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)

Brucella abortus strain RB51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Our ultimate goal is to develop strain RB51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong Th1 type of immune response is needed for effective protection. As a first step in that direction, we studied the expression of a foreign reporter protein, beta-galactosidase of Escherichia coli, and the 65-kDa heat shock protein (HSP65) of Mycobacterium bovis in strain RB51. We cloned the promoter sequences of Brucella sodC and groE genes in pBBR1MCS to generate plasmids pBBSODpro and pBBgroE, respectively. The genes for beta-galactosidase (lacZ) and HSP65 were cloned in these plasmids and used to transform strain RB51. An enzyme assay in the recombinant RB51 strains indicated that the level of beta-galactosidase expression is higher under the groE promoter than under the sodC promoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODpro/lacZ, increased levels of beta-galactosidase expression were observed after subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells. Mice vaccinated with either of the beta-galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation of their splenocytes with beta-galactosidase induced the secretion of gamma interferon (IFN-gamma), but not interleukin-4 (IL-4). A Th1 type of immune response to HSP65, as indicated by the presence of specific serum IgG2a, but not IgG1, antibodies, and IFN-gamma, but not IL-4, secretion by the specific-antigen-stimulated splenocytes, was also detected in mice vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated that expression of beta-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or its vaccine efficacy against B. abortus 2308 challenge.
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PMID:Brucella abortus strain RB51 as a vector for heterologous protein expression and induction of specific Th1 type immune responses. 1081 76

Infectious hematopoietic necrosis virus (IHNV) infection in tissue culture cells has previously been shown to result in the shutdown of host protein synthesis, cell rounding, and cell death. We report here an investigation of the cytopathogenicity of the viral phosphoprotein (P or M1), matrix (M or M2), and nonvirion (NV) proteins in cultured fish cells. The expression of M alone potently inhibited reporter gene expression from a viral and an interferon (IFN)-inducible promoter, whereas P and NV did not produce a similar effect. Northern blot analysis further revealed a reduction in the steady-state level of reporter mRNA when the M gene was cotransfected into cells; conversely, M mRNA was not drastically reduced in the same cells. By immunofluorescence confocal microscopy, fragmented nuclei were found in some cells expressing M protein but not in cells expressing P, NV, or beta-galactosidase protein. Electron microscopy revealed the morphological changes associated with apoptosis in the M-transfected cells. Furthermore, IHNV infection was shown to produce DNA "laddering" in cultured cells. Taken together, these data suggested at least two functions for M protein in an IHNV infection: down regulation of host transcription and the induction of programmed cell death. In the course of these experiments, we also discovered that NV expression was associated with cell rounding, the first biological effect on cells to be attributed to the NV gene.
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PMID:Infectious hematopoietic necrosis virus matrix protein inhibits host-directed gene expression and induces morphological changes of apoptosis in cell cultures. 1090 16

Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by the DNA. The preferred vehicle for plasmid DNA injections has been saline (154 mM sodium chloride) or PBS (154 mM NaCl plus 10 mM sodium phosphate). Here, it is shown that injection of luciferase or beta-galactosidase encoding plasmid DNA in a 150 mM sodium phosphate vehicle into murine muscle resulted in a two- to seven-fold increase in transgene expression compared with DNA injected in saline or PBS. When the DNA encoded secreted alkaline phosphatase, preproinsulin or interferon, sodium phosphate vehicle increased their serum levels by two- to four-fold. When the DNA encoded mouse erythropoietin, sodium phosphate vehicle increased hematocrits by two-fold compared with DNA injected in saline. When the DNA encoded influenza nucleoprotein, sodium phosphate increased anti-nucleoprotein antibody titers by two-fold. The expression of luciferase from plasmid DNA instilled into lung was increased five-fold compared with that in vehicle without sodium phosphate. Incubation of plasmid DNA with muscle extract or serum showed that sodium phosphate protected the DNA from degradation. Thus, a change from sodium chloride to sodium phosphate vehicle can enhance the expression of plasmid DNA in a tissue, possibly by inhibiting DNA degradation. Gene Therapy (2000) 7, 1171-1182.
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PMID:Sodium phosphate enhances plasmid DNA expression in vivo. 1091 85

The Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, open reading frame (ORF) K9 encodes a viral interferon regulatory factor (vIRF) that functions as a repressor for interferon-mediated signal transduction. Consequently, this gene is thought to play an important role in the tumorigenicity of KSHV. To understand the molecular mechanisms underlying vIRF expression, we studied the transcriptional regulation of this gene. Experiments using 5' rapid amplification of cDNA ends and primer extension revealed that vIRF had different transcriptional patterns during the latent and lytic phases. The promoter region of the minor transcript, which was mainly expressed in uninduced BCBL-1 cells, did not contain a canonical TATA box, but a cap-like element and an initiator element flanked the transcription start site. The promoter of the major transcript, which was mainly expressed in tetradecanoyl phorbol acetate-induced BCBL-1 cells, contained a canonical TATA box. A luciferase reporter assay using a deletion mutant of the vIRF promoter and a mutation in the TATA box showed that the TATA box was critical for the lytic activity of vIRF. The promoter activity in the latent phase was eight times stronger than that of the empty vector but was less than 10% of the activity in the lytic phase. Therefore, KSHV may use different functional promoter elements to regulate the expression of vIRF and to antagonize the cell's interferon-mediated antiviral activity. We have also identified a functional domain in the ORF 50 protein, an immediate-early gene product that is mainly encoded by ORF 50. The ORF 50 protein transactivated the vIRF and DNA polymerase promoters in BCBL-1, 293T, and CV-1 cells. Deleting one of its two putative nuclear localization signals (NLSs) resulted in failure of the ORF 50 protein to localize to the nucleus and consequently abrogated its transactivating activity. We further confirmed that the N-terminal region of the ORF 50 protein included an NLS domain. We found that this domain was sufficient to translocate beta-galactosidase to the nucleus. Analysis of deletions within the vIRF promoter suggested that two sequence domains were important for its transactivation by the ORF 50 protein, both of which included putative SP-1 and AP-1 binding sites. Competition gel shift assays demonstrated that SP-1 bound to these two domains, suggesting that the SP-1 binding sites in the vIRF promoter are involved in its transactivation by ORF 50.
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PMID:Transcriptional regulation of the Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor gene. 1095 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

Adenovirus (Adv)-mediated gene transfer requires efficient infection of target cells. The objective of this study was to establish whether alveolar macrophages (AM) and T cells (AT) from sarcoid patients were permissive to infection with Adv vectors and if this property could be used to investigate cytokine gene regulation. Sarcoid and normal bronchoalveolar lavage (BAL) specimens infected with Adv vectors expressing either beta-galactosidase or a green fluorescent protein were analyzed for transgene expression by fluorescence-activated cell sorter (FACS) and direct immunofluorescence, respectively. Expression of surface antigens previously associated with Adv infection, the coxsackie/adenovirus receptor (CAR), alpha v beta 3, and alpha v beta 5 integrins, was also assessed using FACS analysis. Sarcoid AM and AT were found to efficiently express Adv transgenes, unlike AM from normal volunteers, peripheral blood monocytes, and peripheral blood T cells. Cells permissive to Adv infection expressed the CAR and alpha v beta 5 integrin (also alpha v beta 3 integrin for AM). The data indicate that the upregulation of Adv receptors and the ability to infect sarcoid AM and AT are related to the inflammatory environment within the lung. Having demonstrated efficient Adv-mediated transgene delivery to sarcoid AM and AT, a construct encoding porcine I kappa B alpha was then used to investigate the requirement for nuclear factor (NF)-kappa B in the regulation of cytokine gene expression in pulmonary sarcoidosis. Overexpression of I kappa B alpha in sarcoid BAL specimens indicated that tumor necrosis factor-alpha and interleukin (IL)-6 production by AM and interferon (IFN)-gamma production by AT is NF-kappa B dependent, whereas IL-4 production by AT is NF-kappa B independent. This is the first occasion that the requirement for NF-kappa B in IFN-gamma gene expression within primary human T cells has been demonstrated. The results of this study have implications for the future investigation of molecular pathways in inflammatory lung disease.
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PMID:Alveolar macrophages and T cells from sarcoid, but not normal lung, are permissive to adenovirus infection and allow analysis of NF-kappa b-dependent signaling pathways. 1150 22

UBP43 shows significant homology to well characterized ubiquitin-specific proteases and previously was shown to hydrolyze ubiquitin-beta-galactosidase fusions in Escherichia coli. In our assays, the activity of UBP43 toward Ub fusions was undetectable in vitro directing us to investigate the possibility of Ub-like proteins such as SUMO, Nedd8, and ISG15 as probable substrates. We consequently demonstrate that UBP43 can efficiently cleave only ISG15 fusions including native ISG15 conjugates linked via isopeptide bonds. In addition to commonly used methods we introduce a new experimental design featuring ISG15-UBP43 fusion self-processing. Deletion of the UBP43 gene in mouse leads to a massive increase of ISG15 conjugates in tissues indicating that UBP43 is a major ISG15-specific protease. UBP43 is the first bona fide ISG15-specific protease reported. Both ISG15 and UBP43 genes are known to be strongly induced by interferon, genotoxic stress, and viral infection. We postulate that UBP43 is necessary to maintain a critical cellular balance of ISG15-conjugated proteins in both healthy and stressed organisms.
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PMID:UBP43 (USP18) specifically removes ISG15 from conjugated proteins. 1178 88

