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)

Elevated leukotriene (LT)C(4) synthase activity was observed in peripheral blood granulocyte suspensions from patients with chronic myeloid leukemia (CML). Magnetic cell sorting (MACS) with CD16 monoclonal antibodies (mAbs), which were used to fractionate granulocytes from CML patients and healthy individuals, yielded highly purified suspensions of CD16(+) neutrophils. The purity of these cell fractions was verified by extensive morphologic examination. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, demonstrating the absence of interleukin-4 messenger RNA (IL-4 mRNA), further confirmed the negligible contamination of eosinophils in these fractions. Notably, purified CML CD16(+) neutrophils from all tested patients transformed exogenous LTA(4) to LTC(4). These cells also produced LTC(4 )after activation with ionophore A23187 or the chemotactic peptide fMet-LeuPhe (N-formylmethionyl-leucyl-phenylalanine). Subcellular fractionation revealed that the enzyme activity was exclusively distributed to the microsomal fraction. Expression of LTC(4) synthase mRNA in CML CD16(+) neutrophils was confirmed by RT-PCR. Furthermore, Western blot analyses consistently demonstrated expression of LTC(4) synthase at the protein level in CML CD16(+) neutrophils, whereas expression of microsomal glutathione S-transferase 2 occurred occasionally. Expectedly, LTC(4) synthase activity or expression of the protein could not be demonstrated in CD16(+) neutrophil suspensions from any of the healthy individuals. Instead, these cells, as well as CML CD16(+) neutrophils, transformed LTA(4) to LTB(4). The results indicate that aberrant expression of LTC(4) synthase is a regular feature of morphologically mature CML CD16(+) neutrophils. This abnormality, possibly associated with malignant transformation, can lead to increased LTC(4) synthesis in vivo. Such overproduction may be of pathophysiological relevance because LTC(4 )has been demonstrated to stimulate proliferation of human bone marrow-derived myeloid progenitor cells. (Blood. 2000;95:1456-1464)
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PMID:Aberrant expression of active leukotriene C(4) synthase in CD16(+) neutrophils from patients with chronic myeloid leukemia. 1066 25

Using the reverse-transcriptase polymerase chain reaction (RT-PCR), cDNA encoding ovine (Ov) interleukin-4 (OvIL-4) was generated from mitogen-stimulated peripheral blood mononuclear cells (PBMC). Two identical clones generated from separate RT-PCR reactions differed from a published OvIL-4 sequence, although they had a high degree of identity with the bovine and human homologs. We show by sequence analysis that the OvIL-4 cDNA retained the four alpha-helix structure and disulfide bonds identified in human IL-4 (HuIL-4). Moreover, the cDNA encoding OvIL-4 was expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Supernatants from insect cells infected with the recombinant virus secreted an additional protein with a relative molecular mass of 17,000. This protein was recognized by an anticervine IL-4 monoclonal antibody (mAb) in a Western blot and did not react with any proteins in supernatants from uninfected insect cells or cells infected with the wild-type AcMNPV. Supernatants from insect cells infected with the recombinant virus induced the proliferation of activated B cells in a dose-dependent manner and typically demonstrated 5 x 105 dilution U/ml of activity. However, OvIL-4 had no effect on the proliferation of resting T cells isolated from efferent lymph and actually inhibited the ability of a mitogen to stimulate these resting lymphocytes. In contrast, OvIL-4 induced the proliferation of mitogen-activated lymphoblast, demonstrating the complex role(s) OvIL-4 plays in the regulation of B and T cells.
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PMID:The expression and biologic effects of ovine interleukin-4 on T and B cell proliferation. 1080 77

