Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.

Science.gov (United States)

Mega, T; Nishijima, T; Ikenaka, T

1990-04-01

beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.

Alpha fucosidase and beta galactosidase in serum of a Lyme disease patients as a possible marker of accelerated senescence — a preliminary study

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Anna Wasiluk

2012-07-01

Full Text Available Lyme disease (LD is the most prevalent tick-borne disease in Europe. LD is caused by the spirochete Borrelia burgdorferi. LD is a chronic disease which can attack a number of organs: skin, heart, brain, joints. Chronic, low-grade inflammation involves general production of pro-inflammatory cytokines and inflammatory markers and is a typical feature of aging. So far, the best method of diagnosing LD is a time-consuming and expensive two-stage serological method. The aim of our study was to evaluate the activity of two lysosomal exoglycosidases: α-fucosidase (FUC and β-galactosidase (GAL in the serum of patients with Lyme disease, as potential markers of LD. Due to the increasing number of patients with Lyme disease and a number of false results, new ways to diagnose this disease are still being sought. As elevated level of β-galactosidase is a manifestation of residual lysosomal activity in senescent cells, the increase in its activity in serum during chronic Lyme disease might be a marker of a potentially accelerated senescence process. The study was performed on serum taken from cubital veins of 15 patients with Lyme disease and eight healthy subjects (control group. FUC and GAL activity was measured by the method of Chatterjee et al. as modified by Zwierz et al. In the serum of patients with Lyme disease, GAL activity significantly increased (p = 0.029, and the activity of FUC had a tendency to increase (p = 0.153, compared to the control group. A significant increase in GAL activity in the serum of patients with Lyme disease indicates an increased catabolism of glycoconjugates (glycoproteins, glycolipids, proteoglycans and could be helpful in the diagnosis of Lyme disease, although this requires confirmation in a larger group of patients. As GAL is the most widely used assay for detection of senescent cells, an elevated level of β-galactosidase might be a manifestation of accelerated senescence process in the course of Lyme

Melibiose permease and alpha-galactosidase of Escherichia coli: Identification by selective labeling using a T7 RNA polymerase/promoter expression system

International Nuclear Information System (INIS)

Pourcher, T.; Bassilana, M.; Sarkar, H.K.; Kaback, H.R.; Leblanc, G.

1990-01-01

Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested

Characterization of the activity of β-galactosidase from Escherichia coli and Drosophila melanogaster in fixed and non-fixed Drosophila tissues

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Mizuki Tomizawa

2016-12-01

Full Text Available β-Galactosidase encoded by the Escherichia coli lacZ gene, is widely used as a reporter molecule in molecular biology in a wide variety of animals. β-Galactosidase retains its enzymatic activity in cells or tissues even after fixation and can degrade X-Gal, a frequently used colormetric substrate, producing a blue color. Therefore, it can be used for the activity staining of fixed tissues. However, the enzymatic activity of the β-galactosidase that is ectopically expressed in the non-fixed tissues of animals has not been extensively studied. Here, we report the characterization of β-galactosidase activity in Drosophila tissues with and without fixation in various experimental conditions comparing the activity of two evolutionarily orthologous β-galactosidases derived from the E. coli lacZ and Drosophila melanogaster DmelGal genes. We performed quantitative analysis of the activity staining of larval imaginal discs and an in vitro assay using larval lysates. Our data showed that both E. coli and Drosophila β-galactosidase can be used for cell-type-specific activity staining, but they have their own preferences in regard to conditions. E. coli β-galactosidase showed a preference for neutral pH but not for acidic pH compared with Drosophila β-galactosidase. Our data suggested that both E. coli and Drosophila β-galactosidase show enzymatic activity in the physiological conditions of living animals when they are ectopically expressed in a desired specific spatial and temporal pattern. This may enable their future application to studies of chemical biology using model animals.

Study of the transgalactosylation activity of ß-galactosidase from a new strain Kluyveromyces lactis 3

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ILIA ILIEV

2012-01-01

Full Text Available Beta-galactosidase (EC.3.2.1.23 is an important enzyme industrially used for the hydrolysis of lactose from milk and milk whey for several applications. Lately, the importance of this enzyme was enhanced by its galactosyltransferase activity, which is responsible for synthesis of transgalactosylated oligosaccharides that act as prebiotics with several beneficial effects on the consumers. ß-Galactosidase production by Kluyveromyces lactis 3 was studied in shake flask culture. The highest enzymatic activity was obtained at 10-th hour of the fermentation. The optimum temperature for transferase activity was 50°C. When incubated with 30% lactose in 50 mM phosphate buffer (pH 6.0 the enzyme can synthesize up to 41% galacto-oligosaccharides (GalOS. β-Galactosidase from strain Kluyveromyces lactis 3 produces mainly oligosaccharides with degree of polymerization (DP 6 at 40°C and with DP 3 at 50°C.

Senescence-associated β-galactosidase activity in the in vitro ovarian stromal fibroblasts

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Lilian Chuaire-Noack

2011-04-01

Full Text Available A growing biological research field is the cellular senescence, a mechanism that has been associated, under certain circumstances, withmalignant transformation. Given the high incidence of ovarian cancerand its main origin from the ovarian surface epithelium, as well asthe possibility that an epithelial-mesenchymal transition occurs, weevaluated both the in vitro growth of stromal fibroblasts from the ovarian cortex and their β-galactosidase activity at pH 6,enzyme whose expression is considered as a marker of replicativesenescence. Methods: 48 samples of ovarian cortical fibroblasts fromdonors without a history of cancer were serially cultured untilthe end of their replicative life. β-galactosidase activity at pH 6was quantified in each passage by the chemiluminiscent method. Ascontrol, we used ovarian epithelial cell cultures from the samedonors. The enzyme activity was also evaluated in fibroblastspreviously induced to senescence by exposure to hydrogen peroxide.Results: The analysis of the enzyme activity and the replicativecapacity taken together showed that the fibroblast cultures reachedthe senescent state at passages 4-5, as what happened with the control epithelial cells. Fibroblasts induced to senescence showed high variability in the values of enzymatic activity. Conclusions:The similarity between both types of cells in reaching the senescent state deserves to be taken into account in relation to theepithelialmesenchymal transition that has been proposed to explaintheir behavior in the genesis of cancer arising from ovarian surfaceepithelium. Low β-galactosidase activity values at pH 6 would suggestpossible inactivation of the response pathways to oxidative stress.

Purification and properties of beta-galactosidase from Aspergillus nidulans.

Science.gov (United States)

Díaz, M; Pedregosa, A M; de Lucas, J R; Torralba, S; Monistrol, I F; Laborda, F

1996-12-01

Beta-Galactosidase from mycelial extract of Aspergillus nidulans has been purified by substrate affinity chromatography and used to obtain anti-beta-galactosidase polyclonal antibodies. A. nidulans growing in lactose as carbon source synthesizes one active form of beta-galactosidase which seems to be a multimeric enzyme of 450 kDa composed of monomers with 120 and 97 kDa. Although the enzyme was not released to the culture medium, some enzymatic activity was detected in a cell-wall extract, thus suggesting that it can be an extracellular enzyme. Beta-Galactosidase of A. nidulans is a very unstable enzyme with an optimum pH value of 7.5 and an optimum temperature of 30 degrees C. It was only active against beta-galactoside substrates like lactose and p-nitrophenyl-beta-D-galactoside (PNPG).

Purification and properties of a beta-galactosidase from carambola fruit with significant activity towards cell wall polysaccharides.

Science.gov (United States)

Balasubramaniam, Sumathi; Lee, Heng Chin; Lazan, Hamid; Othman, Roohaida; Ali, Zainon Mohd

2005-01-01

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.

Optimization of β-galactosidase production from lactic acid bacteria

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Carević Milica

2015-01-01

Full Text Available β-galactosidase, commonly known as lactase, represents commercially important enzyme that is prevalently used for lactose hydrolysis in milk and whey. To the date, it has been isolated from various sources. In this study different strains of lactic acid bacteria were assessed for their β-galactosidase productivity, and Lactobacillus acidophilus ATCC 4356 resulted with the highest production potential. Thereafter, optimal conditions for accomplishing high yields of β-galactosidase activity were determined. Maximal specific activity (1.01 IU mL-1 was accomplished after 2 days shake flask culture fermentation (150 rpm at 37ºC, with modified Man Rogosa Sharpe culture broth using lactose (2.5% as sole carbon source. Finally, in order to intensify release of intracellular β-galactosidase different mechanical and chemical methods were conducted. Nevertheless, vortexing with quartz sand (150 μm as abrasive was proven to be the most efficient method of cell disruption. The optimum temperature of obtained β-galactosidase was 45°C and the optimum range pH 6.5-7.5.

Inhibitory activity and conformational transition of alpha 1-proteinase inhibitor variants

NARCIS (Netherlands)

Schulze, A.J.; Huber, R.; Degryse, E.; Speck, D.; Bischoff, Rainer

1991-01-01

Several variants of alpha 1-proteinase inhibitor (alpha 1-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in alpha 1-PI containing an Arg residue in position 358 (yielding [Thr345----Arg,

Host-Pathogen Interactions: I. A Correlation Between α-Galactosidase Production and Virulence 1

Science.gov (United States)

English, Patricia D.; Albersheim, Peter

1969-01-01

Resistance or susceptibility of Red Kidney, Pinto and Small White beans (Phaseolus vulgaris) to the alpha, beta, and gamma strains of Colletotrichum lindemuthianum was either confirmed or established. These fungal strains secrete α-galactosidase, β-galactosidase and β-xylosidase when grown on cell walls isolated from the hypocotyls of any of the above bean varieties. These enzymes effectively degrade cell walls isolated from susceptible 5-day old hypocotyls but degrade only slightly the walls isolated from resistant 18-day old hypocotyls. The amounts of the β-galactosidase and β-xylosidase secreted by the 3 fungal strains are relatively low and are approximately equivalent. The secretion of these 2 enzymes is not dependent upon the bean variety from which the hypocotyl cell walls used as a carbon source were isolated. However, the fungal strains secrete greater amounts of α-galactosidase when grown on hypocotyl cell walls isolated from susceptible plants than when grown on walls from resistant plants. Virulent isolates of the fungus, when grown on hypocotyl cell walls isolated from a susceptible plant, secrete more α-galactosidase than do attenuated (avirulent) isolates of the same fungal strain grown under the same conditions. The α-galactosidase secreted by each of the fungal strains is capable of removing galactose from the hypocotyl cell walls of each bean variety tested. Galactose is removed from the cell walls of each variety at the same rate regardless of whether the cell walls were isolated from a susceptible or resistant plant. PMID:16657049

Thermomyces lanuginosus CBS 395.62/b Strain as Rich Source of α-Galactosidase Enzyme

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Quang D. Nguyen

2003-01-01

Full Text Available Seventeen Thermomyces lanuginosus strains, cultivated on raffinose and sucrose, were ranked on the basis of α-galactosidase activities. T. lanuginosus CBS 395.62/b strain showed the highest α-galactosidase activity on both investigated carbohydrates. Several carbon sources were tested as potential inducers for the α-galactosidase synthesis. On melibiose substrate α-galactosidase activity was higher in the intracellular fraction than in the filtrate of the fermentation broth, although both values were very low and did not reach the value of 1 U/mL. Raffinose, sucrose and Lactosucrose® proved to be inducers for α-galactosidase production. The highest titer (about 30 U/mL was achieved on 1 % sucrose and 0.45 % ammonium acetate. The optimum sucrose and ammonium acetate concentrations, at which about 90 U/mL α-galactosidase activity was reached during an 8-day fermentation, were 3 and 0.9 %, respectively.

Potential applicaton of β-galactosidase in food science and nutrition

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Nika ŽIBRAT

2017-10-01

Full Text Available β-galactosidase is an enzyme with hydrolytic and transgalactosylation activity. The origin of the enzyme dictates the balance between both activities. Industrially used β-galactosidases are obtained with recombinant production from filamentus funghi Aspergillus sp. and yeasts Kluyveromyces sp. Recently thermostabile β-galactosidases have been subject of many research. The enzyme can be industrially used in free or immobilized form. Immobilization often provides better stability, reusability and lower expenses. Application of β-galactosidase is most common in food processing and nutrition, it is also used in medicine and ecology. Hydrolytic activity of the enzyme has long been used for reducing lactose content in milk, while transgalactosylitic activity is used for synthesis of products such as galactooligosaccharides, lactosucrose and others. The latter have a great potential in food industry for obtaining products with reduced lactose content and increasing of nutritional value by adding dietetic fibers such as galactooligosaccharides. Despite the potential it is vital that reaction mechanisms become better understood and optimization is in place in order to reach the usability of this enzyme at industrial level.

A network of hydrophobic residues impeding helix alphaC rotation maintains latency of kinase Gcn2, which phosphorylates the alpha subunit of translation initiation factor 2.

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Gárriz, Andrés; Qiu, Hongfang; Dey, Madhusudan; Seo, Eun-Joo; Dever, Thomas E; Hinnebusch, Alan G

2009-03-01

Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix alphaC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2alpha phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of alphaC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of alphaC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.

β-Galactosidase Deficiency in Colombia

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Alfredo Uribe PhD

2015-05-01

Full Text Available β-Galactosidase (BGal is the first enzyme involved in the catabolism of sphingolipids. Two pathologies have been directly associated with its deficiency: GM1 gangliosidosis and Morquio B. Morquio B is among the rarest types of mucopolysaccharidosis (MPS. We aim to document the β-galactosidase deficiency in Colombia. We evaluated leukocytes from 1492 healthy Colombian individuals and 923 patients, referred between 2005 and August 2014. Dried blood spot (DBS samples from the same number of patients were evaluated. β-Galactosidase was measured with 4-methylumbelliferyl-β- d -galactoside. As a control enzyme, the total hexosaminidase activity was also evaluated. We identified 14 patients with GM1 gangliosidosis, 5 patients with Morquio B, and 1 patient with I-cell disease. We could establish a reference value for Bgal in Colombian leukocyte samples. GM1 gangliosidosis is the main pathology associated with a direct deficiency of BGal. The high number of patients found with MPS IVB indicates that there are patients who could be misdiagnosed due to an unawareness of the disease.

A comparison of the activities of three beta-galactosidases in aqueous-organic solvent mixtures

NARCIS (Netherlands)

Yoon, JH; Mckenzie, D

2005-01-01

The hydrolytic activities of beta-galactosidases from three different sources have been determined in various 50% (v/v) organic solvent-buffer mixtures with a view to finding solvent systems of reduced water content suitable for the synthesis of glycosides and oligosaccharides. K. fragilis

A new cold-adapted β-D-galactosidase from the Antarctic Arthrobacter sp. 32c – gene cloning, overexpression, purification and properties

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Kur Józef

2009-07-01

Full Text Available Abstract Background The development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production. Results In this article, we present a new β-D-galactosidase as a candidate to be applied in the above mentioned biotechnological processes. The gene encoding this β-D-galactosidase has been isolated from the genomic DNA library of Antarctic bacterium Arthrobacter sp. 32c, sequenced, cloned, expressed in Escherichia coli and Pichia pastoris, purified and characterized. 27 mg of β-D-galactosidase was purified from 1 L of culture with the use of an intracellular E. coli expression system. The protein was also produced extracellularly by P. pastoris in high amounts giving approximately 137 mg and 97 mg of purified enzyme from 1 L of P. pastoris culture for the AOX1 and a constitutive system, respectively. The enzyme was purified to electrophoretic homogeneity by using either one step- or a fast two step- procedure including protein precipitation and affinity chromatography. The enzyme was found to be active as a homotrimeric protein consisting of 695 amino acid residues in each monomer. Although, the maximum activity of the enzyme was determined at pH 6.5 and 50°C, 60% of the maximum activity of the enzyme was determined at 25°C and 15% of the maximum activity was detected at 0°C. Conclusion The properties of Arthrobacter sp. 32cβ-D-galactosidase suggest that this enzyme could be useful for low-cost, industrial conversion of lactose into galactose and glucose in milk products and could be an interesting alternative for the production of ethanol from lactose-based feedstock.

Effect of Carrot-Juice on Exopolisaccharides and β-D Galactosidase Activity in Yogurt

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Lilik Eka Radiati

2016-09-01

Full Text Available Carrot juice and milk  were  blended and fermented by  culture bacteria Streptococcus thermophillus  and Lactobacillus bulgaricus.  Carrot juice  affect significantly  on   lactic acid content   (1.09± 0.12% – 1.15± 0.01%,   pH value (3.80±0.06 – 4.17± 0.10,   viscosity  (133±2.30 cP–146±2.10cP,  β-carotene (0-173.19±1.02 µg/g, EPS (11.90 ±0.50 - 18.00 ±0.40 mg/100g,   β-D-galactosidase activity (2.27±0.30-192.40±0.48 µ/g yogurt, but did not affect significantly on  bacteria number (9.0± 0.5 – 9.8±0.4 log CFU/g.    Carrot juice increased the yogurt culture activity with increasing acidifying, β-catotene, EPS and β-D-galactosidase, suggesting that yogurt could be fortified with carrot juice.

Activity of enzymes that hydrolyze sucrose and raffinose in the first stages of germination of Lactuca sativa cv. Grand rapids. [Invertase, alpha-galactosidose, and sucrose synthetase were observed

Energy Technology Data Exchange (ETDEWEB)

Slabnik, E.; Calderon, P.; Diaz, H.

1981-01-01

The activities of enzymes capable of metabolizing raffinose and sucrose on achenes of lettuce were studied. During the first stages of germination, evidence was obtained for the occurrence of invertase in the endosperm and embryonic axis. Alpha-galactosidase was localized in the endosperm and cotyledons. Sucrose synthetase was present in the dry seed.

Identification of the gamma subunit-interacting residues on photoreceptor cGMP phosphodiesterase, PDE6alpha '.

Science.gov (United States)

Granovsky, A E; Artemyev, N O

2000-12-29

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory gamma subunit (Pgamma). Chimeric proteins between cone PDE6alpha' and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by Pgamma. Substitution of the segment PDE5-(773-820) by the corresponding PDE6alpha'-(737-784) sequence in the wild-type PDE5 or in a PDE5/PDE6alpha' chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pgamma. The catalytic properties of the chimeric PDEs remained similar to those of PDE5. Ala-scanning mutational analysis of the Pgamma-binding region, PDE6alpha'-(750-760), revealed PDE6alpha' residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired the inhibition of chimeric PDE by Pgamma. The analysis of the catalytic properties of mutant PDEs and a model of the PDE6 catalytic domain suggest that residues Met(758) and Gln(752) directly bind Pgamma. A model of the PDE6 catalytic site shows that PDE6alpha'-(750-760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pgamma to Met(758) would effectively block access of cGMP to the catalytic cavity, providing a structural basis for the mechanism of PDE6 inhibition.

Water quality - Measurement of gross alpha activity in non-saline water - Thick source method. 2. ed.

International Nuclear Information System (INIS)

2007-01-01

This International Standard specifies a method for the determination of gross alpha activity in non-saline waters for alpha-emitting radionuclides which are not volatile at 350 degree Centigrade. It is possible to determine supported volatile radionuclides measured to an extent determined by half-life, matrix retention (of the volatile species) and the duration of measurement (counting time). The method is applicable to raw and potable waters. The range of application depends on the amount of suspended matter in the water and on the performance characteristics (background count rate and counting efficiency) of the counter. Gross alpha radioactivity is determined by using proportional counting or solid scintillation counting on water residue deposited on a planchet. Due to the strong absorption of the residue deposit, it is considered that the alpha emission from the surface is proportional to the alpha activity of the deposit. Gross alpha determination is not an absolute determination of the sample alpha radioactive content, but a relative determination referring to a specific alpha emitter which constitutes the standard calibration source. This type of determination is also known as alpha index. The sample is acidified to stabilize it, evaporated almost to dryness, converted to the sulfate form and then ignited at 350 degree Centigrade. A portion of the residue is transferred to a planchet and the alpha activity measured by counting in an alpha-particle detector or counting system previously calibrated against an alpha-emitting standard and the alpha activity concentration calculated. The paper provides information about scope, normative references, symbols, definitions and units, principle, reagents and equipment, procedure, contamination check, expression of results and test report

Three-residue turns in alpha/beta-peptides and their application in the design of tertiary structures.

Science.gov (United States)

Sharma, Gangavaram V M; Nagendar, Pendem; Ramakrishna, Kallaganti V S; Chandramouli, Nagula; Choudhary, Madavi; Kunwar, Ajit C

2008-06-02

A new three-residue turn was serendipitously discovered in alpha/beta hybrid peptides derived from alternating C-linked carbo-beta-amino acids (beta-Caa) and L-Ala residues. The three-residue beta-alpha-beta turn at the C termini, nucleated by a helix at the N termini, resulted in helix-turn (HT) supersecondary structures in these peptides. The turn in the HT motif is stabilized by two H bonds-CO(i-2)-NH(i), with a seven-membered pseudoring (gamma turn) in the backward direction, and NH(i-2)-CO(i), with a 13-membered pseudoring in the forward direction (i being the last residue)--at the C termini. The study was extended to generalize the new three-residue turn (beta-alpha-beta) by using different alpha- and beta-amino acids. Furthermore, the HT motifs were efficiently converted, by an extension with helical oligomers at the C termini, into peptides with novel helix-turn-helix (HTH) tertiary structures. However, this resulted in the destabilization of the beta-alpha-beta turn with the concomitant nucleation of another three-residue turn, alpha-beta-beta, which is stabilized by 11- and 15-membered bifurcated H bonds. Extensive NMR spectroscopic studies were carried out to delineate the secondary and tertiary structures in these peptides, which are further supported by molecular dynamics (MD) investigations.

Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31.

Science.gov (United States)

Miyazaki, Takatsugu; Ishizaki, Yuichi; Ichikawa, Megumi; Nishikawa, Atsushi; Tonozuka, Takashi

2015-07-01

Glycoside hydrolase family 31 (GH31) proteins have been reportedly identified as exo-α-glycosidases with activity for α-glucosides and α-xylosides. We focused on a GH31 subfamily, which contains proteins with low sequence identity (Pedobacter heparinus and Pedobacter saltans. The enzymes unexpectedly exhibited α-galactosidase activity, but were not active on α-glucosides and α-xylosides. The crystal structures of one of the enzymes, PsGal31A, in unliganded form and in complexes with D-galactose or L-fucose and the catalytic nucleophile mutant in unliganded form and in complex with p-nitrophenyl-α-D-galactopyranoside, were determined at 1.85-2.30 Å (1 Å=0.1 nm) resolution. The overall structure of PsGal31A contains four domains and the catalytic domain adopts a (β/α)8-barrel fold that resembles the structures of other GH31 enzymes. Two catalytic aspartic acid residues are structurally conserved in the enzymes, whereas most residues forming the active site differ from those of GH31 α-glucosidases and α-xylosidases. PsGal31A forms a dimer via a unique loop that is not conserved in other reported GH31 enzymes; this loop is involved in its aglycone specificity and in binding L-fucose. Considering potential genes for α-L-fucosidases and carbohydrate-related proteins within the vicinity of Pedobacter Gal31, the identified Gal31 enzymes are likely to function in a novel sugar degradation system. This is the first report of α-galactosidases which belong to GH31 family. © 2015 Authors; published by Portland Press Limited.

Effect of salivary gland adenocarcinoma cell-derived alpha-N-acetylgalactosaminidase on the bioactivity of macrophage activating factor.

Science.gov (United States)

Matsuura, Takashi; Uematsu, Takashi; Yamaoka, Minoru; Furusawa, Kiyofumi

2004-03-01

The aim of this study was to clarify the effects of alpha-N-acetylgalactosaminidase (alpha-NaGalase) produced by human salivary gland adenocarcinoma (SGA) cells on the bioactivity of macrophage-activating factor (GcMAF). High exo-alpha-NaGalase activity was detected in the SGA cell line HSG. HSG alpha-NaGalase had both exo- and endo-enzyme activities, cleaving the Gal-GalNAc and GalNAc residues linked to Thr/Ser but not releasing the [NeuAc2-6]GalNac residue. Furthermore, GcMAF enzymatically prepared from the Gc protein enhanced the superoxide-generation capacity and phagocytic activity of monocytes/macrophages. However, GcMAF treated with purified alpha-NaGalase did not exhibit these effects. Thus, HSG possesses the capacity to produce larger quantities of alpha-NaGalase, which inactivates GcMAF produced from Gc protein, resulting in reduced phagocytic activity and superoxide-generation capacity of monocytes/macrophages. The present data strongly suggest that HSG alpha-NaGalase acts as an immunodeficiency factor in cancer patients.

Structure of .beta.-galactosidase complexes

Czech Academy of Sciences Publication Activity Database

Dohnálek, Jan; Skálová, Tereza; Dušková, Jarmila; Petroková, Hana; Hašek, Jindřich; Lipovová, P.; Spiwok, V.; Strnad, Hynek; Králová, B.

2006-01-01

Roč. 13, č. 3 (2006), s. 137-138 ISSN 1211-5894. [Czech and Slovak Crystallographic Colloquium. 22.06.2006-24.06.2006, Grenoble] R&D Projects: GA AV ČR KJB500500512 Keywords : .beta.-galactosidase * X-ray diffraction * cold-active enzyme Subject RIV: EB - Genetics ; Molecular Biology http://www. xray .cz/ms/default.htm

Glycosylation of phenolic compounds by the site-mutated β-galactosidase from Lactobacillus bulgaricus L3.

Science.gov (United States)

Lu, Lili; Xu, Lijuan; Guo, Yuchuan; Zhang, Dayu; Qi, Tingting; Jin, Lan; Gu, Guofeng; Xu, Li; Xiao, Min

2015-01-01

β-Galactosidases can transfer the galactosyl from lactose or galactoside donors to various acceptors and thus are especially useful for the synthesis of important glycosides. However, these enzymes have limitations in the glycosylation of phenolic compounds that have many physiological functions. In this work, the β-galactosidase from Lactobacillus bulgaricus L3 was subjected to site-saturation mutagenesis at the W980 residue. The recombinant pET-21b plasmid carrying the enzyme gene was used as the template for mutation. The mutant plasmids were transformed into Escherichia coli cells for screening. One recombinant mutant, W980F, exhibited increased yield of glycoside when using hydroquinone as the screening acceptor. The enzyme was purified and the effects of the mutation on enzyme properties were determined in detail. It showed improved transglycosylation activity on novel phenolic acceptors besides hydroquinone. The yields of the glycosides produced from phenol, hydroquinone, and catechol were increased by 7.6% to 53.1%. Moreover, it generated 32.3% glycosides from the pyrogallol that could not be glycosylated by the wild-type enzyme. Chemical structures of these glycoside products were further determined by MS and NMR analysis. Thus, a series of novel phenolic galactosides were achieved by β-galactosidase for the first time. This was a breakthrough in the enzymatic galactosylation of the challenging phenolic compounds of great values.

Glycosylation of phenolic compounds by the site-mutated β-galactosidase from Lactobacillus bulgaricus L3.

Directory of Open Access Journals (Sweden)

Lili Lu

Full Text Available β-Galactosidases can transfer the galactosyl from lactose or galactoside donors to various acceptors and thus are especially useful for the synthesis of important glycosides. However, these enzymes have limitations in the glycosylation of phenolic compounds that have many physiological functions. In this work, the β-galactosidase from Lactobacillus bulgaricus L3 was subjected to site-saturation mutagenesis at the W980 residue. The recombinant pET-21b plasmid carrying the enzyme gene was used as the template for mutation. The mutant plasmids were transformed into Escherichia coli cells for screening. One recombinant mutant, W980F, exhibited increased yield of glycoside when using hydroquinone as the screening acceptor. The enzyme was purified and the effects of the mutation on enzyme properties were determined in detail. It showed improved transglycosylation activity on novel phenolic acceptors besides hydroquinone. The yields of the glycosides produced from phenol, hydroquinone, and catechol were increased by 7.6% to 53.1%. Moreover, it generated 32.3% glycosides from the pyrogallol that could not be glycosylated by the wild-type enzyme. Chemical structures of these glycoside products were further determined by MS and NMR analysis. Thus, a series of novel phenolic galactosides were achieved by β-galactosidase for the first time. This was a breakthrough in the enzymatic galactosylation of the challenging phenolic compounds of great values.

Purification of microbial b-galactosidase from Kluyveromyces fragilis by bioaffinity partitioning Purificação de b-galactosidase de Kluyveromyces fragilis por partição por bioafinidade

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Maria Estela da Silva

1999-12-01

Full Text Available This work investigated the partitioning of b-galactosidase from Kluyveromyces fragilis in aqueous two-phase systems (ATPS by bioaffinity. PEG 4000 was chemically activated with thresyl chloride, and the biospecific ligand p-aminophenyl 1-thio-b-D-galactopyranoside (APGP was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme b-galactosidase from Kluyveromyces fragilis was developed. In the first step, a system composed of 6% PEG 4000-APGP and 8% dextran 505 was used, where b-galactosidase was strongly partitioned to the top phase (K = 2,330. In the second step, a system formed of 13% PEG-APGP and 9% phosphate salt was used to revert the value of the partition coefficient of b-galactosidase (K = 2 x 10-5 in order to provide the purification and recovery of 39% of the enzyme in the bottom salt-rich phase.Foi desenvolvido um método novo de extração e purificação da ß-galactosidase de Kluyveromyces fragilis em sistema de duas fases aquosas (SDFA. PEG 4000 foi ativado quimicamente com cloreto de tresila e o ligante bioespecífico p-aminofenil-ß-D-tiogalactopiranosídeo (APGP foi acoplado ao PEG 4000 ativado. Na primeira etapa foi usado um sistema composto de 6% PEG-APGP e 8% dextrana 505, onde a partição da ß-galactosidase ocorreu na fase superior (K = 2.330. Na segunda etapa foi usado um sistema composto por 13% PEG-APGP e 9% fosfato para reverter o valor do coeficiente de partição da ß-galactosidase (K = 2,2 x 10-5, obtendo-se recuperação de 39% da enzima na fase salina.

Kinetic Characterization of Galacto-Oligosaccharide (GOS) Synthesis by Three Commercially Important b-Galactosidases

NARCIS (Netherlands)

Warmerdam, A.; Zisopoulos, F.K.; Boom, R.M.; Janssen, A.E.M.

2014-01-01

Many b-galactosidases show large differences in galacto-oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various b-galactosidases is

The alpha-galactosidase type A gene aglA from Aspergillus niger encodes a fully functional alpha-N-acetylgalactosaminidase

Czech Academy of Sciences Publication Activity Database

Kulik, Natallia; Weignerová, L.; Filipi, Tomáš; Pompach, Petr; Novák, Petr; Mrázek, H.; Slámová, Kristýna; Bezouška, K.; Křen, Vladimír; Ettrich, Rüdiger

2010-01-01

Roč. 20, č. 11 (2010), s. 1410-1419 ISSN 0959-6658 R&D Projects: GA MŠk(CZ) LC06010; GA ČR GAP207/10/1934; GA ČR GAP207/10/1040; GA ČR GA303/09/0477; GA ČR GAP207/10/0321 Institutional research plan: CEZ:AV0Z60870520; CEZ:AV0Z50200510; CEZ:AV0Z10750506 Keywords : α-N-acetylgalactosaminidase * α-galactosidase * Aspergillus niger * substrate binding * molecular modeling Subject RIV: CE - Biochemistry Impact factor: 3.791, year: 2010

Analysis of β-galactosidase production and their genes of two strains of Lactobacillus bulgaricus.

Science.gov (United States)

Zhang, Wen; Wang, Chuan; Huang, Cheng-Yu; Yu, Qian; Liu, Heng-Chuan; Zhang, Chao-Wu; Pei, Xiao-Fang; Xu, Xin; Wang, Guo-Qing

2012-06-01

A bacterial β-galactosidase delivery system is a potential therapy for lactose intolerance. Currently, two Lactobacillus bulgaricus strains with different biological characteristics are under consideration as potential sources. However, differences in these β-galactosidase genes and their resulting production levels are poorly characterized. The β-galactosidase ORF of L. bulgaricus yogurt isolate had high variability and was terminated at site 1924 due to a stop codon. However, the full 114 kDa β-galactosidase band was still resolved by SDS-PAGE, which may indicate that the interrupted ORF was translated into more than one peptide, and they together were folded into the complete enzyme protein that showed much higher β-galactosidase activity (6.2 U/mg protein) than the enzyme generated from L. bulgaricus reference strain (2.5 U/mg protein).

Future Applications of Apricot (Prunus Armeniaca Kaisa ß Galactosidase in Dairy Industry

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Ansari Shakeel Ahmed

2014-09-01

Full Text Available The present study demonstrates the immobilization of β galactosidase from apricots (Prunus armeniaca kaisa on an inexpensive concanavalin A layered cellulose-alginate hybrid gel. Immobilized β galactosidase retained 78% of the initial activity after crosslinking by glutaraldehyde. It exhibited greater fraction of activity at both acidic and basic pH, and showed broad spectrum temperature optimum as compared to free enzyme. Moreover, immobilized enzyme exhibited higher thermal stability at 60°C and retained 80% of the original enzyme activity in presence of 3% galactose. The crosslinked immobilized enzyme showed improved hydrolysis of lactose from milk and whey in batch processes at 50°C as well as in continuous reactors operated at fl ow rate of 20 mL/h and 30 mL/h even after one month. Moreover, crosslinked adsorbed β galactosidase retained 76% activity even after its sixth repeated use, thereby promoting its use for lactose hydrolysis in various dairy products even for longer durations.

Purification and chemical characterisation of a cell wall-associated β-galactosidase from mature sweet cherry (Prunus avium L.) fruit.

Science.gov (United States)

Gerardi, Carmela; Blando, Federica; Santino, Angelo

2012-12-01

Using four different chromatographic steps, β-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium β-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and β-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing β-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry β-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple β-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry β-galactosidase exhibited a strict specificity towards p-nitrophenyl β-D-galactopyranoside, a pH optimum of 4.0 and K(m) and V(max) values of 0.42 mM and 4.12 mmol min(-1) mg(-1) of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Matrix Characterization of Plutonium Residues by Alpha-Particle Self-Interrogation

International Nuclear Information System (INIS)

Prettyman, T.H.; Foster, L.A.; Staples, P.

1998-01-01

Legacy plutonium residues often have inadequate item descriptions. Nondestructive characterization can help segregate these items for reprocessing or provide information needed for disposal or storage. Alpha particle-induced gamma-ray spectra contain a wealth of information that can be used for matrix characterization. We demonstrate how this information can be used for item identification. Gamma-ray spectra were recorded at the Los Alamos Plutonium Facility from a variety of legacy, plutonium-processing residues and product materials. The comparison and analysis of these spectra are presented

Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger β-galactosidase

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Becerra Manuel

2006-12-01

Full Text Available Abstract Background The β-galactosidase from Kluyveromyces lactis is a protein of outstanding biotechnological interest in the food industry and milk whey reutilization. However, due to its intracellular nature, its industrial production is limited by the high cost associated to extraction and downstream processing. The yeast-system is an attractive method for producing many heterologous proteins. The addition of a secretory signal in the recombinant protein is the method of choice to sort it out of the cell, although biotechnological success is not guaranteed. The cell wall acting as a molecular sieve to large molecules, culture conditions and structural determinants present in the protein, all have a decisive role in the overall process. Protein engineering, combining domains of related proteins, is an alternative to take into account when the task is difficult. In this work, we have constructed and analyzed two hybrid proteins from the β-galactosidase of K. lactis, intracellular, and its Aspergillus niger homologue that is extracellular. In both, a heterologous signal peptide for secretion was also included at the N-terminus of the recombinant proteins. One of the hybrid proteins obtained has interesting properties for its biotechnological utilization. Results The highest levels of intracellular and extracellular β-galactosidase were obtained when the segment corresponding to the five domain of K. lactis β-galactosidase was replaced by the corresponding five domain of the A. niger β-galactosidase. Taking into account that this replacement may affect other parameters related to the activity or the stability of the hybrid protein, a thoroughly study was performed. Both pH (6.5 and temperature (40°C for optimum activity differ from values obtained with the native proteins. The stability was higher than the corresponding to the β-galactosidase of K. lactis and, unlike this, the activity of the hybrid protein was increased by the presence

Potential Applications of Immobilized β-Galactosidase in Food Processing Industries

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Parmjit S. Panesar

2010-01-01

Full Text Available The enzyme β-galactosidase can be obtained from a wide variety of sources such as microorganisms, plants, and animals. The use of β-galactosidase for the hydrolysis of lactose in milk and whey is one of the promising enzymatic applications in food and dairy processing industries. The enzyme can be used in either soluble or immobilized forms but the soluble enzyme can be used only for batch processes and the immobilized form has the advantage of being used in batch wise as well as in continuous operation. Immobilization has been found to be convenient method to make enzyme thermostable and to prevent the loss of enzyme activity. This review has been focused on the different types of techniques used for the immobilization of β-galactosidase and its potential applications in food industry.

Entrappment of alkaline protease and β-galactosidase in radiation stitched together poly-N-vinylcaprolactam

International Nuclear Information System (INIS)

Davidenko, T.I.; Kravchenko, I.A.

1996-01-01

The gel formations by poly-N-vinylcaprolactam upon its γ-irradiation by the 20-25 kGy dose as a results of partial polymer stitching together is shown, which is confirmed by the CD-and thermogravimetric data. By the alkaline protease and β-galactosidase entrapment in poly-N- vinylcaprolactam stitched together by γ-irradiation, the active preparations are obtained with 90-98 % and 30-35 % activity retained for alkaline protease and β-galactosidase, respectively. The increased stability of alkaline protease at acidic pH values and higher temperature was noted, and for β-galactosidase - the possibility of repeated use of the obtained preparation for lactose hydrolysis

Screening and Characterization of Cold-Active β-Galactosidase Producing Psychrotrophic Enterobacter ludwigii from the Sediments of Arctic Fjord.

Science.gov (United States)

Alikkunju, Aneesa P; Sainjan, Neethu; Silvester, Reshma; Joseph, Ajith; Rahiman, Mujeeb; Antony, Ally C; Kumaran, Radhakrishnan C; Hatha, Mohamed

2016-10-01

Low-temperature-tolerant microorganisms and their cold-active enzymes could be an innovative and invaluable tool in various industrial applications. In the present study, bacterial isolates from the sediment samples of Kongsfjord, Norwegian Arctic, were screened for β-galactosidase production. Among the isolates, KS25, KS85, KS60, and KS92 have shown good potential in β-galactosidase production at 20 °C. 16SrRNA gene sequence analysis revealed the relatedness of the isolates to Enterobacter ludwigii. The optimum growth temperature of the isolate was 25 °C. The isolate exhibited good growth and enzyme production at a temperature range of 15-35 °C, pH 5-10. The isolate preferred yeast extract and lactose for the maximum growth and enzyme production at conditions of pH 7.0, temperature of 25 °C, and agitation speed of 100 rpm. The growth and enzyme production was stimulated by Mn 2+ and Mg 2+ and strongly inhibited by Zn 2+ , Ni 2+ , and Cu + . β-Galactosidases with high specific activity at low temperatures are very beneficial in food industry to compensate the nutritional problem associated with lactose intolerance. The isolate exhibited a remarkable capability to utilize clarified whey, an industrial pollutant, for good biomass and enzyme yield and hence could be well employed in whey bioremediation.

Novel high-performance metagenome β-galactosidases for lactose hydrolysis in the dairy industry.

Science.gov (United States)

Erich, Sarah; Kuschel, Beatrice; Schwarz, Thilo; Ewert, Jacob; Böhmer, Nico; Niehaus, Frank; Eck, Jürgen; Lutz-Wahl, Sabine; Stressler, Timo; Fischer, Lutz

2015-09-20

The industrially utilised β-galactosidases from Kluyveromyces spp. and Aspergillus spp. feature undesirable kinetic properties in praxis, such as an unsatisfactory lactose affinity (KM) and product inhibition (KI) by galactose. In this study, a metagenome library of about 1.3 million clones was investigated with a three-step activity-based screening strategy in order to find new β-galactosidases with more favourable kinetic properties. Six novel metagenome β-galactosidases (M1-M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial β-galactosidase from Kluyveromyces lactis (GODO-YNL2). The best metagenome candidate, called "M1", was recombinantly produced in Escherichia coli BL21(DE3) in a bioreactor (volume 35 L), resulting in a total β-galactosidase M1 activity of about 1100 μkatoNPGal,37 °C L(-1). Since milk is a sensitive and complex medium, it has to be processed at 5-10 °C in the dairy industry. Therefore, the β-galactosidase M1 was tested at 8 °C in milk and possessed a good stability (t1/2=21.8 d), a desirably low apparent KM,lactose,8 °C value of 3.8±0.7 mM and a high apparent KI,galactose,8 °C value of 196.6±55.5 mM. A lactose hydrolysis process (milk, 40 nkatlactose mLmilk,8 °C(-1)) was conducted at a scale of 0.5L to compare the performance of M1 with the commercial β-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (>99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis β-galactosidase after 25 h process time. Thus, M1 was able to achieve the limit of lactose per litre milk, which is recommended for dairy products labelled as "lactose-free". Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Fabry's disease: biochemical and histochemical studies on hair roots for carrier detection.

Science.gov (United States)

Vermorken, A J; Weterings, P J; Spierenburg, G T; vanBennekom, C A; Wirtz, P; deBruyn, C H; Oei, T L

1978-02-01

A method of assay alpha-galactosidase and acid phosphatase activities in single hair roots is described. Enzyme histochemical studies show that the distribution of acid phosphatase in the human hair root matches that of alpha-galactosidase. Histochemically, the main activity is located in the upper part of the sheath near the orifice of the duct of the sebaceous gland. This is confirmed by enzyme assays on different parts of the hair root after dissection. The variation in the values found in individual hair roots is improved by relating alpha-galactosidase to acid phosphatase activities. Storage experiments indicate a remarkable stability of both alpha-galactosidase and acid phosphatase in human hair roots.

Mechanism-based fluorescent labeling of beta-galactosidases. An efficient method in proteomics for glycoside hydrolases.

Science.gov (United States)

Kurogochi, Masaki; Nishimura, Shin-Ichiro; Lee, Yuan Chuan

2004-10-22

(4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-beta-d-galactopyranoside was synthesized and successfully tested on beta-galactosidases from Xanthomonas manihotis (Wong-Madden, S. T., and Landry, D. Glycobiology (1995) 5, 19-28 and Taron, C. H., Benner, J. S., Hornstra, L. J., and Guthrie, E. P. (1995) Glycobiology 5, 603-610), Escherichia coli (Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Nature 369, 761-766), and Bacillus circulans (Fujimoto, H., Miyasato, M., Ito, Y., Sasaki, T., and Ajisaka, K. (1988) Glycoconj. J. 15, 155-160) for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from beta-galactosidases. It was demonstrated by using X. manihotis beta-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42 (Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., and Wakagi, T. (2002) J. Mol. Biol. 322, 79-91). A catalytic glutamic acid (Glu-537) of E. coli beta-galactosidase was proved to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B. circulans beta-galactosidase, indicating that Glu

Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus.

Science.gov (United States)

Yamamoto, N; Naraparaju, V R; Moore, M; Brent, L H

1997-03-01

A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.

Nano-coating of beta-galactosidase onto the surface of lactose by using an ultrasound-assisted technique

DEFF Research Database (Denmark)

Genina, Natalja; Räikkönen, Heikki; Heinämäki, Jyrki

2010-01-01

We nano-coated powdered lactose particles with the enzyme beta-galactosidase using an ultrasound-assisted technique. Atomization of the enzyme solution did not change its activity. The amount of surface-attached beta-galactosidase was measured through its enzymatic reaction product D-galactose us......We nano-coated powdered lactose particles with the enzyme beta-galactosidase using an ultrasound-assisted technique. Atomization of the enzyme solution did not change its activity. The amount of surface-attached beta-galactosidase was measured through its enzymatic reaction product D......-galactose using a standardized method. A near-linear increase was obtained in the thickness of the enzyme coat as the treatment proceeded. Interestingly, lactose, which is a substrate for beta-galactosidase, did not undergo enzymatic degradation during processing and remained unchanged for at least 1 month....... Stability of protein-coated lactose was due to the absence of water within the powder, as it was dry after the treatment procedure. In conclusion, we were able to attach the polypeptide to the core particles and determine precisely the coating efficiency of the surface-treated powder using a simple approach....

Adsorption of β-galactosidase on silica and aluminosilicate adsorbents

Science.gov (United States)

Atyaksheva, L. F.; Dobryakova, I. V.; Pilipenko, O. S.

2015-03-01

It is shown that adsorption of β-galactosidase of Aspergillus oryzae fungi on mesoporous and biporous silica and aluminosilicate adsorbents and the rate of the process grow along with the diameter of the pores of the adsorbent. It is found that the shape of the adsorption isotherms changes as well, depending on the texture of the adsorbent: the Michaelis constant rises from 0.3 mM for the enzyme in solution to 0.4-0.5 mM for the enzyme on a surface in the hydrolysis of o-nitrophenyl-β-D-galactopyranoside. It is concluded that β-galactosidase displays its maximum activity on the surface of biporous adsorbents.

Purification and characterization of α-galactosidase from ...

African Journals Online (AJOL)

α−Galactosidase (α-D-galactoside galactohydrolase [EC 3.2.1.22]) was obtained from Lactobacillus acidofillus which was grown in modified de Man, Rogosa and Sharpe (MRS) medium, supplemented with raffinose. α-Galactosidase was released from the cells by ultrasonic treatment, then precipitated by ...

Enhanced alpha-galactosidase expression in pseudomonas chlororaphis

Science.gov (United States)

Pseudomonas chlororaphis is a non-pathogenic bacterium useful for fermentative production of biopolymer (i.e., poly(hydroxyalkanoates); PHA) and biosurfactant (i.e., rhamnolipid; RhL). In order to enable P. chlororaphis to better fermentatively utilize the residual soy sugars in soy molasses – a lo...

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

Science.gov (United States)

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of Glutaraldehyde in Imparting Stability to Immobilized β-Galactosidase Systems

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Rukhsana Satar

2018-01-01

Full Text Available ABSTRACT This review article highlights the role of glutaraldehyde as a matrix activator/stabilizer in imparting higher operational and thermal stability to β-galactosidase (βG for biotechnological applications. Glutaraldehyde has been used extensively as a crosslinking agent as well as for functionalization of matrices to immobilize β-galactosidase. Immobilized β-galactosidase systems (IβGS obtained as a result of glutaraldehyde treatment has been employed to hydrolyze whey and milk lactose in batch reactors, continuous packed-bed and fluidized bed reactors under various operational conditions. Moreover, these IβGS have also been utilized for the production of galactooligosaccharides in food, dairy and fermentation industries. It was observed that glutaraldehyde provided remarkable stability to immobilize βG against various physical/chemical denaturants, thus enhancing thermal/operational stability and rendering it more suitable for repeated utilization in industrial scale operations.

β-Galactosidase activity of commercial lactase samples in raw and pasteurized milk at refrigerated temperatures.

Science.gov (United States)

Horner, T W; Dunn, M L; Eggett, D L; Ogden, L V

2011-07-01

Many consumers are unable to enjoy the benefits of milk due to lactose intolerance. Lactose-free milk is available but at about 2 times the cost of regular milk or greater, it may be difficult for consumers to afford. The high cost of lactose-free milk is due in part to the added cost of the lactose hydrolysis process. Hydrolysis at refrigerated temperatures, possibly in the bulk tank or package, could increase the flexibility of the process and potentially reduce the cost. A rapid β-galactosidase assay was used to determine the relative activity of commercially available lactase samples at different temperatures. Four enzymes exhibited low-temperature activity and were added to refrigerated raw and pasteurized milk at various concentrations and allowed to react for various lengths of time. The degree of lactose hydrolysis by each of the enzymes as a function of time and enzyme concentration was determined by HPLC. The 2 most active enzymes, as determined by the β-galactosidase assay, hydrolyzed over 98% of the lactose in 24h at 2°C using the supplier's recommended dosage. The other 2 enzymes hydrolyzed over 95% of the lactose in 24h at twice the supplier's recommended dosage at 2°C. Results were consistent in all milk types tested. The results show that it is feasible to hydrolyze lactose during refrigerated storage of milk using currently available enzymes. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in Aspergillus niger.

Science.gov (United States)

Xu, Yue; Wang, Yan-Hui; Liu, Tian-Qi; Zhang, Hui; Zhang, He; Li, Jie

2018-03-31

α-Galactosidases are widely used in many fields. It is necessary to improve the production of enzymes through microbiological processes. The aim of this study was to construct recombinant Aspergillus niger strains with high α-galactosidase production. Two recombinant A. niger strains were constructed: AB and AGB. The recombinant AB strain contained the α-galactosidase aglB gene from A. niger with its native AglB signal peptide regulated by the glucoamylase promoter. In the AGB recombinant strain, the AglB signal peptide was replaced with the glucoamylase (GlaA) signal peptide. The extracellular maximum α-galactosidase activity of the AGB strain was 215.7 U/ml and that of the AB strain was 9.8 U/mL. The optimal conditions for α-galactosidase were pH 3.5 and 35 °C. The GlaA signal peptide substantially increased the yield of secreted α-galactosidase in A. niger. This recombinant strain holds great potential for industrial applications.

