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At least 1,063 records · Page 59Linked to original sources
PubMed1988

Hog cholera virus: identification and characterization of the viral RNA and the virus-specific RNA synthesized in infected swine kidney cells.

Virus-specific RNA in swine kidney cells infected with hog cholera virus and the RNA of gradient-purified virions were identified and characterized. The time curve for virus replication in SK-6 cells was established. The logarithmic phase of virus growth was between 4 and 12 h after virus adsorption. Virus production was maximum at 20 h after virus adsorption. Analysis of cytoplasmic RNA in a neutral agarose gel showed that the time curve for synthesis of a high molecular weight RNA in infected cells closely paralleled that for virus growth. Electrophoresis in an agarose-formaldehyde gel of [3H]uridine-labeled RNA, synthesized with actinomycin-D, showed that this high molecular weight RNA species was approximately 12 kilobases (kb) in length. RNA, extracted from virions purified in a glycerol-tartrate gradient, co-migrated in a neutral agarose gel with the 12 kb RNA species; moreover, it hybridized specifically with a cDNA clone prepared against the intracellular 12 kb RNA. These results confirm the virus-specific nature of the 12 kb RNA. Since no prominent subgenomic virus-specific RNA was identified in infected cells, an RNA species of this size may also act as a messenger RNA.

Animals↗
PubMedDate not supplied

Detection of circulating human immunodeficiency virus type 2 in plasma by reverse transcription polymerase chain reaction.

Genomic RNA was detected using a reverse transcription (RT) nested polymerase chain reaction (PCR) method on plasma from 24 HIV2-infected patients. Results were interpreted based on immune and clinical status and results of plasma and cellular viraemia assays. Amplification of RNA extracted from plasma was positive in 13 of the 24 cases studied (54%). There was a negative correlation between the detection of RNA in plasma and the patients' CD4+ cell counts: all 5 patients with counts below 200 x 10(6)/l were RT-PCR RNA-positive, compared to only 4 of the 12 patients with counts above 500 x 10(6)/l. Cellular viraemia was positive in 12 of the 24 patients, and the results correlated with the CD4+ cell count. HIV2 was isolated from the plasma of 3 of the 24 patients, all of whom had CD4+ cell counts below 200 x 10(6)/l. The small viral load in HIV2-infected patients before the onset of immunodeficiency appeared to be a major limiting factor in the detection of the virus with current tests. The low percentage of RNA-positive plasma samples contrasts with the high rate of positivity in HIV1-infected patients. Differences in viral load and replication between HIV1 and HIV2 correlate with differences in the epidemiology and pathogenicity of the two viruses.

DNA, Viral↗
PubMed1999

Reverse transcription polymerase chain reaction method for the detection of glycopeptide resistance in enterococci.

In this work we have developed reverse transcription polymerase chain reaction (RT-PCR) methods for detecting specific mRNA from enterococci, particularly vanA and vanB genes, responsible for glycopeptide resistance in this genus. mRNA from the two genes was detected immediately after RNA extraction of a midlog phase culture, determined by growth rate analysis. Because of the short half-life associated with many bacterial RNA species (1.5-2 min), time is an important factor in obtaining RNA of good yield and high purity. Our results showed that: (i) the transcription of mRNA related to vanA ligase in enterococci showing Van A phenotype happens only after induction with both vancomycin and teicoplanin; (ii) the transcription of mRNA related to vanB ligase happens only in the presence of vancomycin and (iii) there was no transcription of mRNA in the two strains positive to vanA gene after PCR experiments. RT-PCR methodology can have numerous applications in microbiology for studying gene expression in isolated bacteria and also in nonculturable cells in environmental samples, for studies of mechanisms and/or as an indicator of viability in bacterial communities.

Anti-Bacterial Agents↗
PubMedDate not supplied

Polymerase-chain-reaction-based semi-quantification of hepatitis D viraemia in patients treated with high doses of alpha 2b interferon.

