Thermostability of Probiotics and Their α -Galactosidases and the Potential for Bean Products.

PubMed

Liu, Xiaoli; Champagne, Claude P; Lee, Byong H; Boye, Joyce I; Casgrain, Michel

2014-01-01

Soybeans and other pulses contain oligosaccharides which may cause intestinal disturbances such as flatulence. This study was undertaken to investigate α -galactosidase-producing probiotics added to frozen foods which can survive warming treatments used in thawing and consumption of the pulses. The maximum α -galactosidase activity (1.26 U/mg protein) was found in Bifidobacterium breve S46. Lactobacillus casei had the highest α -galactosidase thermostability among the various strains, with D values of 35, 29, and 9.3 minutes at 50°C, 55°C, and 60°C, respectively. The enzyme activity was less affected than viable cells by heating. However, the D values of two bacterial enzymes were lower than those of three commercial α -galactosidase-containing products. Freshly grown cells and their enzymes were more stable than the rehydrated cultures and their enzymes. Practical Application. Enzymes and cultures can be added to foods in order to enhance the digestibility of carbohydrates in the gastrointestinal tract. However since many foods are warmed, it is important that the thermostability of the enzymes be assessed. This paper provides data on the stability of α -galactosidase, which could potentially be added to food matrices containing stachyose or raffinose, such as beans.

Thermostability of Probiotics and Their α-Galactosidases and the Potential for Bean Products

PubMed Central

Liu, Xiaoli; Champagne, Claude P.; Lee, Byong H.; Boye, Joyce I.; Casgrain, Michel

2014-01-01

Soybeans and other pulses contain oligosaccharides which may cause intestinal disturbances such as flatulence. This study was undertaken to investigate α-galactosidase-producing probiotics added to frozen foods which can survive warming treatments used in thawing and consumption of the pulses. The maximum α-galactosidase activity (1.26 U/mg protein) was found in Bifidobacterium breve S46. Lactobacillus casei had the highest α-galactosidase thermostability among the various strains, with D values of 35, 29, and 9.3 minutes at 50°C, 55°C, and 60°C, respectively. The enzyme activity was less affected than viable cells by heating. However, the D values of two bacterial enzymes were lower than those of three commercial α-galactosidase-containing products. Freshly grown cells and their enzymes were more stable than the rehydrated cultures and their enzymes. Practical Application. Enzymes and cultures can be added to foods in order to enhance the digestibility of carbohydrates in the gastrointestinal tract. However since many foods are warmed, it is important that the thermostability of the enzymes be assessed. This paper provides data on the stability of α-galactosidase, which could potentially be added to food matrices containing stachyose or raffinose, such as beans. PMID:24744923

Two Distinct States of Escherichia coli Cells That Overexpress Recombinant Heterogeneous β-Galactosidase*

PubMed Central

Zhao, Yun; He, Wei; Liu, Wei-Feng; Liu, Chun-Chun; Feng, Li-Kui; Sun, Lei; Yan, Yong-Bin; Hang, Hai-Ying

2012-01-01

The mechanism by which inclusion bodies form is still not well understood, partly because the dynamic processes of the inclusion body formation and its solubilization have hardly been investigated at an individual cell level, and so the important detailed information has not been acquired for the mechanism. In this study, we investigated the in vivo folding and aggregation of Aspergillus phoenicis β-d-galactosidase fused to a red fluorescence protein in individual Escherichia coli cells. The folding status and expression level of the recombinant β-d-galactosidase at an individual cell level was analyzed by flow cytometry in combination with transmission electron microscopy and Western blotting. We found that individual E. coli cells fell into two distinct states, one containing only inclusion bodies accompanied with low galactosidase activity and the other containing the recombinant soluble galactosidase accompanied with high galactosidase activity. The majority of the E. coli cells in the later state possessed no inclusion bodies. The two states of the cells were shifted to a cell state with high enzyme activity by culturing the cells in isopropyl 1-thio-β-d-galactopyranoside-free medium after an initial protein expression induction in isopropyl 1-thio-β-d-galactopyranoside-containing medium. This shift of the cell population status took place without the level change of the β-d-galactosidase protein in individual cells, indicating that the factor(s) besides the crowdedness of the recombinant protein play a major role in the cell state transition. These results shed new light on the mechanism of inclusion body formation and will facilitate the development of new strategies in improving recombinant protein quality. PMID:22303013

Immobilized alpha-Galactosidase in the Biochemistry Laboratory

ERIC Educational Resources Information Center

Mulimani, V. H.; Dhananjay, K.

2007-01-01

This laboratory experiment was designed to demonstrate the application of immobilized galactosidase in food industry to hydrolyze raffinose family oligosaccharides in soymilk. This laboratory experiment was conducted for postgraduate students of biochemistry and developed for graduate and undergraduate students of biochemistry, biotechnology,…

Adsorption of β-galactosidase on silica and aluminosilicate adsorbents

NASA Astrophysics Data System (ADS)

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.

Study on beta-galactosidase enzymatic activity of herbal yogurt.

PubMed

Chowdhury, Banani Ray; Chakraborty, Runu; Raychaudhuri, Utpal

2008-03-01

Different types of herbal yogurts were developed by mixing standardized milk with pretreated herbs, namely tulsi leaf (Ocimum sanctum), pudina leaf (Mentha arvensis) and coriander leaf (Coriandrum sativum), with leaves separately and a 1:1 (v/v) mixture of the strains of lactic starter cultures---Lactobacillus acidophilus (NCIM 2903) and Lactobacillus plantarum (NCIM 2083)-followed by incubation at 40 degrees C for 6 h. The beta-galactosidase enzymatic activity of the abovementioned herbal yogurts was determined and interestingly noted to exhibit higher enzymatic activity compared with the control yogurt (without any herbs). Among all herbal yogurts, tulsi yogurt had the maximum beta-galactosidase activity.

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

PubMed

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.

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

PubMed

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.

Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase

PubMed Central

Shi, Zhen-Dan; Motabar, Omid; Goldin, Ehud; Liu, Ke; Southall, Noel; Sidransky, Ellen; Austin, Christopher P.; Griffiths, Gary L.

2009-01-01

Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in α-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl α-D-galactopyranoside, for a new α-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pKa of resorufin (~6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease. PMID:19521690

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

PubMed

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.

Effect of pH on subunit association and heat protection of soybean alpha-galactosidase

NASA Technical Reports Server (NTRS)

Porter, J. E.; Sarikaya, A.; Herrmann, K. M.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

1992-01-01

Soybeans contain the enzyme alpha-galactosidase, which hydrolyzes alpha-1, 6 linkages in stachyose and raffinose to give sucrose and galactose. We have found that galactose, a competitive product inhibitor of alpha-galactosidase, strongly promotes the heat stability of the tetrameric form of the enzyme at pH 4.0 and at temperatures of up to 70 degrees C for 60 min. Stachyose and raffinose also protect alpha-galactosidase from denaturation at pH 4.0 although to a lesser extent. Glucose and mannose have little effect. At pH 7.0 the enzyme is a monomer, and galactose has no effect on the heat stability of the enzyme. In the absence of heat protection of the enzyme by added sugars, a series deactivation mechanism was found to describe the deactivation data. In comparison, a unimolecular, non-first order deactivation model applies at pH 4.0, where heat protection effects were observed. At a temperature above 60 degrees C, simple deactivation is a suitable model. The results suggest that alpha-galactosidase conformation and heat stability are directly related.

Thermal inactivation kinetics of β-galactosidase during bread baking.

PubMed

Zhang, Lu; Chen, Xiao Dong; Boom, Remko M; Schutyser, Maarten A I

2017-06-15

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 baking at 175 or 205°C. In the wheat flour/water system, the thermostability of β-galactosidase increased with decreased moisture content, and a kinetic model was accurately fitted to the corresponding inactivation data (R 2 =0.99). Interestingly, the residual enzyme activity in the bread crust (about 30%) was hundredfold higher than that in the crumb (about 0.3%) after baking, despite the higher temperature in the crust throughout baking. This result suggested that the reduced moisture content in the crust increased the thermostability of the enzyme. Subsequently, the kinetic model reasonably predicted the enzyme inactivation in the crumb using the same parameters derived from the wheat flour/water system. However, the model predicted a lower residual enzyme activity in the crust compared with the experimental result, which indicated that the structure of the crust may influence the enzyme inactivation mechanism during baking. The results reported can provide a quantitative understanding of the thermal inactivation kinetics of enzyme during baking, which is essential to better retain enzymatic activity in bakery products supplemented with heat-sensitive enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Application of α-N-acetylgalactosaminidase and α-galactosidase in AB to O red blood cells conversion.

PubMed

Gao, Hongwei; Li, Subo; Tan, Yingxia; Ji, Shouping; Wang, Yingli; Bao, Guoqiang; Xu, Lijuan; Gong, Feng

2013-02-01

Enzymatical conversion of A or B RBCs into group O RBCs (ECORBCs) was achieved by using α-N-acetylgalactosaminidase and α-galactosidase, respectively. Now, we initiated AB to O-RBC conversion by using these two enzymes together. But α-N-acetylgalactosaminidase and α-galactosidase's preserving and their reaction buffer were quite different. The aim of this study is to confirm an available system for converting AB to O RBCs, especially to study the maximal permission amount of PCS which was brought to the system-accompanied enzyme addition. Enzyme activity was detected by using GalNAc-pNp or Gal-pNp as substrates. The efficiency of the conversion of A or B antigen was evaluated by routine method and measured by fluorescence-activated cell sorting analysis. The optimal buffer component and the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed according to A and B antigen epitope removal efficiency. The activity of α-N-acetylgalactosaminidase and α-galactosidase was not decreased drastically when they were kept in PCS Buffer in 4°C. The optimal reaction buffer composed of glycine 250 mM and NaCl 3 mM, pH 6.8 and PCS less than 10%(v/v). For converting A(1)B to O RBCs completely, the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed as 0.015 mg/ml packed RBCs(pRBCs) for A(1) antigen epitopes and 0.005 mg/ml pRBCs for B epitopes. Approximately 0.004 mg α-N-acetylgalactosaminidase and 0.005 mg α-galactosidase were required to convert 1 ml pRBCs. Our studies indicated that α-N-acetylgalactosaminidase and α-galactosidase were stable in PCS buffer and a modified protocol which was propitious to converting AB to O RBCs was provided.

Strong inhibitory effect of furanoses and sugar lactones on beta-galactosidase Escherichia coli.

PubMed

Huber, R E; Brockbank, R L

1987-03-24

Various sugars and their lactones were tested for their inhibition of beta-galactosidase (Escherichia coli). L-Ribose, which in the furanose form has a hydroxyl configuration similar to that of D-galactose at positions equivalent to the 3- and 4-positions of D-galactose, was a very strong inhibitor, and D-lyxose, which in the furanose form also resembles D-galactose, was a much better inhibitor than expected. Structural comparisons prelude the pyranose forms of these sugars from being significant contributors to the inhibition, and inhibition at different temperatures (at which there are different furanose concentrations) strongly supported the conclusion that the furanose form is inhibitory. Studies with sugar derivatives that can only be in the furanose form also supported the conclusion. This is the first report of the inhibitory effect of furanose on beta-galactosidase. Lactones were also inhibitory. Every lactone tested was much more inhibitory than was its parent sugar. D-Galactonolactone was especially good. Experiments indicated that it was D-galactono-1,5-lactone rather than D-galactono-1,4-lactone which was inhibitory. Inhibition of beta-galactosidases from mammalian sources by lactones has been reported previously, but this is the first report of the effect of beta-galactosidase from E. coli. Since furanoses in the envelope form are analogous (in some ways) to half-chair or sofa conformations and since lactones with six-membered rings probably have half-chair or sofa conformations, the results indicate that beta-galactosidase probably destabilizes its substrate into a planar conformation of some type and that the galactose in the transition state may, therefore, also be quite planar.(ABSTRACT TRUNCATED AT 250 WORDS)

Disulfide Bond Formation and Activation of Escherichia coli β-Galactosidase under Oxidizing Conditions

PubMed Central

Seras-Franzoso, Joaquin; Affentranger, Roman; Ferrer-Navarro, Mario; Daura, Xavier; Villaverde, Antonio

2012-01-01

Escherichia coli β-galactosidase is probably the most widely used reporter enzyme in molecular biology, cell biology, and biotechnology because of the easy detection of its activity. Its large size and tetrameric structure make this bacterial protein an interesting model for crystallographic studies and atomic mapping. In the present study, we investigate a version of Escherichia coli β-galactosidase produced under oxidizing conditions, in the cytoplasm of an Origami strain. Our data prove the activation of this microbial enzyme under oxidizing conditions and clearly show the occurrence of a disulfide bond in the β-galactosidase structure. Additionally, the formation of this disulfide bond is supported by the analysis of a homology model of the protein that indicates that two cysteines located in the vicinity of the catalytic center are sufficiently close for disulfide bond formation. PMID:22286993

A Family of at Least Seven β-Galactosidase Genes Is Expressed during Tomato Fruit Development

PubMed Central

Smith, David L.; Gross, Kenneth C.

2000-01-01

During our search for a cDNA encoding β-galactosidase II, a β-galactosidase/exogalactanase (EC 3.2.1.23) present during tomato (Lycopersicon esculentum Mill.) fruit ripening, a family of seven tomato β-galactosidase (TBG) cDNAs was identified. The shared amino acid sequence identity among the seven TBG clones ranged from 33% to 79%. All contained the putative active site-containing consensus sequence pattern G-G-P-[LIVM]-x-Q-x-E-N-E-[FY] belonging to glycosyl hydrolase family 35. Six of the seven single-copy genes were mapped using restriction fragment length polymorphisms of recombinant inbred lines. RNA gel-blot analysis was used to evaluate TBG mRNA levels throughout fruit development, in different fruit tissues, and in various plant tissues. RNA gel-blot analysis was also used to reveal TBG mRNA levels in fruit of the rin, nor, and Nr tomato mutants. The TBG4-encoded protein, known to correspond to β-galactosidase II, was expressed in yeast and exo-galactanase activity was confirmed via a quantified release of galactosyl residues from cell wall fractions containing β(1→4)-d-galactan purified from tomato fruit. PMID:10889266

Production of 2-Aminophenoxazin-3-one by Staphylococcus aureus Causes False-Positive Results in β-Galactosidase Assays

PubMed Central

Tse, Herman; Chan, Elaine; Lam, Ching-Wan; Leung, Ka-Fai; Chow, Pat; Lee, Kim-Chung; Sze, Kong-Hung; Cheung, Stanley K. K.; Tse, Man-Kit; Ho, Pak-Leung; Leung, Sze-Pui; Lau, Susanna K. P.; Woo, Patrick C. Y.

2012-01-01

Staphylococcus aureus can be distinguished from similar coagulase-positive staphylococci by its absence of β-galactosidase activity. This is commonly tested using o-nitrophenyl-β-d-galactopyranoside (ONPG) as the substrate. Unexpectedly, 111 and 58 of 123 isolates displayed apparent β-galactosidase activity in the ONPG assay and on the Vitek 2 system, respectively. Compositional analysis showed that the yellow coloration of the positive ONPG assay resulted from production of 2-aminophenoxazin-3-one. Alternative β-galactosidase substrates like X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) should be used for testing staphylococci. PMID:22972831

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

PubMed Central

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

Homodimeric β-Galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: Expression in Lactobacillus plantarum and Biochemical Characterization

PubMed Central

2012-01-01

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. PMID:22283494

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

PubMed

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.

Glycosidases induced in Aspergillus tamarii. Secreted alpha-D-galactosidase and beta-D-mannanase.

PubMed Central

Civas, A; Eberhard, R; Le Dizet, P; Petek, F

1984-01-01

An alpha-D-galactosidase (EC 3.2.1.22) and a beta-D-mannanase (EC 3.2.1.78), which were secreted into the growth medium when Aspergillus tamarii was cultivated in the presence of galactomannan, were purified by a procedure including chromatography on hydroxyapatite and DEAE-cellulose columns. Each of these enzymes showed a single protein band, corresponding to their respective activities, on polyacrylamide-gel electrophoresis. Both enzymes were shown to be glycoproteins containing N-acetylglucosamine, mannose and galactose, with molar proportions of 1:6:1.5 for alpha-D-galactosidase and 1:13:8 for beta-D-mannanase. Mr values as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and by the electrophoretic method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164] were 56000 and 53000 respectively. The alpha-D-galactosidase differed markedly from the mycelial forms I and II studied in the preceding paper [Civas, Eberhard, Le Dizet & Petek (1984) Biochem. J. 219, 849-855] with regard to both its kinetic and structural properties. Images Fig. 1. PMID:6331399

alpha-Galactosidase from Bacillus megaterium VHM1 and its application in removal of flatulence-causing factors from soymilk.

PubMed

Patil, Aravind Goud G; K, Praveen Kumar S; Mulimani, Veerappa H; Veeranagouda, Yaligara; Lee, Kyoung

2010-11-01

A bacterial strain capable of producing extracellular alpha-galactosidase was isolated from sugar cane industrial waste soil sample. Microbiological, physiological, and biochemical studies revealed that isolate belonged to Bacillus sp,. Furthermore, 16S rDNA sequence analysis of new isolates was identified as Bacillus megaterium VHM1. The production of alpha-galactosidase was optimized by various physical culture conditions. Guar gum and yeast extract acted as the best carbon and nitrogen source, respectively for the production of alpha-galactosidase. The enzyme showed an optimum pH at 7.5 and was stable over a pH between 5 and 9. The enzyme was optimally active in 55degreesC and the enzyme was thermostable with half life of 120 minutes at 55 degrees C and lost their 90%, residual activity in 120 minutes at 60 degrees C. alpha-Galactosidase was strongly inhibited by Ag2, Cu2, and Hg2+ at 1mM concentration. The metal ions Fe2, Mn2+, and Mg2+ had no effect on alpha-galactosidase activity, Zn2+,Ni2+, and Ca2+ reduced the enzyme activity slightly. The B megaterium VHM1 enzyme treatment completely hydrolyzed flatulence-causing sugars of soymilk within one and half hour.

Crystallization and preliminary X-ray diffraction data of β-galactosidase from Aspergillus niger.

PubMed

Rico-Díaz, Agustín; Vizoso Vázquez, Ángel; Cerdán, M Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

2014-11-01

β-Galactosidase from Aspergillus niger (An-β-Gal), belonging to the family 35 glycoside hydrolases, hydrolyzes the β-galactosidase linkages in lactose and other galactosides. It is extensively used in industry owing to its high hydrolytic activity and safety. The enzyme has been expressed in yeasts and purified by immobilized metal-ion affinity chromatography for crystallization experiments. The recombinant An-β-Gal, deglycosylated to avoid heterogeneity of the sample, has a molecular mass of 109 kDa. Rod-shaped crystals grew using PEG 3350 as the main precipitant agent. A diffraction data set was collected to 1.8 Å resolution.

Influence of cultivating conditions on the alpha-galactosidase biosynthesis from a novel strain of Penicillium sp. in solid-state fermentation.

PubMed

Wang, C L; Li, D F; Lu, W Q; Wang, Y H; Lai, C H

2004-01-01

The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Penicillium sp. in solid-state fermentation (SSF). Certain fermentation parameters involving incubation temperature, moisture content, initial pH value, inoculum and load size of medium, and incubation time were investigated separately. The optimal temperature and moisture level for alpha-galactosidase biosynthesis was found to be 30 degrees C and 50%, respectively. The range of pH 5.5-6.5 was favourable. About 40-50 g of medium in 250-ml flask and inoculum over 1.0 x 10(6) spores were suitable for enzyme production. Seventy-five hours of incubation was enough for maximum alpha-galactosidase production. Substrate as wheat bran supplemented with soyabean meal and beet pulp markedly improved the enzyme yield in trays. Under optimum culture conditions, the alpha-galactosidase activity from Penicillium sp. MAFIC-6 indicated 185.2 U g(-1) in tray of SSF. The process on alpha-galactosidase production in laboratory scale may have a potentiality of scaling-up.

Alpha-galactosidase versus active charcoal for improving sonographic visualization of abdominal organs in patients with excessive intestinal gas

PubMed Central

Maconi, G.; Bolzacchini, E.; Radice, E.; Marzocchi, M.; Badini, M.

2012-01-01

Background and aims Intestinal gas is a frequent cause of poor visualization during gastrointestinal ultrasound (US). The enzyme alpha-galactosidase may reduce intestinal gas production, thereby improving abdominal US visualization. We compared the efficacies of alpha-galactosidase and active charcoal in improving US visualization in patients with previous unsatisfactory abdominal US scans caused by excessive intestinal gas. Materials and methods: 45 patients with poor visualization of at least one target organ: pancreas, hepatic lobes (score 0–2) or common bile duct (CBD) (score 0–1) were enrolled in a prospective randomized, crossover, observer-blinded study. The patients received alpha-galactosidase (Sinaire Forte, Promefarm, Milan, Italy) 600 GalU t.i.d. for 2 days before abdominal US plus 900 GalU the morning of exam or active charcoal 448 mg t.i.d., for 2 days before the exam plus 672 mg the morning of the exam. Visualization was graded as follows: 0 = none (complete gas interference); 1 = severe interference, 2 = moderate interference, 3 = mild interference; 4 = complete (no gas interference). Results: 42 patients completed the study. Both alpha-galactosidase and active charcoal improved the visualization of target organs. Visualization of the right hepatic lobe, CBD and pancreatic tail was significantly improved (vs. baseline) only by alpha-galactosidase (p < 0.01). Scores ≥3 for all parts of the pancreas and both hepatic lobes were achieved in only 12.5% of the patients after both treatments. Both products were well tolerated. Conclusion: Alpha-galactosidase and active charcoal can improve US visualization of abdominal organs in patients whose scans are frequently unsatisfactory due to excessive intestinal gas. Visualization of the pancreatic tail and right hepatic lobe was significantly improved only by alpha-galactosidase. However, both treatments allowed adequate visualization of all target organs during the same examination only

Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae β-Galactosidase

PubMed Central

Cardelle-Cobas, Alejandra; Olano, Agustin; Irazoqui, Gabriela; Giacomini, Cecilia; Batista-Viera, Francisco; Corzo, Nieves; Corzo-Martínez, Marta

2016-01-01

β-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 β-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with β-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 with DP 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 β-galactosidase immobilized on a support of glutaraldehyde–agarose constitutes an efficient and cost-effective alternative to the use of soluble β-galactosidases for the synthesis of prebiotic OsLu mixtures. PMID:27014684

Association between the organoleptic scores, oral condition and salivary β-galactosidases in children affected by halitosis.

PubMed

Petrini, M; Costacurta, M; Ferrante, M; Trentini, P; Docimo, R; Spoto, G

2014-08-01

The goal of this work is to evaluate the association between organoleptic scores, oral condition and salivary β-galactosidases, to facilitate the differential diagnosis of halitosis in children. Fifty systemically healthy children with a primary complaint of oral malodour were included in this cross-sectional study. The organoleptic evaluation was carried out by two judges, evaluating the intensity of malodour of the air exhaled 5 s through the mouth of the patients, at a distance of approximately 10 cm from their noses; the level of salivary β-galactosidases was quantified spectrophotometrically after a chromatic reaction between a salivary sample of each patient and a specific chromatic substrate of the enzyme. Clinical conditions, such as visible plaque and gingival bleeding index, tongue coating score, localized food stagnation and other oral parameters, were evaluated by qualified dentists through an oral check-up. The β-galactosidase level was significantly related to the organoleptic scores and clinical parameters, such as the tongue coating score and the visible plaque index. Stratifying results with respect to the different phase of the day at which parents complained halitosis in their children, statistical analysis showed that the organoleptic scores and the level of β-galactosidases were significantly higher in children who suffered of halitosis during the whole day, A = 40%, with respect to those without this problem, N = 20% (P = 0.001 and P = 0.006, respectively). Certain oral parameters such as halitosis during the whole day, high tongue coating score and high visible plaque index were particularly associated with an increase in the salivary β-galactosidase level. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

PubMed

Genina, Natalja; Räikkönen, Heikki; Heinämäki, Jyrki; Veski, Peep; Yliruusi, Jouko

2010-06-01

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 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.

High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a Sakacin P-based expression system.

PubMed

Halbmayr, Elisabeth; Mathiesen, Geir; Nguyen, Thu-Ha; Maischberger, Thomas; Peterbauer, Clemens K; Eijsink, Vincent G H; Haltrich, Dietmar

2008-06-25

This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( Sørvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.

Cloning, sequence, and expression of a blood group B active recombinant alpha-D-galactosidase from pinto bean (Phaseolus vulgaris).

PubMed

Davis, M O; Hata, D J; Johnson, S A; Jones, D E; Harmata, M A; Evans, M L; Walker, J C; Smith, D S

1997-07-01

A cDNA encoding pinto bean alpha-D-galactosidase [E.C. 3.2.1.22] was obtained by amplification of cDNA using highly conserved sequences found in eucaryotic alpha-D-galactosidases. Subsequently a full length Phaseolus cDNA clone was obtained that is 1537 nt long and contains untranslated 5' and 3' sequences. The nucleotide sequence of the cDNA has a high degree of homology with other eucaryotic alpha-D-galactosidase genes. The recombinant alpha-D-galactosidase (rGal) was expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Purified rGal was homogeneous by SDS-PAGE and had relative masses of 40.1 and 45.4 kDa under nonreducing and reducing conditions, respectively. The N-terminal sequence of the expressed protein contained the sequence GNGLGQTPPMG corresponding to that deduced from the cDNA sequence. The native molecular weight for rGal was determined to be 32.18 kDa by Sephacryl S-200 chromatography. The specific activity of the rGal was 349 mu moles of PNP-alpha-D-galactopyranoside hydrolyzed per mg of pure rGal per min. rGal was highly specific for alpha-D-galactosyl residues and degraded B oligosaccharide. No detectable hemagglutinin or protease activity was present in the preparations. Furthermore, rGal was active against the blood group B antigen on native human erythrocytes in cell suspension assays. The only detectable RBC phenotypic change was loss of the B and P1 epitopes. Recombinant Phaseolus vulgaris alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of enzymatically converted, universally transfusable type O RBCs. alpha-D-galactosidase [E.C. 3.2.1.22] has been purified from a variety of procaryotic and eucaryotic species. Most alpha-D-galactosidases have similar low molecular weight substrate specificities, but activity against high molecular weight substrates is variable. Terminal alpha-D-galactoside residues are present in glycoproteins and glycolipids. Some alpha-D-galactosidases

Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates.

PubMed

Guerrero, Cecilia; Vera, Carlos; Conejeros, Raúl; Illanes, Andrés

2015-03-01

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular weight of Escherichia coli β-galactosidase in concentrated solutions of guanidine hydrochloride

PubMed Central

Erickson, Robert P.

1970-01-01

The molecular weight of Escherichia coli β-galactosidase was determined in 6m- and 8m-guanidine hydrochloride by meniscus-depletion sedimentation equilibrium, sedimentation velocity and viscosity. Sedimentation equilibrium revealed heterogeneity with the smallest component having a molecular weight of about 50000. At lower speeds, the apparent weight-average molecular weight is about 80000. By use of a calculation based on an empirical correlation for proteins that are random coils in 6m-guanidine hydrochloride, sedimentation velocity gave a molecular weight of 91000, and the intrinsic viscosity indicated a viscosity-average molecular weight of 84000. Heating in 6m-guanidine hydrochloride lowered the viscosity of β-galactosidase in a variable manner. PMID:4924171

The catalytic properties and stability of β-galactosidases from fungi

NASA Astrophysics Data System (ADS)

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).

Use of an alpha-galactosidase gene as a food-grade selection marker for Streptococcus thermophilus.

PubMed

Labrie, S; Bart, C; Vadeboncoeur, C; Moineau, S

2005-07-01

The alpha-galactosidase gene (aga) of Lactococcus raffinolactis ATCC 43920 was previously shown to be an efficient food-grade selection marker in Lactococcus lactis and Pediococcus acidilactici but not in Streptococcus thermophilus. In this study, we demonstrated that the alpha-galactosidase of L. raffinolactis is thermolabile and inoperative at 42 degrees C, the optimal growth temperature of S. thermophilus. An in vitro assay indicated that the activity of this alpha-galactosidase at 42 degrees C was only 3% of that at 30 degrees C, whereas the enzyme retained 23% of its activity at 37 degrees C. Transformation of Strep. thermophilus RD733 with the shuttle-vector pNZ123 bearing the aga gene of L. raffinolactis (pRAF301) generated transformants that were stable and able to grow on melibiose and raffinose at 37 degrees C or below. The transformed cells possessed 6-fold more alpha-galactosidase activity after growth on melibiose than cells grown on lactose. Slot-blot analyses of aga mRNA indicated that repression by lactose occurred at the transcriptional level. The presence of pRAF301 did not interfere with the lactic acid production when the transformed cells of Strep. thermophilus were grown at the optimal temperature in milk. Using the recombinant plasmid pRAF301, which carries a chloramphenicol resistance gene in addition to aga, we showed that both markers were equally efficient at differentiating transformed from nontransformed cells. The aga gene of L. raffinolactis can be used as a highly efficient selection marker in Strep. thermophilus.

A defect in inducible beta-galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.

PubMed Central

Yamamoto, N; Naraparaju, V R

1996-01-01

Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso-Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso-Pc efficiently activated macrophages whereas lyso-Pc-treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso-Pc-inducible beta-galactosidase of B lymphocytes. Lyso-Pc-inducible beta-galactosidase of B lymphocytes was found to be defective in mi mutant mice. PMID:8881764

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

PubMed

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.

Heterologous expression of a recombinant lactobacillal β-galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system.

PubMed

Nguyen, Tien-Thanh; Nguyen, Hoang-Minh; Geiger, Barbara; Mathiesen, Geir; Eijsink, Vincent G H; Peterbauer, Clemens K; Haltrich, Dietmar; Nguyen, Thu-Ha

2015-03-07

Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric β-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri β-galactosidase. Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum β-galactosidase levels obtained were 130 U/mg protein and 35-40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), β-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant β-galactosidase. The over-expression of recombinant L. reuteri β-galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

Statistical approach for the enhanced production of cold-active β-galactosidase from Thalassospira frigidphilosprofundus: a novel marine psychrophile from deep waters of Bay of Bengal.

PubMed

Pulicherla, K K; Kumar, P Suresh; Manideep, K; Rekha, V P B; Ghosh, Mrinmoy; Sambasiva Rao, K R S

2013-01-01

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active β-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for β-galactosidase production. Under optimal conditions, the production of β-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. β-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active β-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active β-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.

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

PubMed Central

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 < 0.0001) but did not differ from the W 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

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

PubMed Central

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. PMID:26053048

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

PubMed

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.

Cloning, expression, and characterization of a peculiar choline-binding beta-galactosidase from Streptococcus mitis.

PubMed

Campuzano, Susana; Serra, Beatriz; Llull, Daniel; García, José L; García, Pedro

2009-09-01

A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative beta-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric beta-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40 degrees C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In S. mitis, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first beta-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.

Two novel mutations in the alpha-galactosidase gene in Japanese classical hemizygotes with Fabry disease.

PubMed

Okumiya, T; Takenaka, T; Ishii, S; Kase, R; Kamei, S; Sakuraba, H

1996-09-01

Four alpha-galactosidase gene mutations were identified in Japanese male patients with Fabry disease who had no detectable alpha-galactosidase activity. Two of them were novel mutations, an 11-bp deletion in exon 2 and a g-1 to t substitution at the 3' end of the splice acceptor site in intron 1. The former caused a frameshift and led to the creation of a new stop codon at codon 118. The latter was predicted to provoke aberrant mRNA splicing followed by accelerated degradation of the mRNA. A nonsense mutation, R301X, and a 2-bp deletion starting at nucleotide position 718, which were reported previously, were also identified in unrelated patients.

Structural features of Aspergillus niger β-galactosidase define its activity against glycoside linkages.

PubMed

Rico-Díaz, Agustín; Ramírez-Escudero, Mercedes; Vizoso-Vázquez, Ángel; Cerdán, M Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

2017-06-01

β-Galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or transgalactosylation of β-galactosides. Among them, the Aspergillus niger β-galactosidase (AnβGal) belongs to the glycoside hydrolase family 35 (GH35) and is widely used in the industry due to its high hydrolytic activity which is used to degrade lactose. We present here its three-dimensional structure in complex with different oligosaccharides, to illustrate the structural determinants of the broad specificity of the enzyme against different glycoside linkages. Remarkably, the residues Phe264, Tyr304, and Trp806 make a dynamic hydrophobic platform that accommodates the sugar at subsite +1 suggesting a main role on the recognition of structurally different substrates. Moreover, complexes with the trisaccharides show two potential subsites +2 depending on the substrate type. This feature and the peculiar shape of its wide cavity suggest that AnβGal might accommodate branched substrates from the complex net of polysaccharides composing the plant material in its natural environment. Relevant residues were selected and mutagenesis analyses were performed to evaluate their role in the catalytic performance and the hydrolase/transferase ratio of AnβGal. Thus, we generated mutants with improved transgalactosylation activity. In particular, the variant Y304F/Y355H/N357G/W806F displays a higher level of galacto-oligosaccharides production than the Aspergillus oryzae β-galactosidase, which is the preferred enzyme in the industry owing to its high transferase activity. Our results provide new knowledge on the determinants modulating specificity and the catalytic performance of fungal GH35 β-galactosidases. In turn, this fundamental background gives novel tools for the future improvement of these enzymes, which represent an interesting target for rational design. Structural data are available in PDB database under the accession numbers 5IFP (native form), 5IHR (in complex with 6

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy.

PubMed

Nakano, Sachie; Tsukimura, Takahiro; Togawa, Tadayasu; Ohashi, Toya; Kobayashi, Masahisa; Takayama, Katsuyoshi; Kobayashi, Yukuharu; Abiko, Hiroshi; Satou, Masatsugu; Nakahata, Tohru; Warnock, David G; Sakuraba, Hitoshi; Shibasaki, Futoshi

2015-01-01

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.

Beta-galactosidase catalyzed selective galactosylation of aromatic compounds.

PubMed

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.

Purification an α-galactosidase from Coriolus versicolor with acid-resistant and good degradation ability on raffinose family oligosaccharides.

PubMed

Du, Fang; Liu, Qin; Wang, Hexiang; Ng, TziBin

2014-04-01

An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg⁻¹. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4-50 °C and at a pH range of 2-5. Among the metal ions tested, Cu²⁺, Cd²⁺ and Hg²⁺ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag⁺ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.

Purification and characterization of thermostable alpha-galactosidase from Aspergillus terreus (GR).

PubMed

Shankar, S K; Dhananjay, S K; Mulimani, V H

2009-02-01

An extracellular thermostable alpha-galactosidase producing Aspergillus terreus (GR) strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 degrees C, respectively, for artificial substrate pNPalphaGal. alpha-Galactosidase from A. terreus (GR) is found to be thermostable, as it was not inactivated after heating at 65 degrees C for 40 min. The K (m) for pNPalphaGal, oNPalphaGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg(2+), Ni(2+), Ca(2+), Co(2+), and Mn(2+) had no effect on enzyme activity, but Ag(+), Hg(2+), and Cu(2+) have inhibited complete activity.

Identification of a Novel α-Galactosidase from the Hyperthermophilic Archaeon Sulfolobus solfataricus†

PubMed Central

Brouns, Stan J. J.; Smits, Nicole; Wu, Hao; Snijders, Ambrosius P. L.; Wright, Phillip C.; de Vos, Willem M.; van der Oost, John

2006-01-01

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable α-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for α-linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable α-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea. PMID:16547025

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

PubMed

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.

Effect of cultivating conditions on alpha-galactosidase production by a novel Aspergillus foetidus ZU-G1 strain in solid-state fermentation.

PubMed

Liu, Cai-qin; Chen, Qi-he; Cheng, Qian-jun; Wang, Jin-ling; He, Guo-qing

2007-05-01

The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Aspergillus foetidus ZU-G1 in solid-state fermentation (SSF). Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of alpha-galactosidase production in SSF were 60% initial moisture of medium, 28 degrees C incubation temperature, 18 h cultivation period of seed, 10% inoculum volume, 5.0 approximately 6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum alpha-galactosidase production was 2 037.51 U/g dry matter near the 144th hour of fermentation.

Effect of cultivating conditions on α-galactosidase production by a novel Aspergillus foetidus ZU-G1 strain in solid-state fermentation

PubMed Central

Liu, Cai-qin; Chen, Qi-he; Cheng, Qian-jun; Wang, Jin-ling; He, Guo-qing

2007-01-01

The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain Aspergillus foetidus ZU-G1 in solid-state fermentation (SSF). Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of α-galactosidase production in SSF were 60% initial moisture of medium, 28 °C incubation temperature, 18 h cultivation period of seed, 10% inoculum volume, 5.0~6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum α-galactosidase production was 2 037.51 U/g dry matter near the 144th hour of fermentation. PMID:17542067

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guenther, Izabela; Zolkiewski, Michal; Kedzierska-Mieszkowska, Sabina, E-mail: kedzie@biotech.ug.gda.pl

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-terminalmore » 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

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

PubMed

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.

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy

PubMed Central

Nakano, Sachie; Tsukimura, Takahiro; Togawa, Tadayasu; Ohashi, Toya; Kobayashi, Masahisa; Takayama, Katsuyoshi; Kobayashi, Yukuharu; Abiko, Hiroshi; Satou, Masatsugu; Nakahata, Tohru; Warnock, David G.; Sakuraba, Hitoshi; Shibasaki, Futoshi

2015-01-01

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy. PMID:26083343

Repeated-batch operation of immobilized β-galactosidase inclusion bodies-containing Escherichia coli cell reactor for lactose hydrolysis.

PubMed

Yeon, Ji-Hyeon; Jung, Kyung-Hwan

2011-09-01

In this study, we investigated the performance of an immobilized β-galactosidase inclusion bodies-containing Escherichia coli cell reactor, where the cells were immobilized in alginate beads, which were then used in repeated-batch operations for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose over the long-term. In particular, in the Tris buffer system, disintegration of the alginate beads was not observed during the operation, which was observed for the phosphate buffer system. The o-nitrophenyl-β-D-galactoside hydrolysis was operated successfully up to about 80 h, and the runs were successfully repeated at least eight times. In addition, hydrolysis of lactose was successfully carried out up to 240 h. Using Western blotting analyses, it was verified that the beta-galactosidase inclusion bodies were sustained in the alginate beads during the repeated-batch operations. Consequently, we experimentally verified that β-galactosidase inclusion bodies-containing Escherichia coli cells could be used in a repeated-batch reactor as a biocatalyst for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose. It is probable that this approach can be applied to enzymatic synthesis reactions for other biotechnology applications, particularly reactions that require long-term and stable operation.

Structural Explanation for Allolactose (lac Operon Inducer) Synthesis by lacZ β-Galactosidase and the Evolutionary Relationship between Allolactose Synthesis and the lac Repressor

PubMed Central

Wheatley, Robert W.; Lo, Summie; Jancewicz, Larisa J.; Dugdale, Megan L.; Huber, Reuben E.

2013-01-01

β-Galactosidase (lacZ) has bifunctional activity. It hydrolyzes lactose to galactose and glucose and catalyzes the intramolecular isomerization of lactose to allolactose, the lac operon inducer. β-Galactosidase promotes the isomerization by means of an acceptor site that binds glucose after its cleavage from lactose and thus delays its exit from the site. However, because of its relatively low affinity for glucose, details of this site have remained elusive. We present structural data mapping the glucose site based on a substituted enzyme (G794A-β-galactosidase) that traps allolactose. Various lines of evidence indicate that the glucose of the trapped allolactose is in the acceptor position. The evidence includes structures with Bis-Tris (2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol) and l-ribose in the site and kinetic binding studies with substituted β-galactosidases. The site is composed of Asn-102, His-418, Lys-517, Ser-796, Glu-797, and Trp-999. Ser-796 and Glu-797 are part of a loop (residues 795–803) that closes over the active site. This loop appears essential for the bifunctional nature of the enzyme because it helps form the glucose binding site. In addition, because the loop is mobile, glucose binding is transient, allowing the release of some glucose. Bioinformatics studies showed that the residues important for interacting with glucose are only conserved in a subset of related enzymes. Thus, intramolecular isomerization is not a universal feature of β-galactosidases. Genomic analyses indicated that lac repressors were co-selected only within the conserved subset. This shows that the glucose binding site of β-galactosidase played an important role in lac operon evolution. PMID:23486479

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

PubMed Central

LeBlanc, Jean Guy; Ledue-Clier, Florence; Bensaada, Martine; de Giori, Graciela Savoy; Guerekobaya, Theodora; Sesma, Fernando; Juillard, Vincent; Rabot, Sylvie; Piard, Jean-Christophe

2008-01-01

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 delivery of digestive enzymes in

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

PubMed

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.

Crystallization and preliminary X-ray diffraction studies of two thermostable α-galactosidases from glycoside hydrolase family 36

DOE Office of Scientific and Technical Information (OSTI.GOV)

Foucault, M.; Watzlawick, H.; Mattes, R.

2006-02-01

The α-galactosidases AgaA, AgaB and AgaA A355E mutant from Geobacillus stearothermophilus have been overexpressed in Escherichia coli. Crystals of AgaB and AgaA A355E have been obtained by the vapour-diffusion method and synchrotron data have been collected to 2.0 and 2.8 Å resolution, respectively. α-Galactosidases from thermophilic organisms have gained interest owing to their applications in the sugar industry. The α-galactosidases AgaA, AgaB and AgaA A355E mutant from Geobacillus stearothermophilus have been overexpressed in Escherichia coli. Crystals of AgaB and AgaA A355E have been obtained by the vapour-diffusion method and synchrotron data have been collected to 2.0 and 2.8 Å resolution,more » respectively. Crystals of AgaB belong to space group I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 87.5, b = 113.3, c = 161.6 Å. Crystals of AgaA A355E belong to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 150.1, c = 233.2 Å.« less

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

PubMed

Benavente, Rocio; Pessela, Benevides C; Curiel, Jose Antonio; de las Rivas, Blanca; Muñoz, Rosario; Guisán, Jose Manuel; Mancheño, Jose M; Cardelle-Cobas, Alejandra; Ruiz-Matute, Ana I; Corzo, Nieves

2015-04-30

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 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.

PubMed

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.

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

PubMed

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.

Cold active β-galactosidase from Thalassospira sp. 3SC-21 to use in milk lactose hydrolysis: a novel source from deep waters of Bay-of-Bengal.

PubMed

Ghosh, Mrinmoy; Pulicherla, K K; Rekha, V P B; Raja, P Kumar; Sambasiva Rao, K R S

2012-09-01

The cold active β-galactosidase from psychrophilic bacteria accelerate the possibility of outperforming the current commercial β-galactosidase production from mesophilic sources. The present study is carried out to screen and isolate a cold active β-galactosidase producing bacterium from profound marine waters of Bay-of-Bengal and to optimize the factors for lactose hydrolysis in milk. Isolated bacterium 3SC-21 was characterized as marine psychrotolerant, halophile, gram negative, rod shaped strain producing an intracellular cold active β-galactosidase enzyme. Further, based upon the 16S rRNA gene sequence, bacterium 3SC-21 was identified as Thalassospira sp. The isolated strain Thalassospira sp. 3SC-21 had shown the enzyme activity between 4 and 20 °C at pH of 6.5 and the enzyme was completely inactivated at 45 °C. The statistical method, central composite rotatable design of response surface methodology was employed to optimize the hydrolysis of lactose and to reveal the interactions between various factors behind this hydrolysis. It was found that maximum of 80.18 % of lactose in 8 ml of raw milk was hydrolysed at pH of 6.5 at 20 °C in comparison to 40 % of lactose hydrolysis at 40 °C, suggesting that the cold active β-galactosidase from Thalassospira sp. 3SC-21 would be best suited for manufacturing the lactose free dairy products at low temperature.

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and Saccharomyces cerevisiae Cells in Dual Matrices.

PubMed

Kokkiligadda, Anusha; Beniwal, Arun; Saini, Priyanka; Vij, Shilpa

2016-08-01

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.

Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda.

PubMed Central

Zabeau, M; Stanley, K K

1982-01-01

Hybrid plasmids carrying cro-lacZ gene fusions have been constructed by joining DNA segments carrying the PR promoter and the start of the cro gene of bacteriophage lambda to the lacZ gene fragment carried by plasmid pLG400 . Plasmids in which the translational reading frames of the cro and lacZ genes are joined in-register (type I) direct the synthesis of elevated levels of cro-beta-galactosidase fusion protein amounting to 30% of the total cellular protein, while plasmids in which the genes are fused out-of-register (type II) produce a low level of beta-galactosidase protein. Sequence rearrangements downstream of the cro initiator AUG were found to influence the efficiency of translation, and have been correlated with alterations in the RNA secondary structure of the ribosome-binding site. Plasmids which direct the synthesis of high levels of beta-galactosidase are conditionally lethal and can only be propagated when the PR promoter is repressed. Deletion of sequences downstream of the lacZ gene restored viability, indicating that this region of the plasmid encodes a function which inhibits the growth of the cells. The different applications of these plasmids for expression of cloned genes are discussed. Images Fig. 6. PMID:6327257

Cooperation between two ClpB isoforms enhances the recovery of the recombinant β-galactosidase from inclusion bodies.

PubMed

Guenther, Izabela; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

2012-10-05

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 (ClpBΔN), 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 β-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 β-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of β-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a synergistic cooperation between the two isoforms of ClpB chaperone. In addition, no significant recovery of the β-galactosidase from IBs in ΔclpB mutant cells suggests that ClpB is a key chaperone in IB protein release. Copyright © 2012 Elsevier Inc. All rights reserved.

Development and Analytical Characterization of Pegunigalsidase Alfa, a Chemically Cross-Linked Plant Recombinant Human α-Galactosidase-A for Treatment of Fabry Disease.

PubMed

Ruderfer, Ilya; Shulman, Avidor; Kizhner, Tali; Azulay, Yaniv; Nataf, Yakir; Tekoah, Yoram; Shaaltiel, Yoseph

2018-05-16

The current treatment of Fabry disease by enzyme replacement therapy with commercially available recombinant human α-Galactosidase A shows a continuous deterioration of the disease patients. Human recombinant α-Galactosidase A is a homodimer with noncovalently bound subunits and is expressed in the ProCellEx plant cell-based protein expression platform to produce pegunigalsidase alfa. The effect of covalent bonding between two α-Galactosidase A subunits by PEG-based cross-linkers of various lengths was evaluated in this study. The results show that cross-linking by a bifunctional PEG polymer of 2000 Da produces a more stable protein with improved pharmacokinetic and biodistribution properties. The chemical modification did not influence the tertiary protein structure but led to an increased thermal stability and showed partial masking of immune epitopes. The developed pegunigalsidase alfa is currently tested in phase III clinical trials and has a potential to show superior efficacy versus the currently used enzyme replacement therapies in the treatment of Fabry disease patients.

Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms and Escherichia coli

PubMed Central

Tryland, I.; Fiksdal, L.

1998-01-01

Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities of Bacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified as Aeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria. PMID:9501441

Electrochemical Measurement of the β-Galactosidase Reporter from Live Cells: A Comparison to the Miller Assay.

PubMed

Tschirhart, Tanya; Zhou, Xinyi Y; Ueda, Hana; Tsao, Chen-Yu; Kim, Eunkyoung; Payne, Gregory F; Bentley, William E

2016-01-15

In order to match our ability to conceive of and construct cells with enhanced function, we must concomitantly develop facile, real-time methods for elucidating performance. With these, new designs can be tested in silico and steps in construction incrementally validated. Electrochemical monitoring offers the above advantages largely because signal transduction stems from direct electron transfer, allowing for potentially quicker and more integrated measurements. One of the most common genetic reporters, β-galactosidase, can be measured both spectrophotometrically (Miller assay) and electrochemically. However, since the relationship between the two is not well understood, the electrochemical methods have not yet garnered the attention of biologists. With the aim of demonstrating the utility of an electrochemical measurement to the synthetic biology community, we created a genetic construct that interprets and reports (with β-galactosidase) on the concentration of the bacterial quorum sensing molecule autoinducer-2. In this work, we provide a correlation between electrochemical measurements and Miller Units. We show that the electrochemical assay works with both lysed and whole cells, allowing for the prediction of one from the other, and for continuous monitoring of cell response. We further present a conceptually simple and generalized mathematical model for cell-based β-galactosidase reporter systems that could aid in building and predicting a variety of synthetic biology constructs. This first-ever in-depth comparison and analysis aims to facilitate the use of electrochemical real-time monitoring in the field of synthetic biology as well as to facilitate the creation of constructs that can more easily communicate information to electronic systems.

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

PubMed

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.

Characterization of a Glycoside Hydrolase Family 27 α-Galactosidase from Pontibacter Reveals Its Novel Salt-Protease Tolerance and Transglycosylation Activity.

PubMed

Zhou, Junpei; Liu, Yu; Lu, Qian; Zhang, Rui; Wu, Qian; Li, Chunyan; Li, Junjun; Tang, Xianghua; Xu, Bo; Ding, Junmei; Han, Nanyu; Huang, Zunxi

2016-03-23

α-Galactosidases are of great interest in various applications. A glycoside hydrolase family 27 α-galactosidase was cloned from Pontibacter sp. harbored in a saline soil and expressed in Escherichia coli. The purified recombinant enzyme (rAgaAHJ8) was little or not affected by 3.5-30.0% (w/v) NaCl, 10.0-100.0 mM Pb(CH3COO)2, 10.0-60.0 mM ZnSO4, or 8.3-100.0 mg mL(-1) trypsin and by most metal ions and chemical reagents at 1.0 and 10.0 mM concentrations. The degree of synergy on enzymatic degradation of locust bean gum and guar gum by an endomannanase and rAgaAHJ8 was 1.22-1.54. In the presence of trypsin, the amount of reducing sugars released from soybean milk treated by rAgaAHJ8 was approximately 3.8-fold compared with that treated by a commercial α-galactosidase. rAgaAHJ8 showed transglycosylation activity when using sucrose, raffinose, and 3-methyl-1-butanol as the acceptors. Furthermore, potential factors for salt adaptation of the enzyme were presumed.

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

PubMed

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.

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

PubMed

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.

Beta-galactosidase deficiency in a Korat cat: a new form of feline GM1-gangliosidosis.

PubMed

De Maria, R; Divari, S; Bo, S; Sonnio, S; Lotti, D; Capucchio, M T; Castagnaro, M

1998-09-01

A 7-month-old Korat cat was referred for a slowly progressive neurological disease. Circulating monocytes and lymphocytes showed the presence of single or multiple empty vacuoles and blood leukocytes enzyme assay revealed a very low beta-galactosidase activity level (4.7 nmol/mg per h) as compared to unaffected parents and relatives. Histologically, the cat, euthanized at the owner request at 21 months of age, presented diffuse vacuolization and enlargement of neurons throughout the brain, spinal cord and peripheral ganglia, severe cerebellar neuronal cell loss, and moderate astrocytosis. Stored material was stained with periodic acid-Schiff on frozen sections and with the lectins Ricinus conmmunis agglutinin-I, concanavalin A and wheat germ agglutinin on paraffin-embedded sections. Ultrastructurally, neuronal vacuoles were filled with concentrically whorled lamellae and small membrane-bound vesicles. In the affected cat, beta-galactosidase activity was markedly reduced in brain (18.9%) and liver (33.25%), while total beta-hexosaminidase activity showed a remarkable increase. Quantitation of total gangliosides revealed a 3-fold increase in brain and 1.7-fold in liver of affected cat. High-performance thin layer chromatography (HPTLC) detected a striking increase of GM1-ganglioside. On densitometric analysis of HPTLC bands, the absorption of GM1-ganglioside band was 98.52% of all stained bands (GD1a, GD1b, GT1b). Based on clinical onset, morphological and histochemical features, and biochemical findings, the Korat cat GM1-gangliosidosis is comparable with the human type II (juvenile) form. However, clinical progression, survival time and level of beta-galactosidase deficiency do not completely fit with those of human type II GM1-gangliosidosis. The disease in the Korat cat is also different from other reported forms of feline GM1-gangliosidosis.

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

PubMed Central

2017-01-01

Summary 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. PMID:28867957

Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered β-galactosidase.

PubMed

Estevinho, Berta N; Samaniego, Nuria; Talens-Perales, David; Fabra, Maria José; López-Rubio, Amparo; Polaina, Julio; Marín-Navarro, Julia

2018-08-01

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C. Copyright © 2018 Elsevier B.V. All rights reserved.

2-(Benzothiazol-2-yl)-phenyl-β-d-galactopyranoside derivatives as fluorescent pigment dyeing substrates and their application for the assay of β-d-galactosidase activities.

PubMed

Otsubo, Tadamune; Minami, Akira; Fujii, Haruna; Taguchi, Risa; Takahashi, Tadanobu; Suzuki, Takashi; Teraoka, Fumiteru; Ikeda, Kiyoshi

2013-04-01

2-(Benzothiazol-2-yl)-phenyl-β-d-galactopyranoside derivatives were synthesized as novel artificial fluorescent pigment dyeing substrates for β-d-galactosidase. The substrates, which exhibited non-fluorescence or weak fluorescence in solution phase, were smoothly hydrolyzed by β-d-galactosidase from Aspergillus oryzae and yielded a water-insoluble strong fluorescent pigment. The difference of fluorescent intensity exhibited a linear relationship with the amount of enzyme. Copyright © 2013 Elsevier Ltd. All rights reserved.

Arrhenius plot for a reaction catalyzed by a single molecule of β-galactosidase.

PubMed

Craig, Douglas B; Chase, Linden N

2012-02-21

The activity of a single enzyme molecule of Escherichia coli β-galactosidase was measured using a capillary electrophoresis continuous flow assay. As the enzyme molecule traversed the capillary the incubation temperature was increased from 27 to 37 °C, providing a continuous record of the change in rate with temperature. This data was used to develop a single enzyme molecule Arrhenius plot, from which the activation energy of the reaction was determined to be 31 kJ mol(-1).

Cloning, expression and structural stability of a cold-adapted ß-Galactosidase from Rahnella sp.R3

USDA-ARS?s Scientific Manuscript database

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...

Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

2016-06-01

Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

A novel mutation of α-galactosidase A gene causes Fabry disease mimicking primary erythromelalgia in a Chinese family.

PubMed

Ge, Wei; Wei, Bin; Zhu, Hao; Miao, Zhigang; Zhang, Weimin; Leng, Cuihua; Li, Jizhen; Zhang, Dan; Sun, Miao; Xu, Xingshun

2017-05-01

Fabry disease is an X-linked genetic disorder caused by the mutations of α-galactosidase A (GLA, MIM 300644) gene presenting with various clinical symptoms including small-fiber peripheral neuropathy and limb burning pain. Here, we reported a Chinese pedigree with the initial diagnosis of primary erythromelalgia in an autosomal dominant (AD)-inherited pattern. Mutation analysis of SCN9A and GLA genes by direct sequencing and functional analysis of a novel mutation of GLA in cells were performed. Our data did not show any pathological mutations in SCN9A gene; however, a novel missense mutation c.139T>C (p.W47R) of GLA was identified in a male proband as well as two female carriers in this family. Enzyme assay of α-galactosidase A activity showed deficient enzyme activity in male patients and female carriers, further confirming the diagnosis of Fabry disease. Finally, a functional analysis indicated that the replacement of the 47th amino acid tryptophan (W47) with arginine (W47R) or glycine (W47G) led to reduced activity of α-galactosidase A in 293T cells. Therefore, these findings demonstrated that the novel mutation p.W47R of GLA is the cause of Fabry disease. Because Fabry disease and primary erythromelalgia share similar symptoms, it is a good strategy for clinical physicians to perform genetic mutation screenings on both SCN9A and GLA genes in those patients with limb burning pain but without a clear inheritant pattern.

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

PubMed

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.

Atomic Resolution Cryo-EM Structure of β-Galactosidase.

PubMed

Bartesaghi, Alberto; Aguerrebere, Cecilia; Falconieri, Veronica; Banerjee, Soojay; Earl, Lesley A; Zhu, Xing; Grigorieff, Nikolaus; Milne, Jacqueline L S; Sapiro, Guillermo; Wu, Xiongwu; Subramaniam, Sriram

2018-05-10

The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for β-galactosidase bound to the inhibitor phenylethyl β-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ∼ 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design. Published by Elsevier Ltd.

A Naturally Occurring Mutation in an Arabidopsis Accession Affects a β-d-Galactosidase That Increases the Hydrophilic Potential of Rhamnogalacturonan I in Seed Mucilage[W

PubMed Central

Macquet, Audrey; Ralet, Marie-Christine; Loudet, Olivier; Kronenberger, Jocelyne; Mouille, Gregory; Marion-Poll, Annie; North, Helen M.

2007-01-01

The Arabidopsis thaliana accession Shahdara was identified as a rare naturally occurring mutant that does not liberate seed mucilage on imbibition. The defective locus was found to be allelic to the mum2-1 and mum2-2 mutants. Map-based cloning showed that MUCILAGE-MODIFIED2 (MUM2) encodes the putative β-d-galactosidase BGAL6. Activity assays demonstrated that one of four major β-d-galactosidase activities present in developing siliques is absent in mum2 mutants. No difference was observed in seed coat epidermal cell structure between wild-type and mutant seed; however, weakening of the outer tangential cell wall by chemical treatment resulted in the release of mucilage from mum2 seed coat epidermal cells, and the mum2 mucilage only increased slightly in volume, relative to the wild type. Consistent with the absence of β-d-galactosidase activity in the mutant, the inner layer of mucilage contained more Gal. The allocation of polysaccharides between the inner and outer mucilage layers was also modified in mum2. Mass spectrometry showed that rhamnogalacturonan I in mutant mucilage had more branching between rhamnose and hexose residues relative to the wild type. We conclude that the MUM2/BGAL6 β-d-galactosidase is required for maturation of rhamnogalacturonan I in seed mucilage by the removal of galactose/galactan branches, resulting in increased swelling and extrusion of the mucilage on seed hydration. PMID:18165330

[Production and partial characterization of beta-galactosidase from Kluyveromyces lactis grown in deproteinized whey].

