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Sample records for acid phosphatase staining

  1. The acid phosphatase test two minute cut-off: an insufficient time to detect some semen stains.

    PubMed

    Redhead, Paul; Brown, Melanie K

    2013-06-01

    The ability to detect semen in sexual offence cases is a crucial first step to locating stains which may be suitable for DNA profiling. Since the development of the acid phosphatase test in the late 1950s by Stuart S. Kind, the process undertaken to perform the test has gone largely unchanged. The method currently accepted by operational forensic science laboratories allows 2 min for a reaction to be obtained, and until relatively recently, this has not been challenged. In this research, samples of semen were obtained from three donors and a range of dilutions for each sample were prepared. Each dilution was subjected to acid phosphatase testing using both direct testing and the 'press test' method. The results showed that semen could be detected in excess of 15 min in dilutions up to 1 in 400 using the press test method and in dilutions up to 1 in 1000 using the direct method. Of further significance was the observation that using the press test method, the two minute cut-off was insufficient to detect the majority of stains and in some cases, semen stains as strong as 1 in 20 dilutions. This research provides compelling evidence for protocols currently utilised in forensic practice to be reviewed in order that forensic scientists do not overlook potential evidential material that may prove suitable for body fluid identification such as DNA STR profiling.

  2. Structure of Acid phosphatases.

    PubMed

    Araujo, César L; Vihko, Pirkko T

    2013-01-01

    Acid phosphatases are enzymes that have been studied extensively due to the fact that their dysregulation is associated with pathophysiological conditions. This characteristic has been exploited for the development of diagnostic and therapeutic methods. As an example, prostatic acid phosphatase was the first marker for metastatic prostate cancer diagnosis and the dysregulation of tartrate resistant acid phosphatase is associated with abnormal bone resorption linked to osteoporosis. The pioneering crystallization studies on prostatic acid phosphatase and mammalian tartrate-resistant acid phosphatase conformed significant milestones towards the elucidation of the mechanisms followed by these enzymes (Schneider et al., EMBO J 12:2609-2615, 1993). Acid phosphatases are also found in nonmammalian species such as bacteria, fungi, parasites, and plants, and most of them share structural similarities with mammalian acid phosphatase enzymes. Acid phosphatase (EC 3.1.3.2) enzymes catalyze the hydrolysis of phosphate monoesters following the general equation. Phosphate monoester + H2O -->/<-- alcohol + phosphate. The general classification "acid phosphatase" relies only on the optimum acidic pH for the enzymatic activity in assay conditions using non-physiological substrates. These enzymes accept a wide range of substrates in vitro, ranging from small organic molecules to phosphoproteins, constituting a heterogeneous group of enzymes from the structural point of view. These structural differences account for the divergence in cofactor dependences and behavior against substrates, inhibitors, and activators. In this group only the tartrate-resistant acid phosphatase is a metallo-enzyme whereas the other members do not require metal-ion binding for their catalytic activity. In addition, tartrate-resistant acid phosphatase and erythrocytic acid phosphatase are not inhibited by L-(+)-tartrate ion while the prostatic acid phosphatase is tartrate-sensitive. This is an important

  3. Differential staining of bacteria: acid fast stain.

    PubMed

    Reynolds, Jackie; Moyes, Rita B; Breakwell, Donald P

    2009-11-01

    Acid-fastness is an uncommon characteristic shared by the genera Mycobacterium (Section 10A) and Nocardia. Because of this feature, this stain is extremely helpful in identification of these bacteria. Although Gram positive, acid-fast bacteria do not take the crystal violet into the wall well, appearing very light purple rather than the deep purple of normal Gram-positive bacteria.

  4. New Grocott Stain without Using Chromic Acid.

    PubMed

    Shiogama, Kazuya; Kitazawa, Kayo; Mizutani, Yasuyoshi; Onouchi, Takanori; Inada, Ken-Ichi; Tsutsumi, Yutaka

    2015-01-01

    We established a new "ecological" Grocott stain for demonstrating fungi, based upon a 4R principle of refusal, reduction, reuse, and recycle of waste management. Conventional Grocott stain employs environmentally harsh 5% chromic acid for oxidization. Initially, we succeeded in reducing the concentration of chromic acid from 5% to 1% by incubating the solution at 60°C and using five-fold diluted chromic acid solution at which point it was reusable. Eventually, we reached the refusal level where 1% periodic acid oxidization was efficient enough, when combined with preheating of sections in the electric jar, microwave oven, or pressure pan. For convenience sake, we recommend pressure pan heating in tap water for 10 min. Stainability of fungi in candidiasis and aspergillosis was comparable with conventional Grocott stain, while Mucor hyphae showed enhanced staining. The modified sequence was further applicable to detecting a variety of mycotic pathogens in paraffin sections. Our environmentally-friendly Grocott stain also has the advantage of avoiding risk of human exposure to hexavalent chromium solution in the histopathology laboratory. The simple stain sequence is can be easily applied worldwide.

  5. Acid phosphatase/phosphotransferases from enteric bacteria.

    PubMed

    Mihara, Y; Utagawa, T; Yamada, H; Asano, Y

    2001-01-01

    We have investigated the enzymatic phosphorylation of nucleosides and found that Morganella morganii phoC acid phosphatase exhibits regioselective pyrophosphate (PP(i))-nucleoside phosphotransferase activity. In this study, we isolated genes encoding an acid phosphatase with regioselective phosphotransferase activity (AP/PTase) from Providencia stuartii, Enterobacter aerogenes, Escherichia blattae and Klebsiella planticola, and compared the primary structures and enzymatic characteristics of these enzymes with those of AP/PTase (PhoC acid phosphatase) from M. morganii. The enzymes were highly homologous in primary structure with M. morganii AP/PTase, and are classified as class A1 acid phosphatases. The synthesis of inosine-5'-monophosphate (5'-IMP) by E. coli overproducing each acid phosphatase was investigated. The P. stuartii enzyme, which is most closely related to the M. morganii enzyme, exhibited high 5'-IMP productivity, similar to the M. morganii enzyme. The 5'-IMP productivities of the E. aerogenes, E. blattae and K. planticola enzymes were inferior to those of the former two enzymes. This result underlines the importance of lower K(m) values for efficient nucleotide production. As these enzymes exhibited a very high degree of homology at the amino acid sequence level, it is likely that local sequence differences in the binding pocket are responsible for the differences in the nucleoside-PP(i) phosphotransferase reaction.

  6. Combination of alkaline phosphatase anti-alkaline phosphatase (APAAP)- and avidin-biotin-alkaline phosphatase complex (ABAP)-techniques for amplification of immunocytochemical staining of human testicular tissue.

    PubMed

    Davidoff, M S; Schulze, W; Holstein, A F

    1991-01-01

    An amplification procedure was developed for the visualization of antigens in human testis using monoclonal antibodies against desmin and vimentin. The technique combines the high sensitive and specific APAAP- and ABAP-methods. Depending on the quality of the antibodies used and the processing of the material prior to the immunocytochemical staining the amplification technique may be applied either as a single APAAP and ABAP- or as a double APAAP and ABAP-combination. Especially after the double amplification reaction a distinct increase of the staining intensity of the vimentin- (in Sertoli cells, myofibroblasts of the lamina propria, and fibroblasts of the interstitium) and desmin- (in myofibroblasts of the lamina propria and smooth muscle cells of the blood vessels) like immunoreactivity was observed. If different diazonium salts were used for the visualization of the alkaline phosphatase activity (e.g. Fast Red TR Salt, Fast Blue BB Salt) desmin- and vimentin-like immunoreactivity can be demonstrated in the same tissue section in a double sequential staining approach. For double staining, the alkaline phosphatase technique may be combined successfully with a technique or a combination that uses peroxidase as a marker.

  7. A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter

    PubMed Central

    2013-01-01

    Background To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein. Direct chromogenic staining for α-sec-EAP activity is non-specific as its NBT/BCIP substrate cross-reacts with cellular phosphatases which can be inhibited with Levulinic acid. However, the parental E(P) strain only exports detectable levels of α-sec-EAP at 69 hours and not within the 36 hour period post-seeding required for effective screening with the consequent absence of a reference for secretion. We substituted the endogenous cellular phosphatase activity as a comparative reference for secretion rate and levels as well as for colony alignment while elevating specificity and sensitivity of detection of the exported protein with other innovative modifications of the immuno-chromogenic staining application for screening protein export mutants. Results Raising the specificity and utility of staining for α-sec-EAP activity required 5 modifications including some to published methods. These included, exploitation of endogenous phosphatase activity, reduction of the cell/protein burden, establishment of the direct relation between concentrations of transcriptional inducer and exported membrane immobilized protein and concentrations of protein exported into growth media, amplification of immuno-specificity and sensitivity of detection of α-sec-EAP reporter enzyme signal and restriction of staining to optimal concentrations of antisera and time periods. The resultant immuno-chromogenic screen allows for the detection of early secretion and as little as 1.3 fold over-secretion of α-sec-EAP reporter protein by E(M) mutants in the presence of 10 fold -216 fold higher concentrations of HSA. Conclusions The modified immuno-chromogenic screen is sensitive, specific and has

  8. Primary structure of rat secretory acid phosphatase and comparison to other acid phosphatases.

    PubMed

    Roiko, K; Jänne, O A; Vihko, P

    1990-05-14

    Overlapping cDNA clones encoding rat prostatic acid phosphatase (rPAP) were isolated by using two human prostatic acid phosphatase (hPAP)-encoding cDNAs to screen rat prostatic cDNA libraries. The isolated cDNAs encompassed a total of 1626 nucleotides (nt), of which 1143 nt corresponded to the protein coding sequence encoding a mature polypeptide of 350 amino acids (aa) and a 31-aa long signal peptide-like sequence. The deduced Mr of the mature rPAP was 40,599. RNA blot analysis indicated the presence of three mRNA species (4.9, 2.3 and 1.5 kb in size) in the rat prostate. The deduced aa sequences of rPAP and hPAP show 75% identity, whereas the similarity between rPAP and human lysosomal acid phosphatase (hLAP) is only 45%. Furthermore, the sequence similarity between rPAP and rat lysosomal acid phosphatase (rLAP) is 46% at the aa level. Similar to hPAP, but unlike hLAP and rLAP, the rPAP sequence lacks a membrane-anchoring domain indicating the secretory character of this phosphatase. All six cysteines present in the overlapping areas of the mature rPAP, hPAP, rLAP and hLAP proteins are positionally conserved, suggesting that these residues are important for the tertiary structure of acid phosphatases (APs). The previously reported active site residues, two arginines and one histidine, are also conserved in these APs.

  9. Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.

    PubMed Central

    Shekels, L. L.; Smith, A. J.; Van Etten, R. L.; Bernlohr, D. A.

    1992-01-01

    We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins. PMID:1304913

  10. Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma.

    PubMed

    Tanaka, Masayuki; Kishi, Yasuhiro; Takanezawa, Yasukazu; Kakehi, Yoshiyuki; Aoki, Junken; Arai, Hiroyuki

    2004-07-30

    Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities and is detected in various biological fluids, including human seminal plasma. Due to its cell proliferation stimulatory and anti-apoptotic activities, LPA has been implicated in the progression of some cancers such as ovarian cancer and prostate cancer. Here, we show that prostatic acid phosphatase, which is a non-specific phosphatase and which has been implicated in the progression of prostate cancer, inactivates LPA in human seminal plasma. Human seminal plasma contains both an LPA-synthetic enzyme, lysoPLD, which converts lysophospholipids to LPA and is responsible for LPA production in serum, and its major substrate, lysophosphatidylcholine. In serum, LPA accumulated during incubation at 37 degrees C. However, in seminal plasma, LPA did not accumulate. This discrepancy is explained by the presence of a strong LPA-degrading activity. Incubation of LPA with seminal plasma resulted in the disappearance of LPA and an accompanying accumulation of monoglyceride showing that LPA is degraded by phosphatase activity present in the seminal plasma. When seminal plasma was incubated in the presence of a phosphatase inhibitor, sodium orthovanadate, LPA accumulated, indicating that LPA is produced and degraded in the fluid. Biochemical characterization of the LPA-phosphatase activity identified two phosphatase activities in human seminal plasma. By Western blotting analysis in combination with several column chromatographies, the major activity was revealed to be identical to prostatic acid phosphatase. The present study demonstrates active LPA metabolism in seminal plasma and indicates the possible role of LPA signaling in male sexual organs including prostate cancer.

  11. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  12. The cytochemistry of tartrate-resistant acid phosphatase. Technical considerations.

    PubMed

    Janckila, A J; Li, C Y; Lam, K W; Yam, L T

    1978-07-01

    Cytochemical demonstration of tartrate-resistant acid phosphatase activity is essential for the diagnosis of leukemic reticuloendotheliosis. In order to perform this test correctly and to interpret the results propertly, it is necessary to understand the technical details of the cytochemical methods thoroughly. The method using naphthol--ASBI phosphoric acid--fast garnet GBC is recommended for this purpose, and factors crucial to the cytochemical study, such as fixation, substrate, coupler, pH and temperature of incubation buffer, counterstains, and mounting media are examined and discussed. Conventional methods for acid phosphatase in the presence and absence of L(+) tartaric acid are also critically examined. The naphthol--ASBI phosphoric acid--fast garnet GBC method is sensitive, technically simple and easily reproducible. Its reaction product is highly chromogenic and is most suitable for cytochemical demonstration of acid phosphatase and tartrate-resistant acid phosphatase activity in cytologic preparations. The naphthol--ASBI phosphoric acid--pararosaniline method is highly specific and is best for histochemical demonstration of acid phosphatase and tartrate-resistant acid phosphatase in tissue sections.

  13. Emerging Roles of Human Prostatic Acid Phosphatase

    PubMed Central

    Kong, Hoon Young; Byun, Jonghoe

    2013-01-01

    Prostate cancer is one of the most prevalent non-skin related cancers. It is the second leading cause of cancer deaths among males in most Western countries. If prostate cancer is diagnosed in its early stages, there is a higher probability that it will be completely cured. Prostatic acid phosphatase (PAP) is a non-specific phosphomonoesterase synthesized in prostate epithelial cells and its level proportionally increases with prostate cancer progression. PAP was the biochemical diagnostic mainstay for prostate cancer until the introduction of prostate-specific antigen (PSA) which improved the detection of early-stage prostate cancer and largely displaced PAP. Recently, however, there is a renewed interest in PAP because of its usefulness in prognosticating intermediate to high-risk prostate cancers and its success in the immunotherapy of prostate cancer. Although PAP is believed to be a key regulator of prostate cell growth, its exact role in normal prostate as well as detailed molecular mechanism of PAP regulation is still unclear. Here, many different aspects of PAP in prostate cancer are revisited and its emerging roles in other environment are discussed. PMID:24009853

  14. Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana.

    PubMed

    Reddy, Venky Sreedhar; Rao, D K Venkata; Rajasekharan, Ram

    2010-04-01

    Lysophosphatidic acid (LPA) acts as a signaling molecule that regulates diverse cellular processes and it can rapidly be metabolized by phosphatase and acyltransferase. LPA phosphatase gene has not been identified and characterized in plants so far. The BLAST search revealed that the At3g03520 is similar to phospholipase family, and distantly related to bacterial phosphatases. The conserved motif, (J)4XXXNXSFD, was identified in both At3g03520 like phospholipases and acid phosphatases. In silico expression analysis of At3g03520 revealed a high expression during phosphate starvation and abiotic stresses. This gene was overexpressed in Escherichia coli and shown to posses LPA specific phosphatase activity. These results suggest that this gene possibly plays a role in signal transduction and storage lipid synthesis.

  15. Moraxella catarrhalis synthesizes an autotransporter that is an acid phosphatase.

    PubMed

    Hoopman, Todd C; Wang, Wei; Brautigam, Chad A; Sedillo, Jennifer L; Reilly, Thomas J; Hansen, Eric J

    2008-02-01

    Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10(-10)) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity.

  16. OBSERVATIONS ON THE ACID PHOSPHATASES OF EUGLENA GRACILIS

    PubMed Central

    Blum, Jacob J.

    1965-01-01

    When a bleached strain of Euglena is maintained in a medium containing very low con centrations of phosphate, the acid phosphatase activity increases. The increase in acid phosphatase activity is prevented by Actinomycin D and by p-fluorophenylalanine (PFA), indicating that the increased activity is due to de novo synthesis of acid phosphatase. When phosphate is replenished, the acid phosphatase activity decreases to the level characteristic of uninduced cells before there is any appreciable cell division. When cell division resumes in the presence of PFA, the level of acid phosphatase activity remains approximately constant. This indicates that there are two different phosphatases: a constitutive enzyme, whose synthesis is insensitive to the presence of PFA, and an induced enzyme, whose synthesis is sensitive to PFA. These enzymes are not equally sensitive to changes in pH and in fluoride concentration, thus permitting them to be assayed individually in whole toluene-treated cells. Induced cells also acquire the ability to remove phosphate from the medium very rapidly. PMID:14326108

  17. Improving acid-fast fluorescent staining for the detection of mycobacteria using a new nucleic acid staining approach.

    PubMed

    Ryan, Gavin J; Shapiro, Howard M; Lenaerts, Anne J

    2014-09-01

    Acid fast staining of sputum smears by microscopy remains the prevalent method for detecting Mycobacterium tuberculosis. The sensitivity of microscopy using acid fast stains requires 10(4) bacilli per ml of sputum. Although fluorescent acid fast stains, such as Auramine-O, show improved sensitivity, almost half of culture-positive TB cases are currently estimated to remain smear-negative. These current diagnosis problems provide impetus for improving staining procedures. We evaluated a novel fluorescent acid-fast staining approach using the nucleic acid-binding dye SYBR(®) Gold on mycobacterial in vitro cultures. The SYBR(®) Gold stain detected 99% of MTB in both actively replicating aerobic and non-replicating hypoxic cultures. Transmission light microscopy with Ziehl-Neelsen fuchsin, and fluorescence microscopy with Auramine-O or Auramine-rhodamine detected only 54%-86% of MTB bacilli. SYBR(®) Gold fluoresces more intensely than Auramine-O, and is highly resistant to fading. The signal to noise ratio is exceptionally high due to a >1000-fold enhanced fluorescence after binding to DNA/RNA, thereby reducing most background fluorescence. Although cost and stability of the dye may perhaps limit its clinical use at this time, these results warrant further research into more nucleic acid dye variants. In the meantime, SYBR(®) Gold staining shows great promise for use in numerous research applications.

  18. Acid phosphatase and lipid peroxidation in human cataractous lens epithelium.

    PubMed

    Vasavada, A R; Thampi, P; Yadav, S; Rawal, U M

    1993-12-01

    The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium) and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium). In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium) and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium). From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.

  19. Properties of nucleic acid staining dyes used in gel electrophoresis.

    PubMed

    Haines, Alicia M; Tobe, Shanan S; Kobus, Hilton J; Linacre, Adrian

    2015-03-01

    Nucleic acid staining dyes are used for detecting nucleic acids in electrophoresis gels. Historically, the most common dye used for gel staining is ethidium bromide, however due to its toxicity and mutagenicity other dyes that are safer to the user and the environment are preferred. This Short Communication details the properties of dyes now available and their sensitivity for detection of DNA and their ability to permeate the cell membrane. It was found that GelRed™ was the most sensitive and safest dye to use with UV light excitation, and both GelGreen™ and Diamond™ Nucleic Acid Dye were sensitive and the safer dyes using blue light excitation.

  20. Acid phosphatase activities during the germination of Glycine max seeds.

    PubMed

    dos Prazeres, Janaina Nicanuzia; Ferreira, Carmen Veríssima; Aoyama, Hiroshi

    2004-01-01

    In this paper, we describe a study concerning the determination of some characteristics of soybean seedlings and the detection of acid phosphatase activities towards different substrates during the germination. Enzyme activities with p-nitrophenylphosphate (pNPP) and inorganic pyrophosphate (PPi) as substrates were detected from the 5th and 7th days after germination, respectively. Acid phosphatase activities with tyrosine phosphate (TyrP), glucose-6-phosphate (G6P) and phosphoenol pyruvate (PEP) were also observed but to a lesser extent. Under the same conditions, no enzyme activity was detected with phytic acid (PhyAc) as substrate. The appearance of phosphatase activity was coincident with the decrease of inorganic phosphate content during germination; over the same period, the protein content increased up to the 5th day, decreased until the 8th day, and remained constant after this period. Relative to phosphatase activity in the cotyledons, the activities detected in the hypocotyl and roots were 82% and 38%, respectively. During storage the enzyme maintained about 63% of its activity for 3 months at 5 degrees C. The specificity constant (Vmax/Km) values for pNPP and PPi were 212 and 64 mu kat mM-1 mg-1, respectively. Amongst the substrates tested, PPi could be a potential physiological substrate for acid phosphatase during the germination of soybean seeds.

  1. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor.

    PubMed

    Dounay, A B; Forsyth, C J

    2002-11-01

    As the first recognized member of the "okadaic acid class" of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine/threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested.

  2. Studies on the catalytic mechanism of pig purple acid phosphatase.

    PubMed

    Wynne, C J; Hamilton, S E; Dionysius, D A; Beck, J L; de Jersey, J

    1995-05-10

    Several independent experiments failed to reveal any evidence in support of the involvement of a phosphoryl-enzyme intermediate in the catalytic mechanism of pig allantoic fluid purple acid phosphatase: (i) attempts to label enzyme with phosphate derived from [32P]p-nitrophenyl phosphate were unsuccessful; (ii) values of kcat for a series of phosphate derivative varied over a wide range, with the enzyme showing a marked preference for activated ester and anhydride substrates over those with a stable leaving group; (iii) burst titrations revealed a "burst" of p-nitrophenol from p-nitrophenyl phosphate only when the enzyme was added after the substrate, suggesting that this result was an artifact of the order of addition of reagents; (iv) transphosphorylation from p-nitrophenyl phosphate to acceptor alcohols could not be detected, even under conditions where a transphosphorylation to hydrolysis ratio as low as 0.015 could have been measured; (v) enzyme-catalyzed exchange of 180 between phosphate and water was demonstrated, although at a rate much slower than that observed for other phosphatases where the involvement of a phosphoryl-enzyme intermediate in the mechanism has been clearly established. The present results are compared with those obtained in similar studies on other phosphatases, particularly the highly homologous beef spleen purple acid phosphatase, and their implications for the catalytic mechanism of the purple acid phosphatases are discussed.

  3. New form of acid phosphatase during lysosome biogenesis.

    PubMed Central

    Rao, G R; Aithal, H N; Toback, F G; Getz, G S

    1981-01-01

    Lysosome formation was induced in cells of the renal medulla by feeding rats on a K+-deficient diet. The role of the endoplasmic reticulum in the production of acid phosphatase, a typical lysosomal enzyme, was examined. Lysosomal and microsomal fractions were prepared for study by differential centrifugation of homogenates of renal papilla and inner stripe of red medulla. Acid phosphatase activity in the microsomal fraction was distinguished from the activity in the lysosomal fraction in normal tissue by differences in pH optima, tartrate inhibition, distribution of multiple forms after polyacrylamide-gel electrophoresis and detergent-sensitivity. During progressive K+ depletion, acid phosphatase activity in both microsomal and lysosomal fractions of the tissue increased 3-fold. In the lysosomes, K+ depletion was associated with the appearance of a new band of acid phosphatase. The neuraminidase-sensitivity of this band on polyacrylamide-gel electrophoresis indicated that the enzyme protein had been modified by the addition of sialic acid residues. K+ depletion also altered the lysosomal enzyme so that thiol compounds were able to stimulate its activity. Images Fig. 4. PMID:7326004

  4. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    ERIC Educational Resources Information Center

    Kulkarni, Suhasini; Tran, Vu; Ho, Maggie K.-M.; Phan, Chieu; Chin, Elizabeth; Wemmer, Zeke; Sommerhalter, Monika

    2010-01-01

    This experiment was performed in an upper-level undergraduate biochemistry laboratory course. Students learned how to immobilize an enzyme in a sol-gel matrix and how to perform and evaluate enzyme-activity measurements. The enzyme acid phosphatase (APase) from wheat germ was encapsulated in sol-gel beads that were prepared from the precursor…

  5. Yeast Acid Phosphatases and Phytases: Production, Characterization and Commercial Prospects

    NASA Astrophysics Data System (ADS)

    Kaur, Parvinder; Satyanarayana, T.

    The element phosphorus is critical to all life forms as it forms the basic component of nucleic acids and ATP and has a number of indispensable biochemical roles. Unlike C or N, the biogeochemical cycling of phosphorus is very slow, and thus making it the growth-limiting element in most soils and aquatic systems. Phosphohydrolases (e.g. acid phosphatases and phytases) are enzymes that break the C-O-P ester bonds and provide available inorganic phosphorus from various inassimilable organic forms of phosphorus like phytates. These enzymes are of significant value in effectively combating phosphorus pollution. Although phytases and acid phosphatases are produced by various plants, animals and micro organisms, microbial sources are more promising for the production on a commercial scale. Yeasts being the simplest eukaryotes are ideal candidates for phytase and phos-phatase research due to their mostly non-pathogenic and GRAS status. They have not, however, been utilized to their full potential. This chapter focuses attention on the present state of knowledge on the production, characterization and potential commercial prospects of yeast phytases and acid phosphatases.

  6. Phosphotyrosine as a substrate of acid and alkaline phosphatases.

    PubMed

    Apostoł, I; Kuciel, R; Wasylewska, E; Ostrowski, W S

    1985-01-01

    A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described. The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9. The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm. The difference absorption coefficients were calculated for several pH values. Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E. coli are inhibited by excess of substrate.

  7. Effect of vanadium compounds on acid phosphatase activity.

    PubMed

    Vescina, C M; Sálice, V C; Cortizo, A M; Etcheverry, S B

    1996-01-01

    The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activity seems to depend on the geometry around the vanadium atom more than on the oxidation state. Our results indicate a correlation between the PTPase activity and the sensitivity to vanadate and vanadyl cation.

  8. Method and apparatus for staining immobilized nucleic acids

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.; Jacobson, Stephen C.

    2000-01-01

    A method for staining immobilized nucleic acids includes the steps of affixing DNA probes to a solid substrate, moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes, and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

  9. Purification and Characterization of Acid Phosphatase V from Aspergillus nidulans

    PubMed Central

    Harsanyi, Zsolt; Dorn, Gordon L.

    1972-01-01

    Acid phosphatase V of Aspergillus nidulans was purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzyme demonstrated a charge microheterogeneity on starch and acrylamide gel electrophoresis, but proved to be homogeneous on ultracentrifugation and gel filtration. Phosphatase V was found to be a classic acid orthophosphoric monoester phosphohydrolase, and it cleaved p-nitrophenylphosphate, glucose-6-phosphate, and uridine-5′-monophosphate at maximal rates. It was inhibited by fluoride, borate, and molybdate ions, and demonstrated end-product inhibition by inorganic phosphate. Metallic ions or cofactors were not required for activity. The molecular weight was estimated to be 100,000, the S20,w was calculated to be 4.1, and the pH optimum was found to be 6.1. Images PMID:4552990

  10. Crystallization of recombinant Haemophilus influenzaee (P4) acid phosphatase

    SciTech Connect

    Ou, Zhonghui; Felts, Richard L.; Reilly, Thomas J.; Nix, Jay C.; Tanner, John J.

    2006-05-01

    Lipoprotein e (P4) is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host–pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. The space group is P4{sub 2}2{sub 1}2, with unit-cell parameters a = 65.6, c = 101.4 Å, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase.

  11. Calcitonin releases acid phosphatase from rat ventral prostate explants.

    PubMed

    Latif, A; Nakhla, A M

    1994-01-01

    Inclusion of salmon calcitonin in the culture medium of rat ventral prostate explants diminished l-tartarate-sensitive acid phosphatase activity in the tissues with a concomitant increment of the enzyme activity in the medium. The effect of the hormone was dose-dependent for a dose range of 10(-12)-10(-6) M. Acid phosphatase activity in prostate explants decreased from 38.6 +/- 3.5 to 20.5 +/- 2.8, whereas it increased from 0.60 +/- 0.15 to 2.80 +/- 0.40 nmol p-nitrophenol liberated/mg protein/30 min in the culture medium. Tissues exposed to 10(-6) M salmon calcitonin had higher acetylcholinesterase activity (8.8 +/- 0.7) than non-exposed ones (6.2 +/- 0.5 mumol substrate hydrolyzed/g tissue/min). These results suggest that locally produced calcitonin causes a release for prostatic acid phosphatase from prostate tissues possibly through its interaction with the cholinergic system.

  12. Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas (Thunberg)

    NASA Astrophysics Data System (ADS)

    Wang, Yiyan; Sun, Hushan; Wang, Yanjie; Yan, Dongchun; Wang, Lei

    2015-03-01

    In this study, a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase, and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules (YGI, YGII, and YGIII) that differed in electron density and acid phosphatase reaction were identified in early cleavage, morula, blastula, gastrula, trochophore, and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation, indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI (free of acid phosphatase reaction) with YGII (rich in acid phosphatase reaction) could be the way by which yolk degradation is triggered.

  13. Inositol phosphatase activity of the Escherichia coli agp-encoded acid glucose-1-phosphatase.

    PubMed

    Cottrill, Michael A; Golovan, Serguei P; Phillips, John P; Forsberg, Cecil W

    2002-09-01

    When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6 with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55 degrees C. while InsP6 and p-nitrophenyl phosphate were hydrolyzed maximally at 60 degrees C. The Agp exhibited Km values of (0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5. D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4 and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase.

  14. Human prostatic acid phosphatase directly stimulates collagen synthesis and alkaline phosphatase content of isolated bone cells

    SciTech Connect

    Ishibe, M.; Rosier, R.N.; Puzas, J.E. )

    1991-10-01

    Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP has no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor.

  15. Characterization of cationic acid phosphatase isozyme from rat liver mitochondria.

    PubMed

    Fujimoto, S; Murakami, K; Hosoda, T; Yamamoto, Y; Watanabe, K; Morinaka, Y; Ohara, A

    1992-05-01

    Acid phosphatase isozyme was highly purified from rat liver mitochondrial fraction. The enzyme showed an isoelectric point value of above 9.5 on isoelectric focusing, and the apparent molecular weight was estimated to be 32000 by Sephadex G-100 gel filtration or 16000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, thiamine pyrophosphate, inorganic pyrophosphate, and phosphoprotein such as casein and phosvitin, but not of several phosphomonoesters, except for p-nitrophenyl phosphate and o-phosphotyrosine. The enzyme was not inhibited by L-(+)-tartrate, and was significantly activated by Fe2+ and reducing agents such as ascorbic acid, L-cysteine,and dithiothreitol. The enzyme was found to be distributed in various rat tissues including liver, spleen, kidney, small intestine, lung, stomach, brain and heart, but not in skeletal muscle.

  16. Direct electrochemistry of porcine purple acid phosphatase (uteroferrin).

    PubMed

    Bernhardt, Paul V; Schenk, Gerhard; Wilson, Gregory J

    2004-08-17

    Cyclic voltammetry of the non-heme diiron enzyme porcine purple acid phosphatase (uteroferrin, Uf) has been reported for the first time. Totally reversible one-electron oxidation responses (FeIII-FeII --> FeII-FeIII) are seen both in the absence and in the presence of weak competitive inhibitors phosphate and arsenate, and dissociation constants of these oxoanion complexes formed with uteroferrin in its oxidized state (Uf(o)) have been determined. The effect of pH on the redox potentials has been investigated in the range 3 < pH < 6.5, enabling acid dissociation constants for Uf(o) and its phosphate and arsenate complexes to be calculated.

  17. Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli

    PubMed Central

    2014-01-01

    Background The genus Burkholderia is widespread in diverse ecological niches, the majority of known species are soil bacteria that exhibit different types of non-pathogenic interactions with plants. Burkholderia species are versatile organisms that solubilize insoluble minerals through the production of organic acids, which increase the availability of nutrients for the plant. Therefore these bacteria are promising candidates for biotechnological applications. Results Burkholderia sp. (R 3.25 isolate) was isolated from agricultural soil in Ponta Grossa-PR-Brazil and identified through analysis of the 16S rDNA as a strain classified as Burkholderia gladioli. The expression of membrane-bound acid phosphatase (MBAcP) was strictly regulated with optimal expression at a concentration of phosphorus 5 mM. The apparent optimum pH for the hydrolysis of p-nitrophenylphosphate (PNPP) was 6.0. The hydrolysis of PNPP by the enzyme exhibited a hyperbolic relationship with increasing concentration of substrate and no inhibition by excess of substrate was observed. Kinetic data revealed that the hydrolysis of PNPP exhibited cooperative kinetics with n = 1.3, Vm = 113.5 U/mg and K0.5 = 65 μM. The PNPPase activity was inhibited by vanadate, p-hydroxymercuribenzoate, arsenate and phosphate, however the activity was not inhibited by calcium, levamisole, sodium tartrate, EDTA, zinc, magnesium, cobalt, ouabain, oligomycin or pantoprazol. Conclusion The synthesis of membrane-bound non-specific acid phosphatase, strictly regulated by phosphate, and its properties suggest that this bacterium has a potential biotechnological application to solubilize phosphate in soils with low levels of this element, for specific crops. PMID:24713147

  18. Stimulation of phosphatidylglycerolphosphate phosphatase activity by unsaturated fatty acids in rat heart.

    PubMed

    Cao, S G; Hatch, G M

    1994-07-01

    Phosphatidylglycerolphosphate (PGP) synthase and PGP phosphatase catalyze the sequential synthesis of phosphatidylglycerol from cytidine-5'-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG) and glycerol-3-phosphate. PGP synthase and PGP phosphatase activities were characterized in rat heart mitochondrial fractions, and the effect of fatty acids on the activity of these enzymes was determined. PGP synthase was observed to be a heat labile enzyme that exhibited apparent Km values for CDP-PG and glycerol-3-phosphate of 46 and 20 microM, respectively. The addition of exogenous oleic acid to the assay mixture did not affect PGP synthase activity. PGP phosphatase was observed to be a heat labile enzyme, and addition of oleic acid to the assay mixture caused a concentration-dependent stimulation of PGP phosphatase activity. Maximum stimulation (1.9-fold) of enzyme activity was observed in the presence of 0.5 mM oleic acid, but the stimulation was slightly attenuated by the presence of albumin in the assay. The presence of oleic acid in the assay mixture caused the inactivation of PGP phosphatase activity to be retarded at 55 degrees C. Stimulation of PGP phosphatase activity was also observed with arachidonic acid, whereas taurocholic, stearic and palmitic acids did not significantly affect PGP phosphatase activity. The activity of mitochondrial phosphatidic acid phosphohydrolase was not affected by inclusion of oleic acid in the incubation mixture. We postulate that unsaturated fatty acids stimulate PGP phosphatase activity in rat heart.

  19. A chemical relaxation study of human prostatic acid phosphatase.

    PubMed

    Shear, D B; Kustin, K

    1968-01-01

    Chemical relaxation methods and a dilution technique were applied to the study of the hydrolysis of p-nitrophenyl phosphate by human prostatic acid phosphatase. Although the reaction mechanism was not elucidated, rate constants and equilibrium constants were obtained for the reaction of enzyme and p-nitrophenol to form a complex. A slow, 2-sec relaxation effect which showed no concentration dependence was observed in various reaction mixtures, including some lacking the substrate and products of the hydrolytic reaction. The conclusion drawn is that there are two forms of the prostatic enzyme, which are normally in equilibrium with each other, but which undergo a relatively slow interconversion when this equilibrium is perturbed. A preliminary calculation indicates that these forms are present in the equilibrium ratio of 2:1.

  20. Comparison of diazo-coupling, formazan, and silver staining techniques for visualizing alkaline phosphatase isoenzymes after electrophoresis in homogeneous-pore and gradient-pore polyacrylamide gels.

    PubMed

    Hodson, A W; Skillen, A W

    1988-03-01

    Three techniques for visualization of alkaline phosphatase after polyacrylamide-gel electrophoresis are compared. These are diazo-dye simultaneous coupling with the substrate sodium naphthyl phosphate and 5-chloro-2-toluene diazonium chloride; formazan precipitation with the substrate 5-bromo-4-chloro-3-indolyl phosphate and 3-[4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; and silver staining with the substrate sodium glycerophosphate. Each staining technique was tested with gradient-pore and homogeneous-pore acrylamide-gel electrophoresis. The main factors assessed are sensitivity; separation of the human serum alkaline phosphatase isoenzymes of the liver, bone, and intestinal types; and differences in substrate affinity, as well as the complexity of each technique. Using the three techniques only minor differences in substrate affinity are evident. There is some nonspecific staining with the diazo-coupling technique but not with the formazan and silver staining techniques. The differences, in the mobility of the liver, bone, and intestinal isoenzymes achieved by homogeneous-pore gel electrophoresis are sufficient to allow them to be clearly distinguished. However, only very small differences in mobility are found with gradient-pore gel electrophoresis, but the sharper bands in this medium allow much smaller amounts of activity to be detected. As little as 160 microU of enzyme can be visualized by the diazo technique. Silver staining gives an approximately fourfold increase in sensitivity over the formazan technique, which in turn gives a fourfold increase over the diazo technique. An important aspect of the silver staining technique is the potential of increasing sensitivity much further by improvements in the photographic physical development stage.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid-phosphatase-encoding genes.

    PubMed

    Thaller, M C; Lombardi, G; Berlutti, F; Schippa, S; Rossolini, G M

    1995-01-01

    The gene encoding a minor phosphate-irrepressible acid phosphatase (named NapA) of Morganella morganii was cloned and sequenced, and its product characterized. NapA is a secreted acid phosphatase composed of four 27 kDa polypeptide subunits. The enzyme is active on several organic phosphate monoesters but not on diesters, and is also endowed with transphosphorylating activity from organic phosphoric acid esters to nucleosides and other compounds with free hydroxyl groups. Its activity is inhibited by EDTA, inorganic phosphate, nucleosides and Ca2+, but not by fluoride or tartrate, and is enhanced by Mg2+, Co2+ and Zn2+. At the sequence level, the NapA enzyme did not show similarities to any other sequenced bacterial phosphatases. However, a search for homologous genes in sequence databases allowed identification of two open reading frames located within sequenced regions of the Escherichia coli and Proteus mirabilis genomes respectively, encoding proteins of unknown function which are highly homologous to the Morganella enzyme. Moreover, the properties of the NapA enzyme are very similar to those reported for the periplasmic nonspecific acid phosphatase II of Salmonella typhimurium (for which no sequence data are available). These data point to the existence of a new family of bacterial acid phosphatases, which we propose designating class B bacterial acid phosphatases.

  2. [Tartrate-resistant acid phosphatase in free-living Amoeba proteus].

    PubMed

    Sopina, V A

    2002-01-01

    Tartrate-resistant acid phosphatase (TRAP) of Amoeba proteus (strain B) was represented by 3 of 6 bands (= electromorphs) revealed after disc-electrophoresis in polyacrylamide gels with the use of 2-naphthyl phosphate as a substrate at pH 4.0. The presence of MgCl2, CaCl2 or ZnCl2 (50 mM) in the incubation mixture used for gel staining stimulated activities of all 3 TRAP electromorphs or of two of them (in the case of ZnCl2). When gels were treated with MgCl2, CaCl2 or ZnCl2 (10 and 100 mM, 30 min) before their staining activity of TRAP electromorphs also increased. But unlike 1 M MgCl2 or 1 M CaCl2, 1 M ZnCl2 partly inactivated two of the three TRAP electromorphs. EDTA and EGTA (5 mM), and H2O2 (10 mM) completely inhibited TRAP electromorphs after gel treatment for 10, 20 and 30 min, resp. Of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+ and Zn2+), only the latter reactivated the TRAP electromorphs previously inactivated by EDTA or EGTA treatment. In addition, after EDTA inactivation, TRAP electromorphs were reactivated better than after EGTA. The resistance of TRAP electromorphs to okadaic acid and phosphatase inhibitor cocktail 1 used in different concentrations is indicative of the absence of PP1 and PP2A among these electromorphs. Mg2+, Ca2+ and Zn2+ dependence of TRAP activity, and the resistance of its electromorphs to vanadate and phosphatase inhibitor cocktail 2 prevents these electromorphs from being classified as PTP. It is suggested that the active center of A. proteus TRAP contains zinc ion, which is essential for catalytic activity of the enzyme. Thus, TRAP of these amoebae is metallophosphatase showing phosphomonoesterase activity in acidic medium. This metalloenzyme differs from both mammalian tartrate-resistant PAPs and tartrate-resistant metallophosphatase of Rana esculenta.

  3. Bovine brain low Mr acid phosphatase: purification and properties.

    PubMed

    Saeed, A; Tremori, E; Manao, G; Camici, G; Cappugi, G; Ramponi, G

    1990-01-01

    Low molecular weight acid phosphatase from bovine brain was purified to homogeneity using affinity chromatography on p-aminobenzylphosphonic acid-agarose to obtain the enzyme with both high specific activity (110 mumol min-1 mg-1 measured at pH 5.5 and 37 degrees C with p-nitrophenyl phosphate as substrate) and good yields. The enzyme was characterized with respect to molecular weight, amino acid composition, pH optimum, Km and Vmax in varying substrates, and to the Ki of varying inhibitors. Furthermore, transphosphorylation to glycerol was demonstrated by measuring the released p-nitrophenol/Pi concentration ratio during the initial phase of the catalyzed reaction. The enzyme was inactivated by iodoacetate and 1,2-cycloexanedione. Inorganic phosphate, a competitive inhibitor, protected the enzyme from being inactivated by the above compounds, demonstrating the involvement of both cysteine(s) and arginine(s) at the active site of the enzyme. Furthermore, the strong inhibition exerted by pyridoxal 5'-phosphate and the low inhibitory capacity possessed by the pyridoxal 5'-phosphate analogues pyridoxamine 5'-phosphate and pyridoxal, indicate that at least one lysine residue is present at the active site.

  4. Vanadate monomers and dimers both inhibit the human prostatic acid phosphatase.

    PubMed

    Crans, D C; Simone, C M; Saha, A K; Glew, R H

    1989-11-30

    A combination of enzyme kinetics and 51V NMR spectroscopy was used to identify the species of vanadate that inhibits acid phosphatases. Monomeric vanadate was shown to inhibit wheat germ and potato acid phosphatases. At pH 5.5, the vanadate dimer inhibits the human prostatic acid phosphatase whereas at pH 7.0 it is the vanadate monomer that inhibits this enzyme. The pH-dependent shift in the affinity of the prostatic phosphatase for vanadate is presumably due to deprotonation of an amino acid side chain in or near the binding site resulting in a conformational change in the protein. pH may be a subtle effector of the insulin-like vanadate activity in biological systems and may explain some of the differences in selectivity observed with the protein phosphatases.

  5. Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells.

    PubMed

    Kent, C; Vagelos, P R

    1976-06-17

    Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.

  6. Purification and characterization of a low-molecular-weight acid phosphatase--a phosphotyrosyl-protein phosphatase from bovine heart.

    PubMed

    Zhang, Z Y; Van Etten, R L

    1990-10-01

    A low-molecular-weight acid phosphatase that is representative of a group recently shown to be phosphotyrosyl protein phosphatases was purified to homogeneity from bovine heart. The enzyme was a monomer with a molecular mass of 18 kDa and had an isoelectric point of 7.0. The absorption coefficient, E1% 1cm was 9.65 at 280 nm. The enzyme had pH optima of 5.3 and 6.0 with the substrates p-nitrophenyl phosphate and tyrosine phosphate, respectively. When measured at pH 5 and 37 degrees C, the enzyme had specific activities of 114 and 86 mumol min-1 mg-1 for p-nitrophenyl phosphate and tyrosine O-phosphate, respectively, while the Km values were 0.38 and 14 mM. The enzyme was highly specific for aryl monophosphate esters and showed little or no activity toward aliphatic phosphate esters, with the remarkable exception of flavin mononucleotide (FMN) and certain of its structural analogs. As shown by 31P NMR data, the activity toward FMN was due to the hydrolysis of one of the eight components present in the (commercial) sample. Both molybdate and vanadate were potent inhibitors, with inhibition constants of 37 and 29 microM, respectively; tartrate and fluoride had little effect on enzymatic activity. A two-stage reversible denaturation of the enzyme by guanidine HCl was observed with midpoints of 0.25 and 1.75 M, respectively. The amino acid composition was homologous to the low-molecular-weight acid phosphatases from other tissue. The enzyme showed immunological cross-reactivity against low-molecular-weight human liver acid phosphatase. There were 7 or 8 accessible cysteines on the monomeric protein and at least one was essential for enzyme activity. The enzyme also had phosphotransferase activity, for example transferring phosphate from p-nitrophenyl phosphate to a wide variety of alcohol acceptors.

  7. The thermal stability of a castor bean seed acid phosphatase.

    PubMed

    Granjeiro, Paulo Afonso; Cavagis, Alexandre Donizeti Martins; de Campos Leite, Luciana; Ferreira, Carmen Veríssima; Granjeiro, José Mauro; Aoyama, Hiroshi

    2004-11-01

    The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45 degrees C using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55 degrees C. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60 degrees C. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60 degrees C, with the reaction products inorganic phosphate-P (10 mM) and p-nitrophenol-p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (Tm) value of 75 degrees C and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.

  8. Monomeric Tartrate Resistant Acid Phosphatase Induces Insulin Sensitive Obesity

    PubMed Central

    Lång, Pernilla; van Harmelen, Vanessa; Rydén, Mikael; Kaaman, Maria; Parini, Paolo; Carneheim, Claes; Cassady, A. Ian; Hume, David A.; Andersson, Göran; Arner, Peter

    2008-01-01

    Background Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP) is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer. Principal Findings Using mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity. Conclusion Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity. PMID:18320034

  9. Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C.

    PubMed

    Ahlemeyer, B; Selke, D; Schaper, C; Klumpp, S; Krieglstein, J

    2001-10-26

    The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.

  10. Synthesis of functionalized fluorescent gold nanoclusters for acid phosphatase sensing

    NASA Astrophysics Data System (ADS)

    Sun, Jian; Yang, Fan; Yang, Xiurong

    2015-10-01

    A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by introducing an alkaline aqueous solution of MUA into the GSH-Au+ complexes or AuNC@GSH solution. Subsequently, a reliable AuNC@GSH/MUA-based real-time assay of acid phosphatase (ACP) is established for the first time, inspired by the selective coordination of Fe3+ with surface ligands of AuNCs, the higher binding affinity between the pyrophosphate ion (PPi) and Fe3+, and the hydrolysis of PPi into orthophosphate by ACP. Our fluorescent chemosensor can also be applied to assay ACP in a real biological sample and, furthermore, to screen the inhibitor of ACP. This report paves a new avenue for synthesizing AuNCs based on either the bottom-up reduction or top-down etching method, establishing real-time fluorescence assays for ACP by means of PPi as the substrate, and further exploring the sensing applications of fluorescent AuNCs.A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by

  11. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    PubMed

    Leis, J F; Kaplan, N O

    1982-11-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

  12. Acid and alkaline phosphatase localization in the digestive tract mucosa of the Hemisorubim platyrhynchos.

    PubMed

    Faccioli, Claudemir Kuhn; Chedid, Renata Alari; Mori, Ricardo Hideo; Amaral, Antônio Carlos do; Franceschini-Vicentini, Irene Bastos; Vicentini, Carlos Alberto

    2016-09-01

    This cytochemical study investigated the acid and alkaline phosphatase of the digestive tract of Hemisorubim platyrhynchos. Acid phosphatase was detected in the lining epithelium throughout the digestive tract, whereas alkaline phosphatase was only observed in the intestine. In the esophagus, an acid phosphatase reaction occurred in the apical cytoplasm of the epithelial cells and was related to epithelial protection and freeing of superficial cells for sloughing. Similar results were also observed in epithelial cells of gastric epithelium. In the gastric glands, acid phosphatase occurred in lysosomes of the oxynticopeptic cells acting in the macromolecule degradation for use as an energy source, whereas in the vesiculotubular system, its presence could be related to secretion processes. Furthermore, acid phosphatase in the intestine occurred in microvilli and lysosomes of the enterocytes and was correlated to absorption and intracellular digestion. However, no difference was reported among the regions of the intestine. However, alkaline phosphatase reaction revealed a large number of reaction dots in the anterior intestine, with the number decreasing toward the posterior intestine. This enzyme has been related to several functions, highlighting its role in the nutrient absorption primarily in the anterior intestine but also being essential in pH regulation because this is a carnivorous species with many gastric glands with secretions that could damage the intestine.

  13. Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase.

    PubMed

    Story, Sandra; Brigmon, Robin L

    2017-03-01

    Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. These results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications.

  14. Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

    PubMed Central

    Hadler, Kieran S; Huber, Thomas; Cassady, A Ian; Weber, Jane; Robinson, Jodie; Burrows, Allan; Kelly, Gregory; Guddat, Luke W; Hume, David A; Schenk, Gerhard; Flanagan, Jack U

    2008-01-01

    Background Tartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics. Findings A new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine. Conlusion The gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism. PMID:18771593

  15. A critical evaluation of a specific radioimmunoassay for prostatic acid phosphatase

    SciTech Connect

    Goldenberg, S.L.; Silver, H.K.; Sullivan, L.D.; Morse, M.J.; Archibald, E.L.

    1982-11-01

    A radioimmunoassay (RIA) method for acid phosphatase detection was compared to a standard enzyme assay using sera from 210 normal volunteers and 285 patients with prostatic disease. Statistical and clinical comparisons were made between defined subgroups. All 55 normal females had RIA detectable serum acid phosphatase, implying that this assay cannot be entirely specific for enzyme of prostatic origin. Urinary catheterization did not affect acid phosphatase levels. In all stages of carcinoma there were more acid phosphatase elevations by the RIA method than enzyme method, but neither assay could differentiate intercapsular cancer from benign prostatic hyperplasia. A small number of patients with biopsy proven negative nodules had marginally elevated values, suggesting an obligation for closer follow-up. The RIA method may be superior for monitoring patients with more advanced malignancy. Additional practical advantages of the RIA include relative simplicity and elimination of the special serum handling required for the enzyme assay.

  16. Optimization of the tartrate-resistant acid phosphatase detection by histochemical method

    PubMed Central

    Galvão, M.J.; Santos, A. R.; Ribeiro, M.D.; Ferreira, A.; Nolasco, F.

    2011-01-01

    According to the new kidney disease improving global outcomes (KDIGO) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling, the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 µm were stained to identify TRACP at different incubation temperatures (37°, 45°, 60°, 70° and 80°C) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60°C and 70°C. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 min, the reaction should be carried out at 60

  17. An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells.

    PubMed

    Yang, T T; Sinai, P; Kain, S R

    1996-10-01

    We describe an acid phosphatase assay for determination of cell growth based on quantification of cytosolic acid phosphatase activity. The assay is based on the hydrolysis of the p-nitrophenyl phosphate by intracellular acid phosphatases in viable cells to produce p-nitrophenol. For all cell types examined, absorbance of p-nitrophenol at 405 nm is directly proportional to the cell number in the range of 10(3)-10(5) cells. The assay can quantify as few as 1000 cells per well in 96-well microtiter plates. The acid phosphatase assay was used to count various adherent and nonadherent cells, including human tumors, L6, and HT-2 cells. We also demonstrate the utility of this assay for analysis of growth factor and cytokine bioactivity on mammalian cells in culture. In comparison to [3H]thymidine incorporation, the acid phosphatase assay has similar sensitivity but a wider linear response range. The method also shows higher sensitivity and reproducibility in comparison to cell proliferation assays based on the reduction of tetrazolium salts. Because of the ease of use, sensitivity, and low cost, the acid phosphatase method is especially suited to applications where a large number of samples are assayed.

  18. Synthesis of repressible acid phosphatase in Saccharomyces cerevisiae under conditions of enzyme instability.

    PubMed Central

    Bostian, K A; Lemire, J M; Halvorson, H O

    1982-01-01

    The synthesis of repressible acid phosphatase in Saccharomyces cerevisiae was examined under conditions of blocked derepression as described by Toh-e et al. (Mol. Gen. Genet. 162:139-149, 1978). Based on a genetic and biochemical analysis of the phenomenon these authors proposed a new regulatory model for acid phosphatase expression involving a simultaneous interaction of regulatory factors in the control of structural gene transcription. We demonstrate here that under growth conditions that fail to produce acid phosphatase the enzyme is readily inactivated. Furthermore, we demonstrate under these conditions the production of acid phosphatase mRNA which is active both in vitro and in vivo in the synthesis of enzyme. This eliminates any step prior to translation of acid phosphatase polypeptide as an explanation for the phenomenon. We interpret our results for the block in appearance of acid phosphatase as a result of both deaccelerated growth and cellular biosynthesis during derepression, accompanied by an enhanced instability of the enzyme. Images PMID:7050664

  19. Improvement of an acid phosphatase/DHAP-dependent aldolase cascade reaction by using directed evolution.

    PubMed

    van Herk, Teunie; Hartog, Aloysius F; Babich, Lara; Schoemaker, Hans E; Wever, Ron

    2009-09-04

    To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild-type enzyme at pH 6.0. This variant is 17 times more active than the wild-type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.

  20. Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus.

    PubMed

    Weinberg, R A; Zusman, D R

    1990-05-01

    One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and

  1. Inhibition of acid, alkaline, and tyrosine (PTP1B) phosphatases by novel vanadium complexes.

    PubMed

    McLauchlan, Craig C; Hooker, Jaqueline D; Jones, Marjorie A; Dymon, Zaneta; Backhus, Emily A; Greiner, Bradley A; Dorner, Nicole A; Youkhana, Mary A; Manus, Lisa M

    2010-03-01

    In the course of our investigations of vanadium-containing complexes for use as insulin-enhancing agents, we have generated a series of novel vanadium coordination complexes with bidentate ligands. Specifically we have focused on two ligands: anthranilate (anc(-)), a natural metabolite of tryptophan, and imidizole-4-carboxylate (imc(-)), meant to mimic naturally occurring N-donor ligands. For each ligand, we have generated a series of complexes containing the V(III), V(IV), and V(V) oxidation states. Each complex was investigated using phosphatase inhibition studies of three different phosphatases (acid, alkaline, and tyrosine (PTP1B) phosphatase) as prima facia evidence for potential use as an insulin-enhancing agent. Using p-nitrophenyl phosphate as an artificial phosphatase substrate, the levels of inhibition were determined by measuring the absorbance of the product at 405nm using UV/vis spectroscopy. Under our experimental conditions, for instance, V(imc)(3) appears to be as potent an inhibitor of alkaline phosphatase as sodium orthovanadate when comparing the K(cat)/K(m) term. VO(anc)(2) is as potent an inhibitor of acid phosphatase and tyrosine phosphatase as the Na(3)VO(4). Thus, use of these complexes can increase our mechanistic understanding of the effects of vanadium in vivo.

  2. Determination of acid phosphatase in biological fluids using a new substrate, 2,6-dichloro-4-nitrophenyl phosphate.

    PubMed

    Teshima, S; Hayashi, Y; Ando, M

    1987-09-30

    A new substrate, 2,6-dichloro-4-nitrophenyl phosphate (DCNP-P), is used for the determination of acid phosphatase (EC 3.1.3.2) in serum and urine. It was hydrolyzed by acid phosphatase to 2,6-dichloro-4-nitrophenol (DCNP) and phosphoric acid. At a pH of 4.5-6.0, the absorption of DCNP liberated by acid phosphatase was much higher than that of p-nitrophenol, which is commonly used as an aglycone in the acid phosphatase assay. By using DCNP-P as a substrate for acid phosphatase activity, determinations can be made without the colour reaction which requires the addition of an alkaline solution, and can be determined by the rate assay that does not require measurement of sample blanks in serum or urine. This method using DCNP-P is highly sensitive and is the most suitable for the rate assay of acid phosphatase activity in biological fluids.

  3. Renal neuroendocrine tumour and synchronous pancreas metastasis: histopathological diagnosis using prostatic acid phosphatase.

    PubMed

    Kawasaki, Keishi; Kawaguchi, Yoshikuni; Suzuki, Yoshio; Tanaka, Nobutaka

    2016-11-01

    A woman aged 56 years developed 2 synchronous tumours: one, 1.2 cm in diameter at the head of the pancreas; and the other, 4.0 cm in diameter, at the left side of her horseshoe kidney. Preoperative differential diagnosis of these hypovascular lesions included pancreatic ductal carcinoma (PDC) with renal metastasis, PDC with renal angiomyolipoma, renal cell carcinoma with pancreatic metastasis or PDC and renal cell carcinoma. Following pancreaticoduodenectomy and left nephrectomy, both specimens were diagnosed as grade 2 neuroendocrine tumours (NETs). Immunohistochemistry revealed that both were positive for prostatic acid phosphatase (PAP), which is specific to hindgut-derived NET, including renal NET. Accordingly, the renal tumour was diagnosed as the primary lesion, and the pancreatic tumour as a metastasis. To the best of our knowledge, this is the first report of a renal NET with a synchronous pancreas metastasis. Immunohistochemical staining for PAP was a useful diagnostic marker for synchronous NETs in the kidney and pancreas.

  4. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

    PubMed Central

    Roth, B L; Poot, M; Yue, S T; Millard, P J

    1997-01-01

    A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility. PMID:9172364

  5. Immobilization of acid phosphatase from Vigna aconitifolia seeds on chitosan beads and its characterization.

    PubMed

    Srivastava, Pramod Kumar; Anand, Asha

    2014-03-01

    Acid phosphatase isolated from Vigna aconitifolia seeds was immobilized onto glutaraldehyde activated chitosan beads by crosslinking method. Chitosan beads activated with 2% of glutaraldehyde have demonstrated maximum immobilization yield (∼ 83%). The immobilized enzyme showed optimum activity at pH 7.0, while soluble form was maximally active in acidic range (pH 5.0). With respect to free form, immobilized acid phosphatase showed better activity in alkaline range. On the other side, immobilization does not affect the optimum temperature range i.e., both, soluble and immobilized acid phosphatase exhibited maximum activity at 60 °C. The Km and Vmax values for the immobilized enzyme were calculated to be 0.37 mM and 13.5 U/mg. The immobilization on chitosan beads enhanced the shelf life of acid phosphatase. The immobilized enzyme retained its more than 50% hydrolytic activity for approximately two months. The immobilized acid phosphatase was reusable for more than 40 cycles of reaction.

