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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aim of this study was to better characterize rabbit proximal kidney tubule cells cultured on collagen IV-coated porous inserts, as compared to the same cells seeded in standard plastic wells. Total protein contents in confluent monolayers on permeable membranes were about twofold higher than those measured in confluent cultures in plastic wells. Microscopy examinations suggested that such a difference was probably due to a higher cell density and to an impressive development of the apical brush-border membrane. Moreover, measurement of unidirectional transport of p-aminohippuric acid and tetraethylammonium bromide confirmed the high polarization level of cultures on porous inserts. Results of methyl(alpha-D-[U-14C]glyco)pyranoside uptake suggested that cell phenotype was probably influenced by culture conditions. Analysis of different markers as a function of time in culture showed decreases of alkaline phosphatase (AP), gamma-glutamyltranspeptidase (GGT), and Na(+)-K(+)-ATPase activities as well as increases in LDH, ATP, and glutathione levels, similar to those formerly reported for cells cultured in standard plastic plates. However, comparative data from 6-d-old monolayers have shown that AP, GGT, Na(+)-K(+)-ATPase, glutathione reductase (GRED), and selenium-dependent glutathione peroxidase (Se-GPX) activities were 2.8-, 2.6-, 1.6-, 1.2-, and 2.1-fold, respectively, better preserved on precoated permeable membranes. On the other hand, this paper reports for the first time in the literature that GRED and SE-GPX, two phase II detoxification enzymes, were well maintained in cultures of rabbit proximal kidney tubule cells. Our results show that culturing rabbit proximal kidney tubule cells on collagen IV-coated porous membranes was accompanied by an improvement of both morphological and biochemical properties of the cells.
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PMID:Morphological and biochemical characterization of primary culture of rabbit proximal kidney tubule cells grown on collagen-IV coated Millicell-CM. 935 85

The purpose of this study was to investigate the effects of 316L stainless steel (SS) corrosion products on the in vitro biomineralization process, because tissue necrosis, bone loss, impaired bone mineralization, and loosening of orthopedic implants are associated with ions and debris resulting from biodegradation. Rat bone marrow cells were cultured in experimental conditions that favored the proliferation and differentiation of osteoblastic cells and were exposed to SS corrosion products obtained by electrochemical means for periods ranging from 1 to 21 days. Quantification of total and ionized Ca and P, as well as Fe, Cr, and Ni, ions in the culture media of control and metal added cultures during the incubation period was performed to study the influence of corrosion products on the Ca and P consumption that occurs during the mineralization process. Control cultures and metal effects on cultures were evaluated concerning DNA content, enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alkaline phosphatase (ALP) activity. Histochemical detection of ALP, Ca, and phosphate deposition, and examination of the cultures by scanning and transmission electron microscopy (SEM and TEM) were also performed. The presence of SS corrosion products resulted in impairment of the normal behavior of rat bone marrow cultures. Levels of Cr and Ni in the medium of cultures exposed to 316L SS corrosion products decreased throughout the incubation period, suggesting a regular deposition of these species; these results were supported by TEM observation of the cultures. Cultures exposed to the corrosion products presented lower DNA content, MTT reduction, and ALP activity and failed to form mineralized areas. These cultures showed negative staining on histochemical reactions for the identification of calcium and phosphate deposition and SEM and TEM examination did not show mineral globular structures or mineralization foci, respectively, which is characteristic of cultures grown in control conditions. These results suggest that metal ions associated with 316L SS are toxic to osteogenic cells, affecting their proliferation and differentiation.
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PMID:Decreased consumption of Ca and P during in vitro biomineralization and biologically induced deposition of Ni and Cr in presence of stainless steel corrosion products. 977 16

Although membrane associated enzymes such as ATPase, alkaline phosphatase, and NTP pyrophosphohydrolase in matrix vesicles (MVs) may underlie the mechanisms of ATP-promoted calcification, prior to the current investigation, the role of the MV membrane in calcification had not been addressed. In this study, various perturbations were introduced to the MV membrane in in vitro calcification systems to determine ideal conditions for ATP-initiated calcification by MVs isolated from rachitic rat epiphyseal cartilage. Membrane integrity appears to be required, since the rupture of the vesicular membrane by vigorously mixing with 10% butanol abolished calcification. In contrast, a mild treatment of MVs with low concentrations (e.g., 0.01%, which is much below the critical concentration for micelle formation) of either neutral Triton X-100 or anionic deoxycholate stimulated calcification by >2-fold, without inducing obvious changes in vesicular appearance. Fourier transform infrared spectroscopic studies were done to identify the mineral phase formed in these experiments. For the first time, rachitic MVs were shown to induce the formation of a calcium pyrophosphate dihydrate-like phase after their exposure to calcifying medium with 1 mM ATP. The integration of spectral areas indicated that calcification was enhanced by Triton X-100. The detergent effect was reversible and appeared to be not mediated through activation of ATPase, alkaline phosphatase, or ATP pyrophosphohydrolase. In contrast to neutral Triton X-100 and anionic deoxycholate, cationic cetyltrimethylammonium bromide inhibited both ATPase activity (I50=10 microM) and ATP-initiated calcification. These observations suggest that membrane perturbations can affect calcification and that the presence of NTP-pyrophosphohydrolase in MVs may play a role in the deposition of CaPPi in rachitic cartilage.
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PMID:Further characterization of ATP-initiated calcification by matrix vesicles isolated from rachitic rat cartilage. Membrane perturbation by detergents and deposition of calcium pyrophosphate by rachitic matrix vesicles. 988 89

