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)

Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 die of vascular overgrowth, and we are interested in how flt-1 normally prevents this outcome. Our results support a model whereby aberrant endothelial cell division is the cellular mechanism resulting in vascular overgrowth, and they suggest that VEGF-dependent endothelial cell division is normally finely modulated by flt-1 to produce blood vessels. Flt-1(-/-) embryonic stem cell cultures had a 2-fold increase in endothelial cells by day 8, and the endothelial cell mitotic index was significantly elevated before day 8. Flt-1 mutant embryos also had an increased endothelial cell mitotic index, indicating that aberrant endothelial cell division occurs in vivo in the absence of flt-1. The flt-1 mutant vasculature of the cultures was partially rescued by mitomycin C treatment, consistent with a cell division defect in the mutant background. Analysis of cultures at earlier time points showed no significant differences until day 5, when flt-1 mutant cultures had increased beta-galactosidase(+) cells, indicating that the expansion of flt-1 responsive cells occurs after day 4. Mitomycin C treatment blocked this early expansion, suggesting that aberrant division of angioblasts and/or endothelial cells is a hallmark of the flt-1 mutant phenotype throughout vascular development. Consistent with this model is the finding that expansion of platelet and endothelial cell adhesion molecule(+) and VE-cadherin(+) vascular cells in the flt-1 mutant background first occurs between day 5 and day 6. Taken together, these data show that flt-1 normally modulates vascular growth by controlling the rate of endothelial cell division both in vitro and in vivo.
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PMID:Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. 1189 72

Incorporation of an internal ribosome entry site (IRES) into the gene therapy vector represents a promising strategy to efficiently co-express several gene products from the same promoter. However, vector systems that utilize the encephalomyocarditis virus IRES express the downstream gene much less efficiently than the upstream gene. In this study, we compared four IRESes isolated from human genes against the EMCV IRES, using beta-galactosidase and chloramphenicol acetyl transferase genes as reporters, to evaluate their potential for providing better expression of the downstream gene. We found that an IRES from the eukaryotic initiation factor 4G gene mediates greater than 100-fold higher expression of the downstream gene compared with the EMCV IRES in four different cell lines tested. Other mammalian IRESes displayed more variable results and smaller enhancement of downstream gene expression in three different cell lines tested. Furthermore, while the efficiency of the IRES from the vascular endothelium growth factor gene was not significantly higher than the EMCV IRES under normoxic conditions, expression was significantly increased under hypoglycemic conditions, suggesting that the VEGF IRES could be exploited in cancer gene therapy to preferentially target expression of therapeutic genes at the relatively hypoglycemic cores of tumors.
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PMID:Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes. 1193 53

The identification of adult-derived stem cells which maintain plasticity throughout the course of a lifetime, has transformed the field of stem cell biology. Bone marrow derived hematopoietic stem cells (HSC) are the most well-characterized population of these multipotential cells. First identified for their ability to reconstitute blood lineages and rescue lethally irradiated hosts, these cells have also been shown to differentiate and integrate into skeletal muscle, cardiac myocytes, vascular endothelium, liver, and brain tissue. Various populations of HSC are being studied, exploiting cell surface marker expression, such as Sca-1, c-kit, CD34, and lin; as well as the abilityto efflux the vital dye Hoecsht 33342. Detection of engrafted donor derived cells into various tissue types in vivo is a laborious process and may involve detection of beta-galactosidase via colorimetric reaction or antibody labeling or green fluorescent protein (GFP) via fluorescence microscopy, as well as in situ hybridization to detect the Y-chromosome. Using these techniques, the search has begun for tissue specific stem cells capable of host tissue regeneration, self renewal, and transdifferentiation. Caution is urged when interpreting these types of experiments because although they are stimulating, limitations of the technologies may provide misleading results.
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PMID:Stem cells: a minireview. 1204 43