PubMed

Ramírez Matheus, Alejandra O; Rivas, Nilo

2003-06-01

The purpose of this work was to optimize the beta-galactosidase production by Kluyveromyces lactis, applying the Surface Response Methodology (SRM) and using deproteinized whey as fermentation medium. An Orthogonal Central Compound Design (OCCD) was used without repetition, with four factors: temperature, pH, agitation speed and fermentation time. Then, enzyme activity (U/ml) as response variable was used. Thirty trials in twenty-five treatments, with six repetitions at the central point, were carried out, in a New Brunswick Bioflo 2000 fermentor with a volume of 2 liters. The deproteinized whey obtained by thermocoagulation was chemically analyzed. The results were: moisture 93.83%, total solids 6.17%, protein 0.44%, lactose 4.85%, acidity 0.43% and pH 4.58. The best conditions in the enzyme production were: temperature 30.3 degrees C, pH 4.68, agitation speed 191 r.p.m. and fermentation time 18.5 h. with an enzyme production of 8.3 U/ml. The degree of purification obtained was 7.4 times and the yield was 50.8%. The purified enzyme had an optimum temperature of 60 degrees C and a pH of 6.2. This work shows that the yeast Kluyveromyces lactis grown in deproteinized whey is able to produce the enzyme beta-galactosidase and SRM can be used in the fermentology processes, specifically in determining the best suitable operation conditions.

Structural analysis of a glycosides hydrolase family 42 cold-adapted ß-galactosidase from Rahnella sp. R3

USDA-ARS?s Scientific Manuscript database

The ß-galactosidase isolated from a psychrotrophic bacterium, Rahnella sp. R3 (R-ß-Gal), exhibits high activity at low temperature. R-ß-Gal is a member of the glycoside hydrolases family 42 (GH42), and forms a 225 kDa trimeric structure in solution. The X-ray crystal structure of R-ß-Gal was determi...

Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion.

PubMed

Condori, Jose; Acosta, Walter; Ayala, Jorge; Katta, Varun; Flory, Ashley; Martin, Reid; Radin, Jonathan; Cramer, Carole L; Radin, David N

2016-02-01

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called "hard-to-treat" tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid β-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid β-galactosidase enzyme were produced using a plant-based bioproduction platform. β-gal:RTB and RTB:β-gal fusion products retained both lectin activity and β-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived β-galactosidase. In contrast, plant-derived β-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native β-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1-4h, both β-gal:RTB and RTB:β-gal were processed to the ~64kDa "activated" β-gal form; the RTB lectin was

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus.

PubMed

Geiger, Barbara; Nguyen, Hoang-Minh; Wenig, Stefanie; Nguyen, Hoang Anh; Lorenz, Cindy; Kittl, Roman; Mathiesen, Geir; Eijsink, Vincent G H; Haltrich, Dietmar; Nguyen, Thu-Ha

2016-12-15

β-Galactosidase from Streptococcus thermophilus was overexpressed in a food-grade organism, Lactobacillus plantarum WCFS1. Laboratory cultivations yielded 11,000 U of β-galactosidase activity per liter of culture corresponding to approximately 170 mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate. The enzyme showed high transgalactosylation activity. When using an initial concentration of whey permeate corresponding to 205 g L -1 lactose, the maximum yield of galacto-oligosaccharides (GOS) obtained at 50°C reached approximately 50% of total sugar at 90% lactose conversion, meaning that efficient valorization of the whey lactose was obtained. GOS are of great interest for both human and animal nutrition; thus, efficient conversion of lactose in whey into GOS using an enzymatic approach will not only decrease the environmental impact of whey disposal, but also create additional value.

The βI-galactosidase of Cicer arietinum is located in thickened cell walls such as those of collenchyma, sclerenchyma and vascular tissue.

PubMed

Martín, I; Jiménez, T; Hernández-Nistal, J; Dopico, B; Labrador, E

2011-09-01

We report localisation of the chickpea βI-Gal, a member of the chickpea β-galactosidase family, which contains at least four members. After generation of specific antibodies, the distribution and cellular immunolocalisation of the protein in different organs and developmental stages of the plant was studied. βI-Gal protein is much longer than the other chickpea β-galactosidases because of the presence of a lectin-like domain in the carboxyl terminus of the protein. Western blot experiments indicated that the active βI-Gal retains this lectin-like domain for its function in the plant. The βI-Gal protein was mainly detected in cell walls of elongating organs, such as seedling epicotyls and stem internodes. An immunolocation study indicated a very good correlation between the presence of this βΙ-galactosidase and cells whose walls are thickening, not only in aged epicotyls and mature internodes in the final phase of elongation, but mostly in cells with a support function, such as collenchyma cells, xylem and phloem fibres and a layer of sclerenchyma cells surrounding the vascular cylinder (perivascular fibres). These results could suggest a function for the βI-Gal in modification of cell wall polymers, leading to thicker walls than the primary cell walls. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

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

PubMed

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.

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

PubMed

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.

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.

PubMed

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.

Substrate specificity and kinetic properties of alpha-galactosidases from Vicia faba.

PubMed

Dey, P M; Pridham, J B

1969-10-01

1. The hydrolysis of a variety of galactosides and other glycosides by alpha-galactosidases I and II of Vicia faba was studied. 2. The effect of temperature on kinetic parameters was also examined. 3. Both enzymes are inhibited by excess of substrate (p-nitrophenyl alpha-d-galactoside); with enzyme I this is competitive and is caused by the galactosyl moiety. 4. Enzyme I is inhibited by oligosaccharides possessing terminal non-reducing galactose residues and to a smaller extent by l-arabinose and d-fucose. 5. The effect of pH on K(m) and V(max.) values suggests that carboxyl and imidazole groups are involved in the catalytic activity of enzyme I. 6. Photo-oxidation experiments with enzyme I also suggest that an imidazole group is present at the active site.

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

PubMed

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.

Partial Purification and Properties of an Alkaline α-Galactosidase from Mature Leaves of Cucurbita pepo1

PubMed Central

Gaudreault, Pierre-Richard; Webb, John A.

1983-01-01

A fourth molecular from of α-galactosidase, designated LIV, an alkaline α-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-α-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. Km determinations in McIlvaine buffer (200 millimolar Na2-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-α-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. LIV was partially inhibited by Ca2+, Mg2+, and Mn2+, more so by Ni2+, Zn2+, and Co2+, and highly so by Cu2+, Ag2+, Hg2+ and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-α-d-galactoside or by myo-inositol, but α-d-galactose was a strong inhibitor. As observed for most other forms of α-galactosidase, LIV only catalyzed the hydrolysis of glycosides possessing the α-d-galactose configuration at C1, C2, and C4, and did not hydrolyze p-nitrophenyl-α-d-fucoside (α-d-galactose substituted at C6). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-α-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of LIV in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of LIVin vivo. It was possible that the

Optimal immobilization of β-galactosidase onto κ-carrageenan gel beads using response surface methodology and its applications.

PubMed

Elnashar, Magdy M; Awad, Ghada E; Hassan, Mohamed E; Mohy Eldin, Mohamed S; Haroun, Bakry M; El-Diwany, Ahmed I

2014-01-01

β-Galactosidase (β-gal) was immobilized by covalent binding on novel κ-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 2(2) full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55 °C compared to the free enzyme. The apparent K(m) after immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocity Vmax was 131.2 μ mol · min(-1) while it was 177.1 μ mol · min(-1) for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of the κ-carrageenan beads loaded with β -galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.

Optimization of the method for α-l-fucosidase, β-d-galactosidase and β-d-glucuronidase determination in serum from hemolyzed blood.

PubMed

Chojnowska, Sylwia; Ptaszyńska-Sarosiek, Iwona; Kępka, Alina; Szajda, Sławomir Dariusz; Waszkiewicz, Napoleon; Zwierz, Krzysztof

2018-06-06

Adaptation of the colorimetric method for the determination of β-d-galactosidase, β-d-glucuronidase and α-l-fucosidase activities in serums from hemolyzed blood, the material currently being discarded. The materials included serums from hemolyzed and non-hemolyzed blood, obtained from 26 healthy volunteers. The adaptation of the method involved precipitation of the proteins with trichloroacetic acid after incubating serums with substrates, but before determining the products of enzymatic reactions. In serums from hemolyzed and non-hemolyzed blood of the same persons, we found high correlations among the results obtained using hemolyzed blood (with adapted) and non-hemolyzed blood (with non-adapted) methods. We are able to determine the β-d-galactosidase, β-d-glucuronidase and α-l-fucosidase activities in serums from hemolyzed blood (with adapted) and non-hemolyzed blood (with non-adapted) methods, with the same accuracy and precision. Copyright © 2018 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

Identification and characterization of a novel β-galactosidase from Victivallis vadensis ATCC BAA-548, an anaerobic fecal bacterium.

PubMed

Temuujin, Uyangaa; Chi, Won-Jae; Park, Jae-Sun; Chang, Yong-Keun; Song, Jae Yang; Hong, Soon-Kwang

2012-12-01

Victivallis vadensis ATCC BAA-548 is a Gram-negative, anaerobic bacterium that was isolated from a human fecal sample. From the genomic sequence of V. vadensis, one gene was found to encode agarase; however, its enzymatic properties have never been characterized. The gene encoding the putative agarase (NCBI reference number ZP_01923925) was cloned by PCR and expressed in E. coli Rosetta-gami by using the inducible T(7) promoter of pET28a(+). The expressed protein with a 6×His tag at the N-terminus was named His6-VadG925 and purified as a soluble protein by Ni(2+)-NTA agarose affinity column chromatography. The purification of the enzyme was 26.8-fold, with a yield of 73.2% and a specific activity of 1.02 U/mg of protein. The purified His6-VadG925 produced a single band with an approximate MW of 155 kDa, which is consistent with the calculated value (154,660 Da) including the 6×His tag. Although VadG925 and many of its homologs were annotated as agarases, it did not hydrolyze agarose. Instead, purified His(6)-VadG925 hydrolyzed an artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for this β-galactosidase activity were pH 7.0 and 40°C, respectively. The K(m) and V(max) of His6-VadG925 towards p-nitrophenyl-β-D-galactopyranoside were 1.69 mg/ml (0.0056 M) and 30.3 U/mg, respectively. His6-VadG925 efficiently hydrolyzed lactose into glucose and galactose, which was demonstrated by TLC and mass spectroscopy. These results clearly demonstrated that VadG925 is a novel β-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated β-galactosidases.

A novel promising strain of Trichoderma evansii (WF-3) for extracellular α-galactosidase production by utilizing different carbon sources under optimized culture conditions.

PubMed

Chauhan, Aishwarya; Siddiqi, Nikhat Jamal; Sharma, Bechan

2014-01-01

A potential fungal strain of Trichoderma sp. (WF-3) was isolated and selected for the production of α-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Basal media selected for the growth of fungal isolate containing different carbon sources like guar gum (GG), soya bean meal (SM), and wheat straw (WS) and combinations of these carbon substrates with basic sugars like galactose and sucrose were used to monitor their effects on α-galactosidase production. The results of this study indicated that galactose and sucrose enhanced the enzyme activity in guar gum (GG) and wheat straw (WS). Maximum α-galactosidase production (213.63 U mL(-1)) was obtained when the basic medium containing GG is supplemented with galactose (5 mg/mL). However, the presence of galactose and sucrose alone in the growth media shows no effect. Soya meal alone was able to support T. evansii to produce maximum enzyme activity (170.36 U mL(-1)). The incubation time, temperature, and pH for the maximum enzyme synthesis were found to be 120 h (5 days), 28°C, and 4.5-5.5, respectively. All the carbon sources tested exhibited maximum enzyme production at 10 mg/mL concentration. Among the metal ions tested, Hg was found to be the strongest inhibitor of the enzyme. Among the chelators, EDTA acted as stronger inhibitor than succinic acid.

A Novel Promising Strain of Trichoderma evansii (WF-3) for Extracellular α-Galactosidase Production by Utilizing Different Carbon Sources under Optimized Culture Conditions

PubMed Central

Chauhan, Aishwarya; Siddiqi, Nikhat Jamal

2014-01-01

A potential fungal strain of Trichoderma sp. (WF-3) was isolated and selected for the production of α-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Basal media selected for the growth of fungal isolate containing different carbon sources like guar gum (GG), soya bean meal (SM), and wheat straw (WS) and combinations of these carbon substrates with basic sugars like galactose and sucrose were used to monitor their effects on α-galactosidase production. The results of this study indicated that galactose and sucrose enhanced the enzyme activity in guar gum (GG) and wheat straw (WS). Maximum α-galactosidase production (213.63 UmL−1) was obtained when the basic medium containing GG is supplemented with galactose (5 mg/mL). However, the presence of galactose and sucrose alone in the growth media shows no effect. Soya meal alone was able to support T. evansii to produce maximum enzyme activity (170.36 UmL−1). The incubation time, temperature, and pH for the maximum enzyme synthesis were found to be 120 h (5 days), 28°C, and 4.5–5.5, respectively. All the carbon sources tested exhibited maximum enzyme production at 10 mg/mL concentration. Among the metal ions tested, Hg was found to be the strongest inhibitor of the enzyme. Among the chelators, EDTA acted as stronger inhibitor than succinic acid. PMID:25126562

Microbial β-Galactosidase of Pediococcus pentosaceus ID-7: Isolation, Cloning, and Molecular Characterization.

PubMed

Lee, Ji-Yeong; Kwak, Mi-Sun; Roh, Jong-Bok; Kim, Kwang; Sung, Moon-Hee

2017-03-28

Pediococcus pentosaceus ID-7 was isolated from kimchi, a Korean fermented food, and it showed high activity for lactose hydrolysis. The β-galactosidase of P. pentosaceus ID-7 belongs to the GH2 group, which is composed of two distinct proteins. The heterodimeric LacLM type of β-galactosidase found in P. pentosaceus ID-7 consists of two genes partially overlapped, lacL and lacM encoding LacL (72.2 kDa) and LacM (35.4 kDa). In this study, Escherichia coli MM294 was used for the production of LacL, LacM, and LacLM. These three types of recombinant proteins were expressed, purified, and characterized. The specific activities of LacLM and LacL were 339 and 31 U/mg, respectively. However, activity was not detected with LacM alone. The optimal pH of LacLM and LacL was pH 7.5 and pH 7.0, and the optimal temperature of LacLM and LacL was 40°C and 50°C, respectively. The optimal temperature changes indicate that LacLM is able to achieve higher activity at a relatively lower temperature. LacLM was strongly activated by Mg 2+ , Mn 2+ , and Zn 2+ , which was not true for LacL. Consistent with this, EDTA strongly inactivated LacLM and LacL, but the presence of reducing agents did not dramatically alter the activity. Taken together, multiple alignment of amino acid sequences and phylogenetic analysis results of LacL and LacM of P. pentosaceus ID-7 suggest the evolution of LacL into LacLM and that the use of divalent metal ions results in higher activity.

A Fungal α-Galactosidase from Tricholoma matsutake with Broad Substrate Specificity and Good Hydrolytic Activity on Raffinose Family Oligosaccharides.

PubMed

Geng, Xueran; Tian, Guoting; Zhao, Yongchang; Zhao, Liyan; Wang, Hexiang; Ng, Tzi Bun

2015-07-24

An acidic α-galactosidase designated as TMG was purified from the fruiting bodies The purification protocol entailed ion exchange chromatography on Q-Sepharose and of Tricholoma matsutake with 136-fold purification and a specific activity of 909 units/mg. Mono-Q and fast protein liquid chromatography on Superdex 75. TMG is a monomeric protein exhibiting a molecular mass of 47 kDa in SDS-PAGE and gel filtration. The purified enzyme was identified by LC-MS/MS and three inner amino acid sequences were obtained. The optimum pH and temperature for TMG with pNPGal as substrate were pH 4.5 and 55 °C, respectively. The α-galactosidase activity was strongly inhibited by K+, Ca2+, Cd2+, Hg2+, Ag+ and Zn2+ ions. The enzyme activity was inhibited by the chemical modification agent N-bromosuccinimide (NBS), indicating the importance of tryptophan residue(s) at or near the active site. Besides hydrolyzing pNPGal, TMG also efficaciously catalyzed the degradation of natural substrates such as stachyose, raffinose, and melibiose. Thus TMG can be exploited commercially for improving the nutritional value of soy milk by degradation of indigestible oligosaccharides.

Comprehensive and differential long-term characterization of the alpha-galactosidase A deficient mouse model of Fabry disease focusing on the sensory system and pain development

PubMed Central

Biko, Lydia; Hose, Dorothea; Hofmann, Lukas; Sommer, Claudia

2016-01-01

Background Fabry disease is an X-linked lysosomal storage disorder due to impaired activity of alpha-galactosidase A with intracellular accumulation of globotriaosylceramide. Associated small fiber pathology leads to characteristic pain in Fabry disease. We systematically assessed sensory system, physical activity, metabolic parameters, and morphology of male and female mice with alpha-galactosidase A deficiency (Fabry ko) from 2 to 27 months of age and compared results with those of age- and gender-matched wild-type littermates of C57Bl/6J background. Results From the age of two months, male and female Fabry mice showed mechanical hypersensitivity (p < 0.001 each) compared to wild-type littermates. Young Fabry ko mice of both genders were hypersensitive to heat stimulation (p < 0.01) and developed heat hyposensitivity with aging (p < 0.05), while cold hyposensitivity was present constantly in young (p < 0.01) and old (p < 0.05) Fabry ko mice compared to wild-type littermates. Stride angle increased only in male Fabry ko mice with aging (p < 0.01) in comparison to wild-type littermates. Except for young female mice, male (p < 0.05) and female (p < 0.01) Fabry ko mice had a higher body weight than wild-type littermates. Old male Fabry ko mice were physically less active than their wild-type littermates (p < 0.05), had lower chow intake (p < 0.001), and lost more weight (p < 0.001) in a one-week treadmill experiment than wild-type littermates. Also, Fabry ko mice showed spontaneous pain protective behavior and developed orofacial dysmorphism resembling patients with Fabry disease. Conclusions Mice with alpha-galactosidase A deficiency show age-dependent and distinct deficits of the sensory system. alpha-galactosidase A-deficient mice seem to model human Fabry disease and may be helpful when studying the pathophysiology of Fabry-associated pain. PMID:27145802

Purification and characterization of Phaseolus vulgaris alpha-D-galactosidase isozymes.

PubMed

Dhar, M; Mitra, M; Hata, J; Butnariu, O; Smith, D

1994-11-01

A highly purified preparation of alpha-D-galactosidase [E.C. 3.2.1.22] isozymes was obtained from Phaseolus vulgaris (pinto bean) seeds by extraction, salt precipitation, ion exchange, and affinity chromatography. The final preparation was homogeneous by SDS-PAGE but revealed isozymes of relative mass of 38.3 and 39.6 kDa. The N-terminal sequence for both isozymes was identical, LANGLAKT (one letter code for amino acids). Relative native molecular mass was estimated at 149.3 kDa by Sephacryl S-200 chromatography. Activity was unaffected by ionic strength at high enzyme concentrations, and was specific for alpha-D-galactoside conjugates. No protease or hemagglutinin activity was detected, and activity was stable at 4 degrees C. Studies with soluble oligosaccharides demonstrated high activity against the selected straight and branched-chain substrates. The enzyme was active against terminal alpha 1-3 galactosyl residues on human and rabbit erythrocyte membranes. Because of its activity against membrane glycoconjugates, these isozymes may have potential utility for modifying membrane epitopes on native erythrocytes.

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

PubMed

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.

Novel Fe3+-Based 1H MRI β-Galactosidase Reporter Molecules**

PubMed Central

Yu, Jian-Xin; Gulaka, Praveen K.; Liu, Li; Kodibagkar, Vikram D.; Mason, Ralph P.

2012-01-01

There is increasing interest in the development of reporter agents to reveal enzyme activity in vivo using small animal imaging. We have previously demonstrated the feasibility of detecting lacZ gene activity using the commercially available 3,4-cyclohexenoesculetin-β-D-galactopyranoside (S-Gal™) as a 1H MRI reporter. Specifically, β-galactosidase (β-gal) releases the aglycone, which forms an MR contrast-inducing paramagnetic precipitate in the presence of Fe3+. Contrast was primarily T2-weighted signal loss, but T1 effects were also observed. Since T1-contrast generally provides signal enhancement as opposed to loss, it appeared attractive to explore whether analogues could be generated with enhanced characteristics. We now report the design and successful synthesis of novel analogues together with characterization of 1H MRI contrast based on both T1 and T2 response to β-gal activity in vitro for the lead agent. PMID:23807909

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

PubMed

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 (<24%) to the previously reported GH31 glycosidases and characterized two enzymes from 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.

Poly(lactic acid) nanoparticles coated with combined WGA and water-soluble chitosan for mucosal delivery of β-galactosidase.

PubMed

Sheng, Yan; He, Hongjun; Zou, Hui

2014-08-01

A combinatorial design, physical adsorption of water-soluble chitosan (WSC) to particle surface and covalent conjugation of wheat germ agglutinin (WGA) to WSC, was applied to surface modification of poly(lactic acid) nanoparticles (NPs) for targeted delivery of β-galactosidase to the intestinal mucosa. All the surface-engineered NPs in the size range of 500-600 nm were prepared by a w/o/w solvent diffusion/evaporation technique. β-Galactosidase encapsulated in these NPs was well protected from external proteolysis and exerted high hydrolytic activity on the permeable lactose. The presence of WSC coating, whether alone or with WGA, highly improved the suspension stability of NPs and tailored the particle surface positively charged. In comparison to NPs modified with WGA or WSC alone, the synergistic action of WGA and WSC greatly enhanced the NP-mucin interactions in vitro. The highest amount of NPs was found in the small intestine at 24 h after oral administration in rats. Notably, calculated half-life of WGA-WSC-NPs in the small intestine was 6.72 h, resulting in 2.1- and 4.3-fold increase when compared to WGA-polyvinylalcohol (PVA)-NPs and WSC-NPs, much longer than that of control PVA-NPs (6.9-fold). These results suggest that NPs with the combined WGA and WSC coating represent promising candidates for efficient mucosal drug delivery as well as biomimetic treatment of lactose intolerance.

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

PubMed

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.

Introducing transgalactosylation activity into a family 42 β-galactosidase.

PubMed

Strazzulli, Andrea; Cobucci-Ponzano, Beatrice; Carillo, Sara; Bedini, Emiliano; Corsaro, Maria Michela; Pocsfalvi, Gabriella; Withers, Stephen G; Rossi, Mosè; Moracci, Marco

2017-05-01

Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a β-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.

PubMed

Takala, T M; Saris, P E J; Tynkkynen, S S H

2003-01-01

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.

Chelation by collagen in the immobilization of Aspergillus oryzae β-galactosidase: A potential biocatalyst to hydrolyze lactose by batch processes.

PubMed

Gennari, Adriano; Mobayed, Francielle Herrmann; Volpato, Giandra; de Souza, Claucia Fernanda Volken

2018-04-01

This work is the first study of the immobilization of Aspergillus oryzae β-galactosidase (Gal) on powdered collagen (Col) that had formed a chelate with aluminum (Col-Al-Gal). Other collagen treatments, including those with acetic acid, glutaraldehyde, and a combination of aluminum and glutaraldehyde (Col-Al-Glu-Gal), were also tested. High-yield (superior to 80%) and high-efficiency (superior to 99%) immobilization was obtained for the derivatives Col-Al-Gal and Col-Al-Glu-Gal, even at high protein loads (500-1,000 mg g -1 of support). The storage stability of Gal immobilized on Col-Al and Col-Al-Glu resulted in Gal retaining approximately 60% of its initial activity after 90 days at 4 °C. The half-life values of derivatives Col-Al-Gal and Col-Al-Glu-Gal were higher than those of soluble enzyme at 65, 68, 70, and 73 °C. The derivatives Col-Al-Gal and Col-Al-Glu-Gal retained high enzyme activity in batch hydrolysis of lactose in permeate and lactose solutions for 50 and 60 cycles, respectively. Our results suggest that powdered collagen treated with aluminum, a low-cost support, is a promising support for the immobilization of β-galactosidase. Copyright © 2017 Elsevier B.V. All rights reserved.

Mutagenesis of the three bases preceding the start codon of the beta-galactosidase mRNA and its effect on translation in Escherichia coli.

PubMed Central

Hui, A; Hayflick, J; Dinkelspiel, K; de Boer, H A

1984-01-01

The effect on the translation efficiency of various mutations in the three bases (the -1 triplet) that precede the AUG start codon of the beta-galactosidase mRNA in Escherichia coli was studied. Of the 39 mutants examined, the level of expression varies over a 20-fold range. The most favorable combinations of bases in the -1 triplet are UAU and CUU. The expression levels in the mutants with UUC, UCA or AGG as the -1 triplet are 20-fold lower than those with UAU or CUU. In general, a U residue immediately preceding the start codon is more favorable for expression than any other base; furthermore, an A residue at the -2 position enhances the translation efficiency in most instances. In both cases, however, the degree of enhancement depends on its context, i.e. the neighboring bases. Although the rules derived from this study are complex, the results show that mutations in any of the three bases preceding the start codon can strongly affect the translational efficiency of the beta-galactosidase mRNA. PMID:6425057

Structure of the beta-galactosidase gene from Thermus sp. strain T2: expression in Escherichia coli and purification in a single step of an active fusion protein.

PubMed

Vian, A; Carrascosa, A V; García, J L; Cortés, E

1998-06-01

The nucleotide sequence of both the bgaA gene, coding for a thermostable beta-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the beta-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric beta-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70 degrees C and a Km value of 1.6 mM with o-nitrophenyl-beta-D-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C.

Lactose-induced cell death of beta-galactosidase mutants in Kluyveromyces lactis.

PubMed

Lodi, Tiziana; Donnini, Claudia

2005-05-01

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.

Synthesis of galactooligosaccharides by CBD fusion β-galactosidase immobilized on cellulose.

PubMed

Lu, Lili; Xu, Shuze; Zhao, Renfei; Zhang, Dayu; Li, Zhengyi; Li, Yumei; Xiao, Min

2012-07-01

The β-galactosidase gene (bgaL3) was cloned from Lactobacillus bulgaricus L3 and fused with cellulose binding domain (CBD) using pET-35b (+) vector in Escherichia coli. The resulting fusion protein (CBD-BgaL3) was directly adsorbed onto microcrystalline cellulose with a high immobilization efficiency of 61%. A gram of cellulose was found to absorb 97.6 U of enzyme in the solution containing 100mM NaCl (pH 5.8) at room temperature for 20 min. The enzymatic and transglycosylation characteristics of the immobilized CBD-BgaL3 were similar to the free form. Using the immobilized enzyme as the catalyst, the yield of galactooligosaccharides (GOS) reached a maximum of 49% (w/w) from 400 g/L lactose (pH 7.6) at 45 °C for 75 min, with a high productivity of 156.8 g/L/h. Reusability assay was subsequently performed under the same reaction conditions. The immobilized enzyme could retain over 85% activity after twenty batches with the GOS yields all above 40%. Copyright © 2012 Elsevier Ltd. All rights reserved.

Ultra-high throughput detection of single cell β-galactosidase activity in droplets using micro-optical lens array

NASA Astrophysics Data System (ADS)

Lim, Jiseok; Vrignon, Jérémy; Gruner, Philipp; Karamitros, Christos S.; Konrad, Manfred; Baret, Jean-Christophe

2013-11-01

We demonstrate the use of a hybrid microfluidic-micro-optical system for the screening of enzymatic activity at the single cell level. Escherichia coli β-galactosidase activity is revealed by a fluorogenic assay in 100 pl droplets. Individual droplets containing cells are screened by measuring their fluorescence signal using a high-speed camera. The measurement is parallelized over 100 channels equipped with microlenses and analyzed by image processing. A reinjection rate of 1 ml of emulsion per minute was reached corresponding to more than 105 droplets per second, an analytical throughput larger than those obtained using flow cytometry.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pourcher, T.; Bassilana, M.; Sarkar, H.K.

1990-01-23

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 initiationmore » 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.« less

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

PubMed Central

Ross, G S; Wegrzyn, T; MacRae, E A; Redgwell, R J

1994-01-01

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening. PMID:7991682

Glucose buffer is suitable for blood group conversion with α-N acetylgalactosaminidase and α-galactosidase.

PubMed

Gao, Hong-Wei; Li, Su-Bo; Bao, Guo-Qiang; Zhang, Xue; Li, Hui; Wang, Ying-Li; Tan, Ying-Xia; Ji, Shou-Ping; Gong, Feng

2014-01-01

It is well known that the buffer plays a key role in the enzymatic reaction involved in blood group conversion. In previous study, we showed that a glycine buffer is suitable for A to O or B to O blood group conversion. In this study, we investigated the use of 5% glucose and other buffers for A to O or B to O blood group conversion by α-N-acetylgalactosaminidase or α-galactosidase. We compared the binding ability of α-N-acetylgalactosaminidase/α-galactosidase with red blood cells (RBC) in different reaction buffers, such as normal saline, phosphate-buffered saline (PBS), a disodium hydrogen phosphate-based buffer (PCS), and 5% commercial glucose solution. The doses of enzymes necessary for the A/B to O conversion in different reaction buffers were determined and compared. The enzymes' ability to bind to RBC was evaluated by western blotting, and routine blood typing and fluorescence activated cell sorting was used to evaluate B/A to O conversion efficiency. The A to O conversion efficiency in glucose buffer was similar to that in glycine buffer with the same dose (>0.06 mg/mL pRBC). B to O conversion efficiency in glucose buffer was also similar to that in glycine buffer with the same dose (>0.005 mg/mL pRBC). Most enzymes could bind with RBC in glycine or glucose buffer, but few enzymes could bind with RBC in PBS, PCS, or normal saline. These results indicate that 5% glucose solution provides a suitable condition for enzymolysis, especially for enzymes combining with RBC. Meanwhile, the conversion efficiency of A/B to O was similar in glucose buffer and glycine buffer. Moreover, 5% glucose solution has been used for years in venous transfusion, it is safe for humans and its cost is lower. Our results do, therefore, suggest that 5% glucose solution could become a novel suitable buffer for A/B to O blood group conversion.

Glucose buffer is suitable for blood group conversion with α-N acetylgalactosaminidase and α-galactosidase

PubMed Central

Gao, Hong-Wei; Li, Su-Bo; Bao, Guo-Qiang; Zhang, Xue; Li, Hui; Wang, Ying-Li; Tan, Ying-Xia; Ji, Shou-Ping; Gong, Feng

2014-01-01

Background It is well known that the buffer plays a key role in the enzymatic reaction involved in blood group conversion. In previous study, we showed that a glycine buffer is suitable for A to O or B to O blood group conversion. In this study, we investigated the use of 5% glucose and other buffers for A to O or B to O blood group conversion by α-N-acetylgalactosaminidase or α-galactosidase. Materials and methods We compared the binding ability of α-N-acetylgalactosaminidase/α-galactosidase with red blood cells (RBC) in different reaction buffers, such as normal saline, phosphate-buffered saline (PBS), a disodium hydrogen phosphate-based buffer (PCS), and 5% commercial glucose solution. The doses of enzymes necessary for the A/B to O conversion in different reaction buffers were determined and compared. The enzymes’ ability to bind to RBC was evaluated by western blotting, and routine blood typing and fluorescence activated cell sorting was used to evaluate B/A to O conversion efficiency. Results The A to O conversion efficiency in glucose buffer was similar to that in glycine buffer with the same dose (>0.06 mg/mL pRBC). B to O conversion efficiency in glucose buffer was also similar to that in glycine buffer with the same dose (>0.005 mg/mL pRBC). Most enzymes could bind with RBC in glycine or glucose buffer, but few enzymes could bind with RBC in PBS, PCS, or normal saline. Conclusion These results indicate that 5% glucose solution provides a suitable condition for enzymolysis, especially for enzymes combining with RBC. Meanwhile, the conversion efficiency of A/B to O was similar in glucose buffer and glycine buffer. Moreover, 5% glucose solution has been used for years in venous transfusion, it is safe for humans and its cost is lower. Our results do, therefore, suggest that 5% glucose solution could become a novel suitable buffer for A/B to O blood group conversion. PMID:24333060

Cloning and High-Level Expression of α-Galactosidase cDNA from Penicillium purpurogenum

PubMed Central

Shibuya, Hajime; Nagasaki, Hiroaki; Kaneko, Satoshi; Yoshida, Shigeki; Park, Gwi Gun; Kusakabe, Isao; Kobayashi, Hideyuki

1998-01-01

The cDNA coding for Penicillium purpurogenum α-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides. PMID:9797312

Synthesis of Stachyobifiose Using Bifidobacterial α-Galactosidase Purified from Recombinant Escherichia coli.

PubMed

Oh, So Young; Youn, So Youn; Park, Myeong Soo; Baek, Nam In; Ji, Geun Eog

2018-02-07

The prebiotic effects of GOS (galactooligosaccharides) are known to depend on the glycosidic linkages, degree of polymerization (DP), and the monosaccharide composition. In this study, a novel form of α-GOS with a potentially improved prebiotic effect was synthesized using bifidobacterial α-galactosidase (α-Gal) purified from recombinant Escherichia coli. The carbohydrate produced was identified as α-d-galactopyranosyl-(1→6)-O-α-d-glucopyranosyl-(1→2)-[α-d-galactopyranosyl-(1→6)-O-β-d-fructofuranoside] and was termed stachyobifiose. Among 17 nonprobiotics, 16 nonprobiotics showed lower growth on stachyobifiose than β-GOS. In contrast, among the 16 probiotics, 6 probiotics showed higher growth on stachyobifiose than β-GOS. When compared with raffinose, stachyobifiose was used less by nonprobiotics than raffinose. Moreover, compared with stachyose, stachyobifiose was used less by Escherichia coli, Enterobacter cloacae, and Clostridium butyricum. The average amounts of total short-chain fatty acids (SCFA) produced were in the order of stachyobifiose > stachyose > raffinose > β-GOS. Taken together, stachyobifiose is expected to contribute to beneficial changes of gut microbiota.

Clinical and genetic investigation of a Japanese family with cardiac fabry disease. Identification of a novel α-galactosidase A missense mutation (G195V).

PubMed

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.

Impact of a multicarbohydrase containing α-galactosidase and xylanase on ileal digestible energy, crude protein digestibility, and ileal amino acid digestibility in broiler chickens.

PubMed

Jasek, A; Latham, R E; Mañón, A; Llamas-Moya, S; Adhikari, R; Poureslami, R; Lee, J T

2018-06-08

Exogenous enzymatic supplementation of poultry feeds, including α-galactosidase and xylanase, has been shown to increase metabolically available energy, although little information has been published on the impact on amino acid digestibility. An experiment was conducted to investigate a multicarbohydrase containing α-galactosidase and xylanase on amino acid digestibility, ileal digestible energy (IDE), and CP in male broiler chicks. The experiment was a 2 × 2 (diet × enzyme) factorial arrangement with 15 replicates of 8 male broilers per replicate raised for 21 d in a battery setting. The 2 dietary treatments included a positive control (PC) and a negative control (NC) diet formulated to contain 2.5% less calculated AME and digestible amino acids. Each of these diets was fed with and without enzyme. Broilers were fed a starter diet from 0-14 d (crumble) and a grower from 14-21 d (pellet). Birds were sampled on day 21 to determine ileal amino acid digestibility, IDE, and CP digestibility. Titanium dioxide (TiO2) was used as an indigestible marker for the determination of digestibility coefficients. Total ileal amino acid digestibility was increased (P = 0.008) by 3.80% with the inclusion of enzyme. Methionine and lysine digestibility was improved (P < 0.05) with the inclusion of enzyme by 3.37% and 2.61%, respectively. Enzyme inclusion increased (P = 0.001) cysteine digestibility by 9.3%. Diet-influenced ileal amino acid digestibility with tryptophan, threonine, isoleucine, and valine digestibility being increased (P < 0.05) in the PC when compared to the NC. IDE was decreased (P = 0.037) in broilers fed the NC diet by 100 kcal/kg feed when compared to broilers fed the PC diet. Enzyme inclusion increased (P = 0.047) IDE value by 90 kcal/kg. Crude protein digestibility was not influenced by diet; however, similar improvements in CP digestibility with enzyme inclusion were observed as with energy. These data support the benefits of a multicarbohydrase containing α-galactosidase

Immobilization of Aspergillus oryzae  β-Galactosidase on Cellulose Acetate-Polymethylmethacrylate Membrane and Its Application in Hydrolysis of Lactose from Milk and Whey.

PubMed

Ansari, Shakeel Ahmed; Satar, Rukhsana; Kashif Zaidi, Syed; Ahmad, Abrar

2014-01-01

The present study demonstrates the immobilization of Aspergillus oryzae β-galactosidase on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane and its application in hydrolyzing lactose in dairy industries. The effect of physical and chemical denaturants like pH, temperature, product inhibition by galactose, storage stability, and reuse number of the enzyme immobilized on CA-PMMA membrane has been investigated. Lactose was hydrolyzed from milk and whey in batch reactors at 50°C by free and immobilized β-galactosidase (IβG). Optimum pH for the free and immobilized enzyme was found to be the same, that is, 4.5. However, IβG retained greater fractions of catalytic activity at lower and higher pH ranges. The temperature optimum for the immobilized enzyme was increased by 10°C. Moreover, Michaelis-Menten constant was increased for IβG as compared to the native one while maximum reaction rate was reduced for the immobilized enzyme. The preserved activity of free and immobilized enzyme was found to be 45% and 83%, respectively, after five weeks of storage at 4°C. Reusability of IβG was observed to be 86% even after fifth repeated use, thereby signifying its application in lactose hydrolysis (as shown in lab-scale batch reactors) in various dairy products including milk and whey.

Transient detection of beta-galactosidase activity in hematopoietic cells, following reinjection of retrovirally marked autologous blood progenitors in patients with breast or ovarian cancer receiving high-dose chemotherapy.

PubMed

Bagnis, Claude; Chabannon, Christian; Gravis, Gwenaelle; Imbert, Anne-Marie; Maroc, Christine; Bardin, Florence; Ladaique, Patrick; Viret, Frédéric; Genre, Dominique; Faucher, Catherine; Stoppa, Anne-Marie; Vey, Norbert; Blaise, Didier; Maraninchi, Dominique; Viens, Patrice; Mannoni, Patrice

2002-02-01

The aim of this report is to demonstrate the feasibility and safety of genetically modifying autologous human blood CD34(+) cells in vitro, with a retroviral vector that encodes a marker gene. The fate of genetically modified cells and their progeny was followed in vivo, after reinfusion in patients treated with high-dose chemotherapy for poor-prognosis breast or ovarian carcinomas. Six patients received genetically modified autologous peripheral blood progenitors, together with unmanipulated aphereses, following high-dose chemotherapy. CD34(+) cells were immunoselected from aphereses, and retrovirally transduced by coculture with the retroviral vector producing cell line, to express a nuclear localized version of E. coli beta-galactosidase, encoded by a defective Moloney-murine leukemia virus-derived retroviral vector. Cells were reinfused to the patients after myeloablation, without prior ex vivo selection. Five out of six patients showed the transient presence of low numbers of beta-galactosidase(+) cells, as detected with an immunocytochemical assay, in the peripheral blood, during the first month following infusion. One patient had beta-galactosidase(+) clonogenic progenitors in her marrow at two months after transplantation, including HPP-CFC; intriguingly, this patient had the lowest percentage of X-gal(+) cells in her graft. Patients experienced side effects that are often observed after high-dose chemotherapy. Feasibility and safety of genetic modification of human hematopoietic stem and progenitor cells are demonstrated by this study. Ex vivo or in vivo selection is not mandatory, even in clinical situations where transduced cells have no survival advantage over wild-type cells; however, significant improvements in gene transfer technology are needed to achieve potentially useful levels of expression in such clinical situations.

Lactose digestion by yogurt beta-galactosidase: influence of pH and microbial cell integrity.

PubMed

Martini, M C; Bollweg, G L; Levitt, M D; Savaiano, D A

1987-02-01

Lactase-deficient subjects more effectively digest lactose in yogurt than lactose in other dairy products, apparently due to yogurt microbial beta-galactosidase (beta-gal) which is active in the GI tract. We evaluated the effects of buffering capacity of yogurt, gastric pH, and microbial cell disruption on beta-gal activity and lactose digestion. Three times more acid was required to acidify yogurt than to acidify milk. Yogurt beta-gal was stable at pH 4.0 but inactivated at lower pH. When yogurt was sonicated to disrupt microbial cell structure, only 20% activity remained after incubation at pH 4.0 for 60 min. In vivo gastric pH remained greater than 2.7 for 3 h after ingestion of yogurt. Acidified milk alone or with disrupted yogurt microorganisms caused twice as much lactose malabsorption as did acidified milk containing intact yogurt microorganisms. The results provide a possible explanation for the survival of beta-gal activity from yogurt in the GI tract.

Interaction of SDS with β-galactosidase. A FT-IR study of the influence of detergent concentration and temperature.

NASA Astrophysics Data System (ADS)

Muga, A.; Castresana, J.; Arrondo, J. L. R.; López, S.; Bernabeu, C.

1988-05-01

The major structure of the enzyme β-galactosidase as studied by FT-IR is β-sheet with maxima in the amide I band at 1639 and 1655 cm -1 in H 2O and 1634 in D 2O. α-helix structure is also present with contribution from β-turns and less-ordered structure. Temperature induces a rearrangement of the structure producing a β-sheet-like conformation. In the presence of the surfactant SDS no big difference in structure is seen at 1% SDS (w:ww) concentration but there is a decrease of 5°C in the midpoint thermal denaturation. In the presence of 10% SDS a different picture is obtained with a higher random structure content.

The specificity of induction of alpha-galactosidase from Saccharomyces carlsbergensis.

PubMed

Flórez, I G; Lazo, P S; Ochoa, A G; Gascón, S

1981-04-17

A number of sugars and derivatives have been tested for their ability to induce the synthesis of alpha-galactosidase from Saccharomyces carlsbergensis. Besides galactose and the substrates of the enzyme melibiose, raffinose and stachyose, D-galacturonic acid, L-arabinose, D-tagatose, methyl-alpha-D-galactoside, lactose and isopropyl-beta-D-thiogalactoside were able to act as inducers. Of these, methyl-alpha-D-galactoside, lactose, isopropyl-beta-D-thiogalactoside and L-arabinose have been shown to be gratuitous inducers with which kinetic studies of induction have been carried out. Lactose was the most efficient inducer, giving a maximal differential rate of synthesis of the enzyme of 110 mU/10(7) cells at a concentration of 180 mM, followed by L-arabinose (60 mU/10(7) cells at 40 mM), isopropyl-beta-D-thiogalactoside (43 mU/10(7) cells at 60 mM) and methyl-alpha-D-galactoside (25 mU/10(7) cells at 150 mM). The concentration of inducer required to obtain half-maximal induction was similar for lactose, L-arabinose and isopropyl-beta-D-thiogalactoside and about 5-fold higher for methyl-alpha-D-galactoside. The property of the compounds to act as inducers was compared to their ability to interact with the enzyme and the results discussed in terms of the molecular structures which are recognized by the enzyme and by the induction machinery.

Immobilization of Aspergillus oryzae   β-Galactosidase on Cellulose Acetate-Polymethylmethacrylate Membrane and Its Application in Hydrolysis of Lactose from Milk and Whey

PubMed Central

Ansari, Shakeel Ahmed; Satar, Rukhsana; Kashif Zaidi, Syed; Ahmad, Abrar

2014-01-01

The present study demonstrates the immobilization of Aspergillus oryzae β-galactosidase on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane and its application in hydrolyzing lactose in dairy industries. The effect of physical and chemical denaturants like pH, temperature, product inhibition by galactose, storage stability, and reuse number of the enzyme immobilized on CA-PMMA membrane has been investigated. Lactose was hydrolyzed from milk and whey in batch reactors at 50°C by free and immobilized β-galactosidase (IβG). Optimum pH for the free and immobilized enzyme was found to be the same, that is, 4.5. However, IβG retained greater fractions of catalytic activity at lower and higher pH ranges. The temperature optimum for the immobilized enzyme was increased by 10°C. Moreover, Michaelis-Menten constant was increased for IβG as compared to the native one while maximum reaction rate was reduced for the immobilized enzyme. The preserved activity of free and immobilized enzyme was found to be 45% and 83%, respectively, after five weeks of storage at 4°C. Reusability of IβG was observed to be 86% even after fifth repeated use, thereby signifying its application in lactose hydrolysis (as shown in lab-scale batch reactors) in various dairy products including milk and whey. PMID:27350979

Galactosidase Alpha p.A143T Variant Fabry Disease May Result in a Phenotype With Multifocal Microvascular Cerebral Involvement at a Young Age.

PubMed

Hauth, Lothar; Kerstens, Jeroen; Yperzeele, Laetitia; Eyskens, François; Parizel, Paul M; Willekens, Barbara

2018-01-01

A 16-year-old male presented with episodic headaches and a brain magnetic resonance imaging (MRI) that showed multifocal punctate to patchy white matter lesions. The diagnosis of Fabry disease (FD) was suggested upon the finding of significantly reduced plasma alpha-galactosidase A activity (0.62 µmol/L or 13% of normal; normal range ≥ 1.65 μmol/L) and genetic investigation confirmed the presence of a hemizygous missense variant in the galactosidase alpha (GLA) gene (p.A143T). Baseline assessment of other systemic involvement showed only a discrete proteinuria. FD is a rare lysosomal storage disorder. Genetic screening studies have revealed over 600 variants in the GLA gene. The p.A143T variant is a genetic variant of unknown significance, with its associated phenotype ranging from classical FD to healthy unaffected patients. Some authors, however, deem this variant non-pathogenic. We describe the case of a 16-year-old male with multifocal white matter lesions on brain MRI, who was diagnosed with FD and carried this genetic variant. The causative p.A143T mutation can be associated with a more severe subclinical phenotype than has been reported to date. Furthermore, a diagnosis of FD should be considered when finding asymptomatic cerebral white matter lesions in a young patient.

Bulk soil and rhizosphere bacterial community PCR-DGGE profiles and beta-galactosidase activity as indicators of biological quality in soils contaminated by heavy metals and cultivated with Silene vulgaris (Moench) Garcke.

PubMed

Martínez-Iñigo, M J; Pérez-Sanz, A; Ortiz, I; Alonso, J; Alarcón, R; García, P; Lobo, M C

2009-06-01

The biological quality of two heavy metal contaminated soils (soil C: Typic Calcixerept, pH 8.3 and soil H: Typic Haploxeraf, pH 7.3) was investigated after growing the metal-tolerant plant Silene vulgaris (Moench) Garcke for two vegetative periods. The activity of the enzyme beta-galactosidase, which is sensitive to the presence of contaminants in soil, and the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of 16S rRNA gene fragments of culturable bacteria from bulk soil and rhizosphere were determined. The microbial enzymatic activity was higher in planted soils than in bare soils at the contamination level of 600 mg of total heavy metals kg(-1) soil. After growing S. vulgaris, beta-galactosidase activity was almost recovered in the calcareous soil. In this soil new bands appeared in the PCR-DGGE profiles of the rhizosphere bacterial community as a response to the exposure to heavy metals.

Enhanced sialylation and in vivo efficacy of recombinant human α-galactosidase through in vitro glycosylation

PubMed Central

Sohn, Youngsoo; Lee, Jung Mi; Park, Heung-Rok; Jung, Sung-Chul; Park, Tai Hyun; Oh, Doo-Byoung

2013-01-01

Human α-galactosidase A (GLA) has been used in enzyme replacement therapy for patients with Fabry disease. We expressed recombinant GLA from Chinese hamster ovary cells with very high productivity. When compared to an approved GLA (agalsidase beta), its size and charge were found to be smaller and more neutral. These differences resulted from the lack of terminal sialic acids playing essential roles in the serum half-life and proper tissue targeting. Because a simple sialylation reaction was not enough to increase the sialic acid content, a combined reaction using galactosyltransferase, sialyltransferase, and their sugar substrates at the same time was developed and optimized to reduce the incubation time. The product generated by this reaction had nearly the same size, isoelectric points, and sialic acid content as agalsidase beta. Furthermore, it had better in vivo efficacy to degrade the accumulated globotriaosylceramide in target organs of Fabry mice compared to an unmodified version. [BMB Reports 2013; 46(3): 157-162] PMID:23527859

Synthesis of novel galactose containing chemicals by beta-galactosidase from Enterobacter cloacae B5.

PubMed

Lu, Lili; Xu, Xiaodong; Gu, Guofeng; Jin, Lan; Xiao, Min; Wang, Fengshan

2010-09-01

The beta-galactosidase from Enterobacter cloacae B5 was employed to synthesize novel galactose containing chemicals (GCCs) using mannitol, sorbose, and salicin as acceptors in the presence of o-nitrophenyl-beta-d-galactopyranoside (oNPGal) as donor. The influences of the process parameters on GCC synthesis using mannitol as an acceptor, including effects of variations in initial substrate concentration, reaction time, and temperature, were studied in detail. The mannitol derivative reached a yield of 14.6% when the enzyme was used in the presence of 30 mM oNPGal and 60mM mannitol at 50 degrees C for 10 min. The sorbose and salicin derivatives reached yields of 19.4% and 25.2%, respectively, under the same conditions except for acceptor concentrations. Through analysis of ESI-MS and NMR spectroscopy, the three derivatives were identified to be beta-D-galactopyranosyl-(1-->1')-D-mannitol, beta-D-galactopyranosyl-(1-->1')-l-sorbose, and 2-(hydroxymethyl) phenyl beta-D-galactopyranosyl-(1-->6')-beta-D-glucopyranoside. (c) 2010 Elsevier Ltd. All rights reserved.

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

PubMed

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.

2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor.

PubMed

Bartesaghi, Alberto; Merk, Alan; Banerjee, Soojay; Matthies, Doreen; Wu, Xiongwu; Milne, Jacqueline L S; Subramaniam, Sriram

2015-06-05

Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli β-galactosidase and the cell-permeant inhibitor phenylethyl β-D-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of ~2.2 angstroms (Å). Besides the PETG ligand, we identified densities in the map for ~800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 Å using single-particle cryo-EM. Copyright © 2015, American Association for the Advancement of Science.

Development of a sensitive chemiluminometric assay for the detection of beta-galactosidase in permeabilized coliform bacteria and comparison with fluorometry and colorimetry.

PubMed Central

Van Poucke, S O; Nelis, H J

1995-01-01

We developed a chemiluminometric assay of beta-galactosidase in coliform bacteria, using a phenylgalactose-substituted 1,2-dioxetane derivative as a substrate. Permeabilization of cells is required to ensure the efficient cellular uptake of this compound. By this method, one coliform seeded in 100 ml of sterile water can be detected after a 6- to 9-h propagation phase followed by a 45-min enzyme assay in the presence of polymyxin B. Compared with fluorometry and colorimetry, chemiluminometry afforded 4- and 1,000-fold increases in sensitivity and 1- and 6-h increases in the speed of detection, respectively. PMID:8534120

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.

PubMed

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

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

PubMed Central

2012-01-01

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 of lactose hydrolysis, the

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

PubMed

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.

Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening

PubMed Central

Paniagua, Candelas; Blanco-Portales, Rosario; Barceló-Muñoz, Marta; García-Gago, Juan A.; Waldron, Keith W.; Quesada, Miguel A.; Muñoz-Blanco, Juan; Mercado, José A.

2016-01-01

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness. PMID:26585222

Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia1

PubMed Central

Pennycooke, Joyce C.; Jones, Michelle L.; Stushnoff, Cecil

2003-01-01

α-Galactosidase (α-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the α-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia (Petunia × hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that α-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of α-Gal mRNAs. α-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating α-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the α-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with α-Gal provides an additional method for improving the freezing tolerance of plants. PMID:14500789

Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening.

PubMed

Paniagua, Candelas; Blanco-Portales, Rosario; Barceló-Muñoz, Marta; García-Gago, Juan A; Waldron, Keith W; Quesada, Miguel A; Muñoz-Blanco, Juan; Mercado, José A

2016-02-01

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Pathological Renal Findings of Chronic Renal Failure in a Patient with the E66Q Mutation in the α-galactosidase A Gene.

PubMed

Satomura, Atsushi; Fujita, Takayuki; Nakayama, Tomohiro; Kusano, Hiroyuki; Takayama, Eiichi; Hamada, Hiroaki; Maruyama, Toshiharu

2015-01-01

A 66-year-old Japanese man was diagnosed with interstitial nephritis on a renal biopsy at 45 years of age and began to receive hemodialysis at 65 years of age. He was suspected of having Fabry disease as a result of a screening study for Fabry disease performed in hemodialysis patients. He had an E66Q mutation in the α-galactosidase A gene. We conducted an electron microscopic examination of a renal biopsy specimen obtained when the patient was diagnosed with chronic renal failure at 45 years of age in order to elucidate the pathogenicity of the E66Q mutation. Interestingly, an electron microscopic examination of the renal biopsy specimen indicated no characteristic findings of Fabry disease.

Crystal structure of β1→6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α-galactose.

PubMed

Godoy, Andre Schutzer; Camilo, Cesar Moises; Kadowaki, Marco Antonio; Muniz, Heloisa Dos S; Espirito Santo, Melissa; Murakami, Mario Tyago; Nascimento, Alessandro S; Polikarpov, Igor

2016-11-01

In a search for better comprehension of β-galactosidase function and specificity, we solved the crystal structures of the GH42 β-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with d-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with d-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme k cat without significant modification in its K m , whereas mutations Tyr289Phe and His371Phe simultaneously decreased k cat and increased K m values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with β1→6 and β1→3 bonds, while binding of the substrates with β1→4 linkages is less favorable. Structural data are available in the PDB under the accession numbers 4UZS and 4UCF. © 2016 Federation of European Biochemical Societies.

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

NASA Astrophysics Data System (ADS)

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.

Production of chlorphenesin galactoside by whole cells of β-galactosidase-containing Escherichia coli.

PubMed

Lee, Sang-Eun; Lee, Hyang-Yeol; Jung, Kyung-Hwan

2013-06-28

We investigated the transgalactosylation reaction of chlorphenesin (CPN) using β-galactosidase (β-gal)-containing Escherichia coli (E. coli) cells, in which galactose from lactose was transferred to CPN. The optimal CPN concentration for CPN galactoside (CPN-G) synthesis was observed at 40 mM under the conditions that lactose and β-gal (as E. coli cells) were 400 g/l and 4.8 U/ml, respectively, and the pH and temperature were 7.0 and 40oC, respectively. The time-course profile of CPN-G synthesis under these optimal conditions showed that CPN-G synthesis from 40 mM CPN reached a maximum of about 27 mM at 12 h. This value corresponded to an about 67% conversion of CPN to CPN-G, which was 4.47-5.36-fold higher than values in previous reports. In addition, we demonstrated by thin-layer chromatography to detect the sugar moiety that galactose was mainly transferred from lactose to CPN. Liquid chromatography-mass spectrometry revealed that CPN-G and CPN-GG (CPN galactoside, which accepted two galactose molecules) were definitively identified as the synthesized products using β-gal-containing E. coli cells. In particular, because we did not use purified β-gal, our β-gal-containing E. coli cells might be practical and cost-effective for enzymatically synthesizing CPN-G. It is expected that the use of β-gal-containing E. coli will be extended to galactose derivatization of other drugs to improve their functionality.

Identification of novel mutations in the α-galactosidase A gene in patients with Fabry disease: pitfalls of mutation analyses in patients with low α-galactosidase A activity.

PubMed

Yoshimitsu, Makoto; Higuchi, Koji; Miyata, Masaaki; Devine, Sean; Mattman, Andre; Sirrs, Sandra; Medin, Jeffrey A; Tei, Chuwa; Takenaka, Toshihiro

2011-05-01

Fabry disease is an X-linked lysosomal storage disorder caused by mutations of the α-galactosidase A (GLA) gene, and the disease is a relatively prevalent cause of left ventricular hypertrophy followed by conduction abnormalities and arrhythmias. Mutation analysis of the GLA gene is a valuable tool for accurate diagnosis of affected families. In this study, we carried out molecular studies of 10 unrelated families diagnosed with Fabry disease. Genetic analysis of the GLA gene using conventional genomic sequencing was performed in 9 hemizygous males and 6 heterozygous females. In patients with no mutations in coding DNA sequence, multiplex ligation-dependent probe amplification (MLPA) and/or cDNA sequencing were performed. We identified a novel exon 2 deletion (IVS1_IVS2) in a heterozygous female by MLPA, which was undetectable by conventional sequencing methods. In addition, the g.9331G>A mutation that has previously been found only in patients with cardiac Fabry disease was found in 3 unrelated, newly-diagnosed, cardiac Fabry patients by sequencing GLA genomic DNA and cDNA. Two other novel mutations, g.8319A>G and 832delA were also found in addition to 4 previously reported mutations (R112C, C142Y, M296I, and G373D) in 6 other families. We could identify GLA gene mutations in all hemizygotes and heterozygotes from 10 families with Fabry disease. Mutations in 4 out of 10 families could not be identified by classical genomic analysis, which focuses on exons and the flanking region. Instead, these data suggest that MLPA analysis and cDNA sequence should be considered in genetic testing surveys of patients with Fabry disease. Copyright © 2011 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

Effect of immobilization conditions on the properties of β-galactosidase immobilized in xanthan/chitosan multilayers

NASA Astrophysics Data System (ADS)

Yovcheva, T.; Vasileva, T.; Viraneva, A.; Cholev, D.; Bodurov, I.; Marudova, M.; Bivolarski, V.; Iliev, I.

2017-01-01

The effect of lactose concentration on the activity of the immobilised enzyme β-galactosidase from Aspergillus niger has been evaluated, considering future applications for the production of galactooligosaccahrides with prebiotic potential. The following enzyme was immobilized in xanthan and chitosan polyelectrolyte multilayers (PEMs) deposited by dip coating method on polylactic acid positively corona charged pads. The pads were charged in a corona discharge system, consisting of a corona electrode (needle), a grounded plate, and a metal grid placed between them. Positive 5 kV voltage was applied to the corona electrode. 1 kV voltage of the same polarity as that of the corona electrode was applied to the grid. The chitosan layers were crosslinked with sodium tripolyphosphate (Na-TPP). The enzyme showed a temperature optimum at 50 °C and a pH optimum at 5.0. The immobilization was carried out over the different adsorption time and optimum conditions were determined. These results give insights for further optimization of transgalactosydase reactions in order to produce galactooligosaccharides with specific structure and having pronounced better prebiotic properties. For the determination of the surface morphology of the investigated samples an atomic force microscope was used and root mean square roughness was obtained.

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

PubMed Central

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

Thermal, Chemical and pH Induced Denaturation of a Multimeric β-Galactosidase Reveals Multiple Unfolding Pathways

PubMed Central

Kishore, Devesh; Kundu, Suman; Kayastha, Arvind M.

