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)

It is believed that long-term cultures of CML marrow cells favor the outgrowth of BCR/ABL negative hematopoietic progenitor cells (HPC) and that this phenomenon may be enhanced with negative hematopoietic regulators which can maintain primitive HPC in a quiescent state. Proliferation of CML marrow CD34+ cells in primary short-term cultures, maintained in the presence or absence of macrophage inhibitory protein-1 alpha (MIP-1 alpha), was tracked with the membrane dye PKH2. After 7 to 10 days it was possible to distinguish between cytokine responsive (CR) CD34+ cells (cells which had divided thus becoming PKH2dim) and cytokine nonresponsive (CNR) CD34+ cells (cells which had not divided and had therefore remained PKH2bright). CR and CNR CD34+ cells were isolated by flow cytometric cell sorting, seeded in secondary long-term cultures, and their progeny cells assayed weekly for their clonogenic progenitor cell content and expression of BCR/ABL by reverse transcriptase polymerase chain reaction (RT-PCR). Whereas CNR cells isolated from control primary cultures (control/CNR) sustained in vitro hematopoiesis, similar cells from cultures treated with MIP-1 alpha (MIP-1 alpha/CNR) supported a higher and, in some patients, a more extended production of clonogenic HPC, indicating that MIP-1 alpha was able to maintain primitive HPC in a quiescent state. Predominance of BCR/ABL negative progenitors in vitro was more evident in secondary cultures initiated with CNR cells than in those initiated with CR cells, especially those established with MIP-1 alpha/CNR cells. Of interest is the observed decline in the percentage of BCR/ABL+ progenitors in these cultures with time. Whereas up to 100% of progenitors were BCR/ABL+ on day 0, by day 14, only 46% of progenitors in MIP-1 alpha/CNR secondary cultures were BCR/ABL+ and by day 28 and beyond, the percentage of BCR/ABL+ progenitors dropped to below 20%. These results suggest that the quiescent nature of normal HPC present in CML marrow may favor their identification via cell tracking and, subsequently, their isolation from the more actively cycling leukemic cells. These studies also confirm the feasibility of employing negative hematopoietic regulators to augment the sequestration of normal HPC among the cytokine nonresponsive fraction of CD34+ cells, an approach that may be clinically feasible for autotransplantation.
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PMID:Preferential sequestration in vitro of BCR/ABL negative hematopoietic progenitor cells among cytokine nonresponsive CML marrow CD34+ cells. 920 15

Chemokines are small proteins that selectively activate and recruit leukocytes to sites of inflammation. Several of them, including the CC chemokines RANTES, MIP-1 alpha, MIP-1 beta, MCP-1, and the CXC chemokines IL-8, GRO-alpha, ENA-78 have been identified in rheumatoid synovium, implicating a potential role for these molecules in rheumatoid arthritis. We have investigated the expression patterns of CC chemokine receptors in the joints of mice with collagen-induced arthritis, a model for human rheumatoid arthritis. In addition, we have investigated the incidence and severity of arthritis in mice receiving administration of MetRANTES, a modified chemokine which is a nanomolar antagonist of certain CC chemokine receptors. The mRNA expression pattern of the chemokines and their receptors in the joints of arthritic mice was investigated using reverse transcriptase-PCR and in situ hybridization. An upregulation of the CC chemokine receptors mCCR1, mCCR2; mCCR3 and mCCR5 was found in the joints from arthritic mice, compared to control animals. In addition, injections of MetRANTES reduced the incidence of disease in a dose dependent manner. Furthermore, in MetRANTES-treated mice that did develop arthritis a significantly lower severity of disease was observed compared with control animals. Our data clearly demonstrate a role for CC chemokines and their receptors in inflammatory joint destruction and support the use of chemokine receptor antagonists as potential tools to control inflammatory diseases such as rheumatoid arthritis.
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PMID:Effect of a CC chemokine receptor antagonist on collagen induced arthritis in DBA/1 mice. 923 36

Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3alpha or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3alpha is expressed predominantly in inflamed and mucosal tissues. MIP-3alpha produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34(+)-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3alpha was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1-5 and CXCR1 and CXCR2, suggesting that MIP-3alpha acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3alpha receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3alpha, and that its activation leads to pertussis toxin-sensitive and phospholipase C-dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.
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PMID:Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3alpha from lung dendritic cells. 929 37

