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Sample records for patients alpha-amylase inhibitors

  1. Proteinaceous alpha-amylase inhibitors.

    PubMed

    Svensson, Birte; Fukuda, Kenji; Nielsen, Peter K; Bønsager, Birgit C

    2004-02-12

    Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous alpha-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha-amylases in complex with inhibitors from five families. These structures indicate major diversity but also some similarity in the structural basis of alpha-amylase inhibition. Mutational analysis of the mechanism of inhibition was performed in a few cases and various protein engineering and biotechnological approaches have been outlined for exploitation of the inhibitory function. PMID:14871655

  2. Zinc oxide nanoparticles as novel alpha-amylase inhibitors

    NASA Astrophysics Data System (ADS)

    Dhobale, Sandip; Thite, Trupti; Laware, S. L.; Rode, C. V.; Koppikar, Soumya J.; Ghanekar, Ruchika-Kaul; Kale, S. N.

    2008-11-01

    Amylase inhibitors, also known as starch blockers, contain substances that prevent dietary starches from being absorbed by the body via inhibiting breakdown of complex sugars to simpler ones. In this sense, these materials are projected as having potential applications in diabetes control. In this context, we report on zinc oxide nanoparticles as possible alpha-amylase inhibitors. Zinc oxide nanoparticles have been synthesized using soft-chemistry approach and 1-thioglycerol was used as a surfactant to yield polycrystalline nanoparticles of size ˜18 nm, stabilized in wurtzite structure. Conjugation study and structural characterization have been done using x-ray diffraction technique, Fourier transform infrared spectroscopy, UV-visible spectroscopy, and transmission electron microscopy. Cytotoxicity studies on human fibrosarcoma (HT-1080) and skin carcinoma (A-431) cell lines as well as mouse primary fibroblast cells demonstrate that up to a dose of 20 μg/ml, ZnO nanoparticles are nontoxic to the cells. We report for the first time the alpha-amylase inhibitory activity of ZnO nanoparticles wherein an optimum dose of 20 μg/ml was sufficient to exhibit 49% glucose inhibition at neutral pH and 35 °C temperature. This inhibitory activity was similar to that obtained with acarbose (a standard alpha-amylase inhibitor), thereby projecting ZnO nanoparticles as novel alpha-amylase inhibitors.

  3. A chimera-like alpha-amylase inhibitor suggesting the evolution of Phaseolus vulgaris alpha-amylase inhibitor.

    PubMed

    Wato, S; Kamei, K; Arakawa, T; Philo, J S; Wen, J; Hara, S; Yamaguchi, H

    2000-07-01

    White kidney bean (Phaseolus vulgaris) contains two kinds of alpha-amylase inhibitors, one heat-stable (alpha AI-s) and one heat-labile (alpha AI-u). alpha AI-s has recently been revealed to be a tetrameric complex, alpha(2)beta(2), with two active sites [Kasahara et al. (1996) J. Biochem. 120, 177-183]. The present study was undertaken to reveal the molecular features of alpha AI-u, which is composed of three kinds of subunits, alpha, beta, and gamma. The gamma-subunit, in contrast to the alpha- and beta-subunits that are indistinguishable from the alpha- and beta-subunits of alpha AI-s, was found to correspond to a subunit of an alpha-amylase inhibitor-like protein, which has been identified as an inactive, evolutionary intermediate between arcelin and the alpha-amylase inhibitor in a P. vulgaris defense protein family. The polypeptide molecular weight of alpha AI-u determined by the light-scattering technique, together with the polypeptide molecular weights of the subunits, suggests that alpha AI-u is a trimeric complex, alpha beta gamma. The inhibition of alpha AI-u by increasing amounts of porcine pancreatic alpha-amylase (PPA) indicates that an inactive 1:1 complex is formed between alpha AI-u and PPA. Molecular weight estimation of the complex by the light-scattering technique confirmed that it is a complex of alpha AI-u with one PPA molecule. Thus it seems probable that alpha AI-u is an evolutionary intermediate of the P. vulgaris alpha-amylase inhibitor. PMID:10876168

  4. Cross-inhibitory activity of cereal protein inhibitors against alpha-amylases and xylanases.

    PubMed

    Sancho, Ana I; Faulds, Craig B; Svensson, Birte; Bartolomé, Begoña; Williamson, Gary; Juge, Nathalie

    2003-08-21

    The purification and characterisation of a xylanase inhibitor (XIP-I) from wheat was reported previously. In our current work, XIP-I is also demonstrated to have the capacity to inhibit the two barley alpha-amylase isozymes (AMY1 and AMY2). XIP-I completely inhibited the activity of AMY1 and AMY2 towards insoluble Blue Starch and a soluble hepta-oligosaccharide derivative. A ternary complex was formed between insoluble starch, a catalytically inactive mutant of AMY1 (D180A), and XIP-I, suggesting that the substrate-XIP-I interaction is necessary for inhibition of barley alpha-amylases. K(i) values for alpha-amylase inhibition, however, could not be calculated due to the nonlinear nature of the inhibition pattern. Furthermore, surface plasmon resonance and gel electrophoresis did not indicate interaction between XIP-I and the alpha-amylases. The inhibition was abolished by CaCl(2), indicating that the driving force for the interaction is different from that of complexation between the barley alpha-amylase/subtilisin inhibitor (BASI) and AMY2. This is the first report of a proteinaceous inhibitor of AMY1. BASI, in addition, was demonstrated to partially inhibit the endo-1,4-beta-D-xylanase from Aspergillus niger (XylA) of glycoside hydrolase family 11. Taken together, the data demonstrate for the first time the dual target enzyme specificity of BASI and XIP-I inhibitors for xylanase and alpha-amylase. PMID:12922177

  5. Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering.

    PubMed

    Nielsen, Peter K; Bønsager, Birgit C; Fukuda, Kenji; Svensson, Birte

    2004-02-12

    Bifunctional alpha-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous alpha-amylase action. The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits the barley alpha-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunitz-type trypsin inhibitor family of the beta-trefoil fold proteins. Diverse approaches including site-directed mutagenesis, hybrid constructions, and crystallography have been used to characterise the structures and contact residues in the AMY2/BASI complex. The three-dimensional structure of the AMY2/BASI complex is characterised by a completely hydrated Ca2+ situated at the protein interface that connects the three catalytic carboxyl groups in AMY2 with side chains in BASI via water molecules. Using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), we have recently demonstrated Ca2+-modulated kinetics of the AMY2/BASI interaction and found that the complex formation involves minimal structural changes. The modulation of the interaction by calcium ions makes it unique among the currently known binding mechanisms of proteinaceous alpha-amylase inhibitors. PMID:14871656

  6. A lectin gene encodes the alpha-amylase inhibitor of the common bean.

    PubMed Central

    Moreno, J; Chrispeels, M J

    1989-01-01

    An alpha-amylase inhibitor that inhibits insect and mammalian alpha-amylases but not plant alpha-amylases, is present in seeds of the common bean (Phaseolus vulgaris). We have purified the alpha-amylase inhibitor by using a selective heat treatment in acidic medium and affinity chromatography with porcine pancreas alpha-amylase coupled to agarose. Under sodium dodecyl sulfate gel electrophoresis, the purified inhibitor gave rise to five bands with mobilities corresponding to molecular masses ranging from 14 to 19 kDa. N-terminal sequencing (up to 15 amino acids) of the polypeptides obtained from these bands resulted in only two different sequences matching two stretches of the amino acid sequence deduced from an already described lectin gene [Hoffman, L. M. (1984) J. Mol. Appl. Gen. 2,447-453]. This gene is different from but closely related to the genes that code for phytohemagglutinin, the major lectin of bean. Further evidence based on amino acid composition, identification of a precursor, and recognition of the product of the gene (expressed in Escherichia coli) by an anti-alpha-amylase inhibitor serum confirms that the inhibitor is encoded by this or a closely related lectin gene. This finding assigns a biological function, which has been described at the molecular level, to a plant lectin gene product and supports the defense role postulated for seed lectins. The lack of homology with other families of enzyme inhibitors suggests that this may be the first member of a new family of plant enzyme inhibitors. Images PMID:2682631

  7. Differentiation of alpha-amylase from various sources: an approach using selective inhibitors.

    PubMed

    Quarino, L; Hess, J; Shenouda, M; Ristenbatt, R R; Gold, J; Shaler, R C

    1993-01-01

    A radial diffusion assay in an agarose/starch gel utilizing crude kidney bean extract and a commercially prepared alpha-amylase inhibitor isolated from wheat seeds was developed and assessed to determine its ability to differentiate alpha-amylase from various sources. Kidney bean extract was found to have a greater inhibitory effect on AMY2, while the wheat lectin inhibitor was found to have a greater inhibitory effect on AMY1. Neither inhibitor was found to have any effect on commercially prepared bacterial alpha-amylase extract in both liquid preparations and dried stains. Mixtures of varying concentrations of pancreatic and salivary extracts also gave interpretable results. Additionally, dried stains prepared from human body fluids having high levels of AMY2 were differentiated from dried stains prepared from human body fluids containing high levels of AMY1. PMID:8360608

  8. The bean. alpha. -amylase inhibitor is encoded by a lectin gene

    SciTech Connect

    Moreno, J.; Altabella, T.; Chrispeels, M.J. )

    1989-04-01

    The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has the same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.

  9. Crystal structures of human pancreatic alpha-amylase in complex with carbohydrate and proteinaceous inhibitors.

    PubMed Central

    Nahoum, V; Roux, G; Anton, V; Rougé, P; Puigserver, A; Bischoff, H; Henrissat, B; Payan, F

    2000-01-01

    Crystal structures of human pancreatic alpha-amylase (HPA) in complex with naturally occurring inhibitors have been solved. The tetrasaccharide acarbose and a pseudo-pentasaccharide of the trestatin family produced identical continuous electron densities corresponding to a pentasaccharide species, spanning the -3 to +2 subsites of the enzyme, presumably resulting from transglycosylation. Binding of the acarviosine core linked to a glucose residue at subsites -1 to +2 appears to be a critical part of the interaction process between alpha-amylases and trestatin-derived inhibitors. Two crystal forms, obtained at different values of pH, for the complex of HPA with the protein inhibitor from Phaseolus vulgaris (alpha-amylase inhibitor) have been solved. The flexible loop typical of the mammalian alpha-amylases was shown to exist in two different conformations, suggesting that loop closure is pH-sensitive. Structural information is provided for the important inhibitor residue, Arg-74, which has not been observed previously in structural analyses. PMID:10657258

  10. Structural studies of wheat monomeric and dimeric protein inhibitors of alpha-amylase.

    PubMed Central

    Petrucci, T; Sannia, G; Parlamenti, R; Silano, V

    1978-01-01

    Two wheat monomeric protein inhibitors of alpha-amylase with mol.wt. 12000, designated inhibitors 0.28 and 0.39 according to their gel-electrophoretic mobilities, showed almost identical circular-dichroism spectra in both the far and near u.v. at different pH values as well as in the presence or absence of dissociating and reducing agents. Both inhibitors (0.28 and 0.39) were readily inactivated by reduction of the five disulphide bridges present in each inhibitor molecule. These properties are very similar to those exhibited by the wheat dimeric protein inhibitor of alpha-amylase with mol.wt. 24000, designated inhibitor 0.19 according to its gel-electrophoretic mobility. The N-terminal sequence of the 0.19 inhibitor was determined without separating its subunits and compared with that of the 0.28 inhibitor reported by Redman [(1976) Biochem. J. 155, 193--195]. Petide 'maps' from tryptic digests of reduced and carboxymethylated inhibitors 0.19 and 0.28 were compared. One molecule of reducing sugar is covalently bound per inhibitor-0.19 protomer and inhibitor-0.28 molecule. The results obtained strongly support previous findings indicating the structural equivalence of inhibitor 0.28 with each inhibitor-0.19 protomer and the common phylogenetic origin of these protein alpha-amylase inhibitors from wheat kernel. PMID:308369

  11. Insecticidal activity of an alpha-amylase inhibitor-like protein resembling a putative precursor of alpha-amylase inhibitor in the common bean, Phaseolus vulgaris L.

    PubMed

    Ishimoto, M; Yamada, T; Kaga, A

    1999-06-15

    alpha-Amylase inhibitor (alphaAI) in the common bean, Phaseolus vulgaris L., protects seeds from insect pests such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (C. chinensis). Cultivars which lack alphaAI still show resistance to both bruchids. These cultivars have a glycoprotein that reacts with anti-alphaAI-1 antibodies. The glycoprotein with a molecular mass of 29 kDa (Gp29) was purified and the encoding gene was isolated. The primary structure of Gp29 is the same as alpha-amylase inhibitor-like protein (AIL) from which the encoding gene has already been isolated. AIL resembles a putative precursor of alphaAI, even though it does not form the active inhibitor. However, AIL has some inhibitory effect on the growth of C. maculatus but not C. chinensis. The presence of AIL alone is insufficient to explain the bruchid resistance of common bean cultivars lacking alpha-AI. Common bean seeds appear to contain several factors responsible for the bruchid resistance. PMID:10366733

  12. Characterization, crystallization and preliminary X-ray crystallographic analysis of the complex between barley alpha-amylase and the bifunctional alpha-amylase/subtilisin inhibitor from barley seeds.

    PubMed

    Vallée, F; Kadziola, A; Bourne, Y; Abe, J; Svensson, B; Haser, R

    1994-02-11

    The complex between a member of the barley malt alpha-amylase isozyme 2 family (AMY2-2), and the endogenous bifunctional alpha-amylase/subtilisin inhibitor, BASI, has been crystallized by the hanging drop vapour diffusion technique at a AMY2-2: BASI molar ratio of 1:1. Crystals have been grown within 4 days from solutions containing polyethylene glycol and calcium chloride. Analysis of single crystals by gel electrophoresis showed the presence of both proteins in the crystal lattice. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 74.5 A, b = 96.9 A, c = 171.3 A and they diffract to 2.0 A resolution. The presence of two molecules of the 1:1 complex in the asymmetric unit gives a solvent content of 45% by volume. The 1:1 stoichiometry of the complex was confirmed by the molecular replacement method, using as a search model the recently determined three-dimensional structure of the barley alpha-amylase. PMID:8107117

  13. Screening alpha-glucosidase and alpha-amylase inhibitors from natural compounds by molecular docking in silico.

    PubMed

    Jhong, Chien-Hung; Riyaphan, Jirawat; Lin, Shih-Hung; Chia, Yi-Chen; Weng, Ching-Feng

    2015-01-01

    The alpha-glucosidase inhibitor is a common oral anti-diabetic drug used for controlling carbohydrates normally converted into simple sugars and absorbed by the intestines. However, some adverse clinical effects have been observed. The present study seeks an alternative drug that can regulate the hyperglycemia by down-regulating alpha-glucosidase and alpha-amylase activity by molecular docking approach to screen the hyperglycemia antagonist against alpha-glucosidase and alpha-amylase activities from the 47 natural compounds. The docking data showed that Curcumin, 16-hydroxy-cleroda-3,13-dine-16,15-olide (16-H), Docosanol, Tetracosanol, Antroquinonol, Berberine, Catechin, Quercetin, Actinodaphnine, and Rutin from 47 natural compounds had binding ability towards alpha-amylase and alpha-glucosidase as well. Curcumin had a better biding ability of alpha-amylase than the other natural compounds. Analyzed alpha-glucosidase activity reveals natural compound inhibitors (below 0.5 mM) are Curcumin, Actinodaphnine, 16-H, Quercetin, Berberine, and Catechin when compared to the commercial drug Acarbose (3 mM). A natural compound with alpha-amylase inhibitors (below 0.5 mM) includes Curcumin, Berberine, Docosanol, 16-H, Actinodaphnine/Tetracosanol, Catechin, and Quercetin when compared to Acarbose (1 mM). When taken together, the implication is that molecular docking is a fast and effective way to screen alpha-glucosidase and alpha-amylase inhibitors as lead compounds of natural sources isolated from medicinal plants. PMID:26154585

  14. Molecular characterization of a bean alpha-amylase inhibitor that inhibits the alpha-amylase of the mexican bean weevil Zabrotes subfasciatus.

    PubMed

    Grossi de Sa, M F; Mirkov, T E; Ishimoto, M; Colucci, G; Bateman, K S; Chrispeels, M J

    1997-01-01

    Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an alpha-amylase inhibitor (alpha AI-1) that inhibits porcine pancreatic alpha-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of alpha AI-1, called alpha AI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant alpha AI-2 inhibits Z. subfasciatus alpha-amylase (ZSA), but not PPA. We purified alpha AI-2 and studied its interaction with ZSA. The formation of the alpha AI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode alpha AI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes alpha AI-2. The inhibitors alpha AI-1 and alpha AI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the alpha AI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction. PMID:9431678

  15. Effects of alpha-amylase and its inhibitors on acid production from cooked starch by oral streptococci.

    PubMed

    Aizawa, S; Miyasawa-Hori, H; Nakajo, K; Washio, J; Mayanagi, H; Fukumoto, S; Takahashi, N

    2009-01-01

    This study evaluated acid production from cooked starch by Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus mitis, and the effects of alpha-amylase inhibitors (maltotriitol and acarbose) and xylitol on acid production. Streptococcal cell suspensions were anaerobically incubated with various carbohydrates that included cooked potato starch in the presence or absence of alpha-amylase. Subsequently, the fall in pH and the acid production rate at pH 7.0 were measured. In addition, the effects of adding alpha-amylase inhibitors and xylitol to the reaction mixture were evaluated. In the absence of alpha-amylase, both the fall in pH and the acid production rate from cooked starch were small. On the other hand, in the presence of alpha-amylase, the pH fell to 3.9-4.4 and the acid production rate was 0.61-0.92 micromol per optical density unit per min. These values were comparable to those for maltose. When using cooked starch, the fall in pH by S. sanguinis and S. mitis was similar to that by S. mutans and S. sobrinus. For all streptococci, alpha-amylase inhibitors caused a decrease in acid production from cooked starch, although xylitol only decreased acid production by S. mutans and S. sobrinus. These results suggest that cooked starch is potentially acidogenic in the presence of alpha-amylase, which occurs in the oral cavity. In terms of the acidogenic potential of cooked starch, S. sanguinis and S. mitis were comparable to S. mutans and S. sobrinus. Alpha-amylase inhibitors and xylitol might moderate this activity. PMID:19136828

  16. Isolation and activity of an alpha-amylase inhibitor from white kidney beans.

    PubMed

    Zhang, Xiao-qi; Yang, Ming-yan; Ma, Yu; Tian, Jia; Song, Ji-Rong

    2007-12-01

    An alpha-amylase inhibitor (alpha-AI) was isolated from white kidney beans (Phaseolus vulgaris L) by ethanol fractional precipitation, ion exchange chromatography and gel filtration column chromatography. It was a homogeneity glycoprotein demonstrated by SDS-PAGE and gel filtration on CL-6B. The glycoprotein contained 88.2% protein and was rich in aspartic acid, glutamic acid, leucine, threonine and serine. The carbohydrate moiety was consisted of Man, Glc, Gal and Xyl in a mole ratio of 2.42: 1.50: 1.52: 1.00. The glycan and the core protein backbone was connected by O-linkage as determined by beta-elimination reaction. The continuous oral administration of the alpha-AI (150 mg x kg(-1) x d(-1)) for 7 days can lower fasting blood glucose and 300 mg x kg(-1) x d(-1) alpha-AI for 7 days can improve the sugar tolerance on alloxan-dependent diabetic model rats. The result showed the alpha-AI obtained from white kidney beans had good hypoglycemic effect on alloxan induced diabetic rats and may have high potential pharmaceutical value as a regulative digestive-starch degradation in patients suffering from diabetes. PMID:18338641

  17. Protein structures of common bean (Phaseolus vulgaris) alpha-amylase inhibitors.

    PubMed

    Lee, Shih-Chieh; Gepts, Paul L; Whitaker, John R

    2002-10-23

    Two nucleotide sequences for genes that encode alpha-amylase inhibitor 4 (alphaAI-4) from white kidney bean (WKB) cv. 858, designated gene alphaAI-4 (Accession No. ), and alpha-amylase inhibitor 5 (alphaAI-5) from black bean (BB), designated gene alphaAI-5 (Accession No. ), were determined. Genes alphaAI-4 and alphaAI-5 encode 244 amino acid prepro-alphaAI-4 and prepro-alphaAI-5 polypeptides that are 93 and 95% identical with alpha-amylase inhibitor l (alphaAI-l; Hoffman, L. M.; Ma, Y.; Barker, R. F. Nucleic Acids Res. 1982, 10, 7819-7828), 40 and 43% identical with red kidney bean lectin, and 52 and 55% identical with arcelin l of wild-type bean. The high degree of sequence similarity indicates the evolutionary relationship among these genes. PCR analysis of genomic DNA purified from six genotypes of Phaseolus vulgaris showed very similar band patterns in 2% agarose gel, another indication of the conserved size homology among these genes. Proteolytic processing sites were located between Asn77 and Ser78 for pro-alphaAI-4 and pro-alphaAI-5. A bend next to Asn77 in three-dimensional model structures of alphaAI-4 and alphaAI-5 proinhibitors indicates that the proteolytic cleavage is necessary to remove the conformational constraint for activation to the mature protein. Mature WKB alphaAI-4 was composed of four subunits (2alpha2beta) and had a molecular weight of 50000 determined by multiangle laser light scattering and 56714 determined by laser-assisted time-of-flight mass spectrometry. PMID:12381161

  18. Specific inhibition of barley alpha-amylase 2 by barley alpha-amylase/subtilisin inhibitor depends on charge interactions and can be conferred to isozyme 1 by mutation.

    PubMed

    Rodenburg, K W; Vallée, F; Juge, N; Aghajari, N; Guo, X; Haser, R; Svensson, B

    2000-02-01

    alpha-Amylase 2 (AMY2) and alpha-amylase/subtilisin inhibitor (BASI) from barley bind with Ki = 0.22 nM. AMY2 is a (beta/alpha)8-barrel enzyme and the segment Leu116-Phe143 in domain B (Val89-Ile152), protruding at beta-strand 3 of the (beta/alpha)8-barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2. In the AMY2-BASI crystal structure [F. Vallée, A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg, B. Svensson & R. Haser (1998) Structure 6, 649-659] Arg128AMY2 forms a hydrogen bond with Ser77BASI, while Asp142AMY2 makes a salt-bridge with Lys140BASI. These two enzyme residues are substituted by glutamine and asparagine, respectively, to assess their contribution in binding of the inhibitor. These mutations were performed in the well-expressed, inhibitor-sensitive hybrid barley alpha-amylase 1 (AMY1)-(1-90)/AMY2-(90-403) with Ki = 0.33 nM, because of poor production of AMY2 in yeast. In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T129R/K130P in the inhibitor-insensitive hybrid AMY1-(1-161)/AMY2-(161-403). The binding energy was reduced by 2.7-3.0 kcal.mol-1 as determined from Ki after the mutations R128Q and D142N. This corresponds to loss of a charged interaction between the protein molecules. In contrast, sensitivity to the inhibitor was gained (Ki = 7 microM) by the mutation T129R/K130P in the insensitive isozyme hybrid. Charge screening raised Ki 14-20-fold for this latter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants. Thus electrostatic stabilization was effectively introduced and lost in the different mutant enzyme-inhibitor complexes and rational engineering using an inhibitor recognition motif to confer binding to the inhibitor mimicking the natural AMY2-BASI complex. PMID:10672010

  19. Stopped-flow kinetic studies of the reaction of barley alpha-amylase/subtilisin inhibitor and the high pI barley alpha-amylase.

    PubMed

    Sidenius, U; Olsen, K; Svensson, B; Christensen, U

    1995-03-20

    The interaction of alpha-amylase/subtilisin inhibitor (BASI) from barley seeds and the high pI barley alpha-amylase (AMY2) de novo synthesized during seed germination, has been studied at pH 8.0, 25 degrees C, using stopped-flow fluorescence spectroscopy, equilibrium fluorescence titration and kinetic analysis of the displacement of BASI from the BASI-AMY2 complex by the substrate blue starch. The results are in accordance with a two-step reaction model: [formula: see text] The resulting values of the kinetic parameters were: k2/K1 = (1.0 +/- 0.2) x 10(6) M-1.s-1, K1 = 0.4 +/- 0.21 mM, k2 = 320 +/- 150 s-1, k-2 = (7.2 +/- 0.6) x 10(-5)s-1, and the overall dissociation constant Kd = (0.7 +/- 0.1) x 10(-10) M. BASI thus is best characterized as a fast reacting, tight-binding inhibitor of AMY2. PMID:7698332

  20. Activation of bean (Phaseolus vulgaris) [alpha]-amylase inhibitor requires proteolytic processing of the proprotein

    SciTech Connect

    Pueyo, J.J.; Hunt, D.C.; Chrispeels, M.J. )

    1993-04-01

    Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[sub r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.

  1. Activation of bean (Phaseolus vulgaris) alpha-amylase inhibitor requires proteolytic processing of the proprotein.

    PubMed Central

    Pueyo, J J; Hunt, D C; Chrispeels, M J

    1993-01-01

    Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the alpha-amylases of mammals and insects. This alpha-amylase inhibitor (alpha AI) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M(r)) 15,000 to 18,000. We report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, we found that antibodies to alpha AI recognize large (M(r) 30,000-35,000) polypeptides as well as typical alpha AI processing products (M(r) 15,000-18,000). Alpha AI activity was found in all extracts that had the typical alpha AI processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant alpha AI in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-alpha AI when the gene is expressed in tobacco. When pro-alpha AI was separated from mature alpha AI by gel filtration, pro-alpha AI was found not to have alpha-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule. PMID:8310064

  2. Complete sequence, subunit structure, and complexes with pancreatic alpha-amylase of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

    PubMed

    Kasahara, K; Hayashi, K; Arakawa, T; Philo, J S; Wen, J; Hara, S; Yamaguchi, H

    1996-07-01

    The complete amino acid sequence of a white kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor (PHA-I), which is composed of two kinds of glycopolypeptide subunits, alpha and beta, was established by conventional methods. The polypeptide molecular weight of PHA-I determined by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that PHA-I has the subunit stoichiometry of (alpha beta)2 complex. Inhibition test of PHA-I with increasing amounts of porcine pancreatic alpha-amylase (PPA) suggested that an inactive 2:1 complex is formed between PPA and PHA-I. In fact, two complexes differing from each other in the molar ratio of PPA to PHA-I were separated by gel filtration, and molecular weight estimation by the light-scattering technique confirmed that they are complexes of PHA-I with one or two PPA molecules. The binding of PPA to PHA-I appeared to follow simple binomial statistics, suggesting that two binding sites on PHA-I are independent and of high affinity for PPA. PMID:8864861

  3. [Inhibitors of alpha-amylase from plants--a possibility to treat diabetes mellitus type II by phytotherapy?].

    PubMed

    Melzig, Matthias F; Funke, Ines

    2007-01-01

    Antidiabetics of plant origin are in common use. A proof of their effectiveness or their mode of action is often missing. The aim of this work was to review the knowledge about inhibitors of alpha-amylase from plants and to comment on the use in anti-diabetic treatment. Herbal alpha-amylase inhibitors are rarely described in the literature, nevertheless they have the ability to lower postprandial blood glucose level and should be used in the supplementary treatment of diabetes. Important constituents for the inhibitory activity against alpha-amylase are mainly polyphenolic compounds. There is a need for further clinical studies to establish a rational therapy with traditional herbal preparations, especially for the leaves from the blueberry, tamarind, lemon balm and rosemary, the hulls from white kidney beans or green tea extract. PMID:17704980

  4. The relationship between the level of salivary alpha amylase activity and pain severity in patients with symptomatic irreversible pulpitis

    PubMed Central

    Shahriari, Shahriar; Goodarzi, Mohammad Taghi; Moghimbeigi, Abbas; Jazaeri, Mina; Babaei, Parisa

    2013-01-01

    Objectives Assessment of dental pain severity is very challenging in dentistry. Previous studies have suggested that elevated salivary alpha amylase may contribute to increased physical stresses. There is a close association between salivary alpha amylase and plasma norepinephrine under stressful physical conditions. The aim of this study was to evaluate the relationship between pain severity and salivary alpha amylase levels in patients with symptomatic irreversible pulpitis. Materials and Methods Thirty-six patients (20 females and 16 males) with severe tooth pain due to symptomatic irreversible pulpitis were selected. The visual analogue scale (VAS) score was used to assess the pain severity in each patient. Unstimulated whole saliva was collected, and the level of alpha amylase activity was assessed by the spectrophotometric method. Statistical analysis was performed using SPSS 13. Results The level of alpha amylase was significantly increased in the saliva in association with pain severity assessed by VAS. The salivary alpha amylase was also elevated with increased age and in males. Conclusions There was a significant correlation between the VAS pain scale and salivary alpha amylase level, which indicates this biomarker may be a good index for the objective assessment of pain intensity. PMID:24010080

  5. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat Butte 86

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complement of genes encoding alpha-amylase/protease inhibitors expressed in Triticum aestivum cv. Butte 86 was characterized by transcript and proteomic analysis. Coding sequences for 18 distinct proteins were identified among a collection of expressed sequence tags (ESTs) from Butte 86 developi...

  6. Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

    PubMed

    Takahashi, T; Hiramoto, S; Wato, S; Nishimoto, T; Wada, Y; Nagai, K; Yamaguchi, H

    1999-11-01

    Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue. PMID:10544275

  7. Porcine pancreatic alpha-amylase inhibition by the kidney bean (Phaseolus vulgaris) inhibitor (alpha-AI1) and structural changes in the alpha-amylase inhibitor complex.

    PubMed

    Santimone, Marius; Koukiekolo, Roger; Moreau, Yann; Le Berre, Véronique; Rougé, Pierre; Marchis-Mouren, Guy; Desseaux, Véronique

    2004-02-12

    Porcine pancreatic alpha-amylase (PPA) is inhibited by the red kidney bean (Phaseolus vulgaris) inhibitor alpha-AI1 [Eur. J. Biochem. 265 (1999) 20]. Inhibition kinetics were carried out using DP 4900-amylose and maltopentaose as substrate. As shown by graphical and statistical analysis of the kinetic data, the inhibitory mode is of the mixed noncompetitive type whatever the substrate thus involving the EI, EI2, ESI and ESI2 complexes. This contrast with the E2I complex obtained in the crystal and with biophysical studies. Such difference very likely depends on the [I]/[E] ratio. At low ratio, the E2I complex is favoured; at high ratio the EI, ESI and EI2 complexes are formed. The inhibition model also differs from those previously proposed for acarbose [Eur. J. Biochem. 241 (1996) 787 and Eur. J. Biochem. 252 (1998) 100]. In particular, with alpha-AI1, the inhibition takes place only when PPA and alpha-AI are preincubated together before adding the substrate. This indicates that the abortive PPA-alphaAI1 complex is formed during the preincubation period. One additional carbohydrate binding site is also demonstrated yielding the ESI complex. Also, a second protein binding site is found in EI2 and ESI2 abortive complexes. Conformational changes undergone by PPA upon alpha-AI1 binding are shown by higher sensitivity to subtilisin attack. From X-ray analysis of the alpha-AI1-PPA complex (E2I), the major interaction occurs with two hairpin loops L1 (residues 29-46) and L2 (residues 171-189) of alpha-AI1 protruding into the V-shaped active site of PPA. The hydrolysis of alpha-AI1 that accounts for the inhibitory activity is reported. PMID:14871659

  8. Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean.

    PubMed Central

    Ishimoto, M; Chrispeels, M J

    1996-01-01

    Alpha-amylase inhibitor (alpha AI) protects seeds of the common bean (Phaseolus vulgaris) against predation by certain species of bruchids such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis), but not against predation by the bean weevil (Acanthoscelides obtectus) or the Mexican bean weevil (Zabrotes subfasciatus), insects that are common in the Americas. We characterized the interaction of alpha AI-1 present in seeds of the common bean, of a different isoform, alpha AI-2, present in seeds of wild common bean accessions, and of two homologs, alpha AI-Pa present in seeds of the tepary bean (Phaseolus acutifolius) and alpha AI-Pc in seeds of the scarlet runner bean (Phaseolus coccineus), with the midgut extracts of several bruchids. The extract of the Z. subfasciatus larvae rapidly digests and inactivates alpha AI-1 and alpha AI-Pc, but not alpha AI-2 or alpha AI-Pa. The digestion is caused by a serine protease. A single proteolytic cleavage in the beta subunit of alpha AI-1 occurs at the active site of the protein. When degradation is prevented, alpha AI-1 and alpha AI-Pc do not inhibit the alpha-amylase of Z. subfasciatus, although they are effective against the alpha-amylase of C. chinensis. Alpha AI-2 and alpha AI-Pa, on the other hand, do inhibit the alpha-amylase of Z. subfasciatus, suggesting that they are good candidates for genetic engineering to achieve resistance to Z. subfasciatus. PMID:8787024

  9. Soyacystatin N inhibits proteolysis of wheat alpha-amylase inhibitor and potentiates toxicity against cowpea weevil.

    PubMed

    Amirhusin, Bahagiawati; Shade, Richard E; Koiwa, Hisashi; Hasegawa, Paul M; Bressan, Ray A; Murdock, Larry L; Zhu-Salzman, Keyan

    2004-12-01

    Genetic engineering may be used to introduce multiple insect resistance genes with different modes of action into crop plants. We explored the possible interactions of two differing gene products fed in the diet of cowpea weevil, Callosobruchus maculates (F.), a stored grain pest. The soybean cysteine protease inhibitor soyacystatin N (scN) and alpha-amylase inhibitor (alphaAI) from wheat have defensive function against this coleopteran. When artificial seeds containing both scN and alpha(AI) were infested with eggs of C. maculatus, the delays in larval development were longer than was predicted by summing the developmental delays seen when larvae were fed a diet containing the individual proteins, indicating that the effects of scN and alpha(AI) are synergistic. Alpha(AI) was readily hydrolyzed when incubated with insect gut extract. This proteolytic degradation was inhibited by scN, but not by Kunitz inhibitor (a serine protease inhibitor). Thus, degradation of alpha(AI) was due to proteolysis by insect digestive cysteine proteases. These data suggest that C. maculatus uses digestive enzymes not only to function in food protein digestion but also to defend the insects themselves by helping reduce the concentration of a toxic dietary protein. PMID:15666770

  10. Capillary electrophoresis as a screening tool for alpha amylase inhibitors in plant extracts

    PubMed Central

    Hamdan, Imad I.; Afifi, Fatima U.

    2010-01-01

    Capillary electrophoresis (CE) method was developed for screening plant extract for potential alpha amylase (AA) inhibitory activity. The method was validated against a well established UV method. Overall, the proposed method was shown able to detect plants with significant alpha amylase inhibitory activity but not those with rather clinically insignificant activities. Fifty plant species were screened using both the proposed CE method and the UV method and seven plant species were found to possess significant AA inhibitory activities. Two plant species were proved to have alpha amylase inhibitory activity for the first time. PMID:24115900

  11. Kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor and barley alpha-amylase 2 analyzed by surface plasmon resonance and isothermal titration calorimetry.

    PubMed

    Nielsen, Peter K; Bønsager, Birgit C; Berland, Carolyn R; Sigurskjold, Bent W; Svensson, Birte

    2003-02-18

    The kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor (BASI) or BASI mutants and barley alpha-amylase 2 (AMY2) were determined using surface plasmon resonance and isothermal titration calorimetry (ITC). Binding kinetics were in accordance with a 1:1 binding model. At pH 5.5, [Ca(2+)] = 5 mM, and 25 degrees C, the k(on) and k(off) values were 8.3 x 10(+4) M(-1) s(-1) and 26.0 x 10(-4) s(-1), respectively, corresponding to a K(D) of 31 nM. K(D) was dependent on pH, and while k(off) decreased 16-fold upon increasing pH from 5.5 to 8.0, k(on) was barely affected. The crystal structure of AMY2-BASI shows a fully hydrated Ca(2+) at the protein interface, and at pH 6.5 increase of [Ca(2+)] in the 2 microM to 5 mM range raised the affinity 30-fold mainly due to reduced k(off). The K(D) was weakly temperature-dependent in the interval from 5 to 35 degrees C as k(on) and k(off) were only increasing 4- and 12-fold, respectively. A small salt dependence of k(on) and k(off) suggested a minor role for global electrostatic forces in the binding and dissociation steps. Substitution of a positively charged side chain in the mutant K140L within the AMY2 inhibitory site of BASI accordingly did not change k(on), whereas k(off) increased 13-fold. ITC showed that the formation of the AMY2-BASI complex is characterized by a large exothermic heat (Delta H = -69 +/- 7 kJ mol(-1)), a K(D) of 25 nM (27 degrees C, pH 5.5), and an unfavorable change in entropy (-T Delta S = 26 +/- 7 kJ mol(-1)). Calculations based on the thermodynamic data indicated minimal structural changes during complex formation. PMID:12578360

  12. A putative precursor protein in the evolution of the bean alpha-amylase inhibitor.

    PubMed

    Finardi-Filho, F; Mirkov, T E; Chrispeels, M J

    1996-09-01

    Seeds of the common bean Phaseolus vulgaris and the tepary bean (P. acutifolius) contain a family of plant defence proteins that includes phytohaemagglutinin (PHA), arcelin and alpha-amylase inhibitor (alpha AI). These homologous proteins differ by the absence of short loops at the surface of the protein and by the presence of a proteolytic processing site (Asn77) that allows alpha AI to be post-translationally cleaved and activated. We now report the derived amino acid sequence of two amylase inhibitor-like (AIL) proteins that are not proteolytically processed, although they have the typical processing site. One protein is from the common bean, and the other from the tepary bean. On a dendrogram, these proteins are grouped with alpha AIs rather than with the arcelins or lectins. alpha AI differs from AIL primarily by the deletion of a 15-amino-acid segment from the middle of the AIL sequence. When alpha AI is expressed in tobacco, it is proteolytically processed to form an active molecule. However, AIL sequences are not processed. We suggest that the AIL proteins may be an intermediate in the evolution of an active alpha AI. PMID:8987505

  13. Characterization and functional properties of the alpha-amylase inhibitor (alpha-AI) from kidney bean (Phaseolus vulgaris) seeds.

    PubMed

    Le Berre-Anton, V; Bompard-Gilles, C; Payan, F; Rougé, P

    1997-11-14

    Alpha-amylase inhibitor (alpha-AI) from kidney bean (Phaseolus vulgaris L. cv Tendergreen) seeds has been purified to homogeneity by heat treatment in acidic medium, ammonium sulphate fractionation, chromatofocusing and gel filtration. Two isoforms, alpha-AI1 and alpha-AI1', of 43 kDa have been isolated which differ from each other by their isoelectric points and neutral sugar contents. The major isoform alpha-AI1 inhibited human and porcine pancreatic alpha-amylases (PPA) but was devoid of activity on alpha-amylases of bacterial or fungal origins. As shown on the Lineweaver-Burk plots, the nature of the inhibition is explained by a mixed non-competitive inhibition mechanism. Alpha-AI1 formed a 1:2 stoichiometric complex with PPA which showed an optimum pH of 4.5 at 30 degrees C. Owing to the low optimum pH found for alpha-AI activity, inhibitor-containing diets such as beans or transgenic plants expressing alpha-AI should be devoid of any harmful effect on human health. PMID:9428656

  14. Bean [alpha]-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.).

    PubMed Central

    Schroeder, H. E.; Gollasch, S.; Moore, A.; Tabe, L. M.; Craig, S.; Hardie, D. C.; Chrispeels, M. J.; Spencer, D.; Higgins, TJV.

    1995-01-01

    Bruchid larvae cause major losses of grain legume crops through-out the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing in the field. We transferred the cDNA encoding the [alpha]-amylase inhibitor ([alpha]-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacterium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The [alpha]-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T5 seed generation, and [alpha]-AI accumulated in the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2% [alpha]-AI. In the T5 seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although [alpha]-AI also inhibits human [alpha]-amylase, cooked peas should not have a negative impact on human energy metabolism. PMID:12228429

  15. Characterization of. alpha. -amylase-inhibitor, a lectin-like protein in the seeds of Phaseolus vulgaris

    SciTech Connect

    Moreno, J.; Altabella, T.; Chrispeels, M.J. )

    1990-03-01

    The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect {alpha}-amylases but not of plant {alpha}-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, {alpha}-amylase inhibitor ({alpha}Al) is present in embryonic axes and cotyledons, but not in other organs of the plant. The protein is synthesized during the same time period that phaseolin and phytohemagglutinin are made and also accumulates in the protein storage vacuoles (protein bodies). All the glycoforms have complex glycans that are resistant to removal by endoglycosidase H, indicating transport of the protein through the Golgi apparatus. The two different polypeptides correspond to the N-terminal and C-terminal halves of a lectin-like protein encoded by an already identified gene or a gene closely related to it. The primary translation product of {alpha}Al is a polypeptide of M{sub r} 28,000. Immunologically cross-reacting glycopolypeptides of M{sub r} 30,000 to 35,000 are present in the endoplasmic reticulum, while the smaller polypeptides (M{sub r} 15,000-19,000) accumulate in protein storage vacuoles (protein bodies). Together these data indicate that {alpha}Al is a typical bean lectin-type protein that is synthesized on the rough endoplasmic reticulum, modified in the Golgi, and transported to the protein storage vacuoles.

  16. Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

    SciTech Connect

    Altabella, T.; Chrispeels, M.J. )

    1990-06-01

    Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{sub r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.

  17. Mutational analysis of target enzyme recognition of the beta-trefoil fold barley alpha-amylase/subtilisin inhibitor.

    PubMed

    Bønsager, Birgit C; Nielsen, Peter K; Abou Hachem, Maher; Fukuda, Kenji; Praetorius-Ibba, Mette; Svensson, Birte

    2005-04-15

    The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits alpha-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the alpha-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of binding to target enzymes by surface plasmon resonance. Generally, the mutations had little effect on k(on), whereas the k(off) values were increased up to 67-fold. The effects on the inhibitory activity, however, were far more pronounced, and the K(i) values of some mutants on the AMY2-binding side increased 2-3 orders of magnitude, whereas mutations on the other side of the inhibitor had virtually no effect. The mutants K140L, D150N, and E168T lost inhibitory activity, revealing the pivotal role of charge interactions for BASI activity on AMY2. A fully hydrated Ca(2+) at the AMY2-BASI interface mediates contacts to the catalytic residues of AMY2. Mutations involving residues contacting the solvent ligands of this Ca(2+) had weaker affinity for AMY2 and reduced sensitivity to the Ca(2+) modulation of the affinity. These results suggest that the Ca(2+) and its solvation sphere are integral components of the AMY2-BASI complex, thus illuminating a novel mode of inhibition and a novel role for calcium in relation to glycoside hydrolases. PMID:15657043

  18. Assessment of the importance of alpha-amylase inhibitor-2 in bruchid resistance of wild common bean.

    PubMed

    Nishizawa, Keito; Teraishi, Masayoshi; Utsumi, Shigeru; Ishimoto, Masao

    2007-02-01

    Both alpha-amylase inhibitor-2 (alphaAI-2) and arcelin have been implicated in resistance of wild common bean (Phaseolus vulgaris L.) to the Mexican bean weevil (Zabrotes subfasciatus Boheman). Near isogenic lines (NILs) for arcelin 1-5 were generated by backcrossing wild common bean accessions with a cultivated variety. Whereas seeds of a wild accession (G12953) containing both alphaAI-2 and arcelin 4 were completely resistant to Z. subfasciatus, those of the corresponding NIL were susceptible to infestation, suggesting that the principal determinant of resistance was lost during backcrossing. Three independent lines of transgenic azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi] expressing alphaAI-2 accumulated high levels of this protein in seeds. The expression of alphaAI-2 in these lines conferred protection against the azuki bean weevil (Callosobruchus chinensis L.), likely through inhibition of larval digestive alpha-amylase. However, although the seed content of alphaAI-2 in these transgenic lines was similar to that in a wild accession of common bean (G12953), it did not confer a level of resistance to Z. subfasciatus similar to that of the wild accession. These results suggest that alphaAI-2 alone does not provide a high level of resistance to Z. subfasciatus. However, alphaAI-2 is an effective insecticidal protein with a spectrum of activity distinct from that of alphaAI-1, and it may prove beneficial in genetic engineering of insect resistance in legumes. PMID:17186215

  19. Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus alpha-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).

    PubMed

    Kim, Young-Hwa; Woloshuk, Charles P; Cho, Eun Hee; Bae, Jung Myung; Song, Young-Sun; Huh, Gyung Hye

    2007-04-01

    Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection. PMID:17149640

  20. Interaction of wheat monomeric and dimeric protein inhibitors with alpha-amylase from yellow mealworm (Tenebrio molitor L. larva).

    PubMed Central

    Buonocore, V; Gramenzi, F; Pace, W; Petrucci, T; Poerio, E; Silano, V

    1980-01-01

    The highly purified alpha-amylase from Tenebrio molitor L. larva (yellow mealworm) reversibly combines with two closely related homogeneous glycoprotein inhibitors, one dimeric (termed 'inhibitor 0.19') and one monomeric (termed 'inhibitor 0.28'), from wheat flour. As established by means of difference spectroscopy and kinetic studies, molar combining ratios for the amylase--inhibitor-0.19 and amylase-inhibitor-0.28 complexes were 1:1 and 1:2 respectively. Two amylase--inhibitor-0.19 complexes with slightly different retention volumes on Bio-Gel P-300 and only one amylase--inhibitor-0.28 complex were observed. Dissociation constants of the amylase--inhibitor-0.19 and amylase--inhibitor-0.28 complexes were 0.85 nM and 0.13 nM respectively. A strong tendency of both complexes to precipitate under an ultracentrifugal field was observed; the minimum molecular weight calculated for the two complexes under such conditions was approx. 95 000. The two complexes showed difference spectra indicating involvement of structurally related or identical tryptophyl side chains in the binding of inhibitors 0.28 and 0.19 to the amylase. A model summarizing the main features of the inhibition of the insect amylase by the two wheat protein inhibitors is proposed. PMID:6985361

  1. Hypoglycaemic and anorexigenic activities of an alpha-amylase inhibitor from white kidney beans (Phaseolus vulgaris) in Wistar rats.

    PubMed

    Tormo, M A; Gil-Exojo, I; Romero de Tejada, A; Campillo, J E

    2004-11-01

    An inhibitor of alpha-amylase was isolated and purified from an extract of white kidney beans (Phaseolus vulgaris). The acute oral administration of the inhibitor (50 mg/kg body weight) to adult Wistar rats together with a starch load (2 g/kg body weight suspended in NaCl (9 g/l)) reduced the increase in glycaemia over the basal value (NaCl, 222 (SEM 49); inhibitor, 145 (SEM 16) mmol/l x 180 min; P<0.05) without modifying the insulin response. On administering the inhibitor orally (50 mg/kg body weight dissolved in NaCl (9 g/l)) for 21 d to rats fed on a standard diet, a decline was observed in the glycaemia values on day 0 (NaCl, 5.53 (SEM 0.12); inhibitor, 5.25 (SEM 0.16) mmol/l) relative to those obtained on days 10 (NaCl, 5.00 (SEM 0.14); inhibitor, 4.60 (SEM 0.08) mmol/l; P<0.05) and 21 (NaCl, 5.22 (SEM 0.22); inhibitor, 4.50 (SEM 0.12) mmol/l; P<0.01) of treatment, without modifying the plasma concentration of insulin. There was found to be a significant anorexigenic action of the inhibitor; there was reduced food intake (NaCl, 23.07 (SEM 0.31); inhibitor, 19.50 (SEM 0.49) g/d; P<0.01), a reduced weight gain (NaCl, 52 (SEM 3); inhibitor, -1.33 (SEM 8.9) g/21 d; P<0.01), as well as changes in the activity of some intestinal enzymes such as maltase (NaCl, 87 (SEM 7); inhibitor, 127 (SEM 11) U/g proteins; P<0.05). The present study has shown, for the first time, that the prolonged administration of an alpha-amylase inhibitor reduces blood glucose levels and body-weight gain in Wistar rats. PMID:15533267

  2. Post-translational processing of two alpha-amylase inhibitors and an arcelin from the common bean, Phaseolus vulgaris.

    PubMed

    Young, N M; Thibault, P; Watson, D C; Chrispeels, M J

    1999-03-01

    Mass spectrometric methods were used to investigate the proteolytic processing and glycopeptide structures of three seed defensive proteins from Phaseolus vulgaris. The proteins were the alpha-amylase inhibitors alphaAI-1 and alphaAI-2 and arcelin-5, all of which are related to the seed lectins, PHA-E and PHA-L. The mass data showed that the proteolytic cleavage required for activation of the amylase inhibitors is followed by loss of the terminal Asn residue in alphaAI-1, and in all three proteins, seven or more residues were clipped from the C-termini, in the manner of the seed lectins. In most instances, individual glycoforms could be assigned at each Asn site, due to the unique masses of the plant glycopeptides. It was found that alphaAI-1 and alphaAI-2 differed significantly in their glycosylation patterns, despite their high sequence homology. These data complement the previous X-ray studies of the alpha1-amylase inhibitor and arcelin, where many of the C-terminal residues and glycopeptide residues could not be observed. PMID:10100643

  3. cDNA cloning, biochemical characterization and inhibition by plant inhibitors of the alpha-amylases of the Western corn rootworm, Diabrotica virgifera virgifera.

    PubMed

    Titarenko, E; Chrispeels, M J

    2000-10-01

    We report the characterization and cDNA cloning of two alpha-amylase isozymes from larvae of the Western corn rootworm (Diabrotica virgifera virgifera LeConte). Larvae raised on artificial media have very low levels of amylase activity, and much higher levels are found in larvae raised on maize seedlings. At pH 5.7, the optimum pH for enzyme activity, the alpha-amylases are substantially but not completely inhibited by amylase inhibitors from the common bean (Phaseolus vulgaris) and from wheat (Triticum aestivum). Using the reverse transcriptase polymerase chain reaction (RT-PCR), we cloned two cDNAs with 83% amino acid identity that encode alpha-amylase-like polypeptides. Expression of one of the two cDNAs in insect cells with a baculovirus vector shows that this cDNA encodes an active amylase with a mobility that corresponds to that of one of the two isozymes present in larval extracts. The expressed enzyme is substantially inhibited by the same two inhibitors. We also show that expression in Arabidopsis of the cDNA that encodes the amylase inhibitor AI-1 of the common bean results in the accumulation of active inhibitor in the roots, and the results are discussed with reference to the possibility of using amylase inhibitors as a strategy to genetically engineer maize plants that are resistant to Western corn rootworm larvae. PMID:10899464

  4. Arg-27, Arg-127 and Arg-155 in the beta-trefoil protein barley alpha-amylase/subtilisin inhibitor are interface residues in the complex with barley alpha-amylase 2.

    PubMed

    Rodenburg, K W; Várallyay, E; Svendsen, I; Svensson, B

    1995-08-01

    Arginine residues in barley alpha-amylase/subtilisin inhibitor (BASI) involved in binding to barely alpha-amylase 2 (AMY2) were differentially labelled using AMY2 as protectant and phenylglyoxal (PGO) and [14C]PGO as modifying agents. Chymotryptic fragments of labelled BASI were purified by reverse-phase HPLC, and we concluded that the radiolabelled Arg-27, Arg-155 and most likely Arg-127, identified by amino acid, sequence and 14C analyses, are protected by AMY2. While Arg-106 and Arg-107 showed intermediate reactivity and apparently were only partly accessible, Arg-15, Arg-41 and Arg-61 reacted with PGO and were thus exposed in the BASI-AMY2 complex. Patterns of arginine modification by [14C]PGO in free or in AMY2-complexed BASI were consistent with the results of differential labelling. The AMY2-protected arginines in BASI are at a distance from each other, as deduced from crystal structures of different beta-trefoil proteins (Erythrina caffra and soybean trypsin inhibitors, interleukin-1 alpha and -1 beta and WASI, the wheat homologue), suggesting that the BASI-AMY2 complex has multiple contacts at a larger interface. Accordingly, 11-16-residue-long BASI oligopeptides synthesized to include Arg-27, Arg-106/Arg-107 or Arg-127 were unable to suppress the formation of BASI-AMY2 or the effect of an inhibitory monoclonal antibody to BASI. Since Arg-27 is not conserved in rice and wheat ASIs, we further propose that Arg-155 in BASI is the kinetically identified PGO-sensitive group that is essential for inhibition [Abe, Sidenius and Svensson (1993) Biochem. J. 293, 151-155]. PMID:7639717

  5. Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli.

    PubMed

    Bønsager, Birgit C; Praetorius-Ibba, Mette; Nielsen, Peter K; Svensson, Birte

    2003-08-01

    Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively. PMID:12880767

  6. cDNA sequence and deduced primary structure of an alpha-amylase inhibitor from a bruchid-resistant wild common bean.

    PubMed

    Suzuki, K; Ishimoto, M; Kitamura, K

    1994-06-12

    alpha-Amylase inhibitor-2 (alpha AI-2), a seed storage protein present in a bruchid-resistant wild common bean (Phaseolus vulgaris), inhibits the growth of bruchid pests. The authors isolated and determined the sequence of an 852 nucleotide cDNA, designated as alpha ai2, and found it to contain a 720 base open reading frame (ORF). This ORF encodes a 240 amino-acid alpha AI-2 polypeptide 75.8% identical with alpha-amylase inhibitor-1 (alpha AI-1) and 50.6-55.6% with arcelin-1, phytohemagglutinin (PHA)-L and PHA-E of common bean. The high degree of sequence homology suggests that there is an evolutionary relationship among these genes. PMID:8003534

  7. Potential of the bean alpha-amylase inhibitor alpha-AI-1 to inhibit alpha-amylase activity in true bugs(Hemiptera)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    True bugs (Hemiptera) are an important pest complex not controlled by Bt crops. An alternative source of resistance includes inhibitors of digestive enzymes. aAI-1, an a-amylase inhibitor from the common bean, has been shown to inhibit a-amylases of bruchid pests of grain legumes. Here we quantify t...

  8. Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding.

    PubMed

    Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Ali, Ishtiaq; Bonfili, Laura; Cecarini, Valentina; Eleuteri, Anna Maria; Angeletti, Mauro

    2016-12-15

    Alpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity. PMID:27451220

  9. The impact of single nucleotide polymorphism in monomeric alpha-amylase inhibitor genes from wild emmer wheat, primarily from Israel and Golan

    PubMed Central

    2010-01-01

    Background Various enzyme inhibitors act on key insect gut digestive hydrolases, including alpha-amylases and proteinases. Alpha-amylase inhibitors have been widely investigated for their possible use in strengthening a plant's defense against insects that are highly dependent on starch as an energy source. We attempted to unravel the diversity of monomeric alpha-amylase inhibitor genes of Israeli and Golan Heights' wild emmer wheat with different ecological factors (e.g., geography, water, and temperature). Population methods that analyze the nature and frequency of allele diversity within a species and the codon analysis method (comparing patterns of synonymous and non-synonymous changes in protein coding sequences) were used to detect natural selection. Results Three hundred and forty-eight sequences encoding monomeric alpha-amylase inhibitors (WMAI) were obtained from 14 populations of wild emmer wheat. The frequency of SNPs in WMAI genes was 1 out of 16.3 bases, where 28 SNPs were detected in the coding sequence. The results of purifying and the positive selection hypothesis (p < 0.05) showed that the sequences of WMAI were contributed by both natural selection and co-evolution, which ensured conservation of protein function and inhibition against diverse insect amylases. The majority of amino acid substitutions occurred at the C-terminal (positive selection domain), which ensured the stability of WMAI. SNPs in this gene could be classified into several categories associated with water, temperature, and geographic factors, respectively. Conclusions Great diversity at the WMAI locus, both between and within populations, was detected in the populations of wild emmer wheat. It was revealed that WMAI were naturally selected for across populations by a ratio of dN/dS as expected. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs. A sharp genetic divergence over very short geographic distances compared to

  10. [Baking ingredients, especially alpha-amylase, as occupational inhalation allergens in the baking industry].

    PubMed

    Wüthrich, B; Baur, X

    1990-03-31

    Baker's asthma is the most frequent occupational lung disease in Switzerland and West Germany. Cereal flours, and more rarely flour parasites, are implicated as the responsible allergens. Based on an observation of a case of baker's asthma due to monovalent sensitization to alpha-amylase used as additive to flour, 31 bakers with occupational asthma and/or rhinitis were routinely tested by skin tests and serological RAST examinations for allergic sensitivity to flour, alpha-amylase and other bakery additives. 17/31 subjects (55%) reacted positively in scratch tests to a commercial powdered alpha-amylase and 13/20 (65%) to a lecithin preparation. 23/31 (74%) and 19/31 (61%) were RAST positive to wheat and to rye flour respectively. 32% had RAST specific IgE to alpha-amylase (from Aspergillus oryzae), 19.3% to soya bean flour and 16% to malt. 7/12 and 5/12 respectively reacted to trypsin inhibitor and lipoxidase, the main allergens in soya bean. In two patients monosensitization to alpha-amylase was present. In accordance with other reports we recommend that baking additives, especially alpha-amylase, should be tested in allergological diagnosis of occupational diseases in flour processing workers. Full declaration of all additives used in the bakery industry is needed. PMID:2326614

  11. Evolutionary relationships among proteins in the phytohemagglutinin-arcelin-alpha-amylase inhibitor family of the common bean and its relatives.

    PubMed

    Mirkov, T E; Wahlstrom, J M; Hagiwara, K; Finardi-Filho, F; Kjemtrup, S; Chrispeels, M J

    1994-11-01

    The common bean, Phaseolus vulgaris, contains a family of defense proteins that comprises phytohemagglutinin (PHA), arcelin, and alpha-amylase inhibitor (alpha AI). Here we report eight new derived amino acid sequences of genes in this family obtained with either the polymerase chain reaction using genomic DNA, or by screening cDNA libraries made with RNA from developing beans. These new sequences are: two alpha AI sequences and arcelin-4 obtained from a wild accession of P. vulgaris that is resistant to the Mexican bean weevil (Zabrotes subfasciatus) and the bean weevil (Acanthoscelides obtectus); an alpha AI sequence from the related species P. acutifolius (tepary bean); a PHA and an arcelin-like sequence from P. acutifolius; an alpha AI-like sequence from P. maculatus; and a PHA sequence from an arcelin-5 type P. vulgaris. A dendrogram of 16 sequences shows that they fall into the three identified groups: phytohemagglutinins, arcelins and alpha AIs. A comparison of these derived amino acid sequences indicates that one of the four amino acid residues that is conserved in all legume lectins and is required for carbohydrate binding is absent from all the arcelins; two of the four conserved residues needed for carbohydrate binding are missing from all the alpha AIs. Proteolytic processing at an Asn-Ser site is required for the activation of alpha AI, and this site is present in all alpha AI-like sequences; this processing site is also found at the same position in certain arcelins, which are not proteolytically processed. The presence of this site is therefore not sufficient for processing to occur. PMID:7811969

  12. Effects of a high-pressure treatment on the wheat alpha-amylase inhibitor and its relationship to elimination of allergenicity

    NASA Astrophysics Data System (ADS)

    Yamamoto, S.; Takanohashi, K.; Hara, T.; Odani, S.; Suzuki, A.; Nishiumi, T.

    2010-03-01

    In this study, the effects of high-pressure treatment on structure and allergeincity of alpha amylase inhibitor (a-AI) were investigated. The pressure-induced structural changes of α-AI were estimated by fluorescence spectra and by fourth derivative UV-spectroscopy for probed tyrosine residues and by circular dichroism (CD) spectroscopy. The changes in the tertiary structure detected by fluorescence spectra and by fourth derivative UV-spectroscopy under high pressure were indicated at over 300 MPa. Measurements of CD spectroscopy suggested that the effects of a high-pressure treatment on changes in the secondary structure of α-AI were little. From our results, pressure-induced changes of the α-AI structure were not apparent. On the other hands, the IgE-specific binding activities of pressurized α-AI to sera from allergic patients against wheat, which is estimated by observations of dot-blotting, were decreased by high-pressure treatment. It is known that the pressure-induced elimination of allergenicity is related to the tertiary structural changes of allergen molecules. This study are suspected that the epitopes of α-AI do not contain tyrosine residues, and thus the decrease of IgE-specific binding activities is probably caused by the tertiary structural changes of these parts of α-AI.

  13. A heterotetrameric alpha-amylase inhibitor from emmer (Triticum dicoccon Schrank) seeds.

    PubMed

    Capocchi, A; Muccilli, V; Cunsolo, V; Saletti, R; Foti, S; Fontanini, D

    2013-04-01

    Plants have developed a constitutive defense system against pest attacks, which involves the expression of a set of inhibitors acting on heterologous amylases of different origins. Investigating the soluble protein complement of the hulled wheat emmer we have isolated and characterized a heterotetrameric α-amylase inhibitor (ETI). Based on mass spectrometry data, it is an assembly of proteins highly similar to the CM2/CM3/CM16 found in durum wheat. Our data indicate that these proteins can also inhibit exogenous α-amylases in binary assemblies. The calculated dissociation constants (K(i)) for the pancreatic porcine amylase- and human salivary amylase-ETI complexes are similar to those found in durum and soft wheat. Homology modeling of the CM subunits indicate structural similarities with other proteins belonging to the cereal family of trypsin/α-amylase inhibitors; a possible homology modeled structure for a tetrameric assembly of the subunits is proposed. PMID:23320956

  14. A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris): a review of clinical studies on weight loss and glycemic control.

    PubMed

    Barrett, Marilyn L; Udani, Jay K

    2011-01-01

    Obesity, and resultant health hazards which include diabetes, cardiovascular disease and metabolic syndrome, are worldwide medical problems. Control of diet and exercise are cornerstones of the management of excess weight. Foods with a low glycemic index may reduce the risk of diabetes and heart disease as well as their complications. As an alternative to a low glycemic index diet, there is a growing body of research into products that slow the absorption of carbohydrates through the inhibition of enzymes responsible for their digestion. These products include alpha-amylase and glucosidase inhibitors. The common white bean (Phaseolus vulgaris) produces an alpha-amylase inhibitor, which has been characterized and tested in numerous clinical studies. A specific and proprietary product named Phase 2® Carb Controller (Pharmachem Laboratories, Kearny, NJ) has demonstrated the ability to cause weight loss with doses of 500 to 3000 mg per day, in either a single dose or in divided doses. Clinical studies also show that Phase 2 has the ability to reduce the post-prandial spike in blood glucose levels. Experiments conducted incorporating Phase 2 into food and beverage products have found that it can be integrated into various products without losing activity or altering the appearance, texture or taste of the food. There have been no serious side effects reported following consumption of Phase 2. Gastro-intestinal side effects are rare and diminish upon extended use of the product. In summary, Phase 2 has the potential to induce weight loss and reduce spikes in blood sugar caused by carbohydrates through its alpha-amylase inhibiting activity. PMID:21414227

  15. A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris): A review of clinical studies on weight loss and glycemic control

    PubMed Central

    2011-01-01

    Obesity, and resultant health hazards which include diabetes, cardiovascular disease and metabolic syndrome, are worldwide medical problems. Control of diet and exercise are cornerstones of the management of excess weight. Foods with a low glycemic index may reduce the risk of diabetes and heart disease as well as their complications. As an alternative to a low glycemic index diet, there is a growing body of research into products that slow the absorption of carbohydrates through the inhibition of enzymes responsible for their digestion. These products include alpha-amylase and glucosidase inhibitors. The common white bean (Phaseolus vulgaris) produces an alpha-amylase inhibitor, which has been characterized and tested in numerous clinical studies. A specific and proprietary product named Phase 2® Carb Controller (Pharmachem Laboratories, Kearny, NJ) has demonstrated the ability to cause weight loss with doses of 500 to 3000 mg per day, in either a single dose or in divided doses. Clinical studies also show that Phase 2 has the ability to reduce the post-prandial spike in blood glucose levels. Experiments conducted incorporating Phase 2 into food and beverage products have found that it can be integrated into various products without losing activity or altering the appearance, texture or taste of the food. There have been no serious side effects reported following consumption of Phase 2. Gastro-intestinal side effects are rare and diminish upon extended use of the product. In summary, Phase 2 has the potential to induce weight loss and reduce spikes in blood sugar caused by carbohydrates through its alpha-amylase inhibiting activity. PMID:21414227

  16. Gender determines cortisol and alpha-amylase responses to acute physical and psychosocial stress in patients with borderline personality disorder.

    PubMed

    Inoue, Ayako; Oshita, Harumi; Maruyama, Yoshihiro; Tanaka, Yoshihiro; Ishitobi, Yoshinobu; Kawano, Aimi; Ikeda, Rie; Ando, Tomoko; Aizawa, Saeko; Masuda, Koji; Higuma, Haruka; Kanehisa, Masayuki; Ninomiya, Taiga; Akiyoshi, Jotaro

    2015-07-30

    Borderline personality disorder (BPD) is characterized by affective instability, unstable relationships, and identity disturbance. We measured salivary alpha-amylase (sAA) and salivary cortisol levels in all participants during exposure to the Trier Social Stress Test (TSST) and an electric stimulation stress. Seventy-two BPD patients were compared with 377 age- and gender- matched controls. The State and Trait versions of the Spielberger Anxiety Inventory test (STAI-S and STAI-T, respectively), the Profile of Mood State (POMS) tests, and the Beck Depression Inventory (BDI), the Depression and Anxiety Cognition Scale (DACS) were administered to participants before electrical stimulation. Following TSST exposure, salivary cortisol levels significantly decreased in female patients and significantly increased in male patients compared with controls. POMS tension-anxiety, depression-dejection, anger-hostility, fatigue, and confusion scores were significantly increased in BPD patients compared with controls. In contrast, vigor scores were significantly decreased in BPD patients relative to controls. Furthermore, STAI-T and STAI-S anxiety scores and BDI scores were significantly increased in BPD patient compared with controls. DACS scores were significantly increased in BPD patient compared with controls. Different stressors (e.g., psychological or physical) induced different responses in the HPA and SAM systems in female or male BPD patients. PMID:25979467

  17. The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

    PubMed

    Ohtsubo, K; Richardson, M

    1992-08-31

    A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds. PMID:1511747

  18. The activity of granulocyte alpha-amylase in acute appendicitis.

    PubMed

    Zakrzewska, I; Gajda, R

    1994-01-01

    The activity of alpha-amylase was measured in isolated granulocytes, serum and urine of 35 patients with acute appendicitis. The measurements were performed before operation and on the 7th day after operation. Slightly increased activity of alpha-amylase was found in the serum and urine of 15 patients. On the 7th day after operation the activity of this enzyme reached normal value. The activity of granulocyte alpha-amylase was elevated in 22 patients. In 2 of them the increased activity still maintained on the 7th day after operation. Positive correlation between the serum and granulocyte alpha-amylase activities was found. These observations allow to conclude that granulocytes are the source of increased alpha-amylase activity in the serum of patients with acute appendicitis. PMID:7497089

  19. Salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in major depressive disorder patients.

    PubMed

    Tanaka, Yoshihiro; Ishitobi, Yoshinobu; Maruyama, Yoshihiro; Kawano, Aimi; Ando, Tomoko; Okamoto, Shizuko; Kanehisa, Masayuki; Higuma, Haruka; Ninomiya, Taiga; Tsuru, Jusen; Hanada, Hiroaki; Kodama, Kensuke; Isogawa, Koichi; Akiyoshi, Jotaro

    2012-03-30

    Major depressive disorder (MDD) is often associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis by chronic stress. In comparison, psychosocial stress-induced activation of salivary α-amylase (sAA) functions as a marker of sympathoadrenal medullary system (SAM) activity. However, in contrast to salivary cortisol, sAA has been less extensively studied in MDD patients. The present study measured sAA and salivary cortisol levels in patients with MDD. The authors determined Profile of Mood State (POMS) and State-Trait anxiety Inventory (STAI) scores, Heart Rate Variability (HRV), and sAA and salivary cortisol levels in 88 patients with MDD and 41 healthy volunteers following the application of electrical stimulation stress. Patients with major depressive disorder were 8 points or more on Hamilton Depression Scale (HAM-D) scores. Tension-Anxiety, Depression-Dejection, Anger-Hostility, Fatigue, and Confusion scores in patients with major depressive disorder were significantly increased compared to healthy controls. In contrast, Vigor scores in patients with MDD were significantly decreased compared with healthy controls. There was no difference in heart rate variability measures between MDD patients and healthy controls. The threshold of electrical stimulation applied in MDD patients was lower than that in healthy controls. SAA levels in female MDD patients were significantly elevated relative to controls both before and after electrical stimulation. Finally, there were no differences in salivary cortisol levels between major depressive patients and controls. In the present study only three time points were explored. Furthermore, the increased secretion of sAA before and after stimulation could allude to an increased responsiveness of novel and uncontrollable situations in patients with MDD. These preliminary results suggest that sAA might be a useful biological marker of MDD. PMID:22063648

  20. Effect of chemicals on fungal alpha-amylase activity.

    PubMed

    Ali, F S; Abdel-Moneim, A A

    1989-01-01

    The effect of 8 growth regulators at concentrations of 1,000, 5,000 and 10,000 ppm on the activity of fungal (Aspergillus flavus var. columnaris) alpha-amylase was studied. Indol acetic acid (IAA) and naphthalene acetic acid (NAA) inhibited alpha-amylase activity by 2% and 7% at 1,000 ppm. The other 6 growth regulators, indol butyric acid (IBA), gibberellic acid, cumarin, cycocel (CCC), atonik-G and kylar, did not inhibit but stimulated alpha-amylase activity (0 to 9%) at 1,000 ppm. All growth regulators studied inhibited alpha-amylase activity at 5,000 and 10,000 ppm concentration except kylar. The effect of organic acids and formaldehyde at 0.01, 0.005, and 0.001 M was studied. Acetic acid stimulated alpha-amylase at all concentrations, but formic acid, oxalic acid, lactic acid and citric acid inhibited alpha-amylase activity by 91, 100, 100 and 79%, respectively, at a concentration of 0.01 M, while by 31, 100, 15 and 20%, respectively, at 0.005 M. Formaldehyde induced 7, 3 and 2% inhibition at 0.01, 0.005 and 0.001 M, respectively. At 0.01 M either sorbitol or fructose inhibited alpha-amylase by 8%, Maltose 7%, sucrose 6%, phenol, glucose and galactose each by 5%, ethanol, glycerol, arabinose and sodium benzoate each by 4%, isopropanol and mannitol 1%, but methanol and ammonium citrate dibasic did not inhibit alpha-amylase. The results indicate that CuCl2, SnCl2, AgNO3 and Fe2(SO4)3 were the strongest inhibitors, followed by Cd(C2H3O2), HgCl2, Na2-EDTA, Na2HPO4, and CaCl2 in decreasing order. NaCl, NaBr and Mn SO4 did not inhibit alpha-amylase at concentrations from 10 mM to 0.01 mM. PMID:2515680

  1. Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

    PubMed

    Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

    2014-03-01

    (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats. PMID:24319124

  2. Novel prediction method of beer foam stability using protein Z, barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and yeast thioredoxin.

    PubMed

    Iimure, Takashi; Takoi, Kiyoshi; Kaneko, Takafumi; Kihara, Makoto; Hayashi, Katsuhiro; Ito, Kazutoshi; Sato, Kazuhiro; Takeda, Kazuyoshi

    2008-09-24

    Foam stability is an important quality trait of beer. Our previous results of two-dimensional gel electrophoresis (2DE) analyses of beer proteins implied a relationship between barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and beer foam stability as judged by the NIBEM-T analyzer. To develop a novel prediction method of beer foam stability under different conditions of barley cultivar and malt modification, multiple linear regression analysis was applied. The spot intensities of major beer proteins on 2DE gel were quantified and used as explanatory variables. The foam stabilities of 25 beer samples each brewed from malt with different malt modification in one of the three cultivars (cultivars A, B, and C) were explained by the spot intensities of BDAI-1 at the 5% significance level ( r = 0.421). Furthermore, two other major protein spots (b0 and b5) were observed on the 2DE gels of Japanese commercial beer samples with different foam stability. Then, multiple regression for foam stability was calculated using these three spot intensities as explanatory variables. As a result, 72.1% of the beer foam stability in 25 beer samples was explained by a novel multiple regression equation calculated using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. To verify the validity of the multiple regression equation and the explanatory variables, the beer foam stability in practical beer samples was analyzed. As a result, 81.5% of the beer foam stability in 10 Japanese commercial beer samples was also explained by using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. Mass spectrometry analyses followed by database searches revealed that protein spots b0 and b5 were identified as protein Z originated from barley and thioredoxin originated from yeast, respectively. These results confirm that BDAI-1 and protein Z are foam-positive factors and identify yeast thioredoxin as a possible novel foam

  3. On the mechanism of alpha-amylase.

    PubMed

    Oudjeriouat, Naïma; Moreau, Yann; Santimone, Marius; Svensson, Birte; Marchis-Mouren, Guy; Desseaux, Véronique

    2003-10-01

    Two inhibitors, acarbose and cyclodextrins (CD), were used to investigate the active site structure and function of barley alpha-amylase isozymes, AMY1 and AMY2. The hydrolysis of DP 4900-amylose, reduced (r) DP18-maltodextrin and maltoheptaose (catalysed by AMY1 and AMY2) was followed in the absence and in the presence of inhibitor. Without inhibitor, the highest activity was obtained with amylose, kcat/Km decreased 103-fold using rDP18-maltodextrin and 10(5) to 10(6)-fold using maltoheptaose as substrate. Acarbose is an uncompetitive inhibitor with inhibition constant (L1i) for amylose and maltodextrin in the micromolar range. Acarbose did not bind to the active site of the enzyme, but to a secondary site to give an abortive ESI complex. Only AMY2 has a second secondary binding site corresponding to an ESI2 complex. In contrast, acarbose is a mixed noncompetitive inhibitor of maltoheptaose hydrolysis. Consequently, in the presence of this oligosaccharide substrate, acarbose bound both to the active site and to a secondary binding site. alpha-CD inhibited the AMY1 and AMY2 catalysed hydrolysis of amylose, but was a very weak inhibitor compared to acarbose.beta- and gamma-CD are not inhibitors. These results are different from those obtained previously with PPA. However in AMY1, as already shown for amylases of animal and bacterial origin, in addition to the active site, one secondary carbohydrate binding site (s1) was necessary for activity whereas two secondary sites (s1 and s2) were required for the AMY2 activity. The first secondary site in both AMY1 and AMY2 was only functional when substrate was bound in the active site. This appears to be a general feature of the alpha-amylase family. PMID:14511369

  4. Salivary alpha-amylase and cortisol responsiveness following electrical stimulation stress in obsessive-compulsive disorder patients.

