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At least 757 records · Page 42Linked to original sources
PubMed1998

Detection of widespread hepatocyte infection in chronic hepatitis C.

The controversial question of the extent of hepatocyte infection in chronic hepatitis C was re-examined in both chimpanzees and humans using a newly modified in situ hybridization (ISH) method for detecting hepatitis C virus (HCV) RNA. The specificity of the methodology for distinguishing positive- and negative-strand synthetic HCV RNA was at least six magnitudes greater than the reverse-transcription polymerase chain reaction (RT-PCR) assay for HCV. The sensitivity of the methodology as determined by cell culture assay was 14 +/- 2 genomic equivalents (gE) of HCV positive strand per cell, which was three magnitudes less sensitive than RT-PCR quantitation of HCV. In contrast to previous studies in both humans and chimpanzees with chronic hepatitis C, a high percentage of hepatocytes positive for both positive- and negative-strand HCV RNA was found in most specimens studied. In humans, the extent of hepatocyte infection varied with histological activity index (HAI). In the two chimpanzees studied, the liver biopsies showed minimal histological disease activity, but high percentages of hepatocytes were HCV-positive by ISH that correlated with hepatocyte ultrastructural changes associated with HCV infection. Hepatocyte infection was confirmed by RNA extraction and RT-PCR techniques for detecting HCV RNA that minimize the false detection of negative strands. In both human and chimpanzee liver biopsies showing minimal HAI, the hepatocyte concentration of HCV was estimated to be very low. These findings suggested the hypothesis that persistent infection in the liver may be caused in part by low-level HCV replication. The theoretical and clinical implications of these findings are discussed.

Adult↗
PubMed2002

Experimental infection of Macaca nemestrina with a Toronto Norwalk-like virus of epidemic viral gastroenteritis.

Norwalk virus (NV) and Norwalk-like viruses (NLVs) are common etiologic agents of viral gastroenteritis. Viral gastroenteritis is a common disease that is highly transmissible, spreading rapidly through families, institutions, and communities. Because methods for in vitro cultivation of Norwalk etiologic agents are not available, information regarding this syndrome has come largely from studies in human volunteers. Sequential passaging of an NLV through an immunoincompetent newborn pigtail macaque (Macaca nemestrina) may allow for the adaptation of a human NLV to a primate host, thus providing an animal model for investigating this disease. A fecal filtrate of human origin containing NLV, Toronto virus P2-A, was obtained from a patient during an epidemic of viral gastroenteritis. The filtrate was administered via nasogastric tube to three newborn pigtailed macaques. Clinical illness, which was characterized by diarrhea, dehydration, and vomiting, occurred in three monkeys. Reverse transcription-polymerase chain reaction (RT-PCR) and oligonucleotide probe analysis of RNA extracted from the stool samples following infection revealed viral RNA in all inoculated monkeys. Infection was also transmitted experimentally by feeding two additional newborn macaques a fecal filtrate prepared from the three previously infected animals. Detection of viral RNA in the stools of animals that received the fecal filtrate indicates that viral replication occurred in association with clinical illness. The susceptibility of Macaca nemestrina to infection with a Norwalk-like agent will facilitate the study of the mechanisms of the pathogenesis of NLV. This system may also have the potential to serve as a vaccine test model for human epidemic viral gastroenteritis.

Animals↗
PubMed1981

Preliminary characterization of the major RNA species from Plasmodium falciparum.

