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)

We describe the characterization of a spontaneously transformed chicken monocytic cell line that developed as a single colony of cells in a heterophil culture that was inadvertently left in the incubator over a period of 25 days. These cells, hitherto named HTC, grow efficiently at both 37 or 41 degrees C in culture medium containing either 5% FBS or 2% chicken serum. The HTC cells are acid phosphatase positive, show expressions of both class I and class II major histocompatibility complex (MHC), CD44, K1, and K55 cell surface antigens, and engulf latex beads, produce nitrite and interleukin-6 on stimulation with bacterial lipopolysaccharide (LPS). Treatment with phorbol myristate acetate (PMA) induces respiratory burst in HTC cells and the secretion of matrix metalloproteinase (MMP) into culture medium. Using gene-specific primers and reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of mRNA trancripts for interferon-gamma (IFN-gamma), interleukin-1 (IL-1), interleukin-6 (IL-6), nitric oxide synthase (NOS), matrix metalloproteinase-2 (MMP-2), and transforming growth factor-beta (TGF-beta) were detected. Lipopolysaccharide (LPS) treatment of HTC cells modulated IL-1, IL-6, IFN-gamma, NOS mRNA levels as detected by RT-PCR analyses. Using different avian tumor virus gene-specific primers and PCR, the HTC cells were positive for the presence of avian leukosis virus (ALV) and Marek's disease virus (MDV) but negative for reticuloendothelial virus (REV), chicken infectious anemia virus (CIAV), and herpes virus of turkeys (HVT). The production of ALV antigens by HTC cells was further confirmed using p27 gag protein ELISA. Collectively, these results show that the HTC cells belong to myeloid/macrophage lineage and were likely transformed by ALV and MDV but retain many interesting and useful biological activities.
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PMID:Characterization of a spontaneously transformed chicken mononuclear cell line. 1452 38

Radical prostatectomy as a primary treatment for clinically localized prostate cancer has increased dramatically over the past decade due to prostate-specific antigen (PSA) screening and the awareness of the increased incidence of localized disease. Despite the stage migration to increase clinically localized disease, there are still vast numbers of men who harbor occult extraprostatic extension and develop recurrence after surgery. The study of molecular markers in the blood or tissue of surgical patients prior to treatment, called " molecular staging, " is the focus of this review. The reverse transcriptase- polymerase chain reaction (RT-PCR) test for PSA gene expression in peripheral blood or bone marrow has received considerable attention since its first report in 1992. The test detects messenger RNA species for prostate-specific/abundant genes such as PSA and prostate-specific membrane antigen. These messenger RNAs were not detected in normal blood or bone marrow, but were detected in some prostate cancer patients presumably due to circulating prostatic epithelial cells. These prostate epithelial cells are thought to be occult metastases cells, and early studies correlated a positive RT-PCR test with surgical pathology adverse features such as positive margins. Despite the many studies over the past few years, there have been inconsistent results, and the most recent studies have not been able to confirm clinical utility. Bone marrow RT-PCR has been more promising; however, it is still a research tool that needs further study. The study of molecular markers in tissue material, ie, prostate biopsy samples prior to radical prostatectomy, is problematic due to the sampling error inherent in a multifocal heterogeneous tumor such as prostate cancer. The tumor suppressor proteins p53 and p27, Bcl-2 oncoprotein, Ki-67 proliferation index protein, E-cadherin, and microvessel density have been assessed in preradical prostatectomy needle biopsy. Results have been conflicting, and none are yet accepted as a clinically useful marker. Current and future work is focusing on analysis of multiple gene expressions or proteins simultaneously via gene chip or proteomics technology. While these expression profiles might be of value in whole prostate surgical specimens where tissues are well characterized, it is unclear how this new technology will be applied to the needle biopsy samples. Although molecular staging of radical prostatectomy patients has been under study for a decade, all assays remain research tools. Still, this area holds great promise for improving the accuracy of staging and providing a more accurate prognosis of individual men with clinically localized prostate cancer.
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PMID:Molecular markers in prostate cancer: the role in preoperative staging. 1504 12

