Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80 kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92 kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the alpha- and beta-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92 kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120 kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92 kDa or 120 kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated beta-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.
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PMID:Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis. 1204 27

Skin and hair colour mostly depend on the activity of melanogenic melanocytes. Numerous proteins involved in melanocyte function have been identified including pMel-17, Mitf-M, Sox10, tyrosinase, tyrosinase related proteins-1 (TRP-1) and -2 (TRP-2). In the hair, melanogenic activity occurs only during the anagen phase of the hair cycle. In order to evaluate the implications of some known melanogenic proteins in human hair pigmentation, we performed immunohistochemical studies to reveal the expression of pMel-17, Mitf-M, tyrosinase, TRP-1 and TRP-2 in active bulb melanocytes of eumelanic brown and black anagen hairs of different ethnic origins, e.g. brown Caucasian, black Asian and African hairs. The labelling was compared with that observed in Caucasian and African scalp epidermis (interfollicular epidermis) melanocytes. We found that while pMel-17, TRP-1 and TRP-2 were expressed in epidermal melanocytes irrespective of ethnic origin and melanin content of the scalp epidermis, Mitf-M and tyrosinase expression were clearly evidenced only in pigmented epidermis, e.g. African scalps. Regarding human hair, pMel-17, Mitf-M, tyrosinase and TRP-1 were detected in a similar manner in active bulb melanocytes of brown and black hairs. In contrast and unexpectedly, TRP-2 could not be detected in hair bulb melanocytes, whatever the hair colour and ethnic origin. The lack of TRP-2 was further confirmed by western blot analyses. Reverse transcriptase-polymerase chain reaction (RT-PCR) performed on hair bulb mRNA demonstrated that Mitf-M, tyrosinase and TRP-1 amplimer signals were easily detected, whereas the TRP-2 amplimer signal was barely detectable. Furthermore Sox10 was not detected in hair bulb. Altogether our results suggest that the absence of detectable level of TRP-2 is due to transcriptional control in active melanocytes of human eumelanic hair bulbs. According to the absence of TRP-2 in melanin-producing melanocytes of brown and black hair bulbs, one must consider that eumelanogenesis as well as brown and black colour do not require TRP-2 expression in human hair.
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PMID:Absence of TRP-2 in melanogenic melanocytes of human hair. 1535 35

Melanoma antigen recognized by T cells 1 (MART-1) and tyrosinase-related protein-2 (TRP-2) are two useful markers for immunohistochemical detection of melanocytic tumors. However, these markers may be passively acquired (phagocytosed) rather than actively synthesized. Reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) can amplify even small amounts of specific mRNA in cells and therefore confirm the cellular source of a marker. We developed a one-step RT in situ PCR procedure in which Thermus thermophilus DNA polymerase synthesizes and amplifies cDNA from mRNA in a single reaction mixture. To examine its practicability and feasibility with formalin-fixed, paraffin-embedded (FFPE) tissue, we compared the results of one-step RT in situ PCR with those of immunohistochemistry (IHC). MART-1 mRNA was identified in the cytoplasm of lesional cells from 23/26 primary melanomas (92%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). MART-1 epitope was detected by IHC in 23/24 primary melanomas (96%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). TRP-2 mRNA was identified in the cytoplasm of lesional cells from 17/26 primary melanomas (65%), 6/9 metastatic melanomas (67%) and 4/6 nevi (67%). TRP-2 epitope was detected by IHC in 20/24 primary melanomas (83%), 9/9 metastatic melanomas (100%) and 4/6 nevi (67%). Both techniques detected MART-1 and TRP-2 in FFPE melanoma cell lines. Neither marker was detected in squamous cell carcinomas or basal cell carcinomas by RT in situ PCR or IHC. We conclude that the RT in situ PCR technique can be successfully applied to FFPE tissue to determine the cellular sources of gene expression observed by conventional PCR approaches.
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PMID:RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi. 1820 35