Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Osteoarthritic lesions were observed in the mandibular condyle cartilage of mice aged 7 months and older. These lesions appeared as fibrillations along the articular surface and were accompanied by reduced extracellular matrix synthesis and chondrocyte proliferation. Explants of mandibular condyle cartilage were cultured in serum-free BGJb medium supplemented with ascorbic acid (300 micrograms/ml), penicillin (100 U/ml) and streptomycin (100 micrograms/ml) for up to 72 h. Cultures were further supplemented with either hTGF-beta 1 (0.1-5.0 ng/ml) or human IL-1 alpha (40 U/ml). [3H]thymidine (2 microCi/ml) and [35S]SO4 (10 microCi/ml) were added to the culture medium for the last 24 h of culture and incorporation into DNA and sulfated proteoglycans, respectively, studied. The results indicated that protein and DNA contents, [3H]thymidine and [35S]SO4 incorporation, as well as the specific activity of alkaline phosphatase, were increased by TGF-beta 1. Addition of 1.0 or 5.0 ng/ml hTGF-beta 1 to the cultures for 48 h, had the most stimulatory effect on matrix synthesis and cell proliferation, whereas 0.1 ng/ml hTGF-beta 1 appeared to be inhibitory when compared to controls. Increased [35S]SO4 labeling of chondrocyte clusters was observed by autoradiography in tissue sections from cultures treated with TGF-beta 1 (1.0 and 5.0 ng/ml). In contrast, IL-1 alpha exerted inhibitory effects on cell proliferation and matrix synthesis. However, it induced the activity of acid phosphatase in these cultures. The results of the present study show that IL-1 alpha had catabolic effect on his tissue, while TGF-beta 1 enhanced proliferation and induced synthetic activity of the cartilage cells. Such action by TGF-beta suggests the existance of a possible repair process in osteoarthritic cartilage of the temporo-mandibular joint of aged mice.
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PMID:Effects of transforming growth factor-beta 1 and interleukin-1 alpha on matrix synthesis in osteoarthritic cartilage of the temporo-mandibular joint in aged mice. 915 64

The temporo-mandibular joint of aged mice develops osteoarthritic (OA) degenerative lesions. Adult chondrocytes have a low rate of cell replication, and cartilage repair potential is very limited. One of the major problems in OA is the low rate of matrix synthesis and the inability of the chondrocytes to exceed the rate of matrix degradation. These combined factors lead to the overall destruction of the cartilage as seen in OA. Cartilage degradation is mediated by elevated proteolytic activity of enzymes. Among the enzymes degrading cartilage are the metalloproteinases, stromelysin and collagenase. Other proteinases that may potentially participate in matrix degradation are the lysosomal enzymes cathepsin B, D, and L, and acid phosphatase. On the other hand, alkaline phosphatase (ALP) is an enzyme that has been shown to be a marker for anabolic activity in skeletal tissues such as bone and cartilage. The cartilage of the mandibular condyle in the T-M-J from aged mice reveals OA lesions. An overall reduction of cell proliferation and sulfated proteoglycan synthesis has been also shown in this joint. In the present study the effects of hTGF-beta on the stimulation of DNA and sulfated GAG synthesis and ALP activity were studied. Mandibular condyle cartilage obtained from 12-month-old ICR male mice were cultured in BGJb serum-free medium for 24-72 hours, supplemented with 0.1-10 ng/ml hTGF-beta 1. 3H-thymidine and 35S-sulfate were added for the last 24 hours of the culture and their incorporation into DNA and sulfated GAGs respectively, as well as the activity of ALP, were determined. Results indicated that hTGF-beta 1 enhanced the incorporation of both 3H-thymidine and of 35S-sulfate into cartilage cultures of aged mice, and also induced ALP activity. It thus appeared that in OA degenerating articular cartilage, the chondrocytes could be stimulated in vitro to proliferate and to synthesize new matrix, thus indicating induced anabolic activity in the tissue.
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PMID:Osteoarthritis in the temporo-mandibular joint (TMJ) of aged mice and the in vitro effect of TGF-beta 1 on cell proliferation, matrix synthesis, and alkaline phosphatase activity. 918 53