Simian/human immunodeficiency virus SHIV(KU2) replicates with extremely high titers in macaques. In order to determine whether the DNA of the viral genome could be used as a vaccine if the DNA were rendered noninfectious, we deleted the reverse transcriptase gene from SHIVKU2 and inserted this DNA (DeltartSHIVKU2) into a plasmid that was then used to test gene expression and immunogenicity. Transfection of Jurkat and human embryonic kidney epithelial (HEK 293) cells with the DNA resulted in production of all of the major viral proteins and their precursors and transient export of a large quantity of the Gag p27 into the supernatant fluid. As expected, no infectious virus was produced in these cultures. Four macaques were injected intradermally with 2 mg of the DNA at 0, 8, and 18 weeks. The animals developed neutralizing antibodies and low enzyme-linked immunospot assay (E-SPOT) titers against SHIVKU2. These four animals and two unvaccinated control animals were then challenged with heterologous SHIV89.6P administered into their rectums. The two control animals developed viral RNA titers exceeding 10(6) copies/ml of plasma, and these titers were accompanied by the loss of CD4+ T cells by 2 weeks after challenge. The two control animals died at weeks 8 and 16, respectively. All four of the immunized animals became infected with the challenge virus but developed lower titers of viral RNA in plasma than the control animals, and the titers decreased over time in three of the four macaques. The fourth animal remained viremic and died at week 47. Whereas the control animals failed to develop E-SPOT responses, all four of the immunized animals developed anamnestic E-SPOT responses after challenge. The animal that died developed the highest E-SPOT response and was the only one that produced neutralizing antibodies against the challenge virus. These results established that noninfectious DNA of pathogenic SHIV could be used as a vaccine to prevent AIDS, even though the immunological assays used did not predict the manner in which the challenge virus would replicate in the vaccinated animals.
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PMID:A noninfectious simian/human immunodeficiency virus DNA vaccine that protects macaques against AIDS. 1573 Dec 36

1-(3,4-dimethoxyphenyl)-3,5-dodecenedione (I6), a gingerdione derivative, was synthesized in our laboratory, has been demonstrated to be an effective anti-tumor agent in human leukemia cells. Gingerdione is one of the components from ginger. In the present study, we found that I6 could inhibit cell proliferation in the time- and dose-dependent manner in human promyelocytic leukemia HL-60 cells. To investigate the anti-proliferation mechanism of I6, cell cycle analysis was performed. Results showed that I6 induced significant G1 arrest and apoptosis in HL-60 cells. It was proved by the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of regulatory on G1 arrest that the levels of p15 and p27 increased after treatment and mRNA levels of cyclin D2, cyclin E, and cdc25A were decreased. The I6-induced apoptosis was further confirmed by DNA fragmentation assay. The DNA gel electrophoresis showed that I6 induced DNA fragmentation, a biochemical hallmark of apoptosis, in HL-60 cells. I6-induced apoptosis was accompanied by an apparent up-regulation of caspase-3, and down-regulation of Bcl-2. Taken together, these results suggest that markedly down-regulation of G1 associated cyclin D2, cyclin E and cdc25A and up-regulation of CDKI, p15 and p27, and may contribute to I6-mediated cell cycle arrest. Furthermore, the Bcl-2 expression decrease and caspase-3 activation may be the plausible mechanism by which I6 induced apoptosis. These results suggest that I6 is a potent anti-HL-60 drug and possess a significant action on cell cycle before commitment for apoptosis occurrence.
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PMID:1-(3,4-dimethoxyphenyl)-3,5-dodecenedione (I6) induces G1 arrest and apoptosis in human promyelocytic leukemia HL-60 cells. 1592 54

