cells. A frequent event in hepatitis B surface antigen-positive and -negative patients with acute and chronic liver disease.

We have investigated 38 hepatitis B surface antigen (HBsAg)-positive and 34-negative patients with acute and chronic liver disease for the presence of hepatitis B virus (HBV) DNA in peripheral mononuclear blood cells. Among the HBsAg-positive subjects HBV DNA was detected in the mononuclear cells of asymptomatic HBV carriers (2/6), patients with acute hepatitis (8/8), chronic active hepatitis (18/21), and with hepatocellular carcinoma (2/3); the viral DNA sequences were also identified in the mononuclear cells of patients with HBsAg-negative acute hepatitis (2/3), chronic active hepatitis (5/15) and hepatocellular carcinoma (5/16), some of these showing no evidence of HBV by conventional serological markers. By contrast HBV DNA was not detected after resolution of the acute viral infection. For 7 patients different mononuclear cell-enriched subpopulations were assayed and the viral DNA was observed in T lymphocytes (both OKT4+ and OKT8+ enriched subsets) and/or in B enriched lymphocytes; the restriction DNA patterns showed in some patients a genetic organisation of the viral DNA similar to those observed in the liver (including free monomeric and oligomeric HBV DNA and results consistent with integrated viral sequences); however, no HBV DNA replicative forms were detected. These results show that the hepatitis B virus infection of mononuclear blood cells (including lymphoid cells) is a frequent event at all stages of the viral infection which might be related to immunological abnormalities observed in HBV carriers; in addition the mononuclear blood cells analysis may provide an insight to the liver cells status.

B-Lymphocytes↗
PubMed1998

Molecular Biology's Lessons about Toxoplasma Development: Stage-specific Homologs.

Within intermediate hosts (such as humans), the protozoan parasite Toxoplasma gondii has two life cycle stages: a rapidly replicating form called a tachyzoite and a slowly growing, quiescent form called a bradyzoite. Recently, molecular biology studies have shown that tachyzoites and bradyzoites express a number of homologs (ie. evolutionary related genes)expressed exclusively in one or the other stage. Here, Laura Knoll and John Boothroyd describe examples of how these stage-specific homologs were discovered, and speculate about their regulation and functional significance.

Journal Article↗
PubMed1986

Persistent viral infection. The carrier state.

A persistent viral infection is one in which the virus in a replicating or non-replicating form persists in the host beyond the normal recovery and elimination period for that particular viral infection. The clinical significance and mechanisms of persistence, when known, are discussed for the important viral infections of dogs and cats. Particular emphasis is given to feline viral rhinotracheitis, feline calicivirus, canine distemper, and feline leukemia.

Animals↗
PubMed1986

Two-stage incorporation of thymidine triphosphate into mammalian DNA as indicated by chromatography on benzoylated DEAE-cellulose.

Replicating DNA contains single-stranded regions as indicated by the extent of its recovery in the final fraction, following stepwise elution of sheared preparations from benzoylated DEAE-cellulose using 0.3 M sodium chloride, 1.0 M sodium chloride and caffeine solutions, respectively. In preparations of hepatic DNA, isolated up to 60 min after administration of [3H]thymidine to rats subjected earlier to partial hepatectomy, the proportion of radioactivity contained in the caffeine-eluted fraction progressively decreased. This change was attributable to migration of incorporated radioactivity from replicating forms to mature (double-stranded) DNA, the latter being recovered from the column in 1.0 M sodium chloride. In terms of the same chromatograms, the relative size of the 0.3 M sodium chloride-eluted fraction also decreased, this fraction of radioactivity co-eluting from benzoylated DEAE-cellulose with thymidine triphosphate. The precursor--intermediate--product relationship between the three products separated by benzoylated DEAE-cellulose chromatography was confirmed using DNA isolated from cultured mammalian cells. Early eluting radioactivity co-chromatographed with thymidine triphosphate on thin-layer cellulose, whilst the intermediate status of caffeine-eluted radioactivity was confirmed following pulse-chase labelling procedures. Utilizing stepwise chromatography of such DNA on benzoylated DEAE-cellulose, the effect of three inhibitors could thus be described as affecting either an early or late stage of DNA synthesis. Such an approach offers a simple quantitative method of monitoring influences on DNA synthesis.

Animals↗
PubMed1997

Different repair of O6-methylguanine occurring in DNA modified by MMS in vivo or in vitro.