To study the antiviral efficacy of high doses of alpha 2b interferon (alpha 2b-IFN) for chronic hepatitis D treatment, we used polymerase-chain-reaction(PCR)-based semi-quantitative detection of HDV RNA. The semi-quantification method used was based on the appearance of a positive amplification signal as a function of the number of PCR cycles. By amplifying dilutions (10(-1)-10(-8)) of an HDV-positive woodchuck liver RNA, we confirmed that exponential amplification efficacy occurred at between 20 and 30 cycles. Positive signals were observed from dilution 10(-2) (gel electrophoresis after 20 cycles of PCR) to dilution 10(-7) (hybridization after 30 cycles of PCR). To characterize the HDV RNA level in sera of 8 patients treated with alpha 2b-IFN (10 MU/3 times a week) for 1 year, we extracted RNA from serum samples taken every 6 months. All samples were amplified in parallel for 20 and 30 PCR cycles. Analysis of HDV cDNA after ethidium bromide/agarose gel electrophoresis and after molecular hybridization (100 times more sensitive than gel analysis), enabled us to grade the signals observed from negative to positive as 1+, 2+, 3+ and 4+, with all results being positive. Three types of evolution of HDV viraemia were evidenced among the 8 treated patients. HDV replication continued to occur at a high level at the 6th and 12th month in 2 patient sera. For 2 other patients, an HDV RNA decrease or disappearance was evidenced in the serum at the 6th month; however, viral replication recurred at a higher level at the 12th month.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1997

Interleukin-12 p40 m-RNA expression in human kidney allograft biopsies.

Interleukin-12 (IL-12) is a heterodimeric cytokine implicated in the early differentiation of naive T-lymphocytes into the Th1 subset. IL-12 is important for induction of the cellular immune response against viruses, intracellular parasites and neoplasms. Its role in alloresponsiveness has not been fully elucidated. Preliminary data in the literature point toward the prevalence of Th1 lymphocytes in processes of allograft rejection. In attempt to further investigate the expression of this cytokine during episodes of cellular rejection of renal allografts, we searched for IL-12 message in human kidney allograft biopsies using the reverse transcriptase-polymerase chain reaction technique. Twenty-three allograft core biopsies from 19 patients were obtained percutaneously for clinical indications in 18 cases, and as part of an investigational protocol in five cases. A portion of the tissue was used for RNA extraction using the guanidium-thiocyanide phenol-chloroform method. Histology was performed on the remaining core material. Ten mg of total RNA were used for reverse transcription. PCR of the c-DNAs was done for 40 cycles using primers for the p40 subunit of IL-12 and GAPDH which was used as a control. PCR products were photographed after electrophoresis, transferred to a nylon membrane and hybridized with a radiolabelled cloned human IL-12 p40 1 kb c-DNA fragment. Autoradiographies were developed after 20-min exposure. All samples were run in triplicate. IL-12 p40 m-RNA was expressed in all 17 biopsies showing acute cellular rejection as well as in all three biopsies showing focal interstitial fibrosis. No message was found in the presence of normal allograft histology. This is the first in vivo report of IL-12 p40 subunit m-RNA expression during renal allograft rejection in humans. The role of this Th1 cytokine in the alloresponse deserves further investigation.

Adult↗
PubMed1968

Polyribonucleotide synthesis by subfractions of microsomes from rat liver.

1. The microsome fraction of rat liver has been fractionated and the ability of the fractions to incorporate ribonucleotides into polyribonucleotides has been studied. Activity was found in the rough-surfaced vesicle (light) fraction and in the free-ribosome fraction and this latter activity has been examined. 2. The free-ribosome fraction contains ribosome monomers, dimers and trimers together with some higher oligomers and ferritin. In addition to catalysing the incorporation of ribonucleotides into acid-insoluble material it contains diesterase activity. It catalyses the incorporation of UMP from UTP, but not UDP, AMP from ATP and CMP from CTP into polyribonucleotide material, and for UTP the product appears to be a homopolymer not more than eight units long attached to the ends of primer polyribonucleotide strands. 3. The activity could not be removed from the free-ribosome fraction by washing or by isolation in the presence of ethylenediaminetetra-acetic acid. 4. Partially hydrolysed polyuridylic acid but not polyadenylic acid could serve as a primer for the incorporation of UMP, but some activity was always associated with an endogenous primer. 5. Analysis of RNA extracted from the free-ribosome fraction after incubation with [(3)H]UTP showed the presence of 28s, 18s, 5s and transfer RNA types, but no radioactivity was associated with any of these RNA fractions.

