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Phosphate triester derivatives of the anti-neoplastic alkyl lyso phospholipid (ALP) have been prepared as novel potential therapeutic agents. In particular, symmetrical phosphate triesters have been prepared, using phosphorochloridate chemistry. The compounds have been fully characterised by a range of techniques, and assayed for their inhibition of DNA synthesis by mammalian cells in culture. The compounds are generally inhibitory towards DNA synthesis in the μM range. However, the magnitude of the effect varies greatly with the phosphate structure; alkynyl and glycol substituted phosphates being especially potent.
The effects of 24R,25-dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ] on alkaline phosphatase activity (ALP) were evaluated in pig kidney LLC-PK1 cells in culture.The vitamin D 3 metabolite increased ALP activity in these cells, whereas no effect of the hormone was observed on γ-glutamyltranspeptidase and acid phosphatase activities.ALP activity was stimulated after 3- to 12-hr incubation in the presence of 10 - 9 mol/l 24,25(OH) 2 D 3 with a maximum after 6 hr.The hormonal induction of ALP activity was prevented by pretreatment of cells by actinomycin D.It is proposed that 24,25(OH) 2 D 3 could increase ALP activity by de novo protein synthesis.
A detection of alkaline phosphatase (ALP, EC activity by the monitoring of pH changes caused by the biocatalytic action of the enzyme has been experimentally examined. Enzymatically catalyzed hydrolysis of monofluorophosphate has been found to be the best basis for such measurements. Protolytic equilibria connected with the developed biosensing system were recognized and the optimal conditions for the assay have been found. Advantages and disadvantages of the developed (bio)sensing scheme have been discussed. The prototype of pH-ALP based enzyme electrode has been demonstrated. Potential utility of such substrate–enzyme–sensor system for the development of a new group of biosensors has been announced.
In this study, we have found that Escherichia coli lipopolysaccharide (LPS) could induce the expression of pentraxin 3 (PTX3) mRNA in osteoblast-like cells (MC 3T3-E1) dose dependently. The relation between this expression and alkaline phosphatase (ALP), mitogen-activated protein kinases (MAPKs), and nuclear factor kappa B (NF-κB) was also analyzed. The results show that the mRNA expression of PTX3 was related to that of ALP. It was also related to the mRNA expression of ERK-1 and p38 but not JNK-1 and NF-κB. These results suggest that ERK-1 and p38 are involved in the regulation of PTX3 expression and PTX3 promotes the differentiation of MC 3T3-E1 cells in response to LPS.
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A one-step carbodiimide method was found to allow covalent binding of enzymes to the inner wall of poly(vinyl chloride) (PVC) tubing. The immobilization is performed under mild conditions without laborious pretreatment or activation of reactor surface. In these preliminary studies, alkaline phosphatase (ALP, EC and p-nitrophenyl phosphate (NPP) were applied as a model enzyme and substrate, respectively. The resulting open-tubular bioreactor exhibits satisfactory operational and storage stability. In addition, a novel and very simple instrumental concept for optical monitoring of the biocatalytic process directly inside the microbioreactor using a system of paired emitter–detector diodes is presented.
Earlier reports have shown that ALP has an internal interaction site. We were able to stablize the structure of this unfolded part to a great extent by aspartic acid, which allowed the backbone assignment. No secondary structure of the polypeptide was observed.
A number of physical processes show some form of bifurcation or periodic splintering of single distributions into two new ones. Recently, it has been noted that cavity searches for interactions between photons and exotic fields may also result in bifurcation [1]. This paper builds on previous simulations of bifurcation of an optical beam in the presence of periodic focusing [2]. Here, however, the focus is on predicting a shifting of the beam's position, defined by the resulting center of the energy density. Mathematical models are described and the formalism for simulating bifurcation under complex conditions is delineated.