Following traumatic injury to the spinal cord, hematogenous inflammatory cells including neutrophils, monocytes, and lymphocytes infiltrate the lesion in a distinct temporal sequence. To examine potential mechanisms for their recruitment, we measured chemokine mRNAs in the contused rat spinal cord, using specific and sensitive reverse transcriptase polymerase chain reaction (RT-PCR) dot-blot hybridization assays. The neutrophil chemoattractant GRO-alpha was 30-fold higher than control values at 6 hr postinjury and decayed rapidly thereafter. LIX, a highly related alpha-chemokine, also was elevated early postinjury. Monocyte chemoattractant peptide (MCP)-1 and MCP-5 mRNAs, potent chemoattractants for monocytes, were significantly elevated at the lesion epicenter at 12 and 24 hr postinjury and declined thereafter. Interferon-gamma-inducible protein, 10 kDa (IP-10), chemoattractant towards activated T-lymphocytes, was significantly elevated at 6 and 12 hr postinjury. The dendritic cell chemoattractant MIP-3alpha also was increased, perhaps contributing to the development of T-cell autoreactivity to neural components after spinal cord injury (SCI) in rats. Other beta-chemokines, including MIP-1alpha and RANTES (regulated on expression normal T-cell expressed and secreted), were minimally affected by SCI. Expression of chemokines, therefore, directly precedes the influx of target neutrophils, monocytes, and T-cells into the spinal cord postinjury, as noted previously. Thus, selective chemokine expression may be integral to inflammatory processes within the injured spinal cord as a mechanism of recruitment for circulating leukocytes.
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PMID:Selective chemokine mRNA accumulation in the rat spinal cord after contusion injury. 969 65

Chemokines play an important role in the selective movement of leucocytes into inflammatory areas and they also activate various cells in inflamed tissues. However, it is unclear which cells are the main sources of chemokines in actual inflammatory diseases, even though both mononuclear cells and non-inflammatory resident cells are able to produce chemokines in vitro and the former cells are also the main target of chemokines. To clarify the roles of chemokines that are produced by mononuclear cells in AD, we measured levels in vivo of mRNA for IL-8 and MIP-1 alpha, as well as the level of regulated upon activation normal T cell expressed and secreted (RANTES) mRNA in freshly isolated peripheral blood mononuclear cells from patients with AD. We compared the results with those from psoriatic patients, and patients without AD who were suffering from other cutaneous diseases and eosinophilia. Levels of mRNAs were determined by semiquantitative reverse transcriptase-polymerase chain reactions. Levels of IL-8 and MIP-1 alpha mRNA were elevated not only in atopic patients but also in non-atopic patients with inflammatory skin disease associated with eosinophilia, compared with levels in psoriatic patients and healthy controls. Levels of RANTES mRNA were similar in atopic patients but they were lower in the other two groups of patients when compared with levels in healthy controls. In atopic patients, the levels of both IL-8 and MIP-1 alpha mRNAs but not of RANTES mRNA decreased with improvements in symptom scores after therapy. These findings suggest that mononuclear cells are not only the target of chemokines but might also play an important role in the pathogenesis of AD by producing IL-8 and MIP-1 alpha.
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PMID:Increased levels in vivo of mRNAs for IL-8 and macrophage inflammatory protein-1 alpha (MIP-1 alpha), but not of RANTES mRNA in peripheral blood mononuclear cells of patients with atopic dermatitis (AD). 1044 53

The CC chemokine macrophage inflammatory protein-3alpha (MIP-3alpha) is the product of recent electronic cloning efforts, however, little characterization of its spectrum of biological effects has been undertaken. Human eosinophils exhibited pertussis-toxin-sensitive migration in response to human recombinant (hr)MIP-3alpha. Messenger RNA for the MIP-3alpha receptor, CCR-6, and low levels of surface expression were demonstrated by reverse transcriptase-polymerase chain reaction and FACS analysis. Analyses of cell signaling revealed dose-dependent increases in intracellular calcium mobilization, calcium transients that were, however, greatly reduced when compared with MCP-3-induced responses. Further investigations of MIP-3alpha-induced signal transduction revealed time- and dose-dependent, partially pertussis toxin-dependent, increases in phosphorylation of the p42/p44 mitogen-activated protein kinases (MAPK) that occurred at 10- to 100-fold lower concentrations, and that were linked to a phosphoinositide 3-kinase pathway. These results suggest that MIP-3alpha can regulate multiple, parallel signal transduction pathways in eosinophils, and suggest that MAPK activation by MIP-3alpha in eosinophils is a significant signaling pathway for migration induction.
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PMID:MIP-3alpha induces human eosinophil migration and activation of the mitogen-activated protein kinases (p42/p44 MAPK). 1053 25

