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)

An in vitro culture system was developed that facilitates detailed studies of the interaction of Human Immunodeficiency Virus (HIV) with dendritic cells (DC). Cultured immature DC were generated from adherent peripheral blood mononuclear cells in the presence of GM-CSF and IL-4. These cells were non-adherent, non-phagocytic and had a veiled surface appearance. They expressed high levels of MHC class I and II proteins, CD1a, B7/BB1 and low levels of CD4, and were known to possess a potent soluble antigen presenting capacity. Upon infection with the HIV-1 strains Lai (lymphocytotropic) and BaL (monocytotropic), the viral RNA was reverse transcribed to complete DNA provirus. However the infection was non-productive as judged from measuring the activity of the virus encoded reverse transcriptase in the culture supernatant. Thus HIV infection was restricted at a step post entry.
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PMID:Infection of cultured immature dendritic cells with human immunodeficiency virus type 1. 852 22

Susceptibility to HIV infection was examined in macrophages differentiated from human monocytes by macrophage colony-stimulating factor (M-CSF) or granulocyte/macrophage colony-stimulating factor (GM-CSF). The replication of macrophage-tropic human immunodeficiency virus type-1 (HIV-1), which was determined by reverse transcriptase (RT) activity, was significantly suppressed in macrophages induced by GM-CSF (GM-type macrophages) but not in those induced by M-CSF (M-type macrophages). Multinucleated giant cells were formed only in M-type macrophages after HIV infection. However, the expression of CD4 molecules on the surface of both types of macrophages was similar and the proviral DNA was detectable in cell lysates of both macrophages, although the amount of proviral DNA in M-type macrophages was higher than that in GM-type macrophages. Many steps have been defined in HIV infection and replication, such as adsorption of HIV to the cell surface, internalization of the viral core into the cytoplasm, uncoating of viral RNA, reverse transcription and integration of proviral DNA into cellular DNA, transcription and translation of proviral DNA, assembly of viral components, and budding of virus particles. Our findings suggested that the suppression of HIV-1 replication in macrophages induced by GM-CSF is mainly due to a disturbance at certain steps of replication after synthesis of the proviral DNA. Thus, the suppression of HIV replication in GM-type macrophages may provide a model of the latency of HIV infection in vivo.
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PMID:Suppression of HIV replication in human monocyte-derived macrophages induced by granulocyte/macrophage colony-stimulating factor. 855

A single tube, reverse transcriptase/polymerase chain reaction (RT-PCR) was developed and evaluated for detecting a 400-bp product of the small RNA of sandfly fever virus, serotype Toscana (TOS). For more sensitive detection of genomic TOS RNA, a nested PCR amplifying a 243-bp cDNA within the RT-PCR product was established. Nucleotide sequence analysis of first- and second-round PCR products using the dideoxy cycle sequencing technique confirmed a previously published sequence of the TOS reference strain (ISS. Phl.3). By nested PCR, genomic TOS RNA was amplified from two consecutive sera taken 3 and 7 weeks after the onset of illness in one patient, and from CSF of a second patient obtained at the onset of meningitis. Authenticity of amplified PCR products was confirmed by nucleotide sequence analysis, revealing a sequence identical to the TOS reference strain. RT-PCR and nested PCR are useful for laboratory diagnosis and studies of the molecular epidemiology of TOS infection.
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PMID:Nested RT-PCR for detection of sandfly fever virus, serotype Toscana, in clinical specimens, with confirmation by nucleotide sequence analysis. 857 9

During the process of placental implantation, sessile villous trophoblast cells migrate from the villi into the decidua as isolated motile extravillous trophoblast cells. There is differential expression of the epidermal growth factor-receptor (EGF-R) and c-erbB2 proteins on villous and extravillous trophoblast populations. Using monoclonal antibodies to EGF-R and c-erbB2, we have obtained highly purified populations of villous and extravillous trophoblast by fluorescence activated cell sorting. These cells were examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) using nested internal primer pairs for the following cytokines: CSF-1, GM-CSF, TNF-alpha, TGF-beta1, IFN-gamma, IL-2, LIF and also for LIF-receptor. TNF-alpha and TGF-beta 1 were present in all trophoblast populations. GM-CSF and CSF-1 were only found in some samples, with preferential expression of CSF-1 in villous populations. IFN-gamma, IL-2 and LIF mRNA were not found, although all samples contained LIF-receptor mRNA. These cytokines (CSF-1, TGF-beta, TNF-alpha and GM-CSF) are likely to influence trophoblast growth and differentiation in an autocrine manner, since their receptors are also present on trophoblast. These results illustrate a quick and simple method to analyse for the presence of cytokine and other transcripts in trophoblast subpopulations during early pregnancy.
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PMID:Screening for cytokine mRNA in human villous and extravillous trophoblasts using the reverse-transcriptase polymerase chain reaction (RT-PCR). 858 67

