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)

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.
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PMID:Electric chips for rapid detection and quantification of nucleic acids. 1468 37

A fully electrical array for voltammetric detection of redox molecules produced by enzyme-labeled affinity binding complexes is shown. The electronic detection is based on ultramicroelectrode arrays manufactured in silicon technology. The 200-microm circular array positions have 800-nm-wide interdigitated gold ultramicroelectrodes embedded in silicon dioxide. Immobilization of oligonucleotide capture probes onto the gold electrodes surfaces is accomplished via thiol-gold self-assembling. Spatial separation of probes at different array positions is controlled by polymeric rings around each array position. The affinity bound complexes are labeled with alkaline phosphatase, which converts the electrochemically inactive substrate 4-aminophenyl phosphate into the active 4-hydroxyaniline (HA). The nanoscaled electrodes are used to perform a sensitive detection of enzyme activity by signal enhancing redox recycling of HA resulting in local and position-specific current signals. Multiplexing and serial readout is realized using a CMOS ASIC module and a computer-controlled multichannel potentiostat. The principle of the silicon-based electrical biochip array is shown for different experimental setups and for the detection of virus DNA in real unpurified multiplex PCR samples. The fast and quantitative electronic multicomponent analysis for all kinds of affinity assays is robust and particle tolerant.
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PMID:Electrical detection of viral DNA using ultramicroelectrode arrays. 1475 Aug 64

Bioactive ceramics developed during the past few decades have interesting properties from the biological standpoint, but their effects on cellular events remain partially unknown. In the current work, we investigated cellular viability, proliferation, morphology changes and metabolic activity of rat primary culture osteoblasts in contact with the ionic products from the dissolution of a bioactive glass with 60% of silica (BG60S) and a biphasic calcium phosphate (BCP). We observed that although osteoblasts cultured with BG60S showed vacuole formation, cell viability was increased when compared to BCP and control. The vacuole formation was not due to the presence of high calcium concentration in the ionic products from the dissolution of BG60S and was not related to nitric oxide production from the osteoblasts. We did find that high silicon concentration could induce cellular vacuole formation. Additionally, energy dispersive spectroscopy analysis indicated that vacuole contained 75% more silicon than other regions in the cell outside the vacuole. We further found that collagen production was higher in osteoblast cultured in the presence of BG60S compared to BCP and control, while alkaline phosphatase production was similar among cells incubated with BG60S, BCP and control. Together, our results indicate that osteoblast vacuole formation was due to high silicon contents in the dissolution of BG60S and we can suggest that despite the vacuole formation, there is no significant alteration in the bioceramic cell interaction.
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PMID:The effect of ionic products from bioactive glass dissolution on osteoblast proliferation and collagen production. 1496 26

A factorial rat experiment using two dietary concentrations each of copper, zinc, and silicon was conducted to identify areas in which interrelationships involving silicon may exist. The concentrations used were (mg/kg of diet): copper, 1 and 5; zinc, 2 and 12; and silicon, 5 and 270. An antagonism between silicon and zinc, whereby increases in dietary levels of either one resulted in a reduction in blood plasma concentrations of the other, was demonstrated. The depressing effect of silicon on plasma concentrations of zinc and on alkaline phosphatase occurred only in zinc-deficient rats. However, silicon had no effect on growth. Effects on aortic composition, interpreted as beneficial, accompanied increases in the silicon content of copper-deficient diets. Silicon-dependent increases in the chloroform-methanol extractable fraction of aorta closely approximated a similar response to copper. High dietary silicon increased aortic elastin in copper-deficient rats when dietary zinc was adequate. The aortic effects of silicon, while mimicking the gross effects of copper, occurred in the absence of any silicon-related changes in blood copper concentrations. Interrelationships of silicon with other elements, particularly copper and zinc, may warrant consideration in future nutritional and metabolic studies.
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PMID:Interactive effects of dietary silicon, copper, and zinc in the rat. 1553 63

