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)

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.
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PMID:On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli. 138 56

A strategy for the purification and cleavage of chimeric recombinant proteins based on a genetically engineered metal-binding peptide and a human renin cleavage site is described. Vectors were constructed to direct the synthesis of chimeric human immunodeficiency virus (HIV) reverse transcriptase (RT) or beta-galactosidase in Escherichia coli. As shown below, two control chimerics without the metal-binding peptide were also included: 1. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 2. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 3. Pro-Ile-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 4. Pro-Ile-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 5. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-beta-galactosidase Both N-terminal sequencing and an enzyme-linked immunosorbent assay utilizing antibodies to the metal-binding peptide were used to characterize the purified chimeric proteins. The relative RT activity of the chimeric protein was indistinguishable from the HIV-1 RT without the fusion sequence, indicating that the metal-binding and renin-cleavage sequences have no effect on the polymerase function of HIV-1 RT. The cleavage by recombinant human renin occurred at the expected site. A future paper will describe results on the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography.
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PMID:Expression and characterization of chimeric rDNA proteins engineered for purification and enzymatic cleavage. 172 60

A double-stranded synthetic oligonucleotide that codes for an amino acid sequence specifically recognized and cleaved by the endopeptidase, renin, was inserted into a plasmid expression vector. The double-stranded oligonucleotide was placed at the junction between the sequences coding for two distinct domains of a fusion protein. The vector used for this analysis expressed a 190-kD Epstein-Barr virus membrane antigen (EBV-MA)-beta-galactosidase (beta-gal) fusion protein (Beisel et al., 1985). The resultant novel protein product expressed by the new construction can be cleaved specifically by renin to yield two distinct polypeptides, EBV-MA and beta-gal, corresponding to the two domains of the original fusion protein product.
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PMID:Site-specific cleavage of a fusion protein by renin. 282 77

We developed a sensitive and specific heterogeneous enzyme immunoassay for measuring angiotensin I (AI) or renin (EC 3.4.99.19) activities. Linking AI under carefully controlled conditions by means of N-succinimidyl-3-(2-pyridyldithio)propionate to highly purified beta-D-galactosidase (EC 3.2.1.23) produced a well-defined conjugate in high yield. The procedure is simple and consists of three steps: (a) incubate 0.5 mL of plasma with 100 microL of inhibitor solution for 1 h to generate renin activity; (b) incubate this with labeled AI for 1 h at 37 degrees C; and (c) do a double-antibody precipitation. Bound beta-D-galactosidase activity is determined, with O-nitrophenyl-beta-D-galactoside as substrate, by measuring the liberated O-nitrophenol at 410-420 nm. The analytical range of the assay extends from 62.5 to 4000 ng/L, and renin activities so measured in plasma correlate well with those measured by an established radioimmunoassay (n = 35, r = 0.97, intercept -0.6, and slope 1.44).
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PMID:Enzyme immunoassay of angiotensin I and renin. 311 31

A patient with early infantile galactosialidosis presenting as congenital adrenal hyperplasia with clitoral hypertrophy and arterial hypertension is reported. Serum 17-alpha-OH-progesterone and plasma renin levels were elevated. Adrenal hyperplasia and thickening of the cardiac septum were detected by sonography; however, progressive hepatosplenomegaly, increasingly coarse features, and vacuolization of bone marrow and liver cells suggested a storage disorder. Combined deficiency of beta-galactosidase and sialidase enzyme activity in both lymphocytes and cultured fibroblasts was detected. This patient with early infantile galactosialidosis is the first reported who presented with congenital adrenal hyperplasia.
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PMID:Infantile galactosialidosis presenting with congenital adrenal hyperplasia and renal hypertension. 821 48

Mesangial cells represent a major target for gene transfer approaches to the kidney. To establish a liposome-based system for transfection of mesangial cells we analyzed the efficacy and toxicity of different cationic liposomes and other nonviral transfection methods in primary cultures of rat and human mesangial cells using the Escherichia coli beta-galactosidase (lacZ) gene as a marker. In addition, an expression vector containing a human renin cDNA under the control of the cytomegalovirus immediate-early promoter/enhancer was generated, introduced into mesangial cells, and assayed in a system of transient gene expression. In vivo, gene transfer was studied after infusion of liposome/DNA complexes in the kidney of rats via the renal artery. Transfection efficiency ranged from 5.5% with DMRIE Liposomes in rat mesangial cells to 1.1% with LipofectAmine liposomes in human mesangial cells. Cytotoxicity following transfection was dependent on the transfection method. Transfection with the human renin expression vector led to the secretion of 11 pg/10(4) cells/48 h human renin in rat mesangial cells, 3,600 pg/10(4) cells/48 h in 293 cells, and 113 pg/10(4) cells/48 h human renin in opossum kidney cells. In vivo, infusion of liposomes was accompanied by nephrotoxicity and did not result in marker gene expression. Together the data demonstrate that cationic liposomes are useful tools for transferring genes into mesangial cells, including human mesangial cells. Cationic liposomes provide a functional system for the synthesis and secretion of human renin in mesangial cells and other mammalian kidney cells. The current limitation of the evaluated liposomes for an efficient in vivo gene transfer to mesangial cells is the toxicity upon intrarenal arterial administration.
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PMID:Efficacy of cationic liposome-mediated gene transfer to mesangial cells in vitro and in vivo. 1140 9

