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At least 541 records · Page 30Linked to original sources
PubMed2003

[Application of one step RT-PCR assay for detection of flavivirus RNA in mosquitoes].

The conditions of one step RT-PCR method for detection of virus RNA in field-collected mosquitoes, and preservation period of infected mosquitoes for one step RT-PCR were examined. We compared several virus RNA extraction methods with artificially contaminated mosquito pools with dengue virus (DV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) with a known amount of plaque forming unit (PFU) to establish the condition of one step RT-PCR. In this study, most effective RNA extraction method was ISOGEN-LS extraction combined with supernatant of centrifuged mosquito homogenates. Detection limit of one step RT-PCR using flavivirus universal primer in ten mosquitoes/tube (pool) was 10 PFU of DV, JEV and YFV, 1 PFU of each viruses using species-specific primer respectively, in one hundred mosquitoes/tube, 100 PFU/tube using universal primer pairs, 10 PFU/tube using species-specific primer pairs respectively. Dengue virus infected single mosquito was mixed with 99 un-infected mosquitoes, and tested by one step RT-PCR. We could detect single infected mosquito in pools containing 99 un-infected mosquitoes. Aedes aegypti and Aedes albopictus were inoculated intrathoracically with a mouse-adapted strain of dengue-1 virus and were kept up to 30 days at different temperature. Then examined by one step RT-PCR to determine the appropriate mosquito handling method and the condition of transportation. Positive result was obtained up to 30 days after the mosquito died naturally. These results suggested that we could detect flavivirus RNA tested not only from live mosquitoes but also dead mosquitoes as well, and could apply one step RT-PCR as a rapid, specific, and highly sensitive tool for flavivirus surveillance.

Animals↗
PubMed1990

Modulation of levels of messenger RNA for tissue-type plasminogen activator in rat Sertoli cells, and levels of messenger RNA for plasminogen activator inhibitor in testis peritubular cells.

Messenger RNA for tissue-type plasminogen activator has been detected in RNA extracts from rat Sertoli cells in culture. Relative levels are increased in Sertoli cells stimulated by follicle-stimulating hormone or by dibutyryl cyclic AMP (dbcAMP) and decreased in cells maintained in the presence of transforming growth factor beta, type 1 (TGF-beta 1). Messenger RNA for plasminogen activator inhibitor, type 1 (PAI-1) has been detected in RNA extracts from rat peritubular myoid cells. Relative levels are increased in peritubular cells stimulated by TGF-beta 1, and decreased by the presence of dbcAMP in the medium. Data are interpreted to indicate that net protease activities in the seminiferous tubule are regulated at transcriptional levels by endocrine and paracrine agents.

Animals↗
PubMed1992

Early expression of cytokine mRNA in mice infected with Listeria monocytogenes.

Protective immunity first becomes evident at 3 to 4 days after inoculation of mice with a sublethal dose of Listeria monocytogenes. Recent evidence suggests that production of gamma interferon (IFN-gamma) occurs earlier (within the first 24 h of infection). The purpose of this study was to define better the sequence of cytokine mRNA expression during the early stages of L. monocytogenes infection. Cytokine mRNA expression was detected by polymerase chain reaction-assisted amplification of RNA extracted from the spleen cells of individual mice euthanized at 0.5 to 120 h after L. monocytogenes challenge. By using this method, mRNAs for tumor necrosis factor alpha, interleukin-1 alpha (IL-1 alpha), IL-2, IL-4, IL-5, and IFN-gamma were detected in RNA from the spleen cells of uninfected mice. The intensity of the bands for IFN-gamma, however, was increased greatly at 16 h after intravenous injection of 5 x 10(4) CFU (nearly 1 50% lethal dose) of L. monocytogenes. IL-6 and granulocyte-macrophage colony-stimulating factor mRNAs were not detected in spleen cell RNA from uninfected mice but were induced within 30 and 60 min, respectively, after inoculation with L. monocytogenes. Increased amounts of mRNAs for IFN-gamma, IL-6, and granulocyte-macrophage colony-stimulating factor were detected after injection of viable, but not killed, L. monocytogenes. IL-3 mRNA was not detected at any time in RNA extracted from the spleen cells of uninfected or L. monocytogenes infected mice. These results suggest that infection with L. monocytogenes elicits a detectable cytokine mRNA response within the first few hours of infection.

