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)

We have studied the distribution of the lysosomal sphingolipid hydrolases beta-glucosaminidase, beta-galactosaminidase, beta-galactosidase, alpha- and beta-glucosidases, and alpha-mannosidase in the bovine and human ocular tissues, choroid, cornea, lens, retina, and sclera using synthetic substrates in the form of the 4-methylumbelliferyl derivatives of the corresponding glycosides. As compared to the bovine ocular tissues, the human ocular tissues possessed higher levels of all the enzyme activities examined with the exception of beta-galactosidase, and alpha-glucosidase than the other bovine ocular tissues. In contrast to the retina, which is primarily a neural tissue, human and bovine lens have minimal or trace levels of all the lysosomal hydrolases examined. Human and bovine retina, cornea, sclera, and choroid possess enzyme activities which are higher than the lens. This would indicate a slow turnover of glycosphingolipids in lens tissue as compared to the other ocular tissues.
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PMID:Distribution of lysosomal hydrolases in human and bovine ocular tissues. 734 Oct 62

To achieve gene delivery to sensory neurons of the trigeminal ganglion, thymidine kinase-negative (TK-) herpes simplex viruses (HSV) containing the reporter gene lacZ (the gene for E. coli beta-galactosidase) downstream of viral (in vectors RH116 and tkLTRZ1) or mammalian (in vector NSE-lacZ-tk) promoters were inoculated onto mouse cornea and snout. Trigeminal ganglia were removed 4, 14, 30, and 60 days after inoculation with vectors and histochemically processed with 5-bromo-4-chloro-3 indolyl-beta-galactoside (X-Gal). With vector tkLTRZ1, large numbers of labeled neurons were observed in rostromedial and central trigeminal ganglion at 4 days after inoculation. A gradual decline in the number of labeled neurons was observed with this vector at subsequent time points. With vectors RH116 and NSE-lacZ-tk, smaller numbers of labeled neurons were seen at 4 days following inoculation than were observed with vector tkLTRZ1. No labeled neurons could be observed at 14 days after inoculation with vectors RH116 and NSE-lacZ-tk. Immunocytochemistry for E. coli beta-galactosidase and in situ hybridization to HSV latency-associated transcripts revealed labeled neurons in regions of the trigeminal ganglion similar to that observed with X-Gal staining. A comparable distribution of labeled neurons in trigeminal ganglion was also observed after application of the retrograde tracer Fluoro-Gold to mouse cornea and snout. These data provide evidence that retrogradely transported tk- herpes virus vectors can be used to deliver a functional gene to sensory neurons in vivo in an anatomically predictable fashion.
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PMID:Comparative efficacy of expression of genes delivered to mouse sensory neurons with herpes virus vectors. 810 60

We have used a mouse model system and the corneal route of inoculation to examine the issue of extra-neuronal persistence of herpes simplex virus type 1 (HSV-1). HSV-1 strain F DNA and inflammatory lesions were detected in corneal tissue of mice at 5, 11, 23, 37 and 60 days post-infection (p.i.) Viral DNA was localized by in situ PCR to epithelial cells and less frequently to cells in the stroma of the cornea. Viral proteins were not detected in the cornea and virus could not be isolated from tissue homogenates after 11 days p.i. even though histopathological lesions became progressively more severe at 37 and 60 days p.i. The DNA-containing cells were usually adjacent to the sites of inflammation or within these sites in the chronic stage (23, 37 and 60 days p.i.). In contrast to strain F, persistence of HSV-1 strain KOS DNA and inflammatory lesions were not detected after 11 days p.i.; this result suggests that the long-term persistence of HSV-1 DNA and the development of inflammatory lesions are virus strain-dependent. We tested for the possibility of transgenic HSV-1 immediate early gene (ICP4) promoter activity in chronically infected corneas of transgenic mice containing the ICP4 promoter fused to the bacterial beta-galactosidase coding sequence. Our results indicated that the chimeric transgene was expressed in the cornea at 5, 11, 23, 37 and 41 days p.i. Possible explanations for these results and mechanisms for the generation of the chronic inflammatory lesions are discussed. The properties of chronic HSV infections in the cornea may be similar to those which have been described for persistent or defective viral infections in other systems.
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PMID:Herpes simplex virus type 1 DNA persistence, progressive disease and transgenic immediate early gene promoter activity in chronic corneal infections in mice. 820 87

