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)

Transglutaminase (TG) enzymes and protein crosslinking have long been implicated in the formation of mineralized tissues. The aim of this study was to analyze the expression, activity and function of TGs in differentiating osteoblasts to gain further insight into the role of extracellular matrix protein crosslinking in bone formation. MC3T3-E1 (subclone 14) pre-osteoblast cultures were treated with ascorbic acid and beta-glycerophosphate to induce cell differentiation and matrix mineralization. Expression of TG isoforms was analyzed by RT-PCR. TG activity was assessed during osteoblast differentiation by in vitro biochemical assays and by in situ labeling of live cell cultures. We demonstrate that MC3T3-E1/C14 osteoblasts express two TG isoforms--TG2 and FXIIIA. Abundant TG activity was observed during cell differentiation which increased significantly after thrombin treatment, a result confirming the presence of FXIIIA in the cultures. Ascorbic acid treatment, which stimulated collagen secretion and assembly, also stimulated externalization of TG activity, likely from FXIIIA which was externalized upon this treatment as analyzed by immunofluoresence microscopy. Inhibition of TG activity in the cultures by cystamine resulted in complete abrogation of mineralization, attributable to decreased matrix accumulation and an arrested state of osteoblast differentiation as measured by decreased levels of bone sialoprotein, osteocalcin and alkaline phosphatase. Additional functional studies and substrate characterization showed that TG activity was required for the formation of a fibronectin-collagen network during the early stages of matrix formation and assembly. This network, in turn, appeared to be essential for further matrix production and progression of the osteoblast differentiation program, and ultimately for mineralization.
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PMID:Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MC3T3-E1 osteoblast cultures. 1646 87

This study analyzed human mesenchymal stem cell (hMSC) behavior in a fibrin sealant. hMSC morphology, proliferation, and osteogenic differentiation were analyzed after up to 28 days of incubation in eight different formulations of fibrin gels (Tisseel) prepared with various concentrations of fibrinogen complex (FC) and thrombin. Cell morphology and distribution within the gels were observed by fluorescence microscopy after cell staining with calcein dye. Cell proliferation was assessed by measuring the fluorescence intensity of the cell suspension stained with calcein dye after dissolution of the gels. A standard alkaline phosphatase (ALP) assay, von Kossa staining, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze hMSC osteogenic differentiation. Cell behavior varied depending on the gel formulation. Proliferation was higher in the formulations containing a low FC concentration, but ALP activity was higher in the formulations containing a high FC concentration. Variations in thrombin concentration had a lesser effect. Small nodules of mineralization were observed at days 21 and 28 in a formulation containing a high FC concentration, in addition to a marked increase in bone sialoprotein (BSP) gene expression level as well as a lower increase in ALP and osteopontin (OPN) levels. However, there was no significant increase in osteocalcin (OCN) expression, a late marker of osteogenic differentiation, up to day 28. In conclusion, this study demonstrated that hMSC morphology, proliferation, and osteogenic differentiation in fibrin gels depended on the FC/thrombin ratio. hMSCs appeared to undergo osteogenic differentiation when seeded in Tisseel fibrin sealant containing a high FC concentration, but they did not fully differentiate into mature osteoblasts.
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PMID:Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro. 1696 77

This study was designed to investigate alterations in coagulation, and in biochemical and haematological parameters in cattle with traumatic reticuloperitonitis (TRP). In the study, 28 dairy cattle with TRP and 10 clinically healthy cattle (control) of different ages and breeds were used. Cattle with TRP had prolonged prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (APTT). Erythrocytopenia, thrombocytopenia and hyperfibrinogenaemia were detected in animals with TRP. Furthermore, the serum concentrations of total protein, globulin and total bilirubin, and the activities of alkaline phosphatase (ALP) and aspartate aminotransferase (AST) were also high in cattle with TRP compared to those of the control group. The serum concentrations of calcium were significantly low in the TRP group. The results of this study, therefore, indicate that TRP causes significant coagulation abnormalities and biochemical and haematological alterations in dairy cattle.
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PMID:Alterations in coagulation profiles and biochemical and haematological parameters in cattle with traumatic reticuloperitonitis. 1722 80

