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)

Skeletal tissues contain, apart from cells of the osteogenic and chondrogenic lineage, cells of hemopoietic origin, e.g., macrophages, osteoclasts, and their precursors. In the present study we examined the sensitivity for extracellular ATP4- of the above-mentioned cell types in freshly isolated, bone-derived cell populations and in explanted fetal metatarsal bones. Cells of hemopoietic origin reacted to the presence of ATP4- with an increased permeability for impermeant cytotoxic molecules, e.g., ethidium bromide (EB), thiocyanate (KSCN), and an increased non-ion selective membrane conductance. As a consequence, these cells could be killed by a short treatment with adenosine-5' triphosphate (ATP)+KSCN. On the other hand, cells of nonhemopoietic origin (e.g., osteoblasts, chondrocytes) were found to be insensitive to ATP4- in this respect. These cells survived the treatment without apparent damage to their alkaline phosphatase activities, osteogenic potentials, and osteoclast induction capacities. The elimination of the endogenous cells of hemopoietic origin from bone tissue or cell populations derived therefrom offers the possibility to study the properties and functions of osteogenic or chondrogenic cells without interference by the presence of cells of hemopoietic origin. It also allows the study of interactions between osteogenic cells and selected cell populations of hemopoietic origin in coculture experiments.
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PMID:Permeabilization of cells of hemopoietic origin by extracellular ATP4-: elimination of osteoclasts, macrophages, and their precursors from isolated bone cell populations and fetal bone rudiments. 795 80

Methicillin resistance in S. aureus is primarily due to the presence of the mec A gene. However, in addition to mec, the phenotypic expression of methicillin resistance requires the presence of an additional gene(s), fem A which is chromosomally encoded. Previous studies suggest an increase in the biochemical function of fem A gene products due to base substitutions in the region upstream of the fem A gene and in its coding frame. The partial nucleotide sequences of fem A regions in reference and clinical strains of S.aureus were therefore analyzed by PCR-direct solid-phase sequencing and suitable DNA probes. Amplified target DNAs of 251, 330 and 271 bp were resolved on ethidium bromide-stained gels and hybridized with DNA probes conjugated to alkaline phosphatase. In ATCC 12600 strain, a palindromic sequence was conserved in the region upstream of fem A. However, it was destroyed by the occurrence of mutations in other reference, and clinical strains tested regardless of whether they are methicillin-susceptible or resistant. Furthermore, in the coding frame of fem A, two missense mutations were present in MSSA and MRSA without any regularity. These findings suggest that mutations in the fem A region may not be a single factor essential for regulation of methicillin resistance, although fem A probably functions cooperatively with mec A.
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PMID:[Detection of methicillin-resistant Staphylococcus aureus using PCR and non-radioactive DNA probes: III. Mutations of the fem A gene in clinical strains of Staphylococcus aureus]. 799 17

A hybrid heterodimeric alkaline phosphatase expressed in KB cells, consisting of placental and intestinal (fetal) subunits, was purified by use of two different immunoaffinity columns using the monoclonal antibodies 2HIMS-1 and HPMS-1. The closely related subunits were found to yield a dimeric active enzyme glycosylated as the mature heterodimeric forms. This enzyme displays intermediate properties to the placental and intestinal (fetal) isozymes with regard to heat stability, inhibition patterns with amino acids and amino acid derivatives, as well as reactivity with monoclonal antibodies specific for human alkaline phosphatase isozymes. Peptide fragments obtained from the hybrid enzyme after cyanogen bromide cleavage belong to either the placental or intestinal (meconial) isozyme as evaluated by SDS polyacrylamid gel electrophoresis, and the N-terminal amino acid sequences, corresponding to the placental and intestinal subunits, can be identified in the peptide fragments. By N-glycanase digestion or tunicamycin treatment, the molecular mass of the subunits was reduced to 62 kDa compared to 69 kDa for the native ones. The results confirm that some cell lines can synthesize hybrid alkaline phosphatases.
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PMID:Expression of a heterodimeric (placental-intestinal) hybrid alkaline phosphatase in KB cells. 801 16