Adenovirus-mediated gene delivery via the intramuscular route efficiently promotes an immune response against the transgene product. In this study, a recombinant adenovirus vector encoding beta-galactosidase (Ad beta Gal) was used to transduce dendritic cells (DC), which are antigen-presenting cells, as well as myoblasts and endothelial cells (EC), neither of which present antigens. C57BL/6 mice received a single intramuscular injection of Ad beta Gal-transduced DC, EC, or myoblasts and were then monitored for anti-beta-galactosidase (anti-beta-Gal) antibody production, induction of gamma interferon-secreting CD8(+) T cells, and protection against melanoma tumor cells expressing beta-Gal. While all transduced cell types were able to elicit an antibody response against the transgene product, the specific isotypes were distinct, with exclusive production of immunoglobulin G2a (IgG2a) antibodies following injection of transduced DC and EC versus equivalent IgG1 and IgG2a responses in mice inoculated with transduced myoblasts. Transduced DC induced a strong ex vivo CD8(+) T-cell response at a level of 50% of the specific response obtained with the Ad beta Gal control. In contrast, this response was 6- to 10-fold-lower in animals injected with transduced myoblasts and EC. Accordingly, only animals injected with transduced DC were protected against a beta-Gal tumor challenge. Thus, in order to induce a strong and protective immune response to an adenovirus-encoded transgene product, it is necessary to transduce cells of dendritic lineage. Importantly, it will be advantageous to block the transduction of DC for adenovirus-based gene therapy strategies.
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PMID:Distinct roles of adenovirus vector-transduced dendritic cells, myoblasts, and endothelial cells in mediating an immune response against a transgene product. 1186 57

Herpes simplex virus type 1 (HSV-1) is resistant to the antiviral effects of interferon (IFN)-alpha, -beta, or -gamma. The fact that ICP0(-) mutants replicate like wild-type virus in IFN-alpha/beta receptor knockout mice (Leib et al., 1999, J. Exp. Med. 189, 663) suggested that ICP0 may serve a direct role in the resistance of HSV-1 to IFN. To test this hypothesis, the effects of IFN-alpha, -beta, and -gamma were compared against wild-type HSV-1 and an ICP0(-) mutant virus, 7134. In Vero cells, 7134 was more sensitive to inhibition by low doses of type I IFN (-alpha/beta) or type II IFN (-gamma) than vesicular stomatitis virus, a well-studied IFN-sensitive virus. At a concentration of 100 U/ml, IFN-alpha, -beta, or -gamma reduced the efficiency of 7134 plaque formation by 120-, 560-, and 45-fold, respectively. In contrast, none of the IFNs reduced wild-type HSV-1 plaque formation by more than 3-fold. Even when Vero cells were infected with 10 pfu per cell, IFN-alpha and -beta inhibited 7134 replication by over 100-fold, but inhibition by IFN-gamma decreased to less than 10-fold. While IFN-beta efficiently inhibited 7134 replication in primary mouse kidney and SK-N-SH cells, IFN-gamma did not inhibit 7134 to a comparable extent in these cells. ICP0 provided in trans from an adenovirus vector allowed 7134 to replicate efficiently in Vero cells in the presence of IFN-alpha, -beta, or -gamma. While IFN-beta or -gamma efficiently repressed the ICP0 promoter-lacZ reporter gene in 7134 (i.e., approximately 60-fold reduction in beta-galactosidase activity), ICP0 provided in trans almost completely reversed IFN-mediated repression of the lacZ gene in 7134. The results suggest that the rate of ICP0 expression in infected cells in vivo may be critical in determining whether host IFNs repress the HSV-1 genome. This concept is discussed in light of its potential relevance to the establishment of latent HSV-1 infections.
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PMID:The immediate-early protein, ICP0, is essential for the resistance of herpes simplex virus to interferon-alpha/beta. 1188 49

Delivery of plasmid DNA by nanoparticles improves the DNA bioavailability, for instance in intramuscular administration, by localizing the DNA in the muscle tissue. Extracellular sustained release of the DNA may lead to more prolonged transgene expression. The present study describes a novel controlled gene delivery system based on a water soluble and biodegradable polyphosphoester, poly(2-aminoethyl propylene phosphate) (PPE-EA). The polymer degraded in PBS at 37 degrees C through the cleavage of the backbone phosphate bonds, and it was synthesized with a relative high molecular weight to ensure a suitable hydrolytic stability as a gene carrier. The tissue response and cytotoxicity study demonstrated a better tissue compatibility of PPE-EA in mouse muscle compared with commonly used polyethylenimine and poly-L-lysine. PPE-EA condensed DNA efficiently and protected DNA from nuclease and serum degradation. Sustained release of plasmid was achieved from PPE-EA/DNA complexes as a result of PPE-EA degradation. The DNA release profiles appear to be predominantly controlled by carrier degradation and the release rate of plasmid could be adjusted by varying the charge ratio of PPE-EA to DNA. At an N/P (amino to phosphate groups) ratio of 1, a 46% burst was observed for the first day, followed by about 4% release per day (24 microg DNA/day/mg of complex) for 12 days. Higher charge ratios reduced both the DNA release rate and the burst effect. The released DNA retained its structural and functional integrity. Intramuscular injection of PPE-EA-p43-LacZ complexes at N/P ratios of 0.5 and 1 resulted in enhanced beta-galactosidase expression in anterior tibialis muscle in Balb/c mice, as compared with naked DNA injections. Similarly, PPE-EA/IFN(alpha)2b DNA complexes generated an increased systemic level of interferon-alpha2b in mouse serum following intramuscular injection, as compared with naked DNA injection.
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PMID:Enhanced gene expression in mouse muscle by sustained release of plasmid DNA using PPE-EA as a carrier. 1221 93


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