Ever-increasing evidence in the literature suggests that the antiinflammatory and cytoprotective properties of activated protein C (APC) are mediated through its endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on endothelial cells. However, recent results monitoring the cleavage rate of PAR-1 on human umbilical vein endothelial cells, transfected with an alkaline phosphatase-PAR-1 fusion reporter construct, have indicated that the catalytic activity of thrombin toward PAR-1 is several orders of magnitude higher than that of APC. Because thrombin is required for generation of APC, and because it also functions in the proinflammatory pathways through the activation of PAR-1, it has been difficult to understand how APC can elicit protective cellular responses through the activation of PAR-1 when thrombin is present. In this study we provide a plausible answer to this question by demonstrating that the critical receptors required for both protein C activation (thrombomodulin and EPCR) and APC cellular signaling (EPCR and PAR-1) pathways are colocalized in the membrane lipid rafts in endothelial cells. We further show that the APC cleavage of PAR-1 on cells transfected with a PAR-1 cleavage reporter construct is not sensitive to the cofactor function of EPCR. Thus, the colocalization of EPCR and PAR-1 in lipid rafts is a key requirement for the cellular signaling activity of APC. Thrombomodulin colocalization with these receptors on the same membrane microdomain can also recruit thrombin to activate the EPCR-bound protein C, thereby eliciting PAR-1 signaling events that are involved in the APC protective pathways.
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PMID:Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells. 1729 37

The cells responsible for bone formation express protease-activated receptors. Although serine protease thrombin has been shown to elicit functional responses in bone cells that impact on cell survival and alkaline phosphatase activity, nothing is known about tissue factor, factor VIIa, and factor Xa, the serine proteases that act upstream of thrombin in the coagulation cascade. This paper demonstrates that tissue factor is expressed in the osteoblast-like cell line SaOS-2 and, that tissue factor in a factor VIIa-bound complex induces a transient intracellular Ca(2+) increase through protease-activated receptor-2. In SaOS-2 cells, factor Xa induced a sustained intracellular Ca(2+) response, as does SLIGRL, a PAR2-activating peptide, and PAR-1-dependent cell viability.
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PMID:Osteosarcoma cell-calcium signaling through tissue factor-factor VIIa complex and factor Xa. 1750 70

Measurements of serum concentrations of des-gamma-carboxy-prothrombin (PIVKA-II) are widely used for diagnosing hepatocellular carcinoma (HCC). Recently, when we evaluated the correlation of PIVKA-II between two commercially available PIVKA-II immunoassay kits (Lumipulse f vs. Picolumi) to introduce it in our hospital, false high values of PIVKA-II were observed in Lumipulse assay. Four(4%) of 100 serum samples showed false high values, and all of them were obtained from patients less than 2 month after curative resection of HCC. Examining additional 7 patients with HCC resection, serum samples from the 5 patients had the same trend. To elucidate the non-specific reaction by Lumipulse assay which utilized alkaline phosphatase (ALP) enzymatic reaction, inhibition assays by various absorbents such as inactive ALP and IgM antibodies were performed. Excess of inactive ALP reduced the high values of PIVKA-II. Note that anti-bleeding sheets (fibrinogen combined drug), which included bovine thrombin, were directly attached on liver of all patients with HCC resection in this study. As the sheets also contaminate ALP and probably produce IgM antibodies to ALP, the IgM may cross-react with anti-PIVKA-II antibodies directly. Taken together, it was suggested that produced antibodies against ALP derived from anti-bleeding sheets led false high values of PIVKA-II in the patients with HCC resection.
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PMID:[False positive serum des-gamma-carboxy prothrombin after resection of hepatocellular carcinoma]. 1751 Dec 63

This study aimed to evaluate the effects of human platelet-rich plasma (hPRP) on the proliferation and osteogenic differentiation of human adipose-derived stromal cells (hADSCs) and to construct a novel injectable tissue-engineered bone (ITB) composed of hPRP and hADSCs. hADSCs were isolated from liposuction tissues of healthy patients. hPRP was obtained by traditional two-step centrifugation. MTT, alkaline phosphatase (ALP) activity and mineralization assays were used to evaluate the effects of different concentrations of hPRP on cell proliferation and osteogenic differentiation in vitro. hADSCs cultured in optimal concentration of activated hPRP were subcutaneously injected into the inguinal groove of nude mice with hPRP and thrombin. X-ray, H&E staining and immunohistochemical analysis were used to examine the bone formation. Studies in vitro revealed that cell proliferation, ALP activity and mineralization were induced by hPRP and 10-12.5% of hPRP seemed to be the optimal concentration. Studies in vivo showed that this ITB formed bone structure in heterotopic site of nude mice. These findings indicate that the ITB composed of hPRP and hADSCs may represent a prologue for the development of a novel biological solution to bone defect. However, further investigations should be done to fully reveal the characteristics of this ITB.
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PMID:Injectable tissue-engineered bone composed of human adipose-derived stromal cells and platelet-rich plasma. 1848 75