We have previously demonstrated that RGD peptides prevent tubular obstruction in ischemic acute renal failure (ARF) suggested that exposed unoccupied integrin receptors represent the target for such therapy. The present study investigated the topography of RGD binding sites and integrin receptors in ischemic rat kidneys. Two RGD peptides were synthesized: a cyclic biotinylated (Bt) RGD peptide and a linear RGD peptide (GRGDSP) labeled with rhodamine green (RhoG). Rats were subjected to 45 minutes of renal artery occlusion kidneys were harvested at different times post-ischemia, and stained with RGD peptides and a panel of antibodies to integrins. In control, Bt-RGD staining was undetectable in alkaline phosphatase histochemistry, whereas immunofluorescence detection with Rho-streptavidin conjugate as well as RhoG-GRGDSP staining faintly decorated the basolateral aspect of the proximal tubular cells in a punctate fashion. In contrast, ischemic kidneys showed binding to the basolateral and apical aspects of proximal tubules, peritubular capillaries, and desquamated cells within tubular lumen. The most conspicuous staining of ischemic kidneys was obtained with antibodies to the beta 1 (labeling of the apical aspect of proximal and distal tubules, as well as desquamated cells obstructing tubular lumen) and the alpha V (glomeruli, tubular epithelia, intima of blood vessels stained faintly, while the obstructing cellular conglomerates showed intense staining) subunits. Double staining with Bt-RGD and antibodies against the beta 1 and alpha V beta 3 integrins showed co-localization of staining within the tubules and vasculature, respectively. In vitro attachment of HL-60 leukocytes to the endothelial cells was inhibited by the cyclic RGD peptide. In conclusion, expression of RGD binding sites and beta 1 integrin subunits along the apical aspect of tubular epithelia and on the surface of desquamated cells is in concert with the hypothesis on the pathogenetic role of RGD-recognizing integrins in tubular obstruction. The expression of RGD binding sites along the intimal surface of blood vessels in ischemic kidneys suggests an additional target for RGD peptides in vascular endothelial cells.
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PMID:Two novel probes reveal tubular and vascular Arg-Gly-Asp (RGD) binding sites in the ischemic rat kidney. 921 51

Ipriflavone (i.p.) positively affects bone density in postmenopausal osteoporosis, primarily by inhibiting bone resorption. Using in vitro models of human osteoblast differentiation, we have observed that i.p. and some of its metabolites stimulate the expression of bone sialoprotein, decorin, and type I collagen, and facilitate the deposition of mineralized matrix. This suggests that i.p. may stimulate bone formation in addition to its antiresorptive activity. To assess whether these effects translate into an improved bone "quality" in vivo, we measured biomechanical properties, mineral composition, and crystallinity of femurs of 12-week-old, male, Sprague-Dawley rats treated with i.p. for 1 month. i.p. significantly decreased vibration damping, an index of strain energy loss. Because vibration damping increases as bone porosity increases, the results indicate that i.p.-treated bones acquired a higher capacity to withstand dynamic stress. In fact, 1.5-fold higher energy was required to fracture femurs of i.p.-treated rats after a single supramaximal impact. i.p. also increased BMD, assessed by both volume displacement and ash analysis, whereas the relative contents of Ca, P, and Mg in the ashes were not affected. Thus, no gross abnormalities in mineral composition of bone occurred after i.p. administration. As a measure of bone crystallinity, X-ray diffraction analysis was performed. The broadening parameter beta 1/2 for the (310) and (002) reflections was not significantly different between i.p.-treated and control animals. Similarly, there were no differences in serum levels of Ca, Mg, alkaline phosphatase, and type I collagen telopeptides between treated and control animals at the end of the study. Therefore, 1-month treatment with i.p. increased bone density and improved the biomechanical properties of adult male rat bones without altering mineral composition or bone crystallinity.
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PMID:In vitro and in vivo effects of ipriflavone on bone formation and bone biomechanics. 926 10

The mechanism of action of thyroid hormones on bone is poorly understood. Thyroid hormones may act on bone cells either indirectly by increasing secretion of growth hormone (GH) and insulin-like growth factor-1 (IGF-1), or directly by influencing target genes via specific nuclear receptors. The presence of thyroid hormone receptors (TRs) has been demonstrated in human and rodent osteoblast-like cells and cell lines and recently in osteoclasts derived from an osteoclastoma in vitro. However, their presence in human bone in situ has not been reported. We have used specific polyclonal antibodies to TR-alpha 1, -alpha 2, and -beta 1 to investigate the expression of these receptors in sections of human osteophytes and heterotopic bone. Osteoblasts and osteoclasts were identified by alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), respectively, whereas chondrocytes were identified morphologically. At sites of endochondral and intramembranous bone formation, TR-beta 1 and the splice variant -alpha 2 were widely expressed by proliferating, mature, and hypertrophic chondrocytes and also in cells within the fibrous tissue and at the bone forming surfaces, respectively. They were also detected in osteoblasts, osteoclasts, and a few osteocytes at sites of bone remodeling. In contrast, TR-alpha 1 was the least expressed and was present mainly in osteoblasts at remodeling sites and in a few mature and undifferentiated chondrocytes. Our results show, for the first time, the presence and distribution of TRs in human bone in situ and suggest that the skeletal actions of thyroid hormones may be mediated via these receptors. Further studies are required to define the role of the individual receptor isoforms in bone metabolism.
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PMID:The expression of thyroid hormone receptors in human bone. 926 88

Retinoic acid induces differentiation of preosteoblastic cells. We have demonstrated that osteoblastic differentiation and down-regulation of transforming growth factor (TGF)-beta receptors requires the interaction of type I collagen with alpha 2 beta 1 integrin (J Biol Chem 271: 3938-3944, 1996). The purpose of this study was to clarify the role of collagen in retinoic acid-induced differentiation and down-regulation of TGF-beta receptors using preosteoblastic RCT-1 cells. Retinoic acid enhanced the expression of alkaline phosphatase and type I collagen, and reduced TGF-beta receptors in these cells. Inhibiting collagen synthesis abolished these changes. Because TGF-beta inhibits osteoblastic differentiation, the changes described here may contribute to the osteoblastic differentiation by retinoic acid.
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PMID:Role of collagen in retinoic acid-induced differentiation and down-regulation of TGF-beta receptors in rat preosteoblastic RCT-1 cells. 927 12

