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:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interference of reverse transcription by heparin was removed by heparinase. When the HIV-1 RNA in the presence of heparin was detected by a combination of reverse transcription and the polymerase chain reaction (PCR), heparinase treatment followed by removal of Ca2+ before the reverse transcription step permitted the efficient detection of HIV-1 RNA. Prior treatment with heparinase revealed HIV-1 RNA in 68% (13/19) of heparinized plasma samples from HIV-1 carriers, whereas only 26% (5/19) of the same specimens were positive without the heparinase step. Heparinase removed the inhibition of reverse transcription by heparin and is highly recommended when detecting low levels of viral RNA in heparinized plasma.
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PMID:Detection of HIV-1 RNA in heparinized plasma of HIV-1 seropositive individuals. 137 11

The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937 promonocytic cells growing in culture. Here we show that Tat enters these cells by adsorptive endocytosis. Tat appears to bind non-specifically to the cell surface, with greater than 10(7) sites per cell. A specific receptor was not detected by protein crosslinking experiments, and uptake was not affected by treating cells with trypsin, heparinase or neuraminidase. Uptake and transactivation could be inhibited by incubation with heparin, dextran sulfate, an anti-Tat monoclonal antibody, or by incubation at 4 degrees C. In contrast, transactivation by Tat was markedly stimulated by the addition of basic peptides, such as Tat 38-58 or protamine. Fluorescence experiments with rhodamine-conjugated Tat show punctate staining on the cell surface and then localization to the cytoplasm and nucleus. The lack of a specific receptor makes it unclear whether Tat uptake is biologically important in HIV infection, however, the efficiency of uptake raises the possibility that Tat may be useful for delivery of protein molecules into cells.
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PMID:Endocytosis and targeting of exogenous HIV-1 Tat protein. 205 Jan 10

Homosexual males often present signs of immune activation and are likely to have increased levels of inflammatory cytokines such as IL-1 beta, TNF-alpha, and IFN-gamma. These individuals develop Kaposi's sarcoma (AIDS-KS) more frequently than other HIV-1-infected groups. Our previous work demonstrated that inflammatory cytokines stimulate the growth of spindle cells derived from AIDS-KS lesions (AIDS-KS cells) and that these cells produce high levels of bFGF that mediate autocrine and paracrine (endothelial) cell growth and angiogenesis. Here we show that AIDS-KS cells constitutively produce and release bioactive bFGF in the absence of cell death, and that extracellular bFGF exist in both a soluble and a bound form; the latter can be released by treatment with trypsin, heparin, or heparinase I. Inflammatory cytokines stimulate both the synthesis and release of biologically active bFGF from KS cells and enhance their ability to induce angiogenic KS-like lesions in nude mice. Because bFGF is highly expressed in primary KS lesions, and is a mediator of KS-like lesion formation, these results suggest that the export of bFGF induced by inflammatory cytokines may play a critical role in the induction and progression of KS in HIV-1-infected homosexual men.
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PMID:Inflammatory cytokines induce AIDS-Kaposi's sarcoma-derived spindle cells to produce and release basic fibroblast growth factor and enhance Kaposi's sarcoma-like lesion formation in nude mice. 789 37

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA or hepatitis C virus (HCV) RNA has been facilitated by adapting a spin column procedure for sample preparation and the use of chemiluminescent detection of polymerase chain reaction (PCR) products in microtiter plate format. All materials were commercially available and relatively inexpensive. By making a single dilution prior to amplification, concentrations of 500 copies to 2.5 million HIV-1 1 RNA copies per mL and 1,000 copies to 50 million HCV RNA copies per mL could be determined on 140-microL samples. Between-run imprecision employing the improved procedure for HIV-1 RNA was 23%. Correlation of HIV-1 RNA concentrations obtained using chemiluminescent detection with values obtained by colorimetric assay of PCR products was 0.98. Correlation of HCV RNA concentration determined by the spin column-chemiluminescent assay procedure with those obtained by branched DNA methodology was 0.91. Spin columns could be used with serum or plasma containing acid-citrate-dextrose or heparin anticoagulant, but heparinized samples required treatment with heparinase prior to amplification.
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PMID:Improved methods for quantification of human immunodeficiency virus type 1 RNA and hepatitis C virus RNA in blood using spin column technology and chemiluminescent assays of PCR products. 898 50