    PubMed

    Kawano, Aimi; Tanaka, Yoshihiro; Ishitobi, Yoshinobu; Maruyama, Yoshihiro; Ando, Tomoko; Inoue, Ayako; Okamoto, Shizuko; Imanaga, Junko; Kanehisa, Masayuki; Higuma, Haruka; Ninomiya, Taiga; Tsuru, Jusen; Akiyoshi, Jotaro

    2013-08-30

    Salivary α-amylase (sAA) serves as a marker of sympathoadrenal medullary system (SAM) activity. Salivary AA has not been extensively studied in obsessive-compulsive disorder (OCD) patients. In the current study, 45 OCD patients and 75 healthy volunteers were assessed with the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS), the Profile of Mood State (POMS), and the State-Trait Anxiety Inventory (STAI). Measures of heart rate variability (HRV), sAA, and salivary cortisol were also obtained following the application of electrical stimulation stress. The Y-BOCS and POMS Tension-Anxiety, Depression-Dejection, Anger-Hostility, Fatigue, and Confusion scores were significantly increased in patients with OCD compared with healthy controls. In contrast, Vigor scores were significantly decreased in patients with OCD relative to scores in healthy controls. There was no difference in HRV between the patients and the controls. Salivary AA levels in female and male OCD patients were significantly elevated relative to controls both before and after electrical stimulation. In contrast, there were no differences in salivary cortisol levels between OCD patients and controls. The elevated secretion of sAA before and after stimulation may suggest an increased responsiveness to novel and uncontrollable situations in patients with OCD. An increase in sAA might be a characteristic change of OCD. PMID:23266021

  5. Different polyphenolic components of soft fruits inhibit alpha-amylase and alpha-glucosidase.

    PubMed

    McDougall, Gordon J; Shpiro, Faina; Dobson, Patricia; Smith, Pauline; Blake, Alison; Stewart, Derek

    2005-04-01

    Polyphenol-rich extracts from soft fruits were tested for their ability to inhibit alpha-amylase and alpha-glucosidase. All extracts tested caused some inhibition of alpha-amylase, but there was a 10-fold difference between the least and most effective extracts. Strawberry and raspberry extracts were more effective alpha-amylase inhibitors than blueberry, blackcurrant, or red cabbage. Conversely, alpha-glucosidase was more readily inhibited by blueberry and blackcurrant extracts. The extent of inhibition of alpha-glucosidase was related to their anthocyanin content. For example, blueberry and blackcurrant extracts, which have the highest anthocyanin content, were the most effective inhibitors of alpha-glucosidase. The extracts most effective in inhibiting alpha-amylase (strawberry and raspberry) contain appreciable amounts of soluble tannins. Other tannin-rich extracts (red grape, red wine, and green tea) were also effective inhibitors of alpha-amylase. Indeed, removing tannins from strawberry extracts with gelatin also removed inhibition. Fractionation of raspberry extracts on Sephadex LH-20 produced an unbound fraction enriched in anthocyanins and a bound fraction enriched in tannin-like polyphenols. The unbound anthocyanin-enriched fraction was more effective against alpha-glucosidase than the original extract, whereas the alpha-amylase inhibitors were concentrated in the bound fraction. The LH-20 bound sample was separated by preparative HPLC, and fractions were assayed for inhibition of alpha-amylase. The inhibitory components were identified as ellagitannins using LC-MS-MS. This study suggests that different polyphenolic components of fruits may influence different steps in starch digestion in a synergistic manner. PMID:15796622

  6. [Microbial alpha-amylases: physicochemical properties, substrate specificity and domain structure].

    PubMed

    Avdiiuk, K V; Varbanets', L D

    2013-01-01

    The current literature data on producers, physico-chemical properties and substrate specificity of a-amylases produced by microbes from different taxonomic groups such as bacteria, fungi and yeasts are discussed in the survey. Synthesis of alpha-amylase majority is an inducible process which is stimulated in the presence of starch or products of its hydrolysis. It is possible to increase enzymes activity level by optimization of cultivation conditions of strains-producers. alpha-Amylases, isolated from different sources are distinguished in their physico-chemical properties, particularly in their molecular weights, pH- and thermooptimums, inhibitors and activators. The enzymes hydrolyse soluble starch, amylose, amylopectin, glycogen, maltodextrins, alpha- and beta3-cyclodextrins and other carbohydrate substrates. It is well known that alpha-amylases belong to GH-13 family of glycosyl-hydrolases, which contain the catalytic domain A as (beta/alpha)8-barrel. In addition to domain A, alpha-amylases contain two other domains: B and C, which are localized approximately on opposite sides of (beta/alpha)8-barrel. Most of the known alpha-amylases contain calcium ion, which is located on the surface between domains A and B and plays an important role in stability and activity of the enzyme. PMID:24319968

  7. Inhibition of growth of Aspergillus flavus and fungal alpha-amylases by a lectin-like protein from Lablab purpureus.

    PubMed

    Fakhoury, A M; Woloshuk, C P

    2001-08-01

    Aspergillus flavus is a fungal pathogen of maize causing an important ear rot disease when plants are exposed to drought and heat stress. Associated with the disease is the production of aflatoxins, which are a series of structurally related mycotoxins known to be carcinogenic. Previous research has suggested that the alpha-amylase of A. flavus promotes aflatoxin production in the endosperm of infected maize kernels. We report here the isolation and characterization of a 36-kDa alpha-amylase inhibitor from Lablab purpureus (AILP). AILP inhibited the alpha-amylases from several fungi but had little effect on those from animal and plant sources. The protein inhibited conidial germination and hyphal growth of A. flavus. The amino acid sequence indicated that AILP is similar to lectin members of a lectin-arcelin-alpha-amylase inhibitor family described in common bean and shown to be a component of plant resistance to insect pests. AILP also agglutinated papain-treated red blood cells from human and rabbit. These data indicate that AILP represents a novel variant in the lectin-arcelin-alpha-amylase inhibitor family of proteins having lectin-like and alpha-amylase inhibitory activity. PMID:11497467

  8. Oligosaccharide binding to barley alpha-amylase 1.

    PubMed

    Robert, Xavier; Haser, Richard; Mori, Haruhide; Svensson, Birte; Aghajari, Nushin

    2005-09-23

    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers." PMID:16030022

  9. Production of alpha-amylase by yeast

    SciTech Connect

    Thomse, K.K.

    1987-01-01

    The enzyme alpha-amylase confers to an organism the enzymatic activity for the degradation of polyglucosides with alpha-1,4 glycosidic bonds such as starch and glycogen which are among the major storage compounds in plants and animals. Most alpha-amylases are single polypeptides of molecular weights around 50,000 dalton. They are generally found in the digestive tract of animals and in germinating seeds. Among the products released upon enzymatic degradation of polyglucosides maltose, a sugar that can be utilized as carbon source by yeast, is a major constituent. A cDNA segment complementary to mouse salivary amylase messenger RNA has been inserted into the yeast expression vector pMA56 behind the promoter of the gene encoding alcohol dehydrogenase I of yeast. Yeast transformants harboring plasmids with the normal orientation of the promoter and the mouse amylase cDNA gene produce amylase and release the enzyme in free form into the culture medium. Approximately 90% of the amylase activity is found in the medium. Yeast strains carrying MAL allele and transformed with a plasmid which directed the synthesis of mouse alpha-amylase were tested on plates containing starch and in batch fermentations using different high molecular weight sugars and oligosaccharides as carbon source. The results of these experiments will be discussed. (Refs. 21).

  10. Intracellular alpha-amylase of Streptococcus mutans.

    PubMed

    Simpson, C L; Russell, R R

    1998-09-01

    Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding alpha-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular alpha-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single alpha-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose. PMID:9721315

  11. alpha-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus.

    PubMed

    Ali, Hasenah; Houghton, P J; Soumyanath, Amala

    2006-10-11

    Extracts of six selected Malaysian plants with a reputation of usefulness in treating diabetes were examined for alpha-amylase inhibition using an in vitro model. Inhibitory activity studied by two different protocols (with and without pre-incubation) showed that Phyllanthus amarus hexane extract had alpha-amylase inhibitory properties. Hexane and dichloromethane extracts of Anacardium occidentale, Lagerstroemia speciosa, Averrhoa bilimbiPithecellobium jiringa and Parkia speciosa were not active when tested without pre-incubation. Extraction and fractionation of Phyllanthus amarus hexane extract led to the isolation of dotriacontanyl docosanoate, triacontanol and a mixture of oleanolic acid and ursolic acid. Dotriacontanyl docosanoate and the mixture of oleanolic acid and ursolic acid are reported from this plant species for the first time. All compounds were tested in the alpha-amylase inhibition assay and the results revealed that the oleanolic acid and ursolic acid (2:1) mixture was a potent alpha-amylase inhibitor with IC(50)=2.01 microg/ml (4.41 microM) and that it contributes significantly to the alpha-amylase inhibition activity of the extract. Three pure pentacyclic triterpenoids, oleanolic acid, ursolic acid and lupeol were shown to inhibit alpha-amylase. PMID:16678367

  12. Method for using a yeast alpha-amylase promoter

    DOEpatents

    Gao, Johnway; Skeen, Rodney S.; Hooker, Brian S.; Anderson, Daniel B.

    2003-04-22

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  13. Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin.

    PubMed

    Lin, Ku-Feng; Lee, Tian-Ren; Tsai, Ping-Hsing; Hsu, Ming-Pin; Chen, Ching-San; Lyu, Ping-Chiang

    2007-08-01

    The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by (1)H nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V. radiate plant defensin 2 protein, comprises an alpha-helix and one triple-stranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses. PMID:17444520

  14. Classification and evolution of alpha-amylase genes in plants.

    PubMed

    Huang, N; Stebbins, G L; Rodriguez, R L

    1992-08-15

    The DNA sequences for 17 plant genes for alpha-amylase (EC 3.2.1.1) were analyzed to determine their phylogenetic relationship. A phylogeny for these genes was obtained using two separate approaches, one based on molecular clock assumptions and the other based on a comparison of sequence polymorphisms (i.e., small and localized insertions) in the alpha-amylase genes. These polymorphisms are called "alpha-amylase signatures" because they are diagnostic of the gene subfamily to which a particular alpha-amylase gene belongs. Results indicate that the cereal alpha-amylase genes fall into two major classes: AmyA and AmyB. The AmyA class is subdivided into the Amy1 and Amy2 subfamilies previously used to classify alpha-amylase genes in barley and wheat. The AmyB class includes the Amy3 subfamily to which most of the alpha-amylase genes of rice belong. Using polymerase chain reaction and oligonucleotide primers that flank one of the two signature regions, we show that the AmyA and AmyB gene classes are present in approximately equal amounts in all grass species examined except barley. The AmyB (Amy3 subfamily) genes in the latter case are comparatively underrepresented. Additional evidence suggests that the AmyA genes appeared recently and may be confined to the grass family. PMID:1502164

  15. Characterization of salivary alpha-amylase binding to Streptococcus sanguis

    SciTech Connect

    Scannapieco, F.A.; Bergey, E.J.; Reddy, M.S.; Levine, M.J. )

    1989-09-01

    The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase was the prominent salivary component eluted from S. sanguis. Studies with {sup 125}I-labeled HSMSL or {sup 125}I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of ({sup 125}I)alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.

  16. Alpha-amylase gene transcription in tissues of normal dog.

    PubMed

    Mocharla, H; Mocharla, R; Hodes, M E

    1990-02-25

    We studied the distribution of alpha-amylase mRNA in normal dog tissues by northern blotting (NB) and reverse transcription-polymerase chain reaction (RT-PCR) with human pancreatic (AMY2) and salivary (AMY1) alpha-amylase cDNA-specific primers. Analysis of poly(A+) RNA from various normal tissues by NB indicated the presence of detectable levels of alpha-amylase mRNA transcripts only in pancreas. Dot-blot analysis of DNA amplified with primers common to both (human) isoamylase mRNAs showed presence of alpha-amylase gene transcripts not only in pancreas but also in liver, small intestine, large intestine and fallopian tube. Traces of amylase gene transcripts were also observed in ovary, uterus and lung. Interestingly, amylase transcripts were not detectable in the parotid gland by NB or RT-PCR. We have also localized alpha-amylase mRNA transcripts to dog pancreas by in situ transcription and in situ hybridization. Our results suggest that there is high degree of homology between the alpha-amylase mRNA sequences in dog and human at least in the exon 3-4 regions of the human gene. PMID:2315015

  17. Characterization of salivary alpha-amylase binding to Streptococcus sanguis.

    PubMed Central

    Scannapieco, F A; Bergey, E J; Reddy, M S; Levine, M J

    1989-01-01

    The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase (EC 3.2.1.1) was the prominent salivary component eluted from S. sanguis. Studies with 125I-labeled HSMSL or 125I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of [125I]alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch. Images PMID:2788139

  18. Some studies of alpha-amylase production using Aspergillus oryzae.

    PubMed

    Esfahanibolandbalaie, Z; Rostami, K; Mirdamadi, S S

    2008-11-15

    The extracellular alpha-amylase production by Aspergillus oryzae was studied in submerged fermentation using an Adlof-Kuhner orbital shaker. The effect of initial pH values in the range of 4 to 7.5 on enzyme production was investigated and initial pH medium of 6.2 +/- 0.1 resulted in enhanced alpha-amylase production. The effect of carbon and nitrogen source and composition was examined and it has been observed that corn starch concentration of 15 g L(-1) has sound effect on enzyme production. The medium containing corn starch, sodium nitrate resulted in considerable higher enzyme production. Further, the yeast extract of 2.5 g L(-1) in the medium produced higher enzyme in view to other organic nitrogen sources. The effect of temperature on alpha-amylase production from 20 to 40 degrees C has been studied and at 35 +/- 1 degrees C higher alpha-amylase has been obtained. The effect of shaker's speed on alpha-amylase production from 50 to 200 rpm was investigated. And at about 180 rpm higher enzyme production has been observed. In the present study, it has been found that glucose has repressing effect on a-amylase production using A. oryzae PTCC5164. PMID:19260332

  19. Alcoholysis reactions from starch with alpha-amylases.

    PubMed

    Santamaría, R I; Del Río, G; Saab, G; Rodríguez, M E; Soberón, X; López-Manguía, A

    1999-06-11

    The ability of alpha-amylases from different sources to carry out reactions of alcoholysis was studied using methanol as substrate. It was found that while the enzymes from Aspergillus niger and Aspergillus oryzae, two well-studied saccharifying amylases, are capable of alcoholysis reactions, the classical bacterial liquefying alpha-amylases from Bacillus licheniformis and Bacillus stearothermophilus are not. The effect of starch and methanol concentration, temperature and pH on the synthesis of glucosides with alpha-amylase from A. niger was studied. Although methanol may inactivate alpha-amylase, a 90% substrate relative conversion can be obtained in 20% methanol at a high starch concentration (15% w/v) due to a stabilizing effect of starch on the enzyme. As the products of alcoholysis are a series of methyl-oligosaccharides, from methyl-glucoside to methyl-hexomaltoside, alcoholysis was indirectly quantified by high performance liquid chromatography analysis of the total methyl-glucoside produced after the addition of glucoamylase to the alpha-amylase reaction products. More alcoholysis was obtained from intact soluble starch than with maltodextrins or pre-hydrolyzed starch. The biotechnological implications of using starch as substrate for the production of alkyl-glucosides is analyzed in the context of these results. PMID:10386619

  20. Salivary Cortisol, Salivary Alpha Amylase, and the Dental Anxiety Scale

    PubMed Central

    Sadi, Hana; Finkelman, Matthew; Rosenberg, Morton

    2013-01-01

    The aim of this study was to investigate the correlation between dental anxiety, salivary cortisol, and salivary alpha amylase (sAA) levels. Furthermore, the aim was to look into individual differences such as age, race, gender, any existing pain, or traumatic dental experience and their effect on dental anxiety. This study followed a cross-sectional design and included a convenience sample of 46. Every patient was asked to complete the Dental Anxiety Scale (DAS) and a basic demographic/dental history questionnaire. A saliva sample, utilizing the method of passive drooling, was then collected in 2-mL cryovials. Samples were analyzed for salivary cortisol and sAA levels by Salimetrics. Significant associations were observed between DAS scores and presence of pain and history of traumatic dental experience. However, no significant correlations were observed between DAS, cortisol, and sAA levels. Our study reconfirms that dental anxiety is associated with presence of pain and a history of traumatic dental experience. On the other hand, our study was the first to our knowledge to test the correlation between the DAS and sAA; nevertheless, our results failed to show any significant correlation between dental anxiety, cortisol, and sAA levels. PMID:23763559

  1. Alpha-amylase Inhibition and Antioxidant Activity of Marine Green Algae and its Possible Role in Diabetes Management

    PubMed Central

    Unnikrishnan, P. S.; Suthindhiran, K.; Jayasri, M. A.

    2015-01-01

    Aim: In the continuing search for safe and efficient antidiabetic drug, marine algae become important source which provide several compounds of immense therapeutic potential. Alpha-amylase, alpha-glucosidase inhibitors, and antioxidant compounds are known to manage diabetes and have received much attention recently. In the present study, four green algae (Chaetomorpha aerea, Enteromorpha intestinalis, Chlorodesmis, and Cladophora rupestris) were chosen to evaluate alpha-amylase, alpha-glucosidase inhibitory, and antioxidant activity in vitro. Materials and Methods: The phytochemical constituents of all the extracts were qualitatively determined. Antidiabetic activity was evaluated by inhibitory potential of extracts against alpha-amylase and alpha-glucosidase by spectrophotometric assays. Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl, hydrogen peroxide (H2O2), and nitric oxide scavenging assay. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out to determine the major compound responsible for its antidiabetic action. Results: Among the various extracts screened, chloroform extract of C. aerea (IC50 − 408.9 μg/ml) and methanol extract of Chlorodesmis (IC50 − 147.6 μg/ml) showed effective inhibition against alpha-amylase. The extracts were also evaluated for alpha-glucosidase inhibition, and no observed activity was found. Methanol extract of C. rupestris showed notable free radical scavenging activity (IC50 – 666.3 μg/ml), followed by H2O2 (34%) and nitric oxide (49%). Further, chemical profiling by GC-MS revealed the presence of major bioactive compounds. Phenol, 2,4-bis (1,1-dimethylethyl) and z, z-6,28-heptatriactontadien-2-one were predominantly found in the methanol extract of C. rupestris and chloroform extract of C. aerea. Conclusion: Our results demonstrate that the selected algae exhibit notable alpha-amylase inhibition and antioxidant activity. Therefore, characterization of active compounds and its in vivo

  2. Optimization of alpha-amylase application in raw sugar manufacture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In recent years there have been warnings by some U.S. refineries that there may be a penalty for high starch concentration sin raw sugar if starch control is not improved. Most commercial alpha-amylases used by the U.S. sugar industry to control starch have intermediate temperature stability (up to...

  3. Genetics of alpha-amylases in hexaploid oat species.

    PubMed

    Sharopova, N R; Portyanko, V A; Sozinov, A A

    1998-06-01

    The inheritance of alpha-amylases was studied in six F2 populations of hexaploid oats (Avena sativa, A. byzantina, A. fatua, A. sterillis) using polyacrylamide gel electrophoresis. A total of 22 loci was identified and described. Three main linkages of four or five loci each and an additional two pairs of linked loci were detected. It seems likely that the three main linkage groups represent homeologous chromosomes. Matching of alpha-amylase profiles of hexaploid (AACCDD), tetraploid (AACC), and diploid (AA) species was made to assign the linkage groups to particular subgenomes in the hexaploid oat. It was proposed that Linkage 1 (Amy12-Amy10-1-Amy4-Amy13-Amy11) belongs to the D-subgenome; Linkage 2 (Amy10-2, Amy9-Amy8-Amy6) belongs to the A-subgenome; and Linkage 3 (Amy7-Amy3-Amy5-Amy2) belongs to the C-subgenome of the hexaploid oat. The "malt" and "green" alpha-amylases in hexaploid and tetraploid oats have been identified. Isozymes of "green" alpha-amylase were lower in electrophoretic mobility than other isozymes and were governed by loci assigned to the A- and D-subgenomes. PMID:9775349

  4. Domain B protruding at the third beta strand of the alpha/beta barrel in barley alpha-amylase confers distinct isozyme-specific properties.

    PubMed

    Rodenburg, K W; Juge, N; Guo, X J; Søgaard, M; Chaix, J C; Svensson, B

    1994-04-01

    alpha-Amylases belong to the alpha/beta-barrel protein family in which the active site is created by residues located at the C-terminus of the beta strands and in the helix-connecting loops extending from these ends. In the alpha-amylase family, a small separate domain B protrudes at the C-terminus of the third beta strand of the (beta/alpha)8-barrel framework. The 80% identical barley alpha-amylase isozymes 1 and 2 (AMY1 and AMY2, respectively) differ in substrate affinity and turnover rate, CaCl2 stimulation of activity, sensitivity to the endogenous 21-kDa alpha-amylase/subtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme-specific variations, AMY1-AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMY1-(1-90)-AMY2-(90-403) and AMY1-(1-161)-AMY2-(161-403) characterized in this study contain the 90-residue and 161-residue N-terminal sequences, respectively, of AMY1 and complementary C-terminal regions of AMY2. AMY1-(1-90)-AMY2-(90-403) comprises the 60-amino-acid domain B of AMY2 and resembles this isozyme in sensitivity to alpha-amylase/subtilisin inhibitor and its low affinity for the substrates p-nitrophenyl alpha-D-maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1-(1-161)-AMY2-(161-403) and AMY1, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaCl2. Domain B thus determines several functional and stability properties that distinguish the barley alpha-amylase isozymes. PMID:8168517

  5. Clinical and immunological responses to occupational exposure to alpha-amylase in the baking industry.

    PubMed Central

    Brisman, J; Belin, L

    1991-01-01

    alpha-Amylase is a starch cleaving enzyme often used in the baking industry as a flour additive. It is usually of fungal origin, produced by Aspergillus oryzae. One previous report has shown IgE antibodies and positive skin prick test against alpha-amylase in asthmatic bakers. This paper describes four alpha-amylase sensitised index cases with occupational asthma or rhinitis and the results of a cross sectional study of 20 workers from the same factory who were also exposed to alpha-amylase powder. Air sampling detected airborne alpha-amylase at a concentration of 0.03 mg/m3. Significantly more work related symptoms such as rhinitis and dermatitis were found among the alpha-amylase exposed workers compared with referents. A skin prick test to alpha-amylase was positive in 30% (6/20) of the exposed workers. Most of the persons showing a positive skin prick test had work related symptoms and were also skin prick test positive to common allergens. Nasal challenge tests with amylase were performed in selected cases and validated three cases of alpha-amylase induced rhinitis. Two non-symptomatic workers had precipitins to alpha-amylase. Specific IgG antibodies were shown by two further serological techniques. The nature and relevance of these antibodies are currently being studied. It is concluded that alpha-amylase powder is a potent occupational sensitiser. Precautions should be taken when handling this allergenic enzyme. PMID:1832939

  6. Clinical and immunological responses to occupational exposure to alpha-amylase in the baking industry.

    PubMed

    Brisman, J; Belin, L

    1991-09-01

    alpha-Amylase is a starch cleaving enzyme often used in the baking industry as a flour additive. It is usually of fungal origin, produced by Aspergillus oryzae. One previous report has shown IgE antibodies and positive skin prick test against alpha-amylase in asthmatic bakers. This paper describes four alpha-amylase sensitised index cases with occupational asthma or rhinitis and the results of a cross sectional study of 20 workers from the same factory who were also exposed to alpha-amylase powder. Air sampling detected airborne alpha-amylase at a concentration of 0.03 mg/m3. Significantly more work related symptoms such as rhinitis and dermatitis were found among the alpha-amylase exposed workers compared with referents. A skin prick test to alpha-amylase was positive in 30% (6/20) of the exposed workers. Most of the persons showing a positive skin prick test had work related symptoms and were also skin prick test positive to common allergens. Nasal challenge tests with amylase were performed in selected cases and validated three cases of alpha-amylase induced rhinitis. Two non-symptomatic workers had precipitins to alpha-amylase. Specific IgG antibodies were shown by two further serological techniques. The nature and relevance of these antibodies are currently being studied. It is concluded that alpha-amylase powder is a potent occupational sensitiser. Precautions should be taken when handling this allergenic enzyme. PMID:1832939

  7. Characterization of the L. manihotivorans alpha-amylase gene.

    PubMed

    Morlon-Guyot, J; Mucciolo-Roux, F; Rodriguez Sanoja, R; Guyot, J P

    2001-07-01

    Primers and probes were established from the sequences of the alpha-amylase genes (amyA) of L. amylovorus CIP 102989 and of L. plantarum A6 (Giraud and Cuny 1997). They were successfully used for the detection of the amyA gene in L. manihotivorans strain LMG 18010T and a 2842 bp region, containing the entire gene (2706 bp) with its putative promoter has been sequenced. More than 98% nucleotide sequence identities was found with L. amylovorus and L. plantarum amyA genes. The deduced amino acid sequence shares more than 96% amino acid sequence identities with L. amylovorus and L. plantarum alpha-amylases, and also 65% and 59% identities with the alpha-amylases of B. subtilis and S. bovis, respectively. The 3' terminal part of L. manihotivorans LMG 18010T amyA gene contained four repeated sequences (SRU). The amyA genes of the three lactobacilli species differed mainly in the number of SRU and in the size of the flanking regions of the SRU. PMID:11697143

  8. Thermal stability of alpha-amylase in aqueous cosolvent systems.

    PubMed

    Yadav, Jay Kant; Prakash, V

    2009-09-01

    The activity and thermal stability of alpha-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 +/- 2 degrees C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (Tm) app and activation energy (Ea) of alpha-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (Tm)app were 20 degrees C, 14 degrees C, 13 degrees C and 9 degrees C, respectively. The Ea of thermal denaturation of alpha-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents. PMID:19805899

  9. Alpha-amylase from the Hyperthermophilic Archaeon Thermococcus thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, E. C. M. J.; Pusey, M. L.; Ng, M. L.; Garriott, O. K.

    2003-01-01

    Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments such as hot springs. The ability of survival at extreme conditions has rendered enzymes from extremophiles to be of interest in industrial applications. One approach to producing these extremozymes entails the expression of the enzyme-encoding gene in a mesophilic host such as E.coli. This method has been employed in the effort to produce an alpha-amylase from a hyperthermophile (an organism that displays optimal growth above 80 C) isolated from a hydrothermal vent at the Rainbow vent site in the Atlantic Ocean. alpha-amylases catalyze the hydrolysis of starch to produce smaller sugars and constitute a class of industrial enzymes having approximately 25% of the enzyme market. One application for thermostable alpha-amylases is the starch liquefaction process in which starch is converted into fructose and glucose syrups. The a-amylase encoding gene from the hyperthermophile Thermococcus thioreducens was cloned and sequenced, revealing high similarity with other archaeal hyperthermophilic a-amylases. The gene encoding the mature protein was expressed in E.coli. Initial characterization of this enzyme has revealed an optimal amylolytic activity between 85-90 C and around pH 5.3-6.0.

  10. THE CELL-BOUND ALPHA-AMYLASES OF STREPTOCOCCUS BOVIS.

    PubMed

    WALKER, G J

    1965-02-01

    1. The cell-bound alpha-amylase of Streptococcus bovis has been isolated from other carbohydrases in the cell extract by chromatography on DEAE-cellulose. The enzyme has been compared with the extracellular alpha-amylase produced by this organism. 2. The two amylases had similar action patterns on amylose, the main product being maltotriose with smaller amounts of maltose and a little glucose. 3. The cell-bound amylase hydrolysed maltopentaose and maltohexaose at a similar rate to the hydrolysis of amylose. Maltotetraose was hydrolysed six times more slowly, and maltotriose 280 times more slowly, than amylose. 4. Studies with end-labelled maltodextrins revealed that the cell-bound alpha-amylase preferentially hydrolysed the third linkage from the non-reducing end, liberating maltotriose. The linkage at the reducing end of maltotriose was more easily hydrolysed than the other. 5. Egg-white lysozyme and the extracellular enzymes of Streptomyces albus lysed the cell walls of Streptococcus bovis, releasing amylase into the medium. In the presence of 0.6 m-sucrose 10% of the maximal amylase activity was released by lysozyme. Suspension of the spheroplasts in dilute buffer caused the rupture of the cytoplasmic membrane and the liberation of amylase. 6. A sensitive method for determining the ability of amylases to degrade starch granules is described. PMID:14346085

  11. Action of human pancreatic and salivary alpha-amylases on maltooligosaccharides: evaluation of kinetic parameters.

    PubMed

    Saito, N; Horiuchi, T; Yoshida, M; Imai, T

    1979-10-01

    The kinetic studies on the reactions of human pancreatic and salivary alpha-amylases with several maltooligosaccharides (maltotetraose, maltopentaose, maltohexaose, and maltoheptaose) were carried out. The susceptibility to hydrolysis with human pancreatic alpha-amylase decreased in the order of maltopentaose, maltohexaose, maltotetraose, and maltoheptaose, while with human salivary alpha-amylase maltopentaose was hydrolysed slightly slower than maltohexaose but fairly faster than maltotetraose or maltoheptaose from a viewpoint of the rates of reactions based on the amount of substrate changed. The relative rates of production of substrates, utilized in the coupled yeast alpha-glucosidase reaction, increased in the order of maltoheptaose, maltohexaose, maltotetraose, and maltopentaose with human pancreatic alpha-amylase, while with human salivary alpha-amylase in the order of maltoheptaose, maltotetraose, maltohexaose, and maltopentaose. Thus, maltopentaose was considered to be the best substrate over maltotetraose, maltohexaose or maltoheptaose for the alpha-glucosidase coupled method of alpha-amylase determination. PMID:385176

  12. A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics.

    PubMed

    Aydin, Suleyman

    2007-01-31

    During the past decade, many salivary parameters have been used to characterize disease states. Ghrelin (GAH) is recently-discovered peptide hormone secreted mainly from the stomach but also produced in a number of other tissues including salivary glands. The aim of this work was to examine the relationship between active (aGAH) and inactive (dGAH) ghrelin in the saliva and other salivary parameters in type II diabetic patients and healthy controls. Salivary parameters were assessed in a single measurement of unstimulated whole saliva from 20 obese and 20 non-obese type II diabetes patients, and in 22 healthy controls. Total protein and alpha-amylase were determined by colorimetric methods, and glucose by the glucose-oxidase method. Saliva aGAH and dGAH levels were measured using a commercial radioimmunoassay (RIA) kit. Salivary concentrations of aGAH and dGAH ghrelin were more markedly decreased in obese diabetic subjects than in the two other groups. Glucose and alpha-amylase levels were higher in diabetic subjects than in controls. Furthermore, there were correlations between GAH levels and BMI, and between GAH and blood pressure. However, there was no marked variability in saliva flow rates among the groups. These results indicate that measurement of salivary GAH and its relationship to other salivary parameters might help to provide insight into the role of ghrelin in diabetes. PMID:17244479

  13. Alpha amylase enzyme inhibitory and anti-inflammatory effect of Lawsonia inermis.

    PubMed

    Imam, Hasan; Mahbub, Nasir Uddin; Khan, Md Forhad; Hana, Humayera Kabir; Sarker, Md Moklesur Rahman

    2013-12-01

    Previously it was reported elsewhere that Lawsonia inermis have anti-inflammatory and analgesic effect in experimental animals. The in vitro porcine alpha amylase inhibitory effect was investigated of this plant methanolic extracts and consequently hypoglycemic effect by quantitatively determining the maltose from the maltose standard curve while the anti-inflammatory effect by acetic acid induced writhing test in mice. Acarbose (10 microg mL(-1)) and Diclofenac sodium (20 mg kg(-1)) were used as reference hypoglycemic and anti-inflammatory drugs, respectively, for this study. The methanolic leaves extract of the plant significantly inhibited (60.97% compared to untreated) enzymatic activity of the amylase at 10 microg mL(-1) dose (p < 0.05) also reduced the chemically induced nociceptive pain stimuli significantly at all doses (p < 0.01). Carbohydrates, glycosides, flavonoids, saponins and tannins were found to have in phytochemical screening of the extract which are thought to bring these effects. For the conclusive purpose, it is suggesting from the result that the pharmacological properties of this Lawsonia inermis can elicit hypoglycemic effect by inhibiting alpha-amylase enzyme and can reduce neurogenic pain stimulus. It gives the notion that how this group of patient would be therapeutically benefitted by decreasing both these effects by the same agent which is easy available. PMID:24506051

  14. Biochemical properties of alpha-amylase from peel of Citrus sinensis cv. Abosora.

    PubMed

    Mohamed, Saleh Ahmed; Drees, Ehab A; El-Badry, Mohamed O; Fahmy, Afaf S

    2010-04-01

    alpha-Amylase activity was screened in the peel, as waste fruit, of 13 species and cultivars of Egyptian citrus. The species Citrus sinensis cv. Abosora had the highest activity. alpha-Amylase AI from Abosora peel was purified to homogeneity using anion and cation-exchange, and gel filtration chromatographies. Molecular weight of alpha-amylase AI was found to be 42 kDa. The hydrolysis properties of alpha-amylase AI toward different substrates indicated that corn starch is the best substrate. The alpha-amylase had the highest activity toward glycogen compared with amylopectin and dextrin. Potato starch had low affinity toward alpha-amylase AI but it did not hydrolyze beta-cyclodextrin and dextran. Apparent Km for alpha-amylase AI was 5 mg (0.5%) starch/ml. alpha-Amylase AI showed optimum activity at pH 5.6 and 40 degrees C. The enzyme was thermally stable up to 40 degrees C and inactivated at 70 degrees C. The effect of mono and divalent metal ions were tested for the alpha-amylase AI. Ba2+ was found to have activating effect, where as Li+ had negligible effect on activity. The other metals caused inhibition effect. Activity of the alpha-amylase AI was increased one and half in the presence of 4 mM Ca2+ and was found to be partially inactivated at 10 mM Ca2+. The reduction of starch viscosity indicated that the enzyme is endoamylase. The results suggested that, in addition to citrus peel is a rich source of pectins and flavanoids, alpha-amylase AI from orange peel could be involved in the development and ripening of citrus fruit and may be used for juice processing. PMID:19941088

  15. Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

    2003-04-01

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  16. High-activity barley alpha-amylase by directed evolution.

    PubMed

    Wong, Dominic W S; Batt, Sarah B; Lee, Charles C; Robertson, George H

    2004-10-01

    Barley alpha-amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae. The gene coding for the wild-type enzyme was subjected to directed evolution. Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate. The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity. After three rounds of directed evolution, one mutant (Mu322) showed 1000 times the total activity and 20 times the specific activity of the wild-type enzyme produced by the same yeast expression system. Comparison of the amino acid sequence of this mutant with the wild type revealed five substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B. Two of these mutations. Q44H and R303K, result in amino acids highly conserved in cereal alpha-amylases. R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule. PMID:15635937

  17. Circadian rhythm of alpha-amylase in rat parotid gland.

    PubMed

    Bellavía, S L; Sanz, E G; Chiarenza, A P; Sereno, R; Vermouth, N T

    1990-01-01

    The circadian rhythm of alpha-amylase, E.C. 3.2.1.1. (alpha-1,4-glucan-4-glucanohydrolase) in parotid gland of 25 day old rats was studied under different experimental conditions (fast, reversed photoperiod, constant light or darkness and treatment with reserpine and alpha-methyl-p-tyrosine). The rhythm of rats fasted or exposed for 7 days to constant darkness did not change. There were modifications in the rhythm of rats submitted to a reversed photoperiod and it disappeared in animals submitted to constant light or darkness for 15 days or treated with reserpine or alpha-methyl-p-tyrosine. The rhythm persisted, with minor changes in the acrophase, in parotids of rats kept during their gestation and post-natal life in constant light or darkness. Results suggest that the circadian rhythm of alpha-amylase in parotid gland of young rats is endogenous, synchronized by the photoperiod, under autonomous nervous system control and maternal coordination. This model appears to be useful in the study of sympathetic nervous system control of target organs and circadian rhythms in general. PMID:2076161

  18. Salivary Alpha-Amylase Reactivity in Breast Cancer Survivors

    PubMed Central

    Wan, Cynthia; Couture-Lalande, Marie-Ève; Narain, Tasha A.; Lebel, Sophie; Bielajew, Catherine

    2016-01-01

    The two main components of the stress system are the hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medullary (SAM) axes. While cortisol has been commonly used as a biomarker of HPA functioning, much less attention has been paid to the role of the SAM in this context. Studies have shown that long-term breast cancer survivors display abnormal reactive cortisol patterns, suggesting a dysregulation of their HPA axis. To fully understand the integrity of the stress response in this population, this paper explored the diurnal and acute alpha-amylase profiles of 22 breast cancer survivors and 26 women with no history of cancer. Results revealed that breast cancer survivors displayed identical but elevated patterns of alpha-amylase concentrations in both diurnal and acute profiles relative to that of healthy women, F (1, 39) = 17.95, p < 0.001 and F (1, 37) = 7.29, p = 0.010, respectively. The average area under the curve for the diurnal and reactive profiles was 631.54 ± 66.94 SEM and 1238.78 ± 111.84 SEM, respectively. This is in sharp contrast to their cortisol results, which showed normal diurnal and blunted acute patterns. The complexity of the stress system necessitates further investigation to understand the synergistic relationship of the HPA and SAM axes. PMID:27023572

  19. Comparison of alpha-amylase activity in larval stages of flour beetles, Tribolium confusum (Coleoptera: Tenebionidae).

    PubMed

    Bandani, A R; Balvasi, A

    2006-01-01

    Flour beetles attack stored grain products such as flour, cereals, meal, dried pet food, dried flowers and even dried museum specimens and other foods in the house. Stored-product insects cause tremendous losses by lowering weight, germination rate, nutritional value and grain grade. These beetles are of the most important pests of stored products in the home and grocery stores. The adult female may live for as long as two years, depositing 300 to 400 eggs. The life cycle requires one to four months when temperatures are favorable. Several methods could be used to control this insect including synthetic insecticides, biological control, physical control and transgenic plant carrying gene of interest. Chemical controls are discouraged due to pesticide residue in the commodities and resistance in insects. The study of insect digestive enzymes seems to make sense in the realization that the gut is the major interface between the insect and its environment. Hence, an understanding of digestive enzyme function is essential when developing methods of insect control such as the use of enzyme inhibitors and transgenic plants to control insect pests. Therefore, the aim of the current study was to get a good understanding from enzyme composition of different larval stages of the insect and finally characterize amylase which is the key enzyme in digestive system of this insect. For alpha-amylase study whole larvae were homogenized in 0.02 M phosphate buffer at pH 7.2. The homogenates were separately transferred to a 1.5 ml of centrifuge tubes and centrifuged at 15000xg for 20 min at 4degrees C. The supernatants were used as enzyme source in assays. alpha-Amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure using 1% soluble starch (Merck) as substrate. The results show that enzyme activity (OD) in the first, second, third and fourth larval stages were 0.5, 1.15, 1.35 and 1.362, respectively. There are significant differences in amylase activity in

  20. alpha-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans.

    PubMed

    Agger, T; Spohr, A B; Nielsen, J

    2001-01-01

    The effect of biomass concentration on the formation of Aspergillus oryzae alpha-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of alpha-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2-12 g dry weight kg(-1). When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzae alpha-amylase gene (creA) was deleted, no significant decrease in the specific rate of alpha-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific alpha-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium. PMID:11234963

  1. Crystal and molecular structure of barley alpha-amylase.

    PubMed

    Kadziola, A; Abe, J; Svensson, B; Haser, R

    1994-05-27

    The three-dimensional structure of barley malt alpha-amylase (isoform AMY2-2) was determined by multiple isomorphous replacement using three heavy-atom derivatives and solvent flattening. The model was refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement to an R-factor of 0.153 based on 18,303 independent reflections with F(o) > sigma(F(o)) between 10 and 2.8 A resolution, with root-mean-square deviations of 0.016 A and 3.3 degrees from ideal bond lengths and bond angles, respectively. The final model consists of 403 amino acid residues, three calcium ions and 153 water molecules. The polypeptide chain folds into three domains: a central domain forming a (beta alpha)8-barrel of 286 residues, with a protruding irregular structured loop domain of 64 residues (domain B) connecting strand beta 3 and helix alpha 3 of the barrel, and a C-terminal domain of 53 residues forming a five stranded anti-parallel beta-sheet. Unlike the previously known alpha-amylase structures, AMY2-2 contains three Ca2+ binding sites co-ordinated by seven or eight oxygen atoms from carboxylate groups, main-chain carbonyl atoms and water molecules, all calcium ions being bound to domain B and therefore essential for the structural integrity of that domain. Two of the Ca2+ sites are located only 7.0 A apart with one Asp residue serving as ligand for both. One Ca2+ site located at about 20 A from the other two was found to be exchangeable with Eu3+. By homology with other alpha-amylases, some important active site residues are identified as Asp179, Glu204 and Asp289, and are situated at the C-terminal end of the central beta-barrel. A starch granule binding site, previously identified as Trp276 and Trp277, is situated on alpha-helix 6 in the central (beta alpha)8-barrel, at the surface of the enzyme. This binding site region is associated with a considerable disruption of the (beta alpha)8-barrel 8-fold symmetry. PMID:8196040

  2. Optimization of alpha-amylase immobilization in calcium alginate beads.

    PubMed

    Ertan, Figen; Yagar, Hulya; Balkan, Bilal

    2007-01-01

    alpha-Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl(2) concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl(2) concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL(-1), and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40 degrees C. PMID:17516249

  3. Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability.

    PubMed

    Svensson, B

    1994-05-01

    Most starch hydrolases and related enzymes belong to the alpha-amylase family which contains a characteristic catalytic (beta/alpha)8-barrel domain. Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme. Crystal structures have been reported in three of these enzyme classes: the alpha-amylases, the cyclodextrin glucanotransferases, and the oligo-1,6-glucosidases. Throughout the alpha-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the beta-->alpha loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of alpha-amylases, changed the distribution of alpha-, beta- and gamma-cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative alpha-1,4:alpha-1,6 dual-bond specificity of neopullulanase. Barley alpha-amylase isozyme hybrids and Bacillus alpha-amylases demonstrate the impact of a small domain B protruding from the (beta/alpha)8-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as alpha-amylase, starch branching and debranching enzymes, and amylomaltase. PMID:8018865

  4. Effect of oilseed cakes on alpha-amylase production by Bacillus licheniformis CUMC305.

    PubMed

    Krishnan, T; Chandra, A K

    1982-08-01

    The effects of oilseed cakes on extracellular thermostable alpha-amylase production by Bacillus licheniformis CUMC305 was investigated. Each oilseed cake was made of groundnut, mustard, sesame, linseed, coconut copra, madhuca, or cotton. alpha-Amylase production was considerably improved in all instances and varied with the oilseed cake concentration in basal medium containing peptone and beef extract. Maximum increases were effected by a low concentration (0.5 to 1.0%) of groundnut or coconut, a high concentration (3%) of linseed or mustard, and an Rintermediate concentration (2%) of cotton, madhuca, or sesame. The oilseed cakes made of groundnut or mustard could completely replace the conventional peptone-beef extract medium as the fermentation base for the production of alpha-amylase by B. licheniformis. The addition of corn steep liquor to cotton, linseed, sesame, or madhuca cake in the medium improved alpha-amylase production. PMID:6181738

  5. Synergistic action of. alpha. -amylase and glucoamylase on hydrolysis of starch

    SciTech Connect

    Fujii, M.; Kawamura, Y.

    1985-03-01

    Synergistic action of ..alpha..-amylase and glucoamylase on hydrolysis of starch is modeled by the kinetic equations presented in this paper. At the early stage of the reaction ..alpha..-amylase acts as a contributor of newly formed non-reducing ends of starch molecules to glucoamylase by splitting the original starch molecules. This is expressed by the simultaneous differential equations which consist of each rate equation for ..alpha.. amylase and glucoamylase. After the molecular weight of the substrate decreases to the value of about 5000, which is obtained experimentally in this work, the action of ..alpha.. amylase can be neglected and the rate of formation of glucose obeys only the rate equation for glucoamylase. 5 references.

  6. Effect of oilseed cakes on alpha-amylase production by Bacillus licheniformis CUMC305.

    PubMed Central

    Krishnan, T; Chandra, A K

    1982-01-01

    The effects of oilseed cakes on extracellular thermostable alpha-amylase production by Bacillus licheniformis CUMC305 was investigated. Each oilseed cake was made of groundnut, mustard, sesame, linseed, coconut copra, madhuca, or cotton. alpha-Amylase production was considerably improved in all instances and varied with the oilseed cake concentration in basal medium containing peptone and beef extract. Maximum increases were effected by a low concentration (0.5 to 1.0%) of groundnut or coconut, a high concentration (3%) of linseed or mustard, and an Rintermediate concentration (2%) of cotton, madhuca, or sesame. The oilseed cakes made of groundnut or mustard could completely replace the conventional peptone-beef extract medium as the fermentation base for the production of alpha-amylase by B. licheniformis. The addition of corn steep liquor to cotton, linseed, sesame, or madhuca cake in the medium improved alpha-amylase production. PMID:6181738

  7. Isozyme hybrids within the protruding third loop domain of the barley alpha-amylase (beta/alpha)8-barrel. Implication for BASI sensitivity and substrate affinity.

    PubMed

    Juge, N; Rodenburg, K W; Guo, X J; Chaix, J C; Svensson, B

    1995-04-24

    Barley alpha-amylase isozymes AMY1 and AMY2 contain three structural domains: a catalytic (beta/alpha)8-barrel (domain A) with a protruding loop (domain B; residues 89-152) that binds Ca2+, and a small C-terminal domain. Different parts of domain B secure isozyme specific properties as identified for three AMY1-AMY2 hybrids, obtained by homeologous recombination in yeast, with crossing-over at residues 112, 116, and 144. The AMY1 regions Val90-Thr112 and Ala145-Leu161 thus confer high affinities for the substrates alpha-D-maltoheptaoside and amylose, respectively. Leu117-Phe144, and to a lesser degree Ala145-Leu161, are critical for the stability at low pH characteristic of AMY1 and for the sensitivity to barley alpha-amylase/subtilisin inhibitor specific to AMY2. PMID:7737421

  8. alpha. -Amylase of Clostridium thermosulfurogenes EM1: Nucleotide sequence of the gene, processing of the enzyme, and comparison to other. alpha. -amylases

    SciTech Connect

    Bahl, H.; Burchhardt, G.; Spreinat, A.; Haeckel, K.; Wienecke, A.; Antranikian, G.; Schmidt, B. )

    1991-05-01

    The nucleotide sequence of the {alpha}-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes Em1 suggested that the {alpha}-amylase is translated form mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature {alpha}-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 {alpha}-amylase with those from other bacterial and eukaryotic {alpha}-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca{sup 2+}-binding site (consensus region I) of this Ca{sub 2+}-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the {alpha}-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the {beta}-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.

  9. Exposure-sensitization relationship for alpha-amylase allergens in the baking industry.

    PubMed

    Houba, R; Heederik, D J; Doekes, G; van Run, P E

    1996-07-01

    Fungal alpha-amylase is an important occupational allergen in the bakery industry. Epidemiologic studies focusing on the relationship between alpha-amylase allergen exposure and work-related respiratory allergy, however, have not been reported yet. In this cross-sectional study, sensitization to occupational allergens and work-related symptoms were studied in 178 bakery workers and related to allergen exposure. Alpha-amylase allergen concentrations were measured in personal dust samples, using a sandwich enzyme immunoassay. All workers were categorized into groups on the basis of their job histories and the alpha-amylase exposure levels of their job titles. Of all workers 25% had one or more work-related symptoms. As much as 9% of the bakery workers showed a positive skin prick test reaction to fungal amylase, and in 8% amylase-specific IgE was demonstrated. Alpha-amylase exposure and atopy appeared to be the most important determinants of skin sensitization, with prevalence ratios for atopy of 20.8 (95% CI, 2.74 to 158) and for medium and high alpha-amylase exposure groups of 8.6 (95% CI, 1.01 to 74) and 15.9 (95% CI, 1.95 to 129), respectively. Furthermore, a positive association was found between positive skin prick tests to alpha-amylase and work-related respiratory symptoms. In conclusion, this study has shown that there is a strong and positive relationship between alpha-amylase allergen exposure levels in bakeries and specific sensitization in bakery workers. PMID:8680668

  10. [Cloning the alpha-amylase gene of Streptococcus bovis and its expression in Bacillus subtilis cells].

    PubMed

    Iakorski, P; Kuntsova, M M; Loseva, E F; Khasanov, F K

    1991-06-01

    The gene coding for alpha-amylase from the ruminant bacterium Streptococcus bovis was cloned on the plasmid pMX39 in Bacillus subtilis cells. An alpha-amylase positive colony was isolated in the initial screening of 3900 colonies on the medium containing insoluble starch. The size of the insert was approximately 2.8 kb. The recombinant plasmid was stably maintained in Bacillus subtilis cells under the nonselective conditions. PMID:1944323

  11. Psychological stress-induced changes in salivary alpha-amylase and adrenergic activity.

    PubMed

    Kang, Younhee

    2010-12-01

    The aim of the study was to examine the relationships among salivary alpha-amylase, plasma catecholamines, blood pressure, and heart rate during psychological stress. This study used a pretest-post-test experimental design with a control group, using repeated measures. A total of 33 participants was divided into the experimental group (n = 16) that underwent a college academic final test as the psychological stress and the control group (n = 17) that did not undergo the test. The levels of salivary alpha-amylase and plasma catecholamines, blood pressure, and heart rate were measured seven times and stress and anxiety were measured once and twice, respectively, as subjective stress markers. Significant changes in the level of salivary alpha-amylase were found in response to psychological stress. However, the correlations of salivary alpha-amylase with the plasma catecholamines, blood pressure, and heart rate were only partially found to be statistically significant. In conclusion, it was shown that salivary alpha-amylase was sensitive to stress throughout this study. Thus, salivary alpha-amylase may be used to measure stress uninvasively in both clinical settings and nursing research where the effects of stress might be scrutinized. Furthermore, the mechanisms of illnesses that are induced by stress could be explored. PMID:21210927

  12. Interparental Aggression and Parent-Adolescent Salivary Alpha Amylase Symmetry

    PubMed Central

    Gordis, Elana B.; Margolin, Gayla; Spies, Lauren; Susman, Elizabeth J.; Granger, Douglas A.

    2010-01-01

    The present study examined salivary alpha-amylase (sAA), a putative marker of adrenergic activity, in family members engaging in family conflict discussions. We examined symmetry among family members' sAA levels at baseline and in response to a conflict discussion. The relation between a history of interparental aggression on parent-adolescent sAA symmetry also was examined. Participants were 62 families with a mother, father, and biological child age 13-18 (n = 29 girls). After engaging in a relaxation procedure, families participated in a 15-minute triadic family conflict discussion. Participants provided saliva samples at post-relaxation/pre-discussion, immediately post-discussion, and at 10 and 20 min post-discussion. Participants also reported on interparental physical aggression during the previous year. Across the sample we found evidence of symmetry between mothers' and adolescents' sAA levels at baseline and around the discussion. Interparental aggression was associated with lower sAA levels among fathers. Interparental aggression also affected patterns of parent-child sAA response symmetry such that families reporting interparental aggression exhibited greater father-adolescent sAA symmetry than did those with no reports of interparental aggression. Among families with no interparental aggression history, we found consistent mother-adolescent symmetry. These differences suggest different patterns of parent-adolescent physiological attunement among families with interparental aggression. PMID:20096715

  13. Regulation of the synthesis of barley aleurone. cap alpha. -amylase by gibberellic acid and calcium ions

    SciTech Connect

    Jones, R.L.; Carbonell, J.

    1984-09-01

    The effects of gibberellic acid (GA/sub 3/) and calcium ions on the production of ..cap alpha..-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA/sub 3/ or CA/sup 2 +/ show qualitative and quantitative changes in hydrolase production following incubation in either GA/sub 3/ or CA/sup 2 +/ or both. In cubation in H/sub 2/O or CA/sup 2 +/ results in the production of low levels of ..cap alpha..-amylase or acid phosphatase. The addition of GA/sub 3/ to the incubation medium causes 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of CA/sup 2 +/ at 10 millimolar causes a further 8- to 9-fold increase in ..cap alpha..-amylase release and a 75% increase in phosphatase release. Production of ..cap alpha..-amylase isoenzymes is also modified by the levels of GA/sub 3/ and CA/sup 2 +/ in the incubation medium. ..cap alpha..-amylase 2 is produced under all conditions of incubation, while ..cap alpha..-amylase 1 appears only when layers are incubated in GA/sub 3/ or GA/sub 3/ plus CA/sup 2 +/. The synthesis of ..cap alpha..-amylases 3 and 4 requires the presence of both GA/sub 3/ and CA/sup 2 +/ in the incubation medium. Laurell rocket immunoelectrophoresis shows that two distinct groups of ..cap alpha..-amylase antigens are present in incubation media of aleurone layers incubated with both GA/sub 3/ and CA/sup 2 +/, while only one group of antigens is found in media of layers incubated in GA/sub 3/ alone. Strontium ions can be substituted for CA/sup 2 +/ in increasing hydrolase production, although higher concentrations of Sr/sup 2 +/ are requried for maximal response. We conclude that GA/sub 3/ is required for the production of ..cap alpha..-amylase 1 and that both GA/sub 3/ and either CA/sup 2 +/ or Sr/sup 2 +/ are required for the production of isoenzymes 3 and 4 of barley aleurone ..cap alpha..-amylase. 22 references, 8

  14. Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148.

    PubMed

    Satoh, E; Uchimura, T; Kudo, T; Komagata, K

    1997-12-01

    An intracellular alpha-amylase from Streptococcus bovis 148 was purified and characterized. The enzyme was induced by maltose and soluble starch and produced about 80% maltotriose from soluble starch. Maltopentaose was hydrolyzed to maltotriose and maltose and maltohexaose was hydrolyzed mainly to maltotriose by the enzyme. Maltotetraose, maltotriose, and maltose were not hydrolyzed. This intracellular enzyme was considered to be a maltotriose-producing enzyme. The enzymatic characteristics and hydrolysis product from soluble starch were different from those of the extracellular raw-starch-hydrolyzing alpha-amylase of strain 148. The deduced amino acid sequence of the intracellular alpha-amylase was similar to the sequences of the mature forms of extracellular liquefying alpha-amylases from Bacillus strains, although the intracellular alpha-amylase did not contain a signal peptide. No homology between the intracellular and extracellular alpha-amylases of S. bovis 148 was observed. PMID:9406414

  15. Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148.

    PubMed Central

    Satoh, E; Uchimura, T; Kudo, T; Komagata, K

    1997-01-01

    An intracellular alpha-amylase from Streptococcus bovis 148 was purified and characterized. The enzyme was induced by maltose and soluble starch and produced about 80% maltotriose from soluble starch. Maltopentaose was hydrolyzed to maltotriose and maltose and maltohexaose was hydrolyzed mainly to maltotriose by the enzyme. Maltotetraose, maltotriose, and maltose were not hydrolyzed. This intracellular enzyme was considered to be a maltotriose-producing enzyme. The enzymatic characteristics and hydrolysis product from soluble starch were different from those of the extracellular raw-starch-hydrolyzing alpha-amylase of strain 148. The deduced amino acid sequence of the intracellular alpha-amylase was similar to the sequences of the mature forms of extracellular liquefying alpha-amylases from Bacillus strains, although the intracellular alpha-amylase did not contain a signal peptide. No homology between the intracellular and extracellular alpha-amylases of S. bovis 148 was observed. PMID:9406414

  16. Decreased shoot stature and grain alpha-amylase activity following ectopic expression of a gibberellin 2-oxidase gene in transgenic wheat.

    PubMed

    Appleford, Nigel E J; Wilkinson, Mark D; Ma, Qian; Evans, Daniel J; Stone, Marlon C; Pearce, Stephen P; Powers, Stephen J; Thomas, Stephen G; Jones, Huw D; Phillips, Andrew L; Hedden, Peter; Lenton, John R

    2007-01-01

    Ectopic expression of a gibberellin 2-oxidase gene (PcGA2ox1) decreased the content of bioactive gibberellins (GAs) in transgenic wheat, producing a range of dwarf plants with different degrees of severity. In at least one case, a single transformation event gave rise to T(1) plants with different degrees of dwarfism, the phenotypes being stably inherited over at least four generations. The dwarf phenotype, which included dark-green leaves, increased tillering and, in severe cases, a prostrate growth habit, was replicated by the application of a GA biosynthesis inhibitor to the wild type. Ear rachis length, grain set, and grain size were also decreased in the wheat transformants, compared with an azygous (null) line. The extent of post-germination alpha-amylase production in grains reflected the severity of the shoot phenotype of the transformants and both developmental processes were restored to normal by the application of gibberellic acid (GA(3)). Expression of two GA biosynthesis genes (TaGA20ox1 and TaGA3ox2) was up-regulated, and that of two alpha-amylase gene families (alpha-Amy1 and alpha-Amy2) down regulated, in scutella of semi-dwarf lines, compared with controls. The marked decline in transcript abundance of both alpha-amylase gene families in aleurone was associated with a decreased content of bioactive GAs in grains of the semi-dwarf lines. PMID:17916639

  17. Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea, Prokaryotes and Eukaryotes.

    PubMed

    Huma, Tayyaba; Maryam, Arooma; Rehman, Shahid Ur; Qamar, Muhammad Tahir Ul; Shaheen, Tayyaba; Haque, Asma; Shaheen, Bushra

    2014-01-01

    Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry. PMID:25187685

  18. Purification and characterization of the extracellular. alpha. -amylase from Clostridium acetobutylicum ATCC 824

    SciTech Connect

    Paquet, V.; Croux, C.; Goma, G.; Soucaille, P. )

    1991-01-01

    The extracellular {alpha}-amylase (1,4-{alpha}-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (Mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying {alpha}-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the {alpha}-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. {alpha}-Amylase activity on soluble starch was optimal at pH 5.6 and 45{degree}C. The {alpha}-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a K{sub m} of 3.6 g {center dot} liter{sup {minus}1} and a K{sub cat} of 122 mol of reducing sugars {center dot} s{sup {minus}1} {center dot} mol{sup {minus}1}. The {alpha}-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the {alpha}-amylase.

  19. Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824.

    PubMed Central

    Paquet, V; Croux, C; Goma, G; Soucaille, P

    1991-01-01

    The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying alpha-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the alpha-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. alpha-Amylase activity on soluble starch was optimal at pH 5.6 and 45 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-1. The alpha-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the alpha-amylase. Images PMID:8967771

  20. Alpha-Amylase Activity in Blood Increases after Pharmacological, But Not Psychological, Activation of the Adrenergic System

    PubMed Central

    Nater, Urs M.; La Marca, Roberto; Erni, Katja; Ehlert, Ulrike

    2015-01-01

    Background & Aim Alpha-amylase in both blood and saliva has been used as a diagnostic parameter. While studies examining alpha-amylase activity in saliva have shown that it is sensitive to physiological and psychological challenge of the adrenergic system, no challenge studies have attempted to elucidate the role of the adrenergic system in alpha-amylase activity in blood. We set out to examine the impact of psychological and pharmacological challenge on alpha-amylase in blood in two separate studies. Methods In study 1, healthy subjects were examined in a placebo-controlled, double-blind paradigm using yohimbine, an alpha2-adrenergic antagonist. In study 2, subjects were examined in a standardized rest-controlled psychosocial stress protocol. Alpha-amylase activity in blood was repeatedly measured in both studies. Results Results of study 1 showed that alpha-amylase in blood is subject to stronger increases after injection of yohimbine compared to placebo. In study 2, results showed that there was no significant effect of psychological stress compared to rest. Conclusions Alpha-amylase in blood increases after pharmacological activation of the adrenergic pathways suggesting that sympathetic receptors are responsible for these changes. Psychological stress, however, does not seem to have an impact on alpha-amylase in blood. Our findings provide insight into the mechanisms underlying activity changes in alpha-amylase in blood in healthy individuals. PMID:26110636

  1. Stress affects salivary alpha-Amylase activity in bonobos.

    PubMed

    Behringer, Verena; Deschner, Tobias; Möstl, Erich; Selzer, Dieter; Hohmann, Gottfried

    2012-01-18

    Salivary alpha-Amylase (sAA) is a starch digesting enzyme. In addition to its function in the context of nutrition, sAA has also turned out to be useful for monitoring sympathetic nervous system activity. Recent studies on humans have found a relationship between intra-individual changes in sAA activity and physical and psychological stress. In studies on primates and other vertebrates, non-invasive monitoring of short-term stress responses is usually based on measurements of cortisol levels, which are indicative of hypothalamic-pituitary-adrenal activity. The few studies that have used both cortisol levels and sAA activity indicate that these two markers may respond differently and independently to different types of stress such that variation in the degree of the activation of different stress response systems might reflect alternative coping mechanisms or individual traits. Here, we present the first data on intra- and inter-individual variation of sAA activity in captive bonobos and compare the results with information from other ape species and humans. Our results indicate that sAA activity in the bonobo samples was significantly lower than in the human samples but within the range of other great ape species. In addition, sAA activity was significantly higher in samples collected at times when subjects had been exposed to stressors (judged by changes in behavioral patterns and cortisol levels) than in samples collected at other times. Our results indicate that bonobos possess functioning sAA and, as in other species, sAA activity is influenced by autonomic nervous system activity. Monitoring sAA activity could therefore be a useful tool for evaluating stress in bonobos. PMID:21945369

  2. RAmy2A; a novel alpha-amylase-encoding gene in rice.

    PubMed

    Huang, N; Reinl, S J; Rodriguez, R L

    1992-02-15

    The structure and expression of the alpha-amylase-encoding gene, RAmy2A, are described. This only representative of the Amy2 subfamily in rice differs from other cereal alpha-amylase-encoding genes in several respects. It contains the largest introns of all the cereal alpha-amylase-encoding genes examined to date. Moreover, the second of three introns in this gene contains a long inverted repeat sequence that can potentially form a large and stable stem-loop structure in the unspliced RNA transcript. Finally, RAmy2A is constitutively expressed at very low levels in germinated seeds, root, etiolated leaves, immature seeds and callus. This is in marked contrast to the Amy2 genes of wheat and barley which are highly expressed in the aleurone layer of the germinated seeds. PMID:1541400

  3. Differential expression of rice alpha-amylase genes during seedling development under anoxia.

    PubMed

    Hwang, Y S; Thomas, B R; Rodriguez, R L

    1999-08-01

    The unique capability of rice (Oryza sativa L.) seedlings to grow under anoxic conditions may result in part from their ability to express alpha-amylase and maintain the supply of sugar needed for energy metabolism. Previous studies have demonstrated that under aerobic conditions the Amy1 and Amy2 subfamily genes are regulated primarily by phytohormones while the Amy3 subfamily genes are induced during sugar starvation. The expression patterns for these alpha-amylase genes were considerably different in anoxic vs. aerobic rice seedlings. The level of total alpha-amylase mRNA under anoxic conditions was decreased in aleurone layers while it increased in the embryo. Anoxic conditions greatly diminished the expression of the Amy1A gene in aleurone. Conversely, expression of many Amy3 subfamily genes was up-regulated and prolonged in embryo tissues under anoxic conditions. PMID:10527416

  4. Thermal stability of alpha-amylase from Aspergillus oryzae entrapped in polyacrylamide gel.

    PubMed

    Raviyan, Patcharin; Tang, Juming; Rasco, Barbara A

    2003-08-27

    To determine the suitability as a time-temperature indicator for dielectric pasteurization processes, the thermal stability (50-75 degrees C) of Aspergillus oryzae alpha-amylase immobilized in polyacrylamide gel in phosphate buffer, mashed potatoes, and minced shrimp was examined. Changing the cross-linking agent concentration from 3.3 to 5.3% and adding 2% salt did not markedly affect the thermal stability of the immobilized alpha-amylase. Thermal inactivation was first order, and immobilization generally improved the thermal stability of alpha-amylase. z values of the immobilized system in test food systems were 10.2 degrees C (phosphate buffer), 8.45 degrees C (minced shrimp), and 7.78 degrees C (mashed potatoes). PMID:12926898

  5. Heterologous expression and secretion of Lactobacillus amylovorus alpha-amylase in Leuconostoc citreum.

    PubMed

    Eom, Hyun-Ju; Moon, Jin-Seok; Seo, Eun-Young; Han, Nam Soo

    2009-11-01

    To develop a gene expression system for Leuconostoc genus, construction of expression vector and expression of a heterologus protein in Leuconostoc was performed. Alpha-amylase gene from Lactobacillus amylovorus was cloned into a Leuconostoc cloning vector, pLeuCM, with its own signal peptide. pLeuCMamy was introduced into Leuconostoc citreum CB2567 and a successful expression of alpha-amy gene was confirmed by enzyme activity assays. About 90% of alpha-amylase activity was detected in the culture broth, revealing most of expressed alpha-amylase was secreted out cells. The signal sequence of alpha-amy gene is a good candidate for the secretion of heterologous protein by using Leuconostoc host-vector system. PMID:19618275

  6. Study on alpha-amylase hydrolysis of potato amylopectin by a quartz crystal microbalance.

    PubMed

    Sasaki, Tomoko; Noel, Timothy R; Ring, Steve G

    2008-02-13

    Potato amylopectin with phosphate groups was immobilized on a quartz crystal microbalance with dissipation monitoring (QCMD) using the attractive interaction between opposite charges, and enzymatic starch hydrolysis was monitored directly. Poly( L-lysine) (PLL) proved to be an appropriate cationic linker between the QCMD silica sensor and potato amylopectin. Increased mass and dissipation were observed when amylopectin was adsorbed onto the PLL layer and reversed when alpha-amylase was added. The effect of chitosan with cationic property on the hydrolysis of amylopectin was studied. Chitosan was observed to be adsorbed onto the amylopectin surface and to suppress hydrolysis by alpha-amylase. The formation of alternating layers of amylopectin and chitosan was monitored by QCMD. Amylopectin-chitosan trilayers increased resistance to digestion by alpha-amylase compared to one layer and to control without chitosan. PMID:18181571

  7. Effect of sexual steroids upon ontogeny of alpha-amylase of rat parotid gland.

    PubMed

    Bellavia, S L; Sanz, E G; Vermouth, N T; Blanco, A

    1982-04-30

    The effect of gonadectomy (at the 10th day of life) and treatment with sexual steroids (during the first month) upon development of alpha-amylase activity in rat parotid gland has been studied. Alpha-amylase specific activity of parotid glands from 20-day-old orchidectomized rats and from 25-day-old ovariectomized animals was significantly higher than that of intact male and female rats of the same age respectively. Spayed males treated with testosterone (10 microgram/day on the 13th, 15th, and 17th day) and ovariectomized rats treated with oestradiol (2.5 microgram/day from the 16th to the 22nd day) showed values of enzymic activity similar to those of normal animals. Results indicate that oestradiol and testosterone have an inhibitory effect upon the increase of alpha-amylase activity in parotid gland during a very defined period of development. PMID:6178953

  8. The synergetic effect of starch and alpha amylase on the biodegradation of n-alkanes.

    PubMed

    Karimi, M; Biria, D

    2016-06-01

    The impact of adding soluble starch on biodegradation of n-alkanes (C10-C14) by Bacillus subtilis TB1 was investigated. Gas chromatography was employed to measure the residual hydrocarbons in the system. It was observed that the efficiency of biodegradation improved with the presence of starch and the obtained residual hydrocarbons in the system were 53% less than the samples without starch. The produced bacterial enzymes were studied through electrophoresis and reverse zymography for explaining the observations. The results indicated that the produced amylase by the bacteria can degrade hydrocarbons and the same was obtained by the application of a commercial alpha amylase sample. In addition, in silico docking of alpha-amylase with n-alkanes with different molecular weights was studied by Molegro virtual docker which showed high negative binding energies and further substantiated the experimental observations. Overall, the findings confirmed the catalytic effect of alpha amylase on n-alkanes degradation. PMID:26971168

  9. General Subject 1. Report to ICUMSA on the determination of commercial alpha-amylase activity by a spectrophotometric method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A report is given on a new industrial method for the determination of the activity or strength of commercial alpha-amylase at a sugarcane factory or refinery, as well as a recommendation. At the present time, the activities or strengths of commercial alpha-amylases cannot be directly compared becau...

  10. Purification and characterization of periplasmic alpha-amylase from Xanthomonas campestris K-11151.

    PubMed Central

    Abe, J; Onitsuka, N; Nakano, T; Shibata, Y; Hizukuri, S; Entani, E

    1994-01-01

    Xanthomonas campestris K-11151, isolated from soil, produced a periplasmic alpha-amylase of a new type. The enzyme was purified to homogeneity, as shown by several criteria. The purified enzyme showed almost the same activities on alpha-, beta-, and gamma-cyclodextrins, soluble starch, and amylose. Moreover, it was active on branched cyclodextrins, pullulan, and maltose but not on glycogen. Kinetic analysis showed that alpha-cyclodextrin was the best substrate among the cyclodextrins. The substrate specificity suggested that this enzyme had the combined activities of alpha-amylase, cyclodextrinase, and neopullulanase. Images PMID:8206836

  11. Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity.

    PubMed

    Maurus, Robert; Begum, Anjuman; Williams, Leslie K; Fredriksen, Jason R; Zhang, Ran; Withers, Stephen G; Brayer, Gary D

    2008-03-18

    A mechanistic study of the essential allosteric activation of human pancreatic alpha-amylase by chloride ion has been conducted by exploring a wide range of anion substitutions through kinetic and structural experiments. Surprisingly, kinetic studies indicate that the majority of these alternative anions can induce some level of enzymatic activity despite very different atomic geometries, sizes, and polyatomic natures. These data and subsequent structural studies attest to the remarkable plasticity of the chloride binding site, even though earlier structural studies of wild-type human pancreatic alpha-amylase suggested this site would likely be restricted to chloride binding. Notably, no apparent relationship is observed between anion binding affinity and relative activity, emphasizing the complexity of the relationship between chloride binding parameters and the activation mechanism that facilitates catalysis. Of the anions studied, particularly intriguing in terms of observed trends in substrate kinetics and their novel atomic compositions were the nitrite, nitrate, and azide anions, the latter of which was found to enhance the relative activity of human pancreatic alpha-amylase by nearly 5-fold. Structural studies have provided considerable insight into the nature of the interactions formed in the chloride binding site by the nitrite and nitrate anions. To probe the role such interactions play in allosteric activation, further structural analyses were conducted in the presence of acarbose, which served as a sensitive reporter molecule of the catalytic ability of these modified enzymes to carry out its expected rearrangement by human pancreatic alpha-amylase. These studies show that the largest anion of this group, nitrate, can comfortably fit in the chloride binding pocket, making all the necessary hydrogen bonds. Further, this anion has nearly the same ability to activate human pancreatic alpha-amylase and leads to the production of the same acarbose product

  12. Validation of an assay for quantification of alpha-amylase in saliva of sheep.

    PubMed

    Fuentes-Rubio, Maria; Fuentes, Francisco; Otal, Julio; Quiles, Alberto; Hevia, María Luisa

    2016-07-01

    The objective of this study was to develop a time-resolved immunofluorometric assay (TR-IFMA) for quantification of salivary alpha-amylase in sheep. For that purpose, after the design of the assay, an analytical and a clinical validation were carried out. The analytical validation of the assay showed intra- and inter-assay coefficients of variation (CVs) of 6.1% and 10.57%, respectively and an analytical limit of detection of 0.09 ng/mL. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution. For clinical validation, a model of acute stress testing was conducted to determine whether expected significant changes in alpha-amylase were picked up in the newly developed assay. In that model, 11 sheep were immobilized and confronted with a sheepdog to induce stress. Saliva samples were obtained before stress induction and 15, 30, and 60 min afterwards. Salivary cortisol was measured as a reference of stress level. The results of TR-IFMA showed a significant increase (P < 0.01) in the concentration of alpha-amylase in saliva after stress induction. The assay developed in this study could be used to measure salivary alpha-amylase in the saliva of sheep and this enzyme could be a possible noninvasive biomarker of stress in sheep. PMID:27408332

  13. ALPHA-AMYLASE ACTIVITY IN VARIOUS CONCENTRATIONS OF THE IONIC LIQUID, 1-BUTYL-3-METHYLIMIDAZOLIUM CHLORIDE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch is an extremely abundant, economical and versatile industrial commodity. Many industrial uses of starch depend on hydrolyzing the polymer for the conversion of glucose and maltodextrins. Starch hydrolysis is frequently achieved by utilizing alpha-amylase, which is an endo-acting enzyme that...