Animals↗
PubMed1995

Search for persistent infection with poliovirus or other enteroviruses in amyotrophic lateral sclerosis-motor neurone disease.

A longstanding hypothesis proposes that amyotrophic lateral sclerosis-motor neurone disease (ALS-MND) is a late consequence of subclinical poliovirus (PV) infection. In this study, RNA extracts of CNS tissue from 28 patients with ALS-MND and 7 controls were assayed by nested polymerase chain reaction (PCR) using primers to the 5'-untranslated region (UTR) of the enterovirus (EV) genome which is highly conserved between EVs including PV, echovirus and coxsackie viruses. The integrity of RNA extracted from either archival paraffin-embedded or frozen CNS tissue was assessed by detection of constitutive Ableson tyrosine kinase (ABL) mRNA by PCR. Of 63 tissue samples assayed, 81% (51/63) were ABL-positive corresponding to 78% (22/28) of the ALS-MND cases and all controls. None of the 27 ALS-MND cases (i.e. 21 ABL+ and 6 ABL-) in which paraffin-embedded tissue was used nor any of the age and sex matched controls were positive for specific PV/EV RNA. Moreover, CNS tissue from 14 different locations obtained from one patient < 2 hrs after death and immediately frozen, showed no evidence of PV/EV at any site by PCR. Disease duration, degree of tissue autolysis and duration of tissue storage were all excluded as factors which may predispose to negative results. The sensitivity of the PV PCR was determined to be 40-400 copies (12.5 - 125 ag) of synthetic EV RNA transcripts in 1 microgram of cellular RNA and the assay was shown to detect all types of PV and and other EVs tested. Thus it seems unlikely that a persistent PV or related EV infection is implicated in ALS-MND unless there has been alteration in the 5'-UTR of the virus genome.

Adult↗
PubMed1993

Lymphokine expression in granulomas of Schistosoma mansoni-infected mice.

Lymphokine mRNA for IL-2, IL-4, IL-5, IL-10, and IFN-gamma was identified in total RNA, extracted from granuloma and spleen cells of Schistosoma mansoni-infected mice. The specific mRNA in these preparations was identified after reverse transcription and polymerase chain reaction amplification. In some experiments, spleen cells were incubated in the presence of soluble egg antigens or mitogen before RNA extraction. Also, the secretion of these lymphokines from cells maintained in vitro was measured by ELISA or bioassay. The spleen cells from either uninfected or infected mice constitutively released no detectable cytokines and expressed only IL-10 mRNA prominently. Mitogen induced spleen cells from either source to express IL-2 and IFN-gamma mRNAs and their corresponding proteins. Also, spleen cells from noninfected mice stimulated with mitogen expressed IL-4 mRNA, but not IL-5 mRNA, and no detectable protein products. Yet, IL-5 was inducible in spleen cells from infected animals. Soluble egg antigens induced spleen cells from infected mice to prominently express a variety of cytokine genes and produced large amounts of cytokine proteins, but spleen cells from uninfected mice did not respond. Unlike the spleen, the granulomas constitutively expressed mRNAs for all the cytokines examined. Yet, there was limited expression of IL-2 mRNA as compared to that of other cytokines. Except for IL-5, unfractionated granuloma cells constitutively released cytokines at low or undetectable levels. Soluble egg antigens or mitogen enhanced the production of all cytokines to different extents. These observations indicate that schistosome granulomas and spleens express both Th1 and Th2 cytokines. The various lymphokines display complex differences in both lymphokine mRNA and product expression. These results suggest that the production of granuloma lymphokines is governed selectively, possibly by a variety of undetermined regulatory pathways.