In this study, we have investigated CD40 expression in human peripheral blood eosinophils and in human chronically inflamed nasal tissues, i.e., nasal polyps. We show by both reverse transcriptase-PCR and Northern blot analysis that eosinophils from allergic subjects express human CD40 mRNA. We also show that constitutive CD40 mRNA expression in eosinophils could be upregulated by exposure to IgA immune complexes and downregulated by IL-10 and the synthetic steroid budesonide. In addition, we demonstrate that eosinophils express CD40 protein by flow cytometry. Such expression is biologically functional as cross-linking CD40 with CD40 mAbs enhances eosinophil survival in a dose-dependent fashion; in addition, CD40 ligation stimulates eosinophils to release GM-CSF. CD40-mediated eosinophil survival was largely inhibited by an anti-GM-CSF neutralizing antibody suggesting GM-CSF involvement in the survival enhancing mechanism. CD40 mRNA was also detected in total RNA extracted from nasal polyp tissues but not in RNA isolated from normal nasal mucosa (inferior turbinate); by immunohistochemistry, we were able to detect immunoreactive CD40 protein in a variety of cell types in the polyp stroma, but primarily in eosinophils. These observations suggest previously unforeseen interactions between eosinophils and cells expressing the CD40 ligand and, thus, novel pathways by which eosinophils may contribute to the regulation of airway inflammation.
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PMID:CD40 expression by human peripheral blood eosinophils. 860 42

The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is composed of at least two chains (alpha and beta). The alpha chain binds GM-CSF specifically with low affinity, and the binding is converted to high affinity when the alpha chain is associated with the beta chain. To date, there are at least six isoforms described for the GM-CSFR alpha, all involving alternative splicing at the 3' end, which alters the coding region and hence the protein produced. To detect variants at the 5' end of the GM-CSFR alpha mRNA, RNAse protection and reverse transcriptase polymerase chain reaction (RT-PCR) assays were performed using a probe spanning nucleotides 102-392 and pairs of primers covering exons 1-4. in addition to the expected full-length transcript, two mRNAs were detected, one containing a deletion of 24 nucleotides by alternative splicing at the 3' end of exon 2 (exon 2b-deleted isoform) and another in which exon 2 was completely deleted (exon 2-deleted isoform). Together, the isoforms were more highly expressed form). Together, the isoforms were more highly expressed than the full-length sequence (TF-1 cells: full-length 36 +/- 2.8% vs. exon 2-deleted isoforms 64 +/- 5.5%). These isoforms were detected in primary hematopoietic cells, blasts from patients with acute myeloid leukemia (AML), and malignant cell lines and the relative mRNA expression for the isoforms, was always similar to that of TF-1 cells. As sequences in the 5'untranslated region can be involved in the modulation of translational efficiency, translation of constructs constructs corresponding to these exon 2 deleted isoforms was assessed using an in vitro reticulocyte lysate system. Deletion of exon 2 resulted in significantly lower in vitro translation of the receptor protein relative to the full-length sequence (53, 56, and 76% in three separate batches of reticulocytes), while deletion of exon 2b resulted in higher translation of the sequence (164, 128, and 305%; p = 0.01). These data suggest a mechanism by which expression of the GM-CSFR alpha protein may be regulated by alternatively spliced transcripts with different translational efficiencies.
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PMID:Expression of two alternatively spliced forms of the 5' untranslated region of the GM-CSF receptor alpha chain mRNA. 863 32

Urokinase-type plasminogen activator (uPA), a proteinase which activates plasminogen by cleaving at -CPGR(arrow downward)V-, was shown to cleave the V3 loop in recombinant gp120 of human immunodeficiency virus type 1 (HIV-1) IIIB and MN strains, as well as a synthetic, cyclized peptide representing the clade B consensus sequence of V3. Proteolysis occurred at the homologous -GPGR(arrow downward)A-, an important neutralizing determinant of HIV-1. It required soluble CD4 and was prevented by inhibitors of uPA but not by inhibitors of likely contaminating plasma proteinases. It was accelerated by heparin, a known cofactor for plasminogen activation. In immune capture experiments, tight binding of uPA to viral particles, which did not depend on CD4, was also demonstrated. Active site-directed inhibitors or uPA diminished this binding, as did a neutralizing antibody to V3. Addition of exogenous uPA to the laboratory-adapted IIIB strain of HIV-1, the macrophage-tropic field strains JR-CSF and SF-162, or a fresh patient isolate of indeterminate tropism, followed by infection of macrophages with the various treated viruses, resulted in severalfold increases in subsequent viral replication, as judged by yields of reverse transcriptase activity and p24 antigen, as well as incorporation, as judged by PCR in situ. These responses were reversible by inhibitors or antibodies targeting the proteinase active site or the V3 loop. We propose that uPA, a transcriptionally regulated proteinase which is upregulated when macrophages are HIV infected, can be bound and utilized by the virus to aid in fusion and may be an endogenous component that is critical to the infection of macrophages by HIV-1.
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PMID:A role for urokinase-type plasminogen activator in human immunodeficiency virus type 1 infection of macrophages. 867 69

