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)

We have developed an enzyme linked immunosorbent assay (ELISA) for antimitochondrial antibody. Polyvinyl microtiter plate wells are coated with partially purified rat kidney mitochondria, and excess protein binding sites are blocked with bovine serum albumin. Human serum, diluted 1:1,000, is incubated for 1 hr. Then beta-galactosidase-goat-anti-human IgG (H + L) is added followed by the substrate, p-nitrophenyl-beta-D-galactopyranoside. The plates are then read at 404 nM in a microelisa autoreader. A positive result was defined as optical density greater than or equal to 0.100, more than 5 standard deviations above the mean of 36 normal individuals. With this technique, 56 of 60 patients with primary biliary cirrhosis were positive for antimitochondrial antibody (93%), mean O.D., 0.456 +/- 0.031 S.E. Seventeen of 17 patients with extrahepatic bile duct obstruction and 14 or 14 patients with alcoholic cirrhosis were negative. Only 1 of 29 patients with chronic active liver disease was positive (4%). Antinuclear antibody and antimicrosomal antibody do not bind in this assay, and activity is absorbed from sera by preincubation with suspensions of rat kidney mitochondria. The ELISA is approximately 20 times more sensitive than a quantitative microtiter complement fixation technique and more convenient than radioimmunoassay. It is rapid, quantitative and uses stable reagents. In contrast to immunofluorescence techniques, it is not affected by observer interpretation.
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PMID:An enzyme-linked immunosorbent assay (ELISA) for detecting antimitochondrial antibody. 637 51

The activities of the lysosomal enzymes beta-hexosaminidase, beta-glucuronidase, and beta-galactosidase were confirmed to be elevated in serum from pregnant women. The increased activity of beta-hexosaminidase and beta-glucuronidase showed the same changes in the isoenzyme pattern as found in serum from patients with some liver affections. In the light of recent findings, the increased activity of beta-hexosaminidase (and some other lysosomal enzymes) in pregnancy and liver disease might depend upon the non-parenchymal liver cells not being able properly to clear these enzymes from the blood.
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PMID:A possible explanation for the occurrence of increased beta-hexosaminidase activity in pregnancy serum. 645 14

Vibrio vulnificus infections have been associated with iron overload and preexisting liver disease. Iron may play a major role in the pathogenesis of V. vulnificus infections. Many virulence genes, as well as genes involved in the transport of iron by bacteria, are regulated by iron, with increased expression under low-iron conditions. In Escherichia coli and Vibrio cholerae, transcriptional regulation by iron depends on the fur gene. We utilized Southern hybridization under low- and high-stringency conditions with both E. coli and V. cholerae fur gene probes to demonstrate that there are fur-homologous sequences in the DNAs of V. vulnificus, Vibrio fischeri, and Aeromonas sp. but not in the DNAs of the other bacterial species tested. We developed a restriction map and cloned the fur-homologous sequence from V. vulnificus. The hybridizing clone of V. vulnificus chromosomal DNA complemented a V. cholerae fur mutant. DNA sequence analysis confirmed the presence of a 149-amino-acid open reading frame that was 77% homologous to E. coli Fur and 93% homologous to V. cholerae Fur. Primer extension localized a single promoter for the V. vulnificus fur gene. Northern (RNA) blot analysis and beta-galactosidase assays of an operon fusion to lacZ suggested that there was not significant regulation of transcription of V. vulnificus fur by iron or the E. coli Fur protein. We used marker exchange to construct a V. vulnificus fur deletion mutant and confirmed its phenotype by observing overexpression of iron-regulated outer membrane proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fur deletion mutant of V. vulnificus will be helpful in future studies of the role of iron in V. vulnificus pathogenesis.
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PMID:Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange. 767 93