2012-01-01

Background In this case study, we analysed the properties of unfolded states and pathways leading to complete denaturation of a multimeric chick pea β-galactosidase (CpGAL), as obtained from treatment with guanidium hydrochloride, urea, elevated temperature and extreme pH. Methodology/Principal Findings CpGAL, a heterodimeric protein with native molecular mass of 85 kDa, belongs to α+β class of protein. The conformational stability and thermodynamic parameters of CpGAL unfolding in different states were estimated and interpreted using circular dichroism and fluorescence spectroscopic measurements. The enzyme was found to be structurally and functionally stable in the entire pH range and upto 50°C temperature. Further increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were irreversible, non-coincidental and sigmoidal. Free energy of protein unfolding (ΔG0) and unfolding constant (Kobs) were also calculated for chemically denatured CpGAL. Significance The protein seems to use different pathways for unfolding in different environments and is a classical example of how the environment dictates the path a protein might take to fold while its amino acid sequence only defines its final three-dimensional conformation. The knowledge accumulated could be of immense biotechnological significance as well. PMID:23185611

Preparation and characterization of gellan gum microspheres containing a cold-adapted β-galactosidase from Rahnella sp. R3.

PubMed

Fan, Yuting; Yi, Jiang; Hua, Xiao; Zhang, Yuzhu; Yang, Ruijin

2017-04-15

R-β-Gal is a cold-adapted β-galactosidase that is able to hydrolyze lactose and has the potential to produce low-lactose or lactose-free dairy products at low temperatures (4°C). Cold-adapted enzymes unfold at moderate temperatures due to the lower intramolecular stabilizing interactions necessary for flexibility at low temperatures. To increase stability and usage-performance, R-β-Gal was encapsulated in gellan gum by injecting an aqueous solution into two different hardening solutions (10mM CaCl 2 or 10mM MgCl 2 ). Enzyme characteristics of both free and encapsulated R-β-Gal were carried out, and the different effects of two cations were investigated. R-β-Gal showed better thermal and pH stability after encapsulation. Ca 2+ gels had higher encapsulation efficiency (71.4%) than Mg 2+ (66.7%) gels, and Ca 2+ formed larger inner and surface pores. R-β-Gal was released from the Ca 2+ hydrogel beads more rapidly than the Mg 2+ hydrogels during storage in aqueous solution due to the larger inner/surface pores of the matrix. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Magini, Alessandro; Department of Medical and Biological Sciences; Polchi, Alice

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,more » 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.« less

A rapid and sensitive fluorimetric β-galactosidase assay for coliform detection using chlorophenol red-β-D-galactopyranoside.

PubMed

Sicard, Clémence; Shek, Norman; White, Dawn; Bowers, Raymond J; Brown, R Stephen; Brennan, John D

2014-09-01

We report on a new fluorimetric assay for β-galactosidase (β-gal) and faecal coliform bacteria that utilizes a long-wavelength dye, chlorophenol red-β-D-galactopyranoside (CPRG), that has been widely used for colorimetric assays. The novel feature of this new assay is the unexpected development of a large fluorescence response from liberated chorophenol red (CPR) upon complexation with poly-L-arginine (pR) in solution. The binding of CPR to pR occurs through the sulphonate group of CPR, causing formation of a charge-transfer complex and up to a 70-fold increase in emission intensity. A major advantage of the assay is the ability to utilize excitation and emission wavelengths in the red end of the spectrum, which avoids common interferences obtained when using UV-absorbing dyes such as 4-methylumbelliferyl-β-D-galactopyranoside. We provide data on the utility of CPRG as a fluorimetric reporter for both β-gal and Escherichia coli ATCC 25922 and demonstrate optimized reaction conditions for rapid and sensitive detection of E. coli at a level of 1 colony-forming unit (cfu)/10 mL after 12 h of culture followed by a 1-h assay, which is below the regulatory limit for testing of recreational water.

Early Cardiac Involvement Affects Left Ventricular Longitudinal Function in Females Carrying α-Galactosidase A Mutation: Role of Hybrid Positron Emission Tomography and Magnetic Resonance Imaging and Speckle-Tracking Echocardiography.

PubMed

Spinelli, Letizia; Imbriaco, Massimo; Nappi, Carmela; Nicolai, Emanuele; Giugliano, Giuseppe; Ponsiglione, Andrea; Diomiaiuti, Tommaso Claudio; Riccio, Eleonora; Duro, Giovanni; Pisani, Antonio; Trimarco, Bruno; Cuocolo, Alberto

2018-04-01

Hybrid 18 F-fluorodeoxyglucose (FDG) positron emission tomography and magnetic resonance imaging may differentiate mature fibrosis or scar from fibrosis associated to active inflammation in patients with Anderson-Fabry disease, even in nonhypertrophic stage. This study was designed to compare the results of positron emission tomography and magnetic resonance cardiac imaging with those of speckle-tracking echocardiography in heterozygous Anderson-Fabry disease females. Twenty-four heterozygous females carrying α-galactosidase A mutation and without left ventricular hypertrophy underwent cardiac positron emission tomography and magnetic resonance using 18 F-FDG for glucose uptake and 2-dimensional strain echocardiography. 18 F-FDG myocardial uptake was quantified by measuring the coefficient of variation (COV) of the standardized uptake value using a 17-segment model. Focal 18 F-FDG uptake with COV >0.17 was detected in 13 patients, including 2 patients with late gadolinium enhancement at magnetic resonance. COV was 0.30±0.14 in patients with focal 18 F-FDG uptake and 0.12±0.03 in those without ( P <0.001). Strain echocardiography revealed worse global longitudinal systolic strain in patients with COV >0.17 compared with those with COV ≤0.17 (-18.5±2.7% versus -22.2±1.8%; P =0.024). For predicting COV >0.17, a global longitudinal strain >-19.8% had 77% sensitivity and 91% specificity and a value >2 dysfunctional segments 92% sensitivity and 100% specificity. In females carrying α-galactosidase A mutation, focal 18 F-FDG uptake represents an early sign of disease-related myocardial damage and is associated with impaired left ventricular longitudinal function. These findings support the hypothesis that inflammation plays an important role in glycosphingolipids storage disorders. © 2018 American Heart Association, Inc.

Anti-α-galactosidase A antibody response to agalsidase beta treatment: data from the Fabry Registry.

PubMed

Wilcox, William R; Linthorst, Gabor E; Germain, Dominique P; Feldt-Rasmussen, Ulla; Waldek, Stephen; Richards, Susan M; Beitner-Johnson, Dana; Cizmarik, Marta; Cole, J Alexander; Kingma, Wytske; Warnock, David G

2012-03-01

Agalsidase beta, a form of recombinant human α-galactosidase A (αGAL), is approved for use as enzyme replacement therapy (ERT) for Fabry disease. An immunogenic response against a therapeutic protein could potentially impact its efficacy or safety. The development of anti-αGAL IgG antibodies was evaluated in 571 men and 251 women from the Fabry Registry who were treated with agalsidase beta. Most men developed antibodies (416 of 571, 73%), whereas most women did not (31 of 251, 12%). Women were also significantly more likely to tolerize than men; whereas 18 of 31 women tolerized (58%, 95%CI: 52%-64%), only 47 of 416 men tolerized during the observation period (11%, 95% CI: 8%-15%). Patients who eventually tolerized had lower median peak anti-αGAL IgG antibody titers than patients who remained seropositive at their most recent assessment (400 versus 3200 in men, 200 versus 400 in women, respectively). Patients with nonsense mutations in the GLA gene were more likely to develop anti-αGAL IgG antibodies than patients with missense mutations. Approximately 26% of men (151 of 571) reported infusion-associated reactions (IARs), compared to 11% of women (27 of 251). Men who developed anti-αGAL IgG antibodies were more likely to experience IARs compared to those who remained seronegative. Nine percent of seronegative men and women (34 of 375) reported IARs. The majority of IARs occurred during the first 6 to 12 months of agalsidase beta treatment and decreased over time, in both seroconverted and seronegative patients. Copyright © 2012 Elsevier Inc. All rights reserved.

Production, recovery and purification of a recombinant β-galactosidase by expanded bed anion exchange adsorption.

PubMed

Boeris, Valeria; Balce, Izabella; Vennapusa, Rami Reddy; Arévalo Rodríguez, Miguel; Picó, Guillermo; Lahore, Marcelo Fernández

2012-07-01

β-Galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides; its major application in the food industry is to reduce the content of lactose in lactic products. The aim of this work is to recover this enzyme from a cell lysate by adsorption onto Streamline-DEAE in an expanded bed, avoiding, as much as possible, biomass deposition onto the adsorbent matrix. So as to achieve less cell debris-matrix interaction, the adsorbent surface was covered with polyvinyl pyrrolidone. The enzyme showed to bind in the same extent to naked and covered Streamline-DEAE (65 mg β-gal/g matrix) in batch mode in the absence of any biomass. The kinetics of the adsorption process was studied and no effect of the polyvinyl pyrrolidone covering was found. The optimal conditions for the recovery were achieved by using a lysate made of 40% wet weight of cells, a polyvinyl pyrrolidone-covered matrix/lysate ratio of 10% and carrying out the adsorption process in expanded bed with recirculation over 2h in 20 mM phosphate buffer pH 7.4. The fraction recovered after the elution contained 65% of the initial amount of enzyme with a 12.6-fold increased specific activity with respect to the lysate. The polyvinyl pyrrolidone content in the eluate was determined and found negligible. The remarkable point of this work is that it was possible to partially purify the enzyme using a feedstock containing an unusually high biomass concentration in the presence of polyvinyl pyrrolidone onto weak anion exchangers. Copyright © 2012 Elsevier B.V. All rights reserved.

Immobilization of β-galactosidase from Kluyveromyces lactis onto polymeric membrane surfaces: effect of surface characteristics.

PubMed

Güleç, Hacı Ali

2013-04-01

The aim of this study was to investigate the effects of surface characteristics of plain and plasma modified cellulose acetate (CA) membranes on the immobilization yield of β-galactosidases from Kluyveromyces lactis (KLG) and its galacto-oligosaccharide (GOS) yield, respectively. Low pressure plasma treatments involving oxygen plasma activation, plasma polymerization (PlsP) of ethylenediamine (EDA) and PlsP of 2-mercaptoethanol were used to modify plain CA membrane surfaces. KLG enzyme was immobilized onto plain and oxygen plasma treated membrane surfaces by simple adsorption. Oxygen plasma activation increased the hydrophylicity of CA membrane surfaces and it improved the immobilization yield of the enzyme by 42%. KLG enzyme was also immobilized onto CA membrane surfaces through amino groups created by PlsP of EDA via covalent binding. Plasma action at 60W plasma power and 15 min. exposure time improved the amount of membrane bounded enzyme by 3.5-fold. The enrichment of the amount of amino groups via polyethyleneimine (PEI) addition enhanced this increase from 3.5-fold to 4.5-fold. Although high enzyme loading was achived (65-83%), both of the methods dramatically decreased the enzyme activity (11-12%) and GOS yield due to probably negative effects of active amino groups. KLG enzyme was more effectively immobilized onto thiolated CA membrane surface created by PlsP of 2-mercaptoethanol with high immobilization yield (70%) and especially high enzyme activity (46%). Immobilized enzymes on the CA membranes treated by PlsP were successively reutilized for 5-8 cycles at 25°C and enzymatic derivatives retained approximately 75-80% of their initial activites at the end of the reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

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

PubMed

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.

Enzyme replacement therapy for Fabry disease: lessons from two alpha-galactosidase A orphan products and one FDA approval.

PubMed

Desnick, Robert J

2004-07-01

Two virtually identical products have been developed for enzyme replacement therapy for the treatment of Fabry disease, which is a rare and debilitating genetic disease caused by decreased activity of the lysosomal enzyme alpha-galactosidase A. Lack of this enzyme results in progressive tissue accumulation of globotriaosylceramide (GL-3), resulting in life-threatening renal, cardiac and cerebrovascular complications. Both enzyme replacement products, agalsidase alfa (Replagal; Transkaryotic Therapies, Cambridge, MA, USA) and agalsidase beta (Fabrazyme; Genzyme Corporation, Cambridge, MA, USA), were approved by the European Agency for the Evaluation of Medicinal Products in 2001; agalsidase alfa at a recommended dose of 0.2 mg/kg and agalsidase beta at a recommended dose of 1 mg/kg. In the US, however, orphan drug laws dictated that only one of these products could be approved. In April 2003, after a rigorous evaluation of both products by the US FDA, this approval was granted to agalsidase beta. This decision reflected clinical trial design, how dosages were determined, antibody effects and the ability of each product to demonstrate either clinical efficacy or reduction of tissue storage of GL-3 in major organs of pathology when administered at the recommended dosage. The process also highlighted important issues in the evaluation of drugs to treat life-threatening genetic diseases for which the pathological basis is well-defined.

Production of galacto-oligosaccharides from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth.

PubMed

Albayrak, Nedim; Yang, Shang-Tian

2002-01-05

The production of galacto-oligosaccharides (GOS) from lactose by A. oryzae beta-galactosidase immobilized on cotton cloth was studied. The total amounts and types of GOS produced were mainly affected by the initial lactose concentration in the reaction media. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOS produced in the reactions. Temperature and pH affected the reaction rate, but did not result in any changes in GOS formation. The presence of galactose and glucose at the concentrations encountered near maximum GOS greatly inhibited the reactions and reduced GOS yield by as much as 15%. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme, suggesting no diffusion limitation in the enzyme carrier. The thermal stability of the enzyme increased approximately 25-fold upon immobilization on cotton cloth. The half-life for the immobilized enzyme on cotton cloth was more than 1 year at 40 degrees C and 48 days at 50 degrees C. Stable, continuous operation in a plugflow reactor was demonstrated for 2 weeks without any apparent problem. A maximum GOS production of 21 and 26% (w/w) of total sugars was attained with a feed solution containing 200 and 400 g/L of lactose, respectively, at pH 4.5 and 40 degrees C. The corresponding reactor productivities were 80 and 106 g/L/h, respectively, which are at least several-fold higher than those previously reported. Copyright 2002 John Wiley & Sons, Inc.

β-galactosidase Production by Aspergillus niger ATCC 9142 Using Inexpensive Substrates in Solid-State Fermentation: Optimization by Orthogonal Arrays Design.

PubMed

Kazemi, Samaneh; Khayati, Gholam; Faezi-Ghasemi, Mohammad

2016-01-01

Enzymatic hydrolysis of lactose is one of the most important biotechnological processes in the food industry, which is accomplished by enzyme β-galactosidase (β-gal, β-D-galactoside galactohydrolase, EC 3.2.1.23), trivial called lactase. Orthogonal arrays design is an appropriate option for the optimization of biotechnological processes for the production of microbial enzymes. Design of experimental (DOE) methodology using Taguchi orthogonal array (OA) was employed to screen the most significant levels of parameters, including the solid substrates (wheat straw, rice straw, and peanut pod), the carbon/nitrogen (C/N) ratios, the incubation time, and the inducer. The level of β-gal production was measured by a photometric enzyme activity assay using the artificial substrate ortho-Nitrophenyl-β-D-galactopyranoside. The results showed that C/N ratio (0.2% [w/v], incubation time (144 hour), and solid substrate (wheat straw) were the best conditions determined by the design of experiments using the Taguchi approach. Our finding showed that the use of rice straw and peanut pod, as solid-state substrates, led to 2.041-folds increase in the production of the enzyme, as compared to rice straw. In addition, the presence of an inducer did not have any significant impact on the enzyme production levels.

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications

PubMed Central

Rico-Díaz, Agustín; Álvarez-Cao, María-Efigenia; Escuder-Rodríguez, Juan-José; González-Siso, María-Isabel; Cerdán, M. Esperanza; Becerra, Manuel

2017-01-01

Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-β-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-β-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-β-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-β-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-β-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis. PMID:28361909

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications.

PubMed

Rico-Díaz, Agustín; Álvarez-Cao, María-Efigenia; Escuder-Rodríguez, Juan-José; González-Siso, María-Isabel; Cerdán, M Esperanza; Becerra, Manuel

2017-03-31

Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-β-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-β-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-β-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-β-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-β-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis.

Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation.

PubMed

Saad, Rawia R; Fawzi, Eman M

2012-03-01

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A

PubMed Central

Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

2015-01-01

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

A new electrochemical substrate for rapid and sensitive in vivo monitoring of β-galactosidase gene expressions.

PubMed

Manibalan, Kesavan; Mani, Veerappan; Huang, Chih-Hung; Huang, Sheng-Tung; Chang, Pu-Chieh

2015-09-07

A 4-Methoxyphenyl-β-galactopyranoside (4-MPGal) substrate incorporating 4-methoxy phenol (4-MP) as an electrochemical reporter is described for the monitoring of β-Galactosidase (β-Gal) gene expressions. β-Gal derived from Escherichia coli (E. coli) and Aspergillus oryzae (A. oryzae) were investigated, while a graphene oxide film modified electrode was employed as the transducer. The electrochemical signal of 4-MPG within 4-MPGal was masked by protecting their hydroxyl group with galactose. The externally added β-Gal triggered the deprotection through specific enzymatic hydrolysis with concomitant release of 4-MP. The apparent Km and Vmax values of 4-MPGal are determined to be 0.21 mM and 0.51 μM min(-1) mg of β-Gal(-1) (E. coli), which is consistent with the previous reports. To detect β-Gal derived from E. coli, cyclic voltammetry (CV) provides linear ranges of 12-1200 ng mL(-1) and 1.2-12 μg mL(-1) with a limit of detection (LOD) of 5 ng mL(-1), while differential pulse voltammetry (DPV) shows a linear range of 1.2-120 ng mL(-1) and LOD of 1 ng mL(-1). To detect β-Gal derived from A. oryzae, CV provides linear ranges of 0.1-100 ng mL(-1) and 0.1-1 μg mL(-1) with a LOD of 0.06 ng mL(-1), while DPV shows a linear range of 10 pg mL(-1)-10 ng mL(-1) with a LOD of 8 pg mL(-1). Moreover, we set up a platform for the real-time in vivo monitoring of β-Gal gene expressions in E. coli cultivated through microbiological culture. The developed sensing platform using 4-MPGal as a substrate is simple, rapid, sensitive, specific and advantageous over its laborious optical analogues.

Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and beta-galactosidase fusions.

PubMed

Sarsero, J P; Pittard, A J

1995-01-01

The mtr gene of Escherichia coli K-12 encodes an inner membrane protein which is responsible for the active transport of trypotophan into the cell. It has been proposed that the Mtr permease has a novel structure consisting of 11 hydrophobic transmembrane spans, with a cytoplasmically disposed amino terminus and a carboxyl terminus located in the periplasmic space (J.P. Sarsero, P. J. Wookey, P. Gollnick, C. Yanofsky, and A.J. Pittard, J. Bacteriol. 173:3231-3234, 1991). The validity of this model was examined by the construction of fusion proteins between the Mtr permease and alkaline phosphatase or beta-galactosidase. In addition to the conventional methods, in which the reporter enzyme replaces a carboxyl-terminal portion of the membrane protein, the recently developed alkaline phosphatase sandwich fusion technique was utilized, in which alkaline phosphatase is inserted into an otherwise intact membrane protein. A cluster of alkaline phosphatase fusions to the carboxyl-terminal end of the Mtr permease exhibited high levels of alkaline phosphatase activity, giving support to the proposition of a periplasmically located carboxyl terminus. The majority of fusion proteins produced enzymatic activities which were in agreement with the positions of the fusion sites on the proposed topological model of the permease. The synthesis of a small cluster of hybrid proteins, whose enzymatic activity did not agree with the location of their fusion sites within putative transmembrane span VIII or the preceding periplasmic loop, was not detected by immunological techniques and did not necessitate modification of the proposed model in this region. Slight alterations may need to be made in the positioning of the carboxyl-terminal end of transmembrane span X.

Propensity approach to nonequilibrium thermodynamics of a chemical reaction network: Controlling single E-coli β-galactosidase enzyme catalysis through the elementary reaction stepsa)

NASA Astrophysics Data System (ADS)

Das, Biswajit; Banerjee, Kinshuk; Gangopadhyay, Gautam

2013-12-01

In this work, we develop an approach to nonequilibrium thermodynamics of an open chemical reaction network in terms of the elementary reaction propensities. The method is akin to the microscopic formulation of the dissipation function in terms of the Kullback-Leibler distance of phase space trajectories in Hamiltonian system. The formalism is applied to a single oligomeric enzyme kinetics at chemiostatic condition that leads the reaction system to a nonequilibrium steady state, characterized by a positive total entropy production rate. Analytical expressions are derived, relating the individual reaction contributions towards the total entropy production rate with experimentally measurable reaction velocity. Taking a real case of Escherichia coli β-galactosidase enzyme obeying Michaelis-Menten kinetics, we thoroughly analyze the temporal as well as the steady state behavior of various thermodynamic quantities for each elementary reaction. This gives a useful insight in the relative magnitudes of various energy terms and the dissipated heat to sustain a steady state of the reaction system operating far-from-equilibrium. It is also observed that, the reaction is entropy-driven at low substrate concentration and becomes energy-driven as the substrate concentration rises.

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

PubMed Central

Karl, Franziska; Sommer, Claudia; Üçeyler, Nurcan

2017-01-01

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. PMID:28662189

Structure-activity relationships on the study of β-galactosidase folding/unfolding due to interactions with immobilization additives: Triton X-100 and ethanol.

PubMed

Soto, Dayana; Escobar, Sindy; Guzmán, Fanny; Cárdenas, Constanza; Bernal, Claudia; Mesa, Monica

2017-03-01

Improving the enzyme stability is a challenge for allowing their practical application. The surfactants are stabilizing agents, however, there are still questions about their influence on enzyme properties. The structure-activity/stability relationship for β-galactosidase from Bacillus circulans is studied here by Circular Dichroism and activity measurements, as a function of temperature and pH. The tendency of preserving the β-sheet and α-helix structures at temperatures below 65°C and different pH is the result of the balance between the large- and short-range effects, respecting to the active site. This information is fundamental for explaining the structural changes of this enzyme in the presence of Triton X-100 surfactant and ethanol. The enzyme thermal stabilization in the presence of this surfactant responds to the rearrangement of the secondary structure for having optimal activity/stability. The effect of ethanol is more related with changes in the dielectric properties of the aqueous solution than with protein structural transformations. These results contribute to understand the effects of surfactant-enzyme interactions on the enzyme behavior, from the structural point of view and to rationalize the surfactant-based stabilizing strategies for β-galactosidades. Copyright © 2016 Elsevier B.V. All rights reserved.

Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification, characterization, and insights towards thermal denaturation.

PubMed

M, Ramadevi; Panwar, Deepesh; Joseph, Juby Elsa; Kaira, Gaurav Singh; Kapoor, Mukesh

2018-06-20

A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal) from germinating black gram (Vigna mungo) seeds was purified to homogeneity (432 U/mg specific activity, 1542-fold purification) using ion-exchange (DEAE-cellulose, CM-sepharose) and affinity (Con-A Sepharose 4B) chromatography. VM-αGal appeared as a single band ~ 45 kDa on SDS-PAGE and showed optimal activity at pH 5 and 55 °C. Hg 2+ and SDS completely inhibited VM-αGal activity. The K m, V max and catalytic efficiency (k cat /K m ) of VM-αGal for pNPG and raffinose was found to be 0.99, 17.23 mM, 0.413 s -1  mM -1 and 1.66, 0.146 mM ml -1  min -1 , 0.0026 s -1  mM -1 , respectively. VM-αGal was competitively inhibited by galactose (K i 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-51 °C showed that the enzyme was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in β-sheet content at 45-65 °C, and above 55 °C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing the flatulence-causing raffinose family oligosaccharides (26.5% and 18.45% decrease in stachyose and raffinose, respectively). Copyright © 2018. Published by Elsevier B.V.

A Pharmacogenetic Approach to Identify Mutant Forms of α-Galactosidase A that Respond to a Pharmacological Chaperone for Fabry Disease

PubMed Central

Wu, Xiaoyang; Katz, Evan; Valle, Maria Cecilia Della; Mascioli, Kirsten; Flanagan, John J; Castelli, Jeffrey P; Schiffmann, Raphael; Boudes, Pol; Lockhart, David J; Valenzano, Kenneth J; Benjamin, Elfrida R

2011-01-01

Fabry disease is caused by mutations in the gene (GLA) that encodes α-galactosidase A (α-Gal A). The iminosugar AT1001 (GR181413A, migalastat hydrochloride, 1-deoxygalactonojirimycin) is a pharmacological chaperone that selectively binds and stabilizes α-Gal A, increasing total cellular levels and activity for some mutant forms (defined as “responsive”). In this study, we developed a cell-based assay in cultured HEK-293 cells to identify mutant forms of α-Gal A that are responsive to AT1001. Concentration-dependent increases in α-Gal A activity in response to AT1001 were shown for 49 (60%) of 81 mutant forms. The responses of α-Gal A mutant forms were generally consistent with the responses observed in male Fabry patient-derived lymphoblasts. Importantly, the HEK-293 cell responses of 19 α-Gal A mutant forms to a clinically achievable concentration of AT1001 (10 µM) were generally consistent with observed increases in α-Gal A activity in peripheral blood mononuclear cells from male Fabry patients orally administered AT1001 during Phase 2 clinical studies. This indicates that the cell-based responses can identify mutant forms of α-Gal A that are likely to respond to AT1001 in vivo. Thus, the HEK-293 cell-based assay may be a useful aid in the identification of Fabry patients with AT1001-responsive mutant forms. Hum Mutat 32:1–13, 2011. © 2011 Wiley-Liss, Inc. PMID:21598360

Modification of Immobead 150 support for protein immobilization: effects on the properties of immobilized Aspergillus oryzae β-galactosidase.

PubMed

Gennari, Adriano; Herrmann Mobayed, Francielle; da Silva Rafael, Ruan; Rodrigues, Rafael C; Sperotto, Raul Antonio; Volpato, Giandra; Volken de Souza, Claucia Fernanda

2018-05-01

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead-Glu) or carboxyl groups through acid solution (Immobead-Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β-galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead-Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10 to 500 mg.g -1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg.g -1 support. Gal immobilized on Immobead-Glu and Immobead-Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half-lifes than the soluble enzyme, where the half-lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Acidic α-galactosidase is the most abundant nectarin in floral nectar of common tobacco (Nicotiana tabacum)

PubMed Central

Zha, Hong-Guang; Flowers, V. Lynn; Yang, Min; Chen, Ling-Yang; Sun, Hang

2012-01-01

Background and Aims To date, most floral nectarins (nectar proteins) are reported to function in nectar defence, particularly for insect-pollinated outcrossing species. We compared nectarin composition and abundance in selfing common tobacco (Nicotiana tobaccum) with outcrossing ornamental tobacco plants to elucidate the functional difference of nectarins in different reproductive systems. Methods Common tobacco (CT) nectarins were separated by SDS-PAGE and the N terminus of the most abundant nectarin was sequenced via Edman degradation. The full-length nectarin gene was amplified and cloned from genomic DNA and mRNA with hiTail-PCR and RACE (rapid amplification of cDNA ends), and expression patterns were then investigated in different tissues using semi-quantitative reverse transcriptase PCR. Additionally, high-performance liquid chromatography and enzymatic analyses of nectar sugar composition, and other biochemical traits and functions of the novel nectarin were studied. Key Results The most abundant nectarin in CT nectar is an acidic α-galactosidase, here designated NTα-Gal. This compound has a molecular mass of 40 013 Da and a theoretical pI of 5·33. NTα-Gal has a conserved α-Gal characteristic signature, encodes a mature protein of 364 amino acids and is expressed in different organs. Compared with 27 other melliferous plant species from different families, CT floral nectar demonstrated the highest α-Gal activity, which is inhibited by d-galactose. Raffinose family oligosaccharides were not detected in CT nectar, indicating that NTα-Gal does not function in post-secretory hydrolysis. Moreover, tobacco plant fruits did not develop intact skin with galactose inhibition of NTα-Gal activity in nectar, suggesting that NTα-Gal induces cell-wall surface restructuring during the initial stages of fruit development. Conclusions α-Gal was the most abundant nectarin in selfing CT plants, but was not detected in the nectar of strictly outcrossing sister tobacco

Water-Tryptophan Interactions: Lone-pair⋅⋅⋅π or O-H⋅⋅⋅π? Molecular Dynamics Simulations of β-Galactosidase Suggest that Both Modes Can Co-exist.

PubMed

Durec, Matúš; Marek, Radek; Kozelka, Jiří

2018-04-17

In proteins, the indole side chain of tryptophan can interact with water molecules either in-plane, forming hydrogen bonds, or out-of-plane, with the water molecule contacting the aromatic π face. The latter interaction can be either of the lone pair⋅⋅⋅π (lp⋅⋅⋅π) type or corresponds to the O-H⋅⋅⋅π binding mode, an ambiguity that X-ray structures usually do not resolve. Here, we report molecular dynamics (MD) simulations of a solvated β-galactosidase monomer, which illustrate how a water molecule located at the π face of an indole side chain of tryptophan can adapt to the position of proximate residues and "select" its binding mode. In one such site, the water molecule is predicted to rapidly oscillate between the O-H⋅⋅⋅π and lp⋅⋅⋅π binding modes, thus gaining entropic advantage. Our MD simulations provide support for the role of lp⋅⋅⋅π interactions in the stabilization of protein structures. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Positive regulation of Leptospira interrogans kdp expression by KdpE as Demonstrated with a novel β-galactosidase reporter in Leptospira biflexa.

PubMed

Matsunaga, James; Coutinho, Mariana L

2012-08-01

Leptospirosis is a potentially deadly zoonotic disease that afflicts humans and animals. Leptospira interrogans, the predominant agent of leptospirosis, encounters diverse conditions as it proceeds through its life cycle, which includes stages inside and outside the host. Unfortunately, the number of genetic tools available for examining the regulation of gene expression in L. interrogans is limited. Consequently, little is known about the genetic circuits that control gene expression in Leptospira. To better understand the regulation of leptospiral gene expression, the L. interrogans kdp locus, encoding homologs of the P-type ATPase KdpABC potassium transporter with their KdpD sensors and KdpE response regulators, was selected for analysis. We showed that a kdpE mutation in L. interrogans prevented the increase in kdpABC mRNA levels observed in the wild-type L. interrogans strain when external potassium levels were low. To confirm that KdpE was a positive regulator of kdpABC transcription, we developed a novel approach for constructing chromosomal genetic fusions to the endogenous bgaL (β-galactosidase) gene of the nonpathogen Leptospira biflexa. We demonstrated positive regulation of a kdpA'-bgaL fusion in L. biflexa by the L. interrogans KdpE response regulator. A control lipL32'-bgaL fusion was not regulated by KdpE. These results demonstrate the utility of genetic fusions to the bgaL gene of L. biflexa for examining leptospiral gene regulation.

Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum.

PubMed

Woods, J P; Heinecke, E L; Goldman, W E

1998-04-01

We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional beta-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption

Progressive renal failure despite long-term biweekly enzyme replacement therapy in a patient with Fabry disease secondary to a new α-galactosidase mutation of Leu311Arg (L311R).

PubMed

Suzuki, Keisuke; Miura, Naoto; Kitagawa, Wataru; Suzuki, Shinkichi; Komatsuda, Atsushi; Nishikawa, Kazuhiro; Watanabe, Daisuke; Imai, Hirokazu

2011-12-01

A 37-year-old Japanese man affected by Fabry disease secondary to a novel mutation of Leu311Arg (L311R) in α-galactosidase demonstrated progressive renal failure despite biweekly enzyme replacement therapy (ERT) for approximately 10 years. Kidney biopsy revealed foamy glomerular epithelial cells, compatible with the typical pathologic features of Fabry disease. The patient entered a phase III study of Replagal (agalsidase alfa) in 2001, allowing him to continue ERT with biweekly dosing for almost 10 years. During 2 years of that period, he was continued on Fabrazyme (agalsidase beta) biweekly dosing. His estimated GFR was calculated to decrease by 9.9 mL/min/1.73 m(2) per year. Patients with Fabry disease have been reported to have a mean decrease in GFR of 12.2 ± 8.1 mL/min/1.73 m(2) per year. This result suggests that biweekly ERT is only mildly effective at preventing loss of kidney function.

The effect of physico-chemical parameters and chemical compounds on the activity of beta-d-galactosidase (B-GAL), a marker enzyme for indicator microorganisms in water.

PubMed

Wutor, V C; Togo, C A; Pletschke, B I

2007-06-01

The presence of coliforms in polluted water was determined enzymatically (in situ) by directly monitoring the activity of beta-d-galactosidase (B-GAL) through the hydrolysis of the yellow chromogenic subtrate, chlorophenol red beta-d-galactopyranoside (CPRG), which produced a red chlorophenol red (CPR) product. The objectives of this study were to monitor the effect of compounds commonly found in the environment and used in water treatment on a B-GAL CPRG assay and to investigate the differences between the environmental B-GAL enzyme and the pure commercial enzyme. Environmental B-GAL was optimally active at pH 7.8. Two temperature optima were observed at 35 and 55 degrees C, respectively. B-GAL activity was strongly inhibited by silver and copper ions. While calcium and ferrous ions at lower concentrations (50-100mgl(-1)) increased the enzyme activity, a reduction was observed at higher concentrations (200mgl(-1)). Sodium hypochlorite, normally used in rural areas to disinfect water gradually decreased B-GAL activity at concentrations between 0 and 5600ppm for both the commercial and environmental enzymes. B-GAL from the environment behaved differently from its commercially available counterpart.

Analysis of β-Galactosidase During Fruit Development and Ripening in Two Different Texture Types of Apple Cultivars

PubMed Central

Yang, Huijuan; Liu, Junling; Dang, Meile; Zhang, Bo; Li, Hongguang; Meng, Rui; Qu, Dong; Yang, Yazhou; Zhao, Zhengyang

2018-01-01

β-galactosidase (β-Gal), one of the cell wall modifying enzymes, plays an important role in fruit ripening and softening. However, its role in apple fruit texture remains unclear. In this study, the role of β-Gal was analyzed in two apple cultivars, ‘Fuji’ and ‘Qinguan,’ which are characterized by different fruit texture types, during fruit development and ripening. The firmness and pectin content of the fruits rapidly decreased and were much lower in ‘Fuji’ than in ‘Qinguan’ from 105 days after full bloom (DAFB). Transmission electron microscopy showed that the pectin-rich middle lamella was substantially degraded from 105 to 180 DAFB in the two apple cultivars. However, the degradation was more severe in ‘Fuji’ than in ‘Qinguan.’ Subcellular localization analysis showed that the Mdβ-Gal1, Mdβ-Gal2, and Mdβ-Gal5 proteins were located in the cell wall. β-Gal activity continuously increased during all fruit developmental stages and was much higher in the mature fruits of ‘Fuji’ than in those of ‘Qinguan,’ indicating that pectin was degraded by β-Gal. Consistent with the enzyme activities, expression levels of β-Gal genes (Mdβ-Gal1, Mdβ-Gal2, and Mdβ-Gal5) showed only slight changes from 60 to 105 DAFB but then dramatically increased until fruit ripening, with higher values in ‘Fuji’ than in ‘Qinguan.’ Furthermore, we found that activities of deletion derivatives in the Mdβ-Gal2 promoter and transcript level of Mdβ-Gal2 were induced by the treatment with methyl jasmonate (MeJA) and ethylene (ETH) hormones. Two ETH and one MeJA hormone-responsive elements were identified by analyzing the promoter sequence. These results suggest that β-Gals, induced by ETH and MeJA, are involved in different fruit texture types of apple cultivars by influencing the degradation of pectin during the mature fruit stage. PMID:29740469

Reduction of soybean oligosaccharides and properties of alpha-D-galactosidase from Lactobacillus curvatus R08 and Leuconostoc mesenteroides [corrected] JK55.

PubMed

Yoon, Mi Young; Hwang, Han-Joon

2008-09-01

This study was undertaken to investigate the potential for reducing non-digestive oligosaccharides (NDO) in soy foods, as well as the influence of exogenous conditions on intracellular alpha-galactosidase (alpha-Gal) producing lactic acid bacteria. Two strains, Lactobacillus curvatus R08 and Leuconostoc mesenteroides [corrected]JK55, showed the highest levels of raffinose degrading activity at over 40 U mL(-1), and presented maximum activities during the stationary phase in a medium where raffinose was the only carbon source. Raffinose was the most effective inducer, followed by melibiose, and galactose; the enzymes were partially inhibited by fructose and sucrose. On the other hand, limited activity was observed in glucose. The strains displayed optimum activity levels at neutral pH and a 35-37 degrees C temperature range. The alpha-Gal activities of L. curvatus R08 and Leu. mesenteroides [corrected] JK55 were maintained at pH 6.5-10.0. The activity of the alpha-Gal enzyme was stable in a relatively broad range of temperatures from 0 to 40 degrees C for 3h. In soymilk, Leu. mesenteroides [corrected] JK55 and L. curvatus R08 completely hydrolyzed the NDO after 18-24h of fermentation. The abilities of L. curvatus R08 and Leu. mesenteroides [corrected] JK55 to degrade raffinose sugars and, particularly, to produce organic acids from sugar, could contribute to reductions in the anti-nutritional properties of soy, and to the accumulation of compounds with beneficial properties during food processing. Furthermore, this study provides the optimum conditions to induce alpha-Gal from these strains.

Enzymatic breakdown of raffinose oligosaccharides in pea seeds.

PubMed

Blöchl, Andreas; Peterbauer, Thomas; Hofmann, Julia; Richter, Andreas

2008-06-01

Both alkaline and acidic alpha-galactosidases (alpha-D: -galactoside galactohydrolases, E.C.3.2.1.22) isolated from various plant species have been described, although little is known about their co-occurrence and functions in germinating seeds. Here, we report on the isolation of two cDNAs, encoding for alpha-galactosidases from maturing and germinating seeds of Pisum sativum. One was identified as a member of the acidic alpha-galactosidase of the family 27 glycosyl hydrolase cluster and the other as a member of the family of alkaline alpha-galactosidases, which are highly homologous to seed imbibition proteins (SIPs). PsGAL1 transcripts, encoding for the ACIDIC alpha-GALACTOSIDASE, were predominately expressed during seed maturation and acidic enzyme activities were already present in dry seeds, showing little changes during seed germination. Compartmentation studies revealed that acidic alpha-galactosidases were located in protein storage vacuoles (PSVs). PsAGA1, encoding for the ALKALINE alpha-GALACTOSIDASE, was only expressed after radicle protrusion, when about 50% of RFOs have already been broken down. RFO breakdown was markedly decreased when the translation of the alkaline enzyme was inhibited, providing evidence that PsAGA1 indeed functioned in RFO degradation. Based on these data, we present an integrated model of RFO breakdown by two sequentially active alpha-galactosidases in pea seeds.

Electrochemical magneto immunosensor based on endogenous β-galactosidase enzyme to determine enterotoxicogenic Escherichia coli F4 (K88) in swine feces using square wave voltammetry.

PubMed

Viviana Tarditto, Lorena; Alicia Zon, María; García Ovando, Hugo; Roberto Vettorazzi, Nelio; Javier Arévalo, Fernando; Fernández, Héctor

2017-11-01

Diseases caused by enterotoxicogenic Escherichia coli F4 (K88) (ETEC F4) are a problem in swine production establishments. Due to the high rate of mortality and morbidity of E. coli infections, a rapid and accurate diagnosis is important in order to choose an appropriate treatment to reduce the economic impact. Therefore, an electrochemical magneto-immunosensor (EMI) was developed to detect and quantify ETEC F4 in swine feces samples through a direct non-competitive immunoassay. ETEC F4 was selectively captured by immunomagnetic separation. The detection principle was based on the activity of β-galactosidase endogenous enzyme (β-gal), which hydrolyses the p-aminophenyl-β-D-galactopyranoside (p-APG) producing p-aminophenol (p-AP), which was oxidized on a carbon screen printed electrode (CSPE) using square wave voltammetry (SWV). All parameters related to construction and electrochemical responses were optimized. The total analysis time to quantify ETEC F4 using the EMI was less than 2h and the limit of detection (LOD) was 33CFUmL -1 . The perceptual relative error (%E r ) was 20%. The magneto-immunosensor was validated versus conventional method of culture and plate count, obtaining a very good agreement. The EMI is simple, fast and economical to detect and quantify ETEC F4 in swine feces samples, being thus a valuable tool in swine production. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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.

Treatment in Fabry disease.

PubMed

López Rodríguez, M

2018-04-13

Fabry disease is an X-linked inborn disease caused by deficit of alpha-galactosidaseA. This results in accumulation of glycosphingolipids in all cells and tissues. All males should receive enzyme replacement treatment in case of very low or undetectable levels of alpha-galactosidaseA. Female carriers and males with marginally levels of alpha-galactosidaseA should be treated in case of renal, neurologic o cardiac manifestations. There are two intravenous formulations of human recombinant enzyme, agalsidase alpha and agalsidase beta, showing similar efficacy and safety. Patients with amenable mutations of alpha-galactosidase can be treated with oral migalastat hydrochloride. Migalastat hydrochloride is a pharmacological chaperone that facilitates trafficking of alpha-galactosidaseA to lysosomes increasing enzyme activity. Patients treated with migalastat hydrochloride had significant improvements in left ventricular mass and gastrointestinal symptoms. Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Medicina Interna (SEMI). All rights reserved.

Structure-function relationships in Gan42B, an intracellular GH42 β-galactosidase from Geobacillus stearothermophilus.

PubMed

Solomon, Hodaya V; Tabachnikov, Orly; Lansky, Shifra; Salama, Rachel; Feinberg, Hadar; Shoham, Yuval; Shoham, Gil

2015-12-01

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Å resolution) and its catalytic mutant E323A (at 2.50 Å resolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/β domain, and the smallest all-β domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Å from each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Å at the wide opening and ∼5 Å at the small opening and ∼40 Å in length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active

Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile red.

PubMed

Sutter, Marc; Oliveira, Sabrina; Sanders, Niek N; Lucas, Bart; van Hoek, Arie; Hink, Mark A; Visser, Antonie J W G; De Smedt, Stefaan C; Hennink, Wim E; Jiskoot, Wim

2007-03-01

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein beta-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of beta-galactosidase below and above the protein's unfolding temperature of 57.4 degrees C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with beta-galactosidase aggregates led to a shift of the emission maximum (lambda (max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated beta-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native beta-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with beta-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.

Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins.

PubMed Central

Van der Vaart, J M; te Biesebeke, R; Chapman, J W; Toschka, H Y; Klis, F M; Verrips, C T

1997-01-01

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag alpha 1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and eightfold lower than that of alpha-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectable. A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production. alpha-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell. PMID:9023939

Digestion in adult females of the leaf-footed bug Leptoglossus zonatus (Hemiptera: Coreidae) with emphasis on the glycoside hydrolases α-amylase, α-galactosidase, and α-glucosidase.

PubMed

Rocha, Ariane A; Pinto, Carlos J C; Samuels, Richard I; Alexandre, Daniel; Silva, Carlos P

2014-03-01

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α-glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane-bound isoforms, being more abundant as a membrane-bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion-exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α-amylases and α-galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α-amylases acting on maize seed starch granules. © 2014 Wiley Periodicals, Inc.

Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses

PubMed Central

Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L.; Merritt, Robert; Hackett, Neil R.; Rovelink, Peter W.; Bruder, Joseph T.; Wickham, Thomas J.; Kovesdi, Imi; Crystal, Ronald G.

2004-01-01

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing β-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the β-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing β-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to β-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-β-galactosidase antibody levels following vector administration. However, cellular responses to β-galactosidase were significantly enhanced, with the frequency of CD4+ as well as the CD8+ β-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing β-galactosidase: BALB/c mice implanted with the CT26 syngeneic β-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These

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.

PubMed

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.

The alpha-galactosidase A p.Arg118Cys variant does not cause a Fabry disease phenotype: data from individual patients and family studies

PubMed Central

Ferreira, Susana; Ortiz, Alberto; Germain, Dominique P.; Viana-Baptista, Miguel; Gomes, António Caldeira; Camprecios, Marta; Fenollar-Cortés, Maria; Gallegos-Villalobos, Ángel; Garcia, Diego; García-Robles, José Antonio; Egido, Jesús; Gutiérrez-Rivas, Eduardo; Herrero, José Antonio; Mas, Sebastián; Oancea, Raluca; Péres, Paloma; Salazar-Martín, Luis Manuel; Solera-Garcia, Jesús; Alves, Helena; Garman, Scott C.; Oliveira, João Paulo

2015-01-01

Summary Lysosomal α-galactosidase A (α-Gal) is the enzyme deficient in Fabry disease (FD), an X-linked glycosphingolipidosis caused by pathogenic mutations affecting the GLA gene. The early-onset, multi-systemic FD classical phenotype is associated with absent or severe enzyme deficiency, as measured by in vitro assays, but patients with higher levels of residual α-Gal activity may have later-onset, more organ-restricted clinical presentations. A change in the codon 118 of the wild-type α-Gal sequence, replacing basic arginine by a potentially sulfhydryl-binding cysteine residue – GLA p.(Arg118Cys) –, has been recurrently described in large FD screening studies of high-risk patients. Although the Cys118 allele is associated with high residual α-Gal activity in vitro, it has been classified as a pathogenic mutation, mainly on the basis of theoretical arguments about the chemistry of the cysteine residue. However its pathogenicity has never been convincingly demonstrated by pathology criteria. We reviewed the clinical, biochemical and histopathology data obtained from 22 individuals of Portuguese and Spanish ancestry carrying the Cys118 allele, including 3 homozygous females. Cases were identified either on the differential diagnosis of possible FD manifestations and on case-finding studies (n=11; 4 males), or on unbiased cascade screening of probands’ close relatives (n=11; 3 males). Overall, those data strongly suggest that the GLA p.(Arg118Cys) variant does not segregate with FD clinical phenotypes in a Mendelian fashion, but might be a modulator of the multifactorial risk of cerebrovascular disease, since the allelic frequency in stroke patients was 0.0087 (p=0.0185 vs the general population). The Cys118 allelic frequency in healthy Portuguese adults (n=696) has been estimated as 0.001, therefore not qualifying for “rare” condition. PMID:25468652

The alpha-galactosidase A p.Arg118Cys variant does not cause a Fabry disease phenotype: data from individual patients and family studies.

PubMed

Ferreira, Susana; Ortiz, Alberto; Germain, Dominique P; Viana-Baptista, Miguel; Caldeira-Gomes, António; Camprecios, Marta; Fenollar-Cortés, Maria; Gallegos-Villalobos, Ángel; Garcia, Diego; García-Robles, José Antonio; Egido, Jesús; Gutiérrez-Rivas, Eduardo; Herrero, José Antonio; Mas, Sebastián; Oancea, Raluca; Péres, Paloma; Salazar-Martín, Luis Manuel; Solera-Garcia, Jesús; Alves, Helena; Garman, Scott C; Oliveira, João Paulo

2015-02-01

Lysosomal α-galactosidase A (α-Gal) is the enzyme deficient in Fabry disease (FD), an X-linked glycosphingolipidosis caused by pathogenic mutations affecting the GLA gene. The early-onset, multi-systemic FD classical phenotype is associated with absent or severe enzyme deficiency, as measured by in vitro assays, but patients with higher levels of residual α-Gal activity may have later-onset, more organ-restricted clinical presentations. A change in the codon 118 of the wild-type α-Gal sequence, replacing basic arginine by a potentially sulfhydryl-binding cysteine residue - GLA p.(Arg118Cys) -, has been recurrently described in large FD screening studies of high-risk patients. Although the Cys118 allele is associated with high residual α-Gal activity in vitro, it has been classified as a pathogenic mutation, mainly on the basis of theoretical arguments about the chemistry of the cysteine residue. However its pathogenicity has never been convincingly demonstrated by pathology criteria. We reviewed the clinical, biochemical and histopathology data obtained from 22 individuals of Portuguese and Spanish ancestry carrying the Cys118 allele, including 3 homozygous females. Cases were identified either on the differential diagnosis of possible FD manifestations and on case-finding studies (n=11; 4 males), or on unbiased cascade screening of probands' close relatives (n=11; 3 males). Overall, those data strongly suggest that the GLA p.(Arg118Cys) variant does not segregate with FD clinical phenotypes in a Mendelian fashion, but might be a modulator of the multifactorial risk of cerebrovascular disease. The Cys118 allelic frequency in healthy Portuguese adults (n=696) has been estimated as 0.001, therefore not qualifying for "rare" condition. Copyright © 2014 Elsevier Inc. All rights reserved.

Beta-Galactosidase Staining in the Nucleus of the Solitary Tract of Fos-Tau-LacZ Mice Is Unaffected by Monosodium Glutamate Taste Stimulation

PubMed Central

Stratford, Jennifer M.; Thompson, John A.

2014-01-01

Fos-Tau-LacZ (FTL) transgenic mice are used to visualize the anatomical connectivity of neurons that express c-Fos, an immediate early gene, in response to activation. In contrast to typical c-Fos protein expression, which is localized to the nucleus of stimulated neurons, activation of the c-Fos gene results in beta galactosidase (β-gal) expression throughout the entire cytoplasm of activated cells in FTL mice; thereby making it possible to discern the morphology of c-Fos expressing cells. This can be an especially important tool in brain areas in which function may be related to cell morphology, such as the primary taste/viscerosensory brainstem nucleus of the solitary tract (nTS). Thus, to further characterize FTL activity in the brain, the current study quantified both β-gal enzymatic activity as well as c-Fos protein expression in the nTS under a variety of experimental conditions (no stimulation, no stimulation with prior overnight food and water restriction, monosodium glutamate taste stimulation, and monosodium glutamate taste stimulation with perfusion 5 h post stimulation). Contrary to previous research, we found that β-gal activity (both labeled cell bodies and overall number of labeled pixels) was unchanged across all experimental conditions. However, traditional c-Fos protein activity (both cell bodies and number of activated pixels) varied significantly across experimental conditions, with the greatest amount of c-Fos protein label found in the group that received monosodium glutamate taste stimulation. Interestingly, although many c-Fos positive cells were also β-gal positive in the taste stimulated group, some c-Fos protein labeled cells were not co-labeled with β-gal. Together, these data suggest that β-gal staining within the nTS reflects a stable population of β-gal- positive neurons whose pattern of expression is unaffected by experimental condition. PMID:25192442

A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase.

PubMed

Selwood, T; Sinnott, M L

1990-06-01

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the

Kidney transplantation from a mother with unrecognized Fabry disease to her son with low α-galactosidase A activity: A 14-year follow-up without enzyme replacement therapy.

PubMed

Odani, Keiko; Okumi, Masayoshi; Honda, Kazuho; Ishida, Hideki; Tanabe, Kazunari

2016-07-01

We report a case of kidney transplantation from mother to son, both of whom were likely to have had an unrecognized renal variant phenotype of Fabry disease. The patient was a 54-year-old man, with an unknown primary cause of end stage renal disease. He had no notable past medical history, other than end stage renal disease. He underwent living-related kidney transplantation from his mother at age 40 years. Foam cells in the glomeruli were identified on histology assessment of a 0-hour allograft biopsy, with zebra bodies identified in the glomerular visceral epithelial cells by electron microscopy. These findings were indicative of Fabry disease in the donated kidney. As a definitive diagnosis of Fabry's disease could not be confirmed, enzyme replacement therapy was not initiated. Thirteen years after kidney transplantation, the patient underwent left nephrectomy for a left renal tumour, with pathological findings of clear cell carcinoma, foam cells and zebra bodies in the native kidney. Detailed examinations identified low α-galactosidase A activity and mutation of the α-Gal A gene, confirming a diagnosis of a renal variant phenotype of Fabry disease. Histology of several allograft biopsies performed over the 14 years from the time of kidney transplantation revealed only moderate interstitial fibrosis and tubular atrophy, with no evidence of disease progression on electron microscopy, despite the presence of zebra bodies in the glomerular visceral epithelial cells. © 2016 Asian Pacific Society of Nephrology.

Induction of bphA, encoding biphenyl dioxygenase, in two polychlorinated biphenyl-degrading bacteria, psychrotolerant Pseudomonas strain Cam-1 and mesophilic Burkholderia strain LB400.

PubMed

Master, E R; Mohn, W W

2001-06-01

We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader Pseudomonas strain Cam-1 and in the mesophilic PCB degrader Burkholderia strain LB400. Using a counterselectable gene replacement vector, we inserted a lacZ-Gm(r) fusion cassette between chromosomal genes encoding the large subunit (bphA) and small subunit (bphE) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of bphA were added to cell suspensions of Cam-10 and LB400-1 incubated at 30 degrees C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7 degrees C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15 degrees C was approximately five times less than the level in LB400-1 grown at 30 degrees C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria.

Two β-Galactosidases from the Human Isolate Bifidobacterium breve DSM 20213: Molecular Cloning and Expression, Biochemical Characterization and Synthesis of Galacto-Oligosaccharides

PubMed Central

Suljic, Jasmina; Kittl, Roman; Pham, Ngoc Hung; Kosma, Paul; Haltrich, Dietmar; Nguyen, Thu-Ha

2014-01-01

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The k cat/K m values for oNPG and lactose hydrolysis are 722 and 7.4 mM−1s−1 for β-gal I, and 543 and 25 mM−1s−1 for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide. PMID:25089712

Two β-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.

PubMed

Arreola, Sheryl Lozel; Intanon, Montira; Suljic, Jasmina; Kittl, Roman; Pham, Ngoc Hung; Kosma, Paul; Haltrich, Dietmar; Nguyen, Thu-Ha

2014-01-01

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The kcat/Km values for oNPG and lactose hydrolysis are 722 and 7.4 mM-1s-1 for β-gal I, and 543 and 25 mM-1s-1 for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.

A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase.

PubMed Central

Selwood, T; Sinnott, M L

1990-01-01

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the

Transition-State Ensembles Navigate the Pathways of Enzyme Catalysis.

PubMed

Mickert, Matthias J; Gorris, Hans H

2018-06-07

Transition-state theory (TST) provides an important framework for analyzing and explaining the reaction rates of enzymes. TST, however, needs to account for protein dynamic effects and heterogeneities in enzyme catalysis. We have analyzed the reaction rates of β-galactosidase and β-glucuronidase at the single molecule level by using large arrays of femtoliter-sized chambers. Heterogeneities in individual reaction rates yield information on the intrinsic distribution of the free energy of activation (Δ G ‡ ) in an enzyme ensemble. The broader distribution of Δ G ‡ in β-galactosidase compared to β-glucuronidase is attributed to β-galactosidase's multiple catalytic functions as a hydrolase and a transglycosylase. Based on the catalytic mechanism of β-galactosidase, we show that transition-state ensembles do not only contribute to enzyme catalysis but can also channel the catalytic pathway to the formation of different products. We conclude that β-galactosidase is an example of natural evolution, where a new catalytic pathway branches off from an established enzyme function. The functional division of work between enzymatic substates explains why the conformational space represented by the enzyme ensemble is larger than the conformational space that can be sampled by any given enzyme molecule during catalysis.

The validation of pharmacogenetics for the identification of Fabry patients to be treated with migalastat.

PubMed

Benjamin, Elfrida R; Della Valle, Maria Cecilia; Wu, Xiaoyang; Katz, Evan; Pruthi, Farhana; Bond, Sarah; Bronfin, Benjamin; Williams, Hadis; Yu, Julie; Bichet, Daniel G; Germain, Dominique P; Giugliani, Roberto; Hughes, Derralynn; Schiffmann, Raphael; Wilcox, William R; Desnick, Robert J; Kirk, John; Barth, Jay; Barlow, Carrolee; Valenzano, Kenneth J; Castelli, Jeff; Lockhart, David J

2017-04-01

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene. Migalastat, a pharmacological chaperone, binds to specific mutant forms of α-galactosidase A to restore lysosomal activity. A pharmacogenetic assay was used to identify the α-galactosidase A mutant forms amenable to migalastat. Six hundred Fabry disease-causing mutations were expressed in HEK-293 (HEK) cells; increases in α-galactosidase A activity were measured by a good laboratory practice (GLP)-validated assay (GLP HEK/Migalastat Amenability Assay). The predictive value of the assay was assessed based on pharmacodynamic responses to migalastat in phase II and III clinical studies. Comparison of the GLP HEK assay results in in vivo white blood cell α-galactosidase A responses to migalastat in male patients showed high sensitivity, specificity, and positive and negative predictive values (≥0.875). GLP HEK assay results were also predictive of decreases in kidney globotriaosylceramide in males and plasma globotriaosylsphingosine in males and females. The clinical study subset of amenable mutations (n = 51) was representative of all 268 amenable mutations identified by the GLP HEK assay. The GLP HEK assay is a clinically validated method of identifying male and female Fabry patients for treatment with migalastat.Genet Med 19 4, 430-438.

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.

PubMed

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.

Oral Migalastat HCl Leads to Greater Systemic Exposure and Tissue Levels of Active α-Galactosidase A in Fabry Patients when Co-Administered with Infused Agalsidase

PubMed Central

Warnock, David G.; Bichet, Daniel G.; Holida, Myrl; Goker-Alpan, Ozlem; Nicholls, Kathy; Thomas, Mark; Eyskens, Francois; Shankar, Suma; Adera, Mathews; Sitaraman, Sheela; Khanna, Richie; Flanagan, John J.; Wustman, Brandon A.; Barth, Jay; Barlow, Carrolee; Valenzano, Kenneth J.; Lockhart, David J.; Boudes, Pol; Johnson, Franklin K.

2015-01-01

Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified. Trial Registration ClinicalTrials.gov NCT01196871 PMID:26252393

Oral Migalastat HCl Leads to Greater Systemic Exposure and Tissue Levels of Active α-Galactosidase A in Fabry Patients when Co-Administered with Infused Agalsidase.

PubMed

Warnock, David G; Bichet, Daniel G; Holida, Myrl; Goker-Alpan, Ozlem; Nicholls, Kathy; Thomas, Mark; Eyskens, Francois; Shankar, Suma; Adera, Mathews; Sitaraman, Sheela; Khanna, Richie; Flanagan, John J; Wustman, Brandon A; Barth, Jay; Barlow, Carrolee; Valenzano, Kenneth J; Lockhart, David J; Boudes, Pol; Johnson, Franklin K

2015-01-01

Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified. ClinicalTrials.gov NCT01196871.

Degradative Enzymes from the Pharmacy or Health Food Store: Interesting Examples for Introductory Biology Laboratories

ERIC Educational Resources Information Center

Deutch, Charles E.

2007-01-01

Degradative enzymes in over-the-counter products from pharmacies and health food stores provide good examples of biological catalysis. These include [beta]-galactosidase in Lactaid[TM], [alpha]-galactosidase in Beano[R], [alpha]-amylase and proteases in digestive aids, and proteases in contact lens cleaners. These enzymes can be studied…

High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein β-galactosidase expression.

PubMed

Lin, Hsiao-Hsien; Lee, Tsung-Yih; Liu, Ting-Wei; Tseng, Ching-Ping

2017-07-01

Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (∼300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27 kip by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PKA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERK phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the abovementioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce β-galactosidase (β-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the β-Gal production (from 2.8- to 4.5-fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. Copyright © 2017. Published by Elsevier B.V.