  14. Production and Partial Purification of Alpha Amylase from Bacillus subtilis (MTCC 121) Using Solid State Fermentation.

    PubMed

    Raul, Dibyangana; Biswas, Tania; Mukhopadhyay, Suchita; Kumar Das, Shrayan; Gupta, Suvroma

    2014-01-01

    Amylase is an enzyme that catalyzes the breakdown of starch into sugars and plays a pivotal role in a variety of areas like use as digestives, for the production of ethanol and high fructose corn syrup, detergents, desiring of textiles, modified starches, hydrolysis of oil-field drilling fluids, and paper recycling. In the present work, solid state fermentation (SSF) for α -amylase production has been used in lieu of submerged fermentation (SmF) due to its simple technique, low capital investment, lower levels of catabolite repression, and better product recovery. Bacillus subtilis has been well known as producer of alpha amylase and was tested using solid state fermentation for 48 hours at 37°C with wheat bran as substrate. Comparison between different fermentation hours demonstrated high yield of alpha amylase after 48 hours. This alpha amylase has optimum pH and temperature at 7.1 and 40°C, respectively. With the goal to purify alpha amylase, 30-70% (NH4)2SO4 cut concentrated the amylase activity threefold with respect to crude fermented extract. This was verified in quantitative DNS assay method as well as in zymogram gel profile. The exact molecular weight of the amylase is yet to be determined with the aid of other protein purification techniques. PMID:24672727

  15. Production of alpha-amylase from Aspergillus oryzae for several industrial applications in a single step.

    PubMed

    Porfirif, María C; Milatich, Esteban J; Farruggia, Beatriz M; Romanini, Diana

    2016-06-01

    A one-step method as a strategy of alpha-amylase concentration and purification was developed in this work. This methodology requires the use of a very low concentration of biodegradable polyelectrolyte (Eudragit(®) E-PO) and represents a low cost, fast, easy to scale up and non-polluting technology. Besides, this methodology allows recycling the polymer after precipitation. The formation of reversible soluble/insoluble complexes between alpha-amylase and the polymer Eudragit(®) E-PO was studied, and their precipitation in selected conditions was applied with bioseparation purposes. Turbidimetric assays allowed to determine the pH range where the complexes are insoluble (4.50-7.00); pH 5.50 yielded the highest turbidity of the system. The presence of NaCl (0.05M) in the medium totally dissociates the protein-polymer complexes. When the adequate concentration of polymer was added under these conditions to a liquid culture of Aspergillus oryzae, purification factors of alpha-amylase up to 7.43 and recoveries of 88% were obtained in a simple step without previous clarification. These results demonstrate that this methodology is suitable for the concentration and production of alpha-amylase from this source and could be applied at the beginning of downstream processing. PMID:27085017

  16. Increased production of alpha-amylase by Bacillus amyloliquefaciens in the presence of glycine

    SciTech Connect

    Zhang, Q.; Tsukagoshi, N.; Miyashiro, S.; Udaka, S.

    1983-07-01

    The production of alpha-amylase by Bacillus amyloliquefaciens increased by a factor of 300 when glycine was added to a chemically defined simple medium at the early-logarithmic phase of growth. Glycine was not metabolized to a significant extent under the conditions used, but it considerably prevented the lowering of the pH of the culture. (Refs. 10).

  17. Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1.

    PubMed

    Freer, S N

    1993-05-01

    The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase. alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C. When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose. PMID:8517735

  18. Optimization of Alpha-Amylase Application in U.S. Factories

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In recent years there have been warnings by some U.S. refineries that there may be a penalty for high starch concentrations in raw sugar if starch control is not improved. Most commercial alpha-amylases used by the U.S. sugar industry to control starch have intermediate temperature stability (up to...

  19. Peer Victimization and Aggression: Moderation by Individual Differences in Salivary Cortisol and Alpha-Amylase

    ERIC Educational Resources Information Center

    Rudolph, Karen D.; Troop-Gordon, Wendy; Granger, Douglas A.

    2010-01-01

    This research examined whether variations in salivary measures of the hypothalamic-pituitary-adrenal axis (cortisol) and autonomic nervous system (alpha amylase [sAA]) contribute to individual differences in the association between peer victimization and aggression. Children (N = 132; M age = 9.46 years, SD = 0.33) completed a measure of peer…

  20. Kinetic study of the irreversible thermal denaturation of Bacillus licheniformis alpha-amylase.

    PubMed Central

    Violet, M; Meunier, J C

    1989-01-01

    The irreversible thermal inactivation of Bacillus licheniformis alpha-amylase was studied. A two-step behaviour in the irreversible denaturation process was found. Our experimental results are consistent only with the two-step model and rule out the two-isoenzyme one. They suggest that the deactivation mechanism involves the existence of a temperature-dependent intermediate form. Therefore the enzyme could exist in a great number of active conformational states. We have shown that Ca2+ is necessary for the structural integrity of alpha-amylase. Indeed, dialysis against chelating agents leads to a reversible enzyme inactivation, though molecular sieving has no effect. Further, the key role of Ca2+ in the alpha-amylase thermostability is reported. The stabilizing effect of Ca2+ is reflected by the decrease of the denaturation constants of both the native and the intermediate forms. Below 75 degrees C, in the presence of 5 mM-CaCl2, alpha-amylase is completely thermostable. Neither other metal ions nor substrate have a positive effect on enzyme thermostability. The effect of temperature on the native enzyme and on one intermediate form was studied. Both forms exhibit the same optimum temperature. Identical activation parameters for the hydrolytic reaction catalysed by these two forms were found. PMID:2597125

  1. Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1.

    PubMed Central

    Freer, S N

    1993-01-01

    The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase. alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C. When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose. Images PMID:8517735

  2. Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal alpha-amylase enzymes.

    PubMed

    van der Kaaij, R M; Janecek, S; van der Maarel, M J E C; Dijkhuizen, L

    2007-12-01

    Currently known fungal alpha-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal alpha-amylases. The phylogenetic analysis shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal alpha-amylases of family GH13_1. Homologues of these intracellular enzymes are present in the genome sequences of all filamentous fungi studied, including ascomycetes and basidiomycetes. One of the enzymes belonging to this new group, Amy1p from Histoplasma capsulatum, has recently been functionally linked to the formation of cell wall alpha-glucan. To study the biochemical characteristics of this novel cluster of alpha-amylases, we overexpressed and purified a homologue from Aspergillus niger, AmyD, and studied its activity product profile with starch and related substrates. AmyD has a relatively low hydrolysing activity on starch (2.2 U mg(-1)), producing mainly maltotriose. A possible function of these enzymes in relation to cell wall alpha-glucan synthesis is discussed. PMID:18048915

  3. Two Strategies for Microbial Production of an Industrial Enzyme-Alpha-Amylase

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, Eva C. M. J.; Garriott, Owen; Pusey, Marc L.; Ng, Joseph D.

    2003-01-01

    Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments including hot springs, soda lakes and arctic water. This ability of survival at extreme conditions has rendered extremophiles to be of interest in astrobiology, evolutionary biology as well as in industrial applications. Of particular interest to the biotechnology industry are the biological catalysts of the extremophiles, the extremozymes, whose unique stabilities at extreme conditions make them potential sources of novel enzymes in industrial applications. There are two major approaches to microbial enzyme production. This entails enzyme isolation directly from the natural host or creating a recombinant expression system whereby the targeted enzyme can be overexpressed in a mesophilic host. We are employing both methods in the effort to produce alpha-amylases from a hyperthermophilic archaeon (Thermococcus) isolated from a hydrothermal vent in the Atlantic Ocean, as well as from alkaliphilic bacteria (Bacillus) isolated from a soda lake in Tanzania. Alpha-amylases catalyze the hydrolysis of internal alpha-1,4-glycosidic linkages in starch to produce smaller sugars. Thermostable alpha-amylases are used in the liquefaction of starch for production of fructose and glucose syrups, whereas alpha-amylases stable at high pH have potential as detergent additives. The alpha-amylase encoding gene from Thermococcus was PCR amplified using carefully designed primers and analyzed using bioinformatics tools such as BLAST and Multiple Sequence Alignment for cloning and expression in E.coli. Four strains of Bacillus were grown in alkaline starch-enriched medium of which the culture supernatant was used as enzyme source. Amylolytic activity was detected using the starch-iodine method.

  4. Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase.

    PubMed

    Agger, Teit; Petersen, Jesper B; O'Connor, Susan M; Murphy, Rachael L; Kelly, Joan M; Nielsen, Jens

    2002-01-18

    The physiology of three strains of Aspergillus nidulans was examined--a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon sources on the alpha-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l(-1) of alpha-amylase was reached after 162 h of cultivation. PMID:11689252

  5. Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

    PubMed Central

    Satoh, E; Niimura, Y; Uchimura, T; Kozaki, M; Komagata, K

    1993-01-01

    The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148. Images PMID:8285674

  6. Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

    PubMed

    Satoh, E; Niimura, Y; Uchimura, T; Kozaki, M; Komagata, K

    1993-11-01

    The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148. PMID:8285674

  7. Studies on alpha-amylase induced degradation of binary polymeric blends of crosslinked starch and pectin.

    PubMed

    Bajpai, A K; Shrivastava, Jyoti

    2007-05-01

    A blend matrix of crosslinked starch and pectin was prepared and characterized by infra-red (IR) spectroscopy, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). The prepared blends were investigated kinetically for water sorption studies and alpha-amylase induced degradation adopting a gravimetric procedure. Based on the experimental findings, a plausible mechanism including both diffusion and surface enhanced degradation was suggested and degradation profiles were interpreted. The influence of various factors such as chemical architecture of the blend, pH and temperature of alpha-amylase solution were examined for the swelling and degradation kinetics of crosslinked starch-pectin blends. The effect of concentration of enzyme solution was also studied on the degradation profile of the blends. A correlation was established between the extent of degradation and water imbibing capacity of the degrading blends. PMID:17143735

  8. Convenience of immobilized Bacillus licheniformis alpha-amylase as time-temperature-integrator (TTI).

    PubMed

    De Cordt, S F; Hendrickx, M E; Maesmans, G J; Tobback, P P

    1994-02-01

    For the immobilization of Bacillus licheniformis alpha-amylase to porous glass beads, the performances of three possible linking agents, glutaric dialdehyde, benzoquinone and s-trichlorotriazine were assessed in respect of the protein yield, the enzymic activity and the thermostability of the immobilized enzyme. These three properties are to be evaluated in view of the possible use of the enzyme preparations as time-temperature-integrators (TTIs) for assessing the severity of heat pasteurization or sterilization processes of food or pharmaceuticals. All three linkers improved the enzyme's resistance to irreversible heat inactivation to a similar extent and in each case biphasic inactivation kinetics were observed, whereas the dissolved B. licheniformis alpha-amylase showed a simple first order decay. The immobilization yield, measured as protein per carrier weight, did not differ markedly for the three linkers, although the enzymic activity of the glutaric dialdehyde-linked enzyme was lower than that of the benzoquinone- and s-trichlorotriazine-linked preparations. PMID:7764538

  9. Expression of a bacterial alpha-amylase gene in transgenic rice seeds.

    PubMed

    Xu, Xiaoli; Fang, Jun; Wang, Wei; Guo, Jianli; Chen, Pinnan; Cheng, Jiaan; Shen, Zhicheng

    2008-08-01

    An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction. PMID:17926139

  10. Alpha-amylase circadian rhythm of young rat parotid gland: an endogenous rhythm with maternal coordination.

    PubMed

    Bellavía, S L; Sanz, E G; Sereno, R; Vermouth, N T

    1992-01-01

    The circadian rhythm of alpha-amylase, E.C. 3.2.1.1. alpha-1,4-glucan-4-glucanohydrolase) in the parotid glands of 25-day-old rats were studied under different experimental designs (fasting, reversed photoperiod, constant lighting conditions and treatment with reserpine and alpha-methyl-p-tyrosine). The rhythm of fasted rats did not change. There were modifications in the rhythm of rats submitted to a reversed photoperiod or treated with reserpine or alpha-methyl-p-tyrosine. The rhythm was present, with changes in the acrophase, in parotids of rats kept during their gestation and postnatal life in constant light or dark. Results suggest that the circadian rhythm of alpha-amylase in parotid gland of young rats is endogenous, synchronized by the photoperiod, and with maternal coordination. PMID:1610312

  11. Ontogenesis of alpha-amylase in rat parotid gland during postnatal development.

    PubMed

    Bellavia, S L; Sanz, E G; Vermouth, N T; Rins, L; Aoki, A

    1981-01-01

    Changes in alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) of parotid gland were investigated during postnatal development of the rat. Modifications in amylase activity after birth allow the distinction of three stages which can be correlated with the morphologic development of the parotid gland. Significant sexual differences in the evolution of alpha-amylase activity were found. During the first stage (from birth to the 20th day) there is a higher increase in females, while males have a more pronounced increment in the second stage (from the 20th to the 30th day). By means of gel electrophoresis of parotid extracts, four molecular forms of amylase can be separated. The slowest migrating band (Form 1) is not detected at the initial stage. PMID:6164673

  12. Production and characterization of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean.

    PubMed

    Stamford, T L; Stamford, N P; Coelho, L C; Araújo, J M

    2001-01-01

    Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications. PMID:11131797

  13. Expression and secretion of alpha-amylase and glucoamylase in Saccharomyces cerevisiae.

    PubMed

    Luo, J; He, M; Li, W; Zhang, T

    1994-01-01

    alpha-Amylase genes of Bacillus licheniformis and glucoamylase cDNA of Aspergillus niger were ligated to a E. coli-yeast shuttle vector. The resultant plasmid was used to transform Saccharomyces cerevisiae to construct starch-degrading yeast strain. The results of enzyme activity assay and enzyme property analysis show that alpha-amylase and glucoamylase genes have been expressed simultaneously in yeast under the control of promoters and terminators of yeast MF-alpha 1 factor and PGK genes and over 99% of enzyme activities were secreted to the medium. The engineered yeast strain hydrolyses 97% of the starch (10%) in the medium after 6 days. The recombinant plasmid exists stably in yeast. PMID:7780020

  14. Polymorphism of salivary esterase and alpha-amylase in the Greek population.

    PubMed

    Petalopoulos, A; Fousteri, M; Kouvatsi, A; Triantaphyllidis, C

    1993-01-01

    The genetic polymorphism of two salivary enzymes (esterase and alpha-amylase) was studied in individuals from eight districts of Greece. The pooled gene frequencies were: SetS = 0.63, SetF = 0.37, AMY1 = 0.87, AMY2 = 0.10, AMY3 = 0.02, and AMY4 = 0.01. There was no intrapopulation heterogeneity, while there was a significant difference between the Greeks and the few other European populations studied. PMID:7507080

  15. The genetic control of the alpha-amylase isozymes of the durum wheat (Triticum durum Desf.).

    PubMed

    Prokopyk, D O; Antonyuk, M Z; Ternovskaya, T K

    2009-01-01

    The hybridological analysis was provided on several durum wheat genotypes with utilizing three F2 populations developed from the crossing between parental forms that differed in the invariable malt-zone triplet on electrophoretic spectrum of alpha-amylase. Three components of this zone are controlled by three genes with an independent way of inheritance: one of them is located on the 6B or 5B chromosome, and two genes are located on the chromosomes of A subgenome. PMID:19938630

  16. Starch-binding domain affects catalysis in two Lactobacillus alpha-amylases.

    PubMed

    Rodríguez-Sanoja, R; Ruiz, B; Guyot, J P; Sanchez, S

    2005-01-01

    A new starch-binding domain (SBD) was recently described in alpha-amylases from three lactobacilli (Lactobacillus amylovorus, Lactobacillus plantarum, and Lactobacillus manihotivorans). Usually, the SBD is formed by 100 amino acids, but the SBD sequences of the mentioned lactobacillus alpha-amylases consist of almost 500 amino acids that are organized in tandem repeats. The three lactobacillus amylase genes share more than 98% sequence identity. In spite of this identity, the SBD structures seem to be quite different. To investigate whether the observed differences in the SBDs have an effect on the hydrolytic capability of the enzymes, a kinetic study of L. amylovorus and L. plantarum amylases was developed, with both enzymes acting on several starch sources in granular and gelatinized forms. Results showed that the amylolytic capacities of these enzymes are quite different; the L. amylovorus alpha-amylase is, on average, 10 times more efficient than the L. plantarum enzyme in hydrolyzing all the tested polymeric starches, with only a minor difference in the adsorption capacities. PMID:15640201

  17. Statistical media optimization and production of ITS alpha-amylase from Aspergillus oryzae in a bioreactor.

    PubMed

    Gigras, Paresh; Sahai, Vikram; Gupta, Rani

    2002-09-01

    The production of an intermediate temperature-stable (ITS) alpha-amylase from Aspergillus oryzae was studied by using a central composite design with three independent variables, viz., starch, yeast extract, and K(2)HPO(4). The model equation provided a suitable model for the response surface for alpha-amylase production, and, from the optimal concentrations of the medium components, a model was predicted, which was then used for enzyme production in a 150-L bioreactor. In the bioreactor studies, the enzyme yields (161 U/ml) were similar to that of the shake flask (133 U/ml); however, the time required for maximum alpha-amylase production in the bioreactor was reduced to 48 h compared with 120 h in shake flask cultures. An increased level of phosphate in the medium and low inoculum size were necessary to control the excessive foaming in the bioreactor; however, control of the pO(2) level and agitation was not mandatory for enzyme production. The peak enzyme production coincided with the increase in pH of the fermentation broth and was maximal when the pH of the system was above 7.5. Thus, in the present study, pH acted as an indicator of the initiation or end of the enzyme synthesis or of the fermentation cycle. PMID:12177743

  18. Coconut oil cake--a potential raw material for the production of alpha-amylase.

    PubMed

    Ramachandran, Sumitra; Patel, Anil K; Nampoothiri, K Madhavan; Francis, Febe; Nagy, Viviana; Szakacs, George; Pandey, Ashok

    2004-06-01

    Solid-state fermentation (SSF) was carried out using coconut oil cake (COC) as substrate for the production of alpha-amylase using a fungal culture of Aspergillus oryzae. Raw COC supported the growth of the culture, resulting in the production of 1372 U/gds alpha-amylase in 24 h. Process optimization using a single parameter mode showed enhanced enzyme titre, which was maximum (1827 U/gds) when SSF was carried out at 30 degrees C for 72 h using a substrate with 68% initial moisture. Supplementation with glucose and starch further enhanced enzyme titre, which was maximum (1911 U/gds) with 0.5% starch. However, maltose inhibited the enzyme production. Studies on the effect of addition of external organic and inorganic nitrogenous compounds further showed a positive impact on enzyme synthesis by the culture. Increase of 1.7-fold in the enzyme activity (3388 U/gds) was obtained when peptone at 1% concentration was added to the fermentation medium. The enzyme production was growth-related, the activity being the maximum when the fungal biomass was at its peak at 72 h. Use of COC as raw material for enzyme synthesis could be of great commercial significance. To the best of our knowledge this is the first report on alpha-amylase production using COC in SSF. PMID:15051078

  19. Production and properties of alpha-amylase from Penicillium chrysogenum and its application in starch hydrolysis.

    PubMed

    Balkan, Bilal; Ertan, Figen

    2005-01-01

    Fungi were screened for their ability to produce alpha-amylase by a plate culture method. Penicillium chrysogenum showed high enzymatic activity. Alpha-amylase production by P. chrysogenum cultivated in liquid media containing maltose (2%) reached its maximum at 6-8 days, at 30 degrees C, with a level of 155 U ml(-1). Some general properties of the enzyme were investigated. The optimum reaction pH and temperature were 5.0 and 30-40 degrees C, respectively. The enzyme was stable at a pH range from 5.0-6.0 and at 30 degrees C for 20 min and the enzyme's 92.1% activity's was retained at 40 degrees C for 20 min without substrate. Hydrolysis products of the enzyme were maltose, unidefined oligosaccharides, and a trace amount of glucose. Alpha-amylase of P. chrysogenum hydrolysed starches from different sources. The best hydrolysis was determined (98.69%) in soluble starch for 15 minute at 30 degrees C. PMID:15881598

  20. [Influence of amaranth on the production of alpha-amylase using Aspergillus niger NRRL 3112].

    PubMed

    Mariani, D D; Lorda, G; Balatti, A P

    2000-01-01

    In this paper the influence of the amaranth seed meal and the aeration conditions on the alpha-amylase production by Aspergillus niger NRRL 3112 were studied. The assays of selection of culture medium were carried out in a rotary shaker at 250 rpm and 2.5 cm stroke. The aeration conditions were studied in a mechanically stirred fermentor New Brunswick type. A concentration of alpha-amylase of 2750 U.Dun/ml was achieved at 120 h with a dry weight of 8.0 g/l, using a base medium with 5.0 g/l Amaranthus cruentus seed meal. In the experiment performed in a New Brunswick fermentor, the highest value was 2806 U.Dun/ml. This result was obtained after 120 h, operating at 300 rpm and an airflow of 1 l/l. min. in a limited dissolved oxygen concentration. It was determined that the increase in the agitation rate was not favorable to the enzyme production, despite that an increase was verified in the dissolved oxygen. The morphology of the microorganism, in long and ramified hyphae, was the critical factor to obtain higher levels of alpha-amylase. PMID:11149149

  1. Ontogeny of alpha-amylase circadian rhythms in rat parotid gland.

    PubMed

    Sanz, E G; Vermouth, N T; Bellavia, S L

    1986-01-01

    The content of alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1.) and total soluble proteins of parotid glands (from rats exposed to a photoperiod of 14 hr light: 10 hr dark), have been determined every 2 or 3 hr over 24 hr periods in 15, 25 and 90-day-old rats. In 35-, 45- and 72-day-old rats, determinations were performed only at 0100 and 1400 hr. The alpha-amylase and total soluble protein contents from 90-day-old rats show a circadian variation, with a maximum value at 2200 hr and a minimum at 1400 hr. Parotids from 15- and 25-day-old rats also show a circadian rhythm. The minimum value is recorded at 0100 hr and the maximum at 1400 hr. At day 35 and after, there is an inversion of the amylase rhythm. In immature rats, it appears that alpha-amylase and soluble protein are under the influence of another synchronizer, whose timing is independent of that imposed by mastication of solid food. PMID:2878787

  2. Grain Development Mutants of Barley ([alpha]-Amylase Production during Grain Maturation and Its Relation to Endogenous Gibberellic Acid Content).

    PubMed Central

    Green, L. S.; Faergestad, E. M.; Poole, A.; Chandler, P. M.

    1997-01-01

    Barley (Hordeum vulgare L. Himalaya) mutants with altered grain morphology were isolated to investigate whether defects in grain development, possibly involving gibberellins (GAs) and abscisic acid, would lead to altered patterns of [alpha]-amylase gene expression. Following treatment with sodium azide, 75 mutants, typically showing grain shriveling, were identified. At grain maturity 15 of the 75 mutants had higher [alpha]-amylase activities in shriveled grains compared with either phenotypically normal grains that developed on the same heterozygous plant or with grains of cv Himalaya. Studies of four of these mutants demonstrated increased levels of both high- and low-isoelectric point [alpha]-amylase isozymes midway through grain development. This category of mutant has been designated pga, for premature grain [alpha]-amylase. One such mutant (M326) showed an endosperm-determined inheritance pattern. When crossed into a (GA-deficient) dwarfing background there was a 10- to 20-fold reduction in [alpha]-amylase activity, suggesting a requirement for GA biosynthesis. Endogenous GAs and abscisic acid were quantified by combined gas chromatography-specific ion monitoring in normal and mutant grains of heterozygous M326 plants during the period of [alpha]-amylase accumulation. Mutant grains had significantly higher (5.8-fold) levels of the bioactive GA1 compared with normal grains but much lower (approximately 10-fold) levels of the 2[beta]-hydroxylated ("inactive") GAs, typical of developing barley grains (e.g. GA8, GA34, GA48). We propose that a reduced extent of 2[beta]-hydroxylation in the mutant grains results in an increased level of GA1, which is responsible for premature [alpha]-amylase gene expression. PMID:12223700

  3. In vitro and in vivo inhibition of alpha-amylases of stored-product mite Acarus siro.

    PubMed

    Hubert, Jan; Dolecková-Maresová, Lucie; Hýblová, Jana; Kudlíková, Iva; Stejskal, Václav; Mares, Michael

    2005-01-01

    The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of alpha-amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mite's gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops. PMID:15969461

  4. A Proposed Mechanism for the Thermal Denaturation of a Recombinant Bacillus Halmapalus Alpha-amylase - the Effect of Calcium Ions

    NASA Technical Reports Server (NTRS)

    Nielsen, Anders D.; Pusey, Marc L.; Fuglsang, Claus C.; Westh, Peter

    2003-01-01

    The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where previous crystallographic studies have identified the existence of 3 calcium binding sites in the structure. Denaturation of BHA is irreversible with a Tm of approximately 89 C, and DSC thermograms can be described using a one-step irreversible model. A 5 C increase in T(sub m) in the presence of 10 fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40 fold excess calcium chelator (EDTA or EGTA) results in a large destabilization of BHA corresponding to about 40 C lower T(sub m), as determined by both CD and DSC. Ten fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which possibly originate from different populations of BHA:calcium complexes. The observations in the present study have, in combination with structural information of homologous alpha-amylases, provided the basis for the proposal of a simple denaturation mechanism of BHA. The proposed mechanism describes the irreversible thermal denaturation of different BHA:calcium complexes and the calcium binding equilibrium involved. Furthermore, the model accounts for a temperature induced reversible structural change associated with calcium binding.

  5. Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger.

    PubMed

    Juge, N; Svensson, B; Williamson, G

    1998-04-01

    Efficient production of recombinant barley alpha-amylase has been achieved in Aspergillus niger. The cDNA encoding alpha-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for alpha-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44,960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley alpha-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol beta-D-maltoheptaoside and amylose (degree of polymerisation = 17). Barley alpha-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence. PMID:9615479

  6. Alpha-amylase inhibitory activity and phytochemical study of Zhumeria majdae Rech. f. and Wendelbo

    PubMed Central

    Mirshafie, Behnaz; Mokhber-Dezfouli, Najmeh; Manayi, Azadeh; Saeidnia, Soodabeh; Ajani, Yousef; Gohari, Ahmad Reza

    2015-01-01

    Background: Zhumeria majdae (Lamiaceae) is an endemic species growing in the South parts of Iran especially Hormozgan province. The plant is so-called Mohrekhosh locally and widely used for medicinal purposes including stomachache and dysmenorrhea. Objective: In order to separation and identification of the main flavonoid glycosides of the plant (aerial parts including leaves, stems, flowers, and fruits were used) and evaluation of its alpha-amylase inhibitory (AAI) activity, methanolic extract was prepared and fractionated to botanolic portion. Materials and Methods: Isolation of the main compounds of the butanol extract of the plant have been performed using different column chromatography methods such as high-performance liquid chromatography (C18 column) and Sephadex LH-20 as well. The isolated compounds were identified by Hydrogen-1 nuclear magnetic resonance and Carbon-13 nuclear magnetic resonance spectra and comparison with those reported in previous literature. Moreover, inhibitory activity of the butanolic extract of the plant against alpha-amylase enzyme was examined in different concentrations (15–30 mg/mL), where acarbose used as a positive control. Results: Three flavonoid glycosides: Linarin (1), hispidulin-7-O-(4-O-acetyl-rutinoside) (2), hispidulin-7-O-rutinoside (3) were successfully identified in the extract. The activity of alpha amylase enzyme was dose-dependently suppressed by the butanol extract. The extract exhibited the highest inhibition at 30 mg/mL toward enzyme (77.9 ± 2.1%), while acarbose inhibited the enzyme at 20 mg/mL by 73.9 ± 1.9%. The inhibitory concentrations of 50% for the extract and acarbose were calculated at 24.5 ± 2.1 and 6.6 ± 3.1 mg/mL, respectively. Conclusion: Z. majdae contains glycosylated flavones and could be a good candidate for anti-diabetic evaluations in animal and clinical trials due to possessing AAI activity. PMID:26692743

  7. Purification by expanded bed adsorption and characterization of an alpha-amylases FORILASE NTL from A. niger.

    PubMed

    Toledo, A L; Severo, J B; Souza, R R; Campos, E S; Santana, J C C; Tambourgi, E B

    2007-02-01

    In this work the purification and biochemistry characterization of alpha-amylases from Aspergillus niger (FORILASE NTL) were studied. The effects of expansion degree of resin bed on enzyme purification by expanded bed adsorption (EBA) have also been studied. Residence time distributions (RTD) studies were done to achieve the optimal conditions of the amylases recovery on ion-exchange resin, and glucose solution was used as a new tracer. Results showed that height equivalent of the theoretical plates (HETP), axial dispersion and the Prandt number increased with bed height, bed voidage and linear velocity. The adsorption capacity of alpha-amylases, on the resin, increased with bed height and the best condition was at four-expansion degree. alpha-Amylase characterization showed that this enzyme has high affinity with soluble starch, good hydrolysis potential and molecular weight of 116 kDa. PMID:16959553

  8. Characteristics of alpha-Amylase during Germination of Two High-Sugar Sweet Corn Cultivars of Zea mays L.

    PubMed

    Sanwo, M M; Demason, D A

    1992-07-01

    The role of the scutellum and the aleurone in alpha-amylase production in the high-sugar sweet corn cultivars Illini X-tra Sweet (shrunken-2, sh2) and Illinois 677a (sugary, sugary enhancer; su se) was compared to that in the starchy (Su) hybrid Funks G4646 with the use of alpha-amylase enzyme assays, isoelectric focusing, electron microscopy, and laser scanning confocal microscopy. The scutellum of Illinois 677a had low levels of alpha-amylase activity compared to that of Funks G4646 through 10 days after imbibition, and the aleurone of Illini X-tra Sweet had negligible activity. On the isoelectric focusing gels, the Illinois 677a scutellum had fewer alpha-amylase isozymes at 7 days compared to the Funks G4646 scutellum. The Illini X-tra Sweet aleurone had no alpha-amylase isozymes. Funks G4646 scutellar epithelial and aleurone cells contained abundant rough endoplasmic reticulum, polysomes, and dictyosomes at 5 and 7 days, respectively. The scutellar epithelial cells of Illinois 677a contained fewer of these structures by 5 days, and the Illini X-tra Sweet aleurone contained mostly lipid bodies through 7 days. Few cytoplasmic membranes and little RNA were detected with laser scanning confocal microscopy in the Illini X-tra Sweet aleurone compared to Funks G4646 at 7 days. These data suggest that the scutellum of Illinois 677a and the aleurone of Illini X-tra Sweet have impaired abilities to produce alpha-amylase. PMID:16668987

  9. Isolation and Partial Characterization of a Factor from Barley Aleurone that Modifies alpha-Amylase in Vitro.

    PubMed

    Sticher, L; Jones, R L

    1991-11-01

    Posttranslational modifications that give rise to multiple forms of alpha-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) were studied. When analyzed by denaturing polyacrylamide gel electrophoresis, barley alpha-amylase has a molecular mass of 43 to 44 kilodaltons, but isoelectric focusing resolves the enzyme into a large number of isoforms. To precisely identify these isoforms, we propose a system of classification based on their isoelectric points (pl). alpha-Amylases with pls of approximately 5, previously referred to as low pl or Amy1 isoforms, have been designated HAMY1, and alpha-amylases with pls of approximately 6, referred to as high pl or Amy2, are designated HAMY2. Individual isoforms of HAMY1 and HAMY2 are identified by their pls. For example, the most acidic alpha-amylase synthesized and secreted by barley aleurone layers is designated HAMY1(4.56). Some of the diversity in the pls of barley alpha-amylases arises from posttranslational modifications of the enzyme. We report the isolation of a factor from barley aleurone layers and incubation media that can modify HAMY1 isoforms in vitro. This factor has a molecular mass between 30 and 50 kilodaltons, and it can catalyze the conversion of HAMY1(4.90) and HAMY1(4.64) to isoforms 4.72 and 4.56, respectively. The in vitro conversion of HAMY1 isoforms by the factor is favored by pH values of approximately 5 and is inhibited at approximately pH 7. The level of this factor in aleurone layers and incubation media is not affected by treatment of the tissue with gibberellic acid. The amylase-modifying activity from barley will also modify alpha-amylases isolated from human saliva and porcine pancreas. An activity that can modify HAMY1 isoforms in vitro has also been isolated from Onozuka R10 cellulase. Because the activity isolated from barley lowers the pl of alpha-amylase from barley, human saliva, and porcine pancreas, we speculate that it is a deamidase. PMID:16668534

  10. Stable yeast transformants that secrete functional. cap alpha. -amylase encoded by cloned mouse pancreatic cDNA

    SciTech Connect

    Filho, S.A.; Galembeck, E.V.; Faria, J.B.; Frascino, A.C.S.

    1986-04-01

    Mouse pancreatic ..cap alpha..-amylase complementary DNA was inserted into a yeast shuttle vector after the Saccharomyces cerevisiae MF..cap alpha..1 promoter and secretion signals coding sequences. When transformed with the recombinant plasmid, S. cerevisiae cells were able to synthesize and secrete functional ..cap alpha..-amylase, efficiently hydrolyzing starch present in the culture medium. Stable amylolytic cells were obtained from different yeast strains. This work represents a significant step towards producing yeast that can convert starchy materials directly to ethanol.

  11. Cloning and characterization of a third type of human alpha-amylase gene, AMY2B.

    PubMed

    Yokouchi, H; Horii, A; Emi, M; Tomita, N; Doi, S; Ogawa, M; Mori, T; Matsubara, K

    1990-06-15

    We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes. PMID:2401405

  12. Dual feeding strategy for the production of alpha-amylase by Bacillus caldolyticus using complex media.

    PubMed

    Schwab, Karima; Bader, Johannes; Brokamp, Christian; Popović, Milan K; Bajpai, Rakesh; Berovic, Marin

    2009-10-01

    In this study, the objective was to investigate an exponential feeding strategy for fed-batch production of thermostable alpha-amylase (E.C. 3.2.1.1.) from the Bacillus caldolyticus (DSM405). The parameters for establishing compositions of feed media and feeding rate were obtained by statistical analysis of batch and continuous shake flask experiments. These parameters were casitone to starch ratio of 2.67g(casitone)g(starch)(-1), maintenance coefficient 0.174g(casitone)g(DW)(-1)h(-1), cell yield 0.62g(DW)g(casitone)(-1) and mu(opt)=0.2h(-1). The exponentially fed fermentation resulted in yield of 120Uml(-1) alpha-amylase that was thermostable up to 105 degrees C. Results of the exponentially fed fermentation have been discussed in the light of a feed-back controlled fed-batch fermentation reported earlier by the authors. A comparison of the temperature and pH effects on amylase produced by B. caldolyticus and on several other commercially available amylases has also been presented. PMID:19439206

  13. Cortisol, salivary alpha-amylase and children's perceptions of their social networks.

    PubMed

    Ponzi, Davide; Muehlenbein, Michael P; Geary, David C; Flinn, Mark V

    2016-04-01

    In recent years there has been a growing interest in the use of social network analysis in biobehavioral research. Despite the well-established importance of social relationships in influencing human behavior and health, little is known about how children's perception of their immediate social relationships correlates with biological parameters of stress. In this study we explore the association between two measures of children's personal social networks, perceived network size and perceived network density, with two biomarkers of stress, cortisol and salivary alpha-amylase. Forty children (mean age = 8.30, min age = 5, and max age = 12) were interviewed to collect information about their friendships and three samples of saliva were collected. Our results show that children characterized by a lower pre-interview cortisol concentration and a lower salivary alpha-amylase reactivity to the interview reported the highest density of friendships. We discuss this result in light of the multisystem approach to the study of children's behavioral outcomes, emphasizing that future work of this kind is needed in order to understand the cognitive and biological mechanisms underlying children's and adolescents' social perceptual biases. PMID:25919481

  14. Cloning, characterization, and expression of two alpha-amylase genes from Aspergillus niger var. awamori.

    PubMed

    Korman, D R; Bayliss, F T; Barnett, C C; Carmona, C L; Kodama, K H; Royer, T J; Thompson, S A; Ward, M; Wilson, L J; Berka, R M

    1990-03-01

    Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional. PMID:2340591

  15. Effect of neohesperidin dihydrochalcone on the activity and stability of alpha-amylase: a comparative study on bacterial, fungal, and mammalian enzymes.

    PubMed

    Kashani-Amin, Elaheh; Ebrahim-Habibi, Azadeh; Larijani, Bagher; Moosavi-Movahedi, Ali Akbar

    2015-10-01

    Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha-amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha-amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha-amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha-amylase surface in domain B. This domain shows differences in various alpha-amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. PMID:25808616

  16. Barley malt-alpha-amylase. Purification, action pattern, and subsite mapping of isozyme 1 and two members of the isozyme 2 subfamily using p-nitrophenylated maltooligosaccharide substrates.

    PubMed

    Ajandouz, E H; Abe, J; Svensson, B; Marchis-Mouren, G

    1992-09-23

    Isoforms AMY1, AMY2-1 and AMY2-2 of barley alpha-amylase were purified from malt. AMY2-1 and AMY2-2 are both susceptible to barley alpha-amylase/subtilisin inhibitor. The action of these isoforms is compared using substrates ranging from p-nitrophenylmaltoside through p-nitrophenylmaltoheptaoside. The kcat/Km values are calculated from the substrate consumption. The relative cleavage frequency of different substrate bonds is given by the product distribution. AMY2-1 is 3-8-fold more active than AMY1 toward p-nitrophenylmaltotrioside through p-nitrophenylmaltopentaoside. AMY2-2 is 10-50% more active than AMY2-1. The individual subsite affinities are obtained from these data. The resulting subsite maps of the isoforms are quite similar. They comprise four and six glucosyl-binding subsites towards the reducing and the non-reducing end, respectively. Towards the non-reducing end, the sixth and second subsites have a high affinity, the third has very low or even lack of affinity and the first (catalytic subsite) has a large negative affinity. The affinity declines from moderate to low for subsites 1 through 4 toward the reducing end. AMY1 has clearly a more negative affinity at the catalytic subsite, but larger affinities at both the fourth subsites, compared to AMY2. AMY2-1 has lower affinity than AMY2-2 at subsites adjacent to the catalytic site, and otherwise mostly higher affinities than AMY2-2. Theoretical kcat/Km values show excellent agreement with experimental values. PMID:1390923

  17. Maltose effects on barley malt diastatic power enzyme activity and thermostability at high isothermal mashing temperature: II. Alpha-amylase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maltose, the primary product of starch degradation during mashing, has the potential as a compatible solute to affect the activity of and increase the thermostability of barley malt alpha-amylase activity at high temperatures used in mashing and temperatures above those normally used in mashing. To ...

  18. A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

    PubMed Central

    Uozumi, N; Sakurai, K; Sasaki, T; Takekawa, S; Yamagata, H; Tsukagoshi, N; Udaka, S

    1989-01-01

    The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes. Images PMID:2464578

  19. Permissive role of the acidification caused by wheat aleurone layers upon. alpha. -amylase induction by GA sub 3

    SciTech Connect

    Rodriguez-Campos, E.; Bernal-Lugo, I.; Hamabata, A. )

    1989-04-01

    Wheat aleurone has the capacity of acidifying the incubation medium in 1 to 2 pH units. The {alpha}-amylase induction by GA{sub 3} in isolated wheat aleurone layers is strongly dependent on acidic pH of the medium (pH < 5). To examine possible mechanisms {sup 35}-Met incorporation into proteins and {alpha}-amylase, in the presence of GA{sub 3} and Ca{sup 2+} at pH, 4, 5 and 6 was studied. Although {sup 35}-Met uptake decreased markedly ({approx} 90%) at pH 4 in thepresence of GA{sub 3}, incorporation into total protein did not change significantly from other conditions. Auto-radiography of SDS-PAGE showed that most of the amino acid was in the {alpha}-amylase band, meaning that the effect of acidic pH is specific for GA{sub 3} actions on aleurone tissue. On the other hand, an increase of protonated GA{sub 3} diffusion could be ruled out. Also, there was not {alpha}-amylase inactivation at pH 6. These findings point out to the important physiological role of the acidification caused by the aleurone.

  20. Effects of metals on {alpha}-amylase activity in the digestive gland of the green mussel, Perna viridis L.

    SciTech Connect

    Yan, T.; Teo, L.H.; Sin, Y.M.

    1996-04-01

    A number of digestive enzymes in the green mussel, Perna viridis L., have been reported, and {alpha}-amylase is believed to have a higher activity than the others. Small plankton, on which the green mussel feeds, may supply plenty of starch and glycogen. They may be an important source of nutrients for the green mussel and the ability of the latter to make good use of them depends mainly on the activities of amylase. The effect of heavy metals on amylase activity is also important as the ability of the mussel`s digestive gland to accumulate these metals is well known. High concentrations of heavy metals, especially lead, have been observed in the water around Singapore. The in vitro inhibition of some metals on the activities of digestive enzymes from the green mussel has been observed, but kinetic properties of the inhibition and the in vivo inhibition of the heavy metals on digestive enzymes are little understood. In the present study, in vitro inhibition of four metals (Pb, Cd, Zn and Hg) on the activity of {alpha}-amylase from the digestive gland of the green mussel will be compared. Their effects on the K{sub M} and V{sub max} values of {alpha}-amylase will also be compared. Finally, lead is either added to the food or water, to see how it affects the activity of {alpha}-amylase and how this effect acts in combination with starvation. 12 refs., 3 figs., 3 tabs.

  1. Daytime Secretion of Salivary Cortisol and Alpha-Amylase in Preschool-Aged Children with Autism and Typically Developing Children

    ERIC Educational Resources Information Center

    Kidd, Sharon A.; Corbett, Blythe A.; Granger, Douglas A.; Boyce, W. Thomas; Anders, Thomas F.; Tager, Ira B.

    2012-01-01

    We examined daytime salivary cortisol and salivary alpha-amylase (sAA) secretion levels and variability in preschool-aged children with autism (AUT) and typically developing children (TYP). Fifty-two subjects (26 AUT and 26 TYP) were enrolled. Salivary samples were obtained at waking, midday, and bedtime on two consecutive days at three phases…

  2. Improved activity and modulated action pattern obtained by random mutagenesis at the fourth beta-alpha loop involved in substrate binding to the catalytic (beta/alpha)8-barrel domain of barley alpha-amylase 1.

    PubMed

    Matsui, I; Svensson, B

    1997-09-01

    The functionality of the sequence Arg183-Gly184-Tyr185 of the substrate binding fourth beta-alpha loop in the (beta/alpha)8-barrel of barley alpha-amylase isozyme 1 (AMY1) was studied by random mutagenesis. A motif of polar Gly184 hydrophobic residues was present in active mutants, selected by starch plate screening of yeast transformants. Gly184 was important, probably due to the carbonyl group binding to Ca2+ and the spatial proximity of Phe181. Mutation of both flanking residues as in Ser183-Gly184-Met185 (SGM-) and TGL-AMY1 decreased the Ca2+ affinity. SGM-AMY1 has 2-fold increased activity for amylose but reduced activity on maltooligosaccharides, whereas KGY-AMY1 has up to 3-fold elevated activity toward the oligosaccharides. TGL-AMY1 has modest activity on all substrates. Shifted action pattern on maltooligosaccharides for NGY-, SGM-, and TGL-AMY1 support that Arg183 in wild type is located at subsites +1 and +2, accommodating two sugar rings toward the reducing end from the site of cleavage. In the crystal structure of barley alpha-amylase 2 (AMY2), Lys182 (equivalent to AMY1 Arg183) is hydrogen-bonded with sugar OH-3 in subsite +2. Higher Ki app for acarbose inhibition of KGY-AMY1 and parent AMY1 compared with the other mutants suggests favorable substrate interactions for Arg/Lys183. KGY-AMY1 was not inhibited by the AMY2-specific proteinaceous barley alpha-amylase/subtilisin inhibitor, although Lys182 of AMY2 is salt-linked to the inhibitor. PMID:9278396

  3. A novel type of human alpha-amylase produced in lung carcinoid tumor.

    PubMed

    Tomita, N; Horii, A; Doi, S; Yokouchi, H; Shiosaki, K; Higashiyama, M; Matsuura, N; Ogawa, M; Mori, T; Matsubara, K

    1989-03-15

    A novel type of alpha-amylase was detected in a lung carcinoid tissue after surveying the cDNA library constructed from this tumor mRNA. Nucleotide sequence analysis showed that the amylase expressed in this carcinoid tumor has 13 and 6 amino acid substitutions when compared with salivary amylase (Amy1) and pancreatic amylase (Amy2), respectively. The nucleotide sequence homologies of cDNAs between this carcinoid amylase and amy1, amy2 are 97.5% and 98.2%, respectively. The nucleotide sequence comparison strongly suggests that this new amylase is the product of the amy3 gene that has been detected in human genome [Emi et al., Gene 62 (1988) 229-235] PMID:2701942

  4. Expression, purification and preliminary crystallographic studies of alpha-amylase isozyme 1 from barley seeds.

    PubMed

    Robert, Xavier; Gottschalk, Tine E; Haser, Richard; Svensson, Birte; Aghajari, Nushin

    2002-04-01

    The germinating barley seed contains two major alpha-amylase isozyme families, AMY1 and AMY2, involved in starch degradation to provide energy used by the plant embryo for growth. Many years of difficulty in growing three-dimensional crystals of natural AMY1 have now been overcome by a nonapeptide truncation of the enzyme C-terminus. The truncated enzyme was overexpressed in Pichia pastoris, purified and crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 8000 as precipitant and 2-propanol as an additive. Crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 88.36, b = 72.82, c = 61.74 A and one molecule per asymmetric unit. PMID:11914496

  5. Effects of calcium ion concentration on starch hydrolysis of barley alpha-amylase isozymes.

    PubMed

    Yuk, Jeong-Bin; Choi, Seung-Ho; Lee, Tae-Hee; Jang, Myoung-Uoon; Park, Jung-Mi; Yi, Ah-Rum; Svensson, Birte; Kim, Tae-Jip

    2008-04-01

    Barley alpha-amylase genes, amy1 and amy2, were separately cloned into the expression vector of pPICZalphaA and recombinant Pichia strains were established by homologous recombination. Both AMYs from Pichia shared almost identical hydrolysis patterns on short maltooligosaccharides to result in glucose, maltose, or maltotriose. Against insoluble blue starch, AMY1 showed the highest activity at 0.1-5 mM calcium concentration, whereas 15-20 mM was optimal for AMY2. On the hydrolysis of soluble starch, unexpectedly, there was no significant difference between AMYs with increase of calcium. However, the relative activity on various starch substrates was significantly different between AMYs, which supports that the isozymes are clearly distinguished from each other on the basis of their unique preferences for substrates. PMID:18467868

  6. Three alpha-amylase genes of Aspergillus oryzae exhibit identical intron-exon organization.

    PubMed

    Wirsel, S; Lachmund, A; Wildhardt, G; Ruttkowski, E

    1989-01-01

    We have cloned three genes (amy1, amy2 and amy3) encoding alpha-amylase in the filamentous fungus Aspergillus oryzae. The established overall sequences have a very high degree of homology, showing divergences mainly in the 3'-untranslated regions. The positions and the sequences of the eight introns were found to be absolutely identical in the three genes. The sequence analysis of the 5'-regions revealed presumptive TATA, CAAT and GC boxes. Primer extension analysis was performed to determine the transcription start. We were able to detect mRNAs from amy1 and amy3 but not from amy2 with gene-specific oligonucleotide probes complementary to the 3'-noncoding regions. PMID:2785629

  7. Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome.

    PubMed Central

    Scheirlinck, T; Mahillon, J; Joos, H; Dhaese, P; Michiels, F

    1989-01-01

    A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria. Images PMID:2679379

  8. Structures of multi-branched dextrins produced by saccharifyiing alpha-amylase from starch.

    PubMed

    Umeki, K; Yamamoto, T

    1975-11-01

    From the digest of beta-limit dextrin (prepared from glutinous rice starch) with saccharifying alpha-amylase of Bacillus subtilis [EC 3.2.1.1] (BSA), two extensibely branched dextrins consisting of nine (No. 6, Fig. 1) and ten (No 7, Fig.1) glucose units were isolated by paper chromatography. Structural analysis using various enzymes revealed that No. 6 and No. 7 were both mixtures of four triply branched dextrins. They had structures which were built up with 63-alpha-glucosylmaltotriose and/or 62-alpha-glucosylmaltose as a linking unit. However, the branching configuration and the minimum alpha-1, 4-glucosidic linkages existing between two branches followed one of the three structures shown below: (see article). PMID:814118

  9. Production and characterization of thermostable alpha-amylase by thermophilic Geobacillus stearothermophilus.

    PubMed

    Al-Qodah, Zakaria

    2006-01-01

    Studies on the alpha-amylase-producing thermophilic bacterium isolated and identified from a hot spring in Jordan and designated as Geobacillus stearothermophilus JT2 were carried out. The optimum conditions for growth and enzyme production were pH 7 and 55 degrees C. The study of the kinetics of cellular growth indicated a mu(max) of 0.22/h, a K(s) of 1.2 g/L, a tau(d) of 3.15 h and a Y(x/s) of 0.43 g cell/g starch. In addition, the activation energy for growth and death were estimated and found to be 30.5 and 210 J/mol, respectively. The effect of different carbon and nitrogen sources on the cellular growth was tested. PMID:16927263

  10. Measurement of microbial alpha-amylases with p-nitrophenyl glycosides as the substrate complex.

    PubMed

    Trepeta, R W; Edberg, S C

    1984-01-01

    The detection of alpha-amylase is commonly used in clinical microbiology laboratories to aid in differentiating Streptococcus bovis from other streptococci. It is also useful in identifying Eikenella corrodens and the gravis subspecies of Corynebacterium diphtheriae and in separating species of the genera Bacteroides, Clostridium, Actinomyces, and Bacillus. Currently, the most frequently used procedure utilizes starch as the substrate and iodine as the indicator. Starch is incorporated into a agar medium, the isolate is inoculated on the surface, and the medium is incubated for 24 to 48 h. A 15-min test containing p-nitrophenyl polyglycosides as the substrate complex was developed to yield results comparable with the agar-based starch test. The reagent was made in liquid form, 0.20 ml per tube, and could be incubated either in ambient air or at 35 degrees C. When dried, the p-nitrophenyl polyglycoside reagent could be stored at 0 degrees C for 4 weeks. PMID:6418764

  11. Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

    PubMed Central

    Glymph, J L; Stutzenberger, F J

    1977-01-01

    Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose. Images PMID:21612

  12. Multiple time courses of salivary alpha-amylase and dimensions of affect in adolescence.

    PubMed

    Doane, Leah D; Van Lenten, Scott A

    2014-11-01

    Previous research has illustrated associations among daily experiences, emotions and stress-responding physiological systems. Recently, investigators have examined salivary alpha-amylase (sAA), a surrogate marker of the autonomic nervous system, and its associations with affect. The current study examined associations among affective valence, arousal and sAA across three different time courses at the momentary, daily and inter-individual level to understand varying influences of adolescents' daily emotional experiences on sAA reactivity and diurnal sAA activity. Adolescents (N=82) provided salivary samples and diary reports of affect and experiences five times a day for three consecutive days. They also completed self-report questionnaires on trait affect. Findings from multilevel growth curves demonstrated that adolescents in our sample displayed typical sAA diurnal rhythms with levels dropping 30 min after waking and then increasing across the day to a peak in the late afternoon. Within person momentary experiences of high arousal positive affect were associated with momentary sAA reactivity. Prior day experiences of high arousal negative affect were associated with a greater amylase awakening response (i.e., greater decrease) and flatter slopes the next day. Trait positive affect was also associated with flatter sAA slopes. Our findings suggest that both affective arousal and valence should be accounted for when examining differences in sAA reactivity and diurnal patterns. Further, our results indicated that emotion-physiology transactions among adolescents occur over varying time scales for salivary alpha-amylase as well as cortisol. PMID:25076484

  13. [Alpha-amylase as an occupational allergen in baking industry employees].

    PubMed

    De Zotti, R; Larese, F; Molinari, S

    1994-01-01

    In a group of 226 bakers and pastry makers and in 88 students of a training school for bakers, we evaluated skin sensitization to the common allergens, wheat and alpha amylase. Skin prick tests were positive to the enzyme in 17 exposed subjects (7.5%) and in one student with previous occupational exposure as a baker; 27 exposed subjects (11.9%) and 2 students were sensitized to wheat. Among the 42 exposed workers who complained of work-related symptoms, 12 (28.6%) cases were skin positive to amylase and 17 (42.9%) to wheat. Among the 17 workers who were positive to amylase, 16 were also sensitized to wheat and/or common allergens, 12 complained of symptoms at work but since in many cases there was a positive response to wheat, too, it is impossible to speculate on the role of each allergen in inducing symptoms. One case, with work-related rhinoconjunctivitis, had skin sensitization only to alpha amylase but no specific IgE in the serum. These findings confirm that bakers are at risk of sensitization not only to wheat allergen but also to amylase from Aspergillus oryzae. The enzyme should be included in the list of substances to be tested among bakers in whom an occupational allergy is suspected, but particular care should be taken in evaluating the cutaneous response, especially if compared to wheat wheals. Further investigations are also needed to identify the source of risk and to better define the characteristics of the enzyme and the relationship between skin reaction to amylase, sensitization to wheat and atopy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8072442

  14. Thermal stability of alpha-amylase from malted jowar (Sorghum bicolor).

    PubMed

    Kumar, R Siva Sai; Singh, Sridevi Annapurna; Rao, A G Appu

    2005-08-24

    Malted cereals are rich sources of alpha-amylase, which catalyzes the random hydrolysis of internal alpha-(1-4)-glycosidic bonds of starch, leading to liquefaction. Amylases play a role in the predigestion of starch, leading to a reduction in the water absorption capacity of the cereal. Among the three cereal amylases (barley, ragi, and jowar), jowar amylase is found to be the most thermostable. The major amylase from malted jowar, a 47 kDa alpha-amylase, purified to homogeneity, is rich in beta structure ( approximately 60%) like other cereal amylases. T(m), the midpoint of thermal inactivation, is found to be 69.6 +/- 0.3 degrees C. Thermal inactivation is found to follow first-order kinetics at pH 4.8, the pH optimum of the enzyme. Activation energy, E(a), is found to be 45.3 +/- 0.2 kcal mol(-)(1). The activation enthalpy (DeltaH), entropy (DeltaS*), and free energy change (DeltaG) are calculated to be 44.6 +/- 0.2 kcal mol(-)(1), 57.1 +/- 0.3 cal mol(-)(1) K(-)(1), and 25.2 +/- 0.2 kcal mol(-)(1), respectively. The thermal stability of the enzyme in the presence of the commonly used food additives NaCl and sucrose has been studied. T(m) is found to decrease to 66.3 +/- 0.3, 58.1 +/- 0.2, and 48.1 +/- 0.5 degrees C, corresponding to the presence of 0.1, 0.5, and 1 M NaCl, respectively. Sucrose acts as a stabilizer; the T(m) value is found to be 77.3 +/- 0.3 degrees C compared to 69.6 +/- 0.3 degrees C in the control. PMID:16104815

  15. Isolation, characterization and inhibition by acarbose of the alpha-amylase from Lactobacillus fermentum: comparison with Lb. manihotivorans and Lb. plantarum amylases.

    PubMed

    Talamond, P; Desseaux, V; Moreau, Y; Santimone, M; Marchis-Mouren, G

    2002-11-01

    Extracellular alpha-amylase from Lactobacillus fermentum (FERMENTA) was purified by glycogen precipitation and ion exchange chromatography. The purification was approximately 28-fold with a 27% yield. The FERMENTA molecular mass (106,000 Da) is in the same range as the ones determined for L. amylovorus (AMYLOA), L. plantarum (PLANTAA) and L. manihotivorans (MANIHOA) alpha-amylases. The amino acid composition of FERMENTA differs from the other lactobacilli considered here, but however, indicates that the peptidic sequence contains two equal parts: the N-terminal catalytic part; and the C-terminal repeats. The isoelectric point of FERMENTA, PLANTAA, MANIHOA are approximately the same (3.6). The FERMENTA optimum pH (5.0) is slightly more acidic and the optimum temperature is lower (40 degrees C). Raw starch hydrolysis catalyzed by all three amylases liberates maltotriose and maltotretaose. Maltose is also produced by FERMENTA and MANIHOA. Maltohexaose FERMENTA catalyzed hydrolysis produces maltose and maltotriose. Finally, kinetics of FERMENTA, PLANTAA and MANIHOA using amylose as a substrate and acarbose as an inhibitor, were carried out. Statistical analysis of kinetic data, expressed using a general velocity equation and assuming rapid equilibrium, showed that: (1) in the absence of inhibitor k(cat)/Km are, respectively, 1x10(9), 12.6x10(9) and 3.2x10(9) s(-1) M(-1); and (2) the inhibition of FERMENTA is of the mixed non-competitive type (K(1i)=5.27 microM; L(1i)=1.73 microM) while the inhibition of PLANTAA and MANIHOA is of the uncompetitive type (L(1i)=1.93 microM and 1.52 microM, respectively). Whatever the inhibition type, acarbose is a strong inhibitor of these Lactobacillus amylases. These results indicate that, as found in porcine and barley amylases, Lactobacillus amylases contain in addition to the active site, a soluble carbohydrate (substrate or product) binding site. PMID:12431403

  16. Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express alpha-amylase and glucoamylase separately or as bifunctional fusion proteins.

    PubMed

    de Moraes, L M; Astolfi-Filho, S; Oliver, S G

    1995-11-01

    Eight constructions involving the Bacillus subtilis alpha-amylase gene (amyE), a mouse pancreatic alpha-amylase cDNA (AMY2) and an Aspergillus awamori glucoamylase cDNA (glaA) were prepared: three fusion genes, involving one alpha-amylase and the glucoamylase, two double-cassette plasmids (expressing one or other alpha-amylase and the glucoamylase) and three single-cassette plasmids, expressing the individual coding sequences. Following transformation of each plasmid into Saccharomyces cerevisiae, a plate test revealed that the largest starch hydrolysis halo was produced by the strain bearing the B. subtilis alpha-amylase/glucoamylase fusion (BsAAase/GAase), and the smallest halo by the one expressing the mouse pancreatic alpha-amylase/glucoamylase fusion (MAAase/GAase). When assayed for enzymatic activity in liquid medium, the strains bearing the fusion and the double-cassette plasmids involving B. subtilis alpha-amylase and the glucoamylase exhibited both enzymic activities. Moreover, the BsAAase/GAase hybrid was able to adsorb and digest raw starch. The MAAse/GAase fusion protein was found to exhibit only alpha-amylase activity. Finally, the capacity to grow on soluble and corn starch was tested in liquid medium for the strains bearing plasmids coding for the fusion proteins and the separate enzymes. The strain carrying the double-cassette BsAAase + GAase, which produced one of the smallest hydrolysis haloes in the place test, showed the best performance, not only in digesting soluble and corn starch but also in using all of the hydrolysis products for growth. The transformant bearing the BsAAase/GAase fusion was able to grow on soluble starch, but not on corn starch. PMID:8590658

  17. Aleurone nuclear proteins bind to similar elements in the promoter regions of two gibberellin-regulated alpha-amylase genes.

    PubMed

    Rushton, P J; Hooley, R; Lazarus, C M

    1992-09-01

    Binding of nuclear proteins from wild oat aleurone protoplasts to the promoter regions of two gibberellin-regulated wheat alpha-amylase genes (alpha-Amy1/18 and alpha-Amy2/54) has been studied by gel retardation and DNase 1 footprinting. Gel retardation studies using 300-430 bp fragments of the promoters showed similar binding characteristics with nuclear extracts from both gibberellin A1-treated and untreated protoplasts. DNase 1 footprints localised binding of nuclear proteins from gibberellin A1-treated aleurone protoplasts to regions in both promoters. Similar sequence elements in the promoter regions of both genes were protected from digestion although the location and number of footprints in each promoter region were different. Each footprint contained either a sequence similar to the cAMP and/or phorbol ester response elements, or a hyphenated palindrome sequence. The presence of cAMP and/or phorbol ester response element-like sequences in the footprints suggests that transcription factors of the bZIP type may be involved in the expression of alpha-amylase genes in aleurone cells. Footprints containing hyphenated palindrome sequences, found in the promoter regions of both genes, suggest the possible involvement of other classes of transcription factor. The conserved alpha-amylase promoter sequence TAA-CAGA was also shown to bind nuclear protein in the alpha-Amy2/54 promoter. These observations are discussed in relation to alpha-amylase gene expression in aleurone and to functional data concerning these genes. PMID:1511135

  18. Cloning and expression of Lipomyces starkeyi alpha-amylase in Escherichia coli and determination of some of its properties.

    PubMed

    Kang, Hee Kyoung; Lee, Jin Ha; Kim, Doman; Day, Donal F; Robyt, John F; Park, Kwan-Hwa; Moon, Tae-Wha

    2004-04-01

    The Lipomyces starkeyi alpha-amylase (LSA) gene encoding soluble starch-degrading alpha-amylase was cloned and characterized from a derepressed and partially constitutive mutant for both dextranase and amylase activities. The nucleotide (nt) sequence of the cDNA fragment reveals an open reading frame of 1944 bp encoding a 619 amino acid (aa) mature protein (LSA) with a calculated molecular weight of 68.709 kDa that was estimated to be about 73 kDa, including His tag (4 kDa) based on SDS-PAGE (10% acrylamide gel), activity staining, and the Western blotting, using anti-amylase-Ab. LSA had a sequence similar to other alpha-amylases in four conserved regions of the alpha-amylase family: (I) (287)DIVVNH(292), (II) (372)GLRIDTVKH(380), (III) (399)GEVFD(403), (IV) (462)FLENQD(467). Polymerase chain reaction and sequence analysis showed one intron of 60 nucleotides in the genomic lsa at positions between 966 and 967 of cDNA. The cloned LSA amylase showed a maximum activity at pH 6 and optimum temperature of 40 (o)C, with greater than 90% stability between pH 5 and pH 8 for 16 h. It was inhibited by Cu(2+) and stimulated by Ca(2+) and Mg(2+). Enzyme activity was not affected by 1 mM EGTA but was inhibited by 1 mM EDTA. LSA did not hydrolyze maltodextrins of G2 to G4, yet formed G2+G3 from G5, G2+G4 or G3+G3 from G6, and G3+G4 from G7. LSA did not hydrolyze soluble starch in the present of 2% (w/v) of acarbose. Kinetics of LSA was carried out by using starch as a substrate and the inhibition type of acarbose was the mixed non-competitive type (ki = 3.4 microM). PMID:15043869

  19. Diurnal patterns of salivary alpha-amylase and cortisol secretion in female adolescent tennis players after 16 weeks of training.

    PubMed

    Filaire, Edith; Ferreira, Jose Pedro; Oliveira, Miguel; Massart, Alain

    2013-07-01

    We examined the effects of 16 weeks of training on diurnal pattern of salivary alpha-amylase (sAA), cortisol, and the ratio of sAA over cortisol (AOC) in 12 national adolescent female tennis players. Stress and recovery were also evaluated using the Recovery-Stress-Questionnaire for Athletes-RESTQ-Sport. Data were collected after a 2-week rest (January, W0), and 4 months after W0 (W16). Subjects collected five saliva samples throughout a day. While all participants displayed the previously shown decrease after awakening in adolescents at W0, they showed a rise in the alpha-amylase awakening response and a higher alpha-amylase activity output (p<0.01) at W16 compared to W0. For the daily rhythm of cortisol we found subjects having a low overall output of salivary cortisol (p<0.01) and a blunted response to awakening at W16. Furthermore, an increase in the ratio AOC at W16, and a negative correlation between this ratio and Sport-specific recovery score. Our findings offer support for the hypothesis that increase of training load during the study period induced asymmetry activation between the two stress systems, in relation to psychological alterations and performance decrease. These results provide encouragement to continue exploring the impact of training program using a psychobiological approach among young athletes in order to prevent fatigue and preserve the health of these athletes. PMID:23200107

  20. A single residue mutation abolishes attachment of the CBM26 starch-binding domain from Lactobacillus amylovorus alpha-amylase.

    PubMed

    Rodríguez-Sanoja, Romina; Oviedo, N; Escalante, L; Ruiz, B; Sánchez, S

    2009-03-01

    Starch is degraded by amylases that frequently have a modular structure composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. The C-terminal domain from the Lactobacillus amylovorus alpha-amylase has an unusual architecture composed of five tandem starch-binding domains (SBDs). These domains belong to family 26 in the carbohydrate-binding modules (CBM) classification. It has been reported that members of this family have only one site for starch binding, where aromatic amino acids perform the binding function. In SBDs, fold similarities are better conserved than sequences; nevertheless, it is possible to identify in CBM26 members at least two aromatic residues highly conserved. We attempt to explain polysaccharide recognition for the L. amylovorus alpha-amylase SBD through site-directed mutagenesis of aromatic amino acids. Three amino acids were identified as essential for binding, two tyrosines and one tryptophan. Y18L and Y20L mutations were found to decrease the SBD binding capacity, but unexpectedly, the mutation at W32L led to a total loss of affinity, either with linear or ramified substrates. The critical role of Trp 32 in substrate binding confirms the presence of just one binding site in each alpha-amylase SBD. PMID:19052787

  1. A novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization.

    PubMed

    Liu, Xu Dong; Xu, Yan

    2008-07-01

    This study reports the purification and characterization of a novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1. Maximum alpha-amylase activity (53 U mL(-1)) was obtained at 45 degrees C after 44 h of incubation. The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography, and showed a molecular weight of 56 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH 5.0, performed stability over a broad range of pH 4.5-11.0, and was optimally active at 40-50 degrees C. The enzyme preparation had a strong digesting ability towards various raw starches and efficiently hydrolyzed raw corn starch at a concentration of 20% and pH 5.0, which were normally used in the starch industries, in a period of 12h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel alpha-amylase. PMID:17920264

  2. Solution of the structure of Aspergillus niger acid alpha-amylase by combined molecular replacement and multiple isomorphous replacement methods.

    PubMed

    Brady, R L; Brzozowski, A M; Derewenda, Z S; Dodson, E J; Dodson, G G

    1991-08-01

    The crystal structure of Aspergillus niger acid alpha-amylase was solved by a combination of multiple isomorphous replacement and molecular replacement methods. The atomic coordinates of Aspergillus oryzae (TAKA) alpha-amylase (entry 2TAA in the Protein Data Bank) and experimental diffraction data from a new monoclinic crystal form of TAKA alpha-amylase, were used during the procedure. Sequence identity between the two proteins is approximately 80%. The atomic parameters derived from the molecular replacement solution were too inaccurate to initiate least-squares crystallographic refinement. The molecular model was extensively revised against the experimental electron density map calculated at 3 A resolution. Subsequent crystallographic refinement of this model using synchrotron data to 2.1 A resolution led to a conventional R factor of 16.8%. The structure conforms well to expected stereochemistry with bond lengths deviating from target values by 0.031 A, and planar groups showing a root-mean-square deviation from ideal planes of 0.025 A. PMID:1930834

  3. Cloning and Characterization of an alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, Eva C. M. J.; Pusey, Mark L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular alpha-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  4. Variation in salivary and pancreatic alpha-amylase genes in Italian horse breeds.

    PubMed

    Coizet, Beatrice; Nicoloso, Letizia; Marletta, Donata; Tamiozzo-Calligarich, Alessandra; Pagnacco, Giulio; Crepaldi, Paola

    2014-01-01

    The dietary demand of the modern horse relies on high-cereal feeding and limited forage compared with natural grazing conditions, predisposing the horse to several important diseases. Salivary and pancreatic alpha-amylases (coded by AMY1 and AMY2 genes, respectively) play a crucial role in carbohydrate digestion in nonruminants, but little is known about these 2 genes in the horse. Aim of this work has been to distinguish genomic sequences of horse AMY1 and AMY2 genes and to analyze any polymorphisms in breeds historically characterized by marked differences in nutritional management. A single nucleotide polymorphism detection was performed and 7 novel single nucleotide polymorphisms were found. Three single nucleotide polymorphisms are in exons and were genotyped in 112 horses belonging to 6 breeds. One single nucleotide polymorphism in AMY1 gene distinguished Haflinger and the Italian native Murgese from the other breeds, whereas both the single nucleotide polymorphisms in AMY2 gene showed different allelic frequencies in Friesian compared with the other breeds. These differences are confirmed by quite high fixation index (Fst) values for these 2 nonsynonymous single nucleotide polymorphisms. These preliminary results highlight marked divergences in allele frequencies of AMY1 and AMY2 genes, involved in starch digestion, between horse breeds characterized by different histories of selection, thus providing first indications of possible relations between genetics and nutritional management. PMID:24558100

  5. Aspergillus oryzae S2 alpha-amylase production under solid state fermentation: optimization of culture conditions.

    PubMed

    Sahnoun, Mouna; Kriaa, Mouna; Elgharbi, Fatma; Ayadi, Dorra-Zouari; Bejar, Samir; Kammoun, Radhouane

    2015-04-01

    Aspergillus oryzae S2 was assayed for alpha-amylase production under solid state fermentation (SSF). In addition to AmyA and AmyB already produced in monitored submerged culture, the strain was noted to produce new AmyB oligomeric forms, in particular a dominant tetrameric form named AmyC. The latter was purified to homogeneity through fractional acetone precipitation and size exclusion chromatography. SDS-PAGE and native PAGE analyses revealed that, purified AmyC was an approximately 172 kDa tetramer of four 42 kDa subunits. AmyC was also noted to display the same NH2-terminal amino acid sequence residues and approximately the same physico-chemical properties of AmyA and AmyB, to exhibit maximum activity at pH 5.6 and 60 °C, and to produce maltose and maltotriose as major starch hydrolysis end-products. Soyabean meal was the best substitute to yeast extract compared to fish powder waste and wheat gluten waste. AmyC production was optimized under SSF using statistical design methodology. Moisture content of 76.25%, C/N substrate ratio of 0.62, and inoculum size of 10(6.87) spores allowed maximum activity of 22118.34 U/g of dried substrate, which was 33 times higher than the one obtained before the application of the central composite design (CCD). PMID:25617840

  6. Effects of simulated firefighting on the responses of salivary cortisol, alpha-amylase and psychological variables.

    PubMed

    Perroni, F; Tessitore, A; Cibelli, G; Lupo, C; D'Artibale, E; Cortis, C; Cignitti, L; De Rosas, M; Capranica, L

    2009-04-01

    The aim of this study was to evaluate the effects of a simulated firefighting intervention on salivary alpha-amylase (sA-A), free cortisol (sC), anxiety (STAI), and profile of mood states (POMS) in 20 male firefighters (age 32 +/- 1 years, VO(2peak): 43 +/- 5 ml/kg per min). During the 12-min firefighting intervention (ambient temperature: 13 +/- 1 degrees C; relative humidity: 63 +/- 1%), individuals spent 63 +/- 28% of the time working at heart rate (HR) >85% of individual HR(max), [La] (peak) 9.2 +/- 2.9 mM and ratings of perceived exertion 16 +/- 2. At 30 min post-intervention significant (p < 0.001) increases in sA-A (174%) and sC (109%) were found with regard to values recorded before and after 90 min of the firefighting intervention. Since no differences emerged between pre-intervention and post intervention for STAI and POMS values, the hormonal changes were attributable to the intense physical stress of the simulated intervention. Further research is needed during real firefighting activities, where high emotional stress may also be present. PMID:19401900

  7. Salivary nitric oxide and alpha-amylase as indexes of training intensity and load.

    PubMed

    Diaz, M M; Bocanegra, O L; Teixeira, R R; Soares, S S; Espindola, F S

    2013-01-01

    This study examined the variation in salivary nitric oxide (NO), alpha-amylase (sAA) and serum markers of muscle injury during 21 weeks of training in elite swimmers. Samples of saliva and blood were collected once a month during 5 months from 11 male professional athletes during their regular training season. The variation in each marker throughout the 21 weeks was compared with the dynamics of training volume, intensity and load. Unstimulated whole saliva was assessed for NO and sAA whereas venous blood was assessed for lactate dehydrogenase, creatine kinase, and γ-glutamyltransferase. Nitric oxide and sAA showed a proportional response to the intensity of training. However, whereas the concentration of NO increased across the 21 weeks, the activity of sAA decreased. Similar variations in the concentration of NO and the markers of muscle injury were also observed. The higher concentration of NO might be attributed to changes in haemodynamics and muscle regenerative processes. On the other hand, autonomic regulation towards parasympathetic predominance might have been responsible for the decrease in sAA activity. These findings provide appealing evidence for the utilization of salivary constituents in sports medicine to monitor training programmes. PMID:22960992

  8. Effects of alpha-amylase on in vitro growth of Legionella pneumophila.

    PubMed Central

    Bortner, C A; Miller, R D; Arnold, R R

    1983-01-01

    Sterile parotid saliva inhibited growth of Legionella pneumophila on solid media, and the salivary component involved in this inhibition has been shown to be amylase. Disk diffusion and well plate assays were used to study possible mechanisms for this effect. The amylolytic activity of saliva copurified with inhibitory activity, and both activities were sensitive to proteinase K digestion and heat treatment. In addition, purified alpha-amylase from several sources (bacteria, fungi, porcine pancreas, and human saliva) exhibited similar activity. Incorporation of charcoal or bovine serum albumin into media blocked inhibition by amylase. Replacement of Bacto-Agar with Noble agar (both from Difco Laboratories) prevented growth inhibition in the absence of starch. However, when corn starch was present with Noble agar, amylase-induced growth inhibition occurred. Purification of starch by washing with methanol eliminated some toxic component. The toxic component from starch could be recovered from the methanol wash and inhibited growth of L. pneumophila in the absence of amylase activity. The results suggest that toxic substances exist in media components which may be unmasked during salivary amylase digestion of starch. This effect may explain, in part, the difficulty in recovery of the organism from clinical specimens containing amylase. PMID:6190756

  9. Production and characterization of alpha-amylase from Aspergillus niger JGI 24 isolated in Bangalore.

    PubMed

    Varalakshmi, K N; Kumudini, B S; Nandini, B N; Solomon, J; Suhas, R; Mahesh, B; Kavitha, A P

    2009-01-01

    Five fungal isolates were screened for the production of alpha-amylase using both solid-state and submerged fermentations. The best amylase producer among them, Aspergillus niger JGI 24, was selected for enzyme production by solid-state fermentation (SSF) on wheat bran. Different carbon and nitrogen supplements were used to enhance enzyme production and maximum amount of enzyme was obtained when SSF was carried out with soluble starch and beef extract (1% each) as supplements. Further attempts to enhance enzyme production by UV induced mutagenesis were carried out. Survival rate decreased with increase in duration of UV exposure. Partial purification of the enzyme using ammonium sulphate fractionation resulted in 1.49 fold increase in the enzyme activity. The enzyme showed a molecular weight of 43 kDa by SDS-PAGE. Metal ions Ca2+ and Co2+ increased the enzyme activity. The enzyme was optimally active at 30 degrees C and pH 9.5. PMID:19469283

  10. Alpha-amylase inhibitory activity and sterol composition of the marine algae, Sargassum glaucescens

    PubMed Central

    Payghami, Nasrin; Jamili, Shahla; Rustaiyan, Abdolhossein; Saeidnia, Soodabeh; Nikan, Marjan; Gohari, Ahmad Reza

    2015-01-01

    Background: Sargassum species (phaeophyceae) are economically important brown algae in southern parts of Iran. Sargassum is mainly harvested as a row material in alginate production industries and is a source of plant foods or plant bio-stimulants even a component of animal foods. Objective: In this study, Sargassum glaucescens, collected from the seashore of Chabahar, was employed for phytochemical and biological evaluations. Materials and Methods: For that purpose, the dried algae was extracted by methanol and subjected to different chromatographic separation methods. Results: Six sterols, fucosterol (1), 24(S)-hydroxy-24-vinylcholesterol (2), 24(R)-hydroxy-24-vinylcholesterol (3), stigmasterol (4), β-sitosterol (5) and cholesterol (6) were identified by spectroscopic methods including 1H-NMR, 13C-NMR and mass spectroscopy. In vitro alpha-amylase inhibitory test was performed on the methanolic extract and the results revealed a potent inhibition (IC50 = 8.9 ± 2.4 mg/mL) of the enzyme compared to acarbose as a positive control. Conclusion: Various biological activities and distribution of sterols in Sargassum genus have been critically reviewed here. The results concluded that these algae are a good candidate for further anti-diabetic investigations in animals and human. PMID:26692744

  11. Cortisol and alpha amylase reactivity and timing of puberty: Vulnerabilities for antisocial behaviour in young adolescents

    PubMed Central

    Susman, Elizabeth J.; Granger, Douglas A; Blades, Keeva T.; Randazzo, William; Heaton, Jodi A.; Dorn, Lorah D.