Lack of the adaptive response effect on the level of GC-->AT transitions induced by methyl methanesulfonate (MMS) in E. coli [Sledziewska-Gójska, E. (1993) The level of GC-->AT transitions induced by MMS is not affected by adaptive response of Escherichia coli K12. Mutation Res., 294, 1-8.] can be explained by MMS inactivation of the ada encoded O6-methylguanine-DNA methyltransferase [Takahashi, K.Y., Kawazoe, K., Sakumi, Nakabeppu Y. and M. Sekiguchi (1988) Activation of Ada protein as a transcriptional regulator by direct alkylation with methylating agents, J. Biol. Chem., 263, 13490-13492; Sledziewska-Gójska, E. (1995) Inactivation of O6-methylguanine-DNA methyltransferase in vivo by SN2 alkylating agents, Mutation Res., 336, 61-67]. To evaluate this explanation and clarify the origin of MMS-induced GC-->AT transitions, we compared the repair of DNA treated by MMS in vivo or in vitro. Replication forms of lacZ mutants of E. coli phage M13mp18 were used to analyse the effect of the adaptive response on the frequency of GC-->AT transitions occurring in control and mismatch repair deficient strains. It was shown that DNA lesions, leading to GC-->AT transitions, induced by MMS in vivo are not repaired in adapted E. coli cells. In contrast, induction of the adaptive response causes efficient repair of these DNA lesions induced by MMS in vitro. This repair is consistent with the assumption that GC-->AT transitions induced by MMS are originated by O6-methylguanine and that MMS treatment of the cells during in vivo mutagenesis interfere with the adaptation mediated repair of the lesion. In agreement with this we have shown that treatment of the adapted cultures with 5 mM MMS completely blocks repair of in vitro modified DNA. Increased level of GC-->AT transitions induced by MMS occurs in mutS- strains. These mutations are avoided in adapted mutS- cells, when induced by MMS in vitro. This confirms that mismatch repair system of E. coli recognises mismatches formed in DNA by O6-methylguanine.

DNA Repair↗
PubMedDate not supplied

Interleukin-6 production by human hepatoma lines is related to a low degree of cell differentiation.

In this study, we showed by Northern blot analysis and bioassays that scarcely differentiated human hepatoma cell line HA22T/VGH constitutively produces interleukin-6 (IL6). This cytokine was produced neither by moderately differentiated Li7A nor by well-differentiated HepG2 human hepatoma cell lines. The finding that transcripts for IL6 were not present in 2.2.15 cells, a HepG2-derived clone transfected with the intracellular replicative form of hepatitis B virus (HBV) DNA, suggests that production of this cytokine is not affected by HBV-encoded gene products, but is more likely related to the dedifferentiated state of hepatoma cells.

Biological Assay↗
PubMed1997

Molecular analysis of a filamentous phage (fsl) of Vibrio cholerae O139.

A filamentous bacteriophage from Vibrio cholerae O139 strain A1-4450 was isolated (fsl). The phage fsl had a ssDNA genome and dsDNA as a replicative form (RF) in lysogenic host cell. The DNA sequence of fsl RF was determined. It consisted of 6340 bp and had a G + C content of 43.5%. Fifteen possible ORFs were found in fsl. One of them, ORF384, was estimated to encode 384 amino acid residues (44.6 kDa) and had homologous regions with the zot gene of V. cholerae and gene I of the coliphage group. ORF104, located upstream of ORF384, was homologous to gene 93 protein of Pf3 (filamentous phage of Pseudomonas sp.) corresponding to gene VI of coliphage. Other than ORF384 and ORF104, the ORF81, ORF44, ORF29, and ORF193 were speculated to correspond to gene V, gene VII, gene IX, and gene III, respectively, in the order as reported on f1 phage.

Amino Acid Sequence↗
PubMed1997

Viral proteins of bovine papillomavirus type 4 during the development of alimentary canal tumours.

In cattle infection of the upper alimentary canal mucosa by bovine papillomavirus type 4 (BPV-4) results in the development of papillomas which can progress to cancer in animals fed on bracken fern. This paper describes a study of the cellular and subcellular distribution of a number of different BPV-4 products in experimentally-induced BPV-4 tumours. E8 and E4 proteins were detected solely as cytoplasmic antigens in the undifferentiated and differentiated layers of the papilloma, respectively; L2 was detected solely as a nuclear antigen in the differentiated layers, whereas E7 was present in either the nucleus or the cytoplasm depending on the differentiation stage of the keratinocyte. Replicative forms of viral DNA were detected from the spinous to the squamous layers. Viral antigens were not detected during papilloma regression or in carcinomas. E8 was most prominent in early developmental stages, while E4 and L2 were most abundant in mature papillomas. E7 was present in large amounts in both early and mature stages, declining at later stages. These results suggest a temporal and spatial requirement for the expression and function of the viral proteins.