Human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma are currently widely used clinically for prognostication and in monitoring antiretroviral therapy. Accurate and reproducible results are critical for patient management. To determine the effects of specimen collection and handling procedures on quantitative measurement of HIV-1 RNA, we compared anticoagulants and sample processing times. Whole blood was collected from 20 HIV-1-infected patients in EDTA, acid citrate dextrose (ACD), and heparin tubes, aliquoted, and stored at room temperature. Plasma was separated from whole-blood aliquots prepared at < or =1, 3, 6, 24, and 48 h postcollection and then stored at -70 degrees C until use. HIV-1 RNA levels were determined by the AMPLICOR HIV-1 MONITOR assay. Heparinized plasma samples, which were pretreated with heparinase prior to analysis, had the lowest baseline HIV-1 RNA levels. In the first 6 h, HIV-1 RNA levels decreased by 10, 20, and 31% in EDTA, ACD, and heparin tubes, respectively. From 6 to 48 h postcollection, HIV-1 RNA levels decreased in all anticoagulants, albeit at a slower, more consistent rate. Our results indicate that EDTA should be the anticoagulant of choice for plasma HIV-1 RNA measurement by reverse transcriptase PCR, but ACD tubes are acceptable if the plasma is separated within 6 h of blood collection. Caution must be applied in the interpretation of absolute HIV-1 RNA copy number values obtained with suboptimal specimen collection and processing procedures.
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PMID:Optimization of specimen-handling procedures for accurate quantitation of levels of human immunodeficiency virus RNA in plasma by reverse transcriptase PCR. 954 39

Basic peptides such as human immunodeficiency virus type 1 (HIV-1) Tat-(48-60) and Drosophila Antennapedia-(43-58) have been reported to have a membrane permeability and a carrier function for intracellular protein delivery. We have shown that not only Tat-(48-60) but many arginine-rich peptides, including HIV-1 Rev-(34-50) and octaarginine (Arg(8)), efficiently translocated through the cell membranes and worked as protein carriers (Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001) J. Biol. Chem. 276, 5836-5840). Quantification and time course analyses of the cellular uptake of the above peptides by mouse macrophage RAW264.7, human cervical carcinoma HeLa, and simian kidney COS-7 cells revealed that Rev-(34-50) and Arg(8) had a comparable translocation efficiency to Tat-(48-60). Internalization of Tat-(48-60) and Rev-(34-50) was saturable and inhibited by the excess addition of the other peptide. Typical endocytosis and metabolic inhibitors had little effect on the internalization. The uptake of these peptides was significantly inhibited in the presence of heparan sulfate or chondroitin sulfates A, B, and C. Treatment of the cells with the anti-heparan sulfate antibody or heparinase III also lowered the translocation of these peptides. These results strongly suggest that the arginine-rich basic peptides share a certain part of the internalization pathway.
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PMID:Possible existence of common internalization mechanisms among arginine-rich peptides. 1171 47