    2009-01-01

    The theoretical framework proposed that cortisol and saliva alpha amylase (sAA) reactivitiy are vulnerabilities for antisocial behaviour. These indices of hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medulary (SAM) components of the stress system, respectively, were considered vulnerabilities that also interact with the putative stressful transition of timing of puberty to predispose adolescents toward antisocial behaviour. The sample consisted of 8- to-13-year-old boys and girls (N=135) and a parent. For boys, timing of puberty moderated the association between cortisol and sAA reactivity and antisocial behaviour. Higher cortisol reactivity in later timing boys was related to a composite index of antisocial behaviour and rule-breaking behaviour problems. In contrast, lower sAA reactivity and earlier timing of puberty in boys was related to rule breaking and conduct disorder symptoms. The interaction between timing of puberty and HPA or SAM regulation and timing of puberty in boys suggests that reproductive, neuroendocrine mechanisms may be involved in the extensively documented adverse consequences of off-time pubertal development. PMID:19819639

  12. Harsh discipline and behavior problems: the moderating effects of cortisol and alpha-amylase.

    PubMed

    Chen, Frances R; Raine, Adrian; Rudo-Hutt, Anna S; Glenn, Andrea L; Soyfer, Liana; Granger, Douglas A

    2015-01-01

    Numerous studies link harsh discipline to adjustment problems in youth, yet not all individuals exposed to harsh discipline develop behavior problems. Contemporary theory suggests that this relationship could be moderated by individual differences in environmentally sensitive biological systems. This study investigated whether the interaction between hypothalamic-pituitary-adrenal (HPA) activity and autonomic nervous system (ANS) arousal moderated the link between harsh discipline and behavior problems. Three saliva samples were collected on a single day from 425 inner city youth (50% male, age 11-12 years, 80% African American) and were later assayed for cortisol (HPA) and alpha-amylase (ANS). Problem behavior was assessed by self- and parent-report using the Child Behavior Checklist. Youth also reported the level of harsh discipline that they experienced. Harsh discipline was positively associated with externalizing and internalizing problems only when there were asymmetrical profiles of HPA activity and ANS arousal. This pattern was evident for boys but not girls. Findings are discussed in relation to prevailing theories suggesting that biological susceptibility translates adversity into risk for behavior problems. PMID:25451383

  13. LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031(T).

    PubMed

    Song, Qinghao; Wang, Yan; Yin, Chong; Zhang, Xiao-Hua

    2016-08-01

    Alpha-amylase is a kind of broadly used industrial enzymes, most of which have been exploited from terrestrial organism. Comparatively, alpha-amylase from marine environment was largely undeveloped. In this study, a novel alkalophilic alpha-amylase with high activity, Luteimonas abyssi alpha-amylase (LaaA), was cloned from deep-sea bacterium L. abyssi XH031(T) and expressed in Escherichia coli BL21. The gene has a length of 1428bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881U/mg at the optimum pH 9.0, which is obvious higher than other reported alpha-amylase. This enzyme can remain active at pH levels ranging from 6.0 to 11.0 and temperatures below 45°C, retaining high activity even at low temperatures (almost 38% residual activity at 10°C). In addition, 1mM Na(+), K(+), and Mn(2+) enhanced the activity of LaaA. To investigate the function of potential active sites, R227G, D229K, E256Q/H, H327V and D328V mutants were generated, and the results suggested that Arg227, Asp229, Glu256 and Asp328 were total conserved and essential for the activity of alpha-amylase LaaA. This study shows that the alpha-amylase LaaA is an alkali-tolerant and high-active amylase with strong potential for use in detergent industry. PMID:27241296

  14. Job categories and their effect on exposure to fungal alpha-amylase and inhalable dust in the U.K. baking industry.

    PubMed

    Elms, Joanne; Beckett, Paul; Griffin, Peter; Evans, Paul; Sams, Craig; Roff, Martin; Curran, Andrew D

    2003-01-01

    Enzymes in flour improver, in particular fungal alpha-amylase, are known to be a significant cause of respiratory allergy in the baking industry. This study measured total inhalable dust and fungal alpha-amylase exposures in U.K. bakeries, mills, and a flour improver production and packing facility and determined whether assignment of job description could identify individuals with the highest exposures to fungal alpha-amylase and inhalable dust. A total of 117 personal samples were taken for workers in 19 bakeries, 2 mills, and a flour improver production and packing facility and were analyzed using a monoclonal based immunoassay. Occupational hygiene surveys were undertaken for each site to assign job description and identify individuals who worked directly with flour improvers. Analysis of exposure data identified that mixers and weighers from large bakeries had the highest exposures to both inhalable dust and fungal alpha-amylase among the different categories of bakery workers (p<.01). Currently, the maximum exposure limit for flour dust in the United Kingdom is 10 mg/m(3) (8-hour time-weighted average reference period). In this study 25% of the total dust results for bakers exceeded 10 mg/m(3), and interestingly, 63% of the individuals with exposure levels exceeding 10 mg/m(3) were weighers and mixers. Individuals who worked directly with flour improvers were exposed to higher levels of both inhalable dust and fungal alpha-amylase (p<.01) than those who were not directly handling these products. Before sensitive immunoassays were utilized for the detection of specific inhalable allergens, gravimetric analysis was often used as a surrogate. There was a weak relationship between inhalable dust and fungal alpha-amylase exposures; however, inhalable dust levels could not be used to predict amylase exposures, which highlights the importance of measuring both inhalable dust and fungal alpha-amylase exposures. PMID:12908861

  15. Elevated Salivary Alpha Amylase in Adolescent Sexual Abuse Survivors with Posttraumatic Stress Disorder Symptoms

    PubMed Central

    Strawn, Jeffrey R.; Out, Dorothee; Granger, Douglas A.; Putnam, Frank W.

    2015-01-01

    Abstract Objective: Little is known regarding neuroendocrine responses in adolescent girls with posttraumatic stress disorder (PTSD) who have experienced sexual abuse. Therefore, we collected saliva samples three times daily for 3 days to assess concentrations of salivary alpha amylase (sAA) – a surrogate marker for autonomic nervous system (ANS) activity and, in particular, sympathetic activity – in sexually abused adolescent girls. Methods: Twenty-four girls (mean age: 15±1.4 years) who had experienced recent sexual abuse (i.e., sexual abuse occurred 1–6 months prior to study enrollment) and 12 healthy comparison subjects (mean age: 14.8±1.3 years) completed a structured interview and assessments to ascertain symptoms of posttraumatic stress, then collected saliva at home upon awakening, 30 minutes after waking, and at 5 p.m. on three consecutive school days. Results: For sexually abused girls, total PTSD symptoms were associated with higher overall morning levels of sAA (r[20]=0.51, p=0.02), a finding driven by intrusive symptoms (r[20]=0.43, p<0.05) and hyperarousal symptoms (r[20]=0.58, p=0.01). There were no significant differences in diurnal sAA secretion between the sexually abused girls and healthy comparison adolescents. Conclusions: Overall morning concentrations of sAA in sexually abused girls are associated with overall PTSD severity as well as symptoms of hyperarousal and intrusive symptoms, possibly reflecting symptom-linked increases in ANS tone. These data raise the possibility that alterations in ANS activity are related to the pathophysiology of sexual abuse-related PTSD in adolescent girls, and may inform therapeutic interventions (e.g., antiadrenergic medications). PMID:25803321

  16. Diacylglycerol pyrophosphate inhibits the alpha-amylase secretion stimulated by gibberellic acid in barley aleurone.

    PubMed

    Racagni, Graciela; Villasuso, Ana L; Pasquaré, Susana J; Giusto, Norma M; Machado, Estela

    2008-11-01

    ABA plays an important regulatory role in seed germination because it inhibits the response to GA in aleurone, a secretory tissue surrounding the endosperm. Phosphatidic acid (PA) is a well-known intermediary in ABA signaling, but the role of diacylglycerol pyrophosphate (DGPP) in germination processes is not clearly established. In this study, we show that PA produced by phospholipase D (E.C. 3.1.4.4) during the antagonist effect of ABA in GA signaling is rapidly phosphorylated by phosphatidate kinase (PAK) to DGPP. This is a crucial fact for aleurone function because exogenously added dioleoyl-DGPP inhibits secretion of alpha-amylase (E.C. 3.2.1.1). Aleurone treatment with ABA and 1-butanol results in normal secretory activity, and this effect is reversed by addition of dioleoyl-DGPP. We also found that ABA decreased the activity of an Mg2+-independent, N-ethylmaleimide-insensitive form of phosphatidate phosphohydrolase (PAP2) (E.C. 3.1.3.4), leading to reduction of PA dephosphorylation and increased PAK activity. Sequence analysis using Arabidopsis thaliana lipid phosphate phosphatase (LPP) sequences as queries identified two putative molecular homologues, termed HvLPP1 and HvLPP2, encoding putative Lpps with the presence of well-conserved structural Lpp domains. Our results are consistent with a role of DGPP as a regulator of ABA antagonist effect in GA signaling and provide evidence about regulation of PA level by a PAP2 during ABA response in aleurone. PMID:18573189

  17. Psychosocial determinants of diurnal alpha-amylase among healthy Quebec workers.

    PubMed

    Marchand, Alain; Juster, Robert-Paul; Lupien, Sonia J; Durand, Pierre

    2016-04-01

    Salivary alpha-amylase (sAA) is a stress-sensitive biomarker the shows promise as an indirect proxy of sympathetic-adrenal-medullary axis activities that are otherwise difficult to discern non-invasively. This comprehensive study investigated diurnal sAA in association with numerous psychosocial characteristics related to mental health, work stress, and non-work stress. Participants included 395 workers (56.1% women, age: M=41.3, SD=10.81) from across 34 distinct workplaces. Diurnal sAA was sampled over two non-consecutive work days at awakening, 30min after awakening, 14h00, 16h00, and bedtime. Well-validated psychometrics and survey items were used to measure mental health (psychological distress, depression, burnout, work characteristics) (task design, demands, social relations, gratifications), and non-work characteristics (marital/parental status, economic statuses, marital and parental stress, work-family conflicts). Preliminary results revealed that men showed occasionally higher sAA concentrations than women. Multilevel regressions were used to analyze sAA concentrations nested according to levels (i) for each time-point, (ii) between workers, and (iii) across workplaces while covarying for time of awakening, sex, age, cigarette smoking, alcohol consumption, regular physical activity, psychotropic drug use, and body mass index. Main results revealed that psychological demands, support from colleagues, interpersonal conflicts, job recognition and job insecurity appear to be associated with diurnal sAA, while non-work factors did not. Our findings showing a distinct diurnal profile for sAA replicate and expand those of Nater et al. (2007, Psychoneuroendocrinology 32, 392-401), providing further evidence that sAA is associated to subjective psychosocial factors. PMID:26799849

  18. Salivary alpha amylase and salivary cortisol response to fluid consumption in exercising athletes.

    PubMed

    Backes, T P; Horvath, P J; Kazial, K A

    2015-11-01

    The objective of the study was to examine salivary biomarker response to fluid consumption in exercising athletes. Exercise induces stress on the body and salivary alpha amylase (sAA) and salivary cortisol are useful biomarkers for activity in the sympathoadrenal medullary system and the hypothalamic pituitary adrenal axis which are involved in the stress response. Fifteen college students were given 150 ml and 500 ml of water on different days and blinded to fluid condition. The exercise protocol was identical for both fluid conditions using absolute exercise intensities ranging from moderate to high. Saliva was collected prior to exercise, post moderate and post high intensities and analyzed by Salimetrics assays. Exercise was significant for sAA with values different between pre-exercise (85 ± 10 U · ml(-1)) and high intensity (284 ± 30 U · ml(-1)) as well as between moderate intensity (204 ± 32 U · ml(-1)) and high intensity. There was no difference in sAA values between fluid conditions at either intensity. Exercise intensity and fluid condition were each significant for cortisol. Cortisol values were different between pre-exercise (0.30 ± 0.03 ug · dL(-1)) and high intensity (0.45 ± 0.05 ug · dL(-1)) as well as between moderate intensity (0.33 ± 0.04 ug · dL(-1)) and high intensity. Moderate exercise intensity cortisol was lower in the 500 ml condition (0.33 ± 0.03 ug · dL(-1)) compared with the 150 ml condition (0.38 ± 0.03 ug · dL(-1)). This altered physiological response due to fluid consumption could influence sport performance and should be considered. In addition, future sport and exercise studies should control for fluid consumption. PMID:26681828

  19. Cell-associated alpha-amylases of butyrate-producing Firmicute bacteria from the human colon.

    PubMed

    Ramsay, Alan G; Scott, Karen P; Martin, Jenny C; Rincon, Marco T; Flint, Harry J

    2006-11-01

    Selected butyrate-producing bacteria from the human colon that are related to Roseburia spp. and Butyrivibrio fibrisolvens showed a good ability to utilize a variety of starches for growth when compared with the Gram-negative amylolytic anaerobe Bacteroides thetaiotaomicron. A major cell-associated amylase of high molecular mass (140-210 kDa) was detected in each strain by SDS-PAGE zymogram analysis, and genes corresponding to these enzymes were analysed for two representative strains. Amy13B from But. fibrisolvens 16/4 is a multi-domain enzyme of 144.6 kDa that includes a family 13 glycoside hydrolase domain, and duplicated family 26 carbohydrate-binding modules. Amy13A (182.4 kDa), from Roseburia inulinivorans A2-194, also includes a family 13 domain, which is preceded by two repeat units of approximately 116 aa rich in aromatic residues, an isoamylase N-terminal domain, a pullulanase-associated domain, and an additional unidentified domain. Both Amy13A and Amy13B have N-terminal signal peptides and C-terminal cell-wall sorting signals, including a modified LPXTG motif similar to that involved in interactions with the cell surface in other Gram-positive bacteria, a hydrophobic transmembrane segment, and a basic C terminus. The overexpressed family 13 domains showed an absolute requirement for Mg2+ or Ca2+ for activity, and functioned as 1,4-alpha-glucanohydrolases (alpha-amylases; EC 3.2.1.1). These major starch-degrading enzymes thus appear to be anchored to the cell wall in this important group of human gut bacteria. PMID:17074899

  20. Alpha-Amylase Reactivity in Relation to Psychopathic Traits in Adults

    PubMed Central

    Glenn, Andrea L.; Remmel, Rheanna J.; Raine, Adrian; Schug, Robert A.; Gao, Yu; Granger, Douglas A.

    2015-01-01

    Recent investigations of the psychobiology of stress in antisocial youth have benefited from a multi-system measurement model. The inclusion of salivary alpha-amylase (sAA), a surrogate marker of autonomic/sympathetic nervous system (ANS) activity, in addition to salivary cortisol, a biomarker of the hypothalamic-pituitary-adrenal (HPA) axis functioning, has helped define a more complete picture of individual differences and potential dysfunction in the stress response system of these individuals. To the authors' knowledge, no studies have examined sAA in relation to antisocial behavior in adults or in relation to psychopathic traits specifically. In the present study, we examined sAA, in addition to salivary cortisol, in a relatively large sample (n = 158) of adult males (M age = 36.81, range = 22-67 years; 44% African-American, 34% Caucasian, 16% Hispanic) recruited from temporary employment agencies with varying levels of psychopathic traits. Males scoring highest in psychopathy were found to have attenuated sAA reactivity to social stress compared to those scoring lower in psychopathy. No differential relationships with the different factors of psychopathy were observed. In contrast to studies of antisocial youth, there were no interactions between sAA and cortisol levels in relation to psychopathy, but there was a significant interaction between pre-stressor levels of sAA and cortisol. Findings reveal potential regulatory deficits in the fast-acting, ‘fight or flight’, component of the stress response in adult males with psychopathic traits, as well as abnormalities in how this system may interact with the HPA axis. PMID:25662339

  1. Salivary alpha amylase and salivary cortisol response to fluid consumption in exercising athletes

    PubMed Central

    Horvath, PJ; Kazial, KA

    2015-01-01

    The objective of the study was to examine salivary biomarker response to fluid consumption in exercising athletes. Exercise induces stress on the body and salivary alpha amylase (sAA) and salivary cortisol are useful biomarkers for activity in the sympathoadrenal medullary system and the hypothalamic pituitary adrenal axis which are involved in the stress response. Fifteen college students were given 150 ml and 500 ml of water on different days and blinded to fluid condition. The exercise protocol was identical for both fluid conditions using absolute exercise intensities ranging from moderate to high. Saliva was collected prior to exercise, post moderate and post high intensities and analyzed by Salimetrics assays. Exercise was significant for sAA with values different between pre-exercise (85 ± 10 U · ml−1) and high intensity (284 ± 30 U · ml−1) as well as between moderate intensity (204 ± 32 U · ml−1) and high intensity. There was no difference in sAA values between fluid conditions at either intensity. Exercise intensity and fluid condition were each significant for cortisol. Cortisol values were different between pre-exercise (0.30 ± 0.03 ug · dL−1) and high intensity (0.45 ± 0.05 ug · dL−1) as well as between moderate intensity (0.33 ± 0.04 ug · dL−1) and high intensity. Moderate exercise intensity cortisol was lower in the 500 ml condition (0.33 ± 0.03 ug · dL−1) compared with the 150 ml condition (0.38 ± 0.03 ug · dL−1). This altered physiological response due to fluid consumption could influence sport performance and should be considered. In addition, future sport and exercise studies should control for fluid consumption. PMID:26681828

  2. Alpha-amylase reactivity in relation to psychopathic traits in adults.

    PubMed

    Glenn, Andrea L; Remmel, Rheanna J; Raine, Adrian; Schug, Robert A; Gao, Yu; Granger, Douglas A

    2015-04-01

    Recent investigations of the psychobiology of stress in antisocial youth have benefited from a multi-system measurement model. The inclusion of salivary alpha-amylase (sAA), a surrogate marker of autonomic/sympathetic nervous system (ANS) activity, in addition to salivary cortisol, a biomarker of the hypothalamic-pituitary-adrenal (HPA) axis functioning, has helped define a more complete picture of individual differences and potential dysfunction in the stress response system of these individuals. To the authors' knowledge, no studies have examined sAA in relation to antisocial behavior in adults or in relation to psychopathic traits specifically. In the present study, we examined sAA, in addition to salivary cortisol, in a relatively large sample (n=158) of adult males (M age=36.81, range=22-67 years; 44% African-American, 34% Caucasian, 16% Hispanic) recruited from temporary employment agencies with varying levels of psychopathic traits. Males scoring highest in psychopathy were found to have attenuated sAA reactivity to social stress compared to those scoring lower in psychopathy. No differential relationships with the different factors of psychopathy were observed. In contrast to studies of antisocial youth, there were no interactions between sAA and cortisol levels in relation to psychopathy, but there was a significant interaction between pre-stressor levels of sAA and cortisol. Findings reveal potential regulatory deficits in the fast-acting, 'fight or flight', component of the stress response in adult males with psychopathic traits, as well as abnormalities in how this system may interact with the HPA axis. PMID:25662339

  3. Salivary alpha-amylase, secretory IgA and free cortisol as neurobiological components of the stress response in the acute phase of anorexia nervosa.

    PubMed

    Paszynska, E; Dmitrzak-Weglarz, M; Tyszkiewicz-Nwafor, M; Slopien, A

    2016-06-01

    Objectives One novel hypothesis of the pathogenesis of anorexia nervosa (AN) is the possible role of mental stress in hyperactivity of the autonomic nervous system (ANS) and of the hypothalamic-pituitary-adrenal (HPA) axis. Two components of stress response - salivary alpha-amylase (sAA) and free cortisol - have been proposed. They can be determined in saliva, which closely reflects their concentrations in plasma. The purpose of this study was to measure salivary free cortisol, sAA and their correlation to secretory IgA (sIgA) of patients with AN in comparison to the average population. Methods A controlled clinical trial was designed for a matched group of 47 AN patients and 54 healthy individuals. After clinical examination, unstimulated salivary samples were taken during the acute stage of AN (BMI < 15 kg/m(2)) in the first week of hospitalisation. An enzyme-linked immunosorbent assay (ELISA) suitable for measuring sAA, sIgA and free cortisol were used. Results Anorexic patients exhibited disturbances in sAA secretion, and significantly increased cortisol and sIgA levels with a distinct correlation between these two parameters. Conclusions The behaviour of cortisol, sAA and sIgA levels can be assessed as an effect of stress reaction among AN patients with hyperactivity of the HPA axis and ANS dysregulation. The effect of stress response can be assessed reliably in saliva. PMID:26983011

  4. The Multiple Forms of alpha-Amylase Enzyme of the Araucaria Species of South America: A. araucana (Mol.) Koch and A. angustifolia (Bert.) O. Kutz : A Comparative Study.

    PubMed

    Salas, E; Cardemil, L

    1986-08-01

    alpha-Amylase is one of the major enzymes present in the seeds of both Araucaria species of South America and it initiates starch hydrolysis during germination and early seedling growth. The pattern of the multiple forms of alpha-amylase of the two Araucaria species was investigated by electrophoresis and isoelectrofocusing of the native enzyme in polyacrylamide gels. The enzyme forms were compared in the embryo and megagametophyte of quiescent seeds and of seeds imbibed for 18, 48, and 90 hours. Specific alpha-amylase enzyme forms appear and disappear during these imbibition periods showing both similarities and differences between tissues and species. Before imbibition, there are five alpha-amylase forms identical in both tissues, but different between species. After 18 hours of imbibition, there are two enzyme forms in both tissues of Araucaria araucana seeds, only one form in the embryo of Araucaria angustifolia but two forms in the megagametophyte of this specie. After 48 hours of seed imbibition, most of the enzyme forms present in quiescent seeds reappear. At 90 hours of imbibition different enzyme forms are detected in the embryo with respect to the gametophyte. The changes in form patterns of alpha-amylase are discussed according to a possible regulation of gene expression by endogenous gibberellins. PMID:16664944

  5. [The contribution of different alpha-amylase isoenzymes of the commodity grain spring wheat in the formation of falling number values].

    PubMed

    Mamytova, N S; Kuzovlev, V A; Khakimzhanov, A A; Fursov, O V

    2014-01-01

    The participation of various isoenzymes of alpha-amylase in the formation of falling number values of the commodity grain of wheat grown in the Republic of Kazakhstan was investigated. It was found that active isoenzymes alpha-AMY1 and alpha-AMY2 of the embryonic shield were present in the grain with an index over 200. A significant decrease in the falling number depended mainly on the synthesis of alpha-AMY1 and alpha-AMY2 isoenzymes in the aleurone layer. In the grain, isoenzymes with high isoelectric points (p1 > or = 7.3) were found; these isoenzymes belong to alpha-amylase or late maturing or alpha-amylase of practically mature grains. It was discovered that the exogenous hormone (gibberellic acid) induced synthesis of alpha-amylase isoenzymes of scutellum, whole caryopses, and aleurone. It was shown that the impact of exogenous gibberellic acid on the activity and structure of alpha-amylase is reduced in grain with a low falling number. PMID:25707111

  6. Putative implication of alpha-amylase loop 7 in the mechanism of substrate binding and reaction products release.

    PubMed

    André, G; Tran, V

    2004-10-01

    Alpha-amylases are widespread endo-enzymes involved in the hydrolysis of internal alpha-(1,4) glycosidic linkages of starch polymers. Molecular modeling of amylose-amylase interactions is a step toward enzymatic mechanism understanding and rational design of new enzymes. From the crystallographic complex of barley alpha-amylase AMY2-acarbose, the static aspects of amylose-amylase docking have been characterized with a model of maltododecaose (DP12) (G. André, A. Buléon, R. Haser, and V. Tran, Biopolymers 1999, Vol. 50, pp. 751-762; G. André and V. Tran, Special Publication no. 246 1999, The Royal Society of Chemistry, H. J. Gilbert, G. J. Davies, B. Henrissat, and B. Svensson, Eds., Cambridge, pp. 165-174). These studies, consistent with the experimental subsite mapping (K. Bak-Jensen, G. André, V. Tran, and B. Svensson, Journal of Biological Chemistry, to be published), propose a propagation scheme for an amylose chain in the active cleft of AMY2. The topographical overview of alpha-amylases identified loop 7 as a conserved segment flanking the active site. Since some crystallographic experiments suspected its high flexibility, its putative motion was explored through a robotic scheme, an alternate route to dynamics simulations that consume CPU time. The present article describes the characteristics of the flexibility of loop 7: location and motion in AMY2. A back-and-forth motion with a large amplitude of more than 0.6 nm was evaluated. This movement could be triggered by two hinge residues. It results in the loop flipping over the active site to enhance the docking of the native helical substrate through specific interactions, it positions the catalytic residues, it distorts the substrate towards its transition state geometry, and finally monitors the release of the products after hydrolysis. The residues involved in the process are now rational mutation points in the hands of molecular biologists. PMID:15356864

  7. Production of L-Lysine from starch by Corynebacterium glutamicum displaying alpha-amylase on its cell surface.

    PubMed

    Tateno, Toshihiro; Fukuda, Hideki; Kondo, Akihiko

    2007-04-01

    We engineered a Corynebacterium glutamicum strain displaying alpha-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of alpha-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed L-lysine fermentation at various temperatures (30-40 degrees C) and pHs (6.0-7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest L-lysine yield was recorded at 30 degrees C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l L-lysine was produced in 24 h. The L-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying alpha-amylase has a potential to directly convert soluble starch to amino acids. PMID:17216452

  8. Heat shock inhibits. alpha. -amylase synthesis in barley aleurone without inhibiting the activity of endoplasmic reticulum marker enzymes

    SciTech Connect

    Sticher, L.; Biswas, A.K.; Bush, D.S.; Jones, R.L. )

    1990-02-01

    The effects of heat shock on the synthesis of {alpha}-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25{degree}C to 40{degree}C for 3 hours, inhibits the accumulation of {alpha}-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca{sup 2+}. When ER is isolated from heat-shocked aleurone layers, less newly synthesized {alpha}-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca{sup 2+} transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.

  9. Monoclinic crystal form of Aspergillus niger alpha-amylase in complex with maltose at 1.8 angstroms resolution.

    PubMed

    Vujicić-Zagar, A; Dijkstra, B W

    2006-08-01

    Aspergillus niger alpha-amylase catalyses the hydrolysis of alpha-1,4-glucosidic bonds in starch. It shows 100% sequence identity to the A. oryzae homologue (also called TAKA-amylase), three crystal structures of which have been published to date. Two of them belong to the orthorhombic space group P2(1)2(1)2(1) with one molecule per asymmetric unit and one belongs to the monoclinic space group P2(1) with three molecules per asymmetric unit. Here, the purification, crystallization and structure determination of A. niger alpha-amylase crystallized in the monoclinic space group P2(1) with two molecules per asymmetric unit in complex with maltose at 1.8 angstroms resolution is reported. Furthermore, a novel 1.6 angstroms resolution orthorhombic crystal form (space group P2(1)2(1)2) of the native enzyme is presented. Four maltose molecules are observed in the maltose-alpha-amylase complex. Three of these occupy active-site subsites -2 and -1, +1 and +2 and the hitherto unobserved subsites +4 (Asp233, Gly234) and +5 (Asp235). The fourth maltose molecule binds at the distant binding sites d1 (Tyr382) and d2 (Trp385), also previously unobserved. Furthermore, it is shown that the active-site groove permits different binding modes of sugar units at subsites +1 and +2. This flexibility of the active-site cleft close to the catalytic centre might be needed for a productive binding of substrate chains and/or release of products. PMID:16880540

  10. Location of the alpha-amylase gene in rumen Streptococcus bovis strains distinguished by unstable amylase activity.

    PubMed

    Mareková, M; Jonecová, Z; Kmeĭ, V

    1995-01-01

    Genetic stability of amylase activity after serial subcultivation experiments with amylolytic ruminal Streptococcus bovis strains was investigated. Two strains Amy+ and Amy- were obtained. Loss of amylase activity connected with the loss of plasmid DNA was not found in these strains. The presence of the gene responsible for the amylase activity in the chromosome of these strains was revealed by hybridization of the alpha-amylase gene on pJK108 against chromosomal DNA of S. bovis and Bacillus subtilis after a complete restriction with EcoRI. PMID:8851562

  11. Electrospray mass spectrometry characterization of post-translational modifications of barley alpha-amylase 1 produced in yeast.

    PubMed

    Søgaard, M; Andersen, J S; Roepstorff, P; Svensson, B

    1993-10-01

    We have used electrospray mass spectrometry (ESMS) in combination with protein chemistry and genetics to delineate post-translational modifications in yeast of barley alpha-amylase 1 (AMY1), a 45 kD enzyme crucial for production of malt, an important starting material in the manufacture of beer and whisky. In addition to signal peptide processing these modifications are: (1) removal of C-terminal Arg-Ser by Kex1p, (2) glutathionylation of Cys95, (3) O-glycosylation, and (4) additional degradation of the C-terminus. PMID:7764097

  12. The structure of barley alpha-amylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs.

    PubMed

    Robert, Xavier; Haser, Richard; Gottschalk, Tine E; Ratajczak, Fabien; Driguez, Hugues; Svensson, Birte; Aghajari, Nushin

    2003-08-01

    Though the three-dimensional structures of barley alpha-amylase isozymes AMY1 and AMY2 are very similar, they differ remarkably from each other in their affinity for Ca(2+) and when interacting with substrate analogs. A surface site recognizing maltooligosaccharides, not earlier reported for other alpha-amylases and probably associated with the different activity of AMY1 and AMY2 toward starch granules, has been identified. It is located in the C-terminal part of the enzyme and, thus, highlights a potential role of domain C. In order to scrutinize the possible biological significance of this domain in alpha-amylases, a thorough comparison of their three-dimensional structures was conducted. An additional role for an earlier-identified starch granule binding surface site is proposed, and a new calcium ion is reported. PMID:12906828

  13. High-efficiency, one-step starch utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse alpha-amylase.

    PubMed Central

    Kim, K; Park, C S; Mattoon, J R

    1988-01-01

    Transformed, hybrid Saccharomyces strains capable of simultaneous secretion of glucoamylase and alpha-amylase have been produced. These strains could carry out direct, one-step assimilation of starch, with conversion efficiency greater than 93% during a 5-day growth period. One of the transformants converted 92.8% of available starch into reducing sugars in only 2 days. Glucoamylase secretion by these strains resulted from expression of one or more chromosomal STA genes derived from Saccharomyces diastaticus. The strains were transformed by a plasmid (pMS12) containing mouse salivary alpha-amylase cDNA in an expression vector containing yeast alcohol dehydrogenase promoter and a segment of yeast 2 micron plasmid. The major starch hydrolysis product produced by crude amylases found in culture broths was glucose, indicating that alpha-amylase and glucoamylase acted cooperatively. PMID:3132104

  14. Salivary cortisol and alpha-amylase reactivity to taekwondo competition in children.

    PubMed

    Capranica, Laura; Lupo, Corrado; Cortis, Cristina; Chiodo, Salvatore; Cibelli, Giuseppe; Tessitore, Antonio

    2012-02-01

    The aim of this study was to evaluate the effects of an official taekwondo competition (three 1-min rounds with a 1-min recovery in-between) on heart rate (HR), salivary alpha-amylase (sAA), and salivary-free cortisol (sC) in children. Parental consent was obtained for 12 young (10.4 ± 0.2 years) male taekwondo athletes. Saliva sample were collected 15 min before and 1 min after an official taekwondo competition, and at 30, 60, and 90 min of the recovery period. To evaluate the exercise intensity during the competition, HR was measured and expressed as a percentage of individuals HR(peak). Athletes spent 78% of the time working at HR > 90% HR(max), with significant increases from round 1 to round 2 and 3. Peak sAA observed at the end of the match (169.6 ± 47.0 U/mL) was different (P = 0.0001) from the other samplings (pre-competition 55.0 ± 14.0 U/mL, 30-min recovery 80.4 ± 17.7 U/mL, 60-min recovery 50.5 ± 7.6 U/ml; 90-min recovery 53.2 ± 9.6 U/mL). Peak sC values observed at 30-min recovery (17.9 ± 3.5 nmol/L) were different (P < 0.0001) from pre-competition (5.6 ± 0.9 nmol/L), post-competition (9.0 ± 2.0 nmol/L), 60-min recovery (10.3 ± 2.6 nmol/L) and 90-min recovery (4.2 ± 0.8 nmol/L) values. These findings confirm that taekwondo competitions pose a high stress on young athletes. The different sAA and sC reactions in response to the physical stressor mirror the faster reactivity of the sympathetic-adrenomedullary system relatively to the hypothalamic-pituitary-adrenocortical system, respectively. This experimental paradigm might represent a useful model for further research on the effects of various stressors (i.e., training and competition) in taekwondo athletes. PMID:21643917

  15. Alpha-amylase from germinating soybean (Glycine max) seeds--purification, characterization and sequential similarity of conserved and catalytic amino acid residues.

    PubMed

    Kumari, Arpana; Singh, Vinay Kumar; Fitter, Jörg; Polen, Tino; Kayastha, Arvind M

    2010-10-01

    Starch hydrolyzing amylase from germinated soybeans seeds (Glycine max) has been purified 400-fold to electrophoretic homogeneity with a final specific activity of 384 units/mg. SDS-PAGE of the final preparation revealed a single protein band of 100 kDa, whereas molecular mass was determined to be 84 kDa by MALDI-TOF and gel filtration on Superdex-200 (FPLC). The enzyme exhibited maximum activity at pH 5.5 and a pI value of 4.85. The energy of activation was determined to be 6.09 kcal/mol in the temperature range 25-85 degrees C. Apparent Michaelis constant (K(m)((app))) for starch was 0.71 mg/mL and turnover number (k(cat)) was 280 s(-1) in 50 mM sodium acetate buffer, pH 5.5. Thermal inactivation studies at 85 degrees C showed first-order kinetics with rate constant (k) equal to 0.0063 min(-1). Soybean alpha-amylase showed high specificity for its primary substrate starch. High similarity of soybean alpha-amylase with known amylases suggests that this alpha-amylase belongs to glycosyl hydrolase family 13. Cereal alpha-amylases have gained importance due to their compatibility for biotechnological applications. Wide availability and easy purification protocol make soybean as an attractive alternative for plant alpha-amylase. Soybean can be used as commercially viable source of alpha-amylase for various industrial applications. PMID:20655076

  16. Coordinate increase in major transcripts from the high pI alpha-amylase multigene family in barley aleurone cells stimulated with gibberellic acid.

    PubMed

    Rogers, J C; Milliman, C

    1984-10-10

    The purpose of this study was to identify specifically genes and transcripts for the high pI isozyme of barley alpha-amylase. From hybridization of coding sequence probes to blots of genomic DNA digested with restriction enzymes that do not cut within our cloned high pI alpha-amylase cDNA, it is estimated that about 7 alpha-amylase genes or pseudogenes exist. No difference could be detected between barley aleurone cell and sprout DNAs. Experiments using probes from the 5' and 3' untranslated sequences of the high pI alpha-amylase cDNA clone identified three HindIII fragments that probably carry high pI sequences. Primer extension experiments used as a primer the terminal 5' coding sequence from our cDNA clone; this primer would not cross-hybridize to low pI alpha-amylase transcripts. Two major transcripts were identified. These shared a conserved 23-base sequence immediately 5' to the ATG start codon, although a C----G transversion and a 3-base deletion were present within this sequence. An unusual 8-base pair GC palindrome was present in the conserved region immediately preceding the ATG start codon. Distal to the conserved sequence there was no apparent homology. One transcript carrying a 97-base untranslated region was identical to our high pI cDNA clone E. The gene for the other was recovered from a lambda phage genomic library. The 5' coding sequence was very similar, but not identical to clone E, demonstrating that these transcripts arise from separate genes. The two transcripts increased coordinately in aleurone cells stimulated with gibberellic acid. These data indicate that there is a high pI alpha-amylase multigene family with at least two active members, both of which are regulated in some manner by the plant hormone gibberellic acid. PMID:6090459

  17. A Pilot Study of Psychotherapist Trainees’ Alpha-Amylase and Cortisol Levels During Treatment of Recently Suicidal Clients With Borderline Traits

    PubMed Central

    Miller, Grant D.; Iverson, Katherine M.; Kemmelmeier, Markus; MacLane, Chelsea; Pistorello, Jacqueline; Fruzzetti, Alan E.; Crenshaw, Katrina Y.; Erikson, Karen M.; Katrichak, Barrie M.; Oser, Megan; Pruitt, Larry D.; Watkins, Melanie M.

    2010-01-01

    Psychotherapists often experience stress while providing psychotherapy, in particular when working with difficult presentations such as suicidality. As part of a larger study on the treatment of recently suicidal college students with borderline traits, 6 therapists in training collected their own salivary samples for alpha-amylase (AA) and cortisol (C) analyses immediately before and after sessions with 2 selected clients. On average, samples were collected for the same therapist–patient dyad throughout the year-long study to ensure that data reflected therapist responses across stages of treatment. Therapists also completed a working alliance questionnaire and rated perceived session difficulty immediately after each selected session. Contrary to expectations, therapists demonstrated elevated levels of stress as measured by AA and C at presession relative to postsession levels. Greater session difficulty was related to more pronounced declines in AA, whereas a stronger working alliance was linked to more pronounced reductions in C. Results suggest that physiological stress responses while working with recently suicidal clients with borderline traits occur primarily in terms of session anticipatory anxiety, whereas AA and C changes may be affected differently by factors such as session difficulty and working alliance. This is a pilot study, limited by its sample size, but the design, findings, and inclusion of physiological measures present an initial step in an essential line of research. PMID:21709772

  18. Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae.

    PubMed

    Eksteen, Jeremy M; Steyn, Andries J C; van Rensburg, Pierre; Cordero Otero, Ricardo R; Pretorius, Isak S

    2003-01-15

    Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active alpha-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases

  19. Application of the extracellular alpha-amylase gene from Streptococcus bovis 148 to construction of a secretion vector for yogurt starter strains.

    PubMed

    Satoh, E; Ito, Y; Sasaki, Y; Sasaki, T

    1997-11-01

    Streptococcus thermophilus ATCC 19258, Lactobacillus delbrueckii subsp. bulgaricus T-11, and Lactococcus lactis subsp. lactis IL1403 were transformed with the alpha-amylase gene (amyA) from Streptococcus bovis 148 by using a wide host-range vector, and all the transformants secreted the alpha-amylase successfully. Since the promoter and the secretion signal of the amyA gene were functional in these strains, we constructed a secretion vector using the expression elements of amyA. Trials to secrete foreign enzymes in yogurt starter strains were performed using this novel secretion vector. PMID:9361445

  20. Application of the extracellular alpha-amylase gene from Streptococcus bovis 148 to construction of a secretion vector for yogurt starter strains.

    PubMed Central

    Satoh, E; Ito, Y; Sasaki, Y; Sasaki, T

    1997-01-01

    Streptococcus thermophilus ATCC 19258, Lactobacillus delbrueckii subsp. bulgaricus T-11, and Lactococcus lactis subsp. lactis IL1403 were transformed with the alpha-amylase gene (amyA) from Streptococcus bovis 148 by using a wide host-range vector, and all the transformants secreted the alpha-amylase successfully. Since the promoter and the secretion signal of the amyA gene were functional in these strains, we constructed a secretion vector using the expression elements of amyA. Trials to secrete foreign enzymes in yogurt starter strains were performed using this novel secretion vector. PMID:9361445

  1. N-terminal sequence of amino acids and some properties of an acid-stable alpha-amylase from citric acid-koji (Aspergillus usamii var.).

    PubMed

    Suganuma, T; Tahara, N; Kitahara, K; Nagahama, T; Inuzuka, K

    1996-01-01

    An acid-stable alpha-amylase (AA) was purified from an acidic extract of citric acid-koji (A. usamii var.). The N-terminal sequence of the first 20 amino acids of the enzyme was identical with that of AA from A. niger, but the two enzymes differed in molecular weight. HPLC analysis for identifying the anomers of products indicated that the AA hydrolyzed maltopentaose (G5) at the third glycoside bond predominantly, which differed from Taka-amylase A and the neutral alpha-amylase (NA) from the citric acid-koji. PMID:8824843

  2. Improvement in lactic acid production from starch using alpha-amylase-secreting Lactococcus lactis cells adapted to maltose or starch.

    PubMed

    Okano, Kenji; Kimura, Sakurako; Narita, Junya; Fukuda, Hideki; Kondo, Akihiko

    2007-07-01

    To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting alpha-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l(-1) h(-1) lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l(-1) h(-1) lactate). Maximum volumetric lactate productivity was further increased (1.57 g l(-1) h(-1) lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of L: -lactate) was achieved. In this study, we propose a new approach to lactate production by alpha-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation. PMID:17384945

  3. Production and characterization of alpha-amylase from mango kernel by Fusarium solani NAIMCC-F-02956 using submerged fermentation.

    PubMed

    Kumar, Devendra; Yadav, Kaushlesh K; Muthukumar, M; Garg, Neelima

    2013-11-01

    Microbial production of enzymes using low valued agro industrial wastes is gaining importance globally. Mango is one of the major fruit processed into a variety of products. During processing 40-50% of solid waste is generated in form of peel and stones. After decortications of mango stone, kernel is obtained which is a rich source of starch (upto 60%). It was utilized as a substrate for alpha-amylase production using Fusarium soloni. Maximum alpha-amylase production (0.889 U g(-1)) was recorded using a substrate concentration of 5% (w/v), pH-4 and temperature 30 degrees C on 9th day of incubation. Supplementation of production medium with micronutrients viz., Ca2+, Fe2+ or Mg2+ improved the enzyme production while, Zn2+, B3+ or Mn2+ ions exhibited inhibitory effect. The extracellular protein was precipitated by ammonium sulphate up to 70% saturation, dialyzed and purified (27.84 fold) by gel-exclusion (Sephadex G-75) chromatography. Protein profiling on 12% SDS-PAGE revealed three bands corresponding to 26, 27 and 30 kDa molecular sizes. The optimum amylase activity was achieved at pH 5.0 at 40 degrees C. The Michaelis constant (KM), Vmax and activation energy (-Ea) were found to be 3.7 mg ml(-1), 0.24 U mg(-1) and 42.39 kJ mole(-1), respectively. PMID:24555336

  4. Structure and molecular model refinement of Aspergillus oryzae (TAKA) alpha-amylase: an application of the simulated-annealing method.

    PubMed

    Swift, H J; Brady, L; Derewenda, Z S; Dodson, E J; Dodson, G G; Turkenburg, J P; Wilkinson, A J

    1991-08-01

    Monoclinic crystals of a neutral alpha-amylase from Aspergillus oryzae, containing three molecules in the asymmetric unit, have been reported previously and studied at 3 A resolution [Matsuura, Kunusoki, Harada & Kakudo (1984). J. Biochem. 95, 697-702]. Here we report the solution of the structure of this enzyme in a different crystal form (space group P2(1)2(1)2(1), a = 50.9, b = 67.2, c = 132.7 A), with only one molecule in the asymmetric unit. The structure was solved by the molecular replacement method, using a model of acid alpha-amylase from a related fungus A. niger [Brady, Brzozowski, Derewenda, Dodson & Dodson (1991). Acta Cryst. B47, 527-535]. Conventional least-squares crystallographic refinement failed to converge in a satisfactory manner, and the technique of molecular dynamics in the form of the XPLOR package [Brunger (1988). XPLOR Manual. Yale Univ., USA] was used to overcome the problem. A large rigid-body type movement of the C-terminal domain was identified and accounted for. The final round of restrained least-squares refinement (at 2.1 A resolution) including 3675 protein atoms and 247 water molecules resulted in a conventional crystallographic R factor of 0.183 and an atomic model which conforms well to standard stereochemical parameters (standard deviation of bond lengths from their expected values is 0.028 A, while that for planar groups is 0.029 A). PMID:1930835

  5. Effects of sorghum (Sorghum bicolor (L.) Moench) tannins on alpha-amylase activity and in vitro digestibility of starch in raw and processed flours

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of condensed tannins (CT) on in vitro starch digestibility in cooked, wholegrain sorghum flours and on corn starch was investigated. CT extracts were also tested for their inhibitory effect on alpha-amylases. Rapidly digestible starch, slowly digestible starch, and resistant starch were n...

  6. MALTOTRIOSE, PRODUCT OF ALPHA-AMYLASE STARCH HYDROLYSIS, SUPPRESSES MALTASE-GLUCOAMYLASE ACTIVITY AND SLOWS TERMINAL STARCH DIGESTION 44.5 FOLD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starches constitute the main caloric source in the average human diet. The digestion of starches is far more complex than sugars and requires six different enzyme activities to produce free glucose before absorption. Salivary and pancreatic alpha-amylase activities initially hydrolyze internal 1-4 g...

  7. Discovering an Accessible Enzyme: Salivary [alpha]-Amylase--"Prima Digestio Fit in Ore"--A Didactic Approach for High School Students

    ERIC Educational Resources Information Center

    Marini, Isabella

    2005-01-01

    Human salivary [alpha]-amylase is used in this experimental approach to introduce biology high school students to the concept of enzyme activity in a dynamic way. Through a series of five easy, rapid, and inexpensive laboratory experiments students learn what the activity of an enzyme consists of: first in a qualitative then in a semi-quantitative…

  8. Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor

    PubMed Central

    Mukhtar, Hamid; Ikram-ul-Haq

    2012-01-01

    We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0–8.0 and 8.0–10, respectively. Maximum production of proteases was observed at an incubation temperature of 37°C while that of alpha amylase was observed at 40°C. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions. PMID:24031930

  9. Salivary Alpha Amylase and Cortisol Levels in Children with Global Developmental Delay and Their Relation with the Expectation of Dental Care and Behavior during the Intervention

    ERIC Educational Resources Information Center

    dos Santos, Marcio Jose Possari; Bernabe, Daniel Galera; Nakamune, Ana Claudia de Melo Stevanato; Perri, Silvia Helena Venturoli; de Aguiar, Sandra Maria Herondina Coelho Avila; de Oliveira, Sandra Helena Penha

    2012-01-01

    The purpose of this study was to analyze the alpha-amylase (sAA) and cortisol levels in children with Global developmental delay (GDD) before and after dental treatment and its association with the children's behavior during treatment. The morning salivary cortisol levels and activity of sAA of 33 children with GDD were evaluated before and after…

  10. General Subject 2. Report to ICUMSA on the determination of carry-over alpha-amylase activity in white and refined sugars by a spectrophotometric method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A report is given on a new industrial method for the determination of carry-over alpha-amylase activity in raw and refined sugars, as well as a recommendation. In recent years, there has been increased concern over carry-over activity of mostly high temperature (HT) and very high temperature (VHT) s...

  11. Bioactive compounds from Carissa opaca roots and xanthine oxidase and alpha-amylase inhibitory activities of their methanolic extract and its fractions in different solvents

    PubMed Central

    Saeed, Ramsha; Ahmed, Dildar

    2015-01-01

    Background: Carissa opaca is known for its many ethnomedicinal uses. There was a need to study its bioactivities and identify its phytochemicals. Objective: The objective was to isolate and identify phytochemicals from roots of C. opaca and to evaluate xanthine oxidase (XO) and alpha-amylase inhibitory activities of their methanolic extract and its fractions. Materials and Methods: Methanolic extract of finely divided powder of roots of C. opaca was obtained by cold maceration, followed by its fractionation to obtain hexane, chloroform, ethyl acetate, n-butanolic, and aqueous fractions. Phytochemicals screening was done by standard protocols. XO and alpha-amylase inhibitory activities of the methanolic extract and its fractions were studied. The most active ethyl acetate fraction was subjected to the column and thin layer chromatography to isolate its compounds, which were identified by gas chromatography-mass spectrometry and high-performance liquid chromatography comparison. Results: Methanolic extract displayed significant activity against both the enzymes with IC50 of 156.0 mg/mL and 5.6 mg/mL for XO and alpha-amylase, respectively. Ethyl acetate fraction showed highest activity against both the enzymes with IC50 of 129 mg/mL and 4.9 mg/mL for XO and alpha-amylase, respectively. Chloroform fraction had IC50 of 154.2 mg/mL and 5.5 mg/mL for XO and alpha-amylase, respectively. Aqueous fraction exhibited significant efficacy against alpha-amylase (IC50 5.0 mg/mL). Hexane fraction showed good activity against alpha-amylase in a dose-dependent manner but exhibited opposite trend against XO. The compounds isolated from ethyl acetate fraction included limonene, vanillin, lupeol, rutin, quercetin, b-sitosterol, Vitamin E, 2-hydroxyacetophenone, naphthalenone, 2,3,3-trimethyl-2-(3-methylbuta-1,3-dienyl)-6-methylenecyclohexanone, and 2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester. Conclusions: Moderately polar phytochemicals of C. opaca roots possess exploitable

  12. Daytime Secretion of Salivary Cortisol and Alpha-Amylase in Preschool-Aged Children with Autism and Typically Developing Children

    PubMed Central

    Corbett, Blythe A.; Granger, Douglas A.; Boyce, W. Thomas; Anders, Thomas F.; Tager, Ira B.

    2013-01-01

    We examined daytime salivary cortisol and salivary alpha-amylase (sAA) secretion levels and variability in preschool-aged children with autism (AUT) and typically developing children (TYP). Fifty-two subjects (26 AUT and 26 TYP) were enrolled. Salivary samples were obtained at waking, midday, and bedtime on two consecutive days at three phases (baseline, 3 months later, 6 months later). There were modest increases in waking cortisol and sAA levels in AUT relative to TYP, but the increases were not statistically significant. Important differences were observed in cortisol and sAA variability between AUT and TYP. There was also a graded response among AUT by functional status—cortisol and sAA secretion levels were higher when IQ was lower. PMID:22477468

  13. Expression, crystallization and preliminary X-ray crystallographic studies of Klebsiella pneumoniae maltohexaose-producing alpha-amylase.

    PubMed

    Momma, Mitsuru; Fujimoto, Zui

    2004-12-01

    A recombinant form of Klebsiella pneumoniae maltohexaose-producing alpha-amylase has been overexpressed in Escherichia coli and purified to homogeneity. Crystals were obtained at 293 K by the microbatch technique using 80 mM sodium/potassium phosphate buffer pH 6.2 containing 8% polyethylene glycol 3000, 4% polyethylene glycol 3350 and 40 mM sodium thiocyanate. Crystals of the overexpressed recombinant enzyme diffracted to better than 2.5 A resolution at 95 K using a synchrotron-radiation source. The crystals belong to the primitive monoclinic space group P2(1), with unit-cell parameters a = 74.8, b = 107.6, c = 82.2 A, beta = 96.2 degrees. Assuming the presence of two molecules per asymmetric unit, the V(M) value for the crystal was 2.3 A(3) Da(-1), indicating a solvent content of 47%. PMID:15583388

  14. Alpha-amylase starch binding domains: cooperative effects of binding to starch granules of multiple tandemly arranged domains.

    PubMed

    Guillén, D; Santiago, M; Linares, L; Pérez, R; Morlon, J; Ruiz, B; Sánchez, S; Rodríguez-Sanoja, R

    2007-06-01

    The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch. PMID:17468268

  15. Self-compassion training modulates alpha-amylase, heart rate variability, and subjective responses to social evaluative threat in women

    PubMed Central

    Arch, Joanna J.; Brown, Kirk Warren; Dean, Derek J.; Landy, Lauren N.; Brown, Kimberley; Laudenslager, Mark L.

    2014-01-01

    A growing body of research has revealed that social evaluative stressors trigger biological and psychological responses that in chronic forms have been linked to aging and disease. Recent research suggests that self-compassion may protect the self from typical defensive responses to evaluation. We investigated whether brief training in self-compassion moderated biopsychological responses to the Trier Social Stress Test (TSST) in women. Compared to attention (placebo) and no-training control conditions, brief self-compassion training diminished sympathetic (salivary alpha-amylase), cardiac parasympathetic, and subjective anxiety responses, though not HPA-axis (salivary cortisol) responses to the TSST. Self-compassion training also led to greater self-compassion under threat relative to the control groups. In that social stress pervades modern life, self-compassion represents a promising approach to diminishing its potentially negative psychological and biological effects. PMID:24636501

  16. Improved alpha-amylase and Helicobacter pylori inhibition by fenugreek extracts derived via solid-state bioconversion using Rhizopus oligosporus.

    PubMed

    Randhir, Reena; Shetty, Kalidas

    2007-01-01

    The present research investigated the enrichment of fenugreek (Trigonella foenum graceum) seed substrate with phenolic antioxidants and L-DOPA via fungal-based solid-state bioconversion (SSB) system. This approach using food grade fungus Rhizopus oligosporus, was chosen because it has been demonstrated to be effective in other seed and food substrates for improving health-relevant functionality and has long history of use for food processing in Asia. The protein content and beta-glucosidase activity of the substrate which reflects fungal growth, increased with incubation time in conjunction with enhanced phenolic content and also suggested its possible involvement in phenolic mobilization. The antioxidant activity assayed by beta-carotene bleaching and DPPH free radical scavenging methods both indicated high activity during early growth stage (days 4-6) followed by reduced activity during later growth stage (days 8-20). A direct association between higher phenolic contents during early growth stage (days 4-6) and antioxidant activity suggested a link to mobilization of polymeric and hydrophobic phenolic forms. The L-DOPA content of the fenugreek extract fluctuated during the course of bioconversion with higher levels during days 6-10 (1.5-1.7 mg/g DW). The SSB process substantially improved the in vitro porcine alpha-amylase inhibition activity by 75 % on day 4 which correlated to higher levels of total phenolics and related antioxidant activity of the extracts. The high alpha-amylase inhibitory activity also coincided with high L-DOPA content on day 6. These results have implications for diet-based diabetes management. The same bioconversion stage had Helicobacter pylori inhibitory activity, which has implications for ulcer management. PMID:17704018

  17. Conversion of starch to ethanol in a recombinant saccharomyces cerevisiae strain expressing rice [alpha]-amylase from a novel Pichia pastoris alcohol oxidase promoter

    SciTech Connect

    Kumagai, M.H.; Sverlow, G.G.; della-Cioppa, G.; Grill, L.K. )

    1993-05-01

    A recombinant Saccharomyces cerevisiae, expressing and secreting rice [alpha]-amylase, converts starch to ethanol. The rice [alpha]-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTG-N[sub 3]-GCTTCCAA-N[sub 5]-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida biodinii S2. The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch. 45 refs., 8 figs.

  18. Calcium binding in. alpha. -amylases: An X-ray diffraction study at 2. 1- angstrom resolution of two enzymes from Aspergillus

    SciTech Connect

    Boel, E.; Jensen, V.J.; Petersen, S.B.; Thim, L. Woldike, H.F. ); Brady, L.; Brzozowski, AM.; Derewenda, Z.; Dodson, G.G.; Swift, H. )

    1990-07-03

    X-ray diffraction analysis (at 2.1-{angstrom} resolution) of an acid alpha-amylase from Aspergillus niger allowed a detailed description of the stereochemistry of the calcium-binding sites. The primary site (which is essential in maintaining proper folding around the active site) contains a tightly bound Ca{sup 2+} with an unusually high number of eight ligands. A secondary binding site was identified at the bottom of the substrate binding cleft; it involves the residues presumed to play a catalytic role (Asp206 and Glu230). This explains the inhibitory effect of calcium observed at higher concentrations. Neutral Aspergillus oryzae (TAKA) {alpha}-amylase was also refined in a new crystal at 2.1-{angstrom} resolution. The structure of this homologous (over 80%) enzyme and addition kinetic studies support all the structural conclusions regarding both calcium-binding sites.

  19. Effect of sympathetic denervation of the pineal gland on maternal co-ordination of the circadian rhythm of alpha-amylase in parotid gland from young rats.

    PubMed

    Bellavía, S L; Sanz, E G; Gallará, R V; Carpentieri, A; Vermouth, N T

    1993-12-01

    Twenty-five-day-old rats maintained in constant darkness since birth and born from mothers kept in the dark since the 14th day of pregnancy showed a circadian rhythm of alpha-amylase content in parotid glands, which may be explained by a mechanism of maternal co-ordination. Rats in the same conditions, except that their mothers had been submitted to bilateral excision of the superior cervical ganglia 30 days before mating, did not show diurnal variations of alpha-amylase activity in the parotid glands. When ganglionectomized mothers were treated with a daily dose of melatonin (1 mg/kg) from the 14th day of gestation up to the 10th day of lactation, their litters showed significant diurnal variations of amylase in the parotid glands, suggesting a role of the maternal pineal gland in the maternal-fetal and/or maternal-neonatal transfer of photoperiodic information. PMID:8141675

  20. Improvement of cloned [alpha]-amylase gene expression in fed-batch culture of recombinant Saccharomyces cerevisiae by regulating both glucose and ethanol concentrations using a fuzzy controller

    SciTech Connect

    Shiba, Sumihisa; Nishida, Yoshio; Park, Y.S.; Iijima, Shinji; Kobayashi, Takeshi . Dept. of Biotechnology)

    1994-11-05

    The effect of ethanol concentration on cloned gene expression in recombinant Saccharomyces cerevisiae strain 20B-12 containing one of two plasmids, pNA3 and pNA7, was investigated in batch cultures. Plasmids pNA3 and pNA7 contain the [alpha]-amylase gene under the control of the SUC2 or PGK promoter, respectively. When the ethanol concentration was controlled at 2 to 5 g/L, the gene expressions were two times higher than those at 20 g/L ethanol. To increase the gene expression by maintaining both the ethanol and glucose concentrations at low levels, a fuzzy controller was developed. The concentrations of glucose and ethanol were controlled simultaneously at 0.15 and 2 g/L, respectively, in the production phase using the fuzzy controller in fed-batch culture. The synthesis of [alpha]-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory [alpha]-amylase activities of cells harboring plasmids pNA3 and pNA7 in fed-batch culture were 175 and 392 U/mL, and their maximal specific activities 7.7 and 12.4 U/mg dry cells, respectively. These values are two to three times higher in activity and three to four times higher in specific activity than those obtained when glucose only was controlled.

  1. An enzymatic method for the alpha-amylase assay which comprises a new procedure for eliminating glucose and maltose in biological fluids.

    PubMed

    Kondo, H; Shiraishi, T; Nagata, K; Tomita, K

    1988-03-15

    An alpha-amylase assay in biological fluids, characterized by a new procedure for eliminating glucose and/or maltose, was developed. The reagent includes thermostable glucokinase, glucosephosphate isomerase and phosphofructokinase obtained from a thermophile Bacillus stearothermophilus. Up to 4,000 mg/dl glucose or 600 mg/dl maltose had no effect on the measured value of alpha-amylase activity when measured at 37 degrees C, even at a serum volume fraction in the reagent of 1/50. Alpha-Amylase activity was monitored by the absorbance increase at 340 nm due to NADPH production. The assay has a high degree of precision, with the within-run and day-to-day coefficients of variation being 2.17% at 75.0 U/l and 2.49% at 112 U/l, respectively, and is linear up to about 2,500 U/l. Regarding interferences, bilirubin, urate, ascorbate, pyruvate, EDTA, sodium fluoride and others were found to have no effect on the assay, and the reagent in solution is stable for about 2 wk at low temperatures (6-8 degrees C). PMID:2967134

  2. Raw-starch-digesting and thermostable alpha-amylase from the yeast Cryptococcus sp. S-2: purification, characterization, cloning and sequencing.

    PubMed

    Iefuji, H; Chino, M; Kato, M; Iimura, Y

    1996-09-15

    A starch-degrading enzyme produced by the yeast Cryptococcus sp. S-2 was purified in only one step by using an alpha-cyclodextrin-Sepharose 6B column, and was characterized as an alpha-amylase (EC 3.2.1.1). The molecular mass and isoelectric point of purified alpha-amylase (AMY-CS2) were estimated to be 66 kDa and 4.2 respectively. AMY-CS2 has raw-starch-digesting and raw-starch-absorbing activities. Furthermore it was shown to be thermostable. An open reading frame of the cDNA specified 611 amino acids, including a putative signal peptide of 20 amino acids. The N-terminal region of AMY-CS2 (from the N-terminus to position 496) had 49.7% similarity with the whole region of alpha-amylase from Aspergillus oryzae (Taka-amylase), whereas the C-terminal region had a sequence that was similar to the C-terminal region of glucoamylase G1 from A. niger. In addition, putative raw-starch-binding motifs exist in some amylolytic enzymes. A mutant AMY-CS2 that lacks the C-terminal domain lost not only its ability to bind or digest raw starch, but also its thermostability. Consequently it is possible that the putative raw-starch-binding domain of AMY-CS2 plays a role not only in the molecule's raw-starch-digesting ability but also in its thermostability. PMID:8836148

  3. Modulation of activity and substrate binding modes by mutation of single and double subsites +1/+2 and -5/-6 of barley alpha-amylase 1.

    PubMed

    Mori, H; Bak-Jensen, K S; Gottschalk, T E; Motawia, M S; Damager, I; Møller, B L; Svensson, B

    2001-12-01

    Enzymatic properties of barley alpha-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites -5/-6 (Cys95-->Ala/Thr) and +1/+2 (Met298-->Ala/Asn/Ser) as well as in the double mutants, Cys95-->Ala/Met298-->Ala/Asn/Ser. Cys95-->Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, kcat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside (Cl-PNPG7), respectively, but fivefold to 20-fold higher Km. The Cys95-->Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95-->Ala and wild-type. Met298-->Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl-PNPG7 are < or = 30% and < or = 10% of wild-type, respectively. The activity of Cys95-->Ala/Met298-->Ala/Asn/Ser is 100-180% towards starch, and the kcat/Km is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG7, respectively, emphasizing the strong impact of the Cys95-->Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG7 and amylose/Cl-PNPG7 are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites -5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6"'-maltotriosyl-maltohexaose thus covering subsites -1 to +5, while productive binding of unbranched substrate involves subsites -3 to +3. PMID:11737209

  4. Fermentation of starch by Klebsiella oxytoca P2, containing plasmids with {alpha}-amylase and pullulanase genes

    SciTech Connect

    Santos, V.L. dos; Araujo, E.F.; Barros, E.G. de; Guimaraes, W.V.

    1999-12-20

    Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12--24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower {alpha}-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.

  5. Direct production of cadaverine from soluble starch using Corynebacterium glutamicum coexpressing alpha-amylase and lysine decarboxylase.

    PubMed

    Tateno, Toshihiro; Okada, Yusuke; Tsuchidate, Takeyuki; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-02-01

    Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 alpha-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli-C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5'-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert L-lysine to cadaverine, as there was no accumulation of L-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources. PMID:18989633

  6. A fluid response: Alpha-amylase reactions to acute laboratory stress are related to sample timing and saliva flow rate.

    PubMed

    Nagy, Tamás; van Lien, René; Willemsen, Gonneke; Proctor, Gordon; Efting, Marieke; Fülöp, Márta; Bárdos, György; Veerman, Enno C I; Bosch, Jos A

    2015-07-01

    Salivary alpha-amylase (sAA) is used as a sympathetic (SNS) stress marker, though its release is likely co-determined by SNS and parasympathetic (PNS) activation. The SNS and PNS show asynchronous changes during acute stressors, and sAA responses may thus vary with sample timing. Thirty-four participants underwent an eight-minute memory task (MT) and cold pressor task (CPT). Cardiovascular SNS (pre-ejection period, blood pressure) and PNS (heart rate variability) activity were monitored continuously. Unstimulated saliva was collected repeatedly during and after each laboratory stressor, and sAA concentration (U/ml) and secretion (U/minute) determined. Both stressors increased anxiety. The MT caused an immediate and continued cardiac SNS activation, but sAA concentration increased at task cessation only (+54%); i.e., when there was SNS-PNS co-activation. During the MT sAA secretion even decreased (-35%) in conjunction with flow rate and vagal tone. The CPT robustly increased blood pressure but not sAA. In summary, sAA fluctuations did not parallel changes in cardiac SNS activity or anxiety. sAA responses seem contingent on sample timing and flow rate, likely involving both SNS and PNS influences. Verification using other stressors and contexts seems warranted. PMID:25976524

  7. Condition stabilization for Aspergillus niger FCBP-198 and its hyperactive mutants to yield high titres of alpha-amylase.

    PubMed

    Shafique, Sobiya; Bajwa, Rukhsana; Shafique, Shazia

    2010-01-01

    A number of substrates were tested for the cultivation of microorganisms to produce a host of enzymes. The effect of different substrates (wheat and rice straw, sugar cane waste, wood waste), incubation temperatures (20-40 degrees C), initial pH levels (3.5-9.0), incubation periods (0-72 hours) and nitrogen sources (ammonium sulfate, urea, peptone, yeast extract, sodium nitrate) on growth and alpha-amylase activity was studied for the native and mutant strains. Maximum enzyme activity was observed at 1.5% wheat straw for Aspergillus niger FCBP-198 and An-Ch-4.7 and at 2% wheat straw for An-UV-5.6, with sodium nitrate as a principle nitrogen source. The optimum temperature for maximum enzyme activity was 30 degrees C for the parental strain, while An-UV-5.6 and An-Ch-4.7 thrived well at 32.5 degrees C. The best conditions of pH and incubation duration were 4.5 and 48 hours, respectively, for all the strains. Mass production under preoptimized growth conditions demonstrated the suitability of wheat straw for swift mycelial colonization and viability. PMID:20734811

  8. Combined impact of Bacillus stearothermophilus maltogenic alpha-amylase and surfactants on starch pasting and gelation properties.

    PubMed

    Van Steertegem, Bénédicte; Pareyt, Bram; Brijs, Kristof; Delcour, Jan A

    2013-08-15

    In baking applications involving starch gelatinisation, surfactants such as sodium stearoyl lactylate (SSL) and monoacylglycerols (MAG) and Bacillus stearothermophilus maltogenic alpha-amylase (BStA) can be used jointly. We here showed that SSL but not MAG delays wheat starch hydrolysis by BStA. The effects were explained in terms of different degrees of adsorption of the surfactants on the starch granule surface, retarded and/or decreased water uptake and delayed availability of gelatinised starch for hydrolysis by BStA. Additional experiments with waxy maize starch led to the conclusion that SSL impacts swelling power and carbohydrate leaching more by covering the starch granule surface than by forming amylose-lipid complexes. SSL postponed starch hydrolysis by BStA, but this did not influence subsequent starch gelation. Finally, when adding SSL or MAG on top of BStA to starch suspensions, the effect of the surfactants on gel strength predominated over that of BStA. PMID:23561216

  9. Effects of Cardiorespiratory Fitness and Obesity on Salivary Secretory IgA and Alpha-Amylase in South African Children.

    PubMed

    Starzak, Dorota E; Konkol, Kristen F; McKune, Andrew J

    2016-01-01

    This study examined whether cardiorespiratory fitness (CRF) and body composition are associated with salivary secretory immunoglobulin A (SIgA), a mucosal immunity marker, and salivary alpha-amylase (sAA), a marker of stress-related sympathetic nervous system (SNS) activity, in South African children. Morning (7:30-8:00 a.m.) saliva samples were collected from 132 children (10.05 ± 1.68 years old, 74 females, 58 males). Body composition, resting blood pressure, and predicted maximal aerobic capacity (VO2max) were determined, and SIgA and sAA were quantified. Obese children had significantly higher sAA compared with overweight and normal weight children (p < 0.01). SIgA secretion rate was significantly lower in obese and overweight vs. normal weight children (p < 0.01). Multiple-linear regression analysis revealed that body mass index (BMI) (p < 0.05) and diastolic blood pressure (DBP) (p < 0.05) were independent predictors of sAA with CRF acting as a mitigator. Age and BMI predicted SIgA secretion rate (p < 0.05) with BMI (p < 0.001) found to be an independent predictor of SIgA secretion rate. Obesity, based on BMI, was associated with elevated SNS activity and lowered mucosal immunity. CRF-mitigated sympathetic activation was not associated with mucosal immunity. PMID:27483329

  10. Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

    PubMed Central

    Galdino, Alexsandro Sobreira; Silva, Roberto Nascimento; Lottermann, Muriele Taborda; Álvares, Alice Cunha Morales; de Moraes, Lídia Maria Pepe; Torres, Fernando Araripe Gonçalves; de Freitas, Sonia Maria; Ulhoa, Cirano José

    2011-01-01

    An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH4+ and inhibited by Cu+2 and Hg+2. Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to Km values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1w, points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels. PMID:21490699

  11. Miconia sp. Increases mRNA Levels of PPAR Gamma and Inhibits Alpha Amylase and Alpha Glucosidase

    PubMed Central

    Ortíz-Martinez, David Mizael; Rivas-Morales, Catalina; de la Garza-Ramos, Myriam Angelica; Verde-Star, Maria Julia; Nuñez-Gonzalez, Maria Adriana

    2016-01-01

    Diabetes mellitus is a public health problem worldwide. For this reason, ethanolic extract of Miconia sp. from Oaxaca, Mexico, was selected in search of an alternative against this disease. The effect of Miconia sp. on mRNA expression of PPARγ on cell line 3T3-L1, its effect on alpha amylase and alpha glucosidase, lipid accumulation during adipogenesis, and cell viability on VERO cells were evaluated. The mRNA levels of PPARγ increased on 1.393 ± 0.008 folds, lipid accumulation was increased by 29.55% with Miconia sp. extract and 34.57% with rosiglitazone, and α-amylase and α-glycosidase were inhibited with IC50 values from 28.23 ± 2.15 μg/mL and 1.95 ± 0.15 μg/mL, respectively; the IC50 on antiproliferative activity on VERO cells was 314.54 ± 45.40 μg/mL. In case of α-amylase and α-glycosidase assays, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of enzymatic activity. On the other hand, on antiproliferative activity, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of cell proliferation. It was concluded that the compounds present in Miconia sp. ethanolic extract increase mRNA expression of PPARγ, inhibit α-amylase and α-glucosidase, and increase lipid accumulation. It constitutes an alternative as adjuvant in diabetes mellitus treatment; therefore, we recommend continuing identifying the compounds responsible for its promising in vivo antidiabetic activity. PMID:27478477

  12. Immediate Effects of Traditional Thai Massage on Psychological Stress as Indicated by Salivary Alpha-Amylase Levels in Healthy Persons.

