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)

Homozygous endothelin B receptor deficiency leads to congenital aganglionosis of the gut in rats and mice, equivalent to human Hirschsprung disease. Homozygous endothelin B receptor deficient rats (spotting lethal rats, sl/sl) are characterized not only by this developmental disorder of the enteric nervous system, which limits their life span to 3-4 weeks, but exhibit an increased rate of apoptosis in the dentate gyrus compared to wildtype (+/+) rats. Recently, endothelin B receptor deficient transgenic rescue rats (sl/sl, tg/tg) were created to further investigate the role of the endothelin B receptor in mature animals. Linkage of the human dopamine-beta-hydroxylase promoter to the rat endothelin B receptor gene and expression of this transgenic construct results in normal development of the enteric nervous system. We investigated the expression pattern of this transgenic construct in the brain by using reverse transcriptase polymerase chain reaction. Unexpectedly, transgene mRNA expression was not restricted to the brain stem where adrenergic and noradrenergic nuclei are known to be present but, in addition, was also detectable in hippocampus and cortex. Using in situ tailing technique, cleaved caspase-3 immunohistochemistry and analysis of hematoxylin-eosin-stained serial sections, we found that all studied transgenic animals were rescued from the increased rate of apoptosis in the dentate gyrus characteristic for non-transgenic sl/sl rats. This finding supports our previous observation that the endothelin B receptor might be an important regulatory element supporting cellular survival in the hippocampus during postnatal development. The endothelin B receptor deficient transgenic rescue rats used here are rescued from developmental disorders both in the gut and in the brain.
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PMID:Endothelin B receptor deficient transgenic rescue rats: a rescue phenomenon in the brain. 1502 12

Immunohistochemical analysis revealed the presence of a gut-brain peptide, neuromedin U (NMU), in the suprachiasmatic nucleus (SCN), which is the site of the master circadian oscillator. The expression of NMU mRNA exhibited a circadian rhythm, with the peak expression in the SCN occurring at CT4-8h. The two NMU-binding receptors (NMU-R1 and NMU-R2) were also expressed in the SCN, but their phase angles were different. Intracerebroventricular injection (ICV) of NMU induced the expression of Fos protein in the SCN cells and caused a phase-dependent phase shift of the circadian locomotor activity rhythm. The magnitude of the phase shift was dose dependent. This NMU-induced phase shift was of the nonphotic type. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed increases in the expression in the SCN of immediate early genes, such as c-fos, NGFI-A, NGFI-B, and JunB. Furthermore, ICV injection of NMU increased the expression of Per1, but not Per2, in the SCN. These results indicate that NMU may play some important role in the circadian oscillator by exerting an autocrine or paracrine action in the SCN.
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PMID:The gut-brain peptide neuromedin U is involved in the mammalian circadian oscillator system. 1511 Jul 67

Orazipone (OR-1384) and OR-1958 are novel anti-inflammatory sulfhydryl reactive compounds with potential applications in the treatment of chronic obstructive lung disease and colitis. Mast cells are potent immune system cells which can be found in abundant numbers in mucosa of lung and gut. We have studied whether the anti-inflammatory effect of these compounds could be mediated through inhibition of the function of mast cells and compared their effects with the glucocorticoid budesonide. Human mast cell line (HMC-1) cells were activated using a combination of a calcium ionophore and a phorbol ester and the production of cytokines was measured using ELISA assay. Tumour necrosis factor-alpha mRNA levels were assessed using a semiquantitative reverse transcriptase polymerase chain reaction assay. Histamine release was studied in rat peritoneal mast cells. Orazipone, OR-1958 and budesonide inhibited significantly and dose dependently tumour necrosis factor-alpha production in HMC-1 cells with IC50-values of 20, 10, and 0.25 microM, respectively. Polymerase chain reaction studies showed that OR-1958 attenuated the activation-induced increase of tumour necrosis factor-alpha mRNA in HMC-1 cells. OR-1958 and, to a lesser extent, orazipone inhibited dose dependently compound 48/80-induced histamine release from rat peritoneal mast cells in a reversible manner. In contrast, budesonide did not appreciably affect the histamine release. Both orazipone and OR-1958 inhibit efficiently mast cell functions and therefore could prove useful in the treatment of diseases associated with inappropriate mast cell activation.
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PMID:Novel sulfhydryl-reactive compounds orazipone and OR-1958 inhibit cytokine production and histamine release in rat and human mast cells. 1558 79