The cellular immune responses against major piroplasm surface protein (MPSP) of Theileria sergenti were characterized. Three cattle were immunized with recombinant MPSP (C type) encapsulated by mannan-coated liposome. The proliferative responses of peripheral blood mononuclear cells (PBMC) against MPSP were detected in all immunized animals. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that T cell lines derived from each animal expressed relatively high levels of interferon (IFN)-gamma mRNA, low levels of interleukin (IL)-2, IL-10, and tumor necrosis factor (TNF)-alpha mRNAs, but no detectable level of IL-4 mRNA. From the results of T cell epitope-mapping, T-cell lines from two animals responded to DTSKFTPTVAHRLKHAEDLF (residues 61 to 80), while other animal responded to GTGKVYDFVGNFKVTKVKFE (residues 141 to 160). The MPSP-type specific response of a T-cell line was observed in the latter region of MPSP. These data suggest that immunization with cocktail vaccine consisting of different types of MPSP may be necessary in the field trial.
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PMID:Epitope-Mapping of antigen-specific T lymphocyte in cattle immunized with recombinant major piroplasm surface protein of Theileria sergenti. 1155 46

Primary mediastinal B-cell lymphoma (PMBL) is a subentity of diffuse large B-cell lymphoma with characteristic clinical, histomorphologic, immunophenotypical, and genetic features. Unlike other B-cell lymphomas, PMBL has not yet been the subject of comprehensive molecular studies on the rearranged immunoglobulin (Ig) gene. Such investigations have proved essential to obtaining information about the differentiation stage of the lymphomagenic B cell. In the present study, the clonally rearranged immunoglobulin heavy-chain gene of 13 PMBL cases is analyzed by polymerase chain reaction (PCR) in conjunction with cloning and DNA sequencing. Twelve of 13 rearrangements were potentially functional. All clonally rearranged immunoglobulin genes bore a high load of somatic mutations (average, 13.0%), which appeared to be selected for a functional antibody in the majority of cases. The comparison of cloned PCR products revealed no evidence of ongoing mutation of the immunoglobulin variable gene. By means of reverse-transcriptase PCR, lymphoma-specific immunoglobulin transcripts were detected in 8 of 13 cases, all of which were of the postswitched type, whereas immunoglobulin protein expression was undetectable except for 1 case. A PMBL cell line, MedB-1, generated from an IgG(-) parental tumor, constitutively expressed IgG protein in a subset of cells, which was moderately suppressed by interleukin-4 and up-regulated in the presence of dexamethasone. PMBL is thus characterized by a heavily mutated, class-switched immunoglobulin gene without evidence of ongoing mutational activity. Moreover, our data indirectly suggest that regulation by extrinsic signals contributes to the immunoglobulin-negative phenotype of PMBL.
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PMID:Isotype-switched immunoglobulin genes with a high load of somatic hypermutation and lack of ongoing mutational activity are prevalent in mediastinal B-cell lymphoma. 1167 49

In the murine model for respiratory syncytial virus (RSV) infection, cytokine patterns induced by vaccinations with either killed (i.e. formalin-inactivated, alum-precipitated) virus (KV) or live virus (LV) have been shown to influence disease expression. To determine the mRNA expression of the cytokines IL-4 and IFN-gamma in BALB/c mice challenged with RSV, a real-time quantitative reverse-transcriptase PCR assay was developed. This assay uses 5'-exonuclease fluorogenic probes and is performed on the ABI PRISM 7700 Sequence Detector System (TaqMan). The relative quantitative levels of mRNA for IL-4 and IFN-gamma were compared with those measured by an RNase protection assay (RPA) and an enzyme immunoassay (EIA), which are methods used to measure the levels of mRNA and protein, respectively. Results obtained by the TaqMan assay showed that mice primed with KV induces increased IL-4 mRNA production while LV induces increased IFN-gamma mRNA, which is in agreement with conventional methods. IL-4 and IFN-gamma relative quantities obtained from TaqMan were highly correlated to those determined by RPA (r=0.96 for IFN-gamma, P<0.01) and EIA (r=0.90 for IL-4 and r=0.75 for IFN-gamma, P<0.01). Assay reproducibility was examined by testing a same sample in triplicate at three experiments. Minimal deviation values were observed in both intra- and inter-assays. TaqMan, which is rapid, sensitive and reproducible, provides an alternative tool for the quantitative analysis of cytokine mRNA expression in the murine model of RSV immunopathogenesis.
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PMID:Cytokine expression in respiratory syncytial virus-infected mice as measured by quantitative reverse-transcriptase PCR. 1250 27

