Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Six human alpha-tubulin and seven human beta-tubulin isotypes, each of which can undergo posttranslational modifications, have been detected by the reverse transcriptase-polymerase chain reaction. This repertoire of tubulin isotypes plays a role in development and in the building of specialized microtubule-based structures. In cell lines, the relationship between resistance to microtubule-interacting drugs and altered tubulin isotype expression profiles is often established by quantitation of cDNA and/or Western blot analysis. Tubulin mutations in major isotypes are detected by sequencing cDNA, but more analysis of expression of tubulin mutations at the protein level, to assess their role in drug resistance, is needed. We utilized a Taxol-based purification and high-resolution isoelectrofocusing combined with a mass spectrometry-based analysis of tubulin. This approach has allowed the separation and relative quantitation of tubulin isotypes having a difference in isoelectric point values of 0.01, without the need for two-dimensional gel electrophoresis. The specificity of tubulin isotype antibodies also has been established. In cell lines resistant to microtubule-stabilizing drugs that express heterozygous tubulin mutations, the relative amount of mutant tubulin expression has been determined. In these cell lines, the absence of betaII- and betaIVa-tubulin has been demonstrated, and an increased level of expression of betaIII-tubulin in resistant cells has been confirmed, indicating that this tubulin isotype is a unique marker of resistance.
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PMID:Analysis of tubulin isotypes and mutations from taxol-resistant cells by combined isoelectrofocusing and mass spectrometry. 1273 76

We describe the successful use of the reverse genetic technique RNA interference (RNAi) to investigate gene function in the human filarial nematode parasite Brugia malayi. We used fluorescently labelled double stranded RNA (dsRNA) to demonstrate that 300 bp molecules are able to enter adult females in culture while they remain excluded from microfilariae (mf). We have developed an optimised microvolume culture system to allow the exposure of parasites to high concentrations of dsRNA for extended periods. Culturing of adult female parasites in this system for 24h does not significantly reduce parasite lifespan or mf release in culture. Three B. malayi genes, beta-tubulin (Bm-tub-1), RNA polymerase II large subunit (Bm-ama-1) and B. malayi mf sheath protein 1/mf22 (Bm-shp-1) were targeted by soaking adult female B. malayi in dsRNA complementary to these transcripts in the optimised culture system. Targeting of the two housekeeping genes Bm-tub-1 and Bm-ama-1 led to a reduction in the levels of their transcripts, as assessed by reverse transcriptase coupled PCR (RT-PCR), and resulted in parasite death in culture. In contrast, targeting of the Bm-shp-1 gene was not lethal to adult females in culture. A marked reduction in mf release was observed for shp-1 RNAi parasites compared to controls and in addition 50% of mf released did not have fully elongated sheaths. This "short" phenotype correlated with the loss of the stockpiled shp-1 transcript from developing mf in treated adult female gonads. From these data we conclude that RNAi may be a useful method for assessment of drug target potential of genes identified in filarial gene discovery projects.
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PMID:Use of RNA interference to investigate gene function in the human filarial nematode parasite Brugia malayi. 1279 5

Although nematodes like Caenorhabditis elegans are incapable of de novo cholesterol biosynthesis, they can utilize nonfunctional sterols by converting them into cholesterol and other sterols for cellular function. The results reported previously and presented here suggest that blocking of sterol conversion to cholesterol in C. elegans by 25-azacoprostane-HCl (azacoprostane) treatment causes a serious defect in germ cell development, growth, cuticle development, and motility behavior. To establish a biochemical basis for these physiological abnormalities, we performed proteomic analysis of mixed stage worms that had been treated with the drug. Our results from a differential display proteomic analysis revealed significant decreases in the levels of proteins involved in collagen and cytoskeleton organization such as protein disulfide isomerase (6.7-fold), beta-tubulin (5.41-fold), and NEX-1 protein (>30-fold). Also reduced were enzymes involved in energy production such as phosphoglycerate kinase (4.8-fold) and phosphoenolpyruvate carboxykinase (8.5-fold), a target for antifilarial drugs such as azacoprostane. In particular, reductions in the expression of lipoprotein families such as vitellogenin-2 (7.7-fold) and vitellogenin-6 (5.4-fold) were prominent in the drug-treated worms, indicating that sterol metabolism disturbance caused by azacoprostane treatment is tightly coupled with suppression of the lipid transfer-related proteins at the protein level. However, competitive quantitative reverse transcriptase polymerase chain reaction showed that the transcriptional levels of vit-2, vit-6, and their receptors (e.g. rme-2 and lrp-1) in drug-treated worms were 3- to 5-fold higher than those in the untreated group, suggesting a presence of a sterol regulatory element-binding protein (SREBP)-like pathway in these genes. In fact, multiple predicted sterol regulatory elements or related regulatory sequences responding to sterols were found to be located at the 5'-flanking regions in vit-2 and lrp-1 genes, and their transcriptional activities fluctuated highly in response to changes in sterol concentration. Thus, many physiological abnormalities caused by azacoprostane-mediated sterol metabolism disturbance appear to be exerted at least in part through SREBP pathway in C. elegans.
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PMID:Proteomic changes during disturbance of cholesterol metabolism by azacoprostane treatment in Caenorhabditis elegans. 1290 48

