Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The capacity of a previously described plasmid vector pAZ to deliver bioactive proteins to targets in vivo has been studied. This vector molecule has a strong constitutive promoter, is extremely stable in cells of vaccinal S. choleraesuis strain, and encodes the synthesis of marker protein beta-galactosidase which helps monitor the vector's fate in the host. The gene encoding hepatitis B virus core antigen (HBcAg) has been inserted into pAZ under its constitutive promoter. The resultant recombinant plasmid p19-24 has been used to transform Enterobacteriaceae (E. coli and S. choleraesuis) cells. Transformed cells produce immunologically active HBcAg. p19-24 was stable in S. choleraesuis cells during their culturing and during this strain persistence in mice. Triple oral immunization of rabbits in a dose of 1 x 10(9) S. choleraesuis cells TC177 induced the production of virus-specific antibodies. Successful transformation of cells of another vaccinal strain S. abortus ovis by this plasmid extends the potentialities of the vector. The results demonstrate good prospects of using pAZ vector for the construction of live oral vaccines.
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PMID:[Construction of an expression plasmid vector for accomplishing in vivo delivery of recombinant biologically active proteins. 2. Synthesis of HBcAG in a vaccine strain of Salmonella choleraesuis]. 1097 75

Hepatitis B virus X protein (HBX) has been implicated in the transactivation of diverse cellular genes and possibly also the pathogenesis of human hepatocellular carcinoma (HCC). We report the characterization of HBX variants from HBV-related human hepatocellular carcinoma (HCC). These HBX variants were integrated into the host chromosomes and also expressed in the HCC tissues. In addition, we report a novel in vitro HBX activity assay based on color changes that were indicative of the beta-galactosidase enzyme activity. Conducted in wheat germ lysates, the transactivating function of either wild type or mutant HBX protein was measured through their interaction with the Early Growth Response factor 1 (Egr-1) that controls the beta-galactosidase gene. Further analysis of these HBX deletion mutants using this assay may shed new insights on the significance of various mutations occurring in HCC-associated HBX.
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PMID:Expression of integrated hepatitis B virus X variants in human hepatocellular carcinomas and its significance. 1102 64

Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with approximately 10(13) particles of an adenovirus type 5 vector expressing beta-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.
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PMID:Combination therapy with lamivudine and adenovirus causes transient suppression of chronic woodchuck hepatitis virus infections. 1109 Jan 75

Two Chinese hamster ovary (CHO) cell lines stably transfected with human insulin receptor cDNA, CHO-wt and CHO-mut, which express an equivalent number of normal and kinase-defective human insulin receptors, respectively, were used to assess the roles of insulin receptor tyrosine kinase activity in insulin-regulated gene expression. The effect of insulin on gene-33-promoter-driven chloramphenicol acetyltransferase (CAT), RSVLTR-driven beta-galactosidase (pRSVLTR-betagal) and SV40 late-promoter-driven hepatitis B surface antigen (pMLSV(2)HBsAg) were examined in CHO-wt and CHO-mut cells. Insulin-stimulated gene 33 promoter is 10- to 50-fold more effective in CHO-wt cells than that in parental CHO cells. However, no enhancement of insulin sensitivity of gene 33 promoter in CHO-mut cells relative to parental CHO cells was found. Similar phenomena were also observed, in that insulin regulated pRSVLTR-betagal and pMLSV(2)HBsAg in these three CHO lines. Our data indicated that the protein kinase activity of the insulin receptor is essential for the stimulatory activity of insulin toward the activities of different promoters. Copyright 1994 S. Karger AG, Basel
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PMID:Protein Kinase Activity of the Insulin Receptor Is Essential for Insulin-Regulated Gene Expression. 1172