Animals↗
PubMed1998

Reverse transcription-PCR analysis of the regulation of the manganese peroxidase gene family.

Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus Phanerochaete chrysosporium is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequenced P. chrysosporium mnp genes, i.e., mnp1, mnp2, and mnp3. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal level in nitrogen-sufficient cultures, with or without Mn, and in nitrogen-limited cultures lacking Mn. However, in 5-day-old, nitrogen-limited, stationary cultures supplemented with 180 microM Mn, the levels of the mnp1 and mnp2 transcripts increased approximately 100- and 1,700-fold, respectively, over basal levels. In contrast, under these conditions, the level of the mnp3 transcript did not increase significantly over the basal level. Quantitative RT-PCR of total RNA extracted from nitrogen-deficient, Mn-supplemented cultures on days 2 through 7 demonstrates that whereas the mnp1 transcript was present at relatively low levels on days 3 through 7, the mnp2 transcript level peaked on day 5 and the mnp3 transcript level peaked on day 3. Comparison of total RNA extracted on day 5 from nitrogen-deficient, Mn-supplemented stationary and agitated cultures indicates that in stationary cultures, mnp2 was the major expressed mnp gene, whereas in large agitated cultures, mnp1 was the major expressed mnp gene.

Basidiomycota↗
PubMed1995

Rapid and sensitive detection of the bovine viral diarrhea virus genome in semen.

A reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed for the detection of bovine viral diarrhea virus (BVDV) in cell culture supernatant and in bovine semen. Several sets of primers, PCR conditions and extraction methods were examined to optimize the procedure. A set of primers designed to amplify a highly conserved portion of the p80 gene from BVDV (corresponding to NADL strain sequence from bp 6668 to 7107), was demonstrated to be the most effective. These oligonucleotide primers consistently amplify a 440-bp fragment from several non-cytopathic and cytopathic biotypes of BVDV. The viral origin of the PCR products was assessed by sequencing. The introduction of a Sephacryl S-400 chromatography step to remove seminal inhibitors prior to RNA extraction permitted RT-PCR detection of BVDV in raw and extended semen samples. A maximum sensitivity of 0.4 TCID50 was achieved with this method using RNA extracted from tissue supernatants. This RT-PCR assay may be a useful tool for the detection of BVDV in semen of persistently infected bulls.

Animals↗
PubMed1981

The measles virus-specific protein synthesis of persistently and lytically infected cells studied in vivo and in vitro.

The virus-specific protein synthesis of the hamster brain cells persistently infected with a wild-type measles virus (M-MB/MVB) was investigated in vivo and in vitro and compared to the protein synthesis of the Vero-cells lytically infected with the wild-type measles virus and the SSPE-measles virus strain LEC. The virus-specific protein synthesis in the M-HB/MVB-cells in vivo was weak. Only two virus-specific polypeptides, corresponding to polypeptide G and nucleocapsid polypeptide NP, were detected. On the other hand, the in vitro translation products, coded by the RNA extracted from the actinomycin D-treated M-HB/MVB-cells, were similar compared to those products coded by the RNAs from the lytically infected Vero-cells. The polypeptides with MWs of 75 000, 61 000, 40 000, 37 000 and 18 000 were coded with all three RNA-extracts. So the similar mRNAs were induced in the actinomycin D-treated M-HB/MVB-cells, but their translation in vivo was limited. In addition to those polypeptides mentioned above the polypeptide with a MW of 42 000 was detected among the in vitro translation products of the M-HB/MVB-cells.

Animals↗
PubMed2001

Cytokine profiles in paraffin-embedded biopsy samples of lepromatous leprosy patients: semi-quantitative measure of cytokine mRNA using RT-PCR.