Diabetic vasculopathy is central to the development of diverse cardiovascular, renal, retinal, and neurological complications of diabetes. We previously demonstrated that growth of endothelial cells on glycated extracellular matrix proteins (collagen and matrigel) results in a significant decrease in cell proliferation. In the present study, we show that early-passage human umbilical vein endothelial cells (HUVECs) grown on glycated collagen (GC) express hallmarks of premature cell senescence, ie, increase in the proportion of cells expressing senescence-associated beta-galactosidase activity, apoptotic rate, and p53 and p14(AFR) expression, but in contrast to replicative senescence, display neither attrition of telomeres nor decrease in telomerase activity. An increased frequency of prematurely senescent cells was similarly observed in vivo in aortae of young Zucker diabetic rats, compared with lean controls. NO production by HUVECs grown on GC was decreased, despite a 3-fold increase in eNOS expression and was associated with the increased nitrotyrosine-modified proteins. Development of premature senescence of HUVECs on GC could be prevented and reversed by treatments with the peroxynitrite scavenger, ebselen, eNOS intermediate N(omega)-hydroxy-L-arginine (NOHA), or superoxide dismutase mimetic Mn-TBAP. Concomitant with the reversal of senescence, ebselen, and NOHA each restored NO production to levels observed with HUVECs grown on unmodified collagen. Our findings indicate that diabetes mellitus in vivo and GC exposure in vitro elicit premature senescence of the vascular endothelium, a process with distinct pathogenetic mechanisms. Premature senescence of the vascular endothelium is hypothesized to be an important contributor to diabetic vasculopathy and a consequence of reduced NO availability, peroxynitrite, and/or superoxide excess.
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PMID:Glycated collagen I induces premature senescence-like phenotypic changes in endothelial cells. 1208 67

We have developed a system utilizing the murine Tie2 promoter/enhancer coupled with the "tetracycline-on" regulatory elements to create a model that allows regulated and selective expression of a beta-galactosidase (betaGal) reporter transgene in the adult murine vascular endothelium. Two independent lines of viable and fertile mice were characterized, and they exhibit minimal betaGal expression under basal conditions. In response to exogenous doxycycline (Dox), selective expression of betaGal was demonstrated in the vascular endothelium of all tissues examined. En face analyses of the aorta and its principle branches indicate that the vast majority of lumenal endothelial cells express the transgene. Inducible betaGal expression also extends to the endocardium and the microvasculature of all organs. There is no evidence of specific transgene expression in nonendothelial cell types. Induction of the betaGal was effectively achieved after 3 days of oral Dox treatment and persisted for over 3 mo with continuous administration. This model can now be widely applied to study the role of specific genes in the phenotype of adult murine vasculature.
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PMID:Inducible and selective transgene expression in murine vascular endothelium. 1238 91

The Cre/loxP transgenic system may be used to achieve temporally and/or spatially regulated gene deletion. The Mx1Cre mouse expresses Cre recombinase under control of the IFN-inducible Mx1 promoter. Mx1Cre mice were crossed with a reporter strain (ROSA26tm1Sor) in which beta-galactosidase activity is expressed only after Cre-mediated recombination to determine the cellular pattern of Cre-mediated genetic recombination in the kidney and other tissues. Widespread recombination was observed in vascular endothelium as well as in the liver and spleen. Recombination was restricted to subsets of stromal cells in uterus, duodenum, colon, aorta, and kidney. In the cortex, chi-galactosidase activity was detected in a subset of tubules and all glomerular cells, including endothelium, mesangium, and podocytes. No chi-galactosidase activity was detected in proximal tubules. Costaining of kidneys with segment-specific markers demonstrated induction of chi-galactosidase activity in collecting duct, with sporadic labeling of the thick ascending limb but no significant labeling of distal convoluted tubules. We conclude that Mx1-driven gene recombination is spatially as well as temporally restricted. The Mx1Cre transgene should prove a useful reagent to achieve temporally regulated recombination in endothelial, glomerular, and distal renal epithelia in mice.
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PMID:Differential, inducible gene targeting in renal epithelia, vascular endothelium, and viscera of Mx1Cre mice. 1252 77