We have developed a system for producing murine leukemia virus (MLV) pseudotyped with human hepatitis B virus (HBV) large (L) and small (S) surface antigens (HBsAg) for targeting primary human hepatocytes. Using the MLV(HBV) pseudotype virus containing a beta-galactosidase reporter gene, we demonstrated that this pseudotype virus exhibits strict tropism for primary human hepatocytes, similar to the natural target cell specificity of HBV. It does not infect any of the established tissue culture cell lines, including human hepatoma cell lines (HepG2 and Huh-7), or rat primary hepatocytes. The infectivity of MLV(HBV) for human hepatocytes was inhibited by anti-HBs antibody. The L form of HBsAg was both necessary and sufficient for virus infectivity, but the presence of both L and S forms enhanced the surface expression of HBsAg and thus increased virus production. The middle form of HBsAg was not necessary. This pseudotype virus bypasses the requirement for the liver-specific transcription factors for HBV replication, enabling direct study of HBV tissue tropism conferred by the viral envelope proteins. This virus also offers a potential liver-specific targeting system for gene therapy.
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PMID:Murine retroviral pseudotype virus containing hepatitis B virus large and small surface antigens confers specific tropism for primary human hepatocytes: a potential liver-specific targeting system. 3166 88

We have tested the ability of bone marrow (BM) cells (BMCs) to form hepatocytes in liver injury models. We used three models: (i) carbon tetrachloride (CCl4) treatment, (ii) albumin-urokinase transgenic mouse [TgN(Alb1Plau)], and (iii) hepatitis B transgenic mouse [TgN(Alb1HBV)]. As a nonselective liver injury model, irradiated C57BL/6 (B6) mice were transplanted with BMCs from GFP transgenic mouse [TgN(ActbEGFP)] or beta-galactosidase transgenic mouse [TgN(MtnLacZ)] followed by the administration of CCl4. Irradiated TgN(Alb1HBV) and TgN(Alb1Plau) were also transplanted with BMCs from TgN(ActbEGFP) or TgN(MtnLacZ). Approximately 1.5 x 106 hepatocytes per liver were analyzed for GFP-positive cells, and the whole livers were inspected for beta-galactosidase expression. No GFP-positive hepatocytes and no gross blue staining of the livers with 5-bromo-4-chloro-3-indolyl beta-d-galactoside in any of the 18 recipient mice analyzed were detected. The livers from female animals with gender-mismatched BM transplantation were also tested with Y chromosome fluorescent in situ hybridization analysis to detect donor-derived cells. A total of five isolated hepatocytes were positive for Y chromosome in 4.1 x 105 hepatocytes analyzed. Our results demonstrate that there is little or no contribution of BMCs to the replacement of injured livers in these models. We conclude that BM-derived cells cannot generally lead to a cure of liver damage.
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PMID:Little evidence of bone marrow-derived hepatocytes in the replacement of injured liver. 1523 8

Self-association of the transactivator HBx protein of hepatitis B virus was investigated using the yeast two-hybrid system. Expression vectors for the full-length HBx (X0) and its truncated mutants (X15 and X16) were constructed by separately ligating the DNA-binding (BD) and transactivation domains (AD) of Gal4. Co-transformants of the BD and AD constructs of HBx were selected using defined minimal medium and analyzed for the reconstitution of beta-galactosidase activity. No two-hybrid interaction was observed either between the full-length HBx molecules or its highly truncated mutant X16. However, a strong functional interaction between X0 and X15, X0 and X16, and X15 and X16 suggested that HBx could self-associate in a cellular environment through its carboxy-terminal region.
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PMID:Self-association of the hepatitis B virus X protein in the yeast two-hybrid system. 1509 70

