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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Peripheral lymphoid tissues contain a fibroblastic cell type referred to as stromal cells or reticulum cells which interact with lymphocytes as part of the lymphoid microenvironment. After isolation from human tonsils and expansion in vitro we analyzed the surface phenotype, extracellular matrix components, cytoskeletal products, cytokine production, binding and functional interaction with B lymphocytes of in vitro cultured stromal cells (HTSC) both in resting condition and after activation with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our results show that HTSC do not express specific myeloid, lymphoid, endothelial or epithelial markers. HTSC express CD54 (ICAM-1), CD49a (VLA-1), CD49b (VLA-2), CD49c (VLA-3), CD49e (VLA-5), CD49f (VLA-6), CD29, CD51, CD44 and produce vinculin, beta-tubulin, alpha-actin, vimentin, fibronectin, laminin and collagen types I, III and IV. Activation of HTSC up-regulated CD54 (ICAM-1) and induced HLA-DR and CD106 (VCAM-1). HTSC constitutively produce interleukin (IL)-6 which is enhanced upon activation with TNF-alpha. IL-8 and granulocyte/macrophage colony-stimulating factor are detected only in the supernatants of activated HTSC. Reverse transcriptase polymerase chain reaction analysis revealed that HTSC display mRNA for IL-1 alpha, leukemia inhibitory factor and IL-7. The adhesion of tonsillar B lymphocytes to activated HTSC is mediated by CD11a/CD18 and CD54. Furthermore, HTSC can induce maximal proliferation of IL-2-activated B lymphocytes cocultured in direct cell-cell contact with HTSC. These results clearly distinguish in vitro cultured HTSC from common fibroblasts and other non-lymphoid elements present in the lymphoid parenchyma, such as follicular dendritic cells, and show that HTSC actively participate in the lymphoid microenvironment. In vitro cultures of HTSC could therefore be a useful model system for detailed analysis of the interactions between stromal cells and lymphocytes under physiological and pathological conditions.
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PMID:In vitro cultured stromal cells from human tonsils display a distinct phenotype and induce B cell adhesion and proliferation. 856 62

A paclitaxel-resistant cell line, KPTA5, was established by co-selecting the parental erythroleukemic cell line K562 with stepwise increased concentrations of paclitaxel (Taxol) in the presence of the cyclosporin D analogue PSC 833 (2 microM), a potent modulator of the multidrug resistance phenotype. KPTA5 cells are 9-fold resistant to paclitaxel and taxotere, but do not exhibit significant resistance to Vinca alkaloids, etoposide, anthracyclines, antimetabolites, or alkylating agents. Doubling time and morphology were similar to the parental K562 cells. Reverse transcriptase-polymerase chain reaction (rt-PCR) analysis revealed no alterations in the expression of the mdr1 and MRP genes. Cellular paclitaxel accumulation was unchanged. Cell cycle analyses showed that at 20 nM there was a significantly higher proportion of K562 cells blocked in G2/M, in comparison with KPTA5 cells. In both cases, disruption of the mitotic spindles and the presence of multiple mitotic asters were comparable but occurred at lower paclitaxel concentrations in K562 cells than in KPTA5 cells. There was no difference in total tubulin content between K562 and KPTA5 cells as analyzed by immunoblotting with an anti-beta-tubulin monoclonal antibody. However, we found that KPTA5 cells presented a 2-fold increase both in 5 beta-tubulin mRNA expression and in the corresponding tubulin protein Class IV isotype content, as evaluated by rt-PCR and immunostaining. In conclusion, the KPTA5 cell line displays a novel mechanism of resistance to paclitaxel which does not involve altered cellular drug accumulation. The data presented suggest that alterations in expression of the 5 beta-tubulin gene may be involved in paclitaxel resistance.
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PMID:Novel mechanism of resistance to paclitaxel (Taxol) in human K562 leukemia cells by combined selection with PSC 833. 886 64

Microtubules play an essential role in cell division. Little is known about possible variations of total tubulin and tubulin isotype expression during the cell cycle. We analyzed the total tubulin content, tubulin polymerization status and tubulin isotype content in resting and dividing human K562 leukemic cells and human MES-SA sarcoma cells. Although the total cellular tubulin content increases as the cells progress toward mitosis, the total tubulin/total protein ratio is stable during the cell cycle. Reverse transcriptase-polymerase chain reaction was applied to analyze the levels of expression of alpha, beta, and gamma-tubulin isotypes. Whereas alpha-tubulin isotype and gamma-tubulin transcripts were found to be expressed at constant levels throughout the cell cycle, some of the beta-tubulin isotype transcripts were found to be more highly expressed in dividing then in resting cells. Both of the class IV beta-tubulin isotype transcripts (human 5 beta and beta 2, Class IVa and IVb, respectively) were expressed in dividing K562 and MES-SA cells at twice the levels found in resting cells. Increased expression of the class IV isotype proteins in dividing cells was confirmed by immunoblotting, both in K562 and in MES-SA cells. A larger fraction of total cell tubulin was found to be polymerized in dividing cells (36-40%) than in resting cells (27-30%). The degree of polymerization of class IV tubulin in dividing and resting cells was similar to that of total tubulin. These results show that total tubulin is expressed as constant levels throughout the cell cycle but that the degree of polymerization is increased as cells are committed to division. The relative overexpression of the two class IV beta-tubulin isotypes in dividing cells suggests functional specificity for these isotypes and a regulatory role of these isotypes on the microtubule network during mitosis.
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PMID:Differential expression of tubulin isotypes during the cell cycle. 887 65

