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)

Known high and low molecular weight (LMW) MAP2 protein isoforms result from alternative splicing of the MAP2 gene. Contrary to previous reports that MAP2 is neural-specific, we recently identified MAP2 mRNA and protein in somatic and germ cells of rat testis, and showed the predominant testicular isoform is LMW. Although cytoplasmic in neural tissue, MAP2 appeared predominantly nuclear in germ cells using immunohistochemistry. We sought to determine whether this unexpected localization was due to the inclusion of exon 10 within novel LMW MAP2 isoforms. Normally excluded from the LMW MAP2c, exon 10 harbors a putative CcN motif, comprising a nuclear localization sequence (NLS) flanked by regulatory phosphorylation sites for protein kinase CK2 and cdc2 kinase. Characterization of MAP2 mRNA in adult and immature brain and testis, by reverse transcriptase-polymerase chain reaction/Southern analysis and Northern blot, identified novel LMW forms containing exons 10 and 11, previously detected only in high molecular weight MAP2a and 2b. The MAP2 NLS targeted a large heterologous protein to the nucleus, as demonstrated using bacterially expressed MAP2-CcN-beta-galactosidase fusion protein and an in vitro nuclear import assay. Antibodies raised against the fusion protein produced a testicular immunohistochemical staining pattern correlating with MAP2 protein distribution in the nucleus of most germ cells, and precipitated both approximately 70-kDa and >220-kDa proteins recognized by the commercial MAP2-specific HM2 monoclonal antibody, supporting our hypothesis of a novel LMW MAP2 isoform. These results demonstrate the presence of a functional NLS in MAP2 and indicate that novel LMW MAP2 isoforms may be targeted to the nucleus in both neural and non-neuronal tissues.
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PMID:Novel low molecular weight microtubule-associated protein-2 isoforms contain a functional nuclear localization sequence. 1038 34

Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint using the adenovirus vector is feasible as an effective in vivo method.
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PMID:Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs. 1047 Nov 54

Esophagitis is a major toxicity of radiation therapy for nonsmall-cell lung cancer. Intraesophageal injection of manganese superoxide dismutase (MnSOD) plasmid/liposome complexes (1 mg of the pRK5-MnSOD plasmid containing the human MnSOD transgene in a 0.15 ml volume of lipofectin) before irradiation was carried out to attempt to prevent irradiation esophagitis. In control noninjected male C3H/HeNsd mice, esophagitis was induced by single fraction 3,500 cGy irradiation. Histopathology at 4 days revealed vacuole formation in squamous lining cells, separation of the squamous layer from the underlying muscle layer, ulceration at 7 days, and dehydration and death by 30 days. MnSOD plasmid/liposome complex-injected mice showed transcription of the human MnSOD transgene message in esophageal squamous lining cells by nested reverse transcriptase-polymerase chain reaction (RT-PCR) increased MnSOD biochemical activity 24 h after injection, decreased vacuole formation at day 4 (P < 0.001) after 3,500 cGy thoracic irradiation, and improved survival (P = 0.0009). In contrast, groups of mice receiving LacZ (bacterial beta-galactosidase gene) plasmid/liposome complexes or liposomes containing no DNA before 3,500 cGy irradiation showed an unaltered irradiation histopathology and decreased survival. Mice receiving intraesophageal MnSOD plasmid/liposomes followed 8 h later by human equivalent doses of Taxol (1.4 mg/kg) and carboplatin (2.5 mg/kg), then 15 h later 3,300 cGy irradiation, showed increased survival, compared with irradiated control or LacZ plasmid/liposome groups. Thus, overexpression of the human MnSOD transgene in the esophagus can prevent irradiation-induced esophagitis in the mouse model.
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PMID:Prevention of irradiation-induced esophagitis by plasmid/liposome delivery of the human manganese superoxide dismutase transgene. 1049 61