There are conflicting data regarding the detection of t(14;18) in reactive lymphoid hyperplasia (RLH) by the polymerase chain reaction (PCR). Although most studies have not detected t(14;18), several groups have definitively shown that a very low number of cells with this translocation (one in 10(5) to 10(6)) are present in a significant proportion of follicular hyperplasias. Review of the methods from these series reveals that modifications of the PCR assay (ie, enhanced sensitivity steps such as seminesting, lengthy autoradiographic exposure times, multiple aliquot reactions of single samples, and/or high concentrations of template DNA) are probably necessary to detect t(14;18) in RLH. We evaluated a diverse set of 111 RLH (85 lymph nodes, 22 tonsils, and four other sites) from patients of different age groups (age range, 9 months to 80 years) to determine if a standard PCR assay would amplify t(14;18). Of these, 61 (55%) specimens had a prominent follicular hyperplastic component. Fifty-seven follicular lymphomas served as a control group. Polymerase chain reaction was performed as a single-run, two-primer-based assay for major breakpoint region bcl-2 translocations (5' major breakpoint region primer and 3' immunoglobulin heavy-chain gene-joining region consensus primer). Two different types of thermocyclers were employed. A metal block thermocycler was used with 35 cycles of amplification on 500 ng to 1 micrograms of genomic DNA, and a separate air thermocycler was used with 45 cycles of amplification on 50 ng of genomic DNA. Product detection was carried out through ethidium bromide staining and UV gel illumination, along with a digoxigenin-alkaline phosphatase-based, internal major breakpoint region oligonucleotide probe system. We found no amplified t(14;18) products in any RLH. In contrast, 36 (63%) of 57 follicular lymphomas showed t(14;18) (published range for detection of major breakpoint region translocations by PCR, 31% to 74%). Moreover, the assay's sensitivity, estimated through dilution studies, was to one in 10(4) to 10(5) cells. Although theoretically possible, our data suggest that there is practically no risk of amplifying a t(14;18) from RLH when utilizing a standard PCR assay.
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PMID:Standard polymerase chain reaction analysis does not detect t(14;18) in reactive lymphoid hyperplasia. 806 Feb 26

Methicillin resistance in staphylococci is primarily due to the presence of a mec A gene which encodes the novel penicillin-binding protein 2'. Some chromosomal factors, fem A and fem B, also participate in the expression of methicillin resistance in S. aureus. This study was designed to detect mec A, fem A and fem B genes for identification of staphylococcal species and for discrimination of methicillin-resistant cells. Three different pairs of DNA primers (PBP2'AF-PBP2'AR, fem AF-fem AR and fem BF-fem BR) complementary to unique regions of mec A, fem A and fem B genes were synthesized for use in polymerase chain reaction with DNAs of methicillin-sensitive S. aureus (MSSA), S. epidermidis, methicillin-resistant S. aureus (MRSA) and S. epidermidis. Amplified target DNAs of 630, 509, and 651 bp were resolved on ethidium bromide-stained gel, and hybridized to DNA probes conjugated to alkaline phosphatase. When applied to pure cultures on the MRSA screen agar, all three DNA probes tested detected MRSA in 47 of 61 culture-positive specimens (77.1%); the detection ratio of MRSA with mec A and either fem A or fem B probes was increased to 95.9%. By contrast, the fem A and fem B probes did not detect S. epidermidis. The result of detecting these species streaked on mannitol-salt agar were similar, while the detection of MSSA with the fem A and fem B probes was incomplete irrespective of the presence or absence of mec A. These findings suggest a good correlation between cultivation and DNA probe assay with respect to MRSA detection.
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PMID:[Detection of methicillin-resistant Staphylococcus aureus using PCR and non-radioactive DNA probes (II)]. 825 62