Bioactive glass (BG) is an effective synthetic bone graft material. BG granules of narrow size range (300-355 mum) have the ability to form new bone tissue inside excavations produced by in vivo resorption. Previously, we demonstrated that BG stimulates the differentiation of cultured osteoblast precursors if the glass surface was biomimetically modified by the formation of bone-like apatite and adsorption of serum proteins. We now report that modified BG can also increase the rate at which multipotential rat bone marrow stromal cells (rMSC) will undergo osteogenesis. BG promoted rMSC osteogenesis both when cells were plated in contact with BG and when cells were not directly in contact with the BG. Alkaline phosphatase activity, a marker of bone cell differentiation, was used as an indicator for osteogenesis. Alkaline phosphatase activity of rMSCs exposed to osteoinducers such as ascorbate, dexamethasone, and BMP-2 was enhanced in the presence of BG. The stimulatory effect of BG was more pronounced in rMSC cultures with low basal alkaline phosphatase activity than in those with higher activity. The enhanced differentiation of rMSCs was associated with both a change in rMSC morphology and altered chemical composition of the cell culture media. rMSCs cultured on BG in the presence of BMP or dexamethasone exhibited a more rounded osteoblast-like appearance as compared with cells grown on tissue culture plastic. In the presence of BG, elevated levels of calcium and silicon in the culture medium were observed throughout the 7-day culture period, suggesting a continuous dissolution of surface-modified BG and resulting release of BG dissolution products. The data suggest that both surface- and solution-mediated events play a role in the osteogenic effect of BG.
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PMID:Osteogenic effects of bioactive glass on bone marrow stromal cells. 1569 19

Osteogenic injectable bone substitutes may be useful for many applications. We developed a novel injectable bone substitute based on osteoblast-fibrin glue suspension and calcium phosphate bone cement (BC). Human osteoblasts were isolated from trabecular bone samples and cultured under standard conditions. Osteoblasts were suspended in fibrinogen solution (FS). BC was cured with thrombin solution. 8 x 4 mm injectable bone discs were prepared using silicon molds and a custom-made applicator device. Discs containing BC, BC/FS, or BC/FS/osteoblasts were implanted subcutaneously into athymic nude mice. After 3, 9 and 24 weeks, specimens were explanted and subjected to morphologic and biomechanical evaluation. In vitro fibrin gel-embedded osteoblasts displayed a differentiated phenotype as evidenced by alkaline phosphatase, collagen type 1 and von Kossa stains. A proportion of osteoblasts appeared morphologically intact over a 3-day in vitro period following application into the BC. BC/FS and BC/FS/osteoblast discs were sparsely infiltrated with vascularized connective tissue. There was no bone formation in implants from all groups. However, positive von Kossa staining only in BC/FS/osteoblast groups suggests engraftment of at least some of the transplanted cells. Biomechanical evaluation demonstrated initial stability of the composites. Young's modulus and maximal load did not differ significantly in the BC/FS and BC/FS/osteoblast groups. The practicability of osteoblast-containing injectable bone could be demonstrated. The dense microstructure and the suboptimal initial vascularization of the composites may explain the lack of bone formation. Modifications with regard to enhanced osteoblast survival are mandatory for a possible application as injectable osteogenic bone replacement system.
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PMID:Fibrin gel-immobilized primary osteoblasts in calcium phosphate bone cement: in vivo evaluation with regard to application as injectable biological bone substitute. 1604 62

A major goal of orthopedic biomaterials research is to design better surface chemistries and configurations to control behavior of bone cells such as osteoblasts. Nanostructured architecture significantly affects the response of several cell lines. In this work, nanostructured surfaces were prepared by vapor liquid solid growth of silicon nanowires from size-controlled gold colloid catalysts deposited on fused silica substrates. The lengths and surface densities of the nanowires were varied to assess the effect of these parameters on bone cell response. Osteoblasts were seeded on nanowire surfaces to investigate both short-term adhesion and proliferation and long-term functionality and matrix production. Cell adhesion and proliferation were characterized using a standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cell counting for up to 4 days of culture. The total protein content, alkaline phosphatase activity, and matrix production were quantified using standard colorimetric assays for up to 4 weeks of culture. Matrix production was also characterized by measuring surface concentrations of calcium and phosphorus using X-ray photoelectron spectroscopy. Further, scanning electron microscopy was used to investigate osteoblast morphology on nanostructured surfaces. Over the 4-week study, the nanostructured surfaces demonstrated improved osteoblast adhesion and proliferation and increased alkaline phosphatase activity and matrix production compared to non-nanostructured control surfaces.
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PMID:Nanostructured surfaces for bone biotemplating applications. 1651 43