Polyacrylamide gel electrophoresis of the total RNA extracted from cultures of Plasmodium falciparum resolved two major RNA components with estimated molecular weights of 1.3 X 10(6) and 0.72 - 0.74 X 10(6). Oligo(dT)-cellulose column chromatography of the total cellular RNA indicates that more than 90% of the total radiolabeled RNA lacks substantial poly(A) sequences (poly(A)-). Resolution of the poly(A)- fraction on polyacrylamide or agarose gels indicates that the majority of the poly(A)- RNAs are the 1.3 X 10(6) and 0.72 - 0.74 X 10(6) species. This observation was confirmed by two-dimensional oligonucleotide fingerprint analysis of the total cellular RNA as well as the poly(A)- RNA components. Oligonucleotide fingerprint analysis confirmed that the small RNA species is not a breakdown or cleavage product of the larger RNA component. Base ratio analysis of the large and small RNA species indicate that they are typically protozoan type with a low guanine + cytosine (G + C) content. The findings that these two RNA species (i) are the major cellular RNA species, (ii) lack substantial poly(A) sequences, (iii) have estimated molecular weights similar to the ribosomal RNAs obtained from other protozoa, (iv) have a low G + C content (approximately equal to 35 - 37%), and (v) are distinct from one another, indicates that the 1.3 X 10(6) and 0.72 - 0.74 X 10(6) RNA components obtained from P. falciparum cultures are the major ribosomal RNAs.

Animals↗
PubMed1977

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine.

Animals↗
PubMed2000

The detection of prostate cells by the reverse transcription-polymerase chain reaction in the circulation of patients undergoing transurethral resection of the prostate.

OBJECTIVE: To determine whether prostate cells are disseminated into the circulation of patients after transurethral resection of the prostate (TURP), as assessed by a reverse transcription-polymerase chain reaction (RT-PCR) assay for prostate specific antigen (PSA) mRNA. PATIENTS AND METHODS: Fifty-one patients, comprising 34 with benign prostatic hyperplasia (BPH) and 17 with prostate cancer who were undergoing routine TURP, had blood samples taken before and 30 min after surgery. The blood mononuclear cell layer was isolated by density-gradient centrifugation and total RNA extracted. Complementary DNA was synthesized by RT of the RNA. The target PSA sequence was amplified by PCR with specific PSA primers and the product detected on agarose-gel electrophoresis with ethidium bromide staining. RESULTS: Five patients (all with prostate cancer) were positive on PSA RT-PCR before surgery and remained positive after TURP. Of the remaining 46 patients, five (11%; three with BPH and two with cancer) changed from a negative to a positive result after TURP. CONCLUSION: In a proportion of patients TURP causes the dissemination of prostate cells into the circulation which are then detectable by RT-PCR of PSA mRNA.

Aged↗
PubMed1983

Characterization of Hantaan virions, the prototype virus of hemorrhagic fever with renal syndrome.

Hantaan virus, strain 76-118, was propagated to high titer in a clone of Vero cells, and infectious virions were successfully concentrated and purified. Infectivity and virus antigenic activity were closely associated with a virus particle that exhibited a sedimentation rate indistinguishable from a representative member of the Bunyaviridae. Purified virions sedimented to a density of 1.16-1.17 in sucrose and 1.20-1.21 in cesium chloride. Detergent disruption of virions resulted in a nucleocapsid structure (density, 1.18 in sucrose and 1.25 in cesium chloride) and soluble protein antigens. Three separate nucleocapsids were resolved by rate-zonal centrifugation and contained a single but common polypeptide of 50,000 daltons. Electrophoresis of radiolabeled RNA extracted from purified virions yielded a profile of three RNA species with apparent molecular weights of 2.7, 1.2, and 0.6 X 10(6). These data support earlier electron microscopy reports which suggested that Hantaan virus has characteristics similar to some members of the virus family Bunyaviridae.

Animals↗
PubMed1976

A rapid method for the quantitative study of RNA from canine distemper virus infected cells.

Centrifugation through CsCl was used to isolate 32P-labelled RNA in a one-step purification procedure. The method is suitable for quantitative as well as preparative studies and appears to have considerable advantages over conventional methods of RNA extraction. We have used this procedure to investigate the RNA synthesized in Vero cells infected with canine distemper virus (CDV). We show that the combination of CsCl centrifugation and affinity chromatography on poly-U Sepharose provides a rapid method for isolating messenger RNA from virus infected cells.

Cell Line↗
PubMed1983

Early RNA synthesis in Bunyamwera virus-infected cells.

