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)

Steroidogenic factor 1, a member of the fushi tarazu factor 1 (FTZ-F1) subfamily of nuclear receptors, is a key regulator in mammalian reproduction. From an embryonic complementary DNA library, the zebrafish homolog of FTZ-F1 (zFF1A) and an alternatively spliced variant (zFF1B) were isolated. zFF1B represented a C-terminally truncated version of zFF1A. Whole mount in situ hybridization and reverse transcriptase-PCR analysis revealed that both zFF1A and B transcripts were present in the developing pituitaries, adult fish brain, gonads, and liver, albeit zFF1B messenger RNA was absent in testis. Comparison of the primary sequences of zFF1 with those of other FTZ-F1 subfamily members showed a close structural relationship between the mouse liver receptor homolog, which activated the alpha1-fetoprotein gene in rodent liver. However, similar to mouse steroidogenic factor 1, zFF1A regulated chinook salmon gonadotropin IIbeta subunit gene expression. On the contrary, zFF1B, which could bind a consensus gonadotrope-specific element with an affinity similar to that of zFF1A, lacked both the trans-activation function and synergistic interaction with the estrogen receptor. Furthermore, cotransfection studies in HeLa cells showed that zFF1B was a strong competitor for the action of zFF1A on the chinook salmon gonadotropin IIbeta subunit gene promoter. Our investigation suggests that 1) zFF1 represents an ancestor protein of the vertebrate FTZ-F1 homologs; 2) the antagonistic relationship between zFF1A and -B may dictate the expression of the FTZ-F1 target genes in a variety of tissues, including the pituitary; and 3) the naturally occurring zFF1B provides evidence that the C-terminal portion of zFF1A (80 amino acid residues) contains a major trans-activation function and a protein-protein interface.
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PMID:Teleost FTZ-F1 homolog and its splicing variant determine the expression of the salmon gonadotropin IIbeta subunit gene. 917 48

This study compared expression of two estrogen receptor (ER alpha and ER beta) genes in the rat upper gastrointestinal tract and the effects of 17 beta-estradiol administration on gastric trefoil factor family (TFF) mRNA steady-state levels in ovariectomized rats. Estrogen receptor alpha and beta cDNA fragments from fundic mucosa were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and sequenced. Both ER subtypes were detected in fundus, antrum and duodenum by RT-PCR. Northern analysis of poly(A)+ mRNA from fundic mucosa showed that ER alpha mRNA is expressed as a single transcript at 6.5 kb and ER beta is expressed as multiple transcripts with major transcripts ranging from 1.1-4.7 kb. ER beta mRNA was expressed in greater abundance than ER alpha mRNA. Fundic TFF2 mRNA steady-state levels were increased by 17 beta-estradiol administration in ovariectomized rats with no significant change in TFF1 mRNA levels. These studies show expression of both ER subtypes in the rat upper gastrointestinal tract with regulation of TFF2 mRNA by 17 beta-estradiol. These results suggest that estrogens, probably acting via ER beta, have a direct role in regulating gastric physiology.
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PMID:Estrogen receptor alpha and beta expression in upper gastrointestinal tract with regulation of trefoil factor family 2 mRNA levels in ovariectomized rats. 938 4

The growth regulatory effects of PRL on the human breast are mediated by its receptor (PRLr), a member of the cytokine receptor family. Recent reverse transcriptase-PCR studies by our laboratory and others have shown PRL expression within breast tissues at the RNA level. To confirm the role of this growth factor-receptor complex in normal and malignant breast tissues, the expression of PRL and PRLr was examined in parallel with the estrogen receptor (ER) and progesterone receptor (PR). Sixty-nine cases of primary invasive breast carcinoma were examined for PRL and PRLr expression by in situ hybridization and immunohistochemical technique, respectively. These data revealed widespread expression of PRL and its receptor in the breast cancers studied (>95%) and in the normal breast tissues (>93%), with no association between the expression of PRL-PRLr and ER or PR. These findings stand in contrast to prior RIA-based studies that detected the PRLr in only 20-60% of breast carcinomas, most commonly in ER-PR-positive cells. These results confirm prior data indicating the presence of an autocrine/paracrine loop for the PRL-PRLr complex within human breast tissues. Given the widespread expression of PRL-PRLr in breast cancer, pharmacological interventions aimed at the inhibition of function of this growth regulatory receptor complex may be of considerable utility in the therapy of this disease.
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PMID:Expression of prolactin and its receptor in human breast carcinoma. 938 44