Bone marrow (BM) mesenchymal stem cells (MSCs) are cells capable of expanding and differentiating in vitro into nonhematopoietic cells. Neurotrophic cytokines, such as human epidermal growth factor (hEGF) and bovine fibroblast growth factor (bFGF) can induce differentiation into neural cells (NCs). When BM MSCs were cultured with hEGF and bFGF, RNA expression of neuronal specific markers Nestin, MAP-2, and tyrosine hydroxylase (TH) were observed. We tested a new cytokine combination to generate mature NCs. The plastic-adherent cells were collected and then split when they were 90% confluent from an enriched mononuclear cell layer. At passage 3, MSCs were cultured in neural differentiation media (dbcAMP, IBMX, FGF-8, BDNF, hEGF, and bFGF in NEUROBASAL media plus B27). Cells were counted on day 6. Immunofluorescent staining and reverse transcriptase (RT)-PCR were performed to evaluate the expression of neural markers. On day 6, 66% of cells developed dendrites and presented typical neural cell morphology. Some cells were positive for early neural markers Nestin and beta-tubulin III. Cells expressing mature neuronal markers (NF, NeuN, Tau, Nurr1, GABA, oligodendryte GalC, and glial GFAP) were also seen. By adding hEGF, bFGF, dbcAMP, IBMX, BDNF, and bFGF-8 into NEUROBASAL media plus B27, BM MSCs were directed toward becoming early and mature NCs.
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PMID:Neural cell differentiation in vitro from adult human bone marrow mesenchymal stem cells. 1572 45

In avian species, the ultimobranchial anlage is populated with neuronal cells derived from the distal vagal ganglion. We found that ultimobranchial C cells of chick embryos cultured in the presence of nicotinamide continued to grow for at least 60 days and exhibited profound morphological changes, resulting in the formation of dense networks of neuronal fibers. Nicotinamide, thus, facilitated the manifestation of neuronal features in C cells. The neuronal phenotypes of cultured C cells were analyzed in detail by both scanning and transmission electron microscopy. Their neural nature was also positively established by immunostaining with monoclonal antibodies to the neuronal markers neuron-specific class III beta-tubulin (TuJ1), microtubule-associated protein (MAP) 2, and synaptophysin. Confocal laser scanning microscopy confirmed that these neuron-specific proteins are colocalized with calcitonin in both the somata and the neuronal processes of C cells. Furthermore, reverse transcriptase-polymerase chain reaction analyses, performed at various times up to 30 days in culture, indicated that the C cells have persistent gene expression of calcitonin, the catecholamine-synthesizing enzyme tyrosine hydroxylase, proenkephalin, proopiomelanocortin, neuron-specific beta-tubulin (cbeta4), SCG10, and Bcl-2. The morphological responses of C cells to nicotinamide treatment were analyzed quantitatively over a period of 60 days. The area of C-cell colonies, number of processes per colony, and length of processes continued to increase until culture day 45. In conclusion, nicotinamide stimulates long-term survival and neuronal differentiation of chick embryo C cells, and this culture system may provide a useful model for studying neuronal differentiation mechanisms.
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PMID:Nicotinamide promotes long-term survival and extensive neurite outgrowth in ultimobranchial C cells cultured from chick embryos. 1621 94

RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, beta-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.
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PMID:Efficacy and specificity of RNA interference in larval life-stages of Ostertagia ostertagi. 1687 64