Murine elongation factor 1 alpha (EF-1 alpha) is posttranslationally modified by novel amide-linked ethanolamine-phosphoglycerol moieties. Addition of ethanolamine-phosphoglycerol to specific glutamic acid residues on EF-1 alpha

International Nuclear Information System (INIS)

Whiteheart, S.W.; Shenbagamurthi, P.; Chen, L.; Cotter, R.J.; Hart, G.W.

1989-01-01

Elongation Factor 1 alpha (EF-1 alpha), an important eukaryotic translation factor, transports charged aminoacyl-tRNA from the cytosol to the ribosomes during poly-peptide synthesis. Metabolic radiolabeling with [ 3 H] ethanolamine shows that, in all cells examined, EF-1 alpha is the major radiolabeled protein. Radiolabeled EF-1 alpha has an apparent Mr = 53,000 and a basic isoelectric point. It is cytosolic and does not contain N-linked oligosaccharides. Trypsin digestion of murine EF-1 alpha generated two major [ 3 H]ethanolamine-labeled peptides. Three peptides were sequenced and were identical to two distinct regions of the human EF-1 alpha protein. Blank sequencing cycles coinciding with glutamic acid in the human cDNA-derived sequence were also found to release [ 3 H]ethanolamine, and compositional analysis of these peptides confirmed the presence of glutamic acid. Dansylation analysis demonstrates that the amine group of the ethanolamine is blocked. These results indicate that EF-1 alpha is posttranslationally modified by the covalent attachment of ethanolamine via an amide bond to at least two specific glutamic acid residues (Glu-301 and Glu-374). The hydroxyl group of the attached ethanolamine was shown by mass spectrometry and compositional analysis, to be further modified by the addition of a phosphoglycerol unit. This novel posttranslational modification may represent an important alteration of EF-1 alpha, comparable to the regulatory effects of posttranslational methylation of EF-1 alpha lysine residues

Residual radioactivity of treated green diamonds.

Science.gov (United States)

Cassette, Philippe; Notari, Franck; Lépy, Marie-Christine; Caplan, Candice; Pierre, Sylvie; Hainschwang, Thomas; Fritsch, Emmanuel

2017-08-01

Treated green diamonds can show residual radioactivity, generally due to immersion in radium salts. We report various activity measurements on two radioactive diamonds. The activity was characterized by alpha and gamma ray spectrometry, and the radon emanation was measured by alpha counting of a frozen source. Even when no residual radium contamination can be identified, measurable alpha and high-energy beta emissions could be detected. The potential health impact of radioactive diamonds and their status with regard to the regulatory policy for radioactive products are discussed. Copyright © 2017. Published by Elsevier Ltd.

Resting alpha activity predicts learning ability in alpha neurofeedback

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Wenya eNan

2014-07-01

Full Text Available Individuals differ in their ability to learn how to regulate the alpha activity by neurofeedback. This study aimed to investigate whether the resting alpha activity is related to the learning ability of alpha enhancement in neurofeedback and could be used as a predictor. A total of 25 subjects performed 20 sessions of individualized alpha neurofeedback in order to learn how to enhance activity in the alpha frequency band. The learning ability was assessed by three indices respectively: the training parameter changes between two periods, within a short period and across the whole training time. It was found that the resting alpha amplitude measured before training had significant positive correlations with all learning indices and could be used as a predictor for the learning ability prediction. This finding would help the researchers in not only predicting the training efficacy in individuals but also gaining further insight into the mechanisms of alpha neurofeedback.

Branching enzyme assay: selective quantitation of the alpha 1,6-linked glucosyl residues involved in the branching points.

Science.gov (United States)

Krisman, C R; Tolmasky, D S; Raffo, S

1985-06-01

Methods previously described for glycogen or amylopectin branching enzymatic activity are insufficiently sensitive and not quantitative. A new, more sensitive, specific, and quantitative one was developed. It is based upon the quantitation of the glucose residues joined by alpha 1,6 bonds introduced by varying amounts of branching enzyme. The procedure involved the synthesis of a polysaccharide from Glc-1-P and phosphorylase in the presence of the sample to be tested. The branched polysaccharide was then purified and the glucoses involved in the branching points were quantitated after degradation with phosphorylase and debranching enzymes. This method appeared to be useful, not only in enzymatic activity determinations but also in the study of the structure of alpha-D-glucans when combined with those of total polysaccharide quantitation, such as iodine and phenol-sulfuric acid.

Homodimeric β-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: expression in Lactobacillus plantarum and biochemical characterization.

Science.gov (United States)

Nguyen, Tien-Thanh; Nguyen, Hoang Anh; Arreola, Sheryl Lozel; Mlynek, Georg; Djinović-Carugo, Kristina; Mathiesen, Geir; Nguyen, Thu-Ha; Haltrich, Dietmar

2012-02-22

The lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081, encoding a β-galactosidase of the glycoside hydrolase family GH2, was cloned into different inducible lactobacillal expression vectors for overexpression in the host strain Lactobacillus plantarum WCFS1. High expression levels were obtained in laboratory cultivations with yields of approximately 53000 U of β-galactosidase activity per liter of medium, which corresponds to ~170 mg of recombinant protein per liter and β-galactosidase levels amounting to 63% of the total intracellular protein of the host organism. The wild-type (nontagged) and histidine-tagged recombinant enzymes were purified to electrophoretic homogeneity and further characterized. β-Galactosidase from L. bulgaricus was used for lactose conversion and showed very high transgalactosylation activity. The maximum yield of galacto-oligosaccharides (GalOS) was approximately 50% when using an initial concentration of 600 mM lactose, indicating that the enzyme can be of interest for the production of GalOS.

Interaction of C-terminal truncated human alphaA-crystallins with target proteins.

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Anbarasu Kumarasamy

2008-09-01

Full Text Available Significant portion of alphaA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH and betaL-crystallin as target proteins, was increased in alphaA(1-172 and decreased in alphaA(1-168 and alphaA(1-162. The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k for ADH and alphaA(1-172 was nearly the same as that of ADH and alphaA-wt, alphaA(1-168 had lower and alphaA(1-162 had the lowest k values. When betaL-crystallin was used as the target protein, alphaA(1-172 had slightly higher k value than alphaA-wt and alphaA(1-168 and alphaA(1-162 had lower k values. As expected from earlier studies, the chaperone activity of alphaA(1-172 was slightly better than that of alphaA-wt, the chaperone activity of alphaA(1-168 was similar to that of alphaA-wt and alphaA(1-162 had substantially decreased chaperone activity.Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.

Cloning, purification and characterization of a thermostable β-galactosidase from Bacillus licheniformis strain KG9.

Science.gov (United States)

Matpan Bekler, F; Stougaard, P; Güven, K; Gül Güven, R; Acer, Ö

2015-06-28

A thermo— and alkalitolerant Bacillus licheniformis KG9 isolated from Taşlıdere hot water spring in Batman/Turkey was found to produce a thermostable β—galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative β—galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding β—galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the β—galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant β—galactosidase was produced in Escherichia coli. Of the four β—galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, β—galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 ºC and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho—nitrophenyl—β—D—galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 μmol/min and 1.55 μmol/min with oNPG and lactose, respectively.

Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and Bacillus amyloliquefaciens

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Shalini RAI

2014-03-01

Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi

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Karan Ram

2013-01-01

Full Text Available Abstract Background Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures. Results Analysis of the genome sequence of H. lacusprofundi showed a gene cluster for carbohydrate utilization containing a glycoside hydrolase family 42 β-galactosidase gene, named bga. In order to study the biochemical properties of the β-galactosidase enzyme, the bga gene was PCR amplified, cloned, and expressed in the genetically tractable haloarchaeon Halobacterium sp. NRC-1 under the control of a cold shock protein (cspD2 gene promoter. The recombinant β-galactosidase protein was produced at 20-fold higher levels compared to H. lacusprofundi, purified using gel filtration and hydrophobic interaction chromatography, and identified by SDS-PAGE, LC-MS/MS, and ONPG hydrolysis activity. The purified enzyme was found to be active over a wide temperature range (−5 to 60°C with an optimum of 50°C, and 10% of its maximum activity at 4°C. The enzyme also exhibited extremely halophilic character, with maximal activity in either 4 M NaCl or KCl. The polyextremophilic β-galactosidase was also stable and active in 10–20% alcohol-aqueous solutions, containing methanol, ethanol, n-butanol, or isoamyl alcohol. Conclusion The H. lacusprofundi β-galactosidase is a polyextremophilic enzyme active in high salt concentrations and low and high temperature. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry. H. lacuprofundi proteins represent a significant biotechnology resource and for developing insights into enzyme catalysis under water limiting conditions. This study provides a system for better understanding how H. lacusprofundi is

Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes.

Science.gov (United States)

Squillaro, Tiziana; Antonucci, Ivana; Alessio, Nicola; Esposito, Anna; Cipollaro, Marilena; Melone, Mariarosa Anna Beatrice; Peluso, Gianfranco; Stuppia, Liborio; Galderisi, Umberto

2017-12-01

Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity. © 2017 Wiley Periodicals, Inc.

b-GALACTOSIDASE IMMOBILIZATION ON CONTROLLED PORE SILICA

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H. C. Trevisan

1997-12-01

Full Text Available The immobilization of b -galactosidase from Kluyveromyces fragilis on controlled pore silica was investigated. Immobilization was performed on amino silica activated with glutaraldehyde and the product was applied to the hydrolysis of lactose of whey. The behaviors of the soluble and immobilized enzyme were compared by using whey and a lactose solution as the substrate. With the aim of optimizing the method, parameters such as the amount of glutaraldehyde and the size of the particles were evaluated by comparing activities and stabilities on batch and continuously fluidized bed reactors

ATP and MO25alpha regulate the conformational state of the STRADalpha pseudokinase and activation of the LKB1 tumour suppressor.

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Elton Zeqiraj

2009-06-01

Full Text Available Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADalpha in complex with MO25alpha. The structure reveals an intricate web of interactions between STRADalpha and MO25alpha involving the alphaC-helix of STRADalpha, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADalpha binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADalpha for MO25alpha, and conversely, binding of MO25alpha promotes interaction of STRADalpha with ATP. Mutagenesis studies reveal that association of STRADalpha with either ATP or MO25alpha is essential for LKB1 activation. We conclude that ATP and MO25alpha cooperate to maintain STRADalpha in an "active" closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADalpha that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE syndrome. We demonstrate this mutation destabilizes STRADalpha and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADalpha to activate LKB1 is dependent on a closed "active" conformation, aided by ATP and MO25alpha binding. Thus, the function of STRADalpha is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.

Thermal inactivation kinetics of β-galactosidase during bread baking

NARCIS (Netherlands)

Zhang, L.; Chen, Xiao Dong; Boom, R.M.; Schutyser, M.A.I.

2017-01-01

In this study, β-galactosidase was utilized as a model enzyme to investigate the mechanism of enzyme inactivation during bread baking. Thermal inactivation of β-galactosidase was investigated in a wheat flour/water system at varying temperature-moisture content combinations, and in bread during

The catalytic properties and stability of β-galactosidases from fungi

Science.gov (United States)

Pilipenko, O. S.; Atyaksheva, L. F.; Poltorak, O. M.; Chukhrai, E. S.

2008-12-01

The catalytic activity of β-galactosidases from fungi Penicillium canescens and Aspergillus oryzae is maximum in a weakly acidic medium and does not depend on the presence of magnesium cations in the reaction medium. The enzyme from Aspergillus oryzae fungi is more active, and that from Penicillium canescens is stabler. One of stability indications is the presence of an induction period in the kinetic curves of thermal inactivation. This period disappears at 54°C for the enzyme from Aspergillus oryzae and at 59°C for the enzyme from Penicillium canescens. The temperature dependences of the effective rate constants for the inactivation of the tetrameric enzyme from Penicillium canescens show that the main reason for enzyme inactivation is the dissociation of oligomeric forms below 66°C ( E act = 85 kJ/mol) and enzyme denaturation at higher temperatures ( E act = 480 kJ/mol). The dissociation stage is absent for monomeric β-galactosidase from Aspergillus oryzae fungi, and the activation energy of inactivation is 450 kJ/mol over the whole temperature range studied (53-60°C).

Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

Science.gov (United States)

Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

2013-01-01

Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

Quantification of activity by alpha-camera imaging and small-scale dosimetry within ovarian carcinoma micrometastases treated with targeted alpha therapy

DEFF Research Database (Denmark)

Chouin, N; Lindegren, S; Jensen, Holger

2012-01-01

Targeted alpha therapy (TAT) a promising treatment for small, residual, and micrometastatic diseases has questionable efficacy against malignant lesions larger than the α-particle range, and likely requires favorable intratumoral activity distribution. Here, we characterized and quantified......% IA/g at 4 h) in the outer tumor layer and a sharp drop beyond a depth of 50 µm. Small-scale dosimetry was performed on a multi-cellular micrometastasis model, using time-integrated activities derived from the experimental data. With injected activity of 400 kBq, tumors exhibiting uniform activity...... distribution received 100 µm might not be effectively treated by the examined regimen....

Crystal structures of β-galactosidase from Penicillium sp. and its complex with galactose

International Nuclear Information System (INIS)

Rojas, A.L.; Nagem, R.A.P.; Garratt, R.C.; Polikarpov, I.; Neustroev, K.N.; Eneyskaya, E.V.; Kulminskaya, A.A.; Golubev, A.M.; Arand, M.; Adamska, M.

2004-01-01

Glycosidase belong to a group of enzymes displaying a great variety of protein folds and substrate specificities. Two critically located acidic residues make up the catalytic machinery of these enzymes, which are responsible for the cleavage of glycosidic bonds. The applications of glycosidase in textile, food, and pulp processing and in catalysts and oligosaccharide synthesis have encouraged the engineering of these proteins to improve their catalytic properties and stability. Furthermore, structural studies broaden our understanding of the catalytic mechanism and the role of glycosidase in the recognition processes of their different substrates. In this work, we describe crystallographic studies of a fungi glycosidase. The crystallographic structures of β-galactosidase from Penicillium sp. and its complex with galactose were solved at 1.90 A and 2.10 A resolution, respectively. The X-ray structure of the enzyme-galactose complex was useful in identifying the residue Glu200 as the proton donor and residue Glu 299 as the nucleophiles involved in catalysis. (author)

Crystal structures of {beta}-galactosidase from Penicillium sp. and its complex with galactose

Energy Technology Data Exchange (ETDEWEB)

Rojas, A.L.; Nagem, R.A.P.; Garratt, R.C.; Polikarpov, I. [Universidade de Sao Paulo, Sao Carlos, SP (Brazil); Neustroev, K.N.; Eneyskaya, E.V.; Kulminskaya, A.A.; Golubev, A.M. [St. Petersburg, Gatchina (Russian Federation); Arand, M.; Adamska, M. [University of Wuerzburg (Germany)

2004-07-01

Glycosidase belong to a group of enzymes displaying a great variety of protein folds and substrate specificities. Two critically located acidic residues make up the catalytic machinery of these enzymes, which are responsible for the cleavage of glycosidic bonds. The applications of glycosidase in textile, food, and pulp processing and in catalysts and oligosaccharide synthesis have encouraged the engineering of these proteins to improve their catalytic properties and stability. Furthermore, structural studies broaden our understanding of the catalytic mechanism and the role of glycosidase in the recognition processes of their different substrates. In this work, we describe crystallographic studies of a fungi glycosidase. The crystallographic structures of {beta}-galactosidase from Penicillium sp. and its complex with galactose were solved at 1.90 A and 2.10 A resolution, respectively. The X-ray structure of the enzyme-galactose complex was useful in identifying the residue Glu200 as the proton donor and residue Glu 299 as the nucleophiles involved in catalysis. (author)

Changes in alpha-L-arabinofuranosidase activity in peel and pulp of banana (Musa sp.) fruits during ripening and softening.

Science.gov (United States)

Zhuang, Jun-Ping; Su, Jing; Li, Xue-Ping; Chen, Wei-Xin

2007-04-01

Arabinose is one of the most dynamic cell wall glycosyl residues released during fruit ripening, alpha-L-arabinofuranosidase (alpha-Arab) are major glycosidases that may remove arabinose units from fruit cell wall polysaccharides. To find out whether alpha-Arab plays important roles in banana fruit softening, the enzyme activities in peel and pulp, fruit firmness, respiration rate and ethylene release rate were assayed during banana softening. The results showed that alpha-Arab activities in banana pulp and peel increased slightly at the beginning of storage and reached their maxima when the fruit firmness decreased drastically, alpha-Arab activity increased by more than ten folds in both pulp and peel during ripening and alpha-Arab activities were higher in pulp than in peel. Treatment of banana fruits with ethylene absorbent postponed the time of reaching of its maxima of respiration and ethylene, enhanced the firmness of pup and decreased alpha-Arab activity in the peel and pulp. These results suggest that alpha-Arab induced the decrease of fruit firmness and played an important role in banana fruit softening, and its activity was regulated by ethylene.

Mini review on role of β-galactosidase in lactose intolerance

Science.gov (United States)

A, Nivetha; V, Mohanasrinivasan

2017-11-01

This review mainly focuses on the role and properties of β-galactosidase in lactose intolerance and its industrial application. β-Galactosidase, hydrolyses the lactose into glucose and galactose and it is most commonly used in food based technology, particularly in the dairy manufacturing industry. This catalyst mainly focus for the improvement of new and novel products with hydrolyzed lactose, which can be appropriate for the lactose-intolerant persons, to improve the technological, texture and scientific properties of non-fermented dairy products. β-Galactosidase derived from the group of saccharides which is a converting enzymes in the family of hydrolases. They are broadly distributed in the several biological living systems. The enzymatic hydrolysis of lactose is also preferred in food based technology due to the low soluble range of lactose. The concentration lactose was found to be high in fermented dairy products such as ice cream, butter, cheese curd, yogurt, etc., can prompt extreme lactose crystallization bringing about items through a coarse, abrasive surface. Lactose hydrolysis in dairy products enhances adaptability also, richness altogether. These products are extra edible. Also for this purpose, the utilization of β-galactosidase enzyme prior to the condensing operation can reduce the lactose content to a point where lactose was no longer a problem industrial application of β-galactosidase. In Industries, due to the positive and constructive effect on intestinal bacterial microflora, different types of applications are possible in β-galactosidase enzyme.

β-galactosidase from Aspergillus lacticoffeatus: A promising biocatalyst for the synthesis of novel prebiotics.

Science.gov (United States)

Cardoso, Beatriz B; Silvério, Sara C; Abrunhosa, Luís; Teixeira, José A; Rodrigues, Lígia R

2017-09-18

β-galactosidase (EC 3.2.1.23) are interesting enzymes able to catalyze lactose hydrolysis and transfer reactions to produce lactose-based prebiotics with potential application in the pharmaceutical and food industry. In this work, Aspergillus lacticoffeatus is described, for the first time, as an effective β-galactosidase producer. The extracellular enzyme production was evaluated in synthetic and alternative media containing cheese whey and corn steep liquor. Although β-galactosidase production occurred in all media (expect for the one composed solely by cheese whey), the highest enzymatic activity values (460U/mL) were obtained for the synthetic medium. Ochratoxin A production in synthetic medium was also evaluated and 9days of fermentation was identified as a suitable fermentation time to obtain a crude extract enzyme with mycotoxin concentration below the legal comparable value established for wine and grape juices (2ng/mL). The optimal pH and temperature for the crude extract enzyme was found in the range of 3.5-4.5 and 50-60°C, respectively. The β-galactosidase activity was reduced in the presence of Ba 2+ , Fe 2+ , Li + , K + and galactose, while additives (except for ascorbic acid) and detergents exhibited a positive effect on enzymatic activity. This enzyme was able to catalyze the synthesis of prebiotics, namely lactulose (2.5g/L) and a galacto-oligosaccharide (trisaccharide, 6.3g/L), either when whole cells or crude enzyme was used as biocatalyst. The lactulose production using fungal whole cells is herein reported for the first time. Additionally, A. lacticoffeatus was also found to produce an enzyme with fructosyltransferase activity and other prebiotics, namely fructo-oligosaccharide 1-kestose (2.4g/L). Copyright © 2017 Elsevier B.V. All rights reserved.

Autoresonant-spectrometric determination of the residual gas composition in the ALPHA experiment apparatus

Energy Technology Data Exchange (ETDEWEB)

Amole, C.; Capra, A.; Menary, S. [Department of Physics and Astronomy, York University, Toronto, Ontario M3J 1P3 (Canada); Ashkezari, M. D.; Hayden, M. E. [Department of Physics, Simon Fraser University, Burnaby, British Columbia V5A 1S6 (Canada); Baquero-Ruiz, M.; Chapman, S.; Little, A.; Povilus, A.; So, C.; Turner, M. [Department of Physics, University of California, Berkeley, California 94720-7300 (United States); Bertsche, W. [Department of Physics, Swansea University, Swansea SA2 8PP (United Kingdom); School of Physics and Astronomy, University of Manchester, Manchester M13 9PL, United Kingdom and The Cockcroft Institute, Daresbury Laboratory, Warrington WA4 4AD (United Kingdom); Butler, E. [Physics Department, CERN, CH-1211 Geneva 23 (Switzerland); Cesar, C. L.; Silveira, D. M. [Instituto de Fisica, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-972 (Brazil); Charlton, M.; Eriksson, S.; Isaac, C. A.; Madsen, N.; Napoli, S. C. [Department of Physics, Swansea University, Swansea SA2 8PP (United Kingdom); Collaboration: ALPHA Collaboration; and others

2013-06-15

Knowledge of the residual gas composition in the ALPHA experiment apparatus is important in our studies of antihydrogen and nonneutral plasmas. A technique based on autoresonant ion extraction from an electrostatic potential well has been developed that enables the study of the vacuum in our trap. Computer simulations allow an interpretation of our measurements and provide the residual gas composition under operating conditions typical of those used in experiments to produce, trap, and study antihydrogen. The methods developed may also be applicable in a range of atomic and molecular trap experiments where Penning-Malmberg traps are used and where access is limited.

Purification of beta-acetylglucosaminase and beta-galactosidase from ram testis.

Science.gov (United States)

Caygill, J C; Roston, C P; Jevons, F R

1966-02-01

1. The presence of beta-galactosidase (EC 3.2.1.23) in an acetic acid extract of ram testis is reported. Some properties of the crude enzyme preparation were studied. 2. The purification of beta-acetylglucosaminase (EC 3.2.1.30) and of beta-galactosidase from the ram-testis extract by ammonium sulphate precipitation and chromatography on a CM-cellulose column is described. 3. The final purifications of the separated enzymes achieved were for the beta-acetylglucosaminase 35 times and for the beta-galactosidase 99 times. 4. The possibility of using DEAE-cellulose and Sephadex G-200 to purify the enzymes was investigated.

Extraction, Purification and Thermodynamic Characterization of Almond (Amygdalus communis β-Galactosidase for the Preparation of Delactosed Milk

Directory of Open Access Journals (Sweden)

Melita Lobo

2013-01-01

Full Text Available Buffer type, pH and ionic strength, as well as the fraction of polyvinylpyrrolidone were optimized for efficient extraction of β-galactosidase from almond seeds. The enzyme was purified up to electrophoretic homogeneity employing (NH42SO4 (15–60 % fractionation, size exclusion and ion-exchange chromatography. Molecular mass of β-galactosidase as estimated by gel filtration and SDS-PAGE was approx. 62 kDa, confirming its monomeric nature. The optimum activity of the enzyme was at pH=5.5, and it was stable within the range of pH=5.0–6.0. Various kinetic parameters of β-galactosidase thermal inactivation were calculated: ΔH°, ΔS° and ΔG° suggested that the enzyme undergoes significant processes of unfolding during denaturation. Using β-galactosidase from almond seed powder, lactose hydrolysis in milk up to approx. 50 % was observed. The findings indicate the potential use of almond seeds for the production of low/delactosed milk for lactose-intolerant population.

Cortical Alpha Activity in Schizoaffective Patients.

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Moeini, Mahdi; Khaleghi, Ali; Mohammadi, Mohammad Reza; Zarafshan, Hadi; Fazio, Rachel L; Majidi, Hamid

2017-01-01

Objective: Electrophysiological studies have identified abnormal oscillatory activities in the cerebral cortex in schizophrenia and mood disorders. Biological and pathophysiological evidence suggests specific deficits in serotonin (5-HT) receptor function in schizoaffective disorder (SA), a clinical syndrome with characteristics of both schizophrenia and bipolar disorder. This study investigated alpha oscillations in patients with SA. Method: Electroencephalography was used to measure ongoing and evoked alpha oscillations in 38 adults meeting Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition (DSM-IV) criteria for SA, and in 39 healthy controls. Results: Spontaneous alpha power of the participants with SA was significantly lower than that of healthy participants [F (1, 75) = 8.81, P < 0.01]. Evoked alpha activity was also decreased in SA compared to controls [F (1, 75) = 5.67, P = 0.025]. Conclusion : A strong reduction of alpha power in the posterior regions may reflect abnormality in the thalamocortical circuits. It is shown that hypoxia and reduced cerebral blood flow is associated with reduced alpha activity among different regions of the brain. Therefore, it can be concluded that greatly decreased alpha activity, particularly in centro-parietal and occipital regions, is related to SA symptoms such as hallucinations.

Production of α-Galactosidase by Aspergillus oryzae through solid-state fermentation and its application in soymilk Galactooligosaccharide hydrolysis

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Shankar Kapnoor

2010-02-01

Full Text Available α-Galactosidase was produced by Aspergillus oryzae on red gram plant waste-wheat bran based media in solid-state fermentation (SSF. Optimum temperature for α-galactosidase production was 35 0C and upto 4 cm of bed height of substrate had no inhibitory effect on enzyme production. Hydrolysis of galactooligosaccharides in soymilk was carried out by α-galactosidase. Optimum temperature and pH for the hydrolysis of raffinose and stachyose of soymilk were 55(0C and 5.2-6.2, respectively. The enzymatic treatment for 3 h completely removed the raffinose oligosaccharides in soymilk. Crude extract also showed considerable amount of invertase activity.

Hydrolysis of whey lactose by immobilized β-Galactosidase

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Marcela Panaro Mariotti

2008-12-01

Full Text Available Hydrolysis of whey lactose to glucose and galactose by immobilized galactosidase comes as an alternative to enlarge the possibilities of commercial use of this feedstock. To be applied at industrial scale, the process should be performed continuously .This work aimed to study the hydrolysis of whey lactose by an immobilized enzyme reactor. b-Galactosidase from Aspergillus oryzae was immobilized on silica and activity and stability were evaluated. The best immobilization results were attained by using glutaraldehyde as support's activator and enzyme stabilizer. The optimized enzyme proportion for immobilization was 15-20 mg g-1 of support. Treatments of whey were performed (microfiltration, thermal treatment and ultrafiltration, seeking the elimination of sludge, and the effects on operating the fixed bed reactor were evaluated. Ultrafiltration was the best treatment towards a proper substrate solution for feeding the reactor.A hidrólise de lactose de soro de leite, resultando em glicose e galactose, apresenta-se como uma alternativa para ampliar as possibilidades de uso comercial deste insumo. Para ser aplicado em escala industrial, o processo deve ser operado de modo contínuo. Reporta-se o estudo de um sistema objetivando hidrólise de lactose de soro de leite através de um reator com enzima imobilizada. b-Galactosidase de Aspergillus oryzae foi imobilizada em sílica, sendo avaliadas a estabilidade e atividade. Os melhores resultados de imobilização foram obtidos usando glutaraldeído como ativante do suporte e estabilizador da enzima. A proporção otimizada entre enzima e suporte foi 15-20 mg.g-1. Foram estudadas formas de tratamento do soro (microfiltração, tratamento térmico e ultrafiltração, objetivando eliminação de material suspenso, e avaliando os efeitos na operação de reator de leito fixo. A ultrafiltração foi o melhor tratamento, na busca de uma solução de substrato apropriada para o reator contínuo.

Purification of β-acetylglucosaminase and β-galactosidase from ram testis

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Caygill, J. C.; Roston, Christine P. J.; Jevons, F. R.

1966-01-01

1. The presence of β-galactosidase (EC 3.2.1.23) in an acetic acid extract of ram testis is reported. Some properties of the crude enzyme preparation were studied. 2. The purification of β-acetylglucosaminase (EC 3.2.1.30) and of β-galactosidase from the ram-testis extract by ammonium sulphate precipitation and chromatography on a CM-cellulose column is described. 3. The final purifications of the separated enzymes achieved were for the β-acetylglucosaminase 35 times and for the β-galactosidase 99 times. 4. The possibility of using DEAE-cellulose and Sephadex G-200 to purify the enzymes was investigated. PMID:5949569

Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and Bacillus amyloliquefaciens

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Shalini RAI

2014-03-01

Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

Recovery of plutonium from the combustion residues of alpha-bearing solid wastes

International Nuclear Information System (INIS)

Gompper, K.; Wieczorek, H.

1991-01-01

Experimental researches on plutonium dioxide dissolution in nitric acid in inactive and alpha-bearing wastes are presented in this report. After a review of the literature published on dissolution methods of PuO 2 combustion residues. Then results obtained in the ALONA plant on the dissolution of plutonium containing ashes in sulfuric acid and nitric acid are presented. Plutonium purification is studied. At last a simplified scheme of processing based on results obtained

Transgalactosylation/Hydrolysis Ratios of Various β-Galactosidases Catalyzing Alkyl-β-Galactoside Synthesis in Single-Phased Alcohol Media

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Eleonora Winkelhausen

2008-01-01

Full Text Available Three microbial galactosidases, Aspergillus oryzae, Escherichia coli and Kluyveromyces marxianus β-galactosidase, were used as catalysts for transgalactosylation synthesis of alkyl-β-galactosides in single-phased alcohol media. Their selectivity towards different alcohol nucleophiles was quantified by determining the transgalactosylation/hydrolysis ratio in the water/alcohol mixtures containing water in concentrations below the level of saturation. p-Nitrophenyl-β-galactoside was used as a glycosyl donor at a concentration of 10 mM. Both the total reaction rate (transgalactosylation+hydrolysis and the ratio between the transgalactosylation (alcoholysis and hydrolysis increased with the increase of water activity. Although the A. oryzae β-galactosidase showed relatively low total activity (3.13 μmol/(min·mg protein, it exhibited the highest selectivity towards the hexanol nucleophile among the examined enzymes (0.65. The selectivity values in all the examined cases were below one, which implies that the hydrolysis, and not the synthesis, was the dominating reaction. The total reaction rate (transgalactosylation+hydrolysis was strongly affected by the water activity, and for the specific water activity in the different alcohols, it increased in the following order: n-octanol, n-hexanol, n-butanol.

Functionalized agarose as an effective and novel matrix for immobilizing Cicer arietinum β-galactosidase and its application in lactose hydrolysis

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Rukhsana Satar

Full Text Available Abstract The present study demonstrates the immobilization of β-galactosidase from Cicer arietinum on a simple and inexpensive matrix, glutaraldehyde functionalized agarose (GFA, to suggest its potential application in hydrolyzing whey lactose in biotechnology industries. The designed matrix provided large surface area for the immobilization of β-galactosidase, apart from exhibiting greater biocatalytic activity in terms of selectivity, loading and stability. GFA retained 83% enzyme activity as a result of immobilization. Soluble and GFA bound Cicer arietinum β-galactosidase showed the same pH and temperature-optima at pH 5.0 and at 50 °C, respectively. However, immobilized enzyme exhibited a greater fraction of activity at both acidic and basic pH, and at higher temperature ranges. GFA bound enzyme lost only 20 % enzyme in the presence of 3% galactose, and retained 70 % activity even after its sixth repeated use. Immobilized enzyme showed pronounced lactose hydrolysis from whey in batch processes at 55 °C as compared to enzyme in solution.

Three-dimensional immobilization of beta-galactosidase on a silicon surface.

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Betancor, Lorena; Luckarift, Heather R; Seo, Jae H; Brand, Oliver; Spain, Jim C

2008-02-01

Many alternative strategies to immobilize and stabilize enzymes have been investigated in recent years for applications in biosensors. The entrapment of enzymes within silica-based nanospheres formed through silicification reactions provides high loading capacities for enzyme immobilization, resulting in high volumetric activity and enhanced mechanical stability. Here we report a strategy for chemically associating silica nanospheres containing entrapped enzyme to a silicon support. beta-galactosidase from E. coli was used as a model enzyme due to its versatility as a biosensor for lactose. The immobilization strategy resulted in a three-dimensional network of silica attached directly at the silicon surface, providing a significant increase in surface area and a corresponding 3.5-fold increase in enzyme loading compared to enzyme attached directly at the surface. The maximum activity recovered for a silicon square sample of 0.5 x 0.5 cm was 0.045 IU using the direct attachment of the enzyme through glutaraldehyde and 0.16 IU when using silica nanospheres. The immobilized beta-galactosidase prepared by silica deposition was stable and retained more than 80% of its initial activity after 10 days at 24 degrees C. The ability to generate three-dimensional structures with enhanced loading capacity for biosensing molecules offers the potential to substantially amplify biosensor sensitivity. (c) 2007 Wiley Periodicals, Inc.

Activation of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) suppresses postprandial lipidemia through fatty acid oxidation in enterocytes

Energy Technology Data Exchange (ETDEWEB)

Kimura, Rino [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Takahashi, Nobuyuki, E-mail: nobu@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murota, Kaeko [Department of Life Science, School of Science and Engineering, Kinki University, Osaka 770-8503 (Japan); Yamada, Yuko [Laboratory of Physiological Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Niiya, Saori; Kanzaki, Noriyuki; Murakami, Yoko [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Moriyama, Tatsuya [Department of Applied Cell Biology, Graduate School of Agriculture, Kinki University, Nara 631-8505 (Japan); Goto, Tsuyoshi; Kawada, Teruo [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

2011-06-24

Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of fatty acid oxidation-related genes in human intestinal epithelial Caco-2 cells. {yields} PPAR{alpha} activation also increased oxygen consumption rate and CO{sub 2} production and decreased secretion of triglyceride and ApoB from Caco-2 cells. {yields} Orally administration of bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and CO{sub 2} production in small intestinal epithelial cells. {yields} Treatment with bezafibrate decreased postprandial serum concentration of triglyceride after oral injection of olive oil in mice. {yields} It suggested that intestinal lipid metabolism regulated by PPAR{alpha} activation suppresses postprandial lipidemia. -- Abstract: Activation of peroxisome proliferator-activated receptor (PPAR)-{alpha} which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPAR{alpha} activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPAR{alpha} activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPAR{alpha} agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and

Activity of β-Galactosidase and Polygalacturonase in Zucchini Squash (Cucurbita pepo L. Stored at Low Temperatures

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René Renato Balandrán-Quintana

2007-01-01

Full Text Available When fruits are exposed to chilling temperatures, enzymatic systems are affected and normal cell metabolism is altered; cell wall enzymes are the least studied in this respect. Our objective is to determine the effect of storage temperature and/or kind of tissue on the activity of the enzymes polygalacturonase (PG and β-galactosidase (β-gal in zucchini squash (Cucurbita pepo L. subjected to 2.5 and/or 12 °C for 16 days. Exocarp and endocarp samples were analyzed every 4 days for PG and β-gal activities. The highest β-gal activity was found in the exocarp at 12 °C; in both tissues β-gal decreased with time at 2.5 °C, but increased at 12 °C. PG activity was higher in the endocarp (highest activity at 2.5 °C than in the exocarp (highest activity at 12 °C. It was concluded that PG activity in the exocarp constitutes an adequate system for studying the relationship between chilling injury and cell wall biological activity.

Characterization of a beta-glycosidase highly active on disaccharides and of a beta-galactosidase from Tenebrio molitor midgut lumen.

Science.gov (United States)

Ferreira, Alexandre H P; Terra, Walter R; Ferreira, Clélia

2003-02-01

The midgut of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae has four beta-glycosidases. The properties of two of these enzymes (betaGly1 and betaGly2) have been described elsewhere. In this paper, the characterization of the other two glycosidases (betaGly3 and betaGly4) is described. BetaGly3 has one active site, hydrolyzes disaccharides, cellodextrins, synthetic substrates and beta-glucosides produced by plants. The enzyme is inhibited by amygdalin, cellotriose, cellotetraose and cellopentaose in high concentrations, probably due to transglycosylation. betaGly3 hydrolyzes beta 1,4-glycosidic linkages with a catalytic rate independent of the substrate polymerization degree (k(int)) of 11.9 s(-1). Its active site is formed by four subsites, where subsites +1 and -1 bind glucose residues with higher affinity than subsite +2. The main role of betaGly3 seems to be disaccharide hydrolysis. BetaGly4 is a beta-galactosidase, since it has highest activity against beta-galactosides. It can also hydrolyze fucosides, but not glucosides, and has Triton X-100 as a non-essential activator (K(a)=15 microM, pH 4.5). betaGly4 has two active sites that can hydrolyze p-nitrophenyl beta-galactoside (NPbetaGal). The one hydrolyzing NPbetaGal with more efficiency is also active against methylumbellipheryl beta-D-galactoside and lactose. The other active site hydrolyzes NPbetaFucoside and binds NPbetaGal weakly. BetaGly4 hydrolyzes hydrophobic substrates with high catalytical efficiency and is able to bind octyl-beta-thiogalactoside in its active site with high affinity. The betaGly4 physiological role is supposed to be the hydrolysis of galactolipids that are found in membranes from vegetal tissues. As the enzyme has a hydrophobic site where Triton X-100 can bind, it might be activated by membrane lipids, thus becoming fully active only at the surface of cell membranes.

Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases

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Michele Dutra Rosolen

2015-01-01

Full Text Available This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C and Kluyveromyces lactis (at temperatures of 10 and 37°C β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C, at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry.

Hooked on α-d-galactosidases: from biomedicine to enzymatic synthesis.

Science.gov (United States)

Bakunina, Irina Yu; Balabanova, Larissa A; Pennacchio, Angela; Trincone, Antonio

2016-01-01

α-d-Galactosidases (EC 3.2.1.22) are enzymes employed in a number of useful bio-based applications. We have depicted a comprehensive general survey of α-d-galactosidases from different origin with special emphasis on marine example(s). The structures of natural α-galactosyl containing compounds are described. In addition to 3D structures and mechanisms of action of α-d-galactosidases, different sources, natural function and genetic regulation are also covered. Finally, hydrolytic and synthetic exploitations as free or immobilized biocatalysts are reviewed. Interest in the synthetic aspects during the next years is anticipated for access to important small molecules by green technology with an emphasis on alternative selectivity of this class of enzymes from different sources.

Discriminating Foot-and-Mouth Disease Virus-Infected and Vaccinated Animals by Use of β-Galactosidase Allosteric Biosensors▿ †

Science.gov (United States)

Sánchez-Aparicio, M. Teresa; Rosas, María Flora; Ferraz, Rosa Maria; Delgui, Laura; Veloso, Juan J.; Blanco, Esther; Villaverde, Antonio; Sobrino, Francisco

2009-01-01

Recombinant β-galactosidases accommodating one or two different peptides from the foot-and-mouth disease virus (FMDV) nonstructural protein 3B per enzyme monomer showed a drastic enzymatic activity reduction, which mainly affected proteins with double insertions. Recombinant β-galactosidases were enzymatically reactivated by 3B-specific murine monoclonal and rabbit polyclonal antibodies. Interestingly, these recombinant β-galactosidases, particularly those including one copy of each of the two 3B sequences, were efficiently reactivated by sera from infected pigs. We found reaction conditions that allowed differentiation between sera of FMDV-infected pigs, cattle, and sheep and those of naïve and conventionally vaccinated animals. These FMDV infection-specific biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected animals. PMID:19553549

Structural prediction and comparative docking studies of psychrophilic β- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes.

Science.gov (United States)

Kumar, Ponnada Suresh; Pulicherla, Kk; Ghosh, Mrinmoy; Kumar, Anmol; Rao, Krs Sambasiva

2011-01-01

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.

Production and Characterization of α-Galactosidase by a Multiple Mutant of Aspergillus niger in Solid-State Fermentation

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Muhammad Siddique Awan

2009-01-01

Full Text Available α-Galactosidase is applied in the sugar industry to enhance sugar recovery from sugar beet syrup and to improve nutritional value of the soymilk. In the present investigation, the influence of process variables on the production of this important enzyme has been explored in a newly isolated multiple mutant strain of Aspergillus niger in solid-state fermentation (SSF. Defined fermentation parameters include substrate type (pure lactose and by-products of rice and flour mills as prime substrates, nitrogen source, incubation time, initial pH of the medium and incubation temperature. Extracellular α-galactosidase reached the value of 135.4 IU/g of dry substrate (IU/g after 96 h of fermentation. Supplementation with 2 g of glucose and 3 g of corn steep liquor significantly increased the enzyme production, and maximum value of product yield (318 IU/g by the mutant strain was significantly higher than that reported by the wild type (this work, or other A. niger mutants, recombinants and yeasts reported in literature as producers of elevated levels of α-galactosidase. Among three α-galactosidases, one possessing high subunit molecular mass proteins (99 and 100 kDa has been characterized in both wild and mutant organisms. Thermal properties of the purified enzymes indicate that the mutation decreased the values of activation energy for the formation of enzyme-substrate (ES complex, enthalpy, Gibbs free energy demand for substrate binding, and transition state stabilization. A thermodynamic study of irreversible inactivation of enzymes suggests that the mutant–derived enzyme is more thermostable than the native enzyme, which is attributable to amino acids involved in active catalysis. Because of these properties, the mutant organism is a novel organism and may be exploited for bulk production of thermostable α-galactosidase for the above industrial and nutritional applications.

Avaliação da atividade das enzimas pectina metilesterase e β-Galactosidase em mamões cv. Golden armazenados sob diferentes concentrações de oxigênio Activity of pectin methylesterase and β-Galactosidase enzymes in 'Golden' papaya stored under different oxygen concentrations

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Luciana Konda de Azevedo Pinto

2013-03-01

Full Text Available Este trabalho foi realizado com o objetivo de avaliar o efeito de atmosferas controladas contendo diferentes concentrações de oxigênio sobre a atividade das enzimas β-galactosidase e pectina metilesterase, e sobre a cor da casca e a firmeza da polpa de mamões 'Golden'. Os frutos foram mantidos por 36 dias, nas seguintes atmosferas controladas: 1% de O2 e 0,03% CO2 com adsorvedor de etileno, 3% de O2 e 0,03% de CO2 com adsorvedor de etileno, 5% O2 e 0,03% de CO2 com adsorvedor de etileno e atmosfera ambiente sem adsorvedor de etileno. A UR e a temperatura foram mantidas entre 85-95% e a 13º C, respectivamente. Os frutos estocados sob atmosfera de 1% de O2 e 0,03% CO2 apresentaram retardamento nas atividades das enzimas β-galactosidase e pectina metilesterase comparado com os frutos estocados nas outras atmosferas avaliadas. Os frutos armazenados sob atmosfera de 1% de O2 e 0,03% O2 também apresentaram atraso no desenvolvimento da cor da casca e amolecimento da polpa.This study was carried out with the objective to evaluate the effect of controlled atmospheres containing different concentration of oxygen on the activity of β-galactosidase and pectinmethylesterase enzymes, on the skin color and pulp firmness in 'Golden' papaya. The fruits were kept for 36 days under the following controlled atmospheres: 1% of O2 and 0.03% CO2 with ethylene absorber, 3% of O2 and 0.03% of CO2 with ethylene absorber, 5% O2 and 0.03% of CO2 with ethylene absorber and environmental atmosphere without ethylene absorber. Relative humidity was set at the range 85-95% and temperature maintained at 13ºC. Fruit stored under atmospheres of 1% of O2 and 0.03% CO2 had a β-galactosidase and pectimethylesterase activities delayed compared with the fruits storage under the other atmospheres evaluated. There was also, a delay in the development of color and pulp softening in fruits stored under atmospheres of 1% of O2 and 0.03% O2.

CARBOXYLATION OF SILVER NANOPARTICLES FOR THE IMMOBILIZATION OF β-GALACTOSIDASE AND ITS EFFICACY IN GALACTO-OLIGOSACCHARIDES PRODUCTION

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Shakeel Ahmed Ansari

2015-03-01

Full Text Available The present study investigated the carboxylation of silver nanoparticles (AgNPs by 1:3 nitric acid-sulfuric acid mixtures for immobilizing Aspergillus oryzae β-galactosidase. Carboxylated AgNPs retained 93% enzyme upon immobilization and the enzyme did not leach out appreciably from the modified nanosupport in the presence of 100 mmol L-1 NaCl. Atomic force micrograph revealed the binding of β-galactosidase on the modified AgNPs. The optimal pH for soluble and carboxylated AgNPs adsorbed β-galactosidase (IβG was observed at pH 4.5 while the optimal operating temperature was broadened from 50 ºC to 60 ºC for IβG. Michaelis constant, Km was increased two and a half fold for IβG while Vmax decreases slightly as compared to soluble enzyme. β-galactosidase immobilized on surface functionalized AgNPs retained 70% biocatalytic activity even at 4% galactose concentration as compared to enzyme in solution. Our study showed that IβG produces greater amount of galacto-oligosaccharides at higher temperatures (50 ºC and 60 ºC from 0.1 mol L-1 lactose solution at pH 4.5 as compared to previous reports.

Deproteinization: an integrated-solution approach to increase efficiency in β-galactosidase production using cheese whey powder (CWP solution

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Leandro Freire dos Santos

2017-08-01

Full Text Available Whey is the liquid that results from the coagulation of milk during cheese manufacture. Cheese whey is also an important environmental pollution source. The present experiment sought to compare β-galactosidase (EC 3.2.1.23 production by Aspergillus oryzae from deproteinized and un-deproteinized CWP solutions. β-galactosidase was produced by submerged fermentation in deproteinized or un-deproteinized CWP solutions. To determine the activity of the enzyme, a reaction mixture containing cell-free extract and ortho Nitrophenyl β galactoside (ONPG was used. The results indicated that β-galactosidase induction was greater when using deproteinized CWP solution compared to the un deproteinized CWP solution. These results may enable an alternative management of cheese whey, thereby decreasing its impact on the environment and producing value-added biomacromolecules.

Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

Energy Technology Data Exchange (ETDEWEB)

Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

2011-04-22

Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

Estabilidade operacional e cinética de hidrólise da lactose pela β-galactosidase de Kluyveromyces fragilis - DOI: 10.4025/actascihealthsci.v25i1.2244 Operational stability and kinetics of lactose hydrolysis by β-galactosidase from Kluyveromyces fragilis - DOI: 10.4025/actascihealthsci.v25i1.2244

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Flávio Faria de Moraes

2003-04-01

Full Text Available A modelagem cinética da hidrólise da lactose e a estabilidade operacional da enzima β-galactosidase de Kluyveromyces fragilis foi determinada utilizando como substrato leite em pó desengordurado e reconstituído de modo a fornecer duas concentrações de lactose: 5% e 10% (p/v. Para a estabilidade operacional, ambas concentrações de lactose foram estudadas na presença e na ausência de tampão pH 6,5. A cada 40 minutos, foi determinada a atividade residual pelo método das velocidades iniciais. Os resultados experimentais mostraram que o tampão provoca inativação da enzima. Para soluções de lactose em água, a atividade residual decaiu apenas 15% em 6 horas e depois decresceu bruscamente. Na modelagem cinética, a reação de hidrólise foi conduzida a 40ºC/7 h, empregando-se concentrações de enzima equivalentes a 1500 e 8100 LAU/L. O modelo ajustado permitiu concluir que é necessário utilizar a quantidade de 3450 LAU/L da enzima para se obter a hidrólise de 70% a 80% da lactose do leite em duas a três horas de reação.The kinetic modeling of the lactose hydrolysis and the operational stability of the enzyme β-galactosidase of Kluyveromyces fragilis was determined using as substrate skimmed powdered milk, reconstituted in a way to supply two lactose concentrations: 5% and 10% (w/v. For the operational stability both lactose concentrations were studied in the presence and in the absence of the buffer pH 6.5. Every 40 minutes the residual activity was determined by the method of the initial velocities. The experimental results showed that the buffer leads to enzyme inactivation. However, for lactose solutions without buffer, the residual activity only declines 15% in 6 hours and later it decreases abruptly. In the kinetic modeling the hydrolysis reaction was led to 40ºC/7h, being used enzyme concentrations equivalent to 1500 and 8100 LAU/L. The adjusted model allowed us to conclude that it is necessary to use the quantity of

Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha and its effect on the respiratory burst activity of phagocytes.

Science.gov (United States)

Kim, Min Sun; Hwang, Yoon Jung; Yoon, Ki Joon; Zenke, Kosuke; Nam, Yoon Kwon; Kim, Sung Koo; Kim, Ki Hong

2009-11-01

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha (rbTNF-alpha) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-alpha on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-alpha was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-alpha gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-alpha, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-alpha was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-alpha might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.