Macrophages have a multifaceted role in wound healing. While their initial activity may be in the degradation and elimination of damaged tissue, macrophages also produce and secrete a variety of mediators that can participate in the repair process as well. To perform these functions, macrophages must be recruited to a wound site. Our purpose was to examine the temporal and spatial expression of macrophage chemoattracting cytokines (chemokines) at a surgical wound site. A surgical wound was prepared on the dorsal aspect of B6AF1/J mice. Biopsies were obtained from the wound and a comparable nonwounded area between 6 and 72 hr after wounding. The presence or absence of various chemokine mRNAs was detected by the reverse transcriptase-polymerase chain reaction (RT-PCR). Immunohistochemical staining and in situ RT-PCR determined localization of cells producing chemokines. In wounded tissue, both macrophage chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1) were detected; however, the time of expression differed for each molecule. MCP-1 mRNA was detected at 6 hr after wounding, with decreased expression at subsequent time periods. In contrast, MIP-1 messages were not observed until 24 hr after wounding, and steadily increased thereafter. MCP-1 and MIP-1 mRNA and protein were localized predominantly in keratinocytes. The rapid and strong expression of MCP-1 and MIP-1 messages within the wound site suggests a pivotal role for these chemokines in the repair process. The differences in appearance and level of expression over time, however, suggest distinctive functions for each chemokine and indicate that the local milieu, rather than a single cytokine, influences macrophage recruitment and/or activation.
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PMID:Differential expression of chemokines in a mouse model of wound healing. 1080 66

Nitric oxide is a crucial mediator of several forms of glomerulonephritis. We examined the effects of NO on the mRNA expression pattern in glomerular mesangial cells by using a low-stringency reverse transcriptase-polymerase chain reaction method and detected a cDNA fragment that was induced by interleukin 1b (IL-1b) and further up-regulated by the NO donor diethylenetriamine-nitric oxide (DETA-NO). Each respective cDNA fragment was found to match with the cDNAs of rat macrophage inflammatory protein 2 (MIP-2) and GRO/cytokine-induced neutrophil chemoattractant 2b (CINC-2b). Further characterization of MIP-2 regulation by Northern blot analysis confirmed an NO- and IL-1b-dependent increase in MIP-2 mRNA levels. Moreover, inhibition of IL-1b-induced endogenous NO formation by the NO-synthase (NOS) inhibitor L-NMMA markedly attenuated MIP-2 protein expression. We cloned 770 bp of the 5'-flanking region of rat MIP-2 and fused this fragment to a luciferase reporter gene. Transfection of the construct into mesangial cells resulted in a 3.5-fold increase in luciferase activity in cells treated with DETA-NO when compared to controls, suggesting a transcriptional mechanism for NO-induced MIP-2 expression. Deletion and mutational analysis identified critical nuclear factor (NF)-kB and NF-IL-6 binding sites required for NO regulation of MIP-2. In vivo, inhibition of NO synthesis in the Thy-1.1 model of mesangioproliferative glomerulonephritis by the specific inducible-NOS inhibitor L-NIL resulted in a marked reduction of MIP-2 mRNA expression. Furthermore, infiltration of neutrophils into the glomerulus was dramatically attenuated in L-NIL-treated rats.
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PMID:Nitric oxide induces MIP-2 transcription in rat renal mesangial cells and in a rat model of glomerulonephritis. 1125 70