  6. Electrophoretic separation of alkaline and acid phosphatase isoenzymes from the pulp of monkey teeth.

    PubMed

    Franzén, A; Hasselgren, G

    1978-01-01

    Monkey pulps were homogenized in a Triton tris solution. After three centrifugation steps (800, 20000, and 105000 g) the supernatant was applied on acryl amide columns at pH 7.5 in a tris-diethyl barbituric acid buffer. Electrophoresis was performed at a constant current of 2.5 mA per gel column at 18--20 degrees C. Incubations for alkaline phosphatase (E.C. 3.1.3.1) were carried out at pH 8.3 using naphthol-AS-MX-phosphate as substrate and Fast Red Violet LB salt as coupler. Incubations for acid phosphatase (E.C. 3.1.3.2) were undertaken at pH 5.0 using alpha-naphtyl phosphate as substrate and hexazotized pararosanilin as coupling agent. After the incubations for alkaline phosphatase as well as acid phosphatase two bands showing enzyme activity were demonstrated. By means of treatment with heat (56 degrees C) prior to incubation or addition of vanadate or pyrophosphate to the incubation medium it was shown that the main part of the fast moving alkaline phosphatase band was sensitive to these procedures. The alkaline phosphatase of the slow moving band appeared to be resistant to heat or the addition of inhibitors.

  7. Partial purification and kinetic characterization of acid phosphatase from garlic seedling.

    PubMed

    Yenigün, Begüm; Güvenilir, Yüksel

    2003-01-01

    The objective of this study was to obtain purer acid phosphatases than produced by prior art by operating under conditions that improve the final product. The study features are the use of a mild nonionic detergent, 40-80% saturation with (NH4)2SOm4, maintained at low temperature to remove impurity, and the use of chromatografic columns to concentrate the acid phosphatase and remove non-acid phosphatase proteins with lower or higher molecular weights. Acid phosphatase was isolated and purified from garlic seedlings by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Grown garlic seedlings of 10- 15 cm height were homogenized with 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. After homogenization, the supernatant was filtered with paper filters. Filtrated supernatant was cooled to 4 degrees C, followed by a threestep fractionation of the proteins with ammonium sulfate. The crude enzyme was isolated as a green precipitate that was dissolved in a small amount of 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. Garlic seedling acid phosphatase was purified with ion-exchange chromatography (DEAE cellulose). The column was equilibrated with 0.1 M acetate buffer. Acid phosphatase was purified 40-fold from the starting material. The specific activity of the pure enzyme was 168 U/mg. A variety of stability and activity profiles were determined for the purified garlic seedling acid phosphatase: optimum pH, optimum temperature, pH stability, temperature stability, thermal inactivation, substrate specificity, effect of enzyme concentration, effect of substrate concentration, activation energy, and effect of inhibitor and activator. The molecular mass of acid phosphatase was estimated to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH was 5.7 and the optimum temperature was 50 degrees C. The enzyme was stable at pH 4.0-10.0 and 40-60 degrees C

  8. [Spectroscopic analysis of the interaction of ethanol and acid phosphatase from wheat germ].

    PubMed

    Xu, Dong-mei; Liu, Guang-shen; Wang, Li-ming; Liu, Wei-ping

    2004-11-01

    Conformational and activity changes of acid phosphatase from wheat germ in ethanol solutions of different concentrations were measured by fluorescence spectra and differential UV-absorption spectra. The effect of ethanol on kinetics of acid phosphatase was determined by using the double reciprocal plot. The results indicate the ethanol has a significant effect on the activity and conformation of acid phosphatase. The activity of acid phosphatase decreased linearly with increasing the concentration of ethanol. Differential UV-absorption spectra of the enzyme denatured in ethanol solutions showed two positive peaks at 213 and 234 nm, respectively. The peaks on the differential UV-absorption spectra suggested that the conformation of enzyme molecule changed from orderly structure to out-of-order crispation. The fluorescence emission peak intensity of the enzyme gradually strengthened with increasing ethanol concentration, which is in concordance with the conformational change of the microenvironments of tyrosine and tryptophan residues. The results indicate that the expression of the enzyme activity correlates with the stability and integrity of the enzyme conformation to a great degree. Ethanol is uncompetitive inhibitor of acid phosphatase.

  9. Vanadate inhibition of fungal phyA and bacterial appA2 histidine acid phosphatases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could also serve as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shutdown by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading ac...

  10. Clostridium stain which produces acetic acid from waste gases

    DOEpatents

    Gaddy, James L.

    1997-01-01

    A method and apparatus for converting waste gases from industrial processes such as oil refining, carbon black, coke, ammonia, and methanol production, into useful products. The method includes introducing the waste gases into a bioreactor where they are fermented to various organic acids or alcohols by anaerobic bacteria within the bioreactor. These valuable end products are then recovered, separated and purified. In an exemplary recovery process, the bioreactor raffinate is passed through an extraction chamber into which one or more non-inhibitory solvents are simultaneously introduced to extract the product. Then, the product is separated from the solvent by distillation. Gas conversion rates can be maximized by use of centrifuges, hollow fiber membranes, or other means of ultrafiltration to return entrained anaerobic bacteria from the bioreactor raffinate to the bioreactor itself, thus insuring the highest possible cell concentration.

  11. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    SciTech Connect

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2006-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  12. Type II phosphoinositide 5-phosphatases have unique sensitivities towards fatty acid composition and head group phosphorylation.

    PubMed

    Schmid, Annette C; Wise, Helen M; Mitchell, Christina A; Nussbaum, Robert; Woscholski, Rüdiger

    2004-10-08

    The catalytic properties of the type II phosphoinositide 5-phosphatases of Lowe's oculocerebrorenal syndrome, INPP5B, Synaptojanin1, Synaptojanin2 and SKIP were analysed with respect to their substrate specificity and enzymological properties. Our data reveal that all phosphatases have unique substrate specificities as judged by their corresponding KM and VMax values. They also possessed an exclusive sensitivity towards fatty acid composition, head group phosphorylation and micellar presentation. Thus, the biological function of these enzymes will not just be determined by their corresponding regulatory domains, but will be distinctly influenced by their catalytic properties as well. This suggests that the phosphatase domains fulfil a unique catalytic function that cannot be fully compensated by other phosphatases.

  13. The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

    PubMed

    Srivastava, Pramod Kumar; Anand, Asha

    2015-01-01

    Sensitivity of acid phosphatase from Vigna aconitifolia seeds to metal ions, fluoride, and phosphate was examined. All the effectors had different degree of inhibitory effect on the enzyme. Among metal ions, molybdate and ferric ion were observed to be most potent inhibitors and both exhibited mixed type of inhibition. Acid phosphatase activity was inhibited by Cu2+ in a noncompetitive manner. Zn and Mn showed mild inhibition on the enzyme activity. Inhibition kinetics analysis explored molybdate as a potent inhibitor for acid phosphatase in comparison with other effectors used in this study. Fluoride was the next most strong inhibitor for the enzyme activity, and caused a mixed type of inhibition. Phosphate inhibited the enzyme competitively, which demonstrates that inhibition due to phosphate is one of the regulatory factors for enzyme activity.

  14. [Effect of acetic acid on adsorption of acid phosphatase by some soil colloids and clay minerals].

    PubMed

    Zhao, Zhenhua; Huang, Qiaoyun; Jiang, Xin; Yu, Guifen; Wang, Fang; Li, Xueyuan

    2004-03-01

    This paper studied the effect of acetic acid with different concentrations and pH values on the adsorption of acid phosphatase by some soil colloids and clay minerals (SCCM). The results showed that the pH values for the maximum adsorption of the enzyme were between the IEP of the enzyme and the PZC of SCCM. In the acetic acid systems, the amount of the enzyme adsorbed by SCCM was in the order of goethite > yellow brown soil > latosol > kaolinite > delta-MnO2. A remarkable influence of acetic acid concentration on the adsorption amount and the binding energy of the enzyme was observed. With the increase of the concentration from 0 to 200 mmol.L-1 in the system, acetic acid exhibited an enhanced effect, followed by an inhibition action on the adsorption of the enzyme on SCCM. The changes of the binding energy (K value) for the enzyme on SCCM were on the contrary to those of the maximum adsorption. The possible mechanisms for the influence of acetic acid on the adsorption of enzyme by SCCM were also discussed.

  15. Acid- and alkaline phosphatase in amniotic fluid in normal and complicated pregnancy.

    PubMed

    Beckman, G; Beckman, L; Löfstrand, T

    1978-01-01

    171 samples of amniotic fluid were obtained by abdominal amniocentesis from 67 women with complicated pregnancies (isoimmunization, diabetes mellitus or toxaemia). The levels of heat-labile alkaline phosphatase (HLAP), heat-stable alkaline phosphatase (HSAP) and acid phosphatase (AcP) were determined and compared to the enzyme levels in 179 samples from women with normal pregnancies of corresponding gestational ages. HLAP showed two "peaks" of activity, one in the 5th-22nd week and the other at term. HSAP and AcP showed increased activity at term. HSAP was decreased (p less than 0.01) in isoimmunization between the 36th and 40th week. 11 cases of toxaemia with placental insufficiency showed no differences in the levels of HLAP and HSAP compared with normal pregnancy. AcP showed no differences between normal and complicated pregnancy. Samples contaminated by blood showed no significant increase in the acid- and alkaline phosphatase levels. Samples contaminated by meconium showed a complex pattern. Some samples had normal enzyme levels, some had high levels of HLAP only and some had high levels of HSAP and AcP. The origin of the enzymes is not known with certainty. HSAP in amniotic fluid is most likely not of placental but intestinal origin. Determinations of acid- and alkaline phosphatase in amniotic fluid seem to be of little values in the clinical management of complicated pregnancy.

  16. Studies on the oligosaccharide heterogeneity of the isoelectric forms of the lower molecular weight acid phosphatase of frog liver.

    PubMed

    Kubicz, A; Szalewicz, A; Chrambach, A

    1991-01-01

    1. The lower molecular weight, heterogeneous acid phosphatase (AcPase) from the frog liver (Rana esculenta) containing AcPase I, II, III and IV was separated into enzymatically active components by isoelectric focusing in an immobilized pH gradient. 2. The blotted enzyme bands were characterized by their different binding patterns obtained with the lectins concanavalin A, wheat germ agglutinin (WGA), Lens culinaris hemagglutinin (LcH) and peanut agglutinin (PNA). 3. In situ neuraminidase treatment reduced the staining intensity of some WGA-bands and increased that of PNA-bands. 4. The finding that AcPases I, II, III and IV differ in their carbohydrate chain composition, together with previous results showing different bioactivities of AcPases III and IV, indicates a correlation between the glycosylation state of enzyme forms and their physiological action.

  17. In vitro effect of agriculture pollutants and their joint action on Pseudokirchneriella subcapitata acid phosphatase.

    PubMed

    Jonsson, Claudio Martín; Aoyama, Hiroshi

    2007-10-01

    Acid phosphatase plays important roles in algae metabolism such as availability and recycling of inorganic phosphate, autophagic digestive processes and fertilization. Chemicals released into the environment from agriculture activities may impair algae phosphatase activity. The aim of this work was to evaluate the in vitro effect of twenty-four organic compounds and six metals used as pesticides, or present as contaminants in sewage sludge, on the acid phosphatase activity extracted from Pseudokirchneriella subcapitata. Results demonstrated that only the linear surfactant alkyl benzenesulphonate (LAS) and the heavy metals Hg(2+), Al(3+) and Cu(2+) markedly altered (50%) the enzyme activity. Join action inhibition studies indicated that Hg(2+) was more potent inhibitor than Al(3+) or LAS, and that the Hg(2+)+Al(3+) and Hg(2+)+LAS mixtures have, respectively, additive and slight antagonism effects. Copper, which demonstrated an activator effect when preincubated with the enzyme, behaved as a slight antagonist for the inhibitor effect of Hg(2+).

  18. Methods of staining target chromosomal DNA employing high complexity nucleic acid probes

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Ol'li-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

    2006-10-03

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  19. Detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining.

    PubMed

    Tabatabaei Shafiei, Mahdieh; Carvajal Gonczi, Catalina M; Rahman, Mohammed Samiur; East, Ashley; François, Jonathan; Darlington, Peter J

    2014-12-23

    Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

  20. [Comparative effects of fluoride on three enzymes, hydrolyzing pyrophosphate - acid and alkaline phosphatases and inorganic pyrophosphatase].

    PubMed

    Kasho, V N; Baĭkov, A A; Avaeva, S M

    1982-08-01

    The effects of fluoride on the activities of acid phosphatase (EC 3.1.3.2) from potato and alkaline phosphatase (EC 3.1.3.1) from E. coli during pyrophosphate and p-nitrophenylphosphate hydrolysis and on the activities of inorganic pyrophosphatase (EC 3.6.1.1) from baker's yeast during pyrophosphate hydrolysis were compared. For both phosphatases the type of interaction was found to be independent on the nature of substrate. For acid phosphatase and inorganic pyrophosphatase the inhibition was of non-competitive and uncompetitive types, respectively. In the case of alkaline phosphatase fluoride increased the rate of p-nitrophenol release during p-nitrophenylphosphate hydrolysis at pH greater than or equal to 7.9 without affecting the rate of phosphate release, which is indicative of fluorophosphate formation in the course of the transphosphorylation reaction. The data obtained suggest the existence of essential differences in the mechanisms of fluoride effects on the three enzymes under study.

  1. Purification of a tartrate-resistant acid phosphatase (TrACP) from bovine cortical bone matrix

    SciTech Connect

    Lau, K.H.W.; Freeman, T.K.; Baylink, D.J.

    1986-05-01

    It has been previously demonstrated that a partially purified bovine skeletal TrACP showed protein phosphatase (P'ase) activity that was specific for phosphotyrosyl (Ptyr) proteins. They have now purified TrACP activity from bovine cortical bone matrix to apparent homogeneity. The purification procedures included CM-Sepharose ion-exchange, cellulose phosphate affinity, sephacryl S-300 gel filtration and phenyl sepharose affinity chromatographies. Overall yield was > 25% and purification was approximately 2000-fold with a specific activity of 8.15 umol pNPP hydrolyzed/min/mg protein at 37/sup 0/C. The purified enzyme was judged to be homogeneous based on: (i) appearance as a single protein band on SDS-PAGE (silver staining technique) and (ii) distribution analysis of radioiodinated purified TrACP after SDS-PAGE revealing one band of radioactivity at the same positions as the TrACP protein band. M.W. of TrACP was 34,600 as assessed by gel filtration and 32,500 by SDS-PAGE, suggesting that bovine skeletal TrACP exists as active monomer. Analysis of the purified TrACP by isoelectric focusing showed at least 9 bands of enzyme activities with pIs between 4 and 5, indicating micro-heterogenecity. Substrate specificity analyses revealed that the purified TrACP also hydrolyzed nucleotide tri- and di-phosphates, but not monophosphates or other low M.W. phosphoryl esters, and was also capable of hydrolyzing phosphotyrosine (Tyr(P)) and Ptyr proteins with little activity toward other phosphoamino acids or phosphoseryl proteins. Optimal pH was 5.5 for TrACP activity, 6.0 for Tyr(P) P'ase activity and 7.0 for Ptyr protein P'ase activity. Results of these studies represent the first purification of a skeletal TrACP to apparent homogeneity.

  2. PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

  3. Acid phosphatase activity: a marker of androgen action in prostate explant cultures.

    PubMed

    Shao, T C; Kong, A Y; Cunningham, G R

    1987-01-01

    Acid phosphatase activity in rat ventral prostate explants has been assayed to determine if this parameter could serve as a specific and quantitative marker of androgen action in this in vitro model. Dihydrotestosterone (10 nM) caused an absolute increase in both total (42.5 +/- 2.9 vs control 27.1 +/- 4.0 nmoles p-nitrophenol generated in 30 min/micrograms DNA, P less than .01) and tartrate-resistant acid phosphatase activity (34.1 +/- 1.5 vs control 17.2 +/- 2.8 U/micrograms DNA, P less than .05), and this effect was maximal on the 4th day of culture. This was the time when explant weight and DNA content tended to fall or only to be maintained by androgen. Similar changes were observed with the potent synthetic androgen, mibolerone. The addition of either the antiandrogen cyproterone acetate or flutamide in a 100-fold excess to that of androgen caused significant inhibition in acid phosphatase activity. No significant change was observed at low concentrations of estradiol or progesterone, and only minimal and inconsistent increases in activity were noted at high concentrations. No increase was noted when cortisol, cyproterone acetate, or flutamide was added to the media. We conclude that measurement of acid phosphatase activity in cultured explants of rat ventral prostate provides a biochemical marker of androgenicity that is more specific than measurement of [3H]-thymidine incorporation.

  4. ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA

    EPA Science Inventory

    An acid phosphatase activity from the aquatic plant Spirodela oligorhiza (duckweed) was isolated and partially characterized. S. oligorhiza was grown in a hydroponic growth medium, harvested, and ground up in liquid nitrogen. The ground plant material was added to a biological ...

  5. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  6. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  7. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  8. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  9. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  10. Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background and Aims Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparat...

  11. Alterations in activities of acid phosphatase, alkaline phosphatase, ATPase and ATP content in response to seasonally varying Pi status in okra (Abelmoschus esculentus).

    PubMed

    Sen, Supatra; Mukherji, S

    2004-04-01

    Phosphorus (P) is the second most important macronutrient for plant growth. Plants exhibit numerous physiological and metabolic adaptations in response to seasonal variations in phosphorus content. Activities of acid and alkaline phosphatases, ATPase and ATP content were studied in summer, rainy and winter seasons at two different developmental stages (28 and 58 days after sowing) in Okra. Activities of both acid and alkaline phosphatases increased manifold in winter to cope up with low phosphorus content. ATP content and ATPase activity were high in summer signifying an active metabolic period. Phosphorus deficiency is characterized by low ATP content and ATPase activity (which are in turn partly responsible for a drastic reduction in growth and yield) and enhanced activities of acid and alkaline phosphatases which increase the availability of P in P-deficient seasons.

  12. Hydrolytic and alcoholytic dephosphorylation of nucleotides by acid phosphatase in the presence of ethanol.

    PubMed

    Tomaszewski, M; Buchowicz, J

    1971-08-01

    The effect of ethanol on the activity of acid phosphatase from wheat germ was studied, by using ribonucleoside monophosphates as the enzyme substrates. The nucleotides were effectively degraded to the corresponding nucleosides in the presence of ethanol at all concentrations tested, including a 96% (v/v) solution. However, the nucleotide dephosphorylation was accompanied by the liberation of orthophosphate only when the concentration of ethanol in the assay mixture did not exceed 15%. No inorganic phosphate was liberated when ethanol was present at higher concentrations. Instead, monoethyl phosphate was formed in quantities expected for orthophosphate. The results are explained in terms of phosphatase-catalysed alcoholysis.

  13. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-09

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  14. Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas).

    PubMed

    Durmus, A; Eicken, C; Spener, F; Krebs, B

    1999-09-14

    The sequence of cDNA fragments of two isozymes of the purple acid phosphatase from sweet potato (spPAP1 and spPAP2) has been determined by 5' and 3' rapid amplification of cDNA ends protocols using oligonucleotide primers based on amino acid information. The encoded amino acid sequences of these two isozymes show an equidistance of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (kbPAP). A three-dimensional model of the active site has been constructed for spPAP2 on the basis of the kbPAP crystallographic structure that helps to explain the reported differences in the visible and EPR spectra of spPAP2 and kbPAP.

  15. Alkaline phosphatase of Physarum polycephalum is insoluble.

    PubMed

    Furuhashi, Kiyoshi

    2008-02-01

    The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg(2+)-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.

  16. Okadaic acid-induced inhibition of B-50 dephosphorylation by presynaptic membrane-associated protein phosphatases.

    PubMed

    Han, Y F; Dokas, L A

    1991-10-01

    The neuronal tissue-specific protein kinase C (PKC) substrate B-50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane-associated B-50 phosphatase activity by using okadaic acid (OA) and purified 32P-labeled substrates. At a low concentration of [gamma-32P]ATP, PKC-mediated [32P]phosphate incorporation into B-50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B-50 at 2 nM, a dose that inhibits PP-2A. At the higher concentration of 1 microM, a dose of OA that inhibits PP-1 as well as PP-2A, a nearly complete blockade of B-50 dephosphorylation was seen. Heat-stable PP inhibitor-2 (I-2) also inhibited dephosphorylation of B-50. The effects of OA and I-2 on B-50 phosphatase activity were additive. Endogenous PP-1- and PP-2A-like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P]phosphorylase a and [32P]casein. With these exogenous substrates, sensitivities of the membrane-bound phosphatases to OA and I-2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP-1- and PP-2A-like enzymes are the major B-50 phosphatases in SPMs.

  17. LEPS2, a Phosphorus Starvation-Induced Novel Acid Phosphatase from Tomato1

    PubMed Central

    Baldwin, James C.; Karthikeyan, Athikkattuvalasu S.; Raghothama, Kashchandra G.

    2001-01-01

    Phosphate (Pi) is one of the least available plant nutrients found in the soil. A significant amount of phosphate is bound in organic forms in the rhizosphere. Phosphatases produced by plants and microbes are presumed to convert organic phosphorus into available Pi, which is absorbed by plants. In this study we describe the isolation and characterization of a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase. LePS2 is a member of a small gene family in tomato. The cDNA is 942 bp long and contains an open reading frame encoding a 269-amino acid polypeptide. The amino acid sequence of LePS2 has a significant similarity with a phosphatase from chicken. Distinct regions of the peptide also share significant identity with the members of HAD and DDDD super families of phosphohydrolases. Many plant homologs of LePS2 are found in the databases. The LePS2 transcripts are induced rapidly in tomato plant and cell culture in the absence of Pi. However, the induction is repressible in the presence of Pi. Divided root studies indicate that internal Pi levels regulate the expression of LePS2. The enhanced expression of LePS2 is a specific response to Pi starvation, and it is not affected by starvation of other nutrients or abiotic stresses. The bacterially (Escherichia coli) expressed protein exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate. The pH optimum of the enzyme activity suggests that LePS2 is an acid phosphatase. PMID:11161030

  18. Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction

    PubMed Central

    Lee, Eunhee; Stafford, III, Walter F.

    2015-01-01

    Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724–837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991–1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998–1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction. PMID:26445108

  19. Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

    PubMed

    Lee, Eunhee; Stafford, Walter F

    2015-01-01

    Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

  20. Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

    PubMed

    Bozzo, Gale G; Raghothama, Kashchandra G; Plaxton, William C

    2002-12-01

    Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension cells. SAP1 and SAP2 were resolved during cation-exchange FPLC of culture media proteins from 8-day-old Pi-starved cells, and purified to homogeneity and final p-nitrophenylphosphate hydrolyzing specific activities of 246 and 940 micro mol Pi produced.min-1 mg.protein-1, respectively. SDS/PAGE, periodic acid-Schiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers. SAP1 and SAP2 are purple acid phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by l-tartrate. The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs. CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct. Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable. Although they exhibited wide substrate specificities, the Vmax of SAP2 with various phosphate-esters was significantly greater than that of SAP1. SAP1 and SAP2 were activated by up to 80% by 5 mm Mg2+, and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively. Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg2+ or molybdate. This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.

  1. An acid phosphatase locus expressed in mouse kidney (Apk) and its genetic location on chromosome 10.

    PubMed

    Womack, J E; Auerbach, S B

    1978-04-01

    A genetic locus controlling the electrophoretic mobility of an acid phosphatase in mouse kidney is described. This locus, called acid phosphatase-kidney (Apk), is not expressed in erythrocytes, liver, spleen, heart, lung, brain, skeletal muscle, stomach, or testes. The product of Apk hydrolyzes the substrate naphthol AS-MX phosphoric acid but is not active on alpha-naphthylphosphate or 4-methylumbelliferylphosphate. It is not inactivated by 50 C for 1 hr, nor is its electrophoretic mobility altered by incubation with neuraminidase. The locus is invariant among 31 inbred strains (Apka), with a variant allele (Apkm) observed only in Mus musculus molossinus. Codominant expression was observed in F1 hybrids of M. m. molossinus and inbred strains. Apk was mapped on Chr 10, near the neurological mutant waltzer (v).

  2. Bioactive enzyme-metal composites: the entrapment of acid phosphatase within gold and silver.

    PubMed

    Ben-Knaz, Racheli; Avnir, David

    2009-03-01

    This paper is concerned with the entrapment of an enzyme within an aggregated metallic matrix and the development of a bioactive enzyme-metal composite. Whereas the use of organic polymers and metal oxides for the preparation of enzymatically active materials is well developed, the third principle enzyme-material combination, namely protein-metal bulk, has not yet been reported. A new methodology for the entrapment of organic molecules and polymers within metals has been employed for the preparation of bioactive acid phosphatase@gold and acid phosphatase@silver, according to which room temperature reduction of the metal cation is carried out in the presence of the enzyme to be entrapped. Protectability of the entrapped enzyme against harsh conditions is shown: the acidic enzyme is kept alive under basic conditions.

  3. Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1.

    PubMed

    Maynes, J T; Bateman, K S; Cherney, M M; Das, A K; Luu, H A; Holmes, C F; James, M N

    2001-11-23

    Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 A. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive beta12-beta13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.

  4. Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

    SciTech Connect

    Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2010-12-08

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  5. Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase.

    PubMed

    Singh, Harkewal; Schuermann, Jonathan P; Reilly, Thomas J; Calcutt, Michael J; Tanner, John J

    2010-12-10

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5'-nucleotides and 3'-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5' substrates in an anti conformation and 3' substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  6. Purification, enzymatic properties, and active site environment of a novel manganese(III)-containing acid phosphatase.

    PubMed

    Sugiura, Y; Kawabe, H; Tanaka, H; Fujimoto, S; Ohara, A

    1981-10-25

    A new manganese-containing acid phosphatase has been isolated and crystallized from sweet potato tubers. The pure enzyme contains one atom of manganese per Mr = 110,000 polypeptide and shows phosphatase activity toward various phosphate substrates. The pH optimum of the enzyme was 5.8 and the enzyme activity was inhibited by Cu2+, Zn2+, Hg2+, AsO43-, and MoO42-. This stable metalloenzyme is red-violet in color with an intense absorption band at 515 nm (epsilon - 2460). Our electronic, circular dichroism, and electron spin resonance findings strongly indicate that the Mn-valence state of the native enzyme is trivalent. When the Mn-enzyme is excited by the 5145 A line of Ar+ laser, prominent Raman lines at 1230, 1298, 1508, and 1620 cm-1 were detected. This Raman spectrum can probably be interpreted in terms of internal vibration of a coordinated tyrosine phenolate anion. The tryptophan-modified enzyme showed a positive Raman band at 370 cm-1, which is preferentially assigned to a Mn(III)-S streching mode. The modification of the Mn-enzyme by N-bromosuccinimide led to a large decrease in the fluorescence intensity of 335 nm which was dominated by its tryptophan residues within a considerable hydrophobic environment. The acid phosphatase activity was significantly decreased by the tryptophan modification. With respect to the active site donor sets, the Mn(III)-containing acid phosphatase is distinctly different from the Zn(II)-containing alkaline phosphatase. Of interest is also the appreciable similarity of some enzymatic and spectroscopic properties between the present enzyme and uteroferrin.

  7. The class C acid phosphatase of Helicobacter pylori is a 5' nucleotidase.

    PubMed

    Reilly, Thomas J; Calcutt, Michael J

    2004-01-01

    The results from purification and characterization studies of the hppA gene product of Helicobacter pylori confirm its identification as a class C acid phosphatase. The hppA gene of H. pylori ATCC strain 49503 was amplified and modified by PCR, cloned into pET21b, and overexpressed in Escherichia coli. The recombinant protein was liberated from membranes and purified (16x) to apparent homogeneity with cation exchange and Ni-chelate chromatography resulting in a recovery of 39% of total starting activity. The recombinant acid phosphatase exhibited a denatured molecular mass of 24 kDa by SDS-PAGE. Phosphatase activity in both crude and purified samples could be renatured and detected after SDS-PAGE. The native molecular mass of recombinant enzyme was approximately 72 kDa by gel filtration chromatography on Superdex 75. While phosphate and tartrate had little effect on phosphatase activity, molybdate, vanadate, and EDTA had significant inhibitory effects on enzymatic activity. Phosphomonoesterase activity for hydrolysis of p-nitrophenylphosphate (pNPP) as well as other substrates was enhanced in the presence of divalent cations including Cu(2+), Ni(2+), Co(2+), and Mg(2+). Recombinant HppA had narrow substrate specificity with highest activity for arylphosphates and significant activity for 5' nucleoside monophosphates. The pH optimum for enzyme activity was 4.6 and 5.2 for purine and pyrimidine 5' monophosphates, respectively. The affinity constants for the 5' nucleoside monophosphates were found to be 0.5-1 mM. Results from this study confirm HppA inclusion in the class C acid phosphatases and led to its identification as a 5' nucleotidase.

  8. Structural basis of the inhibition of class C acid phosphatases by adenosine 5;#8242;-phosphorothioate

    SciTech Connect

    Singh, Harkewal; Reilly, Thomas J.; Tanner, John J.

    2012-01-20

    The inhibition of phosphatases by adenosine 5'-phosphorothioate (AMPS) was first reported in the late 1960s; however, the structural basis for the inhibition has remained unknown. Here, it is shown that AMPS is a submicromolar inhibitor of class C acid phosphatases, a group of bacterial outer membrane enzymes belonging to the haloacid dehalogenase structural superfamily. Furthermore, the 1.35-{angstrom} resolution crystal structure of the inhibited recombinant Haemophilus influenzae class C acid phosphatase was determined; this is the first structure of a phosphatase complexed with AMPS. The conformation of AMPS is identical to that of the substrate 5'-AMP, except that steric factors force a rotation of the thiophosphoryl out of the normal phosphoryl-binding pocket. This conformation is catalytically nonproductive, because the P atom is not positioned optimally for nucleophilic attack by Asp64, and the O atom of the scissile O-P bond is too far from the Asp (Asp66) that protonates the leaving group. The structure of 5'-AMP complexed with the Asp64 {yields} Asn mutant enzyme was also determined at 1.35-{angstrom} resolution. This mutation induces the substrate to adopt the same nonproductive binding mode that is observed in the AMPS complex. In this case, electrostatic considerations, rather than steric factors, underlie the movement of the phosphoryl. The structures not only provide an explanation for the inhibition by AMPS, but also highlight the precise steric and electrostatic requirements of phosphoryl recognition by class C acid phosphatases. Moreover, the structure of the Asp64 {yields} Asn mutant illustrates how a seemingly innocuous mutation can cause an unexpected structural change.

  9. Detection of Ca2+-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin

    PubMed Central

    Petrosyan, Tigran R.; Ter-Markosyan, Anna S.; Hovsepyan, Anna S.

    2016-01-01

    The study aims to confirm the neuroregenerative effects of bacterial melanin (BM) on central nervous system injury using a special staining method based on the detection of Ca2+-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex (group I; n = 12) or unilateral rubrospinal tract transection at the cervical level (C3–4) (group II; n = 12). In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution (BM subgroup) and the remaining six rats were intramuscularly injected with saline (saline subgroup). Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca2+-dependent acid phosphatase activity detection in combination with Chilingarian’s calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca2+-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system. PMID:27630700

  10. Crystal structures and biochemical studies of human lysophosphatidic acid phosphatase type 6.

    PubMed

    Li, Jun; Dong, Yu; Lü, Xingru; Wang, Lu; Peng, Wei; Zhang, Xuejun C; Rao, Zihe

    2013-07-01

    Lysophosphatidic acid (LPA) is an important bioactive phospholipid involved in cell signaling through Gprotein-coupled receptors pathways. It is also involved in balancing the lipid composition inside the cell, and modulates the function of lipid rafts as an intermediate in phospholipid metabolism. Because of its involvement in these important processes, LPA degradation needs to be regulated as precisely as its production. Lysophosphatidic acid phosphatase type 6 (ACP6) is an LPA-specific acid phosphatase that hydrolyzes LPA to monoacylglycerol (MAG) and phosphate. Here, we report three crystal structures of human ACP6 in complex with malonate, L-(+)-tartrate and tris, respectively. Our analyses revealed that ACP6 possesses a highly conserved Rossmann-foldlike body domain as well as a less conserved cap domain. The vast hydrophobic substrate-binding pocket, which is located between those two domains, is suitable for accommodating LPA, and its shape is different from that of other histidine acid phosphatases, a fact that is consistent with the observed difference in substrate preferences. Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis. The structure also indicates a water-supplying channel for substrate hydrolysis. Our structural data are consistent with the fact that the enzyme is active as a monomer. In combination with additional mutagenesis and enzyme activity studies, our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.

  11. Effect of microbial inoculation during vermicomposting of different organic substrates on microbial status and quantification and documentation of acid phosphatase.

    PubMed

    Pramanik, P; Ghosh, G K; Banik, P

    2009-02-01

    In this experiment, three microbial strains were inoculated in two different organic wastes to study their effect on the humic acids content, acid phosphatase activity and microbial properties of the final stabilized products. Pyrophosphate extract of vermicomposts were analyzed through polyacrylamide gel electrophoresis to study the nature of a isozymes in different treatments. Results suggested that vermicomposting increased humic acids content and acid phosphatase activity in organic substrates and microbial inoculation further enhanced the rate of humification and enzyme activity. Although humic acids content in different microorganism-inoculated vermicomposts were statistically at par, acid phosphatase activity in these treatments was significantly (P<0.05) different. Results revealed that microbial respiration was increased due to vermicomposting, but a reduction in microbial biomass was recorded after stabilization of organic wastes. Although vermicomposting increased the value of microbial quotient (qCO(2)), microbial inoculation did not show any significant effect on qCO(2). The zymogram revealed that two isozymes of acid phosphatase (group II and group III) were present in all vermicompost samples and higher acid phosphatase activity in fungi-inoculated vermicomposts might be due to the presence of an additional isozyme (group I) of acid phosphatase.

  12. Lactobacillus plantarum phytase activity is due to non-specific acid phosphatase.

    PubMed

    Zamudio, M; González, A; Medina, J A

    2001-03-01

    Microbial phytases suitable for food fermentations could be obtained from lactic acid bacteria isolated from natural vegetable fermentations. Phytase activity was evaluated for six lactic acid bacteria cultures. Although the highest activity was found for Lactobacillus plantarum, the phytase activity was very low. Further characterization of the enzyme with phytate-degrading activity showed a molecular weight of 52 kDa and an optimum activity at pH 5.5 and 65 degrees C. Enzyme activity was due to a non-specific acid phosphatase which had a higher hydrolysis rate with monophosphorylated compounds such as acetyl phosphate that could explain the low phytase activity.

  13. Acid phosphatase positional correlations in solid surface fungal cultivation: a fractal interpretation of biochemical differentiation.

    PubMed

    Jones, C L; Lonergan, G T; Mainwaring, D E

    1995-03-28

    Colour image analysis was used to measure the positional correlation between acid phosphatase intracellular concentration and hyphal cellular differentiation which leads to concentric circular zonal activity patterns. Acid phosphatase is strongly implicated in the biochemical control of hyphal branching, and exo-enzyme secretion, such as laccase in fungi, occurs predominately via the hyphal tips. Different concentrations of an organic dye were used to effect substrate induction of the enzyme response, which was shown to be statistically correlated according to a fractal power law (H approximately 0.39). A self-organized critical state for the molecular responsivity of dissipative enzyme expression is hypothesized as an efficient mechanism for hyphal adaptation, also accounting for the underlying biophysics of the observed pattern formations.

  14. Position effect variegation of an acid phosphatase gene in Drosophila melanogaster.

    PubMed

    Frisardi, M C; MacIntyre, R J

    1984-01-01

    X-ray mutagenesis has produced a series of deficiencies in a duplication of part of the third chromosome containing the acid phosphatase gene (Acph-1) in Drosophila melanogaster. In one of these deficiencies, Acph-1 is shown to be undergoing position effect variegation. Naturally occurring electrophoretic variants of the enzyme were used to visualize and determine quantitatively the extent of variegation of the allele which is cis to the heterochromatic breakpoint. Alteration of genotypic background and temperature provided further evidence for position effect. Rocket immunoelectrophoresis was used to correlate the levels of acid phosphatase activity and protein in flies containing the deficiency. A novel result indicates that the variegation is not the consequence of an averaging of active and inactive cells, but rather due to a quantitative alteration of gene activity within at least some individual cells.

  15. PP2C gamma: a human protein phosphatase with a unique acidic domain.

    PubMed

    Travis, S M; Welsh, M J

    1997-08-04

    We have cloned a novel cDNA from human skeletal muscle which encodes a protein phosphatase with a unique acidic domain. It is 34% identical to mammalian PP2C alpha and PP2C beta and we call it PP2C gamma. It more closely resembles PP2Cs from Paramecium tetraurelia and Schizosaccharomyces pombe than mammalian PP2Cs. Northern blot analysis shows that PP2C gamma is widely expressed, and is most abundant in testis, skeletal muscle, and heart. Like known PP2Cs, recombinant PP2C gamma requires Mg2+ or Mn2+ for activity. Unlike any other known phosphatase, PP2C gamma has a highly acidic domain: 75% of the 54 residues are glutamate or aspartate.

  16. Phosphatidic acid phosphatase activity in subcellular fractions of normal and dystrophic human muscle.

    PubMed

    Kunze, D; Rüstow, B; Olthoff, D; Jung, K

    1985-03-15

    Biopsy samples from normal and dystrophic human muscle (Duchenne type) were fractionated by differential centrifugation and microsomes, mitochondria and cytosol were assayed for phosphatidic acid phosphatase (EC 3.1.3.4) and marker enzymes of mitochondria and cytosol. The activity of phosphatidic acid phosphatase was significantly lower in microsomes and higher in cytosol and mitochondria of dystrophic muscle than in the corresponding subcellular fractions of normal muscle. The results support an explanation of earlier findings that there is reduced G3P incorporation into diglycerides and phosphatidylcholine and a qualitative and quantitative change in the amount of phosphatidylcholine in dystrophic microsomes. The possible reasons for the reduction in the activity of only microsomal PA-P-ase were discussed.

  17. Evolution of acid phosphatase-1 in the genus Drosophilia. Immunological studies.

    PubMed

    MacIntyre, R J; Dean, M R; Batt, G

    1978-12-29

    The enzyme acid phosphatase-1 was partially purified from 10 Drosophila species. Four antisera were produced and the ten enzymes were reacted against each serum. The method used to quantitate the reactions involved the electrophoretic separation of antigen-antibody complexes from uncomplexed enzyme, followed by densitometry of the free enzyme. Immunological distances were used to obtain correlation coefficients for all pairwise combinations of the 10 species. From these correlation coefficients, a dendrogram was constructed which is very similar to one diagramming the presumed phylogenetic relationships of the ten species. In addition, the data indicate acid phosphatase-1 has evolved at different rates in different lineages within the genus. A preliminary estimate of the unit evolutionary period for this enzyme is 3.25 million years. The method of determining immunological distances which was used in this study is compared to the method of microcomplement fixation in the Discussion.

  18. Identification of Aph1, a Phosphate-Regulated, Secreted, and Vacuolar Acid Phosphatase in Cryptococcus neoformans

    PubMed Central

    Lev, Sophie; Crossett, Ben; Cha, So Young; Desmarini, Desmarini; Li, Cecilia; Chayakulkeeree, Methee; Wilson, Christabel F.; Williamson, P. R.; Sorrell, Tania C.

    2014-01-01

    ABSTRACT Cryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (Δaph1) and showed that Δaph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence. Phosphate starvation induced APH1 expression and secretion of catalytically active acid phosphatase in the WT, but not in the Δaph1 mutant, indicating that Aph1 is the major extracellular acid phosphatase in C. neoformans and that it is phosphate repressible. DsRed-tagged Aph1 was transported to the fungal cell periphery and vacuoles via endosome-like structures and was enriched in bud necks. A similar pattern of Aph1 localization was observed in cryptococci cocultured with THP-1 monocytes, suggesting that Aph1 is produced during host infection. In contrast to Aph1, but consistent with our previous biochemical data, green fluorescent protein (GFP)-tagged phospholipase B1 (Plb1) was predominantly localized at the cell periphery, with no evidence of endosome-mediated export. Despite use of different intracellular transport routes by Plb1 and Aph1, secretion of both proteins was compromised in a Δsec14-1 mutant. Secretions from the WT, but not from Δaph1, hydrolyzed a range of physiological substrates, including phosphotyrosine, glucose-1-phosphate, β-glycerol phosphate, AMP, and mannose-6-phosphate, suggesting that the role of Aph1 is to recycle phosphate from macromolecules in cryptococcal vacuoles and to scavenge phosphate from the extracellular environment. PMID:25227465

  19. Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases.

    PubMed

    Hisanaga, S; Yasugawa, S; Yamakawa, T; Miyamoto, E; Ikebe, M; Uchiyama, M; Kishimoto, T

    1993-06-01

    The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

  20. The Saccharomyces cerevisiae PHM8 gene encodes a soluble magnesium-dependent lysophosphatidic acid phosphatase.

    PubMed

    Reddy, Venky Sreedhar; Singh, Arjun Kumar; Rajasekharan, Ram

    2008-04-04

    Phosphate is the essential macronutrient required for the growth of all organisms. In Saccharomyces cerevisiae, phosphatases are up-regulated, and the level of lysophosphatidic acid (LPA) is drastically decreased under phosphate-starved conditions. The reduction in the LPA level is attributed to PHM8, a gene of unknown function. phm8Delta yeast showed a decreased LPA-hydrolyzing activity under phosphate-limiting conditions. Overexpression of PHM8 in yeast resulted in an increase in the LPA phosphatase activity in vivo. In vitro assays of the purified recombinant Phm8p revealed magnesium-dependent LPA phosphatase activity, with maximal activity at pH 6.5. The purified Phm8p did not hydrolyze any lipid phosphates other than LPA. In silico analysis suggest that Phm8p is a soluble protein with no transmembrane domain. Site-directed mutational studies revealed that aspartate residues in a DXDXT motif are important for the catalysis. These findings indicated that LPA plays a direct role in phosphate starvation. This is the first report of the identification and characterization of magnesium-dependent soluble LPA phosphatase.

  1. Amino-modified tetraphenylethene derivatives as nucleic acid stain: relationship between the structure and sensitivity.

    PubMed

    Xu, Li; Zhu, Zece; Wei, Danqing; Zhou, Xiang; Qin, Jingui; Yang, Chuluo

    2014-10-22

    A series of new amino-functionalized tetraphenylethene (TPE) derivatives were designed and synthesized to study the effect of molecular structures on the detection of nucleic acid. Contrastive studies revealed that the number of binding groups, the length of hydrophobic linking arm and the configuration of TPE molecule all play important roles on the sensitivity of the probes in nucleic acid detection. Z-TPE3 with two binding amino groups, long linking arms, and cis configuration was found to be the most sensitive dye in both solution and gel matrix. Z-TPE3 is able to stain dsDNA with the lowest amount of 1 ng and exclusively stain 40 ng of short oligonucleotide with only 10 nt. This work is of important significance for the further design of TPE probes as biosensors with higher sensitivity.

  2. Androgen metabolism and regulation of rat ventral prostate growth and acid phosphatase during sexual maturation.

    PubMed

    Orlowski, J; Bird, C E; Clark, A F

    1988-01-01

    Androgen metabolism and the regulation of rat ventral prostate cell proliferation and secretory function were examined during sexual maturation. Changes in acid phosphatase (AP) characteristics were measured as a marker of androgen-dependent prostatic secretory function. In immature (21-day-old) rats, total AP activity per cell was low (14.2 +/- 1.3 mol p-nitrophenol phosphate hydrolysed/h per mg DNA); it increased threefold as the weight, protein and DNA contents of the prostate increased to adult (65-day) levels. This corresponded with significant (P less than 0.001) increases in the staining intensities of three of the four bands of secretory AP on isoelectric focusing gels. The extent of inhibition of AP by tartrate decreased at the same time. Secretory AP is known to be relatively tartrate-resistant. The changes in AP activity occurred after prostatic 5 alpha-dihydrotestosterone (5 alpha-DHT) levels increased from 4.6 +/- 0.7 pmol/mg DNA (21 days) to reach a peak of 17.6 +/- 2.3 pmol/mg DNA at 58 days. Prostatic 5 alpha-DHT concentrations were always higher than testosterone levels. Prostatic 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Adiol) levels were lower than 5 alpha-DHT levels except on day 58 when levels peaked dramatically at 26.2 +/- 5.5 pmol/mg DNA. Changes in prostatic 5 alpha-DHT and 3 alpha-Adiol levels corresponded with changes in 5 alpha-reductase and 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities. The oxidative reaction of 3 alpha-HSOR was approximately fourfold higher than the reductive reaction, indicating a preference for the formation of 5 alpha-DHT. The plasma levels of testosterone, 5 alpha-DHT and 3 alpha-Adiol cannot account for their respective prostatic levels, indicating the importance of the steroid-metabolizing enzymes in regulating intracellular androgen levels. Changes in the AP characteristics could be correlated with the androgen status of the prostate.

  3. Endocytosis of lysosomal acid phosphatase; involvement of mannose receptor and effect of lectins.

    PubMed

    Imai, K; Yoshimura, T

    1994-08-01

    Acid phosphatase and beta-glucosidase are unique among lysosomal enzymes in that they have both high mannose and complex type sugasr chains, whereas oligosaccharide chains of lysosomal enzymes in matrix are of high mannose type. We have previously shown that beta-glucosidase was endocytosed into macrophages via an unidentified receptor different from a mannose/fucose receptor (K. Imai, Cell Struct. Funct. 13, 325-332, 1988). Here, we show that uptake of acid phosphatase purified from rat liver lysosomes into rat macrophages was inhibited by ligands for a mannose/fucose receptor and was mediated via an apparently single binding site with Kuptake of 24.7 nM. These results indicate that acid phosphatase and beta-glucosidase recognize different types of receptors even if they have similar sugar chains. Polyvalent concanavalin A which binds both to the enzyme and to macrophages specifically stimulated the uptake in a dose dependent manner, whereas wheat germ agglutinin and phytohaemagglutinin did not.

  4. Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase

    SciTech Connect

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2006-07-01

    Crystallization of a surface-localized acid phosphatase from Bacillus anthracis is reported. Flash annealing increased the high-resolution limit of usable data from 1.8 to 1.6 Å. Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 Å. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6° and a high-resolution limit of 1.8 Å. After flash-annealing, the apparent mosaicity decreased to 0.9° and the high-resolution limit of usable data increased to 1.6 Å. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

  5. Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors.

    PubMed

    Fialho, Eliane; Nakamura, Angelica; Juliano, Luiz; Masuda, Hatisaburo; Silva-Neto, Mário A C

    2005-04-15

    Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods.

  6. Separation of tissue and serum acid phosphatase isoenzymes by ion-exchange column chromatography.

    PubMed

    Mercer, D W

    1977-01-01

    I describe a simple, rapid ion-exchange column-chromatographic technique for separating the acid phosphatase (EC 3.1.3.2) isoenzymes in human serum and tissue. Extracts of platelets, spleen, liver, erythrocytes, and prostate were used to determine optimum conditions for separating these isoenzymes. Samples layered on mini-colunms of DEAE-Sephadex A-50 were eluted stepwise with sodium chloride (100, 200, and 300 mmol/liter, buffered with tris (hydroxymethyl)aminomethane). Activity in column effluents was measured with p-nitrophenol phosphate as substrate, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Comparision of activity patterns so derived for various tissues revealed prostatic tissue to be a rich source of acid phosphatase isoenzyme 2 activity. Evaluation of sera from six patients with prostatic cancer revealed isoenzyme patterns with prominent amount of isoenzyme 2 (3.8 to 27.6 U/liter). sera from 10 healthy laboratory technicians contained isoenzyme 2 in the range of 0.3-0.5 U/liter. Samples from two patients with abnormally high activity owing to nonprostatic conditions (Gaucher's disease and carcinoma of lung) exhibited less than 2 U of isoenzyme 2 per liter and acid phosphatase isoenzymes 3-5 that were 50- to 100-fold the normal range. Quantification of isoenzyme 2 by DEAE-Sephadex column chromatography as described appears to provide a more sensitive and specific approach to diagnosis of prostatic cancer.

  7. Assessment of bioavailable organic phosphorus in tropical forest soils by organic acid extraction and phosphatase hydrolysis.

    PubMed

    Darch, Tegan; Blackwell, Martin S A; Chadwick, David; Haygarth, Philip M; Hawkins, Jane M B; Turner, Benjamin L

    2016-12-15

    Soil organic phosphorus contributes to the nutrition of tropical trees, but is not accounted for in standard soil phosphorus tests. Plants and microbes can release organic anions to solubilize organic phosphorus from soil surfaces, and synthesize phosphatases to release inorganic phosphate from the solubilized compounds. We developed a procedure to estimate bioavailable organic phosphorus in tropical forest soils by simulating the secretion processes of organic acids and phosphatases. Five lowland tropical forest soils with contrasting properties (pH 4.4-6.1, total P 86-429 mg P kg(- 1)) were extracted with 2 mM citric acid (i.e., 10 μmol g(- 1), approximating rhizosphere concentrations) adjusted to soil pH in a 4:1 solution to soil ratio for 1 h. Three phosphatase enzymes were then added to the soil extract to determine the forms of hydrolysable organic phosphorus. Total phosphorus extracted by the procedure ranged between 3.22 and 8.06 mg P kg(- 1) (mean 5.55 ± 0.42 mg P kg(- 1)), of which on average three quarters was unreactive phosphorus (i.e., organic phosphorus plus inorganic polyphosphate). Of the enzyme-hydrolysable unreactive phosphorus, 28% was simple phosphomonoesters hydrolyzed by phosphomonoesterase from bovine intestinal mucosa, a further 18% was phosphodiesters hydrolyzed by a combination of nuclease from Penicillium citrinum and phosphomonoesterase, and the remaining 51% was hydrolyzed by a broad-spectrum phytase from wheat. We conclude that soil organic phosphorus can be solubilized and hydrolyzed by a combination of organic acids and phosphatase enzymes in lowland tropical forest soils, indicating that this pathway could make a significant contribution to biological phosphorus acquisition in tropical forests. Furthermore, we have developed a method that can be used to assess the bioavailability of this soil organic phosphorus.

  8. Crystal structure and tartrate inhibition of Legionella pneumophila histidine acid phosphatase.

    PubMed

    Dhatwalia, Richa; Singh, Harkewal; Reilly, Thomas J; Tanner, John J

    2015-11-01

    Histidine acid phosphatases (HAPs) utilize a nucleophilic histidine residue to catalyze the transfer of a phosphoryl group from phosphomonoesters to water. HAPs function as protein phosphatases and pain suppressors in mammals, are essential for Giardia lamblia excystation, and contribute to virulence of the category A pathogen Francisella tularensis. Herein we report the first crystal structure and steady-state kinetics measurements of the HAP from Legionella pneumophila (LpHAP), also known as Legionella major acid phosphatase. The structure of LpHAP complexed with the inhibitor l(+)-tartrate was determined at 2.0 Å resolution. Kinetics assays show that l(+)-tartrate is a 50-fold more potent inhibitor of LpHAP than of other HAPs. Electrostatic potential calculations provide insight into the basis for the enhanced tartrate potency: the tartrate pocket of LpHAP is more positive than other HAPs because of the absence of an ion pair partner for the second Arg of the conserved RHGXRXP HAP signature sequence. The structure also reveals that LpHAP has an atypically expansive active site entrance and lacks the nucleotide substrate base clamp found in other HAPs. These features imply that nucleoside monophosphates may not be preferred substrates. Kinetics measurements confirm that AMP is a relatively inefficient in vitro substrate of LpHAP.

  9. Structural and functional characterization of the 2H-phosphatase domain of Sts-2 reveals an acid-dependent phosphatase activity.

    PubMed

    Chen, Yunting; Jakoncic, Jean; Carpino, Nick; Nassar, Nicolas

    2009-03-03

    The suppressors of T cell receptor (TCR) signaling 1 and 2 (Sts-1 and -2, respectively) are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including TCR and the epidermal growth factor receptor (EGFR). Sts-1 was recently shown to be a new type of protein tyrosine phosphatase (PTP), with the phosphatase activity located within its C-terminal phosphoglycerate mutase (PGM) homology domain and key for the regulation of TCR signaling in T cells. The activity of the related Sts-2 enzyme is significantly less than that of Sts-1. Here we investigate the phosphatase activity of the PGM domain of Sts-2, Sts-2(PGM). The crystal structure of Sts-2(PGM) is remarkably similar to Sts-1(PGM), including conservation of all catalytic residues. Insight into mechanistic details is provided by the structures of the apo, tungstate-bound, and phosphate-bound enzyme. The active site shows stringent specificity, with the k(cat) optimum at pH 5.0 suggesting that Sts-2 might function as an acid-dependent phosphatase. Mutation of active site residues Gln372, Ala446, Glu481, Ser552, and Ser582 to their equivalents in Sts-1 increases the phosphatase activity of Sts-2(PGM) toward model substrates. Overall, our data demonstrate that Sts-2(PGM) adopts the conformation of an active phosphatase whose activity is fundamentally different from that of Sts-1 despite the strong structural homology. They also demonstrate that nonconserved active site residues are responsible for the difference in activity between the two isoforms. These differences reflect possible distinct physiological substrates.

  10. In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae.

    PubMed

    Krebs, H O; Hoffschulte, H K; Müller, M

    1989-05-01

    We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum. The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA. In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core-glycosylated. The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated. When a wheat-germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core-glycosylated in a signal-recognition-particle-dependent manner. Replacing canine with yeast rough microsomes in the wheat-germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor. Translocation and glycosylation were partially restored by a high-salt extract prepared from yeast ribosomes. The results presented here suggest that yeast-specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.

  11. Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps.

    PubMed

    Henry, Y; Steer, M W

    1985-04-01

    The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.

  12. Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase.

    PubMed

    Waymack, P P; Van Etten, R L

    1991-08-01

    An acid phosphatase (orthophosphoric monoester phosphohydrolase, acid optimum; EC 3.1.3.2) isoenzyme from wheat germ was purified 7000-fold to homogeneity. The effect of wheat germ sources and their relationship to the isoenzyme content and purification behavior of acid phosphatases was investigated. Extensive information about the purification and stabilization of the enzyme is provided. The instability of isoenzymes in the latter stages of purification appeared to be the result of surface inactivation together with a sensitivity to dilution that could be partially offset by addition of Triton X-100 during chromatographic procedures. Added sulfhydryl protecting reagents had no effect on activity or stability, which was greatest in the pH range 4-7. The purified isoenzyme was homogeneous by polyacrylamide gel electrophoresis and exhibited the highest specific activity and turnover number reported for any acid phosphatase. The molecular weights of the pure isoenzyme and of related isoenzymes from wheat germ were found to be identical (58,000). The pure isoenzyme contained a single polypeptide chain and had a negligible carbohydrate content. The amino acid composition was determined. Of the various reasons that were considered to explain isoenzyme occurrence, a genetic basis was considered most likely. The enzyme was found to exhibit substrate inhibition with some substrates below pH 6, while above pH 8 it exhibited downwardly curving Lineweaver-Burk plots of the type that are generally described as "substrate activation". The observation of a phosphotransferase activity was consistent with the formation of a covalent phosphoenzyme intermediate, while inactivation by diethyl pyrocarbonate was consistent with the presence of an active site histidine.

  13. Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

    SciTech Connect

    Singh, Harkewal; Felts, Richard L.; Schuermann, Jonathan P.; Reilly, Thomas J.; Tanner, John J.

    2009-12-01

    Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-{angstrom}-resolution structure of FtHAP complexed with the competitive inhibitor L(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 {angstrom}, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 {angstrom} resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an {alpha}/{beta} core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3'-AMP binds in one end of a 15-{angstrom}-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2'-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K{sub m} by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small

  14. Hydroxyindole Carboxylic Acid-Based Inhibitors for Receptor-Type Protein Tyrosine Protein Phosphatase Beta

    PubMed Central

    Zeng, Li-Fan; Zhang, Ruo-Yu; Bai, Yunpeng; Wu, Li; Gunawan, Andrea M.

    2014-01-01

    Abstract Aims: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. Results: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPβ), a potential target that is implicated in blood vessel development. The representative RPTPβ inhibitor 8b-1 (L87B44) has an IC50 of 0.38 μM and at least 14-fold selectivity for RPTPβ over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. Innovation: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Conclusion: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity. Antioxid. Redox Signal. 20, 2130–2140. PMID:24180557

  15. Is Protein Phosphatase Inhibition Responsible for the Toxic Effects of Okadaic Acid in Animals?

    PubMed Central

    Munday, Rex

    2013-01-01

    Okadaic acid (OA) and its derivatives, which are produced by dinoflagellates of the genera Prorocentrum and Dinophysis, are responsible for diarrhetic shellfish poisoning in humans. In laboratory animals, these toxins cause epithelial damage and fluid accumulation in the gastrointestinal tract, and at high doses, they cause death. These substances have also been shown to be tumour promoters, and when injected into the brains of rodents, OA induces neuronal damage reminiscent of that seen in Alzheimer’s disease. OA and certain of its derivatives are potent inhibitors of protein phosphatases, which play many roles in cellular metabolism. In 1990, it was suggested that inhibition of these enzymes was responsible for the diarrhetic effect of these toxins. It is now repeatedly stated in the literature that protein phosphatase inhibition is not only responsible for the intestinal effects of OA and derivatives, but also for their acute toxic effects, their tumour promoting activity and their neuronal toxicity. In the present review, the evidence for the involvement of protein phosphatase inhibition in the induction of the toxic effects of OA and its derivatives is examined, with the conclusion that the mechanism of toxicity of these substances requires re-evaluation. PMID:23381142

  16. Prostatic acid phosphatase is required for the antinociceptive effects of thiamine and benfotiamine.

    PubMed

    Hurt, Julie K; Coleman, Jennifer L; Fitzpatrick, Brendan J; Taylor-Blake, Bonnie; Bridges, Arlene S; Vihko, Pirkko; Zylka, Mark J

    2012-01-01

    Thiamine (Vitamin B1) is an essential vitamin that must be obtained from the diet for proper neurological function. At higher doses, thiamine and benfotiamine (S-benzoylthiamine O-monophosphate, BT)-a phosphorylated derivative of thiamine-have antinociceptive effects in animals and humans, although how these compounds inhibit pain is unknown. Here, we found that Prostatic acid phosphatase (PAP, ACPP) can dephosphorylate BT in vitro, in dorsal root ganglia (DRG) neurons and in primary-afferent axon terminals in the dorsal spinal cord. The dephosphorylated product S-benzoylthiamine (S-BT) then decomposes to O-benzoylthiamine (O-BT) and to thiamine in a pH-dependent manner, independent of additional enzymes. This unique reaction mechanism reveals that BT only requires a phosphatase for conversion to thiamine. However, we found that the antinociceptive effects of BT, thiamine monophosphate (TMP) and thiamine-a compound that is not phosphorylated-were entirely dependent on PAP at the spinal level. Moreover, pharmacokinetic studies with wild-type and Pap(-/-) mice revealed that PAP is not required for the conversion of BT to thiamine in vivo. Taken together, our study highlights an obligatory role for PAP in the antinociceptive effects of thiamine and phosphorylated thiamine analogs, and suggests a novel phosphatase-independent function for PAP.

