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Results for “Reverse Transcriptase Polymerase Chain Reaction”

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At least 379 records · Page 21Linked to original sources
PubMed2006

The significance of aberrant CHFR methylation for clinical response to microtubule inhibitors in gastric cancer.

BACKGROUND: We studied the correlations between CHFR (checkpoint with FHA and RING finger) gene methylation and responses to microtubule inhibitors (MI) in gastric cancer. METHODS: We examined 9 gastric cancer cell lines and 46 gastric cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by methylation-specific polymerase chain reaction (MSP). CHFR mRNA expression was estimated by quantitative reverse transcription-PCR. The MI-induced growth inhibition was assayed by a standard MTT method. RESULTS: CHFR expression was silenced by aberrant promoter methylation in 3 of 9 gastric cancer cell lines. The level of CHFR mRNA expression was closely correlated with IC(50) in the MI-treated cells (R=0.889, P=0.005). In 46 patients with gastric cancers, 24 (52%) presented aberrant CHFR methylation. Among them, 12 patients had received treatment with MI because of advanced-stage tumor or tumor recurrence after surgery. The responders to the MI treatment were 29% in patients with CHFR methylation and 20% in those without the methylation. However, 6 (86%) of 7 patients with methylated CHFR tumor showed some regression or no progression, whereas 4 (80%) of 5 patients with unmethylated CHFR tumor manifested progressive deterioration. CONCLUSIONS: These observations indicated that CHFR methylation may be a clinically useful approach to predict the responsiveness of gastric cancers to treatment with MI.

Adult↗
PubMed2002

Production of immunogenic VP6 protein of bovine group A rotavirus in transgenic potato plants.

We report here the production of transgenic potato plants expressing the major capsid protein VP6 of bovine group A rotavirus (GAR). Transgenic plants under the control of a cauliflower mosaic virus 35S promoter, or a modified promoter linked to the tobacco mosaic virus 5'-untranslated sequence were positive for GAR antigens by ELISA. The expressed protein was consistent in size with VP6 of GAR by Western blot assay. The presence of the VP6 gene and its transcript was detected by PCR and RT-PCR. Adult BALB/c mice were immunized intraperitoneally with concentrated transgenic potato extracts emulsified in Freund's adjuvant. Sera collected after immunization showed the anti-VP6 response in ELISA and Western blot assay. These results suggest that the immunogenic VP6 protein expressed in plants could be useful for the preparation of diagnostic reagents.

Animals↗
PubMed2004

Application of universal primers for identification of Foot-and-mouth disease virus and Swine vesicular disease virus by PCR and PCR-ELISA.

Two approaches for simultaneous identification of both Foot-and-mouth disease virus (FMDV) and Swine vesicular disease virus (SVDV) are described: (1) a single-step reverse transcription-PCR with three primers and (2) a PCR-ELISA assay with two universal primers for genome amplification and two virus-specific probes for identification. These methods are based on the use of 3D gene universal PCR primers, the structure of which was optimized and refined due to the close relationship between the two viruses belonging to different genera of the Picornaviridae family. In procedure (1), a three-primer PCR containing one universal antisense primer and two virus-specific primers was shown to differentiate between FMDV and SVDV in one reaction, due to the different length of the amplified DNA fragments (600 and 340 base pairs, respectively). In procedure (2), the two viruses were identified by PCR-ELISA, i.e. PCR for the 3D gene followed by two parallel hybridizations with FMDV and SVDV-specific probes in microplate wells and ELISA detection. The application of universal primers could halve the number of PCR experiments in both cases, as compared to the usual virus-specific PCR procedures. Also, we investigated the 3D gene structure of several SVDV strains isolated at different times. No essential changes were detected in the regions coding for conserved motifs of the RNA-dependent RNA polymerase recognized by our universal primers. The multi-primer PCR was successfully tested on 38 FMDV and 15 SVDV strains, and the PCR-ELISA on 32 FMDV and 16 SVDV strains including clinical material from disease cases.

Amino Acid Motifs↗
PubMed2004

Library screening for D-amino-acid oxidase gene: application of real-time PCR.

Quantitative real-time PCR shows the quantity in addition to the presence of the target sequence. This property seemed very useful for library screening. Then, real-time PCR was employed to screen for lambda phages carrying D-amino-acid oxidase gene from mouse genomic library. Using stepwise dilution screening combined with real-time PCR, positive phages were isolated in a short time.

