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)

Conditional transgene expression is a potentially useful approach to investigate complex biological systems in vivo. We recently demonstrated that tetracycline-responsive promoters could be employed to achieve regulated, cardiac-specific expression of target genes in transgenic mice. To more fully define the quantitative and spatial parameters associated with tetracycline-regulated gene expression in the heart, we crossed transgenic mice harboring either a firefly luciferase or a nuclear-localized bacterial lacZ target gene with strains expressing a tetracycline-controlled transactivator (tTA) under the regulatory control of 2.9 kb of 5' flanking sequence from the rat alpha-myosin heavy chain gene. Luciferase activity was induced nearly 300-fold in the hearts of binary-transgenic mice compared with mice carrying only the luciferase reporter gene. No significant transactivation was observed in any other tissues examined. Binary transgenics harboring the lacZ reporter gene showed substantial beta-galactosidase activity throughout the heart, but the response of individual cardiac myocytes was heterogeneous. For both reporter genes, tetracycline treatment fully repressed tTA-dependent transactivation. These data provide important insights into the nature of studies that can be successfully addressed using the tetracycline-regulated gene expression system in the heart.
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PMID:Conditional transgene expression in the heart. 883 92

We have altered the spontaneous contractile activity of neonatal cardiac myocytes in culture to investigate the re-lationship between mechanical forces, myofibril assembly, and the localization and translation of (alpha)-myosin heavy chain mRNA. Immunofluorescence and in situ hybridization techniques revealed that contracting myocytes display well aligned myofibrils and a diffuse distribution of (alpha)-myosin heavy chain mRNA. Inhibition of contractile activity with the calcium channel blocker verapamil (10 microM) resulted in myofibril disassembly and a perinuclear mRNA distribution within six hours. There was a significant decrease (P<0. 05) of mRNA levels, 5 to 15 micron away from the nucleus following 6 hours of verapamil treatment compared with control cells. Inhibition of protein synthesis with cycloheximide (10 microM) also resulted in perinuclear mRNA localization despite having little effect on contractile activity or myofibril assembly. To determine if the 3' untranslated region of (alpha)-myosin heavy chain mRNA was sufficient for localizing the entire message, a chimeric construct composed of beta-galactosidase coding region followed by (alpha)-myosin heavy chain 3' untranslated region sequences was made as a reporter plasmid and transfected into cultured myocytes. A perinuclear accumulation of ss-galactosidase was exhibited in many of the contractile arrested cells (48.3+/-2.4%, n=7). In contrast, significantly fewer (P<0.05) contracting control (29.1+/-3.3%, n=7) and strongly contracting, isoproterenol-treated cells (27.2+/-6.1%, n=3) exhibited a perinuclear localization of protein. The distribution of the reporter protein was not affected by the contractile state in cells transfected with a constitutively translated 3'UTR. We propose that mechanical activity of neonatal cardiac myocytes regulates the intracellular localization of alpha-myosin heavy chain mRNA via the 3' untranslated region mediated by an initial block in translation.
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PMID:Localization of cardiac (alpha)-myosin heavy chain mRNA is regulated by its 3' untranslated region via mechanical activity and translational block. 935 83

Starburst dendrimer, a structurally defined, spherical macromolecule composed of repeating polyamidoamino subunits, was investigated to augment plasmid-mediated gene transfer efficiency in a murine cardiac transplantation model. The grafts were directly injected with naked pCH110, a plasmid encoding beta-galactosidase (beta-Gal), or pCH110-dendrimer complex, and reporter gene expression determined by X-Gal staining. The grafts injected with pCH110-dendrimer demonstrated widespread and extended beta-Gal expression in both myocytes and the graft infiltrating cells from 7 to 28 days, compared to the grafts injected with naked pCH110 that expressed beta-Gal only in myocytes for less than 14 days. p alphaMHC-vIL-10, as plasmid encoding viral interleukin-10 (vIL-10) under the control of alpha-myosin heavy chain promoter, was able to prolong allograft survival from 13.9 +/- 0.9 days to 21.4 +/- 2.3 days (p < 0.005). When dendrimer G5EDA was used with p alphaMHC-vIL-10, 60-fold less DNA resulted in significant prolongation of graft survival to 38.6 +/- 4.7 days (p < 0.0005). The dose of DNA, the charge ratio of DNA to dendrimer, and the size generation of the dendrimers were all determined to be critical variables for prolongation of allograft survival in this model system. Thus, the use of the Starburst dendrimer dramatically increased the efficiency of plasmid-mediated gene transfer and expression. Production of immunosuppressive cytokines at higher amounts for longer periods of time in a greater expanse of tissue enhanced the immunosuppressive effect and prolonged graft survival further.
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PMID:Efficient transfer of genes into murine cardiac grafts by Starburst polyamidoamine dendrimers. 952 16

Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse alpha-tropomyosin cDNA under the control of a cardiac-specific alpha-myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well-organized sarcomeric myofibrils. Transfection of a beta-tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a beta-galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of alpha-tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres.
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PMID:Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts. 985 62