Methicillin resistance in S.aureus and S.epidermidis strains is primarily due to production of a new penicillin-binding protein PBP2' with extremely low binding affinity to most beta-lactam antibiotics. The structural gene for PBP2', mecA, is detectable in clinical specimens by using the polymerase chain reaction (PCR). Amplified target DNA of 630bp can be resolved on ethidium bromide-stained gels, and hybridized with a probe conjugated to alkaline phosphatase. Survey for the mecA gene in 304 staphylococci revealed a good correlation between the presence of mecA and cultivation on agar plates with 4 micrograms/ml of oxacillin, although 3% of sensitive S. aureus strains had the mecA gene. On the other hand, analysis of the regulatory genes (orf 1 and 2) of methicillin resistance was performed on methicillin-resistant S.aureus strains N315 and MR108, demonstrating that the genome of MR108 lacks orf 2 which encodes the repressor protein (Hiramatsu et al., see Ref.5). The regulatory genes of mecA were surveyed for 192 staphylococci by using PCR and allele-specific oligonucleotide probes: 76% of resistant S. aureus strains and 48% of resistant S. epidermidis strains possessed orf 1 corresponding to MR108 (constitutive-type strain), while the remainder of the resistant strains and two strains of sensitive S. epidermidis had two orfs of N315 (inducible-type strain). Furthermore, it appeared that mutation of the femA gene might not be an additional factor for expression of methicillin resistance. These observations suggest that mecA and its regulatory genes should be examined to understand how the genetic background contributed to the phenotypic expression of methicillin resistance in clinical strains.
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PMID:[MRSA--detection of mecA and its regulatory genes]. 828 99

Patterns of alkaline phosphatase (AP)-binding proteins were observed in the alkaline pH range of 6.5-9.5 upon isoelectric focusing and blotting of serum from patients with inflammatory diseases. After isolation using affinity chromatography on protein A or immunoaffinity chromatography on AP coupled to cyanogen bromide (CNBr)-activated Sepharose, the AP-binding protein was identified as IgG on Western blots and in ELISA using human IgG-specific antibodies. It was shown that this IgG binds to AP from both calf (bovine) and human intestine. However, it binds neither to the human liver-bone-kidney (LBK) isoform nor to bacterial AP. Moderate reaction was observed with human placental AP. Comparing patients with various diagnoses (n = 284), AP-binding antibodies were mainly found in severe bacterial infections. They were not detected in serum from healthy blood donors (n = 300). The presence of AP-binding IgG was independent of the infected organ and the bacterial species causing infection. This antibody may be useful for discriminating bacterial from viral infection and for indicating severe bacterial inflammation.
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PMID:Human intestinal alkaline phosphatase-binding IgG in patients with severe bacterial infections. 828 14

The predominant hypothesis for cyclosporine-induced acute renal failure is postulated to be prerenal vasoconstriction with concomitant hemodynamic changes; an alternate hypothesis, however, may be that cyclosporine (CsA) affects intrarenal processes, i.e., direct renal parenchymal cell injury. However, reports on this direct effect of CsA on renal parenchymal cells are contradictory. Therefore, the purpose of this study was to address whether CsA is directly toxic to renal parenchymal cells in a primary culture system of rat renal cortical epithelial cells. The cytotoxicity of Sandimmune, the commercial form of CsA in a polyoxyethylated castor oil vehicle (Cremophor), CsA without vehicle, and the Cremophor vehicle was assessed by plasma membrane integrity (lactate dehydrogenase leakage), mitochondrial metabolic activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and gross morphology (phase-contrast microscopy). The cytotoxicity of Sandimmune was also assessed by lysosomal activity (neutral red uptake), by proximal tubular enzyme activity (alkaline phosphatase), and by three fluorescent probes using a multiwell scanner. The three fluorescent probes were propidium iodide which stains nuclei of nonviable cells; bis-carboxyethyl-carboxyfluorescein which is retained by viable cells; and rhodamine 123, which assesses mitochondrial membrane potential. The results of this study demonstrated that Sandimmune caused dose- (10, 25, and 50 microM) and time- (12, 24, and 48 hr) dependent cytotoxicity, while Cremophor caused cytotoxicity only at high concentrations and long incubations. We conclude that (1) CsA is directly toxic to renal parenchymal cells in vitro and this system potentially represents a sensitive model for further mechanistic studies; (2) CsA plus vehicle (Sandimmune) was more cytotoxic to renal cells than CsA alone (without the polyoxyethylated castor oil vehicle).
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PMID:An in vitro model of cyclosporine-induced nephrotoxicity. 831 63