The transplantation of neural stem cells and predictive factors in hematopoietic recovery in irradiated mice.

PubMed

Filip, S; Mokrý, J; Karbanová, J; Vávrová, J; Vokurková, J; Bláha, M; English, D

2005-04-01

A number of surprising observations have shown that stem cells, in suitable conditions, have the ability to produce a whole spectrum of cell types, regardless, whether these tissues are derived from the same germ layer or not. This phenomenon is called stem cell plasticity, which means that tissue-specific stem cells are mutually interchangeable. In our experiments, as a model, we used neural stem cells (NSCs) harvested from fetal (E14-15) neocortex and beta-galactosidase positive. In the first experiment we found that on days 12 and 30 after sub-lethal irradiation (LD 8.5 Gy) and (beta-galactosidase(+)) NSCs transplantation all mice survived, just as the group with bone marrow transplantation. Moreover, the bone marrow of mice transplanted NSCs contained the number of CFU-GM colonies with beta-galactosidase(+) cells which was as much as 50% higher. These differences were statistically significant, p<0.001. In the second experiment, we studied kinetics of (beta-galactosidase(+)) NSCs after their transplantation to sub-lethally irradiated mice. Histochemistry of tissues was performed on days 12 and 30 post-transplantation, and beta-galactosidase(+) cells were detected with the help of histochemical examination of removed tissues (lung, liver, spleen, thymus, and skeletal muscle). In tissues removed on day 12 post-transplantation, we found a significantly higher number of beta-galactosidase(+) cells in the spleen and thymus on day 30. While we presumed the presence beta-galactosidase(+) cells in the spleen, as spleen and reticuloendothelial system represent an important retaining system for different cell types, the presence of beta-galactosidase(+) cells in the thymus was rather surprising but very interesting. This indicates a certain mutual and close interconnection of transplanted stem cells and immune system in an adult organism. In the third experiment, we verified the mutual interchange of Sca-1 surface antigen in the bone marrow cells and NSCs before

Low levels of citrin (SLC25A13) expression in adult mouse brain restricted to neuronal clusters.

PubMed

Contreras, Laura; Urbieta, Almudena; Kobayashi, Keiko; Saheki, Takeyori; Satrústegui, Jorgina

2010-04-01

The mitochondrial aspartate-glutamate carriers (AGC) aralar (SLC25A12) and citrin (SLC25A13) are components of the malate aspartate shuttle (MAS), a major intracellular pathway to transfer reducing equivalents from NADH to the mitochondrial matrix. Aralar is the main AGC isoform present in the adult brain, and it is expressed mainly in neurons. To search for the other AGC isoform, citrin, in brain glial cells, we used a citrin knockout mouse in which the lacZ gene was inserted into the citrin locus as reporter gene. In agreement with the low citrin levels known to be present in the adult mouse brain, beta-galactosidase expression was very low. Surprisingly, unlike the case with astroglial cultures that express citrin, no beta-galactosidase was found in brain glial cells. It was confined to neuronal cells within discrete neuronal clusters. Double-immunolabelling experiments showed that beta-galactosidase colocalized not with glial cell markers but with the pan-neuronal marker NeuN. The deep cerebellar nuclei and a few midbrain nuclei (reticular tegmental pontine nuclei; magnocellular red nuclei) were the regions where beta-galactosidase expression was highest, and it was up-regulated in fasted mice, as was also the case for liver beta-galactosidase. The results support the notion that glial cells have much lower AGC levels and MAS activity than neurons. (c) 2009 Wiley-Liss, Inc.

Reversal of Myoblast Aging by Tocotrienol Rich Fraction Posttreatment

PubMed Central

Wan Ngah, Wan Zurinah; Mouly, Vincent; Abdul Karim, Norwahidah

2013-01-01

Skeletal muscle satellite cells are heavily involved in the regeneration of skeletal muscle in response to the aging-related deterioration of the skeletal muscle mass, strength, and regenerative capacity, termed as sarcopenia. This study focused on the effect of tocotrienol rich fraction (TRF) on regenerative capacity of myoblasts in stress-induced premature senescence (SIPS). The myoblasts was grouped as young control, SIPS-induced, TRF control, TRF pretreatment, and TRF posttreatment. Optimum dose of TRF, morphological observation, activity of senescence-associated β-galactosidase (SA-β-galactosidase), and cell proliferation were determined. 50 μg/mL TRF treatment exhibited the highest cell proliferation capacity. SIPS-induced myoblasts exhibit large flattened cells and prominent intermediate filaments (senescent-like morphology). The activity of SA-β-galactosidase was significantly increased, but the proliferation capacity was significantly reduced as compared to young control. The activity of SA-β-galactosidase was significantly reduced and cell proliferation was significantly increased in the posttreatment group whereas there was no significant difference in SA-β-galactosidase activity and proliferation capacity of pretreatment group as compared to SIPS-induced myoblasts. Based on the data, we hypothesized that TRF may reverse the myoblasts aging through replenishing the regenerative capacity of the cells. However, further investigation on the mechanism of TRF in reversing the myoblast aging is needed. PMID:24349615

Safety and pharmacodynamic effects of a pharmacological chaperone on α-galactosidase A activity and globotriaosylceramide clearance in Fabry disease: report from two phase 2 clinical studies

PubMed Central

2012-01-01

Background Fabry disease (FD) is a genetic disorder resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A), which leads to globotriaosylceramide (GL-3) accumulation in multiple tissues. We report on the safety and pharmacodynamics of migalastat hydrochloride, an investigational pharmacological chaperone given orally at 150 mg every-other-day. Methods Two open-label uncontrolled phase 2 studies of 12 and 24 weeks (NCT00283959 and NCT00283933) in 9 males with FD were combined. At multiple time points, α-Gal A activity and GL-3 levels were quantified in blood cells, kidney and skin. GL-3 levels were also evaluated through skin and renal histology. Results Compared to baseline, increased α-Gal A activity of at least 50% was demonstrated in blood, skin and kidney in 6 of 9 patients. Patients’ increased α-Gal A activities paralleled the α-Gal A increases observed in vitro in HEK-293 cells transfected with the corresponding mutant form of the enzyme. The same 6 patients who demonstrated increases of α-Gal A activity also had GL-3 reduction in skin, urine and/or kidney, and had α-Gal A mutations that responded in transfected cells incubated with the drug. The 3 patients who did not show a consistent response in vivo had α-Gal A mutations that did not respond to migalastat HCl in transfected cells. Migalastat HCl was well tolerated. Conclusions Migalastat HCl is a candidate pharmacological chaperone that provides a novel genotype-specific treatment for FD. It enhanced α-Gal A activity and resulted in GL-3 substrate decrease in patients with responsive GLA mutations. Phase 3 studies are ongoing. Trial registration Clinicaltrial.gov: NCT00283959 and NCT00283933 PMID:23176611

Direct Real-Time Monitoring of Prodrug Activation by Chemiluminescence.

PubMed

Gnaim, Samer; Scomparin, Anna; Das, Sayantan; Blau, Rachel; Satchi-Fainaro, Ronit; Shabat, Doron

2018-05-22

The majority of theranostic prodrugs reported so far relay information through a fluorogenic response generated upon release of the active chemotherapeutic agent. A chemiluminescence detection mode offers significant advantages over fluorescence, mainly due to the superior signal-to-noise ratio of chemiluminescence. Here we report the design and synthesis of the first theranostic prodrug monitored by a chemiluminescence diagnostic mode. As a representative model, we prepared a prodrug from the chemotherapeutic monomethyl auristatin E, which was modified for activation by β-galactosidase. The activation of the prodrug in the presence of β-galactosidase is accompanied by emission of a green photon. Light emission intensities, which increase with increasing concentration of the prodrug, were linearly correlated with a decrease in the viability of a human cell line that stably expresses β-galactosidase. We obtained sharp intravital chemiluminescent images of endogenous enzymatic activity in β-galactosidase-overexpressing tumor-bearing mice. The exceptional sensitivity achieved with the chemiluminescence diagnostic mode should allow the exploitation of theranostic prodrugs for personalized cancer treatment. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Translational autocontrol of the Escherichia coli ribosomal protein S15.

PubMed

Portier, C; Dondon, L; Grunberg-Manago, M

1990-01-20

When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site.

Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system.

PubMed

Mu, Xiaodong; Peng, Hairong; Pan, Haiying; Huard, Johnny; Li, Yong

2011-02-03

Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

The lysosomal enzyme alpha-Galactosidase A is deficient in Parkinson's disease brain in association with the pathologic accumulation of alpha-synuclein.

PubMed

Nelson, Michael P; Boutin, Michel; Tse, Tonia E; Lu, Hailin; Haley, Emily D; Ouyang, Xiaosen; Zhang, Jianhua; Auray-Blais, Christiane; Shacka, John J

2018-02-01

The aberrant accumulation of alpha-synuclein (α-syn) is believed to contribute to the onset and pathogenesis of Parkinson's disease (PD). The autophagy-lysosome pathway (ALP) is responsible for the high capacity clearance of α-syn. ALP dysfunction is documented in PD and pre-clinical evidence suggests that inhibiting the ALP promotes the pathological accumulation of α-syn. We previously identified the pathological accumulation of α-syn in the brains of mice deficient for the soluble lysosomal enzyme alpha-Galactosidase A (α-Gal A), a member of the glycosphingolipid metabolism pathway. In the present study, we quantified α-Gal A activity and levels of its glycosphingolipid metabolites in postmortem temporal cortex specimens from control individuals and in PD individuals staged with respect to α-syn containing Lewy body pathology. In late-state PD temporal cortex we observed significant decreases in α-Gal A activity and the 46kDa "active" species of α-Gal A as determined respectively by fluorometric activity assay and western blot analysis. These decreases in α-Gal A activity/levels correlated significantly with increased α-syn phosphorylated at serine 129 (p129S-α-syn) that was maximal in late-stage PD temporal cortex. Mass spectrometric analysis of 29 different isoforms of globotriaosylceramide (Gb 3 ), a substrate of α-Gal A indicated no significant differences with respect to different stages of PD temporal cortex. However, significant correlations were observed between increased levels of several Gb 3 isoforms and with decreased α-Gal A activity and/or increased p129S-α-syn. Deacylated Gb 3 (globotriaosylsphingosine or lyso-Gb 3 ) was also analyzed in PD brain tissue but was below the limit of detection of 20pmol/g. Analysis of other lysosomal enzymes revealed a significant decrease in activity for the lysosomal aspartic acid protease cathepsin D but not for glucocerebrosidase (GCase) or cathepsin B in late-stage PD temporal cortex. However, a

Identification of coliform genera recovered from water using different technologies.

PubMed

Fricker, C R; Eldred, B J

2009-12-01

Methods for the detection of coliforms in water have changed significantly in recent years with procedures incorporating substrates for the detection of beta-d-galactosidase becoming more widely used. This study was undertaken to determine the range of coliform genera detected with methods that rely on lactose fermentation and compare them to those recovered using methods based upon beta-d-galactosidase. Coliform isolates were recovered from sewage-polluted water using m-endo, membrane lauryl sulfate broth, tergitol TTC agar, Colilert-18, ChromoCult and ColiScan for primary isolation. Organisms were grouped according to whether they had been isolated based upon lactose fermentation or beta-d-galactosidase production. A wide range of coliform genera were detected using both types of methods. There was considerable overlap between the two groups, and whilst differences were seen between the genera isolated with the two method types, no clear pattern emerged. Substantial numbers of 'new' coliforms (e.g. Raoutella spp.) were recovered using both types of methods. The results presented here confirm that both methods based on lactose fermentation or detection of beta-d-galactosidase activity recover a range of coliform organisms. Any suggestion that only methods which are based upon fermentation of lactose recover organisms of public health or regulatory significance cannot be substantiated. Furthermore, the higher recovery of coliform organisms from sewage-polluted water using methods utilizing beta-d-galactosidase-based methods does not appear to be because of the recovery of substantially more 'new' coliforms.

Effect of postharvest ethylene treatment on sugar content, glycosidase activity and its gene expression in mango fruit.

PubMed

Chidley, Hemangi G; Deshpande, Ashish B; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

2017-03-01

Ripening-associated softening is one of the important attributes that largely determines the shelf-life of mango (Mangifera indica Linn.) fruits. To reveal the effect of pre-climacteric ethylene treatment on ripening-related softening of Alphonso mango, ethylene treatment was given to mature, raw Alphonso fruits. Changes in the pool of reducing and non-reducing sugars, enzymatic activity of three glycosidases: β-d-galactosidase, α-d-mannosidase and β-d-glucosidase and their relative transcript abundance were analysed for control and ethylene treated fruits during ripening. Early activity of all the three glycosidases and accelerated accumulation of reducing and non-reducing sugars on ethylene treatment was evident. β-d-Galactosidase showed the highest activity among three glycosidases in control fruits and marked increase in activity upon ethylene treatment. This was confirmed by the histochemical assay of its activity in control and ethylene treated ripe fruits. Relative transcript abundance revealed high transcript levels of β-d-galactosidase in control fruits. Ethylene-treated fruits showed early and remarkable increase in the β-d-galactosidase transcripts while α-d-mannosidase transcript variants displayed early accumulation. The findings suggest reduction in the shelf-life of Alphonso mango upon pre-climacteric ethylene treatment, a significant role of β-d-galactosidase and α-d-mannosidase in the ripening related softening of Alphonso fruits and transcriptional regulation of their expression by ethylene. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Comparison of immune responses against foot-and-mouth disease virus induced by fusion proteins using the swine IgG heavy chain constant region or beta-galactosidase as a carrier of immunogenic epitopes.

PubMed

Li, Guangjin; Chen, Weizao; Yan, Weiyao; Zhao, Kai; Liu, Mingqiu; Zhang, Jun; Fei, Liang; Xu, Quanxing; Sheng, Zutian; Lu, Yonggan; Zheng, Zhaoxin

2004-10-25

Previously, we demonstrated that a fusion protein (Gal-FMDV) consisting of beta-galactosidase and an immunogenic peptide, amino acids (141-160)-(21-40)-(141-160), of foot-and-mouth disease virus (FMDV) VP1 protein induced protective immune responses in guinea pigs and swine. We now designed a new potential recombinant protein vaccine against FMDV in swine. The immunogenic peptide, amino acids (141-160)-(21-40)-(141-160) from the VP1 protein of serotype O FMDV, was fused to the carboxy terminus of a swine immunoglobulin G single heavy chain constant region and expressed in Escherichia coli. The expressed fusion protein (IgG-FMDV) was purified and emulsified with oil adjuvant. Vaccination twice at an interval of 3 weeks with the emulsified IgG-FMDV fusion protein induced an FMDV-specific spleen proliferative T-cell response in guinea pigs and elicited high levels of neutralizing antibody in guinea pigs and swine. All of the immunized animals were efficiently protected against FMDV challenge. There was no significant difference between IgG-FMDV and Gal-FMDV in eliciting immunity after vaccination twice in swine. However, when evaluating the efficacy of a single inoculation of the fusion proteins, we found that IgG-FMDV could elicit a protective immune response in swine, while Gal-FMDV only elicited a weak neutralizing activity and could not protect the swine against FMDV challenge. Our results suggest that the IgG-FMDV fusion protein is a promising vaccine candidate for FMD in swine.

Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

PubMed Central

Ishii, Satoshi; Chang, Hui-Hwa; Kawasaki, Kunito; Yasuda, Kayo; Wu, Hui-Li; Garman, Scott C.; Fan, Jian-Qiang

2007-01-01

Fabry disease is a lysosomal storage disorder caused by the deficiency of α-Gal A (α-galactosidase A) activity. In order to understand the molecular mechanism underlying α-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal Km and Vmax values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) α-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q α-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant α-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant α-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations. PMID:17555407

The g.1170C>T polymorphism of the 5' untranslated region of the human alpha-galactosidase gene is associated with decreased enzyme expression--evidence from a family study.

PubMed

Oliveira, J P; Ferreira, S; Reguenga, C; Carvalho, F; Månsson, J-E

2008-12-01

Subnormal leukocyte α-galactosidase (α-Gal) activity was found during evaluation of an adolescent male with cryptogenic cerebrovascular small-vessel disease. The only molecular abnormality found was the g.1170C>T single-nucleotide polymorphism (SNP) in the 5' untranslated region of exon 1 in the α-Gal gene (GLA). Historically, this polymorphism has been considered to be biologically neutral. To test the hypothesis that the g.1170T allele might be associated with lower α-Gal expression, we genotyped GLA exon 1 and measured leukocyte and plasma α-Gal in the parents, brother and sister of the index case. The g.1170T allele co-segregated with a subnormal leukocyte α-Gal activity in the three siblings. Although plasma enzyme activities were within the normal range in all five relatives, the ranking of their values suggested a dosage effect of the g.1170T allele. Western blotting assays of leukocyte protein extracts showed that the relative expression of α-Gal in both the patient and his sister was significantly lower than in sex-matched hemizygous or homozygous controls for the g.1170C allele, either normalized to the β-actin immunoblot expression or standardized to a known amount of recombinant human α-Gal. These family data, in combination with results from a recent GLA SNP screening study among healthy Portuguese individuals, suggest that the g.1170C>T SNP may be co-dominantly associated with a relatively decreased GLA expression at the transcription and/or translation level. Larger population studies are needed to confirm these findings and to test the hypothesis that the GLA g.1170C>T may contribute to the multifactorial risk of ischaemic small-vessel cerebrovascular disease.

Catabolite Repression of Tryptophanase in Escherichia coli

PubMed Central

Botsford, James L.; DeMoss, R. D.

1971-01-01

Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of β-glactosidase. Induction of tryptophanase and β-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for β-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3′,5′-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of β-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, β-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either β-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of β-galactosidase in both strains. Addition of 2.5 × 10−3m cAMP appeared partially to

Effects of Environmental Estrogen on Apoptosis in Normal and Cancerous Breast Epithelial Cells

DTIC Science & Technology

1999-05-01

temperature. The cell debris was then pelleted by centrifugation at 15000g for 5 min. The cell extracts were normalized for protein concentration using...the Bio-Rad Reagent following the supplied protocol (Bio-Rad Laboratories, Hercules, CA). For ß- galactosidase assays, the cell extract was placed...galactosidase activity of each reaction was measured at an absorbance of 420 nM. Luciferase activity for the cell extracts were determined using

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.

PubMed

Moeller, Marcus J; Soofi, Abdulsalaam; Hartmann, Inge; Le Hir, Michel; Wiggins, Roger; Kriz, Wilhelm; Holzman, Lawrence B

2004-01-01

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.

Lactose/whey utilization and ethanol production by transformed Saccharomyces cerevisiae cells.

PubMed

Porro, D; Martegani, E; Ranzi, B M; Alberghina, L

1992-04-05

Strains of Saccharomyces cerevisiae transformed with a multicopy expression vector bearing both the Escherichia coli beta-galactosidase gene under the control of the upstream activating sequence of the GAL1-10 genes and the GAL4 activator gene release part of beta-galactosidase in the growth medium. This release is due to cell lysis of the older mother cells; the enzyme maintains its activity in buffered growth media. Fermentation studies with transformed yeast strains showed that the release of beta-galactosidase allowed an efficient growth on buffered media containing lactose as carbon source as well as on whey-based media. The transformed strains utilized up to 95% of the lactose and a high growth yield was obtained in rich media. High productions of ethanol were also observed in stationary phase after growth in lactose minimal media.

Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells

PubMed Central

Hall, Brandon M.; Balan, Vitaly; Gleiberman, Anatoli S.; Strom, Evguenia; Krasnov, Peter; Virtuoso, Lauren P.; Rydkina, Elena; Vujcic, Slavoljub; Balan, Karina; Gitlin, Ilya; Leonova, Katerina; Polinsky, Alexander; Chernova, Olga B.; Gudkov, Andrei V.

2016-01-01

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-galpH6), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-galpH6-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-galpH6-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-galpH6-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-galpH6-positive cells and reconsideration of potential cellular target for anti-aging treatment. PMID:27391570

Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells.

PubMed

Hall, Brandon M; Balan, Vitaly; Gleiberman, Anatoli S; Strom, Evguenia; Krasnov, Peter; Virtuoso, Lauren P; Rydkina, Elena; Vujcic, Slavoljub; Balan, Karina; Gitlin, Ilya; Leonova, Katerina; Polinsky, Alexander; Chernova, Olga B; Gudkov, Andrei V

2016-07-01

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-gal(pH6)), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-gal(pH6)-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-gal(pH6)-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-gal(pH6)-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-gal(pH6)-positive cells and reconsideration of potential cellular target for anti-aging treatment.

Cyclo(dehydroala-L-Leu), an alpha-glucosidase inhibitor from Penicillium sp. F70614.

PubMed

Kwon, O S; Park, S H; Yun, B S; Pyun, Y R; Kim, C J

2000-09-01

A diketopiperazine (1) has been isolated from the culture broth of Penicillium sp. F70614 and its structure has been determined to be cyclo(dehydroala-L-Leu) by various spectroscopic analyses. This compound selectively inhibited yeast alpha-glucosidase and porcine intestinal alpha-glucosidase with IC50 values of 35 and 50 microg/ml, respectively. However, it did not show significant inhibitory effects against almond beta3-glucosidase, Aspergillus alpha-galactosidase, Escherichia coli beta-galactosidase and jack bean alpha-mannosidase.

Simultaneous hydrolysis and co-fermentation of whey lactose with wheat for ethanol production.

PubMed

Jin, Yiqiong; Parashar, Archana; Mason, Beth; Bressler, David C

2016-12-01

Whey permeate was used as a co-substrate to replace part of the wheat for ethanol production by Saccharomyces cerevisiae. The simultaneous saccharification and fermentation was achieved with β-galactosidase added at the onset of the fermentation to promote whey lactose hydrolysis. Aspergillus oryzae and Kluyveromyces lactis β-galactosidases were two enzymes selected and used in the co-fermentation of wheat and whey permeate for the comparison of their effectiveness on lactose hydrolysis. The possibility of co-fermentations in both STARGEN and jet cooking systems was investigated in 5L bioreactors. Ethanol yields from the co-fermentations of wheat and whey permeate were evaluated. It was found that A. oryzae β-galactosidase was more efficient for lactose hydrolysis during the co-fermentation and that whey permeate supplementation can contribute to ethanol yield in co-fermentations with wheat. Copyright © 2016 Elsevier Ltd. All rights reserved.

Carbon Limitation Induces ςS-Dependent Gene Expression in Pseudomonas fluorescens in Soil

PubMed Central

Koch, Birgit; Worm, Jakob; Jensen, Linda E.; Højberg, Ole; Nybroe, Ole

2001-01-01

Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to Pseudomonas are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the ςS-dependent Escherichia coli promoter Pfic and lacZ functions as a reliable reporter for carbon availability in Pseudomonas fluorescens. When P. fluorescens strain DF57(pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of β-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing β-galactosidase from Pfic. Changes in cell size and expression of β-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of β-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain P. fluorescens DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In straw-amended soil β-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted. PMID:11472905

Transduction of cultured fish cells with recombinant baculoviruses.

PubMed

Leisy, Douglas J; Lewis, Teresa D; Leong, Jo-Ann C; Rohrmann, George F

2003-05-01

Five fish cell lines were tested for their ability to be transduced by Ac-CAlacZ, a recombinant baculovirus that is capable of expressing a beta-galactosidase reporter gene from the CAG promoter (consisting of a cytomegalovirus enhancer element, a chicken actin promoter and rabbit beta-globin termination sequences). TO (Tilapia ovary), EPC (carp), CHH-1 (Chum salmon heart fibroblast) and CHSE-214 (chinook salmon embryo) cells were transducible, as demonstrated by an in situ beta-galactosidase assay, whereas RTG-2 (rainbow trout gonad) cells were not. The EPC cell line was used for more detailed studies on baculovirus transduction. The transduction frequency was found to be higher at 28 degrees C than at 21 degrees C. Addition of the histone deacetylase inhibitor sodium butyrate increased the number of blue cells detected 5- to 7-fold. The m.o.i. was positively correlated with transduction frequency, although the relationship did not appear to be strictly linear, as has been observed with mammalian cells. The temperature at which baculoviruses were adsorbed to EPC cells did not affect levels of beta-galactosidase expression. We also examined expression levels of beta-galactosidase in EPC cells after infection with a baculovirus construct that overexpresses the vesicular stomatitis virus G protein and displays it on the virion surface. Expression levels with this virus were approximately 15-fold higher than were observed with Ac-CAlacZ.

[Morphological characteristics and physiological properties of aflatoxin B1 producing and non-producing Aspergillus flavus strains].

PubMed

Kogbo, W; Lemarinier, S; Boutibonnes, P

1985-09-01

Comparison between about 80 strains of Aspergillus flavus, belonging to the series flavus and oryzae, obtained from international collections but also isolated from French or African substrates revealed the following observations: 1. Cultural and morphological characteristics of toxicogenic and atoxicogenic strains of A. flavus are similar. However, the former produce a diffusible yellow pigment in 83% of isolates. 2. The two groups of conidiospores have the same resistance to UV irradiation (254 nm, 5 and 10 min). All the strains are equally sensitive to 4 antifungal antibiotics: nystatine, ketoconazole, clotrimazole and amphotericine. 3. A difference was seen in the capacity to produce enzymes as alpha-galactosidase, beta-galactosidase and beta-glucosidase, implicated in the glucid metabolism. The specific hydrolytic activity has been confirmed by the characterization of a large amount of beta-galactosidase and by a diauxic growth on glucose medium supplemented by lactose. Possible relationship between these characters and aflatoxin B1 production by A. flavus strains is discussed.

Effect of mechanical denaturation on surface free energy of protein powders.

PubMed

Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T; Blagbrough, Ian S; Conway, Barbara R

2016-10-01

Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of a rapid tube assay for presumptive identification of Escherichia coli from veterinary specimens.

PubMed Central

Iritani, B; Inzana, T J

1988-01-01

Three hundred sixty-six isolates of gram-negative, oxidase-negative bacteria from veterinary specimens were tested by a tube test for identification as Escherichia coli by production within 60 min of indole, beta-galactosidase, and beta-glucuronidase. The test correctly identified 255 of 269 isolates of E. coli (95% sensitivity) and correctly indicated that 97 of 97 isolates were not E. coli (100% specificity). We conclude that production of indole, beta-galactosidase, and beta-glucuronidase as measured by a rapid tube test is useful for identification of E. coli from veterinary specimens. PMID:3128581

Small heat shock protein AgsA: an effective stabilizer of enzyme activities.

PubMed

Tomoyasu, Toshifumi; Tabata, Atsushi; Ishikawa, Yoko; Whiley, Robert A; Nagamune, Hideaki

2013-01-01

A small heat shock protein, AgsA, possesses chaperone activity that can reduce the amount of heat-aggregated protein in vivo, and suppress the aggregation of chemical- and heat-denatured proteins in vitro. Therefore, we examined the ability of AgsA to stabilize the activity of several enzymes by using this chaperone activity. We observed that AgsA can stabilize the enzymatic activities of Renilla (Renilla reniformis) luciferase, firefly (Photinus pyralis) luciferase, and β-galactosidase, and showed comparable or greater stabilization of these enzymes than bovine serum albumin (BSA), a well-known stabilizer of enzyme activities. In particular, AgsA revealed better stabilization of Renilla luciferase and β-galactosidase than BSA under disulfide bond-reducing conditions with dithiothreitol. In addition, AgsA also increased the enzymatic performance of β-galactosidase and various restriction enzymes to a comparable or greater extent than BSA. These data indicate that AgsA may be useful as a general stabilizer of enzyme activities. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Spotlight on agalsidase beta in Fabry disease.

PubMed

Keating, Gillian M; Simpson, Dene

2007-01-01

Agalsidase beta (Fabrazyme) is a recombinant human alpha-galactosidase A enzyme approved for intravenous use in the treatment of Fabry disease. Fabry disease is a progressive, multisystemic, potentially life-threatening disorder caused by a deficiency of alpha-galactosidase A. This deficiency results in accumulation of glycosphingolipids, particularly globotriaosylceramide (GL-3), in the lysosomes of various tissues. This accumulation is the underlying driver of disease progression. Agalsidase beta provides an exogenous source of alpha-galactosidase A. Intravenous agalsidase beta is effective and well tolerated in patients with Fabry disease. In a phase III trial, agalsidase beta was shown to clear GL-3 from various target cells and, in a subsequent extension of this trial, prevent GL-3 reaccumulation. In a post-approval trial, agalsidase beta was shown to provide significant clinical benefit by reducing the risk of a major clinical event. Thus, agalsidase beta represents an important advance in the treatment of Fabry disease, and agalsidase beta therapy should be strongly considered in patients with Fabry disease who are suitable candidates.

Agalsidase Beta: a review of its use in the management of Fabry disease.

PubMed

Keating, Gillian M; Simpson, Dene

2007-01-01

Agalsidase beta (Fabrazyme) is a recombinant human alpha-galactosidase A enzyme approved for intravenous use in the treatment of Fabry disease. Fabry disease is a progressive, multisystemic, potentially life threatening disorder caused by a deficiency of alpha-galactosidase A. This deficiency results in accumulation of glycosphingolipids, particularly globotriaosylceramide (GL-3), in the lysosomes of various tissues. This accumulation is the underlying driver of disease progression. Agalsidase beta provides an exogenous source of alpha-galactosidase A.Intravenous agalsidase beta is effective and well tolerated in patients with Fabry disease. In a phase III trial, agalsidase beta was shown to clear GL-3 from various target cells and, in a subsequent extension of this trial, prevent GL-3 reaccumulation. In a post-approval trial, agalsidase beta was shown to provide significant clinical benefit by reducing the risk of a major clinical event. Thus, agalsidase beta represents an important advance in the treatment of Fabry disease, and agalsidase beta therapy should be strongly considered in patients with Fabry disease who are suitable candidates.

Antisense RNA: effect of ribosome binding sites, target location, size, and concentration on the translation of specific mRNA molecules.

PubMed

Daugherty, B L; Hotta, K; Kumar, C; Ahn, Y H; Zhu, J D; Pestka, S

1989-01-01

A series of plasmids were constructed to generate RNA complementary to the beta-galactosidase messenger RNA under control of the phage lambda PL promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda PL promoter and/or the lacZ ribosome binding site in reverse orientation. Fragments of lacZ DNA from the 5' and/or the 3' region were used in these constructions. When these anti-mRNA molecules were produced in Escherichia coli 294, maximal inhibition of beta-galactosidase synthesis occurred when a functional ribosome binding site was present near the 5' end of the anti-mRNA and the anti-mRNA synthesized was complementary to the 5' region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5'-coding sequence. Anti-mRNAs producing maximal inhibition of beta-galactosidase synthesis exhibited an anti-lacZ mRNA:normal lacZ mRNA ratio of 100:1 or higher. Those showing lower levels of inhibition exhibited much lower anti-lacZ mRNA:normal lacZ mRNA ratios. A functional ribosome binding site at the 5'-end was found to decrease the decay rate of the anti-lacZ mRNAs. In addition, the incorporation of a transcription terminator just downstream of the antisense segment provided for more efficient inhibition of lacZ mRNA translation due to synthesis of smaller and more abundant anti-lacZ mRNAs. The optimal constructions produced undetectable levels of beta-galactosidase synthesis.

Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin type II.

PubMed Central

Handl, C; Rönnberg, B; Nilsson, B; Olsson, E; Jonsson, H; Flock, J I

1988-01-01

The gene for Escherichia coli heat-stable enterotoxin type II (STII) was fused to the genes for protein A from Staphylococcus aureus and beta-galactosidase in two different expression systems. Antibodies raised in rabbits against the protein A-STII fusion protein recognized the beta-galactosidase-STII fusion protein. The latter fusion protein was used as the immobilized antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of STII. The correlation between the results of the ELISA and the intestinal loop test in piglets was 95%, suggesting that the ELISA can be used to reliably detect STII. Images PMID:3049659

Floating Droplet Array: An Ultrahigh-Throughput Device for Droplet Trapping, Real-time Analysis and Recovery

PubMed Central

Labanieh, Louai; Nguyen, Thi N.; Zhao, Weian; Kang, Dong-Ku

2016-01-01

We describe the design, fabrication and use of a dual-layered microfluidic device for ultrahigh-throughput droplet trapping, analysis, and recovery using droplet buoyancy. To demonstrate the utility of this device for digital quantification of analytes, we quantify the number of droplets, which contain a β-galactosidase-conjugated bead among more than 100,000 immobilized droplets. In addition, we demonstrate that this device can be used for droplet clustering and real-time analysis by clustering several droplets together into microwells and monitoring diffusion of fluorescein, a product of the enzymatic reaction of β-galactosidase and its fluorogenic substrate FDG, between droplets. PMID:27134760

Effects of Exogenous Enzymatic Treatment During Processing on the Sensory Quality of Summer Tieguanyin Oolong Tea from the Chinese Anxi County

PubMed Central

Zhang, Xue-Bo

2015-01-01

Summary In order to attenuate the bitter taste and improve the aroma of the summer tieguanyin oolong tea from the Chinese Anxi county, the effects of processing treatment with exogenous laccase and α-galactosidase on tea sensory quality and related compounds were investigated. The solutions of laccase and/or α-galactosidase were sprayed on the tea leaves before the first drying process. The sensory evaluation results showed that the sensory quality of the tea was significantly enhanced with the enzymatic treatment. The combined application of laccase at 8.25 and α-galactosidase at 22 U per kg of fresh tea shoots achieved the most satisfying sensory quality. Further analysis of flavour-related constituents was carried out by HPLC and GC-MS. The HPLC analysis showed that the contents of catechins and total polyphenols were reduced, compared to the untreated group, by 11.9 and 13.3% respectively, and the total soluble sugars and water extract content were increased by 19.4 and 6.6% respectively, after the treatment with both enzymes. The decrease of catechins and total polyphenols reduced the bitterness and astringency of the summer tea, while the increase of total soluble sugars and water extract content improved the sweetness and mellow taste. The aromatic compound data from GC-MS showed that the total essential oil content in these tea samples co-treated with laccase and α-galactosidase increased significantly, in which aldehydes, alcohols, esters and alkenes increased by 23.28, 37.05, 20.10 and 38.99%, respectively. Our data suggest that the exogenous enzymatic treatment can enhance the summer oolong tea quality, especially its taste and aroma. PMID:27904347

Ceramic microsystem incorporating a microreactor with immobilized biocatalyst for enzymatic spectrophotometric assays.

PubMed

Baeza, Mireia; López, Carmen; Alonso, Julián; López-Santín, Josep; Alvaro, Gregorio

2010-02-01

Low-temperature cofired ceramics (LTCC) technology is a versatile fabrication technique used to construct microflow systems. It permits the integration of several unitary operations (pretreatment, separation, (bio)chemical reaction, and detection stage) of an analytical process in a modular or monolithic way. Moreover, because of its compatibility with biological material, LTCC is adequate for analytical applications based on enzymatic reactions. Here we present the design, construction, and evaluation of a LTCC microfluidic system that integrates a microreactor (internal volume, 24.28 microL) with an immobilized beta-galactosidase from Escherichia coli (0.479 activity units) and an optical flow cell to measure the product of the enzymatic reaction. The enzyme was immobilized on a glyoxal-agarose support, maintaining its activity along the time of the study. As a proof of concept, the LTCC-beta-galactosidase system was tested by measuring the conversion of ortho-nitrophenyl beta-D-galactopyranoside, the substrate usually employed for activity determinations. Once packed in a monolithically integrated microcolumn, the miniaturized flow system was characterized, the operational conditions optimized (flow rate and injection volume), and its performance successfully evaluated by determining the beta-galactosidase substrate concentration at the millimolar level.

Xyloglucan mobilisation in cotyledons of developing plantlets of Hymenaea courbaril L. (Leguminosae-Caesalpinoideae).

PubMed

Tiné; Cortelazzo; Buckeridge

2000-05-29

Many seeds contain storage compounds that are used by the embryo/plantlet as a source of nutrients after germination. In seeds of Hymenaea courbaril, a leguminous tree, the main reserve consists of a structurally unusual xyloglucan stored in thickened walls of the cotyledon cells. The present work aimed to study H. courbaril xyloglucan metabolism during and after germination in order to compare its degrading system with the other known xyloglucan containing seeds. Polysaccharide degradation occurred after germination between 35 and 55 days after planting. The activities of alpha-xylosidase, beta-glucosidase, beta-galactosidase and XET rose during the period of xyloglucan disassembling but a low level of endo-beta-glucanase activity was detected, suggesting that this XET has high affinity for the oligosaccharides. The pH optimum of beta-galactosidase was different from the alpha-xylosidase, beta-glucosidase and XET optima suggesting that the former may be important in the control of the mobilisation process. A tentative model for xyloglucan disassembling in vivo is proposed, where beta-galactosidase allows the free oligosaccharides to bypass a transglycosylation cycle and be disassembled by the other exo-enzymes. Some ecophysiological comparisons among H. courbaril and other xyloglucan storing seeds are discussed.

Treatment with agalsidase beta during pregnancy in Fabry disease.

PubMed

Politei, Juan M

2010-04-01

Fabry disease is an X-linked lysosomal storage disease caused by a deficiency of alpha-galactosidase A, which leads to excessive accumulation of glycosphingolipids in most tissues in the body, with life-threatening clinical consequences in the kidney, heart, and cerebrovascular system. Enzyme replacement therapy using exogenously produced alpha-galactosidase has been available for treatment of this multisystem progressive disease since 2001. Two different preparations of enzyme replacement therapy for Fabry disease are available outside of the USA: agalsidase alfa and agalsidase beta. Despite being X-linked, Fabry disease affects many female patients, and this report presents a successful pregnancy of a female patient receiving agalsidase beta.

Selective intraarterial gene delivery into a canine meningioma.

PubMed

Chauvet, A E; Kesava, P P; Goh, C S; Badie, B

1998-05-01

The goal of this study was to evaluate gene delivery to a benign brain tumor. A recombinant adenovirus vector bearing the Escherichia coli beta-galactosidase reporter gene was selectively injected into the vascular supply of a spontaneously occurring canine olfactory groove meningioma. The tumor and a small amount of peritumoral brain tissue were removed 5 days after viral injection and stained with X-Gal to assess gene delivery. The authors noted significant beta-galactosidase gene expression by the tumor, but not by surrounding brain tissue. No obvious viral-related cytotoxicity was noted. The authors found that meningiomas can be successfully transduced by adenovirus vectors by using endovascular techniques.

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

PubMed

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.

Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice.

PubMed

Sakuraba, Hitoshi; Murata-Ohsawa, Mai; Kawashima, Ikuo; Tajima, Youichi; Kotani, Masaharu; Ohshima, Toshio; Chiba, Yasunori; Takashiba, Minako; Jigami, Yoshifumi; Fukushige, Tomoko; Kanzaki, Tamotsu; Itoh, Kohji

2006-01-01

We compared two recombinant alpha-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific alpha-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h(-1) mg protein(-1), respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in alpha-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE.

PubMed

Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Abu-Asab, Mones S; Tsokos, Maria; Morris, John C; Kalle, Wouter H J

2007-04-01

We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

Functionalization of paramagnetic nanoparticles for protein immobilization and purification.

PubMed

Carneiro, Lara A B C; Ward, Richard J

2018-01-01

A paramagnetic nanocomposite coated with chitosan and N-(5-Amino-1-carboxy-pentyl) iminodiacetic acid (NTA) that is suitable for protein immobilization applications has been prepared and characterized. The nanoparticle core was synthesized by controlled aggregation of Fe 3 O 4 under alkaline conditions, and Transmission Electron Microscopy revealed a size distribution of 10-50 nm. The nanoparticle core was coated with chitosan and derivatized with glutaraldehyde and NTA, as confirmed by Fourier Transform Infrared Spectroscopy. The final nanoparticles were used as a metal affinity matrix to separate a recombinant polyhistidine-tagged β-galactosidase from Bacillus subtilis directly from E. coli cell lysates with high purity (>95%). After loading with Ni 2+ , nanoparticles demonstrated a binding capacity of 250 μg of a polyhistidine-tagged β-galactosidase per milligram of support. The immobilized enzyme retained 80% activity after 9 cycles of washing, and the immobilized recombinant protein could be eluted with high purity with imidazole. The applications for these nanomagnetic composites extend beyond protein purification, and can also be used for immobilizing enzymes, where the β-galactosidase immobilized on the nanomagnetic support was used in multiple cycles of catalytic reactions with no significant loss of catalytic activity. Copyright © 2017. Published by Elsevier Inc.

Enhanced alpha-galactosidase expression in pseudomonas chlororaphis

USDA-ARS?s Scientific Manuscript database

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...

Fabry disease

PubMed Central

2010-01-01

Fabry disease (FD) is a progressive, X-linked inherited disorder of glycosphingolipid metabolism due to deficient or absent lysosomal α-galactosidase A activity. FD is pan-ethnic and the reported annual incidence of 1 in 100,000 may underestimate the true prevalence of the disease. Classically affected hemizygous males, with no residual α-galactosidase A activity may display all the characteristic neurological (pain), cutaneous (angiokeratoma), renal (proteinuria, kidney failure), cardiovascular (cardiomyopathy, arrhythmia), cochleo-vestibular and cerebrovascular (transient ischemic attacks, strokes) signs of the disease while heterozygous females have symptoms ranging from very mild to severe. Deficient activity of lysosomal α-galactosidase A results in progressive accumulation of globotriaosylceramide within lysosomes, believed to trigger a cascade of cellular events. Demonstration of marked α-galactosidase A deficiency is the definitive method for the diagnosis of hemizygous males. Enzyme analysis may occasionnally help to detect heterozygotes but is often inconclusive due to random X-chromosomal inactivation so that molecular testing (genotyping) of females is mandatory. In childhood, other possible causes of pain such as rheumatoid arthritis and 'growing pains' must be ruled out. In adulthood, multiple sclerosis is sometimes considered. Prenatal diagnosis, available by determination of enzyme activity or DNA testing in chorionic villi or cultured amniotic cells is, for ethical reasons, only considered in male fetuses. Pre-implantation diagnosis is possible. The existence of atypical variants and the availability of a specific therapy singularly complicate genetic counseling. A disease-specific therapeutic option - enzyme replacement therapy using recombinant human α-galactosidase A - has been recently introduced and its long term outcome is currently still being investigated. Conventional management consists of pain relief with analgesic drugs

A Novel Biomedical Device Utilizing Light Emitting Nano-Structures

NASA Technical Reports Server (NTRS)

Varaljay, Vanessa A.

2004-01-01

This paper will discuss the development of a novel biomedical detection device that will be used to detect microorganisms with the use of infrared fluorochrome polymers attached to antibodies in fluids such as water. The fluorochrome polymers emit light in the near inferred region (NIR), approximately 805 nm, when excited by an NIR laser at 778 nm. The device could remarkably change the way laboratory testing is done today. The testing process is usually performed on a time scale of days while our device will be able to detect microorganisms in minutes. This type of time efficient analysis is ideal for use aboard the International Space Station and the Space Shuttle (ISS/SS) and has many useful commercial applications, for instance at a water treatment plant and food processing plants. With more research and experimentation the testing might also one day be used to detect bacteria and viruses in complex fluids such as blood, which would revolutionize blood analysis as it is performed today. My contribution to the project has been to develop a process which will allow an antibody/fluorescent dye pair to be conjugated to a specific bacteria or virus and than to to be separated from a sample body of water for detection. The antibody being used in this experiment is anti beta galactosidase and its complement enzyme is beta galactosidase, a non harmful derivative of E. Coli. The anti beta galactosidase has been conjugated to the fluorochrome polymer, IRDye800, which emits at approximately 806 nm. The dye when excited by the NIR laser emits a signal which is detected by a spectrometer and then is read by state of the art computer software. The state-of-the-art process includes incubating the anti beta galactosidase and beta galactosidase in a phosphate buffer solution in a test tube, allowing the antibody to bind to specific sites on the enzyme. After the antibody is bound to the enzyme, it is centrifuged in specific filters that will allow free antibody to wash away and

[Correlations of telomere length changing and pathogeny of keratoconus].

PubMed

Wang, Jiao-jiao; Li, Shao-wei; Wang, Yi-qiang; Wang, Ye; Zhong, Wen-xian; Zang, Xin-jie

2009-08-01

To study telomere length, senescence-associated-beta-galactosidase (SA-beta-galactosidase) and senescence marker protein-30 (SMP-30) in the stromal cells of keratoconus or normal corneas respectively, aiming finding the association of these indexes with the phenotype of keratoconus. Experiment research. 37 keratoconus lesions corneas were removed from 32 keratoconus patients who were operated in Shangdong Eye Institute between January 2006 and December 2006, and 20 normal corneas were collected from eye bank. The keratoconus corneas ages were from 13 to 34 years [mean ages (19 + or - 5) years] and the control group consists of 20 normal corneas donor ages from 9 to 30 years [mean ages (19 + or - 4) years]. And there was no statistical difference of ages between keratoconus and normal corneas. Southern blot method was utilized to detect telomere length of genomic DNA. SA-beta-galactosidase was detected by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining method respectively in keratoconus and normal corneas. Isolated mRNA from keratoconus and normal corneas were reverse-transcribed to cDNA and SMP-30 was detected using PCR with specific primers (sense: 5' ccg tgg atg cct ttg act at 3'; anti-sense: 5' caa ctt cat gc tgct ttg ga 3'). To compare normal corneas and keratoconus corneas by histopathological study. Statistical analysis by t test. The telomere length in stromal cells in keratoconus corneas were from 10.29 to 14.12 kb, mean (11.54 + or - 1.41) kb, while that of normal corneas were from 12.64 to 15.32 kb, mean (13.45 + or - 0.99) kb. The difference of telomere length in stromal cells of keratoconus and normal corneas reached a statistical significant level (t = 4.753, P < 0.05). That means the telomere length of keratoconus stroma was shorter than that of normal corneal stroma. Light microscopy revealed that collagen fibers in keratoconus corneal stroma were arranged in an irregular manner. Cells density in keratoconus stroma appeared lower

Lactose metabolism by Streptococcus mutans: evidence for induction of the tagatose 6-phosphate pathway.

PubMed Central

Hamilton, I R; Lebtag, H

1979-01-01

Growth on lactose by strains of Streptococcus mutans resulted in the induction of the lactose-phosphoenolpyruvate-phosphotransferase system, phospho-beta-galactosidase, and the enzymes of the tagatose 6-phosphate pathway. PMID:230175

Effects of exogenous lactase administration on hydrogen breath excretion and intestinal symptoms in patients presenting lactose malabsorption and intolerance.

PubMed

Ibba, Ivan; Gilli, Agnese; Boi, Maria Francesca; Usai, Paolo

2014-01-01

To establish whether supplementation with a standard oral dose of Beta-Galactosidase affects hydrogen breath excretion in patients presenting with lactose malabsorption. Ninety-six consecutive patients positive to H2 Lactose Breath Test were enrolled. Mean peak H2 levels, the time to reach the peak H2, the time to reach the cut-off value of 20 ppm, the cumulative breath H2 excretion, the areas under the curve, and a Visual Analogical 10-point Scale for symptoms were calculated. Genotyping of the C/T-13910 variant was carried out. Following the oral administration of Beta-Galactosidase, in 21.88% of the cases, H2 Lactose Breath Test became negative (Group A), while mean peak H2 levels (74.95 ppm versus 7.85), P < 0.0000, in 17.71% (Group B) were still positive, with the H2 level 20 ppm above the baseline, but the peak H2 levels were significantly lower than those observed at the baseline test (186.7 ppm versus 66.64), P < 0.0000, while in 60.41% (Group C) they were still positive with the peak H2 levels similar to those observed at the baseline test (94.43 versus 81.60 ppm). All 96 individuals tested presented the C/C-13910 genotype nonpersistence. The response to oral administration of Beta-Galactosidase in patients with symptoms of lactose malabsorption presents a significant variability.

Superoxide radical-generating compounds activate a predicted promoter site for paraquat-inducible genes of the Chromobacterium violaceum bacterium in a dose-dependent manner.

PubMed

Gabriel, J E; Guerra-Slompo, E P; de Souza, E M; de Carvalho, F A L; Madeira, H M F; de Vasconcelos, A T R

2015-08-21

The purpose of the present study was to functionally evaluate the influence of superoxide radical-generating compounds on the heterologous induction of a predicted promoter region of open reading frames for paraquat-inducible genes (pqi genes) revealed during genome annotation analyses of the Chromobacterium violaceum bacterium. A 388-bp fragment corresponding to a pqi gene promoter of C. violaceum was amplified using specific primers and cloned into a conjugative vector containing the Escherichia coli lacZ gene without a promoter. Assessments of the expression of the β-galactosidase enzyme were performed in the presence of menadione (MEN) and phenazine methosulfate (PMS) compounds at different final concentrations to evaluate the heterologous activation of the predicted promoter region of interest in C. violaceum induced by these substrates. Under these experimental conditions, the MEN reagent promoted highly significant increases in the expression of the β-galactosidase enzyme modulated by activating the promoter region of the pqi genes at all concentrations tested. On the other hand, significantly higher levels in the expression of the β-galactosidase enzyme were detected exclusively in the presence of the PMS reagent at a final concentration of 50 μg/mL. The findings described in the present study demonstrate that superoxide radical-generating compounds can activate a predicted promoter DNA motif for pqi genes of the C. violaceum bacterium in a dose-dependent manner.

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

PubMed Central

Jacinto-Loeza, Eva; Vivanco-Domínguez, Serafín; Guarneros, Gabriel; Hernández-Sánchez, Javier

2008-01-01

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 μg/μl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA. PMID:18583364

One-year calorie restriction impacts gut microbial composition but not its metabolic performance in obese adolescents.

PubMed

Ruiz, Alicia; Cerdó, Tomás; Jáuregui, Ruy; Pieper, Dietmar H; Marcos, Ascensión; Clemente, Alfonso; García, Federico; Margolles, Abelardo; Ferrer, Manuel; Campoy, Cristina; Suárez, Antonio

2017-04-01

Recent evidence has disclosed a connection between gut microbial glycosidase activity and adiposity in obese. Here, we measured microbial α-glucosidase and β-galactosidase activities and sorted fluorescently labeled β-galactosidase containing (βGAL) microorganisms in faecal samples of eight lean and thirteen obese adolescents that followed a controlled calorie restriction program during one year. β-galactosidase is a highly distributed functional trait, mainly expressed by members of Blautia, Bacteroides, Alcaligenes, Acinetobacter and Propionibacterium. Only long-term calorie restriction induced clear changes in the microbiota of obese adolescents. Long-term calorie restriction induced significant shifts in total and βGAL gut microbiota, reducing the Firmicutes:Bacteroidetes ratio and enhancing the growth of beneficial microorganisms such as Bacteroides, Roseburia, Faecalibacterium and Clostridium XIVa. Moreover, the structure and composition of βGAL community in obese after long-term calorie restriction was highly similar to that of lean adolescents. However, despite this high compositional similarity, microbial metabolic performance was different, split in two metabolic states at a body mass index value of 25. Our study shows that calorie restriction is a strong environmental force reshaping gut microbiota though its metabolic performance is linked to host's adiposity, suggesting that functional redundancy and metabolic plasticity are fundamental properties of gut microbial ecosystem. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

PubMed

Agaisse, H; Lereclus, D

1994-08-01

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.

Glycosphingolipid storage in Fabry mice extends beyond globotriaosylceramide and is affected by ABCB1 depletion

PubMed Central

Kamani, Mustafa A; Provençal, Philippe; Boutin, Michel; Pacienza, Natalia; Fan, Xin; Novak, Anton; Huang, Tonny C; Binnington, Beth; Au, Bryan C; Auray-Blais, Christiane; Lingwood, Clifford A; Medin, Jeffrey A

2016-01-01

Aim: Fabry disease is caused by α-galactosidase A deficiency leading to accumulation of globotriaosylceramide (Gb3) in tissues. Clinical manifestations do not appear to correlate with total Gb3 levels. Studies examining tissue distribution of specific acyl chain species of Gb3 and upstream glycosphingolipids are lacking. Material & methods/Results: Thorough characterization of the Fabry mouse sphingolipid profile by LC-MS revealed unique Gb3 acyl chain storage profiles. Storage extended beyond Gb3; all Fabry tissues also accumulated monohexosylceramides. Depletion of ABCB1 had a complex effect on glycosphingolipid storage. Conclusion: These data provide insights into how specific sphingolipid species correlate with one another and how these correlations change in the α-galactosidase A-deficient state, potentially leading to the identification of more specific biomarkers of Fabry disease. PMID:28116130

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

PubMed

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.

Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates

PubMed Central

Garrido, Daniel; Ruiz-Moyano, Santiago; Jimenez-Espinoza, Rogelio; Eom, Hyun-Ju; Block, David E.; Mills, David A.

2013-01-01

Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these dietary interventions. In this study the consumption of galactooligosaccharides was studied for 22 isolates of Bifidobacterium longum subsp. infantis, one of the most representative species in the infant gut microbiota. In general all isolates showed a vigorous growth on these oligosaccharides, but consumption of larger galactooligosaccharides was variable. Bifidobacterium infantis ATCC 15697 has five genes encoding β-galactosidases, and three of them were induced during bacterial growth on commercial galactooligosaccharides. Recombinant β-galactosidases from B. infantis ATCC 15697 displayed different preferences for β-galactosides such as 4′ and 6′-galactobiose, and four β-galactosidases in this strain released monosaccharides from galactooligosaccharides. Finally, we determined the amounts of short chain fatty acids produced by strain ATCC 15697 after growth on different prebiotics. We observed that biomass and product yields of substrate were higher for lactose and galactooligosaccharides, but the amount of acids produced per cell was larger after growth on human milk oligosaccharides. These results provide a molecular basis for galactooligosaccharide consumption in B. infantis, and also represent evidence for physiological differences in the metabolism of prebiotics that might have a differential impact on the host. PMID:23200660

Creation of an In vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular inflammation controls transgene activity.

PubMed

Kitamura, M; Kawachi, H

1997-09-15

Automatic control over exogenous gene expression in response to the activity of disease is a crucial hurdle for gene transfer-based therapies. Towards achieving this goal, we created a "cytosensor" that perceives local inflammatory states and subsequently regulates foreign gene expression. alpha-Smooth muscle actin is known to be expressed in glomerular mesangial cells exclusively in pathologic situations. CArG box element, the crucial regulatory sequence of the alpha-smooth muscle actin promoter, was used as a sensor for glomerular inflammation. Rat mesangial cells were stably transfected with an expression plasmid that introduces a beta-galactosidase gene under the control of CArG box elements. In vitro, the established cells expressed beta-galactosidase exclusively after stimulation with serum. To examine whether the cells are able to automatically control transgene activity in vivo, serum-stimulated or unstimulated cells were transferred into normal rat glomeruli or glomeruli subjected to anti-Thy 1 glomerulonephritis. When stimulated cells were transferred into the normal glomeruli, beta-galactosidase expression was switched off in vivo within 3 d. In contrast, when unstimulated cells were transferred into the nephritic glomeruli, transgene expression was substantially induced. These data indicate the feasibility of using the CArG box element as a molecular sensor for glomerular injury. In the context of advanced forms of gene therapy, this approach provides a novel concept for automatic regulation of local transgene expression where the transgene is required to be activated during inflammation and deactivated when the inflammation has subsided.

A STUDY OF THE INDIGOGENIC PRINCIPLE AND IN VITRO MACROPHAGE DIFFERENTIATION

DTIC Science & Technology

and beta- glucuronidase activities. Moreover, there was a progressive increase in the densities of enzyme reactive centers. Indigo reaction product was...not observed over nuclei; lipid droplets and cell background were free from spurious precipitations. Both galactosidase and glucuronidase were

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing

PubMed Central

2014-01-01

Background The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns. Results A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities. Conclusions Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing.

PubMed

Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

2014-05-20

The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns. A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities. Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to hydrolyze lactose at low

Modeling lactose hydrolysis for efficiency and selectivity: Toward the preservation of sialyloligosaccharides in bovine colostrum whey permeate.

PubMed

de Moura Bell, Juliana M L N; Aquino, Leticia F M C; Liu, Yan; Cohen, Joshua L; Lee, Hyeyoung; de Melo Silva, Vitor L; Rodrigues, Maria I; Barile, Daniela

2016-08-01

Enzymatic hydrolysis of lactose has been shown to improve the efficiency and selectivity of membrane-based separations toward the recovery of bioactive oligosaccharides. Achieving maximum lactose hydrolysis requires intrinsic process optimization for each specific substrate, but the effects of those processing conditions on the target oligosaccharides are not well understood. Response surface methodology was used to investigate the effects of pH (3.25-8.25), temperature (35-55°C), reaction time (6 to 58 min), and amount of enzyme (0.05-0.25%) on the efficiency of lactose hydrolysis by β-galactosidase and on the preservation of biologically important sialyloligosaccharides (3'-siallylactose, 6'-siallylactose, and 6'-sialyl-N-acetyllactosamine) naturally present in bovine colostrum whey permeate. A central composite rotatable design was used. In general, β-galactosidase activity was favored at pH values ranging from 3.25 to 5.75, with other operational parameters having a less pronounced effect. A pH of 4.5 allowed for the use of a shorter reaction time (19 min), lower temperature (40°C), and reduced amount of enzyme (0.1%), but complete hydrolysis at a higher pH (5.75) required greater values for these operational parameters. The total amount of sialyloligosaccharides was not significantly altered by the reaction parameters evaluated, suggesting specificity of β-galactosidase from Aspergillus oryzae toward lactose as well as the stability of the oligosaccharides at pH, temperature, and reaction time evaluated. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Lack of the central nervous system- and neural crest-expressed forkhead gene Foxs1 affects motor function and body weight.

PubMed

Heglind, Mikael; Cederberg, Anna; Aquino, Jorge; Lucas, Guilherme; Ernfors, Patrik; Enerbäck, Sven

2005-07-01

To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-beta-galactosidase reporter gene "knock-in" (Foxs1beta-gal/beta-gal) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a beta-galactosidase reporter gene. Staining for beta-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1beta-gal/beta-gal mice perform significantly better (P < 0.01) on a rotating rod than do wt littermates. We have also noted a lower body weight gain (P < 0.05) in Foxs1beta-gal/lbeta-gal males on a high-fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1beta-gal/beta-gal mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function.

Transplantation of transduced chondrocytes protects articular cartilage from interleukin 1-induced extracellular matrix degradation.