RNA synthesis in Bunyamwera virus-infected cells was analysed either by sedimentation analysis in SDS-containing sucrose gradients or by hybridization procedures involving annealing with viral genome RNA (vRNA) followed by electrophoretic analysis. Using either procedure, none of the virus-specific RNAs from infected cells was found to be polyadenylated when analysed by oligo(dT)-cellulose chromatography. In addition, viral messenger RNA activity was found to be associated only with non-polyadenylated RNA species when assayed in an in vitro translation system. The infected cell RNAs could be partially resolved by sucrose gradient centrifugation, and virus-specific RNAs of each polarity were present in these preparations which indicated that the characteristic amplification of secondary transcription was occurring. In the presence of cycloheximide or puromycin, no detectable primary RNA transcription occurred. The same inhibitors, when used later in the infection cycle, caused a dramatic and almost complete inhibition of secondary RNA transcription. The inhibition of RNA synthesis caused by these drugs appeared to be fully reversible. Thus, these inhibitors of protein synthesis affect both primary and secondary RNA transcription by Bunyamwera virus indicating that this virus employs transcription mechanisms different from those known for other families of negative-stranded viruses. Hybridization of 32P-labelled vRNA from Bunyamwera virus with RNA extracted from virus-infected cells produced four duplex RNA molecules that were resolved by gel electrophoresis. Analysis by hybridization and oligonucleotide mapping showed that the two larger duplexes contained complementary (c)RNAs that were transcribed from the L and M segments of viral RNA while the cRNAs contained in the two smaller duplexes were both transcribed from the S RNA segment. Based on a comparison of their oligonucleotide fingerprints, the two latter cRNAs showed a considerable sequence overlap.

Animals↗
PubMed1987

N-methylisatin-beta-4',4'-diethylthiosemicarbazone, an inhibitor of Moloney leukemia virus protein production: characterization and in vitro translation of viral mRNA.

The mode of inhibition of N-methylisatin-beta-4',4'-diethylthiosemicarbazone (M-IBDET) on Moloney leukemia virus production was studied. Drug treatment of infected cells did not alter the amounts or sizes of the 35S and 22S subgenomic viral RNAs. The translation abilities of poly(A)+ RNA derived from M-IBDET-treated cells was also unaffected, as judged by cell-free translation analysis. Poly(A)+ RNA derived from M-IBDET-treated cells directed translation of equal amounts of viral gag precursors, gPr-80gag and Pr-65gag, as did poly(A)+ RNA prepared from untreated cells. The addition of M-IBDET to a cell-free translation system programmed with either total poly(A)+ RNA extracted from infected cells or hybrid-selected viral RNA inhibited the synthesis of viral protein precursors. An examination of the effect of M-IBDET on polysomes engaged in the translation of viral proteins revealed a fourfold accumulation of polysomal virus-specific RNA in drug-treated cells. These results suggest that the inhibition of Moloney leukemia virus by M-IBDET involves a block in the translation of viral RNA rather than interference with viral RNA transcription.

Gene Products, gag↗
PubMed1973

Relationship of reticuloendotheliosis virus to the avian tumor viruses: nucleic acid and polypeptide composition.

The RNA content and polypeptide composition of reticuloendotheliosis virus (REV) was compared to that of C-type RNA tumor viruses. Two RNA species with approximate sedimentation values of 64S and 4S were observed after sucrose gradient centrifugation of RNA extracted from purified REV. The high-molecular-weight RNA species of REV sedimented slightly faster than that of the Bryan strain of Rous sarcoma virus (RSV). Although these characteristics were consistent with those of other C-type RNA tumor viruses, significant differences were observed when the polypeptide composition of REV was compared with that of RSV possessing envelope determinants of Rous-associated virus RAV-2 and RAV-3. Five polypeptides of which two were glycosylated were resolved by polyacrylamide gel electrophoresis. The major nonglycosylated polypeptide of REV did not comigrate with that of RSV (RAV-2)-RSV(RAV-3). The majority of the group-specific antigen reactivity resides in this major nonglycosylated polypeptide of avian tumor viruses and comigrates when proteins of several avian tumor viruses are subjected to coelectrophoresis. This difference in the migration of the major polypeptide of REV and RSV(RAV-2)-RSV(RAV-3) may explain the absence of avian tumor virus group-specific antigen in REV.