This study examined whether the alkaline phosphatase of human gingival fibroblast is enhanced by recombinant human BMP-4. Gingival fibroblast was obtained from the excised gingival tissue of an implant patient undergoing gingival surgery. The tissue was incubated at 37° in 5% CO2 and 95% humidity, and the cultivating media was changed every 2 days. The 2nd passage gingival fibroblasts were cultured in a medium containing Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 1 X antibiotic antimycotic solution. The control gingival fibroblast was cultured without rhBMP-4. The experimental group 1 were cultured with low-dose rhBMP-4. The experimental group 2 were cultured with high-dose rhBMP-4. This study evaluated the effect of rhBMP-4 on alkaline phosphatase expression of gingival fibroblast using alkaline phosphatase assay. In the experimental group 2 with low dose rhBMP-4, the gingival fibroblast showed abundant positive alkaline phosphatase staining. In the control group, the gingival fibroblast showed weak positive alkaline phosphatase staining. Overall, these results suggest that the alkaline phosphatase expression of human gingival fibroblast can enhanced by low dose rhBMP-4.
W przypadku wykorzystania hydrożeli w inżynierii tkanki kostnej wskazane jest takie ich zmodyfikowanie aby zawierały w swoim składzie składniki mineralne i/lub mogły ulegać samoistnej mineralizacji. W niniejszej pracy podjęto próbę wytworzenia kompozytów hydrożelowych z gumy gellan i różnej zawartości (0,75%, 1,5%, 3%) szkła bioaktywnego (BG) uzyskanego uprzednio metodą zol-żel. Kompozyty dodatkowo wzbogacono w enzym sprzyjający mineralizacji (fosfataza alkaliczna - ALP) oraz poddawano je inkubacji w roztworze glicerofosforanu wapnia (CaGP) przez czas 1, 3 i 7 dni. Oceniono właściwości mechaniczne kompozytów w próbie ściskania oraz dokonano pomiarów ich masy po wysuszeniu, które świadczyły o obecności BG oraz tworzeniu się fazy mineralnej. Morfologię próbek po wysuszeniu analizowano za pomocą mikroskopu stereoskopowego zaś ich mikrostrukturę za pomocą mikroskopu skaningowego elektronowego (SEM). Przeprowadzono też badania za pomocą mikroanalizy rentgenowskiej (EDS). Badania wykazały, że w kompozytach bez dodatku ALP mineralizacja nie zachodziła lub zachodziła w bardzo niewielkim stopniu. Obserwacje SEM i analiza EDS wykazały tworzenie się fosforanów wapnia w próbkach kompozytowych zawierających ALP po inkubacji w CaGP. Najkorzystniejsze właściwości mecha¬niczne wykazywały kompozyty wzbogacone w ALP i zawierające najmniejBG (0.75%) poddane 7-dniowej inkubacji w CaGP.
A human osteoblastic cell line (SV-HFO) established in our laboratory expresses osteoblastic markers, including mineralization in vitro, in response to differentiation-inducing agents such as dexamethasone. In this study, we examined the effects of transforming growth factor β 1 (TGF-β 1 ) on the mineralization of SV-HFO cells and show that TGF-β 1 inhibited the mineralization of the cells via down regulation of tetranectin and alkaline phosphatase without influencing other osteoblastic markers. To examine precisely the effects of TGF-β 1 on the process of mineralization, we tentatively divided the whole process of mineralization into four phases: induced ALP activity (days 0-5), maximal ALP activity (days 5-10), early mineralization (days 10-15), and progressive mineralization (days 15-20). These inhibitory effects of TGF-β 1 on the expression of tetranectin and alkaline phosphatase, like that on mineralization, were observed only when TGF-β 1 was applied in the early phase of the process of mineralization. On the other hand, the other osteoblastic markers were not influenced by treatment with TGF-β 1 . These results suggest that TGF-β 1 may inhibit mineralization of osteoblasts by the downregulation of tetranectin and alkaline phosphatase expression in the early phase. Thus, TGF-β 1 has phase-dependent effects on a human osteoblastic cell line during the process of mineralization.