Non-infectious, envelope protein-free, retrovirus-like particles (VLP) derived from either Moloney murine leukemia virus (MLV) or human HIV are able to bind efficiently to, but not infect, target cells. Upon subsequent addition to the bound particles of the G protein of vesicular stomatitis virus (VSV-G), an efficient surrogate retrovirus envelope protein, the VLP are efficiently taken up by the cells to produce infection. Cell attachment of the VLP is efficiently inhibited by soluble heparin and dextran sulfate and less efficiently abrogated by several other glycosaminoglycans (GAGs) including chondroitin sulfate A and chondroitin sulfate B (dermatan sulfate), as determined by deconvolution microscopic immunodetection of the viral gag protein and by quantitative binding studies of metabolically labeled (35)S-VLP. Enzymatic digestion of heparan sulfate (HS) from the cell surface with heparinase I also reduces VLP binding. Furthermore, VLP adsorption onto several CHO cell lines variably deficient in cell surface GAG is significantly but incompletely abrogated. De-sulfated heparins are less efficient than native heparin in inhibiting the Polybrene-mediated binding of VLP, whereas growth of human cells in the presence of sodium chlorate leads to significant reduction of Polybrene-mediated VLP binding. In addition, specific inhibition of VLP binding and infectivity of mature infectious VSV-G-pseudotyped virus is observed in the presence of heparin and HS under Polybrene-free conditions. We conclude from these studies that the presence of Polybrene, the degree of sulfation of cell surface GAG, and possibly the presence of charged cell surface macromolecules create an electrostatic environment that promotes optimum binding of VLP to cells. Additionally, our results demonstrate that, in the absence of Polybrene, initial attachments of non-infectious, envelope protein-free VLP and probably mature infectious virus particles are mediated by interactions of the virus particles with cell surface heparan sulfate, and possibly with other GAG molecules.
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PMID:Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells. 1199 44

Human immunodeficiency virus type 1 (HIV-1)-associated dementia is observed in 20-30% of patients with acquired immunodeficiency syndrome (AIDS). The epsilon4 allele of the apolipoprotein E (APOE) gene currently is thought to play a role as a risk factor for the development of HIV dementia. The HIV protein Tat is neurotoxic and binds to the same receptor as apoE, the low-density lipoprotein receptor-related protein (LRP). In this study, we investigated the role apoE plays in Tat toxicity. Synaptosomes from wild-type mice treated with Tat had increased reactive oxygen species (ROS), increased lipid and protein oxidation, and decreased mitochondrial membrane potential. Synaptosomes from APOE-knockout mice also had increased ROS, increased protein oxidation, and decreased mitochondrial membrane potential, but to a significantly lesser degree. Treatment of synaptosomes with heparinase and Tat increased Tat-induced oxidative stress, consistent with the notion of Tat requiring interaction with neuronal membranes to induce oxidative damage. Human lipidated apoE3 greatly protected neurons from Tat-induced toxicity, whereas human lipidated apoE4 showed no protection. We demonstrated that human apoE3 has antioxidant properties against Tat-induced toxicity. Taken together, the data suggest that murine apoE and human apoE4 act similarly and do not protect the cell from Tat-induced toxicity. This would allow excess Tat to remain outside the cell and interact with synaptosomal membranes, leading to oxidative stress and neurotoxicity, which could contribute to dementia associated with HIV. We show that the antioxidant properties of apoE3 greatly outweigh the competition for clearance in deterring Tat-induced oxidative stress.
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PMID:Effects of apolipoprotein E on the human immunodeficiency virus protein Tat in neuronal cultures and synaptosomes. 1526 23

Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-beta-D-naphthol and xyloside-beta-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.
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PMID:A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression. 1788 14

Dendritic cells (DCs) efficiently capture HIV-1 and mediate transmission to T cells, but the underlying molecular mechanism is still being debated. The C-type lectin DC-SIGN is important in HIV-1 transmission by DCs. However, various studies strongly suggest that another HIV-1 receptor on DCs is involved in the capture of HIV-1. Here we have identified syndecan-3 as a major HIV-1 attachment receptor on DCs. Syndecan-3 is a DC-specific heparan sulfate (HS) proteoglycan that captures HIV-1 through interaction with the HIV-1 envelope glycoprotein gp120. Syndecan-3 stabilizes the captured virus, enhances DC infection in cis, and promotes transmission to T cells. Removal of the HSs from the cell surface by heparinase III or by silencing syndecan-3 by siRNA partially inhibited HIV-1 transmission by immature DCs, whereas neutralizing both syndecan-3 and DC-SIGN completely abrogated HIV-1 capture and subsequent transmission. Thus, HIV-1 exploits both syndecan-3 and DC-SIGN to mediate HIV-1 transmission, and an effective microbicide should target both syndecan-3 and DC-SIGN on DCs to prevent transmission.
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PMID:Syndecan-3 is a dendritic cell-specific attachment receptor for HIV-1. 1804 49


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