Genetic manipulation of donor cornea prior to transplantation has the potential to modulate the allogeneic response, as well as the endothelial cell function. This study examined the feasibility of gene transfer to corneal endothelial cells using replication-defective recombinant adenoviral vectors. Adult rabbits corneas were infected with recombinant adenovirus RAd35, containing the Escherichia coli beta-galactosidase (lacZ) gene. Localization of gene transfer was assessed by histochemical staining for beta-galactosidase and recombinant protein production was quantified by a soluble assay. In initial experiments, the efficiency of gene transfer and kinetics of expression were studied ex vivo, using organ culture of transfected corneas. Following coculture of whole corneal fragments with RAd35, high levels of gene expression were evident on days 107, diminishing after that time. Gene transfer was found to be almost entirely restricted to corneal endothelial cells, with scattered expression in epithelial cells. Following these ex vivo studies, genetically modified corneas were transplanted as orthotopic allografts in rabbits. Similar kinetics of gene expression were seen after transplantation as in the ex vivo experiment, with maximal levels of gene expression in endothelial cells on days 1-4 after grafting. Corneal function following transplantation was not affected by the gene transfer, with the corneas attaining clarity within 1 day of grafting, and thereafter showing the expected thinning on ultrasonic pachymetry. In the absence of any immunosuppression, no inflammation was evident in graft recipient eyes, with the exception of allograft rejection in 1 animal 23 days after grafting. In this study we show that gene transfer to nonreplicating corneal endothelial cells is feasible using recombinant adenovirus vectors, and so may have potential application in the setting of corneal transplantation.
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PMID:Adenovirus-mediated gene delivery to the corneal endothelium. 861 Mar 41

A number of factors have been implicated in the regulation of tissue-specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta-galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large-diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino-terminal domain of type V collagen was associated with the small-diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.
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PMID:Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal-specific fibril morphology. 894 62

A novel method of in vivo targeted gene transfer to intentionally selected areas of the corneal endothelium was developed. Plasmid DNA with the lacZ gene coding for beta-galactosidase was injected into the anterior chamber of adult Wistar rats, and eight pulses of electricity at intensities ranging from 5 to 40 V/cm were delivered for 50 ms to the cornea with a specially designed electric probe in order to determine the effect of gene transfer on the corneal endothelial cells. Gene expression was visualized by enzymatic color reaction using X-gal in enucleated eyes on days 1, 3, 7, 14 and 21 after gene transfer. The treated eyes were then photographed and the X-gal-positive areas were evaluated by an image analyzer. The ratios of the areas (X-gal-positive area/area of entire corneal endothelium x 100%) were then calculated to determine gene transfection efficiency. The expression of beta-galactosidase was clearly detected in the cytoplasm of the corneal endothelial cells as early as day 1 and lasted until day 21. The most intense gene expression was observed on days 1 and 3 (5.21% on day 1 and 6.45% on day 3). The expression of beta-galactosidase on day 3 was most evident following delivery of 20 V electric pulses (0.09% at 5 V, 0.03% at 10 V, 6.45% at 20 V). beta-Galactosidase expression was limited to the corneal endothelial cells in highly selected areas and no beta-galactosidase expression was detected in any other intra- or axtraocular tissues. In addition, no cell damage was apparent in the cornea and no inflammation was detected in any other intraocular tissues. Thus, low-voltage electric pulses successfully transferred the gene of interest to highly selective areas of the corneal endothelium without inducing any pathological changes. This targeted gene transfer method appears to have great potential for use in gene therapy for ocular diseases.
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PMID:Targeted gene transfer to corneal endothelium in vivo by electric pulse. 993 Mar 40

Endothelial progenitor cells (EPCs) have been isolated from circulating mononuclear cells in human peripheral blood and shown to be incorporated into foci of neovascularization, consistent with postnatal vasculogenesis. We determined whether endogenous stimuli (tissue ischemia) and exogenous cytokine therapy (granulocyte macrophage-colony stimulating factor, GM-CSF) mobilize EPCs and thereby contribute to neovascularization of ischemic tissues. The development of regional ischemia in both mice and rabbits increased the frequency of circulating EPCs. In mice, the effect of ischemia-induced EPC mobilization was demonstrated by enhanced ocular neovascularization after cornea micropocket surgery in mice with hindlimb ischemia compared with that in non-ischemic control mice. In rabbits with hindlimb ischemia, circulating EPCs were further augmented after pretreatment with GM-CSF, with a corresponding improvement in hindlimb neovascularization. There was direct evidence that EPCs that contributed to enhanced corneal neovascularization were specifically mobilized from the bone marrow in response to ischemia and GM-CSF in mice transplanted with bone marrow from transgenic donors expressing beta-galactosidase transcriptionally regulated by the endothelial cell-specific Tie-2 promoter. These findings indicate that circulating EPCs are mobilized endogenously in response to tissue ischemia or exogenously by cytokine therapy and thereby augment neovascularization of ischemic tissues.
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PMID:Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. 1020 35