Detection of Mycobacterium tuberculosis in clinical specimens by the polymerase chain reaction (PCR) was compared with detection by culture. A 317-bp segment within the M. tuberculosis-specific insertion sequence IS6110 was amplified. The detection limit of the PCR assay for cultured mycobacteria was 50 cells per reaction by ethidium bromide-stained agarose gel electrophoresis and 5 cells per reaction by hybridization with an oligonucleotide probe conjugated with either digoxigenin or alkaline phosphatase (AP). This sensitivity was reduced fivefold in sputum specimens seeded with M. tuberculosis. Seventy-six clinical specimens were amplified and examined by the three detection methods. Both the digoxigenin and AP procedures were found to be more sensitive than agarose gel electrophoresis, but they were occasionally associated with a high background. An additional 308 specimens were examined only by agarose gel electrophoresis and the AP procedure. Of 71 specimens found to contain M. tuberculosis, amplified products were detected from 56 (79%) samples by agarose gel electrophoresis and/or the AP procedure. Of the additional 313 specimens that were culture negative for M. tuberculosis, 19 (6%) had amplified products detectable by agarose gel electrophoresis and/or the AP procedure. Compared with culture, PCR showed sensitivities and specificities of 55 and 98%, respectively, for agarose gel electrophoresis and 74 and 95%, respectively, for the AP procedure. Despite this low sensitivity, a rapid positive PCR result was accurate and clinically useful.
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PMID:Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods. 812 99

The differentiating and antitumor activities of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in vitro and 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) in vivo were studied with a human osteosarcoma cell line (OST strain). Anti-tumor activity was estimated with the use of 3-(4,5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide (MTT) assay, colony-forming assay, and athymic mouse assay. The intracellular alkaline phosphatase (ALP) activity of tumor cells and production of bone Gla protein (BGP) in culture media were measured to mark osteoblastic differentiation. In addition, the combination of 1 alpha,25(OH)2D3 and cis-dichlorodiammineplatinum(II) (CDDP) was tested by the colony-forming assay and the measurement of ALP activity and BGP production for differentiating and antitumor effects. The assays revealed that 1 alpha,25(OH)2D3 exerted a dose-related, growth-inhibitory influence. In the colony-forming assay, the 1 alpha,25(OH)2D3-treated colonies were smaller than the untreated colonies. The ALP activity and the BGP production also increased in relation to dose. In the assay in athymic mice, the relative weight of tumors treated with 1 alpha(OH)D3 at 2.5 nmol/kg was significantly smaller than that of the controls, and no side effects were observed in the 1 alpha(OH)D3-treated mice. Marked tumor chondrogenesis was observed in human osteosarcoma treated with 1 alpha(OH)D3 in athymic mice. The combination of 1 alpha,25(OH)2D3 at 10(-8) M and CDDP at 2 micrograms/ml significantly enhanced both the differentiation and the growth inhibition in vitro. Our study apparently is the first demonstration that vitamin D3 metabolites have an antitumor and differentiating effect on human osteosarcoma cells in vitro and in athymic mice.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differentiating and antitumor activities of 1 alpha,25-dihydroxyvitamin D3 in vitro and 1 alpha-hydroxyvitamin D3 in vivo on human osteosarcoma. 842 14


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