The activity and expression of transgene beta-galactosidase (lacZ) by replication-deficient adenoviral vectors (Ad-lacZ) containing prostate-specific promoters were compared using an in vivo canine model. The prostate tissue-specific promoters were prostate-specific antigen, probasin, and mouse mammary tumor virus long-terminal repeat, which were fused separately to an Escherichia coli lacZ gene. Dogs underwent laparotomy, and adenoviral vectors were delivered by direct intraprostatic injection. At 72 hours postinjection, the prostate and various other organs were harvested to evaluate the degree of prostate expression and dissemination of adenoviral vectors. Expression of lacZ in tissues was determined by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining, beta-galactosidase assay, and E. coli lacZ reverse transcriptase-polymerase chain reaction (PCR). The presence of adenoviral DNA sequences in canine tissues was determined by PCR using primers specific for the type 5 adenoviral genome. All three of the prostate-specific adenoviruses tested effectively expressed the lacZ gene in the canine prostate, but expression levels were lower than that of the control viral vector AdRSVlacZ following intraprostatic injection. By PCR, adenoviral vector DNA was detected in other organs and tissues, including the bladder and vas deferens. However, reverse transcriptase-PCR analysis revealed that prostate-specific Ad-lacZ vectors only transcribed lacZ mRNA in the prostate and not in nonprostatic tissues. Thus, these novel prostate-specific adenoviral vectors each have equal in vivo expression exclusively in the prostate and may potentially be used for prostate cancer gene therapy.
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PMID:In vivo expression of prostate-specific adenoviral vectors in a canine model. 1050 56

Introducing recombinant genes into donor hearts may offer a therapeutic intervention that could potentially attenuate the complications of heart transplantation, including rejection, infection and accelerated atherosclerosis. In the cardiovascular system, reduced bioactivity of endothelial nitric oxide is a feature of atherosclerosis and vascular injury. Nitric oxide is an arterial vasodilator that also inhibits proliferation of vascular smooth muscle cells and platelet aggregation. Experiments were designed to determine the distribution of adenoviral-mediated transfer of recombinant endothelial nitric oxide synthase gene (eNOS) and the effect of recombinant gene expression on the function of transplanted hearts. Adenoviral vectors for (a) bovine eNOS (AdeNOS) or (b) beta-galactosidase (AdLacZ; control) were infused into two groups (n = 12, per group) of explanted rat hearts. The transduced hearts were then implanted heterotopically into the abdomen of syngeneic recipient rats. After four days, the hearts were excised and examined for distribution and function of the recombinant genes. Polymerase chain reaction (PCR) verified the presence of the recombinant eNOS gene in eNOS-transduced but not in beta-galactosidase-transduced hearts; reverse transcriptase-PCR identified mRNA for eNOS in AdeNOS-transduced hearts. NOS activity (conversion of tritiated L-arginine to citrulline) was greater in homogenates of AdeNOS-compared to AdLacZ-transduced hearts. Positive immunoreactivity for eNOS was present in cardiomyocytes predominantly in eNOS-transduced hearts. Myocardial contractility and coronary blood flow, as determined using a Langendorff preparation, were not different between hearts transduced with AdeNOS or AdLacZ. These results suggest that, up to four days post transplantation, adenoviral-mediated transfer of eNOS into transplanted hearts is possible. However, expression of the recombinant protein did not result in measurable changes in myocardial contractility or coronary perfusion.
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PMID:Distribution and function of recombinant endothelial nitric oxide synthase in transplanted hearts. 1053 12

Phenotypic drug susceptibility assays of human immunodeficiency virus type 1 (HIV-1) isolates generally use time-consuming, expensive assays with peripheral blood mononuclear cells. A new HIV-1 indicator cell line, MAGI-CCR5, has been developed and applied for this purpose. This cell line expresses human CD4, the two major HIV-1 coreceptors, CCR5 and CXCR4, the reporter gene beta-galactosidase driven by the HIV-1 LTR, and quantitates infection within 48 h. A panel of reference strains and primary HIV-1 isolates were all found to infect this cell line. Susceptibility assays with a nucleoside (zidovudine, ZDV) and a non-nucleoside reverse transcriptase inhibitor (nevirapine, NVP) were performed with reference and primary isolates. The assay was modified into two steps for protease inhibitor (indivinavir, IDV and ritonavir, RTV) susceptibility assays. Primary isolates derived from drug naive patients displayed mean baseline 50% effective concentrations (EC50) of 0.14 microM for ZDV, 0.33 microM for NVP, and 0.02 microM for IDV. Isolates derived from patients under treatment displayed increased EC50 concentrations. The MAGI-CCR5 cell line offers a rapid, efficient, and reproducible method of testing a wide range of HIV-1 isolates for drug susceptibility.
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PMID:Rapid phenotypic drug susceptibility assay for HIV-1 with a CCR5 expressing indicator cell line. 1071 48

Gene transfer was performed using asialo-oroso-mucoid-polylysine (ASOR-PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel-electrophoretic analysis, the ASOR-PL conjugate produced a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR-PL conjugate/pCMV beta-gal plasmid). The gene-transfer efficiency of the ASOR-PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein receptor and NIH 3T3 cells that do not. The expression was assayed by 5-bromo-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chlorophenol Red beta-D-galactopyranoside. When an expression vector for the tumour-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza-virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of beta-galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor-mediated gene-delivery system for hepatic gene therapy using ASOR-PL conjugate in combination with poloxamer 407 may be developed in the future.
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PMID:Improvement of receptor-mediated gene delivery to HepG2 cells using an amphiphilic gelling agent. 1091 34

Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial beta-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1. 3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.
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PMID:Role of murine leukemia virus reverse transcriptase deoxyribonucleoside triphosphate-binding site in retroviral replication and in vivo fidelity. 1104 79

We attempted to determine whether beta1,3-galactosyltransferase beta3Gal-T5 is involved in the biosynthesis of a specific subset of type 1 chain carbohydrates and expressed in a cancer-associated manner. We transfected Chinese hamster ovary (CHO) cells expressing Fuc-TIII with beta3Gal-T cDNAs and studied the relevant glycoconjugates formed. beta3Gal-T5 directs synthesis of Lewis type 1 antigens in CHO cells more efficiently than beta3Gal-T1, whereas beta3Gal-T2, -T3, and -T4 are almost unable to direct synthesis. In the clone expressing Fuc-TIII and beta3Gal-T5 (CHO-FT-T5), sialyl-Lewis a synthesis is strongly inhibited by swainsonine but not by benzyl-alpha-GalNAc, and sialyl-Lewis x is absent, although it is detected in the clones expressing Fuc-TIII and beta3Gal-T1 (CHO-FT-T1) or Fuc-TIII and beta3Gal-T2 (CHO-FT-T2). Endo-beta-galactosidase treatment of N- glycans prepared from clone CHO-FT-T5 releases (+/-NeuAcalpha2-->3)Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->3Gal but not GlcNAcbeta1-->3Gal or type 2 chain oligosaccharides, which are found in CHO-FT-T1 cells. This result indicates that beta3Gal-T5 expression prevents poly-N-acetyllactosamine and sialyl-Lewis x synthesis on N-glycans. Kinetic studies confirm that beta3Gal-T5 prefers acceptors having the GlcNAcbeta1-->3Gal end, including lactotriosylceramide. Competitive reverse transcriptase mediated-polymerase chain reaction shows that the beta3Gal-T5 transcript is expressed in normal colon mucosa but not or poorly in adenocarcinomas. Moreover, recombinant carcinoembryonic antigen purified from a CHO clone expressing Fuc-TIII and beta3Gal-T5 reacts with anti-sialyl-Lewis a and carries type 1 chains on oligosaccharides released by endo-beta-galactosidase. We conclude that beta3Gal-T5 down-regulation plays a relevant role in determining the cancer-associated glycosylation pattern of N-glycans.
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PMID:beta 1,3-Galactosyltransferase beta 3Gal-T5 acts on the GlcNAcbeta 1-->3Galbeta 1-->4GlcNAcbeta 1-->R sugar chains of carcinoembryonic antigen and other N-linked glycoproteins and is down-regulated in colon adenocarcinomas. 1105 88

In the pancreas, ligands of receptor tyrosine kinases (RTKs) are thought to be implicated in the development and function of the islets of Langerhans, which represent the endocrine part of the pancreas. In a previous study, we randomly screened by reverse transcriptase-polymerase chain reaction for RTKs expressed in the embryonic pancreas. One cDNA fragment that was cloned during this screen corresponded to the KIT receptor. The objective of the present study was to analyze the pattern of Kit expression in the pancreas. We demonstrated that Kit is expressed and functional in terms of signal transduction in the insulin-producing cell line INS-1. Indeed, upon treatment with the KIT ligand (KITL), the extracellular signal-regulated protein kinase was phosphorylated, and the expression of early responsive genes was induced. We also demonstrated that Kit mRNAs are present in fetal and adult rat islets. We next used mice that had integrated the lacZ reporter gene into the Kit locus. In these mice, beta-galactosidase (beta-gal) served as a convenient marker for expression of the endogenous Kit gene. Kit was found to be specifically transcribed in beta-cells (insulin-expressing cells), whereas no expression was found in other endocrine cell types or in the exocrine tissue. Interestingly, not all mature beta-cells expressed Kit, indicating that Kit is a marker of a subpopulation of beta-cells. Finally, by following beta-gal expression in the pancreas during fetal life, we found that at E14.5, Kit is expressed in both insulin- and glucagon-expressing cells present at that stage, and also in a specific cell population present in the epithelium that stained negative for endocrine markers. These data suggest that these Kit-positive/endocrine-negative cells could represent a subpopulation of endocrine cell precursors.
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PMID:Expression of the receptor tyrosine kinase KIT in mature beta-cells and in the pancreas in development. 1152 67


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