Purification of beta-galactosidase from Aspergillus Niger and studies of its kinetics

International Nuclear Information System (INIS)

Ali, M.A.A.

1989-01-01

The present study was carried out to extract purity and characterize B-galactosidase which was produced by a local strain of the fungus A. Niger. The obtained results were as follows: 1. The extraction of B-galactosidase by the preparation of acetone powder followed by extraction with 0.1M sodium acetate-2m M PMSC buffer, PH 5.0. 2. B-galactosidase was purified by a combination of techniques. The enzyme was purified 113.6 folds with 27% recovery. 3. The isoelectric point of the purified enzyme was 4.6. 7 tabs.; 37 figs.; 67 refs

Inhibition of β-galactosidase and α-glucosidase synthesis in petroleum refinery effluent bacteria by phenolic compounds

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Gideon C. Okpokwasili

2011-04-01

Full Text Available Inhibition of α-glucosidase (EC 3.2.1.20 and β-galactosidase (EC 3.2.1.23 biosynthesis by phenolic compounds (phenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol in Escherichia coli, Bacillus and Pseudomonas species isolated from petroleum refinery wastewater was assessed. At sufficient concentrations, phenols inhibited the induction of α-glucosidase and β-galactosidase. The patterns of these toxic effects can be mathematically described with logistic and sigmoid dose-response models. The median inhibitory concentrations (IC50 varied among the phenols, the bacteria and enzymes. Quantitative structure–activity relationship (QSAR models based on the logarithm of the octanol–water partition coefficient (log10Kow were developed for each bacterium. The correlation coefficients varied between 0.84and 0.99 for the enzymes. The test results indicated α-glucosidase and β-galactosidase biosynthesis as important microbial indices for evaluation of toxicity of phenolic compounds.

Comparative Studies of Two-Dimensional Electrophoresis on Galactosidase Relating to Bombyx Lectin Activity

OpenAIRE

加藤, 靖夫; カトウ, ヤスオ; Yasuo, Kato

2005-01-01

"Comparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis on the haemolymph of the domesticated silkworm, Bombyx mori and Fraction II obtained by gel filtration from the haemolymph of B. mori was performed using the 2-D mini-slab system (Atto Co.) (the first method of 2-D PAGE) and the Mini-PROTEAN mini tube gel 2-D PAGE system (Bio-Rad Laboratories, Inc.) (the second method). Moreover, two-dimensionnal electrophoresis analysis on standard β-galactosidase, grade III ...

Synthesis of the Galactosyl Derivative of Gluconic Acid With the Transglycosylation Activity of β-Galactosidase

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Aleksandra Wojciechowska

2017-01-01

Full Text Available Bionic acids are bioactive compounds demonstrating numerous interesting properties. They are widely produced by chemical or enzymatic oxidation of disaccharides. This paper focuses on the galactosyl derivative of gluconic acid as a result of a new method of bionic acid synthesis which utilises the transglycosylation properties of β-galactosidase and introduces lactose as a substrate. Products obtained in such a process are characterised by different structures (and, potentially, properties than those resulting from traditional oxidation of disaccharides. The aim of this study is to determine the effect of selected parameters (concentration and ratio of substrates, dose of the enzyme, time, pH, presence of salts on the course of the reaction carried out with the enzymatic preparation Lactozym, containing β-galactosidase from Kluyveromyces lactis. Research has shown that increased dry matter content in the baseline solution (up to 50 %, by mass per volume and an addition of NaCl contribute to higher yield. On the other hand, reduced content of the derivative is a result of increased pH from 7.0 to 9.0 and an addition of magnesium and manganese salts. Moreover, exceeding the β-galactosidase dose over approx. 35 000 U per 100 g of lactose also leads to reduced yield of the process. The most favourable molar ratio of sodium gluconate to lactose is 2.225:0.675. Depending on the conditions of the synthesis, the product concentration ranged between 17.3 and 118.3 g/L of the reaction mixture, which corresponded to the mass fraction of 6.64–23.7 % of dry matter. The data obtained as a result of the present study may be useful for designing an industrial process.

Nutrient Content, Phytonutrient Composition, Alpha Amylase, Alpha Glucosidase Inhibition Activity and Antioxidant Activity of the Stoechospermum Marginatum Collected in Pre Monsoon Season

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Reka Palanivel

2017-03-01

Full Text Available The objective of this study was to investigate the nutrient content, phytonutrient composition, physicochemical properties, alpha amylase and alpha glucosidase inhibition activity and antioxidant activity of the brown algae Stoechospermum marginatum collected from Gulf of Mannar, Tamil Nadu, India in pre monsoon season (June- September, 2015. Six and eight hours of ethanol and aqueous extract of Stoechospermum marginatum were used for phytonutrient screening, alpha amylase, alpha glucosidase inhibition activity and antioxidant activity. From the results of the study it is understood that Stoechospermum marginatum contain a high amount of carbohydrate, protein, crude fiber and phytonutrients like tannin, flavonoid, saponin, alkaloid, terpenoids, steroid and total phenolic content. The physicochemical properties namely Water absorption and Swelling power were very promising. Alpha amylase and alpha glucosidase inhibition activity was recorded to be high in both aqueous and ethanol extracts of eight hour extraction than in extracts taken from six hours extraction. Antioxidant activity was detected using DPPH, FRAP, beta carotene scavenging and H2O2 assay and found to have a high radical scavenging activity. Stoechospermum marginatum possess a valuable amount of total phenolic content and other phytonutrients and physicochemical properties, it may the reason for the potential inhibition of alpha amylase, alpha glucosidase and antioxidant activity. It is concluded from the study that the brown algae may be incorporated into foods to enhance their nutritional and therapeutic value.

5alphaDH-DOC (5alpha-dihydro-deoxycorticosterone) activates androgen receptor in castration-resistant prostate cancer.

Science.gov (United States)

Uemura, Motohide; Honma, Seijiro; Chung, Suyoun; Takata, Ryo; Furihata, Mutsuo; Nishimura, Kazuo; Nonomura, Norio; Nasu, Yasutomo; Miki, Tsuneharu; Shuin, Taro; Fujioka, Tomoaki; Okuyama, Akihiko; Nakamura, Yusuke; Nakagawa, Hidewaki

2010-08-01

Prostate cancer often relapses during androgen-depletion therapy, even under the castration condition in which circulating androgens are drastically reduced. High expressions of androgen receptor (AR) and genes involved in androgen metabolism indicate a continued role for AR in castration-resistant prostate cancers (CRPCs). There is increasing evidence that some amounts of 5alpha-dihydrotestosterone (DHT) and other androgens are present sufficiently to activate AR within CRPC tissues, and enzymes involved in the androgen and steroid metabolism, such as 5alpha-steroid reductases, are activated in CRPCs. In this report, we screened eight natural 5alphaDH-steroids to search for novel products of 5alpha-steroid reductases, and identified 11-deoxycorticosterone (DOC) as a novel substrate for 5alpha-steroid reductases in CRPCs. 11-Deoxycorticosterone (DOC) and 5alpha-dihydro-deoxycorticosterone (5alphaDH-DOC) could promote prostate cancer cell proliferation through AR activation, and type 1 5alpha-steroid reductase (SRD5A1) could convert from DOC to 5alphaDH-DOC. Sensitive liquid chromatography-tandem mass spectrometric analysis detected 5alphaDH-DOC in some clinical CRPC tissues. These findings implicated that under an extremely low level of DHT, 5alphaDH-DOC and other products of 5alpha-steroid reductases within CRPC tissues might activate the AR pathway for prostate cancer cell proliferation and survival under castration.

Entrappment of alkaline protease and {beta}-galactosidase in radiation stitched together poly-N-vinylcaprolactam.; Vklyuchenie shchelochnoj proteazy i {beta}-galaktozidazy v radiatsionno sshityj poli-N-vinilkaprolaktam.

Energy Technology Data Exchange (ETDEWEB)

Davidenko, T I; Kravchenko, I A [AN Ukrainskoj SSR, Odessa (Ukraine). Fiziko-Khimicheskij Inst.

1996-03-01

The gel formations by poly-N-vinylcaprolactam upon its {gamma}-irradiation by the 20-25 kGy dose as a results of partial polymer stitching together is shown, which is confirmed by the CD-and thermogravimetric data. By the alkaline protease and {beta}-galactosidase entrapment in poly-N- vinylcaprolactam stitched together by {gamma}-irradiation, the active preparations are obtained with 90-98 % and 30-35 % activity retained for alkaline protease and {beta}-galactosidase, respectively. The increased stability of alkaline protease at acidic pH values and higher temperature was noted, and for {beta}-galactosidase - the possibility of repeated use of the obtained preparation for lactose hydrolysis.

Enzyme activity of β-galactosidase from Kluyveromyces lactis and Aspergillus oryzae on simulated conditions of human gastrointestinal system

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Alessandra Bosso

2015-09-01

Full Text Available An alternative to relieve the symptoms of lactose intolerance is the intake of the enzyme β-galactosidase in pharmaceutical dosage forms. The ability of β-galactosidase produced by Kluyveromyces lactis and Aspergillus oryzae to hydrolyze lactose in simulated conditions of the human gastrointestinal tract was investigated. The experiment was carried out in the optimum temperature for each enzyme activity, 40 and 55°C, respectively, and at the normal human body temperature (37°C at concentrations of 1.5, 3.0, and 5.0 g/L (enzyme from A. oryzae or mL/L (enzyme from K. lactis. Both enzymes were completely inactivated under simulated gastric conditions (pH 2. When the enzymes were subjected to simulated small intestine conditions (pH 7.4, lactose hydrolysis has occurred, but at 37°C the percentage was lower than that under the optimal temperatures. At concentrations of 1.5, 3.0, and 5.0 mL/L the enzyme from K. lactis hydrolyzed 76.63%, 88.91% and 94.80% of lactose at 40°C, and 55.99%, 80.91% and 81.53% at 37°C, respectively. In contrast, the enzyme from A. oryzae hydrolyzed 7.11%, 16.18% and 21.29% at 55°C, and 8.4%, 11.85% and 16.43% at 37°C. It was observed that under simulated intestinal conditions, the enzyme from K. lactis was more effective on lactose hydrolysis as compared to the enzyme from A. oryzae. Considering the findings of this study, it is extremely necessary to use an enteric coating on β-galactosidase capsules so that this enzyme is released only in the small intestine, which is its site of action, thus not suffering the action of the stomach pH.Keywords: Lactase. Hydrolysis. Lactose intolerance. Gastrointestinal tract. RESUMOAtividade de β-galactosidase de Kluyveromyces lactis e Aspergillus oryzae, em condições simuladas do sistema gastrintestinal humanoUma das alternativas para amenizar os sintomas da intolerância à lactose é a ingestão de β-galactosidase em formas farmacêuticas. Neste trabalho avaliou-se a

Improving Properties of a Novel β-Galactosidase from Lactobacillus plantarum by Covalent Immobilization

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Rocio Benavente

2015-04-01

Full Text Available A novel β-galactosidase from Lactobacillus plantarum (LPG was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl β-d-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50 °C. This β-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35% and oligosaccharides derived from lactulose (30% was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3'-galactosyl lactulose, has been described.

The use of the long-range alpha detector (LRAD) for alpha emission surveys at active and inactive firing sites

International Nuclear Information System (INIS)

Mason, C.F.V.; Allander, K.S.; Bounds, J.A.; Garner, S.E.; Walter, K.J.

1994-01-01

Surveys were carried out at five different firing sites at Los Alamos National Laboratory to measure residual alpha emissions in earth contaminated with natural and depleted uranium. This contamination is caused by controlled experimental explosions during testing of the non fissile components of nuclear weapons. Two conclusions were reached: the first is that post shot clearing of the experimental areas is effective at removing contamination and the second is that the diminution of alpha emissions due to aging is small

Nutrient Content, Phytonutrient Composition, Alpha Amylase, Alpha Glucosidase Inhibition Activity and Antioxidant Activity of the Stoechospermum Marginatum Collected in Pre Monsoon Season

OpenAIRE

Reka Palanivel; Thahira Banu Azeez; Seethalakshmi Muthaya

2017-01-01

The objective of this study was to investigate the nutrient content, phytonutrient composition, physicochemical properties, alpha amylase and alpha glucosidase inhibition activity and antioxidant activity of the brown algae Stoechospermum marginatum collected from Gulf of Mannar, Tamil Nadu, India in pre monsoon season (June- September, 2015). Six and eight hours of ethanol and aqueous extract of Stoechospermum marginatum were used for phytonutrient screening, alpha amylase, alpha glucosidase...

Synthesis of galacto-oligosaccharides with ß-galactosidases

NARCIS (Netherlands)

Warmerdam, A.

2013-01-01

Galacto-oligosaccharides (GOS) are generally enzymatically synthesized with β-galactosidases. GOS are of interest because of their prebiotic effects on human health. They are mainly applied in infant nutrition, because of their resemblance to human milk oligosaccharides, but they are also

a-galactosidase: general aspects and its application on soybean oligosaccharides/ a-galactosidase: aspectos gerais e sua aplicação em produtos a base de soja

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Mara Lucia Luiz Ribeiro

2002-05-01

Full Text Available This literature review has the aim to present topics related to a-galactosidase, considering its general characteristics, sources, functions and methods used in its determination. This enzyme, lacking in the human digestive tract, hydrolises µ-(1,6 bonds from oligosaccharides of the raffinose family. These carbohydrates, called flatulents, are found in leguminoses like soybean, a rich source of protein. However, due to the presence of these flatulents sugars, antinutritional factors are attributed to soyben, decreasing its consumption. a-galactosidase produced by some microorganisms and vegetables can be used in soybean based foods to reduce the interaction of the intestinal flora with the oligosaccharides, decreasing in this way the flatulence.This literature review has the aim to present topics related to a-galactosidase, considering its general characteristics, sources, functions and methods used in its determination. This enzyme, lacking in the human digestive tract, hydrolises µ-(1,6 bonds from oligosaccharides of the raffinose family. These carbohydrates, called flatulents, are found in leguminoses like soybean, a rich source of protein. However, due to the presence of these flatulents sugars, antinutritional factors are attributed to soyben, decreasing its consumption. a-galactosidase produced by some microorganisms and vegetables can be used in soybean based foods to reduce the interaction of the intestinal flora with the oligosaccharides, decreasing in this way the flatulence.

[beta]-hexosaminidase isozymes from cells cotransfected with [alpha] and [beta] cDNA constructs: Analysis of the [alpha]-subunit missense mutation associated with the adult form of Tay-Sachs disease

Energy Technology Data Exchange (ETDEWEB)

Brown, C.A.; Mahuran, D.J. (Univ. of Toronto (Canada))

1993-08-01

In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the [alpha]-subunit of [beta]-hexosaminidase A ([alpha][beta]) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of [alpha]-chain mutations is not straightforward. The authors examine three approaches utilizing previously identified mutations affecting [alpha]-chain folding. These involve transfection of (1) the [alpha] cDNA alone; (2) a [beta] cDNA construct encoding a [beta]-subunit substituted at a position homologous to that of the [alpha]-subunit, and (3) both [alpha] and [beta] cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an [open quotes]active[close quotes] [alpha]Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. The authors conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some [alpha]-chain mutations. Using this technique, they demonstrate that the adult-onset Tay-Sachs mutation, [alpha]Gly[yields]Ser[sup 269], does not directly affect [alpha][beta] dimerization but exerts an indirect effect on the dimer through destabilizing the folded [alpha]-subunit at physiological temperatures. Two other [alpha] mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit. 36 refs., 2 figs., 5 tabs.

Adsorption of β-galactosidase of Alicyclobacillus acidocaldarius on wild type and mutants spores of Bacillus subtilis

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Sirec Teja

2012-08-01

Full Text Available Abstract Background The Bacillus subtilis spore has long been used as a surface display system with potential applications in a variety of fields ranging from mucosal vaccine delivery, bioremediation and biocatalyst development. More recently, a non-recombinant approach of spore display has been proposed and heterologous proteins adsorbed on the spore surface. We used the well-characterized β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius as a model to study enzyme adsorption, to analyze whether and how spore-adsorption affects the properties of the enzyme and to improve the efficiency of the process. Results We report that purified β-galactosidase molecules were adsorbed to purified spores of a wild type strain of B. subtilis retaining ca. 50% of their enzymatic activity. Optimal pH and temperature of the enzyme were not altered by the presence of the spore, that protected the adsorbed β-galactosidase from exposure to acidic pH conditions. A collection of mutant strains of B. subtilis lacking a single or several spore coat proteins was compared to the isogenic parental strain for the adsorption efficiency. Mutants with an altered outermost spore layer (crust were able to adsorb 60-80% of the enzyme, while mutants with a severely altered or totally lacking outer coat adsorbed 100% of the β-galactosidase molecules present in the adsorption reaction. Conclusion Our results indicate that the spore surface structures, the crust and the outer coat layer, have an negative effect on the adhesion of the β-galactosidase. Electrostatic forces, previously suggested as main determinants of spore adsorption, do not seem to play an essential role in the spore-β-galactosidase interaction. The analysis of mutants with altered spore surface has shown that the process of spore adsorption can be improved and has suggested that such improvement has to be based on a better understanding of the spore surface structure

Biosensors based on β-galactosidase enzyme: Recent advances and perspectives.

Science.gov (United States)

Sharma, Shiv K; Leblanc, Roger M

2017-10-15

Many industries are striving for the development of more reliable and robust β-galactosidase biosensors that exhibit high response rate, increased detection limit and enriched useful lifetime. In a newfangled technological atmosphere, a trivial advantage or disadvantage of the developed biosensor may escort to the survival and extinction of the industry. Several alternative strategies to immobilize β-galactosidase enzyme for their utilization in biosensors have been developed in recent years in the quest of maximum utility by controlling the defects seen in the previous biosensors. The overwhelming call for on-line measurement of different sample constituents has directed science and industry to search for best practical solutions and biosensors are witnessed as the best prospect. The main objective of this paper is to serve as a narrow footbridge by comparing the literary works on the β-galactosidase biosensors, critically analyze their use in the construction of best biosensor by showing the pros and cons of the predicted methods for the practical use of biosensors. Copyright © 2017 Elsevier Inc. All rights reserved.

Enhancement of water soluble wheat bran polyphenolic compounds using different steviol glucosides prepared by thermostable β-galactosidase

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Hee-jung Lim

2016-10-01

Full Text Available Background: Production of wheat bran (WB for human consumption is estimated to be about 90 million tons per year. WB contains an abundant source of dietary fiber, minerals, vitamins, and bioactive compounds. WB is a by-product of milling and contains an abundant source of carbohydrate (60%, protein (12%, fat (0.5%, minerals (2%, and bioactive compounds such as phenolic acids, arabinoxylans, flavonoids, caroteinoids alkylresorcinol and phytosterols. These are known for health promoting properties such as controlling glycemic index, reducing plasma cholesterol level, antioxidant, anti-inflammatory, and anticarcinogenic activities. Several terpene glycosides such as mogroside V, paenoiflorin, geniposide, rubusoside (Ru, stevioside (Ste, rebaudioside A (RebA, steviol monoside, and stevioside glucoside have been discovered to enhance the solubility of a number of pharmaceutically and medically important compounds that normally show poor solubility in water. Context and purpose of this study: In this study, in order to increase soluble extraction of polyphenol compounds of WB using Ru, the expression of β-galactosidase from Thermus thermophilus (T. thermophilus was optimized using different E. coli hosts and a different concentration of lactose inducer rather than of isopropyl-1- thio-β-D-galactopyranoside (IPTG for industrial production. Additionally, the effect of different steviol glucosides (Ru, Ste, RebA, and SG on the enhancement of polyphenol compounds extraction from wheat bran was studied. Results: β-galactosidase from T. thermophilus was used for the specific conversion of stevioside (Ste to rubusoside (Ru with 92% productivity. The enzyme was optimized to be expressed in E. coli. With 7 mM lactose, the β-galactosidase activity expressed was 34.3, 14.2, or 34.4 ± 0.5 U/mL in E. coli BL21(DE3pLysS, Rosetta(DE3pLysS, or BL21(DE3 at 37°C, and 9.8 ± 0.2, 7.0 ± 0.5, or 7.4 ± 0.2 U/mL at 28°C respectively. The expression of β-galactosidase

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

Science.gov (United States)

Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-05-01

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

The in vitro synthesis of {beta}-galactosidase induced in a subcellular structure of Escherichia coli (1961); Synthese in vitro de {beta}-galactosidase induite dans une structure subcellulaire d'Escherichia coli (1961)

Energy Technology Data Exchange (ETDEWEB)

Nisman, B; Kayser, A; Demailly, J; Genin, C [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1961-07-01

Isopropyl-thio-galactoside (IPTG), an inducer of 3-galactosidase, makes it possible to synthesise this enzyme in vitro with the subcellular structure (P{sub 1}). The enzyme is isolated from the bacteria Escherichia coli K 12 which are inductive but not induced. The incorporation of radioactive amino-acids, which is stimulated by the presence of an inducer, was studied during the course of the enzyme synthesis. Saccharose suppresses the induction of {beta}-galactosidase. The presence of a specific inhibitor in the structure studied is considered. (authors) [French] L'isopropylthiogalactoside (IPTG), inducteur de la 3-galactosidase, permet la synthese in vitro de cette enzyme dans la structure subcellulaire (P{sub 1}) isolee a partir des bacteries d'Escherichia coli K 12, inductibles mais non induites. L'incorporation d'acides amines radioactifs, stimulee par la presence d'inducteur, a ete etudiee au cours de la synthese de l'enzyme. Le saccharose supprime l'induction de la 3-galactosidase. La presence du represseur specifique dans la structure etudiee est consideree. (auteurs)

Kinetics of β-galactosidase Production by Lactobacillus bulgaricus During pH Controlled Batch Fermentation in Three Commercial Bulk Starter Media

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Saeed Abbasalizadeh

2015-10-01

Full Text Available The potential of bulk starter fermentation strategy for production of a cost-effective and GRAS source of β-galactosidase from a starter culture strain Lactobacillus bulgaricus was investigated. Three different media were selected and the strain, L. bulgaricus DSM 20081 was cultivated in these media under pH-controlled condition (pH = 5.6 at 43°C. The media were: bulk starter medium based on skim milk + whey, bulk starter medium based on whey, and skim milk. Growth and β-lactic acid production parameters were estimated from experimental data with the Garcia and Luedeking-Piret models, respectively. β-galactosidase production kinetics was also simulated using models based on biomass concentration and lactic acid production. Growth in the bulk starter medium based on skim milk + whey resulted in a higher rate of lactic acid production (7.35 ± 0.23  mg lactic acid ml-1 media h-1 and β-galactosidase activity (800.1± 0.7 nmol ONP ml-1 media compared to the other two media (P<0.01. Simulation of β- galactosidase production based on rate of lactic acid production resulted in very good agreement with experimental data for all three tested media. The results revealed the potential of bulk starter fermentation strategy and skim milk + whey based medium for in-house and relatively low cost production of food-grade β-galactosidase by dairy plants.

Standardization and estimation of gross alpha and beta activities for potable water samples in presence of TDS using TDCR based LSA (Hidex 300SL)

International Nuclear Information System (INIS)

Gupta, Anil; Lenka, P.; Sahoo, S.K.; Patra, A.C.; Jha, S.K.; Tripathi, R.M.

2018-01-01

Quality of water is important in environmental studies because of its daily use for human consumption and an important route of intake for various elements and radionuclides. Therefore radiological quality of drinking water must be ensured. The screening parameter to evaluate the radiological safety of potable water is by estimating gross alpha and gross beta activities with a limit of 0.5 Bq/l and 1 Bq/l respectively. These have been traditionally being done by radiochemical co-precipitation of alpha and beta emitters followed by counting by ZnS(Ag) counter for alpha and gas flow proportional counter for beta. Gross alpha estimation in water samples with high TDS by ZnS(Ag) is difficult and often leads to underestimation of result due to self-absorption of alpha within the residue. This study was carried out to standardize a method to provide rapid results by simultaneously estimating both gross alpha and gross beta activity in the presence of TDS with adequate sensitivity, minimum sample processing

Applying neutral drift to the directed molecular evolution of a β-glucuronidase into a β-galactosidase: Two different evolutionary pathways lead to the same variant

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Hale Jennifer R

2011-05-01

Full Text Available Abstract Background Directed protein evolution has been used to modify protein activity and research has been carried out to enhance the production of high quality mutant libraries. Many theoretical approaches suggest that allowing a population to undergo neutral selection may be valuable in directed evolution experiments. Findings Here we report on an investigation into the value of neutral selection in a classical model system for directed evolution, the conversion of the E. coli β-glucuronidase to a β-galactosidase activity. We find that neutral selection, i.e. selection for retaining glucuronidase activity, can efficiently identify the majority of sites of mutation that have been identified as beneficial for galactosidase activity in previous experiments. Each variant demonstrating increased galactosidase activity identified by our neutral drift experiments contained a mutation at one of four sites, T509, S557, N566 or W529. All of these sites have previously been identified using direct selection for beta galactosidase activity. Conclusions Our results are consistent with others that show that a neutral selection approach can be effective in selecting improved variants. However, we interpret our results to show that neutral selection is, in this case, not a more efficient approach than conventional directed evolution approaches. However, the neutral approach is likely to be beneficial when the resulting library can be screened for a range of related activities. More detailed statistical studies to resolve the apparent differences between this system and others are likely to be a fruitful avenue for future research.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

Energy Technology Data Exchange (ETDEWEB)

Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai; Lee, Kyung-Dall; Amidon, Gordon L. (Michigan)

2008-07-08

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

Alfa-Galactosidases and their applications in biotransformations

Czech Academy of Sciences Publication Activity Database

Weignerová, Lenka; Simerská, Pavla; Křen, Vladimír

2009-01-01

Roč. 27, č. 2 (2009), s. 79-89 ISSN 1024-2422 R&D Projects: GA MŠk(CZ) LC06010; GA ČR GA203/05/0172 Institutional research plan: CEZ:AV0Z50200510 Keywords : -Galactosidase * enzyme * transglycosylation Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.017, year: 2009

Effect of 17alpha-ethinylestradiol on activity of rat liver enzymes for synthesis and hydrolysis of cholesterol esters

International Nuclear Information System (INIS)

Nikitin, Yu.P.; Dushkin, M.I.; Dolgov, A.V.; Gordienko, I.A.

1987-01-01

Administration of estrogens is known to lower the concentration of cholesterol esters in the blood vessel wall and may delay the development of arteriosclerosis. It is also known that under the influence of estrogens the redistribution of concentrations of free cholesterol and cholesterol esters takes place in rats between the blood and liver as a result of the intensification of receptor-dependent uptake of low-density lipoproteins by the hepatocytes. The mechanisms of this intracellular redistribution, however, have been inadequately studied. The purpose of this paper is to study the effects of 17alpha-ethinylestradiol on the activity of lysosomal and cytoplasmic cholesterol esterases, acyl-CoA-cholesterol-O-acyltransferase, lysosomal acid phosphatase, and beta-D-galactosidase. The activity was measured by using cholesterol [1-C 14]-oleate as the substrate. The influence of the estradiol is found to be based on cholesterol redistribution between the blood and liver. Accumulation of free cholesterol in the liver under these conditions stimulates bile acid formation. Depression of cholesterol ester synthesis as a result of direct inhibition of the acyltransferase by the estradiol is found to possibly contribute to the fall in the cholesterol level in the body. Liquid scintillation counting was used to measure distribution and accumulation

A Novel Process to Prepare Chitosan Macrospheres without Shrinkage and its Application to Immobilize β-Galactosidase

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Su-Fang Sun

2009-01-01

Full Text Available A new process for the preparation of chitosan macrospheres, which was simple and practicable, was suggested in this paper and various chitosans with different molecular weight were used as materials to immobilize β-galactosidase and the chitosan macrospheres with the lowest molecular weight (500 000 was selected as enzyme immobilization carrier based on the highest enzyme activity. In order to overcome the shrinkage of chitosan during drying, the wet macrospheres obtained was treated by 30% glycerol solution for 1 h before drying and the results showed that the dried chitosan macrospheres obtained could keep almost the same structure as its wet form, which was very important for chitosan as enzyme carrier in industry. Finally, β-galactosidase from Aspergillus oryzae was immobilized on above dry chitosan macrospheres and a satisfactory result of the immobilized enzyme was obtained in enzyme activity yield, pH stability, thermal stability, operational stability, Michaelis constants Km and the maximum velocity (Vm

Activation of the skeletal alpha-actin promoter during muscle regeneration.

Science.gov (United States)

Marsh, D R; Carson, J A; Stewart, L N; Booth, F W

1998-11-01

Little is known concerning promoter regulation of genes in regenerating skeletal muscles. In young rats, recovery of muscle mass and protein content is complete within 21 days. During the initial 5-10 days of regeneration, mRNA abundance for IGF-I, myogenin and MyoD have been shown to be dramatically increased. The skeletal alpha-actin promoter contains E box and serum response element (SRE) regulatory regions which are directly or indirectly activated by myogenin (or MyoD) and IGF-I proteins, respectively. We hypothesized that the skeletal alpha-actin promoter activity would increase during muscle regeneration, and that this induction would occur before muscle protein content returned to normal. Total protein content and the percentage content of skeletal alpha-actin protein was diminished at 4 and 8 days and re-accumulation had largely occurred by 16 days post-bupivacaine injection. Skeletal alpha-actin mRNA per whole muscle was decreased at day 8, and thereafter returned to control values. During regeneration at day 8, luciferase activity (a reporter of promoter activity) directed by -424 skeletal alpha-actin and -99 skeletal alpha-actin promoter constructs was increased by 700% and 250% respectively; however, at day 16, skeletal alpha-actin promoter activities were similar to control values. Thus, initial activation of the skeletal alpha-actin promoter is associated with regeneration of skeletal muscle, despite not being sustained during the later stages of regrowth. The proximal SRE of the skeletal alpha-actin promoter was not sufficient to confer a regeneration-induced promoter activation, despite increased serum response factor protein binding to this regulatory element in electrophoretic mobility shift assays. Skeletal alpha-actin promoter induction during regeneration is due to a combination of regulatory elements, at least including the SRE and E box.

A practical method for screening for beta-galactosidase secreting microbial colonies Método prático de triagem de colônias de microrganismos secretoras de beta-galactosidase

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Eriana S. Barreto

2000-03-01

Full Text Available Microbial colonies were replicated on YNB® agar plates overlaid with soft agar containing the glucose-oxidase/peroxidase (BIOTROL® system. The pink color developed around the colonies was the result of the reaction of the glucose generated by the extracellular hydrolysis of lactose by beta-galactosidase, indicating secretion of this enzyme. This method proved to be very convenient for testing hundreds of colonies grown on agar plates for beta-galactosidase secretion by microbial cells.Colônias microbianas foram replicadas na superfície de ágar YNB® contendo lactose, recoberta com uma camada de ágar semi-sólido contendo o sistema glicose-oxidase/peroxidase (BIOTROL®. Coloração rosa foi desenvolvida ao redor das colônias como resultado da reação de glicose gerada pela hidrólise extracelular de lactose pela beta-galactosidase, indicando secreção da enzima pelo microrganismo.

Spectral, molecular, in vivo cytotoxicity and immobilization of β-galactosidase on poly(o-toluidine)-titanium dioxide nanocomposite

Science.gov (United States)

Shakir, M.; Khan, Mohd Shoeb; Alam, Md Fazle; Younus, H.; Alam, Mahboob; Lee, Dong-Ung

2017-06-01

The nanocomposites of poly(o-toluidine)-titanium dioxide (POT/TiO2) have been synthesized by in-situ oxidative polymerization of o-toluidine incorporating TiO2 nanoparticles. Subsequently, β-Galactosidase (BGAL) has been immobilized on POT/TiO2. The comparative spectral studies like FTIR, SEM, TEM and XRD and TGA revealed the synergistic interaction between POT and TiO2 in POT/TiO2 nanocomposite. The molecular docking simulation predicts the modes of interactions of POT and POT/TiO2 with BGAL. BGAL is successfully immobilized on POT/TiO2 with loading efficiency of 84.51%. The immobilized POT/TiO2 has enhanced its stability, recycling efficiency and residual activity making it an ideal candidate for industrial applications. Furthermore, density functional theory (6-311G (d,p) basic set was used to investigate the structures, theoretical vibrational frequencies, the HOMO-LUMO and other properties like ionization potential and electron affinity of the monomer to pentamer of o-toluidine. The in vivo cytotoxicity studies of POT and POT/TiO2 have also been examined by brine shrimp.

Intraperitoneal alpha-radioimmunotherapy in mice using different specific activities

DEFF Research Database (Denmark)

Elgqvist, Jörgen; Andersson, Håkan; Haglund, Elin

2009-01-01

The aim of this study was to investigate the therapeutic efficacy of the alpha-radioimmunotherapy of ovarian cancer in mice, using different specific activities. This study was performed by using the monoclonal antibody, MX35 F(ab')(2), labeled with the alpha-particle-emitter, 211At.......The aim of this study was to investigate the therapeutic efficacy of the alpha-radioimmunotherapy of ovarian cancer in mice, using different specific activities. This study was performed by using the monoclonal antibody, MX35 F(ab')(2), labeled with the alpha-particle-emitter, 211At....

Biochemical characterization of a novel β-galactosidase from Paenibacillus barengoltzii suitable for lactose hydrolysis and galactooligosaccharides synthesis.

Science.gov (United States)

Liu, Yu; Chen, Zhou; Jiang, Zhengqiang; Yan, Qiaojuan; Yang, Shaoqing

2017-11-01

A β-galactosidase gene (PbBGal2A) was cloned from Paenibacillus barengoltzii and expressed in Escherichia coli. The in silico analysis of the deduced amino acid sequences revealed that PbBGal2A shared the highest identity of 40% with the characterized glycoside hydrolase (GH) family 2 β-galactosidase from Actinobacillus pleuropneumoniae. The recombinant β-galactosidase (PbBGal2A) was purified with a molecular mass of 124.2kDa on SDS-PAGE. The optimal pH and temperature of PbBGal2A were determined to be pH 7.5 and 45°C, respectively. PbBGal2A was stable within pH 6.0-8.0 and up to 45°C. It completely hydrolyzed the lactose in milk and whey powder solution. In addition, PbBGal2A exhibited high transglycosylation activity and a maximum yield of 47.9% (w/w) for galactooligosaccharides (GOS) production was obtained in 8h at a lactose concentration of 350g/L. These properties make PbBGal2A an ideal candidate for commercial use in the production of lactose-free milk and GOS. Copyright © 2017. Published by Elsevier B.V.

Molecular characterization of the alpha subunit of complement component C8 (GcC8alpha) in the nurse shark (Ginglymostoma cirratum).

Science.gov (United States)

Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L

2009-09-01

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of alpha, beta, and gamma subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8alpha and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8gamma-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8alpha respectively. Southern blot analysis showed GcC8alpha exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8alpha orthologs and as a sister taxa to the Xenopus. 2009 Elsevier Ltd.

Relationship between sol-gel conditions and enzyme stability: a case study with β-galactosidase/silica biocatalyst for whey hydrolysis.

Science.gov (United States)

Escobar, Sindy; Bernal, Claudia; Mesa, Monica

2015-01-01

The sol-gel process has been very useful for preparing active and stable biocatalysts, with the possibility of being reused. Especially those based on silica are well known. However, the study of the enzyme behavior during this process is not well understood until now and more, if the surfactant is involved in the synthesis mixture. This work is devoted to the encapsulation of β-galactosidase from Bacillus circulans in silica by sol-gel process, assisted by non-ionic Triton X-100 surfactant. The correlation between enzyme activity results for the β-galactosidase in three different environments (soluble in buffered aqueous reference solution, in the silica sol, and entrapment on the silica matrix) explains the enzyme behavior under stress conditions offered by the silica sol composition and gelation conditions. A stable β-galactosidase/silica biocatalyst is obtained using sodium silicate, which is a cheap source of silica, in the presence of non-ionic Triton X-100, which avoids the enzyme deactivation, even at 40 °C. The obtained biocatalyst is used in the whey hydrolysis for obtaining high value products from this waste. The preservation of the enzyme stability, which is one of the most important challenges on the enzyme immobilization through the silica sol-gel, is achieved in this study.

Lactose hydrolysis in aqueous two-phase system by whole-cell {beta}-galactosidase of Kluyveromyces marxianus. Semicontinuous and continuous processes

Energy Technology Data Exchange (ETDEWEB)

Tomaska, M [Slovak Technical Univ., Bratislava (Slovakia). Dept. of Biochemical Technology; Stredansky, M [Slovak Technical Univ., Bratislava (Slovakia). Dept. of Biochemical Technology; Tomaskova, A [Slovak Technical Univ., Bratislava (Slovakia). Dept. of Biochemical Technology; Sturdik, E [Slovak Technical Univ., Bratislava (Slovakia). Dept. of Biochemical Technology

1995-01-01

Semicontinuous and continuous hydrolysis of lactose in aqueous two-phase systems (polyethylene glycol 20000/ dextran 40) with whole-cell {beta}-galactosidase of K. marxianus were studied. Both phase polymers had no effect on {beta}-galactosidase activity confined in cells. Good operational stability of the biocatalyst during 55 cycles of semicontinuous process was observed without appreciable decrease in product concentration. Continuous hydrolysis of lactose was performed in the stirred bioreactor, connected with the phase separator. The satisfactory degree of hydrolysis (between 82-88%) and volumetric productivity (21.6 g/l/h) were reached during 72 hours of continuous hydrolysis of 5% (w/w) lactose. (orig.)

A novel α-galactosidase from the thermophilic probiotic Bacillus coagulans with remarkable protease-resistance and high hydrolytic activity

Science.gov (United States)

Zhao, Ruili; Zhao, Rui; Tu, Yishuai; Zhang, Xiaoming; Deng, Liping

2018-01-01

A novel α-galactosidase of glycoside hydrolase family 36 was cloned from Bacillus coagulans, overexpressed in Escherichia coli, and characterized. The purified enzyme Aga-BC7050 was 85 kDa according to SDS-PAGE and 168 kDa according to gel filtration, indicating that its native structure is a dimer. With p-nitrophenyl-α-d- galactopyranoside (pNPGal) as the substrate, optimal temperature and pH were 55 °C and 6.0, respectively. At 60 °C for 30 min, it retained > 50% of its activity. It was stable at pH 5.0–10.0, and showed remarkable resistance to proteinase K, subtilisin A, α-chymotrypsin, and trypsin. Its activity was not inhibited by glucose, sucrose, xylose, or fructose, but was slightly inhibited at galactose concentrations up to 100 mM. Aga-BC7050 was highly active toward pNPGal, melibiose, raffinose, and stachyose. It completely hydrolyzed melibiose, raffinose, and stachyose in < 30 min. These characteristics suggest that Aga-BC7050 could be used in feed and food industries and sugar processing. PMID:29738566

A novel α-galactosidase from the thermophilic probiotic Bacillus coagulans with remarkable protease-resistance and high hydrolytic activity.

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Ruili Zhao

Full Text Available A novel α-galactosidase of glycoside hydrolase family 36 was cloned from Bacillus coagulans, overexpressed in Escherichia coli, and characterized. The purified enzyme Aga-BC7050 was 85 kDa according to SDS-PAGE and 168 kDa according to gel filtration, indicating that its native structure is a dimer. With p-nitrophenyl-α-d- galactopyranoside (pNPGal as the substrate, optimal temperature and pH were 55 °C and 6.0, respectively. At 60 °C for 30 min, it retained > 50% of its activity. It was stable at pH 5.0-10.0, and showed remarkable resistance to proteinase K, subtilisin A, α-chymotrypsin, and trypsin. Its activity was not inhibited by glucose, sucrose, xylose, or fructose, but was slightly inhibited at galactose concentrations up to 100 mM. Aga-BC7050 was highly active toward pNPGal, melibiose, raffinose, and stachyose. It completely hydrolyzed melibiose, raffinose, and stachyose in < 30 min. These characteristics suggest that Aga-BC7050 could be used in feed and food industries and sugar processing.

Estudo da produção de beta-galactosidase por fermentação de soro de queijo com Kluyveromyces marxianus Synthesis of beta-galactosidase by fermentation of cheese whey by Kluyveromyces marxianus

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Patrícia A. Santiago

2004-12-01

Full Text Available A hidrólise enzimática da lactose por beta-galactosidase desempenha importante papel no processamento de produtos lácteos, como na obtenção de leite com baixo teor de lactose para consumo por indivíduos intolerantes à mesma e na prevenção da cristalização em produtos de laticínio. Neste trabalho, a enzima beta-galactosidase foi produzida pelo cultivo do microrganismo Kluyveromyces marxianus, em meio de cultura à base de soro de queijo em diferentes concentrações iniciais de lactose e extrato de levedura, de acordo com um planejamento fatorial. As fermentações foram conduzidas em incubador rotativo a 150rpm, a 30°C e pH inicial 5,5. A concentração celular inicial foi de 10(7 células/mL. Para a extração da enzima beta-galactosidase, foi realizada autólise das células utilizando como solvente o clorofórmio em tampão fosfato. No meio de cultura enriquecido com (NH42SO4, KH2PO4 e MgSO4, nas concentrações iniciais de lactose e de extrato de levedura iguais a 50g/L e 12g/L, respectivamente, obteve-se uma atividade de 28,0UGl/mL e uma concentração celular máxima de 5,3g/L.The enzymatic hydrolysis of lactose by beta-galactosidase plays an important role in the processing of milky products such as the production of lactose-hydrolyzed milk for consumption by intolerant person to lactose and the prevention of the crystallization in dairy products. In this work, the influences of nutrient concentrations in the culture medium based on cheese whey were studied with the objective of producing beta-galactosidase from Kluyveromyces marxianus. The fermentations were carried out in a shaker at 30°C and initial pH 5.5 under agitation, starting with an initial cellular concentration of 10(7 cells/mL, varying the initial concentrations of lactose and yeast extract. For extraction of the enzyme of the cells it was used autolysis with chloroform in potassium phosphate buffer. In the medium with a initial lactose concentration of 50g

A cluster of aspartic residues in the extracellular loop II of PAR 4 is important for thrombin interaction and activation of platelets.

Science.gov (United States)

Sánchez Centellas, Daniel; Gudlur, Sushanth; Vicente-Carrillo, Alejandro; Ramström, Sofia; Lindahl, Tomas L

2017-06-01

Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strain MMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased β-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to γ-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development. Copyright © 2017 Elsevier Ltd. All rights reserved.

TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

Energy Technology Data Exchange (ETDEWEB)

Magini, Alessandro [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Polchi, Alice; Urbanelli, Lorena [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Cesselli, Daniela; Beltrami, Antonio [Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Tancini, Brunella [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Emiliani, Carla, E-mail: carla.emiliani@unipg.it [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy)

2013-10-18

Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.

TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

International Nuclear Information System (INIS)

Magini, Alessandro; Polchi, Alice; Urbanelli, Lorena; Cesselli, Daniela; Beltrami, Antonio; Tancini, Brunella; Emiliani, Carla

2013-01-01

Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane

[G-protein potentiates the activation of TNF-alpha on calcium-activated potassium channel in ECV304].

Science.gov (United States)

Lin, L; Zheng, Y; Qu, J; Bao, G

2000-06-01

Observe the effect of tumor necrosis factor-alpha (TNF-alpha) on calcium-activated potassium channel in ECV304 and the possible involvement of G-protein mediation in the action of TNF-alpha. Using the cell-attached configuration of patch clamp technique. (1) the activity of high-conductance calcium-activated potassium channel (BKca) was recorded. Its conductance is (202.54 +/- 16.62) pS; (2) the activity of BKca was potentiated by 200 U/ml TNF-alpha; (3) G-protein would intensify this TNF-alpha activation. TNF-alpha acted on vascular endothelial cell ECV304 could rapidly activate the activity of BKca. Opening of BKca resulted in membrane hyper-polarization which could increase electro-chemical gradient for the resting Ca2+ influx and open leakage calcium channel, thus resting cytoplasmic free Ca2+ concentration could be elevated. G-protein may exert an important regulation in this process.

Activity monitoring of alpha-bearing wastes

International Nuclear Information System (INIS)

Birkhoff, G.; Bondar, L.

1980-01-01

The paper aims at the survey on the actual situation in activity monitoring of alpha-bearing wastes. Homogeneous materials such as liquid-, gaseous- and homogeneous solid wastes are amenable to destructive analyses of representative samples. Available destructive analyses methods are sensitive and precise enough to cope with all requirements in alpha-waste monitoring. The more difficult problems are encountered with alpha-contaminated solids, when representative sampling is not practicable. Non-destructive analysis techniques are applied for monitoring this category of solid wastes. The techniques for nondestructive analysis of alpha-bearing wastes are based on the detection of gamma and/or neutron-emission of actinides. Principles and a theory of non-destructive radiometric assay of plutonium contaminated solid waste streams are explained. Guidelines for the calibration of instruments and interpretation of experimental data are given. Current theoretical and experimental development work in this problem area is reviewed. Evaluations concerning capabilities and limitations of monitoring systems for alpha-bearing solid wastes are very complex and out of the scope of this paper

On alpha activity relationships in 226Ra and U/sub nat/

International Nuclear Information System (INIS)

Krejbichova, Z.

1982-01-01

A gross alpha activity of 0.1 Bq/l is permitted for drinking and surface waters. If gross activity is higher, it is suitable to determine the 226 Ra content which must not exceed 0.1 Bq/l. The permissible content of natural uranium is 0.05 mg/l. In most samples of ground and surface waters gross alpha activity was below the permissible limit. In some samples with a high content of salts a higher Ra+U activity was found which exceeded gross activity. This was caused by the interference of nonradioactive admixtures. In other samples a higher gross alpha activity was found than was the sum Ra+U which may have been caused by alpha sources from radon decay. It was found that the gross alpha activity of waters is rather caused by uranium than by radium. (E.S.)

Kinetic Properties of α-Galactosidase and the Localization of Total Proteins in Erwinia chrysanthemi

Directory of Open Access Journals (Sweden)

John Morgan Brand

2004-01-01

Full Text Available Erwinia chrysanthemi is an enterobacterium that causes soft-rot in plants in general, resulting in enormous economic losses annually. For the pathogen to survive in the host plant, it has to use the readily assimilable compounds from the host fluids and degrade the host tissue. To accomplish this, E. chrysanthemi produces several extracellular and intracellular enzymes. Among the intracellular enzymes there is a special digestive class, the galactosidases, which can be either periplasmic or cytoplasmic. α-Galactosidase is known to degrade melibiose and raffinose into glucose and galactose, and into galactose and sucrose respectively. The aim of the present study was to investigate the kinetic properties of α-galactosidase in E. chrysanthemi, and the localization of total proteins, after culturing it in the presence of raffinose and melibiose. The α-galactosidase that degrades melibiose seems to be the same enzyme that is also responsible for the breakdown of raffinose in E. chrysanthemi. It is localized mainly in the cytoplasm with a fraction of between 2.4 and 5.4 % localized in the periplasm. The majority of E. chrysanthemi proteins have cytoplasmic localization.

The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

Science.gov (United States)

Wieczorek, Anna; McHenry, Charles S

2006-05-05

The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

Low resolution solution structure of HAMLET and the importance of its alpha-domains in tumoricidal activity.

Science.gov (United States)

Ho, C S James; Rydstrom, Anna; Manimekalai, Malathy Sony Subramanian; Svanborg, Catharina; Grüber, Gerhard

2012-01-01

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.

EEG alpha rhythm, ocular activity and basal skin resistance

NARCIS (Netherlands)

Verbaten, M.N.; Beaujon, J.N.R.; Sjouw, W.

Most hypotheses about the origin of the occipital alpha rhythm stress the specific influence of ocular activity. In this study, the influence of eye-movement frequency and extreme upward deviation of the eyeballs (enlarging the corneo-retinal potential) on occipital alpha activity and basal skin

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

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Urs M Nater

Full Text Available 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.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 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.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.

Lactococcus lactis expressing food-grade β-galactosidase alleviates lactose intolerance symptoms in post-weaning Balb/c mice.

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Li, Jingjie; Zhang, Wen; Wang, Chuan; Yu, Qian; Dai, Ruirui; Pei, Xiaofang

2012-12-01

The endogenous β-galactosidase expressed in intestinal microbes is demonstrated to help humans in lactose usage, and treatment associated with the promotion of beneficial microorganism in the gut is correlated with lactose tolerance. From this point, a kind of recombinant live β-galactosidase delivery system using food-grade protein expression techniques and selected probiotics as vehicle was promoted by us for the purpose of application in lactose intolerance subjects. Previously, a recombinant Lactococcus lactis MG1363 strain expressing food-grade β-galactosidase, the L. lactis MG1363/FGZW, was successfully constructed and evaluated in vitro. This study was conducted to in vivo evaluate its efficacy on alleviating lactose intolerance symptoms in post-weaning Balb/c mice, which were orally administered with 1 × 10⁶ CFU or 1 × 10⁸ CFU of L. lactis MG1363/FGZW daily for 4 weeks before lactose challenge. In comparison with naïve mice, the mice administered with L. lactis MG1363/FGZW showed significant alleviation of diarrhea symptoms in less total feces weight within 6 h post-challenge and suppressed intestinal motility after lactose challenge, although there was no significant increase of β-galactosidase activity in small intestine. The alleviation also correlated with higher species abundance, more Bifidobacterium colonization, and stronger colonization resistance in mice intestinal microflora. Therefore, this recombinant L. lactis strain effectively alleviated diarrhea symptom induced by lactose uptake in lactose intolerance model mice with the probable mechanism of promotion of lactic acid bacteria to differentiate and predominantly colonize in gut microbial community, thus making it a promising probiotic for lactose intolerance subjects.