    PubMed

    Sripongngam, Thanarat; Eungpinichpong, Wichai; Sirivongs, Dhavee; Kanpittaya, Jaturat; Tangvoraphonkchai, Kamonwan; Chanaboon, Sutin

    2015-01-01

    BACKGROUND Stress can cause psychological and physiological changes. Many studies revealed that massage can decrease stress. However, traditional Thai massage has not been well researched in this regard. The purpose of this study was to investigate the immediate effects of traditional Thai massage (TTM) on salivary alpha-amylase levels (sAA), heart rate variability (HRV), autonomic nervous system (ANS) function, and plasma renin activity (PRA). MATERIAL AND METHODS Twenty-nine healthy participants were randomly allocated into either a traditional Thai massage (TTM) group or Control (C) group, after which they were switched to the other group with a 2-week wash-out period. Each of them was given a 10-minute mental arithmetic test to induce psychological stress before a 1-hour session of TTM or rest. RESULTS Within-groups comparison revealed that sAA was significantly decreased (p<0.05) in the TTM group but not in the C group. HRV and ANS function were significantly increased (p<0.05) and PRA was significantly decreased (p<0.05) in both groups. However, low frequency per high frequency ratio (LF/HF ratio) and ANS balance status were not changed. Only sAA was found to be significantly different between groups (p<0.05). CONCLUSIONS We conclude that both TTM and rest can reduce psychological stress, as indicated by decreased sAA levels, increased parasympathetic activity, decreased sympathetic activity, and decreased PRA. However, TTM may have a modest effect on stress reduction as indicated by a reduced sAA. PMID:26436433

  13. Miconia sp. Increases mRNA Levels of PPAR Gamma and Inhibits Alpha Amylase and Alpha Glucosidase.

    PubMed

    Ortíz-Martinez, David Mizael; Rivas-Morales, Catalina; de la Garza-Ramos, Myriam Angelica; Verde-Star, Maria Julia; Nuñez-Gonzalez, Maria Adriana; Leos-Rivas, Catalina

    2016-01-01

    Diabetes mellitus is a public health problem worldwide. For this reason, ethanolic extract of Miconia sp. from Oaxaca, Mexico, was selected in search of an alternative against this disease. The effect of Miconia sp. on mRNA expression of PPARγ on cell line 3T3-L1, its effect on alpha amylase and alpha glucosidase, lipid accumulation during adipogenesis, and cell viability on VERO cells were evaluated. The mRNA levels of PPARγ increased on 1.393 ± 0.008 folds, lipid accumulation was increased by 29.55% with Miconia sp. extract and 34.57% with rosiglitazone, and α-amylase and α-glycosidase were inhibited with IC50 values from 28.23 ± 2.15 μg/mL and 1.95 ± 0.15 μg/mL, respectively; the IC50 on antiproliferative activity on VERO cells was 314.54 ± 45.40 μg/mL. In case of α-amylase and α-glycosidase assays, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of enzymatic activity. On the other hand, on antiproliferative activity, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of cell proliferation. It was concluded that the compounds present in Miconia sp. ethanolic extract increase mRNA expression of PPARγ, inhibit α-amylase and α-glucosidase, and increase lipid accumulation. It constitutes an alternative as adjuvant in diabetes mellitus treatment; therefore, we recommend continuing identifying the compounds responsible for its promising in vivo antidiabetic activity. PMID:27478477

  14. Immediate Effects of Traditional Thai Massage on Psychological Stress as Indicated by Salivary Alpha-Amylase Levels in Healthy Persons

    PubMed Central

    Sripongngam, Thanarat; Eungpinichpong, Wichai; Sirivongs, Dhavee; Kanpittaya, Jaturat; Tangvoraphonkchai, Kamonwan; Chanaboon, Sutin

    2015-01-01

    Background Stress can cause psychological and physiological changes. Many studies revealed that massage can decrease stress. However, traditional Thai massage has not been well researched in this regard. The purpose of this study was to investigate the immediate effects of traditional Thai massage (TTM) on salivary alpha-amylase levels (sAA), heart rate variability (HRV), autonomic nervous system (ANS) function, and plasma renin activity (PRA). Material/Methods Twenty-nine healthy participants were randomly allocated into either a traditional Thai massage (TTM) group or Control (C) group, after which they were switched to the other group with a 2-week wash-out period. Each of them was given a 10-minute mental arithmetic test to induce psychological stress before a 1-hour session of TTM or rest. Results Within-groups comparison revealed that sAA was significantly decreased (p<0.05) in the TTM group but not in the C group. HRV and ANS function were significantly increased (p<0.05) and PRA was significantly decreased (p<0.05) in both groups. However, low frequency per high frequency ratio (LF/HF ratio) and ANS balance status were not changed. Only sAA was found to be significantly different between groups (p<0.05). Conclusions We conclude that both TTM and rest can reduce psychological stress, as indicated by decreased sAA levels, increased parasympathetic activity, decreased sympathetic activity, and decreased PRA. However, TTM may have a modest effect on stress reduction as indicated by a reduced sAA. PMID:26436433

  15. Evidence that the glucoamylases and alpha-amylase secreted by Aspergillus niger are proteolytically processed products of a precursor enzyme.

    PubMed

    Dubey, A K; Suresh, C; Kavitha, R; Karanth, N G; Umesh-Kumar, S

    2000-04-14

    A 125-kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673-675] was also found to possess activity towards raw starch. Segregation of these activities in the 71-kDa glucoamylase and a 53-kDa alpha-amylase-like enzyme supported by antibody cross-reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125-kDa starch hydrolysing enzyme as their precursor. N-terminal sequence analysis further revealed that the 71-kDa glucoamylase was the N-terminal product of the precursor enzyme. Immunological cross reactivity of the 53-kDa amylase with antibodies raised against the precursor enzyme but not with the 71- and 61-kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C-terminal fragment of the precursor. The N-terminal sequence of the 53-kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross-reactivity to a 10-kDa non-enzymic peptide and a 61-kDa glucoamylase described these proteins as products of the 71-kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger. PMID:10767433

  16. The burden of inhibitors in haemophilia patients.

    PubMed

    Walsh, Christopher E; Jiménez-Yuste, Víctor; Auerswald, Guenter; Grancha, Salvador

    2016-08-31

    The burden of disease in haemophilia patients has wide ranging implications for the family and to society. There is evidence that having a current inhibitor increases the risk of morbidity and mortality. Morbidity is increased by the inability to treat adequately and its consequent disabilities, which then equates to a poor quality of life compared with non-inhibitor patients. The societal cost of care, or `burden of inhibitors', increases with the ongoing presence of an inhibitor. Therefore, it is clear that successful eradication of inhibitors by immune tolerance induction (ITI) is the single most important milestone one can achieve in an inhibitor patient. The type of factor VIII (FVIII) product used in ITI regimens varies worldwide. Despite ongoing debate, there is in vitro and retrospective clinical evidence to support the use of plasma-derived VWF-containing FVIII concentrates in ITI regimens in order to achieve early and high inhibitor eradication success rates. PMID:27528280

  17. The role of two isoenzymes of alpha-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination.

    PubMed

    Waghorn, Juana J; del Pozo, Talía; Acevedo, Elba A; Cardemil, Liliana A

    2003-03-01

    Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion. PMID:12598561

  18. Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601.

    PubMed Central

    Lee, S P; Morikawa, M; Takagi, M; Imanaka, T

    1994-01-01

    A thermophilic and alkaliphilic Bacillus sp. strain, XAL601, was isolated from soil. It produces a thermostable and alkaline-stable enzyme with both alpha-amylase and pullulanase activities. The alpha-amylase-pullulanase gene (aapT) from this Bacillus strain was cloned, and its nucleotide sequence was determined (GenBank accession number D28467). A very large open reading frame composed of 6,096 bases, which encodes 2,032 amino acid residues with an M(r) of 224,992, was found. The deduced amino acid sequence revealed that the four highly conserved regions that are common among amylolytic enzymes were well conserved. These include an active center and common substrate-binding sites of various amylases. In the C-terminal region, a six-amino-acid sequence (Gly-Ser-Gly-Thr-Thr-Pro) is repeated 12 times. The aapT gene was then subcloned in Escherichia coli and overexpressed under the control of the lac promoter. Purification of AapT from this recombinant E. coli was performed, and it was shown that the aapT gene product exhibits both alpha-amylase and pullulanase activities with one active site. The optimum temperature and pH for enzyme activity were found to be 70 degrees C and pH 9, respectively. Furthermore, AapT was found to strongly adsorb to crystalline cellulose (Avicel) and raw corn starch. Final hydrolyzed products from soluble starch range from maltose (G2) to maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. The enzyme also hydrolyzes raw starch under a broad range of conditions (60 to 70 degrees C and pH 8 to 9). Images PMID:7986049

  19. New insights into the effectiveness of alpha-amylase enzyme presentation on the Bacillus subtilis spore surface by adsorption and covalent immobilization.

    PubMed

    Gashtasbi, Fatemeh; Ahmadian, Gholamreza; Noghabi, Kambiz Akbari

    2014-10-01

    Most of the studies in the field of enzyme immobilization have sought to develop a simple, efficient and cost-effective immobilization system. In this study, probiotic Bacillus spores were used as a matrix for enzyme immobilization, because of their inherent resistance to extreme temperatures, UV irradiation, solvents and drying. Above all, their preparation is cost-effective. The alpha-amylase enzyme was immobilized on the spore surface by the covalent and adsorption methods. For the covalent method, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N hydroxysulfosuccinimide (NHS) were used. The maximum concentration of the alpha-amylase immobilized by the two methods onto the spore surface was 360 μg/1.2×10(11) spore. However, maximum activity was achieved at an enzyme concentration of approximately 60 μg/.4×10(10), corresponding to an estimated activity of 8×10(3) IU mg(-1)/1.2×10(11) spore for covalent immobilization and 8.53×10(3) for the adsorption method. After washing the enzyme with 1M NaCl and 0.5% Triton X-100, the enzyme immobilization yield was estimated to be 77% and 20.07% for the covalent and adsorption methods, respectively. The alpha-amylase immobilized by both methods, displayed improved activity in the basic pH range. The optimum pH for the free enzyme was 5 while it shifted to 8 for the immobilized enzyme. The optimum temperatures for the free and immobilized enzymes were 60 °C and 80 °C, respectively. The covalently-immobilized alpha-amylase retained 65% of its initial activity, even after 10 times of recycling. The Km and Vmax values were determined by the GraphPad Prism software, which showed that the Vmax value decreased moderately after immobilization. This is the first study which reports the covalent immobilization of an enzyme on the spore surface. PMID:25152412

  20. Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase.

    PubMed

    Shigechi, Hisayori; Koh, Jun; Fujita, Yasuya; Matsumoto, Takeshi; Bito, Yohei; Ueda, Mitsuyoshi; Satoh, Eiichi; Fukuda, Hideki; Kondo, Akihiko

    2004-08-01

    Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch. PMID:15294847

  1. Examining multiple sleep behaviors and diurnal salivary cortisol and alpha-amylase: Within- and between-person associations.

    PubMed

    Van Lenten, Scott A; Doane, Leah D

    2016-06-01

    Sleep has been linked to the daily patterns of stress-responsive physiological systems, specifically the hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system (ANS). However, extant research examining sleep and diurnal patterns of cortisol, the primary end product of the HPA axis, has primarily focused on sleep duration with limited attention on other facets of sleep. For example, it is not clear how specific aspects of sleep (e.g., sleep quality, sleep duration variability) are related to specific components of diurnal cortisol rhythms. Salivary alpha-amylase (sAA) has been recognized as a surrogate marker of ANS activity, but limited research has explored relations between sleep and sAA diurnal rhythms. The current study utilized an ecological momentary assessment protocol to examine within- and between-person relations between several facets of sleep behavior using multiple methods (e.g., subjective report, actigraphy) and salivary cortisol and sAA. Older adolescents (N=76) provided saliva samples and diary entries five times per day over the course of three days. Sleep was assessed via questionnaire, through daily diaries, and monitored objectively using actigraphy over a four day period. Between-person results revealed that shorter average objective sleep duration and greater sleep duration variability were related to lower levels of waking cortisol and flatter diurnal slopes across the day. Within-person results revealed that on nights when individuals slept for shorter durations than usual they also had lower levels of waking cortisol the next day. Sleep was not related to the cortisol awakening response (CAR) or diurnal patterns of sAA, in either between-person or within-person analyses. However, typical sleep behaviors measured via questionnaire were related to waking levels of sAA. Overall, this study provides a greater understanding of how multiple components of sleep, measured in naturalistic environments, are related to cortisol and s

  2. Some distinguishable properties between acid-stable and neutral types of alpha-amylases from acid-producing koji.

    PubMed

    Suganuma, Toshihiko; Fujita, Kiyotaka; Kitahara, Kanefumi

    2007-11-01

    The highly humid climate of Japan facilitates the growth of various molds. Among these molds, Aspergillus oryzae is the most important and popular in Japan, and has been used as yellow-koji in producing many traditional fermented beverages and foods, such as Japanese sake, and soy sauce. Taka-amylase A (TAA), a major enzyme produced by the mold, is well known worldwide to be a leading enzyme for industrial utilization and academic study, since many extensive studies have been carried out with TAA. In southern Kyushu, the other koji's of citric acid-producing molds have often been used, such as in the production of a traditional distilled liquor of shochu. The koji molds black-koji and white-koji produce two types of alpha-amylase, namely, acid-stable (AA) and common neutral (NA). The latter enzyme is enzymatically and genetically similar to TAA. In this review, we investigate AA from three molds, Aspergillus niger, A. kawachii and A. awamori, and the yeast Cryptococcus sp. regarding the distinguishable properties between AA and NA. (i) The N-terminus amino acid sequences of AA determined by molecular cloning started with the sequence of L-S-A-, whereas those of NA started with A-T-P-. (ii) Most of the full sequences of AA were composed of, besides a core catalytic domain, an extra domain of a hinge region and a carbohydrate binding domain, which could be responsible for raw-starch-digestibility. The AA from A. niger has no exceptionally extra domain, similarly to NA. (iii) Simple methods for distinguishing AA from NA using CNP-alpha-G3 and G5 as substrates were developed by our group. (iv) The number of subsite in AA on the basis of its cleavage pattern of maltooligosaccharides was estimated to be five, which differs from that of TAA, 7-9. AA has many advantages in industrial applications, such as its acid-stability, thermostability, and raw-starch digesting properties. PMID:18086434

  3. Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley alpha-amylase.

    PubMed

    Kramhøft, Birte; Bak-Jensen, Kristian Sass; Mori, Haruhide; Juge, Nathalie; Nøhr, Jane; Svensson, Birte

    2005-02-15

    Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite -5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites. PMID:15697208

  4. The importance of an extra loop in the B-domain of an alpha-amylase from B. stearothermophilus US100.

    PubMed

    Khemakhem, Bassem; Ben Ali, Mamdouh; Aghajari, Nushin; Juy, Michel; Haser, Richard; Bejar, Samir

    2009-07-17

    To provide insight into the potential role of a loop in domain B of several bacterial alpha-amylases, molecular and structural investigation of Bacillus stearothermophilus alpha-amylase (Amy US100) was used as a model. Combination deletion mutants of G(213), I(214) and G(215), described as a loop-forming on the surface bacterial amylases, were subjected to biochemical and structural investigation. Thermoactivity, thermostability as well calcium requirement were studied for each mutant. Thus, deletion of one residue differently affects only the thermostability. Shortening the loop by deletion of G(213)-I(214) or I(214)-G(215) improved the thermostability and reduces calcium requirement. However, the deletion of three residues has a negative effect on thermostability and reduces the optimal temperature by 17 degrees C. The structural investigation showed that stabilizing deletions contribute to reinforce the architecture of domain B and the active site conformation. The deletion of three residues reduces the flexibility of this region and abolishes a denser hydrogen bond network. PMID:19422796

  5. Safety evaluation of an alpha-amylase enzyme preparation derived from the archaeal order Thermococcales as expressed in Pseudomonas fluorescens biovar I.

    PubMed

    Landry, Timothy D; Chew, Lawrence; Davis, John W; Frawley, Nile; Foley, Holly H; Stelman, Steven J; Thomas, Johnson; Wolt, Jeffrey; Hanselman, David S

    2003-02-01

    BD5088 alpha-amylase derived from archaeal sources has characteristics of pH and temperature tolerance that are well suited to hydrolysis of starch in food processing applications. The production microorganism recipient strain, Pseudomonas fluorescens biovar I, strain MB101, was avirulent after oral administration to mice and does not represent an infectious threat to humans. Repeated dose gavage studies with BD5088 enzyme preparation, up to 13 weeks in duration, showed no systemic toxicity due to the oral route with an NOAEL of 890 mg/kg/day as Total Organic Solids. Some irritation occurred in the respiratory tract, which was considered to be a consequence of reflux and aspiration of test material that contained lipopolysaccharide from the Pseudomonas production strain. A 2-week dietary study (0 and 310 mg/kg/day) confirmed that there were no respiratory tract effects related to oral ingestion. There was no genotoxic activity based on Ames, mouse lymphoma, mouse micronucleus, and rat lymphocyte chromosomal aberration tests. There was no evidence of allergenic potential based on a comparison of the primary sequence of BD5088 with sequences in an allergen database. The enzyme was labile to pepsin digestion. Based on these data, BD5088 alpha-amylase preparation may be considered safe for use in food production such as corn wet milling. PMID:12662916

  6. Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence.

    PubMed

    Tateno, Toshihiro; Fukuda, Hideki; Kondo, Akihiko

    2007-12-01

    Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes alpha-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40 degrees C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37 degrees C, respectively. The alpha-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence. PMID:17891388

  7. Improving production of hyperthermostable and high maltose-forming alpha-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications.

    PubMed

    Uma Maheswar Rao, J L; Satyanarayana, T

    2007-01-01

    By cultivating Geobacillus thermoleovorans in shake flasks containing cane molasses medium at 70 degrees C, the fermentation variables were optimized by 'one variable at a time' approach followed by response surface methodology (RSM). The statistical model was obtained by central composite design (CCD) using three variables (cane-molasses, urea and inoculum density). An overall 1.6- and 2.1-fold increase in enzyme production was achieved in the optimized medium in shake flasks and fermenter, respectively. The alpha-amylase titre increased significantly in cane-molasses medium (60 U ml(-1)) as compared to that in the synthetic medium (26 U ml(-1)). Thus the cost of enzyme produced in cane molasses medium (0.823 euros per million U) was much lower than that produced in the synthetic starch-yeast extract-tryptone medium (18.52 euros per million U). The shelf life of bread was improved by supplementing dough with alpha-amylase, and thus, the enzyme was found to be useful in preventing the staling of bread. Reducing sugars liberated from 20% and 30% raw pearl millet starch were fermented to ethanol; ethanol production levels attained were 35.40 and 28.0 g l(-1), respectively. PMID:16473003

  8. Effect of low oxygen concentrations on growth and alpha-amylase production of Aspergillus oryzae in model solid-state fermentation systems.

    PubMed

    Rahardjo, Yovita S P; Sie, Susana; Weber, Frans J; Tramper, Johannes; Rinzema, Arjen

    2005-02-01

    Oxygen transfer in the fungal mat is a major concern in solid-state fermentation (SSF). Oxygen supply into the mycelial layers is hampered by diffusion limitation. For aerobic fungi, like Aspergillus oryzae, this oxygen depletion can be a severely limiting factor for growth and metabolite production. This paper describes the effects of a low oxygen concentration on growth at the levels of individual hyphae, colonies and overcultures, and on alpha-amylase production in overcultures. PDA medium was used to study the effect of a low oxygen concentration on hyphal elongation rate and branching frequency of hyphae, and radial extension rate of colonies of A. oryzae. We found similar saturation constants (K(O2)) of 0.1% (v/v in the gas phase) for oxygen concentration described with Monod kinetics, for branching frequency of hyphae and colony extension rate. When A. oryzae was grown as an over-culture on wheat-flour model substrate at 0.25% (v/v) oxygen concentration, the reduction in growth was more pronounced than as individual hyphae and a colony on PDA medium. Experimental results also showed that the specific alpha-amylase production rate under the condition of 0.25% (v/v) oxygen was reduced. Because the value of K(O2) is relatively low, it is reasonable to simplify the kinetics of growth of A. oryzae to zero-order kinetics in coupled diffusion/reaction models. PMID:15748690

  9. Asymmetry in children’s salivary cortisol and alpha-amylase in the context of marital conflict: Links to children’s emotional security and adjustment

    PubMed Central

    Koss, Kalsea J.; George, Melissa R.W.; Cummings, E. Mark; Davies, Patrick T.; El-Sheikh, Mona; Cicchetti, Dante

    2013-01-01

    Recent research supports the promise of examining interactive models of physiological processes on children’s adjustment. The present study investigates interactions between children’s autonomic nervous system activity and adrenocortical functioning in the context of marital discord; specifically, testing models of concurrent responses proposed by Bauer, Quas, & Boyce (2002) in the prediction of children’s behavioral responses to conflict and adjustment. Asymmetry and symmetry in children’s salivary alpha-amylase and cortisol were examined in 195 children (M age = 8 years) in response to viewing conflict vignettes. Results were partially consistent with an interactive model in the context of high marital discord; asymmetry among higher alpha-amylase and lower cortisol related to higher emotional insecurity and concurrent and subsequent maladjustment. In contrast, patterns of symmetrical responses were related to greater maladjustment for children exposed to lower levels of marital discord, supporting an additive model. Findings support the importance of a multisystem approach to investigating the adaptiveness of children’s physiological stress responses, while also highlighting the value of considering physiological responses in the context of family risk. PMID:24037991

  10. Calcium binding in alpha-amylases: an X-ray diffraction study at 2.1-A resolution of two enzymes from Aspergillus.

    PubMed

    Boel, E; Brady, L; Brzozowski, A M; Derewenda, Z; Dodson, G G; Jensen, V J; Petersen, S B; Swift, H; Thim, L; Woldike, H F

    1990-07-01

    X-ray diffraction analysis (at 2.1-A resolution) of an acid alpha-amylase from Aspergillus niger allowed a detailed description of the stereochemistry of the calcium-binding sites. The primary site (which is essential in maintaining proper folding around the active site) contains a tightly bound Ca2+ with an unusually high number of eight ligands (O delta 1 and O delta 2 of Asp175, O delta of Asn121, main-chain carbonyl oxygens of Glu162 and Glu210, and three water molecules). A secondary binding site was identified at the bottom of the substrate binding cleft; it involves the residues presumed to play a catalytic role (Asp206 and Glu230). This explains the inhibitory effect of calcium observed at higher concentrations. Neutral Aspergillus oryzae (TAKA) alpha-amylase was also refined in a new crystal at 2.1-A resolution. The structure of this homologous (over 80%) enzyme and additional kinetic studies support all the structural conclusions regarding both calcium-binding sites. PMID:2207069

  11. Enhancement of the alcoholytic activity of alpha-amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by site-directed mutagenesis.

    PubMed

    Damián-Almazo, Juanita Yazmin; Moreno, Alina; López-Munguía, Agustin; Soberón, Xavier; González-Muñoz, Fernando; Saab-Rincón, Gloria

    2008-08-01

    AmyA, an alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal alpha-1,4-glycosidic bonds in various alpha-glucans at 85 degrees C as the optimal temperature. Like other glycoside hydrolases, AmyA also catalyzes transglycosylation reactions, particularly when oligosaccharides are used as substrates. It was found that when methanol or butanol was used as the nucleophile instead of water, AmyA was able to catalyze alcoholysis reactions. This capability has been evaluated in the past for some alpha-amylases, with the finding that only the saccharifying fungal amylases from Aspergillus niger and from Aspergillus oryzae present measurable alcoholysis activity (R. I. Santamaria, G. Del Rio, G. Saab, M. E. Rodriguez, X. Soberon, and A. Lopez, FEBS Lett. 452:346-350, 1999). In the present work, we found that AmyA generates larger quantities of alkyl glycosides than any amylase reported so far. In order to increase the alcoholytic activity observed in AmyA, several residues were identified and mutated based on previous analogous positions in amylases, defining the polarity and geometry of the active site. Replacement of residue His222 by glutamine generated an increase in the alkyl glucoside yield as a consequence of a higher alcoholysis/hydrolysis ratio. The same change in specificity was observed for the mutants H222E and H222D, but instability of these mutants toward alcohols decreased the yield of alkyl glucoside. PMID:18552192

  12. Alpha-Amylase

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Both (Porcine and bacterial) starch degrading enzymes highly valued by the biotechnology industry. (Porcine) A major target for protein engineering and the study of diabetes, obesity and dental care. (Bacterial) Major industrial and biotechnology interest used in brewing, baking, and food processing. World's number one industrial protein.

  13. Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.

    PubMed

    Shiosaki, K; Takata, K; Omichi, K; Tomita, N; Horii, A; Ogawa, M; Matsubara, K

    1990-05-14

    A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes. PMID:2197187

  14. Evolutionary relationships and sequence variation of alpha-amylase variants encoded by duplicated genes in the Amy locus of Drosophila melanogaster.

    PubMed

    Inomata, N; Shibata, H; Okuyama, E; Yamazaki, T

    1995-09-01

    To infer the genealogical relationships of alpha-amylase electromorphs of Drosophila melanogaster, we determined the nucleotide sequences of a collection of electromorphs sampled throughout the world. On average there were 1.0 amino acid substitutions between identical electromorphs and 3.9 between different electromorphs, respectively. We found that the evolution of AMY1 through AMY6 electromorphs occurred by sequential accumulation of single amino acid substitutions each causing one charge difference. The nucleotide diversities at synonymous sites within Amy1,Amy2,Amy3,Amy4 and Amy6 were 0.0321, 0.0000, 0.0355, 0.0059 and 0.0030, respectively. We also obtained evidence of genetic exchanges, such as intrachromosomal recombination, interchromosomal recombination or gene conversion, between the two duplicated Amy genes as well as among the alleles. PMID:8536971

  15. SusG: A Unique Cell-Membrane-Associated [alpha]-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules

    SciTech Connect

    Koropatkin, Nicole M.; Smith, Thomas J.

    2010-09-21

    SusG is an {alpha}-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysis demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.

  16. Use of homologous expression-secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha-amylase and levanase expression.

    PubMed Central

    Hols, P; Ferain, T; Garmyn, D; Bernard, N; Delcour, J

    1994-01-01

    The genuine alpha-amylase gene from Bacillus licheniformis (amyL) is not expressed in Lactobacillus plantarum, but replacement of the amyL promoter by a strong L. plantarum promoter leads to efficient expression of the gene and secretion of more than 90% of the alpha-amylase into the culture supernatant. A series of L. plantarum genetic cassettes (transcription and translation with or without secretion) were cloned by translation fusion of random DNA fragments to the silent amyL coding frame in the pGIP212 probe vector (P. Hols, A. Baulard, D. Garmyn, B. Delplace, S. Hogan, and J. Delcour, Gene 118:21-30, 1992). Five different cassettes were sequenced and found to harbor genetic signals similar to those of other gram-positive bacteria. The functions of the cloned cassettes and the cassettes isolated previously from Enterococcus faecalis were compared in E. faecalis and L. plantarum, respectively. All signals were well recognized in L. plantarum, but cassettes isolated from L. plantarum led to a low level of amylase production in E. faecalis, suggesting that the L. plantarum signals are more species specific. Six transcriptional or translational fusions were constructed to express the Bacillus subtilis levanase gene (sacC) in L. plantarum. All of these constructions were capable of inducing levanase production and secretion in the culture supernatant, and, furthermore, L. plantarum strains harboring the most efficient fusions could grow in MRS medium containing inulin as the major carbon source. Finally, a two-step chromosomal integration procedure was used to achieve efficient stabilization of an amylase construction without any residual resistance marker or vector sequence. Images PMID:8017927

  17. Purification, properties and structural aspects of a thermoacidophilic alpha-amylase from Alicyclobacillus acidocaldarius atcc 27009. Insight into acidostability of proteins.

    PubMed

    Schwermann, B; Pfau, K; Liliensiek, B; Schleyer, M; Fischer, T; Bakker, E P

    1994-12-15

    The alpha-amylase from the thermoacidophilic eubacterium Alicyclobacillus (Bacillus) acidocaldarius strain ATCC 27009 was studied as an example of an acidophilic protein. The enzyme was purified from the culture fluid. On an SDS/polyacrylamide gel, the protein an apparent molecular mass of 160 kDa, which is approximately 15% higher than that predicted from the nucleotide sequence. The difference is due to the enzyme being a glycoprotein. Deglycosylation or synthesis of the enzyme in Escherichia coli gave a product with the mass expected for the mature protein. The amylase hydrolyzed starch at random and from the inside, and its main hydrolysis products were maltotriose and maltose. It also formed glucose from starch (by hydrolysing the intermediate product maltotetraose to glucose and maltotriose) and exhibited some pullulanase activity. the pH and temperature optima were pH3 and 75 degrees C, respectively, characterizing the enzyme as being thermoacidophilic. Alignment of the sequence of the enzyme with that of its closest neutrophilic relatives and with that of alpha-1,4 or alpha-1,6 glycosidic-bond hydrolyzing enzymes of known three-dimensional structure showed that the acidophilic alpha-amylase contains approximately 30% less charged residues than do its closest relatives, that these residues are replaced by neutral polar residues, and that hot spots for these exchanges are likely to be located at the surface of the protein. Literature data show that similar effects are observed in three other acidophilic proteins. It is proposed that these proteins have adapted to the acidic environment by reducing the density of both positive and negative charges at their surface, that this effect circumvents electrostatic repulsion of charged groups at low pH, and thereby contributes to the acidostability of these proteins. PMID:7813489

  18. Salivary cortisol and alpha-amylase levels during an assessment procedure correlate differently with risk-taking measures in male and female police recruits

    PubMed Central

    van den Bos, Ruud; Taris, Ruben; Scheppink, Bianca; de Haan, Lydia; Verster, Joris C.

    2013-01-01

    Recent laboratory studies have shown that men display more risk-taking behavior in decision-making tasks following stress, whilst women are more risk-aversive or become more task-focused. In addition, these studies have shown that sex differences are related to levels of the stress hormone cortisol (indicative of activation of the hypothalamus-pituitary-adrenocortical-axis): the higher the levels of cortisol the more risk-taking behavior is shown by men, whereas women generally display more risk-aversive or task-focused behavior following higher levels of cortisol. Here, we assessed whether such relationships hold outside the laboratory, correlating levels of cortisol obtained during a job-related assessment procedure with decision-making parameters in the Cambridge Gambling Task (CGT) in male and female police recruits. The CGT allows for discriminating different aspects of reward-based decision-making. In addition, we correlated levels of alpha-amylase [indicative of activation of the sympatho-adrenomedullary-axis (SAM)] and decision-making parameters. In line with earlier studies men and women only differed in risk-adjustment in the CGT. Salivary cortisol levels correlated positively and strongly with risk-taking measures in men, which was significantly different from the weak negative correlation in women. In contrast, and less strongly so, salivary alpha-amylase levels correlated positively with risk-taking in women, which was significantly different from the weak negative correlation with risk-taking in men. Collectively, these data support and extend data of earlier studies indicating that risky decision-making in men and women is differently affected by stress hormones. The data are briefly discussed in relation to the effects of stress on gambling. PMID:24474909

  19. A new rice zinc-finger protein binds to the O2S box of the alpha-amylase gene promoter.

    PubMed

    Peng, Rihe; Yao, Quanhong; Xiong, Aisheng; Fan, Huiqin; Li, Xian; Peng, Youliang; Cheng, Zong-Ming; Li, Yi

    2004-07-01

    A putative transcription factor, named RAMY, that binds to the 20-bp O2S sequences of the regulatory region of the Amy2 gene promoter has been identified using the yeast one-hybrid system from a rice library. The full length RAMY cDNA clone encodes a 218-amino acid protein and is homologous to the late embryogenesis-abundant protein (LEA5). In vitro mutagenesis and electrophoretic mobility shift assays confirmed that RAMY can bind with O2S specifically through an unusual zinc finger with a CXCX(4)CX(2)H consensus sequence. Low levels of RAMY mRNAs were detected in rice leaves and roots by Northern blot hybridization. The plant hormone gibberellin (GA) induces expression of both RAMY and Amy2 genes, as performed by Northern blot hybridization, but the increase in RAMY mRNA level occurs prior to that of the Amy2 mRNA level in the GA-treated aleurone tissues. These data suggest that RAMY may act as a trans-acting protein and is probably involved in the GA-induced expression of the rice alpha-amylase gene. PMID:15233790

  20. Amylose chain behavior in an interacting context. III. Complete occupancy of the AMY2 barley alpha-amylase cleft and comparison with biochemical data.

    PubMed

    André, G; Buléon, A; Haser, R; Tran, V

    1999-12-01

    In the first two papers of this series, the tools necessary to evaluate substrate ring deformations were developed, and then the modeling of short amylose fragments (maltotriose and maltopentaose) inside the catalytic site of barley alpha-amylase was performed. In this third paper, this docking has been extended to the whole catalytic cleft. A systematic approach to extend the substrate was used on the reducing side from the previous enzyme/pentasaccharide complex. However, due to the lack of an obvious subsite at the nonreducing side, an alternate protocol has been chosen that incorporates biochemical information on the enzyme and features on the substrate shape as well. As a net result, ten subsites have been located consistent with the distribution of Ajandouz et al. (E. H. Ajandouz, J. Abe, B. Svensson, and G. Marchis-Mouren, Biochimica Biophysica Acta, 1992, Vol. 1159, pp. 193-202) and corresponding binding energies were estimated. Among them, two extreme subsites (-6) and (+4), with stacking residues Y104 and Y211, respectively, have strong affinities with glucose rings added to the substrate. No other deformation has been found for the new glucose rings added to the substrate; therefore, only ring A of the DP 10 fragment has a flexible form when interacting with the inner stacking residues Y51. Global conservation of the helical shape of the substrate can be postulated in spite of its significant distortion at subsite (-1). PMID:10547530

  1. Mind your thoughts: associations between self-generated thoughts and stress-induced and baseline levels of cortisol and alpha-amylase.

    PubMed

    Engert, Veronika; Smallwood, Jonathan; Singer, Tania

    2014-12-01

    Stress is a major health burden in today's society. Research shows that negative cognitive styles are associated with increased stress reactivity, low mood and accelerated cellular aging. Our study sought to unravel the relationship between the content of self-generated thoughts and psychosocial stress measured in terms of hypothalamic-pituitary-adrenal axis and sympathetic activity. Features of self-generated thoughts were assessed using thought sampling while participants performed cognitive tasks following a stress induction or in a baseline condition. More negatively toned emotional thoughts and more social temporal thoughts with a past focus were associated with increased cortisol and alpha-amylase levels, both after stress and at baseline. More social temporal thoughts with a future focus, on the other hand, had an overall attenuating effect on the levels of both stress markers. Our results indicate a fundamental link between the thoughts and stress levels we experience. Understanding the mechanisms governing this mind-body association may have important implications for understanding and counteracting the high incidence of stress-related disorders in today's society. PMID:25457636

  2. Structure of human salivary alpha-amylase at 1.6 A resolution: implications for its role in the oral cavity.

    PubMed

    Ramasubbu, N; Paloth, V; Luo, Y; Brayer, G D; Levine, M J

    1996-05-01

    Salivary alpha-amylase, a major component of human saliva, plays a role in the initial digestion of starch and may be involved in the colonization of bacteria involved in early dental plaque formation. The three-dimensional atomic structure of salivary amylase has been determined to understand the structure-function relationships of this enzyme. This structure was refined to an R value of 18.4% with 496 amino-acid residues, one calcium ion, one chloride ion and 170 water molecules. Salivary amylase folds into a multidomain structure consisting of three domains, A, B and C. Domain A has a (beta/alpha)(8-) barrel structure, domain B has no definite topology and domain C has a Greek-key barrel structure. The Ca(2+) ion is bound to Asnl00, Arg158, Asp167, His201 and three water molecules. The Cl(-) ion is bound to Arg195, Asn298 and Arg337 and one water molecule. The highly mobile glycine-rich loop 304-310 may act as a gateway for substrate binding and be involved in a 'trap-release' mechanism in the hydrolysis of substrates. Strategic placement of calcium and chloride ions, as well as histidine and tryptophan residues may play a role in differentiating between the glycone and aglycone ends of the polysaccharide substrates. Salivary amylase also possesses a suitable site for binding to enamel surfaces and provides potential sites for the binding of bacterial adhesins. PMID:15299664

  3. Refining the multisystem view of the stress response: Coordination among cortisol, alpha-amylase, and subjective stress in response to relationship conflict

    PubMed Central

    Powers, Sally I.; Granger, Douglas A.

    2013-01-01

    This study investigated associations among young adults' hypothalamic-pituitary-adrenal axis activity, autonomic nervous system activity, and subjective stress in response to interpersonal conflict to better characterize coordination across stress systems. Seven saliva samples were collected from 199 young adult opposite-sex couples before, during, and after they discussed an unresolved relationship conflict. Samples were later assayed for cortisol and alpha-amylase (sAA). Couples rated anticipatory stress prior to the conflict and perceived stress immediately following the task. Growth curve modeling was used to examine two possible levels of within-person coordination across physiological systems: alignment between cortisol and sAA responses throughout the sampling period (“matched phase coordination”), and association between overall levels of cortisol and sAA in response to conflict (“average level coordination”). Whereas both partners showed the former type of coordination, only women showed the latter type. Positive anticipation of the stressor predicted stronger cortisol-sAA matched phase coordination for women. Pre-task ratings related to women's sAA, and post-task ratings related to both partners' cortisol responses. Implications for a multisystem interpretation of normal and pathological responses to daily stress are discussed. PMID:23684904

  4. Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids from Neem (Azadirachta indica) as Anti-Diabetic Agents.

    PubMed

    Ponnusamy, Sudha; Haldar, Saikat; Mulani, Fayaj; Zinjarde, Smita; Thulasiram, Hirekodathakallu; RaviKumar, Ameeta

    2015-01-01

    Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (Ki 42.2, 18.6 μM) and starch (Ki' 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia. PMID:26469405

  5. Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids from Neem (Azadirachta indica) as Anti-Diabetic Agents

    PubMed Central

    Zinjarde, Smita; Thulasiram, Hirekodathakallu; RaviKumar, Ameeta

    2015-01-01

    Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (Ki 42.2, 18.6 μM) and starch (Ki′ 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia. PMID:26469405

  6. Role of Electromagnetic Field Exposure in Childhood Acute Lymphoblastic Leukemia and No Impact of Urinary Alpha- Amylase--a Case Control Study in Tehran, Iran.

    PubMed

    Tabrizi, Maral Mazloomi; Hosseini, Seyed Ahmad

    2015-01-01

    Childhood acute lymphoblastic leukemia (ALL) is one of the most common hematologic malignancies which accounts for one fourth of all childhood cancer cases. Exposure to environmental factors around the time of conception or pregnancy can increase the risk of ALL in the offspring. This study aimed to evaluate the influence of prenatal and postnatal exposure to high voltage power lines on the incidence of childhood ALL. It also examines the role of various factors such as environmental factors and alpha-amylase as a marker in the development of leukemia. This cross-sectional case control study was carried out on 22 cases and 100 controls who born and lived in low socioeconomic families in Tehran and were hospitalized for therapeutic purposes in different hospitals of rom 2013-2014. With regard to the underlying risk factors; familial history and parental factors were detected as risk factors of ALL but in this age, socioeconomic and zonal matched case control study, prenatal and childhood exposure to high voltage power lines was considered as the most important environmental risk factor (p=0.006, OR=3.651, CI 95% 1.692-7.878). As the population study was from low socioeconomic state, use of mobiles, computers and microwaves was negligible. Moreover prenatal and postnatal exposure to all indoor electrically charged objects were not detected as significant environmental factors in the present study. This work defined the risk of environmental especially continuous pre and postnatal exposure to high voltage power lines and living in pollutant regions through the parents or children as well as the previously described risk factors of ALL for the first time in low socioeconomic status Iranian population. PMID:26625771

  7. Biased mutagenesis in the N-terminal region by degenerate oligonucleotide gene shuffling enhances secretory expression of barley alpha-amylase 2 in yeast.

    PubMed

    Fukuda, Kenji; Jensen, Malene H; Haser, Richard; Aghajari, Nushin; Svensson, Birte

    2005-11-01

    Recombinant barley alpha-amylase 1 (rAMY1) and 2 (rAMY2), despite 80% sequence identity, are produced in very different amounts of 1.1 and <0.05 mg/l, respectively, by Saccharomyces cerevisiae strain S150-2B. The low yield of AMY2 practically excludes mutational analysis of structure-function relationships and protein engineering. Since different secretion levels of AMY1/AMY2 chimeras were previously ascribed to the N-terminal sequence, AMY1 residues were combinatorially introduced at the 10 non-conserved positions in His14-Gln49 of AMY2 using degenerate oligonucleotide gene shuffling (DOGS) coupled with homologous recombination in S.cerevisiae strain INVSc1. Activity screening of a partial library of 843 clones selected six having a large halo size on starch plates. Three mutants, F21M/Q44H, A42P/A47S and A42P rAMY2, also gave higher activity than wild-type in liquid culture. Only A42P showed wild-type stability and enzymatic properties. The replacement is located to a beta-->alpha loop 2 that interacts with domain B (beta-->alpha loop 3) protruding from the catalytic (beta/alpha)(8)-barrel. Most remarkably Pichia pastoris strain GS115 secreted 60 mg/l A42P compared with 3 mg/l of wild-type rAMY2. The crystal structure of A42P rAMY2 was solved and found to differ marginally from the AMY2 structure, suggesting that the high A42P yield stems from stabilization of the mature and/or intermediate form owing to the introduced proline residue. Moreover, the G to C substitution for the A42P mutation might have a positive impact on protein translation. PMID:16155115

  8. Measurements of Salivary Alpha Amylase and Salivary Cortisol in Hominoid Primates Reveal Within-Species Consistency and Between-Species Differences

    PubMed Central

    Behringer, Verena; Borchers, Claudia; Deschner, Tobias; Möstl, Erich; Selzer, Dieter; Hohmann, Gottfried

    2013-01-01

    Salivary alpha amylase (sAA) is the most abundant enzyme in saliva. Studies in humans found variation in enzymatic activity of sAA across populations that could be linked to the copy number of loci for salivary amylase (AMY1), which was seen as an adaptive response to the intake of dietary starch. In addition to diet dependent variation, differences in sAA activity have been related to social stress. In a previous study, we found evidence for stress-induced variation in sAA activity in the bonobos, a hominoid primate that is closely related to humans. In this study, we explored patterns of variation in sAA activity in bonobos and three other hominoid primates, chimpanzee, gorilla, and orangutan to (a) examine if within-species differences in sAA activity found in bonobos are characteristic for hominoids and (b) assess the extent of variation in sAA activity between different species. The results revealed species-differences in sAA activity with gorillas and orangutans having higher basal sAA activity when compared to Pan. To assess the impact of stress, sAA values were related to cortisol levels measured in the same saliva samples. Gorillas and orangutans had low salivary cortisol concentrations and the highest cortisol concentration was found in samples from male bonobos, the group that also showed the highest sAA activity. Considering published information, the differences in sAA activity correspond with differences in AMY1 copy numbers and match with general features of natural diet. Studies on sAA activity have the potential to complement molecular studies and may contribute to research on feeding ecology and nutrition. PMID:23613746

  9. Expression of cDNAs encoding barley alpha-amylase 1 and 2 in yeast and characterization of the secreted proteins.

    PubMed

    Søgaard, M; Svensson, B

    1990-10-15

    Amylolytic strains of the yeast, Saccharomyces cerevisiae, were constructed by transformation with expression plasmids containing cDNAs encoding either AMY1 (clone E) or AMY2 (clone pM/C). The alpha-amylases were efficiently secreted into the culture medium directed by their own signal peptides. When clone E without its 5'-noncoding region was expressed from the yeast PGK promoter, AMY1 was produced as 1% of total cell protein and was thus the major protein secreted, whereas a similar construct derived from pM/C produced much less AMY2. This level is the highest reported for a plant protein secreted by yeast as mediated by the endogenous signal peptide. Production of AMY1 increased 25-fold when the 5'-noncoding part of clone E which contains a 12-bp dG.dC homopolymer tail had been removed. Moreover, expression was one to two orders of magnitude higher when genes encoding AMY1 or AMY2 were inserted between promoter and terminator of the yeast PGK gene in comparison to expression directed from the ADC1 or GAL1 promoters. Recombinant AMY1 and AMY2 had the same Mr and N-terminal sequence as the corresponding barley malt enzymes. Furthermore, none of the enzymes were found to be N-glycosylated. Isoelectric focusing indicated that transformed yeast cells secreted one major form of AMY2 and four dominant forms of AMY1. One AMY1 form corresponded to one of the major forms found in malt while the others, having either low activity or unusually high pI, probably reflect inefficient/incorrect processing. Enzyme kinetic properties and pH activity-dependence of recombinant AMY2 were essentially identical to those of malt AMY2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2258050

  10. Comparison of barley malt alpha-amylase isozymes 1 and 2: construction of cDNA hybrids by in vivo recombination and their expression in yeast.

    PubMed

    Juge, N; Søgaard, M; Chaix, J C; Martin-Eauclaire, M F; Svensson, B; Marchis-Mouren, G; Guo, X J

    1993-08-25

    Germinating barley produces two alpha-amylase isozymes, AMY1 and AMY2, having 80% amino acid (aa) sequence identity and differing with respect to a number of functional properties. Recombinant AMY1 (re-AMY1) and AMY2 (re-AMY2) are produced in yeast, but whereas all re-AMY1 is secreted, re-AMY2 accumulates within the cell and only traces are secreted. Expression of AMY1::AMY2 hybrid cDNAs may provide a means of understanding the difference in secretion efficiency between the two isozymes. Here, the efficient homologous recombination system of the yeast, Saccharomyces cerevisiae, was used to generate hybrids of barley AMY with the N-terminal portion derived from AMY1, including the signal peptide (SP), and the C-terminal portion from AMY2. Hybrid cDNAs were thus generated that encode either the SP alone, or the SP followed by the N-terminal 21, 26, 53, 67 or 90 aa from AMY1 and the complementary C-terminal sequences from AMY2. Larger amounts of re-AMY are secreted by hybrids containing, in addition to the SP, 53 or more aa of AMY1. In contrast, only traces of re-AMY are secreted for hybrids having 26 or fewer aa of AMY1. In this case, re-AMY hybrid accumulates intracellularly. Transformants secreting hybrid enzymes also accumulated some re-AMY within the cell. The AMY1 SP, therefore, does not ensure re-AMY2 secretion and a certain portion of the N-terminal sequence of AMY1 is required for secretion of a re-AMY1::AMY2 hybrid. PMID:8359683

  11. Measurements of salivary alpha amylase and salivary cortisol in hominoid primates reveal within-species consistency and between-species differences.

    PubMed

    Behringer, Verena; Borchers, Claudia; Deschner, Tobias; Möstl, Erich; Selzer, Dieter; Hohmann, Gottfried

    2013-01-01

    Salivary alpha amylase (sAA) is the most abundant enzyme in saliva. Studies in humans found variation in enzymatic activity of sAA across populations that could be linked to the copy number of loci for salivary amylase (AMY1), which was seen as an adaptive response to the intake of dietary starch. In addition to diet dependent variation, differences in sAA activity have been related to social stress. In a previous study, we found evidence for stress-induced variation in sAA activity in the bonobos, a hominoid primate that is closely related to humans. In this study, we explored patterns of variation in sAA activity in bonobos and three other hominoid primates, chimpanzee, gorilla, and orangutan to (a) examine if within-species differences in sAA activity found in bonobos are characteristic for hominoids and (b) assess the extent of variation in sAA activity between different species. The results revealed species-differences in sAA activity with gorillas and orangutans having higher basal sAA activity when compared to Pan. To assess the impact of stress, sAA values were related to cortisol levels measured in the same saliva samples. Gorillas and orangutans had low salivary cortisol concentrations and the highest cortisol concentration was found in samples from male bonobos, the group that also showed the highest sAA activity. Considering published information, the differences in sAA activity correspond with differences in AMY1 copy numbers and match with general features of natural diet. Studies on sAA activity have the potential to complement molecular studies and may contribute to research on feeding ecology and nutrition. PMID:23613746

  12. Age Differences of Salivary Alpha-Amylase Levels of Basal and Acute Responses to Citric Acid Stimulation Between Chinese Children and Adults

    PubMed Central

    Yang, Ze-Min; Chen, Long-Hui; Zhang, Min; Lin, Jing; Zhang, Jie; Chen, Wei-Wen; Yang, Xiao-Rong

    2015-01-01

    It remains unclear how salivary alpha-amylase (sAA) levels respond to mechanical stimuli in different age groups. In addition, the role played by the sAA gene (AMY1) copy number and protein expression (glycosylated and non-glycosylated) in sAA activity has also been rarely reported. In this study, we analyzed saliva samples collected before and after citric acid stimulation from 47 child and 47 adult Chinese subjects. We observed that adults had higher sAA activity and sAA glycosylated levels (glycosylated sAA amount/total sAA amount) in basal and stimulated saliva when compared with children, while no differences were found in total or glycosylated sAA amount between them. Interestingly, adults showed attenuated sAA activity levels increase over those of children after stimulation. Correlation analysis showed that total sAA amount, glycosylated sAA amount, and AMY1 copy number × total sAA amount were all positively correlated with sAA activity before and after stimulation in both groups. Interestingly, correlation r between sAA levels (glycosylated sAA amount and total sAA amount) and sAA activity decreased after stimulation in children, while adults showed an increase in correlation r. In addition, the correlation r between AMY1 copy number × total sAA amount and sAA activity was higher than that between AMY1 copy number, total sAA amount, and sAA activity, respectively. Taken together, our results suggest that total sAA amount, glycosylated sAA amount, and the positive interaction between AMY1 copy number and total sAA amount are crucial in influencing sAA activity before and after stimulation in children and adults. PMID:26635626

  13. Distinct characteristics of single starch-binding domain SBD1 derived from tandem domains SBD1-SBD2 of halophilic Kocuria varians alpha-amylase.

    PubMed

    Yamaguchi, Rui; Arakawa, Tsutomu; Tokunaga, Hiroko; Ishibashi, Matsujiro; Tokunaga, Masao

    2012-03-01

    Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity from Escherichia coli. The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure. This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw starch granules and amylose resin with 70-80% efficiency compared with binding of equimolar His-SBD12. Although the thermal unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different: His-SBD12 refolded slowly (T(1/2) = ~84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T(1/2) = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5-6 °C increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding were insignificant. PMID:22388479

  14. Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch.

    PubMed

    Narita, Junya; Okano, Kenji; Kitao, Tomoe; Ishida, Saori; Sewaki, Tomomitsu; Sung, Moon-Hee; Fukuda, Hideki; Kondo, Akihiko

    2006-01-01

    We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying alpha-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA. PMID:16391053

  15. The activity of barley alpha-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase.

    PubMed

    Juge, Nathalie; Nøhr, Jane; Le Gal-Coëffet, Marie-Françoise; Kramhøft, Birte; Furniss, Caroline S M; Planchot, Véronique; Archer, David B; Williamson, Gary; Svensson, Birte

    2006-02-01

    High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley alpha-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg x L(-1) and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms of isoelectric points in the range 4.1-5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U x nmol(-1) and K(d) = 0.13 mg x mL(-1), respectively, were both improved with respect to the values 0.015 U x nmol(-1) and 0.67 mg x mL(-1) for rAMY1 (recombinant AMY1 produced in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed barley starch granules 15-fold faster than rAMY1, while higher amounts of AMY-SBD caused molecular overcrowding of the starch granule surface. PMID:16403494

  16. [PCSK9 inhibitors : Recommendations for patient selection].

    PubMed

    Laufs, U; Custodis, F; Werner, C

    2016-06-01

    The 2 or 4‑week subcutaneous therapy with the recently approved antibodies alirocumab and evolocumab for inhibition of proprotein convertase subtilisin-kexin type 9 (PCSK9) reduces low-density lipoprotein cholesterol (LDL-C) in addition to statins and ezetimibe by 50-60 %. The therapy is well-tolerated. The safety profile in the published studies is comparable to placebo. Outcome data and information on long-term safety and the influence on cardiovascular events are not yet available but the results of several large trials are expected in 2016-2018. At present (spring 2016) PCSK9 inhibitors represent an option for selected patients with a high cardiovascular risk and high LDL-C despite treatment with the maximum tolerated oral lipid-lowering therapy. This group includes selected patients with familial hypercholesterolemia and high-risk individuals with statin-associated muscle symptoms (SAMS). PMID:27207595

  17. Salivary alpha amylase diurnal pattern and stress response are associated with body mass index in low-income preschool-aged children.

    PubMed

    Miller, Alison L; Sturza, Julie; Rosenblum, Katherine; Vazquez, Delia M; Kaciroti, Niko; Lumeng, Julie C

    2015-03-01

    Physiological stress responses are proposed as a pathway through which stress can "get under the skin" and lead to health problems, specifically obesity. We tested associations of salivary alpha amylase (sAA) diurnal patterns and stress responses with body mass index (BMI) in young, low-income children (51% male; 54% non-Hispanic white). Diurnal saliva samples were collected three times per day across three days for 269 children (M age 50.8 months, SD 6.3). Individual sAA intercept and slope values were calculated using random effect models to represent morning sAA levels and rate of sAA change across the day. A subset of children (n=195; M age 56.6 months, SD 6.9) participated in a lab-based behavioral stress protocol. Area under the curve increase (AUCI) across four timepoints was calculated to represent increase in sAA output during stress elicitation. Children were weighed and height measured and BMI z-score was calculated. Linear regression was used to evaluate associations of sAA intercept, sAA slope, and sAA AUCI with BMI z-score, controlling for child age, sex, and race/ethnicity; maternal weight status; and family income-to-needs ratio. Diurnal and stress-response sAA patterns were related to child adiposity: for each 1-standard deviation unit (SDU) decrease in morning sAA level, the child's BMI z-score increased by 0.11 (SE 0.05) SDU's (p<.04); for each 1-SDU increase in sAA slope across the day, the child's BMI z-score increased by 0.12 (SE 0.05) SDU's (p<.03); and for each 1-SDU decrease in sAA AUCI during the stress elicitation, the child's BMI z-score increased by 0.14 (SE 0.06) SDU's (p<.03). Blunted stress responses and atypical diurnal patterns of sAA have been found following exposure to chronic life stressors such as poverty. Findings suggest that associations of stress, sAA, and elevated body mass index may develop very early in the lifespan. PMID:25588701

  18. Effect of chronic training on heart rate variability, salivary IgA and salivary alpha-amylase in elite swimmers with a disability.

    PubMed

    Edmonds, Rohan; Burkett, Brendan; Leicht, Anthony; McKean, Mark

    2015-01-01

    The purpose of this study was to a) determine the heart rate variability (HRV) and saliva markers of immunity (salivary immunoglobulin A; sIgA) and stress (salivary alpha-amylase; sAA) responses to chronic training in elite swimmers with a disability; and b) identify the relationships between HRV, sIgA, sAA and training volume. Eight members of a high performance Paralympic swimming program were monitored for their weekly resting HRV, sIgA and sAA levels in the 14 weeks leading up to a major international competition. The 14 week training program included aerobic, anaerobic, power and speed, and taper training phases, while also incorporating two swimming step tests and two swimming competitions. Specific time (root mean square of the successive differences; RMSSD) and frequency (high frequency normalized units [HFnu]) domain measures, along with non-linear indices (standard deviation of instantaneous RR variability; SD1 and short term fractal scaling exponent; α1) of HRV were used for all analyses with effects examined using magnitude-based inferences. Relationships between HRV and saliva markers were identified by Spearman rank rho (ρ) correlation coefficients. Compared with week 1, SD1 was very likely lower (96/4/0, ES = -2.21), while sAA was very likely elevated (100/0/0, ES = 2.32) at the beginning of week 7 for all athletes. The training program did not alter HRV or saliva whereas competition did. There were also no apparent differences observed for HRV, sIgA and sAA between each of the training phases during the 14 week swimming program. Correlations were observed between sAA and SD1 (ρ = -0.212, p<0.05), along with sAA and mean HR (ρ = 0.309, p<0.05). These results show that high level national competition influences depresses HRV (SD1) and increases saliva biomarkers of stress (sAA). It appears that a well-managed and periodised swimming program can maintain these indices within normal baseline levels. The study also highlighted the parasympathetic

  19. Effect of Chronic Training on Heart Rate Variability, Salivary IgA and Salivary Alpha-Amylase in Elite Swimmers with a Disability

    PubMed Central

    Edmonds, Rohan

    2015-01-01

    The purpose of this study was to a) determine the heart rate variability (HRV) and saliva markers of immunity (salivary immunoglobulin A; sIgA) and stress (salivary alpha-amylase; sAA) responses to chronic training in elite swimmers with a disability; and b) identify the relationships between HRV, sIgA, sAA and training volume. Eight members of a high performance Paralympic swimming program were monitored for their weekly resting HRV, sIgA and sAA levels in the 14 weeks leading up to a major international competition. The 14 week training program included aerobic, anaerobic, power and speed, and taper training phases, while also incorporating two swimming step tests and two swimming competitions. Specific time (root mean square of the successive differences; RMSSD) and frequency (high frequency normalized units [HFnu]) domain measures, along with non-linear indices (standard deviation of instantaneous RR variability; SD1 and short term fractal scaling exponent; α1) of HRV were used for all analyses with effects examined using magnitude-based inferences. Relationships between HRV and saliva markers were identified by Spearman rank rho (ρ) correlation coefficients. Compared with week 1, SD1 was very likely lower (96/4/0, ES = -2.21), while sAA was very likely elevated (100/0/0, ES = 2.32) at the beginning of week 7 for all athletes. The training program did not alter HRV or saliva whereas competition did. There were also no apparent differences observed for HRV, sIgA and sAA between each of the training phases during the 14 week swimming program. Correlations were observed between sAA and SD1 (ρ = -0.212, p<0.05), along with sAA and mean HR (ρ = 0.309, p<0.05). These results show that high level national competition influences depresses HRV (SD1) and increases saliva biomarkers of stress (sAA). It appears that a well-managed and periodised swimming program can maintain these indices within normal baseline levels. The study also highlighted the parasympathetic

  20. Physical and psychosocial challenges in adult hemophilia patients with inhibitors

    PubMed Central

    duTreil, Sue

    2014-01-01

    Numerous challenges confront adult hemophilia patients with inhibitors, including difficulty in controlling bleeding episodes, deterioration of joints, arthritic pain, physical disability, emotional turmoil, and social issues. High-intensity treatment regimens often used in the treatment of patients with inhibitors also impose significant scheduling, economic, and emotional demands on patients and their families or primary caregivers. A comprehensive multidisciplinary assessment of the physical, emotional, and social status of adult hemophilia patients with inhibitors is essential for the development of treatment strategies that can be individualized to address the complex needs of these patients. PMID:25093002

  1. Diabetes therapies in hemodialysis patients: Dipeptidase-4 inhibitors.

    PubMed

    Nakamura, Yuya; Hasegawa, Hitomi; Tsuji, Mayumi; Udaka, Yuko; Mihara, Masatomo; Shimizu, Tatsuo; Inoue, Michiyasu; Goto, Yoshikazu; Gotoh, Hiromichi; Inagaki, Masahiro; Oguchi, Katsuji

    2015-06-25

    Although several previous studies have been published on the effects of dipeptidase-4 (DPP-4) inhibitors in diabetic hemodialysis (HD) patients, the findings have yet to be reviewed comprehensively. Eyesight failure caused by diabetic retinopathy and aging-related dementia make multiple daily insulin injections difficult for HD patients. Therefore, we reviewed the effects of DPP-4 inhibitors with a focus on oral antidiabetic drugs as a new treatment strategy in HD patients with diabetes. The following 7 DPP-4 inhibitors are available worldwide: sitagliptin, vildagliptin, alogliptin, linagliptin, teneligliptin, anagliptin, and saxagliptin. All of these are administered once daily with dose adjustments in HD patients. Four types of oral antidiabetic drugs can be administered for combination oral therapy with DPP-4 inhibitors, including sulfonylureas, meglitinide, thiazolidinediones, and alpha-glucosidase inhibitor. Nine studies examined the antidiabetic effects in HD patients. Treatments decreased hemoglobin A1c and glycated albumin levels by 0.3% to 1.3% and 1.7% to 4.9%, respectively. The efficacy of DPP-4 inhibitor treatment is high among HD patients, and no patients exhibited significant severe adverse effects such as hypoglycemia and liver dysfunction. DPP-4 inhibitors are key drugs in new treatment strategies for HD patients with diabetes and with limited choices for diabetes treatment. PMID:26131325

  2. FEIBA versus NovoSeven in hemophilia patients with inhibitors.

    PubMed

    Franchini, Massimo; Coppola, Antonio; Tagliaferri, Annarita; Lippi, Giuseppe

    2013-10-01

    The management of patients with congenital hemophilia who develop alloantibodies that neutralize coagulation factor activity is the most important challenge for hemophilia care providers because this complication renders replacement treatment with factor concentrates partially or completely ineffective, exposing the patients to an increased risk of morbidity and mortality. Development of inhibitors complicates the clinical course of severe hemophilia in up to 30% of patients with hemophilia A and up to 5% of those with hemophilia B. Although the ultimate goal of treatment of patients with alloantibodies against factors VIII and IX is eradication of the inhibitor, the control of bleeding through high doses of factor concentrates (low titer inhibitors) or bypassing agents (high titer inhibitors) is the mainstay of management of these patients. In this review, we summarize the main characteristics of the bypassing agents FEIBA and NovoSeven, briefly discussing available literature data, and in particular, focusing on comparative studies. PMID:24014071

  3. Multimorbidities and Overprescription of Proton Pump Inhibitors in Older Patients

    PubMed Central

    Delcher, Anne; Hily, Sylvie; Boureau, Anne Sophie; Chapelet, Guillaume; Berrut, Gilles; de Decker, Laure

    2015-01-01

    Objectives To determine whether there is an association between overprescription of proton pump inhibitors (PPIs) and multimorbidities in older patients. Design Multicenter prospective study. Setting Acute geriatric medicine at the University Hospital of Nantes and the Hospital of Saint-Nazaire. Participants Older patients aged 75 and over hospitalized in acute geriatric medicine. Measurements Older patients in acute geriatric medicine who received proton pump inhibitors. Variables studied were individual multimorbidities, the burden of multimorbidity evaluated by the Cumulative Illness Rating Scale, age, sex, type of residence (living in nursing home or not), functional abilities (Lawton and Katz scales), nutritional status (Body Mass Index), and the type of concomitant medications (antiaggregant, corticosteroids’, or anticoagulants). Results Overprescription of proton pump inhibitors was found in 73.9% older patients. In the full model, cardiac diseases (odds ratio [OR] = 4.17, p = 0.010), metabolic diseases (OR = 2.14, p = 0.042) and corticosteroids (OR = 5.39, p = 0.028) were significantly associated with overprescription of proton pump inhibitors. Esogastric diseases (OR = 0.49, p = 0.033) were negatively associated with overprescription of proton pump inhibitors. Conclusion Cardiac diseases and metabolic diseases were significantly associated with overprescription of proton pump inhibitors. PMID:26535585

  4. Tissue factor pathway inhibitor levels in patients with homocystinuria.

    PubMed

    Cella, G; Burlina, A; Sbarai, A; Motta, G; Girolami, A; Berrettini, M; Strauss, W

    2000-06-01

    Thrombotic events are a well-recognized complication of homocystinuria. However, the mechanisms involved in the atherogenic and thrombotic effects of homocyst(e)ine remain incompletely understood. The objective of this study was to determine the role of endothelial cell activation/damage as indicated by levels of thrombomodulin, tissue factor and tissue factor pathway inhibitor, and factor VII activity in patients with homocystinuria. Six patients with homocystinuria, nonresponsive to pyridoxine, treated only with trimethylglycine (betaine) were injected with a bolus of 20 IU/kg body weight of unfractionated commercial heparin to induce the release of tissue factor pathway inhibitor from the vascular endothelium. Tissue factor, thrombomodulin, and factor VII activity were measured by enzyme-linked immunosorbent assay and clotting assay before heparin administration. Tissue factor pathway inhibitor antigen and activity were measured before and 5 minutes after the bolus of heparin. Levels of homocyst(e)ine were elevated (patients: 144.2+/-19.2 micromol/L; controls: 10.2+/-0.9 micromol/L); however, levels of thrombomodulin, tissue factor, and tissue factor pathway inhibitor antigen were not statistically different from the control group. In contrast, tissue factor pathway inhibitor activity showed a significantly increased level (patients: 2.09+/-0.34 U/L; controls: 1.14+/-0.20 U/L; p<0.05) that was correlated with homocyst(e)ine. Factor VII activity was significantly decreased (patients: 64.7+/-5.1%; controls: 91.4+/-4.7%; p<0.05) and inversely correlated with homocyst(e)ine. After heparin the patients released higher amounts of tissue factor pathway inhibitor antigen and activity compared with the control group; however, the difference was not statistically significant. Although not treated with antithrombotic drugs, none of the patients had any thromboembolic complications after starting betaine. In addition to betaine treatment, the enhanced factor pathway

  5. Antisense SNF1-related (SnRK1) protein kinase gene represses transient activity of an alpha-amylase (alpha-Amy2) gene promoter in cultured wheat embryos.

    PubMed

    Laurie, Sophie; McKibbin, Rowan S; Halford, Nigel G

    2003-02-01

    A DNA fragment corresponding to part of an SNF1 (sucrose non-fermenting-1)-related protein kinase (SnRK1) transcript was amplified by a polymerase chain reaction (PCR) from a wheat (Triticum aestivum) endosperm cDNA library. It was used to construct a chimaeric gene, pUasSnRKN, comprising a ubiquitin promoter, the SnRK1 PCR product in the antisense orientation and the nopaline synthase (Nos) gene terminator. This construct was used in transient gene expression experiments in cultured wheat embryos together with a series of reporter gene constructs. These included the wheat alpha amylase gene alpha-Amy2 promoter with UidA (Gus) coding region (palpha2GT), rice actin promoter with Gus (pActIDGus), ubiquitin promoter with Gus (pAHC25) and actin promoter with green fluorescent protein (GFP) gene (pAct1Is-GFP1). All of the reporter genes were found to be active when bombarded into scutellae isolated from immature grains at 25 d post-anthesis and incubated on MS medium for 24 h prior to bombardment. However, co-bombardment of palpha2GT with equimolar amounts of pUasSnRKN resulted in no detectable Gus activity, indicating that the antisense SnRK1 construct repressed the alpha-Amy2 promoter. Co-bombardment with pUasSnRKN had no effect on the activity of the other promoters used in the study. A triple bombardment with palpha2GT, pAct1Is-GFP-1 and pUasSnRKN resulted in clear green fluorescence, indicating that the bombarded cells were still viable, but no Gus activity. RT-PCR analysis showed clearly that the antisense SnRK1 gene was expressing. Northern and RT-PCR analyses confirmed that SnRK1 and both alpha-amylase genes, alpha-Amy1 and alpha-Amy2, are expressed in cultured wheat embryos harvested from grain 25 d post-anthesis. Expression of alpha-Amy1 and alpha-Amy2 was up-regulated by sugar starvation. PMID:12554717

  6. New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 alpha-amylase contributes to starch binding and raw starch degrading.

    PubMed

    Sumitani, J; Tottori, T; Kawaguchi, T; Arai, M

    2000-09-01

    The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III. PMID:10947962

  7. Efficient production of optically pure D-lactic acid from raw corn starch by using a genetically modified L-lactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain.

    PubMed

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch. PMID:19011066

  8. Carrier free co-immobilization of alpha amylase, glucoamylase and pullulanase as combined cross-linked enzyme aggregates (combi-CLEAs): a tri-enzyme biocatalyst with one pot starch hydrolytic activity.

