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)

Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neurons in vitro and in vivo; however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time. In vitro, this may be due to direct toxicity of the protein product of the transgene or adenoviral molecules. In vivo, in addition, an immune attack by the host could eliminate the transduced cells. To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conducted in primary neural cultures. Cultures of dissociated murine spinal cord-dorsal root ganglia were exposed to AVR containing the Escherichia coli lacZ (E. coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells. Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1 + E3)-deleted replication-incompetent AVR at high titers [> or = 5 x 10(8) viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, beta-galactosidase, at titers that result in 55-75% transduction efficiency (5 x 10(7)-5 x 10(8) viral particles/ml or m.o.i. 100-1000). Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic.
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PMID:Toxicity of replication-defective adenoviral recombinants in dissociated cultures of nervous tissue. 868 75

Replication-defective (E1-E3-deleted) human adenovirus vectors are a promising means of therapeutic gene delivery to skeletal muscle cells. Since the tropism of adenovirus is nonselective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector. We have generated two replication-defective adenovirus recombinants (AV) in which the reporter gene (either firefly luciferase or E. coli beta-galactosidase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer or by the fast troponin I (TnI) promoter/enhancer. Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at an early age. In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle. The relatively small size, the good efficiency and the muscle specificity of the MCK promoter would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.
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PMID:Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer. 927 24

Direct gene transfer into skeletal muscle in vivo presents a convenient experimental approach for studies of adult muscle gene regulatory mechanisms, including fast vs. slow fiber type specificity. Previous studies have reported preferential expression of fast myosin heavy chain and slow myosin light chain and troponin I (TnIslow) gene constructs in muscles enriched in the appropriate fiber type. We now report a troponin I fast (TnIfast) direct gene transfer study. We injected into the mouse soleus muscle plasmid DNA or recombinant adenovirus carrying a TnIfast/ beta-galactosidase (beta-gal) reporter construct that had previously been shown to be expressed specifically in fast fibers in transgenic mice. Surprisingly, microscopic histochemical analysis 1 and 4 wk postinjection showed similar TnIfast/beta-gal expression in fast and slow fibers. A low but significant level of muscle fiber segmental regeneration was evident in muscles 1 wk postinjection, and TnIfast/beta-gal expression was preferentially targeted to regenerating fiber segments. This finding can explain why TnIfast constructs are deregulated with regard to fiber type specificity, whereas the myosin constructs previously studied are not. The involvement of regenerating fiber segments in transduction by plasmid DNA and recombinant adenoviruses injected into intact normal adult muscle is an unanticipated factor that should be taken into account in the planning and interpretation of direct gene transfer experiments.
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PMID:Skeletal muscle gene transfer: regeneration-associated deregulation of fast troponin I fiber type specificity. 1083 55

The effect of pretreatments of host muscles with metalloproteinases (MMPs) or with notexin on the migration of transplanted myoblasts was investigated. Transgenic TnILacZ mice in which the beta-galactosidase gene is under the control of a quail fast skeletal troponin I gene promoter were used as donors. A polyethylene microtube with four perforations was inserted in the tibialis anterior (TA) of CD1 mice. Both pretreatment substances and cells were slowly injected through that microtube. Muscles were pretreated 2 days before myoblast injection either with a mixture of collagenase, matrilysin, and notexin or with only collagenase and matrilysin or only notexin. As control for our experiments, TnILacZ and C2C12 myoblasts were also injected in TA muscles not pretreated. Comparison of short and long-term myoblast radial migration was performed using a dye (PKH26) and X-gal staining, respectively. The recipient mice were immunosuppressed with FK506. Two days after myoblast transplantation, the cell movement in muscles pretreated with collagenase, matrilysin, and notexin was slightly greater than in muscles pretreated only with collagenase and matrilysin but was about twice that observed in muscles treated with notexin alone. Almost no radial migration of TnILacZ myoblasts was observed in untreated muscles. The C2C12 myoblasts showed a four-to fivefold higher migration capacity than TnILacZ myoblasts. At 15 days after TnILacZ myoblast transplantation, the farthest positive beta-gal muscle fibers show a two- to threefold extension of the initial migration observed at 2 days, demonstrating the ability of myoblasts to continue the migration following all pretreatments and even in the untreated muscles. In addition, more muscle fibers expressed the beta-gal reporter gene in muscles pretreated only with MMPs. Our results clearly demonstrate that muscle pretreatments with MMPs increase myoblast migration and fusion with host muscle fibers after transplantation and that the C2C12 cell line producing MMPs has a higher migratory capacity.
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PMID:Intramuscular migration of myoblasts transplanted after muscle pretreatment with metalloproteinases. 1103 70

Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% beta-galactosidase-positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.
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PMID:Intraarterial injection of muscle-derived CD34(+)Sca-1(+) stem cells restores dystrophin in mdx mice. 1126 50

p38 Mitogen-activated protein kinase (MAPK) is one of the most ancient signaling molecules and is involved in multiple cellular processes, including cell proliferation, cell growth, and cell death. In the heart, enhanced activation of p38 MAPK is associated with ischemia/reperfusion injury and the onset of heart failure. In the present study, we investigated the function of p38 MAPK in regulating cardiac contractility and its underlying mechanisms. In cultured adult rat cardiomyocytes, activation of p38 MAPK by adenoviral gene transfer of an activated mutant of its upstream kinase, MKK3bE, led to a significant reduction in baseline contractility, compared with uninfected cells or those infected with a control adenoviral vector (Adv-beta-galactosidase). The inhibitory effect of MKK3bE on contractility was largely prevented by coexpressing a dominant-negative mutant of p38 MAPK or treating cells with a p38 MAPK inhibitor, SB203580. Conversely, inhibition of endogenous p38 MAPK activity by SB203580 rapidly and reversibly enhanced cell contractility in a dose-dependent manner, without altering L-type Ca(2+) currents or Ca(2+)(i) transients. MKK3bE-induced p38 activation had no significant effect on pH(i), whereas SB203580 had a minor effect to elevate pH(i). Furthermore, activation of p38 MAPK was unable to increase troponin I phosphorylation. Thus, we conclude that the negative inotropic effect of p38 MAPK is mediated by decreasing myofilament response to Ca(2+), rather than by altering Ca(2+)(i) homeostasis and that the reduced myofilament Ca(2+) sensitivity is unlikely attributable to troponin I phosphorylation or alterations in pH(i). These findings reveal a novel function of p38 MAPK and shed a new light on our understanding of the coincidence of p38 MAPK activation and the onset of heart failure.
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PMID:p38 Mitogen-activated protein kinase mediates a negative inotropic effect in cardiac myocytes. 1183 12

High-level tissue-specific expression of recombinant proteins in muscle is an important issue for several therapeutic applications. To achieve this goal, we generated several constructs containing one to five copies of the upstream enhancer (USE) of 160-bp of the human slow troponin I gene, linked to that gene's minimal promoter. We also tested constructs made with one to four copies of a 100-bp deletion of USE (DeltaUSE) reported to drive pan-muscle-specific expression in transgenic mice. These constructs were evaluated by measuring the activity of the reporter gene beta-galactosidase (beta-gal). In cell culture, these multimerized enhancers retained tissue specificity and their transcriptional strength increased with the number of enhancer copies. In myotube cultures (which still contain nondifferentiated cells), constructs containing four and five USE copies were stronger than the cytomegalovirus (CMV) early enhancer/promoter and slightly weaker than the hybrid CMV enhancer/beta-actin (CB) promoter. Those containing three USE, or four DeltaUSE copies were similar in strength to CMV. After electrotransfer of plasmid DNA into the mouse tibialis anterior muscle, the strengths of the two constructs (USEx3 and DeltaUSEx3) were tested; as measured by beta-gal activity in the total muscle lysate and by the number of transduced fibers, they were similar to CMV and CB. Muscle fiber typing, after electrotransfer of the soleus muscle, showed that DeltaUSEx3 and USEx3 were active in slow and fast fibers. The tissue specificity of these two constructs was also evaluated by hydrodynamic plasmid injection through the tail vein. Although significant beta-gal expression was measured in the liver when CMV was tested, no expression above background level was detected with USEx3 and DeltaUSEx3. The strength, muscle specificity, and small size of these transcriptional elements render them very attractive for gene therapy applications.
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PMID:Strong muscle-specific regulatory cassettes based on multiple copies of the human slow troponin I gene upstream enhancer. 1971 87