PubMed Central

Baragi, V M; Renkiewicz, R R; Jordan, H; Bonadio, J; Hartman, J W; Roessler, B J

1995-01-01

Gene therapy used in the context of delivering a therapeutic gene(s) to chondrocytes offers a new approach for treating chondrocyte-mediated cartilage degradation associated with various human arthropathies including osteoarthritis. In this study, gene delivery to human osteoarthritis chondrocytes in monolayer culture was demonstrated using two adenoviral vectors (Ad.CMVlacZ and Ad.RSVntlacZ) carrying the Escherichia coli beta-galactosidase marker gene, and a third vector (Ad.RSV hIL-1ra) containing the cDNA for human interleukin-1 receptor antagonist. At an moi of 10(3) plaque-forming units/chondrocyte, > 90% of the infected cells stained positive for E. coli beta-galactosidase activity, indicating a high efficiency of transduction. Genetically modified chondrocytes were then transplanted onto the articular surface of osteoarthritic cartilage organ cultures with and without the underlying subchondral bone. Both in situ staining of the cartilage organ cultures for E. coli beta-galactosidase activity and examination by scanning electron microscopy indicated that the transplanted chondrocytes adhered and integrated into the articular surface and continued to express transgenic protein. Chondrocytes transduced with Ad.RSV hIL-1ra and seeded onto the surface of osteoarthritic cartilage secreted high levels of biologically active IL-1 receptor antagonist. The Ad.RSV hIL-1ra-treated cartilage samples were resistant to IL1-induced proteoglycan degradation over 10 d of sustained organ culture. These data demonstrate that transplantation of transduced chondrocytes onto the articular surface protects cartilage from IL-1-induced extracellular matrix degradation. Images PMID:7593634

"In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

ERIC Educational Resources Information Center

Higgins, Pamela J.

2005-01-01

This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

Inactivation of the srtA Gene Affects Localization of Surface Proteins and Decreases Adhesion of Streptococcus pneumoniae to Human Pharyngeal Cells In Vitro

PubMed Central

Kharat, Arun S.; Tomasz, Alexander

2003-01-01

Inactivation of sortase gene srtA in Streptococcus pneumoniae strain R6 caused the release of β-galactosidase and neuraminidase A (NanA) from the cell wall into the surrounding medium. Both of these surface proteins contain the LPXTG motif in the C-terminal domain. Complementation with plasmid-borne srtA reversed protein release. Deletion of murM, a gene involved in the branching of pneumococcal peptidoglycan, also caused partial release of β-galactosidase, suggesting preferential attachment of the protein to branched muropeptides in the cell wall. Inactivation of srtA caused decreased adherence to human pharyngeal cells in vitro but had no effect on the virulence of a capsular type III strain of S. pneumoniae in the mouse intraperitoneal model. The observations suggest that—as in other gram-positive bacteria—sortase-dependent display of proteins occurs in S. pneumoniae and that some of these proteins may be involved in colonization of the human host. PMID:12704150

Release of lysosomal enzymes in Candida albicans phagocytosis by rat peritoneal macrophages.

PubMed

Fontenla de Petrino, S E; Sirena, A

1984-02-15

The present paper reports the in vitro release of lysosomal enzymes in the supernatant of cultures of rat peritoneal macrophages, with the addition of Candida albicans cells. Macrophages were taken from the rat peritoneal cavity 72 hr after non-specific activation with Brain-Heart-Infusion (B.H.I.) broth containing 10% proteose-peptone No. 3. They were then cultured in Parker medium No. 199 (TC 199). After 24 hr a suspension of Candida albicans cells, in a determined concentration, was added to the peritoneal macrophage cultures. At that time, and during pre-determined periods, the following enzymes in the culture supernatants were studied using colorimetric methods: beta-glucuronidase, beta-galactosidase and acid phosphatase. It is concluded that, under identical conditions, the release of beta-galactosidase and acid phosphatase is higher than for beta-glucuronidase. The release rate of all three enzymes is the highest at a 6 hr incubation period, after which, a gradual decrease leads to the rate down to 50% at 24 hr.

Safety and efficacy of enzyme replacement therapy in the nephropathy of Fabry disease

PubMed Central

Fervenza, Fernando C; Torra, Roser; Warnock, David G

2008-01-01

Kidney involvement with progressive loss of kidney function (Fabry nephropathy) is an important complication of Fabry disease, an X-linked lysosomal storage disorder arising from deficiency of α-galactosidase activity. Clinical trials have shown that enzyme replacement therapy (ERT) with recombinant human α-galactosidase clears globotriaosylceramide from kidney cells, and can stabilize kidney function in patients with mild to moderate Fabry nephropathy. Recent trials show that patients with more advanced Fabry nephropathy and overt proteinuria do not respond as well to ERT alone, but can benefit from anti-proteinuric therapy given in conjunction with ERT. This review focuses on the use of enzyme replacement therapy with agalsidase-alfa and agalsidase-beta in adults with Fabry nephropathy. The current results are reviewed and evaluated. The issues of dosing of enzyme replacement therapy, the use of adjunctive agents to control urinary protein excretion, and the individual factors that affect disease severity are reviewed. PMID:19707461

Preparation of Gc protein-derived macrophage activating factor (GcMAF) and its structural characterization and biological activities.

PubMed

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

2002-01-01

Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF. Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay. We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity. Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.

Expression of the Pasteurella haemolytica leukotoxin is inhibited by a locus that encodes an ATP-binding cassette homolog.

PubMed Central

Highlander, S K; Wickersham, E A; Garza, O; Weinstock, G M

1993-01-01

Multicopy and single-copy chromosomal fusions between the Pasteurella haemolytica leukotoxin regulatory region and the Escherichia coli beta-galactosidase gene have been constructed. These fusions were used as reporters to identify and isolate regulators of leukotoxin expression from a P. haemolytica cosmid library. A cosmid clone, which inhibited leukotoxin expression from multicopy and single-copy protein fusions, was isolated and found to contain the complete leukotoxin gene cluster plus additional upstream sequences. The locus responsible for inhibition of expression from leukotoxin-beta-galactosidase fusions was mapped within these upstream sequences, by transposon mutagenesis with Tn5, and its DNA sequence was determined. The inhibitory activity was found to be associated with a predicted 440-amino-acid reading frame (lapA) that lies within a four-gene arginine transport locus. LapA is predicted to be the nucleotide-binding component of this transport system and shares homology with the Clp family of proteases. Images PMID:8359916

Non-detergent sulphobetaines: a new class of molecules that facilitate in vitro protein renaturation.

PubMed

Goldberg, M E; Expert-Bezançon, N; Vuillard, L; Rabilloud, T

1996-01-01

Attempts to renature proteins often yield aggregates rather than native protein. To minimize aggregation, low protein concentrations and/or solubilizing agents are used. Here, we test new solubilizing molecules, non-detergent sulphobetaines, to improve the renaturation of two very different enzymes, hen egg white lysozyme and bacterial beta-D-galactosidase. The renaturation was conducted in the presence of five different sulphobetaines and the yield of active enzyme was measured. The five sulphobetaines improved the yield of native lysozyme up to 12-fold. Some sulphobetaines improved the yield of galactosidase up to 80-fold, but one reduced it 100-fold. Non-detergent sulphobetaines strongly affect the balance between aggregation and folding. Their effect depends on their structure and on their interactions with folding intermediates. These results should serve as a basis for designing more efficient sulphobetaines; for designing improved renaturation protocols using existing sulphobetaines; and for characterizing folding intermediates that interact with sulphobetaines.

Association among bad breath, body mass index, and alcohol intake.

PubMed

Rosenberg, M; Knaan, T; Cohen, D

2007-10-01

Bad breath is a common condition, difficult to assess in the general population. In the present study, we tested the hypothesis that a self-administered questionnaire can help identify factors associated with greater risk of oral malodor. Persons (n = 88) undergoing routine medical check-ups completed a questionnaire including 38 questions on general and oral health, dietary habits, and their own oral malodor levels. Oral malodor assessments included odor judge scores, volatile sulfide levels (via a Halimeter, Interscan Corp.), and salivary beta-galactosidase. Among the questionnaire results, 9 responses were significantly associated with odor judge scores (p < 0.05, unpaired t test), including questions on alcohol intake and body mass index (BMI). Predictions of odor judge scores based on these 9 questions (linear multiple regression analysis) yielded R = 0.601; when introduced together with Halimeter and beta-galactosidase scores, the correlation rose to R = 0.843. The results suggest that alcohol intake and BMI may be factors that help predict oral malodor.

Near-infrared fluorescence probes for enzymes based on binding affinity modulation of squarylium dye scaffold.

PubMed

Oushiki, Daihi; Kojima, Hirotatsu; Takahashi, Yuki; Komatsu, Toru; Terai, Takuya; Hanaoka, Kenjiro; Nishikawa, Makiya; Takakura, Yoshinobu; Nagano, Tetsuo

2012-05-15

We present a novel design strategy for near-infrared (NIR) fluorescence probes utilizing dye-protein interaction as a trigger for fluorescence enhancement. The design principle involves modification of a polymethine dye with cleavable functional groups that reduce the dye's protein-binding affinity. When these functional groups are removed by specific interaction with the target enzymes, the dye's protein affinity is restored, protein binding occurs, and the dye's fluorescence is strongly enhanced. To validate this strategy, we first designed and synthesized an alkaline phosphatase (ALP) sensor by introducing phosphate into the squarylium dye scaffold; this sensor was able to detect ALP-labeled secondary antibodies in Western blotting analysis. Second, we synthesized a probe for β-galactosidase (widely used as a reporter of gene expression) by means of β-galactosyl substitution of the squarylium scaffold; this sensor was able to visualize β-galactosidase activity both in vitro and in vivo. Our strategy should be applicable to obtain NIR fluorescence probes for a wide range of target enzymes.

Structural Elucidation of Enzymatically Synthesized Galacto-oligosaccharides Using Ion-Mobility Spectrometry-Tandem Mass Spectrometry.

PubMed

Carević, Milica; Bezbradica, Dejan; Banjanac, Katarina; Milivojević, Ana; Fanuel, Mathieu; Rogniaux, Hélène; Ropartz, David; Veličković, Dušan

2016-05-11

Galacto-oligosaccharides (GOS) represent a diverse group of well-characterized prebiotic ingredients derived from lactose in a reaction catalyzed with β-galactosidases. Enzymatic transgalactosylation results in a mixture of compounds of various degrees of polymerization and types of linkages. Because structure plays an important role in terms of prebiotic activity, it is of crucial importance to provide an insight into the mechanism of transgalactosylation reaction and occurrence of different types of β-linkages during GOS synthesis. Our study proved that a novel one-step method, based on ion-mobility spectrometry-tandem mass spectrometry (IMS-MS/MS), enables complete elucidation of GOS structure. It has been shown that β-galactosidase from Aspergillus oryzae has the highest affinity toward formation of β-(1→3) or β-(1→6) linkages. Additionally, it was observed that the occurrence of different linkages varies during the reaction course, indicating that tailoring favorable GOS structures with improved prebiotic activity can be achieved by adequate control of enzymatic synthesis.

[Fabry's disease and hypoparathyroidism].

PubMed

Misery, Laurent; Gregoire, Madeleine; Prieur, Fabienne; Froissart, Régis; Guffon, Nathalie; Maitre, Séverine; Fond, Laurent; Denis, Laurence; Perrot, Jean-Luc; Cambazard, Frédéric

2002-06-01

Fabry's disease is due to alpha-galactosidase deficiency. This rare lysosomal storage disease is transmitted by recessive X-linked heredity. Sphingolipids (galactosyl-glucosyl-ceramide) accumulate in many organs. A 19-year-old man with known hypoparathyroidism presented with telangiectasia and angiokeratomas on the buttocks, the hips, the hands and around the navel. For many years, he suffered from paroxysmal pain in the hands and feet. From childhood, he had complained of diffuse abdominal pain, associated with diarrhea. Ophthalmological slit lamp fundus examination showed corneal telangiectasia and cornea verticella. There was no kidney or heart involvement. The diagnosis of Fabry's disease was confirmed by very low levels of alpha-galactosidase. We did not find any other association of hypoparathyroidism and Fabry's disease in the literature. Hypoparathyroidism is not a manifestation of Fabry's disease. Idiopathic hypoparathyroidism is very rare and a genetic origin is known. This disease can be recessive X-linked. A co-transmission of idiopathic hypoparathyroidism and Fabry's disease is probable in our patient.

Identification of a Novel GLA Mutation (L206 P) in a Patient with Fabry Disease.

PubMed

Kim, Ji-Hoon; Kim, Gee-Hee; Park, Hoon-Suk; Choi, Jin-A; Bae, Jung-Min; Cho, Uiju

2017-03-01

We report a new α-Galactosidase A (αGal-A) mutation in a 39-year-old Korean born, male Fabry disease patient. Fabry disease is a devastating, progressive inborn error of metabolism caused by X-linked genetic mutations. In this case, the first clinical symptom to occur was in childhood consisting of a burning pain originating in the extremities then radiating inwards to the limbs. This patient also stated to have ringing in his ears, angiokeratomas on his trunk, and cornea verticillata. He visited an outpatient cardiologist due to intermittent and atypical chest discomfort at the age of 39. Electrocardiographic and echocardiographic examination showed left ventricular hypertrophy. A physical examination revealed proteinuria without hematuria. The patient's plasma αGal-A activity was markedly lower than the mean value of the controls. After genetic counseling and obtaining written informed consent, we identified one hemizygous mutation in exon 4 of galactosidase alpha, c.617T>C (p.Leu206 Pro). He was eventually diagnosed as having Fabry disease.

Biomarkers of cell senescence

DOEpatents

Dimri, Goberdhan P.; Campisi, Judith; Peacocke, Monica

1998-01-01

The present invention provides a biomarker system for the in vivo and in vitro assessment of cell senescence. In the method of the present invention, .beta.-galactosidase activity is utilized as a means by which cell senescence may be assessed either in vitro cell cultures or in vivo.

Biomarkers of cell senescence

DOEpatents

Dirmi, Goberdhan P.; Campisi, Judith; Peacocke, Monica

1996-01-01

The present invention provides a biomarker system for the in vivo and in vitro assessment of cell senescence. In the method of the present invention, .beta.-galactosidase activity is utilized as a means by which cell senescence may be assessed either in in vitro cell cultures or in vivo.

A general approach for chemical labeling and rapid, spatially controlled protein inactivation

PubMed Central

Marks, Kevin M.; Braun, Patrick D.; Nolan, Garry P.

2004-01-01

Chemical labeling of proteins inside of living cells can enable studies of the location, movement, and function of proteins in vivo. Here we demonstrate an approach for chemical labeling of proteins that uses the high-affinity interaction between an FKBP12 mutant (F36V) and a synthetic, engineered ligand (SLF′). A fluorescein conjugate to the engineered ligand (FL-SLF′) retained binding to FKBP12(F36V) and possessed similar fluorescence properties as parental fluorescein. FL-SLF′ labeled FKBP12(F36V) fusion proteins in live mammalian cells, and was used to monitor the subcellular localization of a membrane targeted FKBP12(F36V) construct. Chemical labeling of FKBP12(F36V) fusion proteins with FL-SLF′ was readily detectable at low expression levels of the FKBP12(F36V) fusion, and the level of fluorescent staining with FL-SLF′ was proportional to the FKBP12(F36V) expression level. This FL-SLF′-FKBP12(F36V) labeling technique was tested in fluorophore assisted laser inactivation (FALI), a light-mediated technique to rapidly inactivate fluorophore-labeled target proteins. FL-SLF′ mediated FALI of a β-galactosidase-FKBP12(F36V) fusion protein, causing rapid inactivation of >90% of enzyme activity upon irradiation in vitro. FL-SLF′ also mediated FALI of a β-galactosidase fusion expressed in living NIH 3T3 cells, where β-galactosidase activity was reduced in 15 s. Thus, FL-SLF′ can be used to monitor proteins in vivo and to target rapid, spatially and temporally defined inactivation of target proteins in living cells in a process that we call FK-FALI. PMID:15218100

Screening for Fabry disease in left ventricular hypertrophy: documentation of a novel mutation.

PubMed

Baptista, Ana; Magalhães, Pedro; Leão, Sílvia; Carvalho, Sofia; Mateus, Pedro; Moreira, Ilídio

2015-08-01

Fabry disease is a lysosomal storage disease caused by enzyme α-galactosidase A deficiency as a result of mutations in the GLA gene. Cardiac involvement is characterized by progressive left ventricular hypertrophy. To estimate the prevalence of Fabry disease in a population with left ventricular hypertrophy. The patients were assessed for the presence of left ventricular hypertrophy defined as a left ventricular mass index ≥ 96 g/m2 for women or ≥ 116 g/m2 for men. Severe aortic stenosis and arterial hypertension with mild left ventricular hypertrophy were exclusion criteria. All patients included were assessed for enzyme α-galactosidase A activity using dry spot testing. Genetic study was performed whenever the enzyme activity was decreased. A total of 47 patients with a mean left ventricular mass index of 141.1 g/m2 (± 28.5; 99.2 to 228.5 g/m2] were included. Most of the patients were females (51.1%). Nine (19.1%) showed decreased α-galactosidase A activity, but only one positive genetic test - [GLA] c.785G>T; p.W262L (exon 5), a mutation not previously described in the literature. This clinical investigation was able to establish the association between the mutation and the clinical presentation. In a population of patients with left ventricular hypertrophy, we documented a Fabry disease prevalence of 2.1%. This novel case was defined in the sequence of a mutation of unknown meaning in the GLA gene with further pathogenicity study. Thus, this study permitted the definition of a novel causal mutation for Fabry disease - [GLA] c.785G>T; p.W262L (exon 5).

Antibacterial activity and mode of action of ε-polylysine against Escherichia coli O157:H7.

PubMed

Zhang, Xiaowei; Shi, Ce; Liu, Zuojia; Pan, Fengguang; Meng, Rizeng; Bu, Xiujuan; Xing, Heqin; Deng, Yanhong; Guo, Na; Yu, Lu

2018-04-10

Gram-negative Escherichia coli O157:H7 were chosen as model bacteria to evaluate the antimicrobial mechanism of ε-polylysine (ε-PL). The antibacterial activity of ε-PL was detected by measuring the minimum inhibitory concentration values as well as the time-kill curve. The membrane integrity was determined by ultraviolet (UV) absorption, membrane potential (MP) assay and flow cytometry (FCM) experiments. The permeability of the inner membrane was detected by β-galactosidase activity assay. Furthermore, electron microscopy [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] was utilized to observe bacterial morphology. These results demonstrated that ε-PL showed its antibacterial activity by changing the integrity and permeability of cell membranes, leading to rapid cell death. The electron microscopy analysis (SEM and TEM) results indicated that the bacterial cell morphology, membrane integrity and permeability were spoiled when the E. coli O157:H7 cells were exposed to minimum inhibitory concentrations of ε-PL (16 µg ml -1 ). In addition, the bacterial membrane was damaged more severely when the concentration of ε-PL was increased. The present study investigated the antimicrobial mechanism of ε-PL by measuring the content of cytoplasmic β-galactosidase, proteins and DNA. In addition, SEM and TEM were carried out to assess the mechanism. These results show that ε-PL has the ability to decrease the content of large molecules, cellular soluble proteins and nucleic acids associated with increasing the content of cytoplasmic β-galactosidase in supernatant by causing damage to the cell membranes. Consequently, the use of ε-PL as a natural antimicrobial agent should eventually become an appealing method in the field of food preservation.

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

PubMed

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.

Effects of abscisic acid, ethylene and sugars on the mobilization of storage proteins and carbohydrates in seeds of the tropical tree Sesbania virgata (Leguminosae).

PubMed

Tonini, Patricia Pinho; Purgatto, Eduardo; Buckeridge, Marcos Silveira

2010-10-01

Endospermic legumes are abundant in tropical forests and their establishment is closely related to the mobilization of cell-wall storage polysaccharides. Endosperm cells also store large numbers of protein bodies that play an important role as a nitrogen reserve in this seed. In this work, a systems approach was adopted to evaluate some of the changes in carbohydrates and hormones during the development of seedlings of the rain forest tree Sesbania virgata during the period of establishment. Seeds imbibed abscisic acid (ABA), glucose and sucrose in an atmosphere of ethylene, and the effects of these compounds on the protein contents, α-galactosidase activity and endogenous production of ABA and ethylene by the seeds were observed. The presence of exogenous ABA retarded the degradation of storage protein in the endosperm and decreased α-galactosidase activity in the same tissue during galactomannan degradation, suggesting that ABA represses enzyme action. On the other hand, exogenous ethylene increased α-galactosidase activity in both the endosperm and testa during galactomannan degradation, suggesting an inducing effect of this hormone on the hydrolytic enzymes. Furthermore, the detection of endogenous ABA and ethylene production during the period of storage mobilization and the changes observed in the production of these endogenous hormones in the presence of glucose and sucrose, suggested a correlation between the signalling pathway of these hormones and the sugars. These findings suggest that ABA, ethylene and sugars play a role in the control of the hydrolytic enzyme activities in seeds of S. virgata, controlling the process of storage degradation. This is thought to ensure a balanced flow of the carbon and nitrogen for seedling development.

Elements in the murine c-mos messenger RNA 5'-untranslated region repress translation of downstream coding sequences.

PubMed

Steel, L F; Telly, D L; Leonard, J; Rice, B A; Monks, B; Sawicki, J A

1996-10-01

Murine c-mos transcripts isolated from testes have 5'-untranslated regions (5'UTRs) of approximately 300 nucleotides with a series of four overlapping open reading frames (ORFs) upstream of the AUG codon that initiates the Mos ORF. Ovarian c-mos transcripts have shorter 5'UTRs (70-80 nucleotides) and contain only 1-2 of the upstream ORFs (uORFs). To test whether these 5'UTRs affect translational efficiency, we have constructed plasmids for the expression of chimeric transcripts with a mos-derived 5'UTR fused to the Escherichia coli beta-galactosidase coding region. Translational efficiency has been evaluated by measuring beta-galactosidase activity NIH3T3 cells transiently transfected with these plasmids and with plasmids where various mutations have been introduced into the 5'UTR. We show that the 5'UTR characteristic of testis-specific c-mos mRNA strongly represses translation relative to the translation of transcripts that contain a 5'UTR derived from beta-globin mRNA, and this is mainly due to the four uORFs. Each of the four upstream AUG triplets can be recognized as a start site for translation, and no single uAUG dominates the repressive effect. The uORFs repress translation by a mechanism that is not affected by the amino acid sequence in the COOH-terminal region of the uORF-encoded peptides. The very short uORF (AUGUGA) present in ovary-specific transcripts does not repress translation. Staining of testis sections from transgenic mice carrying chimeric beta-galactosidase transgene constructs, which contain a mos 5'UTR with or without the uATGs, suggests that the uORFs can dramatically change the pattern of expression in spermatogenic cells.

The Hematopoietic Stem Cell Therapy for Exploration of Deep Space

NASA Astrophysics Data System (ADS)

Ohi, Seigo; Roach, Allana-Nicole; Ramsahai, Shweta; Kim, Bak C.; Fitzgerald, Wendy; Riley, Danny A.; Gonda, Steven R.

2004-02-01

Astronauts experience severe/invasive disorders caused by space environments. These include hematological and cardiac abnormalities, bone and muscle losses, immunodeficiency, neurological disorders and cancer. Exploiting the extraordinary plasticity of hematopoietic stem cells (HSCs), which differentiate not only to all types of blood cells, but also to various tissues, including muscle, bone, skin, liver, and neuronal cells, we advanced a hypothesis that some of the space-caused disorders might be amenable to hematopoietic stem cell therapy (HSCT) so as to maintain astronauts' homeostasis. If this were achievable, the HSCT could promote human exploration of deep space. Using mouse models of human anemia (β-thalassemia) and spaceflight (hindlimb suspension unloading system), we have obtained feasibility results of HSCT for space anemia, muscle loss, and immunodeficiency. For example, the β-thalassemic mice were successfully transplanted with isologous HSCs, resulting in chimerism of hemoglobin species and alleviation of the hemoglobinopathy. In the case of HSCT for muscle loss, β-galactosidase-marked HSCs, which were prepared from β-galactosidase-transgenic mice, were detected by the X-gal wholemount staining procedure in the hindlimbs of unloaded mice following transplantation. Histochemical and physical analyses indicated structural contribution of HSCs to the muscle. To investigate HSCT for immunodeficiency, β-galactosidase-transformed Escherichia coli was used as the reporter bacteria, and infected to control and the hindlimb suspended mice. Results of the X-gal stained tissues indicated that the HSCT could help eliminate the E. coli infection. In an effort to facilitate the HSCT in space, growth of HSCs has been optimized in the NASA Rotating Wall Vessel (RWV) culture systems, including Hydrodynamic Focusing Bioreactor (HFB).

Control of protein synthesis in Escherichia coli: strain differences in control of translational initiation after energy source shift-down.

PubMed Central

Jacobson, L A; Jen-Jacobson, L

1980-01-01

We have studied the parameters of protein synthesis in a number of Escherichia coli strains after a shift-down from glucose-minimal to succinate-minimal medium. One group of strains, including K-12(lambda) (ATCC 10798) and NF162, showed a postshift translational yield of 50 to 65% and a 2- to 2.5-fold increase in the functional lifetime of general messenger ribonucleic acid. There was no change in the lag time for beta-galactosidase induction in these strains after the shift-down. A second group, including W1 and W2, showed no reduction in translational yield, no change in the functional lifetime of messenger ribonucleic acid, and a 50% increase in the lag time for beta-galactosidase induction. Evidence is presented which indicates that this increased lag time is not the result of a decreased rate of polypeptide chain propagation. A third group of strains, including NF161, CP78, and NF859, showed an intermediate pattern: translational yield was reduced to about 75% of normal, and the messenger ribonucleic acid functional lifetime was increased by about 50%. Calculation of the relative postshift rates of translational initiation gave about 0.2, 1.0, and 0.5, respectively, for the three groups. There was no apparent correlation between the ability to control translation and the genotypes of these strains at the relA, relX, or spoT loci. Measurements of the induction lag for beta-galactosidase during short-term glucose starvation or after a down-shift induced by alpha-methylglucoside indicated that these regimens elicit responses that are physiologically distinct from those elicited by a glucose-to-succinate shift-down. PMID:6155375

Control of protein synthesis in Escherichia coli: strain differences in control of translational initiation after energy source shift-down.

PubMed

Jacobson, L A; Jen-Jacobson, L

1980-06-01

We have studied the parameters of protein synthesis in a number of Escherichia coli strains after a shift-down from glucose-minimal to succinate-minimal medium. One group of strains, including K-12(lambda) (ATCC 10798) and NF162, showed a postshift translational yield of 50 to 65% and a 2- to 2.5-fold increase in the functional lifetime of general messenger ribonucleic acid. There was no change in the lag time for beta-galactosidase induction in these strains after the shift-down. A second group, including W1 and W2, showed no reduction in translational yield, no change in the functional lifetime of messenger ribonucleic acid, and a 50% increase in the lag time for beta-galactosidase induction. Evidence is presented which indicates that this increased lag time is not the result of a decreased rate of polypeptide chain propagation. A third group of strains, including NF161, CP78, and NF859, showed an intermediate pattern: translational yield was reduced to about 75% of normal, and the messenger ribonucleic acid functional lifetime was increased by about 50%. Calculation of the relative postshift rates of translational initiation gave about 0.2, 1.0, and 0.5, respectively, for the three groups. There was no apparent correlation between the ability to control translation and the genotypes of these strains at the relA, relX, or spoT loci. Measurements of the induction lag for beta-galactosidase during short-term glucose starvation or after a down-shift induced by alpha-methylglucoside indicated that these regimens elicit responses that are physiologically distinct from those elicited by a glucose-to-succinate shift-down.

A field release of genetically engineered gypsy moth (Lymantria dispar L.) Nuclear Polyhedrosis Virus (LdNPV)

Treesearch

Vincent D' Amico; Joseph S. Elkinton; John D. Podgwaite; James M. Slavicek; Michael L. McManus; John P. Burand

1999-01-01

The gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus was genetically engineered for nonpersistence by removal of the gene coding for polyhedrin production and stabilized using a coocclusion process. A β-galactosidase marker gene was inserted into the genetically engineered virus (LdGEV) so that infected larvae could be tested for...

Exploring the Ubiquitin-Proteasome Protein Degradation Pathway in Yeast

ERIC Educational Resources Information Center

Will, Tamara J.; McWatters, Melissa K.; McQuade, Kristi L.

2006-01-01

This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme beta-galactosidase (beta-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce beta-gal expression, cycloheximide is added to halt…

Biomarkers of cell senescence

DOEpatents

Dimri, G.P.; Campisi, J.; Peacocke, M.

1998-08-18

The present invention provides a biomarker system for the in vivo and in vitro assessment of cell senescence. In the method of the present invention, {beta}-galactosidase activity is utilized as a means by which cell senescence may be assessed either in vitro cell cultures or in vivo. 1 fig.

Biomarkers of cell senescence

DOEpatents

Dirmi, G.P.; Campisi, J.; Peacocke, M.

1996-02-13

The present invention provides a biomarker system for the in vivo and in vitro assessment of cell senescence. In the method of the present invention, {beta}-galactosidase activity is utilized as a means by which cell senescence may be assessed either in in vitro cell cultures or in vivo. 1 fig.

40 CFR 141.21 - Coliform sampling.

Code of Federal Regulations, 2011 CFR

2011-07-01

... transit. 3 Lactose broth, as commercially available, may be used in lieu of lauryl tryptose broth, if the... total coliform, using lactose broth, is less than 10 percent. 4 If inverted tubes are used to detect gas... negative-rates may be based on lactose fermentation, the rapid test for β-galactosidase and cytochrome...

40 CFR 141.21 - Coliform sampling.

Code of Federal Regulations, 2010 CFR

2010-07-01

... transit. 3 Lactose broth, as commercially available, may be used in lieu of lauryl tryptose broth, if the... total coliform, using lactose broth, is less than 10 percent. 4 If inverted tubes are used to detect gas... negative-rates may be based on lactose fermentation, the rapid test for β-galactosidase and cytochrome...

Carbocysteine counteracts the effects of cigarette smoke on cell growth and on the SIRT1/FoxO3 axis in bronchial epithelial cells.

PubMed

Pace, E; Di Vincenzo, S; Ferraro, M; Bruno, A; Dino, P; Bonsignore, M R; Battaglia, S; Saibene, F; Lanata, L; Gjomarkaj, M

2016-08-01

Cigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. Aim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. 16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. CSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. The study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli.

PubMed

Ihara, Kohei; Sato, Kazuki; Hori, Hatsuhiro; Makino, Yumiko; Shigenobu, Shuji; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

2017-04-01

The alaE gene in Escherichia coli encodes an l-alanine exporter that catalyzes the active export of l-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l-alanyl-l-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less β-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ-deficient and harbored this reporter plasmid showed significant induction of β-galactosidase activity (approximately 17-fold) in the presence of 6 mM l-alanine, l-leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, lrp-deficient cells showed almost no β-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide -161 to -83 with a physiologically relevant affinity (apparent K D , 288.7 ± 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis-element was increased by l-alanine and l-leucine, but not by Ala-Ala and d-alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of l-alanine, which eventually leads to intracellular homeostasis of l-alanine concentrations. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Immunoreactivity of polyclonal antibodies generated against the carboxy terminus of the predicted amino acid sequence of the Huntington disease gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alkatib, G.; Graham, R.; Pelmear-Telenius, A.

1994-09-01

A cDNA fragment spanning the 3{prime}-end of the Huntington disease gene (from 8052 to 9252) was cloned into a prokaryotic expression vector containing the E. Coli lac promoter and a portion of the coding sequence for {beta}-galactosidase. The truncated {beta}-galactosidase gene was cleaved with BamHl and fused in frame to the BamHl fragment of the Huntington disease gene 3{prime}-end. Expression analysis of proteins made in E. Coli revealed that 20-30% of the total cellular proteins was represented by the {beta}-galactosidase-huntingtin fusion protein. The identity of the Huntington disease protein amino acid sequences was confirmed by protein sequence analysis. Affinity chromatographymore » was used to purify large quantities of the fusion protein from bacterial cell lysates. Affinity-purified proteins were used to immunize New Zealand white rabbits for antibody production. The generated polyclonal antibodies were used to immunoprecipitate the Huntington disease gene product expressed in a neuroblastoma cell line. In this cell line the antibodies precipitated two protein bands of apparent gel migrations of 200 and 150 kd which together, correspond to the calculated molecular weight of the Huntington disease gene product (350 kd). Immunoblotting experiments revealed the presence of a large precursor protein in the range of 350-750 kd which is in agreement with the predicted molecular weight of the protein without post-translational modifications. These results indicate that the huntingtin protein is cleaved into two subunits in this neuroblastoma cell line and implicate that cleavage of a large precursor protein may contribute to its biological activity. Experiments are ongoing to determine the precursor-product relationship and to examine the synthesis of the huntingtin protein in freshly isolated rat brains, and to determine cellular and subcellular distribution of the gene product.« less

Hydrolytic enzymes production by Aspergillus section Nigri in presence of butylated hydroxyanisole and propyl paraben on peanut meal extract agar.

PubMed

Barberis, Carla L; Landa, María F; Barberis, Mauricio G; Giaj-Merlera, Guillermo; Dalcero, Ana M; Magnoli, Carina E

2014-01-01

In the last years, food grade antioxidants are used safely as an alternative to traditional fungicides to control fungal growth in several food and agricultural products. In this work, the effect of butylated hydroxyanisole (BHA) and propyl paraben (PP) on two hydrolytic enzyme activity (β-d-glucosidase and α-d-galactosidase) by Aspergillus section Nigri species under different water activity conditions (aW; 0.98, 0.95 and 0.93) and incubation time intervals (24, 48, 72 and 96h) was evaluated on peanut-based medium. The activity of two glycosidases, β-d-glucosidase and α-d-galactosidase, was assayed using as substrates 4-nitrophenyl-β-d-glucopyranosido and 4-nitrophenyl-α-d-galactopyranosido, respectively. The enzyme activity was determined by the increase in optical density at 405nm caused by the liberation of p-nitrophenol by enzymatic hydrolysis of the substrate. Enzyme activity was expressed as micromoles of p-nitrophenol released per minute. The major inhibition in β-d-glucosidase activity of A. carbonarius and A. niger was found with 20mmoll(-1) of BHA or PP at 0.98 and 0.95 aW, respectively, whereas for α-d-galactosidase activity a significant decrease in enzyme activity with respect to control was observed in A. carbonarius among 5 to 20mmoll(-1) of BHA or PP in all conditions assayed. Regarding A. niger, the highest percentages of enzyme inhibition activity were found with 20mmoll(-1) of BHA or PP at 0.95 aW and 96h. The results of this work provide information about the capacity of BHA and PP to inhibit in vitro conditions two of the most important hydrolytic enzymes produced by A. carbonarius and A. niger species. Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Digestive enzymes activity in subsequent generations of Cameraria ohridella larvae harvested from horse chestnut trees after treatment with imidacloprid.

PubMed

Stygar, Dominika; Michalczyk, Katarzyna; Dolezych, Bogdan; Nakonieczny, Miroslaw; Migula, Pawel; Zaak, Maria; Sawczyn, Tomasz; Karcz-Socha, Iwona; Kukla, Michal; Zwirska-Korczala, Krystyna; Buldak, Rafal

2013-01-01

In the present study we describe the effect of chloronicotinoid pesticide (imidacloprid) on the digestive enzymes activity of the Cameraria ohridella larvae after lasting 1 year sublethal exposure to imidacloprid pesticide. Caterpillars - L4 stage (fourth instar, hyperphagic tissue-feeding phase) - were collected from chemically protected white horse chestnut trees 1 year after imidacloprid treatment, and compared with caterpillars collected from non-treated trees in a previous study. Enzymes activity of α-amylase, disaccharidases, glycosidases and proteases was assayed. The presence of pesticide in ingested food changed the digestive enzymes profile of caterpillars. The analysis of correlations between different digestive enzymes showed many significant correlations (P<0.05) among glycolytic activities like β-glucosidase and α-galactosidase activities. Statistically significant correlations for proteolytic activity were found between trypsin and chymotrypsin activity and aminopeptidase activity that occurred only in the 1st generation. PCA distinguished five primary components with eigenvalues higher than 1, from which the first two explain almost 59% of analyzed results. Surprisingly, in the pesticide treated groups significantly higher activities of sucrase and lactase in relation to control were found. In general, glycosidase (α-glucosidase, β-glucosidase and β-galactosidase) activities showed a similar pattern of activity in different generations. These results contrast with those obtained with control larvae, where significant differences in activities of α-glucosidase, β-glucosidase and β-galactosidase may result from the different quantity and quality food intake by subsequent generations of larvae. No inter-generation differences in total proteolytic activity were observed in treated larvae. The absolute value of total proteolytic activity was higher than that in the control group. The pesticide present in the vascular system of the horse chestnut

A beta-l-Arabinopyranosidase from Streptomyces avermitilis is a novel member of glycoside hydrolase family 27.

PubMed

Ichinose, Hitomi; Fujimoto, Zui; Honda, Mariko; Harazono, Koichi; Nishimoto, Yukifumi; Uzura, Atsuko; Kaneko, Satoshi

2009-09-11

Arabinogalactan proteins (AGPs) are a family of plant cell surface proteoglycans and are considered to be involved in plant growth and development. Because AGPs are very complex molecules, glycoside hydrolases capable of degrading AGPs are powerful tools for analyses of the AGPs. We previously reported such enzymes from Streptomyces avermitilis. Recently, a beta-l-arabinopyranosidase was purified from the culture supernatant of the bacterium, and its corresponding gene was identified. The primary structure of the protein revealed that the catalytic module was highly similar to that of glycoside hydrolase family 27 (GH27) alpha-d-galactosidases. The recombinant protein was successfully expressed as a secreted 64-kDa protein using a Streptomyces expression system. The specific activity toward p-nitrophenyl-beta-l-arabinopyranoside was 18 micromol of arabinose/min/mg, which was 67 times higher than that toward p- nitrophenyl-alpha-d-galactopyranoside. The enzyme could remove 0.1 and 45% l-arabinose from gum arabic or larch arabinogalactan, respectively. X-ray crystallographic analysis reveals that the protein had a GH27 catalytic domain, an antiparallel beta-domain containing Greek key motifs, another antiparallel beta-domain forming a jellyroll structure, and a carbohydrate-binding module family 13 domain. Comparison of the structure of this protein with that of alpha-d-galactosidase showed a single amino acid substitution (aspartic acid to glutamic acid) in the catalytic pocket of beta-l-arabinopyranosidase, and a space for the hydroxymethyl group on the C-5 carbon of d-galactose bound to alpha-galactosidase was changed in beta-l-arabinopyranosidase. Mutagenesis study revealed that the residue is critical for modulating the enzyme activity. This is the first report in which beta-l-arabinopyranosidase is classified as a new member of the GH27 family.

Oatmeal porridge: impact on microflora-associated characteristics in healthy subjects.

PubMed

Valeur, Jørgen; Puaschitz, Nathalie G; Midtvedt, Tore; Berstad, Arnold

2016-01-14

Oatmeal porridge has been consumed for centuries and has several health benefits. We aimed to investigate the effect of oatmeal porridge on gut microflora functions. A total of ten healthy subjects ingested 60 g oatmeal porridge daily for 1 week. The following microflora-associated characteristics were assessed before and after the intervention: intestinal gas production following lactulose ingestion, faecal excretion of SCFA and faecal levels of urease and β-galactosidase. In addition, rectal levels of PGE2 were measured. Microbial fermentation as evaluated by intestinal gas production and excretion of SCFA did not change significantly following the dietary intervention. However, faecal levels of β-galactosidase and urease decreased after eating oatmeal porridge (P=0·049 and 0·031, respectively). Host inflammatory state, as measured by rectal levels of PGE2, also decreased, but the change was not significant (P=0·168). The results suggest that oatmeal porridge has an effect on gut microbial functions and may possess potential prebiotic properties that deserve to be investigated further.

Enzymatic degradation of oligosaccharides in pinto bean flour.

PubMed

Song, Danfeng; Chang, Sam K C

2006-02-22

The use of dry edible beans is limited due to the presence of flatulence factors, the raffinose oligosaccharides. Our objective was to investigate the process for the removal of oligosaccharides from pinto bean using enzymatic treatment and to compare it to removal by soaking and cooking methods. Crude enzyme preparation was produced by six fungal species on wheat bran- and okara-based substrates with soy tofu whey. The loss of raffinose oligosaccharides after soaking pinto beans for 16 h at the room temperature was 10%, after cooking for 90 min was 52%, and after autoclaving for 30 min was 58%. On the other hand, the treatment using crude alpha-galactosidase (60 U mL(-1)) produced by Aspergillus awamori NRRL 4869 from wheat bran-based substrate with soy tofu whey on pinto bean flour for 2 h completely hydrolyzed raffinose oligosaccharides. These results supported that the enzymatic treatment was the most effective among various processing methods tested for removing the raffinose oligosaccharides, and hence, crude alpha-galactosidases from fungi have potential use in the food industry.

Enzyme replacement therapy of Fabry disease.

PubMed

Clarke, Joe T R; Iwanochko, R Mark

2005-08-01

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of the enzyme alpha-galactosidase A and results in pain, progressive renal impairment, cardiomyopathy, and cerebrovascular disease. The results of two major randomized, double-blind, placebo-controlled clinical trials and open-label extensions have shown that replacement of the deficient enzyme with either of two preparations of recombinant human alpha-galactosidase A, agalsidase-alfa, and agalsidase-beta is safe. Biweekly i.v. infusions of 0.2 mg/kg of agalsidase-alfa were associated with a significant decrease in pain and stabilization of renal function. Biweekly infusions of 1 mg/kg of agalsidase-beta were associated with virtually complete clearing of accumulated glycolipid substrate from renal and cutaneous capillary endothelial cells. Several smaller, open-label studies, along with observations made in the course of monitoring large numbers of patients on enzyme replacement therapy, indicated that treatment stabilizes renal function and produces significant improvements in myocardial mass and function. Treatment of Fabry disease by enzyme replacement has a significant impact on at least some serious complications of the disease.

Structural definition of a potent macrophage activating factor derived from vitamin D3-binding protein with adjuvant activity for antibody production.

PubMed

Yamamoto, N

1996-10-01

Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.

Hydrogen Treatment Protects against Cell Death and Senescence Induced by Oxidative Damage.

PubMed

Han, A Lum; Park, Seong-Hoon; Park, Mi Sung

2017-02-28

Hydrogen has potential for preventive and therapeutic applications as an antioxidant. However, micro- and macroparticles of hydrogen in water disappear easily over time. In order to eliminate reactive oxygen species (ROS) related with the aging process, we used functional water containing nanoparticle hydrogen. Nanoparticle hydrogen does not disappear easily and collapse under water after long periods of time. We used murine embryonic fibroblasts that were isolated from 12.5-day embryos of C57BL/6 mice. We investigated the ability of nanoparticle hydrogen in water to suppress hydroxyurea-induced ROS production, cytotoxicity, and the accumulation of β-galactosidase (an indicator of aging), and promote cell proliferation. The accumulation of β-galactosidase in the cytoplasm and the appearance of abnormal nuclei were inhibited by daily treatment of cells with hydrogen water. When the aging process was accelerated by hydroxyurea-induced oxidative stress, the effect of hydrogen water was even more remarkable. Thus, this study showed the antioxidant and anti-senescence effects of hydrogen water. Nanoparticle hydrogen water is potentially a potent anti-aging agent.

[Genetic instability of probiotic characteristics in the Bifidobacterium longum subsp. longum B379M strain during cultivation and maintenance].

PubMed

Averina, O V; Nezametdinova, V Z; Alekseeva, M G; Danilenko, V N

2012-11-01

The stability of inheriting several genes in the Russian commercial strain Bifidobacterium longum subsp. longum B379M during cultivation and maintenance under laboratory conditions has been studied. The examined genes code for probiotic characteristics, such as utilization of several sugars (lacA2 gene, encoding beta-galactosidase; ara gene, encoding arabinosidase; and galA gene, encoding arabinogalactan endo-beta-galactosidase); synthesis of bacteriocins (lans gene, encoding lanthionine synthetase); and mobile gene tet(W), conferring resistance to the antibiotic tetracycline. The other gene families studied include the genes responsible for signal transduction and adaptation to stress conditions in the majority of bacteria (serine/threonine protein kinases and the toxin-antitoxin systems of MazEF and RelBE types) and transcription regulators (genes encoding WhiB family proteins). Genomic DNA was analyzed by PCR using specially selected primers. A loss of the genes galA and tet(W) has been shown. It is proposed to expand the requirements on probiotic strains, namely, to control retention of the key probiotic genes using molecular biological methods.

The activity of hydrolases of larval stages of Anisakis simplex (Nematoda).

PubMed

Lopieńska-Biernat, Elzbieta; Zółtowska, Krystyna; Rokicki, Jerzy

2004-01-01

Activity of hydrolases during the third and fourth larval stage of Anisakis simplex was identified by applying the API ZYM test method. In A. simplex larvae the activity of phosphatases was high, particularly that of acid phosphatase (40 nmol/mg(-1)). Among esterases lack of activity of lipase (C14) is worth noticing while the activity of esterases (C4) and (C8) was high. The activity of those later two enzymes was higher in L3 larvae than in L4 larvae. The highest activity in the subclass of glucosidases was recorded for beta-fucosidase and N-acetyl-beta-glucosaminidase. A higher activity in L3 larvae than in L4 larvae was recorded for: beta-glucuronidase and N-acetyl-beta-glucosaminidase (2-fold) and beta-fucosidase (3-fold). Differently the activity of beta-galactosidase and beta-glucosidase was higher in L4 larvae than in L3 larvae. The tests did not show activity of alpha-galactosidase, beta-glucosidase and alpha-mannosidase on both larval forms.

The construction of recombinant industrial yeasts free of bacterial sequences by directed gene replacement into a nonessential region of the genome.

PubMed

Xiao, W; Rank, G H

1989-03-15

The yeast SMR1 gene was used as a dominant resistance-selectable marker for industrial yeast transformation and for targeting integration of an economically important gene at the homologous ILV2 locus. A MEL1 gene, which codes for alpha-galactosidase, was inserted into a dispensable upstream region of SMR1 in vitro; different treatments of the plasmid (pWX813) prior to transformation resulted in 3' end, 5' end and replacement integrations that exhibited distinct integrant structures. One-step replacement within a nonessential region of the host genome generated a stable integration of MEL1 devoid of bacterial plasmid DNA. Using this method, we have constructed several alpha-galactosidase positive industrial Saccharomyces strains. Our study provides a general method for stable gene transfer in most industrial Saccharomyces yeasts, including those used in the baking, brewing (ale and lager), distilling, wine and sake industries, with solely nucleotide sequences of interest. The absence of bacterial DNA in the integrant structure facilitates the commercial application of recombinant DNA technology in the food and beverage industry.

Anderson-Fabry disease in heart failure.

PubMed

Akhtar, M M; Elliott, P M

2018-06-16

Anderson-Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene that result in deficiency of the enzyme alpha-galactosidase A. The worldwide incidence of Fabry's disease is reported to be in the range of 1 in 40,000-117,000, although this value may be a significant underestimate given under recognition of symptoms and delayed or missed diagnosis. Deficiency in alpha-galactosidase A causes an accumulation of neutral glycosphingolipids such as globotriaosylceramide (Gb3) in lysosomes within various tissues including the vascular endothelium, kidneys, heart, eyes, skin and nervous system. Gb3 accumulation induces pathology via the release of pro-inflammatory cytokines, growth-promoting factors and by oxidative stress, resulting in myocardial extracellular matrix remodelling, left ventricular hypertrophy (LVH), vascular dysfunction and interstitial fibrosis. Cardiac involvement manifesting as ventricular hypertrophy, systolic and diastolic dysfunction, valvular abnormalities and conduction tissue disease is common in AFD and is associated with considerable cardiovascular morbidity and mortality from heart failure, sudden cardiac death and stroke-related death.

Development of a pyrG Mutant of Aspergillus oryzae Strain S1 as a Host for the Production of Heterologous Proteins

PubMed Central

Ling, Selina Oh Siew; Storms, Reginald; Zheng, Yun; Rodzi, Mohd Rohaizad Mohd; Mahadi, Nor Muhammad; Illias, Rosli Md

2013-01-01

The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5′-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. β-Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous β-galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus. PMID:24381522

β-galactosidase-catalyzed synthesis of galactosyl chlorphenesin and its characterization.

PubMed

Lee, Sang-Eun; Jo, Tae-Min; Lee, Hyang-Yeol; Lee, Jongsung; Jung, Kyung-Hwan

2013-11-01

We synthesized galactosyl chlorphenesin (CPN-G) using β-gal-containing Escherichia coli (E. coli) cells in which the conversion yield of chlorphenesin (CPN) to CPN-G reached about 64 % during 12 h. CPN-G was identified and characterized using high-performance liquid chromatography, liquid chromatography-mass spectrometry, Fourier transform-infrared spectrometry, and nuclear magnetic resonance analysis ((1)H and (13)C). We verified that a galactose was covalently bound to a CPN alcohol group during CPN-G synthesis throughout these analyses. In particular, by the hydrolysis of CPN-G using β-gal, it was confirmed that a galactose was bound to CPN. The minimal inhibitory concentration (MIC) results showed that the CPN-G MICs were fairly similar to those of CPN. HACAT cell viability was significantly higher in CPN-G-treated cells than in CPN-treated cells at concentrations of 0.0-20.0 mM. Finally, we accomplished the synthesis of less toxic CPN-G, compared with CPN, using β-gal-containing E. coli cells as whole cells without changes in the MICs against microorganisms.

Discovery and Testing of Ricin Therapeutics

DTIC Science & Technology

2012-06-01

variants lung disease ER degradation Fabri disease α -D-galactosidase neurological disease ER degradation Oculocutaneous albinism tyrosinase...against other pathogens and human diseases . 15. SUBJECT TERMS Ricin toxin, dislocation, small weight compounds, chemical inhibitors, high-content screen...the U.S. Departments of Health and Human Services (HHS) and Centers for Disease Control and Prevention (CDC). The toxin’s ability to kill cells and

Atlas of Wnt and R-spondin gene expression in the developing male mouse lower urogenital tract.

PubMed

Mehta, Vatsal; Abler, Lisa L; Keil, Kimberly P; Schmitz, Christopher T; Joshi, Pinak S; Vezina, Chad M

2011-11-01

Prostate development is influenced by β-catenin signaling, but it is unclear which β-catenin activators are involved, where they are synthesized, and whether their mRNA abundance is influenced by androgens. We identified WNT/β-catenin-responsive β-galactosidase activity in the lower urogenital tract (LUT) of transgenic reporter mice, but β-galactosidase activity differed among the four mouse strains we examined. We used in situ hybridization to compare patterns of Wnts, r-spondins (Rspos, co-activators of β-catenin signaling), β-catenin-responsive mRNAs, and an androgen receptor-responsive mRNA in wild type fetal male, fetal female, and neonatal male LUT. Most Wnt and Rspo mRNAs were present in LUT during prostate development. Sexually dimorphic expression patterns were observed for WNT/β-catenin-responsive genes, and for Wnt2b, Wnt4, Wnt7a, Wnt9b, Wnt10b, Wnt11, Wnt16, and Rspo3 mRNAs. These results reveal sexual differences in WNT/β-catenin signaling in fetal LUT, supporting the idea that this pathway may be directly or indirectly responsive to androgens during prostate ductal development. Copyright © 2011 Wiley-Liss, Inc.

A defect in the inflammation-primed macrophage-activation cascade in osteopetrotic rats.

PubMed

Yamamoto, N; Lindsay, D D; Naraparaju, V R; Ireland, R A; Popoff, S N

1994-05-15

Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to wild-type rats but not in osteopetrotic (op) mutant rats. In vitro treatment of wild-type rat peritoneal cells with lyso-Pc or DDG efficiently activated macrophages whereas treatment of op mutant rat peritoneal cells with lyso-Pc or DDG did not activate macrophages. The inflammation-primed macrophage activation cascade in rats requires participation of B lymphocytes and vitamin D binding protein (DBP). Lyso-Pc-inducible beta-galactosidase of wild-type rat B lymphocytes can convert DBP to the macrophage-activating factor (MAF), whereas B lymphocytes of the op mutant rats were shown to be deficient in lyso-Pc-inducible beta-galactosidase. DBP is conserved among mammalian species. Treatment of human DBP (Gc1 protein) with commercial glycosidases yields an extremely high titrated MAF as assayed on mouse and rat macrophages. Because the enzymatically generated MAF (GcMAF) bypasses the role of lymphocytes in macrophage activation, the op mutant rat macrophages were efficiently activated by administration of a small quantity (100 pg/rat) of GcMAF. Likewise, in vitro treatment of op rat peritoneal cells with as little as 40 pg GcMAF/ml activated macrophages.

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

PubMed

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.

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

PubMed Central

Condemine, G; Hugouvieux-Cotte-Pattat, N; Robert-Baudouy, J

1986-01-01

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis. PMID:3949717

Characterization of volatile sulfur compound production by Solobacterium moorei.

PubMed

Tanabe, Shin-ichi; Grenier, Daniel

2012-12-01

Solobacterium moorei is a Gram positive bacterium that has been specifically associated with halitosis. The aim of this study was to characterize volatile sulfur compound (VSC) production by S. moorei. S. moorei was either grown or incubated in the presence of various supplements prior to determining VSC production with a Halimeter sulfide monitor. The effect of exogenous proteases or glycosidase inhibitors on VSC production by S. moorei was examined. We first showed that S. moorei can convert cysteine into hydrogen sulfide. The capacity of S. moorei to produce VSCs from serum, saliva, and mucin was dependent on the presence of an exogenous source of proteases such as pancreatic trypsin or Porphyromonas gingivalis gingipains. VSC production from mucin was inhibited by the presence of a β-galactosidase inhibitor, thus suggesting that deglycosylation of mucin by S. moorei is critical for VSC production. Our study suggests that S. moorei can be a major source of malodorous compounds in halitosis by producing VSCs through a process involving the β-galactosidase activity of the bacterium and an exogenous source of proteases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Construction of the industrial ethanol-producing strain of Saccharomyces cerevisiae able to ferment cellobiose and melibiose.

PubMed

Zhang, L; Guo, Z P; Ding, Z Y; Wang, Z X; Shi, G Y

2012-01-01

The gene mel1, encoding alpha-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and beta-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanol-producing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular alpha-galactosidase and beta-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC 1 co-expressing 2 genes could achieve 0.29 OD600 h(-1) and a biomass yield up to 7.8 g l(-1) dry cell weight on medium containing 10.0 g l(-1) cellobiose and 10.0 g l(-1) melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG 1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification and fermentation process. Approximately 36.5 g l(-1) ethanol was produced from 100 g of cellulose supplied with 5 g l(-1) melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield.

LacI(Ts)-Regulated Expression as an In Situ Intracellular Biomolecular Thermometer▿

PubMed Central

McCabe, K. M.; Lacherndo, E. J.; Albino-Flores, I.; Sheehan, E.; Hernandez, M.

2011-01-01

In response to needs for in situ thermometry, a temperature-sensitive vector was adapted to report changes in the intracellular heat content of Escherichia coli in near-real time. This model system utilized vectors expressing increasing quantities of β-galactosidase in response to stepwise temperature increases through a biologically relevant range (22 to 45°C). As judged by calibrated fluorometric and colorimetric reporters, both whole E. coli cells and lysates expressed significant repeatable changes in β-galactosidase activity that were sensitive to temperature changes of less than 1°C (35 to 45°C). This model system suggests that changes in cellular heat content can be detected independently of the medium in which cells are maintained, a feature of particular importance where the medium is heterogeneous or nonaqueous, or otherwise has a low heat transfer capacity. We report here that the intracellular temperature can be reliably obtained in near-real time using reliable fluorescent reporting systems from cellular scales, with a 20°C range of detection and at least 0.7°C sensitivity between 35 and 45°C. PMID:21378059

Mutational Analysis of a C-Dependent Late Promoter of Bacteriophage Mu

PubMed Central

Chiang, L. W.; Howe, M. M.

1993-01-01

Late transcription of bacteriophage Mu initiates at four promoters, P(lys), P(I), P(P) and P(mom), and requires the Mu C protein and the host RNA polymerase. Promoter-containing DNA fragments extending ~200 bp upstream and downstream of the 5' starts of the lys, I and P transcripts were cloned into a multicopy lacZ-expression plasmid. Promoter activity, assayed by β-galactosidase expression, was determined under two different conditions: (1) with C provided from a compatible plasmid in the absence of other Mu factors and (2) with C provided from an induced Mu prophage. β-galactosidase activities were greatest for P(lys), intermediate for P(I), and lowest for P(P). Similar analysis of plasmids containing nested sets of deletions removing 5' or 3' sequences of P(lys) demonstrated that a 68-bp region was sufficient for full activity. Point mutations were generated within the 68-bp region by mutagenic oligonucleotide-directed PCR (Mod-PCR). Properties of the lys promoter mutants indicated that, in addition to the -10 region, a 19-bp region from -52 to -34 containing the C footprint is required for C-dependent promoter activity. PMID:8293968

Long-Term Outcomes of Kidney Transplantation in Fabry Disease.

PubMed

Ersözlü, Sara; Desnick, Robert J; Huynh-Do, Uyen; Canaan-Kühl, Sima; Barbey, Frédéric; Genitsch, Vera; Müller, Thomas; Cheetham, Marcus; Flammer, Andreas; Schaub, Stefan; Nowak, Albina

2018-04-24

Fabry disease is a rare X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene that obliterate or markedly reduce α-galactosidase A activity. This results in the systemic accumulation of its glycosphingolipid substrates in body fluids and organs, including the kidney. Fabry nephropathy can lead to end-stage renal disease requiring kidney transplantation. Little is known about its long-term outcomes and the overall patient survival after kidney transplantation. Here, we report 17 Fabry patients (15 males, 2 females) who received kidney transplants and their long-term treatment and follow-up at 4 specialized Fabry centers. The posttransplant follow-up ranged to 25 years, with a median of 11.5 [range 0.8-25.5] years. Graft survival was similar and death-censored graft survival was superior to matched controls. Fabry patients died with functioning kidneys, mostly from cardiac causes. In 2 males 14 and 23 years posttransplant, the grafts had a few typical FD lamellar inclusions, presumably originating from invading host macrophages and vascular endothelial cells. We conclude that kidney transplantation has an excellent long-term outcome in Fabry disease.

In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds.

PubMed

Tseng, Jen-Chieh; Kung, Andrew L

2015-06-24

Here we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as β-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry. Efficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular β-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity. In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

A biochemical method for assessing the neurotoxic effects of misonidazole in the rat.

PubMed Central

Rose, G. P.; Dewar, A. J.; Stratford, I. J.