A comprehensive understanding of the mechanisms that underlie hepatic differentiation inside a bioartificial liver (BAL) device is obtained when functional, histological, and gene expression analyses can be combined. We therefore developed a novel cell-sampling technique that enabled us to analyze adherent hepatocytes inside a BAL device during a 5-day culture period, without the necessity of terminating the culture. Biochemical data showed that hepatocyte-specific functions were relatively stable, despite an increase in glycolytic activity. Quantitative reverse transcriptase polymerase chain reaction analysis of hepatic genes cytochrome p450 3A29, albumin, glutamine synthetase, alpha-1 antitrypsin, and carbamoyl-phosphate synthetase, but also de-differentiation marker pi-class glutathione S transferase showed stable messenger ribonucleic acid (mRNA) levels from day 1 to 5. In contrast, mRNA levels of alpha-fetoprotein, pro- and anti-apoptotic genes Bax-alpha and Bcl-X(L), metabolic genes lactate dehydrogenase and uncoupling protein 2, and cytoskeleton genes alpha- and beta-tubulin and beta-actin increased in 5 days. Histological analysis revealed viable tissue-like structures with adaptation to the in vitro environment. We conclude that hepatocytes show a tendency for de-differentiation shortly after seeding but thereafter remain acceptably differentiated during 5 days of culture. Furthermore, partly impaired mitochondrial function is suggestive for local hypoxic regions and may trigger the observed metabolic changes. Anti-apoptotic activity seems to balance pro-apoptotic activity. This new cell-sampling technique facilitates the analysis of dynamic processes of hepatocyte culture inside a BAL.
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PMID:Time-related analysis of metabolic liver functions, cellular morphology, and gene expression of hepatocytes cultured in the bioartificial liver of the Academic Medical Center in Amsterdam (AMC-BAL). 1751 23

In this study, the potential of gold nanoparticles (20 nm) to deliver electrical stimulation to nerve cell cultures in vitro to induce nerve regeneration was evaluated. In order to use these biomaterials to deliver an electrical stimulus, we devised a novel method for the fabrication of a nanostructured 2D substrate comprising gold nanoparticles attached to the surface of a cover glass via an adsorption system. In this strategy, gold nanoparticles are created and then coated onto a positively charged cover glass that has been pretreated with polyethyleneimine (PEI). Scanning electron microscopy (SEM) revealed that the PC 12 cells extended neurites well on the gold nanoparticles in the presence of electrical stimulation. In addition, the neurite outgrowth of PC12 cells in response to pulsed and constant electrical stimulation was evaluated by live/dead cell determination, by reverse transcriptase polymerase chain reaction (RT-PCR) analysis, as well as by beta-tubulin and NF-200 expression. By electrical stimulation (250 mV for 1 h), PC12 cells with their neurite outgrowth length were highly increased, with a mean diameter of 98.5 microm. However, the neurite outgrowth length without electrical stimulation was approximately 10 approximately 20 microm. Moreover, the alternating current stimulation also showed good viability (<90%), while a high amount of cell death (more than 30%) was observed with constant current stimulation. Thus, the gold nanoparticles with pulsed current stimulation may provide suitable tools for the nerve regeneration using neuronal cells.
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PMID:Electrical pulsed stimulation of surfaces homogeneously coated with gold nanoparticles to induce neurite outgrowth of PC12 cells. 1904

The pluripotent nature and proliferative capacity of embryonic stem cells make them an attractive cell source for tissue engineering. In this study, the poly-L-lysine-coated PLGA microspheres which contain retinoic acid (RA) as an inducer factor were prepared by using a water-in-oil-in-water emulsion/solvent evaporation technique. Then, pluripotent P19 embryonic carcinoma cells were seeded on them for differentiating into neural cells. Size and surface morphology of PLGA microspheres were evaluated by scanning electron microscope (SEM). For in vitro examinations, SEM, MTT assay, immunofluorescent staining, histology and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were carried out. SEM micrographs of the scaffolds showed a diameter in range of 13-100 microm. Based on the release profiles obtained, the concentration of RA released from microspheres reached 10(-6) to 10(-7) mg/ml. MTT assay showed that the number of cells attached on coated microspheres were more in comparison with uncoated microspheres. Immunoflourescent staining and RT-PCR analyses for MapII, beta-tubulin III, Nestin and Pax6 indicated differentiation of P19 cells into neural cells on all of the samples. Finally, the counting of positive cells showed 80+/-8.8% and 72+/-6.9% of the cells expressed beta-tubulin III on the surface of coated and uncoated RA-loaded PLGA microspheres, respectively, while the 64+/-1.1% (P < 0.05) cells expressed tubulin III in group with soluble.
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PMID:Preparation and surface characterization of poly-L-lysine-coated PLGA microsphere scaffolds containing retinoic acid for nerve tissue engineering: in vitro study. 1952 May 54

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.
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PMID:The Fungal PCR Initiative's evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay. 3175 73


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