    PubMed

    Talekar, Sachin; Pandharbale, Amol; Ladole, Mayur; Nadar, Shamraja; Mulla, Mosin; Japhalekar, Kshitija; Pattankude, Kishori; Arage, Devika

    2013-11-01

    A tri-enzyme biocatalyst "combi-CLEAs" with starch hydrolytic activity was prepared from commercially available alpha amylase, glucoamylase and pullulanase preparations by aggregating enzymes with ammonium sulphate followed by cross-linking formed aggregates for 4.5h with 40 mM glutaraldehyde. The effects of precipitant type and cross-linking were studied and the biocatalyst was characterized. Scanning electron microscopy analysis showed that tri-enzyme biocatalyst was of spherical structure. For one pot starch hydrolytic activity, shift in optimum pH from 6 to 7 and temperature from 65 to 75 °C were observed after co-immobilization of enzymes. After one pot starch hydrolysis reaction in batch mode, 100%, 60% and 40% conversions were obtained with combi-CLEAs, separate CLEAs mixture and free enzyme mixture, respectively. Co-immobilization also enhanced the thermal stability of enzymes. Finally, the catalytic activity of enzymes in combi-CLEAs during one pot starch hydrolysis was well maintained up to five cycles without performance changes. PMID:23999260

  9. Safety of immune checkpoint inhibitors in Chinese patients with melanoma.

    PubMed

    Wen, Xizhi; Wang, Yao; Ding, Ya; Li, Dandan; Li, Jingjing; Guo, Yiqun; Peng, Ruiqing; Zhao, Jingjing; Zhang, Xing; Zhang, Xiao-Shi

    2016-06-01

    This study aimed to determine the tolerability of Chinese melanoma patients, particularly those with hepatitis B virus (HBV) infection, to immune checkpoint inhibitor therapy. Patients with metastatic melanoma who received anti-cytotoxic T lymphocyte-associated antigen-4 antibody (ipilimumab) or anti-programmed death 1 antibody (pembrolizumab) therapy at our hospital between August 2012 and July 2015 were retrospectively reviewed. Adverse events were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.0. Twenty-three patients with advanced melanoma were included; nine and 10 patients received infusions of ipilimumab and pembrolizumab, respectively, whereas four patients received concurrent ipilimumab and pembrolizumab therapy. There was no cessation of treatment because of agent-related adverse events in any patient. Immune-related adverse events were observed in 44% (4/9), 60% (6/10), 100% (4/4), and 61% (14/23) of patients receiving ipilimumab, pembrolizumab, concomitant ipilimumab and pembrolizumab, and any treatment, respectively. The most frequent immune-related adverse events were endocrine disorders (39%, 9/23), liver function abnormalities (22%, 5/23), and dermatological events (17%, 4/23). There were no gastrointestinal reactions. Toxicities were usually mild and easily managed; only 13% (3/23) of patients had grade 3 adverse events and none experienced grade 4 events or treatment-related death. No additional toxicity nor severe hepatotoxicity was observed in 11 patients who had previous HBV infection. The recommended anti-cytotoxic T lymphocyte-associated antigen-4 and anti-programmed death 1 antibody doses were well tolerated by Chinese patients. Thus, immune checkpoint inhibitors appear to be effective and safe in metastatic melanoma patients, including those with pre-existing HBV infection. PMID:27116334

  10. Elevated Salivary Alpha-Amylase Level, Association Between Depression and Disease Activity, and Stress as a Predictor of Disease Flare in Systemic Lupus Erythematosus

    PubMed Central

    Jung, Ju-Yang; Nam, Jin-Young; Kim, Hyoun-Ah; Suh, Chang-Hee

    2015-01-01

    Abstract Psychological stress has been shown to trigger systemic lupus erythematosus (SLE). However, objective evidence of symptom aggravation due to mental stress is difficult to identify. We aimed to investigate the relationship between SLE disease activity and mental stress, and the usefulness of saliva as an assessment index for stress in patients with SLE. We prospectively assessed the salivary stress hormone and disease-related biomarkers, and questionnaire data regarding stress and depression in 100 patients with SLE and 49 sex- and age-matched normal controls (NCs). Patients with SLE had higher mean salivary α-amylase levels (5.7 ± 4.6 U/mL vs 2.7 ± 2.5 U/mL, P < 0.001), anti-chromatin antibody levels (25.3 ± 22.9 U/mL vs 15.9 ± 10.9 U/mL, P < 0.001), and Beck Depression Index (BDI) scores (11.1 ± 9.2 vs 5.3 ± 5.1, P < 0.001) than NCs. However, salivary cortisol levels and Perceived Stress Scale (PSS) scores did not differ between the groups. The BDI scores correlated with the SLE disease activity index (SLEDAI) scores (r = 0.253, P = 0.011) and erythrocyte sedimentation rates (r = 0.234, P = 0.019). SLE patients with the highest-quartile PSS scores had significantly increased SLEDAI scores compared to those with the lowest-quartile PSS scores after 4 to 5 months’ follow-up. Moreover, SLE patients with elevated SLEDAI scores had higher baseline PSS scores. Patients with SLE showed uncoupling of the sympathetic nervous system and hypothalamic–pituitary–adrenal axis; higher salivary α-amylase and no different cortisol levels compared with NCs. Also, patients with SLE were more depressed, which correlated with disease activity. Furthermore, perceived stress was not correlated with disease activity; however, disease activity worsened several months later with elevated perceived stress levels. PMID:26222848

  11. Nutritional significance of lectins and enzyme inhibitors from legumes.

    PubMed

    Lajolo, Franco M; Genovese, Maria Inés

    2002-10-23

    Legumes have natural components, such as lectins, amylase, and trypsin inhibitors, that may adversely affect their nutritional properties. Much information has already been obtained on their antinutritional significance and how to inactivate them by proper processing. Chronic ingestion of residual levels is unlikely to pose risks to human health. On the other hand, the ability of these molecules to inhibit some enzymes such as trypsin, chymotrypsin, disaccharidases, and alpha-amylases, to selectively bind to glycoconjugates, and to enter the circulatory system may be a useful tool in nutrition and pharmacology. Trypsin inhibitors have also been studied as cancer risk reducing factors. These components seem to act as plant defense substances. However, increased contents may represent an impairment of the nutritional quality of legumes because these glycoproteins and the sulfur-rich protease inhibitors have been shown to be poorly digested and to participate in chemical reactions during processing reducing protein digestibility, a still unsolved question. PMID:12381157

  12. The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora.

    PubMed

    Negreiros, A N; Carvalho, M M; Xavier Filho, J; Blanco-Labra, A; Shewry, P R; Richardson, M

    1991-01-01

    The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin. PMID:1367792

  13. Treatment of young patients with lupus nephritis using calcineurin inhibitors

    PubMed Central

    Tanaka, Hiroshi; Tsuruga, Kazushi; Aizawa-Yashiro, Tomomi; Watanabe, Shojiro; Imaizumi, Tadaatsu

    2012-01-01

    Recent advances in the management of lupus nephritis, together with earlier renal biopsy and selective use of aggressive immunosuppressive therapy, have contributed to a favorable outcome in children and adolescents with systemic lupus erythematosus (SLE). Nevertheless, we believe that a more effective and less toxic treatment is needed to attain an optimal control of the activity of lupus nephritis. Recent published papers and our experiences regarding treatment of young patients with lupus nephritis using calcineurin inhibitors are reviewed. Although it has been reported that intermittent monthly pulses of intravenous cyclophosphamide (IVCY) are effective for preserving renal function in adult patients, CPA is a potent immunosuppressive agent that induces severe toxicity, including myelo- and gonadal toxicity, and increases the risk of secondary malignancy. Thus, treatment for controlling lupus nephritis activity, especially in children and adolescents, remains challenging. Cyclosporine A (CsA) and tacrolimus (Tac) are T-cell-specific calcineurin inhibitors that prevent the activation of helper T cells, thereby inhibiting the transcription of the early activation genes of interleukin (IL)-2 and suppressing T cell-induced activation of tumor necrosis factor-α, IL-1β and IL-6. Therefore, both drugs, which we believe may be less cytotoxic, are attractive therapeutic options for young patients with lupus nephritis. Recently, a multidrug regimen of prednisolone (PDN), Tac, and mycophenolate mofetile (MMF) has been found effective and relatively safe in adult lupus nephritis. Since the mechanisms of action of MMF and Tac are probably complementary, multidrug therapy for lupus nephritis may be useful. We propose as an alternative to IVCY, a multidrug therapy with mizoribine, which acts very similarly to MMF, and Tac, which has a different mode of action, combined with PDN for pediatric-onset lupus nephritis. We also believe that a multidrug therapy including CsA and

  14. Ocular Surface Disease in Breast Cancer Patients Using Aromatase Inhibitors.

    PubMed

    Chatziralli, Irini; Sergentanis, Theodoros; Zagouri, Flora; Chrysikos, Dimosthenis; Ladas, Ioannis; Zografos, George C; Moschos, Marilita

    2016-09-01

    Aromatase inhibitors (AIs) are widely used as adjuvant hormonal therapy in postmenopausal women with hormone receptor-positive breast cancer. The purpose of this study was to investigate the potential impact of AIs on the anterior segment of the eye and especially the ocular surface. Participants in our study were 41 hormone receptor-positive early stage breast cancer patients (80 eyes), treated with AIs, while 80 eyes of 40 age- and gender-matched healthy controls, not previously used AIs for any purpose, were also evaluated. All participants underwent a complete ophthalmological examination, including best corrected visual acuity (BCVA) assessment, slit-lamp biomicroscopy, and dilated fundus examination. Ocular surface disease-related symptoms and signs were also recorded. The most common symptom was found to be blurred vision, while other symptoms included foreign body sensation, tearing, redness, and photophobia. Slit-lamp examination revealed blepharitis and meibomian gland dysfunction in 75% and 42.5% of patients, respectively. Superficial punctate keratitis and conjunctival injection were also present. Our results demonstrated a high prevalence of ocular surface disease-related symptoms and signs in patients receiving AIs compared to healthy controls. This study may raise a flag regarding the use of AIs. However, further and larger prospective longitudinal studies are needed to examine the possible effect of AIs alone or in combination with chemotherapy in the eyes of breast cancer patients. PMID:27296769

  15. Selective serotonin reuptake inhibitor drug interactions in patients receiving statins.

    PubMed

    Andrade, Chittaranjan

    2014-02-01

    Elderly patients commonly receive statin drugs for the primary or secondary prevention of cardiovascular and cerebrovascular events. Elderly patients also commonly receive antidepressant drugs, usually selective serotonin reuptake inhibitors (SSRIs), for the treatment of depression, anxiety, or other conditions. SSRIs are associated with many pharmacokinetic drug interactions related to the inhibition of the cytochrome P450 (CYP) metabolic pathways. There is concern that drugs that inhibit statin metabolism can trigger statin adverse effects, especially myopathy (which can be potentially serious, if rhabdomyolysis occurs). However, a detailed literature review of statin metabolism and of SSRI effects on CYP enzymes suggests that escitalopram, citalopram, and paroxetine are almost certain to be safe with all statins, and rosuvastatin, pitavastatin, and pravastatin are almost certain to be safe with all SSRIs. Even though other SSRI-statin combinations may theoretically be associated with risks, the magnitude of the pharmacokinetic interaction is likely to be below the threshold for clinical significance. Risk, if at all, lies in combining fluvoxamine with atorvastatin, simvastatin, or lovastatin, and even this risk can be minimized by using lower statin doses and monitoring the patient. PMID:24602259

  16. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  17. Different Cholinesterase Inhibitor Effects on CSF Cholinesterases in Alzheimer Patients

    PubMed Central

    Nordberg, Agneta; Darreh-Shori, Taher; Peskind, Elaine; Soininen, Hilkka; Mousavi, Malahat; Eagle, Gina; Lane, Roger

    2014-01-01

    Background The current study aimed to compare the effects of different cholinesterase inhibitors on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and protein levels, in the cerebrospinal fluid (CSF) of Alzheimer disease (AD) patients. Methods and Findings AD patients aged 50–85 years were randomized to open-label treatment with oral rivastigmine, donepezil or galantamine for 13 weeks. AChE and BuChE activities were assayed by Ellman’s colorimetric method. Protein levels were assessed by enzyme-linked immunosorbent assay (ELISA). Primary analyses were based on the Completer population (randomized patients who completed Week 13 assessments). 63 patients were randomized to treatment. Rivastigmine was associated with decreased AChE activity by 42.6% and decreased AChE protein levels by 9.3%, and decreased BuChE activity by 45.6% and decreased BuChE protein levels by 21.8%. Galantamine decreased AChE activity by 2.1% and BuChE activity by 0.5%, but increased AChE protein levels by 51.2% and BuChE protein levels by10.5%. Donepezil increased AChE and BuChE activities by 11.8% and 2.8%, respectively. Donepezil caused a 215.2%increase in AChE and 0.4% increase in BuChE protein levels. Changes in mean AChE-Readthrough/Synaptic ratios, which might reflect underlying neurodegenerative processes, were 1.4, 0.6, and 0.4 for rivastigmine, donepezil and galantamine, respectively. Conclusion The findings suggest pharmacologically-induced differences between rivastigmine, donepezil and galantamine. Rivastigmine provides sustained inhibition of AChE and BuChE, while donepezil and galantamine do not inhibit BuChE and are associated with increases in CSF AChE protein levels. The clinical implications require evaluation. PMID:19199870

  18. Drug Survival Rates of Tumor Necrosis Factor Inhibitors in Patients with Rheumatoid Arthritis and Ankylosing Spondylitis

    PubMed Central

    2014-01-01

    We investigated the compliance of Korean patients using tumor necrosis factor (TNF) inhibitors to treat rheumatoid arthritis (RA) and ankylosing spondylitis (AS), and identified potential predictors associated with treatment discontinuation. The study population comprised 114 RA and 310 AS patients treated with TNF inhibitors at a single tertiary center for at least 1 yr from December 2002 to November 2011. Of the 114 RA patients, 64 (56.1%) discontinued their first TNF inhibitors with a mean duration of 18.1 months. By contrast, 65 of 310 patients (21.0%) with AS discontinued their first TNF inhibitors, with a mean duration of 84 months. Although the survival rate did not differ among the three TNF inhibitors in the AS patients, the etanercept group had a lower discontinuation rate than the infliximab group in the RA patients. In addition, RA patients who received corticosteroids in combination with TNF inhibitors were more likely to discontinue their TNF inhibitors. The independent predictors of drug discontinuation in AS patients were male gender and complete ankylosis on radiographs of the sacroiliac joint. Our results provide further evidence that real-life treatment outcomes of RA and AS patients may be different from those observed in randomized clinical trials. Graphical Abstract PMID:25246737

  19. Factor eight inhibitor bypass activity (FEIBA) in the management of bleeds in hemophilia patients with high-titer inhibitors

    PubMed Central

    Tjønnfjord, Geir E; Andre Holme, Pål

    2007-01-01

    The development of high-titer inhibitors to FVIII and less often to other coagulation factors are the most serious complication of hemophilia therapy and makes treatment of bleeds very challenging. At present, bypassing agents, such as factor eight inhibitor bypass activity (FEIBA) and activated recombinant factor VII (rFVIIa) are the only coagulation factor concentrates available for the treatment of bleeds in inhibitor patients. Both products are effective and safe, and their efficacy has been found to be comparable (approximately 80%) in a recent prospective study. A significant number of patients report a better effect of one or the other of the products, and in a minority of the patients none of the products are particularly effective. The hemostatic efficacy of bypassing agents is not considered equal to that of coagulation factor replacement in patients without inhibitors by most physicians. An improvement in hemostatic efficacy may be achieved by optimizing the dosing of by passing agents. However, the lack of standardized and validated laboratory assays reflecting the hemostatic efficacy of the bypassing agents is an obstacle to this achievement. PMID:17969383

  20. Cyclooxygenase-2 inhibitors in patients with high gastrointestinal risk: are we there yet?

    PubMed

    Wong, Vincent Wai-Sun; Chan, Francis Ka-Leung

    2009-01-01

    Gastrointestinal complications from the use of nonsteroidal antiinflammatory drugs (NSAIDs) are common and costly. Cyclooxygenase (COX)-2 inhibitors cause fewer gastrointestinal events than nonselective NSAIDs but may still result in peptic ulcer complications in a significant proportion of high-risk patients. The risk can be further reduced by prescribing COX-2 inhibitors together with proton pump inhibitors. Small-bowel injury is also commonly seen in long-term users of COX-2 inhibitors. The potential cardiovascular side effects of most COX-2 inhibitors and NSAIDs pose great challenge to physicians. Further studies are required to optimize the treatment regimen in patients with high cardiovascular and gastrointestinal risk and to decide the best treatment options for patients with small- and large-bowel complications. PMID:19148794

  1. How I use bypassing therapy for prophylaxis in patients with hemophilia A and inhibitors.

    PubMed

    Leissinger, Cindy A; Singleton, Tammuella; Kruse-Jarres, Rebecca

    2015-07-01

    Inhibitor development poses a significant challenge in the management of hemophilia because once an inhibitor is present, bleeding episodes can no longer be treated with standard clotting factor replacement therapy. Consequently, patients with inhibitors are at increased risk for difficult-to-control bleeding and complications, particularly arthropathy and physical disability. Three clinical trials in patients with inhibitors have demonstrated that prophylaxis with a bypassing agent reduces joint and other types of bleeding and improves health-related quality of life compared with on-demand bypassing therapy. In hemophilia patients without inhibitors, the initiation of prophylaxis with factor (F) VIII or FIX prior to the onset of recurrent hemarthroses can prevent the development of joint disease. Whether this is also true for bypassing agent prophylaxis remains to be determined. PMID:25827834

  2. Therapeutic management and costs of severe haemophilia A patients with inhibitors in Italy.

    PubMed

    Abbonizio, F; Giampaolo, A; Coppola, A; Arcieri, R; Hassan, H J

    2014-07-01

    Haemophilia A (HA) patients with high responding inhibitors require therapies with bypassing agents to control bleedings or Immune Tolerance Induction (ITI) to attempt inhibitor eradication and restore FVIII therapy. The aim of this study was to assess the therapeutic management and product consumption of HA inhibitor patients and the relative costs in Italy. A retrospective survey was performed utilizing data from the National Registry of Congenital Coagulopathies and from a specific questionnaire on product consumption of HA inhibitor patients over the year 2011. Among HA patients, 10% had currently detectable inhibitors; 24% of patients were undergoing ITI (mostly children) and 76% utilized bypassing agents. Patients on ITI consumed 45,000,000 IU of FVIII (median consumption/patient of 1,200,000 IU year(-1)). Patients receiving bypassing agents utilized 21,000,000 IU of aPCC (median consumption/patient of 360,000 IU year(-1)), and 38,000 mg of rFVIIa (median consumption/patient of 440 mg year(-1)). The annual cost/patient on ITI and on bypassing agents therapy was analysed. Recombinant products represented the product of choice for children therapies in >90% of the cases. FVIII prophylaxis of severe HA patients without inhibitor costs about half than therapy with bypassing agents and is three times less expensive than prophylaxis with such agents. Therefore, the possibility to restore FVIII prophylaxis, having eradicated the inhibitor through ITI, can justify the high costs of ITI treatment needed in the short term. Consistent with this notion, over the last years a 50% increase in the number of patients undergoing ITI in Italy was registered. PMID:24834967

  3. Identification of proteinaceous inhibitors of a cysteine proteinase (an Arg-specific gingipain) from Porphyromonas gingivalis in rice grain, using targeted-proteomics approaches.

    PubMed

    Taiyoji, Mayumi; Shitomi, Yasuyuki; Taniguchi, Masayuki; Saitoh, Eiichi; Ohtsubo, Sadami

    2009-11-01

    Porphyromonas gingivalis is known to be a major etiologic agent in the onset and progression of chronic periodontitis. Among various virulence factors that this bacterium produces, Arg- and Lys-specific cysteine proteinases (gingipains) are believed to be major determinants of the pathogenicity of P. gingivalis. Here, we report on our finding that there are inhibitors of these cysteine proteinases in a rice protein fraction. Comprehensive affinity chromatography and MS analyses resulted in the identification of 17 Arg-gingipain (Rgp)-interacting proteins in the rice endosperm. Of these, four proteins (i.e., a 26 kDa globulin, a plant lipid transfer/trypsin-alpha amylase inhibitor, the RA17 seed allergen, and an alpha amylase/trypsin inhibitor) were estimated to account for 90% of the Rgp inhibitory activity in the rice protein fraction, using a two-dimensional gel system of double-layer reverse zymography. In addition, a synthetic peptide derived from an Rgp-interacting protein, cyanate hydratase, could inhibit the growth of P. gingivalis and showed inhibitory activity against both the Arg- and Lys-gingipains. These results suggest that these rice proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases. PMID:19691286

  4. Economic Impact of Combining Metformin with Dipeptidyl Peptidase-4 Inhibitors in Diabetic Patients with Renal Impairment in Spanish Patients

    PubMed Central

    Navarro-Artieda, Ruth

    2015-01-01

    Background To evaluate resource use and health costs due to the combination of metformin and dipeptidyl peptidase-4 (DPP-4) inhibitors in patients with diabetes and renal impairment in routine clinical practice. Methods An observational, retrospective study was performed. Patients aged ≥30 years treated with metformin who initiated a second oral antidiabetic treatment in 2009 to 2010 were included. Two groups of patients were analysed: metformin+DPP-4 inhibitors and other oral antidiabetics. The main measures were: compliance, persistence, metabolic control (glycosylated hemoglobin< 7%) and complications (hypoglycemia, cardiovascular events) and total costs. Patients were followed up for 2 years. Results We included 395 patients, mean age 70.2 years, 56.5% male: 135 patients received metformin+DPP-4 inhibitors and 260 patients received metformin+other oral antidiabetics. Patients receiving DPP-4 inhibitors showed better compliance (66.0% vs. 60.1%), persistence (57.6% vs. 50.0%), and metabolic control (63.9% vs. 57.3%), respectively, compared with those receiving other oral antidiabetics (P<0.05), and also had a lower rate of hypoglycemia (20.0% vs. 47.7%) and lower total costs (€ 2,486 vs. € 3,002), P=0.001. Conclusion Despite the limitations of the study, patients with renal impairment treated with DPP-4 inhibitors had better metabolic control, lower rates (association) of hypoglycaemia, and lower health costs for the Spanish national health system. PMID:25729716

  5. Three ileus cases associated with the use of dipeptidyl peptidase-4 inhibitors in diabetic patients.

    PubMed

    Kanasaki, Keizo; Konishi, Kazunori; Hayashi, Ranji; Shiroeda, Hisakazu; Nomura, Tomoe; Nakagawa, Atsushi; Nagai, Takako; Takeda-Watanabe, Ai; Ito, Hiroki; Tsuda, Shin-Ichi; Kitada, Munehiro; Fujii, Mizue; Kanasaki, Megumi; Nishizawa, Makoto; Nakano, Yasuharu; Tomita, Yasuto; Ueda, Nobuhiko; Kosaka, Takeo; Koya, Daisuke

    2013-11-27

    Dipeptidyl peptidase (DPP)-4 inhibitors are a new class of antidiabetic drugs that increase incretin hormone levels to enhance blood sugar level-dependent insulinotropic effects, suppress glucagon action, and reduce bowel motility. These incretin effects are ideal for blood sugar control. However, the safety profile of DPP-4 inhibitors is not yet established. Herein, we present three cases of ileus, considered to be closely related to the use of DPP-4 inhibitors, in diabetic patients. Each of the three patients exhibited some risk of a deficiency in bowel movement; the onset of ileus was within 40 days after strengthened inhibition of DPP-4. The use of a DPP-4 inhibitor could be safe, although the cases presented herein enable us to inform the scientific community to some of the potential adverse effects of the use of DPP-4 inhibitors in select populations. PMID:24843724

  6. Management of side effects of mTOR inhibitors in tuberous sclerosis patients.

    PubMed

    Sadowski, Krzysztof; Kotulska, Katarzyna; Jóźwiak, Sergiusz

    2016-06-01

    mTOR inhibitors represent a relatively new therapeutic option in the management of patients affected by tuberous sclerosis complex (TSC). Randomized clinical trials support the use of everolimus in the treatment of subependymal giant cell astrocytomas (SEGA) and renal angiomyolipomas (AML) related to TSC. Accumulating data suggest also systemic disease-modifying potential of mTOR inhibitors. Given that increasing number of patients with TSC receive mTOR inhibitors, the issue of adverse events associated with this therapy becomes practically important. In the present study we provide the overview of clinical manifestations and therapeutic options for the most common adverse events related to mTOR inhibitors in TSC patients. PMID:26891243

  7. Association of Acute Interstitial Nephritis With Programmed Cell Death 1 Inhibitor Therapy in Lung Cancer Patients.

    PubMed

    Shirali, Anushree C; Perazella, Mark A; Gettinger, Scott

    2016-08-01

    Immune checkpoint inhibitors that target the programmed death 1 (PD-1) signaling pathway have recently been approved for use in advanced pretreated non-small cell lung cancer and melanoma. Clinical trial data suggest that these drugs may have adverse effects on the kidney, but these effects have not been well described. We present 6 cases of acute kidney injury in patients with lung cancer who received anti-PD-1 antibodies, with each case displaying evidence of acute interstitial nephritis (AIN) on kidney biopsy. All patients were also treated with other drugs (proton pump inhibitors and nonsteroidal anti-inflammatory drugs) linked to AIN, but in most cases, use of these drugs long preceded PD-1 inhibitor therapy. The association of AIN with these drugs in our patients raises the possibility that PD-1 inhibitor therapy may release suppression of T-cell immunity that normally permits renal tolerance of drugs known to be associated with AIN. PMID:27113507

  8. Prevention of bleeding in hemophilia patients with high-titer inhibitors.

    PubMed

    Leissinger, Cindy A; Konkle, Barbara A; Antunes, Sandra V

    2015-06-01

    Inhibitor development is the most serious adverse event linked to the treatment of hemophilia, as it renders standard hemostatic therapy ineffective. Consequently, inhibitor patients are at increased risk for difficult-to-control bleeding and complications, particularly arthropathy and physical disability. Three randomized clinical trials in patients with inhibitors have demonstrated that compared with on-demand bypassing therapy, prophylaxis with a bypassing agent reduces joint and other types of bleeding and improves health-related quality of life. In hemophilia patients without inhibitors, the initiation of prophylaxis with factor (F) VIII or IX prior to the onset of recurrent hemarthroses can prevent the development of joint disease. Whether this is also true for bypassing agent prophylaxis remains to be determined. PMID:25937074

  9. The incidence and treatment of bleeding episodes in non-severe haemophilia A patients with inhibitors.

    PubMed

    van Velzen, Alice S; Eckhardt, Corien L; Streefkerk, Nina; Peters, Marjolein; Hart, Daniel P; Hamulyak, Karly; Klamroth, Robert; Meijer, Karina; Nijziel, Marten; Schinco, Piercarla; Yee, Thynn T; van der Bom, Johanna G; Fijnvandraat, Karin

    2016-03-01

    The development of an inhibitory antibody in non-severe haemophilia A patients may aggravate the bleeding phenotype considerably. Effective treatment of bleeding episodes may be challenging, with ensuing severe complications. At present, evidence is scarce for optimal treatment of bleeding episodes in this patient group. The aim of this study was to describe the incidence and the treatment of bleeding episodes in inhibitor patients in a population-based unselected cohort of non-severe haemophilia A patients with clinically relevant inhibitors. Data were available for 100 of the 107 non-severe haemophilia A patients (factor VIII (FVIII) baseline, 2-40 IU/dl) from 29 centres in Europe and one centre in Australia who had developed a clinically relevant inhibitor between 1980 and 2011. The majority (89 %) of the patients were treated during the inhibitor period for bleeding episodes or a surgical intervention: 66 % needed treatment for bleeding episodes, at a median annual bleeding rate (ABR) of 1.1 (interquartile range (IQR) 0.1-2.5) and a median total of 2 (IQR 1-6) bleeding episodes. Compared to the median ABR before inhibitor development of 0.095 bleeds per year (IQR 0.02-0.42), the increase in ABR is more than a 10-fold. More than 90 % of the bleeding episodes were treated with only one type of product, most frequently (51 %) FVIII concentrates. This study provides the incidence of bleeding episodes and treatment choices in non-severe haemophilia A patients with inhibitors. The 10-fold increase to a median ABR of 1.1 episodes per year emphasizes the impact of inhibitor development for non-severe haemophilia A patients. PMID:26582077

  10. Variability of Voriconazole Trough Levels in Haematological Patients: Influence of Comedications with cytochrome P450(CYP) Inhibitors and/or with CYP Inhibitors plus CYP Inducers.

    PubMed

    Cojutti, Piergiorgio; Candoni, Anna; Forghieri, Fabio; Isola, Miriam; Zannier, Maria Elena; Bigliardi, Sara; Luppi, Mario; Fanin, Renato; Pea, Federico

    2016-06-01

    Voriconazole plasma exposure greatly varies among haematological patients. The purpose of this study was to identify the magnitude of influence of comedications with CYP inhibitors and/or with CYP inhibitors plus CYP inducers on voriconazole trough level (Cmin ). Voriconazole Cmin was retrospectively assessed among haematological patients who underwent therapeutic drug monitoring (TDM). Univariate and multivariate linear mixed-effect regression analyses were performed to identify the independent predictors of normalized Cmin . Of the 83 included patients, 35 had comedications with CYP inhibitors (omeprazole or pantoprazole) and 21 with CYP inhibitors (omeprazole or pantoprazole) plus CYP inducers (methylprednisolone, dexamethasone, phenobarbital, rifampin or carbamazepine). Median Cmin value (n = 199) was 2.4 mg/L with a wide range of distribution (<0.2-13.5 mg/L). Median (IQR) normalized voriconazole Cmin value was significantly higher in the presence of CYP inhibitors (4.20 mg/L, 3.23-5.51 mg/L) than either in the absence of interacting cotreatments (2.55 mg/L, 1.54-3.47 mg/L) or in the presence of CYP inhibitors plus CYP inducers (2.16 mg/L, 1.19-3.09 mg/L). The presence of CYP inhibitors was highly significantly associated with Cmin >5.5 mg/L (OR: 23.22, 95% CI: 3.01-179.09, p = 0.003). No significant association emerged when CYP inhibitors were coadministered with CYP inducers (OR: 3.53, 95% CI: 0.36-34.95, p = 0.280). The amount of expected Cmin increase was significantly influenced by both the type and the dose of the administered proton pump inhibitor. The study highlights that the benefit from TDM of voriconazole may be maximal in those patients who are cotreated with CYP inhibitors and/or with CYP inhibitors plus CYP inducers, especially when receiving proton pump inhibitors (PPIs) at very high dosages intravenously. PMID:26572687

  11. Enhanced Secretory Leukocyte Protease Inhibitor in Human Immunodeficiency Virus Type 1-Infected Patients

    PubMed Central

    Baqui, A. A. M. A.; Meiller, Timothy F.; Falkler, William A.

    1999-01-01

    Secretory leukocyte protease inhibitor (SLPI) has been found to possess activity against the human immunodeficiency virus type 1 (HIV-1) in vitro at physiological concentrations. A study was undertaken to evaluate SLPI levels in human saliva and plasma among HIV-positive (HIV+) patients with various HIV-1 viral loads in comparison to uninfected controls. Whole blood in EDTA and unstimulated saliva samples were collected from 37 HIV+ patients, of whom 20 had a history of intravenous drug abuse (IVDA). Control samples were collected from 20 appropriate age- and sex-matched HIV-1-negative individuals. SLPI was estimated from both saliva and serum samples by an enzyme-linked immunosorbent assay. HIV viral load was determined using a quantitative reverse transcription-PCR. SLPI levels were increased 16.7% in plasma and 10.3% in saliva among HIV+ patients in comparison to uninfected controls. SLPI levels were increased 5.9% in saliva and 3.9% in plasma among HIV+ patients with a high viral load (>10,000 copies/ml) as compared to patients with a low viral load (<400 copies/ml). Only 23% of patients with a high viral load used combination therapy with protease inhibitor drugs, whereas 92.9% of HIV+ patients with a low viral load used protease inhibitors. SLPI levels did not differ significantly among the IVDA patients, patients with different viral loads, or patients using protease inhibitor drugs. There was a statistically significant increase in SLPI levels in saliva among HIV patients in comparison to non-HIV-infected controls. An increase in SLPI levels among HIV+ patients may be a natural consequence of HIV pathogenesis and an important factor in preventing oral transmission of HIV, but this increase may not be evident during plasma viremia in patients with a high viral load. PMID:10548568

  12. Secretory leukocyte proteinase inhibitor is preferentially increased in patients with acute respiratory distress syndrome.

    PubMed

    Sallenave, J M; Donnelly, S C; Grant, I S; Robertson, C; Gauldie, J; Haslett, C

    1999-05-01

    Inappropriate release of proteases from inflammatory and stromal cells can lead to destruction of the lung parenchyma. Antiproteinases such as alpha-1-proteinase inhibitor (alpha1-Pi), secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (elafin) control excess production of human neutrophil elastase. In the present study, the concentrations of alpha1-Pi, SLPI and elafin found in bronchoalveolar lavage (BAL) fluid from control subjects, patients at risk of developing acute respiratory distress syndrome (ARDS) and patients with established ARDS were determined. Levels of all three inhibitors were raised in patients compared with normal subjects. SLPI was increased in the group of patients who were at risk of ARDS and went on to develop the condition, compared with the "at-risk" group who did not progress to ARDS (p=0.0083). Alpha1-Pi and elafin levels were similar in these two populations. In patients with established ARDS, both alpha1-Pi and SLPI levels were significantly increased, compared to patients at risk of ARDS who did (p=0.0089) or did not (p=0.0003) progress to ARDS. The finding of increased antiproteinases shortly before the development of acute respiratory distress syndrome provide further evidence for enhanced inflammation prior to clinical disease. PMID:10414400

  13. Reversal Strategies for Intracranial Hemorrhages in Patients Taking Oral Factor Xa Inhibitors.

    PubMed

    Karli, Betsy; Bartel, Billie; Pavelko, Rachel

    2015-07-01

    Factor Xa (fXa) inhibitors are becoming more common in clinical practice due to a variety of reasons. Unfortunately, limited data are currently available on the safe and efficacious reversal of these agents. This series presents 3 patient cases of intracranial hemorrhage and illustrates the observed effect of different methodologies undertaken in an attempt to reverse the fXa inhibitors implicated. Additionally, a brief review of the current available literature in reversal strategies is provided. The appropriate reversal for fXa inhibitors at this time is unknown. The cases described indicate that the administration of fresh frozen plasma and 4-factor prothrombin complex concentrate may provide minimal benefit in reversing the coagulation abnormalities caused by fXa inhibitors. However, in a life-threatening situation, the addition of these agents should be considered to prevent further progression of the bleed. PMID:26448667

  14. Reversal Strategies for Intracranial Hemorrhages in Patients Taking Oral Factor Xa Inhibitors

    PubMed Central

    Bartel, Billie; Pavelko, Rachel

    2015-01-01

    Factor Xa (fXa) inhibitors are becoming more common in clinical practice due to a variety of reasons. Unfortunately, limited data are currently available on the safe and efficacious reversal of these agents. This series presents 3 patient cases of intracranial hemorrhage and illustrates the observed effect of different methodologies undertaken in an attempt to reverse the fXa inhibitors implicated. Additionally, a brief review of the current available literature in reversal strategies is provided. The appropriate reversal for fXa inhibitors at this time is unknown. The cases described indicate that the administration of fresh frozen plasma and 4-factor prothrombin complex concentrate may provide minimal benefit in reversing the coagulation abnormalities caused by fXa inhibitors. However, in a life-threatening situation, the addition of these agents should be considered to prevent further progression of the bleed. PMID:26448667

  15. Use of tumor necrosis factor (TNF) inhibitors in patients with HIV/AIDS.

    PubMed

    Gallitano, Stephanie M; McDermott, Laura; Brar, Kanwaljit; Lowenstein, Eve

    2016-05-01

    Patients with HIV and AIDS are living longer because of advancements in antiretroviral therapy. These patients are often susceptible to debilitating inflammatory disorders that are refractory to standard treatment. We discuss the relationship of tumor necrosis factor-alpha and HIV and then review 27 published cases of patients with HIV being treated with tumor necrosis factor-alpha inhibitors. This review is limited because no randomized controlled trials have been performed with this patient population. Regardless, we propose that reliable seropositive patients, who are adherent to medication regimens and frequent monitoring and have failed other treatment modalities, should be considered for treatment with tumor necrosis factor-alpha inhibitors. PMID:26774690

  16. Production of interleukin-2 and interleukin-2 inhibitor in patients with palmoplantar pustulosis.

    PubMed

    Shiohara, T; Kobayashi, M; Ishii, Y; Nagashima, M

    1987-01-01

    We studied production of interleukin-2 (IL-2) and IL-2 inhibitor from peripheral blood of patients with palmoplantar pustulosis (PPP). Nineteen patients were divided into two groups: those with and those without arthro-osteitis. Although IL-2 production in both groups of patients was within normal limits, those with arthro-osteitis showed greater fluctuation in relation to the disease activity. The IL-2 production of five PPP patients with arthro-osteitis was greatly enhanced in the inactive stage compared with the active stage. Sera from two patients treated with a combination of etretinate and colchicine contained extremely low levels of IL-2 inhibitory activity. The increased IL-2 production in the inactive stage may be due in part to the depletion of IL-2 inhibitor-producing cells by the treatment. PMID:2448994

  17. Effect of dipeptidyl peptidase-4 inhibitor, vildagliptin on plasminogen activator inhibitor-1 in patients with diabetes mellitus.

    PubMed

    Tani, Shigemasa; Takahashi, Atsuhiko; Nagao, Ken; Hirayama, Atsushi

    2015-02-15

    Dipeptidyl peptidase-4 (DPP-4) inhibitors may affect the serum levels of plasminogen activator inhibitor-1 (PAI-1) associated with triglyceride (TG) metabolism, which is a prognostic factor for cardiovascular disease, in diabetic patients. We conducted an 8-week, prospective, randomized study in which we assigned type 2 diabetic patients who were inadequately controlled with antidiabetic therapy to the vildagliptin group (50 mg bid, n = 49) or the control group (n = 49). The primary efficacy parameter was the change in the serum level of PAI-1, and the secondary end point was the change in the serum levels of TG-rich lipoproteins. In the vildagliptin group, significant decrease of the serum PAI-1 level by 16.3% (p <0.0001) and significant decreases of the serum TG, remnant-like particle cholesterol, and apolipoprotein B levels by 12.1% (p = 0.002), 13.9% (p = 0.003), and 9.5% (p <0.0001), respectively, were observed. No such changes were observed in the control group. Multivariate regression analyses identified the absolute change from the baseline (Δ) of the PAI-1, but not that of the fasting blood glucose or hemoglobin A1c, as independent predictors of the ΔTG, Δ remnant-like particle cholesterol, and Δ apolipoprotein B. In conclusion, treatment of type 2 diabetes with vildagliptin might prevent the progression of atherosclerotic cardiovascular disease in diabetic patients by decreasing the serum PAI-1 levels and improving TG metabolism. PMID:25637323

  18. Upstream mitogen-activated protein kinase (MAPK) pathway inhibition: MEK inhibitor followed by a BRAF inhibitor in advanced melanoma patients.

    PubMed

    Goldinger, Simone M; Zimmer, Lisa; Schulz, Carsten; Ugurel, Selma; Hoeller, Christoph; Kaehler, Katharina C; Schadendorf, Dirk; Hassel, Jessica C; Becker, Juergen; Hauschild, Axel; Dummer, Reinhard

    2014-01-01

    BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients. BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi. A total of 23 melanoma patients (six females, 17 males, aged 47-80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2-23.2) and subsequent for BRAFi 4.5 (1.2-15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed. Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies. PMID:24183461

  19. Implications of over-the-counter proton pump inhibitors for patient counseling by pharmacists.

    PubMed

    Simonson, William

    2013-01-01

    The article reviews the literature on the role of the pharmacist in patient counseling and discusses how that role may apply to patients with frequent heartburn who are seeking an over-the-counter (OTC) treatment. Searches of the National Library of Medicine PubMed database were conducted using the terms "heartburn," "nonprescription," "therapy," "pharmacist," and "counseling," supplemented by additional searches on counseling for prescription products, and by the author's knowledge of pharmacy practice and the scientific literature. Accurate recognition of the signs of heartburn are an important first step in counseling a patient on the appropriate OTC medication; immediate referral to a health care provider is mandatory if cardiac pain or certain gastrointestinal symptoms are present. When counseling a patient about treatments for heartburn, the pharmacist should practice effective listening in an environment that is conducive to communication by the patient. Proton pump inhibitors are effective for the treatment of heartburn; the histamine2 receptor antagonists and antacids should also be considered for appropriate patients. Adverse events have been noted with proton pump inhibitors; however, overall the benefits significantly outweigh the risks and problems are unlikely to arise during the 2-week duration of OTC treatment of heartburn. Pharmacists can provide valuable services to patients with frequent heartburn, particularly with regard to counseling about the condition and appropriate OTC therapy. The availability of numerous OTC products, including antacids, histamine2 receptor antagonists, and proton pump inhibitors, enables pharmacists to fulfill an important clinical role and improve patient satisfaction. PMID:21642828

  20. Effectiveness and safety of phosphodiesterase 5 inhibitors in patients with cardiovascular disease and hypertension.

    PubMed

    Chrysant, Steven G

    2013-10-01

    Phosphodiesterase 5 (PDE 5) inhibitors are selective inhibitors of the enzyme PDE 5, which catalyzes the hydrolysis of cyclic guanosine monophosphate (cGMP), a potent vasodilator and nitric oxide (NO) donor, to its corresponding metabolites (monophosphates). The enzyme PDE 5 is widely distributed in the body, including the heart and blood vessels. Because of its distribution, it was hypothesized that its inhibition could lead to significant coronary vasodilation, which would benefit patients with coronary artery disease (CAD). This hypothesis led to the development of PDE 5 inhibitors with the first being sildenafil citrate. Subsequent studies with sildenafil in patients with CAD demonstrated a modest cardiovascular effect, but a potent action on penile erection in men, resulting in sildenafil becoming a first-line therapy of erectile dysfunction (ED). Subsequently, two more PDE 5 inhibitors (vardenafil and tadalafil) were developed and approved by the Food and Drug Administration (FDA) for the treatment of ED. Recent studies have shown several pleiotropic beneficial effects of PDE 5 inhibitors in patients with CAD, hypertension, heart failure, pulmonary arterial hypertension, diabetes mellitus and Raynaud's phenomenon. Side effects and interactions of PDE 5 inhibitors with other drugs have been minimal, with the exception of their coadministration with nitrates, which could lead to severe vasodilation and hypotension and therefore, their coadministration is prohibited. All these pleiotropic cardiovascular effects of PDE 5 inhibitors and their drug interactions will be discussed in this concise review in the context of the American College of Cardiology / American Heart Association guidelines and the recent developments in this field. PMID:23917809

  1. Phase II Study of the MEK1/MEK2 Inhibitor Trametinib in Patients With Metastatic BRAF-Mutant Cutaneous Melanoma Previously Treated With or Without a BRAF Inhibitor

    PubMed Central

    Kim, Kevin B.; Kefford, Richard; Pavlick, Anna C.; Infante, Jeffrey R.; Ribas, Antoni; Sosman, Jeffrey A.; Fecher, Leslie A.; Millward, Michael; McArthur, Grant A.; Hwu, Patrick; Gonzalez, Rene; Ott, Patrick A.; Long, Georgina V.; Gardner, Olivia S.; Ouellet, Daniele; Xu, Yanmei; DeMarini, Douglas J.; Le, Ngocdiep T.; Patel, Kiran; Lewis, Karl D.

    2013-01-01

    Purpose BRAF mutations promote melanoma cell proliferation and survival primarily through activation of MEK. The purpose of this study was to determine the response rate (RR) for the selective, allosteric MEK1/MEK2 inhibitor trametinib (GSK1120212), in patients with metastatic BRAF-mutant melanoma. Patients and Methods This was an open-label, two-stage, phase II study with two cohorts. Patients with metastatic BRAF-mutant melanoma previously treated with a BRAF inhibitor (cohort A) or treated with chemotherapy and/or immunotherapy (BRAF-inhibitor naive; cohort B) were enrolled. Patients received 2 mg of trametinib orally once daily. Results In cohort A (n = 40), there were no confirmed objective responses and 11 patients (28%) with stable disease (SD); the median progression-free survival (PFS) was 1.8 months. In cohort B (n = 57), there was one (2%) complete response, 13 (23%) partial responses (PRs), and 29 patients (51%) with SD (confirmed RR, 25%); the median PFS was 4.0 months. One patient each with BRAF K601E and BRAF V600R had prolonged PR. The most frequent treatment-related adverse events for all patients were skin-related toxicity, nausea, peripheral edema, diarrhea, pruritis, and fatigue. No cutaneous squamous cell carcinoma was observed. Conclusion Trametinib was well tolerated. Significant clinical activity was observed in BRAF-inhibitor–naive patients previously treated with chemotherapy and/or immunotherapy. Minimal clinical activity was observed as sequential therapy in patients previously treated with a BRAF inhibitor. Together, these data suggest that BRAF-inhibitor resistance mechanisms likely confer resistance to MEK-inhibitor monotherapy. These data support further evaluation of trametinib in BRAF-inhibitor–naive BRAF-mutant melanoma, including rarer forms of BRAF-mutant melanoma. PMID:23248257

  2. Postanesthetic Severe Oral Angioedema in Patient's Taking Angiotensin-Converting Enzyme Inhibitor

    PubMed Central

    Marques, Acílio; Retroz-Marques, Carla; Mota, Sara; Cabral, Raquel; Campos, Matos

    2014-01-01

    Angiotensin-converting enzyme (ACE) inhibitors are the leading cause of a drug-induced angioedema. This occurrence is frequently underdiagnosed, but its relapse can be life-threatening. The authors' intention in reporting this clinical case is to sound a warning about reviewing attitudes and surveillance to try to improve patient perioperative safety. PMID:25431681

  3. Ticagrelor--a new platelet aggregation inhibitor in patients with acute coronary syndromes. An improvement of other inhibitors?

    PubMed

    Kowalczyk, Mariusz; Banach, Maciej; Mikhailidis, Dimitri P; Hannam, Simon; Rysz, Jacek

    2009-12-01

    Antiplatelet agents play an essential role in the treatment of acute coronary syndrome (ACS). Thienopyridines are a class of drugs that function via inhibition of the adenosine diphosphate (ADP) P2Y12 platelet receptors. Currently, clopidogrel, a second generation thienopyridine, is the main drug of choice and the combination of aspirin and clopidogrel is administered orally for the treatment of ACS. Clopidogrel, is a pro-drug that needs to be metabolized in the liver and intestines to form active metabolites. Prasugrel, a third generation thienopyridine, was approved for use in Europe in February 2009, and is currently available in the United Kingdom. All thienopyridines however, have pharmacological limitations that lead to a search for more effective non-thienopyridine P2Y12 inhibitors. Promising results have been reported with ticagrelor, an oral first reversible, direct-acting inhibitor of the P2Y12 receptor. Ticagrelor is the first oral P2Y12 receptor binding antagonist that does not require metabolic activation. Furthermore, ticagrelor has at last 1 active metabolite, which has very similar pharmacokinetics to the parent compound. Therefore, ticagrelor has more rapid onset and more pronounced platelet inhibition than other antiplatelet agents. The safety and efficacy of ticagrelor compared with clopidogrel in ACS patient has been recently evaluated by the PLATelet inhibition and patient Outcomes (PLATO) trial. Ticagrelor compared with clopidogrel had a significantly greater reduction in the death rate from vascular causes, myocardial infarction, or stroke without major bleeding. There was however, an increase in non-procedure related bleeding, dyspnoea and ventricular pauses in the first week of treatment. Further studies on new antiplatelet agents are needed to establish a new "gold standard" antiplatelet therapy. PMID:19946242

  4. The role of previously untreated patient studies in understanding the development of FVIII inhibitors.

    PubMed

    Carcao, M; Re, W; Ewenstein, B

    2016-01-01

    Development of inhibitors against factor VIII (FVIII), the major complication of haemophilia A treatment today, is influenced by multiple factors. Genetic (F8 mutation, family history, ethnicity, polymorphisms in immune modulating genes) and non-genetic (intensive exposure to FVIII, presence of pro-inflammatory signals as might occur with large bleeds, infections, surgery, or other immune stimulants [e.g. vaccines]) risk factors as well as their complex inter-relationships contribute to the inhibitor risk profile of haemophilia patients, particularly in the previously untreated patient (PUP) population. Studies in PUPs have been fundamental to furthering the understanding of FVIII inhibitor development, as well as discovering previously unappreciated risk factors. The multi-factorial nature of inhibitor development makes it difficult to ascertain the contribution of FVIII products in inhibitor development through individual PUP studies. Sufficiently powered studies of large cohorts may overcome these limitations but interpretations should be conducted cautiously. Proper design and implementation of PUP safety studies will become even more important with the introduction of new molecules, such as extended half-life or human cell-line derived FVIII that propose reduced immunogenicity. Despite these difficulties, carefully performed clinical studies in PUPs may provide important insights into the natural history of the immune response to FVIII and may suggest targets for intervention to reduce immunogenicity. PMID:26315604

  5. Discontinuation rates of biologics in patients with rheumatoid arthritis: are TNF inhibitors different from non-TNF inhibitors?

    PubMed Central

    Ramiro, Sofia; Landewé, Robert; van der Heijde, Désirée; Harrison, David; Collier, David; Michaud, Kaleb

    2015-01-01

    Objective To compare discontinuation rates of first and second biologics in rheumatoid arthritis (RA) by tumour-necrosis factor inhibitor (TNFi) status and identify predictors and reasons for discontinuation. Methods From 1998 to 2011, self-reported medication use for RA was assessed every 6 months via questionnaire in a longitudinal study in the USA. Time-on-drug analyses were conducted for individual biologics and groups, and annual rates reported. Time to discontinuation of TNFi and non-TNFi was compared, unadjusted and adjusted using propensity score analyses. Baseline and time-varying predictors of biologic discontinuation were derived through Cox regression. Results Of 2281 patients initiating their first biologic, 1100 (48%) discontinued and of 1097 initiating a second biologic, 537 (49%) discontinued. The annual discontinuation rate was 17% (median 4 years) for first biologic and 20% (median 3.3 years) for second biologic. TNFi had lower discontinuation rates than non-TNFi after propensity score adjustment: HR for first biologic 0.49 (0.34 to 0.71) and 0.68 (0.51 to 0.90) for second biologic. The annual discontinuation rate was significantly lower in patients starting their first biologic before January 2005 vs after (16 vs 25%, p=0.005). Predictors of discontinuation for the first biologic included smoking, higher comorbidity index, worse overall health and not using concomitant methotrexate. Conclusions In this large cohort, patients with RA tended to remain on their first and second biologics for relatively long periods suggesting the drugs’ effectiveness. Discontinuation rates were lower in patients using TNFi, and all rates increased after January 2005 when the number of biologics available increased. PMID:26629366

  6. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients

    PubMed Central

    Chabon, Jacob J.; Simmons, Andrew D.; Lovejoy, Alexander F.; Esfahani, Mohammad S.; Newman, Aaron M.; Haringsma, Henry J.; Kurtz, David M.; Stehr, Henning; Scherer, Florian; Karlovich, Chris A.; Harding, Thomas C.; Durkin, Kathleen A.; Otterson, Gregory A.; Purcell, W. Thomas; Camidge, D. Ross; Goldman, Jonathan W.; Sequist, Lecia V.; Piotrowska, Zofia; Wakelee, Heather A.; Neal, Joel W.; Alizadeh, Ash A.; Diehn, Maximilian

    2016-01-01

    Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment. PMID:27283993

  7. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients.

    PubMed

    Chabon, Jacob J; Simmons, Andrew D; Lovejoy, Alexander F; Esfahani, Mohammad S; Newman, Aaron M; Haringsma, Henry J; Kurtz, David M; Stehr, Henning; Scherer, Florian; Karlovich, Chris A; Harding, Thomas C; Durkin, Kathleen A; Otterson, Gregory A; Purcell, W Thomas; Camidge, D Ross; Goldman, Jonathan W; Sequist, Lecia V; Piotrowska, Zofia; Wakelee, Heather A; Neal, Joel W; Alizadeh, Ash A; Diehn, Maximilian

    2016-01-01

    Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment. PMID:27283993

  8. Spanish Consensus Guidelines on prophylaxis with bypassing agents in patients with haemophilia and inhibitors.

    PubMed

    López-Fernández, Maria Fernanda; Altisent Roca, Carmen; Álvarez-Román, Maria Teresa; Canaro Hirnyk, Mariana Isabel; Mingot-Castellano, Maria Eva; Jiménez-Yuste, Víctor; Cid Haro, Ana Rosa; Pérez-Garrido, Rosario; Sedano Balbas, Carmen

    2016-05-01

    Prophylaxis with the blood clotting factor, factor VIII (FVIII) is ineffective for individuals with haemophilia A and high-titre inhibitors to FVIII. Prophylaxis with the FVIII bypassing agents activated prothrombin complex concentrates (aPCC; FEIBA® Baxalta) or recombinant activated factor VII (rFVIIa; Novo-Seven®, Novo Nordisk) may be an effective alternative. It was our aim to develop evidence -and expert opinion- based guidelines for prophylactic therapy for patients with high-titre inhibitors to FVIII. A panel of nine Spanish haematologists undertook a systematic review of the literature to develop consensus-based guidance. Particular consideration was given to prophylaxis in patients prior to undergoing immune tolerance induction (ITI) (a process of continued exposure to FVIII that can restore sensitivity for some patients), during the ITI period and for those not undergoing ITI or for whom ITI had failed. These guidelines offer guidance for clinicians in deciding which patients might benefit from prophylaxis with FVIII bypassing agents, the most appropriate agents in various clinical settings related to ITI, doses and dosing regimens and how best to monitor the efficacy of prophylaxis. The paper includes recommendations on when to interrupt or stop prophylaxis and special safety concerns during prophylaxis. These consensus guidelines offer the most comprehensive evaluation of the clinical evidence base to date and should be of considerable benefit to clinicians facing the challenge of managing patients with severe haemophilia A with high-titre FVIII inhibitors. PMID:26842562

  9. Aromatase Inhibitor-Induced Erythrocytosis in a Patient Undergoing Hormonal Treatment for Breast Cancer

    PubMed Central

    Yeruva, Sri Lakshmi Hyndavi; Ogbonna, Onyekachi Henry; Oneal, Patricia

    2015-01-01

    Aromatase inhibitors (AIs) are most commonly used for breast cancer patients with hormone receptor positive disease. Although the side effect profile of aromatase inhibitors is well known, including common side effects like arthralgia, bone pain, arthritis, hot flashes, and more serious problems like osteoporosis, we present a case of an uncommon side effect of these medications. We report the case of a postmenopausal woman on adjuvant hormonal therapy with anastrozole after completing definitive therapy for stage IIIB estrogen receptor-positive breast cancer, who was referred to hematology service for evaluation of persistent erythrocytosis. Primary and known secondary causes of polycythemia were ruled out. On further evaluation, we found that her erythrocytosis began after initiation of anastrozole and resolved after it was discontinued. We discuss the pathophysiology of aromatase inhibitor-induced erythrocytosis and reference of similar cases reported in the literature. PMID:26137331

  10. Activity of thrombin-activatable fibrinolysis inhibitor in the plasma of patients with abdominal aortic aneurysm.

    PubMed

    Dubis, Joanna; Zuk, Natalia; Grendziak, Ryszard; Zapotoczny, Norbert; Pfanhauser, Monika; Witkiewicz, Wojciech

    2014-04-01

    Patients with abdominal aortic aneurysm (AAA) experience impaired balance between fibrinolysis and coagulation, manifested by increased prothrombotic tendency and intensified inflammatory processes. The aim of this study was to evaluate the TAFI activity level (thrombin activatable fibrinolysis inhibitor) in the plasma of AAA patients. Plasma levels of PAI-1 (plasminogen activator inhibitor type 1), urokinase-type plasminogen activator and uPAR (urokinase-type plasminogen activator receptor) were measured as markers of fibrinolytic activity. The study showed that the activity of the thrombin-activatable fibrinolysis inhibitor in the plasma of AAA patients was significantly lower than in the plasma of the control individuals (64.6 ± 10.1 vs. 54.2 ± 10.9%, P < 0.0001). TAFI activity positively correlated with the white blood cell count (r = 0.486, P < 0.005). The uPAR concentration in the AAA patients was statistically significantly higher than in the control group and positively correlated with TAFI activity (r = 0.409, P = 0.02). The levels of PAI-1 and D-dimers (fibrin fragments) were significantly higher in patients with AAA than in the control group (44.3 ± 17.5 vs. 21.7 ± 8.7 ng/ml and 1869.6 ± 1490.1 vs. 181.5 ± 188.6 ng/ml, respectively). Lowered activity of the fibrinolysis inhibitor TAFI may heighten the blood fibrinolytic potential in AAA patients and contribute to the development of comorbidities. Therefore, TAFI participation in AAA pathogenesis cannot be excluded. PMID:24378973

  11. [PCSK9 inhibitors in hypercholesterolemia : New hope for patients with diabetes mellitus?].

    PubMed

    Parhofer, K G

    2016-05-01

    The interrelation between glucose and lipid metabolism is complex and comprises many aspects. In this context diabetic dyslipidemia is of utmost importance as it represents the crucial link between diabetes and cardiovascular disease. Although hypertriglyceridemia is usually the most prominent lipid abnormality in diabetic patients, reduction of low-density lipoprotein (LDL) cholesterol is the most important strategy to prevent cardiovascular disease. Statin trials and more recently the combination of statin with ezetimibe clearly showed that diabetic patients benefit from low LDL cholesterol levels. In this context the newly developed proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors are of great interest as they reduce LDL cholesterol by 50-70 % independent of co-medication and largely independent of the underlying dyslipidemia. Subgroup analyses of phase 2 and phase 3 studies indicated that diabetic patients show a similar response to PCSK9 inhibitors as non-diabetic patients. Furthermore, the overall very low rate of side effects seems to be comparable between diabetic and non-diabetic patients. In contrast to statins PCSK9 inhibitors do not lead to an increased rate of new onset diabetes. Although data from safety studies (post hoc analyses) are very promising concerning the prevention of cardiovascular events, data from outcome studies will only become available in 2016. Until then PCSK9 inhibitors should be restricted to patients with very high risk and a significant distance from the LDL cholesterol target values (despite maximum possible lipid-lowering therapy with statins and ezetimibe). This approach will most likely have to be adapted when outcome data are available. PMID:26961236

  12. Comparative effectiveness of topical calcineurin inhibitors in adult patients with atopic dermatitis.

    PubMed

    Frankel, Hillary C; Qureshi, Abrar A

    2012-04-01

    Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by extreme pruritis and lichenified papules and plaques that may begin in or persist into adulthood. Topical corticosteroids are first-line prescription therapy for AD; they are efficacious and have a well established safety profile. The topical calcineurin inhibitors tacrolimus and pimecrolimus were approved by the US FDA in 2000 and 2001, respectively, as second-line topical therapy for AD. This review evaluates the available studies on the comparative effectiveness, safety, cost, and impact on quality of life of topical corticosteroids and topical calcineurin inhibitors for the treatment of adult AD. Tacrolimus was found to be as effective as class III-V topical corticosteroids for AD of the trunk and extremities, and more effective than low-potency class VI or VII corticosteroids for AD of the face or neck. Pimecrolimus was less effective than both tacrolimus and low-potency topical corticosteroids for moderate to severe AD. The short-term safety studies found that, compared with topical corticosteroid-treated adults, patients treated with topical calcineurin inhibitors had an increased frequency of application-site reactions, an equivalent infection risk, and a decreased risk of skin atrophy. The long-term safety of topical calcineurin inhibitors remains under investigation. Currently published studies that evaluated the comparative cost and quality-of-life effects compared tacrolimus with less potent topical corticosteroids despite the availability of equivalent potency corticosteroids. Further cost and quality-of-life studies are needed that compare topical calcineurin inhibitors with stronger classes of topical corticosteroids over longer time periods. The available clinical trials data do not suggest an efficacy advantage for topical calcineurin inhibitors over topical corticosteroids in adults with AD of the trunk and extremities, and there is not yet adequate evidence to support

  13. JAK2 inhibitors do not affect stem cells present in the spleens of patients with myelofibrosis.

    PubMed

    Wang, Xiaoli; Ye, Fei; Tripodi, Joseph; Hu, Cing Siang; Qiu, Jiajing; Najfeld, Vesna; Novak, Jesse; Li, Yan; Rampal, Raajit; Hoffman, Ronald

    2014-11-01

    Dysregulation of Janus kinase (JAK)-signal transducer and activator of transcription signaling is central to the pathogenesis of myelofibrosis (MF). JAK2 inhibitor therapy in MF patients results in a rapid reduction of the degree of splenomegaly, yet the mechanism underlying this effect remains unknown. The in vitro treatment of splenic and peripheral blood MF CD34(+) cells with the JAK1/2/3 inhibitor, AZD1480, reduced the absolute number of CD34(+), CD34(+)CD90(+), and CD34(+)CXCR4(+) cells as well as assayable hematopoietic progenitor cells (HPCs) irrespective of the JAK2 and calreticulin mutational status. Furthermore, AZD1480 treatment resulted in only a modest reduction in the proportion of HPCs that were JAK2V617F(+) or had a chromosomal abnormality. To study the effect of the drug on MF stem cells (MF-SCs), splenic CD34(+) cells were treated with AZD1480 and transplanted into immunodeficient mice. JAK2 inhibitor therapy did not affect the degree of human cell chimerism or the proportion of malignant donor cells. These data indicate that JAK2 inhibitor treatment affects a subpopulation of MF-HPCs, while sparing another HPC subpopulation as well as MF-SCs. This pattern of activity might account for the reduction in spleen size observed with JAK2 inhibitor therapy as well as the rapid increase in spleen size observed frequently with its discontinuation. PMID:25193869

  14. JAK2 inhibitors do not affect stem cells present in the spleens of patients with myelofibrosis

    PubMed Central

    Wang, Xiaoli; Ye, Fei; Tripodi, Joseph; Hu, Cing Siang; Qiu, Jiajing; Najfeld, Vesna; Novak, Jesse; Li, Yan; Rampal, Raajit

    2014-01-01

    Dysregulation of Janus kinase (JAK)–signal transducer and activator of transcription signaling is central to the pathogenesis of myelofibrosis (MF). JAK2 inhibitor therapy in MF patients results in a rapid reduction of the degree of splenomegaly, yet the mechanism underlying this effect remains unknown. The in vitro treatment of splenic and peripheral blood MF CD34+ cells with the JAK1/2/3 inhibitor, AZD1480, reduced the absolute number of CD34+, CD34+CD90+, and CD34+CXCR4+ cells as well as assayable hematopoietic progenitor cells (HPCs) irrespective of the JAK2 and calreticulin mutational status. Furthermore, AZD1480 treatment resulted in only a modest reduction in the proportion of HPCs that were JAK2V617F+ or had a chromosomal abnormality. To study the effect of the drug on MF stem cells (MF-SCs), splenic CD34+ cells were treated with AZD1480 and transplanted into immunodeficient mice. JAK2 inhibitor therapy did not affect the degree of human cell chimerism or the proportion of malignant donor cells. These data indicate that JAK2 inhibitor treatment affects a subpopulation of MF-HPCs, while sparing another HPC subpopulation as well as MF-SCs. This pattern of activity might account for the reduction in spleen size observed with JAK2 inhibitor therapy as well as the rapid increase in spleen size observed frequently with its discontinuation. PMID:25193869

  15. Treatment of chronic hepatitis C: anticipated impact of resistance in patients treated with protease inhibitors.

    PubMed

    Kronenberger, Bernd; Zeuzem, Stefan

    2009-02-01

    A main target of specifically targeted antiviral therapy for hepatitis C (STAT-C) is the NS3-protease, which has key functions in the hepatitis C virus (HCV) replication cycle. HCV/NS3-protease inhibitors have shown high antiviral activity in vitro and in patients with chronic hepatitis C. Protease-resistant HCV variants occurred rapidly in patients receiving protease-inhibitor monotherapy. The development of resistance can be best explained by selection of preexisting resistant variants, which grow out under selective pressure. Numerous mutations associated with resistance were identified. Clinical trials showed that protease-resistant strains are sensitive to interferon and that a triple combination of protease inhibitors, peginterferon, and ribavirin may improve the sustained virologic response rate compared with standard peginterferon/ribavirin combination therapy. Overall, it can be anticipated that successful treatment with protease inhibitors will require either combination therapy with peginterferon/ribavirin or a combination of STAT-C compounds with distinct modes of action and resistance patterns. PMID:19166654

  16. Results of 4-year analysis of conversion from calcineurin inhibitors to mTOR inhibitors in renal transplant patients: single-center experience.

    PubMed

    Sert, Mehmet; Celik, Ali; Kural, Kemal; Ersan, Sibel; Ataca, Pinar; Atila, Koray; Cavdar, Caner; Sifil, Aykut; Bora, Seymen; Gulay, Hüseyin; Camsari, Taner

    2011-01-01

    In this retrospective study, 83 patients were accepted. Mammalian target of rapamycin (mTOR) group consisting of 37 patients were converted from calcineurin inhibitors (CNI), and the control group included 46 patients (initially CNI-receiving patients). As a control-match of each mTOR inhibitor patient, the succeeding patient with transplantation who continued CNI therapy was chosen. All patients received CNI, MMF, and prednisolone as an immunosuppressive therapy initially. In comparison of two groups, there was no significant difference between sex, donor organ source, donor organ ischemia time, or mismatches. However, mean age between groups was significantly different (mTOR group: 48.3 ± 12, CNI group: 38.6 ± 11, p < 0.001). Decision of conversion to mTOR inhibitors in 30 patients was made by biopsy. The reasons for conversion were determined as CNI nephrotoxicity in 15 patients, chronic allograft nephropathy in 15 patients, malignancy in 6 patients, and renal artery stenosis in 1 patient. Basal glomerular filtration rates (GFRs) were markedly lower in mTOR group than in CNI group (38.8 mL/min vs. 72.7 mL/min). At the end of 48-month follow-ups, GFR increased from 38 mL/min to 54 mL/min in mTOR group; however, it decreased to 53 mL/min from 72 mL/min in CNI group. There was no difference left between the two groups in GFR after 4-year follow-up. Hyperlipidemia was higher in mTOR group. Acute rejection rates were similar. Cytomegalovirus (CMV) disease was more prevalent in CNI group. Graft failure developed due to secondary reasons, causing mortality in both groups. We suggest that conversion to mTOR inhibitors maintains and improves graft functions well. PMID:21787153

  17. Preoperative angiotensin converting enzyme inhibitor usage in patients with chronic subdural hematoma: Associations with initial presentation and clinical outcome.

    PubMed

    Neidert, Marian C; Schmidt, Tobias; Mitova, Tatyana; Fierstra, Jorn; Bellut, David; Regli, Luca; Burkhardt, Jan-Karl; Bozinov, Oliver

    2016-06-01

    The aim of this study is to analyze the association of preoperative usage of angiotensin converting enzyme (ACE) inhibitors with the initial presentation and clinical outcome of patients with chronic subdural hematoma (cSDH). Patients treated for cSDH between 2009 and 2013 at our institution were included in this retrospective case-control study. Medical charts were reviewed retrospectively and data were analyzed using descriptive and inferential statistics. Out of 203 patients (58 females, mean age 73.2years), 53 (26%) patients were on ACE inhibitors before their presentation with cSDH. Median initial hematoma volume in individuals with ACE inhibitors (179.2±standard error of the mean [SEM] 13.0ml) was significantly higher compared to patients without ACE inhibitors (140.4±SEM 6.2ml; p=0.007). There was an increased probability of surgical reintervention in the ACE inhibitor group (12/53, 23% versus 19/153, 12%; p=0.079), especially in patients older than 80years (6/23, 26% versus 3/45, 7%; p=0.026). ACE inhibitors are associated with higher hematoma volume in patients with cSDH and with a higher frequency of recurrences requiring surgery (especially in the very old). We hypothesize that these effects are due to ACE inhibitor induced bradykinin elevation causing increased vascular permeability of the highly vascularized neomembranes in cSDH. PMID:26898577

  18. Off-label use of TNF-alpha inhibitors in a dermatological university department: retrospective evaluation of 118 patients.

    PubMed

    Sand, Freja Lærke; Thomsen, Simon Francis

    2015-01-01

    Tumor necrosis factor-alpha (TNF)-alpha inhibitors are licensed for patients with severe refractory psoriasis and psoriatic arthritis. However, TNF-alpha inhibitors have also been used off-label for various recalcitrant mucocutaneous diseases. This study aimed to evaluate the efficacy and safety of TNF-alpha inhibitors used for off-label dermatological indications. We retrospectively evaluated patient records of 118 patients treated off-label with TNF-alpha inhibitors in a dermatological university department. Patients presented with severe aphthous stomatitis/genital aphthous lesions (26), chronic urticaria (25), hidradenitis suppurativa (29), acne conglobata (11), dissecting cellulitis of the scalp (two), orofacial granulomatosis (four), sarcoidosis (four), granuloma annulare (two), granulomatous rosacea (one), granuloma faciale (one), subcorneal pustulosis (one), pyoderma gangrenosum (four), Sweet's syndrome (four), Well's syndrome (one), benign familial pemphigus (one), lichen planus (one), and folliculitis decalvans (one). A significant number of these patients went into remission during therapy with TNF-alpha inhibitors. A total of 11 patients (9%) experienced severe adverse effects during therapy. Off-label therapy with TNF-alpha inhibitors may be considered for selected patients with severe recalcitrant mucocutaneous diseases. The risk of severe adverse effects signals that a thorough benefit-risk assessment should be performed before initiating off-label treatment with TNF-alpha inhibitors for these conditions. PMID:25731720

  19. Management of BRAF and MEK inhibitor toxicities in patients with metastatic melanoma

    PubMed Central

    Welsh, Sarah J.

    2015-01-01

    Following the discovery that nearly half of all cutaneous melanomas harbour a mutation in the BRAF gene, molecular targeted kinase inhibitors have been developed for the treatment of metastatic melanoma and have dramatically improved outcomes for those patients with BRAF mutant disease, achieving high levels of objective response and prolonging survival. Since 2011, the specific BRAF targeted agents, vemurafenib and dabrafenib, and the MEK inhibitor, trametinib, have been licensed for the treatment of patients with unresectable or metastatic BRAF mutant melanoma. As with other biological targeted agents, these drugs are associated with predictable patterns of adverse events. Proactive toxicity management is important to ensure maximum treatment benefit and avoid unnecessary treatment discontinuation. We review the most common and serious adverse events associated with BRAF targeted agents and suggest management algorithms to guide practitioners in using these drugs effectively in the clinic. PMID:25755684

  20. Risk of Infectious Complications in Hemato-Oncological Patients Treated with Kinase Inhibitors

    PubMed Central

    Reinwald, Mark; Boch, Tobias; Hofmann, Wolf-Karsten; Buchheidt, Dieter

    2015-01-01

    Infectious complications are a major cause of morbidity and mortality in patients with hemato-oncological diseases. Although disease-related immunosuppression represents one factor, aggressive treatment regimens, such as chemotherapy, stem cell transplantation, or antibody treatment, account for a large proportion of infectious side effects. With the advent of targeted therapies affecting specific kinases in malignant diseases, the outcome of patients has further improved. Nonetheless, dependent on the specific pathway targeted or off-target activity of the kinase inhibitor, therapy-associated infectious complications may occur. We review the most common and approved kinase inhibitors targeting a variety of hemato-oncological malignancies for their immunosuppressive potential and evaluate their risk of infectious side effects based on preclinical evidence and clinical data in order to raise awareness of the potential risks involved. PMID:27127405

  1. Acquired factor V inhibitor in a patient with mantle cell lymphoma presenting with hematuria followed by thrombosis: a case report

    PubMed Central

    AlJohani, Naif I; Matthews, John H

    2014-01-01

    Acquired factor V inhibitor is a rare hemostatic disorder that presents with hemorrhagic manifestations in the vast majority of patients. Factor V inhibitor may develop through a variety of mechanisms involving development of alloantibodies or autoantibodies specific to Factor V. Autoantibodies, in particular, have been reported in a number of conditions. In this report, we describe a case of acquired factor V inhibitor in a patient with mantle cell lymphoma who presented with hematuria. Seven weeks after diagnosis and successful management, the patient developed deep vein thrombosis in the right lower extremity. The patient’s factor V levels were normalized, and the inhibitor was successfully eradicated using corticosteroids. Here, we discuss this rare disorder, its unusual manifestation, and provide a mini-review of the current literature regarding factor V inhibitors. PMID:24591851

  2. Cellular HIV-1 DNA quantitation in patients during simplification therapy with protease inhibitor-sparing regimens.

    PubMed

    Sarmati, Loredana; Parisi, Saverio Giuseppe; Nicastri, Emanuele; d'Ettorre, Gabriella; Andreoni, Carolina; Dori, Luca; Gatti, Francesca; Montano, Marco; Buonomini, Anna Rita; Boldrin, Caterina; Palù, Giorgio; Vullo, Vincenzo; Andreoni, Massimo

    2007-07-01

    Simplified regimens containing protease-inhibitors (PI)-sparing combinations were used in patients with virological suppression after prolonged highly active antiretroviral therapy. This study evaluated the total HIV-1 DNA quantitation as a predictor of long-term success for PI-sparing simplified therapy. Sixty-two patients were enrolled in a prospective non-randomized cohort. All patients have been receiving a triple-therapy regimen, two nucleoside reverse transcriptase inhibitors (NRTIs) plus one PI, for at least 9 months and were characterized by undetectable plasma HIV-1 RNA levels (<50 cp/ml) for at least 6 months. Patients were changed to a simplified PI-sparing regimen to overcome PI-associated adverse effects. HIV-DNA levels in peripheral blood mononuclear cells (PBMCs) were evaluated at baseline and at the end of follow-up. Patients with proviral DNA levels below the median value (226 copies/10(6) PBMCs) had a significant higher CD4 cell count at nadir (P = 0.003) and at enrolment (P = 0.001) with respect to patients with HIV-DNA levels above the median value. At month 18, 53 out of 62 (85%) patients on simplified regimen showed virological success, 4 (6.4%) patients experienced virological failure and 5 (8%) patients showed viral blip. At logistic regression analysis, HIV-DNA levels below 226 copies/10(6) PBMCs at baseline were associated independently to a reduced risk of virological failure or viral blip during simplified therapy (OR 0.002, 95% CI 0.001-0.46, P = 0.025). The substitution of PI with NRTI or non-NRTIs may represent an effective treatment option. Indeed, treatment failure or viral blip were experienced by 6% and 8% of the patients on simplified therapy, respectively. In addition, sustained suppression of the plasma viral load was significantly correlated with low levels of proviral DNA before treatment simplification. PMID:17516532

  3. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in sera and tissue of patients with Dupuytren's disease.