Ty3, a member of the Metaviridiae family of long-terminal-repeat retrotransposons found in Saccharomyces cerevisiae, encodes homologs of retroviral Gag and Gag-Pol proteins, which, together with genomic RNA, assemble into virus-like particles (VLPs) that undergo processing and reverse transcription. The Ty3 structural proteins, capsid and nucleocapsid, contain major homology and nucleocapsid motifs similar to retrovirus capsid and nucleocapsid proteins, but Ty3 lacks a matrix-like structural domain amino terminal to capsid. Mass spectrometry analysis of Ty3 Gag3 processing products defined an acetylated Ser residue as the amino terminus of Gag3/p34, p27, and CA/p24 species and supported a model where p34 and p27 occur in phosphorylated forms. Using atomic force microscopy, VLPs were imaged from cells producing wild-type and protease and reverse transcriptase mutant Ty3. Wild-type VLPs were found to have a broad range of diameters, but the majority, if not all of the particles, exhibited arrangements of capsomeres on their surfaces which were consistent with icosahedral symmetry. Wild-type particles were in the range of 25 to 52 nm in diameter, with particles in the 42- to 52-nm diameter range consistent with T=7 symmetry. Both classes of mutant VLPs fell into a narrower range of 44 to 53 nm in diameter and appeared to be consistent with T=7 icosahedral symmetry. The smaller particles in the wild-type population likely correspond to VLPs that have progressed to reverse transcription or later stages, which do not occur in the protease and reverse transcriptase mutants. Ty3 VLPs did not undergo major external rearrangements during proteolytic maturation.
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PMID:Investigation by atomic force microscopy of the structure of Ty3 retrotransposon particles. 1595 49

In a previous study, it was found that even though more male cats were infected by feline leukaemia virus (FeLV), females seemed to progress easier to overt disease. To study the effect of female hormones, 17beta-estradiol and progesterone were added in different concentrations (10(-3) M to 10(-12) M) to a culture of persistently FeLV-infected cells. The effect of both hormones was very similar. After 24 h the cell viability was very low at 10(-3) M and 10(-4) M but similar to controls at the remaining concentrations. Liberation of viral particles was estimated by the reverse transcriptase activity (RT), which was the lowest also at 10(-3) M and 10(-4) M. However, low viability could not account for this low RT, as when cells were lysed with lysis buffer RT was high. Thus, cells were dying without freeing viral particles, suggestive of apoptosis. This possibility was confirmed by staining hormone-treated cells with annexin V and propidium iodide. The FeLV antigen p27 measured in the cultures had a maximum at 10(-3) M and 10(-4) M, higher than controls and lysed cells, so the presence of p27 in the supernatant was not only due to cell lysis but a consequence of hormone effect. In conclusion, 17beta-estradiol and progesterone induce death of FeLV-infected cells at high concentrations, probably through a process of apoptosis, which might limit the spread of the infection, as infective viral particles would be hampered from budding.
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PMID:Effect of 17beta-estradiol and progesterone on the expression of FeLV in chronically infected cells. 1602 97

Viral vectors available for gene therapy are either inefficient or suffer from safety concerns for human applications. Foamy viruses are non-pathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. In this report, we describe the use of simian foamy virus type 1 (SFV-1) vector to examine the efficacy of therapeutic genes. Hairpin short-interfering RNA (siRNA) that targets the simian immunodeficiency virus (SIV) rev/env was placed under the control of the PolIII U6 snRNA promoter for expression and screened for silencing target genes using cognate target-reporter fusions. We have identified an effective siRNA (designated R2) which reduces the rev and env gene expression by 89% and 95%, respectively. Using the simian foamy virus type 1 (SFV-1) based vector, we delivered the PolIII expressed R2 siRNA into cultured cells and challenged with SIV. The results show that the R2 siRNA is a potent inhibitor of SIV replication as determined by p27 expression and reverse transcriptase assays. Vectors based on a non-pathogenic SFV-1 vector may provide a safe and efficient alternative to currently available vectors, and the SIV model will help devise protocols for effective anti-HIV gene therapy.
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PMID:Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs. 1618 54