Representational difference analysis of cDNAs (cDNA-RDA) is a sensitive subtractive hybridization technique capable of isolating rare mRNAs differentially expressed in two cell populations. cDNA-RDA can detect sequences represented at 0.0001% in the starting mRNA. By using reverse transcriptase polymerase chain reaction (PCR), cDNA-RDA also lends itself to studies in which samples are derived from limited numbers of cells. Standard cDNA-RDA protocols depend upon the presence of specific restriction enzyme sites in each cDNA, typically enzymes with four base recognition sequences. These sites are used to reduce the cDNA size range and provide primer sites for subsequent PCR amplification. Consequently, transcripts containing fewer than two of the chosen restriction sites are undetectable by cDNA-RDA. We have developed a restriction enzyme site-independent cDNA-RDA protocol called modified RDA (MRDA). We constructed MRDA test sequences from random hexamer-primed cDNA, thereby increasing the representation of mRNAs which are excluded by cDNA-RDA protocols. MRDA is also more efficient than cDNA-RDA at removing highly expressed housekeeping genes during the subtractive hybridization process, thereby allowing more efficient isolation of preferentially expressed mRNAs. Using MRDA, we isolated cDNAs differentially expressed between limited numbers of human CD4(+) naive and memory T lymphocyte subsets and skin- and gut-homing memory T cell subsets.
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PMID:Modified representational difference analysis: isolation of differentially expressed mRNAs from rare cell populations. 1562 Aug 87

CD4(+) T helper cells are important for the regulation of immune responses in the intestinal mucosa and they exert their effects through the secretion of pro-inflammatory and immunomodulatory cytokines. Human patients with inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have alterations in the normal intestinal cytokine profile. These cytokine abnormalities have been shown at both the protein and messenger RNA (mRNA) level. The role that mucosal cytokines play in the pathogenesis of canine IBD has only been investigated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of gut tissue, as cytokine antisera are not available for this species. Real-time RT-PCR has been recognised to be a more accurate and sensitive method of quantifying mRNA transcripts, so in this study TaqMan real-time RT-PCR assays for the quantification of mRNA encoding IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IFN-gamma, TNF-alpha and TGF-beta in canine intestinal mucosa were developed. The amount of these templates was quantified in normal canine duodenal mucosa (n = 8). IL-18, TGF-beta and TNF-alpha were found to be the most abundant transcripts, with IL-10 and IFN-gamma present at levels approximately 10-fold less. IL-2, IL-4, IL-5, IL-6 and IL-12 were the least abundant templates, with some RNA samples having no detectable mRNA copies. The methods developed in this study will form the basis of further work investigating the expression of mRNA encoding cytokines in mucosa from dogs with chronic enteropathies. In addition, these real-time PCR assays can also be used for the quantification of canine cytokine mRNA in other diseases.
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PMID:Cytokine mRNA quantification in histologically normal canine duodenal mucosa by real-time RT-PCR. 1562 66