    PubMed

    Ulrich, Dietmar; Hrynyschyn, Klaus; Pallua, Norbert

    2003-10-01

    Dupuytren's contracture is a fibroproliferative disorder characterized by progressive deposition of mature collagen fibers. In other fibrotic diseases affecting organs such as the liver, lung, heart, and skin, matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), play an important role. In this study, serum concentrations of MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were determined in 22 patients (five women and 17 men; average age, 67 +/- 11 years) with Dupuytren's disease using an enzyme-linked immunosorbent assay. Tissue samples were obtained for standard histological and immunohistochemical analyses. Sera and samples of palmar fascia from 20 patients (13 women and seven men; average age, 60 +/- 15 years) who had undergone hand surgery for carpal tunnel syndrome were used as the control group. Statistical analysis was performed using the Mann-Whitney test. Patients with Dupuytren's contracture presented with a TIMP-1 concentration of 437 +/- 160 ng/ml, a significantly higher TIMP-1 concentration than that seen in the control patients, who had a concentration of 321 +/- 70 ng/ml (p < 0.05). Patients with a proliferative active disease (n = 14) had a significantly higher TIMP-1 concentration (525 +/- 136 ng/ml) than patients (n = 8) with a contracture in the late involutional and residual phase (286 +/- 41 ng/ml; p < 0.05). There were no significant differences in the TIMP-2, MMP-1, MMP-2, and MMP-9 serum concentrations between patients with palmar fibromatosis and the control group. Patients with Dupuytren's disease had a significantly lower MMP-to-TIMP ratio (1.1 +/- 0.3; p < 0.05) than the control group (1.5 +/- 0.35). Patients with an active palmar fibromatosis presented a significantly (p < 0.05) reduced ratio (1 +/- 0.2) compared with those in later phases (1.4 +/- 0.3). TIMP-1 and TIMP-2 could be detected in tissue of patients with Dupuytren's contracture, with an accumulation in proliferative

  4. Patient adherence to tyrosine kinase inhibitor therapy in chronic myeloid leukemia.

    PubMed

    Jabbour, Elias J; Kantarjian, Hagop; Eliasson, Lina; Cornelison, A Megan; Marin, David

    2012-07-01

    Dramatically improved survival associated with tyrosine kinase inhibitor (TKI) therapy has transformed the disease model for chronic myeloid leukemia (CML) to one of long-term management, but treatment success is challenged with poor medication adherence. Many risk factors associated with poor adherence can be ameliorated by close monitoring, dose modification, and supportive care. Controlling risk factors for poor adherence in combination with patient education that includes direct communication between the health care team and the patient are essential components for maximizing the benefits of TKI therapy. PMID:22473898

  5. A 3H-flunitrazepam binding inhibitor is present in psychiatric patients' sera.

    PubMed

    Marazziti, D; Pietrini, P; Martini, C; Giannaccini, G; Perugi, G; Placidi, G F; Cassano, G B; Lucacchini, A

    1987-01-01

    We investigated the possible existence of endogenous compounds acting on benzodiazepine central receptors in the serum of patients with panic attacks or depression. Our results show the presence of a substance which inhibits the 3H-flunitrazepam binding specifically in the samples taken from the patients' groups, and which is not present in normal controls, in the range of concentrations used. This compound has a molecular weight below 1,000 daltons, is heat-stable, and resistant to proteolytic degradation. The demonstration of this inhibitor opens new perspectives in the study of the biochemistry of anxiety. PMID:2836759

  6. Cough and cold remedies: a potential danger to patients on monoamine oxidase inhibitors.

    PubMed

    Cuthbert, M F; Greenberg, M P; Morley, S W

    1969-02-15

    Experiments have been carried out in healthy volunteers to study the effects of phenylpropanolamine on the blood pressure and possible interactions with monamine oxidase inhibitors.In three subjects 50 mg. of phenylpropanolamine taken orally produced a modest rise of systolic pressure. Two proprietary preparations containing this dose in a slow-release form had no significant effect on the blood pressure. In all three subjects 100 mg. of phenylpropanolamine taken orally caused a more pronounced rise of systolic pressure and a rise of diastolic pressure.In contrast, 50 mg. of phenylpropanolamine orally caused a rapid and potentially dangerous rise of blood pressure in a subject taking the monamine oxidase inhibitor tranylcypromine, and a similar acute rise of blood pressure occurred in this subject when given a proprietary cough linctus containing the same dose of phenylpropanolamine. These and other results suggest that severe hypertensive episodes are more likely to occur when preparations containing phenylpropanolamine in a free form, rather than in slow-release form, are taken by patients being treated with monoamine oxidase inhibitors.The acute rise of blood pressure due to the interaction of phenylpropanolamine and monoamine oxidase inhibitors was reversed by intramuscular injection of phentolamine. PMID:5763957

  7. Efficacy and Tolerability of Integrase Inhibitors in Antiretroviral-Naive Patients.

    PubMed

    D'Abbraccio, Maurizio; Busto, Annunziata; De Marco, Mario; Figoni, Mario; Maddaloni, Adelaide; Abrescia, Nicola

    2015-01-01

    Integrase strand transfer inhibitors are a new class of antiretroviral agents recently licensed for the treatment of both naive and experienced HIV-infected patients. They inhibit the catalytic activity of the HIV-encoded enzyme integrase and prevent the integration of the HIV genome into the host cell genome, so slowing the propagation of the infection. Integrase strand transfer inhibitors cause a rapid drop in viral load, exhibit very low drug interactions (except elvitegravir/cobicistat), and have low pill burden and convenient dosing frequency. Drugs in this class have been compared to others in antiretroviral-naive patients with efavirenz and with protease inhibitors. Final results of the STARTMRK trial highlighted the better virologic and immunologic performance of raltegravir over efavirenz/emtricitabine/tenofovir disoproxil co-formulation. Raltegravir was also superior to atazanavir/ritonavir and darunavir/ritonavir in the ACTG 5257 study for the combined virologic/tolerability endpoint. Elvitegravir/cobicistat/emtricitabine/tenofovir was non-inferior to efavirenz/emtricitabine/tenofovir and to atazanavir/ritonavir plus emtricitabine/tenofovir in terms of confirmed virologic response in the GS-US-236-0102 and GS-US-236-0103 studies, respectively. Finally, dolutegravir showed non-inferiority compared to raltegravir in the SPRING-2 study and was superior to efavirenz and darunavir/ritonavir in the SINGLE and FLAMINGO trials, respectively. The aim of this review is to analyze the data on efficacy and safety of integrase strand transfer inhibitors in antiretroviral-naive HIV patients and discuss the strengths and weaknesses of drugs within this class. PMID:26450805

  8. ITI choice for the optimal management of inhibitor patients - from a clinical and pharmacoeconomic perspective.

    PubMed

    Oldenburg, J; Austin, S K; Kessler, C M

    2014-09-01

    The development of alloantibody inhibitors against factor VIII (FVIII) represents the most significant complication of haemophilia care. Inhibitors tend to develop early in the course of treatment in about 20-30% of patients with severe haemophilia who receive on-demand or prophylactic FVIII therapy. Many factors are associated with inhibitor formation, including disease severity, major FVIII gene defects, family history and non-Caucasian race, as well as age at first treatment, intensity of early treatment, use of prophylaxis and product choice. As these latter treatment-related variables are modifiable, they provide opportunity to minimize inhibitor incidence at the clinical level. Data from the Bonn Centre in Germany have indicated an overall success rate of 78% for immune tolerance induction (ITI) therapy, with a failure rate of 15% and with some treatments either ongoing (3%) or withdrawn (4%). Similarly, data from the G-ITI study, the largest international multicentre ITI study using a single plasma-derived (pd) FVIII/von Willebrand factor (VWF) product, have demonstrated success rates (complete and partial) in primary and rescue ITI of 87% and 74%, respectively, with 85% of poor prognosis patients achieving success. Favourable clinical results based on success rates and time to tolerization continue to be reported for use of pdFVIII/VWF in ITI, with pdFVIII/VWF having a particular role in patients who require rescue ITI and those with a poor prognosis for success. Data from prospective, randomized, controlled clinical studies, such as RES.I.ST (Rescue Immune Tolerance Study), are eagerly awaited. Another factor to consider with ITI therapy is cost; preliminary data from an updated decision analytic model have provided early evidence that ITI has an economic advantage compared with on-demand or prophylactic therapy. PMID:24975701

  9. Use of renin angiotensin system inhibitors in patients with chronic kidney disease.

    PubMed

    Adam, W R; Wright, J R

    2016-05-01

    Current guidelines recommend renin angiotensin system inhibitors (RASI) as key components of treatment of hypertension in patients with chronic kidney disease (CKD), because of their effect on reducing the future rate of loss of glomerular filtration rate (GFR). A common risk of RASI in CKD is a haemodynamically mediated, and reversible, fall in GFR of varying severity and duration, any time after commencement of the Inhibitors. A benefit of the acute reduction in filtration rate with RASI may be a reduction in the future rate of loss in GFR: the greatest benefit likely to be in those patients with a greater rate of loss of GFR prior to, and a lesser acute loss of GFR after, introduction of RASI; and in those patients with significant proteinuria. An acute loss of GFR of >25% following the introduction of RASI is an indication to cease the RASI. An acute loss of GFR < 25% requires consideration of the likely risks of the lower GFR and benefits of any future reduced rate of loss of GFR. A fall in GFR in patients while on RASI is usually associated with a remediable cause. When the cause for the fall in GFR is not revealed, and the fall is less than 25%, hopeful expectancy is recommended. Hyperkalaemia in patients with CKD on RASI is more common with more severe disease, potassium retaining diuretics and hypoaldosteronism. Treatment should be modified to maintain a plasma potassium <6 mmol/L. PMID:27170242

  10. Is the cardiovascular toxicity of NSAIDS and COX-2 selective inhibitors underestimated in patients with haemophilia?

    PubMed

    Boban, Ana; Lambert, Catherine; Hermans, Cedric

    2016-04-01

    Joint pain secondary to chronic arthropathy represents one of the most common and debilitating complications of haemophilia, often requiring analgesic care. When compared with nonselective non-steroidal anti-inflammatory drugs (ns-NSAIDs), selective COX-2 inhibitors (coxibs) offer the major advantage of not increasing the bleeding risk, thus being a better choice of analgesics for haemophilia patients. However, several studies have highlighted the cardiovascular risks posed by coxibs and NSAIDs. Given the assumed protection against thrombosis conferred by the deficiency in coagulation factors VIII or IX, these precautions regarding the use of coxibs and NSAIDs have never really been taken into account in haemophilia management. However, contrary to what has long been suspected, haemophilia patients are indeed affected by the same cardiovascular risk factors as nonhaemophiliac patients. Further studies should be conducted to evaluate the impact of NSAIDs on cardiovascular risks and the prevalence of hypertension in haemophilia patients. PMID:26899022

  11. Pyogenic granuloma in patients treated with selective BRAF inhibitors: another manifestation of paradoxical pathway activation.

    PubMed

    Henning, Benjamin; Stieger, Pascale; Kamarachev, Jivko; Dummer, Reinhard; Goldinger, Simone M

    2016-06-01

    Cutaneous toxicities under therapy with selective BRAF inhibitors such as vemurafenib or encorafenib (LGX818) are frequent, including plantar hyperkeratosis, squamous cell carcinoma, and second primary melanoma. Pyogenic granuloma is a benign, rapidly growing, eruptive hemangioma that often bleeds and ulcerates. Common causes are mechanical trauma and cast immobilization, as well as multiple drugs such as retinoids and antineoplastic agents. However, the development of pyogenic granuloma under treatment with encorafenib (LGX818) has not yet been reported. These three cases might be further examples for paradoxical activation of the mitogen-activated protein kinase pathway. We report three male patients with metastatic BRAFV600E-mutated melanoma who developed pyogenic granulomas 16, 10, and 12 weeks after treatment initiation with the selective BRAF inhibitors vemurafenib or encorafenib (LGX818). Except for one patient receiving retinoids, the clinical history for other frequent causes of pyogenic granuloma was negative. Pyogenic granulomas are not listed in the drugs investigator brochure but seem to be associated with selective BRAF inhibitors and might be a cutaneous phenomenon of paradoxical mitogen-activated protein kinase pathway activation. This correlation has to be confirmed by further observations. PMID:27116335

  12. Tailoring hemostatic therapies to lower inhibitor development in previously untreated patients with severe hemophilia A.

    PubMed

    Mannucci, P M; Mancuso, M E; Franchini, M

    2016-07-01

    After technological progress provided safer therapeutic products for patients with hemophilia A, the development of alloantibodies (inhibitors) neutralizing the coagulant activity of infused factor VIII (FVIII) remains the most serious complication of replacement therapy, predisposing patients to greater morbidity and causing higher treatment costs. The pathogenesis of inhibitors, which develop at a high rate in previously untreated children with severe hemophilia A, is multifactorial, resulting from complex interactions between genetic and environmental factors. Among non-genetic determinants, a key role is played by treatment-related factors, including the source of FVIII product (i.e., plasma derived or recombinant) and the mode of replacement therapy delivery (i.e., intensity, prophylaxis vs. on demand). We review the potential interventions on these modifiable factors that may help to lower the rate of inhibitor development. In addition, interest is currently directed toward the potential for lesser immunogenicity of novel hemostatic agents designed to decrease the dosing frequency or avoid/delay the need of FVIII replacement therapy. PMID:27155314

  13. F8 gene mutation profile in Indian hemophilia A patients: Identification of 23 novel mutations and factor VIII inhibitor risk association.

    PubMed

    Pinto, Patricia; Ghosh, Kanjaksha; Shetty, Shrimati

    2016-04-01

    'FVIII inhibitors', especially in severe hemophilia A (HA) patients, is a serious adverse effect that complicates their clinical management. Many genetic and non-genetic risk factors have been proposed for FVIII inhibitor development, diverse in different population groups. This is the first study in Indian hemophiliacs that analyzes inhibitor risk in relation to the complete F8 mutation profile, in a case-control study that included 145 Indian severe HA patients, i.e. 69 inhibitor positive (with 18 inhibitor concordant/discordant family members), and 58 inhibitor negative patients, after informed consent. While 53.54% (68/127) index cases were positive for intron 22 or intron 1 inversions, 55 causative F8 mutations were detected in the 59 inversion negative patients, of which 23 were novel mutations (in 24 patients) and 32 were reported earlier (in 35 patients). A higher incidence of mutations, in the C1 and C2 domains in inhibitor positive patients, and in the A1 domain in inhibitor negative patients was observed, though not significantly different. The study suggests that large F8 rearrangements (significantly higher in the inhibitor positive patients) pose the highest risk, while missense mutations (significantly higher in the inhibitor negative patients) pose the lowest risk of inhibitor development in Indian hemophilia A patients. PMID:26897466

  14. Management of EGFR-inhibitor associated rash: a retrospective study in 49 patients

    PubMed Central

    2012-01-01

    Background In recent years inhibitors directed against the epidermal growth factor receptor (EGFR) have evolved as effective targeting cancer drugs. Characteristic papulopustular exanthemas, often described as acneiform rashes, are the most frequent adverse effect associated with this class of novel cancer drugs and develop in > 90% of patients. Notably, the rash may significantly compromise the patients' quality of life, thereby potentially leading to incompliance as well as dose reduction or even termination of the anti-EGFR therapy. Yet, an effective dermatologic management of cutaneous adverse effects can be achieved. Whereas various case reports, case series or expert opinions on the management of EGFR-inhibitor (EGFRI) induced rashes have been published, data on systematic management studies are sparse. Methods Here, we present a retrospective, uncontrolled, comparative study in 49 patients on three established regimens for the management of EGFRI-associated rashes. Results Strikingly, patients' rash severity improved significantly over three weeks of treatment with topical mometason furoate cream, topical prednicarbate cream plus nadifloxacin cream, as well as topical prednicarbate cream plus nadifloxacin cream plus systemic isotretinoin. Conclusions In summary our results demonstrate that EGFRI-associated rashes can be effectively managed by specific dermatologic interventions. Whereas mild to moderate rashes should be treated with basic measures in combination with topical glucocorticosteroids or combined regiments using glucocorticosteroids and antiseptics/antibiotics, more severe or therapy-resistant rashes are likely to respond with the addition of systemic retinoids. PMID:22472354

  15. Proton-pump inhibitors in patients requiring antiplatelet therapy: new FDA labeling.

    PubMed

    Johnson, David A; Chilton, Robert; Liker, Harley R

    2014-05-01

    Proton-pump inhibitors (PPIs) are recommended for patients who require antiplatelet therapy and have a history of upper gastrointestinal bleeding. Proton-pump inhibitors should also be considered for patients receiving antiplatelet therapy who have other risk factors for gastrointestinal bleeding, including use of aspirin. Thus, evidence of pharmacokinetic and pharmacodynamic interactions between PPIs and consequent impaired effectiveness of the antiplatelet agent clopidogrel has caused concern. Here, we discuss comparative studies suggesting that the extent to which a PPI reduces exposure to the active metabolite of clopidogrel and attenuates its antithrombotic effect differs among PPIs. Although a clinically meaningful effect of the interaction between PPIs and clopidogrel on cardiovascular outcomes has not been established, these studies provided the basis for recent changes in US Food and Drug Administration (FDA) labeling for several PPIs and clopidogrel. New labeling suggests that PPI use among patients taking clopidogrel be limited to pantoprazole, rabeprazole, lansoprazole, or dexlansoprazole. Because comparative studies indicate that omeprazole and esomeprazole have a greater effect on the CYP2C19-mediated conversion of clopidogrel to its active metabolite and, consequently, clopidogrel's effect on platelet reactivity, FDA labeling recommends avoiding omeprazole and esomeprazole in patients taking clopidogrel. Even a 12-hour separation of dosing does not appear to prevent drug interactions between omeprazole and clopidogrel. PMID:24918808

  16. Effects of different calcineurin inhibitors on sex hormone levels in transplanted male patients.

    PubMed

    Kantarci, G; Sahin, S; Uras, A R; Ergin, H

    2004-01-01

    Hormonal abnormalities in male patients with end-stage renal diseases are primarily organic and related to uremia as well as the other comorbid factors that frequently contribute to chronic renal failure and concomitant drug administration. The restoration of hormonal profiles after successful renal transplantation is still controversial. Immunosuppressive drugs may influence hormonal profiles. Our cross-sectional study of 37 male kidney transplant recipients investigated two groups according to their calcineurin inhibitor therapy, namely 21 cyclosporine versus 16 tacrolimus patients. The two groups were matched for age, graft function, mean duration of dialysis before transplantation, and duration of follow-up after transplantation. There was no statistical significant difference in baseline circulating levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (TTE), and prolactin (PRL) between the two groups. We found that calcineurin inhibitors have favorable effects on sexual hormone levels of male renal transplant patients and that there is no difference in baseline hormone levels between cyclosporine- and tacrolimus-treated male patients. PMID:15013339

  17. Prophylactic proton pump inhibitors in femoral neck fracture patients - A life - and cost-saving intervention.

    PubMed

    Singh, R; Trickett, R; Meyer, Cer; Lewthwaite, S; Ford, D

    2016-07-01

    Introduction Acute gastrointestinal stress ulceration is a common and serious complication of trauma. Prophylactic proton pump inhibitors (PPIs) or histamine receptor antagonists have been used in poly-trauma, burns and head and spinal injuries, as well as on intensive care units, for the prevention of acute gastric stress ulcers. Methods We prospectively studied the use of prophylactic PPIs in with femoral neck fracture patients, gathering data on all acute gastric ulcer complications, including coffee-ground vomiting, malena and haematemesis. We then implemented a treatment protocol in which all patients were given prophylactic PPIs, again prospectively collecting all data. Results Five hundred and fifteen patients were included. Prior to prophylactic PPI, 15% of patients developed gastric stress ulcer complications, with 3% requiring acute intervention with oesophagogastroduodenoscopy (OGD), 5% requiring transfusions and 4% experiencing surgical delays. All patients had delayed discharges. Following PPI implementation, no patients developed gastric stress ulcer complications. Conclusions Femoral neck fracture patients create a substantial workload for orthopaedic units. The increasingly elderly population often have comorbidities, and concomitantly use medications with gastrointestinal side effects. This, combined with the stress of a fracture and preoperative starvation periods increases the risk of gastric ulcers. Here, the use of prophylactic PPIs statistically reduced the incidence of gastric stress ulcers in patients with femoral neck fractures, resulting in fewer surgical delays, reduced length of hospital stay and reduced stress ulcer-related mortality. PMID:27055405

  18. Acquired Factor XIII Inhibitor in Hospitalized and Perioperative Patients: A Systematic Review of Case Reports and Case Series.

    PubMed

    Tone, Kira J; James, Tyler E; Fergusson, Dean A; Tinmouth, Alan; Tay, Jason; Avey, Marc T; Kilty, Shaun; Lalu, Manoj M

    2016-07-01

    Factor XIII (FXIII) cross-links fibrin monomers to support clot stabilization and wound healing. Acquired FXIII deficiency is caused by autoantibodies that inhibit FXIII and can result in bleeding despite normal routine coagulation test results. Given the rarity of this disease, large clinical studies are not feasible. We therefore conducted a systematic review of case reports and case series of acquired FXIII inhibitor to evaluate potential management and treatment strategies for acquired FXIII inhibitor in hospitalized and/or perioperative patients. A systematic search of MEDLINE, Embase, and Web of Science identified reports of hospitalized and perioperative patients with acquired FXIII deficiency. No restrictions were placed on language or publication type. Article screening and data extraction were performed independently by 2 abstractors. Completeness of reporting was evaluated according to modified elements from the CAse REport (CARE) guidelines. A total of 1028 citations were reviewed, with 36 case reports and 3 case series meeting eligibility criteria (63 patients total). The mean age was 60 (range, 9-87) years with balanced sex representation. At presentation, 48 patients (76%) had intramuscular or subcutaneous bleeding, and 34 patients (54%) had external or surgical bleeding. All cases were diagnosed by initially detecting a FXIII deficiency and then identifying the inhibitor. Clinical improvement in bleeding was seen in patients receiving FXIII concentrate (13/17 patients), cryoprecipitate (5/8), and plasma (10/18). Inhibitor reduction was seen in patients who received rituximab (6/6 patients), plasma exchange (2/2), intravenous immunoglobulin (4/5), steroid (15/20), and cyclophosphamide (10/15). Concurrent initiation of multiple therapies and obvious lack of control comparisons made direct association to outcomes difficult to establish. Outcomes were reported for 55 patients, with 25 patients (45%) having complete inhibitor eradication and 15 patients

  19. [Successful treatment of venous thromboembolism with a Factor Xa inhibitor, edoxaban, in patients with lenalidomide-treated multiple myeloma].

    PubMed

    Kawaguchi, Masato; Uchimura, Norio; Okuda, Yuko; Konuma, Satomi; Nehashi, Yoshio

    2015-08-01

    Two multiple myeloma (MM) patients developed venous thromboembolism (VTE) while being treated with lenalidomide and low-dose dexamethasone. Aspirin is recommended for VTE prophylaxis when using lenalidomide/dexamethasone for MM patients with a standard risk of VTE. Despite aspirin administration, however, these two patients experienced VTE. Following VTE development, warfarin and then a Factor Xa inhibitor, edoxaban, were administered. The edoxaban treatment, especially, resulted in favorable and effective control of VTE. Considering these observations, Factor Xa inhibitors may in future become a preferred option for prevention and treatment of VTE when managing MM patients. PMID:26345573

  20. IL-4 in vitro production is upregulated in Alzheimer's disease patients treated with acetylcholinesterase inhibitors.

    PubMed

    Lugaresi, Alessandra; Di Iorio, Angelo; Iarlori, Carla; Reale, Marcella; De Luca, Giovanna; Sparvieri, Eleonora; Michetti, Alessia; Conti, Pio; Gambi, Domenico; Abate, Giuseppe; Paganelli, Roberto

    2004-04-01

    Cytokines appear to be involved in the pathogenesis of Alzheimer's Disease (AD). Their modulation by treatment has been investigated only in a few studies. The aim of our study was to evaluate the effect of acetylcholinesterase inhibitors (AChEI) on Interleukin-4 (IL-4) production in AD patients. IL-4 levels were measured by ELISA on peripheral blood mononuclear cell cultures in the presence or absence of Concanavalin A or Phytohaemagglutinin. Linear regression analysis shows that patients who have been treated, have higher levels of IL-4 independently from age, gender and comorbidity. The increased production of IL-4 in AChEI treated patients might represent an additional mechanism through which AChEI act on AD progression. PMID:15050302

  1. New approaches to hyperkalemia in patients with indications for renin angiotensin aldosterone inhibitors: Considerations for trial design and regulatory approval.

    PubMed

    Zannad, Faiez; Rossignol, Patrick; Stough, Wendy Gattis; Epstein, Murray; Alonso Garcia, Maria de Los Angeles; Bakris, George L; Butler, Javed; Kosiborod, Mikhail; Berman, Lance; Mebazaa, Alexandre; Rasmussen, Henrik S; Ruilope, Luis M; Stockbridge, Norman; Thompson, Aliza; Wittes, Janet; Pitt, Bertram

    2016-08-01

    Hyperkalemia is a common clinical problem, especially in patients with chronic kidney disease, diabetes mellitus, or heart failure. Treatment with renin angiotensin aldosterone system inhibitors exacerbates the risk of hyperkalemia in these patients. Concern about hyperkalemia can result in the failure to initiate, suboptimal dosing, or discontinuation of renin angiotensin aldosterone system inhibitor therapy in patients; effective treatments for hyperkalemia might mitigate such undertreatment. New treatments for hyperkalemia in development may offer better efficacy, tolerability and safety profiles than do existing approved treatments. These compounds might enable more eligible patients to receive renin angiotensin aldosterone system inhibitor therapy or to receive renin angiotensin aldosterone system inhibitors at target doses. The evidence needed to support a treatment claim (reduction in serum potassium) differs from that needed to support a prevention claim (preventing hyperkalemia to allow renin angiotensin aldosterone system inhibitor treatment). Thus, several issues related to clinical trial design and drug development need to be considered. This paper summarizes and expands upon a discussion at the Global Cardiovascular Clinical Trialists 2014 Forum and examines methodologic considerations for trials of new potassium binders for the prevention and management of hyperkalemia in patients with renin angiotensin aldosterone system inhibitor indications. PMID:27140336

  2. Presence of C1-Inhibitor Polymers in a Subset of Patients Suffering from Hereditary Angioedema

    PubMed Central

    Madsen, Daniel Elenius; Hansen, Søren; Gram, Jørgen; Bygum, Anette; Drouet, Christian; Sidelmann, Johannes Jakobsen

    2014-01-01

    Hereditary angioedema (HAE) is a potentially life-threatening disease caused by mutations in the gene encoding the serine protease inhibitor (serpin) C1 inhibitor (C1-inh). The mutations cause decreased functional plasma levels of C1-inh, which triggers unpredictable recurrent edema attacks. Subjects suffering from HAE have been classified in type I patients with decreased functional and antigenic levels of C1-inh, and type II patients with decreased functional but normal antigenic C1-inh levels. However, a few reports have demonstrated that some mutations cause C1-inh polymerization in vitro, and it is speculated that C1-inh polymers may exist in patient plasma, challenging the current classification of HAE patients. To investigate the presence of C1-inh polymers in patient plasma samples, we developed an immunological method, where monoclonal antibodies produced against polymerized C1-inh were applied in native PAGE western blotting. Using this approach we analyzed genuine plasma samples from 31 Danish HAE families, and found that plasma samples from three genotypically distinct HAE type I families (classified upon C1-inh plasma concentrations) contained C1-inh polymers. Identical C1-inh polymerization phenotypes were observed in four affected family members from one of these families. Genotyping of the families revealed that the polymerogenic mutations of two families were located in proximity to the reactive center loop insertion site in C1-inh (p.Ile271Thr and p.Ser258_Pro260del),and one mutation affected helix C (p.Thr167Asn). In conclusion, we demonstrate that C1-inh polymers are present in the plasma of a subgroup of HAE type I patients. PMID:25369003

  3. Treatment of Hypertension in Black Patients With Angiotensin-Converting Enzyme Inhibitors

    PubMed Central

    Shulman, Neil B.

    1988-01-01

    The prevalence of hypertension and the incidence of complications from uncontrolled elevated blood pressure in blacks is much greater than in the white population. In general, blacks have underlying differences in the factors relating to blood pressure level, including low plasma renin, and, in certain instances, a decreased ability to excrete sodium. The stepped-care approach in the management of the black hypertensive patient is similar to that taken with white patients, but racial differences in response to antihypertensive drugs exist that require careful consideration when choosing a treatment regimen. Thiazide diuretics are effective in blacks and are often used as initial therapy. Blacks tend to respond less well to β-blockers, but when combined with a diuretic, they are also effective. Encouraging data are available on the use of calcium channel blockers in blacks. When combined with a diuretic, the angiotensin-converting enzyme (ACE) inhibitors also provide an alternative to therapy for black patients. The use of low doses of ACE inhibitors has reduced the high incidence of adverse effects associated with this group of drugs in earlier studies. PMID:3280812

  4. Risk of liver injury after α-glucosidase inhibitor therapy in advanced chronic kidney disease patients

    PubMed Central

    Kao, Chih-Chin; Wu, Pei-Chen; Wu, Che-Hsiung; Chen, Li-kwang; Chen, Hsi-Hsien; Wu, Mai-Szu; Wu, Vin-Cent

    2016-01-01

    Although α-glucosidase inhibitors (AGIs) are commonly used for controlling postprandial blood glucose, AGIs-induced liver injuries have been reported. However, the relationship between AGIs and liver injuries in advanced chronic kidney disease (CKD) patients remains unexplored. In this nationwide case-control study, we recruited 1765 advanced diabetic CKD patients, who received AGIs therapy from January 1, 2000 to December 31, 2010 as the study sample and 5295 matched controls. Recent and former AGIs users were defined as patients who received the AGIs prescription for 30–60 d and 30–210 d before the event of liver injury. The risk of AGIs-induced liver injury was examined using time-dependent Cox proportional hazards model. Liver injury occurred in 3.9% of patients in the study group and 3.3% of patients in the control group. AGIs use did not increase the risk of liver injury in advanced CKD patients (P = 0.19). The stratified analysis indicated no increased risk of liver injury in all AGIs-using subgroups (all P > 0.05). The available evidence supports extending the use of AGIs without increasing the risk of liver injury in patients with advanced CKD. Additional randomized controlled trials are warranted to confirm our results. PMID:26751038

  5. Acute Kidney Injury in Elderly Patients With Chronic Kidney Disease: Do Angiotensin-Converting Enzyme Inhibitors Carry a Risk?

    PubMed

    Chaumont, Martin; Pourcelet, Aline; van Nuffelen, Marc; Racapé, Judith; Leeman, Marc; Hougardy, Jean-Michel

    2016-06-01

    In contrast to angiotensin receptor blockers (ARBs), mainly excreted by the liver, the dosage of angiotensin-converting enzyme (ACE) inhibitors, cleared by the kidney, must be adapted to account for renal clearance in patients with chronic kidney disease (CKD) to avoid acute kidney injury (AKI). Community-acquired AKI and the use of ACE inhibitors or ARBs in the emergency department were retrospectively assessed in 324 patients with baseline stage 3 or higher CKD. After stepwise regression analysis, the use of ACE inhibitors (odds ratio [OR], 1.9; 95% confidence interval [CI], 1.1-3.1; P=.02) and the presence of dehydration (OR, 30.8; 95% CI, 3.9-239.1) were associated with AKI. A total of 45% of patients using ACE inhibitors experienced overdosing, which causes most of the excess risk of AKI. These results suggest that dosage adjustment of ACE inhibitors to renal function or substitution of ACE inhibitors with ARBs could reduce the incidence of AKI. Moreover, ACE inhibitors and ARBs should be stopped in cases of dehydration. PMID:27080620

  6. Whole-exome sequencing to identify genetic risk variants underlying inhibitor development in severe hemophilia A patients.

    PubMed

    Gorski, Marcin M; Blighe, Kevin; Lotta, Luca A; Pappalardo, Emanuela; Garagiola, Isabella; Mancini, Ilaria; Mancuso, Maria Elisa; Fasulo, Maria Rosaria; Santagostino, Elena; Peyvandi, Flora

    2016-06-01

    The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most problematic and costly complication of FVIII replacement therapy that affects up to 30% of previously untreated patients with severe hemophilia A. The development of inhibitors is a multifactorial complication involving environmental and genetic factors. Among the latter, F8 gene mutations, ethnicity, family history of inhibitors, and polymorphisms affecting genes involved in the immune response have been previously investigated. To identify novel genetic elements underling the risk of inhibitor development in patients with severe hemophilia A, we applied whole-exome sequencing (WES) and data analysis in a selected group of 26 Italian patients with (n = 17) and without (n = 9) inhibitors. WES revealed several rare, damaging variants in immunoregulatory genes as novel candidate mutations. A case-control association analysis using Cochran-Armitage and Fisher's exact statistical tests identified 1364 statistically significant variants. Hierarchical clustering of these genetic variants showed 2 distinct patterns of homozygous variants with a protective or harmful role in inhibitor development. When looking solely at coding variants, a total of 28 nonsynonymous variants were identified and replicated in 53 inhibitor-positive and 174 inhibitor-negative Italian severe hemophilia A patients using a TaqMan genotyping assay. The genotyping results revealed 10 variants showing estimated odds ratios in the same direction as in the discovery phase and confirmed the association of the rs3754689 missense variant (OR 0.58; 95% CI 0.36-0.94; P = .028) in a highly conserved haplotype region surrounding the LCT locus on chromosome 2q21 with inhibitor development. PMID:27060170

  7. Impact on overall survival of the combination of BRAF inhibitors and stereotactic radiosurgery in patients with melanoma brain metastases.

    PubMed

    Wolf, Amparo; Zia, Sayyad; Verma, Rashika; Pavlick, Anna; Wilson, Melissa; Golfinos, John G; Silverman, Joshua S; Kondziolka, Douglas

    2016-05-01

    The aim of this study was to evaluate the impact of BRAF inhibitors on survival outcomes in patients receiving stereotactic radiosurgery (SRS) for melanoma brain metastases. We prospectively collected treatment parameters and outcomes for 80 patients with melanoma brain metastases who underwent SRS. Thirty-five patients harbored the BRAF mutation (BRAF-M) and 45 patients did not (BRAF-WT). Univariate and multivariate analyses were performed to identify predictors of overall survival. The median overall survival from first SRS procedure was 6.7, 11.2 months if treated with a BRAF inhibitor and 4.5 months for BRAF-WT. Actuarial survival rates for BRAF-M patients on an inhibitor were 54 % at 6 months and 41 % at 12 months from the time of SRS. In contrast, BRAF-WT had overall survival rates of 28 % at 6 months and 19 % at 12 months. Overall survival was extended for patients on a BRAF inhibitor at or after the first SRS. The median time to intracranial progression was 3.9 months on a BRAF inhibitor and 1.7 months without. The local control rate for all treated tumors was 92.5 %, with no difference based on BRAF status. Patients with higher KPS, fewer treated intracranial metastases, controlled systemic disease, RPA Class 1 and BRAF-M patients had extended overall survival. Overall, patients with BRAF-M treated with both SRS and BRAF inhibitors, at or after SRS, have increased overall survival from the time of SRS. As patients live longer as a result of more effective systemic and local therapies, close surveillance and early management of intracranial disease with SRS will become increasingly important. PMID:26852222

  8. Patient preference and satisfaction in erectile dysfunction therapy: a comparison of the three phosphodiesterase-5 inhibitors sildenafil, vardenafil and tadalafil

    PubMed Central

    Raheem, Amr Abdel; Kell, Philip

    2009-01-01

    Erectile dysfunction (ED) is a problem that may affect up to 52% of men between the ages of 40 and 70. It can be distressing because of its negative effect on self-esteem, quality of life, and interpersonal relationships. Oral phosphodiesterase-5 inhibitors (PDE5 inhibitors) are now the first choice of treatment in ED. The availability of three (sildenafil citrate, tadalafil, and vardenafil) well tolerated and effective oral PDE5 inhibitors gives treatment options for men with ED. Although the mechanism of action is the same for the three drugs, they differ in their pharmacokinetics. Several preference studies were conducted between the three PDE5 inhibitors but they were not free from bias. Because of the lack of overwhelming reliable data showing that one PDE5 inhibitor is superior to another, current opinion is that the individual patient should have the opportunity to test all three drugs and then select the one that best suits him and his partner. PMID:19936151

  9. Monitoring B-type natriuretic peptide in patients undergoing therapy with neprilysin inhibitors. An emerging challenge?

    PubMed

    Lippi, Giuseppe; Sanchis-Gomar, Fabian

    2016-09-15

    B-type natriuretic peptide (BNP) is primarily synthesized by the ventricles of the heart as a 108-amino acid polypeptide precursor (i.e., proBNP), which is then cleaved into a 76-amino acid biologically inert N-terminal fragment (NT-proBNP) and a biologically active 32-amino acid peptide (BNP). The generation of BNP is considerably enhanced in response to high ventricular filling pressures, so that the measurement of either the active hormone or NT-proBNP has become a mainstay in patients with congestive heart failure. Recent evidence was brought that the enzyme neprilysin efficiently degrades circulating BNP in vivo, whereas proBNP and NT-proBNP are virtually resistant to enzymatic cleavage. Increasing emphasis is currently placed on the fact that that measuring BNP in patients taking the novel and promising neprilysin inhibitors such as LCZ696 may not reliably reflect cardiac dysfunction. Since laboratory monitoring in patients with heart failure should be aimed to define the role of BNP in modulating fluid hemostasis and cardiac remodeling, but natriuretic peptides should also serve as reliable biomarkers of cardiac function and treatment response in these patients, the assessment of neither BNP nor NT-proBNP alone provides a comprehensive biological and clinical picture. Therefore, it seems reasonable to suggest both BNP and the neprilysin-resistant peptide NT-proBNP should be concomitantly assessed in patients with heart failure who take neprilysin inhibitors, so allowing to concomitantly monitor the progression of heart failure and to assess the actual cardiorenal potency of circulating BNP. PMID:27317994

  10. Sclerotherapy Of Esophageal Varices In Severe Hemophilia A Patient And High Titer Inhibitor--Case Report.

    PubMed

    Szczepanik, Andrzej B; Dąbrowski, Wojciech P; Szczepanik, Anna M; Pielaciński, Konrad; Jaśkowiak, Wojciech

    2015-09-01

    In cirrhotic hemophilia patients bleeding from esophageal varices is a serious clinical condition due to congenital deficiency of clotting factors VIII or IX, decreased prothrombin synthesis and hypersplenic thrombocytopenia. In hemophiliac with high-titer inhibitor bypassing therapy is required with activated prothrombin complex concentrates (aPCC) or recombinant activated coagulation factor VII (rFVIIa). Doses and duration treatment with these agents following endoscopic treatment of esophageal varices have not been yet established. Authors report the first case of a severe hemophilia A patient with high titer inhibitor (40 BU) treated with repeated injection sclerotherapy. The patient was admitted with symptoms of massive esophageal variceal hemorrhage ceased with emergency sclerotherapy. Bypassing therapy was administered with aPCC at initial dose of 72.5 U/kg and then with average daily dose of 162 U/kg through 5 days. To achieved a total eradication of esophageal varices the patient was then subjected to four elective sclerotherapy procedures. Two were covered by aPCC with daily dose of 120 U/kg and 145 U/kg for 4 and 3 days respectively and the following two procedures were covered by rFVIIa with the initial dose of 116 µg/kg and the next doses of 87 µg/kg administered every 3 hours in procedure day and every 4 hours on the next two days. During all procedures excellent hemostasis was achieved and no hemorrhagic or thromboembolic complications were observed. Bypassing regimen therapy with aPCC and rFVIIa we applied have been shown to be safe and effective in this patient subjected to sclerotherapy procedures. PMID:26812842

  11. The Tryptophan Hydroxylase Inhibitor LX1031 Shows Clinical Benefit in Patients With Nonconstipating Irritable Bowel Syndrome

    PubMed Central

    Brown, Philip M.; Drossman, Douglas A.; Wood, Alastair J. J.; Cline, Gary A.; Frazier, Kenny S.; Jackson, Jessica I.; Bronner, Johanna; Freiman, Joel; Zambrowicz, Brian; Sands, Arthur; Gershon, Michael D.

    2016-01-01

    BACKGROUND & AIMS Serotonin (5-hydroxytryptamine [5-HT]) has an important role in gastrointestinal function. LX1031 is an oral, locally acting, small molecule inhibitor of tryptophan hydroxylase (TPH). Local inhibition of TPH in the gastrointestinal tract might reduce mucosal production of serotonin (5-HT) and be used to treat patients with nonconstipating irritable bowel syndrome (IBS). METHODS We evaluated 2 dose levels of LX1031 (250 mg or 1000 mg, given 4 times/day) in a 28-day, multicenter, randomized, double-blind, placebo-controlled study of 155 patients with nonconstipating IBS. 5-hydroxyindoleacetic acid (5-HIAA), a biomarker of pharmacodynamic activity, was measured in urine samples at baseline (24 hours after LX1031 administration), and at weeks 4 and 6 (n = 76). RESULTS Each dose of LX1031 was safe and well-tolerated. The primary efficacy end point, relief of IBS pain and discomfort, improved significantly in patients given 1000 mg LX1031 (25.5%), compared with those given placebo, at week 1 (P = .018); with nonsignificant improvements at weeks 2, 3, and 4 (17.9%, 16.3%, and 11.6%, respectively). Symptom improvement correlated with a dose-dependent reduction in 5-HIAA, a marker for TPH inhibition, from baseline until week 4. This suggests the efficacy of LX1031 is related to the extent of inhibition of 5-HT biosynthesis. Stool consistency significantly improved, compared with the group given placebo, at weeks 1 and 4 (P < .01) and at week 2 (P < .001). CONCLUSIONS In a phase 2 study, LX1031 was well tolerated, relieving symptoms and increasing stool consistency in patients with nonconstipating IBS. Symptom relief was associated with reduced levels of 5-HIAA in urine samples. This marker might be used to identify patients with nonconstipating IBS who respond to inhibitors of 5-HT synthesis. PMID:21684281

  12. Binding of ACE-inhibitors to in vitro and patient-derived amyloid-β fibril models

    NASA Astrophysics Data System (ADS)

    Bhavaraju, Manikanthan; Phillips, Malachi; Bowman, Deborah; Aceves-Hernandez, Juan M.; Hansmann, Ulrich H. E.

    2016-01-01

    Currently, no drugs exist that can prevent or reverse Alzheimer's disease, a neurodegenerative disease associated with the presence, in the brain, of plaques that are composed of β-amyloid (Aβ) peptides. Recent studies suggest that angiotensin-converting enzyme (ACE) inhibitors, a set of drugs used to treat hypertension, may inhibit amyloid formation in vitro. In the present study, we investigate through computer simulations the binding of ACE inhibitors to patient-derived Aβ fibrils and contrast it with that of ACE inhibitors binding to in vitro generated fibrils. The binding affinities of the ACE inhibitors are compared with that of Congo red, a dye that is used to identify amyloid structures and that is known to be a weak inhibitor of Aβ aggregation. We find that ACE inhibitors have a lower binding affinity to the patient-derived fibrils than to in vitro generated ones. For patient-derived fibrils, their binding affinities are even lower than that of Congo red. Our observations raise doubts on the hypothesis that these drugs inhibit fibril formation in Alzheimer patients by interacting directly with the amyloids.

  13. Treatment approaches for EGFR-inhibitor-resistant patients with non-small-cell lung cancer.

    PubMed

    Tan, Chee-Seng; Gilligan, David; Pacey, Simon

    2015-09-01

    Discovery of activating mutations in EGFR and their use as predictive biomarkers to tailor patient therapy with EGFR tyrosine kinase inhibitors (TKIs) has revolutionised treatment of patients with advanced EGFR-mutant non-small-cell lung cancer (NSCLC). At present, first-line treatment with EGFR TKIs (gefitinib, erlotinib, and afatinib) has been approved for patients harbouring exon 19 deletions or exon 21 (Leu858Arg) substitution EGFR mutations. These agents improve response rates, time to progression, and overall survival. Unfortunately, patients develop resistance, limiting patient benefit and posing a challenge to oncologists. Optimum treatment after progression is not clearly defined. A more detailed understanding of the biology of EGFR-mutant NSCLC and the mechanisms of resistance to targeted therapy mean that an era of treatment approaches based on rationally developed drugs or therapeutic strategies has begun. Combination approaches-eg, dual EGFR blockade-to overcome resistance have been trialled and seem to be promising but are potentially limited by toxicity. Third-generation EGFR-mutant-selective TKIs, such as AZD9291 or rociletininb, which target Thr790Met-mutant tumours, the most common mechanism of EGFR TKI resistance, have entered clinical trials, and exciting, albeit preliminary, efficacy data have been reported. In this Review, we summarise the scientific literature and evidence on therapy options after EGFR TKI treatment for patients with NSCLC, aiming to provide a guide to oncologists, and consider how to maximise therapeutic advances in outcomes in this rapidly advancing area. PMID:26370354

  14. The Association Between HLA Class II Alleles and the Occurrence of Factor VIII Inhibitor in Thai Patients with Hemophilia A

    PubMed Central

    Nathalang, Oytip; Sriwanitchrak, Pramote; Sasanakul, Werasak; Chuansumrit, Ampaiwan

    2012-01-01

    Objective: This study aimed to investigate the association between HLA class II alleles and the occurrence of FVIIIinhibitor in Thai hemophilia A patients. Material and Methods: The distribution of HLA-DRB1 alleles and DQB1 alleles in 57 Thai hemophilia A patientsand 36 blood donors as controls was determined using the PCR sequence-specific primer (PCR-SSP) method, and theassociation between the occurrence of factor VIII (FVIII) inhibitor and the presence of certain HLA class II alleles wasinvestigated. Results: The frequency of HLA-DRB1*15 was higher in the hemophilia A patients with and without FVIII inhibitor,whereas that of DRB1*14, DRB1*07, and DQB1*02 was lower in the hemophilia A patients with FVIII inhibitor, ascompared to controls. Interestingly, only the frequency of DRB1*15 was significantly higher in the patients with inhibitorthan in the controls (P = 0.021). Moreover, the frequency of DRB1*15 in the patients with inhibitor was higher than inthose without inhibitor (P = 0.198). Conclusion: The study’s findings show that the DRB1*15 allele might have contributed to the occurrence of inhibitorin the Thai hemophilia A patients; however, additional research using larger samples and high-resolution DRB1 typingis warranted. PMID:24744621

  15. Reviewing the controversy surrounding pre-treatment with P2Y12 inhibitors in acute coronary syndrome patients.

    PubMed

    Capodanno, Davide; Angiolillo, Dominick J

    2016-07-01

    Pretreatment with oral P2Y12 inhibitors occurs each time clopidogrel, prasugrel, ticagrelor are given to patients with suspected coronary artery disease before definition of the coronary anatomy. In acute coronary syndromes, the practice of administering oral P2Y12 inhibitors upstream has been the object of significant controversy in recent years, following the publication of two trials of pretreatment in non-ST-segment elevation acute coronary syndromes and ST-segment elevation myocardial infarction, respectively. The introduction in the market of cangrelor - the first intravenous P2Y12 inhibitor - represents a new opportunity but also a new challenge for clinicians. This article reviews current recommendations and supporting evidence surrounding pretreatment with oral and intravenous P2Y12 inhibitors in patients with acute coronary syndromes. PMID:26953527

  16. Musculoskeletal Safety Outcomes of Patients Receiving Daptomycin with HMG-CoA Reductase Inhibitors

    PubMed Central

    Bookstaver, P. Brandon; Lu, Z. Kevin; Dunn, Brianne L.; Rumley, Kathey Fulton

    2014-01-01

    Daptomycin, a cyclic lipopeptide antibiotic, and 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase inhibitors (statins) are commonly administered in the inpatient setting and are associated with creatine phosphokinase (CPK) elevations, myalgias, and muscle weakness. Safety data for coadministration of daptomycin with statins are limited. To determine the safety of coadministration of daptomycin with statin therapy, a multicenter, retrospective, observational study was performed at 13 institutions in the Southeastern United States. Forty-nine adult patients receiving statins concurrently with daptomycin were compared with 171 patients receiving daptomycin without statin therapy. Detailed information, including treatment indication and duration, infecting pathogen, baseline and subsequent CPK levels, and presence of myalgias or muscle complaints, was collected. Myalgias were noted in 3/49 (6.1%) patients receiving combination therapy compared with 5/171 (2.9%) of patients receiving daptomycin alone (P = 0.38). CPK elevations of >1,000 U/liter occurred in 5/49 (10.2%) patients receiving combination therapy compared to 9/171 (5.3%) patients receiving daptomycin alone (P = 0.32). Two of five patients experiencing CPK elevations of >1,000 U/liter in the combination group had symptoms of myopathy. Three patients (6.1%) discontinued therapy due to CPK elevations with concurrent myalgias in the combination group versus 6 patients (3.5%) in the daptomycin-alone group (P = 0.42). CPK levels and myalgias reversed upon discontinuation of daptomycin therapy. Overall musculoskeletal toxicity was numerically higher in the combination group but this result was not statistically significant. Further prospective study is warranted in a larger population. PMID:25022580

  17. Hereditary and acquired C1-inhibitor deficiency: biological and clinical characteristics in 235 patients.

    PubMed

    Agostoni, A; Cicardi, M

    1992-07-01

    Two hundred and twenty-six patients with inherited C1 inhibitor (C1-INH) deficiency, also known as hereditary angioedema (HAE), have been studied. They belonged to 80 unrelated families, and in 11 of them C1-INH was functionally deficient but antigenically normal (type II HAE). Genetic analysis of type 1 families demonstrated restriction fragment length polymorphisms in 11% and abnormal mRNAs in 25%. In type II families, the site of the mutation appeared to determine the rate of catabolism of the dysfunctional C1-INH and its antigenic plasma levels. Clinical symptoms (subcutaneous and mucous swellings) generally first appeared within the second decade of life. The frequency of symptoms was highly variable from patient to patient, but a few patients remained asymptomatic throughout their lives. Prophylactic treatment with attenuated androgens was administered to 59 patients and was totally effective in 57, without significant side effects. Sixty-seven laryngeal and 15 abdominal attacks were treated with C1-INH plasma concentrate, yielding initial regression of symptoms in 30 to 90 minutes. The acquired deficiency of C1-INH, also known as acquired angioedema, was diagnosed in 9 patients. Eight of them had an autoantibody against C1-INH; the only patient without the autoantibody had associated chronic lymphocytic leukemia. Prophylactic treatment with attenuated androgens was effective in this last patient, while those with the autoantibody against C1-INH benefited from prophylaxis with antifibrinolytic agents. Replacement therapy with C1-INH concentrate was necessary only for patients with autoantibodies and required doses 3 or 4 times higher than those used in HAE. PMID:1518394

  18. Musculoskeletal safety outcomes of patients receiving daptomycin with HMG-CoA reductase inhibitors.

    PubMed

    Bland, Christopher M; Bookstaver, P Brandon; Lu, Z Kevin; Dunn, Brianne L; Rumley, Kathey Fulton

    2014-10-01

    Daptomycin, a cyclic lipopeptide antibiotic, and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are commonly administered in the inpatient setting and are associated with creatine phosphokinase (CPK) elevations, myalgias, and muscle weakness. Safety data for coadministration of daptomycin with statins are limited. To determine the safety of coadministration of daptomycin with statin therapy, a multicenter, retrospective, observational study was performed at 13 institutions in the Southeastern United States. Forty-nine adult patients receiving statins concurrently with daptomycin were compared with 171 patients receiving daptomycin without statin therapy. Detailed information, including treatment indication and duration, infecting pathogen, baseline and subsequent CPK levels, and presence of myalgias or muscle complaints, was collected. Myalgias were noted in 3/49 (6.1%) patients receiving combination therapy compared with 5/171 (2.9%) of patients receiving daptomycin alone (P = 0.38). CPK elevations of >1,000 U/liter occurred in 5/49 (10.2%) patients receiving combination therapy compared to 9/171 (5.3%) patients receiving daptomycin alone (P = 0.32). Two of five patients experiencing CPK elevations of >1,000 U/liter in the combination group had symptoms of myopathy. Three patients (6.1%) discontinued therapy due to CPK elevations with concurrent myalgias in the combination group versus 6 patients (3.5%) in the daptomycin-alone group (P = 0.42). CPK levels and myalgias reversed upon discontinuation of daptomycin therapy. Overall musculoskeletal toxicity was numerically higher in the combination group but this result was not statistically significant. Further prospective study is warranted in a larger population. PMID:25022580

  19. Premedication with cyclooxygenase-2 inhibitor meloxicam reduced postoperative pain in patients after oral surgery.

    PubMed

    Aoki, T; Yamaguchi, H; Naito, H; Shiiki, K; Izawa, K; Ota, Y; Sakamoto, H; Kaneko, A

    2006-07-01

    The efficacy of the selective cyclooxygenase-2 (COX-2) inhibitor meloxicam for treatment of postoperative oral surgical pain was assessed in a randomized controlled trial. Patients undergoing unilateral mandibular 3rd molar extraction surgery were allocated to 3 groups, A, B and C. After oral premedication of meloxicam 10 mg in group A, ampiroxicam 27 mg in group B and placebo in group C, surgery was completed within 30 min under local anaesthesia using 2% lidocaine. For postoperative pain relief the patients were allowed to take oral loxoprofen (60 mg per tablet). Postoperative pain was evaluated at the clinic on the 1st, 7th and 14th postoperative day (POD) using a visual analogue scale (VAS), as was the number of loxoprofen tablets consumed, and the results were compared among the 3 groups with statistical significance of P<0.05. VAS scores on 1 POD were significantly lower in group A than in group C. Loxoprofen consumption on the day of surgery and 1 POD was significantly lower in group A than in group C (P<0.01). Total analgesic consumption was significantly lower in groups A and B than in group C (P<0.02). The COX-2 inhibitor, meloxicam 10 mg used for premedication reduced postoperative pain compared with control in oral surgery. PMID:16540287

  20. Switching from Nitrate Therapy to Ranolazine in Patients with Coronary Artery Disease Receiving Phosphodiesterase Type-5 Inhibitors for Erectile Dysfunction

    PubMed Central

    Udeoji, Dioma U; Schwarz, Ernst R

    2014-01-01

    Coronary artery disease (CAD) and erectile dysfunction (ED) frequently coexist. The introduction of phosphodiesterase type-5 (PDE-5) inhibitors has revolutionized medical management of organic ED; however, in patients with angina pectoris, a common symptom of CAD, coadministration of PDE-5 inhibitors and nitrates has been implicated in CAD-related deaths following sexual activity. The mechanism of action of PDE-5 inhibitors results in a potential cumulative drop in blood pressure (BP); thus, these agents are contraindicated in patients receiving nitrates. Beta-blockers and calcium channel antagonists are considered the mainstays of antianginal therapy, but may not be tolerated by all patients. Ranolazine is an antianginal agent that produces minimal reductions in heart rate and BP. Here we report three cases of men with CAD, chronic angina, and concomitant ED. We describe our treatment approach in these patients, using ranolazine as a potential substitute to nitrate therapy. PMID:25452706

  1. [GLP-1 receptor agonists versus SGLT-2 inhibitors in obese type 2 diabetes patients].

    PubMed

    Marques, Ana Rita Forte; Jaafar, Jaafar; de Kalbermatten, Bénédicte; Philippe, Jacques

    2015-06-01

    Who never had a type 2 obese diabetic patient, treated by several oral antidiabetic drugs and insulin, with consequent weight gain associated with the therapeutic escalation and uncontrolled diabetes? The arrival of GLP-1 agonists and SGLT-2 inhibitors allows to reevaluate the management of these patients, with their favorable effects on glycemic control, weight and the risk of hypoglycemia and their complementary mechanisms to conventional treatments. The vicious cycle of weight gain and increased need of insulin is limited. The choice between these two molecules must be based on several factors (glycemic target, weight, comorbidities, route of administration, side effects, etc.), and the balanced enthusiasm of these new treatments with the insufficient data regarding their long-term safety and their impact on micro- and macrovascular complications. PMID:26211282

  2. CHRNA7 Gene and Response to Cholinesterase Inhibitors in an Italian Cohort of Alzheimer's Disease Patients.

    PubMed

    Clarelli, Ferdinando; Mascia, Elisabetta; Santangelo, Roberto; Mazzeo, Salvatore; Giacalone, Giacomo; Galimberti, Daniela; Fusco, Federica; Zuffi, Marta; Fenoglio, Chiara; Franceschi, Massimo; Scarpini, Elio; Forloni, Gianluigi; Magnani, Giuseppe; Comi, Giancarlo; Albani, Diego; Martinelli Boneschi, Filippo

    2016-04-16

    Previous studies suggest that genetic variants in CHRNA7, which encodes for the major subunit of the acetylcholine receptor (α7-nAChR), are associated with the clinical response to cholinesterase inhibitors (ChEI) in Alzheimer's disease (AD) patients. We sought to replicate the association of two SNPs in the CHRNA7 gene, rs6494223 and rs8024987, with response to ChEI treatment in an Italian cohort of 169 AD patients, further extending the study to gene-level analysis. None of the tested variants was associated with clinical response. However, rs6494223 showed a consistent effect direction (OR = 1.4; p = 0.17), which after meta-analysis with previous study yielded a significant result (OR = 1.57, p = 0.02, I2 = 0%). PMID:27104904

  3. Cyclooxygenase-2 Inhibitor Reduces Hepatic Stiffness in Pediatric Chronic Liver Disease Patients Following Kasai Portoenterostomy

    PubMed Central

    Chang, Hye Kyung; Chang, Eun Young; Ryu, Seonae

    2016-01-01

    Purpose The purpose of this study was to define the role of cyclooxygenase-2 inhibitors (COX-2i) in reducing hepatic fibrosis in pediatric patients with chronic liver disease. Materials and Methods From September 2009 to September 2010, patients over 2 years old who visited our outpatient clinic for follow-up to manage their chronic liver disease after Kasai portoenterostomy for biliary atresia, were included in this study. Volunteers were assigned to the study or control groups, according to their preference. A COX-2i was given to only the study group after obtaining consent. The degree of hepatic fibrosis (liver stiffness score, LSS) was prospectively measured using FibroScan, and liver function was examined using serum analysis before and after treatment. After 1 year, changes in LSSs and liver function were compared between the two groups. Results Twenty-five patients (18 females and 7 males) were enrolled in the study group. The control group included 44 patients (26 females and 18 males). After 1 year, the least square mean values for the LSSs were significantly decreased by 3.91±0.98 kPa (p=0.004) only in the study group. Serum total bilirubin did not decrease significantly in either group. Conclusion COX-2i treatment improved the LSS in patients with chronic liver disease after Kasai portoenterostomy for biliary atresia. PMID:27189282

  4. Vaginal estrogen products in hormone receptor-positive breast cancer patients on aromatase inhibitor therapy.

    PubMed

    Sulaica, Elisabeth; Han, Tiffany; Wang, Weiqun; Bhat, Raksha; Trivedi, Meghana V; Niravath, Polly

    2016-06-01

    Atrophic vaginitis represents a major barrier to compliance with aromatase inhibitor (AI) therapy in breast cancer (BC) survivors. While local estrogen therapy is effective for postmenopausal vaginal dryness, the efficacy of such therapies has not been evaluated systematically in hormone receptor-positive (HR+) BC patients on AI therapy. Furthermore, the potential risk of breast cancer recurrence with vaginal estrogen therapy represents a long-term safety concern for the patients with HR + BC. Unfortunately, there is no standardized assay to measure very low concentrations of estradiol (E2) in these women being treated with AI therapy. This makes it difficult to evaluate even indirectly the potential risk of BC recurrence with vaginal estrogen therapy in HR + BC patients on AI therapy. In this review, we describe available assays to measure very low concentrations of E2, discuss the Food and Drug Administration-approved vaginal estrogen products on the market, and summarize published and ongoing clinical trials evaluating the safety and efficacy of vaginal estrogen in HR + BC patients on AI therapy. In the absence of any randomized controlled clinical trials, this review serves as a summary of available clinical data and ongoing studies to aid clinicians in selecting the best available option for their patients. PMID:27178335

  5. Comorbidities and inhibitors in adult patients with haemophilia: issues, costs and management strategies.

    PubMed

    Berntorp, Erik; Mauser-Bunschoten, Evelien; Jiménez-Yuste, Víctor; Spears, Jeffrey B

    2015-11-01

    Along with greater life expectancy in patients with haemophilia has been an increase in associated haemophilia-related (arthropathy, osteoporosis, viral infections) and age-related (cardiovascular disease, renal disease, cancer and others) comorbidities, many of which are only just emerging as the population ages. At present, experience in managing these comorbidities is limited. As the demographic shift continues, haemophilia care centres can expect to encounter more patients with greater levels of complexity. In the absence of evidence-based information to guide the management of adult patients with haemophilia, it is important that the scientific position be reviewed on a regular basis. To this end, several topics relevant to the clinical management of adult patients with haemophilia were examined in a symposium entitled Comorbidities and inhibitors in adult patients with haemophilia: issues, costs and management strategies held on 11 February 2015 in Helsinki, Finland, in conjunction with the 8th Annual Congress of the European Association for Haemophilia and Allied Disorders. This article is a summary of that event. PMID:26492487

  6. Proton Pump Inhibitor Use and Magnesium Concentrations in Hemodialysis Patients: A Cross-Sectional Study

    PubMed Central

    Nakashima, Akio; Ohkido, Ichiro; Yokoyama, Keitaro; Mafune, Aki; Urashima, Mitsuyoshi; Yokoo, Takashi

    2015-01-01

    Magnesium concentration is a proven predictor of mortality in hemodialysis patients. Recent reports have indicated that proton pump inhibitor (PPI) use affects serum magnesium levels, however few studies have investigated the relationship between PPI use and magnesium levels in hemodialysis patients. This study aimed to clarify the association between PPI use and serum magnesium levels in hemodialysis patients. We designed this cross sectional study and included 1189 hemodialysis patients in stable condition. Associations between PPI and magnesium-related factors, as well as other possible confounders, were evaluated using a multiple regression model. We defined hypomagnesemia as a value < 2.0 mg/dL, and created comparable logistic regression models to assess the association between PPI use and hypomagnesemia. PPI use is associated with a significantly lower mean serum magnesium level than histamine 2 (H2) receptor antagonists or no acid-suppressive medications (mean [SD] PPI: 2.52 [0.45] mg/dL; H2 receptor antagonist: 2.68 [0.41] mg/dL; no acid suppressive medications: 2.68 [0.46] mg/dL; P = 0.001). Hypomagnesemia remained significantly associated with PPI (adjusted OR, OR: 2.05; 95% CI: 1.14–3.69; P = 0.017). PPI use is associated with an increased risk of hypomagnesemia in hemodialysis patients. Future prospective studies are needed to explore magnesium replacement in PPI users on hemodialysis. PMID:26618538

  7. Effect of 5-alpha Reductase Inhibitor on Storage Symptoms in Patients with Benign Prostatic Hyperplasia

    PubMed Central

    Cho, Kang Jun; Kang, Se Hee; Kim, Hyo Sin; Koh, Jun Sung

    2011-01-01

    Purpose Many patients with benign prostatic hyperplasia (BPH) have storage symptoms. The aim of this study was to evaluate the effects of treatment with a 5-alpha reductase inhibitor (5ARI) on storage symptoms in patients with BPH. Methods This study was conducted in 738 patients with lower urinary tract symptoms secondary to BPH. Patients with a prostate volume of higher than 30 mL on the transrectal ultrasound were classified into two groups: group A, in which an alpha blocker was solely administered for at least 12 months, and group B, in which a combination treatment regimen of an alpha blocker plus 5ARI was used. This was followed by an analysis of the changes in parameters such as the total International Prostate Symptom Score (IPSS), voiding symptom subscore, and storage symptom subscore between the two groups. In addition, we examined whether there was a significant difference between the two groups in the degree of change in storage symptoms between before and after the pharmacological treatment. Results Of the 738 men, 331 had a prostate volume ≥30 mL, including 150 patients in group A and 181 patients in group B. Total IPSS, the voiding symptom subscore, and the storage symptom subscore were significantly lower after treatment than before treatment in both groups (P<0.05). A comparison of the degree of change between before and after treatment, however, showed no significant differences in the storage symptom subscore between the two groups (P>0.05). Conclusions Alpha blocker and 5ARI combination treatment is effective for patients with BPH including storage symptoms. However, 5ARI does not exert a significant effect on storage symptoms in BPH patients. PMID:22087424

  8. Management of sexual dysfunction in postmenopausal breast cancer patients taking adjuvant aromatase inhibitor therapy

    PubMed Central

    Derzko, C.; Elliott, S.; Lam, W.

    2007-01-01

    Treatment with aromatase inhibitors for postmenopausal women with breast cancer has been shown to reduce or obviate invasive procedures such as hysteroscopy or curettage associated with tamoxifen-induced endometrial abnormalities. The side effect of upfront aromatase inhibitors, diminished estrogen synthesis, is similar to that seen with the natural events of aging. The consequences often include vasomotor symptoms (hot flushes) and vaginal dryness and atrophy, which in turn may result in cystitis and vaginitis. Not surprisingly, painful intercourse (dyspareunia) and loss of sexual interest (decreased libido) frequently occur as well. Various interventions, both non-hormonal and hormonal, are currently available to manage these problems. The purpose of the present review is to provide the practitioner with a wide array of management options to assist in treating the sexual consequences of aromatase inhibitors. The suggestions in this review are based on recent literature and on the recommendations set forth both by the North American Menopause Association and in the clinical practice guidelines of the Society of Gynaecologists and Obstetricians of Canada. The complexity of female sexual dysfunction necessitates a biopsychosocial approach to assessment and management alike, with interventions ranging from education and lifestyle changes to sexual counselling, pelvic floor therapies, sexual aids, medications, and dietary supplements—all of which have been reported to have a variable, but often successful, effect on symptom amelioration. Although the use of specific hormone replacement—most commonly local estrogen, and less commonly, systemic estrogen with or without an androgen, progesterone, or the additional of an androgen in an estrogenized woman (or a combination)—may be highly effective, the concern remains that in patients with estrogen-dependent breast cancer, including those receiving anti-estrogenic adjuvant therapies, the use of these hormones may be

  9. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus.

    PubMed

    Li-Byarlay, Hongmei; Pittendrigh, Barry R; Murdock, Larry L

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant-insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology. PMID:27594789

  10. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus

    PubMed Central

    Li-Byarlay, Hongmei; Pittendrigh, Barry R.; Murdock, Larry L.

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant–insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology. PMID:27594789

  11. Effects of neutrophil elastase inhibitor in patients undergoing esophagectomy: A systematic review and meta-analysis

    PubMed Central

    Wang, Zhi-Qiang; Chen, Long-Qi; Yuan, Yong; Wang, Wen-Ping; Niu, Zhong-Xi; Yang, Yu-Shang; Cai, Jie

    2015-01-01

    AIM: To evaluate the benefit and safety of sivelestat (a neutrophil elastase inhibitor) administration in patients undergoing esophagectomy. METHODS: Online databases including PubMed, EMBASE, the Cochrane Library, Web of Knowledge, and Chinese databases (Wanfang database, VIP and CNKI) were searched systematically up to November 2013. Randomized controlled trials and high-quality comparative studies were considered eligible for inclusion. Three reviewers evaluated the methodological quality of the included studies, and Stata 12.0 software was used to analyze the extracted data. The risk ratio (RR) was used to express the effect size of dichotomous outcomes, and mean difference (MD) or standardized mean difference was used to express the effect size of continuous outcomes. RESULTS: Thirteen studies were included in this systematic review and nine studies were included in the meta-analysis. The duration of mechanical ventilation was significantly decreased in the sivelestat group on postoperative day 5 [I2 = 76.3%, SMD = -1.41, 95%CI: -2.63-(-0.19)]. Sivelestat greatly lowered the incidence of acute lung injury in patients after surgery (I2 = 0%, RR = 0.27, 95%CI: 0.08-0.93). However, it did not decrease the incidence of pneumonia, intensive care unit stay or postoperative hospital stay, and did not increase the incidence of complications such as anastomotic leakage, recurrent nerve palsy, wound infection, sepsis and catheter-related fever. CONCLUSION: A neutrophil elastase inhibitor is beneficial in patients undergoing esophagectomy. More high quality, large sample, multi-center and randomized controlled trials are needed to validate this effect. PMID:25834341

  12. Polymorphism in methylenetetrahydrofolate reductase, plasminogen activator inhibitor-1, and apolipoprotein E in hemodialysis patients.

    PubMed

    Al-Muhanna, Fahad; Al-Mueilo, Samir; Al-Ali, Amein; Larbi, Emmanuel; Rubaish, Abdullah; Abdulmohsen, Mohammed Fakhry; Al-Zahrani, Alhussain; Al-Ateeq, Suad

    2008-11-01

    The methylenetetrahydrofolate reductase (MTHFR) gene polymorphism, apolipoprotein E (apo epsilon4) gene polymorphism and polymorphism of plasminogen activator inhibitor-1 (PAI-1) have been shown to be associated with end-stage renal disease (ESRD). To determine the prevalence of these mutations in Saudi patients with ESRD on hemodialysis, we studied the allelic frequency and genotype distribution in patients receiving hemodialysis and in a control group, all residing in the Eastern Province of Saudi Arabia. The genotypes were determined using allele specific hybridization procedures and were confirmed by restriction fragment length polymorphism. The T allele frequency and homozygous genotype of MTHFR in ESRD patients were 14% and 2.4%, respectively compared to 13.4% and 0%, respectively in the control group. The allele frequency and homozygous genotype of 4G/4G PAI-1 gene polymorphism were 46.4% and 4.8% respectively in ESRD patients compared to 57.1% and 32% respectively in the control group. The apo s4 allele frequency and homozygous genotype distribution in hemodialysis patients were 7% and 2.4%, respectively compared to 13% and 2% in the control group. Although allele frequency of C677T of MTHFR was statistically similar in the hemodialysis patients and in the control group, the homozygotes T allele genotype was over represented in the hemodialysis group compared to normal. The prevalence of PAI-1 4G/4G polymorphism in ESRD patients was lower when compared to the control group. The prevalence of apo s4 allele did not differ significantly between the two groups. The present results demonstrate that all three studied polymorphic mutations are present in our population and that they may contribute to the etiology of the disease in our area. PMID:18974580

  13. Risk of drug-eluting stent thrombosis in patients receiving proton pump inhibitors.

    PubMed

    Sarafoff, Nikolaus; Sibbing, Dirk; Sonntag, Ulrich; Ellert, Julia; Schulz, Stefanie; Byrne, Robert A; Mehilli, Julinda; Schömig, Albert; Kastrati, Adnan

    2010-09-01

    Clopidogrel is a prodrug that is converted via the hepatic cytochrome P450 system into its active thiol metabolite. Evidence is accumulating that proton pump inhibitors (PPIs) inhibit this enzymatic pathway and may therefore attenuate the antiplatelet effect of clopidogrel. The objective of this study was to investigate whether patients on clopidogrel therapy after drug-eluting stent (DES) placement who also receive a PPI are at higher risk of stent thrombosis (ST). This is a retrospective analysis of patients who received dual antiplatelet treatment including clopidogrel after DES placement. Outcomes were compared according to PPI therapy. The primary endpoint was the incidence of definite ST at 30 days. Secondary endpoints were death, combined death or ST and myocardial infarction (MI). The study population included 3,338 patients and 698 patients (20.9%) received PPIs. Patients receiving a PPI had a higher risk profile at baseline. Multivariate analysis showed that PPI treatment was not independently associated with the occurrence of ST [adjusted HR 1.8 (95% CI: 0.7-4.7), p=0.23] or MI [adjusted HR 1.3 (0.8-2.3), p=0.11]. PPI treatment was significantly associated with death [adjusted HR 2.2 (1.1-4.3), p=0.02] and death or ST [adjusted HR 3.3(1.7-6.7), p=0.02]. Concomitant treatment with a PPI in patients receiving dual antiplatelet treatment after coronary stenting is not an independent predictor of ST. The higher mortality is probably due to confounding as patients on PPIs had a higher risk profile at baseline. PMID:20664905

  14. Interaction between clopidogrel and proton-pump inhibitors and management strategies in patients with cardiovascular diseases

    PubMed Central

    Gurbel, Paul A; Tantry, Udaya S; Kereiakes, Dean J

    2010-01-01

    Dual antiplatelet therapy (DAPT) with clopidogrel and aspirin has been successful in reducing ischemic events in a wide range of patients with cardiovascular diseases. However, the anti-ischemic effects of DAPT may also be associated with gastrointestinal (GI) complications including ulceration and bleeding particularly in ‘high risk’ and elderly patients. Current guidelines recommend the use of proton-pump inhibitors (PPIs) to reduce the risk of GI bleeding in patients treated with DAPT. However, pharmacodynamic studies suggest an effect of PPIs on clopidogrel metabolism with a resultant reduction in platelet inhibitory effects. Similarly, several observational studies have demonstrated reduced clopidogrel benefit in patients who coadministered PPIs. Although recent US Food and Drug Administration and European Medicines Agency statements discourage PPI (particularly omeprazole) and clopidogrel coadministration, the 2009 AHA/ACC/SCAI PCI guidelines do not support a change in current practice in the absence of adequately powered prospective randomized clinical trial data. The data regarding pharmacologic and clinical interactions between PPI and clopidogrel therapies are herein examined and treatment strategies are provided. PMID:21701635

  15. Impact of acetylcholinesterase inhibitors on the occurrence of acute coronary syndrome in patients with dementia

    PubMed Central

    Wu, Ping-Hsun; Lin, Yi-Ting; Hsu, Po-Chao; Yang, Yi-Hsin; Lin, Tsung-Hsien; Huang, Chia-Tsuan

    2015-01-01

    The study aimed to investigate the association of acetylcholinesterase inhibitors (AChEIs) use with the risk of acute coronary syndrome (ACS). We conducted a population-based retrospective cohort study of dementia patients during 1 January 1999 to 31 December 2008 using the National Health Insurance Database in Taiwan. New AChEI users during the study period were matched with AChEI nonusers in age-matched and gender-matched cohorts. The risk of ACS associated with use of AChEIs was analyzed using modified Kaplan-Meier analysis and Cox proportional hazard models after adjustment for competing death risk. Use of AChEIs was associated with a lower incidence of ACS (212.8/10,000 person-years) compared to the matched reference cohort (268.7/10,000 person-years). The adjusted hazard ratio for ACS in patients with dementia treated with AChEIs was 0.836 (95% confidence interval, 0.750–0.933; P < 0.001). Further sensitivity analysis of different study populations demonstrated consistent results. A statistical dose–response relationship for AChEI use and ACS risk was significant for the patients with dementia. In patients with dementia, AChEI treatment was associated with decreased risk of ACS. PMID:26577589

  16. Association between acetylcholinesterase inhibitors and risk of stroke in patients with dementia

    PubMed Central

    Lin, Yi-Ting; Wu, Ping-Hsun; Chen, Cheng-Sheng; Yang, Yi-Hsin; Yang, Yuan-Han

    2016-01-01

    Patients with dementia are at increased risk of stroke. Acetylcholinesterase inhibitors (AChEIs) have endothelial function protection effects and anti-inflammatory properties. We investigated the ischemic stroke risk in AChEIs use in dementia patients without stroke history. Using Taiwan National Health Insurance Database from 1999 to 2008, 37,352 dementia patients over 50 years old without stroke history were eligible. The results were analyzed by propensity score–matched Cox proportional hazard models with competing risk adjustment. AChEIs users had lower incidence of ischemic stroke (160.3/10,000 person-years), compared to the propensity score–matched reference (240.8/10,000 person-years). The adjusted hazard ratio for ischemic stroke based on propensity score–matched Cox proportional hazard model was 0.508 (95% confidence interval, 0.434–0.594; P < 0.001). There was no significant difference in all-cause mortality between AChEIs users and nonusers. In conclusion, among dementia patients without previous ischemic stroke history, AChEIs treatment was associated with a decreased risk of ischemic stroke but not greater survival. PMID:27377212

  17. Association between acetylcholinesterase inhibitors and risk of stroke in patients with dementia.

    PubMed

    Lin, Yi-Ting; Wu, Ping-Hsun; Chen, Cheng-Sheng; Yang, Yi-Hsin; Yang, Yuan-Han

    2016-01-01

    Patients with dementia are at increased risk of stroke. Acetylcholinesterase inhibitors (AChEIs) have endothelial function protection effects and anti-inflammatory properties. We investigated the ischemic stroke risk in AChEIs use in dementia patients without stroke history. Using Taiwan National Health Insurance Database from 1999 to 2008, 37,352 dementia patients over 50 years old without stroke history were eligible. The results were analyzed by propensity score-matched Cox proportional hazard models with competing risk adjustment. AChEIs users had lower incidence of ischemic stroke (160.3/10,000 person-years), compared to the propensity score-matched reference (240.8/10,000 person-years). The adjusted hazard ratio for ischemic stroke based on propensity score-matched Cox proportional hazard model was 0.508 (95% confidence interval, 0.434-0.594; P < 0.001). There was no significant difference in all-cause mortality between AChEIs users and nonusers. In conclusion, among dementia patients without previous ischemic stroke history, AChEIs treatment was associated with a decreased risk of ischemic stroke but not greater survival. PMID:27377212

  18. Impact of acetylcholinesterase inhibitors on the occurrence of acute coronary syndrome in patients with dementia.

    PubMed

    Wu, Ping-Hsun; Lin, Yi-Ting; Hsu, Po-Chao; Yang, Yi-Hsin; Lin, Tsung-Hsien; Huang, Chia-Tsuan

    2015-01-01

    The study aimed to investigate the association of acetylcholinesterase inhibitors (AChEIs) use with the risk of acute coronary syndrome (ACS). We conducted a population-based retrospective cohort study of dementia patients during 1 January 1999 to 31 December 2008 using the National Health Insurance Database in Taiwan. New AChEI users during the study period were matched with AChEI nonusers in age-matched and gender-matched cohorts. The risk of ACS associated with use of AChEIs was analyzed using modified Kaplan-Meier analysis and Cox proportional hazard models after adjustment for competing death risk. Use of AChEIs was associated with a lower incidence of ACS (212.8/10,000 person-years) compared to the matched reference cohort (268.7/10,000 person-years). The adjusted hazard ratio for ACS in patients with dementia treated with AChEIs was 0.836 (95% confidence interval, 0.750-0.933; P < 0.001). Further sensitivity analysis of different study populations demonstrated consistent results. A statistical dose-response relationship for AChEI use and ACS risk was significant for the patients with dementia. In patients with dementia, AChEI treatment was associated with decreased risk of ACS. PMID:26577589

  19. Second-Generation Tyrosine Kinase Inhibitors (Tki) as Salvage Therapy for Resistant or Intolerant Patients to Prior TKIs.

