Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.2.2.7 (heparinase)
 1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rab proteins are ras-like low molecular mass GTP-binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. We have previously shown that synthetic peptides, corresponding to the effector domain of Rab3 proteins, stimulate a complete exocytotic response in mast cells. We have used a PCR-cloning strategy to investigate the presence of mRNA encoding Rab3 in mast cells. RNA based PCR was then performed on mast cell RNA using degenerate oligonucleotide primers based on two conserved sequences among Rab3 proteins. However, no PCR products were obtained, even for proteins known to be expressed in high copy numbers in mast cells (beta-actin and Fc receptor). We have found that the problem resides in the presence of mast cell secretory granule derived heparin, that copurifies with the RNA; heparin has been shown to inhibit the activity of reverse transcriptase and Taq polymerase in PCR. After treating the RNA (obtained from about 500 mast cells) with heparinase, several PCR products of varying size were obtained using primers specific for Rab3 proteins. These products were cloned and sequenced. We have found clones containing sequences that had a 100% homology at the deduced amino acid level to a portion of Rab3B and Rab3D (amino acids 16 to 83).
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PMID:RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells. 750 66

The reverse transcriptase polymerase chain reaction (RT-PCR) procedure is markedly inhibited in specimens of blood that contain commercial heparin as an anticoagulant or in cell preparations containing rat or mouse peritoneal mast cells. However, it was not known whether the levels of endogenous, mast cell-associated heparin that are present in some mammalian tissues are sufficient to interfere with the use of RT-PCR in these settings. We show that RT-PCR detects little or no mRNA transcripts for either mast cell-associated products, such as mouse mast cell-associated protease-2 or -4 (MMCP-2 or MMCP-4) or mast cell carboxypeptidase A, or for mast cell-nonspecific products, such as glyceraldehyde 3-phosphate dehydrogenase, in routinely prepared specimens of cells or tissues that include populations of heparin-containing mast cells. However, signals for mast cell-associated or mast cell-nonspecific transcripts can be readily detected in such specimens if they are treated with heparinase before RT-PCR. RT-PCR after heparinase treatment appears to represent an extremely sensitive method for detecting mast cell-associated transcripts in tissue specimens, permitting the identification of transcripts for mast cell-specific proteases in the skin of genetically mast cell-deficient WBB6F1-W/WV mice, a tissue that contains few or no mast cells according to histological analysis.
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PMID:Detection of mouse mast cell-associated protease mRNA. Heparinase treatment greatly improves RT-PCR of tissues containing mast cell heparin. 785 46

To define the molecular regulation of mast cell phenotype and function optimized procedures must be available to study mRNA from mast cells freshly isolated from tissues. However, rat peritoneal mast cells (PMC) contain large amounts of the proteoglycan heparin, and unfortunately, this molecule which is a potent inhibitor of reverse transcriptase (RT) and Taq polymerase and thus RT-PCR, copurifies with RNA. Here we describe an optimized protocol for extracting and amplifying RNA from rat PMC. Mast cells were isolated from rat peritoneum and a method modified from that of Chomczynski and Sacchi (1987) was used to extract the RNA. Following the removal of heparin by heparinase digestion, first strand cDNA synthesis was primed with oligo-dT and the resulting cDNA was quantified by rapid paper chromatography. The use of a detection system for the reverse transcription reaction ensured that the production of cDNA had occurred and allowed subsequent PCR testing to be optimal. cDNA thus produced can be used to detect relatively specific (histidine decarboxylase) and non-specific (beta-actin) mast cell products. Our PCR studies have shown a 300-fold increase in sensitivity over RNA processed by other methods.
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PMID:Optimization of the isolation and effective use of mRNA from rat mast cells. 905 Sep 42