1980-01-01

A proven biochemical method for assessing chemically induced neurotoxicity has been applied to the study of the toxic effects of misonidazole (MISO) in the rat. This involves the fluorimetric measurement of beta-glucuronidase and beta-galactosidase activities in homogenates of rat nervous tissue. The tissues analysed were sciatic/posterior tibial nerve (SPTN) cut into 4 sections, trigeminal ganglia and cerebellum. MISO administered i.p. to Wistar rats in doses greater than 300 mg/kg/day for 7 consecutive days produced maximal increases in both beta-glucuronidase and beta-galactosidase activities in th SPTN at 4 weeks (140-180% of control values). The highest increases were associated with the most distal secretion of the nerve. Significant enzyme-activity changes were also found in the trigeminal ganglia and cerebellum of MISO-dosed rats. The greatest activity occurred 4-5 weeks after dosing, and was dose-related. It is concluded that, in the rat, MISO can produce biochemical changes consistent with a dying-back peripheral neuropathy, and biochemical changes suggestive of cerebellar damage. This biochemical approach would appear to offer a convenient quantitative method for the detection of neurotoxic effects of other potential radio-sensitizing drugs. PMID:7459223

ATF6α regulates morphological changes associated with senescence in human fibroblasts

PubMed Central

Martin, Nathalie; Saas, Laure; Cormenier, Johanna; Malaquin, Nicolas; Huot, Ludovic; Slomianny, Christian; Bouali, Fatima; Vercamer, Chantal; Hot, David; Pourtier, Albin; Chevet, Eric; Abbadie, Corinne; Pluquet, Olivier

2016-01-01

Cellular senescence is known as an anti-tumor barrier and is characterized by a number of determinants including cell cycle arrest, senescence associated β-galactosidase activity and secretion of pro-inflammatory mediators. Senescent cells are also subjected to enlargement, cytoskeleton-mediated shape changes and organelle alterations. However, the underlying molecular mechanisms responsible for these last changes remain still uncharacterized. Herein, we have identified the Unfolded Protein Response (UPR) as a player controlling some morphological aspects of the senescent phenotype. We show that senescent fibroblasts exhibit ER expansion and mild UPR activation, but conserve an ER stress adaptive capacity similar to that of exponentially growing cells. By genetically invalidating the three UPR sensors in senescent fibroblasts, we demonstrated that ATF6α signaling dictates senescence-associated cell shape modifications. We also show that ER expansion and increased secretion of the pro-inflammatory mediator IL6 were partly reversed by silencing ATF6α in senescent cells. Moreover, ATF6α drives the increase of senescence associated-β-galactosidase activity. Collectively, these findings unveil a novel and central role for ATF6α in the establishment of morphological features of senescence in normal human primary fibroblasts. PMID:27563820

ATF6α regulates morphological changes associated with senescence in human fibroblasts.

PubMed

Druelle, Clémentine; Drullion, Claire; Deslé, Julie; Martin, Nathalie; Saas, Laure; Cormenier, Johanna; Malaquin, Nicolas; Huot, Ludovic; Slomianny, Christian; Bouali, Fatima; Vercamer, Chantal; Hot, David; Pourtier, Albin; Chevet, Eric; Abbadie, Corinne; Pluquet, Olivier

2016-10-18

Cellular senescence is known as an anti-tumor barrier and is characterized by a number of determinants including cell cycle arrest, senescence associated β-galactosidase activity and secretion of pro-inflammatory mediators. Senescent cells are also subjected to enlargement, cytoskeleton-mediated shape changes and organelle alterations. However, the underlying molecular mechanisms responsible for these last changes remain still uncharacterized. Herein, we have identified the Unfolded Protein Response (UPR) as a player controlling some morphological aspects of the senescent phenotype. We show that senescent fibroblasts exhibit ER expansion and mild UPR activation, but conserve an ER stress adaptive capacity similar to that of exponentially growing cells. By genetically invalidating the three UPR sensors in senescent fibroblasts, we demonstrated that ATF6α signaling dictates senescence-associated cell shape modifications. We also show that ER expansion and increased secretion of the pro-inflammatory mediator IL6 were partly reversed by silencing ATF6α in senescent cells. Moreover, ATF6α drives the increase of senescence associated-β-galactosidase activity. Collectively, these findings unveil a novel and central role for ATF6α in the establishment of morphological features of senescence in normal human primary fibroblasts.

Senescence as a novel mechanism involved in β-adrenergic receptor mediated cardiac hypertrophy

PubMed Central

Sun, Rongrong; Zhu, Baoling; Sun, Yan; Shi, Dandan; Chen, Li; Zhang, Youyi; Li, Zijian; Xue, Lixiang

2017-01-01

Pathological cardiac hypertrophy used to be elucidated by biomechanical, stretch-sensitive or neurohumoral mechanisms. However, a series of hints have indicated that hypertrophy process simulates senescence program. However, further evidence need to be pursued. To verify this hypothesis and examine whether cardiac senescence is a novel mechanism of hypertrophy induced by isoproterenol, 2-month-old male Sprague Dawley rats were subjected to isoproterenol infusion (0.25mg/kg/day) for 7 days by subcutaneous injection). Key characteristics of senescence (senescence-associated β-galactosidase activity, lipofuscin, expression of cyclin-dependent kinase inhibitors) were examined in cardiac hypertrophy model. Senescence-like phenotype, such as increased senescence-associated β-galactosidase activity, accumulation of lipofuscin and high levels of cyclin-dependent kinase inhibitors (e.g. p16, p19, p21 and p53) was found along the process of cardiac hypertrophy. Cardiac-specific transcription factor GATA4 increased in isoproterenol-treated cardiomyocytes as well. We further found that myocardial hypertrophy could be inhibited by resveratrol, an anti-aging compound, in a dose-dependent manner. Our results showed for the first time that cardiac senescence is involved in the process of pathological cardiac hypertrophy induced by isoproterenol. PMID:28783759

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

PubMed Central

Xuan, J W; Fournier, P; Declerck, N; Chasles, M; Gaillardin, C

1990-01-01

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked. Images PMID:2388625

Optimization of an innovative hollow-fiber process to produce lactose-reduced skim milk.

PubMed

Neuhaus, Winfried; Novalin, Senad; Klimacek, Mario; Splechtna, Barbara; Petzelbauer, Inge; Szivak, Alexander; Kulbe, Klaus D

2006-07-01

The research field for applications of lactose hydrolysis has been investigated for several decades. Lactose intolerance, improvement for technical processing of solutions containing lactose, and utilization of lactose in whey are the main topics for development of biotechnological processes. We report here the optimization of a hollow-fiber membrane reactor process for enzymatic lactose hydrolysis. Lactase was circulated abluminally during luminal flow of skim milk. The main problem, the growth of microorganisms in the enzyme solution, was minimized by sterile filtration, ultraviolet irradiation, and temperature adjustment. Based on previous experiments at 23 +/- 2 degrees C, further characterization was carried out at 8 +/- 2 degrees C, 15 +/- 2 degrees C (beta-galactosidase), and 58 +/- 2 degrees C (thermostable beta-glycosidase) varying enzyme activity and flow rates. For a cost-effective process, the parameters 15 +/- 2 degrees C, 240 U/mL of beta-galactosidase, an enzyme solution flow rate of 25 L/h, and a skim milk flow rate of about 9 L/h should be used in order to achieve an aimed productivity of 360 g/(L x h) and to run at conditions for the highest process long-term stability.

A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

PubMed

York, Joanne; Nunberg, Jack H

2018-01-01

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boustany, R.M.; Qian, W.H.; Suzuki, K.

The authors describe four new mutations in the [beta]-galactosidase gene. These are the first mutations causing infantile and juvenile GM[sub 1]-gangliosidosis to be described in American patients. Cell lines from two patients with juvenile and from six patients with infantile GM[sub 1]-gangliosidosis were analyzed. Northern blot analysis showed the acid [beta]-galactosidase message to be of normal size and quantity in two juvenile and four infantile cases and of normal size but reduced quantity in two infantile cases. The mutations are distinct from the Japanese mutations. All are point mutations leading to amino acid substitutions: Lys[sup 577] [yields] Arg, Arg[sup 590]more » [yields] His, and Glu[sup 632] [yields] Gly. The fourth mutation, Arg[sup 208] [yields] Cys, accounts for 10 of 16 possible alleles. Two infantile cases from Puerto Rico of Spanish ancestry are homozygous for this mutation, suggesting that this allele may have come to South America and North America via Puerto Rico. That these mutations cause clinical disease was confirmed by marked reduction in catalytic activity of the mutant proteins in the Cos-1 cell expression system. 12 refs., 5 figs., 2 tabs.« less

Natural exopolysaccharides enhance survival of lactic acid bacteria in frozen dairy desserts.

PubMed

Hong, S H; Marshall, R T

2001-06-01

Viable lactic acid-producing bacteria in frozen dairy desserts can be a source of beta-galactosidase for persons who absorb lactose insufficiently. However, freezing kills many of the cells, causing loss of enzymatic activity. Cultures selected for high beta-galactosidase activities and high survival rates in the presence of bile were examined for survivability during freezing in reduced-fat ice cream. Encapsulated S. thermophilus strains survived better than their nonencapsulated mutants in reduced-fat ice cream after freezing and frozen storage at -29 degrees C for 16 d (28 vs. 19%). However, a small nonencapsulated strain of Lactobacillus delbrueckii sp. bulgaricus survived better than the large encapsulated strain in reduced-fat ice cream. Factors that improved survival of encapsulated S. thermophilus 1068 in ice cream were 1) harvest of cells in the late-log phase of growth at 37 degrees C rather than at 40, 42.5, or 45 degrees C; 2) overrun at 50% rather than 100%; and 3) storage at -17 degrees C rather than -23 or -29 degrees C. Survival of strain ST1068 was unaffected by 1) neutralization of acid during growth or 2) substitution of nitrogen for air in building overrun.

Amyloid-linked cellular toxicity triggered by bacterial inclusion bodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzalez-Montalban, Nuria; Departament de Genetica i de Microbiologia, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona; Ciber de Bioingenieria, Biomateriales y Nanomedicina

The aggregation of proteins in the form of amyloid fibrils and plaques is the characteristic feature of some pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. The mechanisms by which the aggregation processes result in cell damage are under intense investigation but recent data indicate that prefibrillar aggregates are the most proximate mediators of toxicity rather than mature fibrils. Since it has been shown that prefibrillar forms of the nondisease-related misfolded proteins are highly toxic to cultured mammalian cells we have studied the cytoxicity associated to bacterial inclusion bodies that have been recently described as protein deposits presenting amyloid-likemore » structures. We have proved that bacterial inclusion bodies composed by a misfolding-prone {beta}-galactosidase fusion protein are clearly toxic for mammalian cells but the {beta}-galactosidase wild type enzyme forming more structured thermal aggregates does not impair cell viability, despite it also binds and enter into the cells. These results are in the line that the most cytotoxic aggregates are early prefibrilar assemblies but discard the hypothesis that the membrane destabilization is Key event to subsequent disruption of cellular processes, such as ion balance, oxidative state and the eventually cell death.« less

Translational regulation of sigma 32 synthesis: requirement for an internal control element.

PubMed Central

Kamath-Loeb, A S; Gross, C A

1991-01-01

We have investigated the sequence requirements for the translational regulation of sigma 32 by examining the behavior of a new rpoH-lacZ protein fusion containing a short N-terminal fragment of sigma 32 fused to beta-galactosidase. Although the fusion retains rpoH translational initiation signals, it lacks translational regulation, implicating coding sequences within rpoH in this regulatory process. Images PMID:2050641

Breast Cancer Gene Therapy: Development of Novel Non-Invasive Magnetic Resonance Assay to Optimize Efficacy

DTIC Science & Technology

2007-05-01

inhalation-mouse). HFB had been proposed as a veterinary anesthetic and has been used in many species including ponies, sheep, cats, dogs , rats and...anesthetic and has been used in many species including ponies, sheep, cats, dogs , rats and mice, but was abandoned due to its flammability [265...gene activity detection- specifically, using fluorinated substrates of β-galactosidase to reveal gene activity. Our prototype molecule PFONPG (p

Induction of lactose transport in Escherichia coli during the absence of phospholipid synthesis.

PubMed Central

Weisberg, L J; Cronan, J E; Nunn, W D

1975-01-01

Induction of lactose transport and of beta-galactosidase synthesis was examined in two Escherichia coli strains that require exogenous glycerol for phospholipid synthesis and growth. No preferential inhibition of lactose transport induction was observed when phospholipid synthesis was restricted to 5 to 10% of the normal rate. We conclude that the lactose transport system does not require concurrent phospholipid synthesis for its functional assembly. PMID:1097412

Thio-isoglobotrihexosylceramide, an agonist for activating invariant natural killer T cells.

PubMed

Xia, Chengfeng; Zhou, Dapeng; Liu, Chengwen; Lou, Yanyan; Yao, Qingjia; Zhang, Wenpeng; Wang, Peng George

2006-11-23

Thio-isoglobotrihexosylceramide (S-iGb3) might be resistant to alpha-galactosidases in antigen-presenting cells and have a longer retaining time in the lysosome before being loaded to CD1d. The biological assay showed that S-iGb3 demonstrates a much higher increase as a stimulatory ligand toward invariant natural killer T (iNKT) cells as compared to iGb3. [structure: see text].

Two modes of control of pilA, the gene encoding type 1 pilin in Escherichia coli.

PubMed Central

Orndorff, P E; Spears, P A; Schauer, D; Falkow, S

1985-01-01

Type 1 piliation in Escherichia coli is subject to metastable regulation at the transcriptional level (B. I. Eisenstein, Science 214:337-339, 1981). However, the genes controlling in this fashion are not known. We present evidence that the pilA gene, encoding the structural subunit of type 1 pili, is subject to metastable transcriptional regulation. A pilA'-lacZ fusion, constructed in vitro on a recombinant plasmid, was used in conjunction with a recBC sbcB mutant of E. coli K-12 to introduce the fusion into the chromosomal region encoding Pil. This fusion was found to be subject to metastable transcriptional control. The rate of switching from the Lac+ to the Lac- phenotype was 4 X 10(-4) per cell per generation and 6.2 X 10(-4) in the opposite direction. A ca. 10-fold difference in beta-galactosidase activity was observed between phenotypically "ON" (Lac+) and "OFF" (Lac-) populations. P1 transduction experiments showed that the element determining the ON or OFF phenotype was tightly linked to pilA. In addition to the metastable regulation of pilA, a second type of transcriptional regulation was effected by the product of a gene, hyp, adjacent to pilA. By using a recombinant plasmid containing just a pilA'-lacZ fusion and the putative pilA promoter, we found that a lesion in hyp conferred a beta-galactosidase activity about fivefold higher than that of a strain possessing the parental hyp gene. Mutants constructed to have a pilA'-lacZ fusion and a hyp::Tn5-132 mutation in the chromosome exhibited a frequency of switching from Lac+ to Lac- and vice versa indistinguishable from that of the parental strain. However, in the ON mode, hyp::Tn5-132 mutants showed a twofold-higher beta-galactosidase activity. Thus, hyp does not appear to affect metastable variation but does affect the level of transcription of the pilA gene in the ON (transcribed) mode. Images PMID:3930469

[High-level expression of heterologous protein based on increased copy number in Saccharomyces cerevisiae].

PubMed

Zhang, Xinjie; He, Peng; Tao, Yong; Yang, Yi

2013-11-04

High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering. We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected. mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein. The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering

Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector.

PubMed

Brooks, A I; Halterman, M W; Chadwick, C A; Davidson, B L; Haak-Frendscho, M; Radel, C; Porter, C; Federoff, H J

1998-04-30

To develop a reproducible gene transfer method for the murine CNS we evaluated delivery of various gene vehicles using mechanical or manual stereotaxic intracranial inoculation. A microprocessor controlled microsyringe pump (The World Precision Instruments/UltraMicroPump) programmable for volume, rate and syringe size and designed to dispense nanoliter and picoliter volumes was compared to a standard manual deliver method. Gene transfer efficiency of two viral vectors, two synthetic cationic lipid molecules, and naked DNA were evaluated in mice injected unilaterally in two brain regions. Animals received 1 microl over 10 min. of either HSVlac (1 x 10(5) b.f.u), AdLac (1 x 10(5) p.f.u), Tfx-10 or Tfx-20 (2.6 microg DNA in 2.0 microl Tfx; 1:1 charge ratio of DNA to liposome), or naked DNA (HSVlac plasmid, 10 microg/microl). After 4 days, animals from each group were perfused and tissue prepared for X-gal histochemical detection of beta-galactosidase expression. Blue cells were observed in the HSV, Adenovirus, and Tfx-20 groups only at the injection site in animals injected using the UMP. Animals injected manually exhibited fewer blue cells and positive cells were not restricted to the injection site. To quantify expression, tissue punches harvested from the injection sites as well as other brain regions were analyzed using a chemiluminescent reporter assay to detect beta-galactosidase (Galacto-Light). These data indicated increased activity in all animals injected with a lacZ containing vector via the UMP as compared to manual delivery: A 41% increase in the expression levels of beta-gal in HSVlac infected animals (p = 0.0029); a 29% increase in Adlac infected animals (p = 0.01); a 56% increase in Tfx-10 transduced animals (p = 0.04); a 24% increase in Tfx-20 transduced animals (p = 0.01); and a 69% increase in naked DNA gene transfer (p = 0.05). Total beta-galactosidase activity was greatest in HSVlac infected mice followed by Adlac > Tfx-20 > Tfx-10 = naked DNA.

Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.

PubMed

Matsunaga, James; Schlax, Paula J; Haake, David A

2013-11-01

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double

Genetic transformation of a clinical (genital tract), plasmid-free isolate of Chlamydia trachomatis: engineering the plasmid as a cloning vector.

PubMed

Wang, Yibing; Kahane, Simona; Cutcliffe, Lesley T; Skilton, Rachel J; Lambden, Paul R; Persson, Kenneth; Bjartling, Carina; Clarke, Ian N

2013-01-01

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle" that it is possible to "knock out" selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for

Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus

PubMed Central

Ward, Todd W.; Kimmick, Michael W.; Afanasiev, Boris N.; Carlson, Jonathan O.

2001-01-01

Aedes aegypti densonucleosis virus (AeDNV) has two promoters that have been shown to be active by reporter gene expression analysis (B. N. Afanasiev, Y. V. Koslov, J. O. Carlson, and B. J. Beaty, Exp. Parasitol. 79:322–339, 1994). Northern blot analysis of cells infected with AeDNV revealed two transcripts 1,200 and 3,500 nucleotides in length that are assumed to express the structural protein (VP) gene and nonstructural protein genes, respectively. Primer extension was used to map the transcriptional start site of the structural protein gene. Surprisingly, the structural protein gene transcript began at an initiator consensus sequence, CAGT, 60 nucleotides upstream from the map unit 61 TATAA sequence previously thought to define the promoter. Constructs with the β-galactosidase gene fused to the structural protein gene were used to determine elements necessary for promoter function. Deletion or mutation of the initiator sequence, CAGT, reduced protein expression by 93%, whereas mutation of the TATAA sequence at map unit 61 had little effect. An additional open reading frame was observed upstream of the structural protein gene that can express β-galactosidase at a low level (20% of that of VP fusions). Expression of the AeDNV structural protein gene was shown to be stimulated by the major nonstructural protein NS1 (Afanasiev et al., Exp. parasitol., 1994). To determine the sequences required for transactivation, expression of structural protein gene–β-galactosidase gene fusion constructs differing in AeDNV genome content was measured with and without NS1. The presence of NS1 led to an 8- to 10-fold increase in expression when either genomic end was present, compared to a 2-fold increase with a construct lacking the genomic ends. An even higher (37-fold) increase in expression occurred with both genomic ends present; however, this was in part due to template replication as shown by Southern blot analysis. These data indicate the location and importance of

Genomic and expression analysis of the flax (Linum usitatissimum) family of glycosyl hydrolase 35 genes

PubMed Central

2013-01-01

Background Several β-galactosidases of the Glycosyl Hydrolase 35 (GH35) family have been characterized, and many of these modify cell wall components, including pectins, xyloglucans, and arabinogalactan proteins. The phloem fibres of flax (Linum usitatissimum) have gelatinous-type cell walls that are rich in crystalline cellulose and depend on β-galactosidase activity for their normal development. In this study, we investigate the transcript expression patterns and inferred evolutionary relationships of the complete set of flax GH35 genes, to better understand the functions of these genes in flax and other species. Results Using the recently published flax genome assembly, we identified 43 β-galactosidase-like (BGAL) genes, based on the presence of a GH35 domain. Phylogenetic analyses of their protein sequences clustered them into eight sub-families. Sub-family B, whose members in other species were known to be expressed in developing flowers and pollen, was greatly under represented in flax (p-value < 0.01). Sub-family A5, whose sole member from arabidopsis has been described as its primary xyloglucan BGAL, was greatly expanded in flax (p-value < 0.01). A number of flax BGALs were also observed to contain non-consensus GH35 active sites. Expression patterns of the flax BGALs were investigated using qRT-PCR and publicly available microarray data. All predicted flax BGALs showed evidence of expression in at least one tissue. Conclusion Flax has a large number of BGAL genes, which display a distinct distribution among the BGAL sub-families, in comparison to other closely related species with available whole genome assemblies. Almost every flax BGAL was expressed in fibres, the majority of which expressed predominately in fibres as compared to other tissues, suggesting an important role for the expansion of this gene family in the development of this species as a fibre crop. Variations displayed in the canonical GH35 active site suggest a variety of roles

Genomic and expression analysis of the flax (Linum usitatissimum) family of glycosyl hydrolase 35 genes.

PubMed

Hobson, Neil; Deyholos, Michael K

2013-05-23

Several β-galactosidases of the Glycosyl Hydrolase 35 (GH35) family have been characterized, and many of these modify cell wall components, including pectins, xyloglucans, and arabinogalactan proteins. The phloem fibres of flax (Linum usitatissimum) have gelatinous-type cell walls that are rich in crystalline cellulose and depend on β-galactosidase activity for their normal development. In this study, we investigate the transcript expression patterns and inferred evolutionary relationships of the complete set of flax GH35 genes, to better understand the functions of these genes in flax and other species. Using the recently published flax genome assembly, we identified 43 β-galactosidase-like (BGAL) genes, based on the presence of a GH35 domain. Phylogenetic analyses of their protein sequences clustered them into eight sub-families. Sub-family B, whose members in other species were known to be expressed in developing flowers and pollen, was greatly under represented in flax (p-value < 0.01). Sub-family A5, whose sole member from arabidopsis has been described as its primary xyloglucan BGAL, was greatly expanded in flax (p-value < 0.01). A number of flax BGALs were also observed to contain non-consensus GH35 active sites. Expression patterns of the flax BGALs were investigated using qRT-PCR and publicly available microarray data. All predicted flax BGALs showed evidence of expression in at least one tissue. Flax has a large number of BGAL genes, which display a distinct distribution among the BGAL sub-families, in comparison to other closely related species with available whole genome assemblies. Almost every flax BGAL was expressed in fibres, the majority of which expressed predominately in fibres as compared to other tissues, suggesting an important role for the expansion of this gene family in the development of this species as a fibre crop. Variations displayed in the canonical GH35 active site suggest a variety of roles unique to flax, which will require

Ret Receptor: Functional Consequences of Oncogenic Rearrangements.

DTIC Science & Technology

1996-10-01

incorporation of the thymidine analog 5- bromodeoxyuridine (BrdU) and its subsequent detection by immunostaining (33). Following nuclear ...other LexA- fussions to test for Ret/ptc2 specific interaction. Seventeen of the library plasmids yielded co-transformants which were 3- galactosidase...cellsexpressing the EGFR/Ret chimera and M. Pierotti for the Ret/ptc2 events in papillary thyroid carcinoma (28). In a nuclear micro- clone. injection assay the

Molecular Biology and Functions of Carrier Proteins. Annual Symposium (46th) of the Society of General Physiologists held in Woods Hole, Massachusetts on September 10-13, 1992. Volume 48

DTIC Science & Technology

1993-09-13

grants from the NIH, NSF. and National Foundation for Cancer Research. M. A. Lemmon is supported by a predoctoral fellowship from the Howard Hughes...American Cancer Society Research Professor. References Ahmad. M.. and H. Bussey. 1988. Topology of membrane insertion in vitro and plasma membrane assembly...Beckwith. 1989. Genetic studies on the inabilitv of beta -galactosidase to be translocated across thc E. coli cytoplasmic membrane. Journal of Bactenoloi

Occurrence and diversity of marine yeasts in Antarctica environments

NASA Astrophysics Data System (ADS)

Zhang, Xue; Hua, Mingxia; Song, Chunli; Chi, Zhenming

2012-03-01

A total of 28 yeast strains were obtained from the sea sediment of Antarctica. According to the results of routine identification and molecular characterization, the strains belonged to species of Yarrowia lipolytica, Debaryomyces hansenii, Rhodotorula slooffiae, Rhodotorula mucilaginosa, Sporidiobolus salmonicolor, Aureobasidium pullulans, Mrakia frigida and Guehomyces pullulans, respectively. The Antarctica yeasts have wide potential applications in biotechnology, for some of them can produce β-galactosidase and killer toxins.

Three Dimensional Immobilization of Beta-Galactosidase on a Silicon Surface (Preprint)

DTIC Science & Technology

2006-12-01

initial activity after 10 days at 24°C. The ability to generate three- dimensional structures with enhanced loading capacity for biosensing molecules...dimensional structures for biosensors (Charles et al. 2004). Silicon samples that had been washed but not activated with APTS did not retain any enzyme...preparation. The use of silica particles to build a 3-dimensional structure not only provides an increased capacity for the immobilization of β

Mutually exclusive expression of human red and green visual pigment-reporter transgenes occurs at high frequency in murine cone photoreceptors.

PubMed

Wang, Y; Smallwood, P M; Cowan, M; Blesh, D; Lawler, A; Nathans, J

1999-04-27

This study examines the mechanism of mutually exclusive expression of the human X-linked red and green visual pigment genes in their respective cone photoreceptors by asking whether this expression pattern can be produced in a mammal that normally carries only a single X-linked visual pigment gene. To address this question, we generated transgenic mice that carry a single copy of a minimal human X chromosome visual pigment gene array in which the red and green pigment gene transcription units were replaced, respectively, by alkaline phosphatase and beta-galactosidase reporters. As determined by histochemical staining, the reporters are expressed exclusively in cone photoreceptor cells. In 20 transgenic mice carrying any one of three independent transgene insertion events, an average of 63% of expressing cones have alkaline phosphatase activity, 10% have beta-galactosidase activity, and 27% have activity for both reporters. Thus, mutually exclusive expression of red and green pigment transgenes can be achieved in a large fraction of cones in a dichromat mammal, suggesting a facile evolutionary path for the development of trichromacy after visual pigment gene duplication. These observations are consistent with a model of visual pigment expression in which stochastic pairing occurs between a locus control region and either the red or the green pigment gene promotor.

In vitro evidence for efficacy in food intolerance for the multispecies probiotic formulation Ecologic® Tolerance (Syngut™).

PubMed

Besseling-van der Vaart, I; Heath, M D; Guagnini, F; Kramer, M F

2016-02-01

The beneficial effects of probiotics are currently the subject of extensive studies in health and medical research. The aim of this research was to specifically design a new probiotic formulation for supplementation in people suffering from food intolerance. The selection of strains was focussed on the capacity to influence mechanisms of action that are important in development of food intolerance with the following parameters measure: in vitro capacity to produce β-galactosidase, in vitro strengthening of the epithelial barrier, in vitro stimulation of cytokines produced by regulatory T cells, in addition to assessing fundamental quality criteria (stability, gastrointestinal (GI)-survival, multispecies concept, allergen-free). Ecologic®Tolerance/Syngut™ was subsequently developed consisting of a multispecies concept using 4 different probiotic strains (Bifidobacterium lactis W51, Lactobacillus acidophilus W22, Lactobacillus plantarum W21 and Lactococcus lactis W19). Each of these strains demonstrated ability to survive the GI-tract and strain specific effects in producing β-galactosidase, strengthening the gut barrier function after immunological-induced stress and inhibiting Th2 cytokines (IL-4, IL-5 and IL-13 (≥50%), in addition to stimulating interleukin-10 levels; thus, providing in vitro evidence for the efficacy of the selected strains to provide beneficial effects in patients suffering from food intolerance.

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

PubMed Central

Kirshenbaum, L A; MacLellan, W R; Mazur, W; French, B A; Schneider, M D

1993-01-01

Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells. Images PMID:8326005

Optimization of Milk-Based Medium for Efficient Cultivation of Bifidobacterium pseudocatenulatum G4 Using Face-Centered Central Composite-Response Surface Methodology

PubMed Central

Abdul Khalil, Khalilah; Mustafa, Shuhaimi; Mohammad, Rosfarizan; Bin Ariff, Arbakariya; Shaari, Yamin; Abdul Manap, Yazid; Dahalan, Farrah Aini

2014-01-01

This study was undertaken to optimize skim milk and yeast extract concentration as a cultivation medium for optimal Bifidobacteria pseudocatenulatum G4 (G4) biomass and β-galactosidase production as well as lactose and free amino nitrogen (FAN) balance after cultivation period. Optimization process in this study involved four steps: screening for significant factors using 23 full factorial design, steepest ascent, optimization using FCCD-RSM, and verification. From screening steps, skim milk and yeast extract showed significant influence on the biomass production and, based on the steepest ascent step, middle points of skim milk (6% wt/vol) and yeast extract (1.89% wt/vol) were obtained. A polynomial regression model in FCCD-RSM revealed that both factors were found significant and the strongest influence was given by skim milk concentration. Optimum concentrations of skim milk and yeast extract for maximum biomass G4 and β-galactosidase production meanwhile low in lactose and FAN balance after cultivation period were 5.89% (wt/vol) and 2.31% (wt/vol), respectively. The validation experiments showed that the predicted and experimental values are not significantly different, indicating that the FCCD-RSM model developed is sufficient to describe the cultivation process of G4 using skim-milk-based medium with the addition of yeast extract. PMID:24527457

Diversity of Glycosyl Hydrolases from Cellulose-Depleting Communities Enriched from Casts of Two Earthworm Species▿ †

PubMed Central

Beloqui, Ana; Nechitaylo, Taras Y.; López-Cortés, Nieves; Ghazi, Azam; Guazzaroni, María-Eugenia; Polaina, Julio; Strittmatter, Axel W.; Reva, Oleg; Waliczek, Agnes; Yakimov, Michail M.; Golyshina, Olga V.; Ferrer, Manuel; Golyshin, Peter N.

2010-01-01

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-β-glucanases, β-glucosidases, β-cellobiohydrolases, β-galactosidase, and β-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of β-galactosidases/α-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales). PMID:20622123

Reduction of globotriaosylceramide in Fabry disease mice by substrate deprivation.

PubMed

Abe, A; Gregory, S; Lee, L; Killen, P D; Brady, R O; Kulkarni, A; Shayman, J A

2000-06-01

We used a potent inhibitor of glucosylceramide synthase to test whether substrate deprivation could lower globotriaosylceramide levels in alpha-galactosidase A (alpha-gal A) knockout mice, a model of Fabry disease. C57BL/6 mice treated twice daily for 3 days with D-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidi no-propanol (D-t-EtDO-P4) showed a concentration-dependent decrement in glucosylceramide levels in kidney, liver, and spleen. A single intraperitoneal injection of D-t-EtDO-P4 resulted in a 55% reduction in renal glucosylceramide, consistent with rapid renal glucosylceramide metabolism. A concentration-dependent decrement in renal and hepatic globotriaosylceramide levels was observed in alpha-Gal A(-) males treated for 4 weeks with D-t-EtDO-P4. When 8-week-old alpha-Gal A(-) males were treated for 8 weeks with 10 mg/kg twice daily, renal globotriaosylceramide fell to below starting levels, consistent with an alpha-galactosidase A-independent salvage pathway for globotriaosylceramide degradation. Complications observed with another glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin, including weight loss and acellularity of lymphatic organs, were not observed with D-t-EtDO-P4. These data suggest that Fabry disease may be amenable to substrate deprivation therapy.

Metabolic Flux and Fitness

PubMed Central

Dykhuizen, Daniel E.; Dean, Antony M.; Hartl, Daniel L.

1987-01-01

Studies of Escherichia coli under competition for lactose in chemostat cultures have been used to determine the selective effects of variation in the level of the β-galactoside permease and the β-galactosidase enzyme. The results determine the adaptive topography of these gene products relative to growth in limiting lactose and enable predictions concerning the selective effects of genetic variants found in natural populations. In the terms of metabolic control theory, the β-galactosidase enzyme at wild-type-induced levels has a small control coefficient with respect to fitness (C = 0.018), and hence genetic variants resulting in minor changes in enzyme activity have disproportionately small effects on fitness. However, the apparent control coefficient of the β-galactoside permease at wild-type-induced levels is large (C = 0.551), and hence even minor changes in activity affect fitness. Therefore, we predict that genetic polymorphisms in the lacZ gene are subject to less effective selection in natural populations than are those in the lacY gene. The β-galactoside permease is also less efficient than might be expected, and possible forces resulting in selection for an intermediate optimum level of permease activity are considered. The selective forces that maintain the lactose operon in a regulated state in natural populations are also discussed. PMID:3104135

Statistical investigation of Kluyveromyces lactis cells permeabilization with ethanol by response surface methodology.

PubMed

de Faria, Janaína T; Rocha, Pollyana F; Converti, Attilio; Passos, Flávia M L; Minim, Luis A; Sampaio, Fábio C

2013-12-01

The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as β-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the β-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L(-1) oNP min(-1) g(-1) was obtained by treating cells with 75.0% v/v of ethanol at 20.0 °C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry.

Saccharomyces cerevisiae from Brazilian kefir-fermented milk: An in vitro evaluation of probiotic properties.

PubMed

Lima, Meire Dos Santos Falcão de; Souza, Karoline Mirella Soares de; Albuquerque, Wendell Wagner Campos; Teixeira, José António Couto; Cavalcanti, Maria Taciana Holanda; Porto, Ana Lúcia Figueiredo

2017-09-01

The therapeutic use of probiotics for supporting the antibiotic action against gastrointestinal disorders is a current trend and emerging applications have gained popularity because of their support for various microbiological activities in digestive processes. Microorganisms isolated from kefir with great probiotic properties, in addition to high resistance to harsh environmental conditions, have been widely researched. Administration of probiotic yeasts offers a number of advantages, when compared to bacteria, because of particular characteristics as their larger cell size. In the present study, 28 strains of Saccharomyces cerevisiae were isolated, after in vitro digestion of kefir-fermented milk, and identified by molecular based approaches. A screening was performed to determine important quality requirements for probiotics including: antagonistic and antioxidant activities, β-galactosidase synthesis, autoaggregation, surface hydrophobicity and adhesion to epithelial cells. The results showed strains: with antagonistic activity against microbial pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis; able to produce β-galactosidase; with antioxidant activity levels higher than 90%; with hydrophobicity activity and autoaggregation ability (evaluated by adhesion test, where all the strains presented adhesion to mice ileal epithelial cells). These findings are relevant and the strains are recommended for further in vivo studies as well as for potential therapeutic applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Release of colicin E2 from Escherichia coli.

PubMed

Pugsley, A P; Rosenbusch, J P

1981-07-01

Treatment of Escherichia coli K-12(ColE2.P9) with 500 ng of mitomycin C per ml resulted in rapid and almost synchronous colicin E2 production. Colicin accumulated outside the cytoplasmic membrane, most probably in the periplasmic space. Colicin release occurred during a period in which the turbidity of the culture declined markedly. Periplasmic alkaline phosphatase was released during the same period, but cytoplasmic beta-galactosidase release was delayed.

Inorganic colloidal nanocrystals: Synthesis and bioapplications

NASA Astrophysics Data System (ADS)

Wu, Huimeng

Nanocrystals (NCs) are very small particles, which contain from a few hundred to thousands of atoms depending on the size of NCs. Because of their special properties compared with the bulk materials, NCs have found many promising applications in areas, such as biomedical diagnosis, catalysis, plasmonics, high-density data storage and solar energy conversion. This dissertation presents studies on the syntheses of metal oxide NCs and hybrid NCs, the surface functionalization of NCs by dual-interaction ligands, and gold-NC-based assay for the detection of beta-galactosidase. Monodisperse colloidal uranium dioxide NCs (UO2 NCs) were synthesized by decomposition of uranyl acetylacetonate. By changing the amount of added surfactant, the sizes of the NCs could vary from 2 ˜ 8 nm. Mechanistic studies of the formation of UO2 NCs showed that the condensation product (amide) of oleic acid and oleylamine plays an important role in controlling the particle size. Normally, high-quality NCs are synthesized in organic phase, but most of NC-based bio-applications require water-soluble NCs. To convert these hydrophobic NCs to hydrophilic particles, surface modification is employed. Here dual interaction ligands based on the Tween-derivatives (TDs) were synthesized. Stability tests on TD-capped NCs showed that these dual interaction ligands can significantly increase the stability of NCs compared to single interaction ligands. Further, These TD-capped QDs were further tested as fluorescent labels to detect virusprotein expression in cells. To exploit bio-applications of nanocrystals, gold nanocrystal-based assay to detect enzyme activity was designed. The optical properties of Au-NCs are not only dependent on the particle sizes and shapes, but also the distances between the particles. Here, Lipoic acid-tyramine-beta-galactopyranosyl (LTbeta-gal) was synthesized, as ligands, to cap Au-NCs; and the resultant LTbeta-gal-capped Au-NCs could disperse in water. After the hydrolysis of the

A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut+/pAOX1-lacZ strain

PubMed Central

2013-01-01

Background One of the main challenges for heterologous protein production by the methylotrophic yeast Pichia pastoris at large-scale is related to its high oxygen demand. A promising solution is a co-feeding strategy based on a methanol/sorbitol mixture during the induction phase. Nonetheless, a deep understanding of the cellular physiology and the regulation of the AOX1 promoter, used to govern heterologous protein production, during this co-feeding strategy is still scarce. Results Transient continuous cultures with a dilution rate of 0.023 h-1 at 25°C were performed to quantitatively assess the benefits of a methanol/sorbitol co-feeding process with a Mut+ strain in which the pAOX1-lacZ construct served as a reporter gene. Cell growth and metabolism, including O2 consumption together with CO2 and heat production were analyzed with regard to a linear change of methanol fraction in the mixed feeding media. In addition, the regulation of the promoter AOX1 was investigated by means of β-galactosidase measurements. Our results demonstrated that the cell-specific oxygen consumption (qO2) could be reduced by decreasing the methanol fraction in the feeding media. More interestingly, maximal β-galactosidase cell-specific activity (>7500 Miller unit) and thus, optimal pAOX1 induction, was achieved and maintained in the range of 0.45 ~ 0.75 C-mol/C-mol of methanol fraction. In addition, the qO2 was reduced by 30% at most in those conditions. Based on a simplified metabolic network, metabolic flux analysis (MFA) was performed to quantify intracellular metabolic flux distributions during the transient continuous cultures, which further shed light on the advantages of methanol/sorbitol co-feeding process. Finally, our observations were further validated in fed-batch cultures. Conclusion This study brings quantitative insight into the co-feeding process, which provides valuable data for the control of methanol/sorbitol co-feeding, aiming at enhancing biomass and

A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut⁺/pAOX1-lacZ strain.

PubMed

Niu, Hongxing; Jost, Laurent; Pirlot, Nathalie; Sassi, Hosni; Daukandt, Marc; Rodriguez, Christian; Fickers, Patrick

2013-04-08

One of the main challenges for heterologous protein production by the methylotrophic yeast Pichia pastoris at large-scale is related to its high oxygen demand. A promising solution is a co-feeding strategy based on a methanol/sorbitol mixture during the induction phase. Nonetheless, a deep understanding of the cellular physiology and the regulation of the AOX1 promoter, used to govern heterologous protein production, during this co-feeding strategy is still scarce. Transient continuous cultures with a dilution rate of 0.023 h(-1) at 25°C were performed to quantitatively assess the benefits of a methanol/sorbitol co-feeding process with a Mut+ strain in which the pAOX1-lacZ construct served as a reporter gene. Cell growth and metabolism, including O2 consumption together with CO2 and heat production were analyzed with regard to a linear change of methanol fraction in the mixed feeding media. In addition, the regulation of the promoter AOX1 was investigated by means of β-galactosidase measurements. Our results demonstrated that the cell-specific oxygen consumption (qO2) could be reduced by decreasing the methanol fraction in the feeding media. More interestingly, maximal β-galactosidase cell-specific activity (>7500 Miller unit) and thus, optimal pAOX1 induction, was achieved and maintained in the range of 0.45 ~ 0.75 C-mol/C-mol of methanol fraction. In addition, the qO2 was reduced by 30% at most in those conditions. Based on a simplified metabolic network, metabolic flux analysis (MFA) was performed to quantify intracellular metabolic flux distributions during the transient continuous cultures, which further shed light on the advantages of methanol/sorbitol co-feeding process. Finally, our observations were further validated in fed-batch cultures. This study brings quantitative insight into the co-feeding process, which provides valuable data for the control of methanol/sorbitol co-feeding, aiming at enhancing biomass and heterologous protein productivities

Evaluation of group A1B erythrocytes converted to type as group O: studies of markers of function and compatibility

PubMed Central

Gao, Hong-Wei; Zhuo, Hai-Long; Zhang, Xue; Ji, Shou-Ping; Tan, Ying-Xia; Li, Su-Bo; Jia, Yan-Jun; Xu, Hua; Wu, Qing-Fa; Yun, Zhi-Min; Luo, Qun; Gong, Feng

2016-01-01

Background Enzymatic conversion of blood group A1B red blood cells (RBC) to group O RBC (ECO) was achieved by combined treatment with α-galactosidase and α-N-acetylgalactosaminidase. The aim of this study was to evaluate the function and safety of these A1B-ECO RBC in vitro. Materials and methods A 20% packed volume of A1B RBC was treated with enzymes in 250 mM glycine buffer, pH 6.8. The efficiency of the conversion of A and B antigen was evaluated by traditional typing in test tubes, gel column agglutination technology and fluorescence-activated cell sorting (FACS) analysis. The physiological and metabolic parameters of native and ECO RBC were compared, including osmotic fragility, erythrocyte deformation index, levels of 2,3-diphosphoglycerate, ATP, methaemoglobin, free Na+, and free K+. The morphology of native and ECO RBC was observed by scanning electron microscopy. Residual α-galactosidase or α-N-acetylgalactosaminidase in A1B-ECO RBC was detected by double-antibody sandwich ELISA method. Manual cross-matching was applied to ensure blood compatibility. Results The RBC agglutination tests and FACS results showed that A1B RBC were efficiently converted to O RBC. Functional analysis suggested that the conversion process had little impact on the physiological and metabolic parameters of the RBC. The residual amounts of either α-galactosidase or α-N-acetylgalactosaminidase in the A1B-ECO RBC were less than 10 ng/mL of packed RBC. About 18% of group B and 55% of group O sera reacted with the A1B-ECO RBC in a sensitive gel column cross-matching test. Discussion The conversion process does not appear to affect the morphological, physiological or metabolic parameters of A1B-ECO RBC. However, the A1B-ECO RBC still reacted with some antigens. More research on group O and B sera, which may partly reflect the complexity of group A1 the safety of A1B-ECO RBC is necessary before the application of these RBC in clinical transfusion. PMID:26509826

Purification of N-Acetylgalactosaminidase by Isoelectric Focusing.

DTIC Science & Technology

1983-04-02

aminocaproic acid and histidyl glycine were tried. Focusing in his tidyl glycine separated the galactosidase activity into 2 peaks but did not separate...under acidic conditions. N-acetylgalactosamine was shown to be a competitive inhibitor with a K of 10.1 mM. The KM for p-NP- a-GALNAc was 6.6 mM. In...ampholyte was prepared by the copolymerization of acrylic acid with pentaethylene hexamine (PEHA) as described by Vinogradov et al. (Biochem. Biophys. Res

Evaluating the Significance of CDK2-PELP1 Axis in Tumorigenesis and Hormone Therapy Resistance

DTIC Science & Technology

2011-02-01

reagents an breast cancer cells MCF7, ZR75, IMR-90, NIH3T3, 93T were obtained from the American Type Culture tion. All stable cell lines were generated...galactosidase activity or tal protein concentration. ime PCR and cell cycle microarray s were harvested with Trizol Reagent (Invitrogen), and NA was isolated...Margue, F Huselstein, G, Grignard , W Dippel, M, Nathan, S Giacchi, R Scheiden. ILK as a potential marker gene to ascertain specific adenocarcinoma

Epigenetic Control of Prostate Cancer Metastasis: Role of Runx2 Phosphorylation

DTIC Science & Technology

2014-04-01

prostate cancer cells. In the third budget year, we achieved the following: a. Generation of retrovirus and lentivirus vectors expressing WT RUNX2 and S301A... retrovirus vectors will be developed that express β-galactosidase (negative control), wild type Runx2, S301A/S319A (non-phosphorylated) or S301E/S310E...constitutively active) Runx2 mutants. As described last year, retrovirus and lentivirus vectors were constructed to stably introduce wild type and mutant

Survival of Lactobacillus acidophilus and Bifidobacterium bifidum in ice cream for use as a probiotic food.

PubMed

Hekmat, S; McMahon, D J

1992-06-01

Probiotic ice cream was made by fermenting a standard ice cream mix with Lactobacillus acidophilus and Bifidobacterium bifidum cultures and then freezing the mix in a batch freezer. Survival of the L. acidophilus and B. bifidum, as well as beta-galactosidase activity, was monitored during 17 wk of frozen storage at -29 degrees C. After freezing of the fermented mix, bacterial counts were 1.5 x 10(8) cfu/ml for L. acidophilus and 2.5 x 10(8) cfu/ml for B. bifidum. Seventeen weeks after freezing, these counts had decreased to 4 x 10(6) and 1 x 10(7) cfu/ml, respectively. During the same period, beta-galactosidase activity decreased from 1800 to 1300 units/ml. Probiotic ice cream was prepared at pH 5.0, 5.5, and 6.0 to determine consumer preferences and was compared with standard Utah State University "Aggie" ice cream. All samples were strawberry-flavored and were evaluated by 88 judges. The preferred pH of probiotic ice cream, based on overall acceptance, was pH 5.5. We demonstrated that probiotic ice cream is a suitable vehicle for delivering beneficial microorganisms such as L. acidophilus and B. bifidum to consumers. The bacteria can be grown to high numbers in ice cream mix and remain viable during frozen storage.

Changes in Biochemical Characteristics and Activities of Ripening Associated Enzymes in Mango Fruit during the Storage at Different Temperatures

PubMed Central

Kimura, Yoshinobu

2014-01-01

As a part of the study to explore the possible strategy for enhancing the shelf life of mango fruits, we investigated the changes in biochemical parameters and activities of ripening associated enzymes of Ashwina hybrid mangoes at 4-day regular intervals during storage at −10°C, 4°C, and 30 ± 1°C. Titratable acidity, vitamin C, starch content, and reducing sugar were higher at unripe state and gradually decreased with the increasing of storage time at all storage temperatures while phenol content, total soluble solid, total sugar, and nonreducing sugar contents gradually increased. The activities of amylase, α-mannosidase, α-glucosidase, and invertase increased sharply within first few days and decreased significantly in the later stage of ripening at 30 ± 1°C. Meanwhile polyphenol oxidase, β-galactosidase, and β-hexosaminidase predominantly increased significantly with the increasing days of storage till later stage of ripening. At −10°C and 4°C, the enzymes as well as carbohydrate contents of storage mango changed slightly up to 4 days and thereafter the enzyme became fully dormant. The results indicated that increase in storage temperature and time correlated with changes in biochemical parameters and activities of glycosidases suggested the suppression of β-galactosidase and β-hexosaminidase might enhance the shelf life of mango fruits. PMID:25136564

Improved clinical tolerance to chronic lactose ingestion in subjects with lactose intolerance: a placebo effect?

PubMed Central

Briet, F; Pochart, P; Marteau, P; Flourie, B; Arrigoni, E; Rambaud, J

1997-01-01

Background—Uncontrolled studies of lactose intolerant subjects have shown that symptom severity decreases after chronic lactose consumption. Adaptation of the colonic flora might explain this improvement. 
Aims—To compare the effects of regular administration of either lactose or sucrose on clinical tolerance and bacterial adaptation to lactose. 
Methods—Forty six lactose intolerant subjects underwent two 50 g lactose challenges on days 1 and 15. Between these days they were given 34 g of lactose or sucrose per day, in a double blind protocol. Stool samples were obtained on days 0 and 14, to measure faecal β-galactosidase and pH. Symptoms, breath H2 excretion, faecal weight and electrolytes, and orofaecal transit time were assessed. 
Results—Except for faecal weight, symptoms were significantly milder during the second challenge in both groups, and covariance analysis showed no statistical difference between them. In the lactose group, but not in the sucrose group, faecal β-galactosidase activity increased, pH dropped, and breath H2 excretion decreased. 
Conclusion—Bacterial adaptation occurred when lactose intolerant subjects ingested lactose for 13 days, and all symptoms except diarrhoea regressed. Clinical improvement was also observed in the control group which displayed no signs of metabolic adaptation. This suggests that improved clinical tolerance may be just a placebo effect. 

 Keywords: lactose; lactose intolerance; colonic adaptation; lactase deficiency PMID:9414969

Reduction of elevated plasma globotriaosylsphingosine in patients with classic Fabry disease following enzyme replacement therapy.

PubMed

van Breemen, Mariëlle J; Rombach, Saskia M; Dekker, Nick; Poorthuis, Ben J; Linthorst, Gabor E; Zwinderman, Aeilko H; Breunig, Frank; Wanner, Christoph; Aerts, Johannes M; Hollak, Carla E

2011-01-01

Fabry disease is treated by two-weekly infusions with α-galactosidase A, which is deficient in this X-linked globotriaosylceramide (Gb3) storage disorder. Elevated plasma globotriaosylsphingosine (lysoGb3) is a hallmark of classical Fabry disease. We investigated effects of enzyme replacement therapy (ERT) on plasma levels of lysoGb3 and Gb3 in patients with classical Fabry disease treated with agalsidase alfa at 0.2mg/kg, agalsidase beta at 0.2mg/kg or at 1.0mg/kg bodyweight. Each treatment regimen led to prominent reductions of plasma lysoGb3 in Fabry males within 3 months (P=0.0313), followed by relative stability later on. Many males developed antibodies against α-galactosidase A, particularly those treated with agalsidase beta. Patients with antibodies tended towards smaller correction in plasma lysoGb3 concentration, whereas treatment with high dose agalsidase beta allowed a reduction comparable to patients without antibodies. Pre-treatment plasma lysoGb3 concentrations of Fabry females were relatively low. In all females and with each treatment regimen, ERT gave reduction or stabilisation of plasma lysoGb3. Our investigation revealed that ERT of Fabry patients reduces plasma lysoGb3, regardless of the recombinant enzyme used. This finding shows that ERT can correct a characteristic biochemical abnormality in Fabry patients. Copyright © 2010 Elsevier B.V. All rights reserved.

Evaluation of a low dose, after a standard therapeutic dose, of agalsidase beta during enzyme replacement therapy in patients with Fabry disease.

PubMed

Lubanda, Jean-Claude; Anijalg, Ene; Bzdúch, Vladimír; Thurberg, Beth L; Bénichou, Bernard; Tylki-Szymanska, Anna

2009-04-01

Fabry disease, a genetic deficiency of alpha-galactosidase A, is characterized by pathogenic cellular accumulation of globotriaosylceramide. During clinical trials, recombinant human alpha-galactosidase A (agalsidase beta; Fabrazyme, Genzyme Corporation, Cambridge, MA), infused intravenously at 1.0 mg/kg every 2 weeks for 6 months, cleared or reduced globotriaosylceramide in renal, cardiac, and dermal microvascular endothelia and other cells, with results sustained for up to 5 years in most patients evaluated. This study explored whether a lower dose could maintain globotriaosylceramide clearance achieved with 1.0 mg/kg. Cellular globotriaosylceramide levels were assessed histologically in kidney and skin biopsies from 21 adult Fabry males treated for 6 months at 1.0 mg/kg/2 weeks followed by 18 months at 0.3 mg/kg/2 weeks. In kidney interstitial capillary endothelium, the primary endpoint, globotriaosylceramide clearance was achieved in 100% of patients with 1.0 mg/kg and maintained in 90% with 0.3 mg/kg. In seven other renal cell types and superficial dermal capillary endothelium, globotriaosylceramide reduction or clearance was maintained with 0.3 mg/kg in approximately 70% of patients. A lower dose of agalsidase beta may be sufficient in some, but not all, patients with Fabry disease to maintain the cellular globotriaosylceramide clearance achieved with 1.0 mg/kg/2 weeks. Long-term clinical effects of transitioning to the lower dose have not been evaluated.

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

PubMed

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.

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

PubMed

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.

Investigation of the Germination of Barley and Wheat Grains with a Design of Experiments for the Production of Hydrolases

PubMed Central

Kranz, Bertolt; Koch, Milena; Schapfl, Matthias

2015-01-01

Summary The production of hydrolases from cereals has been examined in order to investigate food-derived enzymes as an alternative source to microbial enzymes for the use in food processes. For that, the influence of temperature on the pretreatment, imbibition and germination of barley and wheat grains was determined by measuring the β-glucosidase, β-galactosidase and lipase activities using a design of experiments. The evaluation of the statistical model showed an increase of the β-glucosidase activity with low imbibition and low germination temperature for barley grains and low imbibition and high germination temperature for wheat grains. The maximum β-glucosidase activity in wheat extracts was (585±151) nkat per g of dry mass (dm), while in barley extracts it was (109±15) nkat per g of dm. The maximum β-galactosidase activities in barley and wheat extracts were (34±12) and (63±23) nkat per g of dm, respectively. The maximum lipase activities of (6.7±0.1) and (4.6±4.4) nkat per g of dm in barley and wheat extracts, respectively, were rather low compared to the glycosidase activities. The extracts were also tested for other hydrolase activities (e.g. peptidase and α-amylase activities). The insights obtained enable the basis for the potential use of cereal hydrolases in food processing, which might be attractive to consumers. PMID:27904341

Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose - an emerging prebiotic.

PubMed

Celebioglu, Hasan Ufuk; Ejby, Morten; Majumder, Avishek; Købler, Carsten; Goh, Yong Jun; Thorsen, Kristian; Schmidt, Bjarne; O'Flaherty, Sarah; Abou Hachem, Maher; Lahtinen, Sampo J; Jacobsen, Susanne; Klaenhammer, Todd R; Brix, Susanne; Mølhave, Kristian; Svensson, Birte

2016-05-01

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Marked heterogeneity in growth characteristics of myoblast clonal cultures and myoblast mixed cultures obtained from the same individual.

PubMed

Maier, Andrea B; Cohen, Ron; Blom, Joke; van Heemst, Diana; Westendorp, Rudi G J

2012-01-01

Sarcopenia is defined as an age-related decrease in skeletal muscle mass and function while adjacent satellite cells are unable to compensate for this loss. However, myoblast cultures can be established even in the presence of sarcopenia. It is yet unknown whether satellite cells from failing muscle in older age are equally affected, as human satellite cells have been assessed using myoblast mixed cultures and not by using myoblast clonal cultures. We questioned to what extent myoblast mixed cultures reflect the in vivo characteristics of single satellite cells from adult skeletal muscle. We established a myoblast mixed culture and three myoblast clonal cultures out of the same muscle biopsy and cultured these cells for 100 days. Replicative capacity and oxidative stress resistance were compared. We found marked heterogeneity between the myoblast clonal cultures that all had a significantly lower replicative capacity when compared to the mixed culture. Replicative capacity of the clonal cultures was inversely related to the β-galactosidase activity after exposure to oxidative stress. Addition of L-carnosine enhanced the remaining replicative capacity in all cultures with a concomitant marginal decrease in β-galactosidase activity. It is concluded that myoblast mixed cultures in vitro do not reflect the marked heterogeneity between single isolated satellite cells. The consequences of the heterogeneity on muscle performance remain to be established. Copyright © 2011 S. Karger AG, Basel.

Acrylamide induces accelerated endothelial aging in a human cell model.

PubMed

Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas

2015-09-01

Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Beware of your Cre-Ation: lacZ expression impairs neuronal integrity and hippocampus-dependent memory.

PubMed

Reichel, J M; Bedenk, B T; Gassen, N C; Hafner, K; Bura, S A; Almeida-Correa, S; Genewsky, A; Dedic, N; Giesert, F; Agarwal, A; Nave, K-A; Rein, T; Czisch, M; Deussing, J M; Wotjak, C T

2016-10-01

Expression of the lacZ-sequence is a widely used reporter-tool to assess the transgenic and/or transfection efficacy of a target gene in mice. Once activated, lacZ is permanently expressed. However, protein accumulation is one of the hallmarks of neurodegenerative diseases. Furthermore, the protein product of the bacterial lacZ gene is ß-galactosidase, an analog to the mammalian senescence-associated ß-galactosidase, a molecular marker for aging. Therefore we studied the behavioral, structural and molecular consequences of lacZ expression in distinct neuronal sub-populations. lacZ expression in cortical glutamatergic neurons resulted in severe impairments in hippocampus-dependent memory accompanied by marked structural alterations throughout the CNS. In contrast, GFP expression or the expression of the ChR2/YFP fusion product in the same cell populations did not result in either cognitive or structural deficits. GABAergic lacZ expression caused significantly decreased hyper-arousal and mild cognitive deficits. Attenuated structural and behavioral consequences of lacZ expression could also be induced in adulthood, and lacZ transfection in neuronal cell cultures significantly decreased their viability. Our findings provide a strong caveat against the use of lacZ reporter mice for phenotyping studies and point to a particular sensitivity of the hippocampus formation to detrimental consequences of lacZ expression. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Encapsulation of lactase in Ca(II)-alginate beads: Effect of stabilizers and drying methods.

PubMed

Traffano-Schiffo, Maria Victoria; Castro-Giraldez, Marta; Fito, Pedro J; Santagapita, Patricio R

2017-10-01

The purpose of the present work was to analyze the effect of trehalose, arabic and guar gums on the preservation of β-galactosidase activity in freeze-dried and vacuum dried Ca(II)-alginate beads. Freezing process was also studied as a first step of freeze-drying. Trehalose was critical for β-galactosidase conservation, and guar gum as a second excipient showed the highest conservation effect (close to 95%). Systems with T g values ~40°C which were stables at ambient temperature were obtained, being trehalose the main responsible of the formation of an amorphous matrix. Vacuum dried beads showed smaller size (with Feret's diameter below 1.08±0.09mm), higher circularity (reaching 0.78±0.06) and large cracks in their surface than freeze-dried beads, which were more spongy and voluminous. Ice crystallization of the beads revealed that the crystallization of Ca(II)-alginate system follows the Avrami kinetics of nucleation and growth. Particularly, Ca(II)-alginate showed an Avrami index of 2.03±0.07, which means that crystal growing is bidimensional. Neither the addition of trehalose nor gums affected the dimension of the ice growing or its rate. These results open an opportunity in the development of new lactic products able to be consumed by lactose intolerance people. Copyright © 2017 Elsevier Ltd. All rights reserved.