  17. Abscisic acid sensor RCAR7/PYL13, specific regulator of protein phosphatase coreceptors.

    PubMed

    Fuchs, Stefan; Tischer, Stefanie V; Wunschel, Christian; Christmann, Alexander; Grill, Erwin

    2014-04-15

    The plant hormone abscisic acid (ABA) acts both as a developmental signal and as an integrator of environmental cues such as drought and cold. ABA perception recruits an ABA-binding regulatory component [regulatory component of ABA receptor (RCAR)/PYR1/PYL] and an associated protein phosphatase 2C (PP2C). Phytohormone binding inactivates the phosphatase activity of the coreceptor, permitting phosphorelay of the ABA signal via downstream protein kinases. RCARs and PP2C coreceptors are represented by small protein families comprising 14 and 9 members in Arabidopsis, respectively. The specificity of the RCAR-PP2C interaction and the constraints contributing to specific combinations are poorly understood. In this contribution, we analyzed RCAR7/PYL13, which is characterized by three variant amino acid residues in the conserved ABA-binding pocket. RCAR7 regulated the phosphatase activity of the PP2Cs ABI1, ABI2, and PP2CA in vitro at nanomolar ABA levels; however, it was unable to regulate the structurally related hypersensitive to ABA 1 (HAB1). Site-directed mutagenesis of HAB1 established ABA-dependent regulation by RCAR7. Conversion of the noncanonical amino acid residues of RCAR7 into the consensus ABA-binding pocket did not perceptibly change receptor function. Ectopic expression of RCAR7 in Arabidopsis resulted in ABA hypersensitivity affecting gene regulation, seed germination, and stomatal closure. The RCAR7 loss-of-function mutant revealed no changes in ABA responses, similar to the RCAR9 knockout line, whereas the combined deficiency of RCAR7 and RCAR9 resulted in ABA-insensitive seed germination. The study shows a role of RCAR7 in early plant development, proves its ABA receptor function, and identifies structural constraints of RCAR7-PP2C interaction.

  18. [Effect of dental alloys on salivary alkaline and acid phosphatase, alpha amylase K+, Na+, and Cl-].

    PubMed

    Todorov, I; Saprjanova, M

    1977-04-01

    Comparative studied were performed in healthy subjects without metals in their oral cavities and in individuals having different metal alloys (gold, steel, amalgam) in their mouths and presenting with various complaints such as xerostomia, burning mucosa, etc. It was found that the contents of alkaline and acid phosphatases, alpha-amylase, K+, Na+ and Cl- in saliva increased significantly with the increase in total corrosion potential when non-precious metal alloys, especially different types of alloys, were present. Parallel to this, the frequency and the intensity of the complaints increased.

  19. Effect of perfluorooctane sulfonate on the conformation of wheat germ acid phosphatase.

    PubMed

    Xu, Dongmei; Jin, Jianchang; Shen, Tong; Wang, Yanhua

    2013-11-01

    Fluorescence spectroscopy was used to study the quenching mechanism, the type of force and the binding sites of perfluorooctane sulfonate (PFOS) on wheat germ acid phosphatase (ACPase). The results showed that the quenching effect of PFOS on ACPase was mainly due to a static quenching mechanism that occurred via the formation of hydrogen bonds and van der Waals forces. The results from synchronous fluorescence spectroscopy demonstrated that PFOS interacts with ACPase close to the tryptophan residues. In addition, synchronous fluorescence spectroscopy also showed that PFOS increases the hydrophobicity of the microenvironment of the tyrosine residues, hence decreasing the local polarity.

  20. Continuous and sensitive acid phosphatase assay based on a conjugated polyelectrolyte.

    PubMed

    Xie, Yonghua; Tan, Ying; Liu, Renxuan; Zhao, Rui; Tan, Chunyan; Jiang, Yuyang

    2012-08-01

    We report a novel continuous and sensitive fluorescence turn-on assay for ACPs, which consists of a cationic conjugated polyelectrolyte (PPE4+) and a commonly used phosphatase substrate p-nitrophenyl phosphate (pNPP). The kinetics of the ACP catalyzed hydrolysis of the substrate pNPP was monitored by the fluorescence change of PPE4+ and corresponding kinetic parameters were derived to be consistent with the literature reports. The applications of PPE4+/pNPP-based ACP assay in high-throughput screening of ACP inhibitors and detection of prostatic acid phosphotase (PAP) in vitro were demonstrated.

  1. Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

    PubMed

    Van Etten, R L; Waymack, P P

    1991-08-01

    The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

  2. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.

    PubMed

    Harris, Thurl E; Huffman, Todd A; Chi, An; Shabanowitz, Jeffrey; Hunt, Donald F; Kumar, Anil; Lawrence, John C

    2007-01-05

    Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.

  3. Unfolding pathway in red kidney bean acid phosphatase is dependent on ligand binding.

    PubMed

    Cashikar, A G; Rao, N M

    1996-03-01

    Structural basis for ligand-induced protein stabilization was investigated in the case of an acid phosphatase (red kidney bean purple acid phosphatase (KBPAP)) from red kidney bean. Phosphate, a physiological ligand, increases the stability against solvent denaturation by 3.5 kcal/mol. Generality of phosphate stabilization was shown by similar effects with other KBPAP ligands viz. adenosine 5'-O-(thiotriphosphate), a nonhydrolyzable ligand, and arsenate, an inhibitor. The dissociation constant of phosphate obtained from denaturation curves matches with the dissociation constant estimated by conventional methods. The guanidinium chloride-mediated denaturation of KBPAP was monitored by several structural and functional parameters viz. activity, tryptophan fluorescence, 8-anilinonaphthalene 1-sulfonic acid binding, circular dichroism, and size exclusion chromatography, in the presence and absence of 10 mm phosphate. In the presence of phosphate, profiles of all the parameters shift to a higher guanidinium chloride concentration. Noncoincidence of these profiles in the absence of phosphate indicates multistate unfolding pathway for KBPAP; however, in the presence of phosphate, KBPAP unfolds with a single intermediate. Based on the crystal structure, we propose that the Arg258 may have an important role to play in stabilization mediated by phosphate.

  4. Design and synthesis of an Fmoc-SPPS-compatible amino acid building block mimicking the transition state of phosphohistidine phosphatase.

    PubMed

    Eerland, Martijn F; Hedberg, Christian

    2012-02-17

    The synthesis of a sulfonamide-based transition-state (TS) analogue of enzymatic phosphohistidine dephosphorylation as an amino acid building block is presented, together with the proof-of-concept of its incorporation into peptides. Key features include final global acidolytic protective group removal as well as full compatibility with standard Fmoc solid-phase peptide synthesis (SPPS). The peptides are designed as inhibitors of phosphohistidine phosphatase and as a pull-down probe for identification of phosphohistidine phosphatases, respectively.

  5. Iron staining of the acquired enamel pellicle after exposure to tannic acid or chlorhexidine: preliminary report.

    PubMed

    Nordbö, H; Eriksen, H M; Rölla, G; Attramadal, A; Solheim, H

    1982-04-01

    Extrinsic discoloration of teeth following a large consumption of tannin-containing beverages or a prolonged use of chlorhexidine mouthrinses is a well known observation. Tannins as well as chlorhexidine are denaturing agents. Based on preliminary studies revealing the presence of iron in chlorhexidine discolored pellicle material, the ability of iron to stain the integument after pretreatment with the two denaturants was studied in a human model. The denaturing effect of an acidic environment was also included. Enamel slabs fixed to acrylic appliances were carried in the oral cavity and alternately exposed to the test solutions in different sequences in vitro. Pretreatment with chlorhexidine or tannic acid led to marked discoloration upon iron application during 5-d tests, whereas the compounds individually had no such effect. A large content of the metal was found in the stained material. Stannous fluoride appeared to reduce the formation of the pigments, and strong oxidation completely bleached the established color. Possible mechanisms underlying the phenomena observed are discussed.

  6. Pre-staining paper chromatography method for quantification of gamma-aminobutyric acid.

    PubMed

    Li, Haixing; Qiu, Ting; Cao, Yusheng; Yang, Jiyan; Huang, Zhibing

    2009-06-19

    The routine method of paper chromatography includes five steps: spotting, separating, drying, spraying/immersing and color development. In this paper, a pre-staining paper chromatography which only consisted of spotting, separating and color development was developed for quantitative analysis of gamma-aminobutyric acid. Compared to the routine paper chromatography, the improved method is clean, rapid, inexpensive and reproducible. The effects of ninhydrin concentration, color temperature, color time and Cu(2+) concentration on the color yield in the ninhydrin reaction were optimized. And then the pre-staining paper chromatography coupled with vis spectrophotometry was applied to gamma-aminobutyric acid quantification. The results indicated that the limit of detection was 0.05 mg mL(-1) and the linear range was from 0.5 to 20.0 mg mL(-1). Furthermore, an excellent correlation coefficient was observed with an R(2)=0.998. The method is accurate (RSD<2.64%), and has good recoveries (102.7-103.9%). The validation of the modified technique was verified by a HPLC method.

  7. Structure of thermotoga maritima stationary phase survival protein SurE : a novel acid phosphatase.

    SciTech Connect

    Zhang, R.-G; Skarina, T.; Katz, J. E.; Khachatryan, A; Vyas, S.; Arrowsmith, C. H.; Clarke, S.; Edwards, A.; Joachimiak, A.; Savchenko, A.; Biosciences Division; Univ. of Toronto; Univ. of California; Clinical Genomics Centre /Proteomics, Univ. Health Network

    2001-11-01

    Background: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase {sigma} subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. Results: The structure of SurE from Thermotoga maritima was determined at 2.0 Angstroms. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability. Conclusions: The structure of SurE provided information about the protein's fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

  8. Inhibition kinetics of acid and alkaline phosphatases by atrazine and methomyl pesticides.

    PubMed

    El-Aswad, Ahmed F; Badawy, Mohamed E I

    2015-01-01

    The main objective of this work was to investigate the kinetic characteristics of acid and alkaline phosphatases isolated from different sources and to study the effects of the herbicide atrazine and insecticide methomyl on the activity and kinetic properties of the enzymes. Acid phosphatase (ACP) was isolated from the tomato plant (Solanum lycopersicum L. var. lycopersicum); alkaline phosphatase (ALP) was isolated from two sources, including mature earthworms (Aporrectodea caliginosa) and larvae of the Egyptian cotton leafworm (Spodoptera littoralis). The specific activities of the enzymes were 33.31, 5.56 and 0.72 mmol substrate hydrolyzed per minute per milligram protein for plant ACP, earthworms ALP and cotton leafworm ALP, respectively. The inhibition kinetics indicated that atrazine and methomyl caused competitive-non-competitive inhibition of the enzymes. The relationships between estimates of K(m) and V(max) calculated from the Michaelis-Menten equation have been explored. The extent of the inhibition was different, as estimated by the values of the inhibition constant Ki that were found to be 3.34 × 10(-3), 1.12 × 10(-2) and 1.07 × 10(-2) mM for plant ACP, earthworms ALP and cotton leafworm ALP, respectively, with methomyl. In the case of atrazine, K(i) were found to be 8.99 × 10(-3), 3.55 × 10(-2) and 1.36 × 10(-2) mM for plant ACP, earthworms ALP and cotton leafworm ALP, respectively.

  9. Photometric and fluorometric continuous kinetic assay of acid phosphatases with new substrates possessing longwave absorption and emission maxima.

    PubMed

    Koller, E; Wolfbeis, O S

    1984-11-15

    A direct and continuous kinetic method for the photometric and fluorometric determination of various acid phosphatases is described. It is based on new coumarin-derived phosphates, which after enzymatic hydrolysis undergo dissociation to form intensely colored and strongly fluorescent phenolate anions. The latter have absorption maxima ranging from 385 to 505 nm, and fluorescence maxima between 470 and 595 nm. The new substrates were compared with respect to their rate of enzymatic hydrolysis, optimum pH, and detection limits of acid phosphatase from potato and wheat germ. Detection limits of 0.001 unit/ml were found by photometry, and as low as 0.00006 unit/ml by fluorometry. The principal advantages of the new substrates over existing ones are longwave absorptions and emissions, large Stokes shifts, and the low pKa values of the corresponding phenols, thus allowing a direct and continuous assay of acid phosphatase even in weakly acidic solutions.

  10. The effectiveness of a modified hydrochloric acid-quartz-pumice abrasion technique on fluorosis stains: a case report.

    PubMed

    Erdogan, G

    1998-02-01

    Endemic dental fluorosis is a form of enamel hypoplasia characterized by moderate-to-severe staining of the tooth surface. Since 1916, numerous investigators have used hydrochloric acid alone on fluorosis stains. More recently, 18% hydrochloric acid-pumice microabrasion has been used to achieve color modification. The main disadvantage of this procedure is the high concentration and low viscosity of hydrochloric acid, which can cause damage to oral and dental tissues. To eliminate this problem, quartz particles can be mixed with the hydrochloric acid. The quartz particles prevent the hydrochloric acid from flowing uncontrollablely by altering it to a gel-like form. A modified 18% hydrochloric acid-quartz-pumice abrasion technique was used to remove fluorine stains from vital teeth in a teenager.

  11. Molecular cloning and characterization of acid phosphatase in venom of the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

    PubMed

    Zhu, Jia-ying; Ye, Gong-yin; Hu, Cui

    2008-06-15

    The present study describes cDNA cloning, sequencing, enzyme activity determination and localization, and mRNA expression of acid phosphatase in the venom apparatus of an endoparasitoid, Pteromalus puparum. This is the first report of cloning a venom acid phosphatase gene which has been described in parasitoid wasps. The cDNA consisted of 1378bp with 1215bp open reading frame and encoded a sequence of 405 amino acids. A 23 residues N-terminal signal peptide was followed by a short 15 residues (25-39) histidine acid phosphatases phosphohistidine signature and a long 302 residues (24-325) acid phosphatase family domain. The deduced amino acid sequence shared 32-88% identity to its counterparts from other insects. Enzyme activity was measured by using p-nitrophenyl phosphate (p-NPP) as substrate, and a high level of acid phosphatase activity in venom was detected. Optimal pH and temperature for this enzyme activity was 4.8 and 45 degrees C, respectively. Ultracytochemical analyses further revealed that strong enzyme activity was located in the nuclei and secretory vesicles of the venom gland secretory cells. Expression of the acid phosphatase gene was observed to be regulated at different developmental stages by RT-PCR analysis as it expressed immediately with low abundance after adult emergence, then increased to the high level at 2-4 days, followed by a drop to the low abundance after 4 days. Compared to the mRNA expression, a time-course-related enzyme activity in an individual venom apparatus was also found.

  12. Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens▿

    PubMed Central

    Reilly, Thomas J.; Chance, Deborah L.; Calcutt, Michael J.; Tanner, John J.; Felts, Richard L.; Waller, Stephen C.; Henzl, Michael T.; Mawhinney, Thomas P.; Ganjam, Irene K.; Fales, William H.

    2009-01-01

    Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ∼31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3′ and 5′ nucleoside monophosphates at pH 6. Calculated Kms ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 μmol of Pi/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class. PMID:19363079

  13. Soybean seed acid phosphatases: unusual optimum temperature and thermal stability studies.

    PubMed

    Ferreira, C V; Granjeiro, J M; Taga, E M; Aoyama, H

    1998-01-14

    In contrast to other acid phosphatases, four cytoplasmic isoforms (AP1, AP2, AP3A, and AP3B) purified from mature soybean seeds presented high activities at temperatures above 80 degrees C, when p-nitrophenylphosphate (p-NPP) was utilized as substrate. However, with tyrosine phosphate and inorganic pyrophosphate as substrates, maximum activities were observed at temperature of 60 degrees C during 10 min reaction. In the absence of substrate, enzymes lost only 20% activity after 60 min at 60 degrees C; the isoforms AP3A and AP3B retained 30% of activity at 70 degrees C after 60 min and all the isoforms were inactivated at 80 degrees C, after 5 min. Thermal inactivation studies indicated that the soybean enzymes showed different temperature dependences in relation to most plant acid phosphatases. A best protective effect was observed when the isoforms were preincubated, at 70 degrees C, with phosphate (10 mM) and p-nitrophenol (10 mM) which indicates that the enzyme inactivation was prevented only in the presence of both reaction products.

  14. Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae.

    PubMed Central

    Bostian, K A; Lemire, J M; Halvorson, H O

    1983-01-01

    We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed. Images PMID:6346058

  15. The Aspergillus nidulans pyrG89 mutation alters glycosylation of secreted acid phosphatase.

    PubMed

    Justino, A; Nozawa, S R; Maccheroni, W; May, G S; Martinez-Rossi, N M; Rossi, A

    2001-03-01

    The glycosylation level of the pacA-encoded acid phosphatase secreted by Aspergillus nidulans was reduced in strains pabaA1 pyroA4and pabaA1 pyroA4 pyrG89, compared to strains carrying these mutations singly. The molecular mass of the enzyme secreted by the triple mutant grown at pH 5.0 was 105 and 45 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. In contrast, the pabaA1 strain secreted acid phosphatases of 119 and 62 kDa. The enzyme also had an altered electrophoretic mobility and glycosylation had a protective effect against its heat inactivation. Thus, this combination of mutants alters glycosylation of the enzyme, leading to changes in their structural properties. In spite of this, no deviation was observed in the apparent optimum pH and Michaelis kinetics for enzymatic hydrolysis of p-nitrophenyl phosphate or alpha-naphthyl phosphate.

  16. Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry

    PubMed Central

    Lee, Jihye; Kim, Jinhee; Son, Kidong; d’Alexandry d’Orengiani, Anne-Laure Pham Humg; Min, Ji-Young

    2017-01-01

    Influenza viruses exploit host factors to successfully replicate in infected cells. Using small interfering RNA (siRNA) technology, we identified six human genes required for influenza A virus (IAV) replication. Here we focused on the role of acid phosphatase 2 (ACP2), as its knockdown showed the greatest inhibition of IAV replication. In IAV-infected cells, depletion of ACP2 resulted in a significant reduction in the expression of viral proteins and mRNA, and led to the attenuation of virus multi-cycle growth. ACP2 knockdown also decreased replication of seasonal influenza A and B viruses and avian IAVs of the H7 subtype. Interestingly, ACP2 depletion had no effect on the replication of Ebola or hepatitis C virus. Because ACP2 is known to be a lysosomal acid phosphatase, we assessed the role of ACP2 in influenza virus entry. While neither binding of the viral particle to the cell surface nor endosomal acidification was affected in ACP2-depleted cells, fusion of the endosomal and viral membranes was impaired. As a result, downstream steps in viral entry were blocked, including nucleocapsid uncoating and nuclear import of viral ribonucleoproteins. Our results established ACP2 as a necessary host factor for regulating the fusion step of influenza virus entry. PMID:28272419

  17. Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application

    PubMed Central

    Souza, Amanda Araújo; Leitão, Vanessa Oliveira; Ramada, Marcelo Henrique; Mehdad, Azadeh; Georg, Raphaela de Castro; Ulhôa, Cirano José; de Freitas, Sonia Maria

    2016-01-01

    Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65°C, respectively, and is stable at 55°C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments. PMID:26938873

  18. An 18 kDa acid phosphatase from chicken heart possesses phosphotransferase activity.

    PubMed

    Naz, Rubina; Saeed, Asma; Saeed, Ahmad

    2006-02-01

    A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K (cat)/K (m), 4.3 x 10(4), found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.

  19. Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application.

    PubMed

    Souza, Amanda Araújo; Leitão, Vanessa Oliveira; Ramada, Marcelo Henrique; Mehdad, Azadeh; Georg, Raphaela de Castro; Ulhôa, Cirano José; de Freitas, Sonia Maria

    2016-01-01

    Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65 °C, respectively, and is stable at 55 °C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0 was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments.

  20. Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae.

    PubMed

    Bostian, K A; Lemire, J M; Halvorson, H O

    1983-05-01

    We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed.

  1. Ultraviolet Light Enhances the Bovine Serum Albumin Fixation for Acid Fast Bacilli Stain

    PubMed Central

    Lai, Pei-Yin; Lee, Shih-Yi; Chou, Yu-Ching; Fu, Yung-Chieh; Wu, Chen-Cheng; Chiueh, Tzong-Shi

    2014-01-01

    The use of a liquid culture system such as MGIT broth has greatly improved the sensitivity of isolating mycobacteria in clinical laboratories. Microscopic visualization of acid fast bacilli (AFB) in the culture positive MGIT broth remains the first routine step for rapidly indicating the presence of mycobacteria. We modified an ultraviolet (UV) light fixation process to increase AFB cells adherence to the slide. The retained haze proportion of a 1-cm circle marked area on the smear slide was quantified after the staining procedure indicating the adherence degree of AFB cells. More AFB cells were preserved on the slide after exposure to UV light of either germicidal lamp or UV crosslinker in a time-dependent manner. We demonstrated both the bovine serum albumin (BSA) in MGIT media and UV light exposure were required for enhancing fixation of AFB cells. While applying to AFB stains for 302 AFB positive MGIT broths in clinics, more AFB cells were retained and observed on smear slides prepared by the modified fixation procedure rather than by the conventional method. The modified fixation procedure was thus recommended for improving the sensitivity of microscopic diagnosis of AFB cells in culture positive MGIT broth. PMID:24586725

  2. Gram stain

    MedlinePlus

    ... Gram stain; Feces - Gram stain; Stool - Gram stain; Joint fluid - Gram stain; Pericardial fluid - Gram stain; Gram ... body to test. This could be from a joint, from the sac around your heart, or from ...

  3. 4-Quinolone-3-carboxylic acids as cell-permeable inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Zhi, Ying; Gao, Li-Xin; Jin, Yi; Tang, Chun-Lan; Li, Jing-Ya; Li, Jia; Long, Ya-Qiu

    2014-07-15

    Protein tyrosine phosphatase 1B is a negative regulator in the insulin and leptin signaling pathways, and has emerged as an attractive target for the treatment of type 2 diabetes and obesity. However, the essential pharmacophore of charged phosphotyrosine or its mimetic confer low selectivity and poor cell permeability. Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent PTP1B inhibitors with low micromolar IC50 values. These 4-quinolone-3-carboxylate based PTP1B inhibitors displayed a 2-10 fold selectivity over a panel of PTP's. Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that the 4-quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B active site in the WPD loop closed conformation. Taken together, our study shows that the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological properties over previously described PTB1B inhibitors and warrant further preclinical studies.

  4. Acid inactivation of and incorporation of phosphate into alkaline phosphatase from Escherichia coli

    PubMed Central

    Pigretti, M. M.; Milstein, C.

    1965-01-01

    1. Alkaline phosphatase of Escherichia coli undergoes below pH 6·0 a reversible acid inactivation that has been studied and related to the extent of uptake of inorganic phosphate occurring below pH 6·0. 2. The rate of inactivation is rapid in the first few minutes but later it decreases markedly. Temperature, pH, composition of buffer and other factors have an important effect on the inactivation. 3. About 60% of the activity lost at pH values above 3·5 is rapidly recovered when the enzyme is taken back to pH 8·0, independently (within certain limits) of the extent of the inactivation. 4. Phosphate and Zn2+, although very good protectors of the inactivation by acid, are not by themselves able to reverse the acid inactivation. 5. Inorganic phosphate seems not to be incorporated into the acid-inactivated enzyme. 6. Incorporation of more than one mole of phosphate/mole of enzyme has been obtained, but the phosphate residues seem to be incorporated to serine residues with a common sequence, suggesting two identical active serine residues/molecule of active enzyme. ImagesFig. 3.Fig. 4. PMID:14342215

  5. Purification and biochemical characterisation of acid phosphatase-I from seeds of Nelumbo nucifera.

    PubMed

    Khan, Sanaullah; Khan, Shahnaz; Batool, Sajida; Ahmed, Mushtaq

    2016-01-01

    Acid phosphatase-I (Apase-I) from seeds of Nelumbo nucifera was purified to electrophoretic homogeneity by combination of ammonium sulfate precipitation, size-exclusion and ion exchange chromatography. SDS-PAGE of purified Apase-I gave a single band with molecular mass of 80 kDa under reducing and non-reducing conditions, indicating that the enzyme was a monomer. The purified enzyme showed maximum activity at 50°C and at pH 5. The Km, Vmax and Kcat for p-nitrophenyl phosphate were 132 μM, 10 μmol/min/mg and 6.7/sec respectively. Apase-I activity was strongly inhibited by Zn(2+), W(2+); weakly inhibited by Cu(2+), Mo(2+) and Cr(6+) and moderately activated by Mg(2+). The enzyme was shown to be thermolabile as it lost 50% of its activity at 50°C after incubation for 1 hour. The amino acid analysis of enzyme revealed high proportion of acidic amino acids, which is very similar to that of tomato Apase-I and lower than potato Apase.

  6. Crystallization and preliminary crystallographic data of purple acid phosphatase from red kidney bean.

    PubMed

    Sträter, N; Fröhlich, R; Schiemann, A; Krebs, B; Körner, M; Suerbaum, H; Witzel, H

    1992-03-20

    Purple acid phosphatase from red kidney bean has been crystallized from ammonium sulfate solutions in the pH range from 3.5 to 5.5. The crystal form is tetragonal bipyramidal and the largest crystals grew up to 2.0 mm long. Systematic absences indicate one of the enantiomorphic space groups P4(1)2(1)2 (92) or P4(3)2(1)2 (96) with cell dimensions a = b = 104.1(1) A and c = 308.7(2) A. The asymmetric unit contains one dimer with Mr of 110,700, determined by ultraviolet-laser desorption mass spectrometry. The crystals, with a salt-free density of 1.12 g/cm3 and a water content of 67%, diffract to 3.5 A.

  7. Regulators of PP2C phosphatase activity function as abscisic acid sensors.

    PubMed

    Ma, Yue; Szostkiewicz, Izabela; Korte, Arthur; Moes, Danièle; Yang, Yi; Christmann, Alexander; Grill, Erwin

    2009-05-22

    The plant hormone abscisic acid (ABA) acts as a developmental signal and as an integrator of environmental cues such as drought and cold. Key players in ABA signal transduction include the type 2C protein phosphatases (PP2Cs) ABI1 and ABI2, which act by negatively regulating ABA responses. In this study, we identify interactors of ABI1 and ABI2 which we have named regulatory components of ABA receptor (RCARs). In Arabidopsis, RCARs belong to a family with 14 members that share structural similarity with class 10 pathogen-related proteins. RCAR1 was shown to bind ABA, to mediate ABA-dependent inactivation of ABI1 or ABI2 in vitro, and to antagonize PP2C action in planta. Other RCARs also mediated ABA-dependent regulation of ABI1 and ABI2, consistent with a combinatorial assembly of receptor complexes.

  8. Ultrastructural localization of acid phosphatase in arbusculate coils of mycorrhizal Phoenix canariensis roots.

    PubMed

    Dreyer, Beatriz; Pérez-Gilabert, Manuela; Olmos, Enrique; Honrubia, Mario; Morte, Asunción

    2008-04-01

    Acid phosphatase (ACP) activity has been detected in roots of mycorrhizal and non-mycorrhizal Phoenix canariensis. This enzyme was ultrastructurally localized in arbusculate coils for the first time. This localization was carried out using a cerium-based method, which minimizes non-specific precipitation. The ACP was localized in inter- and intracellular hyphae, in the fungal cytoplasm as well as at the interface and the fungal cell wall and the periarbuscular membrane limiting it. The novel localization of an ACP in the arbuscular mycorrhizal (AM) interface of arbusculate coils suggests that this enzyme may be involved in the phosphorus efflux from the mycorrhizal fungus to the host. The results presented in this article indicate that the role played by ACP in AM symbiosis may be more important than was previously thought and that arbusculate coils are highly relevant when considering nutrient transfer through AM symbiosis.

  9. Effect of chaotropic agents on reversible unfolding of a soybean (Glycine max) seed acid phosphatase.

    PubMed

    Cavagis, Alexandre Donizeti Martins; Granjeiro, Paulo Afonso; Ferreira, Carmen Veríssima; Aoyama, Hiroshi

    2004-04-01

    In this work we examined the effect of urea and guanidinium chloride on the structural stability of a single isoform of soybean seed acid phosphatase, based on the intensity of tryptophan fluorescence as a function of denaturant concentration. The free energy of unfolding, DeltaGu, was calculated at 25 degrees C as a function of the concentrations of both chaotropic agents; the conformational stability, DeltaG (H2O), was determined to be 2.48 kcal mol(-1). Center of mass, determined from analysis of fluorescence data, was used as a parameter to assess conformational changes. Our results indicate that complete enzyme inactivation occurred before full enzyme unfolding in both cases, and suggest that there are differences between the conformational flexibility of the active-site and that of the macromolecule as a whole.

  10. Deoxycholic acid differentially regulates focal adhesion kinase phosphorylation: role of tyrosine phosphatase ShP2.

    PubMed

    Khare, Sharad; Holgren, Cory; Samarel, Allen M

    2006-12-01

    Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.

  11. Comparative Evaluation of Efficacy of Three Different Herbal Toothpastes on Salivary Alkaline Phosphatase and Salivary Acid Phosphatase - A Randomized Controlled Trial

    PubMed Central

    Dodamani, Arun; Karibasappa, G. N.; Deshmukh, Manjiri; Naik, Rahul

    2016-01-01

    Introduction Very few researches in the past have tried to evaluate the effect of herbal toothpaste on saliva and salivary constituents like alkaline phosphatase and acid phosphatase which play an important role in maintaining oral health. Aim To evaluate and compare the effect of three different herbal toothpastes on Salivary Alkaline Phosphatase (ALP) and salivary Acid Phosphatase (ACP). Material and Methods The present study was a preliminary study conducted among 45 dental students (15 subjects in each group) in the age group of 19-21 years. Subjects in each group were randomly intervened with three different herbal toothpastes respectively (Group A – Patanjali Dant Kanti, Group B - Himalaya Complete Care and Group C – Vicco Vajradanti). Unstimulated saliva sample were collected before and after brushing and salivary ACP and salivary ALP levels were assessed at an interval of one week each for a period of four weeks starting from day one. Compiled data was analyzed using chi square test, paired t-test and ANOVA based on the nature of the obtained data. Results All the three toothpastes showed significant (p<0.001) reduction in ACP and ALP levels at each interval. For patanjali toothpaste, the mean reduction was in the range of 2.55 – 2.62 IU/L for ACP and 2.94 – 2.99 IU/L for ALP. For Himalaya toothpaste, the mean reduction was in the range of 1.39 – 1.47 IU/L for ACP and 1.55 – 1.61 IU/L for ALP. For Vicco toothpaste, the mean reduction was in the range of 2.46 – 2.50 IU/L for ACP and 2.64 – 2.77 IU/L for ALP. Patanjali and Vicco toothpaste were significantly effective in reducing the levels of salivary ACP and ALP more than Himalaya toothpaste (p<0.05). Conclusion Herbal toothpastes, especially Dant Kanti and Vicco Vajradanti, showed significant reduction in levels of ACP and ALP resulting in overall improvement towards the oral health. PMID:27790584

  12. Fine structural and histochemical studies on salivary glands of Peripatoides novae-zealandiae (Onychophora) with special reference to acid phosphatase distribution.

    PubMed

    Nelson, L; van der Lande, V; Robson, E A

    1980-01-01

    The Onychophora feed on small arthropods and produce saliva when ingesting prey. Although saliva undoubtedly helps to liquefy the food its constituents have not yet been fully described. The salivary glands, two long tubes of glandularepithelium, are known to secrete a powerful protease, however, besides other enzymes and mucus. In Peripatoides novae-zealandiae there are protein-secreting cells of three types, referred to here as columnar, cuboidal and modified cells, and mucus cells. The anterior two-thirds of the gland show most cell diversity, while the posterior regionconsists mainly of columnar cells. These are the most numerous elements overall and they probably secrete salivary protease. In thick resin sections the granules of all protein-secreting cells stain strongly with methylene blue. Those of columnar cells are markedly uneven in size and accumulate distally, eventually filling the cytoplasm. More proximal Golgi regions may be discernible. Mucus cells are all of one type and their secretion droplets are stained lightly by methylene blue. The electron microscope shows that distal microvilli, desmosomes and septate junctions are common to all gland cells. In columnar cells, secretory material is contributed by Golgi complexes and by rough endoplasmic reticulum. Early secretory vacuoles containing dense material are seen in the concavity of Golgi regions. They are precursors to larger condensing vacuoles whose contents have a more flocculent appearance, and which may attain 3--4 micrometers in diameter. These evolve into secretory granules, usually of uneven texture, which are up to 2.5 micrometers in diameter. Histochemical tests for acid phosphatase show moderate amounts of enzyme throughout the gland. In whole mounts and sections the strongest reaction is in a band of cuboidal cells along the anterior median border. Columnar cells show a diffuse cytoplasmic reaction towards the base and sometimes distal to the nucleus, and mucus cells may also react

  13. Effect of gingival application of melatonin on alkaline and acid phosphatase, osteopontin and osteocalcin in patients with diabetes and periodontal disease

    PubMed Central

    López-Valverde, Antonio; Gómez-de-Diego, Rafel; Arias-Santiago, Salvador; de Vicente-Jiménez, Joaquín

    2013-01-01

    Objectives: To assess the effect of topical application of melatonin to the gingiva on salivary fluid concentrations of acid phosphatase, alkaline phosphatase, osteopontin, and osteocalcin. Study Design: Cross-sectional study of 30 patients with diabetes and periodontal disease and 30 healthy subjects. Diabetic patients were treated with topical application of melatonin (1% orabase cream formula) once daily for 20 days and controls with a placebo formulation. Results: Before treatment with melatonin, diabetic patients showed significantly higher mean salivary levels of alkaline and acid phosphatase, osteopontin and osteocalcin than healthy subjects (P < 0.01). After treatment with melatonin, there was a statistically significant decrease of the gingival index (15.84± 10.3 vs 5.6 ± 5.1) and pocket depth (28.3 ± 19.5 vs 11.9 ± 9.0) (P < 0.001). Also, use of melatonin was associated with a significant reduction of the four biomarkers. Changes of salivary acid phosphatase and osteopontin correlated significantly with changes in the gingival index, whereas changes of alkaline phosphatase and osteopontin correlated significantly with changes in the pocket depth. Conclusions: Treatment with topical melatonin was associated with an improvement in the gingival index and pocket depth, a reduction in salivary concentrations of acid phosphatase, alkaline phosphatase, osteopontin and osteocalcin. Key words:Melatonin, diabetes mellitus, alkaline phosphatase, acid phosphatase, osteopontin, osteocalcin. PMID:23524437

  14. Iron content and acid phosphatase activity in hepatic parenchymal lysosomes of patients with hemochromatosis before and after phlebotomy treatment

    SciTech Connect

    Cleton, M.I.; de Bruijn, W.C.; van Blokland, W.T.; Marx, J.J.; Roelofs, J.M.; Rademakers, L.H.

    1988-03-01

    Lysosomal structures in liver parenchymal cells of 3 patients with iron overload and of 3 subjects without iron-storage disorders were investigated. A combination of enzyme cytochemistry--with cerium as a captive ion to demonstrate lysosomal acid phosphatase activity--and electron probe X-ray microanalysis (EPMA) was used. We were able (1) to define and quantify lysosomal structures as lysosomes, siderosomes, or residual bodies, (2) to quantify the amount of iron and cerium simultaneously in these structures, and (3) to evaluate a possible relation between iron storage and enzyme activity. With histopathologically increased iron storage, the number of siderosomes had increased at the cost of lysosomes, with a corresponding increase in acid phosphatase activity in both organelles. In histopahtologically severe iron overload, however, acid phosphatase activity was low or not detectable and most of the iron was stored in residual bodies. After phlebotomy treatment, the number of siderosomes had decreased in favor of the lysosomes, approaching values obtained in control subjects, and acid phosphatase activity was present in all iron-containing structures. In this way a relationship between iron storage and enzyme activity was established. The iron content of the individual lysosomal structures per unit area had increased with histopathologically increased iron storage and had decreased after phlebotomy treatment. From this observation, it is concluded that the iron status of the patient is not only reflected by the amount of iron-containing hepatocytes but, as well, by the iron content lysosomal unit area.

  15. Suppression of DS1 phosphatidic acid phosphatase confirms resistance to Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Nakano, Masahito; Nishihara, Masahiro; Yoshioka, Hirofumi; Takahashi, Hirotaka; Sawasaki, Tatsuya; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2013-01-01

    Nicotianabenthamiana is susceptible to Ralstonia solanacearum. To analyze molecular mechanisms for disease susceptibility, we screened a gene-silenced plant showing resistance to R. solanacearum, designated as DS1 (Disease suppression 1). The deduced amino acid sequence of DS1 cDNA encoded a phosphatidic acid phosphatase (PAP) 2. DS1 expression was induced by infection with a virulent strain of R. solanacearum in an hrp-gene-dependent manner. DS1 rescued growth defects of the temperature-sensitive ∆lpp1∆dpp1∆pah1 mutant yeast. Recombinant DS1 protein showed Mg(2+)-independent PAP activity. DS1 plants showed reduced PAP activity and increased phosphatidic acid (PA) content. After inoculation with R. solanacearum, DS1 plants showed accelerated cell death, over-accumulation of reactive oxygen species (ROS), and hyper-induction of PR-4 expression. In contrast, DS1-overexpressing tobacco plants showed reduced PA content, greater susceptibility to R. solanacearum, and reduced ROS production and PR-4 expression. The DS1 phenotype was partially compromised in the plants in which both DS1 and NbCoi1 or DS1 and NbrbohB were silenced. These results show that DS1 PAP may affect plant immune responses related to ROS and JA cascades via regulation of PA levels. Suppression of DS1 function or DS1 expression could rapidly activate plant defenses to achieve effective resistance against Ralstonia solanacearum.

  16. Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea.

    PubMed

    Poirier, T P; Holt, S C

    1983-10-01

    Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS-PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AlP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

  17. A convenient and label-free fluorescence "turn off-on" nanosensor with high sensitivity and selectivity for acid phosphatase.

    PubMed

    Liu, Ziping; Lin, Zihan; Liu, Linlin; Su, Xingguang

    2015-05-30

    In this study, we reported a convenient label-free fluorescence nanosensor for rapid detection of acid phosphatase on the basis of aggregation-caused quenching (ACQ) and enzymolysis approach. The selectivity nanosensor was based on the fluorescence "turn off-on" mode, which possessed high sensitivity features. The original strong fluorescence intensity of CuInS2 QDs was quenched by sodium hexametaphosphate (NaPO3)6. The high efficiency of the quenching was caused by the non-covalent binding of positively charged CuInS2 QDs to the negatively charged (NaPO3)6 through electrostatic interactions, aggregating to form a CuInS2 QDs/(NaPO3)6 complex. Adding acid phosphatase caused intense fluorescence of CuInS2 QDs/(NaPO3)6 to be recovered, and this was because of enzymolysis. (NaPO3)6 was hydrolyzed into small fragments and the high negative charge density decreased, which would weaken the strong electrostatic interactions. As a result, the quenched fluorescence "turned on". Under the optimum conditions, there was a good linear relationship between I/I0 (I and I0 were the fluorescence intensity of CuInS2 QDs/(NaPO3)6 system in the presence and absence of acid phosphatase, respectively) and acid phosphatase concentration in the range of 75-1500 nU mL(-1) with the detection limit of 9.02 nU mL(-1). The proposed nanosensor had been utilized to detect and accurately quantify acid phosphatase in human serum samples with satisfactory results.

  18. The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function

    PubMed Central

    Papadaki, Amalia; Politou, Anastasia S.; Smirlis, Despina; Kotini, Maria P.; Kourou, Konstadina; Papamarcaki, Thomais; Boleti, Haralabia

    2015-01-01

    Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP–mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP–His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP–mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence. PMID:25695743

  19. Characterization of a Soluble Phosphatidic Acid Phosphatase in Bitter Melon (Momordica charantia)

    PubMed Central

    Cao, Heping; Sethumadhavan, Kandan; Grimm, Casey C.; Ullah, Abul H. J.

    2014-01-01

    Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are typically categorized into two subfamilies: Mg2+-dependent soluble PAP and Mg2+-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg2+-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53–60°C and unaffected by up to 0.3 mM MgCl2. The Km and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na3VO4, Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg2+-independent enzyme in plants. PMID:25203006

  20. Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia).

    PubMed

    Cao, Heping; Sethumadhavan, Kandan; Grimm, Casey C; Ullah, Abul H J

    2014-01-01

    Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and P(i). PAPs are typically categorized into two subfamilies: Mg(2+)-dependent soluble PAP and Mg(2+)-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg(2+)-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53-60 °C and unaffected by up to 0.3 mM MgCl2. The K(m) and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na(3)VO(4), Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg(2+)-independent enzyme in plants.

  1. Probing the interaction induced conformation transitions in acid phosphatase with cobalt ferrite nanoparticles: Relation to inhibition and bio-activity of Chlorella vulgaris acid phosphatase.

    PubMed

    Ahmad, Farooq; Zhou, Xing; Yao, Hongzhou; Zhou, Ying; Xu, Chao

    2016-09-01

    The present study explored the interaction and kinetics of cobalt ferrite nanoparticles (NPs) with acid phosphatase (ACP) by utilizing diverse range of spectroscopic techniques. The results corroborate, the CoFe2O4 NPs cause fluorescence quenching in ACP by static quenching mechanism. The negative values of van't Hoff thermodynamic expressions (ΔH=-0.3293Jmol(-1)K(-1) and ΔG=-3.960kJmol(-1)K(-1)) corroborate the spontaneity and exothermic nature of static quenching. The positive value of ΔS (13.2893Jmol(-1)K(-1)) corroborate that major contributors of higher and stronger binding affinity among CoFe2O4 NPs with ACP were electrostatic. In addition, FTIR, UV-CD, UV-vis spectroscopy and three dimensional fluorescence (3D) techniques confirmed that CoFe2O4 NPs binding induces microenvironment perturbations leading to secondary and tertiary conformation changes in ACP to a great extent. Furthermore, synchronous fluorescence spectroscopy (SFS) affirmed the comparatively significant changes in microenvironment around tryptophan (Trp) residue by CoFe2O4 NPs. The effect of CoFe2O4 NPs on the activation kinetics of ACP was further examined in Chlorella vulgaris. Apparent Michaelis constant (Km) values of 0.57 and 26.5mM with activation energy values of 0.538 and 3.428kJmol(-1) were determined without and with 200μM CoFe2O4 NPs. Apparent Vmax value of -7Umml(-1) corroborate that enzyme active sites were completely captured by the NPs leaving no space for the substrate. The results confirmed that CoFe2O4 NPs ceased the activity by unfolding of ACP enzyme. This suggests CoFe2O4 NPs perturbed the enzyme activity by transitions in conformation and hence the metabolic activity of ACP. This study provides the pavement for novel and simple approach of using sensitive biomarkers for sensing NPs in environment.

  2. Vanadyl complexes with dansyl-labelled di-picolinic acid ligands: synthesis, phosphatase inhibition activity and cellular uptake studies.

    PubMed

    Collins, Juliet; Cilibrizzi, Agostino; Fedorova, Marina; Whyte, Gillian; Mak, Lok Hang; Guterman, Inna; Leatherbarrow, Robin; Woscholski, Rudiger; Vilar, Ramon

    2016-04-28

    Vanadium complexes have been previously utilised as potent inhibitors of cysteine based phosphatases (CBPs). Herein, we present the synthesis and characterisation of two new fluorescently labelled vanadyl complexes (14 and 15) with bridged di-picolinic acid ligands. These compounds differ significantly from previous vanadyl complexes with phosphatase inhibition properties in that the metal-chelating part is a single tetradentate unit, which should afford greater stability and scope for synthetic elaboration than the earlier complexes. These new complexes inhibit a selection of cysteine based phosphatases (CBPs) in the nM range with some selectivity. Fluorescence spectroscopic studies (including fluorescence anisotropy) were carried out to demonstrate that the complexes are not simply acting as vanadyl delivery vehicles but they interact with the proteins. Finally, we present preliminary fluorescence microscopy studies to demonstrate that the complexes are cell permeable and localise throughout the cytoplasm of NIH3T3 cells.

  3. The Jasper Ridge elevated CO{sub 2} experiment: Root acid phosphatase activity in Bromus hordeaceus and Avena barbata remains unchanged under elevated [CO{sub 2}

    SciTech Connect

    Cardon, Z.G.; Jackson, R.

    1995-06-01

    Root acid phosphatase activity increases phosphate available to plants by cleaving phosphate esters in soil organic matter. Because of increased plant growth potential under elevated [CO{sub 2}], we hypothesized that high [CO{sub 2}]-grown plants might exhibit higher phosphatase activity than low [CO{sub 2}]-grown plants. We assayed phosphatase activity in two species grown on two substrates (Bromus on serpentine soil and Bromus and Avena on sandstone soil) under high and low [CO{sub 2}] and under several nutrient treatments. Phosphatase activity was expressed per gram fresh weight of roots. Phosphatase activity of Bromus roots (on sandstone) was first assayed in treatments where only P and K, or only N, were added to soil. Bromus roots in this case showed strong induction of phosphatase activity when N only had been added to soil, indicating that Bromus regulated its phosphatase activity in response to phosphate availability. Both Bromus and Avena growing in sandstone, and Bromus growing in serpentine, showed enhanced phosphatase activity at high nutrient (N, P, and K) levels over that at low nutrient levels, but no differences between phosphatase activity were apparent between [CO{sub 2}] treatments. The increased phosphatase activity at high N, P, and K may indicate enhanced {open_quotes}growth demand{close_quotes} (reflected in higher biomass) in both Avena and Bromus. In contrast, though Bromus {open_quotes}growth demand{close_quotes} (biomass) increased under high [CO{sub 2}] on sandstone, phosphatase activity did not increase.

  4. Phosphatidic acid phosphatase and diacylglycerol acyltransferase: potential targets for metabolic engineering of microorganism oil.

    PubMed

    Jin, Hong-Hao; Jiang, Jian-Guo

    2015-04-01

    Oleaginous microorganism is becoming one of the most promising oil feedstocks for biodiesel production due to its great advantages in triglyceride (TAG) accumulation. Previous studies have shown that de novo TAG biosynthesis can be divided into two parts: the fatty acid biosynthesis pathway (the upstream part which generates acyl-CoAs) and the glycerol-3-phosphate acylation pathway (the downstream part in which three acyl groups are sequentially added onto a glycerol backbone). This review mainly focuses on two enzymes in the G3P pathway, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). The former catalyzes a dephosphorylation reaction, and the latter catalyzes a subsequent acylation reaction. Genes, functional motifs, transmembrane domains, action mechanism, and new studies of the two enzymes are discussed in detail. Furthermore, this review also covers diacylglycerol kinase, an enzyme that catalyzes the reverse reaction of diacylglycerol formation. In addition, PAP and DGAT are the conjunction points of the G3P pathway, the Kennedy pathway, and the CDP-diacylglycerol pathway (CDP-DAG pathway), and the mutual transformation between TAGs and phospholipids is discussed as well. Given that both the Kennedy and CDP-diacylglycerol pathways are in metabolic interlock (MI) with the G3P pathway, it is suggested that, via metabolic engineering, TAG accumulation can be improved by the two pathways based on the pivotal function of PAP and DGAT.

  5. Evaluation of serum sialic acid, heat stable alkaline phosphatase and fucose as markers of breast carcinoma.

    PubMed

    Patel, P S; Baxi, B R; Adhvaryu, S G; Balar, D B

    1990-01-01

    Serum levels of total sialic acid (TSA), lipid bound sialic acid (LSA), heat stable alkaline phosphatase (HSAP) and fucose were measured in 39 patients with breast carcinoma, 14 patients with benign breast diseases and 35 healthy female individuals. Elevated levels of the four biomarkers in breast carcinoma were significant when compared with controls (p less than 0.001). Fucose levels were most sensitive (71.8%), while TSA levels were most specific (64.3%) for breast carcinoma. Sensitivity and specificity were 100% when combinations of LSA with fucose and TSA with HSAP were studied respectively. LSA was significantly elevated in infiltrating duct carcinoma patients compared with lobular carcinoma (p less than 0.001). TSA, HSAP and fucose also had lower mean values in lobular carcinoma as compared to infiltrating duct carcinoma. Increase in the levels of LSA and HSAP after surgical removal of the tumor in breast carcinoma occurred prior to the clinical evidence of the recurrence. The results indicate that the combination of the markers studied might be useful in breast cancer diagnosis and treatment monitoring.

  6. Recessive Mutations in ACPT, Encoding Testicular Acid Phosphatase, Cause Hypoplastic Amelogenesis Imperfecta.

    PubMed

    Seymen, Figen; Kim, Youn Jung; Lee, Ye Ji; Kang, Jenny; Kim, Tak-Heun; Choi, Hwajung; Koruyucu, Mine; Kasimoglu, Yelda; Tuna, Elif Bahar; Gencay, Koray; Shin, Teo Jeon; Hyun, Hong-Keun; Kim, Young-Jae; Lee, Sang-Hoon; Lee, Zang Hee; Zhang, Hong; Hu, Jan C-C; Simmer, James P; Cho, Eui-Sic; Kim, Jung-Wook

    2016-11-03

    Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders affecting tooth enamel. The affected enamel can be hypoplastic and/or hypomineralized. In this study, we identified ACPT (testicular acid phosphatase) biallelic mutations causing non-syndromic, generalized hypoplastic autosomal-recessive amelogenesis imperfecta (AI) in individuals from six apparently unrelated Turkish families. Families 1, 4, and 5 were affected by the homozygous ACPT mutation c.713C>T (p.Ser238Leu), family 2 by the homozygous ACPT mutation c.331C>T (p.Arg111Cys), family 3 by the homozygous ACPT mutation c.226C>T (p.Arg76Cys), and family 6 by the compound heterozygous ACPT mutations c.382G>C (p.Ala128Pro) and 397G>A (p.Glu133Lys). Analysis of the ACPT crystal structure suggests that these mutations damaged the activity of ACPT by altering the sizes and charges of key amino acid side chains, limiting accessibility of the catalytic core, and interfering with homodimerization. Immunohistochemical analysis confirmed localization of ACPT in secretory-stage ameloblasts. The study results provide evidence for the crucial function of ACPT during amelogenesis.

  7. Molecular cloning of magnesium-independent type 2 phosphatidic acid phosphatases from airway smooth muscle.

    PubMed

    Tate, R J; Tolan, D; Pyne, S

    1999-07-01

    Members of the type 2 phosphatidic acid phosphatase (PAP2) family catalyse the dephosphorylation of phosphatidic acid (PA), lysophosphatidate and sphingosine 1-phosphate. Here, we demonstrate the presence of a Mg(2+)-independent and N-ethymaleimide-insensitive PAP2 activity in cultured guinea-pig airway smooth muscle (ASM) cells. Two PAP2 cDNAs of 923 and 926 base pairs were identified and subsequently cloned from these cells. The ORF of the 923 base pair cDNA encoded a protein of 285 amino acids (Mr = 32.1 kDa), which had 94% homology with human PAP2a (hPAP2a) and which probably represents a guinea-pig specific PAP2a (gpPAP2a1). The ORF of the 926 base pair cDNA encoded a protein of 286 amino acids (Mr = 32.1 kDa) which had 84% and 91% homology with hPAP2a and gpPAP2a1, respectively. This protein, termed gpPAP2a2, has two regions (aa 21-33 and 51-74) of marked divergence and altered hydrophobicity compared with hPAP2a and gpPAP2a1. This occurs in the predicted first and second transmembrane domains and at the extremes of the first outer loop. Other significant differences between gpPAP2a1/2 and hPAP2a, hPAP2b and hPAP2c occur at the cytoplasmic C-terminal. Transient expression of gpPAP2a2 in Cos-7 cells resulted in an approx. 4-fold increase in Mg(2+)-independent PAP activity, thereby confirming that gpPAP2a2 is another catalytically active member of an extended PAP2 family.

  8. A novel antimicrobial protein isolated from potato (Solanum tuberosum) shares homology with an acid phosphatase.

    PubMed Central

    Feng, Jie; Yuan, Fenghua; Gao, Yin; Liang, Chenggang; Xu, Jin; Zhang, Changling; He, Liyuan

    2003-01-01

    The nucleotide and amino acids sequences for AP(1) will appear in the GenBank(R) and NCBI databases under accession number AY297449. A novel antimicrobial protein (AP(1)) was purified from leaves of the potato ( Solanum tuberosum, variety MS-42.3) with a procedure involving ammonium sulphate fractionation, molecular sieve chromatography with Sephacryl S-200 and hydrophobic chromatography with Butyl-Sepharose using a FPLC system. The inhibition spectrum investigation showed that AP(1) had good inhibition activity against five different strains of Ralstonia solanacearum from potato or other crops, and two fungal pathogens, Rhizoctonia solani and Alternaria solani from potato. The full-length cDNA encoding AP(1) has been successfully cloned by screening a cDNA expression library of potato with an anti-AP(1) antibody and RACE (rapid amplification of cDNA ends) PCR. Determination of the nucleotide sequences revealed the presence of an open reading frame encoding 343 amino acids. At the C-terminus of AP(1) there is an ATP-binding domain, and the N-terminus exhibits 58% identity with an/the acid phosphatase from Mesorhizobium loti. SDS/PAGE and Western blotting analysis suggested that the AP(1) gene can be successfully expressed in Escherichia coli and recognized by an antibody against AP(1). Also the expressed protein showed an inhibition activity the same as original AP(1) protein isolated from potato. We suggest that AP(1) most likely belongs to a new group of proteins with antimicrobial characteristics in vitro and functions in relation to phosphorylation and energy metabolism of plants. PMID:12927022

  9. Prostate cancer derived prostatic acid phosphatase promotes an osteoblastic response in the bone microenvironment

    PubMed Central

    Larson, Sandy R.; Chin, Jessica; Zhang, Xiaotun; Brown, Lisha G.; Coleman, Ilsa M.; Lakely, Bryce; Tenniswood, Martin; Corey, Eva; Nelson, Peter S.; Vessella, Robert L.

    2014-01-01

    Approximately 90 % of patients who die of prostate cancer (PCa) have bone metastases, often promoting osteoblastic lesions. We observed that 88 % of castration-resistant PCa (CRPC) bone metastases express prostatic acid phosphatase (PAP), a soluble secreted protein expressed by prostate epithelial cells in predominately osteoblastic (n = 18) or osteolytic (n = 15) lesions. Additionally, conditioned media (CM) of an osteoblastic PCa xenograft LuCaP 23.1 contained significant levels of PAP and promoted mineralization in mouse and human calvaria-derived cells (MC3T3-E1 and HCO). To demonstrate that PAP promotes mineralization, we stimulated MC3T3-E1 cells with PAP and observed increased mineralization, which could be blocked with the specific PAP inhibitor, phosphonic acid. Furthermore, the mineralization promoted by LuCaP 23.1 CM was also blocked by phosphonic acid, suggesting PAP is responsible for the mineralization promoting activity of LuCaP 23.1. In addition, gene expression arrays comparing osteoblastic to osteolytic CRPC (n = 14) identified betacellulin (BTC) as a gene upregulated during the osteoblastic response in osteoblasts during new bone formation. Moreover, BTC levels were increased in bone marrow stromal cells in response to LuCaP 23.1 CM in vitro. Because new bone formation does occur in osteoblastic and can occur in osteolytic CRPC bone metastases, we confirmed by immunohistochemistry (n = 36) that BTC was highly expressed in osteoblasts involved in new bone formation occurring in both osteoblastic and osteolytic sites. These studies suggest a role for PAP in promoting the osteoblastic reaction in CRPC bone metastases and identify BTC as a novel downstream protein expressed in osteoblasts during new bone formation. PMID:24242705

  10. Behavior of soluble and immobilized acid phosphatase in hydro-organic media.

    PubMed

    Wan, H; Horvath, C

    1975-11-20

    The hydrolysis of p-nitrophenyl phosphate by wheat germ acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) has been investigated in mixtures of aqueous buffers with acetone, dioxane and acetonitrile. The enzyme was either in free solution or immobilized on a pellicular support which consisted of a porous carbonaceous layer on solid glass beads. The highest enzyme activity was obtained in acetone and acetonitrile mixed with citrate buffer over a wide range of organic solvent concentration. In 50% (v/v) acetone both V and Km of the immobilized enzyme were about half of the values in the neat aqueous buffer, but the Ki for inorganic phosphate was unchanged. In 50% (v/v) mixtures of various solvents and citrate buffers of different pH, the enzymic activity was found to depend on the pH of the aqueous buffer component rather than the pH of the hydro-organic mixture as measured with the glass-calomel electrode. The relatively high rates of p-nitrophenol liberation in the presence of glucose even at high organic solvent concentrations suggest that transphosphorylation is facilitated at low water activity.

  11. Characterization of four monoclonal antibodies to recombinant human tartrate-resistant acid phosphatase.

    PubMed

    Miyazaki, Takashi; Matsunaga, Toshiyuki; Miyazaki, Shuichi; Hokari, Shigeru; Komoda, Tsugikazu

    2002-06-01

    In this study we produced a recombinant human Tartrate-resistant acid phosphatase (TRAP) enzyme from baculovirus-infected insect cells, generated four monoclonal antibodies (MAbs) 15A4, 13B9, 1C6 and 3G7, to the enzyme, and characterized these antibodies. In the human serum and lung specimen, all four antibodies appeared to have a high specificity for native TRAP enzyme in western blot analysis, immunohistochemical analysis and enzyme immunoassay. These antibodies may react with respective conformational determinants, therefore, they may be useful for detection of active TRAP. Only one of the antibodies, 15A4 also reacted with a denatured epitope, therefore, it is suitable for western blot analysis, enzyme immunoassay and for immunohistochemistry in the rat. Taken together, having characterized properties of four monoclonal antibodies against recombinant human TRAP enzyme may be useful for development of TRAP specific immunoassays in pathology and hematology of the bone. They will certainly be of use for the study of biosynthesis, regulation and function of the TRAP enzyme.