Basic fibroblast growth factor (bFGF) and its specific receptors have diverse roles on a variety of cell types, such as the induction of vascular smooth-muscle cell proliferation which contributes to restenosis after coronary balloon angioplasty. bFGF is also known to interact with heparan sulphate proteoglycans present on the cell surface or in the extracellular matrix. In this study, the binding of 125I-bFGF to human aortic smooth-muscle cells was investigated. 125I-bFGF binding to these cells was reversible and saturable. Scatchard analysis revealed the presence of two distinct binding sites: a high-affinity receptor (Kd=38+/-7 pM; 1480+/-220 sites/cell) and a low-affinity non-saturable binding site (Kd=8. 0+/-2.0 nM). Pretreatment of the cells with heparinase resulted in a large reduction of 125I-bFGF binding to its low-affinity receptors, suggesting that they are heparin-like molecules. The specificity of the low- and high-affinity binding sites for bFGF was determined with acidic FGF, platelet-derived growth factor-BB and epidermal growth factor, which did not compete for 125I-bFGF binding. Expression of FGF receptor isoforms analysed by reverse transcriptase-PCR revealed the presence of only the type-1 receptor. Binding to low-affinity binding sites was antagonized by heparin, suramin, protamine sulphate and platelet factor 4. Unexpectedly, these molecules also reduced the binding of 125I-bFGF to its high-affinity sites. Consistent with these results, heparin, suramin, protamine sulphate and platelet factor 4 inhibited bFGF-induced proliferation of human aortic smooth-muscle cells. Heparin abrogated bFGF-induced release of tissue-type plasminogen activator by these cells. These observations suggest that the interaction of bFGF with human aortic smooth-muscle cells is different from that described for other cells such as endothelial cells, in which heparin acts as a potentiating factor of the mitogenic activity of bFGF.
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PMID:Heparin inhibits the binding of basic fibroblast growth factor to cultured human aortic smooth-muscle cells. 930 14

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

Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrophages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mast cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alveolar macrophages (NR8383) were studied for the detection of mRNA for beta-actin, TNF-alpha and/or CD8alpha. Each type of cell has unique optimal conditions for RT-in situ PCR. The following parameters were carefully evaluated for optimization: protease digestion, DNAse digestion, heparinase digestion, RT, PCR cycle number and signal development with chromagen. Heparinase digestion was required for PMC mRNA detection because they contain large amounts of heparin proteoglycan, which is a potent inhibitor of RT and Taq polymerase enzymes. Only a few PCR cycles were needed to produce a cytoplasmic signal for mRNA transcripts in RCMC, whereas other types of cells (PMC, BALC and NR8383) needed at least 20 cycles for mRNA detection. The mRNA signal in PMC was localized to the perinuclear region, whereas mRNA in other cell types (RCMC, BALC and NR8383) were detected throughout the cytoplasm. Furthermore, modified Southern blot analysis for TNF-alpha in RCMC treated with RT-in situ PCR demonstrated the specificity of amplification product. The modified and optimized protocols for this procedure were successfully applied to detect and localize several mRNA transcripts in rat MC and AM. The approach is valuable and can be used to further study selected gene expression in these and other cell types.
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PMID:Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages. 1041 Sep 80

The reverse transcriptase polymerase chain reaction (RT-PCR) is a rapid and sensitive method for detecting gene expression. However, when we used this technique to study gene expression of cytokines in ischemic and ex-vivo-reperfused rat lungs as a model for transplantation, significant inhibition of RT-PCR reaction was observed. To optimize RT-PCR conditions, RNA was extracted from rat lungs after flushing, preservation, and reperfusion. RNA was further purified and PCR conditions were modified with various strategies. We found that heparinase I pretreatment completely overcame the inhibitory effects of RT-PCR using RNA extracted from lung tissues after ischemia-reperfusion. With this treatment, a dramatic increase in tumor necrosis factor-a (TNF-a) mRNA was revealed from lung tissues after ischemia-reperfusion. This result suggests that residual heparin in lung tissue interferes with RT-PCR. Because heparinization is routinely used during clinical and experimental organ transplantation, we recommend the treatment of RNA samples with heparinase prior to RT-PCR.
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PMID:Heparin interference with reverse transcriptase polymerase chain reaction of RNA extracted from lungs after ischemia-reperfusion. 1083 52