Two novel transcripts of human estrogen receptor (ER) have been identified that differ in the 5' untranslated sequence. It has previously been determined that an alternate ER transcript is generated from transcription initiated upstream of the main ER cap site (P1), and utilizes a splice acceptor site at +163. Here we report the isolation of 21 ER clones from a MCF7 cDNA library. Eleven of these clones correspond to transcripts that initiate at the P1 cap site, whereas the remaining 10 clones are derived from two previously unidentified ER transcripts (designated E and H) that both utilize the +163 splice acceptor site. A panel of breast and endometrial carcinoma cell lines were screened by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of the E and H transcripts. It was found that all ER-positive cell lines expressed both of the novel transcripts. In addition, 10 primary human breast cancers were analyzed, of which six expressed the E transcript and five abundantly expressed the H transcript. These data indicate that expression of ER in human breast cancers can be dependent upon an alternate promoter at least 20 kb upstream of the primary cap site for ER.
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PMID:Identification of two estrogen receptor transcripts with novel 5' exons isolated from a MCF7 cDNA library. 939 49

Bone morphogenetic proteins (BMPs) induce differentiation of osteoblast and chondroblast lineage cells from uncommitted mesenchymal precursors. Because estrogen has potent osteochondrogenic actions, we investigated its effect on BMP production in two estrogen-responsive, human immortalized cell lines (hFOB/ER3 and hFOB/ER9) that display the mature osteoblast phenotype. These cell lines were produced by stable transfection of the estrogen receptor (ER) gene into immortalized fetal osteoblasts at low ( approximately 800 ER/ nucleus) and at high ( approximately 3, 900 ER/nucleus) levels, respectively. As assessed by reverse transcriptase PCR, treatment with 17beta-estradiol (10(-)10 - 10(-)7 M) increased steady-state levels of BMP-6 mRNA dose dependently by twofold in the hFOB/ER3 cells and by over threefold in the hFOB/ER9 cells. Messenger RNA levels for transforming growth factors-beta1 and -beta2 and BMPs-1 through -5 and -7 levels were unchanged. The results were confirmed by sequence determination of the PCR product and by Northern blot analysis for total RNA. 17beta-estradiol increased BMP-6 protein production sixfold by Western analysis. Cotreatment with antiestrogens (ICI 182,780 or 4-hydroxytamoxifen) antagonized the effects of 17beta-estradiol. These data suggest that some of the skeletal effects of estrogen on bone and cartilage may be mediated by increased production of BMP-6 by osteoblasts.
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PMID:Bone morphogenetic protein-6 production in human osteoblastic cell lines. Selective regulation by estrogen. 943 14

