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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.
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.
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.
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.
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.
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.
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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.
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.
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.
Osteoblast differentiation is tightly regulated by a number of cytokines and growth factors, including bone morphogenetic proteins (BMP) which stimulate osteoblast differentiation by signal transduction via three BMP receptors (BMPR-IA, -IB and -II). Although the mechanisms which regulate osteoblast differentiation are not fully understood, it is possible that endogenous BMPR signaling could play an important part in this process. To test this hypothesis, we have examined the expression and the functional significance of BMPR during osteoblast differentiation of primary human bone cells. The results showed that although the expression of BMPR-IA and -II transcripts were constantly expressed while the bone cells underwent osteoblast differentiation, the level of BMPR-IB mRNA was transiently, but significantly, up-regulated by threefold on day 3. This increase in BMPR-IB expression was found to be associated with the significant up-regulation of core binding factor alpha 1 (Cbfa1) and alkaline phosphatase (ALP) transcripts as well as the ALP activity, the well-established early markers of osteoblast differentiation. Transfection of bone cells with BMPR-IB small interfering RNA (siRNA) was found to significantly ablate the expression of BMPR-IB which subsequently resulted in reduction of Cbfa1 and ALP mRNA as well as the ALP activity. Moreover, exogenously added BMP-2 failed to rescue osteoblast differentiation of BMPR-IB siRNA-transfected bone cells. In conclusion, the present study has shown that endogenous BMPR-IB signaling is required for early phase of osteoblast differentiation of human bone cells in vitro, suggesting that BMPR-IB could be a therapeutic target for initiating bone healing in vivo.
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 .
The purpose of this study was to investigate the effects of electromagnetic pulse (EMP) on bone formation of MC3T3-E1 cells. Proliferation, ALP activity and mineralized nodule formation were examined in MC3T3-E1 cells after EMP exposure (field intensities: 0kV/m, 50kV/m, 400kV/m, 400 pulses once a day for 7 days). After 50 kV/m EMP exposure, all the parameters of MC3T3-E1 cells were not changed significantly. However, the proliferation, ALP activity and mineralized nodule formation of MC3T3-E1 cells in 400 kV/m EMP exposure groups were decreased. In conclusion, the EMP we used was a possibly harmful factor for bone formation. It may suppress bone formation of MC3T3-E1 cells with higher electric field intensity.
Loss of teeth increases with age or after genotoxic treatments, like head and neck radiotherapy, due to periodontium breakdown. Periodontal ligament fibroblasts represent the main cell type in this tissue and are crucial for the maintenance of homeodynamics and for its regeneration. Here, we have studied the characteristics of human periodontal ligament fibroblasts (hPDLF) that became senescent after replicative exhaustion or after exposure to ionizing radiation, as well as their ability for osteoblastic differentiation. We found that senescent hPDLF express classical markers of senescence, as well as a catabolic phenotype, as shown by the decrease in collagen type I and the increase of MMP-2 expression. In addition, we observed a considerably decreased expression of the major transcription factor for osteoblastic differentiation, i.e. Runx2, a down-regulation which was found to be p53-dependent. In accordance to the above, senescent cells have a significantly decreased alkaline phosphatase gene expression and activity, as well as a reduced ability for osteoblastic differentiation, as found by Alizarin Red staining. Interestingly, cells from both type of senescence express similar characteristics, implying analogous functions in vivo. In conclusion, senescent hPDLF express a catabolic phenotype and express a significantly decreased ability towards an osteoblastic differentiation, thus probably affecting tissue development and integrity.
Iron overload has recently been connected with bone mineral density in osteoporosis. However, to date, the effect of iron overload on osteoblasts remains poorly understood. The purpose of this study is to examine osteoblast biological activity under iron overload. The osteoblast cells (hFOB1.19) were cultured in a medium supplemented with different concentrations (50, 100, and 200 μM) of ferric ammonium citrate as a donor of ferric ion. Intracellular iron was measured with a confocal laser scanning microscope. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescin diacetate fluorophotometry. Osteoblast biological activities were evaluated by measuring the activity of alkaline phosphatase (ALP) and mineralization function. Results indicated that iron overload could consequently increase intracellular iron concentration and intracellular ROS levels in a concentration-dependent manner. Additionally, ALP activity was suppressed, and a decline in the number of mineralized nodules was observed in in vitro cultured osteoblast cells. According to these results, it seems that iron overload probably inhibits osteoblast function through higher oxidative stress following increased intracellular iron concentrations.
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.
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.
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.
Optical laboratory-based immunoassays, such as enzyme-linked immunosorbent assay (ELISA) give a high sensitivity and specificity of various fatal diseases. However, these assays are no longer efficient in on-spot diagnostics of wide-spreading and contagious infections. At this point in time, portable and handhold devices play a pivotal role in infectious diseases with quick diagnostics at or near the site of the disease propagation. In this paper, we demonstrated a novel electrical immunoassay of ELISA that was not based on optical signaling but on electrical signaling. This was done by combining an ion-sensitive field-effect transistor (ISFET) with ELISA. By harnessing the catalytic reaction of alkaline phosphatase that precipitated silver particles, we effectively overcame the chronic Debye screening length issue of the ISFET. Ultimately, small signal ranging from 1pg/mL to 10ng/mL was immensely amplified with the ALP label, regardless of buffer conditions. The sensor platform herein surpassed a sensing capability of conventional ELISA that is considered to have a LOD on the order of ~1ng/mL. The results were compared with those of horseradish peroxidase label, which is generally used for optical analyses in ELISA. Our newly developed ISFET-based portable sensor holds a large potential for point-of-care tools in a variety of diseases, without being limited by the need for expensive equipment such as spectrophotometers.
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.
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