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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Quantitative comparisons of the time course of biochemical and morphological changes induced by peroxisome proliferators resulting in low and high incidences of hepatic cancer have not been conducted previously under bioassay conditions. [4-Chloro-6-(2,3-xylidino)-2-pyrimidyl-thio]acetic acid (Wy-14,643) at 0.1% in the diet produced a much higher incidence of hepatic cancer in male rats than 1.2% di(2-ethylhexyl)phthalate (DEHP) in the diet. Both diets, however, caused similar degrees of peroxisome proliferation. To investigate this difference in carcinogenicity, H2O2-detoxification mechanisms and indices of oxidative damage were evaluated in male F-344 rats fed 1.2% DEHP or 0.1% Wy-14,643 for up to one year. DEHP or Wy-14,643 treatment increased hepatic catalase activity approximately 25% from 8 to 365 days. DEHP or Wy-14,643 treatment decreased hepatic glutathione peroxidase activity by 50% from 8 to 365 days. Glutathione concentrations were not affected by 151 days of DEHP or Wy-14,643 feeding. The similar effects of DEHP and Wy on H2O2 detoxification enzymes and glutathione concentrations suggests that these factors are not responsible for the widely different carcinogenicities of Wy-14,643 and DEHP. Hepatic vitamin E concentrations were 50% lower in rats receiving Wy-14,643 for 151 days as compared to rats fed DEHP or control diets. Lipofuscin, which was contained within lysosomes, was increased 3-fold after 39 days of DEHP and remained at this level up to 365 days of treatment. In comparison, lipofuscin was increased 4-fold after 18 days of Wy-14,643 and continued to accumulate in a linear manner reaching values 30-fold over controls after 365 days of treatment. DEHP treatment for 39-365 days increased the activities of the lysosomal enzymes alpha-fucosidase, beta-galactosidase and N-acetylglucosaminidase 50-100%. The same enzyme activities were increased approximately 4-fold after 39-365 days of Wy-14,643. Lysosomal cathepsin B activity was unchanged by DEHP but doubled by 151 and 365 days of Wy-14,643. Acid phosphatase activity was unchanged by DEHP but increased by 50% after 151 and 365 days of Wy-14,643. In addition, conjugated dienes were increased (approximately 45%) only in rats receiving Wy-14,643 for 151 and 365 days. These data show for the first time that the magnitude and time course of lipofuscin deposition, induction of lysosomal enzymes and conjugated diene accumulation, is correlated closely with the degree of carcinogenicity. Wy-14,643-induced decreases in hepatic vitamin E concentrations could contribute to the observed accumulation of conjugated dienes at later time points.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Relationship of oxidative damage to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and Wy-14,643. 292 96

An understanding of how oncogenes affect differentiated liver functions might lead to improved treatments for liver cancer or other disorders where liver-specific functions are compromised. A retroviral vector that coexpressed beta-galactosidase (beta-gal) and activated Ras genes (Ras-gal) was transduced into a small fraction of adult rat hepatocytes in vivo. Hepatocytes from Ras-gal-transduced diethylnitrosamine-untreated livers and hepatocellular carcinomas (HCC) from Ras-gal-transduced diethylnitrosamine-treated rats were analyzed for liver functions by performing histochemical assays on liver sections. Ras-gal-transduced hepatocytes failed to express gluconeogenic, ketogenic, and urea pathway enzymes. In contrast, several enzymes involved in fat synthesis were strongly activated, and microvesicular fat accumulated. These metabolic changes are induced in normal livers by insulin, a hormone that activates p21-ras. The deregulation of p21-ras may inhibit these liver-specific functions and may induce fat synthesis in both malignant and nonmalignant liver diseases. Furthermore, treatment with drugs that inhibit the attachment of p21-ras to the plasma membrane might reverse these changes. The alterations in enzymatic functions in the HCCs were similar to those observed in the hepatocytes, although each of the two cancers had a region that abruptly lost its expression of liver-specific enzymes and acquired the expression of genes that are more characteristic of oval or bile ductule cells. This suggests that a single genetic event in a malignant cell may dramatically alter its apparent phenotype. The identification of this putative gene might lead to insights into the regulation of the phenotype of normal cells in the liver.
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PMID:Alterations in enzymatic functions in hepatocytes and hepatocellular carcinomas from Ras-transduced livers resemble the effects of insulin. 885 86