A reproducible technique for fixation of tissue, RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis from paraffin-embedded leprosy biopsies, has been developed and used to study the mRNA profiles. This approach is valuable in retrospective analysis of gene expression, and the handling of infectious biopsy material is also minimized. Among the methods of RNA extraction compared, the most efficient method was found to be incubation of the tissue sections in digestion buffer followed by extraction with Trizol. The experimental conditions were optimized for first strand cDNA synthesis and PCR, and used to measure the quantity of cytokine transcripts in biopsy materials. Interleukin-10 (IL-10) mRNA was detectable in all cases examined, which correlates well with other earlier reports using frozen tissues. However, IL-5 transcripts were present in 60% of the biopsies, unlike the earlier reports which showed IL-5 mRNA in all LL cases. Transforming growth factor-beta (TGF-beta) mRNA was detected in 80% of the biopsies, and this confirmed earlier immuno-cytochemical data which showed TGF-beta protein in all cases. Tumor necrosis factor-alpha mRNA was found in about 60% of the biopsies; whereas interferon gamma mRNA was detected in 30% of the LL cases. In conclusion, the results obtained in our study confirm and extend earlier observations which examined cytokines in peripheral blood cells and dermal lesions of leprosy. The simplicity of this method would allow the examination of a large number of samples across the spectrum of leprosy.

Biopsy↗
PubMed1996

Characterization of a new high-temperature-induced 66-kDa heat-shock protein, antigenically related to heat-shock protein 72.

M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major "classic" heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45 degrees C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)+ RNA extracted from cells treated at 45 degrees C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)+ RNA extracted from cells heated at 42 degrees C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro. hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer.

Animals↗
PubMed2002

Cloning and expression of a type IX-like collagen in tissues of the ascidian Ciona intestinalis.

Collagens are highly preserved proteins in invertebrates and vertebrates. To identify the collagens in urochordates, the total RNA extracted from the pharynx of the ascidian Ciona intestinalis was hybridized with a heterologous probe specific for the echinoderm Paracentrotus lividus fibrillar type I-like larval collagen. Using this probe, two main bands (i.e. 6 and 2.8 kb mRNA) were observed on Northern blot hybridization. The cDNA library prepared from poly(A)+RNA extracted from pharyngeal tissue was screened and a cDNA that specifies a type IX-like collagen was identified. This molecule presents a conceptual open reading frame for a protein containing 734 amino acids. In particular, we showed a 1 alpha chain type IX-like collagen characterized by three short triple-helical domains interspersed with four non-triple-helical sequences, with structural features of fibril-associated collagens with interrupted triple-helices (FACIT) collagens. Northern blot hybridizations indicate a 2.8 kb transcript size. Sequence comparison indicated homology (47.64%, 48.95%) between the type IX-like collagen of C. intestinalis and mouse and human type IX collagen. In situ hybridization of tunic and pharynx tissues shows the presence of transcripts in connective tissue cells.

Amino Acid Sequence↗
PubMed1980

Expression of viral RNA in Friend virus-induced erythroleukemia cells.

The regulation of expression of the Friend virus complex (FV) was studied in clones of H-2 congenic erythroleukemia cell lines. Molecular probes specific for each of the two FV components were generated and hybridized to RNA extracted from clones differing with respect to virus production or viral envelope antigen expression. Sequences representing the defective component of FV continued to be transcribed in cells regardless of virus production. The presence of helper virus-specific sequences, however, was shown to be directly related to virus expression. Molecular hybridization with cytoplasmic and nuclear RNA extracted from the H-2 congenic clones representing various stages of viral expression demonstrated a direct correlation between the level of transcription of FMuLV-specific sequences and the degree of virus expression. These data suggest that regulation of FV expression is primarily related to the cessation of helper virus transcription in the cell lines studied. In this paper we provide further evidence that the defective viral components is responsible for the rapid malignancy associated with Friend disease.

Animals↗
PubMed1990

Characterization of extracellular matrix proteoglycan transcripts expressed by vascular smooth muscle cells.

