Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A nonradioactive method was employed to detect different cell membrane antigens on human polymorphonuclear granulocytes, monocytes and platelets. We compared the reactivity of one monoclonal antibody, N1III10, assumed to be Fc gamma RII-specific by functional assays, with other well-characterized monoclonal antibodies and human sera. Intact cells were incubated with biotin N-hydroxysulfosuccinimide ester which preferentially reacts with lysine residues in polypeptides. Biotin-labeled cells were lysed and the antigen was isolated from the cell lysate by immunoprecipitation with the antibody bound to Protein A-Sepharose. The precipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and visualized by a streptavidin-alkaline phosphatase system with a suitable substrate. Using this biotin-labeling system we could show that N1III10 detects a 40 kDa antigen on monocytes and platelets, comparable to that expected of Fc gamma RII monoclonal antibodies.
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PMID:Biotinylation: a nonradioactive method for the identification of cell surface antigens in immunoprecipitates. 758 58

At neutral pH, poly-L-lysine-gold complexes labelled the predentine extensively, whereas in dentine the number of gold complexes was reduced by half. Hyaluronidase pretreatment of the section at pH 6.8, prior to labelling, suppressed most of the staining in predentine and did not affect dentine. In contrast, alkaline phosphatase pretreatment at pH 9 enhanced the gold complex labelling in predentine and removed most of the labelling in dentine. This proves that at pH 7.2, the polyanions which are stained include a heterogeneous population of glycosaminoglycans, located in predentine, and phosphoproteins, visualized in dentine. At acidic pH levels (2.9 and 1.1), the number of scored gold complexes decreased, but the ratio between predentine and dentine labelling remained constant. Hyaluronidase pretreatment removed or firmly reduced the gold complex labelling both in predentine and dentine, whereas alkaline phosphatase pretreatment of the sections at pH 9 prior to labelling did not induce any change. This argues in favour of an increased specificity of polylysine-gold complex staining for glycosaminoglycans, stained at low pH in both predentine and dentine. Differential staining of glycosaminoglycans and phosphoproteins according to the pH provides a useful tool for studying the role played respectively by the two matrix components in dentine mineralization.
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PMID:Poly-L-lysine-gold complexes used at different pH are probes for differential detection of glycosaminoglycans and phosphoproteins in the predentine and dentine of rat incisor. 765 59

Pluripotent embryonic stem (ES) cell cultures provide an efficient method for genome manipulation with many applications in marine biotechnology. To develop this technology we have been working to derive fish ES cell lines for in vitro studies of embryo cell growth and differentiation and for the generation of transgenic fish. Zebrafish embryonal cell cultures were derived from blastula-stage embryos in LDF medium supplemented with fetal bovine serum, trout serum, trout embryo extract, selenium, insulin, and leukemia inhibitory factor. Cultures derived under these conditions on feeder layers of zebrafish embryonic fibroblasts possessed a diploid karyotype and exhibited an ES-like morphology with elevated levels of alkaline phosphatase enzyme activity. Injection of primary cell cultures derived from embryos of transgenic fish carrying neo produced chimeric fish detected by polymerase chain reaction analysis. Embryo cells cultured on poly-D-lysine substrate in the presence of retinoic acid or Buffalo rat liver cell-conditioned medium (BRL-CM) and a reduced serum concentration differentiated into neuronal cell types exhibiting elevated levels of acetylcholinesterase enzyme activity and expression of neurofilament and glial fibrillary acidic protein.
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PMID:ES-like cell cultures derived from early zebrafish embryos. 767 May 94

Although lymphocytes are CD-13-negative and therefore should not express the ectoenzyme aminopeptidase N (AP-N), there have been a number of reports suggesting the presence of a cell-surface aminopeptidase with many similarities to AP-N. We have determined aminopeptidase activity with 4-methyl-7-coumarylamide (NMec) derivatives of alanine, leucine, lysine and arginine in Jurkat cells (a human T-cell lymphoma line) and in HL60 cells (a CD-13-positive myeloid leukaemia line) and compared the activities with those of purified pig AP-N and human renal microvillar membranes. Jurkat cell aminopeptidase activity doubled on disrupting the cells and the sensitivity to amastatin increased. When the cells were fractionated only 4% of the activity was recovered in the membrane fraction, compared with 87% recovery for alkaline phosphatase. The profile of activities for intact Jurkat cells was Leu > Ala > Lys > Arg, changing in the cytosolic fraction to Lys > or = Arg > Leu = Ala; the profiles for intact HL60 cells and AP-N were identical, namely Ala > Leu > Arg > Lys. The Km values for the hydrolysis of Ala-NMec and Leu-NMec by Jurkat cells were 65 microM and 11 microM, in each case some 6-fold lower than those for AP-N. The pH-activity curves for the hydrolysis of Ala-NMec by Jurkat cells and human renal microvillar membranes were displaced by almost 1 pH unit and the activity was not sensitive to the anionic composition of the buffers. However, a 3-fold activation of the cytosolic activity by 0.1 M NaCl was observed with Arg-NMec as substrate. With Ala-NMec as substrate, the sensitivity of the aminopeptidase activity to inhibitors increased markedly after disrupting the cells, but still differed from that observed with purified pig AP-N; the concentrations giving 50% inhibition were as follows (values for AP-N in parentheses): amastatin. 28 nM (150 nM); bestatin, 12 microM (43 microM), probestin, 100 nM (< 10 nM), puromycin, 30 microM (> 1 mM). Anion exchange chromatography on Mono Q revealed two activities: that of peak I preferentially hydrolysed Arg-NMec, was activated by NaCl and was insensitive to amastatin; while that of peak II was strongly inhibited by amastatin and had a broad specificity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The aminopeptidase activity in the human T-cell lymphoma line (Jurkat) is not at the cell surface and is not aminopeptidase N (CD-13). 790 64