We demonstrated a novel application of transient coulostatic pulse technique for the detection of label free DNA hybridization on nm-sized gold interdigitated ultramicroelectrode arrays (Au-IDA) made in silicon technology. The array consists of eight different positions with an Au-IDA pair at each position arranged on the Si-based Biochip. Immobilization of capture probes onto the Au-IDA was accomplished by self-assembling of thiol-modified oligonucleotides. Target hybridization was indicated by a change in the magnitude of the time dependant potential relaxation curve in presence of electroactive Fe(CN)(6)(3-) in the phosphate buffer solution. While complementary DNA hybridization showed 50% increase in the relaxation potential, the non-complementary DNA showed a negligible change. A constant behaviour was noted for all positions. The dsDNA specific intercalating molecule, methylene blue, was found to be enhancing the discrimination effect. The changes in the relaxation potential curves were further corroborated following the ELISA like experiments using ExtraAvidine alkaline phosphatase labelling and redox recycling of para-aminophenol phosphate at IDAs. The coulostatic pulse technique was shown to be useful for identifying DNA sequences from brain tumour gene CK20, human herpes simplex virus, cytomegalovirus, Epstein-Barr virus and M13 phage. Compared to the hybridization of short chain ONTs (27 mers), the hybridization of long chain M13 phage DNA showed three times higher increase in the relaxation curves. The method is fast enough to monitor hybridization interactions in milli or microsecond time scales and is well suitable for miniaturization and integration compared to the common impedance techniques for developing capacitative DNA sensors.
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PMID:DNA hybridization detection on electrical microarrays using coulostatic pulse technique. 1657 97

Arginine silicate inositol complex (ASIdagger; arginine 49.5%, silicon 8.2%, inositol 25%) is a novel material which is a bioavailable source of silicon and arginine. ASI offers potential benefits for vascular and bone health. Poor eggshell quality has been a major economic concern to commercial egg producers. Poor egg quality, skeletal abnormalities and architectural deterioration of bone tissue are common problems under hot conditions and in older birds. The effects of ASI supplementation on egg production, egg quality, levels of osteocalcin (OC) and bone mineral content were investigated in heat-stressed Japanese quail during the later part of the laying period. The birds were randomly assigned to six treatment groups consisting of six replicates of five birds each in a 2 x 3 factorial arrangement of treatments (temperatures, ASI levels). The birds were kept in wire cages in a temperature-controlled room at either 22 degrees C (TN) or 34 degrees C (HS) for 8 h/d and fed either a basal (control) diet or the basal diet supplemented with either 500 or 1000 g of ASI/kg. Heat exposure reduced egg production, egg quality and bone mineralisation when the basal diet was fed. ASI supplementation had no effect on feed intake or egg production under TN or HS conditions. However, ASI supplementation increased egg weight, shell thickness, shell weight and Haugh unit in both TN and HS groups during the late laying period. Bone mineral density (BMD) was significantly improved by ASI supplementation in both TN and HS groups. Serum osteocalcin (OC) concentrations and alkaline phosphatase (ALP) activity increased linearly with dietary ASI supplementation during the late laying period. The amount of calcium and phosphorus in the excreta decreased, while ash, mineral content, calcium and phosphorus concentrations in tibia increased in ASI-supplemented quail in both TN and HS groups during the late laying period. ASI supplementation significantly improved egg quality and bone mineralisation in quail during the late laying period and did not affect feed consumption or egg production.
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PMID:Dietary arginine silicate inositol complex during the late laying period of quail at different environmental temperatures. 1664 Oct 32

Calcium silicate ceramics have been proposed as new bone repair biomaterials, since they have proved to be bioactive, degradable, and biocompatible. Beta-tricalcium phosphate ceramic is a well-known degradable material for bone repair. This study compared the effects of CaSiO3 (alpha-, and beta-CaSiO3) and beta-Ca3(PO4)2 (beta-TCP) ceramics on the early stages of rat osteoblast-like cell attachment, proliferation, and differentiation. Osteoblast-like cells were cultured directly on CaSiO3 (alpha-, and beta-CaSiO3) and beta-TCP ceramics. Attachment of a greater number of cells was observed on CaSiO3 (alpha-, and beta-CaSiO3) ceramics compared with beta-TCP ceramics after incubation for 6 h. SEM observations showed an intimate contact between cells and the substrates, significant cells adhesion, and that the cells spread and grew on the surfaces of all the materials. In addition, the proliferation rate and alkaline phosphatase (ALP) activity of the cells on the CaSiO3 (alpha-, and beta-CaSiO3) ceramics were improved when compared with the beta-TCP ceramics. In the presence of CaSiO3, elevated levels of calcium and silicon in the culture medium were observed throughout the 7-day culture period. In conclusion, the results of the present study revealed that CaSiO3 ceramics showed greater ability to support cell attachment, proliferation, and differentiation than beta-TCP ceramic.
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PMID:Comparison of osteoblast-like cell responses to calcium silicate and tricalcium phosphate ceramics in vitro. 1676 35


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