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (delta 459-523) form that lacks the putative PKA Ser468 phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some straining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K+ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of -80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to -40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were -5.7 and -9.9 mV (slope factor) and -35 mV (half activation, V0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and -17.3 and -39.0 mV (V0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from -30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at -30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mM KCl was 1.3 +/- 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni2+ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 +/- 7.3% and 38.7 +/- 9.4% inhibition of the full-length and the truncated channels, respectively, 8-BrcAMP induced a 39.4 +/- 5.4% inhibition of the full-length channel but had no effect (8.6 +/- 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kv beta 1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kv beta 2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kv beta-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced.
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PMID:The sensitivity of the human Kv1.3 (hKv1.3) lymphocyte K+ channel to regulation by PKA and PKC is partially lost in HEK 293 host cells. 943 74

Twenty Polish Landrace gilts were grouped immediately after mating as follows: Experiment I-- Group 1 (5 gilts), control animals and Group 2 (5 gilts), injected i.m. with dexamethasone (30 mg/kg) at 12-h intervals from day 13 to day 22 of pregnancy; Experiment II--Group 3 (5 gilts), injected i.m. with corn oil from day 13 to day 22 of pregnancy and Group 4 (5 gilts), injected i.m. with hydrocortisone acetate (250 mg) at 12-h intervals from day 11 to day 20 of pregnancy. Gilts were placed in metabolic cages on day 7. On days 34-36 of pregnancy gilts were slaughtered and blood samples were collected. Serum was used for analysis of aspartate aminotransferase (S-ASAT), alanine aminotransferase (S-ALAT), alkaline phosphatase (S-ALP), S-cholesterol, S-triglycerides, S-fructosamine, S-urea, S-total protein, and for electrophoretic fractionation of serum proteins, corticosteroid-binding globulin (CBG), cortisol, progesterone, thyroxine (T4) and free T4. There were no significant differences between groups in embryonic survival or in number of viable fetuses after treatment with glucocorticoids. The activity of S-ALP was lower (p < 0.05) in Group 4 than in Group 3 (0.5 vs 1.2 mukat/l). Group 4 had higher (p < 0.05) levels of S-triglycerides (1.17 vs 0.73 mmol/l), S-cholesterol (5.4 vs 2.7 mmol/l), S-total protein (110.5 vs 93.3 g/l), S-albumin (56.3 vs 43.3 g/l) and alpha 2-globulin concentrations (18.0 vs 14.3 g/l) than Group 3. The hydrocortisone-treated gilts had lower (p < 0.05) CBG (6.8 vs 21.3 nmol/l) and beta 1-globulin (3.25 vs 5.0 g/l) concentrations than the oil-treated ones. Concentrations of T4 were lower (p < 0.05) in Groups 2 (61.3 nmo/l) and 4 (49.0 nmol/l) compared with control Groups 1 and 3 (88.2 and 97.0 nmol/l, respectively). Overall, the treatment of early pregnant gilts with hydrocortisone acetate resulted in decreased levels of S-ALP, CBG, beta 1-globulin and T4, and in increased levels of S-cholesterol, S-triglycerides, S-total protein, S-albumin and alpha 2-globulin. The only effect of dexamethasone was a lowering of T4. There were no differences in free T4, S-fructosamine or S-urea between controls and treatments. Furthermore a negative correlation between triglycerides concentrations and the number of embryos (r = -0.76, p < 0.05) was found in control untreated and oil-treated pregnant gilts.
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PMID:Effect of glucocorticoid treatment on biochemical and hormonal blood parameters in early pregnant gilts. 944 80

The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine alpha 8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine alpha 8 and beta1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified alpha 8 beta 1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface alpha 8 beta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands.
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PMID:Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C. 954 28

A partial structure of many glycoproteins, a glycosylated asparagine carrying a complex type undecasaccharide N-glycan (Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man alpha 1-3) [Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4) GlcNAc(beta 1-4)GlcNAc-Asn) was obtained by total synthesis. As a starting material served a chemically synthesized diantennary heptasaccharide azide which was deprotected in a three-step sequence in high yield. The reduction of the anomeric azide was accomplished with propanedithiol in methanol-ethyldiisopropylamine. Coupling of the glycosyl amine to an activated aspartic acid gave the benzyl protected asparagine conjugate. After removal of the six benzyl functions the resulting free heptasaccharide asparagine was elongated enzymatically in the oligosaccharide part. The use of beta-1,4-galactosyltransferase and alpha-2,6-sialytransferase in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor resulting in a full length N-glycan, a sialyated diantennary undecasaccharide-asparagine of the complex type.
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PMID:Chemoenzymatic synthesis of a sialylated diantennary N-glycan linked to asparagine. 964 61


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