  12. Kinetics and optical spectroscopic studies on the purple acid phosphatase from beef spleen.

    PubMed

    Davis, J C; Lin, S S; Averill, B A

    1981-07-07

    A new purification scheme has been developed for the purple acid phosphatase from beef spleen; typical yields are 8 mg of homogeneous enzyme per kg of spleen in only five steps. Kinetics studies have shown that the enzyme is strongly inhibited by fluoride, phosphate, and [p-(acetylamino)-benzyl]phosphonate, a nonhydrolyzable substrate analogue; the last two of these show simple competitive inhibition. In contrast, cyanide, azide, tartrate, and p-nitrophenol show no inhibition at concentrations up to 10 mM. Molecular weight estimations by gel electrophoresis and gel permeation chromatography give a value of 40 000 for the native enzyme, which is shown to consist of two subunits of apparent molecular weight 24 000 and 15 000. Careful metal analyses indicate the presence of 2.1 +/- 0.1 iron atoms per enzyme molecule, and less than 0.1 copper, zinc, nickel, or manganese atom per enzyme. The purple enzyme (lambda max 550 nm) is reversibly converted to a pink, active form (lambda max 505 nm) upon treatment with mild reducing agents (dithioerythritol or ascorbate). Addition of competitive inhibitors to the pink form causes rapid reversion to the purple form. Electron paramagnetic resonance spectroscopy at several temperatures showed only weak g = 4.3 signals (less than 0.1 spin/molecule) for the native, reduced, and inhibited forms of the enzyme.

  13. Serum tissue polypeptide antigen (TPA) and prostatic acid phosphatase (PAP) in patients with prostatic cancer.

    PubMed

    Marczyńska, A; Kulpa, J; Leńko, J; Augustyn, M

    1988-01-01

    Concurrent measurements of serum TPA and PAP concentrations by double antibody radioimmunoassays were done in 49 patients with prostatic cancer in different clinical stages. The reference group comprised patients suffering from BPH. Positive TPA was found in 32.7% of cancer patients, the lowest percentage in stage A (11.1%) and the highest in stage D (55.6%). The additional value as a diagnostic aid of the TPA test was revealed on the basis of examination of the selected group of patients with not increased PAP. Positive TPA was found in 16.7% of patients: none in stage A, 22.2% in stage B, and 33.3% in stage D. Prostatic cancer remains the most common malignancy of the genitourinary tract. The improvement in the results of treatment involves not only a modernization of treatment modalities but also the introduction of laboratory tests which give the most ample information on the stage of tumour development and improve possibilities to control tumour therapy. Besides the refinement of the determination procedures of specific prostatic markers, prostatic acid phosphatase (PAP), through radio- and enzyme-immunological methods, there is a search for additional markers which might be helpful in diagnosis and follow-up of treatment.

  14. Dependence on the Lazaro phosphatidic acid phosphatase for the maximum light response.

    PubMed

    Kwon, Young; Montell, Craig

    2006-04-04

    The Drosophila phototransduction cascade serves as a paradigm for characterizing the regulation of sensory signaling and TRP channels in vivo . Activation of these channels requires phospholipase C (PLC) and may depend on subsequent production of diacylglycerol (DAG) and downstream metabolites . DAG could potentially be produced through a second pathway involving the combined activities of a phospholipase D (PLD) and a phosphatidic acid (PA) phosphatase (PAP). However, a role for a PAP in the regulation of TRP channels has not been described. Here, we report the identification of a PAP, referred to as Lazaro (Laza). Mutations in laza caused a reduction in the light response and faster termination kinetics. Loss of laza suppressed the severity of the phenotype caused by mutation of the DAG kinase, RDGA , indicating that Laza functions in opposition to RDGA. We also showed that the retinal degeneration resulting from overexpression of the PLD was suppressed by elimination of Laza. These data demonstrate a requirement for a PLD/PAP-dependent pathway for achieving the maximal light response. The genetic interactions with both rdgA and Pld indicate that Laza functions in the convergence of both PLC- and PLD-coupled signaling in vivo.

  15. HIV-1 enhancing effect of prostatic acid phosphatase peptides is reduced in human seminal plasma.

    PubMed

    Martellini, Julie A; Cole, Amy L; Svoboda, Pavel; Stuchlik, Olga; Chen, Li-Mei; Chai, Karl X; Gangrade, Bhushan K; Sørensen, Ole E; Pohl, Jan; Cole, Alexander M

    2011-01-20

    We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.

  16. Acid phosphatase activity and color changes in consumer-style griddle-cooked ground beef patties.

    PubMed

    Lyon, B G; Davis, C E; Windham, W R; Lyon, C E

    2001-08-01

    The U.S. Department of Agriculture and the Food and Drug Administration have issued temperature requirements to help consumers cook beef patty products that are free of pathogens. Verification of end-point temperature (EPT) is needed in cooked meat products due to concerns over outbreaks of Escherichia coli O157:H7. Acid phosphatase (ACP) activity was studied as a potential method for determination of EPT in ground beef patties cooked nonfrozen, patties frozen 7 days and thawed at room temperature 4 h in a refrigerator or by microwave, and patties made from ground beef frozen in store packages, then thawed in a refrigerator overnight. Pressed-out meat juices were analyzed from patties (n = 314) cooked to 57.2 degrees C (135 degrees F). 65.6 degrees C (150 degrees F), 71.1 degrees C (160 degrees F), and 79.4 degrees C (175 degrees F) target EPTs. Expressed meat juice and internal meat patty color decreased in redness as EPT increased. Freezing whole packs with slow refrigerator or room temperature thawing caused significantly greater loss of redness in expressed cooked meat juice than did other handling methods. Log10 ACP had a significant linear (R2 = 0.99) response to EPT. Results show that the 3- to 5-min ACP test could be used to verify EPT in griddle-cooked hamburger patties.

  17. The effects of retinoic acid on alkaline phosphatase activity and tissue-non-specific alkaline phosphatase gene expression in human periodontal ligament cells and gingival fibroblasts.

    PubMed

    San Miguel, S M; Goseki-Sone, M; Sugiyama, E; Watanabe, H; Yanagishita, M; Ishikawa, I

    1998-10-01

    Alkaline phosphatase (ALP) in human periodontal ligament (HPDL) cells is classified as a tissue-non-specific alkaline phosphatase (TNSALP) by its enzymatic and immunological properties. Since retinoic acid (RA) has been shown as a potent inducer of TNSALP expression in various osteoblastic and fibroblastic cells, we investigated the effects of RA on the level of ALP activity and expression of TNSALP mRNAs in HPDL cells. Cultured cells were treated with desired RA concentrations (0, 10(-7), 10(-6), 10(-5) M) in medium containing 1% bovine serum albumin without serum. ALP activity was determined by the rate of hydrolysis of p-nitrophenyl phosphate and was also assayed in the presence of specific inhibitors. In order to identify the TNSALP mRNA type expressed by HPDL, a set of oligonucleotide primers corresponding to 2 types of human TNSALP mRNA (i.e. bone-type and liver-type) were designed, and mRNA isolated from HPDL was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). After treatment with RA (10(-6) M) for 4 d, there was a significant increase in the ALP activity of HPDL cells. The use of inhibitors and thermal inactivation experiments showed that the increased ALP activity had properties of the TNSALP type. RT-PCR analysis revealed that bone-type mRNA was highly stimulated in HPDL cells by RA treatment, but the expression of liver-type mRNA was not detected. These results indicated that the upregulation of ALP activity in HPDL cells by RA was due to the increased transcription of bone-type mRNA of the TNSALP gene.

  18. Cloning and characterization of purple acid phosphatase phytases from wheat, barley, maize, and rice.

    PubMed

    Dionisio, Giuseppe; Madsen, Claus K; Holm, Preben B; Welinder, Karen G; Jørgensen, Malene; Stoger, Eva; Arcalis, Elsa; Brinch-Pedersen, Henrik

    2011-07-01

    Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.

  19. Characterization of purple acid phosphatases involved in extracellular dNTP utilization in Stylosanthes

    PubMed Central

    Liu, Pan-Dao; Xue, Ying-Bin; Chen, Zhi-Jian; Liu, Guo-Dao; Tian, Jiang

    2016-01-01

    Stylo (Stylosanthes spp.) is a pasture legume predominant in tropical and subtropical areas, where low phosphorus (P) availability is a major constraint for plant growth. Therefore, stylo might exhibit superior utilization of the P pool on acid soils, particularly organic P. However, little is known about mechanisms of inorganic phosphate (Pi) acquisition employed by stylo. In this study, the utilization of extracellular deoxy-ribonucleotide triphosphate (dNTP) and the underlying physiological and molecular mechanisms were examined for two stylo genotypes with contrasting P efficiency. Results showed that the P-efficient genotype, TPRC2001-1, was superior to the P-inefficient genotype, Fine-stem, when using dNTP as the sole P source. This was reflected by a higher dry weight and total P content for TPRC2001-1 than for Fine-stem, which was correlated with higher root-associated acid phosphatase (APase) activities in TPRC2001-1 under low P conditions. Subsequently, three PAP members were cloned from TPRC2001-1: SgPAP7, SgPAP10, and SgPAP26. Expression levels of these three SgPAPs were up-regulated by Pi starvation in stylo roots. Furthermore, there was a higher abundance of transcripts of SgPAP7 and SgPAP10 in TPRC2001-1 than in Fine-stem. Subcellular localization analysis demonstrated that these three SgPAPs were localized on the plasma membrane. Overexpression of these three SgPAPs could result in significantly increased root-associated APase activities, and thus extracellular dNTP utilization in bean hairy roots. Taken together, the results herein suggest that SgPAP7, SgPAP10, and SgPAP26 may differentially contribute to root-associated APase activities, and thus control extracellular dNTP utilization in stylo. PMID:27194738

  20. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae).

    PubMed

    Shane, Michael W; Stigter, Kyla; Fedosejevs, Eric T; Plaxton, William C

    2014-11-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native 'extremophile' plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors' knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested.

  1. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae)

    PubMed Central

    Shane, Michael W.; Stigter, Kyla; Fedosejevs, Eric T.; Plaxton, William C.

    2014-01-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native ‘extremophile’ plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors’ knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  2. Histopathological periodic acid-schiff stains of nail clippings as a second-line diagnostic tool in onychomycosis.

    PubMed

    Mayer, Eliza; Izhak, Ofer Ben; Bergman, Reuven

    2012-05-01

    The diagnosis of onychomycosis, using direct microscopy and fungal cultures, is often negative despite the presence of disease. Periodic acid-Schiff (PAS) staining of nail clippings, using histopathological processing, may be positive in these cases. It is not always clear, however, whether the fungal elements detected by PAS staining are pathogenic fungi or some are saprophytes. We aimed to study the efficacy of histopathological PAS staining of nail clippings as a second-line diagnostic tool in onychomycosis. The study included 100 consecutive cases in which direct microscopy and fungal cultures from suspected onychomycosis were negative on one occasion or more. The obtained nail clippings were processed for routine histology, stained with hematoxylin and eosin and PAS, and examined microscopically. Of the 100 cases, 38 (38%) showed positive fungal elements. As a result, 9 patients had sought and received oral antifungal therapy and all achieved complete clinical cure. The histological examination also revealed parakeratosis and globules of plasma, which were statistically significantly more common in the fungal infected nail samples. This may indicate an ongoing inflammatory process associated with onychomycosis. Neutrophils and bacteria were not statistically and significantly more common in the fungal infected nails. We conclude that as a second-line diagnostic tool, PAS stain of nail clippings increases markedly the diagnostic yield of onychomycosis and, consequently, the outcome of therapy.

  3. Enhancement of acid phosphatase secretion and Pi acquisition in Suaeda fruticosa on calcareous soil by high saline level.

    PubMed

    Labidi, Nehla; Snoussi, Sana; Ammari, Manel; Metoui, Wissal; Ben Yousfi, N; Hamrouni, Lamia; Abdelly, C

    2010-12-01

    The aim of this study was to identify the relationship between the adaptive processes of Suaeda fruticosa for Pi acquisition and the physic-chemical and biological characteristics of two soil types under moderate and high saline conditions. Four treatments were established in pots: namely SS100, SS600, CS100 and CS600 where SS stood for sandy soil and CS for calcareous soil, and the indexes 100 and 600 were NaCl concentrations (mM) in irrigation distilled water. Assuming that Pi per g of plant biomass is an indicator of plant efficiency for P acquisition, the results showed that Pi acquisition was easiest on SS100 and was difficult on CS100. The differences in Pi acquisition between plants on SS100 and CS100 could be attributed to the low root surface area (-30%) and to the low alkaline phosphatases (Pases) activities (-50%) in calcareous rhizospheric soil. The high salinity level had no effect on the efficiency of P acquisition on SS but increased this parameter on CS (+50%). In the latter soil type, high acid phosphatase activities were observed in rhizospheric soil at high salinity level. Acid phosphatase seemed to be secreted from the roots. The higher secretion of acid phosphatase in this soil was related to the root lipid peroxidation in response to elevated salinity associated with the augmentation of unsaturated acids which might induce an oxidative damage of the root membrane. Thus we can conclude that in deficient soil such as calcareous, the efficiency of P acquisition in S. fruticosa which was difficult at moderate salinity level can be enhanced by high salinity level.

  4. [Effect of aluminium and cAMP on acid phosphatase from the apoplast of barley and maize root cells].

    PubMed

    Fedorovskaia, M D; Tikhaia, N I

    2003-01-01

    Acid phosphatase activity inhibited by 1 mM sodium molybdate was detected at the surface of barley seedling roots and in the cell wall fraction isolated from barley and maize seedling roots. This enzyme hydrolyzed NPP, GP, and PPi at low pH (4.0 and below). NPP hydrolysis was stimulated by magnesium (but not calcium or manganese) ions, while PPi hydrolysis was independent of the presence of bivalent ions. The activity of phosphatase localized in the cell walls of the both crops increased in the presence of 100 microM AlCl3 or CuCl2. Stimulation of NPP hydrolysis by micromolar concentrations of aluminium and copper as well as by millimolar concentrations of magnesium decreased in the presence of 25 microM cAMP. This agrees with the previous data on the enzyme localized at the outer side of the properly oriented vesicles in the microscomal fraction of plasmalemma. The role of the root extracellular acid phosphatase loosely associated with various apoplast structures in plant adaptation to toxic effect of aluminium in the acidic soils as well as possible control of this process by cAMP secretion to the apoplast are discussed.

  5. Differences in sialic acid residues among bone alkaline phosphatase isoforms: a physical, biochemical, and immunological characterization.

    PubMed

    Magnusson, P; Farley, J R

    2002-12-01

    High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P < 0.001, but not Concanavalin A. At 3.0 mg/ml, WGA precipitated approximately 25% of B/I but more than 80% of B1 and B2. Molecular weights were estimated by native gradient gel electrophoresis: B/I, 126 kDa; B1, 136 kDa; and B2, 141 kDa. Desialylation with neuraminidase reduced the apparent sizes of B1 and B2 to 127 kDa (i.e., approximately to that of B/I). The total carbohydrate content was calculated to be 18 kDa, 28 kDa, and 33 kDa (i.e., 14%, 21%, and 23%) for the BALP isofonns, B/I, B1, and B2, respectively. The number of sialic acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P < 0.001). In summary, our data indicate that

  6. Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis

    PubMed Central

    Li, Chengchen; Gui, Shunhua; Yang, Tao; Walk, Thomas; Wang, Xiurong; Liao, Hong

    2012-01-01

    Background and Aims Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparative studies on structure, transcription regulation and responses to phosphate (Pi) deprivation of the soybean PAP gene family should facilitate further insights into the potential physiological roles of GmPAPs. Methods BLAST searches were performed to identify soybean PAP genes at the phytozome website. Bioinformatic analyses were carried out to investigate their gene structure, conserve motifs and phylogenetic relationships. Hydroponics and sand-culture experiments were carried out to obtain the plant materials. Quantitative real-time PCR was employed to analyse the expression patterns of PAP genes in response to P deficiency and symbiosis. Key Results In total, 35 PAP genes were identified from soybean genomes, which can be classified into three distinct groups including six subgroups in the phylogenetic tree. The expression pattern analysis showed flowers possessed the largest number of tissue-specific GmPAP genes under normal P conditions. The expression of 23 GmPAPs was induced or enhanced by Pi starvation in different tissues. Among them, nine GmPAP genes were highly expressed in the Pi-deprived nodules, whereas only two GmPAP genes showed significantly increased expression in the arbuscular mycorrhizal roots under low-P conditions. Conclusions Most GmPAP genes are probably involved in P acquisition and recycling in plants. Also we provide the first evidence that some members of the GmPAP gene family are possibly involved in the response of plants to symbiosis with rhizobia or arbuscular mycorrhizal fungi under P-limited conditions. PMID:21948626

  7. Genetic instability of constitutive acid phosphatase in shochu and sake yeast.

    PubMed

    Takashita, Hideharu; Kajiwara, Yasuhiro; Shimoda, Masahiko; Matsuoka, Masayoshi; Ogawa, Takahira; Ono, Kazuhisa

    2013-07-01

    Genetic instability of constitutive acid phosphatase (cAPase) activity was observed in a shochu brewer's yeast strain (Ko), which consistently produced 0.3-1% progeny without cAPase when it had been subcultured for a long period of time in barley shochu mash or in conventional complete medium. Genetic analysis showed that the cAPase-negative phenotype was associated with a single mutation in the PHO3 gene and that the Ko strain had heteroallelic PHO3/pho3 genes, while the PHO3⁻ mutants had the homoallelic pho3/pho3 defect. Some sake yeast strains that are cAPase negative, such as K6, K7 and K9, also had the same homoallelic defect, whereas another sake yeast strain K3, with heteroallelic PHO3/pho3 genes, displayed similar genetic instability of cAPase activity. In all cases, the pho3-defective genes were generated by deletion of an approximately 1.9 kb region between the PHO5-PHO3 tandem genes on chromosome II, resulting in chimeric PHO5/3 fusion genes with different fusion points. By integrating a lys2 marker, which is linked with the pho3 allele on the arm of chromosome II in the Ko strain, we demonstrated that the pho3/pho3 defect originated either from a loss of heterozygosity at the heteroallelic PHO3/pho3 locus or from a looping out of the PHO3 region. Although fermentation experiments have not yet indicated any correlation between cAPase activity and alcohol production, the PHO3⁻ mutation itself could prove to be a useful selective marker for yeast strains carrying a number of advantageous mutations for fermentation and which display phenotypic diversity and stability.

  8. Secretion and N-linked glycosylation are required for prostatic acid phosphatase catalytic and antinociceptive activity.

    PubMed

    Hurt, Julie K; Fitzpatrick, Brendan J; Norris-Drouin, Jacqueline; Zylka, Mark J

    2012-01-01

    Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6-7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This "cellular" construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia--a yeast strain that is used to generate biologics for therapeutic purposes.

  9. Ontogeny and distribution of alkaline and acid phosphatases in the digestive system of California halibut larvae (Paralichthys californicus).

    PubMed

    Zacarias-Soto, Magali; Barón-Sevilla, Benjamín; Lazo, Juan P

    2013-10-01

    Studies aimed to assess the digestive physiology of marine fish larvae under culture conditions are important to further understand the functional characteristics and digestive capacities of the developing larvae. Most studies to date concentrate on intestinal lumen digestion and little attention to the absorption process. Thus, the objectives of this study were to histochemically detect and quantify some of the enzymes responsible for absorption and intracellular digestion of nutrients in the anterior and posterior intestine of California halibut larvae. Alkaline and acid phosphatases were detected from the first days post-hatch (dph). Alkaline phosphatase maintained a high level of activity during the first 20 dph in both intestinal regions. Thereafter, a clear intestinal regionalization of the activity was observed with the highest levels occurring in the anterior intestine. Acid phosphatase activity gradually increased in both intestinal regions during development, and a regionalization of the activity was not observed until late in development, once the ocular migration began. Highest levels were observed in the anterior intestine at the end of metamorphosis concomitant with the stomach development. The results from this study show some morphological and physiological changes are occurring during larval development and a clear regionalization of the absorption process as the larvae develops. These ontological changes must be considered in the elaboration of diets according to the digestive capacity of the larvae.

  10. Root surface acid phosphatases and their role in phosphorus assimilation by Eriophorum vaginatum

    SciTech Connect

    Kroehler, C.J.; Linkins, A.E.

    1988-01-01

    Eriophorum vaginatum is a dominant plant in much of the arctic tundra ecosystem where phosphorus is frequently a limiting nutrient. The mineralization of this organic phosphorus was thought to be principally controlled by microbial respiration, however, more recent work shows that extracellular soil phosphatases are the principal regulators. The existence of plant root and mycorrhizal surface phosphatases which are capable of hydrolyzing organic phosphorus compounds, suggests that soil organic phosphorus may be directly utilized by plants. Since E. vaginatum is a tussock forming sedge with a very dense annually produced rooting system which can exploit most of the tussock soil volume, its surface phosphatases may play a dominant role in organic phosphorus hydrolysis into inorganic phosphorus. Of equal significance would be the potential for this activity to contribute to the phosphorus nutrition through the coupling of phosphorus hydrolysis on the root and root uptake of the resultant inorganic phosphorus. Phosphatase activity was investigated and found to be uniformly distributed along the surface of the root. Kinetic analysis of the enzyme gave estimates of 9.23 mM for the apparent Km and 1.61 * 10/sup -3/ ..mu..moles mm-2 hr/sup -1/ for the apparent Vmax. Saturation values for E. vaginatum phosphatases are about 3 times higher than average soil solution organic phosphorus concentrations. 12 refs., 4 figs.

  11. Structural and functional assays of AtTLP18.3 identify its novel acid phosphatase activity in thylakoid lumen.

    PubMed

    Wu, Hsin-Yi; Liu, Mao-Sen; Lin, Tsan-Piao; Cheng, Yi-Sheng

    2011-11-01

    The membrane protein AtTLP18.3 of Arabidopsis (Arabidopsis thaliana) contains a domain of unknown function, DUF477; it forms a polysome with photosynthetic apparatuses in the thylakoid lumen. To explore the molecular function of AtTLP18.3, we resolved its crystal structures with residues 83 to 260, the DUF477 only, and performed a series of biochemical analyses to discover its function. The gene expression of AtTLP18.3 followed a circadian rhythm. X-ray crystallography revealed the folding of AtTLP18.3 as a three-layer sandwich with three α-helices in the upper layer, four β-sheets in the middle layer, and two α-helices in the lower layer, which resembles a Rossmann fold. Structural comparison suggested that AtTLP18.3 might be a phosphatase. The enzymatic activity of AtTLP18.3 was further confirmed by phosphatase assay with various substrates (e.g. p-nitrophenyl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate, O-phospho-L-serine, and several synthetic phosphopeptides). Furthermore, we obtained the structure of AtTLP18.3 in complex with O-phospho-L-serine to identify the binding site of AtTLP18.3. Our structural and biochemical studies revealed that AtTLP18.3 has the molecular function of a novel acid phosphatase in the thylakoid lumen. DUF477 is accordingly renamed the thylakoid acid phosphatase domain.

  12. The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.

    PubMed

    Valenti, G; Procino, G; Carmosino, M; Frigeri, A; Mannucci, R; Nicoletti, I; Svelto, M

    2000-06-01

    Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient P(f) by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 microM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 microM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in P(f).

  13. Exploiting Acid Phosphatases in the Synthesis of Phosphorylated Monoalcohols and Diols

    PubMed Central

    Tasnádi, Gábor; Lukesch, Michael; Zechner, Michaela; Jud, Wolfgang; Hall, Mélanie; Ditrich, Klaus; Baldenius, Kai; Hartog, Aloysius F.; Wever, Ron

    2015-01-01

    Abstract A set of phosphatases was evaluated for their potential to catalyze the regio‐ and stereoselective phosphorylation of alcohols using a high‐energy inorganic phosphate donor, such as di‐, tri‐ and polyphosphate. Parameters such as type and amount of phosphate donor and pH of the reaction were investigated in order to minimize the thermodynamically favored hydrolysis of the phosphate donor and the formed phosphate ester. Diols were monophosphorylated with high selectivities. This biocatalytic phosphorylation method provides selectively activated and/or protected synthetic intermediates for further chemical and/or enzymatic transformations and is applicable to a large scale (6.86 g) in a flow setup with immobilized phosphatase.

  14. Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana.

    PubMed

    Oliveira, Danielle M P; Ramos, Isabela B; Reis, Flavia C G; Lima, Ana P C A; Machado, Ednildo A

    2008-05-01

    In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.

  15. A novel class of PTEN protein in Arabidopsis displays unusual phosphoinositide phosphatase activity and efficiently binds phosphatidic acid.

    PubMed

    Pribat, Anne; Sormani, Rodnay; Rousseau-Gueutin, Mathieu; Julkowska, Magdalena M; Testerink, Christa; Joubès, Jerôme; Castroviejo, Michel; Laguerre, Michel; Meyer, Christian; Germain, Véronique; Rothan, Christophe

    2012-01-01

    PTEN (phosphatase and tensin homologue deleted on chromosome ten) proteins are dual phosphatases with both protein and phosphoinositide phosphatase activity. They modulate signalling pathways controlling growth, metabolism and apoptosis in animals and are implied in several human diseases. In the present paper we describe a novel class of PTEN pro-teins in plants, termed PTEN2, which comprises the AtPTEN (Arabidopsis PTEN) 2a and AtPTEN2b proteins in Arabidopsis. Both display low in vitro tyrosine phosphatase activity. In addition, AtPTEN2a actively dephosphorylates in vitro the 3' phosphate group of PI3P (phosphatidylinositol 3-phosphate), PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate) and PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate). In contrast with animal PTENs, PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) is a poor substrate. Site-directed mutagenesis of AtPTEN2a and molecular modelling of protein-phosphoinositide interactions indicated that substitutions at the PTEN2 core catalytic site of the Lys267 and Gly268 residues found in animals, which are critical for animal PTEN activity, by Met267 and Ala268 found in the eudicot PTEN2 are responsible for changes in substrate specificity. Remarkably, the AtPTEN2a protein also displays strong binding activity for PA (phosphatidic acid), a major lipid second messenger in plants. Promoter::GUS (β-glucuronidase) fusion, transcript and protein analyses further showed the transcriptional regulation of the ubiquitously expressed AtPTEN2a and AtPTEN2b by salt and osmotic stress. The results of the present study suggest a function for this novel class of plant PTEN proteins as an effector of lipid signalling in plants.

  16. Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA

    PubMed Central

    Hoang, Ky Van; Chen, Carolyn G.; Koopman, Jacob; Moshiri, Jasmine; Adcox, Haley E.; Gunn, John S.

    2016-01-01

    Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI)-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant) AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease) and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses. PMID:27199935

  17. Cloning, sequence, and developmental expression of a type 5, tartrate-resistant, acid phosphatase of rat bone.

    PubMed

    Ek-Rylander, B; Bill, P; Norgård, M; Nilsson, S; Andersson, G

    1991-12-25

    Tartrate-resistant acid phosphatase (TRAP) is a characteristic constituent of osteoclasts and some mononuclear preosteoclasts and, therefore, used as a histochemical and biochemical marker for osteoclasts and bone resorption. We now report the isolation of a 1397-base pair (bp) full-length TRAP/tartrate-resistant acid ATPase (TrATPase) cDNA clone from a neonatal rat calvaria lambda gt11 cDNA library. The cDNA clone consists of a 92-bp untranslated 5'-flank, an open reading frame of 981 bp and a 324-bp untranslated 3'-poly(A)-containing region. The deduced protein sequence of 327 amino acids contains a putative cleavable signal sequence of 21 amino acids. The mature polypeptide of 306 amino acids has a calculated Mr of 34,350 Da and a pI of 9.18, and it contains two potential N-glycosylation sites and the lysosomal targeting sequence DKRFQ. At the protein level, the sequence displays 89-94% homology to TRAP enzymes from human placenta, beef spleen, and uteroferrin and identity to the N terminus of purified rat bone TRAP/TrATPase. An N-terminal amino acid segment is strikingly homologous to the corresponding region in lysosomal and prostatic acid phosphatases. The cDNA recognized a 1.5-kilobase mRNA in long bones and calvaria, and in vitro translation using, as template, mRNA transcribed from the full-length insert yielded an immunoprecipitated product of 34 kDa. In neonatal rats, TRAP/TrATPase mRNA was highly expressed in skeletal tissues, with much lower (less than 10%) levels detected in spleen, thymus, liver, skin, brain, kidney, brain, lung, and heart. In situ hybridization demonstrated specific labeling of osteoclasts at endostal surfaces and bone trabeculae of long bones. Thus, despite the apparent similarity of this osteoclastic TRAP/TrATPase with type 5, tartrate-resistant and purple, acid phosphatases expressed in other mammalian tissues, this gene appears to be preferentially expressed at skeletal sites.

  18. Toxic impact of aldrin on acid and alkaline phosphatase activity of penaeid prawn, Metapenaeus monoceros: In vitro study

    SciTech Connect

    Reddy, M.S.; Jayaprada, P.; Rao, K.V.R. )

    1991-03-01

    The increasing contamination of the aquatic environment by the indiscriminate and widespread use of different kinds of pesticides is a serious problem for environmental biologists. Organochlorine insecticides are more hazardous since they are not only more toxic but also leave residues in nature. The deleterious effects of aldrin on several crustaceans have been studied. But studies concerning the impact of aldrin on biochemical aspects of crustaceans are very much limited. The present study is aimed at probing the in vitro effects of aldrin on the acid and alkaline phosphatase activity levels in selected tissues of penaeid prawn, Metapenaeus monoceros (Fabricius).

  19. Toxicology of cupric salts on honeybees. V. Gluconate and sulfate action on gut alkaline and acid phosphatases.

    PubMed

    Bounias, M; Kruk, I; Nectoux, M; Popeskovic, D

    1996-10-01

    Some aspects of putative nontarget effects of cupric ions systemically fed to honeybees against their parasite mite Varroa jacobsoni have been investigated on the host phosphatases. The alkaline and acid forms extracted from the guts of worker bees exhibited substrate-inhibition features. Upon detailed kinetic analysis, cupric organic salts indicate activation effects at concentrations of about 1 mM. Concentrations up to 10 mM (alkaline form) and 25 mM (acid form) induced no important changes, except a partial quenching of the substrate-inhibition process, characterized by a wide increase in the constant of apparent inhibitory binding of substrate to the enzyme-substrate complex. Partial purification gave a single alkaline form with quite similar kinetic behavior in the absence of natural ions as in crude extracts. Cupric gluconate and sulfate demonstrated similar patterns, except an increase of the apparent Hill coefficient by sulfate only. The substrate constant of acid phosphatases was decreased at high cupric gluconate doses while its maximum velocity was biphasically increased (with observed maximum at 1 mM), resulting in a sustained activation. Chemiluminescence studies revealed that cupric ion activation is counteracted by oxygen radicals generated by cupric ions and also, in vitro, by the artificial substrate para-nitrophenylphosphate. The para-nitrophenol molecules released from the reaction are therefore responsible for biphasic effects selectively observed with gluconate salts. In apicultural practice, neither blockade of activity nor dramatic changes are to be expected at doses administered to bees against the parasite.

  20. Synergistic salubrious effect of ferulic acid and ascorbic acid on membrane-bound phosphatases and lysosomal hydrolases during experimental myocardial infarction in rats.

    PubMed

    Yogeeta, Surinder Kumar; Gnanapragasam, Arunachalam; Senthilkumar, Subramanian; Subhashini, Rajakannu; Devaki, Thiruvengadam

    2006-12-23

    Altered membrane integrity has been suggested as a major factor in the development of cellular injury during myocardial necrosis. The present study was designed to investigate the effect of the combination of ferulic acid (FA) and ascorbic acid (AA) on lysosomal hydrolases and membrane-bound phosphatases during isoproterenol (ISO) induced myocardial necrosis in rats. Induction of rats with 1SO (150 mg/kg b.wt, i.p.) for 2 days resulted in a significant increase in the activities of lysosomal hydrolases (beta-D-glucuronidase, beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase and cathepsin-D) in the heart and serum. A significant increase in plasma lactate level, cardiac levels of sodium, calcium and a decrease in cardiac level of potassium was also observed, which was paralleled by abnormal activities of membrane-bound phosphatases (Na(+)-K(+) ATPase, Ca(2+) ATPase and Mg(2+) ATPase) in the heart of ISO-administered rats. Pre-co-treatment with the combination of FA (20 mg/kg b.wt) and AA (80 mg/kg b.wt) orally for 6 days significantly attenuated these abnormalities and restored the levels to near normalcy when compared to individual drug treated groups. The combination of FA and AA preserved the membrane integrity by mitigating the oxidative stress and associated cellular damage more effectively when compared to individual treatment groups. In our study, the protection conferred by FA and AA might be through the nitric oxide pathway and by their ability of quenching free radicals. In conclusion, these findings indicate the synergistic modulation of lysosomal hydrolases and membrane phosphatases by the combination of FA and AA.

  1. Inhibition of serine/threonine phosphatase enhances arachidonic acid-induced [Ca2+]i via protein kinase A.

    PubMed

    Saino, Tomoyuki; Watson, Eileen L

    2009-01-01

    Arachidonic acid (AA) regulates intracellular calcium concentration ([Ca2+]i) in a variety of cell types including salivary cells. In the present study, the effects of serine/threonine phosphatases on AA-induced Ca(2+) signaling in mouse parotid acini were determined. Mice were euthanized with CO2. Treatment of acini with the serine/threonine phosphatase inhibitor calyculin A blocked both thapsigargin- and carbachol-induced Ca2+ entry but resulted in an enhancement of AA-induced Ca2+ release and entry. Effects were mimicked by the protein phosphatase-1 (PP1) inhibitor tautomycin but were inhibited by the PP2A inhibitor okadaic acid. The protein kinase A (PKA) inhibitor PKI(14-22) significantly attenuated AA-induced enhancement of Ca2+ release and entry in the presence of calyculin A, whereas it had no effect on calyculin A-induced inhibition of thapsigargin-induced Ca2+ responses. The ryanodine receptor (RyR) inhibitor, tetracaine, and StHt-31, a peptide known to competitively inhibit type II PKA regulatory subunit binding to PKA-anchoring protein (AKAP), abolished calyculin A enhancement of AA-induced Ca2+ release and entry. StHt-31 also abolished forskolin potentiation of 4-chloro-3-ethylphenol (4-CEP) and AA on Ca2+ release but had no effect on 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP potentiation of 4-CEP responses. Results suggest that inhibition of PP1 results in an enhancement of AA-induced [Ca2+]i via PKA, AKAP, and RyRs.

  2. Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme.

    PubMed

    Conklin, Patricia L; Gatzek, Stephan; Wheeler, Glen L; Dowdle, John; Raymond, Marjorie J; Rolinski, Susanne; Isupov, Mikhail; Littlechild, Jennifer A; Smirnoff, Nicholas

    2006-06-09

    In plants, a proposed ascorbate (vitamin C) biosynthesis pathway occurs via GDP-D-mannose (GDP-D-Man), GDP-L-galactose (GDP-L-Gal), and L-galactose. However, the steps involved in the synthesis of L-Gal from GDP-L-Gal in planta are not fully characterized. Here we present evidence for an in vivo role for L-Gal-1-P phosphatase in plant ascorbate biosynthesis. We have characterized a low ascorbate mutant (vtc4-1) of Arabidopsis thaliana, which exhibits decreased ascorbate biosynthesis. Genetic mapping and sequencing of the VTC4 locus identified a mutation (P92L) in a gene with predicted L-Gal-1-P phosphatase activity (At3g02870). Pro-92 is within a beta-bulge that is conserved in related myo-inositol monophosphatases. The mutation is predicted to disrupt the positioning of catalytic amino acid residues within the active site. Accordingly, L-Gal-1-P phosphatase activity in vtc4-1 was approximately 50% of wild-type plants. In addition, vtc4-1 plants incorporate significantly more radiolabel from [2-(3)H]Man into L-galactosyl residues suggesting that the mutation increases the availability of GDP-L-Gal for polysaccharide synthesis. Finally, a homozygous T-DNA insertion line, which lacks a functional At3g02870 gene product, is also ascorbate-deficient (50% of wild type) and deficient in L-Gal-1-P phosphatase activity. Genetic complementation tests revealed that the insertion mutant and VTC4-1 are alleles of the same genetic locus. The significantly lower ascorbate and perturbed L-Gal metabolism in vtc4-1 and the T-DNA insertion mutant indicate that L-Gal-1-P phosphatase plays a role in plant ascorbate biosynthesis. The presence of ascorbate in the T-DNA insertion mutant suggests there is a bypass to this enzyme or that other pathways also contribute to ascorbate biosynthesis.

  3. Evaluation of forensic examination of extremely aged seminal stains.

    PubMed

    Nakanishi, Hiroaki; Hara, Masaaki; Takahashi, Shirushi; Takada, Aya; Saito, Kazuyuki

    2014-09-01

    The results of forensic tests, such as semen identification and short tandem repeat (STR) analysis of extremely aged seminal stains from unsolved sex crimes can provide important evidence. In this study we evaluated whether current forensic methods could be applied to seminal stains that were stored at room temperature for 33-56years (n=2, 33years old; n=1, 41years old; n=1, 44years old; n=1, 56years old). The prostatic acid phosphatase (SM-test reagent), microscopic (Baecchi stain method) and semenogelin (RSID™ Semen Laboratory Kit) tests were performed as discriminative tests for semen. In addition, the mRNA levels of the semen-specific proteins semenogelin 1 (SEMG1) and protamine 2 (PRM2) were investigated. STRs were analyzed using the AmpFlSTR® Identifiler™ PCR Amplification Kit. All samples were positive in the prostatic acid phosphatase and semenogelin tests, and sperm heads were identified in all samples. The staining degree of the aged sperm heads was similar to that of fresh sperm. Although SEMG1 mRNA was not detected in any sample, PRM2 mRNA was detected in three samples. In the STR analysis, all loci were detected in the 33-years-old sample and five loci were detected in the 56-years-old sample. We confirmed that current forensic examinations - including STR analysis - could be applied to extremely aged seminal stains. These results could be useful for forensic practice.

  4. The maize (Zea mays ssp. mays var. B73) genome encodes 33 members of the purple acid phosphatase family

    PubMed Central

    González-Muñoz, Eliécer; Avendaño-Vázquez, Aida-Odette; Montes, Ricardo A. Chávez; de Folter, Stefan; Andrés-Hernández, Liliana; Abreu-Goodger, Cei; Sawers, Ruairidh J. H.

    2015-01-01

    Purple acid phosphatases (PAPs) play an important role in plant phosphorus nutrition, both by liberating phosphorus from organic sources in the soil and by modulating distribution within the plant throughout growth and development. Furthermore, members of the PAP protein family have been implicated in a broader role in plant mineral homeostasis, stress responses and development. We have identified 33 candidate PAP encoding gene models in the maize (Zea mays ssp. mays var. B73) reference genome. The maize Pap family includes a clear single-copy ortholog of the Arabidopsis gene AtPAP26, shown previously to encode both major intracellular and secreted acid phosphatase activities. Certain groups of PAPs present in Arabidopsis, however, are absent in maize, while the maize family contains a number of expansions, including a distinct radiation not present in Arabidopsis. Analysis of RNA-sequencing based transcriptome data revealed accumulation of maize Pap transcripts in multiple plant tissues at multiple stages of development, and increased accumulation of specific transcripts under low phosphorus availability. These data suggest the maize PAP family as a whole to have broad significance throughout the plant life cycle, while highlighting potential functional specialization of individual family members. PMID:26042133

  5. Acid phosphatase test proves superior to standard phenotypic identification procedure for Clostridium perfringens strains isolated from water

    PubMed Central

    Ryzinska-Paier, G.; Sommer, R.; Haider, J.M.; Knetsch, S.; Frick, C.; Kirschner, A.K.T.; Farnleitner, A.H.

    2011-01-01

    Clostridium perfringens is used as an indicator for persistent faecal pollution as well as to monitor the efficacy of water treatment processes. For these purposes, differentiation between C. perfringens and other Clostridia is essential and is routinely carried out by phenotypic standard tests as proposed in the ISO/CD 6461-2:2002 (ISO_LGMN: lactose fermentation, gelatine liquidation, motility and nitrate reduction). Because the ISO_LGMN procedure is time consuming and labour intensive, the acid phosphatase test was investigated as a possible and much more rapid alternative method for confirmation. The aim of our study was to evaluate and compare confirmation results obtained by these two phenotypic methods using genotypically identified strains, what to our knowledge has not been accomplished before. For this purpose, a species specific PCR method was selected based on the results received for type strains and genotypically characterised environmental strains. For the comparative investigation type strains as well as presumptive C. perfringens isolates from water and faeces samples were used. The acid phosphatase test revealed higher percentage (92%) of correctly identified environmental strains (n = 127) than the ISO_LGMN procedure (83%) and proved to be a sensitive and reliable confirmation method. PMID:21872622

  6. Positive cooperativity in substrate binding of human prostatic acid phosphatase entrapped in AOT-isooctane-water reverse micelles.

    PubMed

    Luchter-Wasylewska, Ewa; Iciek, Małgorzata

    2004-05-15

    The kinetics of 1-naphthyl phosphate and phenyl phosphate hydrolysis, catalyzed by human prostatic acid phosphatase (PAP) entrapped in AOT-isooctane-water reverse micelles, has been studied over surfactant hydration degree (w0) range 5 to 35. Continuous spectrophotometric acid phosphatase assays, previously prepared, were employed. PAP was catalytically active over the whole w0 studied range. In order to determine steady-state reaction constants the experimental data were fitted to Hill rate equation. Positive cooperativity in substrate binding was observed, as it was earlier found in aqueous solutions. The extent of cooperativity (expressed as the value of the Hill cooperation coefficient h) increased from 1 to 4, when the micellar water-pool size was growing, at fixed enzyme concentration. In the plots of catalytic activity (kcat) versus w0, the maxima have been found at w0=10 (pH 5.6) and 23 (pH 3.8). It is suggested that catalytically active monomeric and dimeric PAP forms are entrapped in reverse micelles of w0=10 and 23, respectively.

  7. The maize (Zea mays ssp. mays var. B73) genome encodes 33 members of the purple acid phosphatase family.

    PubMed

    González-Muñoz, Eliécer; Avendaño-Vázquez, Aida-Odette; Montes, Ricardo A Chávez; de Folter, Stefan; Andrés-Hernández, Liliana; Abreu-Goodger, Cei; Sawers, Ruairidh J H

    2015-01-01

    Purple acid phosphatases (PAPs) play an important role in plant phosphorus nutrition, both by liberating phosphorus from organic sources in the soil and by modulating distribution within the plant throughout growth and development. Furthermore, members of the PAP protein family have been implicated in a broader role in plant mineral homeostasis, stress responses and development. We have identified 33 candidate PAP encoding gene models in the maize (Zea mays ssp. mays var. B73) reference genome. The maize Pap family includes a clear single-copy ortholog of the Arabidopsis gene AtPAP26, shown previously to encode both major intracellular and secreted acid phosphatase activities. Certain groups of PAPs present in Arabidopsis, however, are absent in maize, while the maize family contains a number of expansions, including a distinct radiation not present in Arabidopsis. Analysis of RNA-sequencing based transcriptome data revealed accumulation of maize Pap transcripts in multiple plant tissues at multiple stages of development, and increased accumulation of specific transcripts under low phosphorus availability. These data suggest the maize PAP family as a whole to have broad significance throughout the plant life cycle, while highlighting potential functional specialization of individual family members.

  8. Relation of fatty acid composition in lead-exposed mallards to fat mobilization, lipid peroxidation and alkaline phosphatase activity

    USGS Publications Warehouse

    Mateo, R.; Beyer, W.N.; Spann, J.W.; Hoffman, D.J.

    2003-01-01

    The increase of n-6 polyunsaturated fatty acids (PUFA) in animal tissues has been proposed as a mechanism of Pb poisoning through lipid peroxidation or altered eicosanoids metabolism. We have studied fatty acid (FA) composition in liver and brain of mallards (Anas platyrhynchos) feeding for three weeks on diets containing combinations of low or high levels of vitamin E (20 or 200 UI/kg) and Pb (0 or 2 g/kg). Saturated FA, n-6 PUFA and total concentrations of FA were higher in livers of Pb-exposed mallards, but not in their brains. The percentage of n-6 PUFA in liver and brain was slightly higher in Pb-exposed mallards. The increase of n-6 PUFA in liver was associated with increased triglycerides and cholesterol in plasma, thus could be in part attributed to feed refusal and fat mobilization. The hepatic ratios between adrenic acid (22:4 n-6) and arachidonic acid (20:4 n-6) or between adrenic acid and linoleic acid (18:2 n-6) were higher in Pb exposed birds, supporting the existing hypothesis of increased fatty acid elongation by Pb. Among the possible consequences of increased n-6 PUFA concentration in tissues, we found increased lipid peroxidation in liver without important histopathological changes, and decreased plasma alkaline phosphatase activity that may reflect altered bone metabolism in birds.

  9. Structural basis for the divergence of substrate specificity and biological function within HAD phosphatases in lipopolysaccharide and sialic acid biosynthesis.

    PubMed

    Daughtry, Kelly D; Huang, Hua; Malashkevich, Vladimir; Patskovsky, Yury; Liu, Weifeng; Ramagopal, Udupi; Sauder, J Michael; Burley, Stephen K; Almo, Steven C; Dunaway-Mariano, Debra; Allen, Karen N

    2013-08-13

    The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases that have been particularly successful at adaptating to novel biological functions relative to members of other phosphatase families. Herein, we examine the structural basis for the divergence of function in two bacterial homologues: 2-keto-3-deoxy-d-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step in KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities toward catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3(-) and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3(-) revealed the substrate-binding residues. The structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in their active-site structures that underlie the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single-domain proteins or fused with the pathway nucleotidyl transferases; the fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue that forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover

  10. Gram Stain

    MedlinePlus

    ... Gram Stain Related tests: Susceptibility Testing , Bacterial Wound Culture , Blood Culture , Body Fluid Analysis , CSF Analysis , Urine Culture , AFB Testing , Gonorrhea Testing , Stool Culture , Fungal Tests , ...

  11. A structure-based proposal for the catalytic mechanism of the bacterial acid phosphatase AphA belonging to the DDDD superfamily of phosphohydrolases.

    PubMed

    Calderone, Vito; Forleo, Costantino; Benvenuti, Manuela; Thaller, Maria Cristina; Rossolini, Gian Maria; Mangani, Stefano

    2006-01-27

    The Escherichia coli gene aphA codes for a periplasmic acid phosphatase called AphA, belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. After our first report about its crystal structure, we have started a series of crystallographic studies aimed at understanding of the catalytic mechanism of the enzyme. Here, we report three crystal structures of the AphA enzyme in complex with the hydrolysis products of nucleoside monophosphate substrates and a fourth with a proposed intermediate analogue that appears to be covalently bound to the enzyme. Comparison with the native enzyme structure and with the available X-ray structures of different phosphatases provides clues about the enzyme chemistry and allows us to propose a catalytic mechanism for AphA, and to discuss it with respect to the mechanism of other bacterial and human phosphatases.

  12. X-ray absorption studies of the purple acid phosphatase from red kidney beans (native enzyme, metal exchanged form)

    NASA Astrophysics Data System (ADS)

    Ahlers, F.; Zippel, F.; Klabunde, T.; Krebs, B.; Löcke, R.; Witzel, H.; Nolting, H.-F.

    1995-02-01

    Purple acid phosphatase from red kidney beans (KBP) catalyzes the hydrolysis of activated phosphoric acid monoesters and contains a heterodinuclear Fe(III)Zn(II) core in its active site. Iron K-edge X-ray absorption data have been obtained for the native enzyme and for a metal exchanged derivative, where Zn(II) was substituted by Fe(III). The environment of the native enzyme consists of 2.5 O/N at 1.91 Å, 3 O/N at 2.09 Å, and 1 Zn at 4.05 Å. For the metal exchanged form we obtained 2.5 O/N at 1.94 Å, 2.5 O/N at 2.09 Å, and 1 Fe at 3.79 Å.

  13. A novel cinnamic acid derivative that inhibits Cdc25 dual-specificity phosphatase activity.

    PubMed

    Aoyagi, Yoshimi; Masuko, Norio; Ohkubo, Shuichi; Kitade, Makoto; Nagai, Kentaro; Okazaki, Shinji; Wierzba, Konstanty; Terada, Tadafumi; Sugimoto, Yoshikazu; Yamada, Yuji

    2005-09-01

    The Cdc25 dual-specificity phosphatases are key regulators of cell cycle progression through activation of cyclin-dependent kinases (Cdk). Three homologs exist in humans: Cdc25A, Cdc25B, and Cdc25C. Cdc25A and Cdc25B have oncogenic properties and are overexpressed in some types of tumors. Compounds that inhibit Cdc25 dual-specificity phosphatase activity might thus be potent anticancer agents. We screened several hundred compounds in a library using an in vitro phosphatase assay, with colorimetric measurement of the conversion of p-nitrophenyl phosphate (pNPP) to p-nitrophenol by the catalytic domain of recombinant human Cdc25, and discovered TPY-835, which inhibits Cdc25A and Cdc25B activity (IC50 = 5.1 and 5.7 microM, respectively). TPY-835 had mixed inhibition kinetics for Cdc25A and Cdc25B. TPY-835 caused cell cycle arrest in the G1 phase in human lung cancer cells (A549 and SBC-5) but not cell cycle arrest in the G2/M phase. After treatment with TPY-835, the activation of Cdk2 was suppressed and phosphorylation of the retinoblastoma (Rb) protein was decreased in SBC-5 cells. In addition, TPY-835 induced an increase of the sub-G1 phase cell population after 48-72 h treatment. The growth inhibitory effects of TPY-835 against cisplatin (CDDP)-, camptothecin- and 5-FU-resistant cell lines are comparable to the growth inhibitory effect on their parental lines, thus indicating that TPY-835 did not show cross-resistance to these cell lines. These results suggest that TPY-835 is a promising candidate for constructing a novel class of antitumor agents that can control the cell cycle progression of cancer cells.

  14. Anisotropic tubular filtering for automatic detection of acid-fast bacilli in Ziehl-Neelsen stained sputum smear samples

    NASA Astrophysics Data System (ADS)

    Raza, Shan-e.-Ahmed; Marjan, M. Q.; Arif, Muhammad; Butt, Farhana; Sultan, Faisal; Rajpoot, Nasir M.

    2015-03-01

    One of the main factors for high workload in pulmonary pathology in developing countries is the relatively large proportion of tuberculosis (TB) cases which can be detected with high throughput using automated approaches. TB is caused by Mycobacterium tuberculosis, which appears as thin, rod-shaped acid-fast bacillus (AFB) in Ziehl-Neelsen (ZN) stained sputum smear samples. In this paper, we present an algorithm for automatic detection of AFB in digitized images of ZN stained sputum smear samples under a light microscope. A key component of the proposed algorithm is the enhancement of raw input image using a novel anisotropic tubular filter (ATF) which suppresses the background noise while simultaneously enhancing strong anisotropic features of AFBs present in the image. The resulting image is then segmented using color features and candidate AFBs are identified. Finally, a support vector machine classifier using morphological features from candidate AFBs decides whether a given image is AFB positive or not. We demonstrate the effectiveness of the proposed ATF method with two different feature sets by showing that the proposed image analysis pipeline results in higher accuracy and F1-score than the same pipeline with standard median filtering for image enhancement.

  15. Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos.

    PubMed

    Onischenko, Evgeny A; Gubanova, Natalia V; Kiseleva, Elena V; Hallberg, Einar

    2005-11-01

    Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

  16. Single laboratory validation of a ready-to-use phosphatase inhibition assay for detection of okadaic acid toxins.

    PubMed

    Smienk, Henry G F; Calvo, Dolores; Razquin, Pedro; Domínguez, Elena; Mata, Luis

    2012-05-01

    A phosphatase inhibition assay for detection of okadaic acid (OA) toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM). Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 µg/kg, respectively; both below the European legal limit of 160 µg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD) calculated was 1.4% at a level of 276 µg/kg and 3.9% at 124 µg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop) on three different days and ranged between 2.4 and 9.5%. The IC(50) values of the phosphatase used in this assay were determined for OA (1.2 nM), DTX-1 (1.6 nM) and DTX-2 (1.2 nM). The accuracy of the method was estimated by recovery testing for OA (mussel, 78-101%; king scallop, 98-114%), DTX-1 (king scallop, 79-102%) and DTX-2 (king scallop, 93%). Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS.

  17. Enhanced degradation of five organophosphorus pesticides in skimmed milk by lactic acid bacteria and its potential relationship with phosphatase production.

    PubMed

    Zhang, Ying-Hua; Xu, Di; Liu, Jia-Qi; Zhao, Xin-Huai

    2014-12-01

    Skimmed milk spiked with five organophosphorus pesticides (OPPs), chlorpyrifos, diazinon, fenitrothion, malathion and methyl parathion, was fermented by ten lactic acid bacteria (LAB) and four strain combinations at 42°C for 24h. OPPs left in the samples at different times were extracted, purified, detected by gas chromatography and calculated for degradation rate constants, based on a first-order reaction model. OPPs degradation was enhanced by the inoculated LAB, resulting in 0.8-225.4% increase in the rate constants. Diazinon and methyl parathion were more stable whereas chlorpyrifos, fenitrothion and malathion were more labile. Lactobacillus brevis 1.0209 showed the strongest acceleration on OPPs degradation while strain combination could bring about a synergy between the strains of lower ability. Phosphatase production of the strains might be one of the key factors responsible for the enhanced OPPs degradation, as the detected phosphatase activities were positively correlated to the measured degradation rate constants of OPPs (r=0.636-0.970, P<0.05).

  18. Differential staining of bacteria: flagella stain.

    PubMed

    Breakwell, Donald P; Moyes, Rita B; Reynolds, Jackie

    2009-11-01

    Bacterial flagella are appendages used for motility. Their presence is a useful tool for identification and differentiation of prokaryotes. Since flagella are too thin to be seen by compound light microscopy, staining methods employ the use of a mordant (often tannic acid) to make them thick enough to see using an oil immersion objective. Two protocols are described. Basic Protocol 1 is a modified Leifson method and is the one that many microbiologists have adapted. Basic Protocol 2 is a wet-mount stain using a Ryu stain and is included because the stain is stable at room temperature. Both of these methods are fairly time-consuming, taking from 15 to as long as 60 min to perform.

  19. The relationship between the acid and alkaline phosphatase activity and the adherence of clinical isolates of Candida parapsilosis to human buccal epithelial cells.

    PubMed

    Fernanado, P H; Panagoda, G J; Samaranayake, L P

    1999-11-01

    Candida parapsilosis is an emerging fungal pathogen implicated in many diseases, especially in compromised hosts. Candidal colonization and infection depends on the initial ability to adhere to host surfaces, which in turn depends upon the cell wall components and the allied structures of both the host and the fungus. Examination of a miscellaneous collection of 24 C. parapsilosis isolates, from both superficial and deep infections, for their potential pathogenic traits displayed a relationship between the phosphatase activity measured with p-nitrophenol phosphate and adhesion of the yeasts to human buccal epithelial cells (BECs). Significant intraspecies differences were seen in both the alkaline and acid phosphatase activity as well as in their adhesion to BECs (p<0.0001). The acid phosphatase activity of the superficial isolates was significantly greater (152%) than that of the systemic isolates (p = 0.0352). A highly significant positive correlation was also established between the yeast adhesion to BECs and both the acid (r = 0.88, p<0.0001) and alkaline (r = 0.9, p<0.0001) phosphatase activity. These relationships, described here for the first time, imply that phosphatases of Candida species may play a crucial role in potentiating their virulence.

  20. Gram staining.

    PubMed

    Coico, Richard

    2005-10-01

    Named after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram-positive and Gram-negative bacteria on the basis of differential staining with a crystal violet-iodine complex and a safranin counterstain. The cell walls of Gram-positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram-negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.

  1. Gram staining.

    PubMed

    Coico, R

    2001-05-01

    Named after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram-positive and Gram-negative bacteria on the basis of differential staining with a crystal violet-iodine complex and a safranin counterstain. The cell walls of Gram-positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram-negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.

  2. Fluorescent staining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Chi, Lisha; Ruan, Dandan; Xuan, Yuanhu; Cong, Weitao; Jin, Litai

    2014-06-07

    A fluorescent detection method for glycoproteins in SDS-PAGE by using 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH) was developed in this study. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be specifically detected by the BH staining method, which is twofold more sensitive than that of the most commonly used Pro-Q Emerald 488 glycoprotein stain. Furthermore, the specificity of the newly developed stain for glycoproteins was demonstrated by 1-D and 2-D SDS-PAGE, deglycosylation, glycoprotein affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that BH stain may provide new choices for convenient, sensitive, specific and economic visualization of gel-separated glycoproteins.

  3. Wood stains

    MedlinePlus

    The harmful substances in wood stains are hydrocarbons, or substances that contain only carbon and hydrogen. Other harmful ingredients may include: Alcohol Alkanes Cyclo alkanes Glycol ether Corrosives, such as sodium ...

  4. Identification of protein tyrosine phosphatase SHP-2 as a new target of perfluoroalkyl acids in HepG2 cells.

    PubMed

    Yang, Yu; Lv, Qi-Yan; Guo, Liang-Hong; Wan, Bin; Ren, Xiao-Min; Shi, Ya-Li; Cai, Ya-Qi

    2016-08-29

    Perfluoroalkyl acids (PFAAs) are widespread environmental contaminants which have been detected in humans and linked to adverse health effects. Previous toxicological studies mostly focused on nuclear receptor-mediated pathways and did not support the observed toxic effects. In this study, we aimed to investigate the molecular mechanisms of PFAA toxicities by identifying their biological targets in cells. Using a novel electrochemical biosensor, 16 PFAAs were evaluated for inhibition of protein tyrosine phosphatase SHP-2 activity. Their potency increased with PFAA chain length, with perfluorooctadecanoic acid (PFODA) showing the strongest inhibition. Three selected PFAAs, 25 μM perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid, and PFODA, also inhibited SHP-2 activity in HepG2 cells and increased paxillin phosphorylation level. PFOA was detected in the immunoprecipitated SHP-2 from the cells exposed to 250 μM PFOA, providing unequivocal evidence for the direct binding of PFOA with SHP-2 in the cell. Molecular docking rationalized the formation of PFAA/SHP-2 complex and chain length-dependent inhibition potency. Our results have established SHP-2 as a new cellular target of PFAAs.