We sought an in vitro primate model for serotonin neurons. Rhesus monkey embryonic stem (ES) cell colonies were isolated and differentiated into embryoid bodies (EBs), then transferred to serum-free medium with 1% insulin-transferrin-selenium for 7 days to induce neural precursor cell (NPC) formation. NPCs were cultured in medium with 1% N-2 neural supplement and human fibroblast growth factor 2 (FGF2, 10 ng/ml) for 7 days to stimulate cell proliferation. Lastly, NPCs were dispersed into single cells and cultured without FGF2 for another 7 days to obtain terminal differentiation. Terminal cells were characterized for neuronal and serotonergic markers. Over 95% of the NPCs were immunopositive for nestin and Musashi1. Terminally differentiated cells appeared in both small and large morphologies. Most (>95%) of the mature cells (both small and large) were immunopositive for neuron-specific nuclear protein (NeuN), synaptophysin, microtubule-associated protein (MAP2C), Tau-1, neurofilament 160 (NF-160), beta-tubulin (TujIII), tryptophan hydroxylase (TPH), serotonin, the serotonin reuptake transporter (SERT), estrogen receptor-beta (ERbeta), and progestin receptor (PR), but not estrogen receptor-alpha (ERalpha). Less than 2-3% of cells were positive for tyrosine hydroxylase (TH). Reverse transcriptase polymerase chain reaction (RT-PCR) detected mRNA transcripts for TPH-1, TPH-2, SERT, 5-HT1A-autoreceptor, ERbeta, and PR in the differentiated population. A low level of expression of ERalpha mRNA was also detected. Quantitative RT-PCR indicated that the relative abundance of TPH-2 mRNA was greater than TPH-1 mRNA. Serotonin as measured by ELISA increased 3-fold in the mature stage compared to the selection and expansion stages. In summary, a remarkably high percentage of cells derived from monkey ES cells exhibited neuronal plus serotonergic markers as well as nuclear steroid receptors similar to primate CNS serotonin neurons, suggesting that these cells may serve as a useful primate model for serotonergic neurons.
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PMID:Serotonin neurons derived from rhesus monkey embryonic stem cells: similarities to CNS serotonin neurons. 1524 35

RNA interference (RNAi) is widely used in Caenorhabditis elegans to identify gene function and has been adapted as a high throughput screening method to identify genes involved in essential processes. We have been examining whether RNAi could also be used on the strongylid parasitic nematode Haemonchus contortus to study gene function. Eleven genes were targeted in L1 and exsheathed L3 H. contortus larvae with RNAi methodologies which have been shown to be effective in C. elegans and parasitic nematodes-feeding, soaking and electroporation. Reverse transcriptase-PCR and, where possible, protein assays were carried out to examine decreases in mRNA and protein levels. RNAi soaking in dsRNA to beta-tubulin and sec-23, a gene involved in vesicle transport, resulted in specific decreases in mRNA levels in exsheathed L3 larvae. No signs of specific decreases in expression levels were observed for the other nine genes tested. Following electroporation of dsRNA in L1 stage larvae, significant decreases were observed for two out of four genes tested. These findings suggest that the RNAi pathway is functional in H. contortus and that, under certain conditions, it is possible to suppress gene expression by RNAi. However, it only works on a limited number of genes and in some cases the effect is small and difficult to reproduce. This indicates that the RNAi approaches established for C. elegans and other nematodes have limited efficacy in H. contortus. This may reflect differences between nematode species in dsRNA uptake and transport into cells and between cells.
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PMID:Testing the efficacy of RNA interference in Haemonchus contortus. 1646 21

Class III beta-tubulin isotype (betaIII-tubulin) is widely regarded as a neuronal marker in developmental neurobiology and stem cell research. To test the specificity of this marker protein, we determined its expression and distribution in primary cultures of glial fibrillary acidic protein (GFAP)-expressing astrocytes isolated from the cerebral hemispheres of 2 human fetuses at 18 to 20 weeks of gestation. Cells were maintained as monolayer cultures for 1 to 21 days without differentiation induction. By immunofluorescence microscopy, coexpression of betaIII-tubulin and GFAP was detected in cells at all time points but in spatially distinct patterns. The numbers of GFAP+ cells gradually decreased from Days 1 to 21 in vitro, whereas betaIII-tubulin immunoreactivity was present in 100% of cells at all time points. beta-III-tubulin mRNA and protein expression were demonstrated in cultured cells by reverse-transcriptase-polymerase chain reaction and immunoblotting, respectively. Glial fibrillary acidic protein+/beta-III-tubulin-positive cells coexpressed nestin and vimentin but lacked neurofilament proteins, CD133, and glutamate-aspartate transporter. Weak cytoplasmic staining was detected with antibodies against microtubule-associated protein 2 isoforms. Confocal microscopy, performed on autopsy brain samples of human fetuses at 16 to 20 gestational weeks, revealed widespread colocalization of GFAP and betaIII-tubulin in cells of the ventricular/subventricular zones and the cortical plate. Our results indicate that in the midgestational human brain, betaIII-tubulin is not neuron specific because it is constitutively expressed in GFAP+/nestin+ presumptive fetal astrocytes.
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PMID:Class III beta-tubulin is constitutively coexpressed with glial fibrillary acidic protein and nestin in midgestational human fetal astrocytes: implications for phenotypic identity. 1837 34

The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), alpha- and beta-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.
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PMID:Association of the influenza virus RNA polymerase subunit PB2 with the host chaperonin CCT. 2057 28

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