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)

The influence of the primary antibody, the fixative, and the antigen unmasking technique on the method sensitivity of immunohistochemistry as a method for the identification of viral hemorrhagic septicemia (VHS) virus in paraffin-embedded specimens of naturally infected rainbow trout (Oncorhynchus mykiss) was examined. Fish (200-300 g) were collected during an outbreak of VHS. Parallel specimens from liver, spleen, kidney, and brain were fixed by immersion in 10% phosphate-buffered formalin, periodate-lysine-paraformaldehyde (PLP), Bouin's fluid, or absolute ethanol. Virus cultivation was also performed on parallel specimens, and the virus titer (TCID50/ml) was determined. Purified nucleocapsid protein (N-protein) of the virus was incorporated in an artificial antigen substrate polymerized bovine serum albumin), fixed as described above, and embedded in paraffin wax. Microwave unmasking was performed on formalin-, PLP-, and Bouin's fluid-fixed specimens. The presence of virus peptides in situ or N-protein in the artificial antigen substrates was visualized using an immunohistochemical method based on alkaline phosphatase or peroxidase and one polyclonal and five monoclonal polypeptide-specific antibodies. VHS virus was identified in situ in specimens with high virus titers (10(7-8) TCID50/ml) regardless of the fixative and without the need of an unmasking procedure. A pronounced masking effect was observed for the cross-linking formalin and PLP fixatives. Regardless of the primary antibodies used, there was a significantly higher epidemiologic sensitivity (the proportion of virus positive samples that tested positive by immunohistochemistry) using ethanol and Bouin's fluid compared with formalin and PLP (P < 0.05). At 10(5) TCID50/ml, the average sensitivity reached 0.5, and at > or = 10(6) TCID50/ml, sensitivity was 0.9. Unmasking procedures showed a moderate effect and did not result in significantly higher epidemiologic sensitivity (P = 0.17), There was great variation for the different monoclonal antibodies/antigens and fixatives. Sensitivity studies on antigen substrates were in accordance with results of in situ studies that showed the highest sensitivity for ethanol and Bouin's fluid. Virus cultivation was more sensitive than immunohistochemistry. This study showed that the fixative and the primary antibody both influence method sensitivity and that VHS virus antigens concealed during fixation are difficult to reexpose. Immunostaining for VHS virus should be performed with monoclonal antibodies specific for the N-protein, and tissue samples should be fixed in either ethanol or Bouin's fluid. Immunohistochemistry is specific but is less sensitive than virus cultivation. Immunostaining for VHS virus can be a valuable supplement to virus cultivation during acute outbreaks of disease.
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PMID:Immunohistochemical detection of VHS virus in paraffin-embedded specimens of rainbow trout (Oncorhynchus mykiss): the influence of primary antibody, fixative, and antigen unmasking on method sensitivity. 916 87

An Escherichia coli expression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA-hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated by in vitro replacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein in E. coli periplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed in E. coli using the PhoA signal sequence for protein export.
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PMID:Amino-terminal charge affects the periplasmic accumulation of recombinant heregulin/EGF hybrids exported using the Escherichia coli alkaline phosphatase signal sequence. 926 80

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress.
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PMID:Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells. 946 58

Recently, a new class of biodegradable PHB-based polyesterurethane (DegraPol/btc) has been prepared and found to exhibit favorable cell and tissue compatibility. The present study has been designed to evaluate the response of primary isolated rat tibia osteoblasts to small crystalline particles of short-chain poly[(R)-3-hydroxybutyric acid] (PHB-P diameter: 2-20 microm), of fluorescent-labeled analogs (DPHP-P), and of lysine methyl ester as possible degradation products of DegraPol/btc. Observations made using confocal microscopy clearly indicate that osteoblasts have the capability of taking up PHB-P particles. Although in single-cell analysis the number of DPHB-P-positive osteoblasts gradually increased up to 16 days, the fluorescence intensity per osteoblast increased only during the first 4 h after DPHB-P incubation, and then it retained the 4 h level up to 16 days. No significant change in the production levels of collagen type I and osteocalcin was detectable after treatment with low concentrations of PHB-P for up to 32 days. In contrast, a time- and dose-dependent alteration of the alkaline phosphatase (ALP) activity was found. Maximal activity was measured after 4 days of treatment with 2 microg of PHB-P/mL (170% of control cells). Rat peritoneal macrophages co-cultured with osteoblasts in a transwell culture system mimicked the observed PHB-P induced ALP elevation. Therefore, the PHB-P-induced ALP increase could be the result of direct or indirect stimulation of osteoblasts, possibly via soluble factors produced by contaminating osteoclasts. Taken collectively, the data demonstrate that osteoblasts are capable of phagocytosing PHB-P and that this process is accompanied at low PHB-P concentrations by dose- and time-dependent alteration of alkaline phosphatase activity but not of collagen type I or osteocalcin.
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PMID:Degradable and highly porous polyesterurethane foam as biomaterial: effects and phagocytosis of degradation products in osteoblasts. 949 21

The values of pKa (10.38) and Hion (10.92 Kcal/mol) have been determined for the ionizing groups controlling activity of green crab alkaline phosphatase. The results suggest that -NH2 of lysine residue responsible for the ionization with pKc = 10.38 and delta H(o)ion = 10.92 Kcal/mol is in the active site of the enzyme. Modification of lysine residues of the enzyme by an excess of 2,4,6-trinitrobenzenesulfonic acid leads to complete inactivation. The two results coincide with each other. Quantitative assessment of the data indicates that among the reactive -NH2 groups modified only one is essential for the activity of the enzyme.
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PMID:An essential lysine residue of green crab (Scylla Serrata) alkaline phosphatase. 980 90

