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)

Knowledge of the number and kinds of differentiation steps that characterize cells of the osteoblast lineage is inadequate. To further analyze osteoblast differentiation, we generated a series of monoclonal antibodies (MAb) to osteogenic cells. Spleen cells from mice immunized with whole-cell populations enriched for expression of osteoblast-associated properties or bone formation in vitro were fused with the SP2/0 myeloma cell line. Supernatants from growing hybridomas were screened by indirect immunofluorescence on frozen sections of a portion of 21-day fetal rat heads that included the calvaria bone, periosteum, muscle, fibrous connective tissue, and skin. Six MAb were selected with bone-associated staining and limited ability to label other tissues. Either cell surface or cytoplasmic molecules were recognized by five of the MAb; one recognized a molecule detectable both in the cytoplasm, on the cell surface, and in the extracellular matrix. Of the antibodies selected, one identified both preosteoblasts and osteoblasts and has been found to be against alkaline phosphatase. The others recognized the mature osteoblasts, osteocytes, and chondrocytic cells. The pattern and distribution of the labeling in vivo extended to primary cells and cell lines in vivo. These results support earlier observations on molecules differentially expressed by cells at different stages of the osteoblast lineage and extend the available cell surface and cytoplasmic epitopes identifiable as marker molecules.
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PMID:Isolation of monoclonal antibodies recognizing rat bone-associated molecules in vitro and in vivo. 150 71

In order to better understand why higher eukaryotic membrane proteins, in contrast to soluble proteins, are not readily expressed in Escherichia coli, the gene encoding the liver mitochondrial phosphate transporter (H+/Pi symporter) (Ferreira, G. C., Pratt, R. D., and Pedersen, P. L. (1989) J. Biol. Chem. 264, 15628-15633), was subcloned into a plasmid (pFOG402) containing the alkaline phosphatase promoter and leader sequence. Although this system is highly efficient in overexpressing soluble mitochondrial proteins in E. coli, e.g. alpha and beta subunits of the liver ATP synthase, it fails to express the H+/Pi transporter. Expression is not obtained by truncation of the transporter gene from either the 3' or 5' end, by fusing the mature transporter gene to genes encoding either the alpha or beta ATP synthase subunits, or by using different expression plasmids. Significantly, the H+/Pi transporter is overexpressed in E. coli provided its cDNA is first truncated at the 3' end (carboxyl-terminal end) and fused to a cDNA fragment derived from the ATP synthase alpha subunit gene. In fact, progressive deletions from the 3' end of the transporter cDNA produce a ladder of increasingly overexpressed fusion proteins which account from the largest to the smallest for approximately 2.5-14% of the total bacterial cell protein. The minimal truncation necessary from the 3' end is 192 base pairs corresponding to 64 COOH-terminal amino acids. This corresponds to 20% of the transporter and involves removal of one of the six predicted membrane-spanning segments. In a variety of additional experiments designed to define the molecular basis for E. coli's inability to express the complete liver H+/Pi transporter, problems related to cell toxicity and transcription were ruled out. However, in vitro transcription-translation assays revealed that the complete transporter is readily expressed when eukaryotic, but not prokaryotic, ribosomes are present. Significantly, the fused transporter gene (i.e. Pi transporter cDNA truncated at the 3' end + ATP synthase alpha subunit cDNA) is expressed when prokaryotic ribosomes are present. These results support the view that the difficulty in expressing higher eukaryotic membrane proteins in bacteria may be related in some cases to a problem at the level of translation.
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PMID:Overexpression of higher eukaryotic membrane proteins in bacteria. Novel insights obtained with the liver mitochondrial proton/phosphate symporter. 153 83

The immortalized rat calvarial bone cell line RCT-1 responds to treatment with retinoic acid (RA) by increased expression of osteoblast phenotype-related features, including the induction of liver/bone/kidney alkaline phosphatase (ALP) activity. ALP mRNA could not be demonstrated in unstimulated cells, but was first detected in cells treated for 6 h with 1 microM RA. Cycloheximide failed to block the RA induction of ALP mRNA, indicating that de novo protein synthesis was not a requirement for the RA effect and that the ALP gene may be a direct target for RA action. This was confirmed by nuclear run-on assays, which demonstrated a 2.5-fold increase in the abundance of ALP transcripts after 6 h of RA treatment. To determine whether the RA responsiveness was mediated by a specific segment of the ALP promoter, RCT-1 cells were transfected with a series of plasmids containing deletions of the 5'-flanking sequence of the human ALP gene fused to the chloramphenicol acetyl transferase (CAT) gene. CAT activity was measured in cells cultured in the presence of RA or vehicle. All but the smallest construct, which contained 44 basepairs up-stream of the initiation of transcription, were found to mediate a 2- to 3-fold increase in the expression of CAT activity in response to RA. Furthermore, when the region -108 to -45 of the human ALP gene was inserted into the expression vector pBLcat2, in a position immediately up-stream of the herpes simplex virus thymidine kinase promoter, the construct was found to mediate a 2-fold enhancement of CAT activity in response to RA. In gel retardation assays, a major band was present corresponding to the formation of a complex between the 32P-labeled probe containing the -108 to -45 sequence and proteins present in nuclear extracts of RCT-1 cells stimulated for 3 h with RA. These data suggest that the sequence of 64 basepairs (-108 to -45) 5' to the transcription start site is involved in the RA inducibility of the human ALP gene.
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PMID:Retinoic acid stimulates transcriptional activity from the alkaline phosphatase promoter in the immortalized rat calvarial cell line, RCT-1. 158 26

