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At least 1,153 records · Page 64Linked to original sources
PubMed2006

Identification of gene networks modulated by activin in LbetaT2 cells using DNA microarray analysis.

Activins, members of the TGFbeta family of proteins, are widely expressed in a variety of tissues. First identified based on their ability to regulate biosynthesis and secretion of follicle-stimulating hormone (FSH), activins have also been shown to modulate development, cell growth, apoptosis, and inflammation. Despite their many known functions, the precise mechanisms and downstream signaling pathways by which activins mediate their diverse effects remain unknown. We have used a DNA microarray assay to identify genes that are regulated by activin, alone or in combination with gonadotropin-releasing hormone (GnRH), another major regulator of FSH, in a murine gonadotrope-derived cell line (LbetaT2). We used mRNA from these cells to screen Affymetrix Mu74av2 mouse Gene Chip oligonucleotide microarrays, representing approximately 12,400 mouse genes. Treatment of LbetaT2 cells with activin A, a gonadotropin-releasing hormone agonist (GnRHA) or activin A plus GnRHA resulted in alterations in levels of gene expression that ranged in magnitude from 15 to 67-fold. Data analysis identified 268 transcripts that were up- or down-regulated by two-fold or more. Distinct sets of genes were affected by treatment with activin, GnRHA and activin plus GnRHA, suggesting interactions between activin and GnRHA. Changes in expression of seven randomly selected representative genes identified by the microarray technique were confirmed by real-time quantitative PCR and semi-quantitative reverse transcription/PCR (RT/PCR). Modulation of expression of genes by activin suggests that activin may mediate its effects through a variety of signaling pathways.

Activin Receptors↗
PubMed2006

Interleukin-1 receptor antagonist (IL-1RA) prevents apoptosis in ex vivo expansion of human limbal epithelial cells cultivated on human amniotic membrane.

Stem cells of the corneal epithelium have been found to be located exclusively at the anatomical junction between the cornea and the conjunctiva, the limbus. Ex vivo expanded limbal epithelial cells on amniotic membrane (AM) are capable of restoring the corneal surface with limbal stem cell deficiency. Recent studies indicate that intact AM preserves the limbal epithelial phenotype and that distinct epithelial morphology is noted among various culture matrix. However, the factors in response to the interaction between limbal epithelial cells and AM were not well understood. Using Annexin V-fluorescein isothiocyanate staining, we found that human limbal epithelial cells expanded on intact human AM demonstrated fewer apoptotic cells as compared with those on plastic dishes. To identify the anti-apoptotic factors, we performed cDNA microarray analysis and showed that interleukin-1 receptor antagonist (IL-1RA) was overexpressed in cultures on intact AM, which was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (Q-PCR) and enzyme-linked immunosorbent assay. In addition, we also noted that the phenomenon of apoptosis detected in cultures on plastic dishes could be reversed by adding recombinant IL-1RA protein into the media, whereas apoptosis of limbal epithelial cells cultivated on intact AM could be induced by exogenous neutralizing IL-1RA neutralizing antibody. These results demonstrated that intact human AM may prevent cultured human limbal epithelial cells from undergoing apoptosis. IL-1RA might be a candidate mediator to exert as an anti-apoptotic molecule during the interaction between human limbal epithelial cells and intact human AM.

Adolescent↗
PubMed1997

Increased prevalence of mitochondrial DNA deletions in skeletal muscle of older individuals with impaired glucose tolerance: possible marker of glycemic stress.

To determine the relationship between mitochondrial DNA (mtDNA) mutations and age-related impaired glucose tolerance (IGT), mtDNA from skeletal muscle of 19 volunteers, ages 55-75 years, with either IGT or diabetes and 17 age- and sex-matched control subjects was analyzed using a long-extension polymerase chain reaction (PCR) combined with a quantitative PCR. We found the common 4,977-bp deletion in 84% of the IGT/diabetes group compared with only 41% in the control group (P < 0.02). Multiple other deletions of different sizes were identified in 13 out of 19 IGT/diabetes patients (68%) compared with 2 out of 17 control subjects (12%) (P < 0.002). Because of the heterogeneity and variation in the mutations identified, we propose that these mtDNA mutations were the result rather than the cause of IGT. The increase in type and frequency of mtDNA deletions in diabetes and IGT patients may be related to oxidative damage by oxygen free radicals. These may be produced in greater amounts as a result of hyperglycemia or may be more abundant because of an abnormality in the scavenging of free radicals by antioxidants.