  5. Bioseparation of Four Proteins from Euphorbia characias Latex: Amine Oxidase, Peroxidase, Nucleotide Pyrophosphatase/Phosphodiesterase, and Purple Acid Phosphatase

    PubMed Central

    Medda, Rosaria; Pintus, Francesca; Spanò, Delia; Floris, Giovanni

    2011-01-01

    This paper deals with the purification of four proteins from Euphorbia characias latex, a copper amine oxidase, a nucleotide pyrophosphatase/phosphodiesterase, a peroxidase, and a purple acid phosphatase. These proteins, very different in molecular weight, in primary structure, and in the catalyzed reaction, are purified using identical preliminary steps of purification and by chromatographic methods. In particular, the DEAE-cellulose chromatography is used as a useful purification step for all the four enzymes. The purification methods here reported allow to obtain a high purification of all the four proteins with a good yield. This paper will give some thorough suggestions for researchers busy in separation of macromolecules from different sources. PMID:22013530

  6. Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.

    PubMed Central

    Vihko, P; Kurkela, R; Porvari, K; Herrala, A; Lindfors, A; Lindqvist, Y; Schneider, G

    1993-01-01

    Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A. Images PMID:8430088

  7. Nucleic Acid Amplification Testing and Sequencing Combined with Acid-Fast Staining in Needle Biopsy Lung Tissues for the Diagnosis of Smear-Negative Pulmonary Tuberculosis

    PubMed Central

    Tian, Panwen; Chen, Xuerong; Liang, Zongan

    2016-01-01

    Background Smear-negative pulmonary tuberculosis (PTB) is common and difficult to diagnose. In this study, we investigated the diagnostic value of nucleic acid amplification testing and sequencing combined with acid-fast bacteria (AFB) staining of needle biopsy lung tissues for patients with suspected smear-negative PTB. Methods Patients with suspected smear-negative PTB who underwent percutaneous transthoracic needle biopsy between May 1, 2012, and June 30, 2015, were enrolled in this retrospective study. Patients with AFB in sputum smears were excluded. All lung biopsy specimens were fixed in formalin, embedded in paraffin, and subjected to acid-fast staining and tuberculous polymerase chain reaction (TB-PCR). For patients with positive AFB and negative TB-PCR results in lung tissues, probe assays and 16S rRNA sequencing were used for identification of nontuberculous mycobacteria (NTM). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy of PCR and AFB staining were calculated separately and in combination. Results Among the 220 eligible patients, 133 were diagnosed with TB (men/women: 76/57; age range: 17–80 years, confirmed TB: 9, probable TB: 124). Forty-eight patients who were diagnosed with other specific diseases were assigned as negative controls, and 39 patients with indeterminate final diagnosis were excluded from statistical analysis. The sensitivity, specificity, PPV, NPV, and accuracy of histological AFB (HAFB) for the diagnosis of smear-negative were 61.7% (82/133), 100% (48/48), 100% (82/82), 48.5% (48/181), and 71.8% (130/181), respectively. The sensitivity, specificity, PPV, and NPV of histological PCR were 89.5% (119/133), 95.8% (46/48), 98.3% (119/121), and 76.7% (46/60), respectively, demonstrating that histological PCR had significantly higher accuracy (91.2% [165/181]) than histological acid-fast staining (71.8% [130/181]), P < 0.001. Parallel testing of histological AFB

  8. Periodic acid-Schiff (PAS) staining of immature platelets in donors.

    PubMed

    Pogorelov, Valery M; Beskorovainova, Victoria Ju; Chanieva, Marem I; Dyagileva, Olga A; Naumova, Iren N; Skedina, Marina A

    2012-01-01

    Glycogen in platelets (PLTs) on smears of peripheral blood of 40 donors was investigated by the periodic acid-Schiff (PAS) method. Three groups were formed. Group 1 was consisted of 21 men undergoing the donor selection procedure. Additionally, 9 first-time donors undergoing plateletpheresis (Group 2) and 10 donors who frequently underwent platelet apheresis (Group 3) were studied as a model of relative thrombocytopenia. Cell sizes were measured with the use of a Image Analyzer "ASPBC" (Russia). The training procedure and classification of PAS-blood PLTs were made on the basis of expert evaluation. In this article, we have established three facts. First, the PAS-positive PLT area was larger than that of the PAS-negative cells (9.5 ± 3.6 sq.mkm vs. 3.9 ± 1.3 sq.mkm, p < 0.001, n = 21). The PAS-positivity of PLTs was 23.1 ± 9.2%. Second, the PAS-positivity correlated (r(S) = 0.63, p < 0.05) with the immature platelets fraction (IPF %), determined using Sysmex XE-2100. The mean IPF was 2.1 ± 1.0% (range 0.3-4.6%). Third, using the IPF% values obtained in Group 1, we found a significantly higher level of IPF in the samples both in Group 2 [mean value 4.2 ± 2.0% (range 1.9-7.0), p < 0.01] and in Group 3 [mean value 5.1 ± 2.5% (range 1.2-8.6), p < 0.004] with relative thrombocytopenia [Group 2: median 198 (95% confidence interval, CI 166-227) vs. median 229 (95% CI 206-267), p < 0.05; Group 3: median 142.5 (95% CI 132-173) vs. median 214.5 (95% CI 196-267), p < 0.01] after plateletpheresis. There was also a significant difference between the pre- and post-plateletpheresis for IPF% in Group 2 and Group 3: median 1.7 (95% CI 1.4-4.0) vs. median 4.0 (95% CI 2.7-5.8), p < 0.05 and median 4.0 (95% CI 2.7-6.0) vs. median 5.1 (95% CI 3.3-6.9), p < 0.01. The increased IPF shows a correlation with the PAS positivity [r(S) = 0.5 (p = 0.14) and r(S) = 0.6 (p = 0.05)] which has a tendency to

  9. Inhibition of hydrolytic enzymes by gold compounds. I. beta-Glucuronidase and acid phosphatase by sodium tetrachloroaurate (III) and potassium tetrabromoaurate (III).

    PubMed

    Lee, M T; Ahmed, T; Friedman, M E

    1989-01-01

    Purified bovine liver beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32) and wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) were inhibited with freshly dissolved and 24 h aquated tetrahaloaurate (III) compounds. Rate and equilibrium inhibition constants were measured. From this data two acid phosphatases species were observed. Equilibrium inhibition constants ranged from 1 to 12.5 microM for the various gold compounds toward both enzymes. The first order rate constants ranged between 0.005 and 0.04 min.-1 for most reactions with the exception of the fast reacting acid phosphatase which had values as high as 2.6 and 2.8 min.-1. It is observed that the beta-glucuronidase is rapidly inhibited during the equilibrium phase before the more slower reaction covalent bond formation takes place. The acid phosphatases form the covalent bonds more rapidly, especially the faster reacting species suggesting a unique difference in the active site geometry to that of the more slowly reacting species. The tightly bonded gold (III)-enzyme complex is probably the reason for its toxicity and non-anti-inflammatory use as a drug.

  10. Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initia...

  11. A comparison of visible wavelength reflectance hyperspectral imaging and Acid Black 1 for the detection and identification of blood stained fingerprints.

    PubMed

    Cadd, Samuel; Li, Bo; Beveridge, Peter; O Hare, William T; Campbell, Andrew; Islam, Meez

    2016-07-01

    Bloodstains are often encountered at scenes of violent crime and have significant forensic value for criminal investigations. Blood is one of the most commonly encountered types of biological evidence and is the most commonly observed fingerprint contaminant. Presumptive tests are used to test blood stain and blood stained fingerprints are targeted with chemical enhancement methods, such as acid stains, including Acid Black 1, Acid Violet 17 or Acid Yellow 7. Although these techniques successfully visualise ridge detail, they are destructive, do not confirm the presence of blood and can have a negative impact on DNA sampling. A novel application of visible wavelength hyperspectral imaging (HSI) is used for the non-contact, non-destructive detection and identification of blood stained fingerprints on white tiles both before and after wet chemical enhancement using Acid Black 1. The identification was obtained in a non-contact and non-destructive manner, based on the unique visible absorption spectrum of haemoglobin between 400 and 500nm. Results from the exploration of the selectivity of the setup to detect blood against ten other non-blood protein contaminants are also presented. A direct comparison of the effectiveness of HSI with chemical enhancement using Acid Black 1 on white tiles is also shown.

  12. Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains.

    PubMed

    Hernández, Martín A; Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo; Alvarez, Héctor M

    2015-03-01

    Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.

  13. Modulation of abscisic acid signaling in vivo by an engineered receptor-insensitive protein phosphatase type 2C allele.

    PubMed

    Dupeux, Florine; Antoni, Regina; Betz, Katja; Santiago, Julia; Gonzalez-Guzman, Miguel; Rodriguez, Lesia; Rubio, Silvia; Park, Sang-Youl; Cutler, Sean R; Rodriguez, Pedro L; Márquez, José A

    2011-05-01

    The plant hormone abscisic acid (ABA) plays a crucial role in the control of the stress response and the regulation of plant growth and development. ABA binding to PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS intracellular receptors leads to inhibition of key negative regulators of ABA signaling, i.e. clade A protein phosphatases type 2C (PP2Cs) such as ABA-INSENSITIVE1 and HYPERSENSITIVE TO ABA1 (HAB1), causing the activation of the ABA signaling pathway. To gain further understanding on the mechanism of hormone perception, PP2C inhibition, and its implications for ABA signaling, we have performed a structural and functional analysis of the PYR1-ABA-HAB1 complex. Based on structural data, we generated a gain-of-function mutation in a critical residue of the phosphatase, hab1(W385A), which abolished ABA-dependent receptor-mediated PP2C inhibition without impairing basal PP2C activity. As a result, hab1(W385A) caused constitutive inactivation of the protein kinase OST1 even in the presence of ABA and PYR/PYL proteins, in contrast to the receptor-sensitive HAB1, and therefore hab1(W385A) qualifies as a hypermorphic mutation. Expression of hab1(W385A) in Arabidopsis (Arabidopsis thaliana) plants leads to a strong, dominant ABA insensitivity, which demonstrates that this conserved tryptophan residue can be targeted for the generation of dominant clade A PP2C alleles. Moreover, our data highlight the critical role of molecular interactions mediated by tryptophan-385 equivalent residues for clade A PP2C function in vivo and the mechanism of ABA perception and signaling.

  14. Acid-fast stain

    MedlinePlus

    ... substance is infected with the bacteria that causes tuberculosis ( TB ) and other illnesses. How the Test is ... 91. Fitzgerald DW, Sterling TR, Haas DW. Mycobacterium tuberculosis. In: Bennett JE, Dolin R, Blaser MJ, eds. ...

  15. Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

    PubMed Central

    Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

    2016-01-01

    The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane. PMID:27330112

  16. Neuro-Sweet disease with positive modified acid-fast staining of the cerebrospinal fluid: A case report.

    PubMed

    Liu, Juan-Fang; Li, Yuan; Li, Kai; Zhang, Xiao; Yang, Yi-Ning; Zhao, Gang; Liu, Zhi-Rong

    2016-04-01

    Neuro-Sweet disease (NSD) is Sweet disease with central nervous system (CNS) involvement. To the best of our knowledge, the present case report is the first to describe NSD complicated by endogenous infection with Mycobacterium tuberculosis. The present case report describes a male patient who developed NSD-induced meningitis, which initially manifested as a fever, headache and neck stiffness. Painful erythematous plaques subsequently developed on his face, neck and upper trunk. Brain magnetic resonance imaging was performed and the results were normal, whereas modified acid-fast stain analysis of the cerebrospinal fluid (CSF) provided a positive result. The patient was thus diagnosed with viral meningitis and tuberculosis. However, subsequent skin biopsy results demonstrated neutrophilic infiltration into the dermis without vasculitis, and subsequent human leukocyte antigen typing was positive for Cw1 and negative for B51 and the patient was diagnosed with NSD. Following treatment with corticosteroids, and antiviral and anti-tuberculotic agents, the clinical symptoms were reduced and the previously abnormal findings in the CSF examinations and associated laboratory data were improved. The present case indicates that the diagnosis of NSD is not easily achieved, and early skin biopsy is vital to ensure a fast and effective diagnosis. In addition to systemic corticosteroids, comprehensive treatment is also recommended for patients with NSD complicated by additional complex medical problems.

  17. [Phosphatase activity in Amoeba proteus at low pH].

    PubMed

    Sopina, V A

    2009-01-01

    In free-living Amoeba proteus (strain B), three forms of tartrate-sensitive phosphatase were revealed using PAGE of the supernatant of ameba homogenates obtained with 1% Triton X-100 or distilled water and subsequent staining of gels with 2-naphthyl phosphate as substrate (pH 4.0). The form with the highest mobility in the ameba supernatant was sensitive to all tested phosphatase activity modulators. Two other forms with the lower mobilities were completely or significantly inactivated not only by sodium L-(+)-tartrate, but also by L-(+)-tartaric acid, sodium orthovanadate, ammonium molybdate, EDTA, EGTA, o-phospho-L-tyrosine, DL-dithiotreitol, H2O2, 2-mercaptoethanol, and ions of heavy metals - Fe2+, Fe3+, and Cu2+. Based on results of inhibitory analysis, lysosome location in the ameba cell, and wide substrate specificity of these two forms, it has been concluded that they belong to nonspecific acid phosphomonoesterases (AcP, EC 3.1.3.2). This AcP is suggested to have both phosphomonoesterase and phosphotyrosyl-protein phosphatase activitis. Two ecto-phosphatases were revealed in the culture medium, in which amebas were cultivated. One of them was inhibited by the same reagents as the ameba tartrate-sensitive AcP and seems to be the AcP released into the culture medium in the process of exocytosis of the content of food vacuoles. In the culture medium, apart from this AcP, another phosphatase was revealed, which was not inhibited by any tested inhibitors of AcP and alkaline phosphatase. It cannot be ruled out that this phosphatase belong to the ecto-ATPases found in many protists; however, its ability to hydrolyze ATP has not yet been proven.

  18. Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities.

    PubMed

    Liu, C; Sanghvi, R; Burnell, J M; Howard, G A

    1987-01-01

    Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Strategies of fluorescence staining for trace total ribonucleic acid analysis by capillary electrophoresis with argon ion laser-induced fluorescence.

    PubMed

    Chung, Yi-An; Chen, Yi-Hsin; Chang, Po-Ling

    2015-08-01

    In this work, five fluorescent dyes (SYTO-9, SYBR Green I, SYBR Green II, SYBR Safe, and SYBR Gold) were used as both on-column and precolumn stains for total RNA analysis by CE-LIF with Ar ion laser excitation. In the on-column RNA stain, the SYTO-9 provided the highest fluorescence intensity and the lowest detectable concentration, as low as 10 pg/μL, while the SYBR Green II and SYBR Gold were adsorbed on the poly(ethylene oxide) thus affected the separation efficiency. As a precolumn stain, SYBR Gold was the most sensitive among the five dyes due to the strong affinity between the dye and RNA molecules. As a result, a single-cell quantity of RNA (10-30 pg per cell) could be detected by CE-LIF with precolumn staining by SYBR Gold. Because of the great savings of fluorescent dye using precolumn stain (one button dye may use for one million stain), this method is the best strategy for RNA staining in terms of cost-effectiveness and sensitivity.

  20. [Acid phosphatase of leukocytes in patients with diffuse toxic goiter in evaluating the stage of the disease].

    PubMed

    Vaiuta, N P; Livshits, A Kh; Mendeleev, I M

    1987-01-01

    Acid phosphatase (AP) of lymphocytes and neutrophils was examined cytochemically in 23 patients with associated diffuse toxic goiter (DTG) and thyrotoxicosis during thiamazol treatment, in 20 persons with a DTG remission, and in 5 patients with postradiation and postoperative hypothyroses. AP activity with multiple-granule distribution of the enzyme was high in the majority of the patients with thyrotoxicosis, decreasing with thiamazol treatment. Thyrotoxicosis recurred in 4 out of 11 patients whose lymphocytes had an increased amount of AP during a DTG remission. In 9 patients with normal AP content, no relapses were noted during a DTG remission. Two patients with iatrogenic hypothyroses associated with high lymphocytic AP activity manifested elevated titres of antithyroglobulin immunoglobulins (ATI). In 3 patients with normal content of cellular AP, the ATI titre was low. Cellular AP, a non-specific marker of immunogenesis activity, makes it possible to presumably differentiate the stages of DTG and to evaluate to a definite degree the character of a remission. The preserved high activity of lymphocytic AP in patients with a DTG remission is a prognostically unfavourable factor as regards thyrotoxicosis relapses. The high titre of ATI and activity of lymphocytic AP attest to the predominant autoimmune component of such hypothyroses in part of DTG patients. The authors stress that such patients should receive combined thyroid and glucocorticoid therapy.

  1. Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2)

    SciTech Connect

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

    2010-08-13

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

  2. pH modulation of transient state kinetics of enzymes. II. Transient state kinetics of plant cell wall acid phosphatase.

    PubMed

    Crasnier, M; Ricard, J

    1984-03-01

    The pre-steady-state kinetics of plant cell wall acid phosphatase has been investigated at different pH values. The approach of the steady stale lasts about 1 or 2 s and may be fitted with two exponential terms. For certain pH values the approach to the steady state exhibits damped oscillations. Plotting the sum and the product of the two time constants of these exponentials as a function of substrate concentration yields two straight lines. From the slopes and intercepts of these lines one may determine the values of rate and ionization constants involved in the reaction scheme. The results obtained are consistent with the view that the binding of the substrate to the enzyme does not induce a 'slow' conformation change of the enzyme. The enzyme reacts with its substrate while being mostly in its ionized form. Release of p-nitrophenol is also favoured by this ionized form of the enzyme. However, the hydrolysis of the phosphoryl-enzyme complex mostly occurs from the protonated form of the enzyme. The ionization constants of the free enzyme and of the various enzyme-ligand complexes are very similar.

  3. Luminescent Aggregated Copper Nanoclusters Nanoswitch Controlled by Hydrophobic Interaction for Real-Time Monitoring of Acid Phosphatase Activity.

    PubMed

    Huang, Yuanyuan; Feng, Hui; Liu, Weidong; Zhou, Yingying; Tang, Cong; Ao, Hang; Zhao, Meizhi; Chen, Guilin; Chen, Jianrong; Qian, Zhaosheng

    2016-12-06

    A reversible luminescence nanoswitch through competitive hydrophobic interaction among copper nanoclusters, p-nitrophenol and α-cyclodextrin is established, and a reliable real-time luminescent assay for acid phosphatase (ACP) activity is developed on the basis of this luminescence nanoswitch. Stable and intensely luminescent copper nanoclusters (CuNCs) were synthesized via a green one-pot approach. The hydrophobic nature of CuNCs aggregate surface is identified, and further used to drive the adsorption of p-nitrophenol on the surface of CuNCs aggregate due to their hydrophobic interaction. This close contact switches off the luminescence of CuNCs aggregate through static quenching mechanism. However, the introduction of α-cyclodextrin switches on the luminescence since stronger host-guest interaction between α-cyclodextrin and p-nitrophenol causes the removal of p-nitrophenol from the surface of CuNCs. This nanoswitch in response to external stimulus p-nitrophenol or α-cyclodextrin can be run in a reversible way. Luminescence quenching by p-nitrophenol is further utilized to develop ACP assay using p-nitrophenyl phosphate ester as the substrate. Quantitative measurement of ACP level with a low detection limit of 1.3 U/L was achieved based on this specific detection strategy. This work reports a luminescence nanoswitch mediated by hydrophobic interaction, and provides a sensitive detection method for ACP level which is capable for practical detection in human serum and seminal plasma.

  4. Red cell acid phosphatase types and GC polymorphisms in Mérida, Oaxaca, León, and Saltillo, Mexico.

    PubMed

    Lisker, R; Ramírez, E; Peñaloza, R; Salamanca, F

    1994-12-01

    Red cell acid phosphatase types and GC polymorphisms were studied in Mérida and Oaxaca, Mexico. GC polymorphisms were also investigated in León and Saltillo. The ACP*A, ACP*B, and ACP*C gene frequencies were 0.215, 0.770, and 0.015 respectively, in Mérida and 0.205, 0.788, and 0.002, respectively, in Oaxaca. In Oaxaca the ACP*R gene had a frequency of 0.005. The results are similar to other Mestizo groups studied in Mexico; it is concluded that the ACP*C and ACP*R genes were introduced by admixture of native Amerindians with whites and blacks, respectively. The GC*1S, GC*1F, and GC*2 gene frequencies were 0.489, 0.289 and 0.222, respectively, in León; 0.500, 0.272, and 0.228, respectively, in Mérida; 0.454, 0.337, and 0.209, respectively, in Oaxaca; and 0.505, 0.356, and 0.139, respectively, in Saltillo. These results are similar to what has been obtained in other Mestizo populations and Indian groups in Mexico, probably because the main ethnic component in both is Amerindian.

  5. The pharmacological role of phosphatases (acid and alkaline phosphomonoesterases) in snake venoms related to release of purines - a multitoxin.

    PubMed

    Dhananjaya, Bhadrapura L; D'Souza, Cletus J M

    2011-02-01

    Snake venom components, acting in concert in the prey, cause their immobilization and initiate digestion. To achieve this, several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as phosphatases (acid and alkaline phosphomonoesterases) are less studied and their pharmacological role in venoms is not clearly defined. Also, they show overlapping substrate specificities and have other common biochemical properties causing uncertainty about their identity in venoms. The near-ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It appears that these enzymes may play a central role in liberating purines (mainly adenosine) - a multitoxin and through the action of purines help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities as venom enzymes have been evolved to interfere in diverse physiological processes. This has not been verified by pharmacological studies using purified enzymes. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established.

  6. Evaluation of different modifications of acid-fast staining techniques and stool enzyme-linked immunosorbent assay in detecting fecal Cryptosporidium in diarrheic HIV seropositive and seronegative patients

    PubMed Central

    Parghi, Ekta; Dash, Lona; Shastri, Jayanthi

    2014-01-01

    Rational: The role of Cryptosporidium as an agent of human diarrhea has been redefined over the past decade following recognition of the strong association between cases of cryptosporidiosis and immune deficient individuals (such as those with AIDS). Purpose: The purpose of this study is to determine the prevalence of enteric parasites and to compare the diagnostic utility of stool enzyme-linked immunosorbent assay (ELISA) with various modifications of acid-fast (AF) staining in detection of Cryptosporidium in stool samples of diarrheic patients. Materials and Methods: Stool samples from 186 cases comprising of 93 HIV seropositive and 93 seronegative patients were included. These were subjected to routine and microscopic examination as well as various modifications of AF staining for detection of coccidian parasites and ELISA for the detection of Cryptosporidium. Results: The prevalence of enteric parasites was 54.8% and of Cryptosporidium was 17.2% in HIV seropositive patients while it was 29.0% and 5.4%, respectively in seronegative patients. Of the 186 cases, 33 cases (17.7%) were positive for Cryptosporidium by stool ELISA as compared to 21 (11.3%) by modified AF staining (gold standard) showing sensitivity and specificity of 100% and 92.7%, respectively. The maximum cases of Cryptosporidium (21; 11.3%) were detected by AF staining using 3% acid alcohol. Conclusion: ELISA is a simple, useful, and rapid tool for detection of Cryptosporidium in stool, especially for large scale population studies. However, the role of modified AF staining in detection of Cryptosporidium and other coccidian parasites is important. Based on the results of various modifications of AF staining, the present study recommends the use of 3% acid alcohol along with 10% H2SO4. PMID:25250230

  7. Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility

    PubMed Central

    2013-01-01

    Background Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. Results Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. Conclusions Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs. PMID:23497261

  8. Protein phosphatase 2A in stretch-induced endothelial cell proliferation

    NASA Technical Reports Server (NTRS)

    Murata, K.; Mills, I.; Sumpio, B. E.

    1996-01-01

    We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.

  9. Analysis of DAPI and SYBR Green I as Alternatives to Ethidium Bromide for Nucleic Acid Staining in Agarose Gel Electrophoresis

    NASA Astrophysics Data System (ADS)

    Bourzac, Kevin M.; Lavine, Lori J.; Rice, Margaret S.

    2003-11-01

    DNA electrophoresis and staining is a common procedure in biochemistry laboratories, but the use of ethidium bromide (EB) for DNA detection is worrisome as EB is a mutagen and probable carcinogen. Five alternative stains were evaluated for DNA detection, safety, cost, and ease of use: BlueView, methylene blue, Carolina Blu, DAPI (4',6-diamidino-2-phenylindole dihydrochloride:hydrate), and SYBR Green I. BlueView, Carolina Blu, and methylene blue are not sensitive enough to detect the microgram amounts of DNA used in many procedures. However, DAPI and SYBR Green I are good staining alternatives to ethidium bromide in that they have similar sensitivity and are both easy to use. SYBR Green I is more expensive than EB or DAPI; however, the limited safety data suggest that SYBR Green I is the safest stain.

  10. [Clinical usefulness of urine-formed elements' information obtained from bacteria detection by flow cytometry method that uses nucleic acid staining].

    PubMed

    Nakagawa, Hiroko; Yuno, Tomoji; Itho, Kiichi

    2009-03-01

    Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.

  11. Isolation of betulinic acid, its methyl ester and guaiane sesquiterpenoids with protein tyrosine phosphatase 1B inhibitory activity from the roots of Saussurea lappa C.B.Clarke.

    PubMed

    Choi, Ji Young; Na, Minkyun; Hyun Hwang, In; Ho Lee, Seung; Young Bae, Eun; Yeon Kim, Bo; Seog Ahn, Jong

    2009-01-08

    Activity-guided fractionation of a MeOH extract of the roots of Saussurea lappa C.B.Clarke (Compositae), using an in vitro protein tyrosine phosphatase 1B (PTP1B) inhibition assay, led to the isolation of four active constituents: betulinic acid (1), betulinic acid methyl ester (2), mokko lactone (3) and dehydrocostuslactone (4), along with nine inactive compounds. Our findings indicate that betulinic acid (1) and its methyl ester 2, as well as the two guaiane sesquiterpenoids 3 and 4 are potential lead moieties for the development of new PTP1B inhibitors.

  12. Phosphatase inhibitors remove the run-down of γ-aminobutyric acid type A receptors in the human epileptic brain

    PubMed Central

    Palma, E.; Ragozzino, D. A.; Di Angelantonio, S.; Spinelli, G.; Trettel, F.; Martinez-Torres, A.; Torchia, G.; Arcella, A.; Di Gennaro, G.; Quarato, P. P.; Esposito, V.; Cantore, G.; Miledi, R.; Eusebi, F.

    2004-01-01

    The properties of γ-aminobutyric acid (GABA) type A receptors (GABAA receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABAA receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat α1β2γ2-GABA receptors exhibited a much less pronounced GABA-current run-down. Moreover, the GABAA receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GABAA receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GABAA-receptor β1, β2, β3, and γ2 subunit mRNAs were significantly overexpressed (8- to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GABAA receptors. Blockage of phosphatases stabilizes the TLE GABAA receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy. PMID:15218107

  13. A preliminary X-ray study of 3-deoxy-D-manno-oct-2-ulosonic acid 8-phosphate phosphatase (YrbI) from Burkholderia pseudomallei.

    PubMed

    Park, Jimin; Lee, Daeun; Kim, Mi Sun; Kim, Dae Yong; Shin, Dong Hae

    2015-06-01

    3-Deoxy-D-manno-oct-2-ulosonic acid 8-phosphate phosphatase (YrbI), the third enzyme in the pathway for the biosynthesis of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO), hydrolyzes KDO 8-phosphate to KDO and inorganic phosphate. YrbI belongs to the haloacid dehalogenase (HAD) superfamily, which is a large family of magnesium-dependent phosphatase/phosphotransferase enzymes. In this study, YrbI from Burkholderia pseudomallei, the causative agent of melioidosis, has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 2.25 Å resolution. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 63.7, b = 97.5, c = 98.0 Å. A full structural determination is in progress to elucidate the structure-function relationship of this protein.

  14. [Some enzymatic activities of the amniotic fluid in human beings (LAP, GGTP, SGOT, SGPT, acid and alkaline phosphatases, 5' nucleotidase, amylase, beta-glucuronidase and aldolase)].

    PubMed

    Galerne, D; Baudon, J; Bruhat, M; Dastugue, G

    1973-10-01

    Quantitative analyses of 10 enzymes (LAP, GGTP, SGOT, SGPT. acid and alkaline phosphatases, 5' nucleotidase, amylase. beta-glucuronidase and aldolase) in a series of 50 samples of amniotic fluid gave widely-scattered results. In some cases, it was possible to relate high enzymatic activity to a pathological condition, in other cases, the amniotic fluid examined seemed to come from normal, full-term or almost full-term pregnancies without particular signs.

  15. In vivo detection of prostatic carcinoma with antibodies against prostatic acid phosphatase

    SciTech Connect

    De Land, F.H.; Goldenberg, D.M.

    1984-01-01

    Serum prostatic acid phosphates (PAP) immunoassay is used to evaluate patients with prostatic carcinoma; however, as with other tumor markers, the enzyme levels do not necessarily reflect the presence or extent of tumor. The authors investigated the use of radiolabeled PAP antibodies for the in vivo detection of prostatic carcinoma by external scintillation imaging. Nine patients with prostatic carcinoma were entered into the study. Each received from 2.0 to 2.5 mCi of I-131 labeled antibody to PAP, administered i.v. The immunogen (PAP) was purified from normal human seminal fluid. Antiserum was prepared in rabbits by injecting the purified PAP. The antibodies were labeled with I-131 by chloramine-T method (10 to 20 Ci/g of IgG). Total body images were obtained at 24 and 48 hrs following administration of the labeled antibody. Nontarget I-131 activity was diminished by computer processing. Tumor sites detected by I-131 antibodies were correlated with other diagnostic procedures. In 7 of 9 patients primary and metastatic sites of cancer were detected by antibody imaging, however, no bone lesions were detected (6 cases). In 3 patients with concomitant pulmonary tumors, one was identified as of prostate origin. The serum PAP was normal in 4 patients; however, the primary tumor was identified in 3 of these. These findings suggest that the localization of prostatic carcinoma by means of in-vivo imaging of labeled antibodies to PAP is feasible and offers diagnostic opportunities based upon the functional characteristics.

  16. Purple Acid Phosphatase5 is required for maintaining basal resistance against Pseudomonas syringae in Arabidopsis

    PubMed Central

    2013-01-01

    Background Plants have evolved an array of constitutive and inducible defense strategies to restrict pathogen ingress. However, some pathogens still manage to invade plants and impair growth and productivity. Previous studies have revealed several key regulators of defense responses, and efforts have been made to use this information to develop disease resistant crop plants. These efforts are often hampered by the complexity of defense signaling pathways. To further elucidate the complexity of defense responses, we screened a population of T-DNA mutants in Colombia-0 background that displayed altered defense responses to virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Results In this study, we demonstrated that the Arabidopsis Purple Acid Phosphatse5 (PAP5) gene, induced under prolonged phosphate (Pi) starvation, is required for maintaining basal resistance to certain pathogens. The expression of PAP5 was distinctly induced only under prolonged Pi starvation and during the early stage of Pst DC3000 infection (6 h.p.i). T-DNA tagged mutant pap5 displayed enhanced susceptibility to the virulent bacterial pathogen Pst DC3000. The pap5 mutation greatly reduced the expression of pathogen inducible gene PR1 compared to wild-type plants. Similarly, other defense related genes including ICS1 and PDF1.2 were impaired in pap5 plants. Moreover, application of BTH (an analog of SA) restored PR1 expression in pap5 plants. Conclusion Taken together, our results demonstrate the requirement of PAP5 for maintaining basal resistance against Pst DC3000. Furthermore, our results provide evidence that PAP5 acts upstream of SA accumulation to regulate the expression of other defense responsive genes. We also provide the first experimental evidence indicating the role PAP5 in plant defense responses. PMID:23890153

  17. Electron micrographic study of precipitates formed by interaction of silicic acid and alkaline phosphatase: contribution to a study of silica urolithiasis in cattle.

    PubMed

    Bailey, C B; Cheng, K J; Costerton, J W

    1982-12-01

    Association of alkaline phosphatase with silicic acid in precipitates formed in dilute solution was studied as a model for the nonspecific reaction between silicic acid and protein. Precipitates contained 68-83% of the silicic acid and 52-83% of the enzyme in the original mixture and were in the form of aggregates of roundish particles 150-800 nm in diameter. Enzyme protein formed a tightly bound layer on the surface of particles formed in solutions of freshly prepared silicic acid. The similarity between the ultrastructural features of precipitates from solutions of silicic acid and of internal portions of siliceous urinary calculi from cattle suggests that deposition of silica during development of such calculi is due, at least in part, to the interaction of protein with silicic acid in urine.

  18. Metal-ion mutagenesis: conversion of a purple acid phosphatase from sweet potato to a neutral phosphatase with the formation of an unprecedented catalytically competent Mn(II)Mn(II) active site.

    PubMed

    Mitić, Natasa; Noble, Christopher J; Gahan, Lawrence R; Hanson, Graeme R; Schenk, Gerhard

    2009-06-17

    The currently accepted paradigm is that the purple acid phosphatases (PAPs) require a heterovalent, dinuclear metal-ion center for catalysis. It is believed that this is an essential feature for these enzymes in order for them to operate under acidic conditions. A PAP from sweet potato is unusual in that it appears to have a specific requirement for manganese, forming a unique Fe(III)-mu-(O)-Mn(II) center under catalytically optimal conditions (Schenk et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 273). Herein, we demonstrate, with detailed electron paramagnetic resonance (EPR) spectroscopic and kinetic studies, that in this enzyme the chromophoric Fe(III) can be replaced by Mn(II), forming a catalytically active, unprecedented antiferromagnetically coupled homodivalent Mn(II)-mu-(H)OH-mu-carboxylato-Mn(II) center in a PAP. However, although the enzyme is still active, it no longer functions as an acid phosphatase, having optimal activity at neutral pH. Thus, PAPs may have evolved from distantly related divalent dinuclear metallohydrolases that operate under pH neutral conditions by stabilization of a trivalent-divalent metal-ion core. The present Mn(II)-Mn(II) system models these distant relatives, and the results herein make a significant contribution to our understanding of the role of the chromophoric metal ion as an activator of the nucleophile. In addition, the detailed analysis of strain broadened EPR spectra from exchange-coupled dinuclear Mn(II)-Mn(II) centers described herein provides the basis for the full interpretation of the EPR spectra from other dinuclear Mn metalloenzymes.

  19. A simple-potentiometric method for determination of acid and alkaline phosphatase enzymes in biological fluids and dairy products using a nitrophenylphosphate plastic membrane sensor.

    PubMed

    Hassan, Saad S M; Sayour, Hossam E M; Kamel, Ayman H

    2009-04-27

    A novel poly(vinyl chloride) matrix membrane sensor responsive to 4-nitrophenylphosphate (4-NPP) substrate is described, characterized and used for the potentiometric assay of acid (ACP) and alkaline (ALP) phosphatase enzymes. The sensor is based on the use of the ion-association complex of 4-NPP anion with nickel(II)-bathophenanthroline cation as an electroactive material and nitrophenyloctyl ether (NPOE) as a solvent mediator. The sensor displays good selectivity and stability and demonstrates a near-Nernstian response for 4-NPP over the concentration range 9.6x10(-6) to 1.0x10(-2) M with an anionic slope of 28.6+/-0.3 mV decade(-1) and a detection limit of 6.3x10(-6) M over the pH range 4.5-10. The sensor is used to measure the decrease of a fixed concentration of 4-NPP substrate as a function of acid and alkaline phosphatase enzyme activities at optimized conditions of pH and temperature. A linear relationship between the initial rate of 4-NPP substrate hydrolysis and enzyme activity holds over 0.05-3.0 and 0.03-3.4 IU L(-1) of ACP and ALP enzymes, respectively. Validation of the method by measuring the lower detection limit, range, accuracy, precision, within-day repeatability and between-day-variability reveals good performance characteristics of the proposed sensor. The sensor is used for the determination of acid and alkaline phosphatase enzyme activities in biological fluids of some patients suffering from alcoholic cirrhosis, acute myelocytic leukemia, pre-eclampsia and prostatic cancer. The sensor is also utilized for assessment of alkaline phosphatase enzyme in milk and dairy products. The results obtained agree fairly well with data obtained by the standard spectrophotometric methods.

  20. Increase in tartrate-resistant acid phosphatase of bone at the early stage of ascorbic acid deficiency in the ascorbate-requiring Osteogenic Disorder Shionogi (ODS) rat.

    PubMed

    Goto, A; Tsukamoto, I

    2003-08-01

    The effect of ascorbic acid deficiency on bone metabolism was evaluated using the ascorbate-requiring Osteogenic Disorder Shionogi (ODS) rat model. Ascorbic acid (Asc)-deficient rats gained body weight in a manner similar to Asc-supplemented rats (control) during 3 weeks, but began to lose weight during the 4th week of Asc deficiency. The tartrate-resistant acid phosphatase (TRAP) activity in serum increased to about 2-fold the control value in the rats fed the Asc-free diet for 2, 3, and 4 weeks (AscD2, AscD3, and AscD4), while a decrease in the alkaline phosphatase (ALP) activity was observed only in AscD4 rats. The serum pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) level significantly increased to 1.3-, 1.4-, and 1.9-fold of that in the controls in AscD2, D3, and D4, respectively. The ALP activity in the distal femur was unchanged in AscD1, D2, and D3, but decreased to 50% of the control level in AscD4 rats. The TRAP activity in the distal femur increased to about 2-fold of that in the controls in the AscD2 and D3 and decreased to the control level in the AscD4 rats. The amount of hydroxyproline in the distal femur significantly decreased to about 80%, 70%, and 60% of the control in AscD2, D3, and D4 rats, respectively. These decreases were associated with a similar reduction in the calcium content of the distal femur. Histochemical analysis of the distal femur showed an increase in TRAP-positive cells in AscD2 and AscD3 rats and a decrease in the trabecular bone in AscD2, D3, and D4 rats. These results suggested that a deficiency of Asc stimulated bone resorption at an early stage, followed by a decrease in bone formation in mature ODS rats which already had a well-developed collagen matrix and fully differentiated osteoblasts.

  1. Purkinje cell compartmentation in the cerebellum of the lysosomal Acid phosphatase 2 mutant mouse (nax - naked-ataxia mutant mouse).

    PubMed

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18-19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22-23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation.

  2. Staphylococcal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener’s granulomatosis?

    PubMed Central

    Brons, R H; Bakker, H I; Van Wijk, R T; Van Dijk, N W; Muller Kobold, A C; Limburg, P C; Manson, W L; Kallenberg, C G M; Cohen Tervaert, J W

    2000-01-01

    The majority of patients with Wegener’s granulomatosis (WG) are chronic nasal carriers of Staphylococcus aureus. Chronic nasal carriage of S. aureus is associated with an increased risk of developing a relapse of the disease. The mechanism by which this occurs is still unknown. We hypothesized that a cationic protein of S. aureus, staphylococcal acid phosphatase (SAcP), acts as a planted antigen and initiates glomerulonephritis and vasculitis in patients with WG. In order to test the hypothesis that SAcP can act as a planted antigen in WG, we studied the ability of SAcP to bind to human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells. We also studied whether this binding can be prevented by preincubation with an anionic protein, and whether binding of SAcP activates endothelial cells. We also evaluated whether antibodies in sera of patients with WG are able to bind to endothelial cell-bound SAcP. The results show that SAcP can act as a planted antigen by binding to both types of endothelial cells in a concentration-dependent manner. Binding of concentrations as low as 4 μ g/ml can be detected on HUVEC within 5 min of incubation. Binding of SAcP to endothelial cells was charge-dependent but did not activate endothelial cells. Finally, endothelial cell-bound SAcP was recognized by sera of patients with WG. The data suggest a possible pathogenic role for SAcP by acting as a planted antigen thereby initiating glomerulonephritis and vasculitis in patients with WG. PMID:10691932

  3. Anti-thyroid and antifungal activities, BSA interaction and acid phosphatase inhibition of methimazole copper(II) complexes.

    PubMed

    Urquiza, Nora M; Islas, María S; Ariza, Santiago T; Jori, Nadir; Martínez Medina, Juan J; Lavecchia, Martín J; López Tévez, Leonor L; Lezama, Luis; Rojo, Teófilo; Williams, Patricia A M; Ferrer, Evelina G

    2015-03-05

    It has been reported that various metal coordination compounds have improved some biological properties. A high activity of acid phosphatase (AcP) is associated to several diseases (osteoporosis, Alzheimer's, prostate cancer, among others) and makes it a target for the development of new potential inhibitors. Anti-thyroid agents have disadvantageous side effects and the scarcity of medicines in this area motivated many researchers to synthesize new ones. Several copper(II) complexes have shown antifungal activities. In this work we presented for a first time the inhibition of AcP and the anti-thyroid activity produced by methimazole-Cu(II) complexes. Cu-Met ([Cu(MeimzH)2(H2O)2](NO3)2·H2O) produces a weak inhibition action while Cu-Met-phen ([Cu(MeimzH)2(phen)(H2O)2]Cl2) shows a strong inhibition effect (IC50 = 300 μM) being more effective than the reported behavior of vanadium complexes. Cu-Met-phen also presented a fairly good anti-thyroid activity with a formation constant value, Kc=1.02 × 10(10)M(-1) being 10(6) times more active than methimazole (Kc = 4.16 × 10(4)M(-1)) in opposition to Cu-Met which presented activity (Kc=9.54 × 10(3)M(-1)) but in a lesser extent than that of the free ligand. None of the complexes show antifungal activity except Cu-phen (MIC = 11.71 μgmL(-1) on Candidaalbicans) which was tested for comparison. Besides, albumin interaction experiments denoted high affinity toward the complexes and the calculated binding constants indicate reversible binding to the protein.

  4. Purification and properties of lectin from potato tubers and leaves; interaction with acid phosphatase from potato tuber.

    PubMed

    Wierzba-Arabska, E; Morawiecka, B

    1987-01-01

    The haemagglutination activity of lectin, (STA) was determined during vegetation of potatoes (Solanum tuberosum var. Uran). Traces of haemagglutination activity were found in young stalks and first leaves. In the next leaves the maximal activity appeared after 6-8 weeks of vegetation and, simultaneously with the decrease in leaves it appeared in young tubers (at the time of their differentiation from stolons), reaching the maximum in the 18th week of vegetation. Lectins from potato tubers and leaves were extracted with acetate buffer, pH 3.6, salted out with ammonium sulphate, and purified alternatively by affinity chromatography on GlcNAc-Spheron or by adsorption on trypsinated and glutaraldehyde-fixed rabbit erythrocytes. Chromatography on Sephadex G-200 was used for preparative separation of isomeric forms. In the tubers four isolectins were found, differeing in molecular mass, pI values, content of sugars and haemagglutination activity. Two of these forms were isolated in homogeneous form: a heavy dimeric form, Mr 130,000-142,000 containing 59.6% of sugars (including 28.8% arabinose, 6.9% galactose, 16.5% glucose, 1.6% xylose and 3.1% mannose), and a light monomeric form. of Mr 21,000-22,000, containing 20% of neutral sugars (2.5% of pentoses and 17.5% of hexoses). From leaves, a single heavy dimeric lectin of Mr 136,000, containing 14.3% of neutral sugars was isolated. The effect of tuber isolectin on the activity, pH optima and heat-stability of the potato acid phosphatase has been described.

  5. Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.

    PubMed

    Eaton, James M; Mullins, Garrett R; Brindley, David N; Harris, Thurl E

    2013-04-05

    The lipin gene family encodes a class of Mg(2+)-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Unlike other enzymes in the Kennedy pathway, lipins are not integral membrane proteins, and they need to translocate from the cytosol to intracellular membranes to participate in glycerolipid synthesis. The movement of lipin 1 within the cell is closely associated with its phosphorylation status. Although cellular analyses have demonstrated that highly phosphorylated lipin 1 is enriched in the cytosol and dephosphorylated lipin 1 is found on membranes, the effects of phosphorylation on lipin 1 activity and binding to membranes has not been recapitulated in vitro. Herein we describe a new biochemical assay for lipin 1 using mixtures of phosphatidic acid (PA) and phosphatidylethanolamine that reflects its physiological activity and membrane interaction. This depends on our observation that lipin 1 binding to PA in membranes is highly responsive to the electrostatic charge of PA. The studies presented here demonstrate that phosphorylation regulates the ability of the polybasic domain of lipin 1 to recognize di-anionic PA and identify mTOR as a crucial upstream signaling component regulating lipin 1 phosphorylation. These results demonstrate how phosphorylation of lipin 1 together with pH and membrane phospholipid composition play important roles in the membrane association of lipin 1 and thus the regulation of its enzymatic activity.

  6. Effects of several low-molecular weight organic acids and phosphate on the adsorption of acid phosphatase by soil colloids and minerals.

    PubMed

    Huang, Qiaoyun; Zhao, Zhenhua; Chen, Wenli

    2003-07-01

    Adsorption of acid phosphatase on goethite, kaolinite and two colloids from the soils in central and south China in the presence of organic acids and phosphate was studied. With the increase of anion concentration, the ability in decreasing enzyme adsorption followed the sequence: phosphate>tartrate>oxalate>acetate. Acetate showed promotive effect on enzyme adsorption at lower anion concentrations whereas oxalate, tartrate and phosphate compete effectively with enzyme in a broad range of anion concentration. The adsorption isotherms of enzyme in most of the anionic systems studied conformed to the Langmuir equation. Phosphate reduced the affinity of enzyme on goethite more significantly than the other anions. However, tartrate decreased the affinity of enzyme on soil colloids and kaolinite to a greater extent than phosphate, oxalate and acetate. This observation suggested that the impact of anions on enzyme adsorption varies with anionic type and the surface characteristics of soil components. The influence of the addition order of ligand on enzyme adsorption was found greater in tartrate and phosphate systems. In general, simultaneous introduction of ligand and enzyme into the system had the lowest enzyme adsorption, showing more competition between ligand and enzyme molecules in this system. Data from this work indicated that the status and activity of enzyme in certain soil microenvironments especially the rhizosphere where various organic and inorganic ligands are active can be altered and may be completely different from the bulk soil.

  7. Removal of Perfluorocarboxylic Acids (PFCAs) from Carpets Treated with Stain-protection Products by Using Carpet Cleaning Machines

    EPA Science Inventory

    PFCAs are found in a variety of consumer products, including, but not limited to, treated clothing and textiles, floor care products, paper containers for food, and carpets. For example, carpet that has been treated with stain-protection, carpet-care solutions, either by the manu...

  8. Short term effects of Glomus claroideum and Azospirillum brasilense on growth and root acid phosphatase activity of Carica papaya L. under phosphorus stress.

    PubMed

    Alarcón, Alejandro; Davies, Frederick T; Egilla, Johnatan N; Fox, Theodore C; Estrada-Luna, Arturo A; Ferrera-Cerrato, Ronald

    2002-01-01

    Arbuscular mycorrhizal fungi (AMF) are able to increase root enzymatic activity of acid and alkaline phosphatases. However, the role of AMF on phosphatase activity has not been reported in papaya (Carica papaya L.), which is frequently established at places with soil phosphorus (P) deficiencies. The goals of this research were to determine the effect of Glomus claroideum (Gc), and plant growth promoting rhizobacterium Azospirillum brasilense strain VS7 [Ab]) on root phosphatase activity and seedling growth of Carica papaya L. cv. Red Maradol under low P conditions. There were four treatments-colonization with: 1) Gc, 2) Ab, 3) Gc+Ab, and 4) non-inoculated seedlings. Plants were established in a coarse sand:sandy loam substrate under P-limitation (11 microg P ml(-1)), supplied with a modified Long Ashton Nutrient Solution. Seedling growth was severely reduced by low P. Gc+Ab inoculated plants had greater total dry matter and leaf area than non-colonized plants. Gc-inoculated plants had greater leaf area than non-colonized plants. Treatments did not differ in leaf area ratio, specific leaf area and, total chlorophyll content. There was a non-significant effect on stem relative growth rate with Gc and Gc+Ab plants. Mycorrhizal colonization enhanced the bacterial population 3.4-fold in the Gc+Ab treatment compared with the population quantified in Ab treatment. Soluble and extractable root acid phosphatase activity (RAPA) was higher in Gc inoculated plants. We discussed on the possible relation among both inoculated microorganisms and also with the P-limitation which plants were established.

  9. The cell wall-targeted purple acid phosphatase AtPAP25 is critical for acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation.

    PubMed

    Del Vecchio, Hernan A; Ying, Sheng; Park, Joonho; Knowles, Vicki L; Kanno, Satomi; Tanoi, Keitaro; She, Yi-Min; Plaxton, William C

    2014-11-01

    Plant purple acid phosphatases (PAPs) belong to a relatively large gene family whose individual functions are poorly understood. Three PAP isozymes that are up-regulated in the cell walls of phosphate (Pi)-starved (-Pi) Arabidopsis thaliana suspension cells were purified and identified by MS as AtPAP12 (At2g27190), AtPAP25 (At4g36350) and AtPAP26 (At5g34850). AtPAP12 and AtPAP26 were previously isolated from the culture medium of -Pi cell cultures, and shown to be secreted by roots of Arabidopsis seedlings to facilitate Pi scavenging from soil-localized organophosphates. AtPAP25 exists as a 55 kDa monomer containing complex NX(S/T) glycosylation motifs at Asn172, Asn367 and Asn424. Transcript profiling and immunoblotting with anti-AtPAP25 immune serum indicated that AtPAP25 is exclusively synthesized under -Pi conditions. Coupled with potent mixed-type inhibition of AtPAP25 by Pi (I50 = 50 μm), this indicates a tight feedback control by Pi that prevents AtPAP25 from being synthesized or functioning as a phosphatase except when Pi levels are quite low. Promoter-GUS reporter assays revealed AtPAP25 expression in shoot vascular tissue of -Pi plants. Development of an atpap25 T-DNA insertion mutant was arrested during cultivation on soil lacking soluble Pi, but rescued upon Pi fertilization or complementation with AtPAP25. Transcript profiling by quantitative RT-PCR indicated that Pi starvation signaling was attenuated in the atpap25 mutant. AtPAP25 exhibited near-optimal phosphatase activity with several phosphoproteins and phosphoamino acids as substrates. We hypothesize that AtPAP25 plays a key signaling role during Pi deprivation by functioning as a phosphoprotein phosphatase rather than as a non-specific scavenger of Pi from extracellular P-monoesters.

  10. Active principle of swine prostate extract: I. Isolation of active principle activating prostatic acid phosphatase and its effect on testosterone uptake of the prostate in castrated rats.

    PubMed

    Yoshida, Y; Mori, H; Inami, K; Koda, A

    1991-07-01

    There have been several reports concerning the therapeutic effect of an extract from animal prostates on benign prostatic hypertrophy. Previously, we reported that the swine prostate extract (PE) had the activity to enhance human prostatic acid phosphatase (PAPase) activity in vitro, and to increase the muscular tonicity of the urinary bladder by directly acting upon vesical muscles, suggesting that PE have an activity to elevate the intravesical voiding pressure in vivo. In the present study, it was attempted to isolate such an active principle of PE as activates human prostatic acid phosphatase (PAPase). The finally purified PE (PPE) was assessed as to some physico-chemical and pharmacological properties. 1) PPE was found to be a peptide with a molecular weight of about 8,800, composed largely of neutral amino acids (approximately 70%) and few of aromatic amino acids. 2) PPE activated PAPase in a dose-dependent fashion, resulting in an increase of the enzyme activity approximately twice in a dose of 2 X 10(-5) g/ml of PPE. Furthermore, PPE recovered PAPase activity dose-dependently from the 50% inhibition by 2 X 10(-3) M L-tartaric acid. 3) In castrated rats, the 3H-testosterone uptake of the prostate was significantly suppressed by the oral administration of PPE. PPE might be one of active principles of PE for the therapeutic effect on prostatic hypertrophy.

  11. Evolutionarily conserved regulatory mechanisms of abscisic acid signaling in land plants: characterization of ABSCISIC ACID INSENSITIVE1-like type 2C protein phosphatase in the liverwort Marchantia polymorpha.

    PubMed

    Tougane, Ken; Komatsu, Kenji; Bhyan, Salma Begum; Sakata, Yoichi; Ishizaki, Kimitsune; Yamato, Katsuyuki T; Kohchi, Takayuki; Takezawa, Daisuke

    2010-03-01

    Abscisic acid (ABA) is postulated to be a ubiquitous hormone that plays a central role in seed development and responses to environmental stresses of vascular plants. However, in liverworts (Marchantiophyta), which represent the oldest extant lineage of land plants, the role of ABA has been least emphasized; thus, very little information is available on the molecular mechanisms underlying ABA responses. In this study, we isolated and characterized MpABI1, an ortholog of ABSCISIC ACID INSENSITIVE1 (ABI1), from the liverwort Marchantia polymorpha. The MpABI1 cDNA encoded a 568-amino acid protein consisting of the carboxy-terminal protein phosphatase 2C (PP2C) domain and a novel amino-terminal regulatory domain. The MpABI1 transcript was detected in the gametophyte, and its expression level was increased by exogenous ABA treatment in the gemma, whose growth was strongly inhibited by ABA. Experiments using green fluorescent protein fusion constructs indicated that MpABI1 was mainly localized in the nucleus and that its nuclear localization was directed by the amino-terminal domain. Transient overexpression of MpABI1 in M. polymorpha and Physcomitrella patens cells resulted in suppression of ABA-induced expression of the wheat Em promoter fused to the beta -glucuronidase gene. Transgenic P. patens expressing MpABI1 and its mutant construct, MpABI1-d2, lacking the amino-terminal domain, had reduced freezing and osmotic stress tolerance, and associated with reduced accumulation of ABA-induced late embryogenesis abundant-like boiling-soluble proteins. Furthermore, ABA-induced morphological changes leading to brood cells were not prominent in these transgenic plants. These results suggest that MpABI1 is a negative regulator of ABA signaling, providing unequivocal molecular evidence of PP2C-mediated ABA response mechanisms functioning in liverworts.

  12. Methods for chromosome-specific staining

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    1995-01-01

    Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

  13. The pH-induced glycosylation of secreted phosphatases is mediated in Aspergillus nidulans by the regulatory gene pacC-dependent pathway.

    PubMed

    Nozawa, S R; Ferreira-Nozawa, M S; Martinez-Rossi, N M; Rossi, A

    2003-08-01

    In this communication, we show that the pacC(c)14 mutation drastically reduced the mannose and N-acetylglycosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans when grown at 22 degrees C, pH 5.0, compared to a control strain. The staining after PAGE was not observed for the pacA-encoded acid phosphatase, while the palD-encoded Pi-repressible alkaline phosphatase had an altered electrophoretic mobility. In addition, the secreted acid phosphatase also had a reduced number of isoforms visualized by staining after IEF and glycosylation had a protective effect against its heat inactivation. We also show that a full-length version of gene pacC-1 cloned from Neurospora crassa complemented the pacC(c)14 mutation of A. nidulans, including the remediation of both the acid and alkaline Pi-repressible phosphatases secreted at pH 5.0, which indicates that glycosylation of secreted phosphatases is mediated in A. nidulans by the conserved PacC pathway that governs pH-responsive gene expression.

  14. Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase.

    PubMed

    Cano, Florencia; Poderoso, Cecilia; Cornejo Maciel, Fabiana; Castilla, Rocío; Maloberti, Paula; Castillo, Fernanda; Neuman, Isabel; Paz, Cristina; Podestá, Ernesto J

    2006-06-01

    The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.

  15. Measuring phosphatidic acid phosphatase (EC 3.1.3.4) activity using two phosphomolybdate-based colorimetric methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), which is also known as PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol (DAG) and inorganic phosphate. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacyl...

  16. The phagocytic function of Sertoli cells: a morphological, biochemical, and endocrinological study of lysosomes and acid phosphatase localization in the rat testis.

    PubMed

    Chemes, H

    1986-10-01

    The lysosomal population of the seminiferous tubules of the rat was studied by conventional electron microscopy and electron microscopic histochemistry. Biochemical determinations of acid phosphatase were carried out in whole cell suspensions of seminiferous tubular cells or in different cell populations purified by sedimentation in albumin gradients. Lysosomes were rarely found in spermatogonia and primary spermatocytes. Young spermatids showed up to six lysosomes per section, and this number increased as spermatid maturation proceeded. Residual bodies had a very heterogeneous lysosomal content. Sertoli cells showed cyclical variations in their lysosomes. These were present in small numbers from stages I-IV of the cycle of the seminiferous epithelium and progressively increased to be numerous in Sertoli cells at stages VI-VIII. After spermiation, their rapidly decreased. Acid phosphatase contents were (nanomoles of nitrophenol formed per mg protein/min): whole cell suspension, 67.5 +/- 7.8; pachytene spermatocytes (72% purity), 76.5 +/- 10.6; round spermatids (73% purity), 95.0 +/- 2.8; residual bodies (88% purity), 96.0 +/- 14.2; and Sertoli cell-enriched fraction, 278.5 +/- 75.7. In a group of rats, endogenous LH and testosterone were lowered by administration of anti-LH antibodies. There was an intense degeneration of meiotic spermatocytes, which were phagocytized and digested by these immature testosterone-depleted Sertoli cells. It is concluded that lysosomes of the seminiferous epithelium show cyclical variations, with an increase toward the time of spermiation and a decrease after the residual bodies have been digested; the acid phosphatase and lysosomal contents of Sertoli cells are higher than those of germ cells, residual body disposal is probably initiated by autophagy and completed by Sertoli cell phagocytosis; and the phagocytic function of Sertoli cells is not hormone (testosterone) dependent.

  17. [Alkaline phosphatase in Amoeba proteus].

    PubMed

    Sopina, V A

    2005-01-01

    In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.

  18. Differential therapeutic responses of thiol compounds in the reversal of methylmercury inhibited acid phosphatase and cathepsin E in the central nervous system of rat

    SciTech Connect

    Vinay, S.D.; Raghu, K.G.; Sood, P.P.

    1992-07-01

    Though considerable headway has been made in elucidating the effect of methylmercury on the biochemical machinery of nervous system, the studies on the alterations in the levels of acid hydrolases received less attention. Being a lysosomal marker, acid phosphatase is one of the most extensively studies enzymes amongst the acid hydrolases. Its significance in various key physiological as well as pathological processes is well preserved in literature. Cathepsin E, an aspartic proteinase, has been demonstrated in a number of cells and tissues within the human body, rat, E. coli where its role is implicated in a number of important metabolic processes. In the present paper, we report the results of the differential levels of inhibition of these enzymes with methylmercury as well as their differential recoveries with two thiols (N-acetyl-DL-homocysteine thiolactone and glutathione) in various neuroanatomical areas (olfactory bulbs, cerebral hemispheres, cerebellum, medulla oblongata and spinal cord) of rat. 22 refs., 5 figs.

