Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple immunocytochemical method using calf intestinal alkaline phosphatase as the enzymatic indicator to demonstrate surface antigens on human blood cells has been developed. The blood cells were labeled with cell specific monoclonal antibodies followed by linkage with an antiimmunoglobulin alkaline phosphatase conjugate. Cytochemical demonstration of alkaline phosphatase activity on the blood cells reflects the presence of surface antigens on these cells. The effects on the cytochemical reaction of fixation, substrates, couplers, activators and inhibitors, and storage of cytologic materials have been examined systematically. The best staining conditions are to incubate labeled smears in a 0.04 M barbital buffer at pH 7.6 containing 30 mg% naphthol AS-TR phosphate, 40 mg% fast red ITR, and 1 mM levamisole. This method is both sensitive and specific and appears most practical for objective identification of the human blood cells.
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PMID:Immunocytochemical characterization of human blood cells. 619 5

Adeno-associated Virus (AAV) has emerged as a promising vector for gene therapy because of its ability to generate high titer recombinant stocks and the potential for site specific integration. However, much of the current knowledge regarding the transduction and integration biology of this virus is based on studies evaluating wild type AAV or recombinant AAV which was unknowingly contaminated with wild type virus. Given the fact that recombinant AAV is replication incompetent, by virtue of deleted viral rep proteins responsible for site specific integration of the wild type virus, the integration process for recombinant AAV may likely be different from its wild type counterpart. To this end, the present study has attempted to elucidate the proviral structure of stably integrated recombinant AAV genomes harboring the alkaline phosphatase reporter gene in 293 and IB3 cell lines. Initial studies attempted to functionally characterize differences in proviral genomes using mobilization assays with assessed both liberated episomal recombinant AAV and infectious virus following transfection with Rep/Cap containing plasmids and/or infection with recombinant adenovirus (Ad). Using Southern and polymerase chain reaction (PCR) analysis, evaluation of genomic DNA from AAV clonal cell lines indicated that head to tail orientations of ITRs were absolutely required for excision of episomal genomes and rescue of infectious recombinant virus. Furthermore, mobilization of proviral DNA could be achieved in the presence of exogenous Rep/Cap without adenovirus, while mobilization of infectious recombinant virus required the addition of both Rep/Cap and Ad. Genomic Southerns suggest that two predominant proviral structures exist for recombinant AAV including head to head and tail to head duplex genomes. A third class of monomer proviral genomes with head to tail oriented ITRs was also observed. No evidence for tail to tail ITR oriented proviral genomes was detected in any of the clonal cell lines. Such findings have begun to lay the foundation for a clearer understanding of the mechanism of recombinant AAV integration and how this process differs from wild type AAV.
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PMID:Structural and functional heterogeneity of integrated recombinant AAV genomes. 914 Jan 93

Itraconazole and fluconazole are oral antifungal drugs, which have a wide spectrum antifungal activity and better efficacy than the older drugs. However, both drugs have been associated with hepatotoxicity in susceptible patients. The mechanism of antifungal drug-induced hepatotoxicity is largely unknown. Therefore, the aim of this present study was to investigate and compare the hepatotoxicity induced by these drugs in vivo. Rats were treated intraperitoneally with itraconazole or fluconazole either single (0, 10, 100 and 200 mg/kg) or subchronic (0, 10, 50 and 100 mg/kg per day for 14 days) doses. Plasma and liver samples were taken at the end of the study. A statistically significant and dose dependent increase of plasma alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities were detected in the subchronic itraconazole-treated group. In addition, dose-dependent hepatocellular necrosis, degeneration of periacinar and mizonal hepatocytes, bile duct hyperplasia and biliary cirrhosis and giant cell granuloma were observed histologically in the same group. Interestingly, fluconazole treated rats had no significant increase in transaminases for both single and subchronic groups. In the subchronic fluconazole treated rats, only mild degenerative changes of centrilobular hepatocytes were observed. These results demonstrated that itraconazole was a more potent hepatotoxicant than fluconazole in vivo in rats.
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PMID:Hepatotoxicity induced by antifungal drugs itraconazole and fluconazole in rats: a comparative in vivo study. 1562 77

Itraconazole and fluconazole are potent wide spectrum antifungal drugs. Both of these drugs induce hepatotoxicity clinically. The mechanism underlying the hepatotoxicity is unknown. The purpose of this study was to investigate the role of phenobarbital (PB), an inducer of cytochrome P450 (CYP), and SKF 525A, an inhibitor of CYP, in the mechanism of hepatotoxicity induced by these two drugs in vivo. Rats were pretreated with PB (75 mg/kg for 4 days) prior to itraconazole or fluconazole dosing (20 and 200 mg/kg for 4 days). In the inhibition study, for 4 consecutive days, rats were pretreated with SKF 525A (50 mg/kg) or saline followed by itraconazole or fluconazole (20 and 200 mg/kg) Dose-dependent increases in plasma alanine aminotransferase (ALT), gamma-glutamyl transferase (gamma-GT), and alkaline phosphatase (ALP) activities and in liver weight were detected in rats receiving itraconazole treatment. Interestingly, pretreatment with PB prior to itraconazole reduced the ALT and gamma-GT activities and the liver weight of rats. No changes were observed in rats treated with fluconazole. Pretreatment with SKF 525A induced more severe hepatotoxicity for both itraconazole and fluconazole. CYP 3A activity was inhibited dose-dependently by itraconazole treatment. Itraconazole had no effects on the activity of CYP 1A and 2E. Fluconazole potently inhibited all three isoenzymes of CYP. PB plays a role in hepatoprotection to itraconazole-induced but not fluconazole-induced hepatotoxicity. SKF 525A enhanced the hepatotoxicity of both antifungal drugs in vivo. Therefore, it can be concluded that inhibition of CYP may play a key role in the mechanism of hepatotoxicity induced by itraconazole and fluconazole.
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PMID:Involvement of phenobarbital and SKF 525A in the hepatotoxicity of antifungal drugs itraconazole and fluconazole in rats. 1677 3

In the present study, cellular tropism and relative transduction efficiency of AAV2/6, AAV2/8 and AAV2/9 vectors have been tested for the cornea using primary cultures of human corneal fibroblasts. The AAV6, AAV8 and AAV9 serotypes having AAV2 ITR plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Primary cultures of human corneal fibroblasts were exposed to AAV infectious particles at two different doses (1 x 10(5) and 2 x 10(5) MOI). Cytochemistry and enzyme assays were used to measure delivered transgene expression in samples collected at 4 and 30 h after AAV infection by counting AP-stained cells or quantifying AP enzyme activity. Cellular toxicity of AAVs was evaluated with TUNEL and trypan blue assays. All three AAV serotypes transduced human corneal fibroblasts. The order of transduction efficiency was AAV2/6>>>AAV2/9>AAV2/8. The transduction efficiency of AAV2/6 was 30-50-fold higher (p < 0.001) for the human corneal fibroblasts compared to the AAV2/8 or AAV2/9 at two tested doses. The level of transgene expression at 4h was considerably low compared to 30 h suggesting that the transgene delivery did not reach its peak at 4h. Cultures exposed to any of the three AAV serotypes showed more than 97% cellular viability and less than 5 TUNEL positive cells suggesting that tested AAV serotypes do not induce significant cell death and are safe for corneal gene therapy.
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PMID:Transduction efficiency of AAV 2/6, 2/8 and 2/9 vectors for delivering genes in human corneal fibroblasts. 1961 80