Adenine Nucleotides↗
PubMed2000

Specific detection of minus-strand hepatitis C virus RNA by reverse-transcription polymerase chain reaction on PolyA(+)-purified RNA.

A full-length complementary DNA (cDNA) clone of the hepatitis C virus (HCV) genome was used to prepare full-length plus- and minus-strand RNA. The minus-strand RNA, which contains a polyA(+) tract complementary to the polyU tract found in the plus strand (genomic) RNA, but not the plus strand RNA, was captured with a commercial polyA(+)-tract isolation system. After elution, the minus strand was amplified by reverse-transcription polymerase chain reaction (RT-PCR). The combination of this procedure and RT-PCR using rTth resulted in an unprecedented level of discrimination of 10 logs(10). HCV minus-strand RNA isolation was unaffected by the addition of an excess of 10(4) of plus strands or by the addition of cellular RNA, and although the polyA(+) isolation step removed 99. 99% of plus strands, there was no loss of minus-strand signal. Minus-strand RNA was detected in RNA extracted from 4/4 liver samples and 4/8 peripheral blood mononuclear cells (PBMC) samples examined. Because the titer of plus-strand HCV RNA in any sample makes a significant contribution to false, random, and self-priming, removal of the plus strand in this manner results in the most accurate method yet devised to confirm the replication of HCV in a population of cells.

Hepatitis C↗
PubMed1985

Identification of proliferin mRNA and protein in mouse placenta.

Proliferin is a recently described, prolactin-related protein whose mRNA appears in several murine cell lines during active growth. We have surveyed a number of mouse organs or tissues for the presence of mRNAs that hybridize to cloned proliferin cDNA. Of the tissues tested, only the placenta yielded proliferin-related mRNA. This placental RNA is about 1 kilobase in length, increases sharply between days 8 and 10 of pregnancy, and then gradually declines through day 18. It is more abundant in RNA extracted from the fetal, compared to the maternal, part of the placenta. From a cDNA plasmid library prepared from poly(A)+ placental RNA, two types of proliferin-related clones were isolated, differing in intensity of hybridization to proliferin cDNA. By nucleotide sequence analysis, a strongly hybridizing clone was found to be nearly identical to the proliferin cDNA clone isolated from a library prepared from mRNA of a growing mouse fibroblastic cell line. Using an antiserum prepared against a synthetic proliferin fusion protein, we show that proliferin is secreted as a glycoprotein by minced placental tissue and that it differs from mouse placental lactogen. We conclude that proliferin is a placental hormone that is synthesized in certain mouse cell lines during active growth. Its function during pregnancy and during the growth of cultured cells is presently unknown.

Amino Acid Sequence↗
PubMed1979

Replication cycle of Newcastle disease virus in three host cells of different permissiveness.

Various degrees of permissiveness for NDV have been described in different cell lines. In the present work three systems were investigated: bovine kidney (MDBK) cells, chick embryo (CE) cells and mouse L cells producing 50 to 100 p.f.u., 2 to 10 p.f.u. and less than 0.1 p.f.u./cell, respectively. Analysis of the radioactive virus messenger RNAs (vmRNAs) accumulating in actinomycin D-treated cells throughout the infection cycle revealed that, except for the absence of one 18S vmRNA species in L cells, all vmRNA components were formed in the three cell types, although variations occurred in their total and relative amounts from one cell type to another. Kinetic studies of vmRNA synthesis confirmed the absence of one 18S vmRNA species in L cells and also showed that the labelling rate of this vmRNA component is higher in CE cells. Virus proteins synthesized in the infected cells were labelled with 14C-amino acids and analysed by polyacrylamide gel electrophoresis. All the major NDV polypeptides were formed as expected in both CE and MDBK cells but only traces were detected in L cells. In contrast in a cell-free translation system from wheat germ, all of the NDV major proteins were synthesized using RNA extracted from the three infected cell types. Moreover, the total radioactivity incorporated into NDV proteins was twice as great with CE cell RNA and five times as great with L cell RNA than with equivalent quantities of RNA from MDBK cells.