Miniature Neutron-Alpha Activation Spectrometer

Science.gov (United States)

Rhodes, E.; Goldsten, J.

2001-01-01

We are developing a miniature neutron-alpha activation spectrometer for in situ analysis of samples including rocks, fines, ices, and drill cores, suitable for a lander or Rover platform, that would meet the severe mass, power, and environmental constraints of missions to the outer planets. In the neutron-activation mode, a gamma-ray spectrometer will first perform a penetrating scan of soil, ice, and loose material underfoot (depths to 10 cm or more) to identify appropriate samples. Chosen samples will be analyzed in bulk in neutron-activation mode, and then the sample surfaces will be analyzed in alpha-activation mode using Rutherford backscatter and x-ray spectrometers. The instrument will provide sample composition over a wide range of elements, including rock-forming elements (such as Na, Mg, Si, Fe, and Ca), rare earths (Sm and Eu for example), radioactive elements (K, Th, and U), and light elements present in water, ices, and biological materials (mainly H, C, O, and N). The instrument is expected to have a mass of about l kg and to require less than 1 W power. Additional information is contained in the original extended abstract.

Alpha-mannosidase activity in goats fed with Sida carpinifolia.

Science.gov (United States)

Bedin, Marisete; Moleta Colodel, Edson; Viapiana, Marli; Matte, Ursula; Driemeier, David; Giugliani, Roberto

2010-03-01

Human alpha-mannosidosis results from alpha-mannosidase deficiency and progressive accumulation of mannose-rich oligosaccharides in lysosomes. Two days before Saanen goats were fed with Sida carpinifolia, alpha-mannosidase activity in leukocytes was 128+/-28 nmoles4-MU/h/mgprotein (first trial) and 104+/-6 nmoles4-MU/h/mgprotein (second trial). At day 5, after the introduction of S. carpinifolia diet, the alpha-mannosidase activity in leukocytes was significantly increased, both in the first (288+/-13 nmoles4-MU/h/mgprotein) and in the second trial (303+/-45 nmoles4-MU/h/mgprotein), and it returned to normal levels 2 days after the withdrawal of the plant from the diet (114+/-7 nmoles4-MU/h/mgprotein in first trial, and 108+/-25 nmoles4-MU/h/mgprotein in the second one). Plasma alpha-mannosidase activity decreased significantly 4 days after animal exposure to the S. carpinifolia diet (769+/-167 nmoles4-MU/h/ml) and returned to normal values 10 days after the withdrawal of the plant from the diet (1289+/-163 nmoles4-MU/h/ml). Thin-layer chromatography showed an abnormal excretion of oligosaccharides in urine as of day 2 after diet exposure, which persisted until one day after the withdrawal of the plant. Animals presented neurological clinical signs beginning at day 37 (in the first trial) and at day 25 (in the second trial) after being fed with the plant. The results obtained herein suggest that oligosaccharides observed in urine are a result of a decrease in alpha-mannosidase activity in plasma. S. carpinifolia seems to have other compounds that act on alpha-mannosidase enzyme in leukocytes in a competitive manner with swainsonine. The increase in alpha-mannosidase enzyme in leukocytes could be attributed to one of these compounds present in S. carpinifolia. Copyright 2009 Elsevier GmbH. All rights reserved.

Release of β-galactosidase from poloxamine/α-cyclodextrin hydrogels

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César A. Estévez

2014-12-01

Full Text Available All mammals lose their ability to produce lactase (β-galactosidase, the enzyme that cleaves lactose into galactose and glucose, after weaning. The prevalence of lactase deficiency (LD spans from 2 to 15% among northern Europeans, to nearly 100% among Asians. Following lactose consumption, people with LD often experience gastrointestinal symptoms such as abdominal pain, bowel distension, cramps and flatulence, or even systemic problems such as headache, loss of concentration and muscle pain. These symptoms vary depending on the amount of lactose ingested, type of food and degree of intolerance. Although those affected can avoid the uptake of dairy products, in doing so, they lose a readily available source of calcium and protein. In this work, gels obtained by complexation of Tetronic 90R4 with α-cyclodextrin loaded with β-galactosidase are proposed as a way to administer the enzyme immediately before or with the lactose-containing meal. Both molecules are biocompatible, can form gels in situ, and show sustained erosion kinetics in aqueous media. The complex was characterized by FTIR that evidenced an inclusion complex between the polyethylene oxide block and α-cyclodextrin. The release profiles of β-galactosidase from two different matrices (gels and tablets of the in situ hydrogels have been obtained. The influence of the percentage of Tetronic in media of different pH was evaluated. No differences were observed regarding the release rate from the gel matrices at pH 6 (t50 = 105 min. However, in the case of the tablets, the kinetics were faster and they released a greater amount of 90R4 (25%, t50 = 40–50 min. Also, the amount of enzyme released was higher for mixtures with 25% Tetronic. Using suitable mathematical models, the corresponding kinetic parameters have been calculated. In all cases, the release data fit quite well to the Peppas–Sahlin model equation, indicating that the release of β-galactosidase is governed by a

Hydrolysis of whey lactose by immobilized β-galactosidase in a bioreactor with a spirally wound membrane.

Science.gov (United States)

Vasileva, Nastya; Ivanov, Yavor; Damyanova, Stanka; Kostova, Iliana; Godjevargova, Tzonka

2016-01-01

The β-galactosidase was covalently immobilized onto a modified polypropylene membrane, using glutaraldehyde. The optimal conditions for hydrolysis of lactose (4.7%) by immobilized β-galactosidase in a batch process were determined 13.6 U enzyme activity, 40°C, pH 6.8 and 10h. The obtained degree of hydrolysis was compared with results received by a free enzyme. It was found, that the lactose hydrolysis by an immobilized enzyme was 1.6 times more effective than the lactose hydrolysis by a free enzyme. It was determined that the stability of the immobilized enzyme was 2 times higher in comparison with the stability of free enzyme. The obtained immobilized system β-galactosidase/polypropylene membrane was applied to produce glucose-galactose syrup from waste whey. The whey characteristics and the different preliminary treatments of the whey were investigated. Then the whey lactose hydrolysis in a bioreactor by an immobilized enzyme on a spirally wound membrane was performed. The optimal membrane surface and the optimal flow rate of the whey through the membrane module were determined, respectively 100 cm(2) and 1.0 mL min(-1). After 10h, the degree of lactose hydrolysis was increased to 91%. The operation stability was studied. After 20th cycle the yield of bioreactor was 69.7%. Copyright © 2015 Elsevier B.V. All rights reserved.

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

DEFF Research Database (Denmark)

Bønsager, Birgit Christine; Nielsen, Per K.; Abou Hachem, Maher

2005-01-01

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...

[Acetylcholine activation of alpha-ketoglutarate oxidation in liver mitochondria].

Science.gov (United States)

Shostakovskaia, I V; Doliba, N M; Gordiĭ, S K; Babskiĭ, A M; Kondrashova, M N

1986-01-01

Activation of alpha-ketoglutarate oxidation in the rat liver mitochondria takes place 15 and 30 min after intraperitoneal injection of acetyl choline. This mediator in doses of 25, 50 and 100 micrograms per 100 g of body weight causes a pronounced stimulation of phosphorylation respiration rate and calcium capacity of mitochondria with alpha-ketoglutarate oxidation. Acetyl choline is found to have a moderate inhibitory action on oxidation of lower (physiological) concentrations of succinate. Its stimulating action on alpha-ketoglutarate oxidation is associated with activation of M-cholinoreceptors; atropine, a choline-blocker, removes completely this effect. It is supposed that alpha-ketoglutarate and succinate are included into the composition of two reciprocal hormonal-substrate nucleotide systems.

Expression and functional importance of collagen-binding integrins, alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

DEFF Research Database (Denmark)

Andreasen, Susanne Ø; Thomsen, Allan R; Koteliansky, Victor E

2003-01-01

decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect......Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using...... this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1...

Expression of a functional cold active β-galactosidase from Planococcus sp-L4 in Pichia pastoris.

Science.gov (United States)

Mahdian, Seyed Mohammad Amin; Karimi, Ehsan; Tanipour, Mohammad Hossein; Parizadeh, Seyed Mohammad Reza; Ghayour-Mobarhan, Majid; Bazaz, Mojtaba Mousavi; Mashkani, Baratali

2016-09-01

Lactase deficiency problem discourages many adults from consuming milk as a major source of micro- and macronutrients. Enzymatic hydrolysis of lactose is an ideal solution for this problem but such processing adds significant costs. In this study, a cold active β-galactosidase from Planococcus sp-L4 (bgal) was optimized for expression of recombinant "BGalP" in Pichia pastoris. As a result of codon optimization, the codon adaptation index was improved from 0.58 to 0.85 after replacing rare codons. After transformation of two P. pastoris strains (KM71H and GS115), the activity of BGalP enzyme was measured in the culture supernatants using ortho-Nitrophenyl-β-galactoside (ONPG). Maximal activity was recorded as 3.7U/ml on day 11 in KM71H clone #2 which was 20% higher than the best GS115 clone. Activity measurements under different conditions indicated optimal activity at pH 6.5. It was active at temperatures ranging from 0 to 55°C with deactivation occurring at or above 60°C. Protein analysis of the crude ultra-filtrate showed the enzyme was ∼75kDa and was the major constituent (85%) of the sample. This enzyme have the potential to find utility for the breakdown of lactose in chilled milk and subsequently can be deactivated by pasteurization. The use of BGalP would minimize energy consumption thus decreasing cost and also help to preserve the nutritional elements of the milk. Copyright © 2015 Elsevier Inc. All rights reserved.

Does alpha 1-acid glycoprotein act as a non-functional receptor for alpha 1-adrenergic antagonists?

Science.gov (United States)

Qin, M; Oie, S

1994-11-01

The ability of a variety of alpha 1-acid glycoproteins (AAG) to affect the intrinsic activity of the alpha 1-adrenergic antagonist prazosin was studied in rabbit aortic strip preparations. From these studies, the activity of AAG appears to be linked to their ability to bind the antagonist. However, a capability to bind prazosin was not the only requirement for this effect. The removal of sialic acid and partial removal of the galactose and mannose residues by periodate oxidation of human AAG all but eliminated the ability of AAG to affect the intrinsic pharmacologic activity of prazosin, although the binding of prazosin was not significantly affected. The presence of bovine AAG, a protein that has a low ability to bind prazosin, reduced the effect of human AAG on prazosin activity. Based upon these results, we propose that AAG is able to bind in the vicinity of the alpha 1-adrenoceptors, therefore extending the binding region for antagonists in such a way as to decrease the ability of the antagonist to interact with the receptor. The carbohydrate side-chains are important for the binding of AAG in the region of the adrenoceptor.

Cooperation between two ClpB isoforms enhances the recovery of the recombinant {beta}-galactosidase from inclusion bodies

Energy Technology Data Exchange (ETDEWEB)

Guenther, Izabela [Department of Biochemistry, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk (Poland); Zolkiewski, Michal [Department of Biochemistry, Kansas State University, Manhattan, KS 66506 (United States); Kedzierska-Mieszkowska, Sabina, E-mail: kedzie@biotech.ug.gda.pl [Department of Biochemistry, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk (Poland)

2012-10-05

Highlights: Black-Right-Pointing-Pointer An important role of synergistic cooperation between the two ClpB isoforms. Black-Right-Pointing-Pointer Both ClpB isoforms are associated with IBs of {beta}-galactosidase. Black-Right-Pointing-Pointer ClpB is a key chaperone in IB protein release. -- Abstract: Bacterial ClpB is a molecular chaperone that solubilizes and reactivates aggregated proteins in cooperation with the DnaK chaperone system. The mechanism of protein disaggregation mediated by ClpB is linked to translocation of substrates through the central channel within the ring-hexameric structure of ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the truncated ClpB80 (ClpB{Delta}N), which does not contain the N-terminal domain. The functional specificity of the two ClpB isoforms and the biological role of the N-terminal domain are still not fully understood. Recently, it has been demonstrated that ClpB may achieve its full potential as an aggregate-reactivating chaperone through the functional interaction and synergistic cooperation of its two isoforms. It has been found that the most efficient resolubilization and reactivation of stress-aggregated proteins occurred in the presence of both ClpB95 and ClpB80. In this work, we asked if the two ClpB isoforms functionally cooperate in the solubilization and reactivation of proteins from insoluble inclusion bodies (IBs) in Escherichia coli cells. Using the model {beta}-galactosidase fusion protein (VP1LAC), we found that solubilization and reactivation of enzymes entrapped in IBs occurred more efficiently in the presence of ClpB95 with ClpB80 than with either ClpB95 or ClpB80 alone. The two isoforms of ClpB chaperone acting together enhanced the solubility and enzymatic activity of {beta}-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of {beta}-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a synergistic

Hydrolysis of lactose with -D-galactosidase

Directory of Open Access Journals (Sweden)

Vesna Stehlik-Tomas

2001-06-01

Full Text Available The conditions of lactose hydrolysis with enzyme preparation of D-galactosidase were investigated. The aim of this work was to considered the use of whey in fermentative processes with yeast Saccharomyces cerevisiae. Enzymatic hydrolysis was conducted at different temperatures, with different lactose concentrations in medium and different concentrations of added enzyme. The results show that optimal temperature for hydrolysis was 40°C. The optimal amount of enzyme preparation was 2 gL-1 in lactose medium with 5-10 % lactose.

Correction of acid beta-galactosidase deficiency in GM1 gangliosidosis human fibroblasts by retrovirus vector-mediated gene transfer: higher efficiency of release and cross-correction by the murine enzyme.

Science.gov (United States)

Sena-Esteves, M; Camp, S M; Alroy, J; Breakefield, X O; Kaye, E M

2000-03-20

Mutations in the lysosomal acid beta-galactosidase (EC 3.2.1.23) underlie two different disorders: GM1 gangliosidosis, which involves the nervous system and visceral organs to varying extents, and Morquio's syndrome type B (Morquio B disease), which is a skeletal-connective tissue disease without any CNS symptoms. This article shows that transduction of human GM1 gangliosidosis fibroblasts with retrovirus vectors encoding the human acid beta-galactosidase cDNA leads to complete correction of the enzymatic deficiency. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. Cross-correction experiments using retrovirus-modified cells as enzyme donors showed, however, that the human enzyme is transferred at low efficiencies. Experiments using a different retrovirus vector carrying the human cDNA confirmed this observation. Transduction of human GM1 fibroblasts and mouse NIH 3T3 cells with a retrovirus vector encoding the mouse beta-galactosidase cDNA resulted in high levels of enzymatic activity. Furthermore, the mouse enzyme was found to be transferred to human cells at high efficiency. Enzyme activity measurements in medium conditioned by genetically modified cells suggest that the human beta-galactosidase enzyme is less efficiently released to the extracellular space than its mouse counterpart. This study suggests that lysosomal enzymes, contrary to the generalized perception in the field of gene therapy, may differ significantly in their properties and provides insights for design of future gene therapy interventions in acid beta-galactosidase deficiency.

Generation and Characterization of Environmentally Sensitive Variants of the β-Galactosidase from Lactobacillus delbrueckii subsp. bulgaricus

Science.gov (United States)

Yoast, Sienna; Adams, Robin M.; Mainzer, Stanley E.; Moon, Keith; Palombella, Anthony L.; Schmidt, Brian F.

1994-01-01

A method is described for generating and screening variants of the β-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus sensitive to several environmental stresses, with potential application in the food industry. Chemical mutagenesis with hydroxylamine or methoxylamine was performed on the β-galactosidase gene carried on an Escherichia coli expression vector. Mutants sensitive to cold, heat, low pH, low magnesium concentration, and the presence of urea were isolated by screening for reduced color development on β-galactosidase indicator plates. The mutations responsible for three variant β-galactosidases were localized, and the base substitutions were determined by DNA sequencing. The amino acid alterations associated with one low-pH-sensitive (pHs) and two urea-sensitive (Us) variants correspond to P584L (pHs1), G400S/R479Q (Us26), and G167E/E168K/E363K/V492M (Us17), respectively. Mutant pHs1 is also heat, cold, low magnesium, and urea sensitive; Us26 is also cold sensitive; and Us17 is also low-pH sensitive. PMID:16349230

Protein backbone motions viewed by intraresidue and sequential H{sup N}-H{sup {alpha}} residual dipolar couplings

Energy Technology Data Exchange (ETDEWEB)

Voegeli, Beat; Yao Lishan; Bax, Ad [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)], E-mail: bax@nih.gov

2008-05-15

Triple resonance E.COSY-based techniques were used to measure intra-residue and sequential H{sup N}-H{sup {alpha}} residual dipolar couplings (RDCs) for the third IgG-binding domain of protein G (GB3), aligned in Pf1 medium. Measurements closely correlate with values predicted on the basis of an NMR structure, previously determined on the basis of a large number of one-bond backbone RDCs measured in five alignment media. However, in particular the sequential H{sup N}-H{sup {alpha}} RDCs are smaller than predicted for a static structure, suggesting a degree of motion for these internuclear vectors that exceeds that of the backbone amide N-H vectors. Of all experimentally determined GB3 structures available, the best correlation between experimental {sup 1}H-{sup 1}H couplings is observed for a GB3 ensemble, previously derived to generate a realistic picture of the conformational space sampled by GB3 (Clore and Schwieters, J Mol Biol 355:879-886, 2006). However, for both NMR and X-ray-derived structures the {sup 1}H-{sup 1}H couplings are found to be systematically smaller than expected on the basis of alignment tensors derived from {sup 15}N-{sup 1}H amide RDCs, assuming librationally corrected N-H bond lengths of 1.041 A.

Overexpression and characterization of a novel transgalactosylic and hydrolytic β-galactosidase from a human isolate Bifidobacterium breve B24.

Science.gov (United States)

Yi, Sung Hun; Alli, Inteaz; Park, Kwan Hwa; Lee, Byonghoon

2011-10-01

After the complete gene of a β-galactosidase from human isolate Bifidobacterium breve B24 was isolated by PCR and overexpressed in E. coli, the recombinant β-galactosidase was purified to homogeneity and characterized for the glycoside transferase (GT) and glycoside hydrolase (GH) activities on lactose. One complete ORF encoding 691 amino acids (2,076 bp) was the structural gene, LacA (galA) of the β-gal gene. The recombinant enzyme shown by activity staining and gel-filtration chromatography was composed of a homodimer of 75 kDa with a total molecular mass of 150 kDa. The K(m) value for lactose (95.58 mM) was 52.5-fold higher than the corresponding K(m) values for the synthetic substrate ONPG (1.82 mM). This enzyme with the optimum of pH 7.0 and 45°C could synthesize approximately 42.00% of GOS from 1M of lactose. About 97.00% of lactose in milk was also quickly hydrolyzed by this enzyme (50 units) at 45°C for 5h to produce 46.30% of glucose, 46.60% of galactose and 7.10% of GOS. The results suggest that this recombinant β-galactosidase derived from a human isolate B. breve B24 may be suitable for both the hydrolysis and synthesis of galacto-oligosaccharides (GOS) in milk and lactose processing. Copyright © 2011 Elsevier B.V. All rights reserved.

Monitoring of β-d-Galactosidase Activity as a Surrogate Parameter for Rapid Detection of Sewage Contamination in Urban Recreational Water

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Ingun Tryland

2016-02-01

Full Text Available Simple, automated methods are required for rapid detection of wastewater contamination in urban recreational water. The activity of the enzyme β-d-galactosidase (GAL can rapidly (<2 h be measured by field instruments, or a fully automated instrument, and was evaluated as a potential surrogate parameter for estimating the level of fecal contamination in urban waters. The GAL-activity in rivers, affected by combined sewer overflows, increased significantly during heavy rainfall, and the increase in GAL-activity correlated well with the increase in fecal indicator bacteria. The GAL activity in human feces (n = 14 was high (mean activity 7 × 107 ppb MU/hour and stable (1 LOG10 variation, while the numbers of Escherichia coli and intestinal enterococci varied by >5 LOG10. Furthermore, the GAL-activity per gram feces from birds, sheep and cattle was 2–3 LOG10 lower than the activity from human feces, indicating that high GAL-activity in water may reflect human fecal pollution more than the total fecal pollution. The rapid method can only be used to quantify high levels of human fecal pollution, corresponding to about 0.1 mg human feces/liter (or 103 E. coli/100 mL, since below this limit GAL-activity from non-fecal environmental sources may interfere.

Continuous Packed Bed Reactor with Immobilized β-Galactosidase for Production of Galactooligosaccharides (GOS

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Barbara Rodriguez-Colinas

2016-11-01

Full Text Available The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal agarose beads and assayed for the continuous production of galactooligosaccharides (GOS in a packed-bed reactor (PBR. The immobilization was fast (1 h and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-d-galactopyranoside (ONPG. The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45 °C in a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and 45 °C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased with time; however, the maximum GOS concentration (24.2 g/L was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS were the trisaccharide Gal-β(1→4-Gal-β(1→4-Glc and the tetrasaccharide Gal-β(1→4-Gal-β(1→4-Gal-β(1→4-Glc. The PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases.

A quick method for estimation of long-lived alpha activity in work atmospheres

International Nuclear Information System (INIS)

Srivastava, G.K.; Ramakrishna Rao, A.; Balbudhe, A.Y.; Sarma, P.S.

2003-01-01

In an operating plant quick reporting of the status of long-lived alpha activity concentrations in the work atmosphere is required. This will help in taking any corrective control measures if required. Radon and thoron progeny concentrations prevalent in the general atmosphere predominantly interfere in measurement of long-lived alpha activity in air. The alpha counts due to radon and thoron progeny vary widely in many atmospheric conditions. Therefore, conventionally, 5 days delay is allowed for all interfering activity to decay completely and true alpha air activity is then estimated. An approach for quick assessment of long-lived alpha activity by eliminating interference due to radon and thoron progeny in air, is made here. Based on the study of the pattern of alpha count rate due to radon and thoron progeny in air, a method for estimation of long-lived alpha activity within 8 hours delay time is suggested in this paper. (author)

HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

Directory of Open Access Journals (Sweden)

Freya M Mowat

2010-06-01

Full Text Available Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF and erythropoietin (Epo by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

Structure and function of small heat shock/alpha-crystallin proteins: established concepts and emerging ideas.

Science.gov (United States)

MacRae, T H

2000-06-01

Small heat shock/alpha-crystallin proteins are defined by conserved sequence of approximately 90 amino acid residues, termed the alpha-crystallin domain, which is bounded by variable amino- and carboxy-terminal extensions. These proteins form oligomers, most of uncertain quaternary structure, and oligomerization is prerequisite to their function as molecular chaperones. Sequence modelling and physical analyses show that the secondary structure of small heat shock/alpha-crystallin proteins is predominately beta-pleated sheet. Crystallography, site-directed spin-labelling and yeast two-hybrid selection demonstrate regions of secondary structure within the alpha-crystallin domain that interact during oligomer assembly, a process also dependent on the amino terminus. Oligomers are dynamic, exhibiting subunit exchange and organizational plasticity, perhaps leading to functional diversity. Exposure of hydrophobic residues by structural modification facilitates chaperoning where denaturing proteins in the molten globule state associate with oligomers. The flexible carboxy-terminal extension contributes to chaperone activity by enhancing the solubility of small heat shock/alpha-crystallin proteins. Site-directed mutagenesis has yielded proteins where the effect of the change on structure and function depends upon the residue modified, the organism under study and the analytical techniques used. Most revealing, substitution of a conserved arginine residue within the alpha-crystallin domain has a major impact on quaternary structure and chaperone action probably through realignment of beta-sheets. These mutations are linked to inherited diseases. Oligomer size is regulated by a stress-responsive cascade including MAPKAP kinase 2/3 and p38. Phosphorylation of small heat shock/alpha-crystallin proteins has important consequences within stressed cells, especially for microfilaments.

Gamma power is phase-locked to posterior alpha activity.

Directory of Open Access Journals (Sweden)

Daria Osipova

Full Text Available Neuronal oscillations in various frequency bands have been reported in numerous studies in both humans and animals. While it is obvious that these oscillations play an important role in cognitive processing, it remains unclear how oscillations in various frequency bands interact. In this study we have investigated phase to power locking in MEG activity of healthy human subjects at rest with their eyes closed. To examine cross-frequency coupling, we have computed coherence between the time course of the power in a given frequency band and the signal itself within every channel. The time-course of the power was calculated using a sliding tapered time window followed by a Fourier transform. Our findings show that high-frequency gamma power (30-70 Hz is phase-locked to alpha oscillations (8-13 Hz in the ongoing MEG signals. The topography of the coupling was similar to the topography of the alpha power and was strongest over occipital areas. Interestingly, gamma activity per se was not evident in the power spectra and only became detectable when studied in relation to the alpha phase. Intracranial data from an epileptic subject confirmed these findings albeit there was slowing in both the alpha and gamma band. A tentative explanation for this phenomenon is that the visual system is inhibited during most of the alpha cycle whereas a burst of gamma activity at a specific alpha phase (e.g. at troughs reflects a window of excitability.

High level production of β-galactosidase exhibiting excellent milk-lactose degradation ability from Aspergillus oryzae by codon and fermentation optimization.

Science.gov (United States)

Zhao, Qianqian; Liu, Fei; Hou, Zhongwen; Yuan, Chao; Zhu, Xiqiang

2014-03-01

A β-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL(-1) with o-nitrophenyl-β-D-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L(-1) in which the target protein (6.24 g L(-1)) occupied approximately 86 %. The recombinant β-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the β-galactosidase may have a good application potential in whey and milk industries.

The alpha activity of soils in relation to landscape development

Energy Technology Data Exchange (ETDEWEB)

Pepper, R G; Quirk, J P [Western Australia Univ., Nedlands. Dept. of Soil Science and Plant Nutrition

1976-02-01

The alpha activity of soils and the degree of the equilibrium of the thorium series has been related to the age of soils developed on a truncated laterite landscape in southwestern Australia. The uplift of the old lateritic plateau has formed a sequence of erosional and depositional surfaces which form the parent materials of the present-day soils. These surfaces because of their different relative ages have been subjected to different degrees of weathering and leaching. The alpha activity of the soils formed on these different landscape surfaces is influenced firstly by the amount of weathering that the surface has undergone, and secondly by the degree of leaching that the soil has undergone as evidenced by profile development. It has been found that the younger soils have higher alpha activities with the thorium series tending more towards equilibrium when compared with older soils, where the alpha activity is lower due to the leaching of the daughter nuclides from the profile.

Investigation of vitreous and crystalline ceramic materials for immobilization of alpha-contaminated residues

International Nuclear Information System (INIS)

Palmer, C.R.; Mellinger, G.B.; Rusin, J.M.

1981-01-01

Experimental investigations of two alternatives for immobilizing dispersible solid wastes contaminated with alpha-emitting radionuclides are reviewed. Borosilicate glasses and sintered silicate ceramics are being studied for such wastes, and results so far indicate both may offer attractive alternatives to waste generators. Waste oxide solubilities, de-vitrification behaviour and effects of residual carbon are examined for glasses incorporating incinerator ash and hydrated ferric oxide sludge. Glasses will accommodate these wastes at loadings of 30-60 wt% while maintaining good performance characteristics. A brief comparative evaluation of cold-pressed and sintered ceramics is also described. The effects on process and product properties of the choice of additives, waste loading and sintering temperature were determined. This approach also appears to promise economic waste loadings while achieving relatively durable waste forms. (author)

Proficiency test on incurred and spiked pesticide residues in cereals

DEFF Research Database (Denmark)

Poulsen, Mette Erecius; Christensen, Hanne Bjerre; Herrmann, Susan Strange

2009-01-01

A proficiency test on incurred and spiked pesticide residues in wheat was organised in 2008. The test material was grown in 2007 and treated in the field with 14 pesticides formulations containing the active substances, alpha-cypermethrin, bifentrin, carbendazim, chlormequat, chlorpyrifos...

Alpha particle losses during sawtooth activity in Tokamaks

International Nuclear Information System (INIS)

Anderson, D.; Lisak, M.

1988-01-01

The time evolution of the direct losses of fusion produced alpha particles in Tokamak plasmas characterized by sawtooth activity is investigated. The alpha particle loss rate during a sawtooth period is predicted to change invertedly with the change in bulk plasma parameters but also to contain a characteristic burst at the sawtooth crash. The spectrum of the lost alpha particles is also discussed. The predictions for the time evolution and the spectrum of the losses are in qualitative agreement with recently obtained losses of 15 MeV fusion produced protons in JET. (authors)

Identification of a Bacillus subtilis secretion mutant using a ß-galactosidase screening procedure

DEFF Research Database (Denmark)

Jacobs, Myra F.; Andersen, Jens Bo; Borchert, Torben V.

1995-01-01

High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent...... mutations in cis, which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis...

Identification of a Bacillus subtilis secretion mutant using a beta-galactosidase screening procedure

DEFF Research Database (Denmark)

Jacobs, M F; Borchert, T V; Kontinen, V P

1995-01-01

High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent...... mutations in cis, which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis...

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia.

Science.gov (United States)

Sims, I M; Craik, D J; Bacic, A

1997-08-25

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xylp, and approximately 0.03 mol acetyl per mol of glucosyl residue. Treatment with alpha- and beta-D-galactosidases showed that the majority of the side-chains were either single Galp-alpha-(1-->residues or the disaccharide Galp-beta-(1-->2)-Galp-alpha-(1-->linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1-->4)-beta-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating-->4)-D-Manp-beta-(1-->and-->4)-D-Glcp-beta-(1-->branch ed at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-beta-(1-->4)-[D-Galp-beta-(1-->2)-D-Galp-alpha-(1-->6)]-D-Man p-beta-(1-->4)-D-Glcp-beta-(1-->4)-[D-Galp-alpha-(1-->6)]-D-Manp -beta-(1-->(27%), and most of the other oligosaccharides produced in significant quantities were based on this structure.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

Science.gov (United States)

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

Gross alpha and beta activities in Tunisian mineral water

International Nuclear Information System (INIS)

Hamrouni Benbelgacem, Samar

2011-01-01

The quality of natural mineral water is a universal health problem seeing its vital importance. This problem is related to the presence of the radionuclides since this water is coming from underground, during their circulation it dissolves and conveys the radionuclides which are present in the earth's crust. This problem which leads to the contamination of the mineral water urged the World Health Organization to set standards and to recommend the respect of the median values of the activities alpha and beta within the framework of the man protection against this internal exhibition. Concerning the radiological quality of Tunisian mineral water studied in this project, we showed, by using the gross alpha and beta activities counting, that this water is specific to human consumption since their gross alpha and beta activities do not forward any risk on health.

Methods for Determination of α-Glycosidase, β-Glycosidase, and α-Galactosidase Activities in Dried Blood Spot Samples.

Science.gov (United States)

Sozmen, Eser Yıldırım; Sezer, Ebru Demirel

2017-01-01

The lysosomal storage diseases (LDSs) are a heterogeneous group of inherited genetic disorders caused by defects of lysosomal proteins. The accumulation of undigested substrates from different catabolic pathways leads to cellular dysfunction. LSDs generally presents during early childhood and have a devastating impact on the families and on public health. Over the years, approaches for treatment of some LSDs have been developed with different strategies. Increasing availability of treatments of these diseases has accelerated the development of new methods and techniques for rapid diagnosis in patients with clinical indication.The use of dried blood spot (DBS) test has been proposed as a first tier test to identify patients with Gaucher, Pompe, and Fabry diseases. DBS usage is advantageous for the purpose of screening as it is non-invasive, sensitive, has low-cost and fast turnaround time compared to measurements in leucocyte and/or fibroblast culture. This chapter focuses on the activity measurement of three lysosomal enzymes (α-glucosidase, β-glucosidase, and α galactosidase) in DBS samples by using fluorescent substrates and by the LC-MS/MS (liquid chromatography-mass spectrometry) method. All steps of the methods, from preparation of the solutions to calculation of the enzyme activity, will be explained in detail.

DMPD: What is disrupting IFN-alpha's antiviral activity? [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 15283983 What is disrupting IFN-alpha's antiviral activity? Mbow ML, Sarisky RT. Tr...ends Biotechnol. 2004 Aug;22(8):395-9. (.png) (.svg) (.html) (.csml) Show What is disrupting IFN-alpha's ant...iviral activity? PubmedID 15283983 Title What is disrupting IFN-alpha's antiviral activity? Authors Mbow ML,

β-Galactosidase treatment is a common first-stage modification of the three major subtypes of Gc protein to GcMAF.

Science.gov (United States)

Uto, Yoshihiro; Yamamoto, Syota; Mukai, Hirotaka; Ishiyama, Noriko; Takeuchi, Ryota; Nakagawa, Yoshinori; Hirota, Keiji; Terada, Hiroshi; Onizuka, Shinya; Hori, Hitoshi

2012-06-01

The 1f1f subtype of the group-specific component (Gc) protein is converted into Gc protein-derived macrophage-activating factor (GcMAF) by enzymatic processing with β-galactosidase and sialidase. We previously demonstrated that preGc(1f1f)MAF, a full Gc(1f1f) protein otherwise lacking a galactosyl moiety, can be converted to GcMAF by treatment with mouse peritoneal fluid. Here, we investigated the effects of the β-galactosidase-treated 1s1s and 22 subtypes of Gc protein (preGc(1s1s)MAF and preGc₂₂MAF) on the phagocytic activation of mouse peritoneal macrophages. We demonstrated the presence of Gal-GalNAc disaccharide sugar structures in the Gc(1s1s) protein by western blotting using peanut agglutinin and Helix pomatia agglutinin lectin. We also found that preGc(1s1s)MAF and preGc₂₂MAF significantly enhanced the phagocytic activity of mouse peritoneal macrophages in the presence and absence of mouse peritoneal fluid. We demonstrate that preGc(1s1s)MAF and preGc₂₂MAF proteins can be used as effective macrophage activators.

Assessment of alpha activity of building materials commonly used in West Bengal, India

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Dipak [School of Studies in Environmental Radiation and Archaeological Sciences, Jadavpur University, Kolkata 700 032 (India); Nuclear and Particle Physics Research Centre, Department of Physics, Jadavpur University, Kolkata 700 032 (India)], E-mail: dipakghosh_in@yahoo.com; Deb, Argha; Bera, Sukumar; Sengupta, Rosalima; Patra, Kanchan Kumar [School of Studies in Environmental Radiation and Archaeological Sciences, Jadavpur University, Kolkata 700 032 (India); Nuclear and Particle Physics Research Centre, Department of Physics, Jadavpur University, Kolkata 700 032 (India)

2008-02-15

This paper, reports for the first time, an extensive study of alpha activity of all widely used building materials (plaster of Paris, stone chips, marble, white cement, mosaic stone, limestone, sand, granite, cement brick, asbestos, red brick, cement tile, ceramic tile and ceramics) in West Bengal, India. The alpha activities have been measured using Solid State Nuclear Track Detector (SSNTD), a very sensitive detector for alpha particles. The samples were collected from local markets of Kolkata. The measured average alpha activities ranged from 22.7 {+-} 2.5 to 590.6 {+-} 16.8 Bq kg{sup -1}. The alpha activity of ceramic tiles was highest and provides additional data to estimate the effect of environmental radiation exposure on human health.

Some problems of residual activity measurements

International Nuclear Information System (INIS)

Katrik, P.; Mustafin, E.; Strasik, I.; Pavlovic, M.

2013-01-01

As a preparatory work for constructing the Facility for Antiproton and Ion Research (FAIR) at GSI Darmstadt, samples of copper were irradiated by 500 MeV/u 238 U ion beam and investigated by gamma-ray spectroscopy. The nuclides that contribute dominantly to the residual activity have been identified and their contributions have been quantified by two different methods: from the whole-target gamma spectra and by integration of depth-profiles of residual activity of individual nuclides. Results obtained by these two methods are compared and discussed in this paper. (authors)

EEG alpha activity reflects motor preparation rather than the mode of action selection

Directory of Open Access Journals (Sweden)

Marie-Pierre eDeiber

2012-08-01

Full Text Available Alpha-band activity (8-13 Hz is suppressed by sensory stimulation and movements, modulated by attention, working memory and mental tasks and may be sensitive to higher motor control functions. The aim of the present study was to examine alpha oscillatory activity during the preparation of simple left or right finger movements, contrasting the external and internal mode of action selection. Three preparation conditions were examined using a precueing paradigm with S1 as the preparatory and S2 as the imperative cue: Full, laterality instructed by S1; Free, laterality freely selected and None, laterality instructed by S2. Time-frequency analysis was performed in the alpha frequency range during the S1-S2 interval, and alpha motor-related amplitude asymmetries (MRAA were also calculated. The significant MRAA during the Full and Free conditions indicated effective external and internal motor response preparation. In the absence of specific motor preparation (None, a posterior alpha power decrease (event-related desynchronization, ERD dominated, reflecting the main engagement of attentional resources. In Full and Free motor preparation, posterior alpha ERD was accompanied by a midparietal alpha power increase (event-related synchronization, ERS, suggesting a concomitant inhibition of task-irrelevant visual activity. In both Full and Free motor preparation, analysis of alpha power according to MRAA amplitude revealed two types of functional activation patterns: 1 a motor alpha pattern, with predominantly midparietal alpha ERS and large MRAA corresponding to lateralized motor activation/visual inhibition and 2 an attentional alpha pattern, with dominating right posterior alpha ERD and small MRAA reflecting visuospatial attention. The present results suggest that alpha oscillatory patterns do not resolve the selection mode of action, but rather distinguish separate functional strategies of motor preparation. 

Assessment of the oestrogenic activity of the contraceptive progestin levonorgestrel and its non-phenolic metabolites.

Science.gov (United States)

Santillán, R; Pérez-Palacios, G; Reyes, M; Damián-Matsumura, P; García, G A; Grillasca, I; Lemus, A E

2001-09-14

Levonorgestrel (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4-gonen-3-one), a potent contraceptive progestin stimulates growth and proliferation of cultured breast cancer cells through a receptor-mediated mechanism, even though levonorgestrel does not bind to the oestrogen receptor (ER). To assess whether the oestrogen-like effects induced by this synthetic progestin are exerted via its metabolic conversion products, we studied the binding affinity of three A-ring levonorgestrel derivatives to the ER and their capability to transactivate an oestrogen-dependent yeast system co-transfected with the human ER gene and oestrogen responsive elements fused to a beta-galactosidase reporter vector. The results demonstrated that the 3beta,5alpha reduced levonorgestrel derivative and to a lesser extent its 3alpha isomer interact with the oestrogen receptor, with a significantly lower relative binding affinity (2.4% and 0.4%, respectively) than that of oestradiol (100%), while levonorgestrel does not. Both levonorgestrel metabolites were able to activate, in a dose-dependent manner, the beta-galactosidase reporter gene in the yeast expression system, an effect that was precluded by a steroidal antioestrogen. The oestrogenic potency of levonorgestrel metabolites was significantly lower (750-fold) than that of oestradiol. Furthermore, high doses of 3beta,5alpha levonorgestrel (2.5 mg/day/6 days) induced an increase of oestrogen-dependent progestin receptor in the anterior pituitary of castrated rats. The overall data offer a plausible explanation for the weak oestrogenic effects induced by high, non-pharmacological doses of levonorgestrel.

Crystallization and preliminary X-ray crystallographic analysis of β-galactosidase from Kluyveromyces lactis

International Nuclear Information System (INIS)

Pereira-Rodríguez, Ángel; Fernández-Leiro, Rafael; González Siso, M. Isabel; Cerdán, M. Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

2010-01-01

β-Galactosidase from K. lactis has been expressed in S. cerevisiae, purified by affinity chromatography and crystallized in its native form. β-Galactosidase from Kluyveromyces lactis catalyses the hydrolysis of the β-galactosidic linkage in lactose. Owing to its many industrial applications, the biotechnological potential of this enzyme is substantial. This protein has been expressed in yeast and purified for crystallization trials. However, optimization of the best crystallization conditions yielded crystals with poor diffraction quality that precluded further structural studies. Finally, the crystal quality was improved using the streak-seeding technique and a complete diffraction data set was collected at 2.8 Å resolution

Synthesis of oligosaccharides derived from lactulose (OsLu using soluble and immobilized Aspergillus oryzae b-galactosidase

Directory of Open Access Journals (Sweden)

ALEJANDRA eCARDELLE COBAS

2016-03-01

Full Text Available b-galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu by transgalactosylation. Oligosaccharides with degree of polymerization (DP ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae b-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with b-galactosidase immobilized onto a glutaraldehyde-agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v of total OsLu and ca. 20% (w/v of OsLu-3, being 6′-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae b-galactosidase immobilized on a support of glutaraldehyde-agarose constitutes an efficient and cost-effective alternative to the use of soluble b-galactosidases for the synthesis of prebiotic OsLu mixtures.

Ability of Lactobacillus fermentum to overcome host α-galactosidase deficiency, as evidenced by reduction of hydrogen excretion in rats consuming soya α-galacto-oligosaccharides

Directory of Open Access Journals (Sweden)

Sesma Fernando

2008-01-01

Full Text Available Abstract Background Soya and its derivatives represent nutritionally high quality food products whose major drawback is their high content of α-galacto-oligosaccharides. These are not digested in the small intestine due to the natural absence of tissular α-galactosidase in mammals. The passage of these carbohydrates to the large intestine makes them available for fermentation by gas-producing bacteria leading to intestinal flatulence. The aim of the work reported here was to assess the ability of α-galactosidase-producing lactobacilli to improve the digestibility of α-galacto-oligosaccharides in situ. Results Gnotobiotic rats were orally fed with soy milk and placed in respiratory chambers designed to monitor fermentative gas excretion. The validity of the animal model was first checked using gnotobiotic rats monoassociated with a Clostridium butyricum hydrogen (H2-producing strain. Ingestion of native soy milk by these rats caused significant H2 emission while ingestion of α-galacto-oligosaccharide-free soy milk did not, thus validating the experimental system. When native soy milk was fermented using the α-galactosidase-producing Lactobacillus fermentum CRL722 strain, the resulting product failed to induce H2 emission in rats thus validating the bacterial model. When L. fermentum CRL722 was coadministered with native soy milk, a significant reduction (50 %, P = 0.019 in H2 emission was observed, showing that α-galactosidase from L. fermentum CRL722 remained active in situ, in the gastrointestinal tract of rats monoassociated with C. butyricum. In human-microbiota associated rats, L. fermentum CRL722 also induced a significant reduction of H2 emission (70 %, P = 0.004. Conclusion These results strongly suggest that L. fermentum α-galactosidase is able to partially alleviate α-galactosidase deficiency in rats. This offers interesting perspectives in various applications in which lactic acid bacteria could be used as a vector for

A polymorphic variant in the human electron transfer flavoprotein alpha-chain (alpha-T171) displays decreased thermal stability and is overrepresented in very-long-chain acyl-CoA dehydrogenase-deficient patients with mild childhood presentation

DEFF Research Database (Denmark)

Bross, P; Pedersen, P; Nyholm, M

1999-01-01

The consequences of two amino acid polymorphisms of human electron transfer flavoprotein (alpha-T/I171 in the alpha-subunit and beta-M/T154 in the beta-subunit) on the thermal stability of the enzyme are described. The alpha-T171 variant displayed a significantly decreased thermal stability, wher....... This is compatible with a negative modulating effect of the less-stable alpha-T171 ETF variant in this group of VLCAD patients that harbor missense mutations in at least one allele and therefore potentially display residual levels of VLCAD enzyme activity. Udgivelsesdato: 1999-Jun...

Amylosucrase, a glucan-synthesizing enzyme from the alpha-amylase family

DEFF Research Database (Denmark)

Skov, L K; Mirza, Osman Asghar; Henriksen, A

2001-01-01

Amylosucrase (E.C. 2.4.1.4) is a member of Family 13 of the glycoside hydrolases (the alpha-amylases), although its biological function is the synthesis of amylose-like polymers from sucrose. The structure of amylosucrase from Neisseria polysaccharea is divided into five domains: an all helical N...... of amylosucrase is at the bottom of a pocket at the molecular surface. A substrate binding site resembling the amylase 2 subsite is not found in amylosucrase. The site is blocked by a salt bridge between residues in the second and eight loops of the (beta/alpha)(8)-barrel. The result is an exo-acting enzyme. Loop......-terminal domain that is not similar to any known fold, a (beta/alpha)(8)-barrel A-domain, B- and B'-domains displaying alpha/beta-structure, and a C-terminal eight-stranded beta-sheet domain. In contrast to other Family 13 hydrolases that have the active site in the bottom of a large cleft, the active site...

Assessment of alpha activity of building materials commonly used in West Bengal, India

International Nuclear Information System (INIS)

Ghosh, Dipak; Deb, Argha; Bera, Sukumar; Sengupta, Rosalima; Patra, Kanchan Kumar

2008-01-01

This paper, reports for the first time, an extensive study of alpha activity of all widely used building materials (plaster of Paris, stone chips, marble, white cement, mosaic stone, limestone, sand, granite, cement brick, asbestos, red brick, cement tile, ceramic tile and ceramics) in West Bengal, India. The alpha activities have been measured using Solid State Nuclear Track Detector (SSNTD), a very sensitive detector for alpha particles. The samples were collected from local markets of Kolkata. The measured average alpha activities ranged from 22.7 ± 2.5 to 590.6 ± 16.8 Bq kg -1 . The alpha activity of ceramic tiles was highest and provides additional data to estimate the effect of environmental radiation exposure on human health

In Patients with an α-Galactosidase A Variant, Small Nerve Fibre Assessment Cannot Confirm a Diagnosis of Fabry Disease

NARCIS (Netherlands)

van der Tol, Linda; Verhamme, Camiel; van Schaik, Ivo N.; van der Kooi, Anneke J.; Hollak, Carla E. M.; Biegstraaten, Marieke

2016-01-01

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by an α-galactosidase A enzyme deficiency due to pathogenic variants in the α-galactosidase A gene (GLA). An increasing number of individuals with a GLA variant, but without characteristic FD features, are identified. A definite

Effect of soil-bound residues of malathion on microbial activities

International Nuclear Information System (INIS)

Hussain, A.; Iqbal, Z.; Asi, M.R.; Tahira, R.; Chudhary, J.A.

2001-01-01

The effect of soil-bound residues of malathion on CO/sub 2/ evolution, dehydrogenase activity and some nitrogen transformations in a loam soil was investigated under laboratory conditions. The soil samples containing bound residues arising from 10 mg g-1 of the applied malathion were mixed in equal quantity with fresh soil and compared with solvent extracted control soil without bound residues (extracted in the same way as soil containing bound residues). Another control comprising un extracted fresh soil without bound residues was also kept to study the effect of solvent extraction on the biological activity. Rate of Carbon mineralization (CO/sub 2/ evolution) was decreased in the presence of soil-bound residues of malathion. Bound residues also affected dehydrogenase activity of soil. Over 40% inhibition of dehydrogenase activity was observed after 4 days and the inhibition persisted at least for 12 days. Nitrogen mineralization was stimulated in soil containing bound residues of malathion and this stimulatory effect increased with time of incubation. Nitrification was partially inhibited in the presence of soil-bound residues of malathion. The inhibitory effect of the soil-bound residues on nitrification did not show much variation with time. The soil-bound residues did not affect denitrification rate (N/sub 2/O evolution). Nitrogen fixation (acetylene reduction) was partially inhibited in soil amended with bound residues of malathion and the inhibitory effect persisted for at least one week. In general, soil bound residues of malathion inhibited CO/sub 2/ evolution, dehydrogenase activity, nitrification and nitrogen fixation while mineralization of nitrogen was stimulated. Denitrification was not affected by the applied insecticide. (author)

The influence of the calibration standard and the chemical composition of the water samples residue in the counting efficiency of proportional detectors for gross alpha and beta counting. Application on the radiologic control of the IPEN-CNEN/SP

International Nuclear Information System (INIS)

Santos, Cecilia Martins

2003-01-01

In this work the efficiency calibration curves of thin-window and low background gas-flow proportional counters were determined for calibration standards with different energies and different absorber thicknesses. For the gross alpha counting we have used 241 Am and natural uranium standards and for the gross beta counting we have used 90 Sr/ 90 Y and 137 Cs standards in residue thicknesses ranging from 0 to approximately 18 mg/cm 2 . These sample thicknesses were increased with a previously determined salted solution prepared simulating the chemical composition of the underground water of IPEN The counting efficiency for alpha emitters ranged from 0,273 +- 0,038 for a weightless residue to only 0,015 +- 0,002 in a planchet containing 15 mg/cm 2 of residue for 241 Am standard. For natural uranium standard the efficiency ranged from 0,322 +- 0,030 for a weightless residue to 0,023 +- 0,003 in a planchet containing 14,5 mg/cm 2 of residue. The counting efficiency for beta emitters ranged from 0,430 +- 0,036 for a weightless residue to 0,247 +- 0,020 in a planchet containing 17 mg/cm 2 of residue for 137 Cs standard. For 90 Sr/ 90 Y standard the efficiency ranged from 0,489 +- 0,041 for a weightless residue to 0,323 +- 0,026 in a planchet containing 18 mg/cm 2 of residue. Results make evident the counting efficiency variation with the alpha or beta emitters energies and the thickness of the water samples residue. So, the calibration standard, the thickness and the chemical composition of the residue must always be considered in the gross alpha and beta radioactivity determination in water samples. (author)

Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

Energy Technology Data Exchange (ETDEWEB)

Serafini, P.; Lobo, R.A.

