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)

Recent studies have shown that the presence of tumor suppressors such as p53 or p16 account for the lack of transformation in primary cells. To investigate a potential role of active Ras in atherosclerosis, we infected bovine aortic endothelial cells with a replication-deficient, recombinant adenovirus containing the activated H-Ras61L gene. Ras overexpression led after 72 hours to G1- and G2/M-cell cycle arrest due to induction of p21(Cip1/Waf1). Treatment of Ras-infected endothelial cells with 40 ng/ml TNF-alpha for 20 hours augmented apoptosis 8-fold in comparison to Ad-Con (control virus with empty expression cassette) infected cells (36.2% vs. 4.3%, p < 0.001), while Ras itself did not cause any cell death. Furthermore, more than 58% of Ras-infected cells stained positive for senescence-associated beta-galactosidase activity as opposed to 2% in control vector-infected cells (p < 0.001), strongly suggesting a senescent phenotype in the Ras-infected population. We found further features of senescence in Ras-transduced endothelial cells, such as growth arrest and the lack of AP-1 serum inducibility. Finally, we evaluated the role of p21(Cip1/Waf1) in this process of senescence. Adenoviral overexpression of p21 led to growth arrest by induction of G1- and G2/M-cell cycle arrest. In addition, p21-overexpressing endothelial cells were highly sensitive for TNF-alpha induced-apoptosis. Surprisingly, senescence-associated beta-galactosidase activity was not apparant in p21-infected endothelial cells, suggesting further signaling events necessary for the senescent morphology of endothelial cells. Our results demonstrate a novel way to render primary endothelial cells senescent by overexpressing oncogenic Ras. Increased sensitivity of senescent endothelial cells for cytotoxic stimuli seemed to be due to Ras-induced upregulation of p21(Cip1/Waf1). Future studies have to investigate a potential role of Ras in human vascular biology.
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PMID:Oncogenic ras induces premature senescence in endothelial cells: role of p21(Cip1/Waf1). 1200 58

Jet-injection has become an applicable technology among other established nonviral delivery systems, such as particle bombardment or in vivo electroporation. The low-volume jet injector employed in this study uses compressed air to inject solutions of 1.5-10 microL containing naked DNA into the desired tissue. The novel design of this prototype makes multiple jet-injections possible. Therefore, repeated jet-injections into one target tissue can be performed easily. This jet-injector hand-held system was used for the direct in vivo gene transfer of plasmid DNA into tumors to achieve efficient expression of reporter genes (beta-galactosidase, green fluorescent protein [GFP]) and of therapeutic genes (TNF-alpha) in different tumor models. The study presented here revealed the key parameters of efficient in vivo jet-injection (jet-injection volume, pressure, jet penetration, DNA stability) to define the optimal conditions for a jet-injection-aided nonviral gene therapy.
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PMID:Intratumoral low-volume jet-injection for efficient nonviral gene transfer. 1205 10