Astrocytes produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and support the survival and proliferation of microglia. To study the functions of GM-CSF in the central nervous system (CNS), we examined the effects of GM-CSF on cytokine production by glial cells. GM-CSF induced interleukin-6 (IL-6) production by microglia, but not by astrocytes, in a dose-dependent manner as assessed by bioassay and the detection of IL-6 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. GM-CSF did not induce tumor necrosis factor (TNF) alpha or IL-1 in microglia and astrocytes, whereas lipopolysaccharide induced all these cytokines. The induction of IL-6 by GM-CSF in microglia was completely inhibited by antibodies to GM-CSF. Neither IL-3 nor macrophage-CSF (M-CSF) induced IL-6 production in microglia. Given that IL-1 and TNF alpha, monokines derived from microglia, induce IL-6 production in astrocytes, but not in microglia, results indicate that astrocytes and microglia may mutually regulate IL-6 production by different cytokines.
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PMID:Selective induction of interleukin-6 in mouse microglia by granulocyte-macrophage colony-stimulating factor. 872 91

Restoration of bone marrow and immune function by means of allogeneic bone marrow transplantation has been attempted in AIDS patients but has not been successful as the donor-derived cells, or their progeny, inevitably became infected. A hairpin ribozyme that specifically cleaves HIV-1 RNA has been developed by F. Wong-Staal et al. and has been demonstrated to confer resistance against HIV-1 infection. Allogeneic transplantation of CD34+ cells or their pluripotent subsets, transduced by vectors bearing this ribozyme gene, can protect the stem cells and their progeny from HIV-1 infection and eventually restores immune function. We have provided evidence that long-term repopulating stem cells can be mobilized into peripheral blood by growth factors. The combination of G-CSF and GM-CSF seems to yield a high frequency of pluripotent stem cells with a CD34+ subset profile that is similar to placental and umbilical cord blood (PUCB). We have then demonstrated a highly efficient transduction of CD34+ cells from PUCB and mobilized leukapheresis products by retroviral vectors bearing the ribozyme gene. Expression of the ribozyme gene, as shown by reverse transcriptase-polymerase chain reaction, was of similar magnitude (70%-90% of cells that grow into colonies). Challenge of the progeny macrophages from such transduced CD34+ cells with monocyte-trophic strains of HIV-1 showed that they were resistant to infection. Thus allogeneic transplantation of CD34+ cells or their pluripotent subsets, transduced with ribozyme gene, can be a promising strategy for the treatment of HIV infection.
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PMID:Stem cells as vehicles for gene therapy: novel strategy for HIV infection. 874 96

Bcl-2 expression has been shown in hematopoietic progenitor cells. Through the use of Bcl-2 specific antisense oligonucleotides we herein report the biologic importance of Bcl-2 expression in primary human CD34+ hematopoietic progenitor cells committed to the myeloid lineage. In bone marrow or peripheral blood derived CD34+ cells Bcl-2 specific antisense decreased cell survival and inhibited the outgrowth of mixed myeloid colonies. A short-term overnight pretreatment of CD34+ cells with 25 mumol/L of Bcl-2 antisense in liquid culture completely ablated the growth of granulocyte-macrophage colony-forming cells (GM-CFC) in a subsequent 14 days methylcellulose colony assay. Control experiments using corresponding Bcl-2 sense or nonsense oligonucleotides did not significantly impair cell survival or growth of GM-colony-forming unit. Western blot analyses revealed the Bcl-2 antisense dependent inhibition of expression of the Bcl-2 protein in CD34+ progenitor cells. Furthermore, regulation of Bcl-2 expression by various cytokines including interleukin-10 (IL-10) was studied. IL-10's effects on the formation of mixed myeloid colonies were examined in the absence or presence of Bcl-2 specific antisense. In the absence of Bcl-2 antisense IL-10 significantly extended the colony forming potential of mixed myeloid colonies to 14 days. In the presence of Bcl-2 antisense rhIL-10 completely restored GM-CSF driven colony growth. Fluorescent microscopy, Western blot analysis, and reverse transcriptase-polymerase chain reaction revealed the IL-10 dependent increase in cellular expression of Bcl-2 protein and Bcl-2 mRNA transcripts in CD34+ cells. Thus these results show that Bcl-2 expression is necessary for the formation of GM-CSF-dependent colony growth in vitro and that rhIL-10 increases Bcl-2 expression and survival in primary human CD34+ hematopoietic progenitor cells that are committed to the myeloid lineage.
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PMID:Interleukin-10 increases Bcl-2 expression and survival in primary human CD34+ hematopoietic progenitor cells. 883 47


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