Soil microbial activities in a constructed soil reed-bed under cheese-dairy farm effluents.

PubMed

Farnet, A M; Prudent, P; Cigna, M; Gros, R

2008-09-01

Soil microbial activities in a reed-bed used for effluent purification of a small cheese-dairy farm under a Mediterranean climate were described and studied. This work aims to demonstrate (i) whether certain enzyme activities used as bioindicators of dairy waste degradation (beta-galactosidase and protease) vary over time, which might influence organic matter degradation and (ii) whether specific microbial communities are selected through contact with the discarded effluent using community level catabolic profiles (CLCPs). beta-galactosidase and protease activities were followed in a 14-month monitoring experiment. These enzyme activities were strongly expressed during the whey-discarding period from February to May. CLCPs using Biolog Ecoplate showed great microbial diversity, as described by Shannon-Weaver index, and no difference was observed in microbial diversity between areas at the receiving end of the reed-bed (where effluent was discarded) and those at the opposite end. This may be explained by successive environmental factors which made enzyme activities vary: whey discarded from February to May and Mediterranean climate conditions (drying-rewetting effects on summer). Microbial enumeration using epifluorescence microscopy also showed a pattern linked to Mediterranean conditions with a drastic decrease in biomass during summer drought. These results on functional biodiversity were correlated with high purification yields: the minimum decrease in Biological Demand in Oxygen was 84% and that in suspended solids was 75%.

Semester-long inquiry-based molecular biology laboratory: Transcriptional regulation in yeast.

PubMed

Oelkers, Peter M

2017-03-04

A single semester molecular biology laboratory has been developed in which students design and execute a project examining transcriptional regulation in Saccharomyces cerevisiae. Three weeks of planning are allocated to developing a hypothesis through literature searches and use of bioinformatics. Common experimental plans address a cell process and how three genes that encode for proteins involved in that process are transcriptionally regulated in response to changing environmental conditions. Planning includes designing oligonucleotides to amplify the putative promoters of the three genes of interest. After the PCR, each product is cloned proximal to β-galactosidase in a yeast reporter plasmid. Techniques used include agarose electrophoresis, extraction of DNA from agarose, plasmid purification from bacteria, restriction digestion, ligation, and bacterial transformation. This promoter/reporter plasmid is then transformed into yeast. Transformed yeast are cultured in conditions prescribed in the experimental design, lysed and β-galactosidase activity is measured. The course provides an independent research experience in a group setting. Notebooks are maintained on-line with regular feedback. Projects culminate with the presentation of a poster worth 60% of the grade. Over the last three years, about 65% of students met expectations for experimental design, data acquisition, and analysis. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):145-151, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

staggerer phenotype in retinoid-related orphan receptor α-deficient mice

PubMed Central

Steinmayr, Markus; André, Elisabeth; Conquet, François; Rondi-Reig, Laure; Delhaye-Bouchaud, Nicole; Auclair, Nathalie; Daniel, Hervé; Crépel, Francis; Mariani, Jean; Sotelo, Constantino; Becker-André, Michael

1998-01-01

Retinoid-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a lacZ reporter gene encoding the enzyme β-galactosidase. In heterozygous RORα+/− mice we found β-galactosidase activity, indicative of RORα protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the RORα gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, RORα is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express RORα. Recently, it was reported that the ataxic mouse mutant staggerer (sg/sg) is caused by a deletion in the RORα gene. The analysis of the cerebellar and the behavioral phenotype of homozygous RORα−/− mice proves identity to sg/sg mice. Although the absence of RORα causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of RORα−/− mice display a normal phenotype. However, the pelage hair of both sg/sg and RORα−/− is significantly less dense and when shaved shows reluctance to regrow. PMID:9520475

Use of yeast spores for microencapsulation of enzymes.

PubMed

Shi, Libing; Li, Zijie; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

2014-08-01

Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose.This suggests that substrate selectivity could be altered by the encapsulation.

A Novel Population of Inner Cortical Cells in the Adrenal Gland That Displays Sexually Dimorphic Expression of Thyroid Hormone Receptor-β1

PubMed Central

Huang, Chen-Che Jeff; Kraft, Cary; Moy, Nicole; Ng, Lily

2015-01-01

The development of the adrenal cortex involves the formation and then subsequent regression of immature or fetal inner cell layers as the mature steroidogenic outer layers expand. However, controls over this remodeling, especially in the immature inner layer, are incompletely understood. Here we identify an inner cortical cell population that expresses thyroid hormone receptor-β1 (TRβ1), one of two receptor isoforms encoded by the Thrb gene. Using mice with a Thrbb1 reporter allele that expresses lacZ instead of TRβ1, β-galactosidase was detected in the inner cortex from early stages. Expression peaked at juvenile ages in an inner zone that included cells expressing 20-α-hydroxysteroid dehydrogenase, a marker of the transient, so-called X-zone in mice. The β-galactosidase-positive zone displayed sexually dimorphic regression in males after approximately 4 weeks of age but persisted in females into adulthood in either nulliparous or parous states. T3 treatment promoted hypertrophy of inner cortical cells, induced some markers of mature cortical cells, and, in males, delayed the regression of the TRβ1-positive zone, suggesting that TRβ1 could partly divert the differentiation fate and counteract male-specific regression of inner zone cells. TRβ1-deficient mice were resistant to these actions of T3, supporting a functional role for TRβ1 in the inner cortex. PMID:25774556

Inhibition of Gene Expression in Escherichia coli by Antisense Phosphorodiamidate Morpholino Oligomers

PubMed Central

Geller, B. L.; Deere, J. D.; Stein, D. A.; Kroeker, A. D.; Moulton, H. M.; Iversen, P. L.

2003-01-01

Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)3KC to the end of PMOs. In strains with an intact outer membrane, (KFF)3KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)3KC-PMO conjugate targeted to lacI mRNA induced β-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)3KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce β-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)3KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets. PMID:14506035

Molecular identification of coliform bacteria isolated from drinking water reservoirs with traditional methods and the Colilert-18 system.

PubMed

Kämpfer, Peter; Nienhüser, Anita; Packroff, Gabriele; Wernicke, Frank; Mehling, Arnd; Nixdorf, Katja; Fiedler, Stefanie; Kolauch, Claudia; Esser, Michael

2008-07-01

The accuracy of a traditional method (lactose utilization with acid and gas production) for the detection of coliform bacteria and E. coli was tested in comparison with method ISO 9308-1 (based on acid formation from lactose) and the Colilert-18 system (detection of beta-galactosidase). A total of 345 isolates were identified after isolation from water samples using API 20E strips. The Colilert-18 led to the highest number of positive findings (95% of the isolates were assigned to coliforms), whereas the ISO-9308-1 method resulted only in 29% coliform findings. With the traditional method only 15% were rated positive. Most of the isolates were identified by the API 20E system as Enterobacter spp. (species of the Enterobacter cloacae complex), Serratia spp., Citrobacter spp.and Klebsiella spp.; but species identification remained vague in several cases. A more detailed identification of 126 pure cultures by using 16S rRNA gene sequence analysis and analysis of the hsp60 gene resulted in the identification of Enterobacter nimipressuralis, E. amnigenus, E. asburiae, E. hormaechei, and Serratia fonticola as predominat coliforms. These species are beta-galactosidase positive, but show acid formation from lactose often after a prolonged incubation time. They are often not of fecal origin and may interfere with the ability to accurately detect coliforms of fecal origin.

Influence of reaction medium during synthesis of Gantrez AN 119 nanoparticles for oral vaccination.

PubMed

Vandamme, Katrien; Melkebeek, Vesna; Cox, Eric; Deforce, Dieter; Lenoir, Joke; Adriaens, Els; Vervaet, Chris; Remon, Jean Paul

2010-02-01

Two synthesis methods of poly(methyl vinyl ether-co-maleic anhydride) (Gantrez AN 119) nanoparticles (NP) (used for oral vaccination) were compared. Wheat germ agglutinin (WGA) was used as ligand to enhance the bioadhesive properties of NP and beta-galactosidase as antigen. The first method encapsulated beta-galactosidase in NP by co-precipitation in an acetone/water mixture containing 44% acetone. In the second method, antigen addition occurred in 100% acetone. To improve stability, NP were crosslinked with 1,3-diaminopropane. The stability of WGA-conjugated NP with encapsulated antigen diminished at lower pH and when decreasing the amount of crosslinker. The binding type between WGA and polymer depended on the synthesis method: predominantly ionic bonds were formed using the 44% acetone method, whereas synthesis via the 100% acetone method resulted in covalent bonds. The biological activity of the WGA coating, evaluated via a pig gastric mucin binding test, was lower in NP prepared via the 100% acetone method. No release of native antigen was detected after hydrolysis of NP, due to the covalent antigen binding during antigen encapsulation and the high reactivity of the polymer. Moreover, the mucosal irritation capacity was evaluated upon nanoparticle hydrolysis using a slug mucosal irritation assay. Herein, hydrolysed NP of the 44% acetone method were classified as mild irritative. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Non-specific gastrointestinal features: Could it be Fabry disease?

PubMed

Hilz, Max J; Arbustini, Eloisa; Dagna, Lorenzo; Gasbarrini, Antonio; Goizet, Cyril; Lacombe, Didier; Liguori, Rocco; Manna, Raffaele; Politei, Juan; Spada, Marco; Burlina, Alessandro

2018-05-01

Non-specific gastrointestinal symptoms, including pain, diarrhoea, nausea, and vomiting, can be the first symptoms of Fabry disease. They may suggest more common disorders, e.g. irritable bowel syndrome or inflammatory bowel disease. The confounding clinical presentation and rarity of Fabry disease often cause long diagnostic delays and multiple misdiagnoses. Therefore, specialists involved in the clinical evaluation of non-specific upper and lower gastrointestinal symptoms should recognize Fabry disease as a possible cause of the symptoms, and should consider Fabry disease as a possible differential diagnosis. When symptoms or family history suggest Fabry disease, in men, low alpha-galactosidase A enzyme levels, and in women, specific Fabry mutations confirm the diagnosis. In addition to symptomatic treatments, disease-specific enzyme replacement therapy with recombinant human alpha-galactosidase A enzyme or chaperone therapy (migalastat) in patients with amenable mutations can improve the disease, including gastrointestinal symptoms, and should be initiated as early as possible after Fabry disease has been confirmed; starting enzyme replacement therapy at as young an age as possible after diagnosis improves long-term clinical outcomes. Improved diagnostic tools, such as a modified gastrointestinal symptom rating scale, may facilitate diagnosing Fabry disease in patients with gastrointestinal symptoms of unknown cause and thus assure timely initiation of disease-specific treatment. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

21 CFR 173.145 - Alpha-Galactosidase derived from Mortierella vinaceae var. raffinoseutilizer.

Code of Federal Regulations, 2013 CFR

2013-04-01

... FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.145... in food in accordance with the following conditions: (a) The food additive is the enzyme alpha... the enzyme. (b) The nonpathogenic microorganism matches American Type Culture Collection (ATCC) No...

21 CFR 173.145 - Alpha-Galactosidase derived from Mortierella vinaceae var. raffinoseutilizer.

Code of Federal Regulations, 2012 CFR

2012-04-01

... FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.145... in food in accordance with the following conditions: (a) The food additive is the enzyme alpha... the enzyme. (b) The nonpathogenic microorganism matches American Type Culture Collection (ATCC) No...

21 CFR 173.145 - Alpha-Galactosidase derived from Mortierella vinaceae var. raffinoseutilizer.

Code of Federal Regulations, 2010 CFR

2010-04-01

... FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.145... in food in accordance with the following conditions: (a) The food additive is the enzyme alpha... the enzyme. (b) The nonpathogenic microorganism matches American Type Culture Collection (ATCC) No...

21 CFR 173.145 - Alpha-Galactosidase derived from Mortierella vinaceae var. raffinoseutilizer.

Code of Federal Regulations, 2011 CFR

2011-04-01

... FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.145... in food in accordance with the following conditions: (a) The food additive is the enzyme alpha... the enzyme. (b) The nonpathogenic microorganism matches American Type Culture Collection (ATCC) No...

Galacto-oligosaccharide hydrolysis by genetically-engineered alpha-galactosidase-producing Pseudomonas chlororaphis strains

USDA-ARS?s Scientific Manuscript database

Various Pseudomonas chlororaphis strains have been shown to produce rhamnolipid (a biosurfactant), poly(hydroxyalkanoate) (PHA; a biopolymer), and/or antifungal compounds for plants. An ability to metabolize galacto-oligosaccharides in soy molasses would allow P. chlororaphis to use the byproduct as...

Looking-glass synergistic pharmacological chaperones: DGJ and L-DGJ from the enantiomers of tagatose.

PubMed

Jenkinson, Sarah F; Fleet, George W J; Nash, Robert J; Koike, Yuriko; Adachi, Isao; Yoshihara, Akihide; Morimoto, Kenji; Izumori, Ken; Kato, Atsushi

2011-08-05

The enantiomers of tagatose are converted to L-DGJ [a noncompetitive inhibitor of human lysosome α-galactosidase A (α-Gal A), K(i) 38.5 μM] and DGJ [a competitive inhibitor of α-Gal A, K(i) 15.1 nM] in 66% yield. L-DGJ and DGJ provide the first examples of pharmacological chaperones that (a) are enantiomeric iminosugars and (b) have synergistic activity with implications for the treatment of lysosomal storage disorders and other protein deficiencies. © 2011 American Chemical Society

Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity: Role of the Ang II/AT1 Receptor/NADPH Oxidase-Mediated Activation of MAPKs and PI3-Kinase Pathways.

PubMed

Abbas, Malak; Jesel, Laurence; Auger, Cyril; Amoura, Lamia; Messas, Nathan; Manin, Guillaume; Rumig, Cordula; León-González, Antonio J; Ribeiro, Thais P; Silva, Grazielle C; Abou-Merhi, Raghida; Hamade, Eva; Hecker, Markus; Georg, Yannick; Chakfe, Nabil; Ohlmann, Patrick; Schini-Kerth, Valérie B; Toti, Florence; Morel, Olivier

2017-01-17

Microparticles (MPs) have emerged as a surrogate marker of endothelial dysfunction and cardiovascular risk. This study examined the potential of MPs from senescent endothelial cells (ECs) or from patients with acute coronary syndrome (ACS) to promote premature EC aging and thrombogenicity. Primary porcine coronary ECs were isolated from the left circumflex coronary artery. MPs were prepared from ECs and venous blood from patients with ACS (n=30) and from healthy volunteers (n=4) by sequential centrifugation. The level of endothelial senescence was assessed as senescence-associated β-galactosidase activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, tissue factor activity using an enzymatic Tenase assay, the level of target protein expression by Western blot analysis, platelet aggregation using an aggregometer, and shear stress using a cone-and-plate viscometer. Senescence, as assessed by senescence-associated β-galactosidase activity, was induced by the passaging of porcine coronary artery ECs from passage P1 to P4, and was associated with a progressive shedding of procoagulant MPs. Exposure of P1 ECs to MPs shed from senescent P3 cells or circulating MPs from ACS patients induced increased senescence-associated β-galactosidase activity, oxidative stress, early phosphorylation of mitogen-activated protein kinases and Akt, and upregulation of p53, p21, and p16. Ex vivo, the prosenescent effect of circulating MPs from ACS patients was evidenced only under conditions of low shear stress. Depletion of endothelial-derived MPs from ACS patients reduced the induction of senescence. Prosenescent MPs promoted EC thrombogenicity through tissue factor upregulation, shedding of procoagulant MPs, endothelial nitric oxide synthase downregulation, and reduced nitric oxide-mediated inhibition of platelet aggregation. These MPs exhibited angiotensin-converting enzyme activity and upregulated AT1 receptors and angiotensin

The influence of soy oligosaccharides on apparent and true ileal amino acid digestibilities and fecal consistency in growing pigs.

PubMed

Smiricky, M R; Grieshop, C M; Albin, D M; Wubben, J E; Gabert, V M; Fahey, G C

2002-09-01

Fourteen ileally cannulated pigs (BW = 35 +/- 2 kg) were randomly allotted to a replicated 7 x 7 Latin square design experiment to evaluate the influence of the soybean oligosaccharides (OS), raffinose and stachyose, on ileal nutrient digestibility and fecal consistency. Semipurified diets containing soy protein concentrate (SPC) or soybean meal (SBM) as the sole protein sources were fed. Soy solubles (SS), a by-product of SBM processing containing 3.5% raffinose and 11.5% stachyose, were used to increase dietary raffinose and stachyose concentrations. The seven dietary treatments were SPC, SPC + 9% SS, SBM, SBM + 9% SS, SBM + 18% SS, SBM + 24,000 U alpha-galactosidase enzyme preparation/kg diet, and a low-protein casein (LPC) diet used to calculate true digestibility. Diets, with the exception of the LPC diet, were formulated to contain 17% CP. All diets contained 0.5% chromic oxide as a marker for ileal digestibility determination. The experimental periods were divided into a 5-d diet adaptation followed by 2-d of ileal digesta collection. Diets and digesta were analyzed for DM, N, Cr, amino acids (AA), raffinose, and stachyose. Fecal consistency was determined on d 6 and 7 of each experimental period. The apparent and true ileal AA digestibilities were not different (P < 0.05) for the SPC and SBM control diets. When SS was added to the SPC diet, apparent and true N and AA digestibilities were depressed (P < 0.05) with the exception of Trp and Pro. The apparent and true ileal N and AA digestibilities were not different (P > 0.05) between the SBM control and SBM + 9% SS diets with the exception of Glu. There was a linear decrease (P < 0.05) in apparent and true DM, Val, Gly, and Tyr digestibilities when increasing levels of SS were added to the SBM diet. The addition of alpha-galactosidase did not improve apparent or true ileal N or AA digestibilities except for apparent and true Val and Tyr. Ileal raffinose digestibility was improved (P < 0.05) by addition of a-galactosidase

The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis.

PubMed

Sato, T; Oeller, P W; Theologis, A

1991-02-25

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.

Studies on the interaction of the Sophora japonica lectin and concanavalin A with erythrocytes and lymphocytes.

PubMed Central

Poretz, R D; Barth, R F

1976-01-01

The agglutinating activity of lectins from the seeds of Sophora japonica and Canavalia ensiformis (concanavalin A) with human and murine erythrocytes and lymphocytes have been compared to one another and related to the mitogenic and immunosuppressive properties of these purified proteins. The S. japonica lectin, which demonstrates blood group specificity, is more active than concanavalin A with human erythrocytes, but has a much lower reactivity than concanavalin A with murine red blood cells. Ficin treatment of human erythrocytes results in an increase in agglutinability by both lectins as well as causing the appearance of S. japonica lectin receptors on type O cells. Treatment of murine reythrocytes with ficin alone or followed by beta-galactosidase causes the cells to be more reactive with concanavalin A. Beta-Galactosidase alone has no observable affect on the cells. In contrast, the agglutinability of cells by the S. japonica lectin increases after ficin treatment but is not affected by beta-galaetosidose treatment either after or in the absence of ficinization. Murine lymphocytes react with both lectins in a manner paralleling the agglutination patterns of murine erythrocytes. The S. japonica lectin appears to be devoid of mitogenic and immuno-suppressive activity, in contrast to concanavalin A which suppresses the T helper-dependent antibody response to sheep erythrocytes. These results are discussed in terms of the types of lectin receptors on lymphocytes related to agglutination, induction of blastogenesis and immuno-suppression. PMID:955676

Probiotic properties of lactic acid bacteria isolated from water-buffalo mozzarella cheese.

PubMed

Jeronymo-Ceneviva, Ana Beatriz; de Paula, Aline Teodoro; Silva, Luana Faria; Todorov, Svetoslav Dimitrov; Franco, Bernadette Dora G Mello; Penna, Ana Lúcia B

2014-12-01

This study evaluated the probiotic properties (stability at different pH values and bile salt concentration, auto-aggregation and co-aggregation, survival in the presence of antibiotics and commercial drugs, study of β-galactosidase production, evaluation of the presence of genes encoding MapA and Mub adhesion proteins and EF-Tu elongation factor, and the presence of genes encoding virulence factor) of four LAB strains (Lactobacillus casei SJRP35, Leuconostoc citreum SJRP44, Lactobacillus delbrueckii subsp. bulgaricus SJRP57 and Leuconostoc mesenteroides subsp. mesenteroides SJRP58) which produced antimicrobial substances (antimicrobial peptides). The strains survived the simulated GIT modeled in MRS broth, whole and skim milk. In addition, auto-aggregation and the cell surface hydrophobicity of all strains were high, and various degrees of co-aggregation were observed with indicator strains. All strains presented low resistance to several antibiotics and survived in the presence of commercial drugs. Only the strain SJRP44 did not produce the β-galactosidase enzyme. Moreover, the strain SJRP57 did not show the presence of any genes encoding virulence factors; however, the strain SJRP35 presented vancomycin resistance and adhesion of collagen genes, the strain SJRP44 harbored the ornithine decarboxylase gene and the strain SJRP58 generated positive results for aggregation substance and histidine decarboxylase genes. In conclusion, the strain SJRP57 was considered the best candidate as probiotic cultures for further in vivo studies and functional food products development.

Repression of choline kinase by inositol and choline in Saccharomyces cerevisiae.

PubMed Central

Hosaka, K; Murakami, T; Kodaki, T; Nikawa, J; Yamashita, S

1990-01-01

The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae. The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity. Additional supplementation of choline caused a further reduction in the activity. The coding frame of the choline kinase gene, CK1, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody. Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CK1 probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance. The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene. The coding region of CK1 was replaced with that of lacZ, and CK1 expression was measured by assaying beta-galactosidase. The expression of beta-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal. The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance. Images FIG. 3 FIG. 4 PMID:2156807

Problems in obtaining perfect images by single-particle electron cryomicroscopy of biological structures in amorphous ice.

PubMed

Henderson, Richard; McMullan, Greg

2013-02-01

Theoretical considerations together with simulations of single-particle electron cryomicroscopy images of biological assemblies in ice demonstrate that atomic structures should be obtainable from images of a few thousand asymmetric units, provided the molecular weight of the whole assembly being studied is greater than the minimum needed for accurate position and orientation determination. However, with present methods of specimen preparation and current microscope and detector technologies, many more particles are needed, and the alignment of smaller assemblies is difficult or impossible. Only larger structures, with enough signal to allow good orientation determination and with enough images to allow averaging of many hundreds of thousands or even millions of asymmetric units, have successfully produced high-resolution maps. In this review, we compare the contrast of experimental electron cryomicroscopy images of two smaller molecular assemblies, namely apoferritin and beta-galactosidase, with that expected from perfect simulated images calculated from their known X-ray structures. We show that the contrast and signal-to-noise ratio of experimental images still require significant improvement before it will be possible to realize the full potential of single-particle electron cryomicroscopy. In particular, although reasonably good orientations can be obtained for beta-galactosidase, we have been unable to obtain reliable orientation determination from experimental images of apoferritin. Simulations suggest that at least 2-fold improvement of the contrast in experimental images at ~10 Å resolution is needed and should be possible.

βIII-Gal is involved in galactan reduction during phloem element differentiation in chickpea stems.

PubMed

Martín, Ignacio; Hernández-Nistal, Josefina; Albornos, Lucía; Labrador, Emilia; Dopico, Berta

2013-06-01

βIII-Gal, a member of the chickpea β-galactosidase family, is the enzyme responsible for the cell wall autolytic process. This enzyme, whose activity increases during epicotyl growth, displays significant hydrolytic activity against cell wall pectins, and its natural substrate has been determined as an arabinogalactan from the pectic fraction of the cell wall. In the present work, the localization of βIII-Gal in different seedling and plant organs was analyzed by using specific anti-βIII-Gal antibodies. Our results revealed that besides its possible role in cell wall loosening and in early events during primary xylem and phloem fiber differentiation βIII-Gal acts on the development of sieve elements. Localization of the enzyme in this tissue, both in epicotyls and radicles from seedlings and in the different stem internodes, is consistent with the reduction in galactan during the maturation of phloem elements, as can be observed with LM5 antibodies. Thus, βIII-Gal could act on its natural substrate, the neutral side chains of rhamnogalacturonan I, contributing to cell wall reinforcement allowing phloem elements to differentiate, and conferring the necessary strengthening of the cell wall to fulfill its function. This work completes the immunolocation studies of all known chickpea β-galactosidases. Taken together, our results reflect the broad range of developmental processes covered by different members of this protein family, and confirm their crucial role in cell wall remodeling during tissue differentiation.

Impact of novel histone deacetylase inhibitors, CHAP31 and FR901228 (FK228), on adenovirus-mediated transgene expression.

PubMed

Taura, Kojiro; Yamamoto, Yuzo; Nakajima, Akio; Hata, Koichiro; Uchinami, Hiroshi; Yonezawa, Kei; Hatano, Etsuro; Nishino, Norikazu; Yamaoka, Yoshio

2004-05-01

Histone deacetylase inhibitors (HDIs) are known to enhance adenovirus (Ad)-mediated transgene expression. Recently, novel HDIs, including cyclic hydroxamic-acid-containing peptide 31 (CHAP31) and FR901228 (FK228), have been developed. The effects of these two novel HDIs on Ad-transduced or endogenous gene expression were investigated. Acetylation of core histones and the expression of the coxsackie and adenovirus receptor (CAR) in HDI-treated cells were examined using Western blot and a quantitative reverse transcription polymerase chain reaction (TaqMan RT-PCR), respectively. Their in vivo effect on adenoviral gene expression was investigated in BALB/c mice. Both compounds enhanced and prolonged Ad-mediated beta-galactosidase expression more effectively than did trichostatin A, a classic HDI. The same effect was observed in Ad-transduced heat shock protein 72 (HSP72), but not in hyperthermia-induced endogenous expression of HSP72, suggesting that the effect is specific for transduced gene expression. Hyperacetylation of core histones induced by HDIs was considered responsible for the augmentative effects of gene expression. Intravenous administration of either CHAP31 or FR901228 enhanced beta-galactosidase expression in mice infected with AdLacZ. CHAP31 and FR901228 amplified Ad-mediated transgene expression. The enhancement of transgene expression by HDIs may result in fewer vector doses for necessary gene expression, helping to alleviate disadvantages caused by Ad vectors. This could be a useful tool in overcoming current limitations of gene therapy using adenovirus vectors. Copyright 2004 John Wiley & Sons, Ltd.

Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi; Karhemo, Piia-Riitta; Räsänen, Kati

Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similarmore » secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.« less

Stereotactic delivery of a recombinant adenovirus into a C6 glioma cell line in a rat brain tumor model.

PubMed

Badie, B; Hunt, K; Economou, J S; Black, K L

1994-11-01

The dismal results of conventional therapy for primary malignant brain tumors has justified exploring gene therapy approaches for this disease. Transduction of animal brain tumor models in vivo has been reported previously with retroviruses and herpes viruses. Because adenoviruses have the advantage of transducing quiescent and actively dividing tumor cells, they may prove to be more effective in such therapy. We used a replication-deficient recombinant adenovirus bearing the Escherichia coli beta-galactosidase gene in a rat C6 glioma tumor model. Transduced cells were detected by X-5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to reveal beta-galactosidase activity. Initial experiments in vitro showed 50% and 90% transduction at vector titers of approximately 10(7) and 10(8) plaque-forming units/ml, respectively. Although no cytopathic effects were seen at 10(7) plaque-forming units/ml, more than 50% reduction in tumor cell growth was noted at 10(8) plaque-forming units/ml both in vitro and in vivo. Stereotactic delivery of the recombinant adenovirus into the frontal lobe of normal rat brains resulted in intense staining of all cell types, that is, neurons, astrocytes, and ependymal cells. Stereotactic injection into C6 glioma brain tumors in rats stained 25 to 30% of the tumor cells. We conclude that adenovirus vectors can be used to transfer genes to central nervous system tumors in vivo. Using stereotactic delivery, adenovirus vectors can transfer genes into the central nervous system intended for tumor therapy.

Analysis of the L1 gene product of human papillomavirus type 16 by expression in a vaccinia virus recombinant.

PubMed

Browne, H M; Churcher, M J; Stanley, M A; Smith, G L; Minson, A C

1988-06-01

The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.

Fabry disease presenting as apical left ventricular hypertrophy in a patient carrying the missense mutation R118C.

PubMed

Caetano, Francisca; Botelho, Ana; Mota, Paula; Silva, Joana; Leitão Marques, António

2014-03-01

Anderson-Fabry disease is an X-linked lysosomal storage disorder caused by abnormalities of the GLA gene, which encodes the enzyme α-galactosidase A. A deficiency of this enzyme leads to the lysosomal accumulation of glycosphingolipids, which may cause left ventricular hypertrophy that is typically concentric and symmetric. We present the case of a 60-year-old woman with symptoms of dyspnea, atypical chest pain and palpitations, in whom a transthoracic echocardiogram revealed an apical variant of hypertrophic cardiomyopathy. Analysis of specific sarcomeric genetic mutations was negative. The patient underwent a screening protocol for Anderson-Fabry disease, using a dried blood spot test, which was standard at our institution for patients with left ventricular hypertrophy. The enzymatic activity assay revealed reduced α-galactosidase A enzymatic activity. Molecular analysis identified a missense point mutation in the GLA gene (p.R118C). This case report shows that Anderson-Fabry disease may cause an apical form of left ventricular hypertrophy. The diagnosis was only achieved because of systematic screening, which highlights the importance of screening for Anderson-Fabry disease in patients with unexplained left ventricular hypertrophy, including those presenting with more unusual patterns, such as apical variants of left ventricular hypertrophy. This case also supports the idea that the missense mutation R118C is indeed a true pathogenic mutation of Anderson-Fabry disease. Copyright © 2012 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

Marek's disease virus protein kinase gene identified within the short unique region of the viral genome is not essential for viral replication in cell culture and vaccine-induced immunity in chickens.

PubMed

Sakaguchi, M; Urakawa, T; Hirayama, Y; Miki, N; Yamamoto, M; Zhu, G S; Hirai, K

1993-07-01

The open reading frame (ORF) of 1206 bp within the short unique region (Us) of Marek's disease virus type 1 (MDV1) shows significant homology with the herpes simplex virus type 1 US3 gene encoding protein kinase (PK). The lacZ gene of Escherichia coli was inserted within the ORF, designated MDV1-US3, of MDV1 K544 strain DNA by homologous recombination. The plaque-purified recombinant MDV1 stably expressed the beta-galactosidase encoded by the inserted lacZ gene in infected cells and replicated well as the parental K544 strain. Antibodies against both MDV1 antigen and beta-galactosidase were detected in the sera of chickens immunized with recombinant MDV1. Chickens vaccinated with the recombinant MDV1 were protected from challenge with virulent MDV1. The MDV1 US3 gene expressed by a baculovirus vector encoded a 44-kDa protein. Mouse antisera against the 44-kDa protein reacted with two proteins of 44 and 45 kDa in extracts of cells infected with MDV1 but not with MDV types 2 or 3. The PK activity was detected in immune complexes of the anti-44-kDa sera with extracts of cells infected with MDV1 but not with the recombinant MDV1. Thus, PK encoded from the MDV1-US3 is not essential for virus replication in cell culture and vaccine-induced immunity.

Regulation of tyramine oxidase synthesis in Klebsiella aerogenes.

PubMed Central

Okamura, H; Murooka, Y; Harada, T

1976-01-01

Tyramine oxidase in Klebsiella aerogenes is highly specific for tyramine, dopamine, octopamine, and norepinephrine, and its synthesis is induced specifically by these compounds. The enzyme is present in a membrane-bound form. The Km value for tyramine is 9 X 10(-4) M. Tyramine oxidase synthesis was subjected to catabolite repression by glucose in the presence of ammonium salts. Addition of cyclic adenosine 3',5'-monophosphate (cAMP) overcame the catabolite repression. A mutant strain, K711, which can produce a high level of beta-galactosidase in the presence of glucose and ammonium chloride, can also synthesize tyramine oxidase and histidase in the presence of inducer in glucose ammonium medium. Catabolite repression of tyramine oxidase synthesis was relieved when the cells were grown under conditions of nitrogen limitation, whereas beta-galactosidase was strongly repressed under these conditions. A cAMP-requiring mutant, MK54, synthesized tyramine oxidase rapidly when tyramine was used as the sole source of nitrogen in the absence of cAMP. However, a glutamine synthetase-constitutive mutant, MK94, failed to synthesize tyramine oxidase in the presence of glucose and ammonium chloride, although it synthesized histidase rapidly under these conditions. These results suggest that catabolite repression of tyramine oxidase synthesis in K. aerogenes is regulated by the intracellular level of cAMP and an unknown cytoplasmic factor that acts independently of cAMP and is formed under conditions of nitrogen limitation. PMID:179974

Influence of lactose hydrolysis on galacto-oligosaccharides, lactose, volatile profile and physicochemical parameters of different yogurt varieties.

PubMed

Vénica, Claudia I; Wolf, Irma V; Bergamini, Carina V; Perotti, María C

2016-12-01

Different types of reduced-lactose yogurt, obtained by lactose hydrolysis using β-galactosidase enzyme, are commercially available. The breakdown of lactose modifies the carbohydrate profile, including the production of prebiotic galacto-oligosaccharides (GOS), which could affect the survival and activity of starter and probiotic cultures and the parameters of yogurt quality. The extension of these changes is dependent on the yogurt matrix composition. This study aimed to evaluate the influence of lactose hydrolysis on GOS, lactose, volatile profile and physicochemical parameters of different yogurt varieties during storage. The presence of β-galactosidase enzyme did not affect either the global composition or the survival of cultures. Overall, the hydrolyzed products had lower acidity than traditional ones. GOS were found at similar levels in fresh hydrolyzed yogurts, whereas in traditional yogurts they were not detected. The proportion of ketones, acids and aldehydes seems to be more dependent on yogurt variety than on addition of the enzyme. Likewise, the storage period affected the volatile fraction to different degree; the increase in acid compounds was more pronounced in hydrolyzed than in traditional yogurts. This work shows that it is possible to obtain different varieties of reduced-lactose yogurt, some of them with additional benefits to health such as reduced fat, reduced calories, added with probiotic/inulin and enriched in GOS, with similar characteristics to traditional products. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

The effect of enzyme replacement therapy on clinical outcomes in paediatric patients with Fabry disease - A systematic literature review by a European panel of experts.

PubMed

Spada, Marco; Baron, Ralf; Elliott, Perry M; Falissard, Bruno; Hilz, Max J; Monserrat, Lorenzo; Tøndel, Camilla; Tylki-Szymańska, Anna; Wanner, Christoph; Germain, Dominique P

2018-04-26

Fabry disease is caused by a deficiency of the lysosomal enzyme α-galactosidase, resulting in progressive accumulation of globotriaosylceramide (GL-3). The disease can manifest early during childhood and adolescence. Enzyme replacement therapy (ERT) with recombinant human α-galactosidase is the first specific treatment for Fabry disease and has been available in Europe since 2001. This paper presents the findings of a systematic literature review of clinical outcomes with ERT in paediatric patients with Fabry disease. A comprehensive systematic review of published literature on ERT in Fabry disease was conducted in January 2017. The literature analysis included all original articles reporting outcomes of ERT in paediatric patients. Treatment-related outcomes in the paediatric population were reported in six publications derived from open-label clinical trials and in 10 publications derived from observational or registry-based studies. ERT was shown to significantly reduce plasma and urine GL-3 levels in paediatric patients with Fabry disease. The effect of ERT on GL-3 clearance from renal podocytes appeared to be agalsidase dose-dependent. ERT relieved pain and improved gastrointestinal symptoms and quality of life. Based on the published literature, the use of ERT in paediatric patients can significantly clear GL-3 accumulation, ameliorate the early symptoms of Fabry disease, and improve quality of life. Treatment with ERT in paediatric patients with Fabry disease may be important to prevent further disease progression and overt organ damage. Copyright © 2018. Published by Elsevier Inc.

Effects of cetyltriethylammonium bromide on the replication of Bombyx mori nucleopolyhedrovirus.

PubMed

Zhou, Yajing; Zhang, Zhifang; He, Jialu; Zhang, Yuanxing

2002-05-01

An experimental study was undertaken to quantify the effects of cetyltriethylammonium bromide (CTAB) on the replication of Bombyx mori nucleopolyhedrovirus (BmNPV) and the transcriptionalactivity of BmNPV ie-1 promoter. The results demonstrated that the budded virus (BV) titer rose about 3.7-fold by adding CTAB to the culture media up to 0.1 mu g ml(-1) in infected Bm-N cells with a wild-type BmNPV. The transient expression level of luciferase driven by BmNPV ie-1 promoter was enhanced by more than 3-fold in the presence of 0.1 mu g ml(-1) of CTAB in uninfected insect cells via a transient expression system. Contrary to the rise in BV titer, the polyhedra inside the nucleus of infected cells dropped linearly from 4.0 x 10(6) ml(-1) down to 2.1 x 10(6) ml(-1) with in a range of CTAB concentrations from 0 to 0.25 mu g ml(-1). The same trend in expression level of beta -galactosidase or phytase was given when the Bm-N cells or fifth-instar silkworm larvae infected with a recombinant BmNPV containing the beta -galactosidase or phytase reporter gene driven by the polyhedrin promoter. We deduced that CTAB appeared to affect the virus bi-phasic life cycle stages and production pathways, resulting in an enhancement in BV production and a suppression of occluded virus (OV) production and expression of foreign genes controlled by the polyhedrin promoter.

Molecular biological enhancement of coal biodesulfurization. [Quarterly] technical report, December 1, 1993--February 28, 1994

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kilbane, J.J. II

1994-06-01

IGT has developed a microbial culture of Rhodococcus rhodochrous, IGTS8, that is capable of specifically cleaving carbon-sulfur bonds in a range of organosulfur model compounds and is capable of removing organic sulfur from coal and petroleum. Although IGTS8 possesses the ability to specifically remove organic sulfur from coal, a major research need is to develop improved strain`s of microorganisms that possess higher levels of desulfurization activity and therefore wall permit more favorable biodesulfurization process conditions: faster rates, mare complete removal, and smaller reactor size. Strain improvement is the single most important aspect to the development of a practical coal biodesulfurizationmore » process and accordingly is the focus of research in this project. Several possible strong promoters have been isolated and are in the process of being analyzed. When these promoters have been characterized for inducibility, strength, transcriptional start sites and other physical properties, they will be placed in front of the desulfurization genes and expression will be monitored. Improved promoter probe vectors have been constructed, allowing a conclusive screen of all putative Rhodococcus promoters. With the improved methodologies in the handling of Rhodococcus RNA, we have begun to gauge promoter expression using Northern blots. During this quarter we have constructed and successfully used a promoter probe vector using the {beta}-galactosidane gene from E. coli. A chromosomal promoter library was constructed upstream from the {beta}-galactosidase gene. Over 200 colonies were isolated that yielded {beta}-galactosidase activity.« less

Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Higgins, M.; Whitworth, G; El Warry, N

2009-01-01

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-{beta}-galactosidase activity on the LewisY antigen. Altered active site topography in themore » other species of GH98 enzyme tune its endo-{beta}-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.« less

Identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus.

PubMed

Smith, A R; Boursnell, M E; Binns, M M; Brown, T D; Inglis, S C

1990-01-01

Nucleotide sequences from the third open reading frame of mRNA D (D3) of infectious bronchitis virus (IBV) were expressed in bacteria as part of a fusion protein with beta-galactosidase. Antiserum raised in rabbits against this fusion protein immunoprecipitated from IBV-infected chick kidney or Vero cells a polypeptide of 12.4K, the size expected for a D3-encoded product. The D3 polypeptide is apparently non-glycosylated, and appears to be associated with the membrane fraction of infected cells, as judged by cell fractionation and immunofluorescence.

Optical Imaging of Targeted β-Galactosidase in Brain Tumors to Detect EGFR Levels

PubMed Central

Broome, Ann-Marie; Ramamurthy, Gopal; Lavik, Kari; Liggett, Alexander; Kinstlinger, Ian; Basilion, James

2015-01-01

A current limitation in molecular imaging is that it often requires genetic manipulation of cancer cells for noninvasive imaging. Other methods to detect tumor cells in vivo using exogenously delivered and functionally active reporters, such as β-gal, are required. We report the development of a platform system for linking β-gal to any number of different ligands or antibodies for in vivo targeting to tissue or cells, without the requirement for genetic engineering of the target cells prior to imaging. Our studies demonstrate significant uptake in vitro and in vivo of an EGFR-targeted β-gal complex. We were then able to image orthotopic brain tumor accumulation and localization of the targeted enzyme when a fluorophore was added to the complex, as well as validate the internalization of the intravenously administered β-gal reporter complex ex vivo. After fluorescence imaging localized the β-gal complexes to the brain tumor, we topically applied a bioluminescent β-gal substrate to serial sections of the brain to evaluate the delivery and integrity of the enzyme. Finally, robust bioluminescence of the EGFR-targeted β-gal complex was captured within the tumor during noninvasive in vivo imaging. PMID:25775241

Optical imaging of targeted β-galactosidase in brain tumors to detect EGFR levels.

PubMed

Broome, Ann-Marie; Ramamurthy, Gopal; Lavik, Kari; Liggett, Alexander; Kinstlinger, Ian; Basilion, James

2015-04-15

A current limitation in molecular imaging is that it often requires genetic manipulation of cancer cells for noninvasive imaging. Other methods to detect tumor cells in vivo using exogenously delivered and functionally active reporters, such as β-gal, are required. We report the development of a platform system for linking β-gal to any number of different ligands or antibodies for in vivo targeting to tissue or cells, without the requirement for genetic engineering of the target cells prior to imaging. Our studies demonstrate significant uptake in vitro and in vivo of an EGFR-targeted β-gal complex. We were then able to image orthotopic brain tumor accumulation and localization of the targeted enzyme when a fluorophore was added to the complex, as well as validate the internalization of the intravenously administered β-gal reporter complex ex vivo. After fluorescence imaging localized the β-gal complexes to the brain tumor, we topically applied a bioluminescent β-gal substrate to serial sections of the brain to evaluate the delivery and integrity of the enzyme. Finally, robust bioluminescence of the EGFR-targeted β-gal complex was captured within the tumor during noninvasive in vivo imaging.

A study of lactose metabolism in Lactococcus garvieae reveals a genetic marker for distinguishing between dairy and fish biotypes.

PubMed

Fortina, Maria Grazia; Ricci, Giovanni; Borgo, Francesca

2009-06-01

Dairy and fish isolates of Lactococcus garvieae were tested for their ability to utilize lactose and to grow in milk. Fish isolates were unable to assimilate lactose, but unexpectedly, they possessed the ability to grow in milk. Genetic studies, carried out constructing different vectorette libraries, provided evidence that in fish isolates, no genes involved in lactose utilization were present. For L. garvieae dairy isolates, a single system for the catabolism of lactose was found. It consists of a lactose transport and hydrolysis depending on a phosphoenolpyruvate-dependent phosphotransferase system combined with a phospho-beta-galactosidase. The genes involved were highly similar at the nucleotide sequence level to their counterparts in Lactococcus lactis; however, while in many L. lactis strains these genes are plasmid encoded, in L. garvieae they are chromosomally located. Thus, in the species L. garvieae, the phospho-beta-galactosidase gene, detectable in all strains of dairy origin but lacking in fish isolates, can be considered a reliable genetic marker for distinguishing biotypes in the two diverse ecological niches. Moreover, we obtained information regarding the complete nucleotide sequence of the gal operon in L. garvieae, consisting of a galactose permease and the Leloir pathway enzymes. This is one of the first reports concerning the determination of the nucleotide sequences of genes (other than the 16S rDNA gene) in L. garvieae and should be considered a step in a continuous effort to explore the genome of this species, with the aim of determining the real relationship between the presence of L. garvieae in dairy products and food safety.

In vitro and in vivo tetracycline-controlled myogenic conversion of NIH-3T3 cells: evidence of programmed cell death after muscle cell transplantation.

PubMed

Del Bo, R; Torrente, Y; Corti, S; D'Angelo, M G; Comi, G P; Fagiolari, G; Salani, S; Cova, A; Pisati, F; Moggio, M; Ausenda, C; Scarlato, G; Bresolin, N

2001-01-01

Ex vivo gene therapy of Duchenne muscular dystrophy based on autologous transplantation of genetically modified myoblasts is limited by their premature senescence. MyoD-converted fibroblasts represent an alternative source of myogenic cells. In this study the forced MyoD-dependent conversion of murine NIH-3T3 fibroblasts into myoblasts under the control of an inducible promoter silent in the presence of tetracycline was evaluated. After tetracycline withdrawal this promoter drives the transcription of MyoD in the engineered fibroblasts, inducing their myogenesis and giving rise to beta-galactosidase-positive cells. MyoD-expressing fibroblasts withdrew from the cell cycle, but were unable to fuse in vitro into multinucleated myotubes. Five days following implantation of engineered fibroblasts in muscles of C57BL/10J mice we observed a sevenfold increase of beta-galactosidase-positive regenerating myofibers in animals not treated with antibiotic compared with treated animals. After 1 week the number of positive fibers decreased and several apoptotic myonuclei were detected. Three weeks following implantation of MyoD-converted fibroblasts in recipient mice, no positive "blue" fiber was observed. Our results suggest that transactivation by tetracycline of MyoD may drive an in vivo myogenic conversion of NIH-3T3 fibroblasts and that, in this experimental setting, apoptosis plays a relevant role in limiting the efficacy of engineered fibroblast transplantation. This work opens the question whether apoptotic phenomena also play a general role as limiting factors of cell-mediated gene therapy of inherited muscle disorders.

Transduction of satellite cells after prenatal intramuscular administration of lentiviral vectors.

PubMed

MacKenzie, Tippi C; Kobinger, Gary P; Louboutin, Jean-Pierre; Radu, Antoneta; Javazon, Elizabeth H; Sena-Esteves, Miguel; Wilson, James M; Flake, Alan W

2005-01-01

We have previously reported long-term expression of lacZ in myocytes after in utero intramuscular injection of Mokola and Ebola pseudotyped lentiviral vectors. In further experiments, we have noted that these vectors also transduce small cells at the periphery of the muscle fibers that have the morphology of satellite cells, or muscle stem cells. In this study we performed experiments to further define the morphology and function of these cells. Balb/c mice at 14-15 days gestation were injected intramuscularly with Ebola or Mokola pseudotyped lentiviral vectors carrying CMV-lacZ. Animals were harvested at various time points, muscles were stained with X-gal, and processed for electron microscopy (EM) and immunofluorescence. To determine whether transduced satellite cells were functionally capable of regenerating injured muscles, animals were injected with notexin in the same area 8 weeks after the in utero injection of viral vector. Transmission EM of transduced cells confirmed the ultrastructural appearance of satellite cells. Double immunofluorescence for beta-galactosidase and satellite cell markers demonstrated co-localization of these markers in transduced cells. In the notexin-injured animals, small blue cells were seen at the areas of regeneration that co-localized beta-galactosidase with markers of regenerating satellite cells. Central nucleated blue fibers were seen at late time points, indicating regenerated muscle fibers arising from a transduced satellite cell. This study demonstrates transduction of muscle satellite cells following prenatal viral vector mediated gene transfer. These findings may have important implications for gene therapy strategies directed toward muscular dystrophy.

Production of Exopolysaccharide by Lactobacillus rhamnosus R and Analysis of Its Enzymatic Degradation during Prolonged Fermentation

PubMed Central

Pham, P. L.; Dupont, I.; Roy, D.; Lapointe, G.; Cerning, J.

2000-01-01

The potential of Lactobacillus rhamnosus R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated. EPS production by L. rhamnosus R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars. The product yield coefficient (YEPS/S) was 3.15 (0.0315 g of EPS [g of lactose]−1) and 2.88 (0.0288 g of EPS [g of glucose]−1). It was clearly shown that the amount of EPS produced declined upon prolonged fermentation. Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities. As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown. The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium. Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (α-d-glucosidase, β-d-glucosidase, α-d-galactosidase, β-d-galactosidase, β-d-glucuronidase, and some traces of α-l-rhamnosidase). These enzymes were localized, two of them (α-d-glucosidase and β-d-glucuronidase) were partially purified and characterized. When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars. Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%. PMID:10831403

Variations in plasma and urinary lipids in response to enzyme replacement therapy for Fabry disease patients by nanoflow UPLC-ESI-MS/MS.

PubMed

Byeon, Seul Kee; Kim, Jin Yong; Lee, Jin-Sung; Moon, Myeong Hee

2016-03-01

A deficiency of α-galactosidase A causes Fabry disease (FD) by disrupting lipid metabolism, especially trihexosylceramide (THC). Enzyme replacement therapy (ERT) is clinically offered to FD patients in an attempt to lower the accumulated lipids. Studies on specific types of lipids that are directly or indirectly altered by FD are very scarce, even though they are crucial in understanding the biological process linked to the pathogenesis of FD. We performed a comprehensive lipid profiling of plasma and urinary lipids from FD patients with nanoflow liquid chromatography electrospray-ionization tandem mass spectrometry (nLC-ESI-MS/MS) and identified 129 plasma and 111 urinary lipids. Among these, lipids that exhibited alternations (>twofold) in patients were selected as targets for selected reaction monitoring (SRM)-based high-speed quantitation using nanoflow ultra-performance LC-ESI-MS/MS (nUPLC-ESI-MS/MS) and 31 plasma and 26 urinary lipids showed significant elevation among FD patients. Higher percentages of sphingolipids (SLs; 48% for plasma and 42% for urine) were highly elevated in patients; whereas, a smaller percentage of phospholipids (PLs; 15% for plasma and 13% for urine) were significantly affected. Even though α-galactosidase A is reported to affect THC only, the results show that other classes of lipids (especially SLs) are changed as well, indicating that FD not only alters metabolism of THC but various classes of lipids too. Most lipids showing significant increases in relative amounts before ERT decreased after ERT, but overall, ERT influenced plasma lipids more than urinary lipids.

Disabled infectious single cycle herpes simplex virus (DISC-HSV) is a candidate vector system for gene delivery/expression of GM-CSF in human prostate cancer therapy.

PubMed

Parkinson, Richard J; Mian, Shahid; Bishop, Michael C; Gray, Trevor; Li, Geng; McArdle, Stephanie E B; Ali, Selman; Rees, Robert C

2003-06-15

DISC-HSV is a replication incompetent herpes simplex virus that is a highly efficient vector for the transduction of genes in vivo and in vitro. We examine the ability of DISC-HSV to infect human prostate cancer cell-lines and xenograft tumor models, and induce expression of reporter and therapeutic cytokine genes. Infection was confirmed by cellular staining for the beta-galactosidase reporter gene product, and by EM. Human GM-CSF production following DISC-hGMCSF infection was measured using ELISA. The metabolic activity of infected cells was determined by NADP/NADPH assay. Cell death was estimated by cell-cycle analysis using flow cytometry with propidium iodide staining. Infection of DU145, PC3 and LNCaP cells with DISC-HSV was dose dependent. Cells infected with DISC-hGM-CSF released significant levels of hGM-CSF for 3 days. NADP/NADPH assay suggested that infected cells continued to be metabolically active for 3 days post-infection, which was consistent with flow cytometry findings that cell death did not occur within 7 days of infection. Tumor xenografts injected with DISC-HSV expressed beta-galactosidase, and intracellular viral particles were demonstrated using EM. We have previously reported the rejection of established tumors following intra-tumoral injection of DISC-GMCSF. This study demonstrates the ability of DISC-HSV to infect prostate cancer and express GMCSF at significant levels. We suggest that prostate cancer is a potential target for therapy using DISC-HSV containing GM-CSF. Copyright 2003 Wiley-Liss, Inc.

A biochemical and pharmacological comparison of enzyme replacement therapies for the glycolipid storage disorder Fabry disease.

PubMed

Lee, Karen; Jin, Xiaoying; Zhang, Kate; Copertino, Lorraine; Andrews, Laura; Baker-Malcolm, Jennifer; Geagan, Laura; Qiu, Huawei; Seiger, Keirsten; Barngrover, Debra; McPherson, John M; Edmunds, Tim

2003-04-01

Fabry disease is a lysosomal storage disease arising from deficiency of the enzyme alpha-galactosidase A. Two recombinant protein therapeutics, Fabrazyme (agalsidase beta) and Replagal (agalsidase alfa), have been approved in Europe as enzyme replacement therapies for Fabry disease. Both contain the same human enzyme, alpha-galactosidase A, but they are produced using different protein expression systems and have been approved for administration at different doses. To determine if there is recognizable biochemical basis for the different doses, we performed a comparison of the two drugs, focusing on factors that are likely to influence biological activity and availability. The two drugs have similar glycosylation, both in the type and location of the oligosaccharide structures present. Differences in glycosylation were mainly limited to the levels of sialic acid and mannose-6-phosphate present, with Fabrazyme having a higher percentage of fully sialylated oligosaccharides and a higher level of phosphorylation. The higher levels of phosphorylated oligomannose residues correlated with increased binding to mannose-6-phosphate receptors and uptake into Fabry fibroblasts in vitro. Biodistribution studies in a mouse model of Fabry disease showed similar organ uptake. Likewise, antigenicity studies using antisera from Fabry patients demonstrated that both drugs were indistinguishable in terms of antibody cross-reactivity. Based on these studies and present knowledge regarding the influence of glycosylation on protein biodistribution and cellular uptake, the two protein preparations appear to be functionally indistinguishable. Therefore, the data from these studies provide no rationale for the use of these proteins at different therapeutic doses.

Chlorogenic acid ameliorates isoproterenol-induced myocardial injury in rats by stabilizing mitochondrial and lysosomal enzymes.

PubMed

Akila, Palaniyandi; Asaikumar, Lourthurani; Vennila, Lakshmanan

2017-01-01

This study was deliberated to aspire the effects of chlorogenic acid (CGA) against myocardial infarction (MI) induced by Isoproterenol (ISO), in a rat model. In the pathology of MI, enzymes released due to the mitochondrial and lysosomal lipid peroxidation play an integral role. Induction of rats with ISO (85mg/kg BW) for 2 consecutive days resulted in a significant decrease in the activities of heart mitochondrial enzymes isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH). The activities of lysosomal enzymes (β- glucosidase, β-glucuronidase, α-galactosidase, β-galactosidase, cathepsin-B and cathepsin-D) were increased significantly in the heart tissue. A prominent expression of LDH 1 and LDH 2 isoenzymes in the serum were observed and changes in the Electrocardiographic (ECG) patterns were also recorded in the ISO-induced rats. The prior administrations of CGA (40mg/kg BW) for 19days markedly ameliorated ISO induced alterations in ECG and significantly restored the activities of all the above enzymes in the heart of ISO-induced rats, which substantiates the stress stabilizing action of CGA. Oral administration of CGA (40mg/kg BW) to normal rats did not show any significant changes. These biochemical functional alterations were supported by the histology of heart (Massion's trichrome and Picrosirius red staining for collagen formation). Thereupon, this study shows that 40mg/kg BW of CGA gives protection against ISO-induced MI and demonstrates that CGA has a significant effect in the protection of heart. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Generation of stable cell line by using chitosan as gene delivery system.

PubMed

Şalva, Emine; Turan, Suna Özbaş; Ekentok, Ceyda; Akbuğa, Jülide

2016-08-01

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.

Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass

DOE PAGES

dos Santos, Hevila Brognaro; Bezerra, Thais Milena Souza; Pradella, Jose G. C.; ...

2016-11-02

Biomass is abundant, renewable and useful for biofuel production as well as chemical priming for plastics and composites. Deconstruction of biomass by enzymes is perceived as recalcitrant while an inclusive breakdown mechanism remains to be discovered. Fungi such as Myceliophthora thermophila M77 appear to decompose natural biomass sources quite well. This work reports on this fungus fermentation property while producing cellulolytic enzymes using natural biomass substrates. Little hydrolytic activity was detected, insufficient to explain the large amount of biomass depleted in the process. Furthermore, this work makes a comprehensive account of extracellular proteins and describes how secretomes redirect their qualitativemore » protein content based on the nature and chemistry of the nutritional source. Fungus grown on purified cellulose or on natural biomass produced secretomes constituted by: cellobiohydrolases, cellobiose dehydrogenase, β-1,3 glucanase, β-glucosidases, aldose epimerase, glyoxal oxidase, GH74 xyloglucanase, galactosidase, aldolactonase and polysaccharide monooxygenases. Fungus grown on a mixture of purified hemicellulose fractions (xylans, arabinans and arabinoxylans) produced many enzymes, some of which are listed here: xylosidase, mixed β-1,3(4) glucanase, β-1,3 glucanases, β-glucosidases, β-mannosidase, β-glucosidases, galactosidase, chitinases, polysaccharide lyase, endo β-1,6 galactanase and aldose epimerase. Secretomes produced on natural biomass displayed a comprehensive set of enzymes involved in hydrolysis and oxidation of cellulose, hemicellulose-pectin and lignin. Furthermore, the participation of oxidation reactions coupled to lignin decomposition in the breakdown of natural biomass may explain the discrepancy observed for cellulose decomposition in relation to natural biomass fermentation experiments.« less

Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass

DOE Office of Scientific and Technical Information (OSTI.GOV)

dos Santos, Hevila Brognaro; Bezerra, Thais Milena Souza; Pradella, Jose G. C.

Biomass is abundant, renewable and useful for biofuel production as well as chemical priming for plastics and composites. Deconstruction of biomass by enzymes is perceived as recalcitrant while an inclusive breakdown mechanism remains to be discovered. Fungi such as Myceliophthora thermophila M77 appear to decompose natural biomass sources quite well. This work reports on this fungus fermentation property while producing cellulolytic enzymes using natural biomass substrates. Little hydrolytic activity was detected, insufficient to explain the large amount of biomass depleted in the process. Furthermore, this work makes a comprehensive account of extracellular proteins and describes how secretomes redirect their qualitativemore » protein content based on the nature and chemistry of the nutritional source. Fungus grown on purified cellulose or on natural biomass produced secretomes constituted by: cellobiohydrolases, cellobiose dehydrogenase, β-1,3 glucanase, β-glucosidases, aldose epimerase, glyoxal oxidase, GH74 xyloglucanase, galactosidase, aldolactonase and polysaccharide monooxygenases. Fungus grown on a mixture of purified hemicellulose fractions (xylans, arabinans and arabinoxylans) produced many enzymes, some of which are listed here: xylosidase, mixed β-1,3(4) glucanase, β-1,3 glucanases, β-glucosidases, β-mannosidase, β-glucosidases, galactosidase, chitinases, polysaccharide lyase, endo β-1,6 galactanase and aldose epimerase. Secretomes produced on natural biomass displayed a comprehensive set of enzymes involved in hydrolysis and oxidation of cellulose, hemicellulose-pectin and lignin. Furthermore, the participation of oxidation reactions coupled to lignin decomposition in the breakdown of natural biomass may explain the discrepancy observed for cellulose decomposition in relation to natural biomass fermentation experiments.« less

An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geller, A.I.; Keyomarsi, K.; Bryan, J.