  19. Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme.

    PubMed Central

    Micanovic, R; Bailey, C A; Brink, L; Gerber, L; Pan, Y C; Hulmes, J D; Udenfriend, S

    1988-01-01

    A carboxyl-terminal chymotryptic peptide from mature human placental alkaline phosphatase was purified by HPLC and monitored by a specific RIA. Sequencing and amino acid assay showed that the carboxyl terminus of the peptide was aspartic acid, representing residue 484 of the proenzyme as deduced from the corresponding cDNA. Further analysis of the peptide showed it to be a peptidoglycan containing one residue of ethanolamine, one residue of glucosamine, and two residues of neutral hexose. The inositol glycan is apparently linked to the alpha carboxyl group of the aspartic acid through the ethanolamine. Location of the inositol glycan on Asp-484 of the proenzyme indicates that a 29-residue peptide is cleaved from the nascent protein during the post-translational condensation with the phosphatidylinositol-glycan. PMID:3422741

  20. Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation.

    PubMed

    Badr, Haitham A; AlSadek, Dina M M; Mathew, Mohit P; Li, Chen-Zhong; Djansugurova, Leyla B; Yarema, Kevin J; Ahmed, Hafiz

    2015-12-01

    This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, "Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show sialylated glycans intracellularly. The impact of sialic acid supplementation under nutrient deprivation was demonstrated by measuring levels of expression and sialylation of two markers, EGFR1 and MUC1. This Data in Brief article complements the main manuscript by providing detailed instructions and representative results for cell-level imaging and Western blot analyses of changes in sialylation during nutrient deprivation and sialic acid supplementation. These methods can be readily generalized for the study of many types of glycosylation and various glycoprotein markers through the appropriate selection of fluorescently-labeled lectins.

  1. Facile fabrication of luminescent organic dots by thermolysis of citric acid in urea melt, and their use for cell staining and polyelectrolyte microcapsule labelling

    PubMed Central

    Zholobak, Nadezhda M; Popov, Anton L; Shcherbakov, Alexander B; Popova, Nelly R; Guzyk, Mykhailo M; Antonovich, Valeriy P; Yegorova, Alla V; Scrypynets, Yuliya V; Leonenko, Inna I; Baranchikov, Alexander Ye

    2016-01-01

    Luminescent organic dots (O-dots) were synthesized via a one-pot, solvent-free thermolysis of citric acid in urea melt. The influence of the ratio of the precursors and the duration of the process on the properties of the O-dots was established and a mechanism of their formation was hypothesized. The multicolour luminescence tunability and toxicity of synthesized O-dots were extensively studied. The possible applications of O-dots for alive/fixed cell staining and labelling of layer-by-layer polyelectrolyte microcapsules were evaluated. PMID:28144539

  2. A New Protein Phosphatase 2C (FsPP2C1) Induced by Abscisic Acid Is Specifically Expressed in Dormant Beechnut Seeds1

    PubMed Central

    Lorenzo, Oscar; Rodríguez, Dolores; Nicolás, Gregorio; Rodríguez, Pedro L.; Nicolás, Carlos

    2001-01-01

    An abscisic acid (ABA)-induced cDNA fragment encoding a putative protein phosphatase 2C (PP2C) was obtained by means of differential reverse transcriptase-polymerase chain reaction approach. The full-length clone was isolated from a cDNA library constructed using mRNA from ABA-treated beechnut (Fagus sylvatica) seeds. This clone presents all the features of plant type PP2C and exhibits homology to members of this family such as AthPP2CA (61%), ABI1 (48%), or ABI2 (47%), therefore it was named FsPP2C1. The expression of FsPP2C1 is detected in dormant seeds and increases after ABA treatment, when seeds are maintained dormant, but it decreases and tends to disappear when dormancy is being released by stratification or under gibberellic acid treatment. Moreover, drought stress seems to have no effect on FsPP2C1 transcript accumulation. The FsPP2C1 transcript expression is tissue specific and was found to accumulate in ABA-treated seeds rather than in other ABA-treated vegetative tissues examined. These results suggest that the corresponding protein could be related to ABA-induced seed dormancy. By expressing FsPP2C1 in Escherichia coli as a histidine tag fusion protein, we have obtained direct biochemical evidence supporting Mg2+-dependent phosphatase activity of this protein. PMID:11299374

  3. Purple acid phosphatase of the human macrophage and osteoclast. Characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells.

    PubMed

    Hayman, A R; Cox, T M

    1994-01-14

    The purple phosphatases catalyze hydrolysis of phosphate esters (optimum pH approximately 5) and are resistant to inhibition by dextro-rotatory tartrate; their distinctive color is due to Fe(III)-phenolate charge-transfer transitions at their active site. Expression of human purple phosphatase, designated type 5 acid phosphatase, is restricted to osteoclasts and other activated cells of monohistiocytic lineage, but its biological rôle in relation to bone resorption and phagocytosis is unknown. To characterize this enzyme further, we have engineered the human type 5 acid phosphatase into a baculovirus vector expression system that enabled milligram quantities of purple protein to be purified from medium containing Sf9 host cells. The phosphatase cDNA was transcribed as a single RNA species of 1.5 kilobases as in human tissues. Tartrate-resistant acid phosphatase activity reacting with uteroferrin antisera appeared in the culture medium, from which up to 8 mg/liter was purified by two-step cation-exchange chromatography at pH 8.0. Two isoforms of approximately 36 kDa were identified by SDS-polyacrylamide electrophoresis and were converted to a single species of apparent molecular size 34 kDa upon treatment with N-glycosidase F, indicating secreted glycoforms of a single polypeptide. Mass spectroscopy showed that the mean molecular mass of the active, secreted glycoprotein was 35849 Da. The recombinant enzyme (specific activity, 190 mumol p-nitrophenol/min/mg at 37 degrees C) contained 2 iron atoms/molecule and formed purple, monoclinic crystals. Exposure to the ferric chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, rapidly inactivated the enzyme, which was not inhibited by alpha, alpha'-bipyridyl, a ferrous chelator. That ferric iron is essential for enzymatic catalysis, was further indicated by the synergistic effects of the reductant, dithiothreitol, and bipyridyl on phosphatase activity. The recombinant purple phosphatase catalyzed the peroxidation of 5

  4. Effect of acidic pH on flow cytometric detection of bacteria stained with SYBR Green I and their distinction from background

    NASA Astrophysics Data System (ADS)

    Baldock, Daniel; Nebe-von-Caron, Gerhard; Bongaerts, Roy; Nocker, Andreas

    2013-12-01

    Unspecific background caused by biotic or abiotic particles, cellular debris, or autofluorescence is a well-known interfering parameter when applying flow cytometry to the detection of microorganisms in combination with fluorescent dyes. We present here an attempt to suppress the background signal intensity and thus to improve the detection of microorganisms using the nucleic acid stain SYBR® Green I. It has been observed that the fluorescent signals from SYBR Green I are greatly reduced at acidic pH. When lowering the pH of pre-stained samples directly prior to flow cytometric analysis, we hypothesized that the signals from particles and cells with membrane damage might therefore be reduced. Signals from intact cells, temporarily maintaining a neutral cytosolic pH, should not be affected. We show here that this principle holds true for lowering background interference, whereas the signals of membrane-compromised dead cells are only affected weakly. Signals from intact live cells at low pH were mostly comparable to signals without acidification. Although this study was solely performed with SYBR® Green I, the principle of low pH flow cytometry (low pH-FCM) might hold promise when analyzing complex matrices with an abundance of non-cellular matter, especially when expanded to non-DNA binding dyes with a stronger pH dependence of fluorescence than SYBR Green I and a higher pKa value.

  5. The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease

    PubMed Central

    Leyria, Jimena; Fruttero, Leonardo L.; Nazar, Magalí; Canavoso, Lilián E.

    2015-01-01

    In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas’ disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia. PMID:26091289

  6. Structure-function relationships of the yeast fatty acid synthase: negative-stain, cryo-electron microscopy, and image analysis studies of the end views of the structure.

    PubMed Central

    Stoops, J K; Kolodziej, S J; Schroeter, J P; Bretaudiere, J P; Wakil, S J

    1992-01-01

    The yeast fatty acid synthase (M(r) = 2.5 x 10(6)) is organized in an alpha 6 beta 6 complex. In these studies, the synthase structure has been examined by negative-stain and cryo-electron microscopy. Side and end views of the structure indicate that the molecule, shaped similar to a prolate ellipsoid, has a high-density band of protein bisecting its major axis. Stained and frozen-hydrated average images of the end views show an excellent concordance and a hexagonal ring having three each alternating egg- and kidney-shaped features with low-protein-density protrusions extending outward from the egg-shaped features. Images also show that the barrel-like structure is not hollow but has a Y-shaped central core, which appears to make contact with the three egg-shaped features. Numerous side views of the structure give good evidence that the beta subunits have an archlike shape. We propose a model for the synthase that has point-group symmetry 32 and six equivalent sites of fatty acid synthesis. The protomeric unit is alpha 2 beta 2. The ends of each of the two archlike beta subunits interact with opposite sides of the two dichotomously arranged disclike alpha subunits. Three such protomeric units form the ring. We propose that the six fatty acid synthesizing centers are composed of two complementary half-alpha subunits and a beta subunit, an arrangement having all the partial activities of the multifunctional enzyme required for fatty acid synthesis. Images PMID:1631160

  7. FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis

    PubMed Central

    Chen, Jia; Yu, Feng; Liu, Ying; Du, Changqing; Li, Xiushan; Zhu, Sirui; Wang, Xianchun; Lan, Wenzhi; Rodriguez, Pedro L.; Liu, Xuanming; Li, Dongping; Chen, Liangbi; Luan, Sheng

    2016-01-01

    Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)–A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals. PMID:27566404

  8. /sup 18/O isotope effect in /sup 13/C nuclear magnetic resonance spectroscopy. Part 9. Hydrolysis of benzyl phosphate by phosphatase enzymes and in acidic aqueous solutions

    SciTech Connect

    Parente, J.E.; Risley, J.M.; Van Etten, R.L.

    1984-12-26

    The /sup 18/O isotope-induced shifts in /sup 13/C and /sup 31/P nuclear magnetic resonance (NMR) spectroscopy were used to establish the position of bond cleavage in the phosphatase-catalyzed and acid-catalyzed hydrolysis reactions of benzyl phosphate. The application of the /sup 18/O-isotope effect in NMR spectroscopy affords a continuous, nondestructive assay method for following the kinetics and position of bond cleavage in the hydrolytic process. The technique provides advantages over most discontinuous methods in which the reaction components must be isolated and converted to volatile derivatives prior to analysis. In the present study, (..cap alpha..-/sup 13/C,ester-/sup 18/O)benzyl phosphate and (ester-/sup 18/O)benzyl phosphate were synthesized for use in enzymatic and nonenzymatic studies. Hydrolysis reactions catalyzed by the alkaline phosphatase from E. coli and by the acid phosphatases isolated from human prostate and human liver were all accompanied by cleavage of the substrate phosphorus-oxygen bond consistent with previously postulated mechanisms involving covalent phosphoenzyme intermediates. An extensive study of the acid-catalyzed hydrolysis of benzyl phosphate at 75/sup 0/C revealed that the site of bond cleavage is dependent on pH. At pH less than or equal to 1.3, the hydrolysis proceeds with C-O bond cleavage; at 1.3 < pH < 2.0, there is a mixture of C-O and P-O bond scission, the latter progressively predominating as the pH is raised; at pH greater than or equal to 2.0, the hydrolysis proceeds with exclusive P-O bond scission. (S)-(+)-(..cap alpha..-/sup 2/H)Benzyl phosphate was also synthesized. Hydrolysis of this chiral benzyl derivative demonstrated that the acid-catalyzed C-O bond scission of benzyl phosphate proceeds by an A-1 (S/sub N/1) mechanism with 70% racemization and 30% inversion at carbon. 37 references, 4 figures, 2 tables.

  9. Effect of okadaic acid and calyculin-A, two protein phosphatase inhibitors, on thyrotropin-stimulated triiodothyronine secretion in cultured sheep thyroid cells.

    PubMed

    Arufe, M C; Beckett, G J; Durán, R; Alfonso, M

    1999-12-01

    We have studied the effect of two protein phosphatase inhibitors on thyrotropin (TSH)-stimulated triiodothyronine (T3) production by sheep thyroid cells grown in primary culture. Incubation of sheep thyrocytes with okadaic acid (OA) and calyculin-A (CL-A), two potent inhibitors of type 1 (PP1) and type 2A (PP2A) protein phosphatases, resulted in an increase of TSH-stimulated T3 production. This effect was detected using concentrations as low as 0.1 pM with OA and 1 fM with CL-A. An inhibitory effect on T3 production, due to cellular death, was observed with 6 nM OA and 1 nM CL-A. In the absence of TSH, OA or CL-A had no effect on T3 production by thyrocytes. Forskoline (10 microM), an activator of adenylate cyclase, increased the basal and TSH-stimulated T3 release by sheep thyroid cells; this effect was increased by OA in cells grown in the basal state but not in the presence of TSH. These results suggest that the marine toxins OA and CL-A, two potent inhibitors of PP-1 and PP-2A, have significant stimulatory effects on T3 secretion promoted by TSH and FK. These observations indicate that these proteins could be important mediators of thyroid hormone production.

  10. [Immunohistochemical staining of the astrocytic expression of glial fibrillary acidic protein and vimentin in the central nervous system of dogs with canine distemper].

    PubMed

    Orsini, Heloísa; Bondan, Eduardo Fernandes; Sanchez, Melissa; Lallo, Maria Anete; Maiorka, Paulo César; Dagli, Maria Lúcia Zaidan; Graça, Dominguita Luthers

    2007-12-01

    Considering that many aspects involved in the pathogenesis of the central nervous system (CNS) demyelinating diseases are still poorly understood and that astrocytes seem to mediate such processes, this study analyzed the participation of astrocytes in the demyelinating processes of CNS by using immunohistochemical staining of two astrocytic proteins--glial fibrillary acidic protein (GFAP) and vimentin (VIM)--comparing samples of cerebellum and brainstem from eight dogs with canine distemper and from two healthy dogs, from different breeds and ages varying from 1 to 4 years old. Histological sections were submitted to the avidin-biotin-peroxidase indirect method of immunohistochemical staining (ABC) and the astrocytic reactivity, observed in light microscopy, was quantified in a computer system for image analysis. It was possible to notice, on most of the sections from sick animals, degenerative lesions that indicate demyelination. The immunostaining for GFAP and VIM was more intense on animals with canine distemper, specially around the ventricules and near degenerated sites. There was no significant difference between the immunostaining (GFAP and VIM) of animals with canine distemper with and without inflammatory infiltrate of the cerebellar white matter. The increased immunoreactivity of astrocytes for GFAP and the VIM reexpression in injured areas indicate the astrocytic involvement on nervous tissue response to the demyelinating lesions induced by the canine distemper virus (CDV) in the CNS.

  11. A STRESS-RESPONSIVE NAC1-Regulated Protein Phosphatase Gene Rice Protein Phosphatase18 Modulates Drought and Oxidative Stress Tolerance through Abscisic Acid-Independent Reactive Oxygen Species Scavenging in Rice1[W][OPEN

    PubMed Central

    You, Jun; Zong, Wei; Hu, Honghong; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

    2014-01-01

    Plants respond to abiotic stresses through a complexity of signaling pathways, and the dephosphorylation mediated by protein phosphatase (PP) is an important event in this process. We identified a rice (Oryza sativa) PP2C gene, OsPP18, as a STRESS-RESPONSIVE NAC1 (SNAC1)-regulated downstream gene. The ospp18 mutant was more sensitive than wild-type plants to drought stress at both the seedling and panicle development stages. Rice plants with OsPP18 suppressed through artificial microRNA were also hypersensitive to drought stress. Microarray analysis of the mutant revealed that genes encoding reactive oxygen species (ROS) scavenging enzymes were down-regulated in the ospp18 mutant, and the mutant exhibited reduced activities of ROS scavenging enzymes and increased sensitivity to oxidative stresses. Overexpression of OsPP18 in rice led to enhanced osmotic and oxidative stress tolerance. The expression of OsPP18 was induced by drought stress but not induced by abscisic acid (ABA). Although OsPP18 is a typical PP2C with enzymatic activity, it did not interact with SNF1-RELATED PROTEIN KINASE2 protein kinases, which function in ABA signaling. Meanwhile, the expression of ABA-responsive genes was not affected in the ospp18 mutant, and the ABA sensitivities of the ospp18 mutant and OsPP18-overexpressing plants were also not altered. Together, these findings suggest that OsPP18 is a unique PP2C gene that is regulated by SNAC1 and confers drought and oxidative stress tolerance by regulating ROS homeostasis through ABA-independent pathways. PMID:25318938

  12. Differential staining of bacteria: capsule stain.

    PubMed

    Breakwell, Donald P; Moyes, Rita B; Reynolds, Jackie

    2009-11-01

    Bacterial capsules are composed of high-molecular-weight polysaccharides and/or polypeptides, and are associated with virulence and biofilm formation. Unfortunately, capsules do not stain well with crystal violet, methylene blue, or other simple stains. This unit describes two methods of capsule staining. The first is a wet-mount method using india ink; the capsule is visualized as a refractile zone surrounding a cell. The second is a direct-staining dry-mount method that precipitates copper sulfate and leaves the capsule as a pale blue zone. Both methods are easily performed within approximately 5 min.

  13. Differential staining of bacteria: gram stain.

    PubMed

    Moyes, Rita B; Reynolds, Jackie; Breakwell, Donald P

    2009-11-01

    In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.

  14. A Novel Phosphatidic Acid-Protein-tyrosine Phosphatase D2 Axis Is Essential for ERBB2 Signaling in Mammary Epithelial Cells*

    PubMed Central

    Ramesh, Mathangi; Krishnan, Navasona; Muthuswamy, Senthil K.; Tonks, Nicholas K.

    2015-01-01

    We used a loss-of-function screen to investigate the role of classical protein-tyrosine phosphatases (PTPs) in three-dimensional mammary epithelial cell morphogenesis and ERBB2 signaling. The study revealed a novel role for PTPD2 as a positive regulator of ERBB2 signaling. Suppression of PTPD2 attenuated the ERBB2-induced multiacinar phenotype in three-dimensional cultures specifically by inhibiting ERBB2-mediated loss of polarity and lumen filling. In contrast, overexpression of PTPD2 enhanced the ERBB2 phenotype. We also found that a lipid second messenger, phosphatidic acid, bound PTPD2 in vitro and enhanced its catalytic activity. Small molecule inhibitors of phospholipase D (PLD), an enzyme that produces phosphatidic acid in cells, also attenuated the ERBB2 phenotype. Exogenously added phosphatidic acid rescued the PLD-inhibition phenotype, but only when PTPD2 was present. These findings illustrate a novel pathway involving PTPD2 and the lipid second messenger phosphatidic acid that promotes ERBB2 function. PMID:25681440

  15. Complex coordinated extracellular metabolism: Acid phosphatases activate diluted human leukocyte proteins to generate energy flow as NADPH from purine nucleotide ribose

    PubMed Central

    Hibbs, John B.; Vavrin, Zdenek; Cox, James E.

    2016-01-01

    Complex metabolism is thought to occur exclusively in the crowded intracellular environment. Here we report that diluted enzymes from lysed human leukocytes produce extracellular energy. Our findings involve two pathways: the purine nucleotide catabolic pathway and the pentose phosphate pathway, which function together to generate energy as NADPH. Glucose6P fuel for NADPH production is generated from structural ribose of purine ribonucleoside monophosphates, ADP, and ADP-ribose. NADPH drives glutathione reductase to reduce an oxidized glutathione disulfide-glutathione redox couple. Acid phosphatases initiate ribose5P salvage from purine ribonucleoside monophosphates, and transaldolase controls the direction of carbon chain flow through the nonoxidative branch of the pentose phosphate pathway. These metabolic control points are regulated by pH. Biologically, this energy conserving metabolism could function in perturbed extracellular spaces. PMID:26895212

  16. Enumeration and Cell Cycle Analysis of Natural Populations of Marine Picoplankton by Flow Cytometry Using the Nucleic Acid Stain SYBR Green I

    PubMed Central

    Marie, D.; Partensky, F.; Jacquet, S.; Vaulot, D.

    1997-01-01

    The novel dye SYBR Green I binds specifically to nucleic acids and can be excited by blue light (488-nm wavelength). Cell concentrations of prokaryotes measured in marine samples with this dye on a low-cost compact flow cytometer are comparable to those obtained with the UV-excited stain Hoechst 33342 (bis-benzimide) on an expensive flow cytometer with a water-cooled laser. In contrast to TOTO-1 and TO-PRO-1, SYBR Green I has the advantage of clearly discriminating both heterotrophic bacteria and autotrophic Prochlorococcus cells, even in oligotrophic waters. As with TOTO-1 and TO-PRO-1, two groups of heterotrophic bacteria (B-I and B-II-like types) can be distinguished. Moreover, the resolution of DNA distribution obtained with SYBR Green I is similar to that obtained with Hoechst 33342 and permits the analysis of the cell cycle of photosynthetic prokaryotes over the whole water column. PMID:16535483

  17. Methods for chromosome-specific staining

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1995-09-05

    Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogeneous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include ways for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes. 3 figs.

  18. Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining

    PubMed Central

    Grégori, Gérald; Citterio, Sandra; Ghiani, Alessandra; Labra, Massimo; Sgorbati, Sergio; Brown, Spencer; Denis, Michel

    2001-01-01

    The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes. PMID:11571170

  19. The purple acid phosphatase GmPAP21 enhances internal phosphorus utilization and possibly plays a role in symbiosis with rhizobia in soybean.

    PubMed

    Li, Chengchen; Li, Caifeng; Zhang, Haiyan; Liao, Hong; Wang, Xiurong

    2017-02-01

    Induction of secreted and intracellular purple acid phosphatases (PAPs; EC 3.1.3.2) is widely recognized as an adaptation of plants to phosphorus (P) deficiency. The secretion of PAPs plays important roles in P acquisition. However, little is known about the functions of intracellular PAP in plants and nodules. In this study, we identified a novel PAP gene GmPAP21 in soybean. Expression of GmPAP21 was induced by P limitation in nodules, roots and old leaves, and increased in roots with increasing duration of P starvation. Furthermore, the induction of GmPAP21 in nodules and roots was more intensive than in leaves in both P-efficient genotype HN89 and P-inefficient genotype HN112 in response to P starvation, and the relative expression in the leaves and nodules of HN89 was significantly greater than that of HN112 after P deficiency treatment. Further functional analyses showed that over-expressing GmPAP21 significantly enhanced both acid phosphatase activity and growth performance of hairy roots under P starvation condition, indicating that GmPAP21 plays an important role in P utilization. Moreover, GUS expression driven by GmPAP21 promoter was shown in the nodules besides roots. Overexpression of GmPAP21 in transgenic soybean significantly inhibited nodule growth, and thereby affected plant growth after inoculation with rhizobia. This suggests that GmPAP21 is also possibly involved in regulating P metabolism in nodules. Taken together, our results suggest that GmPAP21 is a novel plant PAP that functions in the adaptation of soybean to P starvation, possibly through its involvement in P recycling in plants and P metabolism in nodules.

  20. Staining sectioned biological specimens for transmission electron microscopy: conventional and en bloc stains.

    PubMed

    Ellis, E Ann

    2014-01-01

    Post-staining of ultrathin sections and/or en bloc staining of specimens is necessary for differential contrast and improved resolution of cellular structures. Often specimens are fixed and stained with osmium tetroxide during fixation, but additional contrast is the result of additional heavy metal stains on the sections. The most common post-staining of sections is done on grids by aqueous uranyl acetate followed by lead citrate. When it is apparent that simple, aqueous uranium and lead post-staining is not adequate, other stains are invoked. These procedures can be as simple as en bloc staining with uranyl acetate after primary fixation and osmication. Over the years, several other treatments have been developed for use with the primary fixation or during dehydration. Tannic acid, paraphenylenediamine (PPD), and malachite green can all serve as en bloc stains and can contribute to overall improved visualization of ultrastructural details in biological specimens. Tannic acid and PPD improve membrane preservation, and malachite green is a phospholipid stain. All of these stains are compatible with aqueous fixatives and should be considered when the usual stains are not satisfactory. Marinozzi rings and microwave-assisted post-staining offer alternatives to traditional grid staining. In addition, stain precipitates on grids often can be removed by treatment with 10 % (v/v) acetic acid.

  1. Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia.

    PubMed

    Hoff, R H; Hengge, A C; Wu, L; Keng, Y F; Zhang, Z Y

    2000-01-11

    General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.

  2. Spatial structure of heptapeptide Glu-Ile-Leu-Asn-His-Met-Lys, a fragment of the HIV enhancer prostatic acid phosphatase, in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Bloсhin, Dmitri S.; Aganova, Oksana V.; Yulmetov, Aidar R.; Filippov, Andrei V.; Gizatullin, Bulat I.; Afonin, Sergii; Antzutkin, Oleg N.; Klochkov, Vladimir V.

    2013-02-01

    Prostatic acid phosphatase (PAP) is a protein abundantly present in human seminal fluid. PAP plays important role in fertilization. Its 39-amino-acid fragment, PAP(248-286), is effective in enhancing infectivity of HIV virus. In this work, we determined the spatial structure in aqueous solution of a heptapeptide within the PAP fragment, containing amino acid residues 266-272 (Glu-Ile-Leu-Asn-His-Met-Lys). We also report the structure of the complex formed by this heptapeptide with sodium dodecyl sulfate micelles, a model of a biological membrane, as determined by 1H NMR spectroscopy and 2D NMR (TOCSY, HSQC-HECADE, NOESY) spectroscopy. Complex formation was confirmed by chemical shift alterations in the 1H NMR spectra of the heptapeptide, as well as by the signs and values of NOE effects. We also present a comparison of the spatial structure of Glu-Ile-Leu-Asn-His-Met-Lys in water and in complex with sodium dodecyl sulfate.

  3. Tartrate-Resistant Acid Phosphatase 5b in Young Patients With Sickle Cell Disease and Trait Siblings: Relation to Vasculopathy and Bone Mineral Density.

    PubMed

    Mokhtar, Galila Mohamed; Tantawy, Azza Abdel Gawad; Hamed, Ahmed Al-Saeed; Adly, Amira Abdel Moneam; Ismail, Eman Abdel Rahman; Makkeyah, Sara Mostafa

    2017-01-01

    Bone involvement is a frequent cause of acute morbidity in sickle cell disease (SCD). Tartrate-resistant acid phosphatase 5b (TRACP 5b), a bone resorption marker, is produced specifically by activated osteoclasts. We assessed bone mineral density (BMD) in 30 young patients with SCD and 17 asymptomatic patients with sickle cell trait (SCT) compared with 32 healthy controls and determined TRACP 5b levels in relation to vascular complications. Serum ferritin, alkaline phosphatase (ALP), and TRACP 5b were measured. Echocardiography was performed with assessment of BMD using dual energy X-ray absorptiometry (DXA). The BMD was decreased in patients with SCD compared with SCT and controls (P = .005), with no significant difference between the latter 2 groups. Patients with SCD had higher incidence of bone complications than SCT group and controls (P = .03). The SCD group with abnormal DXA scan had higher ferritin and ALP than normal BMD. Serum TRACP 5b was significantly higher in patients with SCD than SCT and controls (P = .003). The TRACP 5b levels were associated with severe vaso-occlusive crisis (P = .022). Patients treated with hydroxyurea and those on chelation therapy had lower TRACP 5b levels than untreated patients. The TRACP 5b level was positively correlated with lactate dehydrogenase, while there was no relation with ferritin, ALP, or BMD. We suggest that bone complications frequently occur in SCD as reflected by low BMD and high ALP and TRACP 5b. Hemolysis and iron overload may be involved in the occurrence of these complications. The lack of correlation between abnormal DXA scan and high TRACP 5b suggests that bone disease in SCD is multifactorial.

  4. The N-Acetylmuramic Acid 6-Phosphate Phosphatase MupP Completes the Pseudomonas Peptidoglycan Recycling Pathway Leading to Intrinsic Fosfomycin Resistance

    PubMed Central

    Borisova, Marina; Gisin, Jonathan

    2017-01-01

    ABSTRACT Bacterial cells are encased in and stabilized by a netlike peptidoglycan (PGN) cell wall that undergoes turnover during bacterial growth. PGN turnover fragments are frequently salvaged by the cells via a pathway referred to as PGN recycling. Two different routes for the recycling of the cell wall sugar N-acetylmuramic acid (MurNAc) have been recognized in bacteria. In Escherichia coli and related enterobacteria, as well as in most Gram-positive bacteria, MurNAc is recovered via a catabolic route requiring a MurNAc 6-phosphate etherase (MurQ in E. coli) enzyme. However, many Gram-negative bacteria, including Pseudomonas species, lack a MurQ ortholog and use an alternative, anabolic recycling route that bypasses the de novo biosynthesis of uridyldiphosphate (UDP)-MurNAc, the first committed precursor of PGN. Bacteria featuring the latter pathway become intrinsically resistant to the antibiotic fosfomycin, which targets the de novo biosynthesis of UDP-MurNAc. We report here the identification and characterization of a phosphatase enzyme, named MupP, that had been predicted to complete the anabolic recycling pathway of Pseudomonas species but has remained unknown so far. It belongs to the large haloacid dehalogenase family of phosphatases and specifically converts MurNAc 6-phosphate to MurNAc. A ΔmupP mutant of Pseudomonas putida was highly susceptible to fosfomycin, accumulated large amounts of MurNAc 6-phosphate, and showed lower levels of UDP-MurNAc than wild-type cells, altogether consistent with a role for MupP in the anabolic PGN recycling route and as a determinant of intrinsic resistance to fosfomycin. PMID:28351914

  5. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

    PubMed

    Ivanov, Delyan P; Parker, Terry L; Walker, David A; Alexander, Cameron; Ashford, Marianne B; Gellert, Paul R; Garnett, Martin C

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

  6. Abscisic Acid Promotion of Arbuscular Mycorrhizal Colonization Requires a Component of the PROTEIN PHOSPHATASE 2A Complex1[W][OPEN

    PubMed Central

    Charpentier, Myriam; Sun, Jongho; Wen, Jiangqi; Mysore, Kirankumar S.; Oldroyd, Giles E.D.

    2014-01-01

    Legumes can establish intracellular interactions with symbiotic microbes to enhance their fitness, including the interaction with arbuscular mycorrhizal (AM) fungi. AM fungi colonize root epidermal cells to gain access to the root cortex, and this requires the recognition by the host plant of fungus-made mycorrhizal factors. Genetic dissection has revealed the symbiosis signaling pathway that allows the recognition of AM fungi, but the downstream processes that are required to promote fungal infection are poorly understood. Abscisic acid (ABA) has been shown to promote arbuscule formation in tomato (Solanum lycopersicum). Here, we show that ABA modulates the establishment of the AM symbiosis in Medicago truncatula by promoting fungal colonization at low concentrations and impairing it at high concentrations. We show that the positive regulation of AM colonization via ABA requires a PROTEIN PHOSPHATASE 2A (PP2A) holoenzyme subunit, PP2AB′1. Mutations in PP2AB′1 cause reduced levels of AM colonization that cannot be rescued with permissive ABA application. The action of PP2AB′1 in response to ABA is unlinked to the generation of calcium oscillations, as the pp2aB′1 mutant displays a normal calcium response. This contrasts with the application of high concentrations of ABA that impairs mycorrhizal factor-induced calcium oscillations, suggesting different modes of action of ABA on the AM symbiosis. Our work reveals that ABA functions at multiple levels to regulate the AM symbiosis and that a PP2A phosphatase is required for the ABA promotion of AM colonization. PMID:25293963

  7. Staining tomato fruit cuticle and exocarp tissues.

    PubMed

    Graham, E T

    1997-05-01

    Immature fruit of tomato, Lycopersicon esculentum (Celebrity), was examined to observe the cuticle, its interface with the epidermis, and the general histology of the outer exocarp. Paraffin sections were stained first with Bismarck brown Y. Structures already stained in various hues of brown were stained again with either azure B, aluminum hematoxylin and alcian blue SGX, or the periodic acid-Schiff (PAS) reaction. Bismarck brown-azure B displayed the cuticle in strong contrast with subjacent tissue; however, nuclei were not easily identified at low magnification. Bismarck brown-hematoxylin-alcian blue produced a sharply contrasted combination of yellow cuticle, bright blue cell walls and purple nuclei. Nuclei stained purple with hematoxylin were easily identified at x100. Bismarck brown-PAS stained the cuticle golden brown and subjacent tissues mageta red. Surprisingly, epidermal cells stained specifically and intensely with PAS while pretreatment with an aldehyde blockade and omission of periodic acid prevented staining of all other tissues.

  8. Sac phosphatase domain proteins.

    PubMed Central

    Hughes, W E; Cooke, F T; Parker, P J

    2000-01-01

    Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. PMID:10947947

  9. Biological activities of Zn(II)-S-methyl-cysteine complex as antiradical, inhibitor of acid phosphatase enzyme and in vivo antidepressant effects.

    PubMed

    Escudero, Graciela E; Martini, Nancy; Jori, Khalil; Jori, Nadir; Maresca, Nahuel R; Laino, Carlos H; Naso, Luciana G; Williams, Patricia A M; Ferrer, Evelina G

    2016-12-01

    The antidepressant effect of simple Zn(II) salts has been proved in several animal models of depression. In this study, a coordination metal complex of Zn(II) having a sulfur containing ligand is tested as antidepressant for the first time. Forced swimming test method on male Wistar rats shows a decrease in the immobility and an increase in the swimming behavior after treatment with [Zn(S-Met)2] (S-Met=S-methyl-l-cysteine) being more effective and remarkable than ZnCl2. The thiobarbituric acid and the pyranine consumption (hydroxyl and peroxyl radicals, respectively) methods were applied to evaluate the antioxidant activity of S-Met and [Zn(S-Met)2] showing evidence of attenuation of hydroxyl but not peroxyl radicals activities. UV-vis studies on the inhibition of acid phosphatase enzyme (AcP) demonstrated that S-methyl-l-cysteine did not produce any effect but, in contrast, [Zn(S-Met)2] complex behaved as a moderate inhibitor. Finally, bioavailability studies were performed by fluorescence spectroscopy denoting the ability of the albumin to transport the complex.

  10. [The morphologic basis for the mechanism of increased serum acid phosphatase activity in patients with viral hepatitis].

    PubMed

    Drozd, T N; Luchshev, V I; Zaĭtsev, V V; Berezina, T A

    1975-01-01

    The activity of acid phosphotase in the blood serum and punctates of the liver was studied in 49 patients with acute cyclic form of viral hepatitis of slight and medium-grave course with the use of histological and histochemical methods. The degree of manifestation of necrotic changes in the punctate tissue was determined with the help of the stereometric method. As a result of the studies conducted, it was established that the degree of hyperfermentemia was the highest at the period when the disease was in full swing, and that it did not depend on the form of its course and extent of necrotic changes observed in the liver punctates. The authors consider a morphological evidence of high activity of acid phosphotase in the blood serum aggregations of large hepatocytes, cytoplasm of which was loaded with mature lipofuscin, the topographic identity of the latter with acid phosphotase at present being proved.

  11. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    SciTech Connect

    Einstein, R.; Gabel, C.A. )

    1991-01-01

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated.

  12. Improvement of Student Understanding of How Kinetic Data Facilitates the Determination of Amino Acid Catalytic Function through an Alkaline Phosphatase Structure/Mechanism Bioinformatics Exercise

    ERIC Educational Resources Information Center

    Grunwald, Sandra K.; Krueger, Katherine J.

    2008-01-01

    Laboratory exercises, which utilize alkaline phosphatase as a model enzyme, have been developed and used extensively in undergraduate biochemistry courses to illustrate enzyme steady-state kinetics. A bioinformatics laboratory exercise for the biochemistry laboratory, which complements the traditional alkaline phosphatase kinetics exercise, was…

  13. Staining proteins in gels with silver nitrate.

    PubMed

    Simpson, Richard J

    2007-07-01

    INTRODUCTIONSilver staining is one of the commonly used procedures for visualizing proteins in acrylamide gels. All silver staining methods rely on the reduction of ionic to metallic silver to provide metallic silver images; the selective reduction at gel sites occupied by proteins compared to nonprotein sites is dependent on differences in the oxidation-reduction potentials at these sites. There are two broad methodologies for silver staining. One approach (nondiamine silver nitrate stains) uses silver nitrate as the silvering agent and formaldehyde in alkaline carbonate solution as the developing agent, whereas the other approach (diamine or ammoniacal stains) uses ammoniacal silver as the silvering agent and formaldehyde in dilute citric acid as the developing agent. Although protocols using ammoniacal silver are arguably more sensitive and give darker hues than those based on silver nitrate, they are more prone to negative staining, resulting in hollow or "doughnut" spots, give unacceptable backgrounds with tricine-based gel systems, and are not very robust because of their reliance on the ammonia-silver ratio. Additionally, ammoniacal silver staining is more sensitive for basic proteins but less so for very acidic proteins. This protocol describes a silver nitrate staining approach. Its sensitivity is in the low-nanogram range, which is 50-100 times more sensitive than classical Coomassie Blue staining, ~10 times better than colloidal Coomassie Blue staining, and at least twice as sensitive as the zinc/imidazole negative staining method.

  14. Teaching resources. Protein phosphatases.

    PubMed

    Salton, Stephen R

    2005-03-01

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein phosphatases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the importance of phosphatases in physiology, recognized by the award of a Nobel Prize in 1992, and then proceeds to describe the two types of protein phosphatases: serine/threonine and tyrosine phosphatases. The information covered includes the structure, regulation, and substrate specificity of protein phosphatases, with an emphasis on their importance in disease and clinical settings.

  15. Studies on the properties of myo-inositol-1,4,5-trisphosphate 5-phosphatase and myo-inositol monophosphatase in bovine iris sphincter smooth muscle: effects of okadaic acid and protein phosphorylation.

    PubMed

    Wang, X L; Akhtar, R A; Abdel-Latif, A A

    1994-05-26

    In bovine iris sphincter, myo-inositol 1,4,5-trisphosphate (IP3) 5-phosphatase and myo-inositol 1-phosphate (IP1) monophosphatase are mainly localized in the microsomal and soluble fractions, respectively. Studies on the properties of these enzymes can be summarized as follows. (1) The microsomal IP3 5-phosphatase hydrolyzed IP3 to myo-inositol 1,4-bisphosphate with an apparent Km of 28 microM and Vmax of 32 nmol/min per mg protein. The IP1 monophosphatase in the soluble fraction hydrolyzed IP1 into free inositol with an apparent Km of 89 microM and Vmax of 7 nmol/min per mg protein. (2) IP3 5-phosphatase and IP1 monophosphatase had optimal pH values at 8.0 and 7.0, respectively. (3) Both enzymes required Mg2+ and their highest specific activities were at a cation concentration of 2 mM. (4) Ca2+ (> 0.5 microM) exerted an inhibitory effect on IP3 5-phosphatase activity, and marked inhibition (47%) was observed at a concentration of 10 microM. Higher concentrations of the cation (> 100 microM) were required to inhibit IP1 monophosphatase. (5) IP1 monophosphatase, but not IP3 5-phosphatase, was inhibited by Li+. Li+ had no effect on the contractile response in this smooth muscle. (6) Both enzymes were inhibited by ATP and by the thiol-blocking agent, disulfiram. In addition, thimerosal, a thiol reagent, also inhibited the IP3 5-phosphatase activity. (7) Protein phosphorylation of the microsomal and soluble fractions with PKA or PKC had no effect on the activities of these enzymes. (8) Okadaic acid, a protein phosphatase inhibitor, had no effect on the activity of IP3 5-phosphatase. However, in the intact iris sphincter the toxin significantly reduced the carbachol-induced IP3 production, 1,2-diacylglycerol formation, measured as phosphatidic acid, and caused muscle relaxation.

  16. Short-chain fatty acid production from different biological phosphorus removal sludges: the influences of PHA and Gram-staining bacteria.

    PubMed

    Wang, Dongbo; Chen, Yinguang; Zheng, Xiong; Li, Xiang; Feng, Leiyu

    2013-03-19

    Recently, the reuse of waste activated sludge to produce short-chain fatty acids (SCFA) has attracted much attention. However, the influences of sludge characteristics, especially polyhydroxyalkanoates (PHA) and Gram-staining bacteria, on SCFA production have seldom been investigated. It was found in this study that during sludge anaerobic fermentation not only the fermentation time but also the SCFA production were different between two sludges, which had different PHA contents and Gram-negative bacteria to Gram-positive bacteria (GNB/GPB) ratios and were generated respectively from the anaerobic/oxic (AO) and aerobic/extended-idle (AEI) biological phosphorus removal processes. The optimal fermentation time for the AEI and AO sludges was respectively 4 and 8 d, and the corresponding SCFA production was 304.6 and 231.0 mg COD/g VSS (volatile suspended solids) in the batch test and 143.4 and 103.9 mg COD/g VSS in the semicontinuous experiment. The mechanism investigation showed that the AEI sludge had greater PHA content and GNB/GPB ratio, and the increased PHA content accelerated cell lysis and soluble substrate hydrolysis while the increased GNB/GPB ratio benefited cell lysis. Denaturing gradient gel electrophoresis profiles revealed that the microbial community in the AEI sludge fermentation reactor was dominated by Clostridium sp., which was reported to be SCFA-producing microbes. Further enzyme analyses indicated that the activities of key hydrolytic and acids-forming enzymes in the AEI sludge fermentation reactor were higher than those in the AO one. Thus, less fermentation time was required, but higher SCFA was produced in the AEI sludge fermentation system.

  17. Characterization of the egg vesicular components in the seaweed, Fucus serratus L. (Fucales, Phaeophyta), using enzyme histochemistry and vital staining: the search for a lysosome-like body.

    PubMed

    Holland, R D; Pitt, D; Moore, M N; Brownlee, C

    1997-03-01

    Fucus serratus eggs were examined for evidence of the existence of a lysosome-like body using enzyme histochemical and vital staining techniques. Simultaneous coupling azo-dye techniques for lysosomal acid phosphatase proved inappropriate owing to endogenous phenolic binding artefacts. The large number of alginate polysaccharide and polyphenolic egg vesicles interfered with vital staining techniques for lysosomes. Lysosomal esterase activity was detected in the abundant egg lipid bodies. The role of the egg lipid body as an equivalent lysosome-like body of higher plants, the spherosome, is discussed in relation to egg fertilization and early zygote development.

  18. Technique and staining optimization leucoconcentration.

    PubMed

    Pierrez, J; Guerci, A; Guerci, O

    1987-09-01

    In cytometric clinical application, it is important to obtain cell suspensions rapidly with as little cytological alteration as possible. A procedure has been achieved to prepare cell suspensions for flow cytometric analysis. The leucoconcentration technique, first described by Herbeuval for cytologic analysis, has been modified to be applied in cytometry. This technique involves Saponin lysis of red cells of peripheral blood or bone marrow samples that have been previously fixed with picric acid alcohol solution. Cells in suspension are not shifted and tinctorial affinity is not modified. Then cells have been stained with Mithramycin. Each parameter defined by Crissman has been analyzed to define the best staining conditions. The availability of Leucoconcentration with Mithramycin-DNA-staining permits determination of cell cycle with a fine resolution.

  19. An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles.

    PubMed

    Terasaki, Yasunobu; Shichijo, Shigeki; Niu, Yamei; Komatsu, Nobukazu; Noguchi, Masanori; Todo, Satoru; Itoh, Kyogo

    2009-11-01

    We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8+ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.

  20. Inhibition of several enzymes by gold compounds. II. beta-Glucuronidase, acid phosphatase and L-malate dehydrogenase by sodium thiomalatoraurate (I), sodium thiosulfatoaurate (I) and thioglucosoaurate (I).

    PubMed

    Lee, M T; Ahmed, T; Haddad, R; Friedman, M E

    1989-01-01

    Bovine liver beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32), wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) and bovine liver L-malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) were inhibited by a series of gold (I) complexes that have been used as anti-inflammatory drugs. Both sodium thiosulfatoaurate (I) (Na AuTs) and sodium thiomalatoraurate (NaAuTM) effectively inhibited all three enzymes, while thioglucosoaurate (I) (AuTG) only inhibited L-malate dehydrogenase. The equilibrium constants (K1) ranged from nearly 4000 microM for the NaAuTM-beta-glucuronidase interaction to 24 microM for the NaAuTS-beta-glucuronidase interaction. The rate of covalent bond formation (kp) ranged from 0.00032 min-1 for NaAuTM-beta-glucuronidase formation to 1.7 min-1 for AuTG-L-malate dehydrogenase formation. The equilibrium data shows that the gold (I) drugs bind by several orders lower than the gold (III) compounds, suggesting a significantly stronger interaction between the more highly charged gold ion and the enzyme. Yet the rate of covalent bond formation depends as much on the structure of the active site as upon the lability of the gold-ligand bond. It was also observed that the more effective the gold inhibition the more toxic the compound.

  1. Serial lectin affinity chromatography with concavalin A and wheat germ agglutinin demonstrates altered asparagine-linked sugar-chain structures of prostatic acid phosphatase in human prostate carcinoma.

    PubMed

    Yoshida, K I; Honda, M; Arai, K; Hosoya, Y; Moriguchi, H; Sumi, S; Ueda, Y; Kitahara, S

    1997-08-01

    Differences between human prostate carcinoma (PCA, five cases) and benign prostatic hyperplasia (BPH, five cases) in asparagine-linked (Asn) sugar-chain structure of prostatic acid phosphatase (PAP) were investigated using lectin affinity chromatography with concanavalin A (Con A) and wheat germ agglutinin (WGA). PAP activities were significantly decreased in PCA-derived PAP, while no significant differences between the two PAP preparations were observed in the enzymatic properties (Michaelis-Menten value, optimal pH, thermal stability, and inhibition study). In these PAP preparations, all activities were found only in the fractions which bound strongly to the Con A column and were undetectable in the Con A unbound fractions and in the fractions which bound weakly to the Con A column. The relative amounts of PAP which bound strongly to the Con A column but passed through the WGA column, were significantly greater in BPH-derived PAP than in PCA-derived PAP. In contrast, the relative amounts of PAP which bound strongly to the Con A column and bound to the WGA column, were significantly greater in PCA-derived PAP than in BPH-derived PAP. The findings suggest that Asn-linked sugar-chain structures are altered during oncogenesis in human prostate and also suggest that studies of qualitative differences of sugar-chain structures of PAP might lead to a useful diagnostic tool for PCA.

  2. Spinal cord projections of the rat main forelimb nerves, studied by transganglionic transport of WGA-HRP and by the disappearance of acid phosphatase.

    PubMed

    Castro-Lopes, J M; Coimbra, A

    1991-03-01

    The spinal cord projections of the 3 main forelimb nerves-median, radial and ulnar, were studied in the rat dorsal horn with transganglionic transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP), or using the disappearance of fluoride resistant acid phosphatase (FRAP) after nerve section. The projection patterns in lamina II were similar following the two procedures. The median and the radial nerve fibers projected to the medial and the intermediate thirds, respectively, of the dorsal horn lamina II in spinal cord segments C4-C8. The ulnar nerve projected to segments C6-C8 between the areas occupied by the other two nerves. The FRAP method also showed that the lateral part of lamina II, which was not filled by radial nerve fibers, received the projections from the dorsal cutaneous branches of cervical spinal nerves. In addition, FRAP disappeared from the medial end of segment T1 after skin incisions extending from the medial brachium to the axilla, which seemed due to severance of the cutaneous branchlets of the lateral anterior thoracic nerve. The FRAP procedure is thus sensitive enough to detect fibers in lamina II arising from small peripheral nerves, and may be used as an alternative to the anterograde tracing methods whenever there are no overlapping projections.

  3. Acid phosphatase activity in liver macrophage aggregates as a marker for pollution-induced immunomodulation of the non-specific immune response in fish

    NASA Astrophysics Data System (ADS)

    Broeg, Katja

    2003-10-01

    The activity of acid phosphatase in liver macrophage aggregates (MA-AP) of different fish species was used as a marker for a pollution-induced modulation of the digestive capacity of phagocytes, since functions of the non-specific immune response play a central role in the maintenance of animals' health. Based upon the investigation of more than 900 individual flounders (Platichthys flesus) and mullets (Liza aurata), natural variations, gender-specific differences and pollution-induced alterations in AP activity are demonstrated in this study. MA-AP activity was dependent on temperature and season but, nevertheless, distinctions between differently polluted areas were visible in all sampling campaigns with lowest MA-AP activity in fish from the polluted areas of the German Bight and the Israeli coast of the Mediterranean Sea. For organochlorine contaminants, as well as for mercury and copper, a significant correlation could be observed between residue concentrations in fish tissues and MA-AP activity. In all cases, except mercury which showed a positive correlation, AP activity was suppressed in animals with a high contaminant burden. MA-AP activity turned out to give reliable and consistent results for a quantification of immunomodulation in both fish species.

  4. Phosphatidic acid phosphatase in neonatal rat lung: effects of prenatal dexamethasone or terbutaline treatment on basal activity and on responsiveness to beta adrenergic stimulation.

    PubMed

    Kudlacz, E M; Navarro, H A; Slotkin, T A

    1989-07-01

    Phosphatidic acid phosphatase (PAPase) is a key enzyme in the synthesis of lung surfactant. This study compares the effects of prenatal exposure (gestational days 17, 18 and 19) to two drugs which enhance surfactant synthesis: dexamethasone (0.2 mg/kg) and terbutaline (2 or 10 mg/kg). Maternal dexamethasone treatment did not cause an initial stimulation of lung PAPase but did eventually evoke a small increase after the 1st postnatal week. The effect was selective in that brain PAPase activity was generally unaffected; liver PAPase was stimulated during the early neonatal period only. Dexamethasone also prolonged the developmental period of peak reactivity of lung PAPase to beta developmental period of peak reactivity of lung PAPase to beta adrenergic stimulation (tested with acute isoproterenol challenge), which ordinarily accompanies genesis of alveoli in the 2nd to 3rd postnatal week. Significant growth retardation was present even at this low dose of dexamethasone. In contrast, maternal administration of the beta adrenergic agonist, terbutaline, resulted in a large increase in basal enzyme activity in the lung during the immediate perinatal period and enhanced the responsiveness to isoproterenol challenge. The effect of terbutaline was accompanied by little or no growth impairment. Thus, although prenatal administration of either glucocorticoids or beta adrenergic agonists can enhance lung PAPase activity and reactivity to stimulation, the two classes of drugs differ substantially in time course of effect and in the propensity to retard growth.

  5. [The importance of studying the acid phosphatase of the blood serum and bone marrow lymphoblasts and polymorphonuclear neutrophils in the prognosis of the course of acute lymphoblastic leukemia].

    PubMed

    Vaiuta, N P; Khaĭfets, L M; Mendeleev, I M

    1988-01-01

    The activity of serum acid phosphatase (AP), bone marrow lymphoblasts and polymorphonuclear neutrophils was studied in 45 ALL patients. Cytochemical coefficients (CCC) and the percentage of positively reacting bone marrow cells were determined. All the patients received programmed polychemotherapy. They were investigated before the start of therapy, during recurrence and at different time of remission (from 1 to 60 mos) during each reinduction cycle. At the climax of ALL the activity of serum AP was increased 2.8-fold, a CCC value for lymphoblastic AP--10-fold, for polymorphonuclear neutrophils--3-fold as compared with normal values. A tendency toward the reduction of indices was noted at different time of remission, the approximation to normal values was noted on the 40th-46th months of remission only. In recurrence development the level of the serum and cellular enzyme as well as the percentage of positively reacting cells significantly exceeded normal values and were close to indices at the climax of disease. The above tendency permitted the use of these tests to evaluate the completeness of remission and to predict recurrences during a follow-up of ALL patients.

  6. Phosphatase-Stable Phosphoamino Acid Mimetics That Enhance Binding Affinities with the Polo-Box Domain of Polo-like Kinase 1.

    PubMed

    Hymel, David; Burke, Terrence R

    2017-02-03

    (2S,3R)-2-Amino-3-methyl-4-phosphonobutanoic acid (Pmab) is a phosphatase-stable analogue of phosphothreonine (pThr), which has been used in a variety of biological contexts. Among these applications are peptidomimetic ligands that bind to the polo-box domain (PBD) of polo-like kinase 1 (Plk1) with affinities approaching that of the corresponding pThr-containing peptides. However, Pmab is not widely used, because there are no direct, high-yield preparations of suitably protected reagent. We have now achieved an efficient synthesis of protected Pmab, as well as variants with different substituents at the 3R center. When incorporated into our peptidomimetic scaffold, these new Pmab analogues exhibit Plk1 PBD-binding affinities that are several-fold higher than Pmab, yet retain good selectivity for Plk1 relative to the PBDs of Plk2 and Plk3. These findings will significantly impact the future development of PBD-binding inhibitors, as well as ligands directed against a broad spectrum of pThr-dependent processes.

  7. A comparative study on phosphotransferase activity of acid phosphatases from Raoultella planticola and Enterobacter aerogenes on nucleosides, sugars, and related compounds.

    PubMed

    Médici, Rosario; Garaycoechea, Juan I; Valino, Ana L; Pereira, Claudio A; Lewkowicz, Elizabeth S; Iribarren, Adolfo M

    2014-04-01

    Natural and modified nucleoside-5'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.

  8. Heterologous Protein Secretion Directed by a Repressible Acid Phosphatase System of Kluyveromyces lactis: Characterization of Upstream Region-Activating Sequences in the KIPHO5 Gene

    PubMed Central

    Fermiñán, Encarnación; Domínguez, Angel

    1998-01-01

    Transcription of the repressible acid phosphatase gene (KIPHO5) in Kluyveromyces lactis is strongly regulated in response to the level of inorganic phosphate (Pi) present in the growth medium. We have begun a study of the promoter region of this gene in order to identify sequences involved in the phosphate control of KIPHO5 expression and to design new expression-secretion systems in K. lactis. Deletion analysis and directed mutagenesis revealed two major identical upstream activating sequences (UAS) CACGTG at positions −430 (UAS1) and −192 (UAS2) relative to the ATG initiation codon. These sequences are identical to those described for Saccharomyces cerevisiae for the binding of Pho4p. Deletion or directed mutagenesis of either one or both UAS reduce KIPHO5 expression by the same amount (approximately 80%). When fused to the coding region of trout growth hormone cDNA (tGH-II), the promoter and signal peptide-encoding region of the phosphate-repressible KIPHO5 gene drives the expression of this gene and the secretion of the tGHII protein. Synthesis of tGHIIp in K. lactis transformants carrying this construct was found to be regulated by the Pi present in the medium; derepression of heterologous protein expression can therefore be achieved by lowering the Pi concentration. PMID:9647807

  9. Terpenes and sterols from the fruits of Prunus mume and their inhibitory effects on osteoclast differentiation by suppressing tartrate-resistant acid phosphatase activity.

    PubMed

    Yan, Xi-Tao; Lee, Sang-Hyun; Li, Wei; Jang, Hae-Dong; Kim, Young-Ho

    2015-02-01

    The fruits of Prunus mume are a common commercial product and a valuable source of food and medicinal material in Eastern Asian countries. Our phytochemical investigation of the P. mume fruit led to the isolation of nine terpenes, including three ursane-type triterpenes (1-3), two cycloartane-type triterpenes (4 and 5), and four tocopherols (10-13), as well as four sterols (6-9). Their structures were elucidated based on extensive spectroscopic analysis, including 1D and 2D NMR and ESI-MS, and the majority of these compounds were isolated from this plant for the first time. The anti-osteoporosis activities of 1-13 were evaluated by measuring their inhibitory effects on tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor κB ligand-induced osteoclastic RAW 264.7 macrophage cells. Compounds 2-7 and 9-12 significantly suppressed TRAP activity down to 47.96 ± 2.45-86.45 ± 3.07 % relative to the control at a concentration of 1 μM. These results suggest that the fruits of P. mume could be an excellent source of anti-osteoporosis phytochemicals that may be developed as natural nutraceuticals and functional foods.

  10. Expression of a Human Prostatic Acid Phosphatase (PAP)-IgM Fc Fusion Protein in Plants Using In vitro Tissue Subculture.

    PubMed

    Kang, Yang J; Kim, Deuk-Su; Myung, Soon-Chul; Ko, Kisung

    2017-01-01

    In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2, and SG3 leaf section (top, middle, and base). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins.

  11. Expression of a Human Prostatic Acid Phosphatase (PAP)-IgM Fc Fusion Protein in Plants Using In vitro Tissue Subculture

    PubMed Central

    Kang, Yang J.; Kim, Deuk-Su; Myung, Soon-Chul; Ko, Kisung

    2017-01-01

    In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2, and SG3 leaf section (top, middle, and base). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins. PMID:28293250

  12. Acute effects of microcystins MC-LR and MC-RR on acid and alkaline phosphatase activities and pathological changes in intraperitoneally exposed tilapia fish (Oreochromis sp.).

    PubMed

    Atencio, Loyda; Moreno, Isabel; Prieto, Ana I; Moyano, Rosario; Molina, Ana M; Cameán, Ana M

    2008-04-01

    Microcystins (MC) are frequently present in cyanobacterial blooms in rivers and lakes, increasing the risk of toxicity to both animals and humans. There more than eighty reported microcystins, and the present study was undertaken to determine whether MC-LR and MC-RR can induce different enzyme alterations and histopathological changes in tilapia fish (Oreochromis sp.) exposed to a single intraperitoneal (i.p.) injection of the pure standards (MC-LR and MC-RR) at a dose of 500 mug/kg; the tilapia fish were then observed for seven days. The two MC variants caused significant changes in the activities of acid and alkaline phosphatases (ACP and ALP) in vital organs, showing a different response pattern. The livers and kidneys of fish injected with MC-LR were particularly affected. MC-RR induced a very pronounced increase of ACP in the kidney and a significant increase of ALP in the liver. Both MC variants caused pathological lesions in hepatic tissues, such as megalocytosis, necrotic process, and microvesicular steatosis, particularly in fish treated with MC-LR, and degenerative renal changes, glomerulopathy, were more severe in tilapias exposed to MC-RR. In addition, both microcystins also caused significant myopathy in the heart. In contrast, the gills did not show any change in enzyme activity or histopathological injury.