Aged↗
PubMed1999

Monitoring antibody titers to recombinant Core-NS3 fusion polypeptide is useful for evaluating hepatitis C virus infection and responses to interferon-alpha therapy.

To evaluate the clinical feasibility of the antibody titer against a chimeric polypeptide (named Core 518), in which a domain of Core and NS3 of hepatitis C virus (HCV) was fused, ELISA was performed in a total of 76 serum samples. Each serum was serially diluted using two-fold dilution method with distilled water into 10 concentrations. They were all positive for second generation anti-HCV assay (HCV EIA II; Abbott Laboratories). Genotyping RT-PCR, quantitative competitive RT-PCR, and RIBA (Lucky Confirm; LG Biotech) were also assayed. Anti-Core 518 antibody was detected in x 12800 or higher dilutions of sera from 35 of 43 chronic hepatitis C (81.4%) and nine of 16 hepatocellular carcinoma sera (56.3%), one of four cirrhosis (25%), 0 of four acute hepatitis C, and one of nine healthy isolated anti-HCV-positive subjects (p=0.0000). The anti-Core 518 antibody titers were well correlated with the presence of HCV RNA in serum (p=0.002). The anti-Core 518 antibody titers decreased significantly in nine of ten responders to IFN-alpha treatment. Monitoring anti-Core 518 titers may be helpful not only for differentiating the status of HCV infection among patients with various type C viral liver diseases, but also for predicting responses to IFN-alpha treatment.

Adult↗
PubMed1997

Reductions in viral load and increases in T lymphocyte numbers in treatment-naive patients with advanced HIV-1 infection treated with ritonavir, zidovudine and zalcitabine triple therapy.

In order to test the hypothesis that a combination of protease inhibitors with nucleoside analogues-agents known to inhibit different steps of the human immunodeficiency virus (HIV) life cycle--is likely to prove more effective in reducing viral loads than either of those modalities alone, we performed a 60 week, open-label trial in 32 HIV-positive patients with depressed CD4 T lymphocyte cell counts but no active AIDS-defining illnesses. For the first 2 weeks, patients received 600 mg twice daily of liquid ritonavir, a protease inhibitor; then zidovudine 200 mg three times daily and zalcitabine 0.75 mg three times daily were added to the treatment regimen. Mononuclear blood cell fractions were analysed for infected cell levels, using a co-culture system. HIV-1 RNA in plasma was measured both by reverse transcriptase-polymerase chain reaction (RT-PCR) and reverse transcriptase quantitative PCR (QcRT-PCR); lymphocyte counts were determined by standard laboratory methods. In the 2 weeks of ritonavir therapy, both the mean count of infectious blood cells and plasma HIV RNA levels decreased dramatically. Mean CD4 cell counts increased from 173 cells/mm3 at baseline to 286 cells/mm3; CD8 cell counts rose from 951 cells/mm3 to 1,141 cells/mm3. With the introduction of the nucleoside analogues, infectious cell counts and plasma virus dropped another log unit to a nadir at 8 weeks, while CD4 T lymphocyte counts continued to rise slowly. By week 28, 12 patients had withdrawn due to adverse events, none of which were life-threatening. At week 36, infectious material could not be detected in the cells of 10 of the 17 remaining patients; by week 60, four of the seven patients with residual viraemia at week 24 had undergone viral relapse. After the introduction of a more palatable capsule formulation of ritonavir at week 52, infectious cells and plasma virus were undetectable in 50-60% of patients. The combination of protease inhibitors and nucleoside analogues significantly reduces HIV load, and in some patients may suppress viral activity for sustained periods.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Quantification of HER2/neu gene amplification by competitive pcr using fluorescent melting curve analysis.

BACKGROUND: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. METHODS: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. RESULTS: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. CONCLUSION: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.

DNA↗
PubMed2001

[Mammaglobin mRNA measurement in the detection of micrometastasis in peripheral blood of breast cancer patients].