Demineralized bone matrix gelatin (BMG) was implanted into the skeletal muscle of Sprague-Dawley (S.D.) rats, and histological changes were examined 3, 5, 7, 10 and 15 days later. Before bone formation, a specific calcification process was found in most of the BMG from day 5 and 7 after implantation. The heterotopic calcified sites were not always consistent with the sites of the alkaline phosphatase activity. It was considered that this calcification progresses without any cellular components, and we distinguished this type of calcification as "acellular mineral deposition" from the calcification which occurs in new bone formation. This "acellular mineral deposition" was first observed as small spherical calcified deposits in the BMG on day 7 after implantation; these deposits then gradually grew and fused with each other. Some multinucleated cells appeared near the site of calcification on day 7 after implantation, but osteoblasts or osteoblast-like cells were scarcely observed around the calcified deposits in BMG until day 7. Vascularization was often observed near the "acellular mineral deposition" and the new bone formation. Fourier transform infrared spectroscopy showed that the calcified deposits in BMG were composed of hydroxyapatite, carbonateapatite and other calcium phosphate components, and that the first two components became prominent with time. It is believed that the "acellular mineral deposition" is due to the deposition of calcium and phosphate into the BMG by a process of heterogenic nucleation that does not involve osteoblasts or matrix vesicles. Bone formation induced by the BMG occurred after the "acellular mineral deposition." The experimental calcification shown in this paper seems a useful model for the study of biocalcification.
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PMID:Calcification preceding new bone formation induced by demineralized bone matrix gelatin. 158 70

A modular vector system has been developed for the extracellular production of heterologous proteins in Bacillus subtilis. This modular vector system consists of four secretion vectors which are based upon the genes encoding the Bacillus amyloliquefaciens extracellular alkaline protease, neutral protease, barnase and levansucrase. The modular vectors contain compatible restriction sites downstream from the signal peptide-coding region. Three reporter proteins (staphylococcal protein A, levansucrase and Escherichia coli alkaline phosphatase) that offer complementary advantages for cloning, genetic manipulations and media optimization have been fused to the various signal peptides. These secretion vectors function in E. coli and hence can be used to compare the mechanisms of protein secretion in E. coli and B. subtilis.
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PMID:Modular expression and secretion vectors for Bacillus subtilis. 158 74

Lipopeptides are potential vaccine candidates with a built-in adjuvant property. To circumvent the present chemical route of synthesis for lipopeptide-antigen conjugates, the lipoprotein property of the pColE2-P9-encoded lysis protein, CelB, was used to create the bacterial fusion plasmid, pKLY3, to produce lipopeptide-antigen chimeras in Escherichia coli. Plasmid pKLY3 is a derivative of pKK233-2 with the origin of replication of the single-stranded DNA phage, fl. Under control of the promoter, ptrc, is the 5' end of the celB gene coding for a lipoprotein signal peptide and the first five amino acids (aa) (CQANY) of the mature lysis protein. As model systems for the synthesis of small and large lipopeptide-antigens, DNA sequences coding for the P2 peptide and E. coli alkaline phosphatase (PhoA) were fused in frame to the region of celB coding for a lipoprotein signal peptide and CQANY. P2 is a 12-aa peptide including a tyrosine phosphorylation site of the epidermal growth factor receptor (EGF-R). Inducible expression of stable lipohexapeptide CQANYV, lipo-CQANY-P2, and lipo-CQANYA-PhoA, was demonstrated. Similar expression was obtained for lipo-CIEGR-P2 and lipo-CIEGRA-PhoA in which IEGR is a cleavage recognition site for the blood coagulation factor, Xa. Like QANY, IEGR is predicted to form a beta-turn structure. The presence of a lipid moiety on the products was confirmed by demonstrating the incorporation of radioactive palmitic acid and inhibition of processing by globomycin. The lipid-modified peptides were also identified by incorporation of radioactive tyrosine, and the nature of the P2 peptide was verified immunologically.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A fusion plasmid for the synthesis of lipopeptide-antigen chimeras in Escherichia coli. 162 39