The effects of acupuncture on the disorders elicited by abnormalities of endocrine system were investigated in ovariectomized mice. Female mice (strain; C57BL/6) were ovariectomized (OVX) and acupuncture points, Shenshu ([Japanese pictograph see text] : BL23) on both side of the back were continuously stimulated by subcutaneous needles for 20 days. After completion of experimental sessions, animals were sacrificed and specific brain regions were assayed for catecholamine contents by high performance liquid chromatography with electro chemical detector (ECD-HPLC). The mitogenic activities of splenic lymphocytes were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTS) assay and alkaline phosphatase (ALP) assay. Furthermore, the effects of needle stimulation on learning and memory ability were studied by the step-through type passive avoidance test. Norepinephrine and dopamine contents in the frontoparietal cerebral cortex, ventral hippocampus and olfactory bulb were decreased in the OVX group, and both MTS activity and ALP activity were decreased 20 days after ovariectomy. The mean latent period was also shortened in the passive avoidance test in the OVX group. However, applying needle stimulation increased norepinephrine and dopamine contents in the brain regions, and enhanced mitogenic activities of splenic lymphocytes. The stimulation also improved memory-related behavior. It was concluded from this study that after mice were stimulated by subcutaneous needle insertion, overall changes were observed in central nervous system (including retention of memory) and immune functions. The study suggests that acupuncture improves the memory loss and decrease of immune responses accompanying aging and/or menopause, and the that it may have an important role in medical care for the elderly.
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PMID:Acupuncture inhibits the decrease in brain catecholamine contents and the impairment of passive avoidance task in ovariectomized mice. 1047 21

Nonstructural protein 3 (Nsp3) is an essential subunit of the alphavirus RNA replication complex, although its specific function(s) has yet to be well defined. Previously, it has been shown that Semliki Forest virus Nsp3 (482 amino acids) is a phosphoprotein, and, in the present study, we have mapped its major phosphorylation sites. Mass spectrometric methods utilized included precursor ion scanning, matrix-assisted laser desorption/ionization mass spectrometry used in conjunction with on-target alkaline phosphatase digestions, and tandem mass spectrometry. Two-dimensional peptide mapping was applied to separate tryptic (32)P-labeled phosphopeptides of Nsp3. Radiolabeled peptides were then subjected to Edman sequencing, and phosphoamino acid analysis. In addition, radiolabeled Nsp3 was cleaved successively with cyanogen bromide and trypsin, and microscale iron-chelate affinity chromatography was used to enrich phosphopeptides. By combining these methods, we showed that Nsp3 is phosphorylated on serine residues 320, 327, 332, 335, 356, 359, 362, and 367, and is heavily phosphorylated on peptide Gly(338)-Lys(415), which carries 7-12 phosphates distributed over its 13 potential phosphorylation sites. These analytical findings were corroborated by constructing a Nsp3 derivative devoid of phosphorylation. The results represent the first determination of phosphorylation sites of an alphavirus nonstructural protein, but the approach can be utilized in phosphoprotein analysis in general.
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PMID:Phosphorylation site analysis of Semliki forest virus nonstructural protein 3. 1085 Dec 34

Several activated derivatives of 9-acridinecarboxylic acid were prepared in order to investigate their utility for detection of hydrogen peroxide. One of these derivatives, 9-acridinecarbonylimidazole (I), is especially stable and is a useful reagent for measuring, by chemiluminescence, the activity of a number of enzymes that directly produce peroxide, including glucose oxidase. Other enzymes can also be assayed if an appropriate intermediate substrate exists that ultimately produces hydrogen peroxide after being acted upon by the enzyme. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and 3-indolyl phosphate (3-IP) are such substrates for alkaline phosphatase. The detection limits for both of these enzymes are in the 1-10 amol range. Other enzymes that can potentially be assayed using I include oxidases, hydrolases and dehydrogenases. Negative assays for compounds that consume or bind peroxide such as reducing agents, antioxidants, catalases and peroxidases are also feasible.
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PMID:Chemiluminescent determination of hydrogen peroxide with 9-acridinecarbonylimidazole and use in measurement of glucose oxidase and alkaline phosphatase activity. 1086 46