  13. The spatial distribution of acid phosphatase activity in ectomycorrhizal tissues depends on soil fertility and morphotype, and relates to host plant phosphorus uptake.

    PubMed

    Alvarez, Maricel; Huygens, Dries; Díaz, Leila Milena; Villanueva, Claudia Añazco; Heyser, Wolfgang; Boeckx, Pascal

    2012-01-01

    Acid phosphatase (ACP) enzymes are involved in the mobilization of soil phosphorus (P) and polyphosphate accumulated in the fungal tissues of ectomycorrhizal roots, thereby influencing the amounts of P that are stored in the fungus and transferred to the host plant. This study evaluated the effects of ectomycorrhizal morphotype and soil fertility on ACP activity in the extraradical mycelium (ACP(myc)), the mantle (ACP(mantle)) and the Hartig net region (ACP(Hartig)) of ectomycorrhizal Nothofagus obliqua seedlings. ACP activity was quantified in vivo using enzyme-labelled fluorescence-97 (ELF-97) substrate, confocal laser microscopy and digital image processing routines. There was a significant effect of ectomycorrhizal morphotype on ACP(myc), ACP(mantle) and ACP(Hartig), while soil fertility had a significant effect on ACP(myc) and ACP(Hartig). The relative contribution of the mantle and the Hartig net region to the ACP activity on the ectomycorrhizal root was significantly affected by ectomycorrhizal morphotype and soil fertility. A positive correlation between ACP(Hartig) and the shoot P concentration was found, providing evidence that ACP activity at the fungus:root interface is involved in P transfer from the fungus to the host. It is concluded that the spatial distribution of ACP in ectomycorrhizas varies as a function of soil fertility and colonizing fungus.

  14. Novel prostate acid phosphatase-based peptide vaccination strategy induces antigen-specific T-cell responses and limits tumour growth in mice.

    PubMed

    Saif, Jaimy M S; Vadakekolathu, Jayakumar; Rane, Shraddha S; McDonald, Danielle; Ahmad, Murrium; Mathieu, Morgan; Pockley, A Graham; Durrant, Lindy; Metheringham, Rachael; Rees, Robert C; McArdle, Stephanie E B

    2014-04-01

    Treatment options for patients with advanced prostate cancer remain limited and rarely curative. Prostatic acid phosphatase (PAP) is a prostate-specific protein overexpressed in 95% of prostate tumours. An FDA-approved vaccine for the treatment of advanced prostate disease, PROVENGE® (sipuleucel-T), has been shown to prolong survival, however the precise sequence of the PAP protein responsible for the outcome is unknown. As the PAP antigen is one of the very few prostate-specific antigens for which there is a rodent equivalent with high homology, preclinical studies using PAP have the potential to be directly relevant to clinical setting. Here, we show three PAP epitopes naturally processed and presented in the context of HHDII/DR1 (114-128, 299-313, and 230-244). The PAP-114-128 epitope elicits CD4(+) and CD8(+) T-cell-specific responses in C57BL/6 mice. Furthermore, when immunised in a DNA vector format (ImmunoBody®), PAP-114-128 prevents and reduces the growth of transgenic adenocarcinoma of mouse prostate-C1 prostate cancer cell-derived tumours in both prophylactic and therapeutic settings. This anti-tumour effect is associated with infiltration of CD8(+) tumour-infiltrating lymphocytes and the generation of high avidity T cells secreting elevated levels of IFN-γ. PAP-114-128 therefore appears to be a highly relevant peptide on which to base vaccines for the treatment of prostate cancer.

  15. AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

    SciTech Connect

    Liu, Xin; Zhu, Yanming; Zhai, Hong; Cai, Hua; Ji, Wei; Luo, Xiao; Li, Jing; Bai, Xi

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

  16. Comparison of alkaline phosphatase activity of MC3T3-E1 cells cultured on different Ti surfaces: modified sandblasted with large grit and acid-etched (MSLA), laser-treated, and laser and acid-treated Ti surfaces

    PubMed Central

    Li, Lin-Jie; Kim, So-Nam

    2016-01-01

    PURPOSE In this study, the aim of this study was to evaluate the effect of implant surface treatment on cell differentiation of osteoblast cells. For this purpose, three surfaces were compared: (1) a modified SLA (MSLA: sand-blasted with large grit, acid-etched, and immersed in 0.9% NaCl), (2) a laser treatment (LT: laser treatment) titanium surface and (3) a laser and acid-treated (LAT: laser treatment, acid-etched) titanium surface. MATERIALS AND METHODS The MSLA surfaces were considered as the control group, and LT and LAT surfaces as test groups. Alkaline phosphatase expression (ALP) was used to quantify osteoblastic differentiation of MC3T3-E1 cell. Surface roughness was evaluated by a contact profilometer (URFPAK-SV; Mitutoyo, Kawasaki, Japan) and characterized by two parameters: mean roughness (Ra) and maximum peak-to-valley height (Rt). RESULTS Scanning electron microscope revealed that MSLA (control group) surface was not as rough as LT, LAT surface (test groups). Alkaline phosphatase expression, the measure of osteoblastic differentiation, and total ALP expression by surface-adherent cells were found to be highest at 21 days for all three surfaces tested (P<.05). Furthermore, ALP expression levels of MSLA and LAT surfaces were significantly higher than expression levels of LT surface-adherent cells at 7, 14, and 21 days, respectively (P<.05). However, ALP expression levels between MSLA and LAT surface were equal at 7, 14, and 21 days (P>.05). CONCLUSION This study suggested that MSLA and LAT surfaces exhibited more favorable environment for osteoblast differentiation when compared with LT surface, the results that are important for implant surface modification studies. PMID:27350860

  17. Overexpression of a Protein Phosphatase 2C from Beech Seeds in Arabidopsis Shows Phenotypes Related to Abscisic Acid Responses and Gibberellin Biosynthesis1

    PubMed Central

    Reyes, David; Rodríguez, Dolores; González-García, Mary Paz; Lorenzo, Oscar; Nicolás, Gregorio; García-Martínez, José Luis; Nicolás, Carlos

    2006-01-01

    A functional abscisic acid (ABA)-induced protein phosphatase type 2C (PP2C) was previously isolated from beech (Fagus sylvatica) seeds (FsPP2C2). Because transgenic work is not possible in beech, in this study we overexpressed this gene in Arabidopsis (Arabidopsis thaliana) to provide genetic evidence on FsPP2C2 function in seed dormancy and other plant responses. In contrast with other PP2Cs described so far, constitutive expression of FsPP2C2 in Arabidopsis, under the cauliflower mosaic virus 35S promoter, produced enhanced sensitivity to ABA and abiotic stress in seeds and vegetative tissues, dwarf phenotype, and delayed flowering, and all these effects were reversed by gibberellic acid application. The levels of active gibberellins (GAs) were reduced in 35S:FsPP2C2 plants, although transcript levels of AtGA20ox1 and AtGA3ox1 increased, probably as a result of negative feedback regulation, whereas the expression of GASA1 was induced by GAs. Additionally, FsPP2C2-overexpressing plants showed a strong induction of the Responsive to ABA 18 (RAB18) gene. Interestingly, FsPP2C2 contains two nuclear targeting sequences, and transient expression assays revealed that ABA directed this protein to the nucleus. Whereas other plant PP2Cs have been shown to act as negative regulators, our results support the hypothesis that FsPP2C2 is a positive regulator of ABA. Moreover, our results indicate the existence of potential cross-talk between ABA signaling and GA biosynthesis. PMID:16815952

  18. Methods and compositions for chromosome-specific staining

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    2003-07-22

    Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

  19. Negative staining of thinly spread biological samples.

    PubMed

    Harris, J Robin

    2007-01-01

    Negative staining is widely applicable to isolated viruses, protein molecules, macro-molecular assemblies and fibrils, subcellular membrane fractions, liposomes and artificial membranes, synthetic DNA arrays, and also to polymer solutions. In this chapter, techniques are provided for the preparation of the necessary support films (continuous carbon and holey/perforated carbon). The range of suitable negative stains is presented, with some emphasis on the benefit of using ammonium molybdate and of negative stain-trehalose combinations. Protocols are provided for the single-droplet negative staining technique (on continuous and holey carbon support films), the negative staining-carbon film technique, for randomly dispersed fragile molecules, 2D crystallization of proteins, and for cleavage of cells and organelles. The newly developed cryonegative staining procedure also is included. Immunonegative staining and negative staining of affinity labeled complexes (e.g., biotin-streptavidin) are discussed in some detail. The formation of immune complexes in solution for droplet negative staining is presented, as is the use of carbon-plastic support films as an adsorption surface on which to perform immunolabeling or affinity experiments, before negative staining. Dynamic biological systems can be investigated by negative staining, where the time period is in excess of a few minutes, but there are possibilities to greatly reduce the time by rapid stabilization of molecular systems with uranyl acetate or tannic acid.

  20. Detection of bacterial phosphatase activity by means of an original and simple test.

    PubMed Central

    Satta, G; Grazi, G; Varaldo, P E; Fontana, R

    1979-01-01

    A new test for the detection of bacterial phosphatase activity has been devised. The test is performed using agar media containing both methyl green (MG) and phenolphthalein diphosphate (PDP); in these media phosphatase-producing strains grow deep-green-stained colonies whereas non-producing strains do not. A total of 739 different strains were tested, including 593 staphylococci, 95 micrococci, 11 streptococci, 10 corynebacteria, 14 enterobacteria, and 16 candidae. All strains found phosphatase-positive according to the conventional phosphatase test displayed deep-green-stained colonies on MG-PDP media, whereas all phosphatase-negative strains showed unstained colonies on the same media. The main advantages of the present phosphatase test as compared with other conventional ones are that it is more simple to perform, it can reveal the phosphatase activity of colonies grown in deep agar, and can be incorporated into commercial multitest kits. PMID:87403

  1. Bodian's Silver Method Stains Neurofilament Polypeptides

    NASA Astrophysics Data System (ADS)

    Gambetti, P.; Autilio-Gambetti, L.; Papasozomenos, S. Ch.

    1981-09-01

    Bodian's silver method was used to stain polypeptides of rat spinal cord or peripheral nerve separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands corresponding to the three polypeptide subunits of the neurofilaments were intensely impregnated. Two other polypeptides were stained inconsistently and less intensely. The tubulin band was stained weakly or not at all; other polypeptides, including glial fibrillary acidic protein, actin, and vimentin, remained unstained. This novel application of Bodian's method provides indirect proof that neurofilaments are the neuronal subcellular structure stained by the technique.

  2. Dramatic Stained Glass.

    ERIC Educational Resources Information Center

    Prater, Michael

    2002-01-01

    Describes an art project that is appropriate for students in fifth through twelfth grade in which they create Gothic-style stained-glass windows. Discusses how college students majoring in elementary education created stained-glass windows. Addresses how to adapt this lesson for younger students. (CMK)

  3. Effect of Bacteria and Amoebae on Rhizosphere Phosphatase Activity

    PubMed Central

    Gould, W. Douglas; Coleman, David C.; Rubink, Amy J.

    1979-01-01

    The contributions of various components of soil microflora and microfauna to rhizosphere phosphatase activity were determined with hydroponic cultures. Three treatments were employed: (i) plants alone (Bouteloua gracilis (H.B.K.) Lag. ex Steud.) (ii) plants plus bacteria (Pseudomonas sp.), and (iii) plants plus bacteria plus amoebae (Acanthamoeba sp.). No alkaline phosphatase was detected, but an appreciable amount of acid phosphatase activity (120 to 500 nmol of p-nitrophenylphosphate hydrolyzed per h per plant) was found in the root culture solutions. The presence of bacteria or bacteria and amoebae increased the amount of acid phosphatase in solution, and properties of additional activity were identical to properties of plant acid phosphatase. The presence of bacteria or bacteria and amoebae increased both solution and root phosphatase activities at most initial phosphate concentrations. PMID:16345390

  4. [Phosphatase activity in Amoeba proteus at pH 9.0].

    PubMed

    Sopina, V A

    2007-01-01

    In the free-living amoeba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as a substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called "fast", "intermediate", and "slow" phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be both acid phosphatase and protein tyrosine phosphatase (PTP). Based on data of inhibitor analysis, large substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, other than in the fast and intermediate phosphatases localization in the amoeba cell, it is concluded that only slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).

  5. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

    PubMed Central

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

    2015-01-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  6. [The diagnostic value of the radioimmunological estimation of prostatic acid phosphatase. Comparative value of the measurement of enzyme activity (author's transl)].

    PubMed

    Tellier, J L; Chatal, J F; Bourdin, S; Auvigne, J; Etienne, P; Faye, R

    1981-01-01

    Radioimmunological estimation of prostatic acid phosphatase was carried out in 72 reference subjects, 46 patients with benign prostatic hypertrophy, 106 patients with untreated prostatic carcinoma and 25 patients with a carcinoma of some other origin. The mean concentration in non-acidified serum was 1.3 +/- 0.4 (M +/- SD) ng/ml for the reference group and 1.6 +/- 0.8 ng/ml for the benign hypertrophy group. The upper limit of discriminatory values for the diagnosis of prostatic carcinoma was fixed at 3 ng/ml. Taking this value, the overall percentage of positive results for carcinoma of the prostate was 61% (65/106). The number of cases with a value greater than 3 ng/ml was 3/18 (17%) for stage A, 8/27 (30%) for stage B, 7/13 (54%) for stage C and 47/48 (98%) for stage D. 8% (2/25) of carcinomas of another origin gave a positive result. The results of estimation using the radioimmunological technique were compared with those obtained by the measurement of enzyme activity using para nitro-phenyl phosphate as a substrate in 34 untreated prostatic carcinomas (all stages mixed together). When measurements by both techniques were carried out under the same ideal conditions using fresh sera as soon as possible after the blood was drawn, the result was abnormal in 10 cases out of 12 (83%) for the radioimmunological method and in 8 cases out of 12 (67%) for the measurement of enzyme activity. By contrast, under routine conditions, the positive percentage figures were 77% (17/22) for the radioimmunological technique and only 36% (8/22) for the measurement of enzyme activity. It would thus appear that radioimmunological measurement is more reliable than the measurement of enzyme activity.

  7. Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action.

    PubMed

    Brandt, Benjamin; Brodsky, Dennis E; Xue, Shaowu; Negi, Juntaro; Iba, Koh; Kangasjärvi, Jaakko; Ghassemian, Majid; Stephan, Aaron B; Hu, Honghong; Schroeder, Julian I

    2012-06-26

    The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca(2+)-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.

  8. High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene

    PubMed Central

    Brinch-Pedersen, Henrik

    2013-01-01

    The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye. This is notably more than other cereals. For instance, rice, maize, and oat have MGPAs below 100 FTU/kg. The cloning and characterization of the PAPhy gene complement from wheat, barley, rye, einkorn, and Aegilops tauschii is reported here. The Triticeae PAPhy genes generally consist of a set of paralogues, PAPhy_a and PAPhy_b, and have been mapped to Triticeae chromosomes 5 and 3, respectively. The promoters share a conserved core but the PAPhy_a promoter have acquired a novel cis-acting regulatory element for expression during grain filling while the PAPhy_b promoter has maintained the archaic function and drives expression during germination. Brachypodium is the only sequenced Poaceae sharing the PAPhy duplication. As for the Triticeae, the duplication is reflected in a high MGPA of ~4200 FTU/kg in Brachypodium. The sequence conservation of the paralogous loci on Brachypodium chromosomes 1 and 2 does not extend beyond the PAPhy gene. The results indicate that a single-gene segmental duplication may have enabled the evolution of high MGPA by creating functional redundancy of the parent PAPhy gene. This implies that similar MGPA levels may be out of reach in breeding programs for some Poaceae, e.g. maize and rice, whereas Triticeae breeders should focus on PAPhy_a. PMID:23918958

  9. Activation of lysophosphatidic acid receptor by gintonin inhibits Kv1.2 channel activity: involvement of tyrosine kinase and receptor protein tyrosine phosphatase α.

    PubMed

    Lee, Jun-Ho; Choi, Sun-Hye; Lee, Byung-Hwan; Hwang, Sung-Hee; Kim, Hyeon-Joong; Rhee, Jeehae; Chung, Chihye; Nah, Seung-Yeol

    2013-08-26

    Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. The primary action of gintonin is to elicit a transient increase in [Ca(2+)]i via activation of LPA receptor subtypes. Voltage-gated potassium (Kv) channels play important roles in synaptic transmission in nervous systems. The previous reports have shown that Kv channels can be regulated by Gαq/11 protein-coupled receptor ligands. In the present study, we examined the effects of gintonin on Kv1.2 channel activity expressed in Xenopus oocytes after injection of RNA encoding the human Kv1.2 α subunit. Gintonin treatment inhibited Kv1.2 channel activity in reversible and concentration-dependent manners. The inhibitory effect of gintonin on Kv1.2 channel activity was blocked by active phospholipase C inhibitor, inositol 1,4,5-triphosphate receptor antagonist, and intracellular Ca(2+) chelator. The co-expression of active receptor protein tyrosine phosphatase α (RPTPα) with Kv1.2 channel greatly attenuated gintonin-mediated inhibition of Kv1.2 channel activity, but attenuation was not observed with catalytically inactive RPTPα. Furthermore, neither genistein, a tyrosine kinase inhibitor, nor site-directed mutation of a tyrosine residue (Y132 to Y132F), which is phosphorylated by tyrosine kinase of the N-terminal of the Kv1.2 channel α subunit, significantly attenuated gintonin-mediated inhibition of Kv1.2 channel activity. These results indicate that the gintonin-mediated Kv1.2 channel regulation involves the dual coordination of both tyrosine kinase and RPTPα coupled to this receptor. Finally, gintonin-mediated regulation of Kv1.2 channel activity might explain one of the modulations of gintonin-mediated neuronal activities in nervous systems.

  10. Evidence for nonbridged coordination of p-nitrophenyl phosphate to the dinuclear Fe(III)-M(II) center in bovine spleen purple acid phosphatase during enzymatic turnover.

    PubMed

    Merkx, M; Pinkse, M W; Averill, B A

    1999-08-03

    The pH dependence of the catalytic parameters k(cat) and K(M) has been determined for the Fe(III)Fe(II)- and Fe(III)Zn(II)-forms of bovine spleen purple acid phosphatase (BSPAP). The parameter k(cat) was found to be maximal at pH 6.3, and a pK(a) of 5.4-5.5 was obtained for the acidic limb of the k(cat) vs pH profile. Two different EPR spectra were detected for the phosphate complex of the mixed-valent diiron enzyme; their relative amounts depended on the pH, with an apparent pK(a) of 6. The EPR spectra of Fe(III)Fe(II)-BSPAP.PO(4) and Fe(III)Zn(II)-BSPAP.PO(4) at pH 5.0 are similar to those previously reported for Fe(III)Fe(II)-Uf.PO(4) and Fe(III)Zn(II)-Uf.PO(4) complexes at pH 5.0. At higher pH, a new Fe(III)Fe(II)-BSPAP.PO(4) species is formed, with apparent g-values of 1.94, 1.71, and 1.50. The EPR spectrum of Fe(III)Zn(II)-BSPAP does not show significant changes upon addition of phosphate up to 30 mM at pH 6.5, suggesting that phosphate binds only to the spectroscopically silent Zn(II). To determine whether the phosphate complexes were good structural models for the enzyme substrate complexes, these complexes were studied using rapid-freeze EPR and stopped-flow optical spectroscopy. The stopped-flow studies showed the absence of burst kinetics at pH 7.0, which indicates that substrate hydrolysis is rate limiting, rather than phosphate release. The EPR spectrum of Fe(III)Fe(II)-BSPAP.p-NPP is similar, but not identical, to that of the corresponding phosphate complex, both at pH 5 and pH 6.5. We propose that both phosphate and p-NPP bridge the two metal ions at low pH. At higher pH where the enzyme is optimally active, we propose that hydroxide competes with phosphate and p-NPP for coordination to Fe(III) and that both phosphate and p-NPP coordinate only to the divalent metal ion.

  11. Morphology, cytochemical staining, and ultrastructural characteristics of the blood cells of the giant lizard of El Hierro (Gallotia simonyi).

    PubMed

    Martínez-Silvestre, A; Marco, I; Rodriguez-Dominguez, M A; Lavín, S; Cuenca, R

    2005-04-01

    The object of this study was to examine the erythrocytes, leukocytes and thrombocytes of the giant lizard of El Hierro (Gallotia simonyi) by light and electron (TEM) microscopy, and cytochemical staining. Smears were prepared from blood from the ventral coccygeal vein of 10 healthy adult lizards (five males and five females) from the Giant Lizard of El Hierro Reproduction and Research Centre, Canary Islands, Spain. The cytochemical stains used were: benzidine peroxidase (BP), chloroacetate esterase (CAE), alpha-naphthyl acetate esterase (ANAE), acid phosphatase (AP), periodic acid-Schiff (PAS), toluidine blue (TB) and May-Grünwald-Giemsa (MGG). Electron microscopy was also performed on all samples. Heterophils had granules that were heterogeneous in both size and electron density, and stained with BP, PAS and ANAE. Eosinophil granules were homogeneously electron-dense and stained for AP, CAE and ANAE. Basophils had both highly and moderately electron-dense granules, and stained with TB and ANAE. Azurophil granules were of low electron-density and stained for AP, CAE and ANAE. Azurophil cytoplasm was vacuolated on TEM. The cytoplasm of lymphocytes contained many ribosomes and was positive for AP. Monocytes had a large nucleus and a vacuolated cytoplasm but did not stain by any of the cytochemical methods used. Thrombocytes had a relatively large nucleus but little cytoplasm; they did not stain cytochemically. The blood cells of the giant lizards of El Hierro differ from those of other members of the Order Squamata both morphologically and cytochemically. The variation in cytochemical responses in the blood of reptiles makes it necessary to study species individually if meaningful clinical decisions are to be made.

  12. Candida, fluorescent stain (image)

    MedlinePlus

    This microscopic film shows a fluorescent stain of Candida. Candida is a yeast (fungus) that causes mild disease, but in immunocompromised individuals it may cause life-threatening illness. (Image ...

  13. Joint fluid Gram stain

    MedlinePlus

    A normal result means no bacteria are present on the Gram stain. Normal value ranges may vary slightly among different laboratories. Some labs use different measurements or test different samples. Talk to your doctor about the meaning ...

  14. Port-Wine Stains

    MedlinePlus

    ... in healing and help prevent infection. Helping Kids Cope As with any birthmark, port-wine stains (especially ... these situations and take cues about how to cope with others' reactions. Practice responses so your child ...

  15. Sputum gram stain

    MedlinePlus

    ... cough very deeply. The Gram stain method is one of the most commonly used methods to rapidly detect a bacterial infection, including pneumonia. How the Test is Performed A sputum sample is needed. You will be asked to cough ...

  16. Pericardial fluid Gram stain

    MedlinePlus

    ... a bacterial infection. The Gram stain method is one of the most commonly used techniques for the rapid diagnosis of bacterial infections. How the Test is Performed A sample of fluid will be taken from the sac ...

  17. Port-Wine Stains

    MedlinePlus

    ... their own, they can be treated. In fact, laser therapies can make many port-wine stains much ... mark might be. The good news is that lasers (highly concentrated light energy) can make many port- ...

  18. Pleural fluid gram stain

    MedlinePlus

    ... a Gram stain). A laboratory specialist uses a microscope to look for bacteria on the slide. If bacteria are present, the color, number, and structure of the cells are used to identify the type of bacteria. This test will be ...

  19. Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B.

    PubMed

    Fan, Junping; Jiang, Daohua; Zhao, Yan; Liu, Jianfeng; Zhang, Xuejun Cai

    2014-05-27

    Membrane-integrated type II phosphatidic acid phosphatases (PAP2s) are important for numerous bacterial to human biological processes, including glucose transport, lipid metabolism, and signaling. Escherichia coli phosphatidylglycerol-phosphate phosphatase B (ecPgpB) catalyzes removing the terminal phosphate group from a lipid carrier, undecaprenyl pyrophosphate, and is essential for transport of many hydrophilic small molecules across the membrane. We determined the crystal structure of ecPgpB at a resolution of 3.2 Å. This structure shares a similar folding topology and a nearly identical active site with soluble PAP2 enzymes. However, the substrate binding mechanism appears to be fundamentally different from that in soluble PAP2 enzymes. In ecPgpB, the potential substrate entrance to the active site is located in a cleft formed by a V-shaped transmembrane helix pair, allowing lateral movement of the lipid substrate entering the active site from the membrane lipid bilayer. Activity assays of point mutations confirmed the importance of the catalytic residues and potential residues involved in phosphate binding. The structure also suggests an induced-fit mechanism for the substrate binding. The 3D structure of ecPgpB serves as a prototype to study eukaryotic PAP2 enzymes, including human glucose-6-phosphatase, a key enzyme in the homeostatic regulation of blood glucose concentrations.

  20. Assessment and kinetics of soil phosphatase in Brazilian Savanna systems.

    PubMed

    Ferreira, Adão S; Espíndola, Suéllen P; Campos, Maria Rita C

    2016-05-31

    The activity and kinetics of soil phosphatases are important indicators to evaluate soil quality in specific sites such as the Cerrado (Brazilian Savanna). This study aimed to determine the activity and kinetic parameters of soil phosphatase in Cerrado systems. Soil phosphatase activity was assessed in samples of native Cerrado (NC), no-tillage (NT), conventional tillage (CT) and pasture with Brachiaria brizantha (PBb) and evaluated with acetate buffer (AB), tris-HCl buffer (TB), modified universal buffer (MUB) and low MUB. The Michaelis-Menten equation and Eadie-Hofstee model were applied to obtain the kinetic parameters of soil phosphatase using different concentrations of p-nitrophenol phosphate (p-NPP). MUB showed the lowest soil phosphatase activity in all soils whereas AB in NC and NT presented the highest. Low MUB decreased interferences in the assessment of soil phosphatase activity when compared to MUB, suggesting that organic acids interfere on the soil phosphatase activity. In NC and NT, soil phosphatase activity performed with TB was similar to AB and low MUB. Km values from the Michaels-Menten equation were higher in NC than in NT, which indicate a lower affinity of phosphatase activity for the substrate in NC. Vmax values were also higher in NC than in NT. The Eadie-Hofstee model suggests that NC had more phosphatase isoforms than NT. The study showed that buffer type is of fundamental importance when assessing soil phosphatase activity in Cerrado soils.

  1. Gallium nitrate inhibits alkaline phosphatase activity in a differentiating mesenchymal cell culture.

    PubMed

    Boskey, A L; Ziecheck, W; Guidon, P; Doty, S B

    1993-02-01

    The effect of gallium nitrate on alkaline phosphatase activity in a differentiating chick limb-bud mesenchymal cell culture was monitored in order to gain insight into the observation that rachitic rats treated with gallium nitrate failed to show the expected increase in serum alkaline phosphatase activity. Cultures maintained in media containing 15 microM gallium nitrate showed drastically decreased alkaline phosphatase activities in the absence of significant alterations in total protein synthesis and DNA content. However, addition of 15 microM gallium nitrate to cultures 18 h before assay for alkaline phosphatase activity had little effect. At the light microscopic and electron microscopic level, gallium-treated cultures differed morphologically from gallium-free cultures: with gallium present, there were fewer hypertrophic chondrocytes and cartilage nodules were flatter and further apart. Because of altered morphology, staining with an antibody against chick cartilage alkaline phosphatase appeared less extensive; however, all nodules stained equivalently relative to gallium-free controls. Histochemical staining for alkaline phosphatase activity was negative in gallium-treated cultures, demonstrating that the alkaline phosphatase protein present was not active. The defective alkaline phosphatase activity in cultures maintained in the presence of gallium was also evidenced when cultures were supplemented with the alkaline phosphatase substrate, beta-glycerophosphate (beta GP). The data presented suggest that gallium inhibits alkaline phosphatase activity in this culture system and that gallium causes alterations in the differentiation of mesenchymal cells into hypertrophic chondrocytes.

  2. Apparatus Would Stain Microscope Slides

    NASA Technical Reports Server (NTRS)

    Breeding, James D.

    1993-01-01

    Proposed apparatus meters specific amounts of fluid out of containers at specific times to stain microscope slides. Intended specifically for semiautomated staining of microbiological and hematological samples in microgravity, leakproof apparatus used in other environments in which technicians have little time to allocate to staining procedures and/or exposure to toxic staining agents or to micro-organisms to be stained hazardous. Apparatus adapted to perform almost any staining procedure and accommodates multiple staining reagents, useful for small or remote clinical laboratories.

  3. Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases.

    PubMed

    Romero-Montoya, Lydia; Martínez-Rodríguez, Hugo; Pérez, Miguel Antonio; Argüello-García, Raúl

    2011-03-20

    In the forensic laboratory the biological analyses for rape investigation commonly include vaginal swabs as sample material combined to biochemical tests including sperm cytology (SC) and detection of acid phosphatase activity (AP) and prostate-specific antigen (PSA, p30) for the conclusive identification of semen components. Most reports comparing these tests relied on analysis of semen samples or donor swabs taken under controlled conditions; however their individual or combined efficacy under real live sampling conditions in different laboratories is largely unknown. We carried out SC, APA and PSA analyses in vaginal swabs collected from casework rapes submitted to Mexican Forensic Laboratories at Texcoco and Toluca. On the basis of positive and negative results from each assay and sample, data were classified into eight categories (I-VIII) and compared with those obtained in the two only similar studies reported in Toronto, Canada and Hong Kong, China. SC and APA assays had the higher overall positivity in Toluca and Texcoco samples respectively and otherwise PSA had a lower but very similar positivity between these two laboratories. When compared to the previous studies some similarities were found, namely similar frequencies (at a ratio of approximately 1 out of 3) of samples being positive or negative by all techniques (Categories I and VI respectively) and a comparable overall positivity of APA and SC but higher than that of PSA. Indeed the combined results of using SC, APA and PSA tests was considered as conclusive for semen detection from approximately 1 out of 3 cases (Category I) to approximately 1 out of 2 cases in a scenario where at least SC is positive, strongly presumptive in 2 out of 3 cases (with at least one test positive) and the remainder 1 out of 3 cases (Category VI) suggested absence of semen. By determining Y-STR polymorphisms (12-loci) in additional samples obtained at Toluca laboratory, complete DNA profiles were determined from all

  4. Biogeochemical drivers of phosphatase activity in salt marsh sediments

    NASA Astrophysics Data System (ADS)

    Freitas, Joana; Duarte, Bernardo; Caçador, Isabel

    2014-10-01

    Although nitrogen has become a major concern for wetlands scientists dealing with eutrophication problems, phosphorous represents another key element, and consequently its biogeochemical cycling has a crucial role in eutrophication processes. Microbial communities are a central component in trophic dynamics and biogeochemical processes on coastal systems, since most of the processes in sediments are microbial-mediated due to enzymatic action, including the mineralization of organic phosphorus carried out by acid phosphatase activity. In the present work, the authors investigate the biogeochemical sediment drivers that control phosphatase activities. Authors also aim to assess biogeochemical factors' influence on the enzyme-mediated phosphorous cycling processes in salt marshes. Plant rhizosediments and bare sediments were collected and biogeochemical features, including phosphatase activities, inorganic and organic phosphorus contents, humic acids content and pH, were assessed. Acid phosphatase was found to give the highest contribution for total phosphatase activity among the three pH-isoforms present in salt marsh sediments, favored by acid pH in colonized sediments. Humic acids also appear to have an important role inhibiting phosphatase activity. A clear relation of phosphatase activity and inorganic phosphorous was also found. The data presented reinforces the role of phosphatase in phosphorous cycling.

  5. Structure-Function Analysis of the 3' Phosphatase Component of T4 Polynucleotide Kinase/phosphatase

    SciTech Connect

    Zhu,H.; Smith, P.; Wang, L.; Shuman, S.

    2007-01-01

    T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity.

  6. Structure-function analysis of the 3' phosphatase component of T4 polynucleotide kinase/phosphatase.

    PubMed

    Zhu, Hui; Smith, Paul; Wang, Li Kai; Shuman, Stewart

    2007-09-15

    T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity.

  7. Action at a distance: amino acid substitutions that affect binding of the phosphorylated CheY response regulator and catalysis of dephosphorylation can be far from the CheZ phosphatase active site.

    PubMed

    Freeman, Ashalla M; Mole, Beth M; Silversmith, Ruth E; Bourret, Robert B

    2011-09-01

    Two-component regulatory systems, in which phosphorylation controls the activity of a response regulator protein, provide signal transduction in bacteria. For example, the phosphorylated CheY response regulator (CheYp) controls swimming behavior. In Escherichia coli, the chemotaxis phosphatase CheZ stimulates the dephosphorylation of CheYp. CheYp apparently binds first to the C terminus of CheZ and then binds to the active site where dephosphorylation occurs. The phosphatase activity of the CheZ(2) dimer exhibits a positively cooperative dependence on CheYp concentration, apparently because the binding of the first CheYp to CheZ(2) is inhibited compared to the binding of the second CheYp. Thus, CheZ phosphatase activity is reduced at low CheYp concentrations. The CheZ21IT gain-of-function substitution, located far from either the CheZ active site or C-terminal CheY binding site, enhances CheYp binding and abolishes cooperativity. To further explore mechanisms regulating CheZ activity, we isolated 10 intragenic suppressor mutations of cheZ21IT that restored chemotaxis. The suppressor substitutions were located along the central portion of CheZ and were not allele specific. Five suppressor mutants tested biochemically diminished the binding of CheYp and/or the catalysis of dephosphorylation, even when the suppressor substitutions were distant from the active site. One suppressor mutant also restored cooperativity to CheZ21IT. Consideration of results from this and previous studies suggests that the binding of CheYp to the CheZ active site (not to the C terminus) is rate limiting and leads to cooperative phosphatase activity. Furthermore, amino acid substitutions distant from the active site can affect CheZ catalytic activity and CheYp binding, perhaps via the propagation of structural or dynamic perturbations through a helical bundle.

  8. Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein.

    PubMed Central

    Takai, A; Sasaki, K; Nagai, H; Mieskes, G; Isobe, M; Isono, K; Yasumoto, T

    1995-01-01

    Several groups have reported that okadaic acid (OA) and some other tight-binding protein phosphatase inhibitors including microcystin-LR (MCLR), calyculin-A and tautomycin prevent each other from binding to protein phosphatase 2A (PP2A). In this paper, we have introduced an improved procedure for examining to what extent the affinity of an enzyme for a labelled tight-binding ligand is reduced by binding of an unlabelled tight-binding, ligand to the enzyme. Using this procedure, we have analysed the dose-dependent reduction of PP2A binding of [24-3H]OA by addition of OA, MCLR, calyculin-A and tautomycin. The results indicate that the binding of the unlabelled inhibitors to the PP2A molecule causes a dramatic (10(6)-10(8)-fold) increase in the dissociation constant associated with the interaction of [24-3H]OA and PP2A. This suggests that OA and the other inhibitors bind to PP2A in a mutually exclusive manner. The protein phosphatase inhibitors may share the same binding site on the PP2A molecule. We have also measured values of the dissociation constant (Ki) for the interaction of these toxins with protein phosphatase 1 (PP1). For MCLR and calyculin-A, the ratio of the Ki value obtained for PP1 to that for PP2A was in the range 4-9, whereas it was 0.01-0.02 for tautomycin. The value of tautomycin is considerably smaller than that (0.4) calculated from previously reported Ki values. PMID:7702557

  9. [Protein phosphatases: structure and function].

    PubMed

    Bulanova, E G; Budagian, V M

    1994-01-01

    The process of protein and enzyme systems phosphorylation is necessary for cell growth, differentiation and preparation for division and mitosis. The conformation changes of protein as a result of phosphorylation lead to increased enzyme activity and enhanced affinity to substrates. A large group of enzymes--protein kinases--is responsible for phosphorylation process in cell, which are divided into tyrosine- and serine-threonine-kinases depending on their ability to phosphorylate appropriate amino acid residues. In this review has been considered the functional importance and structure of protein phosphatases--enzymes, which are functional antagonists of protein kinases.

  10. N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites.

    PubMed

    Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong; Yao, Bin

    2015-12-04

    N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.

  11. N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

    PubMed Central

    Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong

    2015-01-01

    N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. PMID:26637601

  12. Functional size of the thylakoid phosphatases determined by radiation inactivation.

    PubMed

    Hsu, L H; Tzeng, C M; Pan, R L

    1993-02-22

    Radiation inactivation technique was employed to determine the functional size of phosphatases from thylakoid membrane. The enzymatic activities of phosphatases decayed in a simple function with the increase of radiation dosage. D37 values of 18.8 +/- 2.4-14.1 +/- 1.5 Mrad were obtained, using phosphoserine, phosphothreonine, p-nitrophenol phosphate, and phospho-histone V-S, respectively, as substrates. The molecular masses of 48.2 +/- 6.3-61 +/- 5.7 kDa were yielded by target theory analysis. We thus speculate that the thylakoid alkaline phosphatase is probably a monomer while acid phosphatase is functionally a dimer in situ.

  13. Port-wine stain

    MedlinePlus

    Early-stage port-wine stains are usually flat and pink. As the child gets older, the color may deepen to a dark red or purplish color. They occur most often on the face, but can appear anywhere on the body. Over time, ...

  14. "Stained Glass" Landscape Windows

    ERIC Educational Resources Information Center

    Vannata, Janine

    2008-01-01

    Both adults and children alike marvel at the grand vivid stained-glass windows created by American artist Louis Comfort Tiffany. Today he is commonly recognized as one of America's most influential designers and artists throughout the last nineteenth and early twentieth century. In the lesson described in this article, students created their own…

  15. Shimmering Stained Glass.

    ERIC Educational Resources Information Center

    Simon, Gail Murray

    1998-01-01

    Presents an art lesson for fifth- and sixth-graders where they create a translucent design of colored cellophane on black paper inspired by the stained-glass windows of the Middle Ages and the artwork of Lewis Comfort Tiffany. Enables the students to become crafts people rather than just observers of the past. (CMK)

  16. Stained-Glass Pastels

    ERIC Educational Resources Information Center

    Laird, Shirley

    2009-01-01

    The author has always liked the look of stained-glass windows. Usually the designs are simplified and the shapes are easier for younger students to draw. This technique seemed to be the perfect place for her fifth-graders to try their hand at color mixing. The smaller spaces and simple shapes were just what she needed for this group. Her students…

  17. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    SciTech Connect

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  18. Pisiferdiol and pisiferic acid isolated from Chamaecyparis pisifera activate protein phosphatase 2C in vitro and induce caspase-3/7-dependent apoptosis via dephosphorylation of Bad in HL60 cells.

    PubMed

    Aburai, N; Yoshida, M; Ohnishi, M; Kimura, K

    2010-08-01

    Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates and regulates apoptosis, stress response and growth-related pathways. In the course of screening for PP2C activators from natural sources, we isolated abietane-type diterpenes, pisiferdiol and pisiferic acid from Chamaecyparis pisifera. Pisiferdiol having a unique seven-membered ring showed more specific PP2C activation activity (1.3-fold at 100 microM) than pisiferic acid having a normal six-membered ring and oleic acid, which is known to activate PP2C. Pisiferdiol and pisiferic acid showed mixed-type activation with respect to alpha-casein, and this differed from the non-competitive activation of oleic acid in vitro. In vivo, the cytotoxicity of pisiferdiol toward human promyelocytic leukemia cell line HL60 with an IC(50) value of 18.3 microM was 2-fold and 7-fold stronger than those of pisiferic acid and oleic acid, and pisiferdiol induced apoptosis through a caspase 3/7-dependent mechanism involving the dephosphorylation of Bad(1), which is a PP2C substrate. We thus conclude that pisiferdiol and pisiferic acid are novel PP2C activators, and the more specific activator, pisiferdiol, may be a useful chemical probe to study PP2C-mediated signaling pathways, and a lead compound for pharmaceutical agents.

  19. Redox regulation of protein tyrosine phosphatase 1B (PTP1B): Importance of steric and electronic effects on the unusual cyclization of the sulfenic acid intermediate to a sulfenyl amide

    NASA Astrophysics Data System (ADS)

    Sarma, Bani Kanta

    2013-09-01

    The redox regulation of protein tyrosine phosphatase 1B (PTP1B) via the unusual transformation of its sulfenic acid (PTP1B-SOH) to a cyclic sulfenyl amide intermediate is studied by using small molecule chemical models. These studies suggest that the sulfenic acids derived from the H2O2-mediated reactions o-amido thiophenols do not efficiently cyclize to sulfenyl amides and the sulfenic acids produced in situ can be trapped by using methyl iodide. Theoretical calculations suggest that the most stable conformer of such sulfenic acids are stabilized by nO → σ*S-OH orbital interactions, which force the -OH group to adopt a position trans to the S⋯O interaction, leading to an almost linear arrangement of the O⋯S-O moiety and this may be the reason for the slow cyclization of such sulfenic acids to their corresponding sulfenyl amides. On the other hand, additional substituents at the 6-position of o-amido phenylsulfenic acids that can induce steric environment and alter the electronic properties around the sulfenic acid moiety by S⋯N or S⋯O nonbonded interactions destabilize the sulfenic acids by inducing strain in the molecule. This may lead to efficient the cyclization of such sulfenic acids. This model study suggests that the amino acid residues in the close proximity of the sulfenic acid moiety in PTP1B may play an important role in the cyclization of PTP1B-SOH to produce the corresponding sulfenyl amide.

  20. Modified Field's staining--a rapid stain for Trichomonas vaginalis.

    PubMed

    Afzan, M Yusuf; Sivanandam, S; Kumar, G Suresh

    2010-10-01

    Trichomonas vaginalis, a flagellate protozoan parasite commonly found in the human genitourinary tract, is transmitted primarily by sexual intercourse. Diagnosis is usually by in vitro culture method and staining with Giemsa stain. There are laboratories that use Gram stain as well. We compared the use of modified Field's (MF), Giemsa, and Gram stains on 2 axenic and xenic isolates of T. vaginalis, respectively. Three smears from every sediment of spun cultures of all 4 isolates were stained, respectively, with each of the stains. We showed that MF staining, apart from being a rapid stain (20 s), confers sharper staining contrast, which differentiates the nucleus and the cytoplasm of the organism when compared to Giemsa and Gram staining especially on parasites from spiked urine samples. The alternative staining procedure offers in a diagnostic setting a rapid stain that can easily visualize the parasite with sharp contrasting characteristics between organelles especially the nucleus and cytoplasm. Vacuoles are more clearly visible in parasites stained with MF than when stained with Giemsa.

  1. A synthetic pathway for an unsymmetrical N(5)O(2) heptadentate ligand and its heterodinuclear iron(III)zinc(II) complex: a biomimetic model for the purple acid phosphatases.

    PubMed

    Xavier, Fernando R; Bortoluzzi, Adailton J; Neves, Ademir

    2012-09-01

    One major field of interest in bioinorganic chemistry is the design and synthesis of inorganic compounds with low molecular mass, showing structural, spectroscopic, and reactivity properties that mimic enzymes, such as purple acid phosphatases (PAPs). In this study, the unsymmetrical heptadentate ligand 2-[(4,7-diisopropyl-1,4,7-triazacyclonon-1-yl)methyl]-6-{[(2-hydroxybenzyl)(pyridin-2-ylmethyl)-amino]methyl}-4-methylphenol (H(2)L) and its first mixed-valence complex [Fe(III)Zn(II)(L)(μ-OAc)(2)]ClO(4)(1) were synthesized. Physical and chemical measurements (crystal structure, conductometry, IR and UV/VIS spectroscopy, and electrochemistry) were performed for 1, and these properties are compared with those presented by the kbPAPs active sites. Potentiometric titration studies of 1 have confirmed its acid/base properties that are crucial for the understanding of the phosphodiester and DNA catalytic cleavage in future studies.

  2. The histidine phosphatase superfamily: structure and function.

    PubMed

    Rigden, Daniel J

    2008-01-15

    The histidine phosphatase superfamily is a large functionally diverse group of proteins. They share a conserved catalytic core centred on a histidine which becomes phosphorylated during the course of the reaction. Although the superfamily is overwhelmingly composed of phosphatases, the earliest known and arguably best-studied member is dPGM (cofactor-dependent phosphoglycerate mutase). The superfamily contains two branches sharing very limited sequence similarity: the first containing dPGM, fructose-2,6-bisphosphatase, PhoE, SixA, TIGAR [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator], Sts-1 and many other activities, and the second, smaller, branch composed mainly of acid phosphatases and phytases. Human representatives of both branches are of considerable medical interest, and various parasites contain superfamily members whose inhibition might have therapeutic value. Additionally, several phosphatases, notably the phytases, have current or potential applications in agriculture. The present review aims to draw together what is known about structure and function in the superfamily. With the benefit of an expanding set of histidine phosphatase superfamily structures, a clearer picture of the conserved elements is obtained, along with, conversely, a view of the sometimes surprising variation in substrate-binding and proton donor residues across the superfamily. This analysis should contribute to correcting a history of over- and mis-annotation in the superfamily, but also suggests that structural knowledge, from models or experimental structures, in conjunction with experimental assays, will prove vital for the future description of function in the superfamily.

  3. Negative staining and cryo-negative staining: applications in biology and medicine.

    PubMed

    Harris, J Robin; De Carlo, Sacha

    2014-01-01

    Negative staining is widely applicable to isolated viruses, protein molecules, macromolecular assemblies and fibrils, subcellular membrane fractions, liposomes and artificial membranes, synthetic DNA arrays, and also to polymer solutions and a variety of nanotechnology samples. Techniques are provided for the preparation of the necessary support films (continuous carbon and holey/perforated carbon). The range of suitable negative stains is presented, with some emphasis on the benefit of using ammonium molybdate and of negative stain-trehalose combinations. Protocols are provided for the single droplet negative staining technique (on continuous and holey carbon support films), the floating and carbon sandwich techniques in addition to the negative staining-carbon film (NS-CF) technique for randomly dispersed fragile molecules, 2D crystallization of proteins and for cleavage of cells and organelles. Immuno-negative staining and negative staining of affinity labeled complexes (e.g., biotin-streptavidin) are presented in some detail. The formation of immune complexes in solution for droplet negative staining is given, as is the use of carbon-plastic support films as an adsorption surface on which to perform immunolabeling or affinity experiments, prior to negative staining. Dynamic biological systems can be investigated by negative staining, where the time period is in excess of a few minutes, but there are possibilities to greatly reduce the time by rapid stabilization of molecular systems with uranyl acetate or tannic acid. The more recently developed cryo-negative staining procedures are also included: first, the high concentration ammonium molybdate procedure on holey carbon films and second, the carbon sandwich procedure using uranyl formate. Several electron micrographs showing examples of applications of negative staining techniques are included and the chapter is thoroughly referenced.

  4. Ganglioside contained in the neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) fraction prepared from the detergent-resistant membrane microdomain of rat brain inhibits the phosphatase activity of calcineurin.

    PubMed

    Kobayashi, Yuumi; da Silva, Ronan; Kumanogoh, Haruko; Miyata, Shinji; Sato, Chihiro; Kitajima, Ken; Nakamura, Shun; Morita, Mistuhiro; Hayashi, Fumio; Maekawa, Shohei

    2015-09-01

    Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). Neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) is one of the major protein components of neuronal DRM. To determine the cellular function of NAP-22, interacting proteins were screened with an immunoprecipitation assay, and calcineurin (CaN) was detected. Further studies with NAP-22 prepared from DRM and CaN expressed in bacteria showed the binding of these proteins and a dose-dependent inhibitory effect of the NAP-22 fraction on the phosphatase activity of CaN. On the other hand, NAP-22 expressed in bacteria showed low binding to CaN and a weak inhibitory effect on phosphatase activity. To solve this discrepancy, identification of a nonprotein component that modulates CaN activity in the DRM-derived NAP-22 fraction was attempted. After lyophilization, a lipid fraction was extracted with chloroform/methanol. The lipid fraction showed an inhibitory effect on CaN without NAP-22, and further fractionation of the extract with thin-layer chromatography showed the presence of several lipid bands having an inhibitory effect on CaN. The mobility of these bands coincided with that of authentic ganglioside (GM1a, GD1a, GD1b, and GT1b), and authentic ganglioside showed an inhibitory effect on CaN. Treatment of lipid with endoglycoceramidase, which degrades ganglioside to glycochain and ceramide, caused a diminution of the inhibitory effect. These results show that DRM-derived NAP-22 binds several lipids, including ganglioside, and that ganglioside inhibits the phosphatase activity of CaN.

  5. Compositions for chromosome-specific staining

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    1998-01-01

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

  6. Compositions for chromosome-specific staining

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1998-05-26

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. The methods produce staining patterns that can be tailored for specific cytogenetic analyses. The probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. The invention provides for automated means to detect and analyze chromosomal abnormalities. 17 figs.

  7. The involvement of glucose-6-phosphatase in mucilage secretion by root cap cells of Zea mays

    NASA Technical Reports Server (NTRS)

    Moore, R.; McClelen, C. E.

    1985-01-01

    In order to determine the involvement of glucose-6-phosphatase in mucilage secretion by root cap cells, we have cytochemically localized the enzyme in columella and peripheral cells of root caps of Zea mays. Glucose-6-phosphatase is associated with the plasmalemma and cell wall of columella cells. As columella cells differentiate into peripheral cells and begin to produce and secrete mucilage, glucose-6-phosphatase staining intensifies and becomes associated with the mucilage and, to a lesser extent, the cell wall. Cells being sloughed from the cap are characterized by glucose-6-phosphatase staining being associated with the vacuole and plasmalemma. These changes in enzyme localization during cellular differentiation in root caps suggest that glucose-6-phosphatase is involved in the production and/or secretion of mucilage by peripheral cells of Z. mays.

  8. Distinct phosphatase activity profiles in two strains of Trypanosoma cruzi.

    PubMed

    Morales-Neto, R; Hulshof, L; Ferreira, C V; Gadelha, F R

    2009-12-01

    Phosphorylation of parasite proteins plays a key role in the process of cell invasion by Trypanosoma cruzi, the etiologic agent of Chagas' disease. In this sense, characterization of parasite kinases and phosphatases could open new possibilities for the rational design of chemotherapeutic agents for the treatment of Chagas' disease. In this work, we analyzed phosphatase activities in T. cruzi homogenates from 2 strains belonging to different lineages and with different resistance to oxidative stress. Tulahuen 2 cells (Lineage I) showed higher phosphatase activities and specificity constants when compared to the Y strain (Lineage II). Tulahuen 2 had an optimum phosphatase activity at pH 4.0 and the Y strain at pH 7.0. In both cases, neutral–basic, but not acid, phosphatase activities were increased in the presence of Mg2+. Although calcium had an inhibitory effect at a pH of 7.0 and 8.0 in the Y strain, this inhibition was restricted to pH 8.0 in the other strain. Different substrates and acid phosphotyrosine and alkaline phosphatase inhibitors exhibited distinct effects on the phosphatase activity of both strains. Our results provide a better understanding of T. cruzi phosphatases and reinforce the notion of heterogeneity among T. cruzi populations.

  9. Highly chlorinated Escherichia coli cannot be stained by propidium iodide.

    PubMed

    Phe, M-H; Dossot, M; Guilloteau, H; Block, J-C

    2007-05-01

    Several studies have shown that the staining by fluorochromes (DAPI, SYBR Green II, and TOTO-1) of bacteria is altered by chlorination. To evaluate the effect of chlorine (bleach solution) on propidium iodide (PI) staining, we studied Escherichia coli in suspension and biomolecules in solution (DNA, RNA, BSA, palmitic acid, and dextran) first subjected to chlorine and then neutralized by sodium thiosulphate. The suspensions and solutions were subsequently stained with PI. The fluorescence intensity of the PI-stained DNA and RNA in solution dramatically decreased with an increase in the chlorine concentration applied. These results explain the fact that for chlorine concentrations higher than 3 micromol/L Cl2, the E. coli cells were too damaged to be properly stained by PI. In the case of highly chlorinated bacteria, it was impossible to distinguish healthy cells (with a PI-impermeable membrane and undamaged nucleic acids), which were nonfluorescent after PI staining, from cells severely injured by chlorine (with a PI-permeable membrane and damaged nucleic acids) that were also nonfluorescent, as PI penetrated but did not stain chlorinated nucleic acids. Our results suggest that it would be prudent to be cautious in interpreting the results of PI staining, as PI false-negative cells (cells with compromised membranes but not stained by PI because of nucleic acid damage caused by chlorine) are obtained as a result of nucleic acid damage, leading to an underestimation of truly dead bacteria.

  10. Length of stain dosimeter

    NASA Technical Reports Server (NTRS)

    Lueck, Dale E. (Inventor)

    1994-01-01

    Payload customers for the Space Shuttle have recently expressed concerns about the possibility of their payloads at an adjacent pad being contaminated by plume effluents from a shuttle at an active pad as they await launch on an inactive pad. As part of a study to satisfy such concerns a ring of inexpensive dosimeters was deployed around the active pad at the inter-pad distance. However, following a launch, dosimeters cannot be read for several hours after the exposure. As a consequence factors such as different substrates, solvent systems, and possible volatilization of HCl from the badges were studied. This observation led to the length of stain (LOS) dosimeters of this invention. Commercial passive LOS dosimeters are sensitive only to the extent of being capable of sensing 2 ppm to 20 ppm if the exposure is 8 hours. To map and quantitate the HCl generated by Shuttle launches, and in the atmosphere within a radius of 1.5 miles from the active pad, a sensitivity of 2 ppm HCl in the atmospheric gases on an exposure of 5 minutes is required. A passive length of stain dosimeter has been developed having a sensitivity rendering it capable of detecting a gas in a concentration as low as 2 ppm on an exposure of five minutes.

  11. Blood stain pattern analysis.

    PubMed

    Peschel, O; Kunz, S N; Rothschild, M A; Mützel, E

    2011-09-01

    Bloodstain pattern analysis (BPA) refers to the collection, categorization and interpretation of the shape and distribution of bloodstains connected with a crime. These kinds of stains occur in a considerable proportion of homicide cases. They offer extensive information and are an important part of a functional, medically and scientifically based reconstruction of a crime. The following groups of patterns can essentially be distinguished: dripped and splashed blood, projected blood, impact patterns, cast-off stains, expirated and transferred bloodstains. A highly qualified analysis can help to estimate facts concerning the location, quality and intensity of an external force. A sequence of events may be recognized, and detailed questions connected with the reconstruction of the crime might be answered. In some cases, BPA helps to distinguish between accident, homicide and suicide or to identify bloodstains originating from a perpetrator. BPA is based on systematic training, a visit to the crime scene or alternatively good photographic documentation, and an understanding and knowledge of autopsy findings or statements made by the perpetrator and/or victim. A BPA working group has been established within the German Society of Legal Medicine aiming to put the knowledge and practical applications of this subdiscipline of forensic science on a wider basis.

  12. Simple Protocol for Secondary School Hands-On Activity: Electrophoresis of Pre-Stained Nucleic Acids on Agar-Agar Borate Gels

    ERIC Educational Resources Information Center

    Britos, Leticia; Goyenola, Guillermo; Orono, Silvia Umpierrez

    2004-01-01

    An extremely simple, inexpensive, and safe method is presented, which emulates nucleic acids isolation and electrophoretic analysis as performed in a research environment, in the context of a secondary school hands-on activity. The protocol is amenable to an interdisciplinary approach, taking into consideration the electrical and chemical…

  13. Phosphatase activities as biosignatures of extant life

    NASA Astrophysics Data System (ADS)

    Kobayashi, K.; Itoh, Y.; Edazawa, Y.; Moroi, A.; Takano, Y.

    It has been recognized that terrestrial biosphere expands to such extreme environments as deep subsurface lithosphere high temperature hot springs and stratosphere Possible extraterrestrial biospheres in Mars Europa and Titan are being discussed Many biosignatures or biomarkers have been proposed to detect microbial activities in such extreme environments Phosphate esters are essential for the terrestrial life since they are constituents of nucleic acids and cell mebranes Thus all the terrestrial organisms have phosphatases that are enzymes catalyzing hydrolysis of phosphate esters We analyzed phosphatase activities in the samples obtained in extreme environments such as submarine hydrothermal systems and discussed whether they can be used as biosignatures for extant life Core samples and chimney samples were collected at the Suiyo Seamount Izu-Bonin Arc the Pacific Ocean in 2001 and 2002 and in South Mariana hydrothermal systems the Pacific Oceanas in 2003 both in a part of the Archaean Park Project Phosphatase activity in solid rock samples was measured spectrometrically by using 25 mM p-nitrophenyl phosphate pH 8 0 or pH 6 5 as a substrate as follows Pulverized samples were incuvated with substrate solution for an hour and then production rate of p-nitrophenol was calculated with absorbance at 410 nm Phosphatase activity in extracts was measured fluorometrically by using 4-methylumberyferryl phosphate as a substrate Concentration of amino acids and their enantiomeric ratio were determined by HPLC after HF digestion of the

  14. Selective staining of CdS on ZnO biolabel for ultrasensitive sandwich-type amperometric immunoassay of human heart-type fatty-acid-binding protein and immunoglobulin G.

    PubMed

    Qin, Xiaoli; Xu, Aigui; Liu, Ling; Sui, Yuyun; Li, Yunlong; Tan, Yueming; Chen, Chao; Xie, Qingji

    2017-05-15

    We report on an ultrasensitive metal-labeled amperometric immunoassay of proteins, which is based on the selective staining of nanocrystalline cadmium sulfide (CdS) on ZnO nanocrystals and in-situ microliter-droplet anodic stripping voltammetry (ASV) detection on the immunoelectrode. Briefly, antibody 1 (Ab1), bovine serum albumin (BSA), antigen and ZnO-multiwalled carbon nanotubes (MWCNTs) labeled antibody 2 (Ab2-ZnO-MWCNTs) were successively anchored on a β-cyclodextrin-graphene sheets (CD-GS) nanocomposite modified glassy carbon electrode (GCE), forming a sandwich-type immunoelectrode (Ab2-ZnO-MWCNTs/antigen/BSA/Ab1/CD-GS/GCE). CdS was selectively grown on the catalytic ZnO surfaces through chemical reaction of Cd(NO3)2 and thioacetamide (ZnO-label/CdS-staining), due to the presence of an activated cadmium hydroxide complex on ZnO surfaces that can decompose thioacetamide. A beforehand cathodic "potential control" in air and then injection of 7μL of 0.1M aqueous HNO3 on the immunoelectrode allow dissolution of the stained CdS and simultaneous cathodic preconcentration of atomic Cd onto the electrode surface, thus the following in-situ ASV detection can be used for immunoassay with enhanced sensitivity. Under optimized conditions, human immunoglobulin G (IgG) and human heart-type fatty-acid-binding protein (FABP) are analyzed by this method with ultrahigh sensitivity, excellent selectivity and small reagent-consumption, and the limits of detection (LODs, S/N=3) are 0.4fgmL(-1) for IgG and 0.3fgmL(-1) for FABP (equivalent to 73 FABP molecules in the 6μL sample employed).