    PubMed

    Breccia, Massimo; Alimena, Giuliana

    2014-01-01

    With the advent of target therapies, imatinib became the mainstay for treatment of chronic myeloid leukemia. However, despite the brilliant results obtained with this drug, more than 30% of patients discontinue therapy in long-term due to several reasons, including failure and/or intolerance. Second-generation tyrosine kinase inhibitors (TKIs) are more potent drugs and have expanded inhibition against a broad spectrum of mutations resistant to imatinib. Both nilotinib and dasatinib have demonstrated in vitro and in vivo clinical activity against different types of mutations and various forms of resistance. However, patients with T315I mutation do not obtain an advantage from these drugs and a third generation inhibitor ponatinib, a pan-BCR drug, was tested with significant results. In this review, we report the results of second-and third-generation TKIs tested as second or third line therapy in patients resistant and/or intolerant to previous inhibitors. PMID:24455112

  20. Second-Generation Tyrosine Kinase Inhibitors (Tki) as Salvage Therapy for Resistant or Intolerant Patients to Prior TKIs

    PubMed Central

    Breccia, Massimo; Alimena, Giuliana

    2014-01-01

    With the advent of target therapies, imatinib became the mainstay for treatment of chronic myeloid leukemia. However, despite the brilliant results obtained with this drug, more than 30% of patients discontinue therapy in long-term due to several reasons, including failure and/or intolerance. Second-generation tyrosine kinase inhibitors (TKIs) are more potent drugs and have expanded inhibition against a broad spectrum of mutations resistant to imatinib. Both nilotinib and dasatinib have demonstrated in vitro and in vivo clinical activity against different types of mutations and various forms of resistance. However, patients with T315I mutation do not obtain an advantage from these drugs and a third generation inhibitor ponatinib, a pan-BCR drug, was tested with significant results. In this review, we report the results of second-and third-generation TKIs tested as second or third line therapy in patients resistant and/or intolerant to previous inhibitors. PMID:24455112

  1. Elective surgery in patients with congenital coagulopathies and inhibitors: experience of the National Haemophilia Centre of Venezuela.

    PubMed

    Boadas, A; Fernández-Palazzi, F; De Bosch, N B; Cedeño, M; Ruiz-Sáez, A

    2011-05-01

    Elective surgery in patients with congenital haemophilia with inhibitors carries a high risk of bleeding. However, inhibitor patients also have a high risk of haemarthroses and other orthopaedic complications, and surgery could improve their quality of life. Successful elective surgery has been reported in inhibitor patients under haemostatic cover with plasma-derived activated prothrombin complex concentrate (pd-aPCC) or recombinant activated factor VII (rFVIIa). Recombinant FVIIa has recently become available in Venezuela and, unlike pd-aPCC, has not been associated with an anamnestic response. The aim of this study was to assess our experience using rFVIIa as a first-line and sustained treatment in elective invasive surgical procedures at the National Haemophilia Centre in Venezuela. Surgical procedures were classified as major or minor, under haemostatic cover with rFVIIa. A total of 13 patients (12 with haemophilia A with high-responding inhibitors and one with von Willebrand's disease type 3) underwent a total of 19 surgical procedures under rFVIIa cover. Thirteen procedures were classified as major surgeries. Intraoperative haemostasis was achieved in the majority of patients. Only two patients required an additional dose of rFVIIa, at 30 min and 75 min, respectively, with good results. Postoperative haemostasis was considered effective in 16 of 18 (89%) of the procedures in haemophilia A patients. Treatment was considered to be ineffective in two patients because of excessive postoperative bleeding. Data from the study provide no safety concerns, and demonstrate that rFVIIa provides effective haemostatic cover in elective surgery in patients with inhibitors; research is ongoing to determine the optimal dose for such procedures. PMID:21118333

  2. Efficacy of levocarnitine for tyrosine kinase inhibitor-induced painful muscle cramps in patients with chronic myelogenous leukemia.

    PubMed

    Yamada, Michiko; Kuroda, Hiroyuki; Shimoyama, Saori; Ito, Ryo; Sugama, Yusuke; Sato, Ken; Yamauchi, Natsumi; Horiguchi, Hiroto; Nakamura, Hajime; Hamaguchi, Kota; Abe, Tomoyuki; Fujii, Shigeyuki; Maeda, Masahiro; Kato, Junji

    2016-04-01

    Muscle cramps are side effects commonly associated with tyrosine kinase inhibitor (TKI) treatment. Patients suffering from muscle cramps are treated with various medications such as calcium, magnesium and vitamin supplements, but these therapies are often ineffective. We report two patients with chronic myelogenous leukemia who developed muscle cramps caused by TKI. These patients were treated successfully with levocarnitine. Both of our cases revealed the beneficial effects of levocarnitine treatment on TKI-induced muscle cramps. PMID:27169456

  3. DPP-4 Inhibitor Reduces Central Blood Pressure in a Diabetic and Hypertensive Patient: A Case Report.

    PubMed

    Cosenso-Martin, Luciana Neves; Giollo-Junior, Luiz Tadeu; Vilela-Martin, José Fernando

    2015-07-01

    Hypertension and type 2 diabetes mellitus (DM) are among the main risk factors for the development of cardiovascular disease. Pharmacotherapy for DM should not only improve blood glucose control, but also provide beneficial glucose-independent cardiovascular effects. The central systolic blood pressure (SBP) has become more important than the brachial SBP in the assessment of cardiovascular risk.This case report describes the effect of vildagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, on the central SBP in a 54-year-old woman with hypertension and DM. She was submitted to applanation tonometry (AT) before and after vildagliptin association. AT of the radial artery is a non-invasive method that indirectly assesses arterial stiffness by calculating the central SBP and the augmentation index (AIx).After 3 months of follow-up using vildagliptin, central SBP and AIx were improved. Moreover, she presented better glycemic control.This case suggests an effect of DPP-4 inhibitor on arterial stiffness parameter (central SBP) in a hypertensive and diabetic patient, which shows a glucose-independent beneficial cardiovascular effect of this group of drugs. PMID:26166078

  4. Association Between Ischemic Stroke and Tumor Necrosis Factor Inhibitor Therapy in Patients With Rheumatoid Arthritis

    PubMed Central

    Low, Audrey S. L.; Lunt, Mark; Mercer, Louise K.; Watson, Kath D.; Dixon, William G.; Symmons, Deborah P. M.

    2016-01-01

    Objective Patients with rheumatoid arthritis (RA) are at an increased risk of ischemic stroke. Tumor necrosis factor inhibitors (TNFi) may influence risk and mortality after ischemic stroke by reducing inflammation. This study was undertaken to examine the association of TNFi with the risk of incident ischemic stroke and with 30‐day and 1‐year mortality after ischemic stroke. Methods Patients with RA starting therapy with TNFi and a biologics‐naive comparator group treated with synthetic disease‐modifying antirheumatic drugs (DMARDs) only were recruited to the British Society for Rheumatology Biologics Register for Rheumatoid Arthritis from 2001 to 2009. Patients were followed up via clinical and patient questionnaires as well as the national death register. Incident strokes were classified as ischemic if brain imaging reports suggested ischemia or if ischemic stroke was reported as the underlying cause of death on a death certificate. Patients with a previous stroke were excluded. Risk of ischemic stroke was compared between patients receiving synthetic DMARDs only and those ever‐exposed to TNFi using a Cox proportional hazards regression model adjusted for potential confounders. Mortality after ischemic stroke was compared between synthetic DMARD–treated patients and TNFi‐treated patients using logistic regression, adjusted for age and sex. Results To April 2010, 127 verified incident ischemic strokes (21 in 3,271 synthetic DMARD–treated patients and 106 in 11,642 TNFi‐treated patients) occurred during 11,973 and 61,226 person‐years of observation, respectively (incidence rate 175 versus 173 per 100,000 person‐years). After adjustment for confounders, there was no association between ever‐exposure to TNFi and ischemic stroke (hazard ratio 0.99 [95% confidence interval (95% CI) 0.54–1.81]). Mortality 30 days or 1 year after ischemic stroke was not associated with concurrent TNFi exposure (odds ratio 0.18 [95% CI 0.03–1.21] and 0.60 [95

  5. A systematic review on randomized control trials on rennin angiotensin aldosterone system inhibitors role in managing hypertension among hemodialysis patients.

    PubMed

    Aftab, Raja Ahsan; Khan, Amer Hayat; Adnan, Azreen Syazril; Jannah, Nurul

    2016-01-01

    Randomized control trials (RCTs) are considered as most rigors way of determining the cause-effect relationship of a treatment and outcome. Activation of rennin angiotensin aldosterone system (RAAS) is an important contributor to hypertension in hemodialysis patients. The prevalence of hypertension in hemodialysis patients varies from 60% to 80% and hypertension management alone with conventional hemodialysis is insufficient. Hence, the current review was aimed to investigate the effect of RAAS inhibitors in managing hypertension among hemodialysis patients in a randomized control trial. Using PUBMED and EMBASE databases, randomized control trial with primary or secondary outcomes related to the effect of RAAS inhibitors on blood pressure among hemodialysis patients were included for analysis. The current review also assessed the quality of reporting of RCT. A total of eight RCT met inclusion criteria for current review. According to modified jaded scale, one (12.5%) study scored four points for quality reporting, whereas two (25%) studies scored one point that was the least score. The mean score for all included studies was 2.25. Six (75%) of the eight RCT included, involved ARB in hypertension management among hemodialysis patients, whereas two (25%) studies involved angiotensin-converting enzyme (ACE) inhibitors. Of the siz RCT involving ARB, two (33.3%) RCT also included ACE inhibitors comparison group. Altogether six (75%) studies report a reduction in blood pressure with the use of RAAS inhibitors compared to control group; however, of the six studies, two (33.3%) reported that the reduction in blood pressure was not significant. Whereas, two (25%) studies reported no reduction in blood pressure compared to the control group. The findings from current review do not indicate a clear pattern for a role of RAAS inhibitors for hypertension control among hemodialysis patients. PMID:26853680

  6. Interaction of europium and curium with alpha-amylase.

    PubMed

    Barkleit, Astrid; Heller, Anne; Ikeda-Ohno, Atsushi; Bernhard, Gert

    2016-06-01

    The complexation of Eu(iii) and Cm(iii) with the protein α-amylase (Amy), a major enzyme in saliva and pancreatic juice, was investigated over wide ranges of pH and concentration at both ambient and physiological temperatures. Macroscopic sorption experiments demonstrated a strong and fast binding of Eu(iii) to Amy between pH 5 and 8. The protein provides three independent, non-cooperative binding sites for Eu(iii). The overall association constant of these three binding sites on the protein was calculated to be log K = 6.4 ± 0.1 at ambient temperature. With potentiometric titration, the averaged deprotonation constant of the carboxyl groups (the aspartic and glutamic acid residues) of Amy was determined to be pKa = 5.23 ± 0.14 at 25 °C and 5.11 ± 0.24 at 37 °C. Time-resolved laser-induced fluorescence spectroscopy (TRLFS) revealed two different species for both Eu(iii) and Cm(iii) with Amy. In the case of the Eu(iii) species, the stability constants were determined to be log β11 = 4.7 ± 0.2 and log β13 = 12.0 ± 0.4 for Eu : Amy = 1 : 1 and 1 : 3 complexes, respectively, whereas the values for the respective Cm(iii) species were log β11 = 4.8 ± 0.1 and log β13 = 12.1 ± 0.1. Furthermore, the obtained stability constants were extrapolated to infinite dilution to make our data compatible with the existing thermodynamic database. PMID:26866402

  7. ACE Inhibitor and Angiotensin Receptor Blocker Use and Mortality in Patients with Chronic Kidney Disease

    PubMed Central

    Molnar, Miklos Z; Kalantar-Zadeh, Kamyar; Lott, Evan H; Lu, Jun Ling; Malakauskas, Sandra M; Ma, Jennie Z; Quarles, Darryl L; Kovesdy, Csaba P

    2014-01-01

    Objective To assess the association between ACEI/ARB use and mortality in CKD patients. Background There is insufficient evidence about the association of angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARBs) with mortality in chronic kidney disease (CKD) patients. Methods A logistic regression analysis was used to calculate the propensity of ACEI/ARB initiation in 141,413 US veterans with non-dialysis CKD previously unexposed to ACEI/ARB treatment. We examined the association of ACEI/ARB administration with all-cause mortality in patients matched by propensity scores, using the Kaplan-Meier method and Cox models in “intention-to-treat” analyses, and in generalized linear models with binary outcomes and inverse probability treatment weighing (IPTW) in “as-treated” analyses. Results The mean±SD age of the patients at baseline was 75±10 years, 8% of patients were black, and 22% were diabetic. ACEI/ARB administration was associated with significantly lower risk of mortality both in the intention-to-treat analysis (HR=0.81; 95%CI: 0.78-0.84, p<0.001) and in the as-treated analysis with IPTW (OR=0.37; 95%CI: 0.34-0.41, p<0.001). The association of ACEI/ARB treatment with lower risk of mortality was present in all examined subgroups. Conclusions In this large contemporary cohort of non-dialysis dependent CKD patients, ACEI/ARB administration was associated with greater survival. PMID:24269363

  8. Inequity of access to ACE inhibitors in Swedish heart failure patients: a register-based study

    PubMed Central

    Lindahl, Bertil; Hanning, Marianne; Westerling, Ragnar

    2016-01-01

    Background Several international studies suggest inequity in access to evidence-based heart failure (HF) care. Specifically, studies of ACE inhibitors (ACEIs) point to reduced ACEI access related to female sex, old age and socioeconomic position. Thus far, most studies have either been rather small, lacking diagnostic data, or lacking the possibility to account for several individual-based sociodemographic factors. Our aim was to investigate differences, which could reflect inequity in access to ACEIs based on sex, age, socioeconomic status or immigration status in Swedish patients with HF. Methods Individually linked register data for all Swedish adults hospitalised for HF in 2005–2010 (n=93 258) were analysed by multivariate regression models to assess the independent risk of female sex, high age, low employment status, low income level, low educational level or foreign country of birth, associated with lack of an ACEI dispensation within 1 year of hospitalisation. Adjustment for possible confounding was made for age, comorbidity, Angiotensin receptor blocker therapy, period and follow-up time. Results Analysis revealed an adjusted OR for no ACEI dispensation for women of 1.31 (95% CI 1.27 to 1.35); for the oldest patients of 2.71 (95% CI 2.53 to 2.91); and for unemployed patients of 1.59 (95% CI 1.46 to 1.73). Conclusions Access to ACEI treatment was reduced in women, older patients and unemployed patients. We conclude that access to ACEIs is inequitable among Swedish patients with HF. Future studies should include clinical data, as well as mortality outcomes in different groups. PMID:26261264

  9. Effects of acetylcholinesterase inhibitors and memantine on resting-state electroencephalographic rhythms in Alzheimer's disease patients.

    PubMed

    Babiloni, Claudio; Del Percio, Claudio; Bordet, Regis; Bourriez, Jean-Luis; Bentivoglio, Marina; Payoux, Pierre; Derambure, Philippe; Dix, Sophie; Infarinato, Francesco; Lizio, Roberta; Triggiani, Antonio Ivano; Richardson, Jill C; Rossini, Paolo M

    2013-05-01

    Acetylcholinesterase inhibitors (AChEIs) are the most widely used symptomatic treatment for mild to severe Alzheimer's disease (AD) patients, while N-methyl-d-aspartic acid (NMDA) receptor antagonist memantine is licensed for use in moderate to severe AD patients. In this article, the effect of these compounds on resting state eyes-closed electroencephalographic (EEG) rhythms in AD patients is reviewed to form a knowledge platform for the European Innovative Medicine Initiative project "PharmaCog" (IMI Grant Agreement No. 115009) aimed at developing innovative translational models for drug testing in AD. Indeed, quite similar EEG experiments and the same kind of spectral data analysis can be performed in animal models of AD and in elderly individuals with prodromal or manifest AD. Several studies have shown that AChEIs affect both resting state EEG rhythms and cognitive functions in AD patients. After few weeks of successful treatment, delta (0-3 Hz) or theta (4-7 Hz) rhythms decrease, dominant alpha rhythms (8-10 Hz) increase, and cognitive functions slightly improve. Beneficial effects of these rhythms and cognitive functions were also found in AD responders to the long-term successful treatment (i.e. 6-12 months). In contrast, only one study has explored the long-term effects of memantine on EEG rhythms in AD patients, showing reduced theta rhythms. The present review enlightens the expected effects of AChEIs on resting state EEG rhythms in AD patients as promising EEG markers for the development of translational protocols both within the PharmaCog project and for wider use. PMID:23098644

  10. Fatal serotonin syndrome precipitated by oxcarbazepine in a patient using an selective serotonin reuptake inhibitor.

    PubMed

    Dardis, Christopher; Omoregie, Eghosa; Ly, Vanthanh

    2012-07-01

    Oxcarbazepine, a metabolite of carbamazepine, is used as an antiepileptic, analgesic for neuropathic pain and in the treatment of affective disorders. It has been approved by the Food and Drug Administration for partial seizures in adults as both adjunctive and monotherapy, and as adjunctive therapy in children aged from 2 to 16 years (http://www.fda.gov/ohrms/dockets/ac/06/briefing/2006-4254b_07_05_KP%20OxcarbazepineFDAlabel102005.pdf). We present a case of serotonin syndrome, which was precipitated by this medicine in a patient who had been predisposed by long-term treatment with sertraline, a selective serotonin reuptake inhibitor. This is the first reported fatality due to this drug interaction and only the second case of serotonin syndrome reported with oxcarbazepine. Physicians should consider this risk when prescribing the above combination. PMID:22735246

  11. Therapists' and patients' stress responses during graduated versus flooding in vivo exposure in the treatment of specific phobia: A preliminary observational study.

    PubMed

    Schumacher, Sarah; Miller, Robert; Fehm, Lydia; Kirschbaum, Clemens; Fydrich, Thomas; Ströhle, Andreas

    2015-12-15

    Exposure therapy is considered an effective treatment strategy for phobic anxiety, however, it is rarely applied in clinical practice. The under-usage might be due to various factors of which heightened stress levels not only in patients but also in therapists are presumed to be of particular relevance. The present study aimed to investigate whether different forms of exposure might lead to varying physiological and psychological stress responses in therapists and phobic patients. 25 patients with specific phobia underwent individual cognitive behavioural therapy, performed by 25 psychotherapist trainees, applying exposure sessions in graduated form or the flooding technique. Patients and therapists provided subjective evaluations of stress and five saliva samples for analysis of salivary cortisol and alpha-amylase either during two graduated exposure sessions or during one flooding session, while a regular therapy session served as control condition. Therapists displayed heightened salivary alpha-amylase release during exposure of the flooding, but not the graduated, type. Patients showed elevated salivary cortisol during flooding exposure numerically, however, not on a statistically significant level. Therapists reported more pronounced subjective stress during flooding compared to graduated exposure. Elevated stress levels should be addressed in clinical training in order to improve application of exposure in routine practice. PMID:26545614

  12. Early eradication of factor VIII inhibitor in patients with congenital hemophilia A by immune tolerance induction with a high dose of immunoglobulin.

    PubMed

    Mizoguchi, Yoko; Furue, Aya; Kagawa, Reiko; Chijimatsu, Ikue; Tomioka, Keita; Shimomura, Maiko; Imanaka, Yusuke; Nishimura, Shiho; Saito, Satoshi; Miki, Mizuka; Ono, Atsushi; Konishi, Nakao; Kawaguchi, Hiroshi; Kobayashi, Masao

    2016-04-01

    The production of factor VIII (FVIII) inhibitory antibodies is a serious problem in patients with hemophilia A. Immune tolerance induction (ITI) is the only strategy proven to eradicate persistent inhibitors and has been shown to be successful in 70 % of patients with hemophilia A. However, a minority of hemophilia patients present life-long inhibitors. To eliminate such inhibitors, we designed an intravenous immunoglobulin (IVIG) strategy in combination with high dose recombinant FVIII for ITI in hemophilia A children with inhibitors. Four previously untreated patients produced inhibitors within 16 exposures to FVIII. The peak inhibitor titers in these patients ranged from 3 to 14 BU/mL. The patients received ITI combined with IVIG within 1.5 months after the inhibitors were detected. All patients showed a negative titer for inhibitors by 28 days, with no anamnestic responses. The recovery of FVIII in the plasma concentration was normalized within three months after initiation of ITI. An additional course of IVIG administration led to induction of complete tolerance by 20 months after initiation of ITI therapy in all patients. ITI treatment with high-dose FVIII combined with IVIG may be effective for the early elimination of inhibitors. PMID:26830966

  13. Prevalence and incidence of diabetes in HIV-infected minority patients on protease inhibitors.

    PubMed Central

    Salehian, Behrouz; Bilas, Josephine; Bazargan, Mohsen; Abbasian, Mohammad

    2005-01-01

    In HIV-infected patients, the use of protease inhibitors (PIs) is associated with a constellation of abdominal obesity; buffalo hump; decreased facial and subcutaneous fat; hyperlipidemia and type-2 diabetes mellitus, a so-called HAART-associated dysmetabolic syndrome. The incidence and prevalence of one of its components, the type-2 diabetes mellitus, among minority population is unknown. In August and September 1999, we reviewed 101 charts of HIV-infected patients who visited an inner-city HIV outpatient clinic. The age, gender, ethnicity, BMI, fasting plasma glucose, random serum glucose, triglycerides, CD4 counts, and the type and duration of antiretroviral drugs were recorded. Three years later (2002), the same patient charts were reviewed for evidence of new-onset diabetes. Ten percent of the subjects were identified as diabetic at baseline. The prevalence of diabetes was 12% among those who were taking PIs, compared to 0% among those who were not taking PIs. The incidence of newly diagnosed diabetes during this three-year period was 7.2%. Diabetes occurred only in the group taking PIs. Diabetic subjects were older than their nondiabetic counterparts. All were African Americans. Our study suggests that PIs increase the likelihood of diabetes developing with increasing age in African Americans infected with HIV. PMID:16173323

  14. Proton Pump Inhibitor Use Is not Associated with Cardiac Arrhythmia in Critically Ill Patients

    PubMed Central

    Chen, Kenneth P.; Lee, Joon; Mark, Roger G.; Feng, Mornin; Celi, Leo A.; Danziger, John

    2016-01-01

    Hypomagnesemia can lead to cardiac arrhythmias. Recently, observational data has linked chronic proton pump inhibitor (PPI) exposure to hypomagnesemia. Whether PPI exposure increases the risk for arrhythmias has not been well studied. Using a large, single center inception cohort of critically ill patients, we examined whether PPI exposure was associated with admission electrocardiogram (ECG) readings of a cardiac arrhythmia in over 8000 patients. There were 24.5% PPI users while 6% were taking a histamine 2 antagonist. 14.3% had a cardiac arrhythmia. PPI use was associated with a 1.18 (95% CI=1.02–1.36, p=0.02) unadjusted and 0.96 (95% CI=0.83–1.12, p=0.62) adjusted risk of arrhythmia. Amongst diuretic users (n=2468), PPI use was similarly not associated with an increased risk of cardiac arrhythmia. In summary, in a large cohort of critically ill patients, PPI exposure is not associated with an increased risk of cardiac arrhythmia. PMID:25655574

  15. Management of Chronic Myeloid Leukemia Patients Resistant to Tyrosine Kinase Inhibitors Treatment

    PubMed Central

    Wieczorek, Agnieszka; Uharek, Lutz

    2015-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with a characteristic chromosomal translocation called the Philadelphia chromosome. This oncogene is generated by the fusion of breakpoint cluster region (BCR) and Abelson leukemia virus (ABL) genes and encodes a novel fusion gene translating into a protein with constitutive tyrosine kinase activity. The discovery and introduction of tyrosine kinase inhibitors (TKIs) irreversibly changed the landscape of CML treatment, leading to dramatic improvement in long-term survival rates. The majority of patients with CML in the chronic phase have a life expectancy comparable with that of healthy age-matched individuals. Although an enormous therapeutic improvement has been accomplished, there are still some unresolved issues in the treatment of patients with CML. One of the most important problems is based on the fact that TKIs can efficiently target proliferating mature cells but do not eradicate leukemic stem cells, allowing persistence of the malignant clone. Owing to the resistance mechanisms arising during the course of the disease, treatment with most of the approved BCR-ABL1 TKIs may become ineffective in a proportion of patients. This article highlights the different molecular mechanisms of acquired resistance being developed during treatment with TKIs as well as the pharmacological strategies to overcome it. Moreover, it gives an overview of novel drugs and therapies that are aiming in overcoming drug resistance, loss of response, and kinase domain mutations. PMID:26917943

  16. Evaluating Drug Cost per Response with SGLT2 Inhibitors in Patients with Type 2 Diabetes Mellitus

    PubMed Central

    Lopez, Janice M.S.; Macomson, Brian; Ektare, Varun; Patel, Dipen; Botteman, Marc

    2015-01-01

    Background The sodium-glucose cotransporter 2 (SGLT2) inhibitors, which include canagliflozin, dapagliflozin, and empagliflozin, represent a new class of antihyperglycemic agents. Few studies have assessed their cost per response, with “cost per response” being the total cost of a select drug, divided by the resulting change in glycated hemoglobin (HbA1c) levels. Objective To examine the drug cost of SGLT2 inhibitors per a reduction in placebo-adjusted 1% HbA1c in patients with type 2 diabetes mellitus who received treatment during 26 weeks with canagliflozin, dapagliflozin, or empagliflozin. Methods The drug cost per response for each of the 3 agents individually was assessed based on data from a subset of clinical trials discussed in the prescribing information for each drug that were all placebo-controlled studies evaluating each drug as monotherapy, dual therapy (combined with metformin), and triple therapy (combined with metformin and a sulfonylurea) in patients with uncontrolled, type 2 diabetes mellitus. The US 2015 wholesale acquisition cost for each drug was used to calculate each drug's treatment costs over 26 weeks. The average cost per response for each drug was defined as the prescription drug cost of each SGLT2 inhibitor, divided by the average, placebo-adjusted HbA1c reduction at 26 weeks. Results The drug cost per unit dose was the same for canagliflozin (100 mg or 300 mg), dapagliflozin (5 mg or 10 mg), and empagliflozin (10 mg or 25 mg), at $11.43. The drug cost per placebo-adjusted 1% HbA1c reduction varied by agent and by dose, as a result of the differences in the treatment responses for each of the 3 drugs. The costs per response for canagliflozin 100 mg as monotherapy, dual therapy, and triple therapy regimens ranged from $2286 to $3355, and for canagliflozin 300 mg, from $1793 to $2702. The costs per response for dapagliflozin 5 mg as monotherapy and dual therapy (triple therapy was not available at the time of the study) ranged from

  17. Treatment progression in sulfonylurea and dipeptidyl peptidase-4 inhibitor cohorts of type 2 diabetes patients on metformin

    PubMed Central

    Peng, Xiaomei; Jiang, Dingfeng; Liu, Dongju; Varnado, Oralee J; Bae, Jay P

    2016-01-01

    Background Metformin is an oral antidiabetic drug (OAD) widely used as first-line therapy in type 2 diabetes (T2D) treatments. Numerous treatment pathways after metformin failure exist. It is important to understand how treatment choices influence subsequent therapy progressions. This retrospective study compares adherence to, persistence with, and treatment progression in sulfonylurea (SU) and dipeptidyl peptidase-4 (DPP-4) inhibitor patient cohorts with T2D on metformin. Methods Using health insurance claims data, matched patient cohorts were created and OAD use was compared in patients with T2D initiating SU or DPP-4 inhibitors (index drugs) since January 1, 2010, to December 31, 2010, with background metformin therapy. Propensity score matching adjusted for possible selection bias. Persistence was measured via Cox regression as days to a ≥60-day gap in index drug possession; adherence was defined as proportion of days covered (PDC) ≥80%. Evolving treatment patterns were traced at 6-month intervals for 24 months following index drug discontinuation. Results From among 19,621 and 7,484 patients in the SU and DPP-4 inhibitor cohorts, respectively, 6,758 patient pairs were matched. Persistence at 12 months in the SU cohort was 48.0% compared to 52.5% for the DPP-4 inhibitor cohort. PDC adherence (mean [SD]) during the 12-month follow-up period was 63.3 (29.7) for the SU cohort and 65.5 (28.7) for the DPP-4 inhibitor cohort. PDC ≥80% was 40.5% and 43.4% in the SU and DPP-4 inhibitor cohorts, respectively. A higher percentage of patients in the SU cohort remained untreated. Following index drug discontinuation, monotherapy was more common in the SU cohort, while use of two or three OADs was more common in the DPP-4 inhibitor cohort. Insulin therapy initiation was higher in the SU cohort. Conclusion Slightly better adherence and persistence were seen in the DPP-4 inhibitor cohort. Adherence and persistence remain a challenge to many patients; understanding

  18. Merkel cell polyomavirus and human papilloma virus in proliferative skin lesions arising in patients treated with BRAF inhibitors.

    PubMed

    Falchook, G S; Rady, P; Konopinski, J C; Busaidy, N; Hess, K; Hymes, S; Nguyen, H P; Prieto, V G; Bustinza-Linares, E; Lin, Q; Parkhurst, K L; Hong, D S; Sherman, S; Tyring, S K; Kurzrock, R

    2016-07-01

    The potential role of oncogenic viruses mediating development of proliferative skin lesions in patients treated with RAF inhibitors is poorly understood. The objective of this study was to investigate human papilloma virus (HPV) and Merkel cell polyomavirus (MCPyV) in skin lesions among patients treated with RAF inhibitors with the help of a case series describing prevalence of HPV, MCPyV, and RAS mutations in skin biopsies obtained from patients receiving RAF inhibitors and developing cutaneous lesions. HPV-DNA was amplified by PCR utilizing multiple nested primer systems designed for detection of a broad range of HPV types. MCPyV copy number determination with real time PCR technology was performed by a "Quantification of MCPyV, small t region" kit. Thirty-six patients were tested (squamous cell carcinoma (SCC) = 14; verruca vulgaris = 15; other = 11). Nine of 12 SCCs (75 %) and eight of 13 verruca vulgaris lesions (62 %) tested positive for MCPyV whereas none of the normal skin biopsies obtained from nine of these patients tested positive for MCPyV (p = 0.0007). HPV incidence in cutaneous SCCs was not different compared to normal skin (50 vs. 56 %, p = 0.86). The association between MCPyV and proliferative skin lesions after RAF inhibitor therapy merits further investigation. PMID:27098388

  19. Mammalian Target of Rapamycin Inhibitor Induced Complete Remission of a Recurrent Subependymal Giant Cell Astrocytoma in a Patient Without Features of Tuberous Sclerosis Complex.

    PubMed

    Appalla, Deepika; Depalma, Andres; Calderwood, Stanley

    2016-07-01

    The majority of patients with subependymal giant cell astrocytoma (SEGA) have tuberous sclerosis complex (TSC). In such patients, the mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to induce responses. Isolated SEGA have been reported in patients without clinical or genetic features of TSC. The treatment of these patients with everolimus has not previously been reported. We treated a patient with a recurrent isolated SEGA with an mTOR inhibitor. The patient tolerated therapy well and had a sustained complete remission. MTOR inhibitors may be useful for the treatment of isolated SEGA. Further study is warranted. PMID:26929034

  20. Whole-exome tumor sequencing study in biliary cancer patients with a response to MEK inhibitors

    PubMed Central

    Ahn, Daniel H.; Ozer, Hatice Gulcin; Hancioglu, Baris; Lesinski, Gregory B.; Timmers, Cynthia; Bekaii-Saab, Tanios

    2016-01-01

    We previously conducted a phase-II study with selumetinib (AZD6244), a small molecule inhibitor of MEK1/2, in advanced biliary tract cancers (BTC), where the primary endpoint was response rate. Several patients experienced objective response. These findings were confirmed with MEK162 in a similar patient population. To assess for tumor-specific genetic variants that mediate sensitivity to MEK inhibition in BTC, we performed whole-exome sequencing in patients with an objective response to selumetinib. Normal and tumor DNA from FFPE tissue from two patients who experienced an objective response underwent whole-exome sequencing. Raw sequence reads were processed with GATK workflow and tumor specific variants were identified using MuTect and VarScan2. Ensemble Variant Effect Predictor was used to determine functional consequences of these variants. Copy number changes and potential gene fusion events were also screened. Findings were compared to assess for any commonality between the two tumor samples, and whether the identified variants were intrinsic to the MAPK pathway. 1169 and 628 tumor-specific variants were identified in the two samples. Further analysis demonstrated 60 and 53 functional and novel variants, respectively. Of the identified tumor-specific variants, fusion events or copy number changes, no commonality was seen. Several variants in genes associated with ERK signaling were present in each tumor sample. Although there were no common tumor-specific variants in the two patients who exhibited an objective response to selumetinib, several genes associated with ERK signaling were identified. Confirmatory studies investigating the role of the identified genes and other potential tumor independent factors need further investigation. PMID:26683364

  1. Mechanisms of resistance in patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors.

    PubMed

    Perekhrestenko, T; Diachenko, M; Sviezhentseva, I; Gordienko, A; Bilko, D

    2015-03-01

    Up to date, two major mechanisms have been proposed as an explanation for myeloid cells expansion in chronic myeloid leukemia (CML). One is a reduced susceptibility of hematopoietic stem or progenitor cells to apoptosis, while the other one is an increased activity within the hematopoietic progenitor cell population. The aim of the study was to identify specific features of functional activity, proliferation rates and differentiation potential of CML hematopoietic progenitor cells of patients treated with tyrosine kinase inhibitors (TKI) by identifying number of Ki-67, Bcl-2 and CD34 positive cells in bone marrow, as well as in vitro colony-forming unit assay in patients with different response to the TKI therapy. Our results indicated that there was a significant decline in proliferation activity of HSCs and HPCs in group of patients with optimal response to the TKI therapy. Correlation analysis, performed on individual basis for patients independently of response to the TKI therapy demonstrated that there was a negative correlation (ρ=0.7648) between the number of Ki67+ and CD34+ cells. As to colony to cluster ratio our results showed, that there is a correlation (ρ=0.6783) between CCR index and number of bone marrow cells with Philadelphia chromosome. It was indicated, that index of maturation correlates with level of bone marrow cells, containing Philadelphia chromosome, so as with percentage of CD34+, Bcl-2+, Pgp-170+ and Ki67+ cells in bone marrow of CML patients. In summary, obtained results suggest that different mechanisms (bcr-abl dependent and independent) may be involved in CML progression process in the same time. Disease prognosis should be preferably carried out on an individual basis. PMID:25879558

  2. Pericardial effusions with tamponade and visceral constriction in patients with rheumatoid arthritis on tumour necrosis factor (TNF)-inhibitor therapy.

    PubMed

    Soh, May Ching; Hart, Hamish H; Corkill, Michael

    2009-04-01

    Tumour necrosis factor-inhibitor (TNF-inhibitor) therapy is increasingly used for the treatment of rheumatoid arthritis. While it is effective for the articular manifestations of rheumatoid arthritis we have reason to believe that it is less effective for extra-articular disease. We present two cases of life-threatening cardiac tamponade in two patients with well-controlled rheumatoid arthritis on adalimumab. An extensive literature search was carried out and three other patients were found. We believe that these cases highlight the need for rheumatologists to be vigilant for extra-articular manifestations of rheumatoid arthritis even in the presence of quiescent joint disease while on TNF-inhibitors. PMID:20374322

  3. [Side effects of COX-2 selective inhibitors. Critic related with its administration in patients with rheumatoid arthritis and osteoarthritis].

    PubMed

    Carrillo Gutiérrez, Ofmara Y; Pérez Sánchez, Adriana G; Medina Serriteño, Nicolás; Rodríguez Orozco, Alain R

    2007-01-01

    At the end of 2000 the new age of AINEs was introduced, specially the selective inhibitors of the COX-2, whose main function is to block the production of the prostaglandins and the acute tissue inflammation. These inhibitors have analgesic, antithermal and antiinflammatory effects similar to traditional AINEs; they are prescribed specifically to diminish pain and inflammation in patients with rheumatoid arthritis and osteoarthritis. After them introduction, it was reported that they can produce cardiovascular effects, mainly infarcts. This revision exposes the adverse effects that selective inhibitors of the COX-2 produce when elevated doses are administered, during prolonged time, in patients with rheumatoid arthritis and osteoarthritis; in addition, it comments present recommendations for them prescription. PMID:18297851

  4. Efficacy of different dipeptidyl peptidase-4 (DPP-4) inhibitors on metabolic parameters in patients with type 2 diabetes undergoing dialysis.

    PubMed

    Park, Se Hee; Nam, Joo Young; Han, Eugene; Lee, Yong-Ho; Lee, Byung-Wan; Kim, Beom Seok; Cha, Bong-Soo; Kim, Chul Sik; Kang, Eun Seok

    2016-08-01

    Hyperglycemia is associated with increased mortality and morbidity in patients with type 2 diabetes mellitus (T2DM) who are undergoing dialysis. Although dipeptidyl peptidase-4 (DPP-4) inhibitors have been widely used in end-stage renal disease (ESRD) patients with T2DM, there are few studies on their efficacy in this population. We studied the effect of 3 different DPP-4 inhibitors on metabolic parameters in ESRD patients with T2DM.Two hundred ESRD patients with T2DM who were treated with DPP-4 inhibitors (sitagliptin, vildagliptin, or linagliptin) were enrolled and analyzed retrospectively. The changes in glycated hemoglobin (HbA1c), fasting plasma glucose, and lipid profiles were assessed before and after 3 months of treatment with DPP-4 inhibitors. Subgroup analysis was done for each hemodialysis (HD) and peritoneal dialysis (PD) group.There was no significant difference in the decrease in the HbA1c level among sitagliptin, vildagliptin, and linagliptin treatment groups (-0.74 ± 1.57, -0.39 ± 1.45, and -0.08 ± 1.40, respectively, P = 0.076). The changes in fasting blood glucose and lipid profiles were also not significantly different. In HD patients (n = 115), there was no difference in the HbA1c level among the 3 groups. In contrast, in PD patients (n = 85), HbA1c was reduced more after 3 months of treatment with sitagliptin compared with vildagliptin and linagliptin (-1.58 ± 0.95, -0.46 ± 0.98, -0.04 ± 1.22, respectively, P = 0.001).There was no significant difference in the glucose-lowering effect between the different DPP-4 inhibitors tested in ESRD patients. In PD patients, sitagliptin tends to lower the HbA1c level more than the other inhibitors. The glucose-lowering efficacy of the 3 DPP-4 inhibitors was comparable. PMID:27512877

  5. Efficacy of different dipeptidyl peptidase-4 (DPP-4) inhibitors on metabolic parameters in patients with type 2 diabetes undergoing dialysis

    PubMed Central

    Park, Se Hee; Nam, Joo Young; Han, Eugene; Lee, Yong-ho; Lee, Byung-Wan; Kim, Beom Seok; Cha, Bong-Soo; Kim, Chul Sik; Kang, Eun Seok

    2016-01-01

    Abstract Hyperglycemia is associated with increased mortality and morbidity in patients with type 2 diabetes mellitus (T2DM) who are undergoing dialysis. Although dipeptidyl peptidase-4 (DPP-4) inhibitors have been widely used in end-stage renal disease (ESRD) patients with T2DM, there are few studies on their efficacy in this population. We studied the effect of 3 different DPP-4 inhibitors on metabolic parameters in ESRD patients with T2DM. Two hundred ESRD patients with T2DM who were treated with DPP-4 inhibitors (sitagliptin, vildagliptin, or linagliptin) were enrolled and analyzed retrospectively. The changes in glycated hemoglobin (HbA1c), fasting plasma glucose, and lipid profiles were assessed before and after 3 months of treatment with DPP-4 inhibitors. Subgroup analysis was done for each hemodialysis (HD) and peritoneal dialysis (PD) group. There was no significant difference in the decrease in the HbA1c level among sitagliptin, vildagliptin, and linagliptin treatment groups (−0.74 ± 1.57, −0.39 ± 1.45, and −0.08 ± 1.40, respectively, P = 0.076). The changes in fasting blood glucose and lipid profiles were also not significantly different. In HD patients (n = 115), there was no difference in the HbA1c level among the 3 groups. In contrast, in PD patients (n = 85), HbA1c was reduced more after 3 months of treatment with sitagliptin compared with vildagliptin and linagliptin (−1.58 ± 0.95, −0.46 ± 0.98, −0.04 ± 1.22, respectively, P = 0.001). There was no significant difference in the glucose-lowering effect between the different DPP-4 inhibitors tested in ESRD patients. In PD patients, sitagliptin tends to lower the HbA1c level more than the other inhibitors. The glucose-lowering efficacy of the 3 DPP-4 inhibitors was comparable. PMID:27512877

  6. Protease inhibitors partially overcome the interferon nonresponse phenotype in patients with chronic hepatitis C.

    PubMed

    Duarte-Rojo, A; Fischer, S E; Adeyi, O; Zita, D; Deneke, M G; Selzner, N; Chen, L; Malespin, M; Cotler, S J; McGilvray, I D; Feld, J J

    2016-05-01

    The outcome of triple therapy with protease inhibitors (PI) depends on the intrinsic response to interferon. Interferon-stimulated gene (ISG) expression differs by cell type in the liver and is a strong predictor of interferon responsiveness. Patients who respond well to interferon have low/absent ISG expression in hepatocytes but significant ISG expression in macrophages. Nonresponders (NRs) show the opposite pattern. We aimed to determine the association between cell-type-specific ISG staining and treatment outcome with PI-based triple therapy. Liver biopsy tissue from consecutive patients treated with boceprevir or telaprevir with peginterferon and ribavirin was stained for myxovirus A (MxA). Staining was scored 0-3 in macrophages (M-MxA) and hepatocytes (H-MxA), and IL28B genotyping was performed. Of 56 patients included 41 achieved SVR (73%) (sustained virological response), 2 (4%) relapsed, 10 (18%) were NRs, and 3 (5%) were lost to follow-up. Median M-MxA staining was stronger and H-MxA staining was weaker in patients who achieved SVR. MxA staining correlated with IL28B genotype and with the HCV RNA decline during lead-in phase. However, unlike with dual therapy, the negative predictive value (NPV) of absent or weak M-MxA staining was poor (42%), while the positive predictive value improved (93%). Although by multivariable logistic regression M-MxA staining was significantly associated with SVR (OR 4.35, 1.32-14.28, P = 0.012), the predictive ability was inadequate to withhold therapy. The interaction between macrophages and hepatocytes plays a critical role in interferon responsiveness; however, the addition of a PI at least partially overcomes the interferon nonresponse phenotype making the predictive ability of ISG staining less clinically useful. PMID:26710754

  7. Long-Term Use of Selective Serotonin Reuptake Inhibitors and Risk of Glaucoma in Depression Patients

    PubMed Central

    Chen, Hsin-Yi; Lin, Cheng-Li; Kao, Chia-Hung

    2015-01-01

    Abstract This study investigated whether the long-term use of selective serotonin reuptake inhibitors (SSRIs) influences the risk of primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG) in the Chinese ethnic population in Taiwan. The authors retrieved the data under analysis from the National Health Insurance Research Database in Taiwan and identified 26,186 newly diagnosed depression patients without preexisting glaucoma. The study cohort included 13,093 patients with over 1 year of SSRI use, and a comparison cohort of 13,093 patients who had never used SSRIs. The main outcome was a diagnosis of POAG or PACG during follow-up. The authors used univariable and multivariable Cox proportional hazards regression models to assess the effects of SSRIs on the risk of POAG and PACG. The cumulative incidences of POAG and PACG between the SSRI and comparison cohorts exhibited nonsignificant differences (log-rank test P = .52 for POAG, P = .32 for PACG). The overall incidence of POAG in the SSRI cohort was nonsignificantly higher than that in the comparison cohort (1.51 versus 1.39 per 1000 person-years), with an adjusted hazard ratio of 1.07 (95% confidence interval = 0.82–1.40). The overall incidence of PACG in the SSRI cohort was nonsignificantly lower than that in the comparison cohort (0.95 versus 1.11 per 1000 person-years), with an adjusted hazard ratio of 0.85 (95% confidence interval = 0.62–1.18). The long-term use of SSRIs does not influence the risk of POAG or PACG in depression patients. PMID:26559311

  8. An Acquired Factor VIII Inhibitor in a Patient with HIV and HCV: A Case Presentation and Literature Review

    PubMed Central

    Zeichner, S. B.; Harris, A.; Turner, G.; Francavilla, M.; Lutzky, J.

    2013-01-01

    Introduction. Despite its low incidence, acquired factor VIII inhibitor is the most common autoantibody affecting the clotting cascade. The exact mechanism of acquisition remains unclear, but postpartum patients, those with autoimmune conditions or malignancies, and those with exposure to particular drugs appear most susceptible. There have been several case reports describing acquired FVIII inhibitors in patients receiving interferon alpha for HCV treatment and in patients being treated for HIV. To our knowledge, this is the first case of a patient with HCV and HIV who was not actively receiving treatment for either condition. Case Presentation. A 57-year-old Caucasian male with a history of HIV and HCV was admitted to our hospital for a several day history of progressively worsening right thigh bruising and generalized weakness. CTA of the abdominal arteries revealed large bilateral retroperitoneal hematomas. Laboratory studies revealed the presence of a high titer FVIII inhibitor. Conclusion. Our case of a very rare condition highlights the importance of recognizing and understanding the diagnosis of acquired FVIII inhibitor. Laboratory research and clinical data on the role of newer agents are needed in order to better characterize disease pathogenesis, disease associations, genetic markers, and optimal disease management. PMID:24198984

  9. Tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1 Levels in Patients with Acute Paraquat Intoxication

    PubMed Central

    Seok, Su-Jin; Kim, Su-Ji; Gil, Hyo-Wook; Yang, Jong-Oh; Lee, Eun-Young

    2011-01-01

    To investigate the effects of reactive oxygen species (ROS) on tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) plasma levels, and their possible implications on clinical outcome, we measured tPA and PAI-1 levels in 101 patients with acute paraquat (PQ) intoxication. The control group consisted of patients who ingested non-PQ pesticides during the same period. tPA and PAI-1 levels were higher in the PQ group than in the controls. PQ levels were significantly correlated with ingested amount, timelag to hospital, tPA level, and hospitalization duration. tPA levels were correlated with PAI-1, fibrin degradation product (FDP), and D-dimer. D-dimer levels were lower in the PQ group than in the controls. Univariate analysis indicated the following significant determinants of death: age, ingested amount, PQ level, timelag to hospital, serum creatinine, lipase, pH, pCO2, HCO3-, WBC, FDP, PAI-1, and tPA. However, multivariate analysis indicated that only PQ level was significant independent factor predicting death. In conclusion, tPA and PAI-1 levels were higher, while D-dimer levels were lower in the PQ group than in the controls, implying that ROS stimulate tPA and PAI-1, but PAI-1 activity overrides tPA activity in this setting. Decreased fibrinolytic activity appears to be one of the clinical characteristics of acute PQ intoxication. PMID:21468253

  10. Rapid rFVIIa enhanced on-demand dosing in haemophilia inhibitor patients.

    PubMed

    Morfini, Massimo

    2016-02-01

    Recombinant factor VII activated (rFVIIa) is a bypassing agent widely used in haemophilia A and B patients with antibodies against coagulation factors VIII or IX. When used according to the correct doses, rFVIIa may control bleeding, subclinical bleeding and rebleeding, avoiding the effect of neutralising inhibitors. Because of the fast action of the rFVIIa, haemostasis occurs promptly and enables a fast bleeding control with on-demand treatment in home or in surgical setting. Rapidity is also a distinguishing feature in preparation and injection of rFVIIa to cope the restraining times of busy patients and parents. The effective haemostatic activity of rFVIIa enables a sustained bleeding control, which is implemented with every other day (eod) administration and suited for enhanced on-demand therapy and short-term repeated infusions use of rFVIIa to prevent microhaemorrhages or rebleeding. Comprehensive appreciation of these pharmacological and pharmacodynamic' characteristics will likely be a further stimulus to the wider enhanced on-demand use of rFVIIa. PMID:26172449

  11. INHALED ALPHA1-PROTEINASE INHIBITOR THERAPY IN PATIENTS WITH CYSTIC FIBROSIS

    PubMed Central

    Gaggar, Amit; Chen, Junliang; Chmiel, James F; Dorkin, Henry L; Flume, Patrick A; Griffin, Rhonda; Nichols, David; Donaldson, Scott H

    2016-01-01

    Background Inhaled alpha1-proteinase inhibitor (PI) is known to reduce neutrophil elastase burden in some patients with CF. This phase 2a study was designed to test inhaled Alpha-1 HC, a new aerosolized alpha1-PI formulation, in CF patients. Methods We performed a randomized, double-blind, placebo-controlled study and evaluated the safety of 100 or 200 mg of inhaled Alpha-1 HC once daily for 3 weeks in subjects with CF. Thirty adult subjects were randomized in a 2:1 ratio to receive Alpha-1 HC or placebo. Results Drug delivery was confirmed by a dose-dependent increase in the sputum alpha1-PI. Seven (20.0%) of the 35 adverse events in the 100-mg dose group, 3 (13.0%) of 23 in the 200-mg dose group, and 4 (14.3%) of 28 in the placebo group were drug-related in these subjects. One serious adverse event occurred in 1 subject within each group. Conclusions Alpha-1 HC inhalation was safe and well tolerated. PMID:26321218

  12. Liver Fibrosis in HCV Monoinfected and HIV/HCV Coinfected Patients: Dysregulation of Matrix Metalloproteinases (MMPs) and Their Tissue Inhibitors TIMPs and Effect of HCV Protease Inhibitors.

    PubMed

    Latronico, Tiziana; Mascia, Claudia; Pati, Ilaria; Zuccala, Paola; Mengoni, Fabio; Marocco, Raffaella; Tieghi, Tiziana; Belvisi, Valeria; Lichtner, Miriam; Vullo, Vincenzo; Mastroianni, Claudio Maria; Liuzzi, Grazia Maria

    2016-01-01

    An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may contribute to liver fibrosis in patients with hepatitis C (HCV) infection. We measured the circulating levels of different MMPs and TIMPs in HCV monoinfected and HIV/HCV coinfected patients and evaluated the potential for anti-HCV therapy to modulate MMP and TIMP levels in HCV subjects. We analyzed 83 plasma samples from 16 HCV monoinfected patients undergoing dual or triple anti-HCV therapy, 15 HIV/HCV coinfected patients with undetectable HIV load, and 10 healthy donors (HD). Levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, TIMP-1, and TIMP-2 were measured by a SearchLight Multiplex Immunoassay Kit. MMP-2 and MMP-9 were the highest expressed MMPs among all the analyzed samples and their levels significantly increased in HCV monoinfected and HIV/HCV coinfected subjects compared to HD. TIMP-1 levels were significantly higher in HCV and HIV/HCV subjects compared to HD and were correlated with liver stiffness. These findings raise the possibility of using circulating TIMP-1 as a non-invasive marker of liver fibrosis in HCV infection. A longitudinal study demonstrated that MMP-9 levels significantly decreased (40% reduction from baseline) in patients receiving dual as well as triple direct-acting antivirals (DAA) anti-HCV therapy, which had no effect on MMP-2, TIMP-1, and TIMP-2. As the dysregulation of MMP-2 and MMP-9 may reflect inflammatory processes in the liver, the decrease of MMP-9 following HCV protease inhibitor treatment suggests a positive effect on the reduction of liver inflammation. PMID:27023536

  13. Liver Fibrosis in HCV Monoinfected and HIV/HCV Coinfected Patients: Dysregulation of Matrix Metalloproteinases (MMPs) and Their Tissue Inhibitors TIMPs and Effect of HCV Protease Inhibitors

    PubMed Central

    Latronico, Tiziana; Mascia, Claudia; Pati, Ilaria; Zuccala, Paola; Mengoni, Fabio; Marocco, Raffaella; Tieghi, Tiziana; Belvisi, Valeria; Lichtner, Miriam; Vullo, Vincenzo; Mastroianni, Claudio Maria; Liuzzi, Grazia Maria

    2016-01-01

    An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may contribute to liver fibrosis in patients with hepatitis C (HCV) infection. We measured the circulating levels of different MMPs and TIMPs in HCV monoinfected and HIV/HCV coinfected patients and evaluated the potential for anti-HCV therapy to modulate MMP and TIMP levels in HCV subjects. We analyzed 83 plasma samples from 16 HCV monoinfected patients undergoing dual or triple anti-HCV therapy, 15 HIV/HCV coinfected patients with undetectable HIV load, and 10 healthy donors (HD). Levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, TIMP-1, and TIMP-2 were measured by a SearchLight Multiplex Immunoassay Kit. MMP-2 and MMP-9 were the highest expressed MMPs among all the analyzed samples and their levels significantly increased in HCV monoinfected and HIV/HCV coinfected subjects compared to HD. TIMP-1 levels were significantly higher in HCV and HIV/HCV subjects compared to HD and were correlated with liver stiffness. These findings raise the possibility of using circulating TIMP-1 as a non-invasive marker of liver fibrosis in HCV infection. A longitudinal study demonstrated that MMP-9 levels significantly decreased (40% reduction from baseline) in patients receiving dual as well as triple direct-acting antivirals (DAA) anti-HCV therapy, which had no effect on MMP-2, TIMP-1, and TIMP-2. As the dysregulation of MMP-2 and MMP-9 may reflect inflammatory processes in the liver, the decrease of MMP-9 following HCV protease inhibitor treatment suggests a positive effect on the reduction of liver inflammation. PMID:27023536

  14. The role of chemoprevention by selective cyclooxygenase-2 inhibitors in colorectal cancer patients - a population-based study

    PubMed Central

    2012-01-01

    Background There are limited population-based studies focusing on the chemopreventive effects of selective cyclooxygenase-2 (COX-2) inhibitors against colorectal cancer. The purpose of this study is to assess the trends and dose–response effects of various medication possession ratios (MPR) of selective COX-2 inhibitor used for chemoprevention of colorectal cancer. Methods A population-based case–control study was conducted using the Taiwan Health Insurance Research Database (NHIRD). The study comprised 21,460 colorectal cancer patients and 79,331 controls. The conditional logistic regression was applied to estimate the odds ratios (ORs) for COX-2 inhibitors used for several durations (5 years, 3 years, 1 year, 6 months and 3 months) prior to the index date. Results In patients receiving selective COX-2 inhibitors, the OR was 0.51 (95% CI=0.29~0.90, p=0.021) for an estimated 5-year period in developing colorectal cancer. ORs showing significant protection effects were found in 10% of MPRs for 5-year, 3-year, and 1-year usage. Risk reduction against colorectal cancer by selective COX-2 inhibitors was observed as early as 6 months after usage. Conclusion Our results indicate that selective COX-2 inhibitors may reduce the development of colorectal cancer by at least 10% based on the MPRs evaluated. Given the limited number of clinical reports from general populations, our results add to the knowledge of chemopreventive effects of selective COX-2 inhibitors against cancer in individuals at no increased risk of colorectal cancer. PMID:23217168

  15. Comparison of benefit–risk preferences of patients and physicians regarding cyclooxygenase-2 inhibitors using discrete choice experiments

    PubMed Central

    Byun, Ji-Hye; Kwon, Sun-Hong; Lee, Ji-Eun; Cheon, Ji-Eun; Jang, Eun-Jin; Lee, Eui-Kyung

    2016-01-01

    Purpose To elucidate and compare benefit–risk preferences among Korean patients and physicians concerning cyclooxygenase-2 (Cox-2) inhibitor treatments for arthritis. Materials and methods Subjects included 100 patients with arthritis and 60 board-certified orthopedic surgeon physicians in South Korea. Through a systematic review of the literature, beneficial attributes of using Cox-2 inhibitors were defined as a decrease in the Western Ontario and McMaster Universities Arthritis Index for pain score and improvement in physical function. Likewise, risk attributes included upper gastrointestinal (GI) complications and cardiovascular (CV) adverse events. Discrete choice experiments were used to determine preferences for these four attributes among Korean patients and physicians. Relative importance and maximum acceptable risk for improving beneficial attributes were assessed by analyzing the results of the discrete choice experiment by using a conditional logit model. Results Patients ranked the relative importance of benefit–risk attributes as follows: pain reduction (35.2%); physical function improvement (30.0%); fewer CV adverse events (21.5%); fewer GI complications (13.4%). The physicians’ ranking for the same attributes was as follows: fewer CV (33.5%); pain reduction (32.4%); fewer GI complications (18.1%); physical function improvement (16.0%). Patients were more willing than physicians to accept risks when pain improved from 20% or 45% to 55% and physical function improved from 15% or 35% to 45%. Conclusion We confirmed that patients and physicians had different benefit–risk preferences regarding Cox-2 inhibitors. Patients with arthritis prioritized the benefits of Cox-2 inhibitors over the risks; moreover, in comparison with the physicians, arthritis patients were more willing to accept the trade-off between benefits and risks to achieve the best treatment level. To reduce the preference gap and achieve treatment goals, physicians must better

  16. Use of mTOR inhibitors in the treatment of breast cancer: an evaluation of factors that influence patient outcomes

    PubMed Central

    Jerusalem, Guy; Rorive, Andree; Collignon, Joelle

    2014-01-01

    Many systemic treatment options are available for advanced breast cancer, including endocrine therapy, chemotherapy, anti-human epidermal growth factor receptor 2 (HER2) therapy, and other targeted agents. Recently, everolimus, a mammalian target of rapamycin (mTOR) inhibitor, combined with exemestane, an aromatase inhibitor, has been approved in Europe and the USA for patients suffering from estrogen receptor-positive, HER2-negative advanced breast cancer previously treated by a nonsteroidal aromatase inhibitor, based on the results of BOLERO-2 (Breast cancer trials of OraL EveROlimus). This study showed a statistically significant and clinically meaningful improvement in median progression-free survival. Results concerning the impact on overall survival are expected in the near future. This clinically oriented review focuses on the use of mTOR inhibitors in breast cancer. Results reported with first-generation mTOR inhibitors (ridaforolimus, temsirolimus, everolimus) are discussed. The current and potential role of mTOR inhibitors is reported according to breast cancer subtype (estrogen receptor-positive HER2-negative, triple-negative, and HER2-positive ER-positive/negative disease). Everolimus is currently being evaluated in the adjuvant setting in high-risk estrogen receptor-positive, HER2-negative early breast cancer. Continuing mTOR inhibition or alternatively administering other drugs targeting the phosphatidylinositol-3-kinase/protein kinase B-mTOR pathway after progression on treatments including an mTOR inhibitor is under evaluation. Potential biomarkers to select patients showing a more pronounced benefit are reviewed, but we are not currently using these biomarkers in routine practice. Subgroup analysis of BOLERO 2 has shown that the benefit is consistent in all subgroups and that it is impossible to select patients not benefiting from addition of everolimus to exemestane. Side effects and impact on quality of life are other important issues discussed

  17. Advances in bypassing agent therapy for hemophilia patients with inhibitors to close care gaps and improve outcomes.

    PubMed

    Shapiro, Amy D; Hedner, Ulla

    2011-10-01

    In the past, patients with hemophilia and inhibitors have had less-than-optimal treatment and have experienced more orthopedic complications than patients without inhibitors. Bypassing agents offer the potential to close treatment gaps between inhibitor and noninhibitor patients by helping the former better attain key treatment goals, including: facilitating early initiation of treatment and hemostatic control in hemarthroses; providing effective treatment in serious hemorrhagic episodes; and performance of major surgery. Effective treatment with a bypassing agent minimizes joint and/or muscle damage and potentially can serve as an effective prophylactic agent to minimize the number of hemarthroses experienced per year, thereby mitigating the development of arthropathy. The reported efficacy of the currently available bypassing agents ranges from approximately 50-80% (50-64% in controlled studies) for plasma-derived activated prothrombin complex concentrate (pd-aPCC) and 81-91% (in controlled studies) for recombinant activated factor VII (rFVIIa), including use in major orthopedic surgery. Both bypassing agents have undergone key improvements in their formulation and/or properties in recent years. The nanofiltered, vapor-heated formulation of pd-aPCC has diminished the risk of acquiring blood-borne viral infections and the room temperature stable formulation of rFVIIa allows more convenient storage, increased ease to dissolve and inject, and smaller volumes, thereby increasing overall ease of administration. Use of recommended dosing has been demonstrated to provide effective hemostasis with a minimal number of injections for both agents. In this paper, we review the individual characteristics of pd-aPCC and rFVIIa and discuss clinical data from studies conducted in inhibitor patients that demonstrate the potential benefits of these bypassing agents in this difficult-to-treat population, and underscore the potential opportunities to close the gap in care between

  18. The role of B-cell receptor inhibitors in the treatment of patients with chronic lymphocytic leukemia

    PubMed Central

    Wiestner, Adrian

    2015-01-01

    Chronic lymphocytic leukemia is a malignancy of mature auto-reactive B cells. Genetic and functional studies implicate B-cell receptor signaling as a pivotal pathway in its pathogenesis. Full B-cell receptor activation requires tumor-microenvironment interactions in lymphoid tissues. Spleen tyrosine kinase, Bruton’s tyrosine kinase, and the phosphatidylinositol 3-kinase (PI3K) δ isoform are essential for B-cell receptor signal transduction but also mediate the effect of other pathways engaged in chronic lymphocytic leukemia cells in the tissue-microenvironment. Orally bioavailable inhibitors of spleen tyrosine kinase, Bruton’s tyrosine kinase, or PI3Kδ, induce high rates of durable responses. Ibrutinib, a covalent inhibitor of Bruton’s tyrosine kinase, and idelalisib, a selective inhibitor of PI3Kδ, have obtained regulatory approval in chronic lymphocytic leukemia. Ibrutinib and idelalisib are active in patients with high-risk features, achieving superior disease control in difficult-to-treat patients than prior best therapy, making them the preferred agents for chronic lymphocytic leukemia with TP53 aberrations and for patients resistant to chemoimmunotherapy. In randomized trials, both ibrutinib, versus ofatumumab, and idelalisib in combination with rituximab, versus placebo with rituximab improved survival in relapsed/refractory chronic lymphocytic leukemia. Responses to B-cell receptor inhibitors are mostly partial, and within clinical trials treatment is continued until progression or occurrence of intolerable side effects. Ibrutinib and idelalisib are, overall, well tolerated; notable adverse events include increased bruising and incidence of atrial fibrillation on ibrutinib and colitis, pneumonitis and transaminase elevations on idelalisib. Randomized trials investigate the role of B-cell receptor inhibitors in first-line therapy and the benefit of combinations. This review discusses the biological basis for targeted therapy of chronic lymphocytic

  19. The role of B-cell receptor inhibitors in the treatment of patients with chronic lymphocytic leukemia.

    PubMed

    Wiestner, Adrian

    2015-12-01

    Chronic lymphocytic leukemia is a malignancy of mature auto-reactive B cells. Genetic and functional studies implicate B-cell receptor signaling as a pivotal pathway in its pathogenesis. Full B-cell receptor activation requires tumor-microenvironment interactions in lymphoid tissues. Spleen tyrosine kinase, Bruton's tyrosine kinase, and the phosphatidylinositol 3-kinase (PI3K) δ isoform are essential for B-cell receptor signal transduction but also mediate the effect of other pathways engaged in chronic lymphocytic leukemia cells in the tissue-microenvironment. Orally bioavailable inhibitors of spleen tyrosine kinase, Bruton's tyrosine kinase, or PI3Kδ, induce high rates of durable responses. Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase, and idelalisib, a selective inhibitor of PI3Kδ, have obtained regulatory approval in chronic lymphocytic leukemia. Ibrutinib and idelalisib are active in patients with high-risk features, achieving superior disease control in difficult-to-treat patients than prior best therapy, making them the preferred agents for chronic lymphocytic leukemia with TP53 aberrations and for patients resistant to chemoimmunotherapy. In randomized trials, both ibrutinib, versus ofatumumab, and idelalisib in combination with rituximab, versus placebo with rituximab improved survival in relapsed/refractory chronic lymphocytic leukemia. Responses to B-cell receptor inhibitors are mostly partial, and within clinical trials treatment is continued until progression or occurrence of intolerable side effects. Ibrutinib and idelalisib are, overall, well tolerated; notable adverse events include increased bruising and incidence of atrial fibrillation on ibrutinib and colitis, pneumonitis and transaminase elevations on idelalisib. Randomized trials investigate the role of B-cell receptor inhibitors in first-line therapy and the benefit of combinations. This review discusses the biological basis for targeted therapy of chronic lymphocytic

  20. Clinical Effects of Discontinuing 5-Alpha Reductase Inhibitor in Patients With Benign Prostatic Hyperplasia

    PubMed Central

    Kim, Won; Jung, Jae Hung; Kang, Tae Wook; Song, Jae Mann

    2014-01-01

    Purpose To assess changes in lower urinary tract symptoms (LUTS), prostate volume, and serum prostate-specific antigen (PSA) after discontinuation of 5-alpha reductase inhibitor (5ARI) combination therapy in patients with benign prostatic hyperplasia (BPH). Materials and Methods From December 2003 to December 2012, data were collected retrospectively from 81 men more than 40 years of age with moderate to severe BPH symptoms (International Prostate Symptom Score [IPSS]≥8). The men were classified into group 1 (n=42) and group 2 (n=39) according to the use of 5ARI therapy. A combination of dutasteride 0.5 mg with tamsulosin 0.2 mg was given daily to all patients for 1 year. For the next 1 year, group 1 (n=42) received the combination therapy and group 2 (n=39) received tamsulosin 0.2 mg monotherapy only. The IPSS, prostate volume, and PSA level were measured at baseline and at 12 and 24 months according to the use of dutasteride. Results Discontinuation of dutasteride led to significant deterioration of LUTS, increased prostate volume, and increased PSA level. The repeated-measures analysis of variance showed that the changes in IPSS, prostate volume, and PSA level over time also differed significantly between groups 1 and 2 (p<0.001). Conclusions Withdrawal of 5ARI during combination therapy resulted in prostate regrowth and deterioration of LUTS. The PSA level is also affected by the use of 5ARI. Therefore, regular check-up of the IPSS and PSA level may be helpful for all patients who either continue or discontinue the use of 5ARI. PMID:24466398

  1. Immunosuppression in irradiated breast cancer patients: In vitro effect of cyclooxygenase inhibitors

    SciTech Connect

    Wasserman, J.; Blomgren, H.; Rotstein, S.; Petrini, B.; Hammarstroem, S.

    1989-01-01

    We have documented in previous studies that local irradiation therapy for breast cancer caused severe lymphopenia with reduction of both T and non-T lymphocytes. Non-T cells were relatively more depressed but recovered within six months. The recovery of T cells, on the other hand, remained incomplete 10-11 years after irradiation. Several lymphocyte functions were also severely impaired. An association was found between prognosis and postirradiation mitogen reactivity of lymphocytes from these patients. Mortality up to eight years after irradiation was significantly higher in patients with low postirradiation phytohemagglutinin and PPD reactivity. The radiation induced decrease in mitogenic response seemed mainly to be caused by immunosuppressive monocytes, which suggests that the underlying mechanism might be mediated by increased production of prostaglandins by monocytes. For this reason we examined the effect of some cyclooxygenase products on different lymphocyte functions and found that prostaglandins A2, D2, and E2 inhibited phytohemagglutinin response in vitro. Natural killer cell activity was also reduced by prostaglandins D2 and E2. The next step was to examine various inhibitors of cyclooxygenase in respect to their capacity to revert irradiation-induced suppression of in vitro mitogen response in lymphocytes from breast cancer patients. It was demonstrated that Diclofenac Na (Voltaren), Meclofenamic acid, Indomethacin, and lysin-mono-acetylsalicylate (Aspisol) could enhance mitogen responses both before and after radiation therapy. This effect was most pronounced at completion of irradiation. On a molar basis, Diclofenac Na was most effective followed by Indomethacin, Meclofenamic acid, and lysin-monoacetylsalicylate.

  2. Can ACE inhibitors and angiotensin receptor blockers be detrimental in CKD patients?

    PubMed

    Onuigbo, Macaulay A C

    2011-01-01

    Current epidemiological data from the USA, Europe, Asia and the Indian subcontinent, Africa, the Far East, South America, the Middle East and Eastern Europe all point to the increasing incidence of renal failure encompassing acute kidney injury (AKI), chronic kidney disease (CKD) and end-stage renal disease (ESRD). While the explanations for these worldwide epidemics remain speculative, it must be acknowledged that these increases in AKI, CKD and ESRD, happening worldwide, have occurred despite the universal application of strategies of renoprotection over the last 2 decades, more especially the widespread use of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs). We note that many of the published large renin-angiotensin-aldosterone system (RAAS) blockade randomized controlled trials, upon which current evidence-based practice for the increasing use of ACEIs and ARBs for renoprotection derived from, have strong deficiencies that have been highlighted over the years. From reports in the literature, there is an increasing association of exacerbations of renal failure with ACEIs and ARBs, more so in the older hypertensive patient, >65 years old. The biological plausibility for ACEI and ARB to protect the kidneys against a background of potential multiple pathogenetic pathways to account for CKD progression appears to be not very defensible. We reviewe