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antiphospholipid antibodies (aPLs) and recurrent thrombosis or fetal loss. The thrombophilic state has been partially related to the induction of a proinflammatory and procoagulant endothelial cell (EC) phenotype induced by anti-beta(2)-glycoprotein I (beta(2)-GPI) antibodies that bind beta(2)-GPI expressed on the EC surface. Anti-beta(2)-GPI antibody binding has been shown to induce nuclear factor-kappa B (NF-kappa B) translocation leading to a proinflammatory EC phenotype similar to that elicited by interaction with microbial products (lipopolysaccharide [LPS]) and proinflammatory cytokines (interleukin 1 beta [IL-1 beta], tumor necrosis factor alpha [TNF-alpha]). However, the upstream signaling events are not characterized yet. To investigate the endothelial signaling cascade activated by anti-beta(2)-GPI antibodies, we transiently cotransfected immortalized human microvascular endothelial cells (HMEC-1) with dominant-negative constructs of different components of the pathway (Delta TRAF2, Delta TRAF6, Delta MyD88) together with reporter genes (NF-kappa B luciferase and pCMV-beta-galactosidase). Results showed that both human anti-beta(2)-GPI IgM monoclonal antibodies as well as polyclonal affinity-purified anti-beta(2)-GPI IgG display a signaling cascade comparable to that activated by LPS or IL-1. Delta TRAF6 and Delta MyD88 significantly abrogate antibody-induced as well as IL-1- or LPS-induced NF-kappa B activation, whereas Delta TRAF2 (involved in NF-kappa B activation by TNF) does not affect it. Moreover, anti- beta(2)-GPI antibodies and LPS followed the same time kinetic of IL-1 receptor-activated kinase (IRAK) phosphorylation, suggesting an involvement of the toll-like receptor (TLR) family. Our findings demonstrate that anti-beta(2)-GPI antibodies react with their antigen likely associated to a member of the TLR/IL-1 receptor family on the EC surface and directly induce activation.
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PMID:Role of the MyD88 transduction signaling pathway in endothelial activation by antiphospholipid antibodies. 1253 7

The development and implementation of direct gene transfer technologies for the study and treatment of chronic CNS disorders inherently requires consideration of vector safety. Virus-based vectors represent the most efficient modalities but harbor the potential to induce vigorous innate and adaptive immune responses when administered in vivo. These responses can arise because of virus particle components, resultant viral gene expression, and/or transgene expression. In the current study, we describe the innate responses elicited upon stereotactic delivery of herpes simplex virus type 1-based amplicon vectors. C57BL/6 mice were injected with sterile saline, beta-galactosidase-expressing amplicon (HSVlac) packaged by a conventional helper virus-based technique, or helper virus-free HSVlac. After killing the mice at either 1 or 5 days after transduction, we analyzed them by immunocytochemistry and quantitative RT-PCR for various chemokine, cytokine, and adhesion molecule gene transcripts. All injections induced inflammation, with blood/brain barrier opening on day 1 that was enhanced with both amplicon preparations as compared with saline controls. By day 5, mRNA levels for the pro-inflammatory cytokines (IL-1beta, TNF-alpha, IFN-gamma), chemokines (MCP-1, IP-10), and an adhesion molecule (ICAM-1) had returned to baseline in saline-injected mice and to near-baseline levels in helper virus-free amplicon groups. In contrast, mice injected with helper virus-packaged amplicon stocks elicited elevated inflammatory molecule expression and immune cell infiltration even at day 5. In aggregate, we demonstrate that helper virus-free amplicon preparations exhibit a safer innate immune response profile, presumably as a result of the absence of helper virus gene expression, and provide support for future amplicon-based CNS gene transfer strategies.
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PMID:Helper-free HSV-1 amplicons elicit a markedly less robust innate immune response in the CNS. 1259 10

TGF-beta1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets alphavbeta3 and alpha5beta1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-beta1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or beta-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-beta1 for 24 h, but not with PDGF-BB, interferon-gamma, TNF-alpha, nor PMA, increased alphavbeta3 and alpha5beta1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus beta-galactosidase activity increased from 35 +/- 5% (controls) to 75 +/- 5% after TGF-beta1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered beta-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-beta1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases.
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PMID:Targeting alphavbeta3 and alpha5beta1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-beta1. 1291 91