1985-01-01

In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

Blocking of irrelevant memories by posterior alpha activity boosts memory encoding.

Science.gov (United States)

Park, Hyojin; Lee, Dong Soo; Kang, Eunjoo; Kang, Hyejin; Hahm, Jarang; Kim, June Sic; Chung, Chun Kee; Jensen, Ole

2014-08-01

In our daily lives, we are confronted with a large amount of information. Because only a small fraction can be encoded in long-term memory, the brain must rely on powerful mechanisms to filter out irrelevant information. To understand the neuronal mechanisms underlying the gating of information into long-term memory, we employed a paradigm where the encoding was directed by a "Remember" or a "No-Remember" cue. We found that posterior alpha activity increased prior to the "No-Remember" stimuli, whereas it decreased prior to the "Remember" stimuli. The sources were localized in the parietal cortex included in the dorsal attention network. Subjects with a larger cue-modulation of the alpha activity had better memory for the to-be-remembered items. Interestingly, alpha activity reflecting successful inhibition following the "No-Remember" cue was observed in the frontal midline structures suggesting preparatory inhibition was mediated by anterior parts of the dorsal attention network. During the presentation of the memory items, there was more gamma activity for the "Remember" compared to the "No-Remember" items in the same regions. Importantly, the anticipatory alpha power during cue predicted the gamma power during item. Our findings suggest that top-down controlled alpha activity reflects attentional inhibition of sensory processing in the dorsal attention network, which then finally gates information to long-term memory. This gating is achieved by inhibiting the processing of visual information reflected by neuronal synchronization in the gamma band. In conclusion, the functional architecture revealed by region-specific changes in the alpha activity reflects attentional modulation which has consequences for long-term memory encoding. Copyright © 2014 Wiley Periodicals, Inc.

Different responses of spontaneous and stimulus-related alpha activity to ambient luminance changes.

Science.gov (United States)

Benedetto, Alessandro; Lozano-Soldevilla, Diego; VanRullen, Rufin

2017-12-04

Alpha oscillations are particularly important in determining our percepts and have been implicated in fundamental brain functions. Oscillatory activity can be spontaneous or stimulus-related. Furthermore, stimulus-related responses can be phase- or non-phase-locked to the stimulus. Non-phase-locked (induced) activity can be identified as the average amplitude changes in response to a stimulation, while phase-locked activity can be measured via reverse-correlation techniques (echo function). However, the mechanisms and the functional roles of these oscillations are far from clear. Here, we investigated the effect of ambient luminance changes, known to dramatically modulate neural oscillations, on spontaneous and stimulus-related alpha. We investigated the effect of ambient luminance on EEG alpha during spontaneous human brain activity at rest (experiment 1) and during visual stimulation (experiment 2). Results show that spontaneous alpha amplitude increased by decreasing ambient luminance, while alpha frequency remained unaffected. In the second experiment, we found that under low-luminance viewing, the stimulus-related alpha amplitude was lower, and its frequency was slightly faster. These effects were evident in the phase-locked part of the alpha response (echo function), but weaker or absent in the induced (non-phase-locked) alpha responses. Finally, we explored the possible behavioural correlates of these modulations in a monocular critical flicker frequency task (experiment 3), finding that dark adaptation in the left eye decreased the temporal threshold of the right eye. Overall, we found that ambient luminance changes impact differently on spontaneous and stimulus-related alpha expression. We suggest that stimulus-related alpha activity is crucial in determining human temporal segmentation abilities. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

DEFF Research Database (Denmark)

Su, J; Muranjan, M; Sap, J

1999-01-01

of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from...... these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect...

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

Energy Technology Data Exchange (ETDEWEB)

Yamamoto, S [Food Science Center, Niigata University, Ikarashi, Niigata, 950-2181 (Japan); Takanohashi, K; Nishiumi, T [Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata, 950-2181 (Japan); Hara, T [Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarashi, Niigata, 950-2181 (Japan); Odani, S [Department of Living Science and Technology, Faculty of Education and Human Science, Ikarashi, Niigata, 950-2181 (Japan); Suzuki, A, E-mail: shuyama@agr.niigata-u.ac.j [Department of Health and Nutrition, Faculty of Medical Science for Health, Teikyo Heisei University, Ikebukuro, Tokyo, 170-0013 (Japan)

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 {alpha}-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 {alpha}-AI were little. From our results, pressure-induced changes of the {alpha}-AI structure were not apparent. On the other hands, the IgE-specific binding activities of pressurized {alpha}-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 {alpha}-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 {alpha}-AI.

Fabry disease in children: a federal screening programme in Russia.

Science.gov (United States)

Namazova-Baranova, Leyla Seymurovna; Baranov, Alexander Alexandrovich; Pushkov, Aleksander Alekseevich; Savostyanov, Kirill Victorovich

2017-10-01

Our objective was to examine the prevalence of Fabry disease in Russian children with chronic pain in the distal limbs. This non-interventional, multi-centre study included children 2-18 years of age with chronic recurrent unilateral or bilateral pain, burning, or acroparesthesia in the hands or feet. The presence of Fabry disease was defined by abnormal alpha-galactosidase A activity in males or alpha-galactosidase gene (GLA) mutation in females. Among 214 patients (110 males), 84.1% had bilateral limb pain and 31.8% had unilateral limb pain recorded at some time point; 61 (28.5%) patients had a positive family history possibly associated with Fabry disease. Alpha-galactosidase A activity was within the normal range in all 109 of the male patients tested. One female patient had a GLA mutation (C937G > T) and alpha-galactosidase A activity within the normal range. We did not find definitive evidence of Fabry disease in these children with a history of chronic recurrent unilateral or bilateral limb pain or acroparesthesia. The presence of chronic limb pain does not appear to be highly predictive of a diagnosis of Fabry disease in Russian children and adolescents, suggesting that key early signs and symptoms of Fabry disease are not specific to the disease. What is Known: • Signs and symptoms of Fabry disease are seen in children < 10 years of age; pain in the distal limbs is a common early symptom. What is New: • Fabry disease was not diagnosed in this population of Russian children with a history of chronic limb pain. • The presence of acroparesthesia or chronic limb pain does not appear to be highly predictive of a diagnosis of Fabry disease in Russian children and adolescents, suggesting that these early symptoms of Fabry disease are not specific to the disease.

Daily and seasonal variations of outdoor alpha-activity concentration in Salzburg city/Austria

International Nuclear Information System (INIS)

Lettner, H.; Hubmer, A.K.; Rolle, R.; Winkler, R.; Steinhaeusler, F.

1996-01-01

Long-term continuous measurements of the atmospheric outdoor alpha-activity concentration have been performed by using a radioaerosol-monitor, roof-mounted 20 m above ground. The alpha-activity concentration was identified to be predominantly attributed to radon progeny. The total alpha-activity covers a range of two orders of magnitude. Three different components of variations could be identified with regard to temporal variations: Short-term diurnal component (daily variation), mid-term component (days to weeks) and long-term component (seasonal variation). Continuous measurements have been recorded since the end of 1993. The results of continuous measurements of the outdoor alpha-activity concentration over a time span from January 1994 to June 1995 are presented. (author)

Correlation between protein kinase C alpha activity and membrane phase behavior.

Science.gov (United States)

Micol, V; Sánchez-Piñera, P; Villalaín, J; de Godos, A; Gómez-Fernández, J C

1999-02-01

Lipid activation of protein kinase C alpha (PKC alpha) was studied by using a model mixture containing 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), and 1, 2-dimyristoyl-sn-glycerol (1,2-DMG). This lipid mixture was physically characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and 31P-nuclear magnetic resonance (31P-NMR). Based on these techniques, a phase diagram was constructed by keeping a constant DMPC/DMPS molar ratio of 4:1 and changing the concentration of 1,2-DMG. This phase diagram displayed three regions and two compounds: compound 1 (C1), with 45 mol% 1,2-DMG, and compound 2 (C2), with 60 mol% 1,2-DMG. When the phase diagram was elaborated in the presence of Ca2+ and Mg2+, at concentrations similar to those used in the PKC alpha activity assay, the boundaries between the regions changed slightly and C1 had 35 mol% 1,2-DMG. The activity of PKC alpha was studied at several temperatures and at different concentrations of 1,2-DMG, with a maximum of activity reached at 30 mol% 1,2-DMG and lower values at higher concentrations. In the presence of Ca2+ and Mg2+, maximum PKC alpha activity occurred at concentrations of 1,2-DMG that were close to the boundary in the phase diagram between region 1, where compound C1 and the pure phospholipid coexisted in the gel phase, and region 2, where compounds C1 and C2 coexisted. These results suggest that the membrane structure corresponding to a mixture of 1,2-DMG/phospholipid complex and free phospholipid is better able to support the activity of PKC alpha than the 1,2-DMG/phospholipid complex alone.

Frontal alpha EEG asymmetry before and after behavioral activation treatment for depression.

Science.gov (United States)

Gollan, Jackie K; Hoxha, Denada; Chihade, Dietta; Pflieger, Mark E; Rosebrock, Laina; Cacioppo, John

2014-05-01

Mid-frontal and mid-lateral (F3/F4 and F7/F8) EEG asymmetry has been associated with motivation and affect. We examined alpha EEG asymmetry in depressed and healthy participants before and after Behavioral Activation treatment for depression; examined the association between alpha EEG asymmetry and motivational systems and affect; and evaluated the utility of alpha EEG asymmetry in predicting remission. Depressed (n=37) and healthy participants (n=35) were assessed before and after treatment using a clinical interview, a task to measure baseline EEG, and questionnaires of behavioral activation and inhibition, avoidance, and affect. Alpha EEG asymmetry was significantly higher in depressed than healthy participants at pre-treatment, positively correlated with negative affect and behavioral inhibition, and inversely correlated with lower behavioral activation sensitivity. Heightened alpha EEG asymmetry in depressed participants was significantly associated with increased behavioral inhibition and negative emotion and was independent of clinical remission. Copyright © 2014 Elsevier B.V. All rights reserved.

A blended- rather than whole-lentil meal with or without α-galactosidase mildly increases healthy adults' appetite but not their glycemic response.

Science.gov (United States)

Anguah, Katherene O-B; Wonnell, Brittany S; Campbell, Wayne W; McCabe, George P; McCrory, Megan A

2014-12-01

Disrupting the physical structure of pulses by blending them or by using a digestive supplement (α-galactosidase) to reduce intestinal discomfort could potentially negate the previously observed beneficial effects of whole pulses of lowering appetitive and glycemic responses because of more rapid digestion. We hypothesized that blended lentils, α-galactosidase, or both increase postprandial appetite and blood glucose responses vs. whole lentils. Men and women [n = 12; means ± SDs body mass index (kg/m(2)): 23.3 ± 3.1; aged 28 ± 10 y] consumed breakfast meals containing whole (W), blended (B), or no lentils [control (C)], each with 3 α-galactosidase or placebo capsules in a randomized, crossover, double-blind placebo-controlled trial. Between each test day there was a 3- to 5-d washout period. Mixed-model ANOVA showed effects of meal on postprandial appetite and glucose (P = 0.0001-0.031). The B meal resulted in higher postprandial appetite ratings than did the W meal but not the C meal for hunger, desire to eat, and prospective consumption (Δ = 0.4-0.5 points; P = 0.002-0.044). Postprandial glucose concentration was 4.5 mg/dL lower for the B meal than for the C meal (P meal. There were no main effects of α-galactosidase, but there were meal × α-galactosidase interaction effects, with a greater postprandial desire to eat and lower postprandial fullness with the B meal than with the 2 other meals in the placebo condition but not in the α-galactosidase condition. Blending lentils increased appetite (∼6%), but not glycemic response, compared with whole lentils, whereas α-galactosidase did not. Both B and W meals may be consumed (with or without an α-galactosidase supplement) with little impact on appetite, without increasing glycemic response. This trial was registered at clinicaltrials.gov as NCT02110511. © 2014 American Society for Nutrition.

Receptor-mediated endocytosis of α-galactosidase A in human podocytes in Fabry disease.

Directory of Open Access Journals (Sweden)

Thaneas Prabakaran

Full Text Available Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs.

The in vitro synthesis of β-galactosidase induced in a subcellular structure of Escherichia coli (1961)

International Nuclear Information System (INIS)

Nisman, B.; Kayser, A.; Demailly, J.; Genin, C.

1961-01-01

Isopropyl-thio-galactoside (IPTG), an inducer of 3-galactosidase, makes it possible to synthesise this enzyme in vitro with the subcellular structure (P 1 ). The enzyme is isolated from the bacteria Escherichia coli K 12 which are inductive but not induced. The incorporation of radioactive amino-acids, which is stimulated by the presence of an inducer, was studied during the course of the enzyme synthesis. Saccharose suppresses the induction of β-galactosidase. The presence of a specific inhibitor in the structure studied is considered. (authors) [fr

Prevention of Bacterial Contamination of a Silica Matrix Containing Entrapped β-Galactosidase through the Action of Covalently Bound Lysozymes

Directory of Open Access Journals (Sweden)

Heng Li

2017-02-01

Full Text Available β-galactosidase was successfully encapsulated within an amino-functionalised silica matrix using a “fish-in-net” approach and molecular imprinting technique followed by covalent binding of lysozyme via a glutaraldehyde-based method. Transmission electron microscopy (TEM, X-ray diffraction (XRD, scanning electron microscopy (SEM, and Fourier transform infrared (FTIR spectroscopy were used to characterise the silica matrix hosting the two enzymes. Both encapsulated β-galactosidase and bound lysozyme exhibited high enzymatic activities and outstanding operational stability in model reactions. Moreover, enzyme activities of the co-immobilised enzymes did not obviously change relative to enzymes immobilised separately. In antibacterial tests, bound lysozyme exhibited 95.5% and 89.6% growth inhibition of Staphylococcus aureus ATCC (American type culture collection 653 and Escherichia coli ATCC 1122, respectively. In milk treated with co-immobilised enzymes, favourable results were obtained regarding reduction of cell viability and high lactose hydrolysis rate. In addition, when both co-immobilised enzymes were employed to treat milk, high operational and storage stabilities were observed. The results demonstrate that the use of co-immobilised enzymes holds promise as an industrial strategy for producing low lactose milk to benefit people with lactose intolerance.

Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

Energy Technology Data Exchange (ETDEWEB)

Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

1998-06-29

Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

A method of alpha-radiating nuclide activity measuring in aerosol filters

International Nuclear Information System (INIS)

Ignatov, V.P.; Galkina, V.N.

1992-01-01

Scintillation method of determination of alpha-radiating nuclide activity in aerosol filters was suggested. The method involves dissolution of the filter in organic solvent, introduction of luminophore into solution prepared, drying of the preparation and measurement of radionuclide activity. Dependences of alpha-radiation detection efficiency on the content of luminophore, filter material, colourless and coloured substances in preparations analyzed were considered

Study on indium leaching from mechanically activated hard zinc residue

Directory of Open Access Journals (Sweden)

Yao J.H.

2011-01-01

Full Text Available In this study, changes in physicochemical properties and leachability of indium from mechanically activated hard zinc residue by planetary mill were investigated. The results showed that mechanical activation increased specific surface area, reaction activity of hard zinc residue, and decreased its particle size, which had a positive effect on indium extraction from hard zinc residue in hydrochloric acid solution. Kinetics of indium leaching from unmilled and activated hard zinc residue were also investigated, respectively. It was found that temperature had an obvious effect on indium leaching rate. Two different kinetic models corresponding to reactions which are diffusion controlled, [1-(1- x1/3]2=kt and (1-2x/3-(1-x2/3=kt were used to describe the kinetics of indium leaching from unmilled sample and activated sample, respectively. Their activation energies were determined to be 17.89 kJ/mol (umilled and 11.65 kJ/mol (activated within the temperature range of 30°C to 90°C, which is characteristic for a diffusion controlled process. The values of activation energy demonstrated that the leaching reaction of indium became less sensitive to temperature after hard zinc residue mechanically activated by planetary mill.

Left temporal alpha band activity increases during working memory retention of pitches

NARCIS (Netherlands)

Van Dijk, H.; Nieuwenhuis, I.L.C.; Jensen, O.

2010-01-01

The functional role and regional specificity of similar to 10 Hz alpha band activity remains of debate. Alpha band activity is strongly modulated in visual working memory tasks and it has been proposed to subserve resource allocation by disengaging task-irrelevant regions. It remains unknown if

Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity

International Nuclear Information System (INIS)

Chen, Jin-Yan; Chen, Wan-Nan; Jiao, Bo-Yan; Lin, Wan-Song; Wu, Yun-Li; Liu, Ling-Ling; Lin, Xu

2014-01-01

The risk of hepatocellular carcinoma (HCC) increases in chronic hepatitis B surface antigen (HBsAg) carriers who often have concomitant increase in the levels of benzo[alpha]pyrene-7,8-diol-9,10-epoxide(±) (BPDE)-DNA adduct in liver tissues, suggesting a possible co-carcinogenesis of Hepatitis B virus (HBV) and benzo[alpha]pyrene in HCC; however the exact mechanisms involved are unclear. The interaction between hepatitis B spliced protein (HBSP) and microsomal epoxide hydrolase (mEH) was confirmed using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assay; the effects of HBSP on mEH-mediated B[alpha]P metabolism was examined by high performance liquid chromatography (HPLC); and the influences of HBSP on B[alpha]P carcinogenicity were evaluated by bromodeoxyuridine cell proliferation, anchorage-independent growth and tumor xenograft. HBSP could interact with mEH in vitro and in vivo, and this interaction was mediated by the N terminal 47 amino acid residues of HBSP. HBSP could greatly enhance the hydrolysis activity of mEH in cell-free mouse liver microsomes, thus accelerating the metabolism of benzo[alpha]pyrene to produce more ultimate carcinnogen, BPDE, and this effect of HBSP requires the intact HBSP molecule. Expression of HBSP significantly increased the formation of BPDE-DNA adduct in benzo[alpha]pyrene-treated Huh-7 hepatoma cells, and this enhancement was blocked by knockdown of mEH. HBSP could enhance the cell proliferation, accelerate the G1/S transition, and promote cell transformation and tumorigenesis of B[alpha]P-treated Huh-7 hepatoma cells. Our results demonstrated that HBSP could promote carcinogenic effects of B[alpha]P by interacting with mEH and enhancing its hydrolysis activity

Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

Science.gov (United States)

Borges, Chad R; Rehder, Douglas S

2016-09-15

Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Chemical activation of gasification carbon residue for phosphate removal

Science.gov (United States)

Kilpimaa, Sari; Runtti, Hanna; Lassi, Ulla; Kuokkanen, Toivo

2012-05-01

Recycling of waste materials provides an economical and environmentally significant method to reduce the amount of waste. Bioash formed in the gasification process possesses a notable amount of unburned carbon and therefore it can be called a carbon residue. After chemical activation carbon residue could be use to replace activated carbon for example in wastewater purification processes. The effect of chemical activation process variables such as chemical agents and contact time in the chemical activation process were investigated. This study also explored the effectiveness of the chemically activated carbon residue for the removal of phosphate from an aqueous solution. The experimental adsorption study was performed in a batch reactor and the influence of adsorption time, initial phosphate concentration and pH was studied. Due to the carbon residue's low cost and high adsorption capacity, this type of waste has the potential to be utilised for the cost-effective removal of phosphate from wastewaters. Potential adsorbents could be prepared from these carbonaceous by-products and used as an adsorbent for phosphate removal.

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in hepatocellular carcinoma.

Science.gov (United States)

Liu, Rui; Zhang, Haiyang; Zhang, Yan; Li, Shuang; Wang, Xinyi; Wang, Xia; Wang, Cheng; Liu, Bin; Zen, Ke; Zhang, Chen-Yu; Zhang, Chunni; Ba, Yi

2017-04-01

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha plays a crucial role in regulating the biosynthesis of mitochondria, which is closely linked to the energy metabolism in various tumors. This study investigated the regulatory role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha in the pathogenesis of hepatocellular carcinoma. In this study, the changes of peroxisome proliferator-activated receptor gamma coactivator-1 alpha messenger RNA levels between normal human liver and hepatocellular carcinoma tissue were examined by quantitative reverse transcription polymerase chain reaction. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by RNA interference in the human liver cell line L02, while overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha complementary DNA in the human hepatocarcinoma cell line HepG2. Cellular morphological changes were observed via optical and electron microscopy. Cellular apoptosis was determined by Hoechst 33258 staining. In addition, the expression levels of 21,400 genes in tissues and cells were detected by microarray. It was shown that peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression was significantly downregulated in hepatocellular carcinoma compared with normal liver tissues. After knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression in L02 cells, cells reverted to immature and dedifferentiated morphology exhibiting cancerous tendency. Apoptosis occurred in the HepG2 cells after transfection by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Microarray analysis showed consistent results. The results suggest that peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor

In-house method validation and occurrence of alpha-, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone and trifluralin residues in strawberry

Directory of Open Access Journals (Sweden)

Alexandre Augusto Soares

2013-12-01

Full Text Available A method for determination of organohalogen pesticides in strawberry by gas chromatography with electron capture detection was validated and applied in a monitoring program. Linearity, matrix effects, and day effect were evaluated for the analytes alpha-endosulfan, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone, and trifluralin. The linear range varied according to the chromatographic response of the analyte. Significant matrix effects were observed. The mean recoveries ranged from 74.6 to 115.4%, with repeatability standard deviations between 1.6 and 21.0% and intermediate precision between 5.9 and 21.0%. Detection, quantification and decision limit, and detection capacity ranged from 0.003 to 0.007 mg/kg, 0.005 to 0.013 mg/kg; 0.003 to 3.128 mg/kg; and 0.005 to 3.266 mg/kg, respectively. The method was fit for the purpose of monitoring organohalogen residues in strawberries. Residues of these pesticides were detected in 124 of the 186 samples analyzed between 2009 and 2011 in the state of Minas Gerais. Nine of them did not comply with the current legislation requirements; among them, seven (3.8% had residues of unauthorized pesticide for the culture of strawberry, one (0.5% had residues above the maximum residue limit, and another one (0.5% exhibited both non-conformities.

A Blended- Rather Than Whole-Lentil Meal with or without α-Galactosidase Mildly Increases Healthy Adults’ Appetite but Not Their Glycemic Response123

Science.gov (United States)

Anguah, Katherene O-B; Wonnell, Brittany S; Campbell, Wayne W; McCabe, George P; McCrory, Megan A

2014-01-01

Background: Disrupting the physical structure of pulses by blending them or by using a digestive supplement (α-galactosidase) to reduce intestinal discomfort could potentially negate the previously observed beneficial effects of whole pulses of lowering appetitive and glycemic responses because of more rapid digestion. Objective: We hypothesized that blended lentils, α-galactosidase, or both increase postprandial appetite and blood glucose responses vs. whole lentils. Methods: Men and women [n = 12; means ± SDs body mass index (kg/m2): 23.3 ± 3.1; aged 28 ± 10 y] consumed breakfast meals containing whole (W), blended (B), or no lentils [control (C)], each with 3 α-galactosidase or placebo capsules in a randomized, crossover, double-blind placebo-controlled trial. Between each test day there was a 3- to 5-d washout period. Results: Mixed-model ANOVA showed effects of meal on postprandial appetite and glucose (P = 0.0001–0.031). The B meal resulted in higher postprandial appetite ratings than did the W meal but not the C meal for hunger, desire to eat, and prospective consumption (Δ = 0.4–0.5 points; P = 0.002–0.044). Postprandial glucose concentration was 4.5 mg/dL lower for the B meal than for the C meal (P meal. There were no main effects of α-galactosidase, but there were meal × α-galactosidase interaction effects, with a greater postprandial desire to eat and lower postprandial fullness with the B meal than with the 2 other meals in the placebo condition but not in the α-galactosidase condition. Conclusions: Blending lentils increased appetite (∼6%), but not glycemic response, compared with whole lentils, whereas α-galactosidase did not. Both B and W meals may be consumed (with or without an α-galactosidase supplement) with little impact on appetite, without increasing glycemic response. This trial was registered at clinicaltrials.gov as NCT02110511. PMID:25411033

A fragment of alpha-actinin promotes monocyte/macrophage maturation in vitro.

Science.gov (United States)

Luikart, S; Wahl, D; Hinkel, T; Masri, M; Oegema, T

1999-02-01

Conditioned media (CM) from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix contains a factor that induces macrophage-like maturation of HL-60 cells. This factor was purified from the CM of HL-60 cells grown on bone marrow stroma by ammonium sulfate precipitation, then sequential chromatography on DEAE, affi-gel blue affinity, gel exclusion, and wheat germ affinity columns, followed by C-4 reverse phase HPLC, and SDS-PAGE. The maturation promoting activity of the CM was identified in a single 31 kD protein. Amino acid sequence analysis of four internal tryptic peptides of this protein confirmed significant homology with amino acid residues 48-60, 138-147, 215-220, and 221-236 of human cytoskeletal alpha-actinin. An immunoaffinity purified rabbit polyclonal anti-chicken alpha-actinin inhibited the activity of HL-60 conditioned media. A 27 kD amino-terminal fragment of alpha-actinin produced by thermolysin digestion of chicken gizzard alpha-actinin, but not intact alpha-actinin, had maturation promoting activity on several cell types, including blood monocytes, as measured by lysozyme secretion and tartrate-resistant acid phosphatase staining. We conclude that an extracellular alpha-actinin fragment can promote monocyte/macrophage maturation. This represents the first example of a fragment of a cytoskeletal component, which may be released during tissue remodeling and repair, playing a role in phagocyte maturation.

Utilização da β-galactosidase para prevenção da cristalização em doce de leite Use of β-galactosidase in milk sweet: avoiding lactose crystallization

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Manuela Poletto Klein

2010-12-01

Full Text Available Este estudo avaliou o efeito da utilização da enzima β-galactosidase sobre a cristalização da lactose no doce de leite. As concentrações de lactase testadas variaram de 0 a 0,4 g/L. O grau de cristalização do produto foi avaliado sensorialmente (teste de ordenação em função de uma escala específica, após 30, 60, 90 e 180 dias de armazenamento à temperatura ambiente, por provadores previamente treinados. O teste de ordenação indicou não haver diferença estatística significativa (P>0,05 entre as amostras em relação à cristalização, tanto aos 30 quanto aos 60 dias de armazenamento, porém aos 90 e 180 dias essa diferença foi significativa entre o controle (T0 e os tratamentos. A utilização de 0,2 g/L de β-galactosidase (23,16% de hidrólise da lactose foi suficiente para que a arenosidade no doce de leite não fosse percebida sensorialmente pelos provadores, durante todo o período considerado.The effect of using β-galactosidase on the crystallization of lactose in milk sweet has been studied. The concentrations of lactase ranged from 0 to 0.4 g/L of processed milk. The product was subjected to sensorial analysis (crystallization degree ranking test using a specific scale by trained tasters after 30, 60, 90 e 180 days of storage. The ranking test showed no statistically significant difference between the samples in relation to crystallization, both at 30 and 60 days of storage, but at 90 and 180 days the difference was significant among control (T0 and the treatments. Usage of 0.2 g/L of β-galactosidase (23.16% lactose hydrolysis in milk was enough to prevent sandiness in milk sweet from being noticed throughout the studied period.

Diagnostic dilemma: a young woman with Fabry disease symptoms, no family history, and a "sequencing cryptic" α-galactosidase a large deletion

DEFF Research Database (Denmark)

Feldt-Rasmussen, Ulla; Dobrovolny, Robert; Nazarenko, Irina

2011-01-01

Fabry disease, an X-linked lysosomal storage disorder, results from the deficient activity of a-galactosidase A (a-Gal A). In affected males, the clinical diagnosis is confirmed by the markedly decreased a-Gal A activity. However, in female heterozygotes, the a-Gal A activity can range from low t...... on enzyme replacement therapy. Thus, gene dosage analyses can detect large deletions (>50bp) in suspect heterozygotes for X-linked and autosomal dominant diseases that are "sequencing cryptic," resolving molecular diagnostic dilemmas....

Bi209 alpha activity

International Nuclear Information System (INIS)

Araujo Penna, M.M. de.

1970-01-01

The study for measuring Bi 209 alpha activity is presented. Ilford L4 nuclear emulsion pellicles loaded with bismuth citrate to obtain a load of 100 mg/cm 3 of dry emulsion, were prepared. Other pellicles were prepared with the same. Ilford L4 gel to estimate the background radiation. To observe 'fading' effect, pellicles loaded with bismuth were submitted to neutrons of high energy, aiming to record recoil proton tracks. The pellicles were confined in nitrogen atmosphere at temperature lower than -10 0 C. The Bi 209 experimental half-life was obtained and compared with the estimated theoretical data. (M.C.K.) [pt

6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation.

Science.gov (United States)

Yoo, Y-G; Na, T-Y; Yang, W-K; Kim, H-J; Lee, I-K; Kong, G; Chung, J-H; Lee, M-O

2007-05-31

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. Previously, we reported that the orphan nuclear receptor Nur77 functions in stabilizing HIF-1alpha. Here, we demonstrate that 6-mercaptopurine (6-MP), an activator of the NR4A family members, enhances transcriptional activity of HIF-1. 6-MP enhanced the protein-level of HIF-1alpha as well as vascular endothelial growth factor (VEGF) in a dose- and time-dependent manner. The induction of HIF-1alpha was abolished by the transfection of either a dominant-negative Nur77 mutant or si-Nur77, indicating a critical role of Nur77 in the 6-MP action. The HIF-1alpha protein level remained up to 60 min in the presence of 6-MP when de novo protein synthesis was blocked by cycloheximide, suggesting that 6-MP induces stabilization of the HIF-1alpha protein. The fact that 6-MP decreased the association of HIF-1alpha with von Hippel-Lindau protein and the acetylation of HIF-1alpha, may explain how 6-MP induced stability of HIF-1alpha. Further, 6-MP induced the transactivation function of HIF-1alpha by recruiting co-activator cyclic-AMP-response-element-binding protein. Finally, 6-MP enhanced the expression of HIF-1alpha and VEGF, and the formation of capillary tubes in human umbilical vascular endothelial cells. Together, our results provide a new insight for 6-MP action in the stabilization of HIF-1alpha and imply a potential application of 6-MP in hypoxia-associated human vascular diseases.

Coexpression of β-D-galactosidase and L-arabinose isomerase in the production of D-tagatose: a functional sweetener.

Science.gov (United States)

Zhan, Yijing; Xu, Zheng; Li, Sha; Liu, Xiaoliu; Xu, Lu; Feng, Xiaohai; Xu, Hong

2014-03-19

The functional sweetener, d-tagatose, is commonly transformed from galactose by l-arabinose isomerase. To make use of a much cheaper starting material, lactose, hydrolization, and isomerization are required to take place collaboratively. Therefore, a single-step method involving β-d-galactosidase was explored for d-tagatose production. The two vital genes, β-d-galactosidase gene (lacZ) and l-arabinose isomerase mutant gene (araA') were extracted separately from Escherichia coli strains and incorporated into E. coli simultaneously. This gave us E. coli-ZY, a recombinant producing strain capable of coexpressing the two key enzymes. The resulted cells exhibited maximum d-tagatose producing activity at 34 °C and pH 6.5 and in the presence of borate, 10 mM Fe(2+), and 1 mM Mn(2+). Further monitoring showed that the recombinant cells could hydrolyze more than 95% lactose and convert 43% d-galactose into d-tagatose. This research has verified the feasibility of single-step d-tagatose fermentation, thereby laying down the foundation for industrial usage of lactose.

Reaction kinetics and galactooligosaccharide product profiles of the β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae.

Science.gov (United States)

Yin, Huifang; Bultema, Jelle B; Dijkhuizen, Lubbert; van Leeuwen, Sander S

2017-06-15

β-Galactosidase enzymes are used in the dairy industry to convert lactose into galactooligosaccharides (GOS) that are added to infant formula to mimic the molecular sizes and prebiotic functions of human milk oligosaccharides. Here we report a detailed analysis of the clearly different GOS profiles of the commercial β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae. Also the GOS yields of these enzymes differed, varying from 48.3% (B. circulans) to 34.9% (K. lactis), and 19.5% (A. oryzae). Their incubation with lactose plus the monosaccharides Gal or Glc resulted in altered GOS profiles. Experiments with 13 C 6 labelled Gal and Glc showed that both monosaccharides act as acceptor substrates in the transgalactosylation reactions. The data shows that the lactose isomers β-d-Galp-(1→2)-d-Glcp, β-d-Galp-(1→3)-d-Glcp and β-d-Galp-(1→6)-d-Glcp are formed from acceptor reactions with free Glc and not by rearrangement of Glc in the active site. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural modification of serum vitamin D3-binding protein and immunosuppression in AIDS patients.

Science.gov (United States)

Yamamoto, N; Naraparaju, V R; Srinivasula, S M

1995-11-01

A serum glycoprotein, vitamin D3-binding protein (Gc protein), can be converted by beta-galactosidase of stimulated B lymphocytes and sialidase of T lymphocytes to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is a precursor for MAF. Treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high-titered MAF (GcMAF). When peripheral blood monocytes/macrophages of 46 HIV-infected patients were treated with GcMAF (100 pg/ml), the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of plasma Gc protein was low in 16 (35%) of of these patients. Loss of the MAF precursor activity appeared to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase found in the patient blood stream. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Thus, precursor activity of Gc protein and alpha-N-acetylgalactosaminidase activity in patient blood can serve as diagnostic and prognostic indices.

Novel dense CO2 technique for beta-galactosidase immobilization in polystyrene microchannels.

Science.gov (United States)

Leclair Ellis, Jeffrey; Tomasko, David L; Dehghani, Fariba

2008-03-01

In this study we design new fabrication techniques and demonstrate the potential of using dense CO2 for facilitating crucial steps in the fabrication of polymeric lab-on-a-chip microdevices by embedding biomolecules at temperatures well below the polymer's glass transition temperature (T(g)). These new techniques are environmentally friendly and done without the use of a clean room. Carbon dioxide at 40 degrees C and between 4.48 and 6.89 MPa was used to immobilize the biologically active molecule, beta-galactosidase (beta-gal), on the surface of polystyrene microchannels. To our knowledge, this is the first time dense CO2 has been used to directly immobilize an enzyme in a microchannel. beta-gal activity was maintained and shown via a fluorescent reaction product, after enzyme immobilization and microchannel capping by the designed fabrication steps at 40 degrees C and pressures up to 6.89 MPa.

A carrier for non-covalent delivery of functional beta-galactosidase and antibodies against amyloid plaques and IgM to the brain.

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Gobinda Sarkar

Full Text Available BACKGROUND: Therapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB. The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary. APPROACH: A peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated. PRINCIPAL FINDINGS: The transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease. SIGNIFICANCE: The results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the

Hydrolysis of lactose by β-galactosidase from Kluyveromyces fragilis: characterization of the enzyme Hidrólise da lactose pela β-galactosidase de Kluyveromyces fragilis: caracterização da enzima

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Graciette Matioli

2002-03-01

Full Text Available The β-galactosidase enzyme from Kluyveromyces fragilis was characterized in the soluble form using lactose 5% w/v found in skimmed powdered milk as substrate. Enzyme diluted 50 times hydrolyzed the lactose in batch reactor of 50 mL capacity. Enzyme activity and its activation energy were determined as a function of temperature and pH. Temperature ranged from 20 to 55ºC and pH from 5.5 to 8.0. Activation energy was 9.50 kcal/mol. The energy of deactivation was 33.74 kcal/mol. Although the enzyme presented a high specific activity at 45ºC and pH 6.5 (3.312 U/mg protein, values indicate that the best use of the enzymatic activity occur at 40ºC or below, with half-life higher than 12 hours. The activation energy increased proportionally to pH increase. Therefore, the activation energy depends on pH and varies according to the origin of the enzyme.A enzima β-galactosidase de Kluyveromyces fragilis foi caracterizada na forma solúvel, utilizando como substrato, lactose 5% p/v presente no leite em pó desengordurado. A enzima, diluída 50 vezes, hidrolisou a lactose em reator batelada de 50 ml de capacidade. A atividade da enzima e sua energia de ativação foram determinadas em função da temperatura e pH. A faixa de temperatura analisada foi de 20 a 55ºC e de pH de 5,5 a 8,0. A energia de ativação foi de 9,50 kcal/mol. A energia de desativação foi de 33,74 kcal/mol. Embora a enzima tenha apresentado uma atividade específica alta a 45ºC e pH 6,5 (3,312 U/mg proteína, os valores obtidos indicam que o melhor aproveitamento da atividade enzimática se dá a 40ºC ou abaixo, com um tempo de meia-vida superior a 12 horas. A energia de ativação aumentou proporcionalmente com o aumento de pH. Portanto, a energia de ativação depende diretamente do pH da solução e varia com a origem da enzima.

Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing.

Science.gov (United States)

Yang, Wenxia; Zhang, Yuhong; Zhou, Xiaojin; Zhang, Wei; Xu, Xiaolu; Chen, Rumei; Meng, Qingchang; Yuan, Jianhua; Yang, Peilong; Yao, Bin

2015-01-01

Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50 °C) and lower stability over alkaline pH range, but better thermal stability at 60 °C to 70 °C and resistance to feed pelleting inactivation (80 °C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing.

Utilization of whey powder as substrate for low-cost preparation of β-galactosidase as main product, and ethanol as by-product, by a litre-scale integrated process.

Science.gov (United States)

You, Shengping; Chang, Hongxing; Yin, Qingdian; Qi, Wei; Wang, Mengfan; Su, Rongxin; He, Zhimin

2017-12-01

Whey powder, a by-product of dairy industry, is an attractive raw material for value-added products. In this study, utilization of whey powder as substrate for low-cost preparation of β-galactosidase as main product and ethanol as by-product were investigated by a litre-scale integrated strategy, encompassing fermentation, isolation, permeabilization and spray drying. Firstly, through development of low-cost industrial culture and fed-batch strategies by Kluyveromyces lactis, 119.30U/mL β-galactosidase activity and 16.96mg/mL by-product ethanol were achieved. Afterward, an up-dated mathematic model for the recycling permeabilization was established successfully and 30.4g cells sediment isolated from 5L fermentation broth were permeabilized completely by distilled ethanol from broth supernatant. Then β-galactosidase product with 5.15U/mg from protection of gum acacia by spray drying was obtained. Furthermore, by-product ethanol with 31.08% (v/v) was achieved after permeabilization. Therefore, the integrated strategy using whey powder as substrate is a feasible candidate for industrial-scale implementation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cell motility in chronic lymphocytic leukemia: defective Rap1 and alphaLbeta2 activation by chemokine.

Science.gov (United States)

Till, Kathleen J; Harris, Robert J; Linford, Andrea; Spiller, David G; Zuzel, Mirko; Cawley, John C

2008-10-15

Chemokine-induced activation of alpha4beta1 and alphaLbeta2 integrins (by conformational change and clustering) is required for lymphocyte transendothelial migration (TEM) and entry into lymph nodes. We have previously reported that chemokine-induced TEM is defective in chronic lymphocytic leukemia (CLL) and that this defect is a result of failure of the chemokine to induce polar clustering of alphaLbeta2; engagement of alpha4beta1 and autocrine vascular endothelial growth factor (VEGF) restore clustering and TEM. The aim of the present study was to characterize the nature of this defect in alphaLbeta2 activation and determine how it is corrected. We show here that the alphaLbeta2 of CLL cells is already in variably activated conformations, which are not further altered by chemokine treatment. Importantly, such treatment usually does not cause an increase in the GTP-loading of Rap1, a GTPase central to chemokine-induced activation of integrins. Furthermore, we show that this defect in Rap1 GTP-loading is at the level of the GTPase and is corrected in CLL cells cultured in the absence of exogenous stimuli, suggesting that the defect is the result of in vivo stimulation. Finally, we show that, because Rap1-induced activation of both alpha4beta1 and alphaLbeta2 is defective, autocrine VEGF and chemokine are necessary to activate alpha4beta1 for ligand binding. Subsequently, this binding and both VEGF and chemokine stimulation are all needed for alphaLbeta2 activation for motility and TEM. The present study not only clarifies the nature of the alphaLbeta2 defect of CLL cells but is the first to implicate activation of Rap1 in the pathophysiology of CLL.

ALPHA/AMPU, Radionuclide Radioactivity from Alpha Spectrometer Measurements

International Nuclear Information System (INIS)

Sill, D.S.

1990-01-01

1 - Description of program or function: The two computer programs, ALPHA and AMPU, take raw data obtained from alpha spectrometry and from these calculate activities and uncertainties of the radionuclides present in the sample. ALPHA determines activities of any alpha emitter in a sample that has been directly precipitated with NdF 3 . AMPU determines the Pu-239, Pu-238,and Am-241 activities using Pu-236 and Am-243 tracers. 2 - Method of solution: These programs propagate all random and systematic uncertainties, found anywhere in the experimental process, to the final result. The result is rounded and is in decimal agreement with the uncertainty. 3 - Restrictions on the complexity of the problem: In ALPHA, a chemical yield of 98% is assumed

Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients.

Science.gov (United States)

Yamamoto, N; Naraparaju, V R; Asbell, S O

1996-06-15

Serum vitamin D3-binding protein (Gc protein) can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor of the macrophage activating factor (MAF). Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high titered MAF, Gc-MAF. When peripheral blood monocytes/macrophages of 52 patients bearing various types of cancer were incubated with 100 pg/ml of GcMAF, the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of patient plasma Gc protein was found to be severely reduced in about 25% of this patient population. About 45% of the patients had moderately reduced MAF precursor activities. Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase detected in the patient's bloodstream. The source of the enzyme appeared to be cancerous cells. Radiation therapy decreased plasma alpha-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting alpha-N-acetylgalactosaminidase. Both alpha-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient bloodstream can serve as diagnostic and prognostic indices.

Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor-{alpha}

Energy Technology Data Exchange (ETDEWEB)

Lee, Ji Hae; Jun, Hee-jin; Hoang, Minh-Hien; Jia, Yaoyao [Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Department of Biotechnology, Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Han, Xiang Hua [College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763 (Korea, Republic of); Lee, Dong-Ho [Department of Biotechnology, Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Lee, Hak-Ju [Division of Green Business Management, Department of Forest Resources Utilization, Korean Forest Research Institute, Seoul 130-712 (Korea, Republic of); Hwang, Bang Yeon, E-mail: byhwang@chungbuk.ac.kr [College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763 (Korea, Republic of); Lee, Sung-Joon, E-mail: junelee@korea.ac.kr [Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Department of Biotechnology, Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of)

2012-06-15

Highlights: Black-Right-Pointing-Pointer Catalposide is a novel ligand for PPAR{alpha}. Black-Right-Pointing-Pointer Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid {beta}-oxidation and synthesis. Black-Right-Pointing-Pointer Catalposdie reduces hepatic triacylglycerides. Black-Right-Pointing-Pointer Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPAR{alpha}) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPAR{alpha} agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPAR{alpha} agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPAR{alpha}. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPAR{alpha} via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

Determination of total alpha activity index in samples of radioactive wastes

International Nuclear Information System (INIS)

Galicia C, F. J.

2015-01-01

This study aimed to develop a methodology of preparation and quantification of samples containing radionuclides beta and/or alpha emitters, to determine the rates of alpha and beta total activity of radioactive waste samples. For this, a device of planchettes preparer was designed, to assist the planchettes preparation in a controlled environment and free of corrosive vapors. Planchettes were prepared in three means: nitrate, carbonate and sulfate, to different mass thickness, natural uranium (alpha and beta emitter) and in case of Sr-90 (beta emitter pure) only in half nitrate; and these planchettes were quantified in an alpha/beta counter, in order to construct the self-absorption curves for alpha and beta particles. These curves are necessary to determine the rate of alpha-beta activity of any sample because they provide the self-absorption correction factor to be applied in calculating the index. Samples with U were prepared with the help of the device of planchettes preparer and subsequently were analyzed in the proportional counter Mpc-100 Pic brand. Samples with Sr-90 were prepared without the device to see if there was a different behavior with respect to obtaining mass thickness. Similarly they were calcined and carried out count in the Mpc-100. To perform the count, first the parameters of counter operating were determined: operating voltages for alpha and beta particles 630 and 1500 V respectively, a count routine was generated where the time and count type were adjusted, and counting efficiencies for alpha and beta particles, with the aid of calibration sources of 210 Po for alphas and 90 Sr for betas. According to the results, the counts per minute will decrease as increasing the mass thickness of the sample (self-absorption curve), adjusting this behavior to an exponential function in all cases studied. The minor self-absorption of alpha and beta particles in the case of U was obtained in sulfate medium. The self-absorption curves of Sr-90 follow the

Unexpected tolerance of alpha-cleavage of the prion protein to sequence variations.

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José B Oliveira-Martins

Full Text Available The cellular form of the prion protein, PrP(C, undergoes extensive proteolysis at the alpha site (109K [see text]H110. Expression of non-cleavable PrP(C mutants in transgenic mice correlates with neurotoxicity, suggesting that alpha-cleavage is important for PrP(C physiology. To gain insights into the mechanisms of alpha-cleavage, we generated a library of PrP(C mutants with mutations in the region neighbouring the alpha-cleavage site. The prevalence of C1, the carboxy adduct of alpha-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the alpha-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, alpha-cleavage was size-dependently impaired by deletions within the domain 106-119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that alpha-cleavage is executed by an alpha-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrP(C.

Affective and cognitive behavior in the alpha-galactosidase A deficient mouse model of Fabry disease.

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Lukas Hofmann

Full Text Available Fabry disease is an X-linked inherited lysosomal storage disorder with intracellular accumulation of globotriaosylceramide (Gb3 due to α-galactosidase A (α-Gal A deficiency. Fabry patients frequently report of anxiety, depression, and impaired cognitive function. We characterized affective and cognitive phenotype of male mice with α-Gal A deficiency (Fabry KO and compared results with those of age-matched male wildtype (WT littermates. Young (3 months and old (≥ 18 months mice were tested in the naïve state and after i.pl. injection of complete Freund`s adjuvant (CFA as an inflammatory pain model. We used the elevated plus maze (EPM, the light-dark box (LDB and the open field test (OF to investigate anxiety-like behavior. The forced swim test (FST and Morris water maze (MWM were applied to assess depressive-like and learning behavior. The EPM test revealed no intergroup difference for anxiety-like behavior in naïve young and old Fabry KO mice compared to WT littermates, except for longer time spent in open arms of the EPM for young WT mice compared to young Fabry KO mice (p<0.05. After CFA injection, young Fabry KO mice showed increased anxiety-like behavior compared to young WT littermates (p<0.05 and naïve young Fabry KO mice (p<0.05 in the EPM as reflected by shorter time spent in EPM open arms. There were no relevant differences in the LDB and the OF test, except for longer time spent in the center zone of the OF by young WT mice compared to young Fabry KO mice (p<0.05. Complementary to this, depression-like and learning behavior were not different between genotypes and age-groups, except for the expectedly lower memory performance in older age-groups compared to young mice. Our results indicate that genetic influences on affective and cognitive symptoms in FD may be of subordinate relevance, drawing attention to potential influences of environmental and epigenetic factors.

Miniature neutron-alpha activation spectrometer

International Nuclear Information System (INIS)

Rhodes, Edgar; Goldsten, John; Holloway, James Paul; He, Zhong

2002-01-01

We are developing a miniature neutron-alpha activation spectrometer for in-situ analysis of chem-bio samples, including rocks, fines, ices, and drill cores, suitable for a lander or Rover platform for Mars or outer-planet missions. In the neutron-activation mode, penetrating analysis will be performed of the whole sample using a γ spectrometer and in the α-activation mode, the sample surface will be analyzed using Rutherford-backscatter and x-ray spectrometers. Novel in our approach is the development of a switchable radioactive neutron source and a small high-resolution γ detector. The detectors and electronics will benefit from remote unattended operation capabilities resulting from our NEAR XGRS heritage and recent development of a Ge γ detector for MESSENGER. Much of the technology used in this instrument can be adapted to portable or unattended terrestrial applications for detection of explosives, chemical toxins, nuclear weapons, and contraband

Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

Science.gov (United States)

Saher, G; Hildt, E

1999-09-24

Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

An enzymatic glycosylation of nucleoside analogues using beta-galactosidase from Escherichia coli

Czech Academy of Sciences Publication Activity Database

Blažek, Jiří; Jansa, Petr; Baszczyňski, Ondřej; Kaiser, Martin Maxmilian; Otmar, Miroslav; Krečmerová, Marcela; Dračínský, Martin; Holý, Antonín; Králová, B.