Alpharetrovirus↗
PubMed2005

RNA expression profiling of normal and tumor cells following photodynamic therapy with 5-aminolevulinic acid-induced protoporphyrin IX in vitro.

Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX synthesis as a photosensitizing reagent is an encouraging modality for cancer treatment. Understanding the mechanism of tumor phototoxicity is important to provide a basis for combinatory therapy regimens. A normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with cell line-specific LD50 doses of light after exposure to 5-aminolevulinic acid (100 microg/mL), and harvested for RNA extraction 0, 10, and 30 minutes after irradiation. The RNA was hybridized to the metg001A Affymetrix GeneChip containing 2,800 genes, focusing on cancer-related and growth regulatory targets. Comparing the gene expression profiles between the different samples, 40 genes (e.g., SOD2, LUC7A, CASP8, and DUSP1) were identified as significantly altered in comparison with the control samples, and grouped according to their gene ontology. We selected caspase-8 (CASP8) and dual specificity phosphatase 1 (DUSP1) for further validation of the array findings, and compared their expression with the expression of the immediate early gene FOS by quantitative reverse transcription-PCR. RNA expression of CASP8 stayed unchanged whereas DUSP1 RNA was up-regulated in normal and tumor cells starting 30 minutes after irradiation. In contrast, FOS RNA was found continuously up-regulated over time in all three cell lines. Induction of DUSP1 protein expression was clearly shown after 1 hour using Western blot analysis. Interestingly, no changes of caspase-8 protein expression but activation of catalytic activity was detected only in UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. According to caspase-8, the active caspase 3 fragment was found only in the normal urothelial cell line (UROtsa) 1 hour after photodynamic therapy. Combined data analysis suggests that photodynamic therapy in vitro (LD50) leads to apoptosis in UROtsa and to necrosis in the tumor cell lines, respectively. RNA expression profiling of normal and tumor cell lines following photodynamic therapy with 5-aminolevulinic acid gave insight into the major molecular mechanisms induced by photodynamic therapy.

Aminolevulinic Acid↗
PubMed1990

Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus.

Polyadenylated RNA, extracted from rat hypothalami, cross-hybridized with a RNA probe complementary in sequence to rat PTH (rPTH) messenger RNA (mRNA). Amplification of complementary DNA (cDNA) by the polymerase chain reaction also demonstrated the presence of rPTH mRNA in the rat hypothalamus and parathyroid gland. rPTH mRNA was localized by in situ hybridization in the paraventricular and supraoptic nuclei of the rat hypothalamus. These results demonstrate the expression of the PTH gene in the central nervous system of the rat in areas which suggest roles for PTH in neuroendocrine function.

Animals↗
PubMed1981

Structure and expression of human globin genes introduced into mouse fibroblasts.

The human delta- and beta-globin genes, contained in a recombinant bacteriophage (lambda H beta G1), were introduced into mouse fibroblasts by cotransformation with a plasmid (chi 1) containing the herpes simplex thymidine kinase gene using the calcium phosphate precipitation technique. A molar ratio of lambda H eta G1 to chi 1 DNA of 3:1 was used. Four of the eleven stable transformants obtained contained intact delta- and beta-globin genes as determined by Southern blot analysis. To assess methylation in the segment of human DNA introduced into mouse cells, digestion with Hpa II or Msp I alone or with a second restriction enzyme was performed. The sites examined near the human delta- and beta-globin genes in transformed cells were not methylated. RNA extracted from the transformed cells was analyzed by RNA-cDNA hybridization; no more than 100 copies of human beta-globin mRNA/cell were found. Although hypomethylation of sites surrounding expressed globin genes in erythroid cells has been described, this property is not sufficient to ensure a high level of expression in fibroblasts.