Ideal methods for human gene therapy will eventually include direct gene transfer to defective tissues in a patient in vivo. Toward that goal, we have used high titer, pseudotyped retroviral vectors expressing genes for the Escherichia coli beta-galactosidase (lacZ) or hepatitis B virus surface antigen (HBsAg) to infect mouse liver by in vivo direct injection into the liver parenchyma. We have found that a single percutaneous injection of small volumes of vectors into the newborn mouse liver leads to transduction of at least 25-30% of the hepatocytes throughout the liver, as judged by in situ staining of liver sections for beta-gal activity at 4 weeks after injection. We have demonstrated that stable levels of HBsAg were also detected in the circulation of injected mice up to 4 months after HBsAg-vector injection. We suggest that the high efficiency of in vivo transduction in the neonatal liver and subsequent stable transgene expression by high-titer pseudotyped retroviral vectors in the absence of an invasive partial hepatectomy may effectively be applied to gene therapy studies in a number of human liver disease [corrected].
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PMID:Efficient in vivo transduction of the neonatal mouse liver with pseudotyped retroviral vectors. 771 30

One of several postulated roles for tissue transglutaminase (tTG) is the stabilization and assembly of extracellular matrix via peptide cross-linking. We previously determined that tTG activity increased in an animal model of hepatic fibrogenesis and in human liver disease. To further study the role of tTG in liver disease, we initiated investigations into the effect of a proinflammatory mediator, tumor necrosis factor (TNF)-alpha, on tTG activity in cultured liver cells. Treatment of human Hep G2 cells with 1 ng/ml TNF-alpha increased [14C]putrescine cross-linking to cellular proteins. An increase in tTG mRNA content was observed 1 h after addition of TNF-alpha, and levels of tTG mRNA remained elevated after 24 h. Hep G2 cells, transiently transfected with a luciferase reporter containing 1.67 kb of the human tTG promoter, showed an increase in reporter activity after addition of TNF-alpha. Gel shift experiments using nuclear extracts from TNF-alpha-treated cells and oligonucleotides containing the tTG nuclear factor (NF)-kappa B motif revealed increased binding, concordant with mRNA data. Transient transfections with a truncated reporter construct lacking the tTG NF-kappa B sequence showed an attenuated response to TNF-alpha treatment. Similar responses were seen in stably transfected HeLa cells. Primary hepatocytes isolated from a transgenic mouse line containing the mouse tTG promoter driving the beta-galactosidase reporter, show similar time-dependent increases in promoter activity when treated with TNF-alpha. Furthermore, Hep G2 cells are incapable of upmodulating tTG promoter reporter activity in the presence of TNF-alpha when those cells overexpress a transdominant, negative mutant NF-kappa B subunit. Because TNF-alpha expression is upregulated in hepatic inflammation, the data suggest TNF-alpha-mediated increases in tTG expression may play an important role in the process of hepatic fibrogenesis.
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PMID:TNF-alpha modulates expression of the tissue transglutaminase gene in liver cells. 948 75

Hepatitis C virus (HCV) is a major human pathogen causing mild to severe liver disease worldwide. This positive strand RNA virus is remarkably efficient at establishing chronic infections. Although a high rate of genetic variability may facilitate viral escape and persistence in the face of Ag-specific immune responses, HCV may also encode proteins that facilitate evasion of immunological surveillance. To address the latter possibility, we examined the influence of specific HCV gene products on the host immune response to vaccinia virus in a murine model. Various vaccinia/HCV recombinants expressing different regions of the HCV polyprotein were used for i.p. inoculation of BALB/c mice. Surprisingly, a recombinant expressing the N-terminal half of the polyprotein (including the structural proteins, p7, NS2, and a portion of NS3; vHCV-S) led to a dose-dependent increase in mortality. Increased mortality was not observed for a recombinant expressing the majority of the nonstructural region or for a negative control virus expressing the beta-galactosidase protein. Examination of T cell responses in these mice revealed a marked suppression of vaccinia-specific CTL responses and a depressed production of IFN-gamma and IL-2. By using a series of vaccinia/HCV recombinants, we found that the HCV core protein was sufficient for immunosuppression, prolonged viremia, and increased mortality. These results suggest that the HCV core protein plays an important role in the establishment and maintenance of HCV infection by suppressing host immune responses, in particular the generation of virus-specific CTLs.
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PMID:Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence. 991 17