Coxsackievirus B3 (CVB3)-induced myocarditis in NMRI mice represents a model for studying the pathogenesis of this chronic heart disease. Previously, we reported on specific cytokine patterns during the acute stage of myocarditis since cytokines are thought to play the important role in this cardiomyopathy. In this study, the expression of various cytokine mRNAs and CVB3-RNA kinetics was examined with particular emphasis on the late phase of myocarditis, by using reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC). In addition, replicating and persisting CVB3-RNAs were semiquantified by PCR-ELISA. Distinct histopathological changes responsible for ongoing heart disease were found and characterized by increased fibrosis, persistent cellular infiltration and degenerated necrotic myocytes. One of the most important findings of this study was that the mRNA-expression of TNF- alpha, IL-1 alpha, interferon- gamma, IL-10, IL-18, macrophage inflammatory protein-1 alpha (MIP-1 alpha), transforming growth factor- beta (TGF- beta) and inducible nitric oxide synthase (iNOS) persisted as long as 98 days after the virus infection. The induction of IL-10 as well as IFN- gamma mRNAs was also verified by ISH and IHC at days 28 and 98 p.i. The clearly apparent persistence of the viral genomes in the myocardium of infected mice was confirmed by seminested PCR, ISH, and PCR-enzyme linked immunoabsorbent assay (ELISA), showing the highest amount of viral RNA in myocardial cells at day 7 after infection. These data indicate that the persistence of viral RNA is associated with persistently high levels of cytokine mRNAs which, when translated, could severely contribute to pathological changes and injury of connective tissue in the chronic stage of myocarditis.
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PMID:Persistent expression of cytokines in the chronic stage of CVB3-induced myocarditis in NMRI mice. 1154 41

Virtually all the compounds that are currently used, or under advanced clinical trial, for the treatment of HIV infections, belong to one of the following classes: (i) nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs): i.e., zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), abacavir (ABC), emtricitabine [(-)FTC], tenofovir (PMPA) disoproxil fumarate; (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs): i.e., nevirapine, delavirdine, efavirenz, emivirine (MKC-442); and (iii) protease inhibitors (PIs): i.e., saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, and lopinavir. In addition to the reverse transcriptase and protease step, various other events in the HIV replicative cycle are potential targets for chemotherapeutic intervention: (i) viral adsorption, through binding to the viral envelope glycoprotein gp120 (polysulfates, polysulfonates, polyoxometalates, zintevir, negatively charged albumins, cosalane analogues); (ii) viral entry, through blockade of the viral coreceptors CXCR4 and CCR5 [bicyclams (i.e. AMD3100), polyphemusins (T22), TAK-779, MIP-1 alpha LD78 beta isoform]; (iii) virus-cell fusion, through binding to the viral glycoprotein gp41 [T-20 (DP-178), T-1249 (DP-107), siamycins, betulinic acid derivatives]; (iv) viral assembly and disassembly, through NCp7 zinc finger-targeted agents [2,2'-dithiobisbenzamides (DIBAs), azadicarbonamide (ADA) and NCp7 peptide mimics]; (v) proviral DNA integration, through integrase inhibitors such as L-chicoric acid and diketo acids (i.e. L-731,988); (vi) viral mRNA transcription, through inhibitors of the transcription (transactivation) process (fluoroquinolone K-12, Streptomyces product EM2487, temacrazine, CGP64222). Also, in recent years new NRTIs, NNRTIs and PIs have been developed that possess respectively improved metabolic characteristics (i.e. phosphoramidate and cyclosaligenyl pronucleotides of d4T), or increased activity against NNRTI-resistant HIV strains [second generation NNRTIs, such as capravirine and the novel quinoxaline, quinazolinone, phenylethylthiazolylthiourea (PETT) and emivirine (MKC-442) analogues], or, as in the case of PIs, a different, non-peptidic scaffold [i.e. cyclic urea (DMP 450), 4-hydroxy-2-pyrone (tipranavir)]. Given the multitude of molecular targets with which anti-HIV agents can interact, one should be cautious in extrapolating from cell-free enzymatic assays to the mode of action of these agents in intact cells. A number of compounds (i.e. zintevir and L-chicoric acid, on the one hand; and CGP64222 on the other hand) have recently been found to interact with virus-cell binding and viral entry in contrast to their proposed modes of action targeted at the integrase and transactivation process, respectively.
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PMID:New developments in anti-HIV chemotherapy. 1156 82


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