Hookworm infection persists as a public health problem in developing nations. Vaccine-based strategies offer the best chance of long-term control. Aspartyl protease inhibitors from parasitic nematodes are highly immunogenic, and have been suggested as potential vaccine antigens. An aspartyl protease inhibitor, API-1, was cloned and characterised from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. Using sequence from the hookworm expressed sequence tag project, specific primers were designed and used to amplify Ac-api-1 from A. caninum infective L3 cDNA by PCR. Amplicons from the 5' and 3' ends were cloned, sequenced, and combined to create an 874-bp full-length composite sequence of the Ac-api-1 gene. The A. ceylanicum api-1 cDNA of 878 bp was cloned from L3 cDNA using the A. caninum primers. The amino acid sequences of hookworm orthologues were nearly identical, and database searching indicated they belonged to the aspin family, a group of nematode specific aspartyl protease inhibitors that includes the Ascaris pepsin inhibitor PI-3. Ac-api-1 mRNA was detected by reverse transcriptase PCR in eggs, L1, L3 and adult life cycle stages. A polyclonal antiserum against Escherichia coli expressed recombinant Ac-API-1 detected the protein in adult A. caninum excretory/secretory products, but not in those from activated infective larvae. Immunolocalisation experiments using the antiserum indicated that Ac-API-1 is present primarily in the pseudocoelomic fluid in adult hookworms. Soluble, yeast-expressed Ac-API-1 failed to inhibit pepsin or a hookworm gut aspartyl protease in vitro, but inhibited approximately 30% of the proteolytic activity of adult excretory/secretory products. The pseudocoleomic location, presence in all life cycle stages, lack of inhibitory activity against pepsin, and inhibitory activity against excretory/secretory products suggest that Ac-API-1 inhibits an unidentified, putative aspartyl protease secreted by adult hookworms, and may be released as an enzyme-inhibitor complex. The highly immunogenic properties of nematode aspins suggest that Ac-API-1 represents a promising target for a recombinant hookworm vaccine.
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PMID:Cloning and characterisation of an aspartyl protease inhibitor (API-1) from Ancylostoma hookworms. 1572 82

Apart from fusion inhibitors, 'conventional' antiretrovirals such as nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) act on intracellular targets. Intracellular concentrations of these agents may be an important determinant of antiviral activity, and the pharmacokinetics of intracellular drug accumulation (including binding to cytosolic proteins, intracellular-free fraction, influx and efflux kinetics and intracellular drug metabolism) are likely to impact upon efficacy and toxicity. To date, intracellular drug accumulation has been poorly studied in vivo, due to methodological difficulties and the relatively large volumes of blood required. NRTIs require intracellular conversion to their active phosphorylated metabolites: interactions between these agents, or with other drugs may impact upon efficacy. PIs are metabolised by cytochrome P450 enzymes in gut and liver; some intracellular metabolism by P450 isoforms is also possible. PIs are also substrates for drug efflux transporters such as P-gp and MRP1. We have previously observed a hierarchy of intracellular accumulation of PIs, most probably related to physiochemical characteristics of these drugs such as lipophilicity and plasma protein binding. Comparatively, little is known about the intracellular pharmacokinetics of NNRTIs. These drugs probably do not accumulate inside cells to any significant degree. The study of intracellular pharmacokinetics of HIV drugs is central to investigating putative sanctuary sites where HIV may replicate with little selective pressure. However, stringent methodological procedures need to be applied, and techniques for measuring intracellular drug are in their infancy. Moreover, failure to differentiate between truly intracellular drug and drug bound to cell membranes render results difficult to interpret.
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PMID:Intracellular pharmacokinetics of antiretroviral agents. 1573 42

An amphioxus complementary DNA, AmphiCL, encoding cathepsin L proteinase was isolated from the gut cDNA library of Branchiostoma belcheri tsingtauense. It is 1480 bp long, and its longest open reading frame codes for a precursor protein, which consists of 327 amino acid residues including a signal peptide (preregion), a propeptide, and a mature proteinase. Northern blot showed that AmphiCL was expressed in the gill, testis, hepatic cecum, and hind-gut with a molecular size of about 1480 bp. AmphiCL was also expressed at low level in the muscle, notochord, and ovary as revealed by the more sensitive reverse transcriptase polymerase chain reaction techniques. Semiquantitative RT-PCR also showed that although AmphiCL expression in the gut was significantly downregulated by feeding Arthrospira platensis powder, a protein-rich food, its expression in the same tissue was upregulated by exposure to lipopolysaccharide, an integral component of the outer membrane of gram-negative bacteria. This suggests that although the involvement of AmphiCL in food digestion remains to be confirmed, AmphiCL may play a role in inflammatory reaction in amphioxus.
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PMID:Characterization and expression of AmphiCL encoding cathepsin l proteinase from amphioxus Branchiostoma belcheri tsingtauense. 1577 12