Amino Acid Oxidoreductases↗
PubMed2001

Cloning and sequencing of a novel human gene that encodes a putative target protein of Nesh-SH3.

By using a conventional two-hybrid technique with an Src homology 3 (SH3) domain of Nesh as the bait protein, a novel full-length cDNA was isolated and sequenced from a human placenta cDNA library. This cDNA consists of 3023 bp and has a predicted open reading frame that encodes 486 amino acids. It possesses an SH3 binding motif, a nuclear targeting sequence, and no catalytic domain. Overall, it has no similarity to known molecules involved in a signaling cascade. Polymerase Chair reaction-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels localized the gene on human chromosome 3q12 near the marker D3S1271.

Amino Acid Sequence↗
PubMed2000

Developmental profile of Sry transcripts in mouse brain.

Transient activation of the gene Sry in the gonadal ridge during a brief period of embryonic development is believed to function as a key signal for sex determination. However, a number of reports suggest that Sry expression is not as restricted in space and time as one would expect if its role was confined to directing male-specific differentiation in the early gonadal anlage. We have previously reported the occurrence of Sry/SRY transcripts in adult murine and human brain. The present communication is concerned with the study of the ontogenetic time course of Sry transcripts in mouse brain as detected by reverse transcription-polymerase chain reaction (RT-PCR). Particular emphasis was placed on the identification of two different forms of Sry mRNA, which can be linear or circular. To this aim, we used specific RT-PCR strategies to distinguish between both. Sry transcripts were found in male brain tissue of all ontogenetic stages investigated. Circular, presumably untranslatable, transcripts were found in embryonic brains of day 11 through 19. In contrast, postnatal Sry transcripts were linear, and thus translatable, and were found in diencephalon, midbrain, and cortex. The change from one transcript form to the other suggests that expression of the Sry gene in mouse brain is developmentally regulated, presumably by a switch in promoter selection. This supports the notion that Sry expression in brain is biologically significant.

Adult↗
PubMed2000

Swiss Army Survey in Switzerland to determine the prevalence of Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and tick-borne encephalitis virus in ticks.

A total of 6071 Ixodes ricinus ticks were collected on Swiss Army training grounds in five regions of Switzerland. The aim of the survey was to assess the prevalence of ticks infected with the human pathogens Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and the European tick-borne encephalitis virus. TaqMan PCR (PE Biosystems, USA) and TaqMan RT-PCR (PE Biosystems) analyses were performed on DNA and RNA extracted from pools of ten ticks grouped by gender. Here, for the first time, it is shown that ticks may harbor Francisella tularensis in Switzerland, at a rate of 0.12%. Furthermore, 26.54% of the ticks investigated harbored Borrelia burgdorferi sensu lato, 1.18% harbored members of the Ehrlichia phagocytophila genogroup, and 0.32% harbored the European tick-borne encephalitis virus. A new instrumentation was applied in this study to carry out and analyze more than 2300 PCR reactions in only 5 days. Furthermore, the results reveal that people working in outdoor areas, including army personnel on certain training grounds contaminated with ticks containing tick-borne pathogens, are at risk for different tick-borne diseases.

Animals↗
PubMed2003

Innovative tools for detection of plant pathogenic viruses and bacteria.

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.

Bacteria↗
PubMed2004

Suppression subtractive hybridization cDNA libraries to identify differentially expressed genes from contrasting fish habitats.

Suppression subtractive hybridization complementary DNA libraries identified differentially expressed genes in liver tissue of winter flounder collected from the highly impacted Raritan-Hudson estuary versus those from less industrialized estuaries farther south in New Jersey. Distinct transcript profiles emerged in the fish from these different habitats. A total of 251 clones from the forward (upregulated with anthropogenic impact) and reverse (downregulated with anthropogenic impact) subtracted libraries were sequenced. In the upregulated library immune response transcripts, including complement C-3, C-7, factor H, factor Bf/C2, differentially regulated trout protein 1, and the antimicrobial hepcidin, indicated the pollution-impacted fish were under a high viral or bacterial load. Transcripts for cytochrome P450 1A, P450 3A, and glutathione S-transferase, important components of phase I and II metabolism of xenobiotics, were found in the upregulated-with-pollution library. Vitellogenins I and II and egg envelope protein (zp) appeared to be downregulated. A homologue of the tumor suppressor p33(ING1) (down) and hepatocyte growth factor-like protein (up) may indicate liver damage or hepatocellular carcinoma or hepatoma. These expression patterns, confirmed by quantitative polymerase chain reaction, indicate that transcript analysis is a useful method for assessing the health of local habitats and the organisms therein.