Tetrahydrobiopterin (BH4) is an essential co-factor for endothelial nitric oxide synthase enzymatic activity. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme in BH4 synthesis. This study set out to test the hypothesis that in vivo gene transfer of GTPCH I to endothelial cells could increase bioavailability of BH4, enhance biosynthesis of nitric oxide and thereby enhance endothelium-dependent relaxations mediated by nitric oxide. In vivo gene transfer was carried out by adenovirus (Ad)-mediated delivery into rabbit carotid arteries. Each artery was transduced by 20-min intraluminal incubation of 10(9) plaque-forming units of Ad-encoding GTPCH I (AdGTPCH) or beta-galactosidase as a control. The rabbits were euthanized 72 h later, and vasomotor function of isolated arteries was assessed by isometric force recording. GTPCH I enzymatic activity, BH4, and oxidized biopterin levels were detected with the use of HPLC, and cGMP was measured with the use of radioimmunoassay. Expression of recombinant proteins was detected predominantly in endothelial cells. Both GTPCH I activity and BH4 levels were increased in arteries transduced with AdGTPCH. However, contraction to phenylephrine (10(-5) to 10(-9) M), endothelium-dependent relaxation to acetylcholine (10(-5) to 10(-9) M) and cGMP levels were not significantly affected by increased expression of GTPCH I. Our results suggest that expression of GTPCH I in vascular endothelium in vivo increases intracellular concentration of BH4. However, under physiological conditions, it appears that this increase does not affect nitric oxide production in endothelial cells of the carotid artery.
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PMID:In vivo expression and function of recombinant GTPCH I in the rabbit carotid artery. 1455 Oct 46

Current concerns over insertional mutagenesis by retroviral vectors mitigate investigations into alternative, potentially persistent gene therapy vector systems not dependent on genomic integration, such as Sendai virus vectors (SeVV). Prenatal gene therapy requires efficient gene delivery to several tissues, which may not be achievable by somatic gene transfer to the adult. Initially, to test the potential and tropism of the SeVV for gene delivery to fetal tissues, first-generation (replication- and propagation-competent) recombinant SeVV, expressing beta-galactosidase was introduced into late gestation immunocompetent mice via the amniotic and peritoneal cavities and the yolk sac vessels. At 2 days, this resulted in very high levels of expression particularly in the airway epithelium, mesothelium and vascular endothelium, respectively. However, as expected, substantial vector toxicity was observed. The efficiency of gene transfer and the level of gene expression were then examined using a second-generation SeVV. The second generation was developed to be still capable of cytoplasmic RNA replication and therefore high-level gene expression, but incapable of vector spread due to lack of the gene for viral F-protein. Vector was introduced into the fetal amniotic and peritoneal cavities, intravascularly, intramuscularly and intraspinally; at 2 days, expression was observed in the airway epithelia, peritoneal mesothelia, unidentified cells in the gut wall, locally at the site of muscle injection and in the dorsal root ganglia, respectively. Mortality was dramatically diminished compared with the first-generation vector.
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PMID:Reduced toxicity of F-deficient Sendai virus vector in the mouse fetus. 1472 76

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

Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits with the tissue of origin. Microvasculature was localized in situ by immunohistochemistry in breast samples. From this tissue, collagen-rich stroma and adipose tissue were dissected mechanically and further disaggregated to release microvessel organoids. BRENCs were cultured from these organoids in endothelial specific medium and characterized by staining for endothelial markers. Microvessels were a prominent feature of intralobular tissue as evidenced by immunostaining against endothelial specific markers such as CD31, VE-cadherin, and von Willebrand factor (VWF). Double staining against VE-cadherin and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) showed that blood and lymphatic vessels could be distinguished. An antibody against CD31 was used to refine protocols for isolation of microvasculature from reduction mammoplasties. BRENCs retained critical traits even at high passage, including uptake of low-density lipoprotein, and had E-selectin induced upon treatment with tumor necrosis factor-alpha. The first signs of senescence in passage 14 were accompanied by gain of trisomy 11. At passage 18 cells showed chromosomal aberrations and growth arrest as revealed by beta-galactosidase staining. We demonstrate here that breast microvasculature may serve as a large-scale source for expansion of BRENCs with molecular and functional traits preserved. These cells will form the basis for studies on the role of endothelial cells in breast morphogenesis.
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PMID:Human breast microvascular endothelial cells retain phenotypic traits in long-term finite life span culture. 1731 68


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