The effects of estrogen on bone are possibly mediated by several cell types. In the present study, the effect of 17beta-estradiol (E2) on osteoblast-like cells was investigated by using mouse bone marrow cultures. Bone marrow cells were harvested from the shafts of femurs of 10-week-old NMRI mice and cultured. On day 6, confluent primary cultures were trypsinized and subcultured. Under the conditions used (Keila, S., Pitaru, S., Grosskopf, A., and Wernreb, M. Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro. J Bone Miner Res 9:321-327; 1994), the bone marrow cultures showed differentiation towards the osteoblastic phenotype. This was demonstrated by the appearance of osteoblastic markers such as alpha1(I) collagen (COL1), alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OP), and transforming growth factor-beta1 (TGFbeta1), which were detected by using reverse transcriptase polymerase chain reaction (RT-PCR). Bone nodule formation, including deposition of collagen fibers and matrix mineralization, was also studied at several time points of the 3-week culture period. The effect of E2 on the appearance of osteoblastic markers was studied by incubating cultures in the presence or absence of the hormone. The messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) was found to be expressed at all time points as demonstrated by RT-PCR. When grown with E2, the rate of cell proliferation was increased in the early phase of cultures, but not after day 6. The addition of E2 in subcultures resulted in an increase of levels of mRNA for COL1, ALP, OCN, OP, and TGF-beta1. ALP activity was also increased. Bone nodule formation, as well as calcium contents, were significantly increased in the cultures grown in the presence of E2. All E2 concentrations used (0.01-10 nmol/L) were effective but the maximum response was obtained with 0.1 nmol/L E2. Addition of the antiestrogen ICI 182,780 abolished the E2-induced stimulation of proliferation and later an increase in ALP activity. Addition of ICI 182,780 without the hormone did not cause any changes when compared to control cultures. In conclusion, our results demonstrate that E2 stimulates sequential differentiation of osteoblasts and increases deposition and mineralization of matrix in mouse bone marrow cultures in an estrogen receptor-dependent manner.
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PMID:Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture. 951 12

The bcl-2 family of proteins includes some important regulators of apoptosis. Among these, bcl-2 and bcl-xL prevent cells from entering apoptosis, whereas bax and bcl-xS can induce cell death. Alterations in the control of this process can lead to a decrease in cell death, thus contributing to neoplastic growth. Diminished susceptibility to chemotherapy has also been attributed, in in vitro systems, to alterations in the levels of bcl-2, bax, or bcl-x. We analyzed the expression of bcl-2, bax, bcl-xL, and bcl-xS in normal and neoplastic ovarian tissues by reverse transcriptase-PCR and Western blotting. The RNA and protein levels were significantly correlated for all genes. Interestingly, the levels of these genes in normal and neoplastic tissues were significantly different: bcl-2 was higher in normal tissue (P < 0.002), whereas bax and bcl-xL were higher in carcinoma (P < 0.018 and P < 0.030, respectively). bcl-xS was present at low levels in 83% of neoplastic samples and was undetectable in normal tissue. Reverse transcriptase-PCR analysis of 74 tumors showed no major correlation with clinicopathological parameters or with response to chemotherapy. Only bax and bcl-xL were correlated with progesterone receptor levels (n = 29, r = +0.44, P < 0.0189, and r = -0.40, P < 0.035, respectively). No correlation was found with estrogen receptor levels or with p53 immunostaining. Our data indicate that the regulation of the bcl-2 family of proteins differs between normal and neoplastic ovarian tissues. Moreover, the modulation of these genes in ovarian carcinoma is different compared to other tissues; therefore, tissue specificity is very important in regulation of the bcl-2 family of proteins.
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PMID:bcl-2, bax, bcl-XL, and bcl-XS expression in normal and neoplastic ovarian tissues. 951 44

To evaluate the clinical significance of drug resistance mechanisms in breast cancer, we examined the expression of MDR1 and MRP in primary breast carcinoma and normal adjacent tissue using a highly quantitative and reproducible reverse transcription-PCR assay. Expression of both genes was observed in all specimens examined, both tumor (n = 74) and normal adjacent tissue (n = 55). The expression of MDR1, however, was low, with the level of expression being 25 times less than the drug-resistant control cell line KB 8-5. Immunohistochemical analysis of P-glycoprotein corroborated the PCR results; only 6% (2 of 31) were positive for JSB1 staining, and 0 of 32 were positive for for UIC2. MRP expression did not exceed control cell line levels, and immunohistochemistry detected moderate levels of expression. MDR1 expression was independent of grade, stage, tumor size, nodal status, metastasis, and estrogen receptor and progesterone receptor status. There was, however, a significant correlation of MDR1 expression with age and histology. Approximately twice the expression of MDR1 was observed in the < 50 age group compared to the > 50 age group, and lobular carcinoma had 4 times the expression of MDR1 of other histological types. MRP expression was independent of all other clinical parameters. Thus, these results show that although MDR1 expression is detectable in primary breast carcinoma by PCR, this expression as measured by quantitative reverse transcriptase-PCR is extremely low. The significance of these low levels is yet to be determined. MDR1 expression was higher in < 50 age group and lobular carcinoma, which may contribute to poor prognosis associated with young age and lobular histology.
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PMID:Quantitative reverse transcriptase-polymerase chain reaction measured expression of MDR1 and MRP in primary breast carcinoma. 962 74