Animals↗
PubMed1974

Isoleucine and valine metabolism in Escherichia coli K-12: detection and measurement of ilv-specific messenger ribonucleic acid.

Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of messenger RNA transcribed from the ilv gene cluster of Escherichia coli K-12. Strains with derepressed levels of the isoleucine and valine biosynthetic enzymes owing to linked or unlinked genetic lesions were found to exhibit ilv messenger RNA levels from 1.5- to 4-fold higher than did their isogenic parents. When grown under conditions that specifically repressed the synthesis of isoleucine- and valine-forming enzymes, most strains exhibited drastically reduced ilv messenger RNA levels. Hybridization performed with the separated strands of ilv DNA showed that all the ilv genes are transcribed from the same strand, the "l strand" of lambdaphi80CI857St68dilv DNA. Sucrose gradient analyses of RNA extracted from cells starved for isoleucine, valine, or leucine resulted in the detection of at least two distinct types of ilv messenger RNA.

Centrifugation, Density Gradient↗
PubMed1975

Characterization of the mRNA of influenza virus.

The kinetics of the appearance of influenza mRNA, the distribution of mRNA between free and membrane-associated polyribosomes, its poly(A) content, and the extent to which the genome was transcribed into mRNA early in infection were determined. Polyribosomes were prepared from influenza virus-infected cells labeled for 30-min periods at various times after infection with [3H]uridine. Most of the 3H-labeled RNA extracted from these polyribosomes sedimented as a heterogeneous 8S to 20S peak in sucrose gradients, and it was largely complementary to virion RNA. By the following criteria, the complementary RNA had properties normally ascribed to mRNA: (i) it labeled rapidly with [3H]uridine; (ii) after glutaraldelyde treatment, it banded with polyribosomes in CsCl density gradients; and (iii) it contained poly(A). In chick cells at 37 C, virus mRNA was first detectable at 45 min postinfection and reached its maximal rate of appearance at 2 to 2.5 h postinfection. The free and membrane-bound polyribosomes of infected cells were separated and were found to contain the same classes of mRNA. There was no absolute segregation of mRNA sequences into either polyribosome class although each probably contained distinct ratios of the different mRNA's. From 45 min postinfection onwards, both membrane-bound and free polysomal poly(A)-containing RNA contained sequences complementary to at least 80% of the genome RNA, whereas poly(A)-minus RNA contained sequences complementary to 90 to 100% of the genome. There was no evidence for the temporal control of transcription of influenza mRNA. At 31 C, when virus development was slowed relative to 37 C,complementary RNA first appeared at 1 h postinfection. At this time, total polysomal RNA contained sequences complementary to the whole genome.

Base Sequence↗
PubMed1988

Detection and preliminary characterization of herpes simplex virus type 1 transcripts in latently infected human trigeminal ganglia.

RNA extracted from human trigeminal ganglia was examined for the presence of herpes simplex virus type 1 (HSV-1) transcripts by Northern hybridization (RNA blot) analysis. By using cloned DNA and single-stranded RNA probes, two abundant colinear HSV-1 transcripts (1.85 and 1.35 kilobases) were detected in ganglia from 9 of 17 individuals. These RNAs overlap the 3' end of the transcript for immediate-early gene ICP0 but are transcribed from the opposite strand; thus, they are antisense relative to the ICP0 mRNA. We also report evidence by in situ hybridization that these latently infected ganglia contain HSV-1 RNA homologous to the BamHI SP region of the genome which is transcribed in the same direction as the other latency transcripts. In Vero cells productively infected with laboratory strain HSV-1 KOS and in cultures infected with different low-passage clinical isolates, only the 1.85-kilobase transcript was detected, and there was variation in the size of this larger latency transcript. These novel transcripts may play a role in maintaining HSV-1 latency in human ganglia.

Animals↗
PubMed2006

Identification of squamous cell carcinoma associated proteins by proteomics and loss of beta tropomyosin expression in esophageal cancer.