Xenotransplantation of hepatocytes appears to be a novel promising therapy for some forms of liver disease, and may well overcome the problem of donor shortage. We have previously reported that hepatocytes with a spheroidal shape (spheroids) are ideal for cell transplantation. The application of gene transfer techniques to this hepatocyte transplantation could possibly regulate the xenogeneic rejection reaction and, therefore, result in prolongation of the survival of the transplanted hepatocytes. In this study, we chose the adenovirus as a vector and an immunosuppressive cytokine named viral IL-10 (vIL-10) for transfection. A series of experiments was performed to elucidate the efficacy of transfection to the spheroids with adenovirus vectors and the effect of transfected vIL-10 on the survival of xenogeneic hepatocytes. We examined the cell survival quantitatively by evaluating beta-galactosidase (beta-gal) activity, which was transfected into the hepatocytes in the xenogeneic spleen, and semiquantitatively by the histological findings. The results of in-vitro studies identified an efficient expression of the beta-gal gene within the spheroids infected with Ad-CMVLacZ (LacZ-encoding adenovirus vector with CMV promotor) and the presence of BCRF1 mRNA within the spheroids transfected with AdCMVvIL-10 (vIL-10-expressing adenovirus vector with CMV promotor) under the condition of 1 MOI, for 1 h. Xenogeneic hepatocytes with a spheroidal shape showed comparable survival to syngeneic hepatocytes for up to 4 days after transplantation with co-transplantation of the vIL-10-transfected hepatocytes. From this study, we concluded that adenovirus-mediated vIL-10 gene transfer prolongs the survival of xenogeneic hepatocyte spheroids. Furthermore, spheroids possess ideal properties for gene transfection, as well as cell transplantation.
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PMID:Adenovirus-mediated viral IL-10 gene transfer prolongs survival of xenogeneic spheroidal aggregate-cultured hepatocytes. 1111 60

We determined whether extrahepatic biliary epithelial cells can differentiate into cells with phenotypic features of hepatocytes. Gallbladders were removed from transgenic mice expressing hepatocyte-specific beta-galactosidase (beta-Gal) and cultured under standard conditions and under experimental conditions designed to induce differentiation into a hepatocyte-like phenotype. Gallbladder epithelial cells (GBEC) cultured under standard conditions exhibited no beta-Gal activity. beta-Gal expression was prominent in 50% of cells cultured under experimental conditions. Similar morphological changes were observed in GBEC from green fluorescent protein transgenic mice cultured under experimental conditions. These cells showed higher levels of mRNA for genes expressed in hepatocytes, but not in GBEC, including aldolase B, albumin, hepatocyte nuclear factor-4alpha, aldehyde dehydrogenase 1, and glutamine synthetase, and they synthesized bile acids. Additional functional evidence of a hepatocyte-like phenotype included LDL uptake and enhanced benzodiazepine metabolism. Connexin-32 expression was evident in murine hepatocytes and in cells cultured under experimental conditions, but not in cells cultured under standard conditions. Notch 1, 2, and 3 and Notch ligand Jagged 1 mRNAs were downregulated in these cells compared with cells cultured under standard conditions. CD34, alpha-fetoprotein, and Sca-1 mRNA were not expressed in cells cultured under standard conditions, suggesting that the hepatocyte-like cells did not arise from hematopoietic stem cells or oval cells. These results point to future avenues for investigation into the potential use of GBEC in the treatment of liver disease.
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PMID:Murine gallbladder epithelial cells can differentiate into hepatocyte-like cells in vitro. 1771 44