This study analyzed the ability of fibrin gels to deliver added recombinant transforming growth factor beta-1 (TGF-beta1) in a controlled manner and biologically active form. First, the effects of the amount of TGF-beta1 on the release kinetics were analyzed using a single fibrin gel formulation (fibrinogen complex (FC) at 25 mg/mL, thrombin at 2 IU/mL). Then, the effects of FC and thrombin concentrations were analyzed. Finally, to test the biological activity of the released TGF-beta1 from the gels, medium supernatants taken from gels at day 3 were used as culture medium for human mesenchymal stem cell (HMSC) monolayers. Cell proliferation was analyzed after staining with calcein dye, and changes in cell morphology were observed under fluorescence microscopy at days 1, 4, and 7. At day 7, HMSC chondrogenic differentiation was assessed by Alcian Blue staining and osteogenic differentiation by alkaline phosphatase activity and Alizarin Red staining. Results showed that TGF-beta1 added to fibrin gels was gradually released from the gels and increased with the amount of TGF-beta1 initially seeded, with a total of approximately 50% of the initial amount released by day 10 (with gels containing 25 mg/mL of FC and 2 IU/mL of thrombin). The release was lower with increasing FC concentrations, suggesting a binding affinity of TGF-beta1 with the FC component. Varying the thrombin concentration had a lesser effect. HMSC monolayers cultured with medium supernatants collected from gels at day 3 and containing released TGF-beta1 showed a change in morphology (squared to polygonal), lower cell proliferation, positive Alcian Blue staining but low levels of osteogenic differentiation markers. These results demonstrated that released TGF-beta1 was still bioactive and tended to induce mainly chondrogenic differentiation of the HMSC. Overall, the present study demonstrated that fibrin gels could be used as a carrier matrix for controlled release of bioactive TGF-beta1 by adjusting the concentrations of FC and thrombin in the gels.
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PMID:Controlled release of bioactive transforming growth factor beta-1 from fibrin gels in vitro. 1854 28

We propose a novel method to prepare a DNA-protein conjugate using histidine-tag (His-tag) chemistry. Oligo-DNA was modified with nitrilotriacetate (NTA), which has high affinity to a His-tag on recombinant protein via the complexation of Ni(2+). Investigations using a microplate which displayed a complementary DNA-strand revealed that a NTA-modified DNA-protein conjugate was formed and immobilized in the presence of Ni(2+) on the microplate. We then adopted alkaline phosphatase (AP) as a model protein, and application of the DNA-AP conjugate was demonstrated in a thrombin aptamer-based detection system with a detection limit of approximately 10 nM.
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PMID:Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system. 1860 79

An 80 years old male patient was admitted in our hospital with massive haematomas in the left forearm, chest and abdominal wall accompanied by intense back pain symptoms. Laboratory evaluation showed anemia, mild thrombocytopenia and elevated lactate dehydrogenase and alkaline phosphatase levels and normal concentrations of all the other biochemical parameters. Study of the coagulation status demonstrated prolonged thrombin time (TT), low fibrinogen levels--0.98 g/l while plasminogen, antithrombin III (AT III) and protein C levels were found to be within normal range. Computed tomography scans of the head, chest and abdomen showed an enlarged infiltrative prostate, osteolytic bone lesions in vertebras L5-S1 and a large haematoma of the abdominal wall as the only pathologic findings. Very high levels of the prostate specific antigen indicated the possible existence of a prostate carcinoma with metastases to the vertebral column that resulted in elevated alkaline phosphate and lactate dehydrogenase levels. There were no signs of liver involvement and impaired hepatic synthetic function. Based on the results of the laboratory tests we concluded that the cause of the bleeding disorder in our patient was an acquired hypofibrinogenemia, which is a very rare paraneoplastic phenomenon. The patient was treated with daily transfusions of cryoprecipitate with no long-term improvement. Then the specific anti-tumor therapy (ciproteron acetate) was initiated, and two weeks later, fibrinogen concentration and TT returned to normal values.
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PMID:Paraneoplastic bleeding disorder due to isolated hypofibrinogenemia: a case report. 1924 Aug 23


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