Interferon-alpha (IFN-alpha) is a potent suppressor of hepatitis B virus (HBV) replication in the HBV-transgenic mouse, depleting virus replication intermediates from infected hepatocytes via pathways mediated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). It has also been hypothesized that cytokines induce curing of infected hepatocytes via non-cytolytic pathways during resolution of transient hepadnavirus infections. We have therefore evaluated therapy of chronic woodchuck hepatitis virus (WHV) infections using treatment with the nucleoside analog clevudine [L-FMAU; 1-(2-fluoro-5-methyl-b-L-arabinofuranosyl) uracil] and therapy with adenovirus vectors expressing INF-gamma, TNF-alpha, and beta-galactosidase. Before their use in vivo, expression of IFN-gamma and TNF-alpha from the adenovirus vectors was evaluated in vitro. Conditioned media from adenovirus-infected WC-3 cells was shown to inhibit WHV replication in baculovirus-transduced cells. Adenovirus super-infection of the liver in woodchucks led to declines in the percentage of hepatocytes with detectable core antigen and nucleic acids, and in levels of covalently closed circular DNA (cccDNA) and total WHV DNA, but a major long-term benefit of adenovirus super-infection during clevudine treatment was not demonstrated. Moreover, the effect took at least 2 weeks to develop suggesting that the declines in the percentage of WHV-infected cells, ccc, and total WHV DNA resulted from induction of the adaptive immune response by the adenovirus super-infection, and only indirectly from the expression of cytokines by the vectors.
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PMID:Adenovirus-based gene therapy during clevudine treatment of woodchucks chronically infected with woodchuck hepatitis virus. 1532 95

In this paper, different cationic polymers were investigated as a DNA delivery system both in vitro in dendritic and muscle cells and in vivo, in a murine model. Expression of the reporter gene beta-galactosidase was used in order to determine the in vitro transfection efficiency of these polymer-DNA complexes (polyplexes) and both specific mRNA and protein expression were monitored in parallel with polyplex toxicity on the cells. Interestingly, the enhancing expression activities of the different polyplexes were tissue-dependent, implying that they may gain entrance to the cells through specific receptors. Subsequently, complexes of polymers and DNA plasmid (pCMV-S) encoding the human hepatitis B virus (HBV) surface antigen (HBsAg) were injected into the skeletal muscles of BALB/c mice. Higher levels of both HBsAg local expression in the tibial anterior muscles and systemic humoral immune responses were detected when the selected polymers complexed with pCMV-S were compared to those complexed with pCMV-S alone. Induction of immunoglobulin G2a (IgG2a) against HbsAg in the serum of pCMV-S-polyplex vaccinated mice varied with the polymer used, suggesting that polyplex-mediated DNA vaccination can potentially modulate the type of helper T cell immunity (Th). The effect of some polyplexes to switch the host immune response more towards a Th1 response may be associated with their differential efficiency to transfect dendritic cells and/or other antigen-presenting cells (APC) as was observed in vitro. These results suggest that the investigated cationic polymers can be effective as delivery/adjuvant compounds for DNA.
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PMID:Cationic polymers that enhance the performance of HbsAg DNA in vivo. 1553 Jun 94

Genetic immunization through skin is highly desirable as skin has plenty of antigen presenting cells (APCs) and is easily accessible. The purpose of this study was to investigate the effects of electroporation pulse amplitude, pulse length and number of pulses on cutaneous plasmid DNA vaccine delivery and immune responses, following intradermal injection in vivo in rabbits. Expression of the delivered plasmid was studied using a reporter plasmid, coding for beta-galactosidase. The efficiency of DNA vaccine delivery was investigated using a DNA vaccine against Hepatitis B, coding for Hepatitis B surface antigen (HBsAg). Serum samples and peripheral blood mononuclear cells (PBMC) were analyzed for humoral and cellular immunity, respectively, following immunization. The expression of transgene in the skin was transient and reached its peak in 2 days post-delivery with 200 and 300 V pulses. The expression levels with 200 and 300 V pulses were 48- and 129-fold higher, respectively, compared with the passive on day 2. In situ histochemical staining of skin with X-gal demonstrated the localized expression of beta-galactosidase with electroporation pulses of 200 and 300 V. Electroporation mediated cutaneous DNA vaccine delivery significantly enhanced both humoral and cellular immune responses (p<0.05) to Hepatitis B compared to passive delivery. The present study demonstrates the enhanced DNA vaccine delivery to skin and immune responses by topical electroporation. Hence, electroporation mediated cutaneous DNA vaccine delivery could be developed as a potential alternative for DNA vaccine delivery.
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PMID:Skin targeted DNA vaccine delivery using electroporation in rabbits. I: efficacy. 1581 30


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