Recognition of repeat CpG motifs, which are common in bacterial, but not in mammalian, DNA, through Toll-like receptor (TLR)9 is an integral part of the innate immune system. As the role of TLR9 in the human gut is unknown, we determined the spectrum of TLR9 expression in normal and inflamed colon and examined how epithelial cells respond to specific TLR9 ligand stimulation. TLR9 expression was measured in human colonic mucosal biopsies, freshly isolated human colonic epithelial cells and HT-29 cells by reverse transcriptase-polymerase chain reaction or Western blotting. Colonic epithelial cell cultures were stimulated with a synthetic CpG-oligodeoxynucleotide (ODN), exhibiting strong immunostimulatory effects in B cells. Interleukin (IL)-8 secretion was determined by enzyme-linked immunosorbent assay, nuclear factor-kappaB (NF-kB) activity by electrophoretic mobility shift assay and IkB phosphorylation by Western blotting. TLR9 mRNA was equally expressed in colonic mucosa from controls (n = 6) and patients with ulcerative colitis or Crohn's disease disease (n = 13). HT-29 cells expressed TLR9 mRNA and protein and responded to CpG-ODN (P < 0.01), but not to non-CpG-ODN stimulation, by secreting IL-8, apparently in the absence of NF-kB activation. Primary epithelial cells isolated from normal human colon expressed TLR9 mRNA, but were completely unresponsive to CpG-ODN stimulation in vitro. In conclusion, differentiated human colonic epithelial cells are unresponsive to TLR9 ligand stimulation in vitro despite spontaneous TLR9 gene expression. This suggests that the human epithelium is able to avoid inappropriate immune responses to luminal bacterial products through modulation of the TLR9 pathway.
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PMID:Expression of Toll-like receptor 9 and response to bacterial CpG oligodeoxynucleotides in human intestinal epithelium. 1599 94

Summary CD4(+)CD25(+) regulatory T cells (T(regs)) are involved in the maintenance of peripheral tolerance and ensure a balanced immune response competent of fighting pathogens and at the same time recognizing commensals as harmless. This feature is lost in Crohn's disease (CD). The forkhead/winged helix transcription factor FoxP3 is a master gene for T(reg) function and defects in the FoxP3 gene lead to a clinical picture similar to inflammatory bowel disease (IBD). Murine colitis can be cured by adoptive transfer of T(regs) and ex vivo-generated gut-specific T(regs) represent an attractive option for therapy in CD. Thus, defective T(regs) could contribute to the development of CD. We cultured biopsies of colonic mucosa in the presence of high concentrations of interleukin (IL)-2 and IL-4 to overcome the anergic nature of naturally occurring CD4(+)CD25(+) T(regs) in the mucosa. We investigated the expression of FoxP3 and regulatory potential of gut-derived CD4(+)CD25(+) T cells cultured from patients with CD and healthy individuals. The FoxP3 expression was analysed by reverse transcriptase polymerase chain reaction (RT-PCR), and the suppressive effect of FoxP3(+)CD4(+)CD25(+) T cells on proliferation and cytokine production of autologous CD4(+) T cells was assessed by flow cytometry. Cultured gut-derived T cells with CD4(+)CD25(+) phenotype expressed FoxP3 and were able as the freshly isolated T(regs) from peripheral blood to suppress proliferation and cytokine production of autologous CD4(+) T cells. Thus, we demonstrate that FoxP3(+)CD4(+)CD25(+) T cells with regulatory properties can be propagated in vitro from inflamed mucosa of CD patients, which may be of interest in adoptive immunotherapy.
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PMID:FoxP3(+)CD4(+)CD25(+) T cells with regulatory properties can be cultured from colonic mucosa of patients with Crohn's disease. 1604 46


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