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)

Bone matrix gelatin induces bone formation in muscle, and when implanted orthotopically it improves bone repair. Co-60 sterilization of bone gelatin impairs the protein-bound induction mechanisms. Gelatin samples nonirradiated or irradiated by 25 or 50 kGy were implanted into a pouch in the abdominal wall of Sprague-Dawley rats, as well as into a 7-mm calvarial defect. Evaluation was done by histologic studies, histomorphometry of orthotopic implants, and determination of alkaline phosphatase in ectopic implants. Gelatin irradiated with 50 kGy was absorbed in the muscle bed without evidence of any specific host reaction. Irradiation of 25 kGy led to histologically confirmed ectopic bone formation, but the wet weight of the explants was only half that of the nonirradiated control samples. Alkaline phosphatase activity was equal in both of these groups. With orthotopic implantation, neither a histologic nor a morphometric effect was seen with 25 kGy. Loss of osteoinduction with 25-kGy irradiation is apparently masked by osteoconductive mechanisms with orthotopic implantation.
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PMID:Irradiation-sterilization of rat bone matrix gelatin. 336 86

Abnormalities in extracellular matrix degradation may play a pathogenetic role in diabetic nephropathy. Cultured renal mesangial cells are known to synthesize increased amounts of matrix proteins when incubated in high glucose media (e.g., 30 mmol/l). However, the effect of glucose loading on degradative enzymes is unknown. Primary cultures of rat mesangial cells were grown until confluent in the presence of fetal calf serum (FCS) and insulin (0.67 U/ml). Cells were then cultured for 7 days in plastic wells in either 10 or 30 mmol/l glucose media containing neither FCS nor insulin. Collagenase activity in media were determined by zymography and quantitative spectrofluorometry. Cathepsin B and D activities in cell extracts were measured by spectrofluorometry (using the fluorescent substrate Z-Arg-Arg-7-amido-4-methylcoumarin) and 125I-labeled hemoglobin digestion, respectively. Gelatin-degrading activity of live mesangial cells was also determined. mRNA levels for collagenase IV, cathepsin B, and cathepsin D were determined by Northern analysis. A major band of collagenase activity with a molecular size of 72 kDa was observed in all mesangial cell media. Exposure of cells to high glucose media resulted in significant reductions in collagenase and cathepsin B activities as well as impairment in gelatin-degrading activity. Collagenase IV and cathepsin B and D mRNA levels were also decreased by glucose loading. To exclude the possibility that glucose loading was injurious to cells, 3H-leucine uptake (as a measure of protein synthesis) and membrane alkaline phosphatase activity (as a biochemical marker of viability) were not affected by the high glucose condition.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Decreased degradative enzymes in mesangial cells cultured in high glucose media. 762 99

This study investigated the temporary encapsulation of rat marrow stromal osteoblasts in surface crosslinked gelatin microparticles. Cells were encapsulated in uncrosslinked gelatin microparticles of average diameter of 630 microm containing approximately 53 cells. Gelatin microparticles were crosslinked to shell thicknesses of approximately 75 microm via exposure to 1 mM dithiobis(succinimidylpropionate) (DSP) solution for 15 min or 5 mm DSP solution for 5 min for the production of microparticles dispersing approximately 60 min after placement into a physiologic fluid at 37 degrees C. Formed microparticles were placed into culture wells at a cell seeding density of 5.3 x 10(4) cells/cm2 and, following the degradation and/or dissolution of gelatin, the cells were cultured in the presence of osteogenic supplements for 28 days. Samples were taken at specified time points and analyzed by a DNA assay for cell number and a 3H-thymidine incorporation assay for proliferative potential. Samples were also obtained and analyzed at several time points by alkaline phosphatase, osteocalcin, and mineralization assays for early and late phenotypic expression markers of osteoblastic differentiation. The measurements from the different assays for encapsulated cells (EC) in uncrosslinked and crosslinked gelatin microparticles were normalized with the cell numbers from the DNA assay and compared with those for nonencapsulated control cells. The results demonstrated that the marrow stromal cells survived the encapsulation procedure in uncrosslinked gelatin microparticles and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, although at a slightly lower level than the nonencapsulated cells. The results further showed that the marrow stromal cells survived the encapsulation in crosslinked gelatin microparticles prepared via exposure to 5mm DSP for 5 min and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, but at a slightly lower level than the EC in uncrosslinked gelatin microparticles. In contrast, exposure to 1 mM DSP for 15 min led to severely limited cell viability and phenotypic expression probably due to the increased crosslinking time. These results suggest that temporary encapsulation of cells in gelatin microparticles may protect cells from short-term environmental effects such as those associated with the crosslinking of an injectable polymeric carrier for bone tissue engineering.
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PMID:Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 1. Encapsulation of marrow stromal osteoblasts in surface crosslinked gelatin microparticles. 1221 26