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabbit eye after various experimental injuries were performed using the same sensitive fluorogenic substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC). The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme activity in the tear fluid. As until recently the substrate beta-galactoside-4-HFC had not been used for the histochemical detection of acid beta-galactosidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained using the substrate 5-bromo-4-chloro-3-indoxyl beta-galactoside in the indigogenic method (previously shown to be very sensitive for the detection of acid beta-galactosidase activity in the cornea). Both methods revealed similar localization and changes in enzyme activity; using beta-galactoside-4-HFC an acceptable cellular localization was achieved. For the measurement of acid beta-galactosidase activity in the tear fluid a semiquantitative biochemical method was elaborated using filter paper punches with the substrate (beta-galactoside-4-HFC) soaked with tears and incubated at 37 degrees C. The time of the first appearance of a greenish-yellow fluorescence (enzyme positivity) was recorded by UV lamp and compared with the appearance of fluorescence in calibrated punches containing known acid beta-galactosidase activities. The results show that beta-galactoside-4-HFC is useful for the biochemical assessment of acid beta-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that increased enzymatic activity in tears parallels the severity of corneal damage. Further studies are necessary to evaluate whether the detection of acid beta-galactosidase activity in tears might be useful for diagnostic purposes in humans.
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PMID:Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the experimentally injured rabbit cornea and tear fluid using the sensitive substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC). 1021 8

We have previously shown in a transgenic mouse line, in which 5.2 kb of the elastin promoter was linked to the reporter enzyme chloramphenicol acetyltransferase (CAT), that the highest levels of expression were found in embryonic lungs and aorta, while lower levels were detected in other elastin-containing tissues. Furthermore, in general, expression of the transgene showed developmental regulation similar to that of the endogenous gene. However, the precise location of cellular expression could not be determined in this model. To overcome this limitation, we have developed a similar model, but replaced CAT with the reporter enzyme beta-galactosidase. Enzyme activity was readily detected in the transgenic mouse embryos in expected regions of tissue forming elastic fibers, including the dermis and elastic cartilage. Of considerable interest, however, was the novel finding of expression in specific areas of neuroepithelium of the brain and in the perichondrium surrounding areas destined to form hyaline cartilage in endochondral bone formation. These latter areas included all the bones of the limbs, the spine and rib cage. It appeared that these segments of elastin expression demarcated the border between the developing cartilage and the surrounding mesenchymal tissue. Elastin promoter expression was also found in developing somites, in the mesenchymal layer of the forming cornea of the eye, in the genital tubercle and in the epithelium destined to form the olfactory epithelium. These findings indicate that the elastin promoter is activated during embryonic development in a variety of tissues, suggesting that elastin gene expression may play a role in organizing cutaneous, skeletal and neural structures.
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PMID:Expression of the elastin promoter in novel tissue sites in transgenic mouse embryos. 1076 40

The mouse keratocan gene (Ktcn) expression tracks the corneal morphogenesis during eye development and becomes restricted to keratocytes of the adult, implicating a cornea-specific gene regulation of the mouse Ktcn [J. Biol. Chem., 273 (1998) 22584-22588]. To examine the functionality of the mouse Ktcn promoter, we have cloned and sequenced a 3.2kb genomic DNA fragment 5' of the mouse Ktcn gene, which was used to prepare a reporter gene construct that contained the 3.2kb 5' flanking sequence, exon 1 and 0.4kb of intron 1 of Ktcn, and beta-geo hybrid reporter gene. The beta-galactosidase (betaGal) activity was assayed in tissues of two of five transgenic mouse lines obtained via microinjection. In adult transgenic mice, betaGal activity was detected only in cornea, not in other tissues (e.g. lens, retina, sclera, lung, heart, liver, diaphragm, kidney, and brain). During ocular development, the spatial-temporal expression patterns of the betaGal recapitulated that of endogenous Ktcn in transgenic mice. Using XGal staining, strong betaGal activity was first detected in periocular tissues of E13.5 embryos, and restricted to corneal keratocytes at E14.5 and thereafter. Interestingly, in addition to cornea, betaGal activity was transiently found in some non-ocular tissues, i.e. ears, snout, and limbs of embryos of E13.5 and E14.5 but was no longer detected in those tissues of E16.5 embryos. The transient expression of endogenous keratocan in non-ocular tissues during embryonic development was confirmed by in situ hybridization. Taken together, our results suggest that the 3.2kb Ktcn promoter contains sufficient cis-regulatory elements to drive heterologous minigene expression in cells expressing keratocan. The identification of keratocyte-specific expression of betaGal reporter gene in the adult transgenic mice is an important first step in characterizing the Ktcn promoter in order to use it to drive a foreign gene expression in corneal stroma.
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PMID:Identification of a 3.2 kb 5'-flanking region of the murine keratocan gene that directs beta-galactosidase expression in the adult corneal stroma of transgenic mice. 1085 82


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