Cardiac-restricted expression of Cre recombinase can provoke lineage-specific gene excision in the myocardium. However, confounding early lethality may still preclude using loss-of-function models to study the postnatal heart. Here, we have tested whether inducible, heart-specific recombination can be triggered after birth by transgenic expression of a Cre fusion protein that incorporates a mutated progesterone receptor ligand binding domain (PR1) that is activated by the synthetic antiprogestin, RU486, but not by endogenous steroid hormones. CrePR1 driven by the alpha-myosin heavy chain (alphaMHC) promoter was expressed specifically in heart. Translocation of CrePR1 from cytoplasm to nuclei in ventricular myocytes was induced by RU486. To establish whether this approach can mediate cardiac-specific, drug-dependent excision between loxP sites in vivo, we mated alphaMHC-CrePR1 mice with a ubiquitously expressed (ROSA26) Cre reporter line. Offspring harboring alphaMHC-CrePR1 and/or the floxed allele were injected with RU486 versus vehicle, and the prevalence of beta-galactosidase (beta-gal)-positive cells was determined, indicative of Cre-mediated excision. Little or no baseline recombination was seen 1 week after birth. Cardiac-restricted, RU486-inducible recombination was demonstrated in bigenic mice at age 3 and 6 weeks, using each of 3 independent CrePR1 lines. Recombination in the absence of ligand paralleled the levels of CrePR1 protein expression and was more evident at 6 weeks. Thus, conditional, posttranslational activation of a Cre fusion protein can bypass potential embryonic and perinatal effects on the heart and permits inducible recombination in cardiac muscle. High levels of the chimeric Cre protein, in particular, were associated with progressive recombination in the absence of drug.
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PMID:Inducible gene targeting in postnatal myocardium by cardiac-specific expression of a hormone-activated Cre fusion protein. 1128 85

Nitric oxide (NO) functions principally as a diffusible paracrine effector. The exception is in cardiomyocytes where both NO synthases (NOS) and target proteins coexist, allowing NO to work in an autocrine/intracrine fashion. However, the most abundant myocyte isoform (NOS3) is far more expressed in vascular endothelium; thus, the in vivo contribution of myocyte-NOS3 remains less clear. The present study tested this role by transfecting whole hearts of NOS3-null (NOS3(-/-)) mice with adenovirus-expressing NOS3 coupled to a alpha-MHC promoter (AdV(NOS3)), comparing results to hearts transfected with marker-gene beta-galactosidase (AdVbeta(gal)). Total myocardial NOS3 protein and activity were restored to near wild-type (WT) levels in NOS3(-/-)+AdV(NOS3) hearts, and NOS3 relocalized normally with caveolin-3. Ejection function by pressure-volume analysis was enhanced in NOS3(-/-)+AdVbeta(gal) over WT or NOS3(-/-)+AdV(NOS3). More prominently, isoproterenol (ISO)-stimulated systolic and diastolic function in WT was amplified in NOS3(-/-)+AdVbeta(gal), whereas NOS3(-/-)+AdV(NOS3) returned the response to control. ISO-activated systolic function was inhibited 85% by concomitant muscarinic stimulation (carbachol) in NOS3(-/-)+AdV(NOS3) but not NOS3(-/-)+AdVbeta(gal) hearts. Lastly, NOS3(-/-)+AdVbeta(gal) mice displayed enhanced inotropy and lusitropy over WT at slower heart rates but a blunted rate augmentation versus controls. A more positive rate response was restored in NOS3(-/-)+AdV(NOS3) (P<0.001). Thus, myocyte autocrine/intracrine NOS3 regulation in vivo can underlie key roles in beta-adrenergic, muscarinic, and frequency-dependent cardiac regulation.
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PMID:Modulation of in vivo cardiac function by myocyte-specific nitric oxide synthase-3. 1475 30

Because pluripotent embryonic stem cells (ESCs) are able to differentiate into any tissue, they are attractive agents for tissue regeneration. Although improvement of cardiac function has been observed after transplantation of pluripotent ESCs, the extent to which these effects reflect ESC-mediated remuscularization, revascularization, or paracrine mechanisms is unknown. Moreover, because ESCs may generate teratomas, the ability to predict the outcome of cellular differentiation, especially when transplanting pluripotent ESCs, is essential; conversely, a requirement to use predifferentiated ESCs would limit their application to highly characterized subsets that are available in limited numbers. In the experiments reported here, we transplanted low numbers of two murine ESC lines, respectively engineered to express a beta-galactosidase gene from either a constitutive (elongation factor) or a cardiac-specific (alpha-myosin heavy chain) promoter, into infarcted mouse myocardium. Although ESC-derived tumors formed within the pericardial space in 21% of injected hearts, lacZ histochemistry revealed that engraftment of ESC was restricted to the ischemic myocardium. Echocardiographic monitoring of ESC-injected hearts that did not form tumors revealed functional improvements by 4 weeks postinfarction, including significant increases in ejection fraction, circumferential fiber shortening velocity, and peak mitral blood flow velocity. These experiments indicate that the infarcted myocardial environment can support engraftment and cardiomyogenic differentiation of pluripotent ESCs, concomitant with partial functional recovery.
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PMID:Improved cardiac function in infarcted mice after treatment with pluripotent embryonic stem cells. 1700 46