2012-01-01

Roč. 20, č. 9 (2012), s. 3111-3118 ISSN 0968-0896 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : glycosylation * galactosylation * beta-galactosidase * enzymatic synthesis * nucleoside * acyclic nucleoside analogues Subject RIV: CC - Organic Chemistry Impact factor: 2.903, year: 2012

Survival of freeze-dried microcapsules of α-galactosidase producing probiotics in a soy bar matrix.

Science.gov (United States)

Chen, Mo; Mustapha, Azlin

2012-05-01

Soy oligosaccharides, mainly α-galactosides, are prevalently present in soy protein products, and can result in unfavorable digestive effects when consumed. The aim of this research was to investigate the efficiency of α-galactoside reduction by probiotic bacterial hydrolysis and if such bacteria could be maintained in a high number in a soy protein product in a microencapsulated and freeze-dried form. The probiotic bacterium, Lactobacillus acidophilus LA-2, when induced by raffinose, exhibited a high level of α-galactosidase activity at 5.0 U/mg. To preserve probiotics with high viability, cells were microencapsulated and freeze-dried. Optimization of microencapsulation presented that a combination of κ-carrageenan and inulin at a proportion of 1.9:0.1 (w:w) as capsule wall materials, significantly retained the viability of the probiotics through freeze-drying (P ≤ 0.05). Scanning electron microscopic images confirmed that the morphology of the microcapsules was well preserved after freeze-drying. Upon incorporation into soy protein bars, the freeze-dried microcapsules of L. acidophilus LA-2 remained in high numbers throughout 14 weeks of storage at 4 °C. Results of this work with the support of other studies on microencapsulation benefits indicate a promising use of freeze-dried α-galactosidase positive microencapsulated probiotics in a soy food. Copyright © 2011 Elsevier Ltd. All rights reserved.

Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

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Bin Shan

Full Text Available Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+ and negative (Thy-1- subsets of mouse embryonic fibroblasts (MEF. TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

Beta-galactosidase catalyzed selective galactosylation of aromatic compounds.

Science.gov (United States)

Bridiau, Nicolas; Taboubi, Selma; Marzouki, Nejib; Legoy, Marie Dominique; Maugard, Thierry

2006-01-01

A new approach to galacto-oligosaccharides and galacto-conjugates synthesis performed by the beta-galactosidase from Kluyveromyces lactis is reported. The enzymatic galactosylation of eight kinds of adsorbed aromatic primary alcohols, in particular the two drugs guaifenesin and chlorphenesin, gave the corresponding beta-D-galacto-pyranosides in yields ranging between approximately 10% and 96%. For the first time, we have showed that the adsorption of acceptor substrates onto solid supports such as silica gel influences the yield and the selectivity of galacto-conjugates synthesis. In particular, we observed that adsorption of acceptor favored the synthesis of digalactosylated compounds.

Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres.

Science.gov (United States)

Lin, Jiandie; Wu, Hai; Tarr, Paul T; Zhang, Chen-Yu; Wu, Zhidan; Boss, Olivier; Michael, Laura F; Puigserver, Pere; Isotani, Eiji; Olson, Eric N; Lowell, Bradford B; Bassel-Duby, Rhonda; Spiegelman, Bruce M

2002-08-15

The biochemical basis for the regulation of fibre-type determination in skeletal muscle is not well understood. In addition to the expression of particular myofibrillar proteins, type I (slow-twitch) fibres are much higher in mitochondrial content and are more dependent on oxidative metabolism than type II (fast-twitch) fibres. We have previously identified a transcriptional co-activator, peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1 alpha), which is expressed in several tissues including brown fat and skeletal muscle, and that activates mitochondrial biogenesis and oxidative metabolism. We show here that PGC-1 alpha is expressed preferentially in muscle enriched in type I fibres. When PGC-1 alpha is expressed at physiological levels in transgenic mice driven by a muscle creatine kinase (MCK) promoter, a fibre type conversion is observed: muscles normally rich in type II fibres are redder and activate genes of mitochondrial oxidative metabolism. Notably, putative type II muscles from PGC-1 alpha transgenic mice also express proteins characteristic of type I fibres, such as troponin I (slow) and myoglobin, and show a much greater resistance to electrically stimulated fatigue. Using fibre-type-specific promoters, we show in cultured muscle cells that PGC-1 alpha activates transcription in cooperation with Mef2 proteins and serves as a target for calcineurin signalling, which has been implicated in slow fibre gene expression. These data indicate that PGC-1 alpha is a principal factor regulating muscle fibre type determination.

Synthesis and antimicrobial evaluation of new 3-alkyl/aryl-2-[((alpha,alpha-diphenyl-alpha-hydroxy)acetyl)hydrazono]-5-methyl-4-thiazolidinones.

Science.gov (United States)

Güzeldemirci, Nuray Ulusoy; Ilhan, Eser; Küçükbasmaci, Omer; Satana, Dilek

2010-01-01

New 4-thiazolidinone derivatives of benzilic acid (alpha,alpha-diphenyl-alpha-hydroxyacetic acid) have been synthesized and evaluated for antibacterial and antifungal activities. The reaction of 1- (alpha,alpha-diphenyl-alpha-hydroxy)acetyl-4-alkyl/arylthiosemicarbazides with ethyl 2-bromopropionate gave 3-alkyl/aryl-2-[((alpha,alpha-diphenyl-alpha-hydroxy)acetyl)hydrazono]-5-methyl-4-thiazolidinone derivatives. Their antibacterial and antifungal activities were evaluated against S. aureus ATCC 29213, P. aeruginosa ATCC 27853, E. coli ATCC 25922, C. albicans ATCC 10231, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, T. mentagrophytes var. erinacei NCPF 375, M. gypseum NCPF 580 and T. tonsurans NCPF 245. 3e, 3f, 3g and 3h showed the highest antibacterial activity. Particularly 3a and 3e showed the highest antifungal activities against C. parapsilosis ATCC 22019, T. tonsurans NCPF 245 and M. gypseum NCPF 580.

The determination of plutonium alpha activity in urine, faeces and biological materials

International Nuclear Information System (INIS)

Bains, M.E.D.

1963-07-01

Methods have been developed for the determination of plutonium alpha activity in urine, faeces and biological materials. The chemical stages involved give practically complete separation of all extraneous material from the plutonium, which is electrodeposited on to a 0.5 inch stainless steel disc to produce a thin high resolution source. The limit of detection is 0.025 μμc/sample (sixteen-hour count) when the sources are counted in a small scintillator counter, but is lowest when counted in a counter which counts particles of energy 5.05-5.25 MeV only, and which therefore discriminates against small quantities of α-active materials introduced with the reagents in the final electrodeposition stage of the process. (Any such alpha activity may readily be identified by alpha pulse height analysis). (author)

Nitric oxide production and monoamine oxidase activity in cancer patients during interferon-alpha therapy.

Science.gov (United States)

Fekkes, Durk; Van Gool, Arthur R; Bannink, Marjolein; Sleijfer, Stefan; Kruit, Wim H J; van der Holt, Bronno; Eggermont, Alexander M M; Hengeveld, Michiel W; Stoter, Gerrit

2009-10-01

Both increased and decreased nitric oxide (NO) synthesis have been reported in patients treated with interferon-alpha (IFN-alpha). Animal studies showed that IFN-alpha administration results in increased levels of biogenic amines, subsequent activation of monoamine oxidases (MAOs), and finally in a change in NO production due to the H(2)O(2) generated by MAOs. We examined the potential relationship between NO production in plasma and MAO-B activity in platelets of 43 cancer patients during 8 weeks of treatment with IFN-alpha. NO synthesis was quantitated by measuring both the ratio of citrulline and arginine (CIT/ARG-ratio) and total nitrite/nitrate (NOx) levels. Compared to baseline, MAO activity and NOx increased, while the CIT/ARG-ratio decreased. No associations were found between NOx, MAO and CIT/ARG-ratio. Only few associations were observed between changes in the biochemical parameters and changes in psychopathology induced by IFN-alpha, of which the association between changes in CIT and lassitude was the most consistent. The results suggest that peripheral NO production and MAO activity are unrelated to each other, and that peripheral changes in these biochemical parameters induced by IFN-alpha are unlikely to contribute to definite psychiatric disturbance.

Thioesterase activity and acyl-CoA/fatty acid cross-talk of hepatocyte nuclear factor-4{alpha}.

Science.gov (United States)

Hertz, Rachel; Kalderon, Bella; Byk, Tamara; Berman, Ina; Za'tara, Ghadeer; Mayer, Raphael; Bar-Tana, Jacob

2005-07-01

Hepatocyte nuclear factor-4alpha (HNF-4alpha) activity is modulated by natural and xenobiotic fatty acid and fatty acyl-CoA ligands as a function of their chain length, unsaturation, and substitutions. The acyl-CoA site of HNF-4alpha is reported here to consist of the E-F domain, to bind long-chain acyl-CoAs but not the respective free acids, and to catalyze the hydrolysis of bound fatty acyl-CoAs. The free acid pocket, previously reported in the x-ray structure of HNF-4alpha E-domain, entraps fatty acids but excludes acyl-CoAs. The acyl-CoA and free acid sites are distinctive and noncongruent. Free fatty acid products of HNF-4alpha thioesterase may exchange with free acids entrapped in the fatty acid pocket of HNF-4alpha. Cross-talk between the acyl-CoA and free fatty acid binding sites is abrogated by high affinity, nonhydrolyzable acyl-CoA ligands of HNF-4alpha that inhibit its thioesterase activity. Hence, HNF-4alpha transcriptional activity is controlled by its two interrelated acyl ligands and two binding sites interphased in tandem by the thioesterase activity. The acyl-CoA/free-acid and receptor/enzyme duality of HNF-4alpha extends the paradigm of nuclear receptors.

Real-time MEG neurofeedback training of posterior alpha activity modulates subsequent visual detection performance

NARCIS (Netherlands)

Okazaki, Y.O.; Horschig, J.; Luther, L.M.; Oostenveld, R.; Murakami, I.; Jensen, O.

2015-01-01

It has been demonstrated that alpha activity is lateralized when attention is directed to the left or right visual hemifield. We investigated whether real-time neurofeedback training of the alpha lateralization enhances participants' ability to modulate posterior alpha lateralization and causes

Lactose hydrolysis potential and thermal stability of commercial β-galactosidase in UHT and skimmed milk

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Alessandra BOSSO

2016-03-01

Full Text Available Abstract The commercial enzyme (E.C. = 3.2.1.23 from Kluyveromyces lactis (liquid and Aspergillus oryzae(lyophilized was investigated for its hydrolysis potential in lactose substrate, UHT milk, and skimmed milk at different concentrations (0.7; 1.0 and 1.5%, pH values (5.0; 6.0; 6.5 and 7.0, and temperature (30; 35; 40 and 55 ºC. High hydrolysis rates were observed for the enzyme from K. lactis at pH 7.0 and 40 ºC, and from A. oryzae at pH 5.0 and 55 ºC. The enzyme from K. lactis showed significantly higher hydrolysis rates when compared to A. oryzae. The effect of temperature and β-galactosidase concentration on the lactose hydrolysis in UHT milk was higher than in skimmed milk, for all temperatures tested. With respect to the thermal stability, a decrease in hydrolysis rate was observed at pH 6.0 at 35 ºC for K. lactisenzyme, and at pH 6.0 at 55 ºC for the enzyme from A. oryzae. This study investigate the hydrolysis of β-galactosidase in UHT and skimmed milk. The knowledge about the characteristics of the β-galactosidase fromK. lactis and A. oryzae enables to use it most efficiently to control the enzyme concentration, temperature, and pH in many industrial processes and product formulations.

Loss in photosynthesis during senescence is accompanied by an increase in the activity of β-galactosidase in leaves of Arabidopsis thaliana: modulation of the enzyme activity by water stress.

Science.gov (United States)

Pandey, Jitendra Kumar; Dash, Sidhartha Kumar; Biswal, Basanti

2017-07-01

The precise nature of the developmental modulation of the activity of cell wall hydrolases that breakdown the wall polysaccharides to maintain cellular sugar homeostasis under sugar starvation environment still remains unclear. In this work, the activity of β-galactosidase (EC 3.2.1.23), a cell-wall-bound enzyme known to degrade the wall polysaccharides, has been demonstrated to remarkably enhance during senescence-induced loss in photosynthesis in Arabidopsis thaliana. The enhancement in the enzyme activity reaches a peak at the terminal phase of senescence when the rate of photosynthesis is at its minimum. Although the precise nature of chemistry of the interface between the decline in photosynthesis and enhancement in the activity of the enzyme could not be fully resolved, the enhancement in its activity in dark and its suppression in light or with exogenous sugars may indicate the involvement of loss of photosynthetic production of sugars as a key factor that initiates and stimulates the activity of the enzyme. The hydrolase possibly participates in the catabolic network of cell wall polysaccharides to produce sugars for execution of energy-dependant senescence program in the background of loss of photosynthesis. Drought stress experienced by the senescing leaves accelerates the decline in photosynthesis with further stimulation in the activity of the enzyme. The stress recovery of photosynthesis and suppression of the enzyme activity on withdrawal of stress support the proposition of photosynthetic modulation of the cell-wall-bound enzyme activity.

Structural basis for alpha fetoprotein-mediated inhibition of caspase-3 activity in hepatocellular carcinoma cells.

Science.gov (United States)

Lin, Bo; Zhu, Mingyue; Wang, Wenting; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Guo, Junli; Li, Mengsen

2017-10-01

Alpha-fetoprotein (AFP) is an early serum growth factor in the foetal liver development and hepatic carcinogenesis; However, the precise biological role of cytoplasmic AFP remains elusive. Although we recently demonstrated that cytoplasmic AFP might interact with caspase-3 and inhibit the signal transduction of apoptosis in human hepatocellular carcinoma (HCC) cells, the details of this interaction are not clear. To reveal the molecular relationship between AFP and caspase-3, we performed molecular docking, co-immunoprecipitation (Co-IP), laser confocal microscopy, site-directed mutagenesis and functional experiments to analyse the key amino acid residues in the binding site of caspase-3. The results of Co-IP, laser confocal microscopy and functional analyses were consistent with the computational model. We also used the model to explain why AFP cannot bind to caspase-8. These results provide the molecular basis for the AFP-mediated inhibition of caspase-3 activity in HCC cells. Altogether, we found that AFP interacts with caspase-3 through precise amino acids, namely loop-4 residues Glu-248, Asp-253 and His-257. The results further demonstrated that AFP plays a critical role in the inhibition of the apoptotic signal transduction that mediated by caspase-3. Thus, AFP might represent a novel biotarget for the therapy of HCC patients. © 2017 UICC.

Measurement of conformational constraints in an elastin-mimetic protein by residue-pair selected solid-state NMR

Energy Technology Data Exchange (ETDEWEB)

Hong, Mei [Iowa State University, Department of Chemistry (United States)], E-mail: mhong@iastate.edu; McMillan, R. Andrew; Conticello, Vincent P. [Emory University, Department of Chemistry (United States)

2002-02-15

We introduce a solid-state NMR technique for selective detection of a residue pair in multiply labeled proteins to obtain site-specific structural constraints. The method exploits the frequency-offset dependence of cross polarization to achieve {sup 13}CO{sub i} {sup {yields}} {sup 15}N{sub i} {sup {yields}} {sup 13}C{alpha}{sub i} transfer between two residues. A {sup 13}C, {sup 15}N-labeled elastin mimetic protein (VPGVG){sub n} is used to demonstrate the method. The technique selected the Gly3 C{alpha} signal while suppressing the Gly5 C{alpha} signal, and allowed the measurement of the Gly3 C{alpha} chemical shift anisotropy to derive information on the protein conformation. This residue-pair selection technique should simplify the study of protein structure at specific residues.

2-Azido-( sup 32 P)NAD+, a photoactivatable probe for G-protein structure: Evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of alpha interacts with both alpha and gamma subunits

Energy Technology Data Exchange (ETDEWEB)

Vaillancourt, R.R.; Dhanasekaran, N.; Johnson, G.L.; Ruoho, A.E. (Univ. of Wisconsin Medical School, Madison (USA))

1990-05-01

A radioactive and photoactivatable derivative of NAD+, 2-azido-(adenylate-32P)NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-(adenylate-32P)ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-(adenylate-32P)ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.

Adsorption behavior of alpha -cypermethrin on cork and activated carbon.

Science.gov (United States)

Domingues, Valentina F; Priolo, Giuseppe; Alves, Arminda C; Cabral, Miguel F; Delerue-Matos, Cristina

2007-08-01

Studies were undertaken to determine the adsorption behavior of alpha -cypermethrin [R)-alpha -cyano-3-phenoxybenzyl(1S)-cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate, and (S)-alpha-cyano-3-phenoxybenzyl (1R)-cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate] in solutions on granules of cork and activated carbon (GAC). The adsorption studies were carried out using a batch equilibrium technique. A gas chromatograph with an electron capture detector (GC-ECD) was used to analyze alpha -cypermethrin after solid phase extraction with C18 disks. Physical properties including real density, pore volume, surface area and pore diameter of cork were evaluated by mercury porosimetry. Characterization of cork particles showed variations thereby indicating the highly heterogeneous structure of the material. The average surface area of cork particles was lower than that of GAC. Kinetics adsorption studies allowed the determination of the equilibrium time - 24 hours for both cork (1-2 mm and 3-4 mm) and GAC. For the studied alpha -cypermethrin concentration range, GAC revealed to be a better sorbent. However, adsorption parameters for equilibrium concentrations, obtained through the Langmuir and Freundlich models, showed that granulated cork 1-2 mm have the maximum amount of adsorbed alpha-cypermethrin (q(m)) (303 microg/g); followed by GAC (186 microg/g) and cork 3-4 mm (136 microg/g). The standard deviation (SD) values, demonstrate that Freundlich model better describes the alpha -cypermethrin adsorption phenomena on GAC, while alpha -cypermethrin adsorption on cork (1-2 mm and 3-4 mm) is better described by the Langmuir. In view of the adsorption results obtained in this study it appears that granulated cork may be a better and a cheaper alternative to GAC for removing alpha -cypermethrin from water.

Monitoring of gross alpha, gross beta and tritium activities in portuguese drinking waters

International Nuclear Information System (INIS)

Lopes, I.; Madruga, M.J.; Ferrador, G.O.; Sequeira, M.M.; Oliveira, E.J.; Gomes, A.R.; Rodrigues, F.D.; Carvalho, F.P.

2006-01-01

The gross beta and tritium activities in the forty Portuguese drinking waters analyzed using the ISO standard methods (Portuguese Guidelines) are below the guidance levels proposed in the Portuguese Drinking Water Quality Guidelines. In what concerns the gross alpha activity only 18% exceeded the recommended level. In general, it can be concluded that the ingestion of these drinking waters does not create a radiological hazard to the human consumption, however, more detailed analyses will be necessary mainly the determinations of the individual alpha emitters radionuclide concentrations. The minimum gross alpha and gross beta detectable activities by L.S.C. methodology are higher than for the proportional counting technique (ISO method). Higher concentration factors will be needed to reach lower required detection limits. (authors)

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

Science.gov (United States)

Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

Tumor necrosis factor alpha of teleosts: in silico characterization and homology modeling

Directory of Open Access Journals (Sweden)

Tran Ngoc Tuan

2016-10-01

Full Text Available Tumor necrosis factor alpha (TNF- is known to be crucial in many biological activities of organisms. In this study, physicochemical properties and modeling of TNF- protein of fish was analyzed using in silico approach. TNF- proteins selected from fish species, including grass carp (Ctenopharyngodon idella, zebra fish (Danio rerio, Nile tilapia (Oreochromis niloticus, goldfish (Carassius auratus, and rainbow trout (Oncorhynchus mykiss were used in this study. Physicochemical characteristics with molecular weight, theoretical isoelectric point, extinction coefficient, aliphatic index, instability index, total number of negatively charged residues and positively charged residues, and grand average of hydropathicity were computed. All proteins were classified as transmembrane proteins. The “transmembrane region” and “TNF” domain were identified from protein sequences. The function prediction of proteins was also performed. Alpha helices and random coils were dominating in the secondary structure of the proteins. Three-dimensional structures were predicted and verified as good structures for the investigation of TNF- of fish by online server validation.

Measurement of gross alpha and gross beta activity concentrations in human tooth

International Nuclear Information System (INIS)

Soeguet, Omer; Aydin, Mehmet Fatih; Kuecuekoender, Erdal; Zorer, Ozlem Selcuk; Dogru, Mahmut

2010-01-01

The gross alpha and gross beta activity concentrations were measured in human tooth taken from 3 to 6 age-groups to 40 and over ones. Accumulated teeth samples are investigated in two groups as under and above 18 years. The gross alpha and beta radioactivity of human tooth samples was measured by using a gas-flow proportional counter (PIC-MPC 9604-α/β counter). In tooth samples, for female age-groups, the obtained results show that the mean gross alpha and gross beta activity concentrations varied between 0.534-0.203 and 0.010-0.453 Bq g -1 and the same concentrations for male age-groups varied between 0.009-1.168 and 0.071-0.204 Bq g -1 , respectively.

Evidence that 17alpha-estradiol is biologically active in the uterine tissue: Antiuterotonic and antiuterotrophic action

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Navarrete Erika

2005-07-01

Full Text Available Abstract Background 17alpha-Estradiol has been considered as the hormonally inactive isomer of 17beta-estradiol. Recently, nongenomic (smooth muscle relaxation and genomic (light estrogenic activity effects of 17alpha-estradiol have been reported, but no reports have yet determined its possible antiestrogenic activity. Therefore, this study investigated: the nongenomic action of 17alpha-estradiol on uterine contractile activity and its potential agonist-antagonist activity on uterine growth. Methods Uterine rings from rats were isometrically recorded. Different concentrations (0.2–200 microM of 17alpha-estradiol were tested on spontaneous contraction and equimolarly compared with 17beta-estradiol. To examine the mechanism of 17alpha-estradiol action, its effect was studied in presence of beta2-antagonist (propranolol, antiestrogens (tamoxifen and ICI 182,780 or inhibitors of protein synthesis (cycloheximide and transcription (actinomycin D. Moreover, contractions induced by high potassium (KCl solution or calcium in depolarized tissues by KCl-calcium free solution were exposed to 17alpha-estradiol. Collaterally, we performed an uterotrophic assay in adult ovariectomized rats measuring the uterine wet weight. The administration for three days of 0.3 microM/day/Kg 17beta-estradiol was equimolarly compared with the response produced by 17alpha-estradiol. Antiuterotrophic activity was assayed by administration of 0.3 microM/day/Kg 17beta-estradiol and various doses ratios (1:1, 1:3, 1:5, and 1:100 of 17alpha-estradiol. Results The estradiol isomers elicited an immediate relaxation, concentration-dependent and reversible on spontaneous contraction. 17alpha-Estradiol presented lower potency than 17beta-estradiol although it did not antagonize 17beta-estradiol-induced relaxation. Relaxation to 17alpha-estradiol was not inhibited by propranolol, tamoxifen, ICI 182,780, cycloheximide or actinomycin D. The KCl contractions were also sensitive to 17alpha

A pilot trial on subjects with lactose and/or oligosaccharides intolerance treated with a fixed mixture of pure and enteric-coated α- and ß-galactosidase

Directory of Open Access Journals (Sweden)

Di Pierro F

2015-02-01

Full Text Available Francesco Di Pierro,1 Alexander Bertuccioli,2 Eleonora Marini,3 Leandro Ivaldi4 1Velleja Research, Milan, Italy; 2Italian Association Fitness and Medicine, Fano, PU, Italy; 3Pharmextracta, Pontenure, Piacenza, Italy; 4Digestive Endoscopic Department, Ceva Hospital, Ceva, Cuneo, Italy Aim: Lactose and complex carbohydrates maldigestion, common food intolerances due to low gut content of α- and ß-galactosidase, lead to abdominal symptoms including pain, diarrhea, bloating, flatulence, and cramping. Commonly, intolerant patients are advised by physicians to avoid the offending foods (dairy foods, cereals, beans, etc. This food-limiting option, however, has possible nutritional risks. We have therefore evaluated the impact of using pure, enteric-coated α- plus ß-galactosidase on gut symptoms in intolerant subjects instead of avoidance of the offending foods. Methods: Sixteen subjects intolerant to lactose and/or complex carbohydrates were enrolled and evaluated in terms of gut symptoms with 1 uncontrolled diet, 2 diet devoid of offending foods, and 3 uncontrolled diet along with pure, enteric-coated α- and ß-galactosidase (DDM Galactosidase®. Results: Even with the uncontrolled diet, intolerant subjects treated with DDM Galactosidase® exhibited reduced gut symptoms (bloating, flatulence, diarrhea, and constipation significantly better than the control treatment as well as having a diet devoid of offending foods. Conclusion: DDM Galactosidase® is a valid and safe optional treatment to counteract lactose and complex carbohydrate intolerance in subjects who prefer not to avoid, at least partially, offending foods. Keywords: lactase, lactose intolerance, complex carbohydrate intolerance

The T alpha 2 nuclear protein binding site from the human T cell receptor alpha enhancer functions as both a T cell-specific transcriptional activator and repressor

OpenAIRE

1990-01-01

T cell-specific expression of the human T cell receptor alpha (TCR- alpha) gene is regulated by the interaction of variable region promoter elements with a transcriptional enhancer that is located 4.5 kb 3' of the TCR-alpha constant region (C alpha) gene segment. The minimal TCR- alpha enhancer is composed of two nuclear protein binding sites, T alpha 1 and T alpha 2, that are both required for the T cell-specific activity of the enhancer. The T alpha 1 binding site contains a consensus cAMP ...

Alpha-amylase activity in wheat flour and breadmaking properties in relation to different climatic conditions

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Rakita Slađana M.

2015-01-01

Full Text Available The aim of the present paper was to evaluate the influence of different climatic conditions on the activity of alpha-amylase in wheat samples and bread quality parameters. Three wheat varieties grown in three different localities in three years were chosen for this study. Commonly used methods for estimation of alpha-amylase activity in wheat grain were employed. The obtained results indicated that harvest year 2013, which was characterized with the excessive amount of rainfall, exhibited the highest level of alpha-amylase activity and the lowest values of the peak viscosity. The lowest alpha-amylase level and the highest peak viscosity and FN value were observed for samples harvested in 2012 which was characterized with the greatest number of days with an average daily temperature above 30 and 35°C. In addition, a decrease in Mixolab parameter torque C3 and specific bread loaf volume, as well as increase in the breakdown torque (C3-C4 of samples harvested in 2013 were observed, which could be attributed to rainy weather influencing increase in alpha-amylase activity. It is found that specific bread loaf volume of wheat samples is highest in 2012. Moreover, a negative correlation between alpha-amylase activity and specific bread volume for all the samples grown in three years was determined.

Measurement of natural radioactivity in chemical fertilizer and agricultural soil: evidence of high alpha activity.

Science.gov (United States)

Ghosh, Dipak; Deb, Argha; Bera, Sukumar; Sengupta, Rosalima; Patra, Kanchan Kumar

2008-02-01

People are exposed to ionizing radiation from the radionuclides that are present in different types of natural sources, of which phosphate fertilizer is one of the most important sources. Radionuclides in phosphate fertilizer belonging to 232Th and 238U series as well as radioisotope of potassium (40K) are the major contributors of outdoor terrestrial natural radiation. The study of alpha activity in fertilizers, which is the first ever in West Bengal, has been performed in order to determine the effect of the use of phosphate fertilizers on human health. The data have been compared with the alpha activity of different types of chemical fertilizers. The measurement of alpha activity in surface soil samples collected from the cultivated land was also performed. The sampling sites were randomly selected in the cultivated land in the Midnapore district, which is the largest district in West Bengal. The phosphate fertilizer is widely used for large agricultural production, mainly potatoes. The alpha activities have been measured using solid-state nuclear track detectors (SSNTD), a very sensitive detector for alpha particles. The results show that alpha activity of those fertilizer and soil samples varies from 141 Bq/kg to 2,589 Bq/kg and from 109 Bq/kg to 660 Bq/kg, respectively. These results were used to estimate environmental radiation exposure on human health contributed by the direct application of fertilizers.

Simultaneous knock-down of six β-galactosidase genes in petunia petals prevents loss of pectic galactan but decreases petal strength.

Science.gov (United States)

O'Donoghue, Erin M; Somerfield, Sheryl D; Deroles, Simon C; Sutherland, Paul W; Hallett, Ian C; Erridge, Zoë A; Brummell, David A; Hunter, Donald A

2017-04-01

Galactose (Gal) is incorporated into cell wall polysaccharides as flowers open, but then is lost because of β-galactosidase activity as flowers mature and wilt. The significance of this for flower physiology resides in the role of galactan-containing polysaccharides in the cell wall, which is still largely unresolved. To investigate this, transcript accumulation of six cell wall-associated β-galactosidases was simultaneously knocked down in 'Mitchell' petunia (Petunia axillaris x (P. axillaris x P. hybrida)) flower petals. The multi-PhBGAL RNAi construct targeted three bud- and three senescence-associated β-galactosidase genes. The petals of the most down-regulated line (GA19) were significantly disrupted in galactose turnover during flower opening, and at the onset of senescence had retained 86% of their galactose compared with 20% in the controls. The Gal content of Na 2 CO 3 -soluble cell wall extracts and the highly insoluble polysaccharides associated with cellulose were particularly affected. Immunodetection with the antibody LM5 showed that much of the cell wall Gal in GA19 was retained as galactan, presumably the side-chains of rhamnogalacturonan-I. The flowers of GA19, despite having retained substantially more galactan, were no different from controls in their internal cell arrangement, dimensions, weight or timing of opening and senescence. However, the GA19 petals had less petal integrity (as judged by force required to cause petal fracture) after opening and showed a greater decline in this integrity with time than controls, raising the possibility that galactan loss is a mechanism for helping to maintain petal tissue cohesion after flower opening. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Nucleophilic behavior of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase consistent with a role in binding adenosine triphosphate

International Nuclear Information System (INIS)

Xu, K.Y.; Kyte, J.

1989-01-01

An immunoadsorbent specific for the carboxy-terminal sequence -GAPER, which comprises residues 502-506 of the alpha-polypeptide of ovine sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase], was used to isolate the products of the reaction between the lysine immediately preceding this sequence in the intact protein and either [3H]acetic anhydride or fluorescein 5'-isothiocyanate. Changes in the apparent nucleophilicity of this lysine, Lys501, were observed with both reagents when ATP was bound by the intact, native enzyme poised in the E1 conformation or when the structure of the enzyme was changed from the E1 conformation into the E2-P conformation. With both reagents, a decrease of more than 4-fold in the yield of incorporation occurred during the former change, but a decrease of only 2-fold occurred during the latter. Because a much larger decrease occurred when ATP was bound in the absence of a conformational change than occurred when a major conformational change took place in the absence of the occupation of the active site, these changes in the incorporation of [3H]acetyl suggest that Lys501 from the alpha polypeptide is directly involved in binding ATP within the active site of (Na+ + K+)-ATPase. The immunochemical reactions between the specific polyclonal antibodies raised against the sequence-GAPER and denatured or enzymically active (Na+ + K+)-ATPase were also investigated. Western blots and the inhibition of enzymic activity caused by the antibody have shown that it can bind to both the denatured and the native form of the alpha-polypeptide, respectively

alpha-Tubulin of Histriculus cavicola (Ciliophora; Hypotrichea).

Science.gov (United States)

Pérez-Romero, P; Villalobo, E; Díaz-Ramos, C; Calvo, P; Santos-Rosa, F; Torres, A

1997-03-01

An alpha-tubulin gene fragment amplified by PCR from the hypotrichous ciliate Histriculus cavicola has been sequenced. This fragment, 1,182 bp long, contains an in-frame "stop" codon (UAA), which in other hypotrichous species codes for a glutamine residue. The comparison of the alpha-tubulin genes from several ciliates classes have revealed amino acid positions which could serve to distinguish these taxonomic groups.

Conceptual design report for alpha waste incinerator

International Nuclear Information System (INIS)

1979-04-01

The Alpha Waste Incinerator, a new facility in the SRP H-Area, will process transuranic or alpha-contaminated combustible solid wastes. It will seal the radioactive ash and scrubbing salt residues in cans for interim storage in drums on site burial ground pads. This report includes objectives, project estimate, schedule, standards and criteria, excluded costs, safety evaluation, energy consumption, environmental assessment, and key drawings

Properties of a new fungal b-galactosidase with potential application in the dairy industry Propriedades de uma nova b-galactosidade fúngica com potencialidade para emprego na indústria de latícinios

Directory of Open Access Journals (Sweden)

Rubens Cruz

1999-07-01

Full Text Available b-Galactosidase or b-D-galactoside-galactohydrolase (EC. 3.2.1.23 is an important enzyme industrially used for the hydrolysis of lactose from milk and milk whey for several applications. Lately, the importance of this enzyme was enhanced by its galactosyltransferase activity, which is responsible for the synthesis of transgalactosylated oligosaccharides (TOS that act as functional foods, with several beneficial effects on consumers. Penicillium simplicissimum, a strain isolated from soil, when grown in semi-solid medium showed good productivity of b-galactosidase with galactosyltransferase activity. The optimum pH for hydrolysis was in the 4.04.6 range and the optimum pH for galactosyltransferase activity was in the 6.07.0 range. The optimum temperature for hydrolysis and transferase activity was 55-60°C and 50°C, respectively, and the enzyme showed high thermostability for the hydrolytic activity. The enzyme showed a potential for several industrial applications such as removal of 67% of the lactose from milk and 84% of the lactose from milk whey when incubated at their original pH (4.5 and 6.34, respectively under optimum temperature conditions. When incubated with a 40% lactose solution in 150 mM McIlvaine buffer, pH 4.5, at 55°C the enzyme converted 86.5% of the lactose to its component monosaccharides. When incubated with a 60% lactose solution in the same buffer but at pH 6.5 and 50°C, the enzyme can synthetize up to 30.5% TOS, with 39.5% lactose and 30% monosaccharides remaining in the preparation.A b-galactosidase ou b-D-galactosídeo-galactohidrolase (EC. 3.2.1.23 é uma importante enzima utilizada industrialmente na hidrólise da lactose do leite e soro de leite para diversas aplicações. Ultimamente, sua importância foi realçada por sua atividade de galactosiltransferase responsável pela síntese de oligossacárides transgalactosilados (TOS que atuam como alimentos funcionais, trazendo diversos efeitos benéficos para seus

Discovery of Intermediates of lacZ β-Galactosidase Catalyzed Hydrolysis Using dDNP NMR

DEFF Research Database (Denmark)

Kjeldsen, Christian; Ardenkjær-Larsen, Jan Henrik; Duus, Jens Ø.

2018-01-01

Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state 13C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ β-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction...

Discovery of Intermediates of lacZ beta-Galactosidase Catalyzed Hydrolysis Using dDNP NMR

DEFF Research Database (Denmark)

Kjeldsen, Christian; Ardenkjær-Larsen, Jan Henrik; Duus, Jens Øllgaard

2018-01-01

Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state C-13 NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ beta-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction...

Alpha coma in an adolescent with diabetic ketoacidosis.

Science.gov (United States)

Ostojic, Slavica; Vukovic, Rade; Milenkovic, Tatjana; Mitrovic, Katarina; Djuric, Milena; Nikolic, Ljubica

2017-01-01

Ostojic S, Vukovic R, Milenkovic T, Mitrovic K, Djuric M, Nikolic L. Alpha coma in an adolescent with diabetic ketoacidosis. Turk J Pediatr 2017; 59: 318-321. This is the first report of alpha coma (AC) caused by brain edema in a patient with diabetic ketoacidosis (DKA). A previously healthy 15-year-old girl was admitted to the intensive care unit due to altered state of consciousness during the course of treatment for DKA. Patient was in a coma, intubated and had tachycardia with poor peripheral perfusion. Results of laboratory analyses indicated severe DKA and computed tomography scan indicated diffuse brain edema. The EEG pattern showed uniform alpha activity. Treatment with intravenous fluids, insulin and mannitol was started. Patient`s state of consciousness gradually improved and on the third day she was extubated. On the fifth day, her neurologic status and EEG findings were completely normal with no residual neurological deficits. In conclusion, although AC is associated with a high fatality rate, favorable outcome can be achieved with prompt recognition and treatment of cerebral edema in pediatric patients with DKA.

Alpha-in-air monitor for continuous monitoring based on alpha to beta ratio

International Nuclear Information System (INIS)

Somayaji, K.S.; Venkataramani, R.; Swaminathan, N.; Pushparaja

1997-01-01

Measurement of long-lived alpha activity collected on a filter paper in continuous air monitoring of ambient working environment is difficult due to interference from much larger concentrations of short-lived alpha emitting daughter products of 222 Rn and 220 Rn. However, the ratio between the natural alpha and beta activity is approximately constant and this constancy of the ratio is used to discriminate against short-lived natural radioactivity in continuous air monitoring. Detection system was specially designed for the purpose of simultaneous counting of alpha and beta activity deposited on the filter paper during continuous monitoring. The activity ratios were calculated and plotted against the monitoring duration up to about six hours. Monitoring was carried out in three facilities with different ventilation conditions. Presence of any long-lived alpha contamination on the filter paper results in increase in the alpha to beta ratio. Long-lived 239 Pu contamination of about 16 DAC.h could be detected after about 45 minutes of commencement of the sampling. The experimental results using prototype units have shown that the approach of using alpha to beta activity ratio method to detect long-lived alpha activity in the presence of short-lived natural activity is satisfactory. (author)

Presence of fucosyl residues on the oligosaccharide antennae of membrane glycopeptides of human neuroblastoma cells

International Nuclear Information System (INIS)

Santer, U.V.; Glick, M.C.

1983-01-01

Fucosyl residues linked alpha 1 leads to 3 or 4 to N-acetylglucosamine were found in large amounts on glycopeptides from the membranes of human tumor cells of neurectodermal origin but not on membrane glycopeptides from human fibroblasts. The fucosyl residues were detected by release of radioactive fucose from the glycopeptides with an almond alpha-L-fucosidase specific for fucosyl alpha 1 leads to 3(4)-N-acetylglucosamine. In other studies, the linkage was shown to be alpha 1 leads to 3 by nuclear magnetic resonance analysis. Glycopeptides containing these fucosyl residues from four human neuroblastoma cell lines were defined by binding to immobilized lectins. In addition, the glycopeptides from one human neuroblastoma cell line, CHP-134, were further characterized by enzyme degradation and columns calibrated for size and charge. The antennary position of fucosyl alpha 1 leads to 3-N-acetylglucosamine on the glycopeptides was demonstrated by the use of exoglycosidases and endoglycosidase D, since complete degradation to yield fucosyl-N-acetylglucosaminylasparagine was obtained only after treatment with almond alpha-L-fucosidase prior to the sequential degradation. Fucosyl alpha 1 leads to 3-N-acetylglucosamine was present on most size and charge classes of membrane glycopeptides and therefore was not limited to a few glycoproteins. Since the almond alpha-L-fucosidase cleaves fucosyl residues from glycoproteins, the physiological effects of the increased specific fucosylation on human tumors of neurectodermal origin can be examined

A survey of gross alpha and gross beta activity in soil samples in Kinta District, Perak, Malaysia

International Nuclear Information System (INIS)

Lee, Siak Kuan; Wagiran, Husin; Ramli, Ahmad Termizi

2014-01-01

The objective of this study was to determine the gross alpha and gross beta activity concentrations from the different soil types found in the Kinta District, Perak, Malaysia. A total of 128 soil samples were collected and their dose rates were measured 1 m above the ground. Gross alpha and gross beta activity measurements were carried out using gas flow proportional counter, Tennelec Series 5 LB5500 Automatic Low Background Counting System. The alpha activity concentration ranged from 15 to 9634 Bq kg -1 with a mean value of 1558±121 Bq kg -1 . The beta activity concentration ranged from 142 to 6173 Bq kg -1 with a mean value of 1112±32 Bq kg -1 . High alpha and beta activity concentrations are from the same soil type. The results of the analysis show a strong correlation between the gross alpha activity concentration and dose rate (R = 0.92). The data obtained can be used as a database for each soil type. (authors)

Estimation of low level gross alpha activities in the radioactive effluent using liquid scintillation counting technique

International Nuclear Information System (INIS)

Bhade, Sonali P.D.; Johnson, Bella E.; Singh, Sanjay; Babu, D.A.R.

2012-01-01

A technique has been developed for simultaneous measurement of gross alpha and gross beta activity concentration in low level liquid effluent samples in presence of higher activity concentrations of tritium. For this purpose, alpha beta discriminating Pulse Shape Analysis Liquid Scintillation Counting (LSC) technique was used. Main advantages of this technique are easy sample preparation, rapid measurement and higher sensitivity. The calibration methodology for Quantulus1220 LSC based on PSA technique using 241 Am and 90 Sr/ 90 Y as alpha and beta standards respectively was described in detail. LSC technique was validated by measuring alpha and beta activity concentrations in test samples with known amount of 241 Am and 90 Sr/ 90 Y activities spiked in distilled water. The results obtained by LSC technique were compared with conventional planchet counting methods such as ZnS(Ag) and end window GM detectors. The gross alpha and gross beta activity concentrations in spiked samples, obtained by LSC technique were found to be within ±5% of the reference values. (author)

Alpha activity of 190 Pt isotope measured with CR-39 track detector

International Nuclear Information System (INIS)

Tavares, O.A.P.; Terranova, M.L.

1996-11-01

A new method to measure alpha activity of long-lived radioisotopes is reported. The method consists basically in using CR-39 track detectors in close contact with thick samples of the radioelement to be investigated. Accordingly, a long-term exposure experiment has been performed using metallic sheets of natural platinum to measure alpha activity of platinum 190 isotope. The half-life of platinum 190 has been obtained in good agreement with two recent theoretical half-life predictions. (author). 21 refs., 3 figs., 2 tabs

Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions

DEFF Research Database (Denmark)

Jensen, Torbjørn Ølshøj; Pogrebnyakov, Ivan; Falkenberg, Kristoffer Bach

2017-01-01

Use of thermophilic organisms has a range of advantages, but the significant lack of engineering tools limits their applications. Here we show that β-galactosidase from Geobacillus stearothermophilus (BgaB) can be applicable in a range of conditions, including different temperatures and oxygen...... and encompassed fivefold variation. The experimental pipeline allowed construction and measurement of expression levels of the library in just 4 days. This β-galactosidase provides a promising tool for engineering of aerobic, anaerobic, and thermophilic production organisms such as Geobacillus species....

Determination of total alpha and beta activities on vegetable samples by LSC

International Nuclear Information System (INIS)

Nogueira, Regina Apolinaria; Santos, Eliane Eugenia dos; Bakker, Alexandre Pereira; Vavassori, Giullia

2011-01-01

Gross alpha and beta analyses are screening techniques used for environmental radioactivity monitoring. The present study proposes to determine the gross alpha and beta activities in vegetable samples by using LSC - liquid scintillation spectrometry. The procedure was applied to vegetable foods. After ashing vegetable samples in a muffle furnace, 100 mg of ash were added to gel mixture of scintillation cocktails, Water - Instagel - Ultima Gold AB (6:10:4) ml, in polyethylene vial. Am-241 standard solution and a KCl (K-40) solution were used to determine the counting configuration, alpha/beta efficiencies and spillover

Activity, splice variants, conserved peptide motifs, and phylogeny of two new alpha1,3-fucosyltransferase families (FUT10 and FUT11).

Science.gov (United States)

Mollicone, Rosella; Moore, Stuart E H; Bovin, Nicolai; Garcia-Rosasco, Marcela; Candelier, Jean-Jacques; Martinez-Duncker, Iván; Oriol, Rafael

2009-02-13

We report the cloning of three splice variants of the FUT10 gene, encoding for active alpha-l-fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related alpha-l-fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other alpha1,3-fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH(2) termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link alpha-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic alpha1,3-fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an alpha1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude alpha1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta alpha-N-acetylglucosaminidase.

Location of 3-hydroxyproline residues in collagen types I, II, III, and V/XI implies a role in fibril supramolecular assembly.

Science.gov (United States)

Weis, Mary Ann; Hudson, David M; Kim, Lammy; Scott, Melissa; Wu, Jiann-Jiu; Eyre, David R

2010-01-22

Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro(986) in A-clade chains alpha1(I), alpha1(II), and alpha2(V). Two partially modified sites (A2 and A3) were found at Pro(944) in alpha1(II) and alpha2(V) and Pro(707) in alpha2(I) and alpha2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro(470) in alpha2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian alpha1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.

Traditionally used plants in diabetes therapy: phytotherapeutics as inhibitors of alpha-amylase activity

Directory of Open Access Journals (Sweden)

Ingrid Funke

Full Text Available Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycaemia. There are many and diverse therapeutic strategies in the management of Type 2 diabetes. The inhibition of alpha-amylase activity is only one possibility to lower postprandial blood glucose levels. In our in-vitro studies we could demonstrate that different plants, mostly traditionally used in common diabetic therapy in Africa or Europe, are able to inhibit alpha-amylase, which is responsible for the breakdown of oligosaccharides into monosaccharides which are absorbed. An inhibition of alpha-amylase activity of 90% was seen with the extract of the leaves of Tamarindus indica. To quantify inhibtion rates, acarbose was used (IC50: 23.2 µM. Highest inhibition level of acarbose in our testmodel was about 85%. Additionally tests with pure polyphenolic compounds might explain the biological activity of the selected plants.

Probing the dynamic interface between trimethylamine dehydrogenase (TMADH) and electron transferring flavoprotein (ETF) in the TMADH-2ETF complex: role of the Arg-alpha237 (ETF) and Tyr-442 (TMADH) residue pair.

Science.gov (United States)

Burgess, Selena G; Messiha, Hanan Latif; Katona, Gergely; Rigby, Stephen E J; Leys, David; Scrutton, Nigel S

2008-05-06

We have used multiple solution state techniques and crystallographic analysis to investigate the importance of a putative transient interaction formed between Arg-alpha237 in electron transferring flavoprotein (ETF) and Tyr-442 in trimethylamine dehydrogenase (TMADH) in complex assembly, electron transfer, and structural imprinting of ETF by TMADH. We have isolated four mutant forms of ETF altered in the identity of the residue at position 237 (alphaR237A, alphaR237K, alphaR237C, and alphaR237E) and with each form studied electron transfer from TMADH to ETF, investigated the reduction potentials of the bound ETF cofactor, and analyzed complex formation. We show that mutation of Arg-alpha237 substantially destabilizes the semiquinone couple of the bound FAD and impedes electron transfer from TMADH to ETF. Crystallographic structures of the mutant ETF proteins indicate that mutation does not perturb the overall structure of ETF, but leads to disruption of an electrostatic network at an ETF domain boundary that likely affects the dynamic properties of ETF in the crystal and in solution. We show that Arg-alpha237 is required for TMADH to structurally imprint the as-purified semiquinone form of wild-type ETF and that the ability of TMADH to facilitate this structural reorganization is lost following (i) redox cycling of ETF, or simple conversion to the oxidized form, and (ii) mutagenesis of Arg-alpha237. We discuss this result in light of recent apparent conflict in the literature relating to the structural imprinting of wild-type ETF. Our studies support a mechanism of electron transfer by conformational sampling as advanced from our previous analysis of the crystal structure of the TMADH-2ETF complex [Leys, D. , Basran, J. , Sutcliffe, M. J., and Scrutton, N. S. (2003) Nature Struct. Biol. 10, 219-225] and point to a key role for the Tyr-442 (TMADH) and Arg-alpha237 (ETF) residue pair in transiently stabilizing productive electron transfer configurations. Our work

Causality analysis of alpha activities by multidimensional directed coherence; Tajigen yuko coherence ni yoru {alpha}ritsudo no ingasei kaiseki

Energy Technology Data Exchange (ETDEWEB)

Sakata, O.; Shimada, N.; Shiina, T. [University of Tsukuba, Tsukuba (Japan); Saito, Y.; Imanishi, N.

1998-07-01

Alpha activities as a basic component of EEG (electroencephalogram) are mainly observed with eye-closed and reported state, and indicates rhythmic and diffused pattern on the scalp. Therefore analysis of the relation among many sequences of alpha activities measured at different positions on the scalp is expected to be useful not only for diagnosing psychiatric but also for investigating mechanism of brain information processing by means of causality analysis that is, macroscopic estimation of flow pattern within brain. Although coherence analysis has been proposed as a method for estimating the direction and magnitude of information flow between two sequences, superposition of results for each pair of sequences can not represent true relation among the whole sequences. In this paper, we proposed the multidimensional directed coherence analysis by modifying two-channel formula in order to apply it to the analysis of multi-channel sequence of alpha activities. Results of simulation revealed that multidimensional directed coherence can indicate more quantitatively the relation among the multi-channel sequences compared with conventional two-channel formula. Moreover, the proposed method was applied to the analysis of EEG data of normal volunteer and patient. Results show the method can provide a useful diagnostic information by assessment of the signal flow pattern within brain. 16 refs., 10 figs.