We fabricated an on-chip capillary electrophoresis device for blood analysis. An on-chip capillary electrophoresis device was photolithographically fabricated on a glass chip. Alkaline phosphatase (ALP) was employed as a sample enzyme. Small amounts of enzyme in the mixture of other proteins were detected with the electrophoretically mediated microanalysis (EMMA) method. Fluorescein diphosphate was used as fluorogenic substrate. The detection of ALP activity was achieved with laser-induced fluorescence monitoring fluorescein that was produced in enzyme reaction in capillary. Several methods to reduce the adhesion of protein are also discussed.
The aluminium-induced neurotoxic consequences include, among other factors, dephosphorylation, phosphorylation as well as hyperphosphorylation of specific macromolecules. Accordingly, activities of phosphoesterases were measured in different regions of rat brain, maintained with either adequate or inadequate protein diet, following aluminium exposure. Male Wistar rats weighing 80-100 g were treated with aluminium chloride at a dose of 15% of the LD 5 0 for 4 weeks. In different regions of the brain of aluminium-exposed rats, significant variation in both phosphomonoesterase and phosphodiesterase activities have been recorded. These alterations were found to be varied when the rats were subjected to dietary protein insufficiency. These findings demonstrate the specificity of aluminium on different phosphoesterases. These regional variations may be attributed to the accumulated level of aluminium or may be due to cellular localization of these enzymes and linked to whether the enzymes are compartmentalized with different aluminium hydration species.
Enzymatic hydrolysis of p-nitrophenylphosphate by alkaline phosphatase in binary mixtures of water and 1-ethyl-3-methylimidazolium tetrafluoroborate (emimBF 4 ) was monitored with Raman microspectroscopy. Concentrations of emimBF 4 in the studied ionic liquid/water solvent systems ranged from 0 to 75% v/v. Multivariate curve resolution-alternating least squares (MCR-ALS) was successfully applied to the recorded Raman spectra in order to retrieve the concentration profiles and pure Raman spectra of the different species involved in the reaction. Michaelis–Menten constant (K M ) and maximum rate (V max ) of the reaction were calculated from the initial reaction phase for the different solvent systems studied, to investigate the effect of increasing concentration of the ionic liquid on the kinetic behavior. From this study, it was found that the ionic liquid inhibits the reaction under study decreasing both V max and K M .
Preparation, characterization and cellular biocompatibility study of a series of calcium polyphosphate containing 0–100mol% of Ca 2+ replaced by Sr 2+ were reported. The osteoblastic ROS17/2.8 cell line was used and seeded on the strontium-doped calcium polyphosphate (SCPP) scaffolds to estimate its optimal dose and to study its potential to support the growth of osteoblastic cells for bone tissue engineering. The effects of SCPP on cells’ proliferation and differentiation were evaluated by MTT and ALP activity assay. The results showed that porous SCPP did not exert cytotoxic effect on the cells. In addition, the proliferation and differentiation of the growth of ROS17/2.8 cells on the SCPP containing a low dose of strontium showed a higher level compared to the control, and the SCPP containing 1% strontium was optimal according to the results of MTT and ALP activity assay. The cells on the porous SCPP formed a continuous layer on the outer and inner surface observed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The bunchy collagens were excreted from the cells and the calcium granules wrapped by collagens were sedimentated on the surface of cells. The results suggested that the biodegradable SCPP could stimulate the proliferation and differentiation of ROS17/2.8 cells in vitro after addition of proper dose of strontium. The porous SCPP may be a promising material for the bone tissue engineering.
The actinin-associated LIM protein (ALP) is co-localized with α-actinin at the Z-discs and plays a critical role in the integration of cytoskeletal architecture and transcriptional regulation. Here we report that five isoforms of the porcine ALP were generated in skeletal muscle by alternative splicing. All of ALP isoforms were predominantly expressed in skeletal muscle except for ALP2. These isoforms had different expression profiles during the prenatal and postnatal period of the porcine skeletal muscle development and between the two breeds. Moreover, ALP1 and ALP3 were expressed at higher levels in soleus and masseter muscles compared with longissimus dorsi and bicepsfemoris muscles in Yorkshire pigs. Expression analysis in porcine satellite cells showed that all isoforms were induced in differentiated porcine satellite cells, suggesting a role of them in myogenic differentiation. These results provide new insight into roles of regulation at level of splicing of the ALP in governing porcine skeletal muscle development.
Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis.
Polymer sponge replication method was used in this study to prepare the macroporous hydroxyapatite scaffolds with interconnected oval shaped pores of 100–300μm with pore wall thickness of ∼50μm. The compression strength of 60wt.% HA loaded scaffold was 1.3MPa. The biological response of the scaffold was investigated using human osteoblast like SaOS2 cells. The results showed that SaOS2 cells were able to adhere, proliferate and migrate into pores of scaffold. Furthermore, the cell viability was found to increase on porous scaffold compared to dense HA. The expression of alkaline phosphate, a differentiation marker for SaOS2 cells was enhanced as compared to nonporous HA disc with respect to number of days of culture. The enhanced cellular functionality and the ability to support osteoblast differentiation for porous scaffolds in comparison to dense HA has been explained in terms of higher protein absorption on porous scaffold.
Arsenic trioxide (ATO) is widely used in tumor treatment, but excessive arsenic exposure can have adverse health effects. This study was to examine the association between ATO treatment and bone remodeling. The effects of ATO on osteoblast function were investigated in primary cell cultures and in an in vivo study in rats. Sprague–Dawley rats (n=30) were randomly assigned to 3 groups which were injected intraperitoneally with saline or 5 or 10mg/kg of ATO for 4weeks. In cell culture, ATO decreased osteoblast mineralization by decreasing alkaline phosphatase (ALP) expression and this effect was prevented by co-addition of inorganic phosphate (Pi). Moreover, levels of mRNAs for the transcription factors runt-related transcription factor 2 (Runx2) and osterix, the osteoblast osteogenic gene osteocalcin, and the adherence molecule vascular cell adhesion molecule-1 (VCAM-1) were decreased by ATO. Levels of mRNAs for the cytokine IL-6 were also decreased, whereas GM-CSF mRNA levels were increased. Similar effects of ATO on osteoblasts were seen in in vivo experiments in the rat. Moreover, decreases of bone turnover markers of osteocalcin, Procollagen type I N-terminal propeptide (PINP), and C-terminal cross-linked telopeptide (CTX) as well as bone mineral density (BMD) and trabecular bone volume of femur were observed in ATO-treated rats. These results suggest that ATO interferes with bone remodeling mostly through changes in osteoblast differentiation and function.
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In traditional Chinese medicine, the cause of weak bones or bone loss is generally regarded as a result of kidney deficiency. Fructus Cnidii (FC), which is also known as She-Chuang-Zi, is a traditional herb that has been claimed to have kidney warming effects that invigorate Yang. In this study, we tried to determine the bone production-inducing effect of FC on osteoblastic cells in vitro using osthole, the main component of FC. Osteoblasts were isolated from neonatal Sprague-Dawley rat calvaria using the tissue piece culture method and treated with various concentrations of osthole ranging from 2.5 to 640 μg/mL, together with a blank control. Cell proliferation, alkaline phosphatase (ALP) activity, and bone nodules were measured. The cells were examined by hematoxylin-eosin staining, the Gomori Calcium-Cobalt method and immunofluorescent staining. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (or MTT) assay, ALP assay, and bone nodule results indicated significantly enhanced osteoblastic proliferation and differentiation at concentrations of osthole ranging from 40 to 320 μg/mL. Concentrations lower than 40 μg/ mL seemed less effective, and cytotoxicity to osteoblasts was observed at concentrations higher than 320 μg/mL. These results indicate that osthole is effective at inducing osteoblastic bone formation through the up-regulation of ALP activity. FC is a Chinese herb used to treat lumbar pain in clinical practice. Further studies concerning the effects and mechanism of osthole on osteoporotic patients and animals should be performed, as these studies may lead to the development of a drug treatment for osteoporosis in the future.
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