Diet is the cornerstone of diabetes management, but nutritional interventions in diabetes are still being developed; hence, it is important to understand the effects of diet on nutrient metabolism. Dietary sugars stimulate intestinal sugar absorption in diabetic mice, but the effect of dietary protein on amino acid absorption in diabetes is unknown. We fed streptozotocin-diabetic (> 60 d diabetic) and nondiabetic mice high protein (70% casein) or low protein (15% casein) diets designed to elicit adaptation in amino acid uptake by the small intestine. A high protein diet significantly enhanced uptake per milligram of small intestine of the nonessential amino acids proline and aspartate in both diabetic and nondiabetic mice. Uptake per milligram of small intestine of the essential amino acids leucine and lysine and of the nonessential amino acid alanine which shares transporters with essential amino acids was independent of dietary protein. There was no effect of diabetes on uptake per milligram of any amino acid studied. Because weight per centimeter was greater in diabetic mice, uptake per centimeter of all amino acids tended to be greater in diabetics. Specific activity of alkaline phosphatase in the proximal and distal jejunum was independent of diabetes but varied with dietary protein. Changes in levels of dietary protein induce reversible adaptation of the intestinal uptake rate of nonessential but not of essential amino acids, an adaptive pattern typical of nondiabetics and apparently maintained in diabetics as well.
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PMID:Intestinal amino acid transport in mice is modulated by diabetes and diet. 791 22

Numerous osteometabolic factors are implicated in the bone mass loss which occurs with ageing. Among these a significant role is played by the impairment of intestinal calcium absorption which may be attributed in the elderly to various factors such as the reduction of chlorhydro-peptic secretion, the correlated deficiency of vitamin D and their relative duodenal receptors. In order to evaluate the clinical efficacy of an arginine-lysine-lactose preparation a group of 40 subjects with senile involutional osteoporosis was studied. The subjects were divided into two groups using random criteria and were treated with carbocalcitonin alone (40 UMRC day i.m. on alternative days) or carbocalcitonin association complex. The following parameters were evaluated in basal conditions and after six months of treatment: bone mass density (BMD) using computerised bone mineralometry, bone pain, intake of analgesics, serum levels of calcium, phosphorus, alkaline phosphatase, osteocalcin, parathormone, as well as calciuria and hydroxyprolinuria. The comparison between the two groups shows a more marked increment in BMD in subjects treated with arginine-lysine-lactose, a greater reduction in painful symptoms, and a more evident and significant reduction of parathormone and hydroxyprolinuria levels. These effects appear to be due to a distinct improvement in intestinal calcium absorption mediated by lysine and lactose, and probably to a positive action played by the amino acid at the level of support structures.
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PMID:[The effects of the carbocalcitonin + arginine-lysine-lactose combination in senile involutional osteoporosis]. 802 55

Two matched groups of postmenopausal patients were treated respectively with calcitonin or calcitonin and an arginine-lysine-glycerophosphoric acid-lactose association. The rationale underlying this therapy took the form of data in the literature which indicated an action of these amino acids and lactose on calcium absorption and on the metabolism of protein components in the skeletal structure. The following tests were performed: mineralometric evaluation, evaluation of painful symptoms and intake of pain-relieving drugs, serum levels of calcium, phosphorus, alkaline phosphatase, osteocalcin, parathormone, and calciuria and hydroxyproline. These parameters were assayed at the beginning and end of treatment which lasted six months. The results, or in other words the comparison between the two groups, basal or after treatment, and the values recorded before and after treatment in each group, enable the authors to affirm that the administration of the arginine-lysine-glycerophosphoric acid-lactose association leads to an increase in bone density and plasma osteocalcin, a reduction in painful symptoms and analgesic intake, and a reduction in the serum levels of parathromone and hydroxyproline. Data reported in the literature support the conclusion that the results obtained are the consequence of an improved intestinal absorption calcium. It is highly probable that the protein components of the association administered, arginine-lysine-glycerophosphoric acid-lactose, also exercise a direct action on osteoblasts and on the metabolism of bone matrix protein components.
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PMID:[Experience regarding the use of arginine-lysine-lactose treatment in menopausal osteoporosis]. 808 36