Characterization of ß-Galactosidase Isoforms from Bacillus circulans and Their Contribution to GOS Production

NARCIS (Netherlands)

Warmerdam, A.; Paudel, E.; Wanqing, J.; Boom, R.M.; Janssen, A.E.M.

2013-01-01

A ß-galactosidase preparation from Bacillus circulans consists of four isoforms called ß-gal-A, ß-gal-B, ß-gal-C, and ß-gal-D. These isoforms differ in lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis at low substrate concentrations. For this reason, using a selection of the isoforms

Uranium trace and alpha activity characterization of coal and fly ash using particle track etch technique

International Nuclear Information System (INIS)

Chakravarti, S.K.

1991-01-01

Uranium is extensively found in carbonaceous components of sedimentary rocks and is considered to be accumulated in coals during the coalification process through the geological times. Burning of coal is mainly responsible for a manifold increase in the concentration of radioactive nuclides in atmosphere precipitates. Fly ash being an incombustible residue and formed from 90% of the inorganic material in coal, escapes into the atmosphere and constitutes a potential hazard. Also its use as one of the pozzolanic materials in the products of concrete, bricks etc and filling of ground cavities is even more hazardous because of the wall radioactivity, besides emission and diffusion of radon. This paper reports a simple method called Particle Track Etch (PTE) technique, for trace determination of uranium content in coal and fly ash samples by making use of low cost and versatile plastic detectors known as Solid State Nuclear Track Detectors (SSNTDs). Total alpha activity has also been estimated using these SSNTDs. The values of uranium concentration in coal samples are found to range from 1.1 to 3.6 ppm (uniform component) and 33 to 46 ppm (non-uniform part) whereas in fly ash, it varies from 8 to 11 ppm (uniform) and 55 to 71 ppm in non-uniform range. It is also observed that the alpha activity is a function of uranium concentration for most of the natural samples of coal studied except for mixtures of fly ash samples where relationship is found to be on higher side. (author). 13 refs., 2 tabs., 1 fig

Effects of central imidazolinergic and alpha2-adrenergic activation on water intake

Directory of Open Access Journals (Sweden)

Sugawara A.M.

2001-01-01

Full Text Available Non-adrenergic ligands that bind to imidazoline receptors (I-R, a selective ligand that binds to alpha2-adrenoceptors (alpha2-AR and mixed ligands that bind to both receptors were tested for their action on water intake behavior of 24-h water-deprived rats. All drugs were injected into the third cerebral ventricle. Except for agmatine (80 nmol, mixed ligands binding to I-R/alpha2-AR such as guanabenz (40 nmol and UK 14304 (20 nmol inhibited water intake by 65% and up to 95%, respectively. The selective non-imidazoline alpha2-AR agonist, alpha-methylnoradrenaline, produced inhibition of water intake similar to that obtained with guanabenz, but at higher doses (80 nmol. The non-adrenergic I-R ligands histamine (160 nmol, mixed histaminergic and imidazoline ligand and imidazole-4-acetic acid (80 nmol, imidazoline ligand did not alter water intake. The results show that selective, non-imidazoline alpha2-AR activation suppresses water intake, and suggest that the action on imidazoline sites by non-adrenergic ligands is not sufficient to inhibit water intake.

A Trp474Cys mutation in the alpha-subunit of beta-hexosaminidase causes a subacute encephalopathic form of G{sub M2} gangliosidosis, type 1

Energy Technology Data Exchange (ETDEWEB)

Petroulakis, E.; Cao, Z.; Salo, T. [Univ. of Manitoba, Winnipeg (Canada)] [and others

1994-09-01

Mutations in the HEXA gene that encodes the {alpha}-subunit of the heterodimeric lysosomal enzyme {beta}-hexosaminidase A, or Hex A ({alpha}{beta}), cause G{sub M2} gangliosidosis, type 1. The infantile form (Tay-Sachs disease) results when there is no residual Hex A activity, while less severe and more variable clinical phenotypes result when residual Hex A activity is present. A non-Jewish male who presented with an acute psychotic episode at age 16 was diagnosed with a subacute encephalopathic form of G{sub M2} gangliosidosis. At age 19, chronic psychosis with intermittent acute exacerbations remains the most disabling symptom in this patient and his affected brother although both exhibit some ataxia and moderately severe dysarthria. We have found a 4 bp insertion (+TATC 1278) associated with infantile Tay-Sachs disease on one allele; no previously identified mutation was found on the second allele. SSCP analysis detected a shift in exon 13 and sequencing revealed a G1422C mutation in the second allele that results in a Trp474Cys substitution. The presence of the mutation was confirmed by the loss of HaeIII and ScrFI sites in exon 13 PCR products from the subjects and their father. The mutation was introduced into the {alpha}-subunit cDNA and Hex S ({alpha}{alpha}) and Hex A ({alpha}{beta}) were transiently expressed in monkey COS-7 cells. The Trp474Cys mutant protein had approximately 5% and 12% of wild-type Hex S and Hex A activity, respectively. Western blot analysis revealed a small amount of residual mature {alpha}-subunit and a normal level of precursor protein. We conclude that the Trp474Cys mutation is the cause of the Hex A deficiency associated with a subacute (juvenile-onset) phenotype in this patient. Like other mutations in exon 13 of HEXA, it appears to affect intracellular processing. Studies of the defect in intracellular processing are in progress.

Chaperone-like activity of β-casein and its effect on residual in vitro activity of horseradish peroxidase

DEFF Research Database (Denmark)

Sulewska, Anna Maria; Olsen, Karsten; Sørensen, Jens Christian

2014-01-01

, as similar experiment with bovine serum albumin resulted in residual activity of horseradish peroxidase that was significantly lower than without any addition. The effect of β-casein on HRP disappears when pH is below the isoelectric point of β-casein. It was also proven by light scattering studies that β...... proteins. Incubating HRP (0.1 mg mL-1) for 10 min at 72 °C resulted in residual activity of 59 ± 5%, while addition of 1 mg mL-1 β-casein resulted in increase in residual activity up to 85 ± 1%. Increased residual activity is not merely attributed to an effect of higher total protein concentration......-casein interacts with horseradish peroxidase when the temperature was increased from 25 to 70 °C whereas interactions seem to cease when temperature was lowered back to 25 °C. This study highlights how specific proteins can influence enzyme activity, which is of potential importance for various industries...

Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

Energy Technology Data Exchange (ETDEWEB)

McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

1986-03-05

A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

Gas lantern mantle: a low activity alpha particle source

International Nuclear Information System (INIS)

Mukherjee, B.; Manzoor, S.

1991-01-01

Commercially available gas lantern mantles contain a substantial amount of radioactive ThO 2 . Gas lantern mantles purchased from a Sydney camping shop were incinerated, deposited as a thin layer on a aluminium planchette, and the emitted alpha spectrum was measured with a silicon surfacer barrier detector. The specific activity of the samples was estimated by high resolution gamma spectroscopy using a high purity germanium detector as well as CR-39 solid state nuclear track detectors. The micro-morphology of the incinerated powder was analysed by scanning electron microscopy. The depth dose and LET distribution of alpha particles in soft tissue were calculated from the energy spectrum. 12 refs., 2 tabs., 5 figs

Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding.

Science.gov (United States)

Navenot, J M; Wang, Z X; Trent, J O; Murray, J L; Hu, Q X; DeLeeuw, L; Moore, P S; Chang, Y; Peiper, S C

2001-11-09

Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an

Sequential nearest-neighbor effects on computed {sup 13}C{sup {alpha}} chemical shifts

Energy Technology Data Exchange (ETDEWEB)

Vila, Jorge A. [Cornell University, Baker Laboratory of Chemistry and Chemical Biology (United States); Serrano, Pedro; Wuethrich, Kurt [The Scripps Research Institute, Department of Molecular Biology (United States); Scheraga, Harold A., E-mail: has5@cornell.ed [Cornell University, Baker Laboratory of Chemistry and Chemical Biology (United States)

2010-09-15

To evaluate sequential nearest-neighbor effects on quantum-chemical calculations of {sup 13}C{sup {alpha}} chemical shifts, we selected the structure of the nucleic acid binding (NAB) protein from the SARS coronavirus determined by NMR in solution (PDB id 2K87). NAB is a 116-residue {alpha}/{beta} protein, which contains 9 prolines and has 50% of its residues located in loops and turns. Overall, the results presented here show that sizeable nearest-neighbor effects are seen only for residues preceding proline, where Pro introduces an overestimation, on average, of 1.73 ppm in the computed {sup 13}C{sup {alpha}} chemical shifts. A new ensemble of 20 conformers representing the NMR structure of the NAB, which was calculated with an input containing backbone torsion angle constraints derived from the theoretical {sup 13}C{sup {alpha}} chemical shifts as supplementary data to the NOE distance constraints, exhibits very similar topology and comparable agreement with the NOE constraints as the published NMR structure. However, the two structures differ in the patterns of differences between observed and computed {sup 13}C{sup {alpha}} chemical shifts, {Delta}{sub ca,i}, for the individual residues along the sequence. This indicates that the {Delta}{sub ca,i} -values for the NAB protein are primarily a consequence of the limited sampling by the bundles of 20 conformers used, as in common practice, to represent the two NMR structures, rather than of local flaws in the structures.

Phase I Final Report: Ultra-Low Background Alpha Activity Counter

International Nuclear Information System (INIS)

Warburton, W.K.

2005-01-01

processor we easily distinguish between these two risetimes and thereby count only alpha particles emitted by the sample. Alpha particles emitted from the sample tray are absorbed in the rear of the sample, so the tray's emissivity does not contribute to the background either. Extensions of the method to the counter's sidewalls similarly allow us to reject alpha particles emitted from the sidewalls. We can thus able obtain background rates over a factor of 1000 lower than in conventional instruments without active background rejection. Extending this principle to count at the 0.00001 alpha/cm 2 /hour, level encounters difficulties because there will typically be only 2.4 alpha particles per square meter per day. Since about 6 counts are required to measure activity at the 95% confidence level, large sample areas are required to make measurements in reasonable times. Unfortunately, increasing the counter's anode area to a square meter raises its capacitance so much that the preamplifier noise levels swamp the alpha particle signals and make counting impossible. In this SBIR we worked to solve this dilemma by segmenting the single large area electrode into several smaller, lower capacitance electrodes that could still detect the alpha particles reliably. Each electrode would have its own electronic and we would capture signals from all of them in coincidence (since an alpha track might well deposit charge on more than one electrode), a technique in which XIA is experienced. Therefore, in Phase I we worked to show proof of principle by subdividing our original 1,800 cm 2 electrode into 4 square segments, each 625 cm 2 and demonstrating that signal noise on individual channels reduced as expected. Because the Phase II counter with a 1 m 2 segmented anode would require 16 segments plus a segmented guard as well, we also designed low cost signal processing electronics to instrument it in Phase II. Our Phase I effort met our major proof of principle goals. In particular, reducing

Discovery of an Oxybenzylglycine Based Peroxisome Proliferator Activated Receptor Alpha Selective

Energy Technology Data Exchange (ETDEWEB)

Li, J.; Kennedy, L; Shi, Y; Tao, S; Ye, X; Chen, S; Wang, Y; Hernandez, A; Wang, W; et al.

2010-01-01

An 1,3-oxybenzylglycine based compound 2 (BMS-687453) was discovered to be a potent and selective peroxisome proliferator activated receptor (PPAR) {alpha} agonist, with an EC{sub 50} of 10 nM for human PPAR{alpha} and {approx}410-fold selectivity vs human PPAR{gamma} in PPAR-GAL4 transactivation assays. Similar potencies and selectivity were also observed in the full length receptor co-transfection assays. Compound 2 has negligible cross-reactivity against a panel of human nuclear hormone receptors including PPAR{delta}. Compound 2 demonstrated an excellent pharmacological and safety profile in preclinical studies and thus was chosen as a development candidate for the treatment of atherosclerosis and dyslipidemia. The X-ray cocrystal structures of the early lead compound 12 and compound 2 in complex with PPAR{alpha} ligand binding domain (LBD) were determined. The role of the crystal structure of compound 12 with PPAR{alpha} in the development of the SAR that ultimately resulted in the discovery of compound 2 is discussed.

Structural and functional analysis of lysosomal ss-galactosidase and its relation to the protective protein.

NARCIS (Netherlands)

H. Morreau (Hans)

1992-01-01

textabstractLysosomal B-galactosidase is the glycosidase, that cleaves B-linked galactosyl mmenes from a variety of natural and synthetic substrates. In normal tissues of various species this enzyme appears to associate with two other hydrolases, N-acetyl-o:-neuraminidase and the protective protein.

Right prefrontal TMS disrupts interregional anticipatory EEG alpha activity during shifting of visuospatial attention

Directory of Open Access Journals (Sweden)

Paul eSauseng

2011-10-01

Full Text Available Visual attention can be shifted in space without moving the eyes. Amplitude decrease of rhythmical brain activity around 10 Hz (so called alpha activity at contralateral posterior sites has been reported during covert shifts of visuospatial attention to one visual hemifield. Alpha amplitude increase, on the other hand, can be found at ipsilateral visual cortex. There is some evidence suggesting an involvement of prefrontal brain areas during the control of attention-related anticipatory alpha amplitude asymmetry. However, the exact neural mechanism by which prefrontal cortex influences visual processing has not been completely clear yet. This open question has been studied in detail using a multimodal approach combining transcranial magnetic stimulation (TMS and multichannel electroencephalography (EEG in healthy humans. Slow (1 Hz repetitive TMS inducing an inhibitory effect at the stimulation site was delivered either to right frontal eye field or a control site (vertex. Subsequently, participants had to perform a spatial cueing task in which covert shifts of attention were required to either the left or the right visual hemi-field. After stimulation at the vertex (control condition a pattern of anticipatory, attention-related ipsilateral alpha increase / contralateral alpha decrease over posterior recording sites could be obtained. Additionally, there was pronounced coupling between (in particular right FEF and posterior brain sites. When, however, the right prefrontal cortex had been virtually lesioned preceding the task, these EEG correlates of visuospatial attention were attenuated. Notably, the effect of TMS at the right FEF on interregional fronto-parietal alpha coupling predicted the effect on response times. This suggests that visual attention processes associated with posterior EEG alpha activity are at least partly top-down controlled by the prefrontal cortex.

Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

DEFF Research Database (Denmark)

Meggio, F; Boldyreff, B; Issinger, O G

1994-01-01

to substitute for wild-type beta-subunit as a suppressor of activity toward calmodulin. The only mutations that reduced the ability of the beta-subunit to suppress calmodulin phosphorylation activity, though being compatible with normal reconstitution of CK2 holoenzyme, were those affecting Asp55, Glu57...... are conversely ineffective. The latent "calmodulin kinase" activity of CK2 can also be specifically unmasked by a peptide (alpha[66-86]) reproducing a basic insert of the catalytic subunit. This effect is reversed by equimolar addition of a peptide (beta[55-71]) including the 55-64 acidic stretch of the beta......-subunit. Comparable polylysine stimulation was observed with the holoenzymes reconstituted with either beta wt or the beta mutants capable of assembling with the alpha-subunit, with the notable exception of those bearing Ala substitutions for acidic residues at positions 55, 57, and 59-61. These were nearly...

The expression of β-galactosidase during long-term cultured goat skin fibroblasts and the effect of donor cell passage on in vitro development of nuclear transfer embryos.

Science.gov (United States)

Liu, Haijun; Peng, Hui; Liu, Fang; Ma, Qun; Zhang, Wenchang

2016-05-01

The present study aimed to detect the expression of β-galactosidase during long-term cultured goat skin fibroblasts and investigate the effects of donor goat age, sex, and cell passage on senescence and the effects of donor cell passage on in vitro development of nuclear transfer embryos. The results showed that, in the same cell passage, more β-galactosidase-positive cells were detected in cells from older donors than younger donors. Irrespective of the donor age, the number of positive cells was higher in later passages from passages 20 to 50. In the same passage from 20 to 50, the β-galactosidase-positive rate was higher in cells from 5-yr female goat than 5-yr male goat. Using fibroblasts from male goats at various passages as donor cells, reconstructed embryos had similar fusion and cleavage rates, but the blastocyst rate was higher for cells at passages 10 and 20 than passage 30. In conclusion, donor goat age and cell passage had significant effects on the β-galactosidase-positive rate; also, cells from 5-yr female goat had a higher β-galactosidase-positive rate than those from 5-yr male goat, and the donor cell passage affected the developmental potential of nuclear transfer embryos.

Naturally-occurring alpha activity

Energy Technology Data Exchange (ETDEWEB)

Mayneord, W V

1960-12-01

In view of the difficulties of assessing the significance of man-made radioactivity it is important to study for comparison the background of natural radioactivity against which the human race has evolved and lives. It is also important to define the present levels of activity so that it will be possible to detect and study as quickly as possible any changes which may occur owing to the release into the environment of new radioactive materials. Moreover, by the study of the behaviour of natural radioactivity light may be shed upon that of the artificially produced isotopes and a number of analogies traced between the two groups. These concepts have led to studies of naturally-occurring radioactive materials alongside a programme of research into fission products in food, water and air, as well as studies of the metabolism of both sets of materials in the human body. Since the last report there has been a useful increase in our knowledge of natural radioactivity in the biosphere, and its levels relative to the new man-made activities. These studies have necessitated technical developments, particularly in the methods of measuring and identifying alpha-ray emitters, to which group many of the more important natural radioactive materials belong.

Maximization of beta-galactosidase production: a simultaneous investigation of agitation and aeration effects.

Science.gov (United States)

Alves, Fernanda Germano; Filho, Francisco Maugeri; de Medeiros Burkert, Janaína Fernandes; Kalil, Susana Juliano

2010-03-01

In this work, the agitation and aeration effects in the maximization of the beta-galactosidase production from Kluyveromyces marxianus CCT 7082 were investigated simultaneously, in relation to the volumetric enzyme activity and the productivity, as well as the analysis of the lactose consumption and production of glucose, and galactose of this process. Agitation and aeration effects were studied in a 2 L batch stirred reactor. A central composite design (2(2) trials plus three central points) was carried out. Agitation speed varied from 200 to 500 rpm and aeration rate from 0.5 to 1.5 vvm. It has been shown in this study that the volumetric enzyme production was strongly influenced by mixing conditions, while aeration was shown to be less significant. Linear models for activity and productivity due to agitation and aeration were obtained. The favorable condition was 500 rpm and 1.5 vvm, which lead to the best production of 17 U mL(-1) for enzymatic activity, 1.2 U mL(-1) h(-1) for productivity in 14 h of process, a cellular concentration of 11 mg mL(-1), and a 167.2 h(-1) volumetric oxygen transfer coefficient.

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Science.gov (United States)

Avni, A; Avital, S; Gromet-Elhanan, Z

1991-04-25

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.

Antecedent occipital alpha band activity predicts the impact of oculomotor events in perceptual switching

Directory of Open Access Journals (Sweden)

Hironori eNakatani

2013-05-01

Full Text Available Oculomotor events such as blinks and saccades transiently interrupt the visual input and, even though this mostly goes undetected, these brief interruptions could still influence the percept. In particular, both blinking and saccades facilitate switching in ambiguous figures such as the Necker cube. To investigate the neural state antecedent to these oculomotor events during the perception of an ambiguous figure, we measured the human scalp electroencephalogram (EEG. When blinking led to perceptual switching, antecedent occipital alpha band activity exhibited a transient increase in amplitude. When a saccade led to switching, a series of transient increases and decreases in amplitude was observed in the antecedent occipital alpha band activity. Our results suggest that the state of occipital alpha band activity predicts the impact of oculomotor events on the percept.

Studies on chemical modification of cold agglutinin from the snail Achatina fulica.

Science.gov (United States)

Sarkar, M; Mitra, D; Sen, A K

1987-01-01

The cold agglutinin isolated from the albumin gland of the snail Achatina fulica was modified with various chemical reagents in order to detect the amino acids and/or carbohydrate residues present in its carbohydrate-binding sites. Treatment with reagents considered specific for modification of lysine, arginine and tryptophan residues of the cold agglutinin did not affect the carbohydrate-binding activity of the agglutinin. Modification of tyrosine residues showed some change. However, modification with carbodiimide followed by alpha-aminobutyric acid methyl ester causes almost complete loss of its binding activity, indicating the involvement of aspartic acid and glutamic acid in its carbohydrate-binding activity. The carbohydrate residues of the cold agglutinin were removed by beta-elimination reaction, indicating that the sugars are O-glycosidically linked to protein part of the molecule. Removal of galactose residues from the cold agglutinin by the action of beta-galactosidase indicated that the galactose molecules are beta-linked. These carbohydrate-modified glycoproteins showed a marked change in agglutination property, i.e. they agglutinated rabbit erythrocytes at both 10 degrees C and 25 degrees C, indicating that the galactose residues of the glycoprotein play an important role in the cold-agglutination property of the glycoprotein. The c.d. data showed the presence of an almost identical type of random-coil conformation in the native cold agglutinin at 10 degrees C and in the carbohydrate-modified glycoprotein at 10 degrees C and 25 degrees C. This particular random-coil conformation is essential for carbohydrate-binding property of the agglutinin. Images Fig. 1. PMID:3118867

The 2.0-A resolution structure of soybean beta-amylase complexed with alpha-cyclodextrin.

Science.gov (United States)

Mikami, B; Hehre, E J; Sato, M; Katsube, Y; Hirose, M; Morita, Y; Sacchettini, J C

1993-07-13

New crystallographic findings are presented which offer a deeper understanding of the structure and functioning of beta-amylase, the first known exo-type starch-hydrolyzing enzyme. A refined three-dimensional structure of soybean beta-amylase, complexed with the inhibitor alpha-cyclodextrin, has been determined at 2.0-A resolution with a conventional R-value of 17.5%. The model contains 491 amino acid residues, 319 water molecules, 1 sulfate ion, and 1 alpha-cyclodextrin molecule. The protein consists of a core with an (alpha/beta)8 supersecondary structure, plus a smaller globular region formed by long loops (L3, L4, and L5) extending from beta-strands beta 3, beta 4, and beta 5. Between the two regions is a cleft that opens into a pocket whose floor contains the postulated catalytic center near the carboxyl group of Glu 186. The annular alpha-cyclodextrin binds in (and partly projects from) the cleft with its glucosyl O-2/O-3 face abutting the (alpha/beta)8 side and with its alpha-D(1 --> 4) glucosidic linkage progression running clockwise as viewed from that side. The ligand does not bind deeply enough to interact with the carboxyl group of Glu 186. Rather, it occupies most of the cleft entrance, strongly suggesting that alpha-cyclodextrin inhibits catalysis by blocking substrate access to the more deeply located reaction center. Of the various alpha-cyclodextrin interactions with protein residues in loops L4, L5, L6, and L7, most notable is the shallow inclusion complex formed with Leu 383 (in L7, on the core side of the cleft) through contacts of its methyl groups with the C-3 atoms of four of the ligand's D-glucopyranosyl residues. All six residues of the bound alpha-cyclodextrin are of 4C1 conformation and are joined by alpha-1,4 linkages with similar torsional angles to form a nearly symmetrical torus as reported for crystalline inclusion complexes with alpha-cyclodextrin. We envision a significant role for the methyl groups of Leu 383 at the cleft entrance

Peroxisome Proliferator-Activated Receptor-alpha Gene Level Differently Affects Lipid Metabolism and Inflammation in Apolipoprotein E2 Knock-In Mice

NARCIS (Netherlands)

Lalloyer, Fanny; Wouters, Kristiaan; Baron, Morgane; Caron, Sandrine; Vallez, Emmanuelle; Vanhoutte, Jonathan; Bauge, Eric; Shiri-Sverdlov, Ronit; Hofker, Marten; Staels, Bart; Tailleux, Anne

Objective-Peroxisome proliferator-activated receptor-alpha (PPAR alpha) is a ligand-activated transcription factor that controls lipid metabolism and inflammation. PPAR alpha is activated by fibrates, hypolipidemic drugs used in the treatment of dyslipidemia. Previous studies assessing the influence

Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3.

Science.gov (United States)

He, Xi; Han, Ning; Wang, Yan-Ping

2016-01-01

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

Recovery of transuranics from process residues

International Nuclear Information System (INIS)

Gray, J.H.; Gray, L.W.

1987-01-01

Process residues are generated at both the Rocky Flats Plant (RFP) and the Savannah River Plant (SRP) during aqueous chemical and pyrochemical operations. Frequently, process operations will result in either impure products or produce residues sufficiently contaminated with transuranics to be nondiscardable as waste. Purification and recovery flowsheets for process residues have been developed to generate solutions compatible with subsequent Purex operations and either solid or liquid waste suitable for disposal. The ''scrub alloy'' and the ''anode heel alloy'' are examples of materials generated at RFP which have been processed at SRP using the developed recovery flowsheets. Examples of process residues being generated at SRP for which flowsheets are under development include LECO crucibles and alpha-contaminated hydraulic oil

A study of excess H-alpha emission in chromospherically active M dwarf

International Nuclear Information System (INIS)

Young, A.; Skumanich, A.; Stauffer, J.R.; Harlan, E.; Bopp, B.W.

1989-01-01

Spectroscopic observations from three observatories are combined to study the properties of the excess H-alpha emission which characterizes the most chromospherically active subset of the M dwarf stars, known as the dMe stars. It is demonstrated that the excess H-alpha luminosity from these stars is a monotonically decreasing function of their (R-I) color, and evidence is presented which suggests that the product of the mean surface brightness and the mean filling factor of the emissive regions is essentially constant with color. Another significant result of the study is a linear correlation between the excess luminosity in H-alpha and the coronal X-ray luminosity. 39 refs

Escherichia coli LacZ beta-galactosidase inhibition by monohydroxy acetylated glycopyranosides: Role of the acetyl groups

Czech Academy of Sciences Publication Activity Database

Brabcová, Jana; Carrasco-Lopez, C.; Bavaro, T.; Hermoso, J. A.; Palomo, J. M.

2014-01-01

Roč. 107, Sep (2014), s. 31-38 ISSN 1381-1177 Grant - others:GA AV ČR(CZ) M20055123 Institutional support: RVO:61388963 Keywords : biotransformation * galactosidase * inhibition * glycopyranosides * regioselectivity Subject RIV: CE - Biochemistry Impact factor: 2.128, year: 2014

Structure based protein engineering of Bacillus stearothermophilus {alpha}-amylase: toward a new substrate specificity

Energy Technology Data Exchange (ETDEWEB)

Rasera, Ana Claudia [Sao Paulo Univ., SP (Brazil). Inst. de Ciencias Biomedicas; Iulek, Jorge [Universidade Estadual de Ponta Grossa, PR (Brazil). Inst. de Quimica; Delboni, Luis Fernando; Barbosa, Valma Martins Barbosa [Parana Univ., Curitiba, PR (Brazil). Dept. de Bioquimica

1997-12-31

Full text. Structural similarity is observed in all members of {alpha}-amylase family but different products are generated during hydrolysis of starch due to different affinities for intermediate dextrins. In order to understand the structural determinants for this property and to introduce different specificity to {alpha}-amylase of Bacillus stearothermophilus we intend to solve the three dimensional structure by X-ray crystallography of the native protein by using synchrotron radiation at Brazilian National Synchrotron Light Laboratory (LNLS). Protein was over expressed in E. coli, purified and crystallization experiments were carried out by using sparse matrix Crystal Screen and Crystal Screen II from Hampton Research (Laguna Hills, CA, USA). Several condition have produced crystals with some defined characteristic: MDP seems to be important to the crystallization: the preferential pH is around 7.5 with organic buffer (HEPES); organic solvent as 2-propanol seems to be also important for the crystallization. On those condition crystals appeared as cluster of needles or small crystals with high number of nucleation. New conditions are being prepared to improve the site and quality of crystals. Data collection of native of Bacillus stearothermophilus {alpha}-amylase will e done at Protein Crystallography Station at LNLS. Crystal structure of mutated {alpha}-amylase bu site direct mutagenesis of residues suggested by the native crystal structure will be obtained. Co-crystallization of Bacillus stearothermophilus {alpha}-amylase and oligosaccharide will be carried out to identify residues involved in the binding site to plan new mutation. In another series of mutation the putative binding site residues, once identified, will be mutated to residues observed in TAKA amylase to confer different specificity to {alpha}-amylase. Based on the available TAKA amylase structure, in the primary sequence homology and in the three dimensional model of Bacillus stearothermophilus

Different features of the MHC class I heterodimer have evolved at different rates. Chicken B-F and beta 2-microglobulin sequences reveal invariant surface residues

DEFF Research Database (Denmark)

Kaufman, J; Andersen, R; Avila, D

1992-01-01

molecules and the MHC-encoded nonclassical molecules more than CD1 or the class I-like FcR. In contrast, the chicken alpha 3 domain is equally homologous to all alpha 3 domains, to beta 2m and to class II beta 2 domains. For each pair of extracellular domains (alpha 1 vs alpha 2, alpha 3 vs beta 2m...... of small exons in the cytoplasmic region. The cDNA sequences were compared to turkey beta 2m, the apparent allele B-F12 alpha and other vertebrate homologs, using the 2.6 A structure of the human HLA-A2 molecule as a model. Both chicken alpha 1 and alpha 2 domains resemble mammalian classical class I...... the ends of the peptide, two residues that bind CD8, and three residues that are phosphorylated. The positions of the allelic residues are conserved. There are other patches of invariant residues on alpha 1, alpha 2, and beta 2m; these might bind TCR or other molecules involved in class I function...

Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

Energy Technology Data Exchange (ETDEWEB)

Ikeda, Kazuhiro [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Tsukui, Tohru [Experimental Animal Laboratory, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Imazawa, Yukiko; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Inoue, Satoshi, E-mail: INOUE-GER@h.u-tokyo.ac.jp [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan)

2012-09-07

Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively active mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

Absolute measurements of the alpha-gamma emitters activities by a sum-coincidence method

International Nuclear Information System (INIS)

Tobias, C.C.B.

1982-01-01

The absolute activity of U-235 contained in a UO 2 sample, using a sum-coincidence circuit which selected only the alpha particles which were simultaneous with the well known 184 Kev gamma radiation from Th-231. The alpha particles were detected by ZnS(Ag) scintillator specially designed to show its maximun efficiency for U-235 alpha particles, whereas the gamma radiation was detected by NaI(Tl) scintillation detector. The values obtained for the half-life of U-235 was compared with data from various observers using different experimental techniques. (Author) [pt

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

Directory of Open Access Journals (Sweden)

Vidal M.S.

2002-01-01

Full Text Available Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22. Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

Innovative procedure for the determination of gross-alpha/gross-beta activities in drinking water

International Nuclear Information System (INIS)

Wisser, S.; Frenzel, E.; Dittmer, M.

2006-01-01

An alternative sample preparation method for the determination of gross-alpha/beta activity concentrations in drinking water is introduced in this paper. After the freeze-drying of tap water samples, determination by liquid scintillation counting can be applied utilizing alpha/beta separation. It has been shown that there is no adsorption or loss of solid radionuclides during the freeze-drying procedure. However, the samples have to be measured quickly after the preparation since the ingrowth of daughter isotopes negatively effects the measurement. The limits of detection for gross-alpha and gross-beta activity are in the range 25-210 mBq/l, respectively, for a measurement time of only 8-9 h

Identification of galactose as the immunodominant sugar of leishmanial excreted factor and subsequent labeling with galactose oxidase and sodium boro[3H]hydride.

Science.gov (United States)

Slutzky, G M; Greenblatt, C L

1982-01-01

Inhibition by low-molecular-weight sugars of precipitin line formation between a polysaccharide (EF) excreted by Leishmania tropica subsp. major, Leishmania enriettii, and rabbit antileishmanial antibodies on double gel diffusion plates revealed that galactose residues, possibly as components of lactosyl groups, were the critical immunodominant sugars mediating antibody recognition of EF. The galactose residues of the EF of L. tropica subsp. major were specifically labeled with tritium via galactose oxidase and sodium boro[3H]hydride. The radioactive EF had an apparent molecular weight of about 85,000 on sodium dodecyl sulfate-polyacrylamide gels and was precipitated by antileishmanial antibodies as well as Ricinus communis lectins I and II (galactose specific). Lectins specific for glucose-mannose residues, fucose, N-acetylglucosamine, and N-acetylgalactosamine did not precipitate the labeled EF. Treatment of [3H]EF with proteolytic (trypsin, papain, protease) or glycosidic (alpha-amylase, beta-galactosidase) enzymes had no effect on either the electrophoretic pattern of the material or on its recognition by antileishmanial antibodies or R. communis lectin. This resistance to enzyme activity suggests that EF may be a useful marker for the presence of the parasite in vivo if it can be detected in minute quantities. PMID:6179874

Calcination/dissolution residue treatment

International Nuclear Information System (INIS)

Knight, R.C.; Creed, R.F.; Patello, G.K.; Hollenberg, G.W.; Buehler, M.F.; O'Rourke, S.M.; Visnapuu, A.; McLaughlin, D.F.

1994-09-01

Currently, high-level wastes are stored underground in steel-lined tanks at the Hanford site. Current plans call for the chemical pretreatment of these wastes before their immobilization in stable glass waste forms. One candidate pretreatment approach, calcination/dissolution, performs an alkaline fusion of the waste and creates a high-level/low-level partition based on the aqueous solubilities of the components of the product calcine. Literature and laboratory studies were conducted with the goal of finding a residue treatment technology that would decrease the quantity of high-level waste glass required following calcination/dissolution waste processing. Four elements, Fe, Ni, Bi, and U, postulated to be present in the high-level residue fraction were identified as being key to the quantity of high-level glass formed. Laboratory tests of the candidate technologies with simulant high-level residues showed reductive roasting followed by carbonyl volatilization to be successful in removing Fe, Ni, and Bi. Subsequent bench-scale tests on residues from calcination/dissolution processing of genuine Hanford Site tank waste showed Fe was separated with radioelement decontamination factors of 70 to 1,000 times with respect to total alpha activity. Thermodynamic analyses of the calcination of five typical Hanford Site tank waste compositions also were performed. The analyses showed sodium hydroxide to be the sole molten component in the waste calcine and emphasized the requirement for waste blending if fluid calcines are to be achieved. Other calcine phases identified in the thermodynamic analysis indicate the significant thermal reconstitution accomplished in calcination

A study on the neoasozine residues in rice grain by neutron activation method

International Nuclear Information System (INIS)

Kim, Y.H.; Lee, K.J.; Lee, S.R.

1981-01-01

Residues of neoasozine in rice grain were determined by neutron activation and colorimetric techniques. Twice application of the chemical before flowering did not lead to any increased residue level while 4-times application resulted in significant increase in the residue level up to 0.54 - 0.75 mg As 2 O 3 /kg. The partition ratio of arsenic residues into polished rice grain and bran was 73 : 27 in 100 % polishing while most of the residues in the bran was transferred to oil cake fraction during solvent extraction, reaching up to 2.9 mg As 2 O 3 /kg. The neutron activation technique was advantageous because of its high sensitivity and the smaller sample amounts required for analysis. (author)

HNF1 alpha activates the aminopeptidase N promoter in intestinal (Caco-2) cells

DEFF Research Database (Denmark)

Olsen, Jørgen; Laustsen, Lotte; Troelsen, J

1994-01-01

The importance of HNF1 binding proteins for intestinal aminopeptidase N expression was investigated using the Caco-2 cell-line. Aminopeptidase N promoter activity in Caco-2 cells depends on the HNF1 element (positions -85 to -58) and co-transfection with an HNF1 alpha expression vector demonstrates...... a direct activation of the promoter by HNF1 alpha through this element. Electrophoretic mobility shift assays using nuclear extracts from Caco-2 cells show the presence of high amounts of HNF1 binding proteins irrespective of their state of differentiation....

Distinct neural pathways mediate alpha7 nicotinic acetylcholine receptor-dependent activation of the forebrain

DEFF Research Database (Denmark)

Thomsen, Morten S; Hay-Schmidt, Anders; Hansen, Henrik H

2010-01-01

alpha(7) nicotinic acetylcholine receptor (nAChR) agonists are candidates for the treatment of cognitive deficits in schizophrenia. Selective alpha(7) nAChR agonists, such as SSR180711, activate neurons in the medial prefrontal cortex (mPFC) and nucleus accumbens shell (ACCshell) in rats, regions...

Cecal parameters of rats fed diets containing grapefruit polyphenols and inulin as single supplements or in a combination.

Science.gov (United States)

Zduńczyk, Zenon; Juśkiewicz, Jerzy; Estrella, Isabel

2006-09-01

We compared the effects of grapefruit flavonoids and inulin, as single dietary components or in a combination, on cecal fermentation in rats adapted to a semipurified diet. The experimental diets contained 0.3% flavonoid extract and 5% or 10% inulin and a combination of both supplements. The large bowel metabolism assessment was based on cecal parameters: bulk effect, pH, microbial enzymes activity, and short-chain fatty acid production. Both supplements induced significant enlargement of the cecal digesta weight. Acidification of cecal digesta was more pronounced, with a higher inulin addition to the diet. Cecal pH was the highest with the flavonoid-rich diets and lowest in the case of a simultaneous addition of flavonoids and a high content of inulin. The flavonoid extract applied as a single dietary supplement was observed to decrease the activity of bacterial beta-glucosidase and beta- and alpha-galactosidases in the cecal digesta. In contrast, addition of the grapefruit extract to inulin-containing diets increased the activity of alpha-glucosidase, alpha-galactosidase, and beta-galactosidase. Great accumulation of cecal digesta in rats consuming the flavonoid-diet caused a considerable increase in the short-chain fatty acid pool, mainly acetic acid. Inulin added to the diet decreased the excessive enlargement of digesta caused by dietary flavonoids. Dietary addition of inulin to the flavonoid-diet also normalized hydration of cecal digesta and significantly decreased the pH of digesta. The presence of polyphenols in the inulin-containing diets did not change total short-chain fatty acid production in the cecum of rats. Our results suggested that simultaneous intake of inulin and polyphenols can decrease the detrimental effects of the latter on cecal fermentation.

Resting-State Alpha in Autism Spectrum Disorder and Alpha Associations with Thalamic Volume

Science.gov (United States)

Edgar, J. Christopher; Heiken, Kory; Chen, Yu-Han; Herrington, John D.; Chow, Vivian; Liu, Song; Bloy, Luke; Huang, Mingxiong; Pandey, Juhi; Cannon, Katelyn M.; Qasmieh, Saba; Levy, Susan E.; Schultz, Robert T.; Roberts, Timothy P. L.

2015-01-01

Alpha circuits (8-12 Hz), necessary for basic and complex brain processes, are abnormal in autism spectrum disorder (ASD). The present study obtained estimates of resting-state (RS) alpha activity in children with ASD and examined associations between alpha activity, age, and clinical symptoms. Given that the thalamus modulates cortical RS alpha…

Studies of the Gly482Ser polymorphism of the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) gene in Danish subjects with the metabolic syndrome

DEFF Research Database (Denmark)

Ambye, L; Rasmussen, S; Fenger, Mogens

2005-01-01

The peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) is a novel transcriptional co-activator that holds an important role in lipid and glucose metabolism. PGC-1alpha is a candidate gene for the metabolic syndrome (MS) as well as type 2 diabetes. Recent studies...... related to this syndrome. The variant was examined, using PCR-RFLP, in the DanMONICA cohort comprising a population-based sample of 2349 subjects. MS was defined using the National Cholesterol Education Program -- Adult Treatment Panel III (NCEP-ATPIII) criteria. The allelic frequency of the Ser482 allele...... and insulin secretion, 24-ambulatory blood pressure or left ventricular mass index. In conclusion, the Gly482Ser polymorphism of the PGC-1alpha gene is not associated with the metabolic syndrome, related quantitative traits or cardiac hypertrophy among Danish Caucasian subjects...

Comparison of the level of residual coagulant activity in different cheese varieties.

Science.gov (United States)

Bansal, Nidhi; Fox, Patrick F; McSweeney, Paul L H

2009-08-01

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify residual coagulant in 9 cheese varieties by measuring its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) assayed using reversed-phase HPLC. The level of residual coagulant activity was highest in Camembert cheese, probably due to its low pH at whey drainage and the high moisture content of the cheese, followed in order by Feta=Port du Salut=Cheddar>Gouda>Emmental=Parmigiano Reggiano=low-moisture part-skim Mozzarella=Mozzarella di Bufala Campana. The high cooking temperature (50-54 degrees C) used during the manufacture of Emmental and Parmigiano Reggiano cheeses and the cooking and stretching step in hot water during the manufacture of Mozzarella cheese may be the reasons for the lowest residual coagulant activity in these cheeses. The level of residual coagulant activity was higher in Feta cheese made from milk concentrated by ultrafiltration than in conventional Feta.

Determination of alpha activity and fissile mass content in solid waste by systems using neutron interrogation

International Nuclear Information System (INIS)

Romeyer Dherbey, J.; Lacruche, G.; Berne, R.; Auge, J.; Martin Deidier, L.; Butez, M.

1990-01-01

The Quantitative control (determination of heavy nuclides and alpha activity) of alpha radioactive wastes is necessary, particularly to determine if the waste is in accordance with the surface storage limits. In order to reduce the uncertainty on the alpha activity resulting from unknown isotopic composition, inhomogeneity of heavy nuclides in the matrix, combination of several methods is necessary. In the paper we present the Cadarache development work in the NDA of solid waste using the Californium shuffler, 14 Mev neutron generator, and also passive techniques such as neutron emission measurement and gamma spectrometry. Experimental systems combining active and passive methods are presented (COSAC, BANCO, DANAIDE, PROMETHEE)

Alkylation of amide linkages and cleavage of the C chain in the enzyme-activated-substrate inhibition of alpha-chymotrypsin with N-nitrosamides

International Nuclear Information System (INIS)

Donadio, S.; Perks, H.M.; Tsuchiya, K.; White, E.H.

1985-01-01

Active-site-directed N-nitrosamides inhibit alpha-chymotrypsin through an enzyme-activated-substrate mechanism. In this work, the activation results in the release--in the active site--of benzyl carbonium ions, which alkylate and inhibit the enzyme. The final ratio of benzyl groups to enzyme molecules is 1.0, but the alkyl groups are scattered over a number of sites. Reduction and alkylation of the inhibited enzyme generate peptides insoluble in most media. Guanidine hydrochloride at 6 M proved a good solvent, and its use as an eluant on G-75 Sephadex permitted separation of the peptides. In the case of 14 C-labeled enzyme, such an approach has shown that all of the alkylation occurs on the C chain of the enzyme, the chain of which the active site is constructed. Chemical modification of the peptides with ethylenediamine and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide rendered them soluble in dilute acid, permitting high-performance liquid chromatographic separation. Model studies have shown that the benzyl carbonium ions are highly reactive, alkylating amide linkages at both oxygen and nitrogen. Chromatography of this mixture and also 13 C NMR spectroscopy of the intact inhibited enzyme have shown that three major N-alkylations have occurred. Tryptic digestion of the C chain of chymotrypsin, which contains all of the alkylation sites, provides evidence that the stable N sites are principally located between residue 216 and residue 230

Interleukin 2 and 15 activate Stat3alpha in human T lymphocytes

DEFF Research Database (Denmark)

Nielsen, M; Nordahl, M; Svejgaard, A

1998-01-01

Signal transducer and activator of transcription 3 (Stat3) has recently been shown to exist in two alternatively spliced isoforms, a short form, Stat3beta, and a longer form, Stat3alpha, displaying differences in transcriptional activity. It is unknown which Stat3 isoform(s) is activated in respo...

Effect of washing on pesticide residues in olives.

Science.gov (United States)

Guardia-Rubio, M; Ayora-Cañada, M J; Ruiz-Medina, A

2007-03-01

The present work aims at contributing to the knowledge of the fate of 5 pesticides in olives in order to evaluate how washing may affect the presence of these residues in this fruit (and consequently in olive oil). For this purpose, olives were sprayed with commercial formulations containing the active ingredients and a series of analyses were performed for 64 d by using gas chromatography with mass spectrometric detection. Selected pesticides, ranked by their importance, were diuron, terbuthylazine, simazine, alpha-endosulfan, and beta-endosulfan. The pesticide fraction, which was not removable from olives by washing, increased with time after treatment until their degradation started at week 6. Washing performed 1 d after treatment was the most effective in reducing residues, especially for simazine. Consequently, the washing step performed in olive mills could be effective in removing those herbicide residues present in olives as a consequence of contact with contaminated soil for a short time. This happens when olives are dropped and harvested off the ground by means of brushes or suction equipment.

Increase in posterior alpha activity during rehearsal predicts successful long-term memory formation of word sequences.

Science.gov (United States)

Meeuwissen, Esther B; Takashima, Atsuko; Fernández, Guillén; Jensen, Ole

2011-12-01

It is becoming increasingly clear that demanding cognitive tasks rely on an extended network engaging task-relevant areas and, importantly, disengaging task-irrelevant areas. Given that alpha activity (8-12 Hz) has been shown to reflect the disengagement of task-irrelevant regions in attention and working memory tasks, we here ask if alpha activity plays a related role for long-term memory formation. Subjects were instructed to encode and maintain the order of word sequences while the ongoing brain activity was recorded using magnetoencephalography (MEG). In each trial, three words were presented followed by a 3.4 s rehearsal interval. Considering the good temporal resolution of MEG this allowed us to investigate the word presentation and rehearsal interval separately. The sequences were grouped in trials where word order either could be tested immediately (working memory trials; WM) or later (LTM trials) according to instructions. Subjects were tested on their ability to retrieve the order of the three words. The data revealed that alpha power in parieto-occipital regions was lower during word presentation compared to rehearsal. Our key finding was that parieto-occipital alpha power during the rehearsal period was markedly stronger for successfully than unsuccessfully encoded LTM sequences. This subsequent memory effect demonstrates that high posterior alpha activity creates an optimal brain state for successful LTM formation possibly by actively reducing parieto-occipital activity that might interfere with sequence encoding. Copyright © 2010 Wiley Periodicals, Inc.

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

Directory of Open Access Journals (Sweden)

Wanarska Marta

2012-08-01

Full Text Available Abstract Background D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. Results In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c.

Science.gov (United States)

Wanarska, Marta; Kur, Józef

2012-08-23

D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization

OPERATIONAL LIMITATIONS FOR DEMOLITION OF A HIGHLY ALPHA CONTAMINATED BUILDING MODLES VERSUS MEASURED AIR & SURFACE ACTIVITY CONCENTRATIONS

Energy Technology Data Exchange (ETDEWEB)

LLOYD, E.R.

2006-11-02

The demolition of a facility historically used for processing and handling transuranic materials is considered. Residual alpha emitting radionuclide contamination poses an exposure hazard if released to the local environment during the demolition. The process of planning for the demolition of this highly alpha contaminated building, 232-Z, included a predemolition modeling analysis of potential exposures. Estimated emission rates were used as input to an air dispersion model to estimate frequencies of occurrence of peak air and surface exposures. Postdemolition modeling was also conducted, based on the actual demolition schedule and conditions. The modeling results indicated that downwind deposition is the main operational limitation for demolition of a highly alpha-contaminated building. During the demolition of 232-Z, airborne radiation and surface contamination were monitored. The resultant non-detect monitoring results indicate a significant level of conservatism in the modeled results. This comparison supports the use of more realistic assumption in the estimating emission rates. The resultant reduction in modeled levels of potential exposures has significant implications in terms of the projected costs of demolition of such structures.

Functional analysis of the Escherichia coli genome for members of the alpha/beta hydrolase family.

Science.gov (United States)

Zhang, L; Godzik, A; Skolnick, J; Fetrow, J S

1998-01-01

Database-searching methods based on sequence similarity have become the most commonly used tools for characterizing newly sequenced proteins. Due to the often underestimated functional diversity in protein families and superfamilies, however, it is difficult to make the characterization specific and accurate. In this work, we have extended a method for active-site identification from predicted protein structures. The structural conservation and variation of the active sites of the alpha/beta hydrolases with known structures were studied. The similarities were incorporated into a three-dimensional motif that specifies essential requirements for the enzymatic functions. A threading algorithm was used to align 651 Escherichia coli open reading frames (ORFs) to one of the members of the alpha/beta hydrolase fold family. These ORFs were then screened according to our three-dimensional motif and with an extra requirement that demands conservation of the key active-site residues among the proteins that bear significant sequence similarity to the ORFs. 17 ORFs from E. coli were predicted to have hydrolase activity and their putative active-site residues were identified. Most were in agreement with the experiments and results of other database-searching methods. The study further suggests that YHET_ECOLI, a hypothetical protein classified as a member of the UPF0017 family (an uncharacterized protein family), bears all the hallmarks of the alpha/beta hydrolase family. The novel feature of our method is that it uses three-dimensional structural information for function prediction. The results demonstrate the importance and necessity of such a method to fill the gap between sequence alignment and function prediction; furthermore, the method provides a way to verify the structure predictions, which enables an expansion of the applicable scope of the threading algorithms.