Animals↗
PubMed1993

Biologically active thrombomodulin is synthesized by adherent synovial fluid cells and is elevated in synovial fluid of patients with rheumatoid arthritis.

Thrombomodulin (TM) is a transmembrane glycoprotein that interacts with thrombin, thereby serving as a cofactor in the activation of protein C, a major physiologically relevant natural anticoagulant. Although initially described as a vascular endothelial cell receptor, TM has also been reported to be synthesized by several cells, including megakaryocytes, platelets, monocytes, neutrophils (PMN), mesothelial cells, and synovial lining cells. A prominent feature of rheumatoid arthritis (RA) is infiltration of PMN into the joint space. To determine whether TM might play a role in the inflammatory process, we examined synovial fluid for the presence of TM in 10 patients with RA and five patients with osteoarthritis (OA). We determined that the mean synovial fluid and plasma TM levels in the OA group were 23.5 ng/mL and 24.2 ng/mL, respectively, whereas those with RA had a significantly elevated mean synovial fluid TM level of 136.2 ng/mL as compared with the plasma TM concentration of 43.9 ng/mL (P < .05). Synovial fluid TM levels did not correlate with PMN counts (r = .261). Purified TM from synovial fluid was identical in molecular weight to plasma-derived TM and was biologically functional with respect to protein C cofactor activity. Using direct immunofluorescence, we determined that adherent cultured synovial fluid cells that are not monocytoid in origin express surface and cytoplasmic TM, thereby providing an alternative source of the protein. Biologic activity of the cell-surface TM was confirmed by acceleration of thrombin-dependent protein C activation. Northern analysis of RNA extracted from the cultured cells indicated that TM messenger RNA was present, suggesting local synthesis. Our results indicate that in RA-associated synovial effusions, biologically active TM is increased, the source of which may be from plasma, PMN, and/or synovial lining cells. TM may play a regulatory role either in fibrin deposition in the inflamed joint and/or in the progression of the inflammatory process.

Arthritis, Rheumatoid↗
PubMedDate not supplied

Detection of circulating cancer cells by nested reverse transcription-polymerase chain reaction of cytokeratin-19 (K19)--possible clinical significance in advanced gastric cancer.

Intermediate filament cytokeratin-19 (K19) protein is expressed in normal and malignant gastrointestinal epithelial cells, but not in peripheral blood (PB). Small amount of circulating gastric cancer cells can be detected by a sensitive nested reverse transcription-polymerase chain reaction (RTPCR) with primers specific for K19 mRNA. Thirty-four PB samples obtained from patients with inoperable/metastatic gastric cancer were examined. The mononuclear cell (MNC) fraction was collected by Ficoll centrifugation, and followed by total RNA extraction by acid guanidinium thiocyanate-phenol-chloroform method. RNA from 8 gastric cancer cell lines and the mononuclear cells of 33 healthy adults were used as positive and negative controls, respectively. DNA fragment of 774 bp amplified by the internal primers was found to be a highly reliable marker for K19 mRNA expression. The sensitivity of detection was between 1 and 10 cells/10(6) normal MNCs. The K19 transcripts were detected in 20.6% (7/34; 8-37%, 95% C.I.) of PB samples. None of the other pertinent clinicopathological features, including the disease extent and the histopathologic types of the tumors, were related to the expression of K19 in PB. All 34 patients had been treated by systemic chemotherapy. Among the 17 non-responders to chemotherapy, the survival of the 4 patients with detectable K19 was significantly shorter than that of 13 patients without detectable K19 in their circulating blood (p = 0.014). However, the survival impact of K19 was less significant in the other 17 patients whose tumors had responded to systemic chemotherapy. Of the whole group of patients, the median survival of the 7 and 27 patients with and without detectable K19 in their circulating blood was 1 and 3.5 months, respectively (p = 0.368). We concluded that detecting circulating cancer cells by K19 nested RT-PCR is associated with poor prognosis of gastric cancer, particularly in those patients who are not responsive to systemic chemotherapy.