We investigated the effects of Toki-shakuyaku-san (TSS, Tang-Kuei-Shao-Yao-San in Chinese), Japanese traditional herbal medicine, on the nervous and immune systems in ovariectomized mice as a climacteric disorder model. Female C57BL/6 mice were ovariectomized (OVX) and TSS was given daily through the drinking water for either 10 or 20 days from the day after ovariectomy. After completion of experimental sessions, animals were sacrificed and specific brain regions were assayed for choline acetyltransferase (ChAT) activity and norepinephrine contents. The mitogenic activities, alkaline phosphatase activity and 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H terazolium bromide (MTT) activity, in splenic lymphocytes has also measured. Furthermore, the effects of TSS on learning and memory ability were studied by the step-through type passive avoidance test. As the results, the administration of TSS significantly suppressed the decrease of ChAT activity in the cerebral cortex (CC) and the dorsal hippocampus (DH) of ovariectomized mice at 10 days after ovariectomy, however no significant effect was observed at 20 days after ovariectomy. Norepinephrine contents in OVX group were decreased at 10 and 20 days after ovariectomy in the CC and the ventral hippocampus (VH). The administration of TSS significantly suppressed the decrease of norepinephrine contents at 20 days after ovariectomy. The mitogenic activities of lymphocyte in spleen were increased at 10 days after ovariectomy, and decreased at 20 days after ovariectomy. However, the suppression of these changes was observed in the group given TSS. The mean latent period was also shortened in the passive avoidance test in the OVX group, but TSS treated group improved mean latency. From these observations, it is inferred that administration of TSS brings on the synthesis of acetylcholine and norepinephrine in the CC and hippocampus, and may improve the memory related behavior and the abnormalities in lymphocytes in the models of the climacteric disorder.
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PMID:Effects of Kampo medicine, Toki-shakuyaku-san (Tang-Kuei-Shao-Yao-San), on choline acetyltransferase activity and norepinephrine contents in brain regions, and mitogenic activity of splenic lymphocytes in ovariectomized mice. 1090 56

Nitric oxide (NO) is synthesised by a group of enzymes called nitric oxide synthases (NOS) and oxidizes to its stable end-products nitrite (NO2-) and nitrate (NO3-) We have previously reported in an in vivo rat model that NO is an important regulator for rat bone fracture healing. This study examines the effects of NO on alkaline phosphatase (ALP) activity in a rat fracture callus explant culture system. Explants of rat femoral fracture callus from days 4, 7, 14 and 28 post fracture induced NO2 release and ALP activity in a biphasic temporal manner, with the highest activity on day 7 and the lowest activity on day 14. Inhibition of NOS by co-incubation with an NOS inhibitor, S-(2-aminoethyl) isothiouronium bromide hydrobromide (AETU), inhibited ALP activity by an average of 50% at each time point (P <0.01). Supplementation with NO donor 3-morpholinosydnonomine hydrochloride (SIN-1) at low doses (25 and 0.025 microM) increased ALP activity by 20% (P < 0.01). ALP mRNA and histochemical ALP activity were localised to osteoblast-like and chondrocyte-like cells within fracture callus. The current study provides evidence that NO plays a regulatory role in ALP activity during rat fracture healing.
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PMID:Nitric oxide regulates alkaline phosphatase activity in rat fracture callus explant cultures. 1093 91

The effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) on cell growth were studied in three human osteosarcoma cell lines, NOS-1, HuO9, and HuO-3N1; one human prostate cancer cell line, PC-3; and one human breast cancer cell line, OCUB-1M. The growth of these cell lines was not promoted by rhBMP-2 at concentrations of 50, 100, 250, and 500 ng/ml, as evaluated by colorimetric 3 (4,5-dimethyl-thiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. Furthermore, the protein induced osteogenic differentiation, characterized by increased alkaline phosphatase activity, and increased production of type I collagen and gamma-carboxylated osteocalcin in NOS-1 cells. The results of this study may suggest the feasibility of using rhBMP-2 for the reconstruction of bone defects caused by malignant tumors, although the data are still preliminary and require further investigation.
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PMID:Effects of bone morphogenetic protein-2 on human tumor cell growth and differentiation: a preliminary report. 1118 Sep 25

We have investigated the effect of changes in the gravity vector on osteoblast behaviour, using the clinostat set at 8 rpm. Two sources of osteoblasts were used: secondary cultures of fetal rat bone cells, and the rat osteosarcoma line 17/2.8 (ROS). Cell number was determined by incubation with 3-(4,dimethyl-2yl)-2,3 diphenyl) tetrazolium bromide (MTT) and measurement of optical density at 570 nm (OD). Alkaline phosphatase activity was detected by standard cytochemical methods. Dividing cells were localised by labelling dividing nuclei with Bromodeoxyuridine (BrdU), detected by immunofluorescence. Cell culture was initiated at densities between 1-4x10(4) cells ml-1. Growth rates in all cultures during the first 48 hours exposure to clinostat rotation were less than in stationary controls. After 3 days, ROS cell numbers were 35% lower, and calvarial cells 39% lower than their respective controls. Alkaline phosphatase activity in calvarial control cultures was uniformly present in characteristically polygonal cells, but after culture in the clinostat the enzyme was present sporadically, and the cells were cuboid. There was also no BrdU uptake in nuclei, but it was present in cell cytoplasms. We conclude that the clinostat decreases cell numbers and cell division. Both cell shape and the distribution of alkaline phosphatase activity in calvarial cell cultures were also affected. This implies that changes in the gravity vector can affect osteoblasts directly, without interaction with other cell types.
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PMID:Effect of clinostat rotation on differentiation of embryonic bone in vitro. 1153 15


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