We have developed a miniviral vector, pH300, based on the human herpesviruses 1 and 4, herpes simplex virus type 1 (HSV-1), and Epstein-Barr virus (EBV), carrying EBV sequences for plasmid episomal maintenance and HSV-1 sequences for amplification and packaging in multimeric form into HSV-1 capsids in the presence of a helper virus and helper cell line. A reporter gene, the bacterial lacZ gene, which expressed beta-galactosidase, was inserted into the multiple cloning site of pH300 to make pH300-lac. The packaged pH300-lac DNA was very efficient in infecting human cells in tissue culture. The pH300-lac miniviral stock was used to infect in vitro various human cell types derived from breast cancer, lung cancer, and liver cancer. Up to 95% of cells were infected and expressed beta-galactosidase activity after exposure to viral stock at a multiplicity of infection of 3. There was essentially no apparent cytotoxicity after infection of cultured cells in vitro. To test in vivo gene delivery, human liver tumor cells preimplanted subcutaneously in nude mice and injected in situ with pH300-lac showed high efficiency of ectopic gene expression. The pH300 miniviral vector is a simple and effective gene transfer system which shows potential for gene therapy of cancer and inherited diseases.
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PMID:A hybrid herpesvirus infectious vector based on Epstein-Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo. 897 Sep 63

Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the beta-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of beta-galactosidase-positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.
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PMID:In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes. 1206 89

Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.
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PMID:HCV NS3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen. 1578 58

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a class of well-studied DNA-interactive agents with a potential for use in the treatment of cancer. The clinical utility of these molecules is limited because of the lack of selectivity for tumor tissues, high reactivity of the pharmacophoric imine functionality, low water solubility, and stability. To address the shortcomings, especially the lack of selectivity, associated with the molecules, two new beta-galactoside prodrugs of PBDs have been synthesized and evaluated for their potential use in selective therapy of solid tumors by ADEPT and PMT protocols. The preliminary studies reveal the prodrugs to be much less toxic compared to the parent moieties. These prodrugs are activated by E. coli beta-galactosidase (EC 3.2.1.23) to form the active cytotoxic moiety signifying their utility in ADEPT of cancer. One of the significant outcomes of the present study is the toxification of the prodrug 1 a by the endogenous beta-galactosidase of human liver cancer cells (Hep G2) to form the cytotoxic moiety, enabling selective therapy of hepatocellular carcinoma. Another important property of these molecules is their enhanced water solubility and stability, which are essential for a molecule to be an effective drug.
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PMID:Development of pyrrolo[2,1-c][1,4]benzodiazepine beta-galactoside prodrugs for selective therapy of cancer by ADEPT and PMT. 1824 36

Neonatal injection of recombinant adeno-associated virus serotype 8 (rAAV8) vectors results in widespread transduction in multiple organs and therefore holds promise in neonatal gene therapy. On the other hand, insertional mutagenesis causing liver cancer has been implicated in rAAV-mediated neonatal gene transfer. Here, to better understand rAAV integration in neonatal livers, we investigated the frequency and spectrum of genomic integration of rAAV8 vectors in the liver following intraperitoneal injection of 2.0 x 10(11) vector genomes at birth. This dose was sufficient to transduce a majority of hepatocytes in the neonatal period. In the first approach, we injected mice with a beta-galactosidase-expressing vector at birth and quantified rAAV integration events by taking advantage of liver regeneration in a chronic hepatitis animal model and following partial hepatectomy. In the second approach, we performed a new, quantitative rAAV vector genome rescue assay by which we identified rAAV integration sites and quantified integrations. As a result, we find that at least approximately 0.05% of hepatocytes contained rAAV integration, while the average copy number of integrated double-stranded vector genome per cell in the liver was approximately 0.2, suggesting concatemer integration. Twenty-three of 34 integrations (68%) occurred in genes, but none of them were near the mir-341 locus, the common rAAV integration site found in mouse hepatocellular carcinoma. Thus, rAAV8 vector integration occurs preferentially in genes at a frequency of 1 in approximately 10(3) hepatocytes when a majority of hepatocytes are once transduced in the neonatal period. Further studies are warranted to elucidate the relationship between vector dose and integration frequency or spectrum.
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PMID:Frequency and spectrum of genomic integration of recombinant adeno-associated virus serotype 8 vector in neonatal mouse liver. 1861 41

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