Signal transducer and activator of transcription 3 (STAT3) has oncogenic potential. The biological effects of STAT3 have not been studied extensively in the pathogenesis of colon cancer, nor has the role of Janus kinase 3 (JAK3), the physiological activator of STAT3, been evaluated. Here, we demonstrate that activated STAT3 (pSTAT3) and activated JAK3 (pJAK3) are expressed constitutively in two colon cancer cell lines, SW480 and HT29. To evaluate the significance of JAK3/STAT3 signaling, we inhibited JAK3 with AG490 and STAT3 with a dominant-negative construct. Inhibition of JAK3 down-regulated pSTAT3. The blockade of JAK3/STAT3 signaling significantly decreased viability of colon cancer cells due to apoptosis and cell-cycle arrest through down-regulation of Bcl-2, Bcl-X(L), Mcl-1, and cyclin D2 and up-regulation of p21(waf1/cip1) and p27(kip1). We also examined histological sections from 22 tumors from patients with stage II or stage IV colon cancer and found STAT3, JAK3, and their activated forms to be frequently expressed. Furthermore, quantitative reverse transcriptase-polymerase chain reaction identified JAK3 mRNA in colon cancer cell lines and primary tumors. Our findings illustrate the biological importance of JAK3/STAT3 activation in the oncogenesis of colon cancer and provide novel evidence that JAK3 is expressed and contributes to STAT3 activation in this malignant neoplasm.
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PMID:Constitutive activation of JAK3/STAT3 in colon carcinoma tumors and cell lines: inhibition of JAK3/STAT3 signaling induces apoptosis and cell cycle arrest of colon carcinoma cells. 1619 33

Neurodegeneration in Alzheimer's disease and various experimental lesion paradigms are associated with an unscheduled upregulation of cell cycle-related proteins, indicating a link between cell cycle reactivation and neuronal death. Recent evidence, however, suggests that at least some of the canonical cell cycle regulators are constitutively expressed in differentiated neurons of the adult brain. Systematic investigations on the constitutive expression of cell cycle regulators in differentiated neurons in vivo, providing the basis for further insights into their potential role under pathological conditions, however, have not been carried out. Here, we demonstrate a constitutive neuronal expression of Cdks 1, 2, and 4; their activators cyclins D, A, B, and E; and their inhibitors p15(Ink4b), p16(Ink4a), p18(Ink4c), p19(Ink4d), p21(Waf1/Cip1), p27(Kip1), and p57(Kip2) within the neocortex of adult mice by western blot and immunocytochemistry. Expression was verified by single-cell reverse transcriptase-polymerase chain reaction applied to individual microscopically identified neurons captured with laser dissection. Immunoprecipitation and in vitro kinase assays revealed that Cdks 1, 2, and 4 are properly complexed to cyclins and exhibit kinase activity. This physiological expression of positive cell cycle regulators in adult neurons is clearly not related to neuronal proliferation. Taken together, our findings demonstrate a constitutive expression of functionally active cyclin-dependent kinases and their regulators in differentiated neurons suggesting a noncanonical role of cell cycle regulators potentially linked to neuronal plasticity and/or stability.
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PMID:Constitutive expression of functionally active cyclin-dependent kinases and their binding partners suggests noncanonical functions of cell cycle regulators in differentiated neurons. 1705 Jun 46

Cats exposed to feline leukemia virus (FeLV) may develop different outcomes of the infection. However, during acute infection blood proviral and viral RNA loads of cats with progressive and regressive infection are not significantly different. Thus, not the overall loads but rather those of specific leukocyte subsets may influence the infection outcome. By combining fluorescence activated cell sorting (FACS) with sensitive real-time TaqMan PCR and reverse transcriptase (RT) PCR, we established in the present study the methods to determine FeLV proviral and viral RNA loads in specific leukocyte subsets. In addition, they were applied to analyze long-term persistently FeLV-infected (p27-positive) and FeLV exposed but nonantigenemic (p27-negative), nonviremic cats. In the latter animals, CD4(+) and B lymphocytes exhibited the highest proviral loads, whereas in p27-positive cats, all leukocyte subsets showed similar high loads. In p27-positive cats, monocytes and granulocytes bore the highest viral RNA loads, whereas only one p27-negative cat was positive for viral RNA in T lymphocytes. To our knowledge, this is the first study to investigate FeLV proviral and viral RNA loads in leukocyte subsets of FeLV exposed cats. The herein described methods are important prerequisites to gain a deeper insight into the pathogenesis of FeLV infection.
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PMID:Cellular segregation of feline leukemia provirus and viral RNA in leukocyte subsets of long-term experimentally infected cats. 1743 24


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