  15. Phosphatidyl glycerophosphate phosphatase.

    PubMed

    Chang, Y Y; Kennedy, E P

    1967-09-01

    An enzyme (phosphatidyl glycerophosphate phosphatase) that catalyzes the formation of phosphatidyl glycerol from phosphatidyl glycerophosphate has been rendered soluble by treatment of the particulate fraction of E. coli with Triton X-100 in the presence of EDTA, and has been partially purified. The enzyme is specific for phosphatidyl glycerophosphate and does not catalyze the hydrolysis of other simple phosphomonoesters. It requires Mg(++) for activity and is inhibited by sulfhydryl agents. Some other properties of the enzyme are also described.

  16. Evidence for a conserved binding motif of the dinuclear metal site in mammalian and plant purple acid phosphatases: 1H NMR studies of the di-iron derivative of the Fe(III)Zn(II) enzyme from kidney bean.

    PubMed

    Battistuzzi, G; Dietrich, M; Löcke, R; Witzel, H

    1997-05-01

    The di-iron core of mammalian purple acid phosphatases has been reproduced in the plant enzyme from kidney bean (Mr 111000) upon insertion of an Fe(II) ion in place of the native zinc(II) in the dinuclear Fe(III)Zn(II) core. The shortening of the electronic relaxation time of the metal centre allows detection of hyperfine-shifted 1H NMR resonances, although severe broadening due to Curie relaxation prevents independent signal assignment. Nevertheless, comparison of the spectral features of the structurally characterized plant enzyme with those of the mammalian species, which were previously extensively assigned, is consistent with a close similarity of the metal-binding sites, also suggested by previous sequence-alignment studies. Some differences appear to be mainly localized at the M(II) site. Spectral comparison was also carried out on the Fe(III)Co(II) derivatives.

  17. Uniform staining of Cyclospora oocysts in fecal smears by a modified safranin technique with microwave heating.

    PubMed

    Visvesvara, G S; Moura, H; Kovacs-Nace, E; Wallace, S; Eberhard, M L

    1997-03-01

    Cyclospora, a coccidian protist, is increasingly being identified as an important, newly emerging parasite that causes diarrhea, flatulence, fatigue, and abdominal pain leading to weight loss in immunocompetent persons with or without a recent travel history as well as in patients with AIDS. Modified Kinyoun's acid-fast stain is the most commonly used stain to identify the oocyst of this parasite in fecal smears. Oocysts of Cyclospora stain variably by the modified acid-fast procedure, resulting in the possible misidentification of this parasite. We examined fecal smears stained by six different procedures that included Giemsa, trichrome, chromotrope, Gram-chromotrope, acid-fast, and safranin stains. We report on safranin-based stain that uniformly stains oocysts of Cyclospora a brilliant reddish orange, provided that the fecal smears are heated in a microwave oven prior to staining. This staining procedure, besides being superior to acid-fast staining, is fast, reliable, and easy to perform in most clinical laboratories.

  18. Violacein cytotoxicity on human blood lymphocytes and effect on phosphatases.

    PubMed

    Bromberg, N; Justo, G Z; Haun, M; Durán, N; Ferreira, C V

    2005-10-01

    Given the importance of protein phosphorylation in the context of cellular functions, abnormal protein phosphatase activity has been implicated in several diseases, including cancer. These critical roles of protein phosphatases qualify them as potential targets for the development of medicinal compounds that possess distinct modes of action such as violacein. In this work, studies with this natural indolic pigment at a concentration of 10.0 micromol L(-1) demonstrated a 20% activation of total protein phosphatase extracted from human lymphocytes. Although no alteration was observed on protein tyrosine phosphatase (CD45), 30% of inhibition was achieved in cytoplasmatic protein phosphatase activity after incubation with 10.0 micromol L(-1) violacein. Additionally, 5.0 micromol L(-1) of violacein inhibited by 50% the serum tartrate-resistant acid phosphatase activity. Violacein presented toxic effect on lymphocytes with IC50 values of 3 and 10 micromol L(-1) for protein content and protein phosphatase activity, respectively. These findings suggest an important role for protein phosphatases in the mechanisms controlling proliferation and cell death.

  19. Characterization of a tyrosine phosphatase activity in the oogenesis of Periplaneta americana.

    PubMed

    Oliveira, D M P; Machado, E A

    2006-09-01

    In this work, phosphatase activity was characterized in the ovary and the haemolymph of Periplaneta americana. The optimum pH for these activities was 4.0, and a temperature of 44 degrees C was ideal for the maximal enzyme activity. The phosphatase activities were inhibited by NaF, sodium tartrate, Pi, sodium orthovanadate, and ammonium molybdate. The ovarian phosphatase activity at pH 4.0 was almost exclusive against phosphotyrosine, with little or no effect on the residues of phosphoserine or phosphothreonine. These results indicate that this phosphatase activity is due to the presence of an acid tyrosine phosphatase. The phosphatase activities of acid extracts from P. americana ovaries (OEX) and an acid extract from P. americana haemolymph (HEX) were analyzed in non-denaturant gel electrophoresis using an analog substrate beta-naphtyl phosphate. The gel revealed two bands with phosphatase activity in the ovary and one band in the haemolymph; these bands were excised and submitted to a 10% SDS-PAGE showing a single 70-kDa polypeptide in both samples. Histochemistry of the ovary with alpha-naphtyl phosphate for localization of acid phosphatase activity showed mainly labeling associated to the oocyte peripheral vesicles, basal lamina, and between follicle cells. Electron microscopy analysis showed that acid phosphatase was localized in small peripheral vesicles in the oocyte, but not inside yolk granules. The possible role of this phosphatase during oogenesis and embryogenesis is also discussed in this article.

  20. Proteomic analysis of different period excretory secretory products from Clonorchis sinensis adult worms: molecular characterization, immunolocalization, and serological reactivity of two excretory secretory antigens-methionine aminopeptidase 2 and acid phosphatase.

    PubMed

    Zheng, Minghui; Hu, Kunhua; Liu, Wei; Li, Hongyu; Chen, Jingfang; Yu, Xinbing

    2013-03-01

    The excretory secretory products (ESP) of Clonorchis sinensis are the causative agents of clonorchiasis and biliary diseases. The parasites' ESP play important roles in host-parasite interactions. The protein compositions of ESP at different secretory times are different and have not been systemically investigated so far. In this study, we collected ESP from six different periods (0-3 h, 3-6 h, 6-12 h, 12-24 h, 24-36 h, and 36-48 h) from C. sinensis adults. Using a shotgun LC-MS/MS analysis, we found 187, 80, 103, 58, 248, and 383 proteins, respectively. Among these proteins, we selected methionine aminopeptidase 2 (MAP-2, presented in 24-36 h and 36-48 h ESP) and acid phosphatase (AP, presented in 3-6 h, 12-24 h, 24-36 h, and 36-48 h ESP) for further study. Bioinformatics analysis showed that CsMAP-2 has metallopeptidase family M24, unique lysine residue-rich and acidic residue-rich domain, SGTS motif, and auto-cleavage point; and that CsAP has possible signal sequence cleavage site, acid phosphate domain, and two histidine acid phosphatases active regions. CsMAP-2 and CsAP's cDNA have 1,425 bp and1,410 bp ORF, encoding 475 and 470 amino acid proteins and weighing 55.3840 kDa and 55.2875 kDa, respectively. MAP-2 and AP were identified as antigens present in the ESP and circulating antigens by immunoblot analysis, which were also found expressing in the eggs, metacercaria, and adult stages of C. sinensis. Immunofluorescence analysis showed that they were located in tegument and intestinal cecum of adult. MTT assay showed that they could inhibit hepatic stellate cell line (LX-2) proliferation. These findings presented the compositions of different period excretory secretary products from C. sinensis adults.

  1. A bifunctional kinase-phosphatase in bacterial chemotaxis.

    PubMed

    Porter, Steven L; Roberts, Mark A J; Manning, Cerys S; Armitage, Judith P

    2008-11-25

    Phosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, CheA(3), is not only a phosphodonor for the principal CheY protein, CheY(6), but is also is a specific phosphatase for CheY(6)-P. This phosphatase activity accelerates CheY(6)-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. CheA(3) possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (P5) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of CheA(3) is phosphorylated by CheA(4), and it subsequently acts as a phosphodonor for the response regulators. A CheA(3) mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of CheA(3) is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, CheA(3) lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases.

  2. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae.

  3. Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium.

    PubMed Central

    Kier, L D; Weppelman, R; Ames, B N

    1977-01-01

    A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems. Images PMID:192712

  4. Giardia lamblia: Characterization of ecto-phosphatase activities.

    PubMed

    Amazonas, Juliana Natal; Cosentino-Gomes, Daniela; Werneck-Lacerda, Aline; Pinheiro, Ana Acácia de Sá; Lanfredi-Rangel, Adriana; De Souza, Wanderley; Meyer-Fernandes, José R

    2009-01-01

    Ecto-phosphatase activities of Giardia lamblia were characterized in intact cells, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 8.4+/-0.8 nmol p-NP/h/10(7) cells. The ecto-phosphatase activities were inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium fluoride and sodium molybdate and by inorganic phosphate, the final product of the reaction. Experiments using a classical inhibitor of phosphotyrosine phosphatase, sodium orthovanadate, also showed that the ecto-phosphatase activity was inhibited in a dose-dependent manner. Different phosphorylated amino acids were used as substrates for the G. lamblia ecto-phosphatase activities the highest rate of phosphate release was achieved using phosphotyrosine. Not only p-NPP hydrolysis but also phosphotyrosine hydrolysis was inhibited by sodium orthovanadate. Phosphotyrosine but not phospho-serine or phospho-threonine inhibited the p-nitrophenylphosphatase activity. We also observed a positive correlation between the ecto-phosphatase activity and the capacity to encystation of G. lamblia trophozoites.

  5. Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase.

    PubMed

    Douglas, Timothy E L; Messersmith, Philip B; Chasan, Safak; Mikos, Antonios G; de Mulder, Eric L W; Dickson, Glenn; Schaubroeck, David; Balcaen, Lieve; Vanhaecke, Frank; Dubruel, Peter; Jansen, John A; Leeuwenburgh, Sander C G

    2012-08-01

    Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel-protein-inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG/fumaric acid copolymer OPF. After incubation in Ca-GP solution, FTIR, EDS, SEM, XRD, SAED, ICP-OES, and von Kossa staining confirm CaP formation. The amount of mineral formed decreases in the order cPEG > collagen > OPF. The mineral:polymer ratio decreases in the order collagen > cPEG > OPF. Mineralization increases Young's modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel/CaP composites may find application as bone regeneration materials.

  6. Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase

    PubMed Central

    Douglas, Timothy E.L.; Messersmith, Philip B.; Chasan, Safak; Mikos, Antonios G.; de Mulder, Eric L.W.; Dickson, Glenn; Schaubroeck, David; Balcaen, Lieve; Vanhaecke, Frank; Dubruel, Peter; Jansen, John A.

    2013-01-01

    Alkaline Phosphatase (ALP), an enzyme involved in mineralization of bone, was incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These were collagen type I, a mussel protein-inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG-fumaric acid copolymer OPF. After incubation in calcium glycerophosphate (Ca-GP) solution, FTIR, EDS, SEM, XRD, SAED, ICP-OES and von Kossa staining confirmed CaP formation. The amount of mineral formed decreased in the order cPEG > collagen > OPF. Mineral:polymer ratio decreased in the order collagen > cPEG > OPF. Mineralization increased Young’s modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel-CaP composites could find application as bone regeneration materials. PMID:22648976

  7. Changes of serum alkaline phosphatase isoenzymes in fasted rats.

    PubMed

    Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

    1996-10-01

    Changes of serum alkaline phosphatase (sALP) isoenzymes under fasting conditions were examined using polyacrylamide gel electrophoresis (PAGE), amino-acids (L-phenylalanine (L-Phe), L-homoarginine (L-HArg)) inhibition and wheat germ agglutinin (WGA) treatment. The sALP of non-fasted rats was separated into three bands (S1, S2, S3) by PAGE. The molecular weight (M.W.) of S1 corresponded to that of an isoenzyme found in the ileum. By the addition of L-Phe, the staining intensity of S1 was weakened, S2 and S3 remained unchanged and the total activity of the isoenzymes extracted from intestine decreased. On the other hand, the activity of isoenzymes extracted from kidney and bone decreased by the addition of L-HArg. Therefore, S1 was judged to be derived from intestine. The activities of total sALP and S1 decreased from 16 h of fasting. Total sALP activity and sALP activity of the supernatant prepared by WGA treatment decreased, whereas the ALP activity of the precipitate (difference between total sALP activity and supernatant sALP activity) did not change. The activity band of the precipitate corresponded to that of S3 by PAGE. Therefore, S3 was judged to be derived from bone. In conclusion, under fasting conditions, the activity of S1 decreased while the activities of S2 and S3 remained unchanged.

  8. Simplified preparation of a phosphatase inhibitor and further studies of its action.

    PubMed

    Coburn, S P; Schaltenbrand, W E

    1978-05-01

    1-Pyrrolidinecarbothioic acid (2-pyridylmethylene) hydrazide chelates Zn2+ but not Mg2+. This compound is about twice as effective as EDTA for inhibiting alkaline phosphatase from calf mucosa, and approx. 1000-fold more effective than EDTA for inhibiting acid phosphatase from wheat germ. The compound did not inhibit pyridoxine kinase activity in human leucocytes at the highest concentration tested (33 micron). Therefore it may be a useful tool for either examining or eliminating the effects of phosphatases in complex enzyme systems.

  9. Direct determination of phosphatase activity from physiological substrates in cells.

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  10. Enhancing Potato System Sustainability: Crop Rotation Impacts on Soil Phosphatase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato is a species with a low efficiency of acquiring soil P. Rotation crops may potentially influence P uptake by potato by increasing soil organic acids, phosphatase activity, and microbial biomass. However, this kind of information is very limited. We measured the activities of acid phosphatase,...

  11. Cracking the phosphatase code: docking interactions determine substrate specificity.

    PubMed

    Roy, Jagoree; Cyert, Martha S

    2009-12-08

    Phosphoserine- and phosphothreonine-directed phosphatases display remarkable substrate specificity, yet the sites that they dephosphorylate show little similarity in amino acid sequence. Studies reveal that docking interactions are key for the recognition of substrates and regulators by two conserved phosphatases, protein phosphatase 1 (PP1) and the Ca2+-calmodulin-dependent phosphatase calcineurin. In each case, a small degenerate sequence motif in the interacting protein directs low-affinity binding to a docking surface on the phosphatase that is distinct from the active site; several such interactions combine to confer overall binding specificity. Some docking surfaces are conserved, such as a hydrophobic groove on a face opposite the active site that serves as a major recognition surface for the "RVxF" motif of proteins that interact with PP1 and the "PxIxIT" motif of substrates of calcineurin. Secondary motifs combine with this primary targeting sequence to specify phosphatase binding. A comprehensive interactome for mammalian PP1 was described, analysis of which defines several PP1-binding motifs. Studies of "LxVP," a secondary calcineurin-binding sequence, establish that this motif is a conserved feature of calcineurin substrates and that the immunosuppressants FK506 and cyclosporin A inhibit the phosphatase by interfering with LxVP-mediated docking.

  12. Silver stain for electron microscopy

    NASA Technical Reports Server (NTRS)

    Corbett, R. L.

    1972-01-01

    Ammoniacal silver stain used for light microscopy was adapted advantageously for use with very thin biological sections required for electron microscopy. Silver stain can be performed in short time, has more contrast, and is especially useful for low power electron microscopy.

  13. Separation of Alkaline Phosphatase Isoenzymes from Various Rat Tissues Using Flat-Bed Acrylamide Gel Isoelectric Focusing,

    DTIC Science & Technology

    1981-12-22

    Technical Bulletin No. 104. Alkaline phosphatase activity was expressed as 1 micromole of p- nitrophenol hydrolyzed per hour and specific alkaline... phosphatase activity was defined as the number of micromoles of p- nitrophenol hydrolyzed per hour per microgram of protein. The total protein was determined... phosphatase activity is known or suspected. Importantly, the procedure is easily adapted for acid phosphatase examination by merely changing the pH and

  14. Factors influencing extract of Hibiscus sabdariffa staining of rat testes.

    PubMed

    Bassey, R B; Bakare, A A; Peter, A I; Oremosu, A A; Osinubi, A A

    2012-08-01

    Some plant extracts can be used in biology and medicine to reveal or identify cellular components and tissues. We investigated the effects of time and concentration on staining of histological sections of rat testes by an acidified extract of Hibiscus sabdariffa. An ethanolic extract of H. sabdariffa was diluted using 1% acetic acid in 70% ethanol to stain histological sections of testes at concentrations of 0.2, 0.1 and 0.05 g/ml for 5, 10, 15, 30, 45 and 60 min. The sections of testes were stained deep red. The staining efficiency of H. sabdariffa was greater at a high concentration and required less time to achieve optimal staining. H. sabdariffa is a strongly basic dye that can be used for various diagnostic purposes. Staining time and concentration must be considered to achieve optimal results.

  15. Myelin staining of archival brain tissue.

    PubMed

    Sheaffer, S; Rosoklija, G; Dwork, A J

    1999-01-01

    To evaluate the feasibility of staining for myelin in archival materials, paraffin blocks were prepared from brain tissue that had been in formalin for intervals ranging from 7 months to over 53 years. Verhoeff and Luxol fast blue stains of the resulting sections yielded staining whose quality was unaffected by duration of fixation. Myelinated and unmyelinated areas were clearly distinguished, and the morphology of individual myelin sheaths was well-preserved. No changes to conventional protocols were required, but it was necessary carefully to monitor the progress of differentiation. With antigen retrieval, it was possible to display immunoreactivity for myelin basic protein. While this persisted even after prolonged fixation, fine detail was lost from the myelin sheaths, and there was staining of oligodendroglial cytoplasm and nuclei, which was not seen in recently fixed tissue. In contrast to this loss of detail in myelin sheaths, immunohistochemistry for glial fibrillary acidic protein displayed astrocytic morphology clearly, even in the oldest tissue. We conclude that archival, formalin-fixed material can be adequately examined for myelin loss and astrocytosis.

  16. Transmission electron microscopy staining methods for the cortex of human hair: a modified osmium method and comparison with other stains.

    PubMed

    Harland, D P; Vernon, J A; Walls, R J; Woods, J L

    2011-08-01

    For wool, superior staining of a wide range of ultrastructural components is achieved by en bloc treatment of fibres with a chemical reductant followed by osmium tetroxide. For human scalp hair, although staining quality is similar, the penetration of reagents is poor, resulting in large parts of the fibre cortex remaining unstained. Here we describe a modification to the reduction-osmication method in which reagents penetrate through a cut fibre end, allowing visualization of a wide range of features across the cortex. We compare the staining quality, artefacts and range of structure rendered visible using transmission electron microscopy for en bloc reduction-osmication to other staining alternatives including en bloc silver nitrate and section stains based on uranyl acetate and lead citrate, phosphotungstic acid, potassium permanganate, ammoniacal silver nitrate and some combinations of these stains. The effects of hair-care treatments are briefly examined.

  17. A method for staining pollen tubes in pistil.

    PubMed

    Alexander, M P

    1987-03-01

    A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsin, 6 ml; 1% aqueous aniline blue, 4 ml; 1% orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45 +/- 2 C. They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45 +/- 2 C, hydrolyzed in the clearing and softening fluid at 58 +/- 1 C for 30 min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.

  18. Biochemical changes of the synovial liquid of corpses with regard to the cause of death. 2: Alkaline phosphatase, lactic acid dehydrogenase (LDH), and glutamic oxalacetic transaminase (GOT).

    PubMed

    More, D S; Arroyo, M C

    1985-04-01

    We studied the activity of various enzymes in the synovial liquid of 100 corpses with regard to the cause of death finding that the alkaline phospatase and glutamic oxalacetic transaminase (GOT) are increased in cranioencephalic trauma, possibly as a result of the important cellular lysis which goes with them; and lactic acid dehydrogenase (LDH) is increased in the pulmonary processes, almost certainly with relation to the great quantity of this enzyme in the lung.

  19. A chronoamperometric screen printed carbon biosensor based on alkaline phosphatase inhibition for W(IV) determination in water, using 2-phospho-L-ascorbic acid trisodium salt as a substrate.

    PubMed

    Alvarado-Gámez, Ana Lorena; Alonso-Lomillo, María Asunción; Domínguez-Renedo, Olga; Arcos-Martínez, María Julia

    2015-01-22

    This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 µM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 µM. This study was performed by means of a Lineweaver-Burk plot, showing a mixed kinetic inhibition.

  20. Selective hydrolysis of phosphate monoester by a supramolecular phosphatase formed by the self-assembly of a bis(Zn(2+)-cyclen) complex, cyanuric acid, and copper in an aqueous solution (cyclen = 1,4,7,10-tetraazacyclododecane).

    PubMed

    Zulkefeli, Mohd; Suzuki, Asami; Shiro, Motoo; Hisamatsu, Yosuke; Kimura, Eiichi; Aoki, Shin

    2011-10-17

    In Nature, organized nanoscale structures such as proteins and enzymes are formed in aqueous media via intermolecular interactions between multicomponents. Supramolecular and self-assembling strategies provide versatile methods for the construction of artificial chemical architectures for controlling reaction rates and the specificities of chemical reactions, but most are designed in hydrophobic environments. The preparation of artificial catalysts that have potential in aqueous media mimicking natural enzymes such as hydrolases remains a great challenge in the fields of supramolecular chemistry. Herein, we describe that a dimeric Zn(2+) complex having a 2,2'-bipyridyl linker, cyanuric acid, and a Cu(2+) ion automatically assembles in an aqueous solution to form a 4:4:4 complex, which is stabilized by metal-ligand coordination bonds, π-π-stacking interactions, and hydrogen bonding and contains μ-Cu(2)(OH)(2) cores analogous to the catalytic centers of phosphatase, a dinuclear metalloenzyme. The 4:4:4 complex selectively accelerates the hydrolysis of a phosphate monoester, mono(4-nitrophenyl)phosphate, at neutral pH.

  1. Spatial structure of oligopeptide PAP(248-261), the N-terminal fragment of the HIV enhancer prostatic acid phosphatase peptide PAP(248-286), in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Blokhin, Dmitriy S.; Filippov, Andrei V.; Antzutkin, Oleg N.; Karataeva, Farida Kh.; Klochkov, Vladimir V.

    2014-07-01

    Prostatic acid phosphatase (PAP) is an enzyme that facilitates infection of cells by HIV. Its peptide fragment PAP(248-286) forms amyloid fibrils known as SEVI, which enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. The secondary structure of PAP(248-286) in aqueous and SDS solutions can be divided into an N-terminal disordered region, an α-helical central part and an α/310-helical C-terminal region (Nanga et al., 2009). In this work, we used NMR spectroscopy to study the spatial structure of the isolated N-terminal fragment of PAP(248-286), PAP(248-261) (GIHKQKEKSRLQGG), in aqueous and SDS micelle solutions. Formation of a PAP(248-261)-SDS complex was confirmed by chemical shift alterations in the 1H NMR spectra of the peptide, as well as by the signs and values of Nuclear Overhauser Effect (NOE). In addition, the PAP(248-261) peptide does not form any specified secondary structure in either aqueous or SDS solutions.

  2. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

    PubMed

    Solberg, L B; Stang, E; Brorson, S-H; Andersson, G; Reinholt, F P

    2015-02-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis.

  3. High performance liquid chromatography with an electrochemical detector in the cathodic mode as a tool for the determination of p-nitrophenol and assay of acid phosphatase in urine samples.

    PubMed

    Yamauchi, Yuji; Ido, Megumi; Ohta, Merime; Maeda, Hatsuo

    2004-05-01

    Utilizing a commercially available helium-purging device and PEEK tubes for all tubing, especially for connection between the mobile phase and pump, high performance liquid chromatography with an electrochemical detector (ECD/HPLC) at the cathodic mode is a simple and precise method for the determination of p-nitrophenol (NP). Studies with cyclic and hydrodynamic voltammetry indicated that 25% aqueous MeOH containing 0.1% (v/v) CF(3)CO(2)H and -0.8 V vs. Ag/AgCl are the best mobile phase and detection potential for cathodic ECD/HPLC. With the present system, the limits of detection and determination were 0.2 and 0.25 microM, respectively, and up to 50 microM, a linear calibration curve was afforded. Within-day precisions for the analysis of 5 and 50 microM NP were 0.8 and 0.7% (n=6), respectively, and between-day precisions (n=6) for these samples were 3.5 and 2.2%, respectively. Compared with the commonly used Bessey-Lowry-Brock method, cathodic ECD/HPLC was useful for the assay of acid phosphatase in urine samples with p-nitrophenyl phosphate disodium salt as a substrate.

  4. A Chronoamperometric Screen Printed Carbon Biosensor Based on Alkaline Phosphatase Inhibition for W(VI) Determination in Water, Using 2-Phospho-l-Ascorbic Acid Trisodium Salt as a Substrate

    PubMed Central

    Alvarado-Gámez, Ana Lorena; Alonso-Lomillo, María Asunción; Domínguez-Renedo, Olga; Arcos-Martínez, María Julia

    2015-01-01

    This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 μM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 μM. This study was performed by means of a Lineweaver–Burk plot, showing a mixed kinetic inhibition. PMID:25621602

  5. Tartrate-resistant acid phosphatase (TRACP 5b): a biomarker of bone resorption rate in support of drug development: modification, validation and application of the BoneTRAP kit assay.

    PubMed

    Wu, Yuling; Lee, Jean W; Uy, Lennie; Abosaleem, Bassam; Gunn, Han; Ma, Mark; DeSilva, Binodh

    2009-07-12

    A commercial kit assay of tartrate-resistant acid phosphatase (TRACP 5b) used for the diagnosis of bone resorption was modified with a 'Fit-For-Purpose' approach for drug development of anti-resorptive therapeutics. The modifications included changing the standard matrix from buffer to serum, using a consistent bulk reference material to prepare standards and quality controls (QC), and adding sample controls (SC) prepared from authentic sample pools. Method validation experiments were conducted for: inter- and intra-assay accuracy and precision, establishment of SC, range finding of different population groups, selectivity tests, parallelism and stability. The analytical range was 1.00-10.0 U/L and the total errors of lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) validation samples were 8% and 21%, respectively. Data of range finding experiment showed that serum samples should be collected in tubes instead of bags. Selectivity results showed accurate spike recovery among the majority of test samples from target populations. Samples were demonstrated to be stable for up to four freeze/thaw cycles and for 24 months at -70+/-10 degrees C. Our results show that the modified TRACP 5b method is reliable for the quantification of TRACP 5b in human serum samples to support clinical trials of bone resorptive effect reflected by TRACP 5b activities. The method was robust with similar assay performance characteristics shown in three bioanalytical laboratories.

  6. Cloning and characterization of a novel human phosphatidic acid phosphatase type 2, PAP2d, with two different transcripts PAP2d_v1 and PAP2d_v2.

    PubMed

    Sun, Liyun; Gu, Shaohua; Sun, Yaqiong; Zheng, Dan; Wu, Qihan; Li, Xin; Dai, Jianfeng; Dai, Jianliang; Ji, Chaoneng; Xie, Yi; Mao, Yumin

    2005-04-01

    This study reports the cloning and characterization of a novel human phosphatidic acid phosphatase type 2 isoform cDNAs (PAP2d) from the foetal brain cDNA library. The PAP2d gene is localized on chromosome 1p21.3. It contains six exons and spans 112 kb of the genomic DNA. By large-scale cDNA sequencing we found two splice variants of PAP2d, PAP2d_v1 and PAP2d_v2. The PAP2d_v1 cDNA is 1722 bp in length and spans an open reading frame from nucleotide 56 to 1021, encoding a 321aa protein. The PAP2d_v2 cDNA is 1707 bp in length encoding a 316aa protein from nucleotide 56-1006. The PAP2d_v1 cDNA is 15 bp longer than the PAP2d_v2 cDNA in the terminal of the fifth exon and it creates different ORF. Both of the proteins contain a well-conserved PAP2 motif. The PAP2d_v1 is mainly expressed in human brain, lung, kidney, testis and colon, while PAP2d_v2 is restricted to human placenta, skeletal muscle, and kidney. The two splice variants are co-expressed only in kidney.

  7. Gram stain of skin lesion

    MedlinePlus

    ... Names Skin lesion gram stain Images Viral lesion culture References Hall GS, Woods GL. Medical bacteriology. In: McPherson RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods . 22nd ed. Philadelphia, PA: Elsevier ...

  8. Two potential fish glycerol-3-phosphate phosphatases.

    PubMed

    Raymond, James A

    2015-06-01

    Winter-acclimated rainbow smelt (Osmerus mordax Mitchill) produce high levels of glycerol as an antifreeze. A common pathway to glycerol involves the enzyme glycerol-3-phosphate phosphatase (GPP), but no GPP has yet been identified in fish or any other animal. Here, two phosphatases assembled from existing EST libraries (from winter-acclimated smelt and cold-acclimated smelt hepatocytes) were found to resemble a glycerol-associated phosphatase from a glycerol-producing alga, Dunaliella salina, and a recently discovered GPP from a bacterium, Mycobacterium tuberculosis. Recombinant proteins were generated and were found to have GPP activity on the order of a few μMol Pi/mg enzyme/min. The two enzymes have acidic pH optima (~5.5) similar to that previously determined for GPP activity in liver tissue, with about 1/3 of their peak activities at neutral pH. The two enzymes appear to account for the GPP activity of smelt liver, but due to their reduced activities at neutral pH, their contributions to glycerol production in vivo remain unclear. Similar enzymes may be active in a glycerol-producing insect, Dendroctonus ponderosae.

  9. DAPI Staining of Drosophila Embryos.

    PubMed

    Rothwell, Wendy F; Sullivan, William

    2007-10-01

    INTRODUCTIONDrosophila embryos can be stained with specific fluorescent probes or antibodies through either direct or indirect immunofluorescence. In particular, several effective probes exist for visualizing DNA. 4',6-diamidino-2-phenylindole (DAPI) is a commonly used DNA-binding dye. Because it is specific for double-stranded DNA, no prior RNase treatment is required. While the embryo staining method described here uses DAPI, other fluorescent DNA probes can be processed similarly.

  10. An ecto-protein tyrosine phosphatase of Entamoeba histolytica induces cellular detachment by disruption of actin filaments in HeLa cells.

    PubMed

    Anaya-Ruiz, M; Pérez-Santos, J L M; Talamás-Rohana, P

    2003-07-01

    Actin cytoskeleton disruption in host cells has been demonstrated for PTPases from pathogenic microorganisms. In this work, we analysed whether the secreted acid phosphatase from Entamoeba histolytica has phosphotyrosine phosphatase activity and the possibility that this activity may participate in damaging host cells. The secreted acid phosphatase of E. histolytica, which catalyses p-nitrophenyl phosphate hydrolysis at acid pH values, was found to have phosphotyrosine phosphatase activity. The enzymatic properties of phosphotyrosine phosphatase and acid phosphatase were virtually identical and included: Km values of 10 x 10(-4) M, no requirement for divalent cations, and sensitivity to molybdate, vanadate, and tungstate. The phosphotyrosyl phosphatase activity caused significant levels of cell rounding and detachment correlating with disruption of the actin stress fibres in HeLa cells. Thus, our data suggest that secreted phosphotyrosine phosphatase could play a cytotoxic role during amoebic infection.

  11. Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing.

    PubMed

    Palić, Dušan; Beck, Linda S; Palić, Jelena; Andreasen, Claire B

    2011-02-01

    Studies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diff-quick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of lymphocytes, monocytes, platelets/thrombocytes and granulocytes was determined in all fish species. Arctic grayling, June sucker, and cutthroat trout had MPO positive granulocytes, while shovelnose sturgeon heterophils had positive reaction for leukocyte AP, but not MPO. Presence of MPO indicated potential to measure oxidative burst and degranulation of neutrophil primary granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO in shovelnose sturgeon suggested use of different enzyme marker (AP) in degranulation assay for this species. Standardization of cytochemical techniques allowed for rapid screening of leukocyte types, reducing the number of fish, time and effort to select adequate neutrophil function assays to be used in studies of health status in species of special concern.

  12. When one plus one equals more than two--a novel stain for renal biopsies is a combination of two classical stains.

    PubMed

    Brodsky, Sergey V; Albawardi, Alia; Satoskar, Anjali A; Nadasdy, Gyongyi; Nadasdy, Tibor

    2010-11-01

    Histologic evaluation of renal biopsies includes multiple ancillary stains, including Periodic acid-Schiff's (PAS) and Masson's trichrome (Trichrome). Herein we report an innovative double-stain, derived from two standard stains (PAS and Trichrome). This novel stain not only has advantages of both ancestor stains, but became more distinguishable and colorful, when basement membranes stain dark-violet, whereas the interstitial collagen remains blue. This allows the pathologist immediate estimation of the amount of collagen, tubular atrophy and the degree of interstitial fibrosis in one section. Using computer-based analysis, we confirmed that our innovative double-stain highlights interstitial collagen better than Trichrome stain alone. We strongly recommend renal pathologists to try this innovative stain in their practice.

  13. Structural Genomics of Protein Phosphatases

    SciTech Connect

    Almo,S.; Bonanno, J.; Sauder, J.; Emtage, S.; Dilorenzo, T.; Malashkevich, V.; Wasserman, S.; Swaminathan, S.; Eswaramoorthy, S.; et al

    2007-01-01

    The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.

  14. Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins.

    PubMed

    Holmes, C F B; Maynes, J T; Perreault, K R; Dawson, J F; James, M N G

    2002-11-01

    The protein serine/threonine phosphatases constitute a unique class of enzymes that are critical for cell regulation, as they must counteract the activities of thousands of protein kinases in human cells. Uncontrolled inhibition of phosphatase activity by toxic inhibitors can lead to widespread catastrophic effects. Over the past decade, a number of natural product toxins have been identified that specifically and potently inhibit protein phosphatase-1 and 2A. Amongst these are the cyanobacteria-derived cyclic heptapeptide microcystin-LR and the polyether fatty acid okadaic acid from dinoflagellate sources. The molecular mechanism underlying potent inhibition of protein phosphatase-1 by these toxins is becoming clear through insights gathered from diverse sources. These include: 1. Comparison of structure-activity relationships amongst the different classes of toxins. 2. Delineation of the structural differences between protein phosphatase-1 and 2A that account for their differing sensitivity to toxins, particularly okadaic acid and microcystin-LR. 3. Determination of the crystal structure of protein phosphatase-1 with microcystin-LR, okadaic acid and calyculin bound. 4. Site-specific mutagenesis and biochemical analysis of protein phosphatase-1 mutants. Taken together, these data point to a common binding site on protein phosphatase-1 for okadaic acid, microcystin-LR and the calyculins. However, careful analysis of these data suggest that each toxin binds to the common binding site in a subtly different way, relying on distinct structural interactions such as hydrophobic binding, hydrogen bonding and electrostatic interactions to different degrees. The insights derived from studying the molecular enzymology of protein phosphatase-1 may help explain the different sensitivities of other structurally conserved protein serine/theonine phosphatases to toxin inhibition. Furthermore, studies on the binding of structurally diverse toxins at the active site of protein

  15. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  16. Spatial Patterns of Alkaline Phosphatase Expression within Bacterial Colonies and Biofilms in Response to Phosphate Starvation

    PubMed Central

    Huang, Ching-Tsan; Xu, Karen D.; McFeters, Gordon A.; Stewart, Philip S.

    1998-01-01

    The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 μmol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor. PMID:9546188

  17. [A duplicate staining method for permanent specimen of Trichinella spiralis encapsulated larvae].

    PubMed

    Li, Dan; Yang, Ding; Pi, Ben-Wei; Niu, Li-Na; Zhang, Ying; Wang, Guo-Ying

    2012-04-30

    With single staining method, Trichinella spiralis encapsulated larvae specimens were fixed with formaldehyde alcohol acetic acid fixative solution, and stained with alcohol borax-carmine staining solution (4% borax solution 100 ml, carmine 1 g, and 70% alcohol 100 ml). With duplicate staining, the encapsulated larvae specimens were fixed with formaldehyde alcohol acetic acid fixative solution, and double stained with alcohol borax carmine staining solution and fast green staining solution (fast green 0.1 g, 95% alcohol 100 ml). The results showed that with single staining, it was not clear-cut between the cyst and muscle cells although the larva was differentiable, while with duplicate staining, the larva, cyst and muscle cells were distinguished more clearly.

  18. Desialylated alkaline phosphatase: activation by 4-nitrophenol.

    PubMed

    Nayudu, P R

    1984-01-01

    Mouse ileal alkaline phosphatase is a sialyl enzyme (12-14 moles per mole of enzyme). When partially desialylated by treatment with neuraminidase, the enzyme loses most of its activity, associated with reduced apparent Vmax and Km. Part of that loss, however, is recovered as the product 4-nitrophenol's concentration builds up in the cuvette. Experimental results are presented to demonstrate that the activation is due to the binding of 4-nitrophenol as a ligand by the partially desialylated enzyme and that both the loss of activity by sialic acid removal and activation by ligand-binding are correlated with changes in protein conformation.

  19. Leishmania amazonensis: characterization of an ecto-phosphatase activity.

    PubMed

    de Almeida-Amaral, Elmo Eduardo; Belmont-Firpo, Rodrigo; Vannier-Santos, Marcos André; Meyer-Fernandes, José Roberto

    2006-12-01

    We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.

  20. Sudan stain of fecal fat: new insight into an old test.

    PubMed

    Khouri, M R; Huang, G; Shiau, Y F

    1989-02-01

    The 72-h fecal fat determination is used as the gold standard to document the presence of steatorrhea. Although the Sudan stain for fecal fat is advocated as a sensitive screening test, a quantitative correlation between the 72-h fecal fat quantitation and the fecal Sudan stain is lacking. This study was designed to examine the staining properties of different classes of purified lipids in an experimentally defined artificial matrix, and to elucidate the reasons for the lack of quantitative correlation between these two tests. Our results indicate that the "neutral fat" stain without acidification or heating identifies triglyceride; and at an appropriate pH, the "neutral stain" also identifies fatty acid. The "split fat" stain with acidification and heating identifies both triglyceride and fatty acid. After acidification, fatty acid soaps are converted to the nonionized fatty acid. Thus, fatty acid soaps can be identified indirectly as fat droplets that are stained by the split fat stain. Although cholesterol is stained with Sudan stain after heating, upon cooling, cholesterol forms crystals of anhydrous cholesterol, making its staining pattern distinct. Neither the neutral fat nor the split fat stain can detect phospholipid or cholesteryl ester. The 72-h fecal fat determination is a measure of the total fatty acid content after a specimen is saponified. The resulting fatty acids are derived from a variety of endogenous and exogenous sources, including free fatty acids, soaps of fatty acids, triglycerides, cholesterol esters, and phospholipids. Therefore, the 72-h fecal fat quantitation does not differentiate between the primary sources of the measured fatty acid. It is concluded that the 72-h fecal fat determination is not specific for documenting triglyceride (fat) malabsorption. Until new methods are developed that specifically measure fecal triglyceride and fatty acid, the Sudan stain of fecal fat appears to be a more specific method for detecting the presence

  1. Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components.

    PubMed

    Belaldavar, C; Hallikerimath, S; Angadi, P V; Kale, A D

    2014-11-01

    The components of hard tissues including dentin, enamel, cementum, bone and other calcified deposits, and mature and immature collagen pose problems for identification in routine hematoxylin and eosin (H & E) stained sections. Use of combinations of stains can demonstrate the components of hard tissues and soft tissues distinctly. We assessed the efficacy of the Verde Luz-orange G-acid fuchsin (VOF) stain for differentiating hard and soft connective tissues and compared results with other histochemical staining techniques. Eighty tissue sections comprising developing tooth (30), ossifying fibroma (30) and miscellaneous pathologies (20) expected to contain varying types of calcified tissues were stained with H & E, VOF, and Masson's trichrome (MT). In developing tooth, VOF demonstrated better differentiation of hard tissues, while it was comparable to MT for ossifying fibroma and miscellaneous pathologies. The intensity of staining was greater with VOF than with the other stains studied. VOF stains hard tissue components distinctly and gives good contrast with the surrounding connective tissue. VOF is comparable to MT, but has added advantages including single step staining, rapid and easy procedures, and it distinguishes the maturity of the tissues.

  2. The five glucose-6-phosphatase paralogous genes are differentially regulated by insulin alone or combined with high level of amino acids and/or glucose in trout hepatocytes.

    PubMed

    Lucie, Marandel; Weiwei, Dai; Stéphane, Panserat; Sandrine, Skiba-Cassy

    2016-04-01

    A recent analysis of the newly sequenced rainbow trout (Oncorhynchus mykiss) genome suggested that duplicated gluconeogenic g6pc paralogues, fixed in this genome after the salmonid-specific 4th whole genome duplication, may have a role in the setting up of the glucose-intolerant phenotype in this carnivorous species. This should be due to the sub- or neo-functionalization of their regulation. In the present short communication we thus addressed the question of the regulation of these genes by insulin, hormone involved in the glucose homeostasis, and its interaction with glucose and amino acids in vitro. The stimulation of trout hepatocytes with insulin revealed an atypical up-regulation of g6pcb2 ohnologues and confirmed the sub- or neo-functionalization of the five g6pc genes at least at the regulatory level. Intriguingly, when hepatocytes were cultured with high levels of glucose and/or AAs in presence of insulin, most of the g6pc paralogues were up-regulated. It strongly suggested a cross-talk between insulin and nutrients for the regulation of these genes. Moreover these results strengthened the idea that g6pc duplicated genes may significantly contribute to the setting up of the glucose-intolerant phenotype in trout via their atypical regulation by insulin alone or in interaction with nutrients. These findings open new perspectives to better understand in vivo glucose-intolerant phenotype in trout fed a high carbohydrate diet.

  3. Array tomography: imaging stained arrays.

    PubMed

    Micheva, Kristina D; O'Rourke, Nancy; Busse, Brad; Smith, Stephen J

    2010-11-01

    Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are imaged using conventional wide-field fluorescence microscopy. Images can be captured manually or, with the appropriate software and hardware, the process can be automated.

  4. Phosphatase of Chlamydomonas reinhardi: biochemical and cytochemical approach with specific mutants.

    PubMed Central

    Matagne, R F; Loppes, R; Deltour, R

    1976-01-01

    The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate. Images PMID:4437

  5. MDP-1: A novel eukaryotic magnesium-dependent phosphatase.

    PubMed

    Selengut, J D; Levine, R L

    2000-07-18

    We report here the purification, cloning, expression, and characterization of a novel phosphatase, MDP-1. In the course of investigating the reported acid phosphatase activity of carbonic anhydrase III preparations, several discrete phosphatases were discerned. One of these, a magnesium-dependent species of 18.6 kDa, was purified to homogeneity and yielded several peptide sequences from which the parent gene was identified by database searching. Although orthologous genes were identified in fungi and plants as well as mammalian species, there was no apparent homology to any known family of phosphatases. The enzyme was expressed in Escherichia coli with a fusion tag and purified by affinity methods. The recombinant enzyme showed magnesium-dependent acid phosphatase activity comparable to the originally isolated rabbit protein. The enzyme catalyzes the rapid hydrolysis of p-nitrophenyl phosphate, ribose-5-phosphate, and phosphotyrosine. The selectivity for phosphotyrosine over phosphoserine or phosphothreonine is considerable, but the enzyme did not show activity toward five phosphotyrosine-containing peptides. None of the various substrates assayed (including various nucleotide, sugar, amino acid and peptide phosphates, phosphoinositides, and phosphodiesters) exhibited K(M) values lower than 1 mM, and many showed negligible rates of hydrolysis. The enzyme is inhibited by vanadate and fluoride but not by azide, cyanide, calcium, lithium, or tartaric acid. Chemical labeling, refolding, dialysis, and mutagenesis experiments suggest that the enzymatic mechanism is not dependent on cysteine, histidine, or nonmagnesium metal ions. In recognition of these observations, the enzyme has been given the name magnesium-dependent phosphatase-1 (MDP-1).

  6. Human pyridoxal phosphatase. Molecular cloning, functional expression, and tissue distribution.

    PubMed

    Jang, Young Min; Kim, Dae Won; Kang, Tae-Cheon; Won, Moo Ho; Baek, Nam-In; Moon, Byung Jo; Choi, Soo Young; Kwon, Oh-Shin

    2003-12-12

    Pyridoxal phosphatase catalyzes the dephosphorylation of pyridoxal 5'-phosphate (PLP) and pyridoxine 5'-phosphate. A human brain cDNA clone was identified to the PLP phosphatase on the basis of peptide sequences obtained previously. The cDNA predicts a 296-amino acid protein with a calculated Mr of 31698. The open reading frame is encoded by two exons located on human chromosome 22q12.3, and the exon-intron junction contains the GT/AG consensus splice site. In addition, a full-length mouse PLP phosphatase cDNA of 1978 bp was also isolated. Mouse enzyme encodes a protein of 292 amino acids with Mr of 31512, and it is localized on chromosome 15.E1. Human and mouse PLP phosphatase share 93% identity in protein sequence. A BLAST search revealed the existence of putative proteins in organism ranging from bacteria to mammals. Catalytically active human PLP phosphatase was expressed in Escherichia coli, and characteristics of the recombinant enzyme were similar to those of erythrocyte enzyme. The recombinant enzyme displayed Km and kcat values for pyridoxal of 2.5 microM and 1.52 s(-1), respectively. Human PLP phosphatase mRNA is differentially expressed in a tissue-specific manner. A single mRNA transcript of 2.1 kb was detected in all human tissues examined and was highly abundant in the brain. Obtaining the molecular properties for the human PLP phosphatase may provide new direction for investigating metabolic pathway involving vitamin B6.

  7. A conserved phosphatase cascade that regulates nuclear membrane biogenesis.

    PubMed

    Kim, Youngjun; Gentry, Matthew S; Harris, Thurl E; Wiley, Sandra E; Lawrence, John C; Dixon, Jack E

    2007-04-17

    A newly emerging family of phosphatases that are members of the haloacid dehalogenase superfamily contains the catalytic motif DXDX(T/V). A member of this DXDX(T/V) phosphatase family known as Dullard was recently shown to be a potential regulator of neural tube development in Xenopus [Satow R, Chan TC, Asashima M (2002) Biochem Biophys Res Commun 295:85-91]. Herein, we demonstrate that human Dullard and the yeast protein Nem1p perform similar functions in mammalian cells and yeast cells, respectively. In addition to similarity in primary sequence, Dullard and Nem1p possess similar domains and show similar substrate preferences, and both localize to the nuclear envelope. Additionally, we show that human Dullard can rescue the aberrant nuclear envelope morphology of nem1Delta yeast cells, functionally replacing Nem1p. Finally, Nem1p, has been shown to deposphorylate the yeast phosphatidic acid phosphatase Smp2p [Santos-Rosa H, Leung J, Grimsey N, Peak-Chew S, Siniossoglou S (2005) EMBO J 24:1931-1941], and we show that Dullard dephosphorylates the mammalian phospatidic acid phosphatase, lipin. Therefore, we propose that Dullard participates in a unique phosphatase cascade regulating nuclear membrane biogenesis, and that this cascade is conserved from yeast to mammals.

  8. Assay of phosphotyrosyl protein phosphatase using synthetic peptide 1142-1153 of the insulin receptor.

    PubMed

    King, M J; Sale, G J

    1988-09-12

    Synthetic peptide 1142-1153 of the insulin receptor was phosphorylated on tyrosine by the insulin receptor and found to be a potent substrate for dephosphorylation by rat liver particulate and soluble phosphotyrosyl protein phosphatases. Apparent Km values were approximately 5 microM. Vm values (nmol phosphate removed/min per mg protein) were 0.62 (particulate) and 0.2 (soluble). This corresponds to 80% of total activity being membrane-associated, indicating that membrane-bound phosphatases are important receptor phosphatases. The phosphatase activities were distinct from acid and alkaline phosphatase. In conclusion peptide 1142-1153 provides a useful tool for the further study and characterization of phosphotyrosyl protein phosphatases.

  9. Stain-Decolorize-Stain (SDS): a new technique for multiple staining.

    PubMed

    Li, Jing; Zhou, Yan; Gu, Jiang

    2014-03-01

    Multiple staining of more than one gene/antigen on a single tissue section is an indispensable tool in cell and tissue research. However, most of the available multiple staining techniques have limitations, and there has been no technique to simultaneously visualize and distinguish tissue antigens, nucleotide sequences and other chemical compounds on the same slide. Here, we present a practical and economic multiple stain technique, with which multiple cellular components including mRNA (with in situ hybridization), antigen epitope (with immunohistochemistry) and chemical molecules (with histochemistry) can be stained on a single tissue section to study their relationship. In addition, this technique also offers the possibility to evaluate morphology with an H&E staining on the same sections. We used the placenta, pancreas, breast ductal carcinoma, colon adenocarcinoma, cerebellum, tonsil and heart tissue sections to evaluate the applicability of this new technique. The sensitivity and specificity of the technique have been tested, and an optimal protocol is recommended. Its applications in surgical pathology and research are discussed. This technique offers a novel tool to evaluate the relationship among multiple components at the same or adjacent locations to meet the needs of pathology diagnosis and research.

  10. F-actin staining of Drosophila testes.

    PubMed

    Bonaccorsi, Silvia; Giansanti, Maria G; Cenci, Giovanni; Gatti, Maurizio

    2012-01-01

    Preparations of Drosophila testes fixed with paraformaldehyde can be stained for F-actin according to the protocol described here. This staining procedure is particularly suitable for staining the male fusome and the cytokinetic contractile ring.

  11. Automated single-slide staining system

    NASA Technical Reports Server (NTRS)

    Mills, S. M.; Wilkins, J. R.

    1974-01-01

    Apparatus developed to Gram-stain single slides automatically is flexible enough to accommodate other types of staining procedures. Method frees operator and eliminates necessity for subjective evaluations as to length of staining or decolorizing time.

  12. Staining of intracellular deposits of uranium in cultured murine macrophages.

    PubMed

    Kalinich, J F; McClain, D E

    2001-01-01

    In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylenediaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.

  13. The PYL4 A194T Mutant Uncovers a Key Role of PYR1-LIKE4/PROTEIN PHOSPHATASE 2CA Interaction for Abscisic Acid Signaling and Plant Drought Resistance1[C][W][OPEN

    PubMed Central

    Pizzio, Gaston A.; Rodriguez, Lesia; Antoni, Regina; Gonzalez-Guzman, Miguel; Yunta, Cristina; Merilo, Ebe; Kollist, Hannes; Albert, Armando; Rodriguez, Pedro L.

    2013-01-01

    Because abscisic acid (ABA) is recognized as the critical hormonal regulator of plant stress physiology, elucidating its signaling pathway has raised promise for application in agriculture, for instance through genetic engineering of ABA receptors. PYRABACTIN RESISTANCE1/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS ABA receptors interact with high affinity and inhibit clade A phosphatases type-2C (PP2Cs) in an ABA-dependent manner. We generated an allele library composed of 10,000 mutant clones of Arabidopsis (Arabidopsis thaliana) PYL4 and selected mutations that promoted ABA-independent interaction with PP2CA/ABA-HYPERSENSITIVE3. In vitro protein-protein interaction assays and size exclusion chromatography confirmed that PYL4A194T was able to form stable complexes with PP2CA in the absence of ABA, in contrast to PYL4. This interaction did not lead to significant inhibition of PP2CA in the absence of ABA; however, it improved ABA-dependent inhibition of PP2CA. As a result, 35S:PYL4A194T plants showed enhanced sensitivity to ABA-mediated inhibition of germination and seedling establishment compared with 35S:PYL4 plants. Additionally, at basal endogenous ABA levels, whole-rosette gas exchange measurements revealed reduced stomatal conductance and enhanced water use efficiency compared with nontransformed or 35S:PYL4 plants and partial up-regulation of two ABA-responsive genes. Finally, 35S:PYL4A194T plants showed enhanced drought and dehydration resistance compared with nontransformed or 35S:PYL4 plants. Thus, we describe a novel approach to enhance plant drought resistance through allele library generation and engineering of a PYL4 mutation that enhances interaction with PP2CA. PMID:23864556

  14. Lily pollen alkaline phytase is a histidine phosphatase similar to mammalian multiple inositol polyphosphate phosphatase (MINPP).

    PubMed

    Mehta, Bakul Dhagat; Jog, Sonali P; Johnson, Steven C; Murthy, Pushpalatha P N

    2006-09-01

    Phytic acid is the most abundant inositol phosphate in cells; it constitutes 1-5% of the dry weight of cereal grains and legumes. Phytases are the primary enzymes responsible for the hydrolysis of phytic acid and thus play important roles in inositol phosphate metabolism. A novel alkaline phytase in lily pollen (LlALP) was recently purified in our laboratory. In this paper, we describe the cloning and characterization of LlALP cDNA from lily pollen. Two isoforms of alkaline phytase cDNAs, LlAlp1 and LlAlp2, which are 1467 and 1533 bp long and encode proteins of 487 and 511 amino acids, respectively, were identified. The deduced amino acid sequences contains the signature heptapeptide of histidine phosphatases, -RHGXRXP-, but shares < 25% identity to fungal histidine acid phytases. Phylogenetic analysis reveals that LlALP is most closely related to multiple inositol polyphosphate phosphatase (MINPP) from humans (25%) and rats (23%). mRNA corresponding to LlAlp1 and LlAlp2 were expressed in leaves, stem, petals and pollen grains. The expression profiles of LlAlp isoforms in anthers indicated that mRNA corresponding to both isoforms were present at all stages of flower development. The expression of LlAlp2 cDNA in Escherichia coli revealed the accumulation of the active enzyme in inclusion bodies and confirmed that the cDNA encodes an alkaline phytase. In summary, plant alkaline phytase is a member of the histidine phosphatase family that includes MINPP and exhibits properties distinct from bacterial and fungal phytases.

  15. Mechanism of the phosphatase component of Clostridium thermocellum polynucleotide kinase-phosphatase.

    PubMed

    Keppetipola, Niroshika; Shuman, Stewart

    2006-01-01

    Polynucleotide kinase-phosphatase (Pnkp) from Clostridium thermocellum catalyzes ATP-dependent phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and Ni(2+)/Mn(2+)-dependent dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp is an 870-amino-acid polypeptide composed of three domains: an N-terminal module similar to bacteriophage T4 polynucleotide kinase, a central module that resembles the dinuclear metallo-phosphoesterase superfamily, and a C-terminal ligase-like adenylyltransferase domain. Here we conducted a mutational analysis of CthPnkp that identified 11 residues required for Ni(2+)-dependent phosphatase activity with 2'-AMP and 3'-AMP. Eight of the 11 CthPnkp side chains were also required for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. The ensemble of essential side chains includes the conserved counterparts (Asp187, His189, Asp233, Arg237, Asn263, His264, His323, His376, and Asp392 in CthPnkp) of all of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. Three residues (Asp236, His264, and Arg237) required for activity with 2'-AMP or 3'-AMP were dispensable for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. Our findings, together with available structural information, provide fresh insights to the metallophosphoesterase mechanism, including the roles of His264 and Asp236 in proton donation to the leaving group. Deletion analysis defined an autonomous phosphatase domain, CthPnkp-(171-424).

  16. Whole Blood Cell Staining Device

    NASA Technical Reports Server (NTRS)

    Sams, Clarence F.; Clift, Vaughan L.; McDonald, Kelly E.

    2000-01-01

    An apparatus and method for staining particular cell markers is disclosed. The apparatus includes a flexible tube that is reversibly pinched into compartments with one or more clamps. Each compartment of the tube contains a separate reagent and is in selective fluid communication with adjoining compartments.

  17. Phosphatase activity on the cell wall of Fonsecaea pedrosoi.

    PubMed

    Kneipp, L F; Palmeira, V F; Pinheiro, A A S; Alviano, C S; Rozental, S; Travassos, L R; Meyer-Fernandes, J R

    2003-12-01

    The activity of a phosphatase was characterized in intact mycelial forms of Fonsecaea pedrosoi, a pathogenic fungus that causes chromoblastomycosis. At pH 5.5, this fungus hydrolyzed p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 12.78 +/- 0.53 nmol p-NP per h per mg hyphal dry weight. The values of Vmax and apparent Km for p-NPP hydrolyses were measured as 17.89 +/- 0.92 nmol p-NP per h per mg hyphal dry weight and 1.57 +/- 0.26 mmol/l, respectively. This activity was inhibited at increased pH, a finding compatible with an acid phosphatase. The enzymatic activity was strongly inhibited by classical inhibitors of acid phosphatases such as sodium orthovanadate (Ki = 4.23 micromol/l), sodium molybdate (Ki = 7.53 micromol/l) and sodium fluoride (Ki = 126.78 micromol/l) in a dose-dependent manner. Levamizole (1 mmol/l) and sodium tartrate (10 mmol/l), had no effect on the enzyme activity. Cytochemical localization of the acid phosphatase showed electrondense cerium phosphate deposits on the cell wall, as visualized by transmission electron microscopy. Phosphatase activity in F. pedrosoi seems to be associated with parasitism, as sclerotic cells, which are the fungal forms mainly detected in chromoblastomycosis lesions, showed much higher activities than conidia and mycelia did. A strain of F. pedrosoi recently isolated from a human case of chromoblastomycosis also showed increased enzyme activity, suggesting that the expression of surface phosphatases may be stimulated by interaction with the host.

  18. Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

    PubMed

    Scollar, M P; Sigal, G; Klibanov, A M

    1985-03-01

    Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

  19. Tyrosine phosphatases as key regulators of StAR induction and cholesterol transport: SHP2 as a potential tyrosine phosphatase involved in steroid synthesis.

    PubMed

    Cooke, Mariana; Mele, Pablo; Maloberti, Paula; Duarte, Alejandra; Poderoso, Cecilia; Orlando, Ulises; Paz, Cristina; Cornejo Macie