Escherichia coli alkaline phosphatase catalyzes both the nonspecific hydrolysis of phosphomonoesters and a transphosphorylation reaction in which phosphate is transferred to an alcohol via a phosphoseryl intermediate. The rate-determining step for the wild-type enzyme is pH dependent. At alkaline pH, release of the product phosphate from the noncovalent enzyme-phosphate complex determines the reaction rate, whereas at acidic pH hydrolysis of the covalent enzyme-phosphate complex controls the reaction rate. When the lysine at position 328 was substituted with a cysteine (K328C), the rate-determining step at pH 8.0 of the mutant enzyme was altered so that hydrolysis of the covalent intermediate became limiting rather than phosphate release. The transphosphorylation activity of the K328C enzyme was selectively enhanced, while the hydrolysis activity was reduced compared to that of the wild-type enzyme. The ratio of the transphosphorylation to the hydrolysis activities increased 28-fold for the K328C enzyme in comparison with the wild-type enzyme. Several other mutant enzymes for which a positive charge at the active center is removed by site-specific mutagenesis share this characteristic of the K328C enzyme. These results suggest that the positive charge at position 328 is at least partially responsible for maintaining the balance between the hydrolysis and transphosphorylation activities and plays an important role in determining the rate-limiting step of E. coli alkaline phosphatase.
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PMID:Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center. 1005 56

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. In this study, we have used multipotential murine C3H10T1/2 cells to analyze the effect and mechanism of action of bone morphogenetic protein 2 (BMP-2) on chondrogenesis. C3H10T1/2 cells have been previously shown to undergo multiple differentiation pathways. While chondrogenesis, osteogenesis, myogenesis and adipogenesis have been observed, chondrocytes appear significantly less frequently than the other cell types, and the appearance of chondrocytes exclusive of the other cell types has not been observed. We report here that the appearance of chondrocytes in C3H10T1/2 cells is markedly enhanced as a result of culture under conditions favorable for chondrogenesis, i.e. plating as high-density micromass and treatment with BMP-2. Such cultures contain chondrocyte-like cells, elaborate an Alcian blue stained cartilage-like matrix, express link protein and type II collagen, both cartilage matrix markers, and show increased [35S]sulfate incorporation. The appearance of Alcian blue positive material and increased sulfate incorporation are dependent on the dose of BMP-2, culture time, and cell plating density of the micromass cultures. Differentiation of cells within the micromass was specific to the chondrogenic lineage, as alkaline phosphatase staining revealed only faint staining in the micromass at the highest BMP-2 concentration. The importance of enhanced cell-cell interaction in the chondroinductive effects of BMP-2 on high-density C3H10T1/2 cultures was further implicated by the additional promotion of chondrogenesis in the presence of the polycationic compound, poly-L-lysine, which has been previously reported to enhance cellular interactions and chondrogenesis in embryonic limb mesenchymal cells. Taken together, these findings suggest that chondrogenesis in C3H10T1/2 cells is inducible by BMP-2 and requires cell-cell interaction.
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PMID:Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures. 1023 4

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60 degrees C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.
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PMID:Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus. 1039 69

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2 mM sodium fluoride, whereas for osteoclasts 50-100 mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralizing zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.
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PMID:Association of matrix acid and alkaline phosphatases with mineralization of cartilage and endochondral bone. 1040 23

The pharmacokinetics of carprofen, a propionic acid-derived nonsteroidal anti-inflammatory (NSAID), and its effect on gastrointestinal mucosa, complete blood counts (CBC) and biochemical indicators of liver and renal function were investigated in healthy cats using a randomized crossover design. A single dose of 4 mg/kg of carprofen (Zenecarp(R) Injection), normal saline, or 20 mg/kg of DL-lysine acetyl salicylate (Vetalgine(R)) was given intravenously (i.v.) to each of five cats with a washout period of 2 weeks between treatments. Endoscopy of the stomach and duodenum 8 h postinjection revealed one acetyl salicylate-(aspirin)-treated cat with minor pinpoint erosions. None of the other cats in the three treatment groups had evidence of bleeding or ulceration. Serum biochemistry measurements of blood urea nitrogen (BUN), alanine transferase (ALT) and alkaline phosphatase (ALP) and complete blood counts (CBC) were not significantly altered from pretreatment values by the single dose of salicylate or carprofen (P < 0.05). Early and extended sample time points suggest that the pharmacokinetics of carprofen in the cat fit a 2-compartment model, with a long elimination half-life (t1/2) of 20.1 +/- 16.6 h, an area under the plasma concentration-time curve (AUC) of 637 (+/- 237) microgram.mL/h and a volume of distribution (Vdss) of 0.14 +/- 0.05 L/kg. Intravenously administered aspirin fit a 2-compartment model and had a long elimination half-life (t1/2) of 22.2 +/- 3.1 h, an AUC of 3824.2 +/- 506.7 microgram.mL/h and a volume of distribution (Vdss) of 0.17 +/- 0. 01 L/kg.
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PMID:The pharmacokinetics and effects of intravenously administered carprofen and salicylate on gastrointestinal mucosa and selected biochemical measurements in healthy cats. 1084 51


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