Animals↗
PubMed2004

Occult hepatitis B virus infection does not affect liver histology or response to therapy with interferon alpha and ribavirin in intravenous drug users with chronic hepatitis C.

BACKGROUND: the frequency and the impact of occult HBV infection in patients with chronic hepatitis C infection is still a matter of some controversy. OBJECTIVES: our aim was to evaluate the prevalence of occult HBV infection and assess its impact on liver biochemistry, HCV viral titre, liver histology and on outcome of therapy in patients with chronic hepatitis C. STUDY DESIGN: paired liver biopsies and serum samples were collected from 51 patients (84% IVDUS) with HBsAg negative chronic hepatitis C, and tested for HBV-DNA with nested PCR. Liver biopsies were further studied histologically, with morphometric analyses and immunostaining techniques. Twenty-five were treated with alpha Interferon and ribavirin and followed for at least 18 months. RESULTS: HBV DNA was detected in 29.4% of liver tissue specimens and in only one (1.9%) serum sample. Three liver specimens were positive for surface gene, nine for core gene, three for both and none for the X gene. No significant difference in mean transaminase values, HCV viral titre, HCV genotype, or grading and staging and morphometric analysis was observed in patients with or without HBV DNA. Moreover, all 51 liver specimens were negative for both HBsAg and HBcAg. Sustained response to combination therapy was achieved in 40% of patients with and in 53% of patients without HBV DNA in the liver specimens (P=NS). CONCLUSIONS: HBV DNA is frequently found in the liver of patients with chronic hepatitis C. However, the lack of any significant impact on HCV viral titre, liver enzymes, histological parameters and response to therapy, suggests that in most cases HBV DNA detected in the liver by PCR may be either an integrated or low level replicative form.

Adult↗
PubMed1991

Differences in secondary structure between packaged and unpackaged single-stranded DNA of bacteriophage phi X174 determined by Raman spectroscopy: a model for phi X174 DNA packaging.

The single-stranded packaged genome (ssDNA) of bacteriophage phi X174 is shown by Raman spectroscopy to lack both the ordered phosphodiester backbone and base stacking, which are demonstrated for unpackaged, protein-free ssDNA. In solutions of moderate ionic strength, unpackaged ssDNA contains 36 +/- 7% of deoxyribosyl phosphate groups with conventional B-type backbone geometry [i.e., gauche- and trans orientations, respectively, for the 5'O-P (alpha) and 3'O-P (zeta) torsions], indicative of hairpin formation and intramolecular base pairing. Additionally, the bases of unpackaged ssDNA are extensively stacked. Estimates from Raman band hypochromic effects indicate that unpackaged ssDNA contains approximately 70% of the maximal base stacking exhibited in the linear, double-stranded, replicative form III of phi X174 DNA. Conversely, for the packaged phi X174 genome, ordered (B-type) phosphodiester groups are not present, and only 40% of the base stacking in RFIII DNA is observed. These results are interpreted as evidence that the substantial hairpin-forming potential of ssDNA is eliminated by specific and extensive ssDNA-protein interactions within the phi X174 virion. Comparison of the present results with studies of other packaged single-stranded nucleic acids suggests that proteins of the capsid shell (gpF + gpG + gpH) do not fully account for the conformational constraints imposed on ssDNA of phi X174. Accordingly, we propose a model for ssDNA packaging in which the small basic gpJ protein, which is packaged along with the genome, is involved stoichiometrically in binding to the ssDNA (approximately 90 nucleotides per subunit). The proposed gpJ-DNA interactions could prevent helical hairpin formation, restrict base stacking, and disfavor fortuitous base pairing within the capsid. The present analysis is based upon use of model nucleic acids of known conformation for calibration of the Raman intensity in the region 810-860 cm-1 in terms of specific secondary structures. The calibration curve allows quantitative determination of the percentage of ssDNA nucleotides for which the 5'O-P-O3' group is configured (g-,t) as in the B-form of DNA. The method proposed here is analogous to that employed by Thomas and Hartman (1973) for ssRNA and should be applicable to single-stranded DNA and to partially denatured forms of double- and multiple-stranded DNAs.

Bacteriophage phi X 174↗