Elastin degeneration and calcification occur in many cardiovascular diseases, including medial arterial elastocalcinosis, atherosclerosis, and bioprosthetic heart valve mineralization. In the present study, we tested the hypothesis that the onset and progression of elastin-oriented calcification is associated with matrix remodeling and elastin degradation events. We studied whether aluminum ions inhibit elastin calcification by reducing elastin degradation and altering remodeling events. Subdermal implantation of pure elastin in juvenile rats resulted in a time-dependent calcification of elastin, reaching high levels 21 days after implantation. In situ hybridization showed that elastin calcification was associated with an up-regulation of matrix metalloproteinase (MMP) mRNA expression, specifically MMP-9 and MMP-2. Gelatin zymography demonstrated increased MMP-9 and MMP-2 enzyme activities in early stages of elastin calcification. Calcified elastin displayed a time-dependent pattern of tenascin-C (TN-C) and alkaline phosphatase (AP) expression. Pretreatment of pure elastin with aluminum ions prior to implantation resulted in complete inhibition of elastin calcification. Aluminum ion binding to elastin was found to protect elastin against MMP-mediated degradation in vitro. Noncalcified, explanted aluminum-pretreated elastin exhibited reduced activities of MMPs. TN-C expression in elastin implants exhibited a time-dependent pattern that was also affected by pretreatment of elastin with aluminum ions. In conclusion, elastin calcification is accompanied by matrix remodeling events, and the efficacy of aluminum pretreatment in inhibiting elastin calcification may be related in part to its effects on elastin remodeling.
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PMID:Involvement of matrix metalloproteinases and tenascin-C in elastin calcification. 1508 71

The objective of this study is to enhance in vivo ectopic bone formation by combination of plasmid DNA impregnation into three-dimensional (3-D) cell scaffolds and a developed in vitro culture method. Gelatin was cationized by introducing spermine (Sm) to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, collagen sponge reinforced by incorporation of poly(glycolic acid) (PGA) fibers was used. A complex of the cationized gelatin and plasmid DNA of BMP-2 was impregnated into the scaffold. MCS were seeded into each scaffold and cultured by a static and perfusion methods. When MSC were cultured in the PGA-reinforced collagen sponge, the level of BMP-2 expression was significantly enhanced by the perfusion culture compared with static method. When the osteoinduction activity of the PGA-reinforced collagen sponges seeded with PBS, MSC, naked plasmid DNA-BMP-2, cationized gelatin-plasmid DNA-BMP-2 complex, and transfected MSC by static and perfusion method, were studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, homogeneous bone formation was histologically observed throughout the sponges seeded with cationized gelatin-plasmid DNA of BMP-2 complex and transfected MSC by perfusion method, although the extent of bone formation was higher for the later one. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges seeded with transfected MSC by perfusion method were significantly high compared with those seeded with other agents. We conclude that combination of plasmid DNA-impregnated PGA-reinforced collagen sponge and the perfusion method was promising to promote the in vitro gene expression for MSC and in vivo ectopic bone formation.
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PMID:Enhanced ectopic bone formation using a combination of plasmid DNA impregnation into 3-D scaffold and bioreactor perfusion culture. 1613 84

This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.
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PMID:Impregnation of plasmid DNA into three-dimensional scaffolds and medium perfusion enhance in vitro DNA expression of mesenchymal stem cells. 1625 1

Calcification of vascular elastin occurs in patients with arteriosclerosis, renal failure, diabetes, and vascular graft implants. We hypothesized that pathological elastin calcification is related to degenerative and osteogenic mechanisms. To test this hypothesis, the temporal expression of genes and proteins associated with elastin degradation and osteogenesis was examined in the rat subdermal calcification model by quantitative real-time reverse transcription-polymerase chain reaction and specific protein assays. Purified elastin implanted subdermally in juvenile rats exhibited progressive calcification in a time-dependent manner along with fibroblast and macrophage infiltration. Reverse transcription-polymerase chain reaction analysis showed that relative gene expression levels of matrix metalloproteinases (MMP-2 and MMP-9) and transforming growth factor-beta1 were increased in parallel with calcification. Gelatin zymography showed strong MMP activities at early time points, which were associated with high levels of soluble elastin peptides. Gene expression of core binding factor alpha-1, an osteoblast-specific transcription factor, increased in parallel with elastin calcification and attained approximately 9.5-fold higher expression at 21 days compared to 3 days after implantation. Similarly, mRNA levels of the bone markers osteopontin and alkaline phosphatase also increased progressively, but osteocalcin levels remained unchanged. We conclude that degenerative and osteogenic processes may be involved in elastin calcification.
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PMID:Elastin calcification in the rat subdermal model is accompanied by up-regulation of degradative and osteogenic cellular responses. 1643 63