The expression of the messenger RNA of interleukin-12 (IL-12), interferon-gamma, interleukin-4, interleukin-6, and interleukin-10 was examined by reverse-transcriptase polymerase chain reaction in peripheral blood lymphocytes of calves that were orally inoculated with Cryptosporidium parvum oocysts. In all of the calves, gene expression of interleukin-12, interleukin-6, and interferon-gamma was observed at delivery and this expression was repressed within the next 24h. In calves inoculated with C. parvum, mRNA expression of interleukin-12 and interferon-gamma was noticed on day 3 post-inoculation (p.i.) and increased in the convalescent phase of the infection, whereas in non-inoculated calves no mRNA expression was detectable up to the end of the experiment. No mRNA expression of interleukin-4 or 6 was detected during the experiment. Our observations suggest that systemic Th1 type immune responses are induced in calves infected with C. parvum and may be available for evaluation of the control of the infection.
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PMID:Increase of Th1 type cytokine mRNA expression in peripheral blood lymphocytes of calves experimentally infected with Cryptosporidium parvum. 1271 45

Photopheresis has been claimed to have immune-modulating effects, but the mechanisms of action are unknown. This study investigated the immune effect of photopheresis in children with type 1 diabetes, with a focus on the balance of Th1- and Th2-like cytokines. Ten children with newly diagnosed type 1 diabetes (10-17 y) were treated with five double treatments of photopheresis and 10 children matched for disease, age, and gender were given placebo tablets and sham pheresis. Expression of IFN-gamma and IL-4 mRNA was determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and secretion of IFN-gamma, IL-10, and IL-13 in cell-culture supernatants by ELISA after stimulation with glutamic acid decarboxylase (GAD65) (a.a. 247-279), the ABBOS peptide (a.a. 152-169), insulin, phytohemagglutinin (PHA), and keyhole limpet hemocyanin (KLH). Photopheresis changed antigen-stimulated immune balance in line with a Th2-like shift. Thus, the ratio of IFN-gamma/IL-4 mRNA expression after in vitro stimulation with a peptide of the autoantigen GAD65 was reduced after treatment in the photopheresis group. The IFN-gamma/IL-4 mRNA expression ratio after in vitro stimulation with insulin was also lower in children treated with photopheresis compared with the placebo group. Photopheresis has an immune-modulating effect in children with type 1 diabetes, causing a Th2-like deviation.
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PMID:The immunological effect of photopheresis in children with newly diagnosed type 1 diabetes. 1614 57