AIM: To assess the proteome of normal versus tumor tissue in squamous cell carcinoma of the esophagus (SCCE) in Iranian patients and compare our results with former reports by using proteomics. METHODS: Protein was extracted from normal and tumor tissues. Two dimensional electrophoresis was carried out and spots with differential expression were identified with mass spectrometry. RNA extraction and RT-PCR along with immunodetection were performed. RESULTS: Fourteen proteins were found whose expression levels differed in tumor compared to normal tissues. Mass spectrometric analysis resulted in the identification of beta-tropomyosin (TMbeta), myosin light chain 2 (and its isoform), myosin regulatory light chain 2, peroxyredoxin 2, annexin I and an unknown polypeptide as the down regulated polypeptides in tumor tissue. Heat shock protein 70 (HSP70), TPM4-ALK fusion oncoprotein 2, myosin light polypeptide 6, keratin I, GH16431p and calreticulin were the up-regulated polypeptides found in tumor tissue. Several of these proteins, such as TMbeta, HSP70, annexin I, calreticulin, TPM4-ALK and isoforms of myosins, have been well recognized in tumorigenesis of esophageal or other types of cancers. CONCLUSION: Our study not only supports the involvement of some of the formerly reported proteins in SCCE but also introduces additional proteins found to be lost in SCCE, including TMbeta.

Adult↗
PubMed2002

[Standardization of rabies virus genome amplification for its use in molecular epidemiology studies].

In order to improve the diagnosis and typification of rabies viruses at the Instituto Nacional de Salud National Reference Laboratory for rabies, we standardized techniques for the amplification of a 902 nucleotide DNA fragment, complementary to a selected region of the rabies virus genomic RNA. This region codes for a segment of both the glycoprotein and protein L, and contains the G-L intergenic noncoding region known as Pseudogen Psi. The standardized techniques included: 1) biological amplification of rabies viruses by intracerebral mouse inoculation; 2) total RNA extraction from the brains of infected mice, 3) RT-PCR amplification of a 902 nucleotide DNA fragment complementary to the selected RNA region. The study sample consisted of 30 rabies virus strains isolated from dogs and selected from the virus bank of the Virology Laboratory. Due to their simplicity the methods described have several advantages when compared to methods reported in previous papers. The technology proposed is a precise complement to rabies diagnosis techniques, and can be applied to the identification of phylogenetic relations among rabies isolates, and, therefore, it is also used to identify rabies transmission dynamics and geographical distribution.

Animals↗
PubMed2006

Identification of microRNAs and other small RNAs from the adult newt eye.

PURPOSE: MicroRNAs (miRNAs) are capable of controlling gene expression by targeting complimentary sequences in many mRNAs. Thus, a small number of miRNAs are capable of regulating expression of many different genes. miRNAs have been found in all animals from Drosophila to human and they are highly conserved. This work was undertaken in order to identify such RNAs in the newt eye. METHODS: Cloning of these RNAs was attempted after isolating and fractionating total RNA from the adult newt eye. A gel slice ranging from about 15 to 30 nucleotides in length was cut and the extracted RNA was cloned after several processes involving reverse transcription and linker addition. For expression analysis and verification during the process of lens regeneration we used as a probe mir-124a. RESULTS: Several microRNAs, piRNAs and other small RNAS were identified. Some of them have eye specific gene targets in other species, but for many a function in the eye remains to be attributed. Expression of miR-124a showed an interesting regulation in the lens regeneration-competent dorsal iris. CONCLUSIONS: The cloned miRNAs and other small RNAs are the first to be reported for this animal and might bear significance in regulating processes that are unique to the newt eye, i.e., regeneration of the lens and retina.

Animals↗
PubMed1988

Purification and analysis of RNA from paraffin-embedded tissues.

We have developed methods for the purification and analysis of RNA from formalin-fixed and paraffin-embedded tissue. The methods allow retrospective analysis of gene expression or viral infection. RNA extraction uses prolonged solubilization of tissue with detergent and protease in the presence of high concentrations of a ribonuclease inhibitor. The purified RNA is moderately degraded but its hybridization behavior is essentially unaffected. We were able to quantify specific mRNAs by dot-blot hybridization.