Northern blot analysis using intraspecies and cross-species cDNA probes encoding a variety of proteoglycan (PG) core proteins was employed to examine in vitro and in vivo vascular smooth muscle cell (VSMC) extracellular matrix (ECM) PG gene expression. Similar studies were performed using a cDNA probe encoding the rat chondrosarcoma link protein. VSMCs maintained in vitro as well as in vivo expressed a 1.8 kb transcript homologous to a cDNA encoding the bovine bone small PGII. Densitometric analysis revealed an 11-fold increase in the in vivo level of this transcript when compared to an equivalent amount of RNA extracted from rat VSMCs maintained in vitro. Rat aorta medial tissue contained approximately a 10-fold higher level of the bovine bone PGII homologous transcript when compared to rat chondrosarcoma tissue. A 2.9 kb transcript homologous to a cDNA encoding the human bone small PGI (biglycan) was detected in poly(A)+ RNA isolated from rat VSMCs maintained in vitro as well as within rat thoracic aorta medial tissue. Densitometric analysis revealed a 0.55-fold increase in the in vitro level of the PGI transcript when compared to in vivo levels. Rat chondrosarcoma expressed approximately a 3-fold higher level of a human bone PGI homologous transcript when compared to rat aorta medial tissue. A 7.3 kb and an 8.4 kb transcript homologous to a cDNA encoding the rat chondrosarcoma large hyaluronic acid (HA) binding chondroitin-sulfate (CS) PG core protein was detected in poly(A)+ RNA extracted from rat VSMCs maintained in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Up-regulation of type I procollagen C-proteinase enhancer protein messenger RNA in rats with CCl4-induced liver fibrosis.

Using a polyclonal antibody raised against a liver stellate cell (LSC) line derived from a rat CCl4-cirrhotic liver, we isolated 14 clones from a complementary DNA library prepared with total RNA extracted from the same cell line, with nucleotide sequences homologous to that of the type I procollagen C-proteinase enhancer protein (PCPE) gene. The longest PCPE insert of 1,530 base pairs contained an open reading frame coding for 468 amino acids. PCPE cDNA recognized by Northern blot a 1.7-kilobase messenger RNA (mRNA) in total RNA extracted from freshly isolated and early passaged LSC, LSC lines derived from normal (NFSC) and cirrhotic (CFSC) rat livers, and various LSC clones derived from CFSC. The expression of PCPE mRNA was increased threefold in CFSC compared with NFSC. PCPE mRNA was not detected in total rat liver, freshly isolated hepatocytes, or endothelial or Kupffer cells. However, the expression of PCPE mRNA was induced in fibrotic livers of rats treated with CCl4. PCPE mRNA expression in LSC was up-regulated by transforming growth factor beta1 (TGF-beta1) and down-regulated by tumor necrosis factor alpha (TNF-alpha), similar to the changes in alpha1 (1) procollagen mRNA induced by these cytokines. PCPE was not detectable in liver biomatrix proteins obtained from normal liver. However, PCPE was increased in liver biomatrix proteins from cirrhotic livers and was proportional to the amount of collagen. These data suggest that PCPE may play an important role in the processing of type I collagen during liver fibrogenesis, and that TGF-beta1 and TNF-alpha regulate its expression.

Amino Acid Sequence↗
PubMed1991

Specific transcription of an Acanthamoeba castellanii 5S RNA gene in homologous nuclear extracts.

An RNA polymerase III in vitro transcription system has been developed from the protist Acanthamoeba castellanii. The system is dependent on a cloned 5S RNA gene and utilizes a nuclear extract which contains all the necessary protein components. The system is assembled from completely homologous components. Primer extension and RNA sequencing analysis confirm that the in vitro 5S RNA transcript is identical to the 5S RNA isolated from cells. The transcription complex forms unusually rapidly on the 5S RNA gene and is stable to challenge by excess competitor templates. Several 5' deletion mutants were constructed and indicate that the region upstream of -33 is dispensable. Deletion to +16 show the region between -33 and +16 to be required for transcription, a region outside the canonical internal control region.

Acanthamoeba↗