Systemic chemotherapy has limited success in treating liver metastasis of colorectal cancer. Alternative approaches such as hepatic arterial infusion or trans arterial chemoembolisation aim to deliver the chemotherapy locally to address the predominant liver disease. Chemoembolisation with drug eluting beads (DEB) designed to deliver drug at the target over a protracted period of time is a new strategy to reduce the tumor burden of liver metastases. To test this hypothesis, DEB possessing anionic groups capable of ionically complexing with cationic drugs were synthesised by a suspension polymerisation method and were fractionated to produce an average size of 75 microm. The DEB were loaded with the desired concentration of either doxorubicin hydrochloride or irinotecan hydrochloride prior to administration by immersion in the drug solution, yielding essentially 100% loading efficiency. To determine their effect in vivo, a transplantable orthotopic and isogenic rat liver metastasis model was used which is based on intraportal injection of 4 x 10(6) beta-galactosidase transfected CC531 rat colorectal cancer cells into male WAG/Rij rats. By MTT assay, the cells were shown to be sensitive to both drugs in vitro with the IC(50) being by two orders of magnitude lower for doxorubicin (110 nM after 72 h) compared to irinotecan (25 microM after 72 h). For the in vivo phase, a differential expression of the ERK MAP kinase between tumor cells cultured in vitro and those inoculated in vivo was noted using Western blotting techniques. This was considered to be indicative of passage-induced cell senescence that reduced the sensitivity of the tumor cells to DEB chemoembolisation. This notwithstanding, administration of DEB loaded with irinotecan or doxorubicin by single injection into the hepatic artery showed significant anticancer activity, as measured by a reduction in the tumor burden of the liver and a corresponding reduction in liver weight. Comparing the two agents, irinotecan appears more advantageous because of its significant activity and excellent tolerability following administration at two dosages of either 20 or 30 mg/kg. Doxorubicin showed a narrower window of activity, being effective at 4 mg/kg but ineffective at the lower dose of 2 mg/kg. We conclude that chemoembolisation with DEB with either agent may have potential for treating patients with colorectal liver metastasis, although irinotecan DEB appeared to have a more favourable safety profile.
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PMID:Chemoembolisation of rat colorectal liver metastases with drug eluting beads loaded with irinotecan or doxorubicin. 1825 82

BACKGROUND Cholesteryl ester storage disease (CESD), also known as lysosomal acid lipase deficiency (LAL-D), is a rare autosomal-recessive inheritable lysosomal storage disease. Since 2015, a causal treatment with sebelipase alfa, which replaces the missing LAL enzyme, has been approved. We report a fatal course of LAL-D in a female patient. CASE REPORT In 1979, CESD was first diagnosed in a 13-year-old female with marked hepatomegaly. At that time, no specific treatment for CESD was available and the spontaneous course of the disease had to be awaited. In 2013, a laparoscopic cholecystectomy for symptomatic gallstones was performed. The patient's CESD had caused a Child-Pugh A/B and Lab-MELD 14 cirrhosis with esophageal varices (grade III), a solitary fundal varix, as well as hepatosplenomegaly with thrombocytopenia. In 2016, the patient was admitted with compensated cirrhosis and splenomegaly for a ligature of esophageal varices which was complicated by vomiting of blood followed by severe coagulopathy and hemorrhagic shock. The dried blood test showed reduced acid lipase (0.03 nmol/spot*3 hours; reference range 0.2-2) and beta-galactosidase (0.08 nmol/spot*21 hours; reference range 0.5-3.2). Then 15 days after the esophageal varices bleed, the patient died due to multiorgan failure as a sequelae of advanced liver disease. CONCLUSIONS LAL-D should be included in the differential diagnosis of lipid metabolism disorder, hepatomegaly, and non-alcoholic fatty liver disease with fibrosis or cirrhosis. Causal treatment with sebelipase alfa should be introduced even in patients who have LAL-D and many years of clinically mild symptoms of this disease to prevent the serious sequelae of cirrhosis or cardiovascular complications.
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PMID:Cholesteryl Ester Storage Disease: Fatal Outcome without Causal Therapy in a Female Patient with the Preventable Sequelae of Progressive Liver Disease after Many Years of Mild Symptoms. 2977 83


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