Estrogens may influence the expression of various cytokines, adhesion molecules, von Willebrand factor and prostacyclin produced by endothelial cells. However, reports concerning expression of the estrogen receptor in endothelial cells are controversial. Primary human umbilical vein endothelial cells (HUV-EC), the non continuous human umbilical vein endothelial cell line HUV-EC-C (ATCC CRL 1730) and endothelial cells from 10 frozen umbilical cords were analyzed for the expression of the estrogen receptor. Immunological studies using estrogen receptor specific antibodies failed to detect the expression of the receptor in all human umbilical vein endothelial cells tested. No estrogen receptor transcripts were found in primary HUV-EC or HUV-EC-C by reverse transcriptase-polymerase chain reaction. Weak hybridization signals were detected when the PCR amplicons were hybridized with estrogen receptor cDNA sequences as a probe. In vitro protein-DNA interaction studies revealed no complexes between a fully consensus estrogen response element and HUV-EC-C extracts. Finally, transient transfection studies in HUV-EC-C could not demonstrate 17beta-estradiol-induced transcription of the beta-galactosidase reporter gene linked to a consensus estrogen response element. These observations suggest that human umbilical vein endothelial cells lack the estrogen receptor.
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PMID:Human umbilical vein endothelial cells lack expression of the estrogen receptor. 983 29

Several environmental chemicals are known to have estrogenic activity by interacting with development and functions of endocrine systems in nearly all classes of vertebrates. In order to get a better insight of potential estrogenic effects on amphibians caused by environmental pollution this study aims to develop a model for investigating endocrine disruptors using the amphibian Xenopus laevis. In that model the potential estrogenic activity of endocrine disruptors is determined at several levels of investigation: (I) binding to liver estrogen receptor; (II) estrogenicity in vitro by inducing vitellogenin synthesis in primary cultured hepatocytes; and (III) in vivo effects on sexual development. Here we deal with establishing methods to assay estrogenic activity of environmental chemicals in vitro and in vivo. In vitro we used a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique to determine mRNA-induction of the estrogenic biomarker vitellogenin in primary cultured hepatocytes of male Xenopus laevis. Time courses of vitellogenin-mRNA in the presence and absence of 10(-6) M 17 beta-estradiol (E2) resulted in a marked loss of mRNA from controls after 2 days while E2 treatment kept vitellogenin-mRNA at a relatively stable level. After 36 h of incubation estrogenic activities of E2, 4-nonylphenol (NP), and 2,2-bis-(4-hydroxyphenyl)-propan (bisphenol A) at concentrations ranging from 10(-10) to 10(-5) M were assayed by RT-PCR of vitellogenin-mRNA and showed the following ranking of dose-dependent potency: E2 > NP > bisphenol A. These in vitro results were confirmed further by in vivo experiments determining sexual differentiation of Xenopus laevis after exposure to E2 and environmental chemicals during larval development. Concentrations of 10(-7) and 10(-8) M E2 as well as 10(-7) M of NP or bisphenol A caused a significant higher number of female phenotypes compared to controls indicating a similar ranking of estrogenic potencies in vivo as in vitro. In addition, butylhydroxyanisol and octylphenol, both showed feminization at 10(-7) M while octylphenol was also effective at 10(-8) M. In summary these results demonstrate for the first time the use of a semiquantitative RT-PCR technique for screening estrogenicity by assaying mRNA induction of the estrogenic biomarker vitellogenin in vitro. The combination of this newly developed method with classical exposure experiments is necessary for determination of the biological significance of estrogenic chemicals.
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PMID:Amphibians as a model to study endocrine disruptors: II. Estrogenic activity of environmental chemicals in vitro and in vivo. 1002 3


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