Mechanical stimuli are known to have major influences on chondrocyte function. The molecular events that regulate chondrocyte responses to mechanical stimulation have been the subject of much study. Using an in vitro experimental system we have identified mechanotransduction pathways that control molecular and biochemical responses of human articular chondrocytes to cyclical mechanical stimulation, and how these responses differ in cells isolated from diseased cartilage. We have previously shown that mechanical stimulation of normal articular chondrocytes leads to a cell membrane hyperpolarisation. Within 1 hour following mechanical stimulation there is an increase in aggrecan mRNA levels. These responses are mediated via alpha5beta1 integrins, the neuropeptides substance P and NMDA, and the cytokine interleukin-4. In OA chondrocytes mechanical stimulation leads to cell membrane depolarisation, but no change in aggrecan mRNA at 1 hour. The depolarisation response is mediated via alpha5beta1 integrins, substance P and interleukin-4, but the cells show an altered response to NMDA. Having identified that the NMDA receptor is present in human articular cartilage and may play an important role in a chondroprotective mechanotransduction pathway, we were interested in whether other components associated with NMDA signalling may be involved in the chondrocyte mechanotransduction pathways. One such component is calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII mediates many cellular responses to elevated Ca2+ in a wide variety of cells and tissues. It is involved in the regulation of ion channels, cytoskeletal dynamics, gene transcription, neurotransmitter synthesis, insulin secretion, and cell division. CaMKII also shows a broad substrate specificity and is abundant in brain tissue, indicating that this kinase may play a number of roles in the functioning of the central nervous system. This kinase has been studied extensively in brain, but there is only a limited understanding of CaMKII in other tissues. CAMKII has four subunit isoforms (alpha,beta,gamma,delta). The alpha- and beta-isoforms have narrow distributions restricted mainly to neuronal tissues, but the gamma- and delta-isoforms are ubiquitously expressed within neuronal and non-neuronal tissues. The aim of this study was to investigate the expression of CaMKII in normal and OA cartilage and chondrocytes, and whether this enzyme is involved in the response of chondrocytes to cyclical mechanical stimuli. Reverse transcriptase-polymerase chain reaction (RT-PCR), using primers specific for the different CaMKII isoforms, was carried out to assess which isoforms are expressed in human articular chondrocytes. To assess whether CaMKII is expressed in human articular chondrocytes at the protein level, cultured chondrocytes were extracted and analysed by Western blotting using a pan-CaMKII antibody. Immunohistochemistry was carried out to investigate whether CaMKII is expressed by human articular chondrocytes in vivo. Frozen sections of normal, OA and ankle cartilage were incubated for one hour with CaMKII antibody and visualised using ABC and DAB. To assess the role of CaMKII in the mechanotransduction responses of normal and OA chondrocytes, human normal and OA articular chondrocytes were mechanically stimulated at 0.33 Hz, or by addition of recombinant IL-4 for 20 minutes. Cell responses to these stimuli, in the absence or presence of an inhibitor of CaMKII were assessed by measuring changes in cell membrane potential or changes in relative levels of aggrecan mRNA compared with the housekeeping gene GAPDH. Normal, OA, and ankle chondrocytes expressed the gamma and delta isoforms of CaMKII mRNA, but not the alpha and beta isoforms as demonstrated by RT-PCR. Western blotting showed a band at approximately 60 kDa consistent with the expression of CaMKII. Immunohistochemistry revealed the positive staining in the middle and deep zones, but not the superficial zone, of normal, OA, and ankle cartilage. The presence of a CaMKII inhibitor inhibits the membrane hyperpolarisation response and upregulation of aggrecan mRNA in normal chondrocytes following mechanical stimulation, but has no effect on the hyperpolarisation response to recombinant IL4. The depolarisation response of OA chondrocytes to mechanical stimulation is unaffected by the presence of the CaMKII inhibitor. The CaMKII isoforms gamma and delta are expressed in both normal and OA chondrocytes, both in vitro and in vivo, but are only involved in the response of normal chondrocytes to mechanical stimulation. This response is upstream of the effect of IL4. These findings are consistent with previous findings for the NMDA receptor, and suggest that dysregulation of NMDA-CaMKII signalling may be important in onset and progression of osteoarthritis.
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PMID:Calcium/calmodulin-dependent protein kinase II in human articular chondrocytes. 1691 96

In order to investigate the expression of Th1 and Th2 cytokines in the vaginal candidiasis caused by Candida, the fungal vaginitis model was established in female ICR mice by intravaginal inoculation of suspension of C. albicans after the animals were pretreated with estradiol. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of IL-2, IL-4, IL-10 and TGF-beta1 in the vagina in the mice of different groups at different time points after the beginning of the experiment. The average expression level of IL-2 mRNA in group D (estrogen-treated mice) was significantly higher than that in groups H (estrogen-untreated mice) and I (control group) on the day 2. The average expression level of IL-4 mRNA in group D was significantly higher than that in groups I and H on the day 5. The average expression level of IL-10 mRNA in group D was significantly higher than that in groups H and I from day 7 to 11. The average expression level of TGF-beta1 mRNA in group D was significantly higher than that in groups H and I at all time points. It was concludes that the high-level expression of IL-2 mRNA during early infection was associated with clearance of mucosal C. albicans, and the high-level expression of IL-10 mRNA during late stage of the infection was related to susceptibility to infection. TGF-beta1 may play a predominant role when the virtual absence of changes in other Th-type cytokines during infection.
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PMID:Local Th1/Th2 cytokine expression in experimental murine vaginal candidiasis. 1856 41


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