Animals↗
PubMed1994

Entactin gene expression in normal and fibrotic rat liver and in rat liver cells.

BACKGROUND: Entactin, a constituent of basement membranes, is a sulfated glycoprotein with binding sites for laminin, collagen type IV, fibronectin, and cell surfaces. As it is known that excess matrix deposition and sinusoidal basement membrane formation is a central characteristic of liver fibrogenesis, we investigated whether the entactin gene is expressed in normal and in damaged rat liver and which cell types are able to express this gene. In addition, we were interested in the cellular origin and time course of laminin synthesis, a matrix protein closely associated with entactin. EXPERIMENTAL DESIGN: Entactin gene expression was analyzed in normal, acutely and chronically damaged rat livers (CCl4-model) by immunohistochemistry and in situ detection of specific transcripts. Rat fat-storing cells (FSC) (Ito), hepatocytes, Kupffer cells, liver endothelial cells, arterial smooth muscle cells (SMC), and skin fibroblasts (SF) were isolated according to standard techniques. Entactin gene expression in cultured cells was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitates, Northern blot analysis, and immunocytochemistry. RESULTS: In normal liver, entactin was detected in the vessel walls as continuous deposits and in a spot-like fashion along the sinusoids. Entactin was detectable among the cells of the inflammatory infiltrates of acutely damaged liver and in connective tissue septa, in the walls of newly formed vessels and bile ducts of fibrotic liver. Laminin distribution in the vessels was similar, but it was additionally present in the space of Dissé of damaged liver. By in situ hybridization, few entactin-positive cells were found in normal liver sections. Strongly positive cells were scattered over the injured parenchyma of acutely and chronically damaged livers. Northern blot analysis of total RNA extracted from normal and damaged liver tissue showed a distinct increase of entactin specific transcripts during development of fibrosis. Hybridization of total RNA from cultured FSC, hepatocytes, Kupffer cells, endothelial cells, SMC, and SF revealed entactin specific mRNA in FSC, SMC, SF, and endothelial cells; laminin mRNA was found in FSC and SF. Synthesis and secretion of both proteins were found in FSC, SMC and SF. Entactin and laminin gene expression increased in parallel to FSC during time in culture. CONCLUSIONS: Among the main liver cells, FSC show the highest entactin gene expression and might therefore play the dominant role in the synthesis of this protein in normal and fibrotic liver. However, endothelial cells and liver myofibroblasts could also contribute to entactin production.

Animals↗
PubMed1995

Elevated tissue inhibitor of metalloproteinase 1 RNA in colorectal cancer stroma correlates with lymph node and distant metastases.

Tissue inhibitor of metalloproteinase (TIMP) inhibits the proteolytic activity of several matrix metalloproteinases centrally involved in tumor invasion and metastases. The purpose of this study was to determine the origin of TIMP-1 mRNA production in both human colorectal cancer (CRC) and metastatic liver lesions as well as define the relationships between TIMP-1 RNA expression and standard clinicopathological variables of CRC. Total cellular RNA, extracted from 56 CRC and 10 liver metastases, were examined by Northern blot hybridization. The mean/normal mucosa fold increase of TIMP-1 RNA was significantly elevated in both CRC (12.1 +/- 1.7) and liver metastases (10.0 +/- 3.6). No relationship was noted between TIMP-1 expression and tumor size, location nor differentiation. Based on lymph node metastases status, significantly higher TIMP-1 RNA levels were found in CRC with metastases than in those without metastases (15.6 +/- 3.3 versus 7.9 +/- 1.3) (P = 0.04). Similarly, TIMP-1 RNA levels were higher in primary CRC with distant metastases than those without distant metastases (17.6 +/- 4.1 versus 9.3 +/- 1.9) (P = 0. 04). In situ hybridization localized TIMP-1 mRNA predominantly in tumor stroma within spindle fibroblast-like cells rather than in cancer cells themselves. The correlation between the increased TIMP-1 mRNA level and advanced CRC stage noted in this study reflects a possible growth-promoting function for TIMP-1 in human CRC.

Aged↗