Adult↗
PubMed1997

Detection of swine vesicular disease virus RNA by reverse transcription-polymerase chain reaction.

Two polymerase chain reaction (PCR) assays are described for the detection of swine vesicular disease virus (SVDV) RNA, a reverse transcription PCR (RT-PCR) and a reverse transcription nested PCR (RT-nPCR). Both the RT-PCR and RT-nPCR were able to detect representative members of each of seven phylogenetically distinct groups of SVDV and gave negative results with a range of porcine enteroviruses and of viruses responsible for vesicular conditions in pigs. When combined with a commercial kit for rapid RNA extraction, the RT-PCR was useful for the detection of SVDV in samples of epithelium and faeces from animals with clinical SVD. The addition of a second amplification step to create a nested PCR (RT-nPCR) increased the sensitivity of the technique for the detection of viral RNA (vRNA) in SVDV infected tissue culture fluid by a factor of approximately 1,000, for 100 TCID50 for the RT-PCR to 0.1 TCID50 for RT-nPCR. When combined with a more elaborate extraction procedure for RNA, the RT-nPCR was considerably more sensitive than virus isolation in tissue culture for detecting SVDV in nasal swabs, tissues, and faeces collected from pigs between 7 days and 176 days after infection with a recent European isolate of SVDV. However, stringent conditions are necessary for carrying out the RT-nPCR to minimise the possibility of contamination.

Animals↗
PubMed1992

Down-regulation of Leydig cell insulin-like growth factor-I gene expression by interleukin-1.

We have reported previously that insulin-like growth factor-I (IGF-I) messenger RNA (mRNA) is expressed in Leydig cells and that IGF-I can enhance androgen production, whereas interleukin-1 (IL-1) is a potent inhibitor of Leydig cell steroidogenesis. Molecular cloning studies have confirmed the existence of at least two species of IL-1: IL-1 alpha and IL-1 beta. Both IL-1 alpha and beta bind to the same receptors and have the same spectrum of biological activities. The purpose of the present study was to elucidate the molecular mechanisms of the interaction between IGF-I and IL-1 both in vivo and in vitro. Adult Sprague-Dawley rats (55-65 days old) were treated with three injections of human recombinant IL-1 beta (1 microgram/rat ip) at 12-h intervals. Rats were killed 2 h after the last injection of IL-1 beta. Purified Leydig cells were isolated and RNA extracted for Northern blot analyses. For in vitro studies, highly purified Leydig cells were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 0.1% fetal calf serum with or without IL-1 beta (1-100 ng/ml) for 24 h. RNA was then extracted from these cells. For time course studies, purified Leydig cells were initially cultured for 24 h. Fresh medium was then added with or without IL-1 beta (10 ng/ml), and the cultures were continued for an additional 2, 4, or 6 h. In vivo administration of IL-1 beta inhibited IGF-I mRNA expression in Leydig cells (a 40% reduction, P less than 0.05). IL-1 beta also suppressed IGF-I mRNA expression in Leydig cells in vitro in a time- and dose-dependent fashion. Inhibitory effects of IL-1 beta (10 ng/ml) could be demonstrated as early as 2 h and reached a nadir at 6 h (a 60% reduction, P less than 0.05). IL-1 beta (100 ng/ml) inhibited IGF-I mRNA expression to about 10% of the controls (P less than 0.01). Moreover, the inhibitory effect of IL-1 beta could be reversed by the addition of IL-1 receptor antagonist. In conclusion, IL-1 beta could directly inhibit the mRNA expression in Leydig cells for IGF-I, an important autocrine modulator of Leydig cell function. This suggests that the effect of IL-1 beta on Leydig cell function is, at least in part, achieved by down-regulation of IGF-I mRNA levels.

Animals↗