Ligand-mediated approaches to gene transfer offer an alternative to viral vectors for both in vivo and in vitro applications. Although a significant percentage of the plasmid-based DNA complex is lost to lysosomal degradation following receptor-mediated endocytosis, simultaneous infection with adenovirus has been shown to increase the level of transgene expression [Curiel, Agarwal, Wagner and Cotten (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8850-8854; Wagner, Zatloukal, Cotten, Kirlappos, Mechtler, Curiel and Birnstiel (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6099-6103]. In this study we describe an adenovirus-based ligand complex where the plasmid DNA, polycation-ligand conjugate and adenovirus are contained within a single particle structure. At the core of the transfection particle is a replication-defective recombinant adenovirus encoding a cDNA minigene for human placenta alkaline phosphatase that was chemically modified with poly(L-lysine) (Ad-pLys). Electron microscopy of an adenovirus-based ligand complex formed by successively adding plasmid DNA and an asialo-orosomucoid-poly(L-lysine) conjugate to Ad-pLys revealed structures that appeared as intact viral particles coated with a dense biomolecular layer. Adenovirus-based ligand complexes containing either a luciferase or beta-galactosidase reporter plasmid were shown to efficiently deliver the plasmid transgene to cells that express the hepatic asialoglycoprotein receptor. Furthermore, the poly(L-lysine) modification greatly reduced the infectivity potential of the virus without causing a concomitant loss of augmented gene transfer. As an alternative to infectious virions, incomplete products of viral assembly were also considered as a source for endosomalytic activity. However, these defective virions were unable to significantly enhance plasmid transgene delivery.
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PMID:Biochemical and functional analysis of an adenovirus-based ligand complex for gene transfer. 816 59

The interactions of bone cells with their surrounding extracellular microenvironment may be mediated by integrins, a family of heterodimeric glycoproteins consisting of alpha and beta subunits that noncovalently interact to form cell-substratum adhesion receptors. We previously described the integrins on calvarial bone cells in rats with use of polyclonal antibodies against some integrin subunits. In the present study, we expanded this initial characterization by employing a more complete panel of monoclonal antibodies to identify integrins on human bone cells. Minced fragments of trabecular bone obtained during total knee arthroplasty were grown in culture until bone cells became confluent. The cells then were dissociated, plated again, grown to confluence, and assayed for alkaline phosphatase activity, response of cyclic adenosine monophosphate to stimulation with parathyroid hormone, and osteocalcin content. The percentage of the cells that adhered to various substrates was measured; 60-70% adhered to type-I collagen, fibronectin, vitronectin, and poly-D-lysine; 40-50% adhered to type-IV collagen, laminin, and gelatin; and only 10% adhered to fibrinogen. Flow cytometric analysis with anti-integrin monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitates of the human bone cells revealed high levels of alpha 1 beta 1, alpha 3 beta 1, alpha 5, beta 1 and alpha v beta 5 integrins and much lower levels of alpha 2 beta 1, alpha 4 beta 1, alpha v beta 1, and alpha v beta 3 integrins. This description of the integrin repertoire of cultured human bone cells represents the first step toward an understanding of the role played by integrins in the growth, maintenance, and repair of bone.
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PMID:Identification of integrin receptors on cultured human bone cells. 820 92

The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway and is induced by oxygen limitation. The arcD gene specifies a 53-kDa protein with arginine: ornithine exchange activity. The ArcD protein of P. aeruginosa, like the LysI lysine transporter of Corynebacterium glutamicum, has 13 hydrophobic regions which could span the cytoplasmic membrane. Fusion of a Caa (colicin A) epitope to the N-terminal part of ArcD permitted the localization, by immunoblotting, of the hybrid protein in the inner membrane of P. aeruginosa. Fusion of PhoA (alkaline phosphatase) to the very C terminus of ArcD produced another hybrid protein, which exhibited PhoA activity. Both ArcD hybrid proteins retained arginine transport activity and served to support a topological model which proposes that the N terminus is oriented toward the cytoplasm and the C terminus faces the periplasm. Further ArcD-PhoA fusions were consistent with this model. When the Caa epitope was fused to a C-terminal ArcD fragment consisting of only 5 hydrophobic domains, the resulting hybrid protein could be recovered intact from the inner membrane, suggesting that the C-terminal part of ArcD contains sufficient information for insertion into the membrane. This study illustrates the utility of the Caa epitope to tag membrane proteins.
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PMID:Characterization of the arcD arginine:ornithine exchanger of Pseudomonas aeruginosa. Localization in the cytoplasmic membrane and a topological model. 844 2


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