The systemic renin-angiotensin system (RAS) plays a critical role in cardiovascular (CV) homeostasis. All components of the RAS are also known to be produced cell-specifically within specific brain regions, although the role of the brain RAS relative to the systemic RAS has remained a puzzle due to the difficulty of dissecting these two systems. Selectively targeting these regions with genes that modify the RAS could help unravel this puzzle. We compared the ability of adenovirus (Ad) and lentivirus (feline immunodeficiency virus, FIV) vectors to mediate gene delivery in vivo to the supraoptic nucleus (SON) and subfornical organ (SFO), two important CV control regions known to express the various RAS genes. SON or SFO of adult C57BL/6 mice (n=37) were stereotaxically injected with replication-deficient recombinant Ad or FIV harboring a beta-galactosidase (beta-gal) reporter gene. At 1, 3, or 8 weeks post-injection, brain sections were processed for beta-Gal activity, double immunofluorescence to verify cell-type specificity of viral transduction, or immunohistochemical detection of inflammatory mediators. Our results demonstrate that: (1) murine SFO and SON can be selectively targeted for gene transfer in vivo;(2) FIV mediated neuron-specific gene delivery, whereas Ad transduced both neuronal and glial cell types in SFO and SON; (3) Ad injected into the SON transduced neurons within the SFO through retrograde transport, whereas FIV did not; (4) beta-gal activity remained stable for 3 weeks but then declined by 8 weeks with Ad, while minimal decline occurred with FIV; (5) FIV did not cause inflammatory responses, whereas infiltrate was detectable in Ad-injected SFO and SON. These vectors are potentially important tools for dissecting the cell- and site-specific components of the brain RAS and other important CV regulatory systems within this circuitry, and may have therapeutic applications for centrally mediated CV diseases.
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PMID:Selective gene transfer to key cardiovascular regions of the brain: comparison of two viral vector systems. 1188 16

1. The renin-angiotensin system plays a major role in blood pressure regulation and electrolyte homeostasis through the action of angiotensin (Ang) II. The first and rate-limiting step in the production of AngII is the conversion of angiotensinogen into AngI, which is catalysed by the aspartyl protease renin (EC 3.4.23.15). Circulating active renin is mainly synthesized, processed and secreted by the juxtaglomerular cells within the kidney. 2. To determine the renin 5'-flanking sequences involved in cell and tissue specificity, ex vivo and in vivo studies were performed. Several constructs of various lengths of renin promoter linked to the luciferase gene were first tested ex vivo by transfection in primary cultures of human chorionic cells. The constructs giving a high and specific expression in renin-producing cells were then tested in vivo in a transgenic mice model. 3. The reporter gene chosen to generate transgenic mice was LacZ and the screening was performed in embryos at the embryonic day (E) 15 stage, at which mouse renin is expressed in the developing vessels of the kidney. 4. Only constructs containing more than 5.7 kb of the human renin promoter lead to specific expression of beta-galactosidase in the kidney. 5. Our results demonstrate that the human renin distal promoter region allows a more restricted expression of LacZ in the renin-expressing cells in transgenic mice.
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PMID:New elements in human renin promoter involved in cell-specific expression. 1190 15

Renin plays a central role in controlling blood pressure as it catalyzes the first step in the production of angiotensin II. The aim of this study was to isolate fragments of the human renin (hREN) promoter able to direct tissue-specific and regulated expression of a LacZ reporter gene mimicking endogenous renin. We screened several hREN promoter/LacZ constructs for transgene expression in transient embryos at E15 when renin expression begins. We found that a 12-kb hREN promoter conferred high expression in the kidney at both embryonic and adult stages and that the transgene was expressed in the same cells as endogenous renin. We explored two pathophysiological models in which renin is stimulated and showed concomitant increases in beta-galactosidase and renin activities. In situ beta-galactosidase staining showed renin/transgene-expressing cells are recruited in the juxtaglomerular apparatus and in the afferent arterioles as well as in larger arteries outside the kidney. Using our model, renin expression in interlobular arteries was confirmed as being striped and, for the first time, expression of renin in larger arteries outside the kidney was shown. Therefore, this strain is a suitable model to investigate renin gene pathophysiological regulations in vivo.
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PMID:Expression of renin in large arteries outside the kidney revealed by human renin promoter/LacZ transgenic mouse. 1216 96

Although the renin angiotensin system (RAS) is a major regulator of vascular homeostasis, the role of the RAS in tumor angiogenesis is little understood. Here we show that host angiotensin II (ATII) type 1 (AT1) receptor plays an important role in angiogenesis and growth of tumor cells engrafted in mice. Subcutaneous B16-F1 melanoma-induced angiogenesis as assessed by tissue capillary density and microangiography was prominent in WT mice but was reduced in AT1a receptor-deficient (AT1a-/-) mice. Consequently, tumor growth rate was significantly slower, and the mouse survival rate was greater, in AT1a-/- mice than in WT mice. Tumor growth was also reduced in WT mice treated with TCV-116, a selective blocker of AT1 receptor. Because the beta-galactosidase gene was inserted into the AT1a gene locus in AT1a-/- mice, the site of beta-galactosidase expression represents the AT1a receptor expression in these mutant mice. In tumor-implanted AT1a-/- mice, the major site of the beta-galactosidase expression was macrophages in tissues surrounding tumors. Moreover, the number of infiltrated macrophages was significantly lower in AT1a-/- mice than in WT mice, and double-immunofluorescence staining revealed that these macrophages expressed VEGF protein intensively. Therefore, the host ATII-AT1 receptor pathway supports tumor-associated macrophage infiltration, which results in enhanced tissue VEGF protein levels. The host ATII-AT1 receptor pathway thereby plays important roles in tumor-related angiogenesis and growth in vivo.
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PMID:Role of host angiotensin II type 1 receptor in tumor angiogenesis and growth. 1284 60


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