Changes in the functional and plasma membrane organizational states of human neutrophils were examined using two isolation procedures, which may simulate altered physiological states in vivo. A gelatin-based method of blood-neutrophil isolation was used to model in vivo priming, and neutrophils isolated by this method were compared with control populations prepared by a pyrogen-free, dextran-based method. Gelatin-prepared neutrophils were functionally primed for adherence and agonist-stimulated superoxide generation relative to unprimed, control neutrophils. The organizational state of the membrane cortex was examined by mapping the subcellular distribution of select cortical and transmembrane proteins by several methods, including subcellular fractionation, indirect immunofluorescence, and compositional analysis of Triton X-100-insoluble membrane skeleton preparations. Filamentous actin, fodrin, and the fodrin anchor, CD45, were largely cytoplasmic in unprimed neutrophils but translocated to plasma membranes upon priming, whereas CD43 and ezrin were exclusively surface-associated in both populations. Isopycnic sucrose density gradient analysis of N(2)-cavitated neutrophils revealed a major shift in the distribution of surface-associated transmembrane and membrane cortical components relative to the plasma membrane marker alkaline phosphatase in primed but not unprimed neutrophils. Similar results were obtained after neutrophil stimulation with known priming agents, LPS, TNF-alpha, or GM-CSF. Together, these results may suggest that priming of suspended, circulating neutrophils is associated with a large-scale reorganization of the plasma membrane and associated membrane cortex in a process that is independent of cellular adhesion and gross morphologic polarization.
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PMID:Reorganization of the human neutrophil plasma membrane is associated with functional priming: implications for neutrophil preparations. 2935 Aug 10

We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC.
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PMID:Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation. 1732 18

This study evaluated whether the murine leukemia virus (MLV)-based cyclooxygenase-2 (Cox-2) ex vivo gene-transfer strategy promotes healing of calvarial defects and/or synergistically enhances bone morphogenetic protein (BMP) 4-mediated bone regeneration. Gelatin scaffolds impregnated with mouse marrow stromal cells (MSCs) transduced with MLV-expressing BMP4, Cox-2, or a control gene were implanted into mouse calvarial defects. Bone regeneration was assessed by X-ray, dual-energy X-ray absorptiometry, and histology. In vitro, Cox-2 or prostanglandin E(2) enhanced synergistically the osteoblastic differentiation action of BMP4 in mouse MSCs. In vivo, implantation of BMP4-expressing MSCs yielded massive bone regeneration in calvarial defects after 2 weeks, but the Cox-2 strategy surprisingly did not promote bone regeneration even after 4 weeks. Staining for alkaline phosphatase (ALP)-expressing osteoblasts was strong throughout the defect of animals receiving BMP2/4-expressing cells, but defects receiving Cox-2-expressing cells displayed weak ALP staining along the edge of original intact bone, indicating that the Cox-2 strategy lacked bone-regeneration effects. The Cox-2 strategy not only lacked bone-regeneration effects but also suppressed the BMP4-induced bone regeneration. In vitro coculture of Cox-2-expressing MSCs with BMP4-expressing MSCs in gelatin scaffolds reduced BMP4 mRNA transcript levels, suggesting that Cox-2 may promote BMP4 gene silencing in BMP4-expressing cells, which may play a role in the suppressive action of Cox-2 on BMP4-mediated bone formation. In summary, the Cox-2 ex vivo gene-transfer strategy not only lacks bone-regeneration effects but also suppresses the bone-regeneration action of BMP4 in healing of calvarial defects.
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PMID:Marrow stromal cell-based cyclooxygenase 2 ex vivo gene-transfer strategy surprisingly lacks bone-regeneration effects and suppresses the bone-regeneration action of bone morphogenetic protein 4 in a mouse critical-sized calvarial defect model. 1976 74


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