Our previous report of predominant activation of nuclear transcription factor NF-kappaB in the bladder urothelium of interstitial cystitis (IC) patients suggests a potential role for this nuclear factor in the pathogenesis of the disease. Although NF-kappaB has been implicated in the pathogenesis of several inflammatory diseases, the downstream mechanism(s) by which it can mediate its effects are still fragmentary. In this study, we examined the role of this nuclear factor on the induction of proinflammatory cytokine gene expression in human bladder carcinoma T24 cells and further examined their corresponding protein levels in the urine of IC patients. T24 cells transduced with a dominant-negative super-repressor IkappaB mutant (pAxCAmIkappaB-M) or wild-type adenoviral vectors in the presence or absence of rhTNF-alpha. Transduction efficiency and ability of pAxCAmIkappaB-M to inhibit NF-kappaB activation were monitored by in situ reporter beta-galactosidase and gel mobility shift assays, respectively. Expression profile analysis of proinflammatory cytokines was measured in cells and urine of IC patients using RT-PCR and ELISA, respectively. The activation of NF-kappaB by rhTNF-alpha was associated with 27, eight, ten and sevenfold increases in the TNF-alpha, IL-1beta, IL-6 and IL-8 transcripts, respectively. In contrast, abrogation of the TNF-alpha-induced cytokine gene expression by an adenovirus super-repressor IkappaB mutant vector demonstrate that these effects were NF-kappaB-dependent. Interestingly, the NF-kappaB-induced expression of these transcripts correlates with increased protein levels of NF-kappaB-regulated proinflammatory factors in the urine of IC patients in comparison to controls. That these factors are capable of activating NF-kappaB in urothelial cells suggests a pivotal role for this nuclear transcription factor in the pathophysiology of the disease, possibly by inducing aberrant immune and inflammatory responses within the bladder of IC patients.
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PMID:NF-kappaB-dependent gene expression of proinflammatory cytokines in T24 cells: possible role in interstitial cystitis. 1457 33

This study describes a tumor vaccine-induced secondary in vivo T cell response to an immunodominant epitope of beta-galactosidase (Gal) as a model tumor-associated antigen. DBA/2 mice are primed with lacZ transfected live ESbL tumor cells (ESbL-Gal) in the ear pinna, a site which had previously been shown to be non-tumorigenic and immunogenic. Intraperitoneal challenge of such mice with tumor vaccine (i.e., 10(7) irradiation-inactivated ESbL-Gal cells) leads to the production of a powerful CD8+ CTL response and to the establishment of immune memory. Using peptide/MHC-tetrameric complexes, clonal expansion of antigen-specific T cells could be detected during the primary response in bone marrow (BM) and during the secondary response in the peritoneal cavity and BM. The secondary response in the peritoneal cavity involved a >80-fold enrichment of epitope specific CD8+ T cells and the release of various cytokines, including IL-12 and TNF-alpha. The recruitment and/or expansion of Gal specific T cells within the peritoneal cavity could be inhibited by anti-IL-12 and anti-TNF-alpha monoclonal antibody (mAb) treatment. Interestingly, the secondary CTL response was inhibited by anti-IL-12 but not by anti-TNF-alpha mAb. The results characterize a strong systemic CD8+ memory T cell response to a cell bound antigen without the use of adjuvant.
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PMID:Characteristics of a potent tumor vaccine-induced secondary anti-tumor T cell response. 1513 84

Transgene expression from helper-dependent adenoviral (HD-Ad) vectors is effective and long lasting, but not permanent. Their use is also limited by the host response against capsid proteins that precludes successful gene expression upon readministration. In this report, we test the hypothesis that PEGylation of HD-Ad reduces its toxicity and promotes transgene expression upon readministration. PEGylation did not compromise transduction efficiency in vitro and in vivo and reduced peak serum IL-6 levels two-fold. IL-12 and TNF-alpha levels were reduced three- and seven-fold, respectively. Thrombocytopenia was not detected in mice treated with the PEGylated vector. Serum transaminases were not significantly elevated in mice treated with either vector. Mice immunized with 1 x 10(11) particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 x 10(10) particles of unmodified or PEG-conjugated vector expressing beta-galactosidase. Trace levels of beta-galactosidase (52.23+/-19.2 pg/mg protein) were detected in liver homogenates of mice that received two doses of unmodified HD-Ad. Mice rechallenged with PEGylated HD-Ad produced significant levels of beta-galactosidase (5.1+/-0.4 x 10(5) pg/mg protein, P=0.0001). This suggests that PEGylation of HD-Ad vectors may be appropriate for their safe and efficient use in the clinic.
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PMID:PEGylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile. 1564 65