Animals↗
PubMed2003

Expression of a bacterial aroA mutant, aroA-M1, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants.

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. We have previously reported a strategy for engineering glyphosate-resistant class I EPSPS based on staggered-PCR technology. Selected mutant enzymes exhibited high K(i)[glyphosate] and low K(m)[PEP] values compared to the parental enzymes from Escherichia coli ( EcaroA) and Salmonella typhimurium ( StaroA). One mutant, aroA-M1, was further engineered with a tobacco chloroplast leader sequence, and then placed in the binary vector pCAMBIA1300 for Agrobacterium-mediated gene transfer to tobacco ( Nicotiana tabacum cv. Xanthi). Transgenic plants with increased resistance to glyphosate were generated.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2005

Identification of differentially expressed genes in leaf and root between wheat hybrid and its parental inbreds using PCR-based cDNA subtraction.

Heterosis was defined as the advantage of hybrid performance over its parents in terms of growth and productivity. Previous studies showed that differential gene expression between hybrids and their parents is responsible for the heterosis; however, information on systematic identification and characterization of the differentially expressed genes are limited. In this study, an interspecific hybrid between common wheat (Triticum aestivum. L., 2n = 6x = 42, AABBDD) line 3338 and spelt (Triticum spelta L. 2n = 6x = 42, AABBDD) line 2463 was found to be highly heterotic in both aerial growth and root related traits, and was then used for expression assay. A modified suppression subtractive hybridization (SSH) was used to generate four subtracted cDNA libraries, and 748 nonreduandant cDNAs were obtained, among which 465 had high sequence similarity to the GenBank entries and represent diverse of functional categories, such as metabolism, cell growth and maintenance, signal transduction, photosynthesis, response to stress, transcription regulation and others. The expression patterns of 68.2% SSH-derived cDNAs were confirmed by reverse Northern blot, and semi-quantitative RT-PCR exhibited the similar results (72.2%). And it was concluded that the genes differentially expressed between hybrids and their parents involved in diverse physiological process pathway, which might be responsible for the observed heterosis.

Blotting, Northern↗
PubMed2004

Adipocyte differentiation of multipotent cells established from human adipose tissue.

In this study multipotent adipose-derived stem cells isolated from human adipose tissue (hMADS cells) were shown to differentiate into adipose cells in serum-free, chemically defined medium. During the differentiation process, hMADS cells exhibited a gene expression pattern similar to that described for rodent clonal preadipocytes and human primary preadipocytes. Differentiated cells displayed the key features of human adipocytes, i.e., expression of specific molecular markers, lipolytic response to agonists of beta-adrenoreceptors (beta2-AR agonist > beta1-AR agonist >> beta3-AR agonist) and to the atrial natriuretic peptide, insulin-stimulated glucose transport, and secretion of leptin and adiponectin. hMADS cells were able to respond to drugs as inhibition of adipocyte differentiation was observed in the presence of prostaglandin F2alpha, tumour necrosis factor-alpha, and nordihydroguaiaretic acid, a natural polyhydroxyphenolic antioxidant. Thus, for the first time, human adipose cells with normal karyotype and indefinite life span have been established. They represent a novel and valuable tool for studies of fat tissue development and metabolism.

Adipocytes↗
PubMed2005

Nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory.

BACKGROUND: Recent advances in nucleic acid-based methods to detect bacteria offer increased sensitivity and specificity over traditional microbiological techniques. The potential benefit of nucleic acid-based testing to the clinical laboratory is reduced time to diagnosis, high throughput, and accurate and reliable results. METHODS: Several PCR and hybridization tests are commercially available for specific organism detection. Furthermore, hundreds of nucleic acid-based bacterial detection tests have been published in the literature and could be adapted for use in the clinical setting. Contamination potential, lack of standardization or validation for some assays, complex interpretation of results, and increased cost are possible limitations of these tests, however, and must be carefully considered before implementing them in the clinical laboratory. CONCLUSIONS: A major area of advancement in nucleic acid-based assay development has been for specific and broad-range detection of bacterial pathogens.