Incineration of alpha-active solid waste by microwaves

Energy Technology Data Exchange (ETDEWEB)

Mallik, G K; Bhargava, V K; Kamath, H S; Purushotham, D S.C. [Bhabha Atomic Research Centre, Tarapur (India). Advanced Fuel Fabrication Facility

1996-12-31

The conventional techniques for treatment of alpha-active compressible solid waste involve incineration using electrically heated incinerators and subsequent recovery of special nuclear materials (SNM) from the ash by acid leaching. A microwave incineration followed by microwave digestion and SNM recovery from ash has specific advantages from maintenance and productivity consideration. The paper describes a preliminary work carried out with simulated uranium containing compressible solid waste using microwave heating technique. (author). 3 refs., 1 tab.

Purification of the labeled cyanogen bromide peptides of the. cap alpha. polypeptide from sodium and potassium ion-activated adenosinetriphosphatase modified with N-(/sup 3/H)ethylmaleimide

Energy Technology Data Exchange (ETDEWEB)

Le, D.T.

1985-01-01

Sodium and potassium ion-activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-(/sup 3/H)ethylmaleimide under three different conditions, defined by particular concentrations of ligands for the enzyme, such that after the same amount of time the remaining activity of then enzyme varied from 90% to 30%. The conformation of the enzyme also differed among the three conditions. In all cases, the ..cap alpha..-polypeptide was purified and subjected to cyanogen bromide digestion. Two distinct, radioactive peptides were separated by gel filtration of the cyanogen bromide digest on a column of Sephadex LH-60 equilibrated with 95% ethanol: 88% formic acid:4:1. One of the radioactive peptides was shown to contain the sulfhydryl residue whose reaction with N-ethylmaleimide inactivates the enzyme. The other radioactive peptide contained a sulfhydryl residue that seems to react with N-ethylmaleimide only when the binding site for ATP is not occupied. Alkylation of this residue, however, does not result in inactivation of enzyme. Both peptides were purified further by high-pressure liquid chromatography, and their amino-terminal sequences were determined by the manual dansyl-Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluorescein-5'-isothiocyanate.

Pollution Of Insecticide Residues In PPTN Pasar Jumat Area

International Nuclear Information System (INIS)

Syahrir, Ulfa T.; Chairul, Sofnie M.

2000-01-01

Measurement of insecticide residue pollution from some organochlorin and organo-phosphat in soil and water samples were carried out 1999-2000 periode. The aim of the measurement was to get information about impact of laboratorium activity on insecticide contents in PPTN PASAR JUMAT. Gas chromatograph with electron capture and flame ionization detector were used to measure the pesticide content. Result of the measurement in PPTN area showed that organo-chlorin were alpha BHC, endosulfan band DDT and organo-phosphat were klorphyriphos and malation and were lower than tolerance level

Characteristics of chemical binding to alpha 2u-globulin in vitro--evaluating structure-activity relationships

International Nuclear Information System (INIS)

Borghoff, S.J.; Miller, A.B.; Bowen, J.P.; Swenberg, J.A.

1991-01-01

alpha 2u-Globulin (alpha 2u) has been shown to accumulate in the kidneys of male rats treated with 2,2,4-trimethylpentane (TMP). 2,4,4-Trimethyl-2-pentanol (TMP-2-OH), a metabolite of TMP, is found reversibly bound to alpha 2u isolated from the kidneys of these treated rats. The objectives of the following study were to characterize the ability of [3H]TMP-2-OH to bind to alpha 2u in vitro and to determine whether other compounds that cause this protein to accumulate have the same binding characteristics. Although compounds that have been shown to cause the accumulation of alpha 2u in male rat kidneys compete in vitro with [3H]TMP-2-OH for binding to alpha 2u, they do so to varying degrees. The binding affinity (Kd) of the [3H]TMP-2-OH-alpha 2u complex was calculated to be on the order of 10(-7) M. The inhibition constant values (Ki) determined for d-limonene, 1,4-dichlorobenzene, and 2,5-dichlorophenol were all in the range 10(-4) M, whereas the Ki values for isophorone, 2,4,4- or 2,2,4-trimethyl-1-pentanol, and d-limonene oxide were determined to be in the range 10(-6) and 10(-7) M, respectively. TMP and 2,4,4- and 2,2,4-trimethylpentanoic acid did not compete for binding. This suggests that other factors, besides binding, are involved in the accumulation of alpha 2u. In this study the ability of a chemical to bind to alpha 2u was used as a measure of biological activity to assess structure-activity relationships among the chemicals tested and known to cause the accumulation of alpha 2u. The results so far suggest that binding is dependent on both hydrophobic interactions and hydrogen bonding

Interaction of the alpha-toxin of Staphylococcus aureus with the liposome membrane.

Science.gov (United States)

Ikigai, H; Nakae, T

1987-02-15

When the liposome membrane is exposed to the alpha-toxin of Staphylococcus aureus, fluorescence of the tryptophan residue(s) of the toxin molecule increases concomitantly with the degree of toxin-hexamer formation (Ikigai, H., and Nakae, T. (1985) Biochem. Biophys. Res. Commun. 130, 175-181). In the present study, the toxin-membrane interaction was distinguished from the hexamer formation by the fluorescence energy transfer from the tryptophan residue(s) of the toxin molecule to the dansylated phosphatidylethanolamine in phosphatidylcholine liposome. Measurement of these two parameters yielded the following results. The effect of the toxin concentration and phospholipid concentration on these two parameters showed first order kinetics. The effect of liposome size on the energy transfer and the fluorescence increment of the tryptophan residue(s) was only detectable in small liposomes. Under moderately acidic or basic conditions, the fluorescence energy transfer always preceded the fluorescence increment of the tryptophan residue(s). The fluorescence increment at 336 nm at temperatures below 20 degrees C showed a latent period, whereas the fluorescence energy transfer did not. These results were thought to indicate that when alpha-toxin damages the target membrane, the molecule interacts with the membrane first, and then undergoes oligomerization within the membrane.

Primary structure of human alpha 2-macroglobulin. V. The complete structure

DEFF Research Database (Denmark)

Sottrup-Jensen, Lars; Stepanik, Terrence M; Kristensen, Torsten

1984-01-01

The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven......-SH group of Cys-949 is thiol esterified to the gamma-carbonyl group of Glx-952, thus forming an activatable reactive site which can mediate covalent binding of nucleophiles. A putative transglutaminase cross-linking site is constituted by Gln-670 and Gln-671. The primary sites of proteolytic cleavage......-macroglobulin subunit is discussed. A comparison of stretches of sequences from alpha 2-macroglobulin with partial sequence data for complement components C3 and C4 indicates that these proteins are evolutionary related. The properties of alpha 2-macroglobulin are discussed within the context of proteolytically...

Rational design, synthesis, biologic evaluation, and structure-activity relationship studies of novel 1-indanone alpha(1)-adrenoceptor antagonists.

Science.gov (United States)

Li, Minyong; Xia, Lin

2007-11-01

In the present report, a novel series of 1-indanone alpha(1)-adrenoceptor antagonists were designed and synthesized based on 3D-pharmacophore model. Their in vitro alpha(1)-adrenoceptor antagonistic assay showed that three compounds (2a, 2m, and 2o) had similar or improved alpha(1)-adrenoceptor antagonistic activities relative to the positive control prazosin. Based on these results, a three-dimensional quantitative structure-activity relationship study was performed using a Self-Organizing Molecular Field Analysis method to provide insight for the future development of alpha(1)-adrenoceptor antagonists.

Tumor necrosis factor-alpha increases myocardial microvascular transport in vivo

DEFF Research Database (Denmark)

Hansen, P R; Svendsen, Jesper Hastrup; Høyer, S

1994-01-01

Tumor necrosis factor-alpha (TNF-alpha) is a primary mediator in the pathogenesis of tissue injury, and high circulating levels of TNF-alpha are found in a variety of pathological conditions. In open-chest anesthetized dogs, the effects of intracoronary recombinant human TNF-alpha (rTNF-alpha; 100...... in cardiac output and was associated with the appearance of areas with myocardial necrosis in the regional left ventricular wall. The myocardial plasma flow rate and maximum plasma flow rate in response to a 30-s coronary occlusion were not influenced by rTNF-alpha, although a decrease in the myocardial...... ng/kg for 60 min) on myocardial microvascular transport of a small hydrophilic indicator was examined by the single-injection, residue-detection method. Intracoronary infusion of rTNF-alpha increased myocardial microvascular transport after 120 min. This increase was preceded by a sustained decline...

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha

DEFF Research Database (Denmark)

Hwang, C S; Mandrup, S; MacDougald, O A

1996-01-01

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob...... gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes...... to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C...

Correlated alpha activity with the facial expression processing network in a simultaneous EEG-fMRI experiment.

Science.gov (United States)

Simoes, Marco; Direito, Bruno; Lima, Joao; Castelhano, Joao; Ferreira, Carlos; Couceiro, Ricardo; Carvalho, Paulo; Castelo-Branco, Miguel

2017-07-01

The relationship between EEG and fMRI data is poorly covered in the literature. Extensive work has been conducted in resting-state and epileptic activity, highlighting a negative correlation between the alpha power band of the EEG and the BOLD activity in the default-mode-network. The identification of an appropriate task-specific relationship between fMRI and EEG data for predefined regions-of-interest, would allow the transfer of interventional paradigms (such as BOLD-based neurofeedback sessions) from fMRI to EEG, enhancing its application range by lowering its costs and improving its flexibility. In this study, we present an analysis of the correlation between task-specific alpha band fluctuations and BOLD activity in the facial expressions processing network. We characterized the network ROIs through a stringent localizer and identified two clusters on the scalp (one frontal, one parietal-occipital) with marked alpha fluctuations, related to the task. We then check whether such power variations throughout the time correlate with the BOLD activity in the network. Our results show statistically significant negative correlations between the alpha power in both clusters and for all the ROIs of the network. The correlation levels have still not met the requirements for transferring the protocol to an EEG setup, but they pave the way towards a better understand on how frontal and parietal-occipital alpha relates to the activity of the facial expressions processing network.

AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG.

Science.gov (United States)

Senga, Yukako; Imamura, Hiroshi; Miyafusa, Takamitsu; Watanabe, Hideki; Honda, Shinya

2017-09-29

Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.

A role for α-galactosidase in the degradation of the endosperm cell walls of lettuce seeds, cv. Grand Rapids.

Science.gov (United States)

Leung, D W; Bewley, J D

1983-04-01

Isolated endosperms of Grand Rapids lettuce (Lactuca sativa L.) seeds undergo extensive cell-wall degradation and sugars are released into the surrounding incubation medium. One sugar so released is galactose. α-Galactosidase (EC 3.2.122) is present at the same level in both dry and imbibed isolated endosperms and is responsible for the release of galactose. However, this enzyme does not act upon the native endosperm cell wall, but requires first its partial hydrolysis and the production of oligomers by the action of endo-β-mannanase (EC 3.2.1.787). Galactose is then cleaved from these oligomers, allowing their further subsequent hydrolysis by endo-β-mannanase. Thus α-galactosidase and endo-β-mannanase act cooperatively to effect the hydrolysis of the lettuce endosperm cell walls.

Improving the limits of detection of low background alpha emission measurements

Science.gov (United States)

McNally, Brendan D.; Coleman, Stuart; Harris, Jack T.; Warburton, William K.

2018-01-01

Alpha particle emission - even at extremely low levels - is a significant issue in the search for rare events (e.g., double beta decay, dark matter detection). Traditional measurement techniques require long counting times to measure low sample rates in the presence of much larger instrumental backgrounds. To address this, a commercially available instrument developed by XIA uses pulse shape analysis to discriminate alpha emissions produced by the sample from those produced by other surfaces of the instrument itself. Experience with this system has uncovered two residual sources of background: cosmogenics and radon emanation from internal components. An R&D program is underway to enhance the system and extend the pulse shape analysis technique further, so that these residual sources can be identified and rejected as well. In this paper, we review the theory of operation and pulse shape analysis techniques used in XIA's alpha counter, and briefly explore data suggesting the origin of the residual background terms. We will then present our approach to enhance the system's ability to identify and reject these terms. Finally, we will describe a prototype system that incorporates our concepts and demonstrates their feasibility.

Determination of Activated Carbon Residual Life using a Microwave Cavity Resonator

International Nuclear Information System (INIS)

Mason, A; Wylie, S; Shaw, A; Al-Shamma'a, A I; Thomas, A; Keele, H

2011-01-01

This paper presents the continuation of work conducted jointly between Dstl and LJMU. This unique body of work has been, largely, concerned with detecting the residual life of high performance filter materials using electromagnetic (EM) waves within a resonant cavity. Past work has considered both HEPA [1] and ASZM-TEDA[2] activated carbon filter materials. This paper continues the later work, considering the response of ASZM-TEDA activated carbon through the co-ageing of two distinct batches of the material. The paper briefly introduces activated carbon, discusses theory relevant to the work and the methodology used for investigation. A comprehensive set of results is included which seek to validate this technique for determining the residual lifespan of activated carbon.

Conditioning of high activity solid waste: fuel claddings and dissolution residues

International Nuclear Information System (INIS)

Anon.

1982-01-01

This chapter reports on experimental studies of embedding into matrix material, the melting and conversion of zircaloy, and waste properties and characterization. Methods are developed for embedding the waste scrap into a solid and resistant matrix material in order to confine the radioactivity and to prevent it from dispersion. The matrix materials investigated are lead alloys, ceramics and compacted graphite or aluminium powder. The treatment of fuel hulls by melting or chemical conversion is described. Cemented hulls are characterized and the pyrophoricity of zircaloy fines is investigated. Topics considered include the embedding of hulls into graphite and aluminium, the embedding of hulls and dissolution residues into alumino-ceramics, the solidification of alpha-bearing wastes into a ceramic matrix, and the conditioning of cladding waste by eutectoidic melting and by embedding in glass

[(35)S]-GTPgammaS autoradiography reveals alpha(2) adrenoceptor-mediated G-protein activation in amygdala and lateral septum.

Science.gov (United States)

Newman-Tancredi, A; Chaput, C; Touzard, M; Millan, M J

2000-04-03

alpha(2)-adrenoceptor-mediated G-protein activation was examined by [(35)S]-GTPgammaS autoradiography. In alpha(2)-adrenoceptor-rich regions (amygdala, lateral septum), noradrenaline stimulated [(35)S]-GTPgammaS binding. These actions were abolished by the selective alpha(2) antagonist, atipamezole. Conversely, in caudate nucleus, which expresses few alpha(2) receptors, noradrenaline-induced stimulation was not inhibited by atipamezole, suggesting that it is not mediated by alpha(2)-adrenoceptors.

Peroxisome Proliferator-Activated Receptor Alpha Target Genes

Directory of Open Access Journals (Sweden)

Maryam Rakhshandehroo

2010-01-01

Full Text Available The peroxisome proliferator-activated receptor alpha (PPARα is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

Kinetics and Thermal Properties of Crude and Purified β-Galactosidase with Potential for the Production of Galactooligosaccharides

Directory of Open Access Journals (Sweden)

Anna Rafaela Cavalcante Braga

2013-01-01

Full Text Available β-Galactosidase is an enzyme that catalyzes the hydrolysis of lactose. It has potential importance due to various applications in the food and dairy industries, involving lactose-reduced ingredients. The properties of two β-galactosidase enzymes, crude and purified, from different sources, Kluyveromyces marxianus CCT 7082 and Kluyveromyces marxianus ATCC 16045, were analyzed. The pH and temperature optima, deactivation energy, thermal stability and kinetic and thermodynamic parameters were determined, as well as the ability to hydrolyze lactose and produce galactooligosaccharides. Purification process improved the properties of the enzymes, and the results showed that purified enzymes from both strains had a higher optimum temperature, and lower values of Km, thus showing greater affinity for o-nitrophenyl-β-D-galactopiranoside than the crude enzymes. The production of galactooligosaccharides was also greater when using purified enzymes, increasing the synthesis by more than 30 % by both strains.

Instruments used to measure or check {alpha}, {beta}, {gamma} activity and neutron emission in the course of processing ore or irradiated fuel; Appareils de mesure ou de controle {alpha}, {beta}, {gamma}, n, des circuits des usines de traitement du minerai ou du combustible irradie

Energy Technology Data Exchange (ETDEWEB)

Blanc, A; Brunet, M; Kermagoret, M; Labeyrie, J; Roux, G; Vasseur, J; Weil, J [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1959-07-01

One of the methods checking ores in the course of treatment is the rapid quantitative determination of thorium. This measurement is carried out by means of a scintillation instrument which shows the {beta} and {alpha} coincidences of ThC and ThC'. The treatment of irradiated fuel is accompanied by a large number of radioactive checks relative to the performance of the fixation and elution operations of uranium in the ion exchangers, to the concentration of radioactivity of effluent sent from the plant into watercourses. The operations of fixation and elution of the uranium are checked automatically by an instrument which takes a sample of 5 cm{sup 3} of solution, evaporates it and measures its activity every 10 or 20 minutes. Plutonium concentrations are measured: - in the presence of strong {beta} {gamma} activities, by means of rotating cylinder detectors; - in the presence of weak {beta} {gamma} activities, by means of {alpha} detectors scanning a constant level liquid surface; - by means of fission chambers relatively insensitive to {gamma}. Fission product concentrations are measured by chambers, counters or scintillators, according to the amount of {gamma} activity present. Finally, the activity of effluent to be emptied into watercourses is checked by means of a scintillation instrument, which measures the {alpha} activity on the one hand, and on the other hand the {beta} {gamma} activity of residue from a 100 cm{sup 3} sample taken and evaporated in 20 minutes. (author) [French] Parmi les controles relatifs au minerai en cours de traitement, figure le dosage rapide de thorium. Cette mesure est realisee au moyen d'un appareillage a scintillation qui met en evidence la coincidence des emissions {beta} et {alpha} du ThC et du ThC'. Le traitement des combustibles irradies s'accompagne d'un grand nombre de controles radioactifs portant sur le fonctionnement des operations de fixation et d'elution de l'uranium dans les echangeurs d'ions, sur la concentration du

Salivary type alpha-amylase activity in serum and in urine of patients with lung adenocarcinoma

International Nuclear Information System (INIS)

Zakrzewska, I.; Wolska, K.; Koput, A.

1993-01-01

Total alpha-amylase activity in sera and urine of 30 patients with lung adenocarcinoma has been tested. The results were compared with control group of 30 healthy voluntaries. The activity of pancreatic type was differentiated from salivary alpha amylase. Salivary type was inhibited selectively by Triticum aestivum. Higher levels of total and salivary type amylase were noted in patients with lung adenocarcinoma in comparison to healthy control. The increase was significant (p<0.005). Correlation was observed between the activity of salivary type amylase and the stage of adenocarcinoma. (author)

Residual Stress Distribution and Microstructure of a Multiple Laser-Peened Near-Alpha Titanium Alloy

Science.gov (United States)

Umapathi, A.; Swaroop, S.

2018-04-01

Laser peening without coating (LPwC) was performed on a Ti-2.5 Cu alloy with multiple passes (1, 3 and 5), using a Nd:YAG laser (1064 nm) at a constant overlap rate of 70% and power density of 6.7 GW cm-2. Hardness and residual stress profiles indicated thermal softening near the surface (hardness (235 HV at 500 μm) and maximum residual stress (- 890 MPa at 100 μm) were observed for LPwC with 1 pass. Surface roughness and surface 3-D topography imaging showed that the surface roughness increased with the increase in the number of passes. XRD results indicated no significant β phases. However, peak shifts, broadening and asymmetry were observed and interpreted based on dislocation activity. Microstructures indicated no melting or resolidification or refinement of grains at the surface. Twin density was found to increase with the increase in the number of passes.

An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

DEFF Research Database (Denmark)

Lynagh, Timothy Peter; Lynch, Joseph W

2010-01-01

The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity...... conductance, homomeric expression, and human origin may render the F207A/A288G alpha1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue...

cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei.

Science.gov (United States)

Hwang, Jae-Ho; Yokoyama, Yoshihiro; Mizuta, Shoshi; Yoshinaka, Reiji

2006-05-01

A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.

Galactosylation of steroidal saponins by β-galactosidase from Lactobacillus bulgaricus L3.

Science.gov (United States)

Zhang, Jie; Lu, Lili; Lu, Li; Zhao, Yang; Kang, Liping; Pang, Xu; Liu, Jingyuan; Jiang, Tengchuan; Xiao, Min; Ma, Baiping

2016-02-01

The galactosylation of furostanosides and spirostanosides were investigated by using β-galactosidase from Lactobacillus bulgaricus L3 as a catalyst and lactose as a sugar donor. Four novel galactosylated furostanoside products (compounds 1-4) from compound F, compound G, compound I, and compound H were obtained and their structures were identified by HR-ESI-MS, 1D and 2D NMR spectra. The result showed that galactosyl moiety was found to be added to the 6-OH of the 26-O-glucosyl in these four furostanoside substrates.

The Effect of Kaffir Lime Leaves Distillation Residue Oleoresin Concentration on Active Paper Packaging Characteristics

Science.gov (United States)

Kawiji; Utami, R.; Ulum, S.; Khasanah, L. U.; Manuhara, G. J.; Atmaka, W.

2018-03-01

Oleoresin of kaffir lime leaves distillation residue still contains some active compounds such as Citronellal, β-Citronellol, and Linalool which potential to incorporated on the active paper packaging. The purposes of this study were to determine the effect of kaffir lime leaves distillation residue oleoresin concentration on the physical characteristics, sensory characteristics, and antimicrobial activity of the active paper packaging incorporated with kaffir lime leaves distillation residue oleoresin and to determine the functional groups of active paper packaging. The concentration of kaffir lime leaves distillation residue oleoresin were varied at 0%, 2%, 4% and 6%. The result showed that the addition of kaffir lime leaves distillation residue oleoresin increased the thickness and moisture content of the paper and decreased the tensile strengths and folding endurances of active paper packaging. The microbial inhibition tends to increase along with the higher oleoresin concentration addition. Aromatic CH group were found at a wavelength of 897.90 cm-1 of on paper packaging with 2% oleoresin indicated as functional aromatic functional group allegedly obtained from the kaffir lime leaves oleoresin.

In-vitro activity of S. lavandulaefolia (Spanish sage) relevant to treatment of Alzheimer's disease.

Science.gov (United States)

Perry, N S; Houghton, P J; Sampson, J; Theobald, A E; Hart, S; Lis-Balchin, M; Hoult, J R; Evans, P; Jenner, P; Milligan, S; Perry, E K

2001-10-01

Salvia lavandulaefolia Vahl. (Spanish sage) essential oil and individual monoterpenoid constituents have been shown to inhibit the enzyme acetylcholinesterase in-vitro and in-vivo. This activity is relevant to the treatment of Alzheimer's disease, since anticholinesterase drugs are currently the only drugs available to treat Alzheimer's disease. Other activities relevant to Alzheimer's disease include antioxidant, anti-inflammatory and estrogenic effects. Results of in-vitro tests for these activities are reported here for S. lavandulaefolia extracts, the essential oil and its major constituents. Antioxidant activity (inhibition of bovine brain liposome peroxidation) was found in the EtOH extract of the dried herb (5 mg mL(-1)) and the monoterpenoids (0.1 M) alpha- and beta-pinene and 1,8-cineole. Thujone and geraniol had lower antioxidant effects, while camphor had no antioxidant effects. Possible anti-inflammatory activity (eicosanoid inhibition in rat leucocytes) was found in the EtOH extract (50 microg mL(-1)) and was shown by the monoterpenoids alpha-pinene and geraniol (0.2 mM), but not 1,8-cineole, thujone or camphor. Possible estrogenic activity (via induction of beta-galactosidase activity in yeast cells) was found in the essential oil (0.01 mg mL(-1)) and the monoterpenoid geraniol (0.1-2 mM). 1,8-Cineole, alpha- and beta-pinene and thujone did not exhibit estrogenic activity in this analysis. These results demonstrate that S. lavandulaefolia, its essential oil and some chemical constituents have properties relevant to the treatment of Alzheimer's disease and provide further data supporting the value of carrying out clinical studies in patients with Alzheimer's disease using this plant species.

Peroxisome proliferator-activated receptor {alpha} agonists modulate Th1 and Th2 chemokine secretion in normal thyrocytes and Graves' disease

Energy Technology Data Exchange (ETDEWEB)

Antonelli, Alessandro, E-mail: a.antonelli@med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Ferrari, Silvia Martina, E-mail: sm.ferrari@int.med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Frascerra, Silvia, E-mail: lafrasce@gmail.com [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Corrado, Alda, E-mail: dala_res@hotmail.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Pupilli, Cinzia, E-mail: c.pupilli@dfc.unifi.it [Endocrinology Unit, Azienda Ospedaliera Careggi and University of Florence, Viale Morgagni 85, I-50134, Florence (Italy); Bernini, Giampaolo, E-mail: g.bernini@int.med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Benvenga, Salvatore, E-mail: s.benvenga@me.nettuno.it [Department of Clinical and Experimental Medicine, Section of Endocrinology, University of Messina, Piazza Pugliatti 1, I-98122, Messina (Italy); Ferrannini, Ele, E-mail: eferrannini@med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Fallahi, Poupak, E-mail: poupak@int.med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy)

2011-07-01

Until now, no data are present about the effect of peroxisome proliferator-activated receptor (PPAR){alpha} activation on the prototype Th1 [chemokine (C-X-C motif) ligand (CXCL)10] (CXCL10) and Th2 [chemokine (C-C motif) ligand 2] (CCL2) chemokines secretion in thyroid cells. The role of PPAR{alpha} and PPAR{gamma} activation on CXCL10 and CCL2 secretion was tested in Graves' disease (GD) and control primary thyrocytes stimulated with interferon (IFN){gamma} and tumor necrosis factor (TNF){alpha}. IFN{gamma} stimulated both CXCL10 and CCL2 secretion in primary GD and control thyrocytes. TNF{alpha} alone stimulated CCL2 secretion, while had no effect on CXCL10. The combination of IFN{gamma} and TNF{alpha} had a synergistic effect both on CXCL10 and CCL2 chemokines in GD thyrocytes at levels comparable to those of controls. PPAR{alpha} activators inhibited the secretion of both chemokines (stimulated with IFN{gamma} and TNF{alpha}) at a level higher (for CXCL10, about 60-72%) than PPAR{gamma} agonists (about 25-35%), which were confirmed to inhibit CXCL10, but not CCL2. Our data show that CCL2 is modulated by IFN{gamma} and TNF{alpha} in GD and normal thyrocytes. Furthermore we first show that PPAR{alpha} activators inhibit the secretion of CXCL10 and CCL2 in thyrocytes, suggesting that PPAR{alpha} may be involved in the modulation of the immune response in the thyroid.

The glycosylation and characterization of the candidate Gc macrophage activating factor

DEFF Research Database (Denmark)

Ravnsborg, Tina; Olsen, Dorthe T; Thysen, Anna Hammerich

2010-01-01

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common...... isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc...... globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF...

Purificação de três diferentes beta-galactosidades microbianas por partição em sistemas de duas fases aquosas Purification of three different microbial beta-galactosidases by partitioning in aqueous two-phase systems

Directory of Open Access Journals (Sweden)

Maria Estela SILVA

1997-12-01

Full Text Available Este trabalho tratou da investigação do efeito do peso molecular de polietilenoglicol (PEG sobre a partição de enzimas beta-galactosidases de diferentes origens microbianas: Escherichia coli, Klueveromyces lactis e Aspergillus orizae em sistemas de duas fases aquosas (SDFA.Foi observado que os melhores sistemas para purificação da enzima de E. coli foram os formados por PEG 4000, 6000 e 8000/fosfato, fornecendo os mais elevados fatores de purificação da enzima. As enzimas de Klueveromyces lactis e Aspergillus orizae não foram eficientemente purificadas nestes sistemas sendo insensíveis à alterações do peso molecular do PEG. Portanto, um outro sistema de duas fases aquosas foi desenvolvido contendo um ligante específico, p-aminofenil 1-tio-beta-D-galactopiranosídeo (APGP, acoplado ao PEG para purificar a enzima de Klueveromyces lactis. Uma etapa simples de partição no SDFA formado por 6% APGP-PEG4000 + 12% dextrana T505.000 foi capaz de recuperar 83% da enzima na fase superior do sistema e de aumentar 1,6 vezes o fator de purificação.This work investigated the effect of the molecular weight of polyethyleneglycol (PEG upon the partition coefficient of beta-galactosidases from three different microorganisms: Escherichia coli, Klueveromyces lactis and Aspergillus orizae. It was found that PEG 6,000 and PEG 8,000/phosphate were the best systems for achieving the highest purification factors of E. coli beta-galactosidase. However, the other two yeast beta-galactosidases were not efficiently separated from their contaminants in any of the PEG/salt systems. In order to improve the separation of Klueveromyces lactis beta-galactosidase from the main protein contaminants, the biospecific ligand p-aminophenyl 1-thio-beta-D-galactopyranoside (APGP was attached to activated PEG 4000. The affinity PEG having APGP bound to its backbone was synthesized in two steps. The partitioning of Klueveromyces lactis beta-galactosidase in aqueous two

Raffinose family oligosaccharide utilisation by probiotic bacteria: insight into substrate recognition, molecular architecture and diversity of GH36 alpha-galactosidases

DEFF Research Database (Denmark)

Abou Hachem, Maher; Fredslund, Folmer; Andersen, Joakim Mark

2012-01-01

The organisation of genes conferring utilisation of raffinose family oligosaccharides (RFOs) has been analysed in several probiotic bacteria from the Bifidobacterium and Lactobacillus genera. Glycoside hydrolase family 36 (GH36) alpha-galatosidase encoding genes occur together with sugar transpor...

Investigations to determine whether viable microorganisms are required during intestinal lactose hydrolysis of fermented milk products by microbial ß-galactosidase using gnotobiotic Göttingen minipigs

OpenAIRE

Winchenbach, Andrea

2010-01-01

The most common reason worldwide for the indigestibility of milk is the lack of ß-galactosidases in the small intestine, leading to the malabsorbtion of lactose. Fermented dairy products are very often much better tolerated than raw (not fermented) milk, because of the microbial ß-galactosidases they contain. The aim of this thesis was to elucidate the question as to weather lactose hydrolysis in the small intestine requires the presence of living bacteria (with their microbial ß-galac...

Gross alpha and beta activities in drinking water from Goias State, Brazil

Energy Technology Data Exchange (ETDEWEB)

Mingote, Raquel M. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN), Belo Horizonte, MG (Brazil); Nogueira, Regina A.; Costa, Heliana F. da, E-mail: raquel.mingote@cdtn.br, E-mail: rnogueira@cnen.gov.br, E-mail: heliana@cnen.gov.br [Centro Regional de Ciencias Nucleares do Centro-Oeste (CRCN-CO/CNEN), Abadia de Goias, GO (Brazil). Parque Estadual Telma Ortegal

2017-07-01

Detection of gross alpha and beta radioactivity is important for a quick surveying of both natural and anthropogenic radioactivity in water. Furthermore, gross alpha and gross beta parameters are included in Brazilian legislation on quality of drinking water. In this work, a low background liquid scintillation spectrometer was used to simultaneously determine gross alpha and gross beta in samples of the public water supplies in the state of Goias, Brazil, during 2010-2015. Sample preparation involved evaporation to concentrate the sample ten-fold. The results indicate that the water meets the radioactivity standards required by the regulations MS 2914/2011 of the Brazilian Department of Health. Concerning the high level of censored observations, a statistical treatment of data was conducted by using analysis methods of censored data to provide a reference value of the gross alpha and beta radioactivity in drinking water from the state of Goias. The estimated typical activities are very low, 0.030 Bq•L{sup -1} and 0.058 Bq•L{sup -1}, respectively. (author)

Effect of radioactive isotope 32P upon alpha amylase activity and glucose concentration in chickens

International Nuclear Information System (INIS)

Kraljevic, P.; Emanovic, D.; Simpraga, M.; Nejedli, S.; Stojevic, Z.

1996-01-01

An attempt has been made to investigate whether alpha amylase activity and glucose concentration in blood plasma can serve as the help in establishing on early diagnosis of organic or functional damage caused by ionizing radiation in chickens. Fifty day old hybrid chickens of heavy 'Jata' breeds of both sexes, were treated by 32 P administered intramusculary as sodium orthophosphate in a single dose of 333 MBq per kilogram of body weight. Blood samples was taken from the wing vein on day 1, 3, 5, 7 and 10 after administration of 32 P. Alpha amylase activity and glucose concentration were determined spectrophotometrically using kits produced by 'Radonja', Sisak. Alpha amylase activity was decreased and glucose concentration was increased during investigated period. Yet, the further investigations are needed to find out whether these two parameters can be used for early diagnosis of injury in chicken organism by ionizing radiation. (author)

Roles of s3 site residues of nattokinase on its activity and substrate specificity.

Science.gov (United States)

Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

2007-09-01

Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

Synthesis of tritiated 1-alpha-methadol and 1-alpha-acetylmethadol

Energy Technology Data Exchange (ETDEWEB)

Thang, D.C.; Nam, N.H.; Pontikis, R. (Institut National de la Sante et de la Recherche Medicale (INSERM), Hopital Fernand Widal, 75 - Paris (France)); Pichat, L. (CEA Centre d' Etudes Nucleaires de Saclay, 91 - Gif-sur-Yvette (France). Service des Molecules Marquees)

1982-04-01

dl-Methadone was resolved by crystallization of its ammonium d- ..cap alpha.. -bromocamphor-..pi..-sulfonate salt to give d-methadone. The latter in ethyl acetate solution was reduced with tritium gas to 1-..cap alpha..-methadol /sup 3/H in presence of Adams platinum oxide at normal temperature and pressure. Acetylation of 1-..cap alpha..-carbinol hydrochloride by means of acetyl chloride afforded 1-..cap alpha..-acetylmethadol /sup 3/H, specific activity: 20 Ci/mMole. The positions and extent of tritium labelling were determined by /sup 3/H NMR spectroscopy.

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

Science.gov (United States)

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. © 2015 International Union of Biochemistry and Molecular Biology.

Testosterone 5alpha-reductase inhibitory active constituents of Piper nigrum leaf.

Science.gov (United States)

Hirata, Noriko; Tokunaga, Masashi; Naruto, Shunsuke; Iinuma, Munekazu; Matsuda, Hideaki

2007-12-01

Previously we reported that Piper nigrum leaf extract showed a potent stimulation effect on melanogenesis and that (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2) were isolated as active constituents. As a part of our continuous studies on Piper species for the development of cosmetic hair-care agents, testosterone 5alpha-reductase inhibitory activity of aqueous ethanolic extracts obtained from several different parts of six Piper species, namely Piper nigrum, P. methysticum, P. betle, P. kadsura, P. longum, and P. cubeba, were examined. Among them, the extracts of P. nigrum leaf, P. nigrum fruit and P. cubeba fruit showed potent inhibitory activity. Activity-guided fractionation of P. nigrum leaf extract led to the isolation of 1 and 2. Fruits of P. cubeba contain 1 as a major lignan, thus inhibitory activity of the fruit may be attributable to 1. As a result of further assay on other known constituents of the cited Piper species, it was found that piperine, a major alkaloid amide of P. nigrum fruit, showed potent inhibitory activity, thus a part of the inhibitory activity of P. nigrum fruit may depend on piperine. The 5alpha-reductase inhibitory activities of 1 and piperine were found for the first time. In addition, the P. nigrum leaf extract showed in vivo anti-androgenic activity using the hair regrowth assay in testosterone sensitive male C57Black/6CrSlc strain mice.

rTMS Induced Tinnitus Relief Is Related to an Increase in Auditory Cortical Alpha Activity

Science.gov (United States)

Müller, Nadia; Lorenz, Isabel; Langguth, Berthold; Weisz, Nathan

2013-01-01

Chronic tinnitus, the continuous perception of a phantom sound, is a highly prevalent audiological symptom. A promising approach for the treatment of tinnitus is repetitive transcranial magnetic stimulation (rTMS) as this directly affects tinnitus-related brain activity. Several studies indeed show tinnitus relief after rTMS, however effects are moderate and vary strongly across patients. This may be due to a lack of knowledge regarding how rTMS affects oscillatory activity in tinnitus sufferers and which modulations are associated with tinnitus relief. In the present study we examined the effects of five different stimulation protocols (including sham) by measuring tinnitus loudness and tinnitus-related brain activity with Magnetoencephalography before and after rTMS. Changes in oscillatory activity were analysed for the stimulated auditory cortex as well as for the entire brain regarding certain frequency bands of interest (delta, theta, alpha, gamma). In line with the literature the effects of rTMS on tinnitus loudness varied strongly across patients. This variability was also reflected in the rTMS effects on oscillatory activity. Importantly, strong reductions in tinnitus loudness were associated with increases in alpha power in the stimulated auditory cortex, while an unspecific decrease in gamma and alpha power, particularly in left frontal regions, was linked to an increase in tinnitus loudness. The identification of alpha power increase as main correlate for tinnitus reduction sheds further light on the pathophysiology of tinnitus. This will hopefully stimulate the development of more effective therapy approaches. PMID:23390539

Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases.

NARCIS (Netherlands)

Asfaw, B.; Ledvinova, J.; Dobrovolny, R.; Bakker, H.; Desnick, R.J.; Diggelen, O.P. van; Jong, J.G.N. de; Kanzaki, T.; Chabas, A.; Maire, I.; Conzelmann, E.; Schindler, D.

2002-01-01

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B

Fusion alpha loss diagnostic for ITER using activation technique

Czech Academy of Sciences Publication Activity Database

Bonheure, G.; Hult, M.; González de Orduña, R.; Vermaercke, P.; Murari, A.; Popovichev, S.; Mlynář, Jan

2011-01-01

Roč. 86, 6-8 (2011), s. 1298-1301 ISSN 0920-3796. [Symposium on Fusion Technology (SOFT) /26th./. Port o, 27.09.2010-01.10.2010] Institutional research plan: CEZ:AV0Z20430508 Keywords : ITER * fusion product * burning plasma diagnostics * alpha losses * activation technique Subject RIV: BL - Plasma and Gas Discharge Physics Impact factor: 1.490, year: 2011 http://www.sciencedirect.com/science/article/pii/S0920379611002778

Evidence for a prolonged role of alpha 4 integrin throughout active experimental allergic encephalomyelitis.

Science.gov (United States)

Keszthelyi, E; Karlik, S; Hyduk, S; Rice, G P; Gordon, G; Yednock, T; Horner, H

1996-10-01

The leukocyte integrin receptor, alpha 4 beta 1, and its endothelial cell ligand, vascular cell adhesion molecule 1, appear to be of critical importance in the leukocyte trafficking that accompanies CNS damage in experimental allergic encephalomyelitis (EAE). In this study, the persistence of the role for alpha 4 beta 1/VCAM-1 in EAE was established by observing antibody-mediated disease reversal up to 1 month following disease onset. Limited treatment with a monoclonal antibody against alpha 4 integrin, GG5/3, resulted in a significant decrease in both clinical and histopathologic signs. This was not observed in isotype control experiments. In the latter phase of progressive disease, widespread demyelination occurred in the animals that did not respond to 6 days of anti-alpha 4 treatment. These results demonstrate an essential role for alpha 4 beta 1 interactions throughout active EAE and illustrate the difference between reversible clinical deficits caused by edema and irreversible deficits associated with demyelination.

Cow's milk increases the activities of human nuclear receptors peroxisome proliferator-activated receptors alpha and delta and retinoid X receptor alpha involved in the regulation of energy homeostasis, obesity, and inflammation.

Science.gov (United States)

Suhara, W; Koide, H; Okuzawa, T; Hayashi, D; Hashimoto, T; Kojo, H

2009-09-01

The nuclear peroxisome proliferator-activated receptors (PPAR) have been shown to play crucial roles in regulating energy homeostasis including lipid and carbohydrate metabolism, inflammatory responses, and cell proliferation, differentiation, and survival. Because PPAR agonists have the potential to prevent or ameliorate diseases such as hyperlipidemia, diabetes, atherosclerosis, and obesity, we have explored new natural agonists for PPAR. For this purpose, cow's milk was tested for agonistic activity toward human PPAR subtypes using a reporter gene assay. Milk increased human PPARalpha activity in a dose-dependent manner with a 3.2-fold increase at 0.5% (vol/vol). It also enhanced human PPARdelta activity in a dose-dependent manner with an 11.5-fold increase at 0.5%. However, it only slightly affected human PPARgamma activity. Ice cream, butter, and yogurt also increased the activities of PPARalpha and PPARdelta, whereas vegetable cream affected activity of PPARdelta but not PPARalpha. Skim milk enhanced the activity of PPAR to a lesser degree than regular milk. Milk and fresh cream increased the activity of human retinoid X receptor (RXR)alpha as well as PPARalpha and PPARdelta, whereas neither affected vitamin D3 receptor, estrogen receptors alpha and beta, or thyroid receptors alpha and beta. Both milk and fresh cream were shown by quantitative real-time PCR to increase the quantity of mRNA for uncoupling protein 2 (UCP2), an energy expenditure gene, in a dose-dependent manner. The increase in UCP2 mRNA was found to be reduced by treatment with PPARdelta-short interfering (si)RNA. This study unambiguously clarified at the cellular level that cow's milk increased the activities of human PPARalpha, PPARdelta, and RXRalpha. The possible role in enhancing the activities of PPARalpha, PPARdelta, and RXRalpha, and the health benefits of cow's milk were discussed.

Antihypertensive Activity of Residue From “Gebto Arekei”. Locally ...

African Journals Online (AJOL)

Antihypertensive Activity of Residue From “Gebto Arekei”. Locally Distilled Medicinal Spirit From a Brew Containing Lupinus albus Seeds in Renovascular Hypertensive Guines-Pigs. Cherinet Ambaye, Tesfaye Tolessa, Abebe Abera, Hassen Taha Sherief, Dawit Abebe, Kelbessa Urga ...

Immunostimulatory effects of natural human interferon-alpha (huIFN-alpha) on carps Cyprinus carpio L.

Science.gov (United States)

Watanuki, Hironobu; Chakraborty, Gunimala; Korenaga, Hiroki; Kono, Tomoya; Shivappa, R B; Sakai, Masahiro

2009-10-15

Human interferon-alpha (huIFN-alpha) is an important immunomodulatory substance used in the treatment and prevention of numerous infectious and immune-related diseases in animals. However, the immunostimulatory effects of huIFN-alpha in fish remain to be investigated. In the current study, the immune responses of the carp species Cyprinus carpio L. to treatment with huIFN-alpha were analyzed via measurement of superoxide anion production, phagocytic activity and the expression of cytokine genes including interleukin-1beta, tumor necrosis factor-alpha and interleukin 10. Low doses of huIFN-alpha were administered orally once a day for 3 days, and sampling was carried out at 1, 3 and 5 days post-treatment. Our results indicate that a low dose of huIFN-alpha significantly increased phagocytic activity and superoxide anion production in the carp kidney. The huIFN-alpha-treated fish also displayed a significant upregulation in cytokine gene expression. The current study demonstrates the stimulatory effects of huIFN-alpha on the carp immune system and highlights the immunomodulatory role of huIFN-alpha in fish.

Cloning, expression and structural stability of a cold-adapted ß-Galactosidase from Rahnella sp.R3

Science.gov (United States)

A novel gene was isolated for the first time from a psychrophilic gram-negative bacterium Rahnella sp.R3. It encoded a cold-adapted ß-galactosidase (R-ß-Gal). Recombinant R-ß-Gal was expressed in Escherichia coli BL21 (DE3), purified, and characterized. R-ß-Gal belongs to the glycosyl hydrolase fami...

Clinical and genetic investigation of a Japanese family with cardiac fabry disease. Identification of a novel α-galactosidase A missense mutation (G195V).

Science.gov (United States)

Nakagawa, Naoki; Maruyama, Hiroki; Ishihara, Takayuki; Seino, Utako; Kawabe, Jun-ichi; Takahashi, Fumihiko; Kobayashi, Motoi; Yamauchi, Atsushi; Sasaki, Yukie; Sakamoto, Naka; Ota, Hisanobu; Tanabe, Yasuko; Takeuchi, Toshiharu; Takenaka, Toshihiro; Kikuchi, Kenjiro; Hasebe, Naoyuki

2011-01-01

Fabry disease is an X-linked lysosomal storage disorder caused by mutations of the α-galactosidase A gene (GLA), and the disease is a relatively prevalent cause of left ventricular hypertrophy mimicking idiopathic hypertrophic cardiomyopathy. We assessed clinically 5 patients of a three-generation family and also searched for GLA mutations in 10 family members. The proband had left ventricular hypertrophy with localized thinning in the basal posterior wall and late gadolinium enhancement (LGE) in the near-circumferential wall in cardiovascular magnetic resonance images and her sister had vasospastic angina pectoris without organic stenosis of the coronary arteries. LGE notably appeared in parallel with decreased α-galactosidase A activity and increased NT-pro BNP in our patients. We detected a new GLA missense mutation (G195V) in exon 4, resulting in a glycine-to-valine substitution. Of the 10 family members, 5 family members each were positive and negative for this mutation. These new data extend our clinical and molecular knowledge of GLA gene mutations and confirm that a novel missense mutation in the GLA gene is important not only for a precise diagnosis of heterozygous status, but also for confirming relatives who are negative for this mutation.

The combination of IL-21 and IFN-alpha boosts STAT3 activation, cytotoxicity and experimental tumor therapy

DEFF Research Database (Denmark)

Eriksen, Karsten W; Søndergaard, Henrik; Woetmann, Anders

2008-01-01

such as IFN-alpha and IL-2 have multiple and severe side effects. Accordingly, they are generally used at sub-optimal doses, which limit their clinical efficacy. Here we hypothesized that a combination of IFN-alpha and IL-21, a novel cytokine of the IL-2 family with anti-cancer effects, will increase the anti......-cancer efficacy at sub-optimal cytokine doses. We show that the combined stimulation of target-cells with IFN-alpha and IL-21 triggers an increased STAT3 activation whereas the activation of other STATs including STAT1/2 is unaffected. In parallel, the combined stimulation with IFN-alpha and IL-21 triggers...... a selective increase in MHC class I expression and NK- and CD8(+) T-cell-mediated cytotoxicity. In an experimental in vivo model of renal carcinoma, the combined treatment of IFN-alpha and IL-21 also produces a significant anti-cancer effect as judged by an inhibition of tumor growth and an increased survival...

Determination of alpha activity produced by Indian tea samples using solid state nuclear track detection technique

International Nuclear Information System (INIS)

Azam, Ameer; Naqvi, A.H.; Srivastava, D.S.

2001-01-01

India has first place in the world in the production, consumption and export of tea. The average production of tea in India is about 780 million kg annually. Indian population uses tea as a favourite beverage specially with the breakfast. Tea leaves are perennial plants and they have extension root system spread over deeper layers in the soil. Soil contains uranium as trace element. Hence, uranium is transferred to the leaves as a result of uptake from the soil over the years. Since many of the decay products of uranium are alpha emitters hence tea is a source of alpha radioactivity and can cause damaging effects. Therefore, data on alpha activity of tea samples will be useful in estimating these harmful effects at least qualitatively. In the present study the alpha activity produced by thirteen samples of tea available in the local market was measured

Alpha-momorcharin: a ribosome-inactivating protein from Momordica charantia, possessing DNA cleavage properties.

Science.gov (United States)

Wang, Shuzhen; Zheng, Yinzhen; Yan, Junjie; Zhu, Zhixuan; Wu, Zhihua; Ding, Yi

2013-11-01

Ribosome-inactivating proteins (RIPs) function to inhibit protein synthesis through the removal of specific adenine residues from eukaryotic ribosomal RNA and rending the 60S subunit unable to bind elongation factor 2. They have received much attention in biological and biomedical research due to their unique activities toward tumor cells, as well as the important roles in plant defense. Alpha-momorcharin (α-MC), a member of the type I family of RIPs, is rich in the seeds of Momordica charantia L. Previous studies demonstrated that α-MC is an effective antifungal and antibacterial protein. In this study, a detailed analysis of the DNase-like activity of α-MC was conducted. Results showed that the DNase-like activity toward plasmid DNA was time-dependent, temperature-related, and pH-stable. Moreover, a requirement for divalent metal ions in the catalytic domain of α-MC was confirmed. Additionally, Tyr(93) was found to be a critical residue for the DNase-like activity, while Tyr(134), Glu(183), Arg(186), and Trp(215) were activity-related residues. This study on the chemico-physical properties and mechanism of action of α-MC will improve its utilization in scientific research, as well as its potential industrial uses. These results may also assist in the characterization and elucidation of the DNase-like enzymatic properties of other RIPs.