Chronic inflammation in tibialis anterior muscles of mdx mice was produced by a single injection of a recombinant adenovirus vector (AV) expressing an immunogenic beta-galactosidase (beta-gal). In regions of intense beta-gal staining, mononuclear infiltrates abounded, and muscle fibers showed strong extrasynaptic utrophin immunostaining, restoration of dystrophin-associated protein complex, and a marked reduction of the prevalence of centronucleation. Immunoblot analysis confirmed an increase of endogenous utrophin without an increase of the mRNA of the major muscle isoform utrA. Significantly better maximal tetanic force values were demonstrated in the inflammatory versus control mdx muscles. The resistance to lengthening contraction- induced damage was also significantly increased in the former. In muscles of mice lacking TNF-alpha gene, AV vector did not induce inflammation and extrajunctional utrophin increase did not occur. In the inflammatory mdx muscles, proteolytic activity of calcium-activated calpain was reduced, and in mdx myotubes in vitro, incubation with NO donors also reduced calpain-mediated utrophin proteolysis. Since utrophin was shown to be a natural substrate of calpain and known inhibitors of calpain in cultured mdx myotubes increased utrophin levels, the above results were consistent with the following conclusions: (1) extrasynaptic utrophin increase is mainly responsible for the antidystrophic effect; (2) extrasynaptic utrophin increase is a result of posttranscriptional mechanism(s) related to proinflammatory factors; and (3) reduction of endogenous muscle calpain activity by inflammatory cytokines has an important role in the stabilization and increase of the extrasynaptic utrophin.
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PMID:Factors associated with induced chronic inflammation in mdx skeletal muscle cause posttranslational stabilization and augmentation of extrasynaptic sarcolemmal utrophin. 1587 80

Our previous work showed that tumor necrosis factor (TNF)-alpha and FasL induce apoptosis of anterior pituitary cells. To further analyze the effect of these proapoptotic factors, we infected primary cultures from rat anterior pituitary, GH3 and AtT20 cells with first-generation adenoviral vectors encoding TNF-alpha, FasL or, as a control, beta-galactosidase (beta-Gal), under the control of the human cytomegalovirus promoter. Successful expression of the encoded transgenes was determined by immunocytochemistry. Although we observed basal expression of TNF-alpha and FasL in control cultures of anterior pituitary cells, fluorescence-activated cell sorting (FACS) cell cycle analysis showed that the overexpression of TNF-alpha or FasL increases the percentage of hypodiploid lactotropes and somatotropes. Nuclear morphology and TUNEL staining revealed that the cells undergo an apoptotic death process. We detected strong immunoreactivity for TNFR1 and Fas in the somatolactotrope cell line GH3. TNF-alpha, but not FasL, was expressed in control cultures of GH3 cells. The infection of GH3 cells with adenovirus encoding TNF-alpha or FasL increased the percentages of hypodiploid and TUNEL-positive cells. TNF-alpha or FasL immunoreactivity was not observed in the corticotrope cell line AtT20. However, adenovirus encoding TNF-alpha or FasL efficiently transduced these cells and increased the percentages of hypodiploid and TUNEL-positive cells. The expression of beta-Gal was detected in all these cultures but did not affect cell viability. In conclusion, these results suggest that death signaling cascades triggered by TNF receptor 1 (TNFR1) and Fas are present in both normal and tumoral pituitary cells. Therefore, overexpression of proapoptotic factors could be a useful tool in the therapy of pituitary adenomas.
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PMID:Adenoviral vectors encoding tumor necrosis factor-alpha and FasL induce apoptosis of normal and tumoral anterior pituitary cells. 1673 98


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