Bacterial Infections↗
PubMed2007

Molecular cloning, sequencing and phylogenetic analysis of inflammatory cytokines of swamp type buffalo contrasting with other bubaline breeds.

The current research concerned in the cloning, sequencing and phylogenetic analysis of inflammatory cytokine (IL-1alpha, IL-1beta, IL-6 and TNF-alpha) genes from swamp buffalo and two bubaline breeds, CB (cross between swamp and riverine type buffalo) and the Bulgarian Murrah buffalo. Multiple sequence comparison showed a high homology between the bubaline breeds, which ranged from 99.3% to 100.0% similarity, whereas from 98.6% to 99.0% compared to cattle. The phylogenetic analysis had confirmed and justified the degree of relationship between these bubaline species and their distinctness to each other by the bootstrap value (%) generated. These findings were discussed with particular attention to the diversity of the inflammatory cytokine proteins within closely related species. The result of this study concluded that a small difference in the cytokine structures might be the reason behind or has a contributory factor on the previous reports about the existence of disease resistance. However, in-depth study is necessary to further qualify these findings.

Amino Acid Sequence↗
PubMed2006

Tumor-associated antigenic pattern in squamous cell carcinomas of the head and neck--analysed by SEREX.

Most immuno-therapeutic approaches are based on tumor-associated antigens and many newly identified proteins have led to trials exploiting their possible therapeutic applicability. So far only limited information on the antigenic profile of head and neck squamous cell carcinomas (HNSCC) exists. Serological analysis of tumor antigens by recombinant cDNA expression cloning (SEREX) was used to identify the immunogenic patterns in our HNSCC patient collective. A cDNA expression library derived from a pharynx HNSCC case was screened with autologous and heterologous sera. Thirty-seven positive clones coding for 17 immunoreactive gene products, which elicited an in vivo tumor response, were found. Results were confirmed and extended by expression analysis using RT-PCR and in situ-hybridisation. The protein-sequence of five clones exists so far only hypothetically. Of all identified proteins only KIAA0530 has previously been associated with a HNSCC related immune response. All other proteins have not yet been described in a context with HNSCC antigenic patterns. Antibodies against a heat shock transcription factor 2 (HSF2) were found in 2 out of 10 sera from HNSCC patients. In summary, using the SEREX technique, we isolated 17 immunogenic antigens in HNSCC's and confirmed the clinical relevance of KIAA0530. Further analysis concerning their feasibility as target structures for an immunotherapy approach is currently conducted.

Antibodies, Neoplasm↗
PubMed2005

Schistosoma japonicum: a method for transformation by electroporation.

Despite of our knowledge of genetic make up of schistosomes, a number of genes have not been characterized largely due to lack of effective transformation protocols. Here we present electroporation as a strategy for effective introduction of plasmids DNA into schistosomula and adults. Using plasmids of pEGFP-C1 as an expression vector, we first verified that the CMV promoter could direct EGFP to express in primary culture cells from Schistosoma japonicum. Subsequently, the plasmids were introduced into schistosomula and adults by electroporation and EGFP expression was demonstrated using molecular and microscopical methods. Our findings indicate that electroporation is an effective method for transformation of S. japonicum.

Animals↗
PubMed2004

Stress-dependent regulation of the gene encoding thioredoxin reductase from the fission yeast.

The unique putative gene for thioredoxin reductase (TrxR) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The determined DNA sequence carries 3125 bp, and encodes the plausible 322 amino acid sequence of TrxR with a molecular mass of 34,618 Da. The S. pombe cells harboring the cloned TrxR gene contain increased TrxR activity, and shows higher survivals on solid media with mercuric chloride or aluminum chloride. The 1526 bp upstream region was fused into promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid. The synthesis of beta-galactosidase from the fusion plasmid pYUTR10 was enhanced by menadione, mercuric chloride, hydrogen peroxide, aluminium chloride and sodium selenite. Menadione significantly enhanced the TrxR mRNA level in the S. pombe cells, which was detected by RT-PCR. Induction of the S. pombe TrxR gene by menadione and mercuric chloride occurs through the mediation of the transcription factor Pap1. These results suggest that the S. pombe TrxR gene is one of the stress response-related genes.

Amino Acid Sequence↗