OBJECTIVE: To investigate mammanglobin (hMAM) mRNA as a marker for the detection of carcinoma cells by reverse transcriptase chain reaction (RT-PCR) in the peripheral blood of breast cancer patients. METHODS: Blood samples from 63 breast cancer patients obtained at various stages of their disease, blood samples from 8 breast hyperplasia, 5 breast adenofibroma, 25 other cancers (stomach, colon, esophagus, lung, ovary) and 31 healthy volunteers were screened for hMAM mRNA by a nested RT-PCR combined with fluorescence quantitative PCR (FQ-PCR) assay. RESULTS: Among 63 breast carcinoma patients, 19(30.2%) were RT-PCR positive for hMAM mRNA which was elevated with the tumor stage. None of the other cancer patients or those suffering from benign breast diseases were positive but only 1 of the 31 healthy volunteers gave detectable hMAM mRNA findings. hMAM mRNA was not detectable in the peripheral blood after operation in 6(32%) of 19 patients whose pre-operative test had been positive even though the hMAM mRNA expression increased with tumor stage. These results did not correlate with patients' stage, estrogen or pregnant receptor status. CONCLUSION: hMAM transcripts are detectable in the peripheral blood of some breast cancer patients. hMAM may be a novel candidate and a clinically useful breast tumor marker, especially in detecting micrometastasis.

Breast Neoplasms↗
PubMed2006

Effects of induced systemic hypothyroidism upon the retina: regulation of thyroid hormone receptor alpha and photoreceptor production.

PURPOSE: Investigate the effects of systemic hypothyroidism upon the differentiated, growing, and regenerating retina of postmetamorphic winter flounder, a vertebrate that experiences a thyroid hormone (TH) induced metamorphosis during development. METHODS: A loss-of-signal strategy was utilized in which TH signaling was disrupted by inhibiting TH synthesis. Induced hypothyroidism was confirmed by radioimmunoassay. Reverse transcriptase PCR (RT-PCR), real-time quantitative PCR (qPCR), molecular cloning, non-isotopic in situ hybridization, western blot analysis, and indirect immunohistochemistry techniques were performed to analyze retinal thyroid hormone receptors (TR), photoreceptor production, and the phenotypic repertoire of differentiated retinal cells as a function of TH signaling status. RESULTS: Molecular bases for TH signaling were supported by retinal expression of TH receptors alpha and beta. TH-dependent transcriptional regulation of TRalpha but not TRbeta was indicated, with induced hypothyroidism producing an increase in TRalpha expression. Evidence for post-transcriptional regulation of retinal TRalpha was observed. The repertoire of inner retinal cell types in premetamorphic fish (a naturally low TH condition) matched that observed in the central retinas of both normal postmetamorphic fish (a naturally elevated TH condition) and postmetamorphic fish rendered hypothyroidic. In differentiated postmetamorphic retina there was no evidence for significant differences in opsin expression between normal and hypothyroidic animals. Induced hypothyroidism did, however, significantly affect the types of photoreceptors that were produced in postmetamorphic retina: as a hypothyroidic postmetamorphic retina grew or regenerated following injury, the phenotypic repertoire of newly-produced photoreceptors matched that observed for premetamorphic retina, in which rods, SWS2-expressing "blue" cones, and LWS-expressing "red" cones are absent, and only the RH2-expressing "green" cone type is present. The effects of induced hypothyroidism upon photoreceptor specification (manifestation of the rod lineage) and differentiation (expression of a particular opsin by specified cones) were apparently reversible. CONCLUSIONS: The results suggest a TH-dependent regulation of retinal TRalpha, a lack of TH-dependent regulation of the phenotypic identity of differentiated retinal cells, and the operation of similar cytogenic mechanisms during retinal growth and regeneration. The principal conclusion is that TH signaling significantly affects, in a targeted manner, the production of both rod and cone photoreceptors during retinal growth and regeneration.

Animals↗
PubMed2005

Automated validation of polymerase chain reactions using amplicon melting curves.

PCR, the polymerase chain reaction, is a fundamental tool of molecular biology. Quantitative PCR is the gold-standard methodology for determination of DNA copy numbers, quantitating transcription, and numerous other applications. A major barrier to large-scale application of PCR for quantitative genomic analyses is the current requirement for manual validation of individual PCR reactions to ensure generation of a single product. This typically requires visual inspection either of gel electrophoreses or temperature dissociation ("melting") curves of individual PCR reactions - a time-consuming and costly process. Here we describe a robust computational solution to this fundamental problem. Using a training set of 10,080 reactions comprising multiple quantitative PCR reactions from each of 1,728 unique human genomic amplicons, we developed a support vector machine classifier capable of discriminating single-product PCR reactions with better than 99% accuracy. This approach has broad utility, and eliminates a major bottleneck to widespread application of PCR for high-throughput genomic applications.

Algorithms↗
PubMed2005

Real-time PCR for mRNA quantitation.

Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.

Algorithms↗