The construction of four vectors for high-level expression in Escherichia coli of the phosphatidylinositol-specific phospholipase C from Bacillus cereus or Bacillus thuringiensis is described. In all constructs the coding sequence for the mature phospholipase is precisely fused to the E. coli heat-stable enterotoxin II signal sequence for targeting of the protein to the periplasm. In one set of plasmids expression of the B. cereus or B. thuringiensis enzyme is under control of the E. coli alkaline phosphatase promoter, while in a second set of plasmids expression is under control of a lac-tac-tac triple tandem promoter. A simple and rapid procedure for complete purification of the phospholipase C overproduced in E. coli, involving isolation of the periplasmic proteins by osmotic shock followed by a single column chromatography step, is described. The largest quantity of purified enzyme, 40-60 mg per liter culture, is obtained with the plasmid expressing the B. cereus enzyme under control of the lac-tac-tac promoter. Lower quantities are obtained with the plasmids containing the alkaline phosphatase promoter (15-20 and 4-6 mg/liter for the B. cereus and B. thuringiensis enzymes, respectively) and with the plasmid expressing the B. thuringiensis phospholipase under control of the lac-tac-tac promoter (15-20 mg/liter). A comparison of the functional properties of the recombinant phospholipases with the native enzymes isolated from B. cereus or B. thuringiensis culture supernatant shows that they are identical with respect to their catalytic functions, viz., cleavage of phosphatidylinositol and cleavage of the glycosyl-phosphatidylinositol membrane anchor of bovine erythrocyte acetylcholinesterase.
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PMID:High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis. 166 69

A variety of signal transduction pathways contribute to the regulation of transcription in mammalian cells. Several of these pathways ultimately rely upon the interaction of transcription factors with genetic sequences termed response elements in the promoter regions of some genes. The biochemical mechanisms that control the levels and state of activation of transcription factors are poorly understood. However, specific phosphorylation events mediated by protein kinase C, growth factor receptor-linked tyrosine kinases, and protein kinase A clearly participate in the regulation of these signal transduction pathways. To understand the relationship between activation and/or inhibition of these pathways and regulation of gene expression controlled by specific response elements, cell lines were prepared containing the TPA response element (TRE), serum response element (SRE), or cyclic AMP response element (CRE) fused to a gene encoding a secretable form of alkaline phosphatase (SEAP). These TRE-SEAP, SRE-SEAP, and CRE-SEAP cells exhibit dramatic increases in alkaline phosphatase (AP) activity following exposure to TPA, PDGF, or forskolin. Down regulation of protein kinase C or inhibition of tyrosine kinase activity blocked the stimulation of AP activity caused by TPA or PDGF. These cell lines can be used to characterize existing inhibitors, and to identify new agents that affect specific signal transduction pathways in mammalian cells.
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PMID:Mammalian cell lines engineered to identify inhibitors of specific signal transduction pathways. 171 Nov 89

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.
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PMID:Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13. 172 85

Lymph node cells from a patient with Hodgkin's disease (HD) were cultured without Epstein-Barr virus (EBV) or leukine adjuvant. A cell line (719-AB) emerged from the culture after four weeks. The cell line express CD20 (79%), CD 21 (30%), CD30 (63%), CD 35 (61%) antigens and weakly CD25 (19%). using Southern Blot technique, the existence of specific EBV DNA and polyclonal immunoglobulin genes rearrangement were observed in the cell line. In order to obtain a monoclonal antibodies (MoAb), mice Balb/C were immunized with this cell line. The splenic cells suspension of immunized animals were fused with the mouse myeloma NS1. Antibody IgM kappa from secreting clones 2B44 was studied using both indirect immunofluorescence with labeled anti-mouse immunoglobulin and immunohistochemistry based on alkaline phosphatase/antiphosphatase complex (APAAP) and ModAMeX technique on a panel of normal or pathological cells. Normal peripheral lymphocytes, monocytes, polymorphonuclear cells, and erythrocytes, did not react. The MoAb 2B44 recognized the dendritic reticulum cells and the smooth muscle cells of vessels on frozen section and paraffin section from HD or reactive lymph nodes. On specially processed paraffin sections (ModAMeX) Reed-Sternberg cells (RSC) were reactive with 2B44 MoAb (in 2 cases out of 5 tested). The molecular weight of the antigen recognized by 2B44 MoAb is of 37 kd. The description of a new epitope shared by different histological components might be of interest for defining a new cluster and better understanding the nature of RSC.
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PMID:Production of a monoclonal antibody (2B44) reactive on a shared epitope on dendritic reticulum cells, smooth muscle cells of vessels and Reed-Sternberg cells. 172 34


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