1990-11-01

The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli {beta}-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; tsmore » mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses {beta}-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.« less

Delivery of Plasmid DNA to Vascular Tissue in vivo using Catheter Balloons Coated with Polyelectrolyte Multilayers

PubMed Central

Saurer, Eric M.; Yamanouchi, Dai; Liu, Bo; Lynn, David M.

2010-01-01

We report an approach for the localized delivery of plasmid DNA to vascular tissue from the surfaces of inflatable embolectomy catheter balloons. Using a layer-by-layer approach, ultrathin multilayered polyelectrolyte films were fabricated on embolectomy catheter balloons by alternately adsorbing layers of a hydrolytically degradable poly(β-amino ester) and plasmid DNA. Fluorescence microscopy revealed that the films coated the surfaces of the balloons uniformly. Coated balloons that were incubated in phosphate-buffered saline at 37 °C released ~25 μg DNA/cm2 over 24 hours. Analysis of the DNA by gel electrophoresis showed that the DNA was released in open-circular (‘nicked’) and supercoiled conformations, and in vitro cell transfection assays confirmed that the released DNA was transcriptionally active. Arterial injury was induced in the internal carotid arteries of Sprague-Dawley rats using uncoated balloons, followed by treatment with film-coated balloons for 20 minutes. X-gal, immunohistochemical, and immunofluorescence staining of sectioned arteries indicated high levels of β-galactosidase or enhanced green fluorescent protein (EGFP) expression in arteries treated with film-coated balloons. β-galactosidase and EGFP expression were observed throughout the medial layers of arterial tissue, and around approximately two-thirds of the circumference of the treated arteries. The layer-by-layer approach reported here provides a general platform for the balloon-mediated delivery of DNA to vascular tissue. Our results suggest the potential of this approach to deliver therapeutically relevant DNA to prevent complications such as intimal hyperplasia that arise after vascular interventions. PMID:20933275

The Transposable Element Mariner Mediates Germline Transformation in Drosophila Melanogaster

PubMed Central

Lidholm, D. A.; Lohe, A. R.; Hartl, D. L.

1993-01-01

A vector for germline transformation in Drosophila melanogaster was constructed using the transposable element mariner. The vector, denoted pMlwB, contains a mariner element disrupted by an insertion containing the wild-type white gene from D. melanogaster, the β-galactosidase gene from Escherichia coli and sequences that enable plasmid replication and selection in E. coli. The white gene is controlled by the promoter of the D. melanogaster gene for heat-shock protein 70, and the β-galactosidase gene is flanked upstream by the promoter of the transposable element P as well as that of mariner. The MlwB element was introduced into the germline of D. melanogaster by co-injection into embryos with an active mariner element, Mos1, which codes for a functional transposase and serves as a helper. Two independent germline insertions were isolated and characterized. The results show that the MlwB element inserted into the genome in a mariner-dependent manner with the termini of the inverted repeats inserted at a TA dinucleotide. Both insertions exhibit an unexpected degree of germline and somatic stability, even in the presence of an active mariner element in the genetic background. These results demonstrate that the mariner transposable element, which is small (1286 bp) and relatively homogeneous in size among different copies, is nevertheless capable of promoting the insertion of the large (13.2 kb) MlwB element. Because of the widespread phylogenetic distribution of mariner among insects, these results suggest that mariner might provide a wide hostrange transformation vector for insects. PMID:8394264

Endothelial Cell Implantation and Survival within Experimental Gliomas

NASA Astrophysics Data System (ADS)

Lal, Bachchu; Indurti, Ravi R.; Couraud, Pierre-Olivier; Goldstein, Gary W.; Laterra, John

1994-10-01

The delivery of therapeutic genes to primary brain neoplasms opens new opportunities for treating these frequently fatal tumors. Efficient gene delivery to tissues remains an important obstacle to therapy, and this problem has unique characteristics in brain tumors due to the blood-brain and blood-tumor barriers. The presence of endothelial mitogens and vessel proliferation within solid tumors suggests that genetically modified endothelial cells might efficiently transplant to brain tumors. Rat brain endothelial cells immortalized with the adenovirus E1A gene and further modified to express the β-galactosidase reporter were examined for their ability to survive implantation to experimental rat gliomas. Rats received 9L, F98, or C6 glioma cells in combination with endothelial cells intracranially to caudate/putamen or subcutaneously to flank. Implanted endothelial cells were identified by β-galactosidase histochemistry or by polymerase chain reaction in all tumors up to 35 days postimplantation, the latest time examined. Implanted endothelial cells appeared to cooperate in tumor vessel formation and expressed the brain-specific endothelial glucose transporter type 1 as identified by immunohistochemistry. The proliferation of implanted endothelial cells was supported by their increased number within tumors between postimplantation days 14 and 21 (P = 0.015) and by their expression of the proliferation antigen Ki67. These findings establish that genetically modified endothelial cells can be stably engrafted to growing gliomas and suggest that endothelial cell implantation may provide a means of delivering therapeutic genes to brain neoplasms and other solid tumors. In addition, endothelial implantation to brain may be useful for defining mechanisms of brain-specific endothelial differentiation.

Towards complete hydrolysis of soy flour carbohydrates by enzyme mixtures for protein enrichment: A modeling approach.

PubMed

Loman, Abdullah Al; Ju, Lu-Kwang

2016-05-01

Soy protein is a well-known nutritional supplement in proteinaceous food and animal feed. However, soybeans contain complex carbohydrate. Selective carbohydrate removal by enzymes could increase the protein content and remove the indigestibility of soy products for inclusion in animal feed. Complete hydrolysis of soy flour carbohydrates is challenging due to the presence of proteins and different types of non-structural polysaccharides. This study is designed to guide complex enzyme mixture required for hydrolysis of all types of soy flour carbohydrates. Enzyme broths from Aspergillus niger, Aspergillus aculeatus and Trichoderma reesei fermentations were evaluated in this study for soy carbohydrate hydrolysis. The resultant hydrolysate was measured for solubilized carbohydrate by both total carbohydrate and reducing sugar analyses. Conversion data attained after 48h hydrolysis were first fitted with models to determine the maximum fractions of carbohydrate hydrolyzable by each enzyme group, i.e., cellulase, xylanase, pectinase and α-galactosidase. Kinetic models were then developed to describe the increasing conversions over time under different enzyme activities and process conditions. The models showed high fidelity in predicting soy carbohydrate hydrolysis over broad ranges of soy flour loading (5-25%) and enzyme activities: per g soy flour, cellulase, 0.04-30 FPU; xylanase, 3.5-618U; pectinase, 0.03-120U; and α-galactosidase, 0.01-60U. The models are valuable in guiding the development and production of optimal enzyme mixtures toward hydrolysis of all types of carbohydrates present in soy flour and in optimizing the design and operation of hydrolysis reactor and process. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell wall glycosidase activities and protein content variations during fruit development and ripening in three texture contrasted tomato cultivars

PubMed Central

Konozy, Emadeldin H.E.; Causse, Mathilde; Faurobert, Mireille

2012-01-01

Excessive softening is the main factor limiting fruit shelf life and storage. It is generally acceptable now that softening of fruit which occurs during the ripening is due to synergistic actions of several enzymes on cell wall polysaccharides. As a subject for this study, we have assayed some glycosidase activities using three tomato species (Lycopersicon esculentum) contrasted for their texture phenotypes; the cherry tomato line Cervil (Solanum lycopersicum var. cerasiforme), a common taste tomato line Levovil (S. lycopersicum Mill.) and VilB a modern line, large, firmer and with good storage capability. Four glycosidase activities namely α-galactosidase, β-galactosidase, β-mannosidase and β-glucosidase were extracted from tomato’s cell wall of the three species. Cell wall protein from fruits pericarp was extracted and compared among the three cultivars at the following stages; 14 days post anthesis (14DPA) fruit; 21 days post anthesis (21DPA), turning (breaker), red and over ripe. When glycolytic activities were also compared among these cultivars at the precited development stages, gross variations were noticed from stage to stage and also from species to species in accordance with the fruit firmness status. Interestingly, VilB cultivar, the firmer among the other two, though possessed the highest total protein content, exhibited the lowest enzymatic activities. Taken together, these results may therefore allow us to conclude that studies of glycolytic activities in a single tomato cultivar cannot be generalized to all species. On the other hand, relating fruit development to glycosidase activities should logically be coupled to these enzymes from cell wall compartment. PMID:23961187

A thermodynamic assay to test pharmacological chaperones for Fabry disease.

PubMed

Andreotti, Giuseppina; Citro, Valentina; Correra, Antonella; Cubellis, Maria Vittoria

2014-03-01

The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs. The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells. Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Isolation and characterization of a mutant defective in triacylglycerol accumulation in nitrogen-starved Chlamydomonas reinhardtii.

PubMed

Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

2016-09-01

Triacylglycerol (TAG), a major source of biodiesel production, accumulates in nitrogen-starved Chlamydomonas reinhardtii. However, the metabolic pathway of starch-to-TAG conversion remains elusive because an enzyme that affects the starch degradation is unknown. Here, we isolated a new class of mutant bgal1, which expressed an overaccumulation of starch granules and defective photosynthetic growth. The bgal1 was a null mutant of a previously uncharacterized β-galactosidase-like gene (Cre02.g119700), which decreased total β-galactosidase activity 40% of the wild type. Upon nitrogen starvation, the bgal1 mutant showed decreased TAG accumulation mainly due to the reduced flux of de novo TAG biosynthesis evidenced by increased unsaturation of fatty acid composition in TAG and reduced TAG accumulation by additional supplementation of acetate to the culture media. Metabolomic analysis of the bgal1 mutant showed significantly reduced levels of metabolites following the hydrolysis of starch and substrates for TAG accumulation, whereas metabolites in TCA cycle were unaffected. Upon nitrogen starvation, while levels of glucose 6-phosphate, fructose 6-phosphate and acetyl-CoA remained lower, most of the other metabolites in glycolysis were increased but those in the TCA cycle were decreased, supporting TAG accumulation. We suggest that BGAL1 may be involved in the degradation of starch, which affects TAG accumulation in nitrogen-starved C. reinhardtii. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulation of Serratia marcescens ompF and ompC porin genes in response to osmotic stress, salicylate, temperature and pH.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2006-02-01

Serratia marcescens is a Gram-negative enterobacterium that has become an important opportunistic pathogen, largely due to its high degree of natural antibiotic resistance. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, which is controlled in part by OmpC and OmpF porin proteins. OmpF expression is regulated by micF, an RNA transcript encoded upstream of the ompC gene, which hybridizes with the ompF transcript to inhibit its translation. Regulation of S. marcescens porin gene expression, as well as that of micF, was investigated using beta-galactosidase reporter gene fusions in response to 5, 8 and 10 % sucrose, 1, 5 and 8 mM salicylate, and different pH and temperature values. beta-Galactosidase activity assays revealed that a lower growth temperature (28 degrees C), a more basic pH (pH 8), and an absence of sucrose and salicylate induce the transcription of the ompF gene, whereas the induction of ompC is stimulated at a higher growth temperature (42 degrees C), acidic pH (pH 6), and maximum concentrations of sucrose (10 %) and salicylate (8 mM). In addition, when multiple conditions were tested, temperature had the predominant effect, followed by pH. In this study, it was found that the MicF regulatory mechanism does not play a role in the osmoregulation of the ompF and ompC genes, whereas MicF does repress OmpF expression in the presence of salicylate and high growth temperature, and under low pH conditions.

Cytoplasmic p21(CIP1/WAF1), ERK1/2 activation, and cytoskeletal remodeling are associated with the senescence-like phenotype after airborne particulate matter (PM(10)) exposure in lung cells.

PubMed

Sánchez-Pérez, Yesennia; Chirino, Yolanda I; Osornio-Vargas, Álvaro Román; Herrera, Luis A; Morales-Bárcenas, Rocío; López-Saavedra, Alejandro; González-Ramírez, Imelda; Miranda, Javier; García-Cuellar, Claudia María

2014-02-10

The exposure to particulate matter with a mean aerodynamic diameter ≤10 μm (PM10) from urban zones is considered to be a risk factor in the development of cancer. The aim of this work was to determine if PM10 exposure induces factors related to the acquisition of a neoplastic phenotype, such as cytoskeletal remodeling, changes in the subcellular localization of p21(CIP1/WAF1), an increase in β-galactosidase activity and changes in cell cycle. To test our hypothesis, PM10 from an industrial zone (IZ) and a commercial zone (CZ) were collected, and human adenocarcinoma lung cell cultures (A549) were exposed to a sublethal PM10 concentration (10 μg/cm(2)) for 24 h and 48 h. The results showed that PM10 exposure induced an increase in F-actin stress fibers and caused the cytoplasmic stabilization of p21(CIP1/WAF1) via phosphorylation at Thr(145) and Ser(146) and the phosphorylation of ERK1/2 on Thr(202). Changes in the cell cycle or apoptosis were not observed, but an increase in β-galactosidase activity was detected. The PM10 from CZ caused more dramatic effects in lung cells. We conclude that PM10 exposure induced cytoplasmic p21(CIP1/WAF1) retention, ERK1/2 activation, cytoskeleton remodeling and the acquisition of a senescence-like phenotype in lung cells. These alterations could have mechanistic implications regarding the carcinogenic potential of PM10. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Central Projections of Melanopsin-Expressing Retinal Ganglion Cells in the Mouse

PubMed Central

HATTAR, SAMER; KUMAR, MONICA; PARK, ALEXANDER; TONG, PATRICK; TUNG, JONATHAN; YAU, KING-WAI; BERSON, DAVID M.

2010-01-01

A rare type of ganglion cell in mammalian retina is directly photosensitive. These novel retinal photoreceptors express the photopigment melanopsin. They send axons directly to the suprachiasmatic nucleus (SCN), intergeniculate leaflet (IGL), and olivary pretectal nucleus (OPN), thereby contributing to photic synchronization of circadian rhythms and the pupillary light reflex. Here, we sought to characterize more fully the projections of these cells to the brain. By targeting tau-lacZ to the melanopsin gene locus in mice, ganglion cells that would normally express melanopsin were induced to express, instead, the marker enzyme β-galactosidase. Their axons were visualized by X-gal histochemistry or anti-β-galactosidase immunofluorescence. Established targets were confirmed, including the SCN, IGL, OPN, ventral division of the lateral geniculate nucleus (LGv), and preoptic area, but the overall projections were more widespread than previously recognized. Targets included the lateral nucleus, peri-supraoptic nucleus, and subparaventricular zone of the hypothalamus, medial amygdala, margin of the lateral habenula, posterior limitans nucleus, superior colliculus, and periaqueductal gray. There were also weak projections to the margins of the dorsal lateral geniculate nucleus. Co-staining with the cholera toxin B subunit to label all retinal afferents showed that melanopsin ganglion cells provide most of the retinal input to the SCN, IGL, and lateral habenula and much of that to the OPN, but that other ganglion cells do contribute at least some retinal input to these targets. Staining patterns after monocular enucleation revealed that the projections of these cells are overwhelmingly crossed except for the projection to the SCN, which is bilaterally symmetrical. PMID:16736474

Cloning of a methanol-inducible moxF promoter and its analysis in moxB mutants of Methylobacterium extorquens AM1rif.

PubMed Central

Morris, C J; Lidstrom, M E

1992-01-01

In Methylobacterium extorquens AM1, gene encoding methanol dehydrogenase polypeptides are transcriptionally regulated in response to C1 compounds, including methanol (M. E. Lidstrom and D. I. Stirling, Annu. Rev. Microbiol. 44:27-57, 1990). In order to study this regulation, a transcriptional fusion has been constructed between a beta-galactosidase reporter gene and a 1.55-kb XhoI-SalI fragment of M. extorquens AM1rif DNA encoding the N terminus of the methanol dehydrogenase large subunit (moxF) and 1,289 bp of upstream DNA. The fusion exhibited orientation-specific promoter activity in M. extorquens AM1rif but was expressed constitutively when the transcriptional fusion was located on the plasmid. However, correct regulation was restored when the construction was inserted in the M. extorquens AM1rif chromosome. This DNA fragment was shown to contain both the moxFJGI promoter and the sequences necessary in cis for its transcriptional regulation by methanol. Transcription from this promoter was studied in the M. extorquens AM1rif moxB mutant strains UV4rif and UV25rif, which have a pleiotropic phenotype with regard to the components of methanol oxidation. In these mutants, beta-galactosidase activity from the fusion was reduced to a level equal to that of the vector background when the fusion was present in both plasmid and chromosomal locations. Since both constitutive and methanol-inducible promoter activities were lost in the mutants, moxB appears to be required for transcription of the genes encoding the methanol dehydrogenase polypeptides. Images PMID:1624436

Lumbar spine intervertebral disc gene delivery: a pilot study in lewis rats.

PubMed

Damle, Sheela R; Rawlins, Bernard A; Boachie-Adjei, Oheneba; Crystal, Ronald G; Hidaka, Chisa; Cunningham, Matthew E

2013-02-01

Basic research toward understanding and treating disc pathology in the spine has utilized numerous animal models, with delivery of small molecules, purified factors, and genes of interest. To date, gene delivery to the rat lumbar spine has only been described utilizing genetically programmed cells in a matrix which has required partial disc excision, and expected limitation of treatment diffusion into the disc. This study was designed to develop and describe a surgical technique for lumbar spine exposure and disc space preparation, and use of a matrix-free method for gene delivery. Naïve or genetically programmed isogeneic bone marrow stromal cells were surgically delivered to adolescent male Lewis rat lumbar discs, and utilizing quantitative biochemical and qualitative immunohistological assessments, the implanted cells were detected 3 days post-procedure. Statistically significant differences were noted for recovery of the β-galactosidase marker gene comparing delivery of naïve or labeled cells (10(5) cells per disc) from the site of implantation, and between delivery of 10(5) or 10(6) labeled cells per disc at the site of implantation and the adjacent vertebral body. Immunohistology confirmed that the β-galactosidase marker was detected in the adjacent vertebra bone in the zone of surgical implantation. The model requires further testing in larger cohorts and with biologically active genes of interest, but the observations from the pilot experiments are very encouraging that this will be a useful comparative model for basic spine research involving gene or cell delivery, or other locally delivered therapies to the intervertebral disc or adjacent vertebral bodies in rats.

Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term

NASA Technical Reports Server (NTRS)

Jiao, S.; Williams, P.; Safda, N.; Schultz, E.; Wolff, J. A.

1993-01-01

We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

Detection of Protein Interactions in T3S Systems Using Yeast Two-Hybrid Analysis.

PubMed

Nilles, Matthew L

2017-01-01

Two-hybrid systems, sometimes termed interaction traps, are genetic systems designed to find and analyze interactions between proteins. The most common systems are yeast based (commonly Saccharomyces cerevisae) and rely on the functional reconstitution of the GAL4 transcriptional activator. Reporter genes, such as the lacZ gene of Escherichia coli (encodes β-galactosidase), are placed under GAL4-dependent transcriptional control to provide quick and reliable detection of protein interactions. In this method the use of a yeast-based two-hybrid system is described to study protein interactions between components of type III secretion systems.

Influence of Pig Slurry on Microbial and Biochemical Characteristics of Soil in Albacete Region, SE Spain

NASA Astrophysics Data System (ADS)

Halil Yanardaǧ, Ibrahim

2013-04-01

Soil quality is very important in terms of agricultural sustainability, ecosystem and terrestrial carbon (C) cycle. In turn, soil microbial and biochemical characteristics are indicative of nutrient cycling and soil organic matter dynamics. We investigated the effects of the pig slurries (raw pig slurry (RPS) and treated pig slurry (TPS) from liquid and solid feeding diets) on microbial and biochemical characteristics of soil under barley cropping system. Application doses of slurries are identified with legal doses of Castilla La Mancha Region, which is 210 kg N ha-1 year-1. Microbial biomass C, soluble C, black C and three soil enzymes (β-Glucosidase, β-galactosidase and Arylesterase enzymes) are studied to determine effect slurry on soil biochemical characteristics, which are very important in terms of C cycle in soil. Black carbon content and β-Glucosidase enzyme activities are increased with all pig slurry applications from liquid and traditional feeding diet, as well as microbial biomass and organic carbon content and β-galactosidase enzyme activities are increased with slurry from liquid feeding diet doses. However, pig slurry application from liquid feeding diet doses have increased yield, quality, length and total biomass content of barley. Bioavailable metal contents are increased with all slurry application and with using high doses of slurry can be caused soil pollution. Pig slurries from liquid feeding diet had positive impacts on microbial and biochemical characteristics in terms of soil quality in comparison to the different feeding diets. PS addition to soil had a very significant stimulating effect on the enzyme activities, microbial biomass, soluble and black C compared with different kind of PS and control plots on Mediterranean soil in barley monoculture. This effect may originate from the organic C, N, P and S compounds added with PS. The highest enzyme activity and microbial biomass were observed on the soil samples from the RPS treatment

Clinical Features, Diagnosis, and Management of Patients With Anderson-Fabry Cardiomyopathy.

PubMed

Yogasundaram, Haran; Kim, Daniel; Oudit, Omar; Thompson, Richard B; Weidemann, Frank; Oudit, Gavin Y

2017-07-01

Anderson-Fabry disease (AFD) is an X-linked recessive, multisystem disease of lysosomal storage. A mutation in the gene encoding the hydrolase enzyme α-galactosidase A results in its deficiency, or complete absence of activity. Subsequent progressive intracellular accumulation of glycosphingolipids, predominantly globotriaosylceramide, in various tissues, results in progressive organ dysfunction and failure, most commonly affecting the kidneys, nervous system, skin, eyes, vascular endothelium, and the heart. Cardiac involvement in AFD represents a leading cause of morbidity and mortality. Globotriaosylceramide accumulation affects cardiomyocytes, smooth muscle cells, vascular endothelial cells, and fibroblasts leading to various pathologies including valvular regurgitation, conduction disease and arrhythmias, coronary microvascular dysfunction, and right and left ventricular hypertrophy (LVH) leading to early diastolic dysfunction and late-stage systolic impairment. Diagnosis is on the basis of decreased plasma α-galactosidase activity in men and positive genetic testing in women. Contemporary large-scale screening studies have revealed a prevalence of 1%-5% in patients with unexplained LVH in multiple cohorts. Cardiac magnetic resonance imaging, with its unique tissue characterization capabilities, is the most important imaging modality to assess for cardiomyopathy in patients with AFD. Enzyme replacement therapy is indicated in AFD patients with significant organ involvement, and has been shown to clear sphingolipids from endothelial cells in other organs, as well as to reduce left ventricular mass as early as 6 months after starting treatment. There is increasing evidence that enzyme replacement therapy might be more effective if given at earlier stages of disease, before the development of LVH and myocardial fibrosis. Copyright © 2017 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Characterization of two trpE genes encoding anthranilate synthase {alpha}-subunit in Azospirillum brasilense

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ge Shimei; Xie Baoen; Chen Sanfeng

2006-03-10

The previous report from our laboratory has recently identified a new trpE gene (termed trpE {sub 2}) which exists independently in Azospirillum brasilense Yu62. In this study, amplification of trpE(G) (termed trpE {sub 1}(G) here) confirmed that there are two copies of trpE gene, one trpE being fused into trpG while the other trpE existed independently. This is First report to suggest that two copies of the trpE gene exist in this bacterium. Comparison of the nucleotide sequence demonstrated that putative leader peptide, terminator, and anti-terminator were found upstream of trpE {sub 1}(G) while these sequence features did not existmore » in front of trpE {sub 2}. The {beta}-galactosidase activity of an A. brasilense strain carrying a trpE {sub 2}-lacZ fusion remained constant at different tryptophan concentrations, but the {beta}-galactosidase activity of the same strain carrying a trpE {sub 1}(G)-lacZ fusion decreased as the tryptophan concentration increased. These data suggest that the expression of trpE {sub 1}(G) is regulated at the transcriptional level by attenuation while trpE {sub 2} is constantly expressed. The anthranilate synthase assays with trpE {sub 1}(G){sup -} and trpE {sub 2} {sup -} mutants demonstrated that TrpE{sub 1}(G) fusion protein is feedback inhibited by tryptophan while TrpE{sub 2} protein is not. We also found that both trpE {sub 1}(G) and trpE {sub 2} gene products were involved in IAA synthesis.« less

Comparative genome analysis of Bacillus velezensis reveals a potential for degrading lignocellulosic biomass.

PubMed

Chen, Long; Gu, Wei; Xu, Hai-Yan; Yang, Gui-Lian; Shan, Xiao-Feng; Chen, Guang; Kang, Yuan-Huan; Wang, Chun-Feng; Qian, Ai-Dong

2018-05-01

Genomes of 24 sequenced Bacillus velezensis strains were characterized to identity shared and unique genes of lignocellulolytic enzymes and predict potential to degrade lignocellulose. All 24 strains had genes that encoded lignocellulolytic enzymes, with potential to degrade cellulose and hemicelluloses. Several lignocellulosic genes related to cellulose degradation were universally present, including one GH5 (endo-1,4-β-glucanase), one GH30 (glucan endo-1,6-β-glucosidase), two GH4 (6-phospho-β-glucosidase, 6-phospho-α-glucosidase), one GH1 (6-phospho-β-galactosidase), one GH16 (β-glucanase) and three GH32 (two sucrose-6-phosphate hydrolase and levanase). However, in the absence of gene(s) for cellobiohydrolase, it was predicted that none of the 24 strains would be able to directly hydrolyse cellulose. Regarding genes for hemicellulose degradation, four GH43 (1,4-β-xylosidase; except strain 9912D), one GH11 (endo-1,4-β-xylanase), three GH43 (two arabinan endo-1,5-α-L-arabinosidase and one arabinoxylan arabinofuranohydrolase), two GH51 (α-N-arabinofuranosidase), one GH30 (glucuronoxylanase), one GH26 (β-mannosidase) and one GH53 (arabinogalactan endo-1,4-β-galactosidase) were present. In addition, two PL1 (pectate lyase) and one PL9 (pectate lyase) with potential for pectin degradation were conserved among all 24 strains. In addition, all 24 Bacillus velezensis had limited representation of the auxiliary activities super-family, consistent with a limited ability to degrade lignin. Therefore, it was predicted that for these bacteria to degrade lignin, pretreatment of lignocellulosic substrates may be required. Finally, based on in silico studies, we inferred that Bacillus velezensis strains may degrade a range of polysaccharides in lignocellulosic biomasses.

Sustained, long-term renal stabilization after 54 months of agalsidase beta therapy in patients with Fabry disease.

PubMed

Germain, Dominique P; Waldek, Stephen; Banikazemi, Maryam; Bushinsky, David A; Charrow, Joel; Desnick, Robert J; Lee, Philip; Loew, Thomas; Vedder, Anouk C; Abichandani, Rekha; Wilcox, William R; Guffon, Nathalie

2007-05-01

Fabry disease, an inherited deficiency of the lysosomal enzyme alpha-galactosidase A, causes progressive intralysosomal accumulation of globotriaosylceramide (GL-3) and premature death from renal, cardiac, and cerebrovascular manifestations. To determine the long-term safety and efficacy of recombinant human alpha-galactosidase A, an open-label, phase III extension study was conducted, involving 58 patients who had classic Fabry disease and completed a 20-wk, double-blind, randomized, placebo-controlled, phase III study of agalsidase beta and were transitioned to an extension trial to receive biweekly 1 mg/kg agalsidase beta for up to an additional 54 mo. GL-3 accumulation was evaluated in the capillary endothelia of the skin, kidney, and heart. Renal function was assessed. By month 54, all patients with optional kidney biopsies (n = 8) maintained complete GL-3 clearance in renal capillary endothelial cells and multiple cell types. Continued, complete clearance of skin (31 of 36) and heart (six of eight) capillary endothelium was demonstrated. Mean plasma GL-3 levels remained decreased in the normal range. Median serum creatinine and estimated GFR remained stable (normal) in patients with renal data at month 54 (n = 41). Six patients had renal disease progression; most (four of six) were older than 40 yr and had significant proteinuria at baseline and evidence of sclerotic glomeruli pretreatment. Adverse events were generally mild and unrelated to treatment. The most common treatment-related adverse events were infusion-associated reactions, which decreased over time. Long-term agalsidase beta therapy stabilizes renal function in patients without renal involvement at baseline, maintains reduction of plasma GL-3, and sustains GL-3 clearance in capillary endothelial cells and multiple renal cell types.

Cardiomyopathy and response to enzyme replacement therapy in a male mouse model for Fabry disease.

PubMed

Nguyen Dinh Cat, Aurelie; Escoubet, Brigitte; Agrapart, Vincent; Griol-Charhbili, Violaine; Schoeb, Trenton; Feng, Wenguang; Jaimes, Edgar; Warnock, David G; Jaisser, Frederic

2012-01-01

Fabry disease is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, (predominately globotriaosylceramide; GL-3) in lysosomes, as well as other cellular compartments and the extracellular space. Our aim was to characterize the cardiac phenotype of male knock-out mice that are deficient in alpha-galactosidase A activity, as a model for Fabry disease and test the efficacy of Enzyme Replacement Therapy with agalsidase-beta. Male mice (3-4 months of age) were characterized with awake blood pressure and heart rate measurements, cardiac echocardiography and electrocardiography measurements under light anesthesia, histological studies and molecular studies with real-time polymerase chain reaction. The Fabry knock-out mouse has bradycardia and lower blood pressure than control wild type (CB7BL/6J) mice. In Fabry knock-out mice, the cardiomyopathy associated mild hypertrophy at echography with normal systolic LV function and mild diastolic dysfunction. Premature atrial contractions were more frequent in without conduction defect. Heart weight normalized to tibial length was increased in Fabry knock-out mice. Ascending aorta dilatation was observed. Molecular studies were consistent with early stages of cardiac remodeling. A single dose of agalsidase-beta (3 mg/kg) did not affect the LV hypertrophy, function or heart rate, but did improve the mRNA signals of early cardiac remodeling. In conclusion, the alpha-galactosidase A deficient mice at 3 to 4 months of age have cardiac and vascular alterations similar to that described in early clinical stage of Fabry disease in children and adolescents. Enzyme replacement therapy affects cardiac molecular remodeling after a single dose.

The Hematopoietic Stem Cell Therapy for Exploration of Space

NASA Technical Reports Server (NTRS)

Roach, Allana Nicole; Brezo, Jelena

2002-01-01

Astronauts experience severe/invasive disorders caused by space environments. These include hematological/cardiac abnormalities, bone and muscle losses, immunodeficiency, neurological disorders and cancer. While the cause of these symptoms are not yet fully delineated, one possible explanation could be the inhibition of hematopoietic stem cell (HSC) growth and hematopoiesis in space. HSCs differentiate into all types of blood cells, and growing evidence indicates that the HSCs also have the ability to transdifferentiate to various tissues, including muscle, skin, liver, neuronal cells and possibly bone. Therefore, a hypothesis was advanced in this laboratory that the hematopoietic stem cell-based therapy, herein called the hematopoietic stem cell therapy (HSCT), could mitigate some of the disorders described above. Due to the magnitude of this project our laboratory has subdivided it into 3 sections: a) HSCT for space anemia; b) HSCT for muscle and bone losses; and c) HSCT for immunodeficiency. Toward developing the HSCT protocol for space anemia, the HSC transplantation procedure was established using a mouse model of beta thalassemia. In addition, the NASA Rotating Wall Vessel (RWV) culture system was used to grow HSCs in space condition. To investigate the HSCT for muscle loss and bone loss, donor HSCs were genetically marked either by transfecting the beta-galactosidase-containing plasmid, pCMV.SPORT-beta-gal or by preparing from b-galactosidase transgenic mice. The transdifferentiation of HSCs to muscle is traced by the reporter gene expression in the hindlimb suspended mice with some positive outcome, as studied by the X-gal staining procedure. The possible structural contribution of HSCs against muscle loss is being investigated histochemically.

Involvement of Sp1 elements in the promoter activity of genes affected in keratoconus.

PubMed

Maruyama, Y; Wang, X; Li, Y; Sugar, J; Yue, B Y

2001-08-01

Keratoconus is a progressive disease that thins and scars the corneal stroma. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of the inhibitors alpha1-proteinase inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-M) are reduced, especially in the epithelial layer. An increased expression of the transcription factor Sp1 was also demonstrated. The role of Sp1 in regulation of the genes affected in keratoconus was examined in this study. DNA segments, containing 5'-flanking promoter sequences of the alpha 1-PI, LAP, cathepsin B, and alpha 2-M genes were ligated into the secreted alkaline phosphatase (SEAP) reporter gene vector. These constructs, along with the pSV beta-galactosidase control vector, were transfected into cultured human corneal epithelial and stromal cells and skin fibroblasts. Cotransfection with the Sp1 expression vector was performed in parallel. SEAP and beta-galactosidase enzyme activities were assayed. In corneal epithelial cells, as in stromal cells, alpha 1-PI promoter activity was suppressed by cotransfection of pPacSp1. The LAP, cathepsin B, and alpha 2-M promoters were functional in corneal cells, whereas activities of these promoters were much lower in skin fibroblasts. Cotransfection experiments indicated that the up- or downregulation of LAP, cathepsin B, and alpha 2-M observed in keratoconus-affected corneas was not mediated by Sp1. These results support the theory that the corneal epithelium, along with the stroma, is involved in keratoconus. An upstream role of Sp1 is indicated and the Sp1-mediated downregulation of the alpha 1-PI gene may be a key event in the disease development.

Degradation Signals Recognized by the Ubc6p-Ubc7p Ubiquitin-Conjugating Enzyme Pair

PubMed Central

Gilon, Tamar; Chomsky, Orna; Kulka, Richard G.

2000-01-01

Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of β-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Δ and sec61-2) did not inhibit the degradation of the β-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p. PMID:10982838

Cardiomyopathy and Response to Enzyme Replacement Therapy in a Male Mouse Model for Fabry Disease

PubMed Central

Nguyen Dinh Cat, Aurelie; Escoubet, Brigitte; Agrapart, Vincent; Griol-Charhbili, Violaine; Schoeb, Trenton; Feng, Wenguang; Jaimes, Edgar; Warnock, David G.; Jaisser, Frederic

2012-01-01

Fabry disease is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, (predominately globotriaosylceramide; GL-3) in lysosomes, as well as other cellular compartments and the extracellular space. Our aim was to characterize the cardiac phenotype of male knock-out mice that are deficient in alpha-galactosidase A activity, as a model for Fabry disease and test the efficacy of Enzyme Replacement Therapy with agalsidase-beta. Male mice (3–4 months of age) were characterized with awake blood pressure and heart rate measurements, cardiac echocardiography and electrocardiography measurements under light anesthesia, histological studies and molecular studies with real-time polymerase chain reaction. The Fabry knock-out mouse has bradycardia and lower blood pressure than control wild type (CB7BL/6J) mice. In Fabry knock-out mice, the cardiomyopathy associated mild hypertrophy at echography with normal systolic LV function and mild diastolic dysfunction. Premature atrial contractions were more frequent in without conduction defect. Heart weight normalized to tibial length was increased in Fabry knock-out mice. Ascending aorta dilatation was observed. Molecular studies were consistent with early stages of cardiac remodeling. A single dose of agalsidase-beta (3 mg/kg) did not affect the LV hypertrophy, function or heart rate, but did improve the mRNA signals of early cardiac remodeling. In conclusion, the alpha-galactosidase A deficient mice at 3 to 4 months of age have cardiac and vascular alterations similar to that described in early clinical stage of Fabry disease in children and adolescents. Enzyme replacement therapy affects cardiac molecular remodeling after a single dose. PMID:22574107

Regulation of lac Operon Expression: Reappraisal of the Theory of Catabolite Repression

PubMed Central

Wanner, Barry L.; Kodaira, Ryoji; Neidhardt, Frederick C.

1978-01-01

The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of β-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3′,5′-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of β-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism. PMID:214424

Sclerostin alters serum vitamin D metabolite and fibroblast growth factor 23 concentrations and the urinary excretion of calcium

PubMed Central

Ryan, Zachary C.; Ketha, Hemamalini; McNulty, Melissa S.; McGee-Lawrence, Meghan; Craig, Theodore A.; Grande, Joseph P.; Westendorf, Jennifer J.; Singh, Ravinder J.; Kumar, Rajiv

2013-01-01

Inactivating mutations of the SOST (sclerostin) gene are associated with overgrowth and sclerosis of the skeleton. To determine mechanisms by which increased amounts of calcium and phosphorus are accreted to enable enhanced bone mineralization in the absence of sclerostin, we measured concentrations of calciotropic and phosphaturic hormones, and urine and serum calcium and inorganic phosphorus in mice in which the sclerostin (sost) gene was replaced by the β-D-galactosidase (lacZ) gene in the germ line. Knockout (KO) (sost−/−) mice had increased bone mineral density and content, increased cortical and trabecular bone thickness, and greater net bone formation as a result of increased osteoblast and decreased osteoclast surfaces compared with wild-type (WT) mice. β-Galactosidase activity was detected in osteocytes of sost KO mice but was undetectable in WT mice. Eight-week-old, male sost KO mice had increased serum 1α,25-dihydroxyvitamin D, decreased 24,25-dihydroxyvitamin D, decreased intact fibroblast growth factor 23, and elevated inorganic phosphorus concentrations compared with age-matched WT mice. 25-Hydroxyvitamin D 1α-hydroxylase cytochrome P450 (cyp27B1) mRNA was increased in kidneys of sost KO mice compared with WT mice. Treatment of cultured proximal tubule cells with mouse recombinant sclerostin decreased cyp27B1 mRNA transcripts. Urinary calcium and renal fractional excretion of calcium were decreased in sost KO mice compared with WT mice. Sost KO and WT mice had similar serum calcium and parathyroid hormone concentrations. The data show that sclerostin not only alters bone mineralization, but also influences mineral metabolism by altering concentrations of hormones that regulate mineral accretion. PMID:23530237

Active Immunotherapy of Cancer with a Nonreplicating Recombinant Fowlpox Virus Encoding a Model Tumor-Associated Antigen

PubMed Central

Wang, Michael; Bronte, Vincenzo; Chen, Pauline W.; Gritz, Linda; Panicali, Dennis; Rosenberg, Steven A.; Restifo, Nicholas P.

2007-01-01

Some tumor cells express Ags that are potentially recognizable by T lymphocytes and yet do not elicit significant immune responses. To explore new immunotherapeutic strategies aimed at enhancing the recognition of these tumor-associated Ags (TAA), we developed an experimental mouse model consisting of a lethal clone of the BALB/c tumor line CT26 designated CT26.WT, which was transduced with the lacZ gene encoding β-galactosidase, to create CT26.CL25. The growth rate and lethality of CT26.CL25 and CT26.WT were virtually identical despite the expression by CT26.CL25 of the model tumor Ag in vivo. A recombinant fowlpox virus (rFPV), which is replication incompetent in mammalian cells, was constructed that expressed the model TAA, β-galactosidase, under the influence of the 40-kDa vaccinia virus early/late promoter. This recombinant, FPV.bg40k, functioned effectively in vivo as an immunogen, eliciting CD8+ T cells that could effectively lyse CT26.CL25 in vitro. FPV.bg40k protected mice from both subcutaneous and intravenous tumor challenge by CT26.CL25, and most surprisingly, mice bearing established 3-day pulmonary metastasis were found to have significant, Ag-specific decreases in tumor burden and prolonged survival after treatment with the rFPV. These observations constitute the first reported use of rFPV in the prevention and treatment of an experimental cancer and suggest that changing the context in which the immune system encounters a TAA can significantly and therapeutically alter the host immune response against cancer. PMID:7722321

Changes in Soil Carbon and Enzyme Activity As a Result of Different Long-Term Fertilization Regimes in a Greenhouse Field

PubMed Central

Zhang, Lili; Chen, Wei; Burger, Martin; Yang, Lijie; Gong, Ping; Wu, Zhijie

2015-01-01

In order to discover the advantages and disadvantages of different fertilization regimes and identify the best management practice of fertilization in greenhouse fields, soil enzyme activities involved in carbon (C) transformations, soil chemical characteristics, and crop yields were monitored after long-term (20-year) fertilization regimes, including no fertilizer (CK), 300 kg N ha-1 and 600 kg N ha-1 as urea (N1 and N2), 75 Mg ha-1 horse manure compost (M), and M with either 300 or 600 kg N ha-1 urea (MN1 and MN2). Compared with CK, fertilization increased crop yields by 31% (N2) to 69% (MN1). However, compared with CK, inorganic fertilization (especially N2) also caused soil acidification and salinization. In the N2 treatment, soil total organic carbon (TOC) decreased from 14.1±0.27 g kg-1 at the beginning of the long-term experiment in 1988 to 12.6±0.11 g kg-1 (P<0.05). Compared to CK, N1 and N2 exhibited higher soil α-galactosidase and β-galactosidase activities, but lower soil α-glucosidase and β-glucosidase activities (P<0.05), indicating that inorganic fertilization had different impacts on these C transformation enzymes. Compared with CK, the M, MN1 and MN2 treatments exhibited higher enzyme activities, soil TOC, total nitrogen, dissolved organic C, and microbial biomass C and N. The fertilization regime of the MN1 treatment was identified as optimal because it produced the highest yields and increased soil quality, ensuring sustainability. The results suggest that inorganic fertilizer alone, especially in high amounts, in greenhouse fields is detrimental to soil quality. PMID:25706998

Sclerostin alters serum vitamin D metabolite and fibroblast growth factor 23 concentrations and the urinary excretion of calcium.

PubMed

Ryan, Zachary C; Ketha, Hemamalini; McNulty, Melissa S; McGee-Lawrence, Meghan; Craig, Theodore A; Grande, Joseph P; Westendorf, Jennifer J; Singh, Ravinder J; Kumar, Rajiv

2013-04-09

Inactivating mutations of the SOST (sclerostin) gene are associated with overgrowth and sclerosis of the skeleton. To determine mechanisms by which increased amounts of calcium and phosphorus are accreted to enable enhanced bone mineralization in the absence of sclerostin, we measured concentrations of calciotropic and phosphaturic hormones, and urine and serum calcium and inorganic phosphorus in mice in which the sclerostin (sost) gene was replaced by the β-D-galactosidase (lacZ) gene in the germ line. Knockout (KO) (sost(-/-)) mice had increased bone mineral density and content, increased cortical and trabecular bone thickness, and greater net bone formation as a result of increased osteoblast and decreased osteoclast surfaces compared with wild-type (WT) mice. β-Galactosidase activity was detected in osteocytes of sost KO mice but was undetectable in WT mice. Eight-week-old, male sost KO mice had increased serum 1α,25-dihydroxyvitamin D, decreased 24,25-dihydroxyvitamin D, decreased intact fibroblast growth factor 23, and elevated inorganic phosphorus concentrations compared with age-matched WT mice. 25-Hydroxyvitamin D 1α-hydroxylase cytochrome P450 (cyp27B1) mRNA was increased in kidneys of sost KO mice compared with WT mice. Treatment of cultured proximal tubule cells with mouse recombinant sclerostin decreased cyp27B1 mRNA transcripts. Urinary calcium and renal fractional excretion of calcium were decreased in sost KO mice compared with WT mice. Sost KO and WT mice had similar serum calcium and parathyroid hormone concentrations. The data show that sclerostin not only alters bone mineralization, but also influences mineral metabolism by altering concentrations of hormones that regulate mineral accretion.

Characterization of a thermostable glycoside hydrolase (CMbg0408) from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167.

PubMed

Letsididi, Rebaone; Hassanin, Hinawi Am; Koko, Marwa Yf; Ndayishimiye, Jean B; Zhang, Tao; Jiang, Bo; Stressler, Timo; Fischer, Lutz; Mu, Wanmeng

2017-05-01

Hyperthermophilic archaea capable of functioning optimally at very high temperatures are a good source of unique and industrially important thermostable enzymes. A glycoside hydrolase family 1 β-galactosidase gene (BglB) from a hyperthermophilic archaeon Caldivirga maquilingensis IC-167 was cloned and expressed in Escherichia coli. The recombinant enzyme (CMbg0408) displayed optimum activity at 110 °C and pH 5.0. It also retained 92% and 70% of its maximal activity at 115 and 120 °C, respectively. The enzyme was completely thermostable and active after 120 min of incubation at 80 and 90 °C. It also showed broad substrate specificity with activities of 8876 ± 185 U mg -1 for p-nitrophenyl-β-d-galactopyranoside, 4464 ± 172 U mg -1 for p-nitrophenyl-β-d-glucopyranoside, 1486 ± 68 U mg -1 for o-nitrophenyl-β-d-galactopyranoside, 2250 ± 86 U mg -1 for o-nitrophenyl-β-d-xylopyranoside and 175 ± 4 U mg -1 for lactose. A catalytic efficiency (k cat /K m ) of 3059 ± 122 mmol L -1  s -1 and K m value of 8.1 ± 0.08 mmol L -1 were displayed towards p-nitrophenyl-β-d-galactopyranoside. As a result of its remarkable thermostability and high activity at high temperatures, this novel β-galactosidase may be useful for food and pharmaceutical applications. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding.

PubMed

Vincent, S F; Bell, P J; Bissinger, P; Nevalainen, K M

1999-02-01

Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (alpha-galactosidase) activity. To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker's yeast by both classical breeding and recombinant DNA technology. Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture. Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.

Safe and Successful Treatment With Agalsidase Beta During Pregnancy in Fabry Disease.

PubMed

Senocak Tasci, Elif; Bicik, Zerrin

2015-09-01

Fabry disease, an X-linked lysosomal storage disorder, is caused by α-galactosidase A deficiency and leads to accumulation of glycospinhgolipids in most tissues, with life-theratening consequences in the kidney, heart, and cerebrovascular system. Enzyme replacement therapy is available as 2 different preparations: agalsidase alfa and agalsidase beta. Enzyme replacement therapy is started as soon as the diagnosis is confirmed, but there is no data available in the literature about its safety during preganacy. Herein, we described 2 patients with Fabry disease who received agalsidase beta during their pregnancy. This report is important as the data about enzyme replacement therapy during pregnancy is restricted with case reports.

A series of vectors to construct lacZ fusions for the study of gene expression in Schizosaccharomyces pombe.

PubMed

Lafuente, M J; Petit, T; Gancedo, C

1997-12-22

We have constructed a series of plasmids to facilitate the fusion of promoters with or without coding regions of genes of Schizosaccharomyces pombe to the lacZ gene of Escherichia coli. These vectors carry a multiple cloning region in which fission yeast DNA may be inserted in three different reading frames with respect to the coding region of lacZ. The plasmids were constructed with the ura4+ or the his3+ marker of S. pombe. Functionality of the plasmids was tested measuring in parallel the expression of fructose 1,6-bisphosphatase and beta-galactosidase under the control of the fbp1+ promoter in different conditions.

Adenoviral vector gene delivery via the round window membrane in guinea pigs.

PubMed

Suzuki, Mitsuya; Yamasoba, Tatsuya; Suzukawa, Keigo; Kaga, Kimitaka

2003-10-27

We have found that damage from a local anesthetic solution containing phenol permitted beta-galactosidase (beta-gal) gene delivery to the guinea pig inner ear via the round window membrane (RWM). RWM damage was evident as degeneration of the outer epithelium. After adenovirus lacZ vector was applied to the damaged RWM, immunohistochemistry showed strong beta-gal expression in the RWM, mesothelial cells, organ of Corti, spiral limbus, spiral ligament and spiral ganglion. In the vestibular labyrinth, expression was seen in the sensory and supporting cells, transitional cells, and the dark-cell area. Thus, adenovirus can transfect a variety of inner ear cells in the guinea pig through a damaged RWM.

TASK-2 Channels Contribute to pH Sensitivity of Retrotrapezoid Nucleus Chemoreceptor Neurons

PubMed Central

Wang, Sheng; Benamer, Najate; Zanella, Sébastien; Kumar, Natasha N.; Shi, Yingtang; Bévengut, Michelle; Penton, David; Guyenet, Patrice G.; Lesage, Florian

2013-01-01

Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO2/H+ via an unidentified pH-sensitive background K+ channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K+ channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or β-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n = 58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2−/− mice (n = 49 of 88) could be classified as pH sensitive (>30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2−/− mice selected based on β-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K+ currents were reduced in amplitude in RTN neurons from TASK-2−/− mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart–brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2−/− mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold. PMID:24107938

Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf.

PubMed

Hornyak, T J; Hayes, D J; Chiu, L Y; Ziff, E B

2001-03-01

A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing beta-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants, Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitf(mi), with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of beta-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitf(mi)/+ embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quantification and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitf(mi)/Mitf(mi) embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro.

PubMed

Griffiths, Sarah; Baraniak, Priya R; Copland, Ian B; Nerem, Robert M; McDevitt, Todd C

2013-12-01

Multipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown. MSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to "rescue" the proliferative capacity of MSCs. hPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS. hPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Mechanism of killing of streptococcus mutans by light-activated drugs

NASA Astrophysics Data System (ADS)

Burns, Tracy; Wilson, Michael; Pearson, G. J.

1996-01-01

Recent studies have shown that cariogenic bacteria can be killed when exposed to low power laser light in the presence of a photosensitizing agent. The purpose of this study was to determine the mechanism by which the cariogenic bacterium Streptococcus mutans can be killed by toluidine blue O and helium neon laser light. To determine whether membrane damage occurred, suspensions of sensitized S. mutans were exposed to a 7.3 mW HeNe laser for 30 mins and samples removed every 5 mins. Survivors were enumerated by viable counting on tryptone soya agar plates and cell free filtrates were assayed for phosphate and (beta) -galactosidase. Lipid peroxidation was assessed by assaying for malondialdehyde, a by- product of lipid peroxidation. The role of oxygen and reactive oxygen species was studied by exposing sensitized bacteria to laser light (1) under different atmospheric conditions, (2) in the presence of deuterium oxide, and (3) in the presence of inhibitors of reactive oxygen species. Following exposure of sensitizede S. mutans to 13.2 J of HeNe laser light, 2.6 nmoles of phosphate and 228 nmoles of (beta) -galactosidase were detected in the cell free filtrates. Ten micrometers oles of malondialdehyde were also detected. When the sensitized bacteria were exposed to laser light under anaerobic conditions there was no significant decrease in the viable count compared to a 60% kill in the presence of oxygen. In the presence of D2O there was a 15-fold increase in the numbers of bacteria killed. O.1 M methionine and 0.5 M sodium azide each afforded 98% protection from lethal photosensitization. These results imply that lethal photosensitization results from membrane damage due to lipid peroxidation and that reactive oxygen species are mediators of this process.

Probiotic dahi containing Lactobacillus casei protects against Salmonella enteritidis infection and modulates immune response in mice.

PubMed

Jain, Shalini; Yadav, Hariom; Sinha, P R

2009-06-01

In the present study, effect of dahi containing probiotic Lactobacillus casei (probiotic dahi) was evaluated to modulate immune response against Salmonella enteritidis infection in mice. Animals were fed with milk products along with standard diet for 2 and 7 days prior to the S. enteritidis challenge and continued on the respective dairy food-supplemented diets during the postchallenge period. Translocation of S. enteritidis in spleen and liver, beta-galactosidase and beta-glucuronidase enzymatic activities and secretory IgA (sIgA) in intestinal fluid, lymphocyte proliferation, and cytokine (interleukin [IL]-2, IL-4, IL-6, and interferon-gamma [IFN-gamma]) production in cultured splenocytes were assessed on day 2, 5, and 8 of the postchallenge period. Colonization of S. enteritidis in liver and spleen was remarkably low in probiotic dahi-fed mice than mice fed milk and control dahi. The beta-galactosidase and beta-glucuronidase activities in intestinal fluid collected from mice prefed for 7 days with probiotic dahi were significantly lower at day 5 and 8 postchallenge than in mice fed milk and control dahi. Levels of sIgA and lymphocyte proliferation rate were also significantly increased in probiotic dahi-fed mice compared with the other groups. Production of IL-2, IL-6, and IFN-gamma increased, whereas IL-4 decreased in splenic lymphocytes collected from probiotic dahi-fed mice. Data showed that dahi prefed for 7 days before S. enteritidis challenge was more effective than when mice were prefed for 2 days with dahi. Moreover, probiotic dahi was more efficacious in protecting against S. enteritidis infection by enhancing innate and adaptive immunity than fermented milk and normal dahi. Results of the present study suggest that prefeeding of probiotic dahi may strengthen the consumer's immune system and may protect infectious agents like S. enteritidis.

Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

PubMed Central

Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A.; Ligon, G. F.; Perry, C. A.

1995-01-01

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking. Images Figure 7 Figure 8 Figure 11 PMID:7543732

Proteomic Discovery and Development of a Multiplexed Targeted MRM-LC-MS/MS Assay for Urine Biomarkers of Extracellular Matrix Disruption in Mucopolysaccharidoses I, II, and VI.

PubMed

Heywood, Wendy E; Camuzeaux, Stephane; Doykov, Ivan; Patel, Nina; Preece, Rhian-Lauren; Footitt, Emma; Cleary, Maureen; Clayton, Peter; Grunewald, Stephanie; Abulhoul, Lara; Chakrapani, Anupam; Sebire, Neil J; Hindmarsh, Peter; de Koning, Tom J; Heales, Simon; Burke, Derek; Gissen, Paul; Mills, Kevin

2015-12-15

The mucopolysaccharidoses (MPS) are lysosomal storage disorders that result from defects in the catabolism of glycosaminoglycans. Impaired muscle, bone, and connective tissue are typical clinical features of MPS due to disruption of the extracellular matrix. Markers of MPS disease pathology are needed to determine disease severity and monitor effects of existing and emerging new treatments on disease mechanisms. Urine samples from a small cohort of MPS-I, -II, and -VI patients (n = 12) were analyzed using label-free quantative proteomics. Fifty-three proteins including many associated with extracellular matrix organization were differently expressed. A targeted multiplexed peptide MRM LC-MS/MS assay was used on a larger validation cohort of patient samples (MPS-I n = 18, MPS-II n = 12, MPS-VI n = 6, control n = 20). MPS-I and -II groups were further subdivided according to disease severity. None of the markers assessed were altered significantly in the mild disease groups compared to controls. β-galactosidase, a lysosomal protein, was elevated 3.6-5.7-fold significantly (p < 0.05) in all disease groups apart from mild MPS-I and -II. Collagen type Iα, fatty-acid-binding-protein 5, nidogen-1, cartilage oligomeric matrix protein, and insulin-like growth factor binding protein 7 concentrations were elevated in severe MPS I and II groups. Cartilage oligomeric matrix protein, insulin-like growth factor binding protein 7, and β-galactosidase were able to distinguish the severe neurological form of MPS-II from the milder non-neurological form. Protein Heg1 was significantly raised only in MPS-VI. This work describes the discovery of new biomarkers of MPS that represent disease pathology and allows the stratification of MPS-II patients according to disease severity.

Enzyme replacement therapy in Fabry disease: influence on cardiac manifestations.

PubMed

Caballero, L; Climent, V; Hernández-Romero, D; Quintanilla, M A; de la Morena, G; Marín, F

2010-01-01

Fabry disease (FD) is an X-linked glycosphingolipid storage disorder caused by deficient activity of the lysosomal enzyme alpha-galactosidase A. This leads to a progressive accumulation of globotriaosylceramide (Gb3) in the lysosomes of different cells and tissues, causing principally ventricular hypertrophy, renal failure and cerebrovascular accidents, reducing lifespan both in hemizygous males and heterozygous females. Residual enzyme activity might lead to slow progression of the disease and result in the so-called cardiac or renal variants with delayed presentation. Two different forms of alpha-galactosidase A enzyme replacement therapies (ERT) are available for the treatment of FD, one genetically engineered in human cell line (agalsidase alfa, Replagal, Shire) and the other produced in a Chinese hamster ovary cell line (agalsidase beta, Fabrazyme, Genzyme). Although both proteins are structurally and functionally very similar, with the same amino acid sequence as the native human enzyme, they differ in the pattern of glycosilation of the protein depending on the originating cell line. Studies with both preparations have described a reduction in plasma, urinary sediment and tissue levels of Gb3, a decrease in the frequency of pain crisis and a reduction in left ventricular mass and improvement or stabilization of renal function. Studies have generally shown the greatest benefit when treatment is started at an early stage of the disease before extensive fibrosis or other irreversible tissue damage takes place. However, more data are needed to document long-term treatment outcomes. The aim of the present review is to provide an update overview of the two different forms of ERT for FD, their clinical effects in cardiac manifestations and their possible differences in terms of efficacy, side effects and safety profiles.

Ten-year outcome of enzyme replacement therapy with agalsidase beta in patients with Fabry disease.

PubMed

Germain, Dominique P; Charrow, Joel; Desnick, Robert J; Guffon, Nathalie; Kempf, Judy; Lachmann, Robin H; Lemay, Roberta; Linthorst, Gabor E; Packman, Seymour; Scott, C Ronald; Waldek, Stephen; Warnock, David G; Weinreb, Neal J; Wilcox, William R

2015-05-01

Fabry disease results from deficient α-galactosidase A activity and globotriaosylceramide accumulation causing renal insufficiency, strokes, hypertrophic cardiomyopathy and early demise. We assessed the 10-year outcome of recombinant α-galactosidase A therapy. The outcomes (severe clinical events, renal function, cardiac structure) of 52/58 patients with classic Fabry disease from the phase 3 clinical trial and extension study, and the Fabry Registry were evaluated. Disease progression rates for patients with low renal involvement (LRI, n=32) or high renal involvement (HRI, n=20) at baseline were assessed. 81% of patients (42/52) did not experience any severe clinical event during the treatment interval and 94% (49/52) were alive at the end of the study period. Ten patients reported a total of 16 events. Patients classified as LRI started therapy 13 years younger than HRI (mean 25 years vs 38 years). Mean slopes for estimated glomerular filtration rate for LRI and HRI were -1.89 mL/min/1.73 m(2)/year and -6.82 mL/min/1.73 m(2)/year, respectively. Overall, the mean left ventricular posterior wall thickness and interventricular septum thickness remained unchanged and normal. Patients who initiated treatment at age ≥ 40 years exhibited significant increase in left ventricular posterior wall thickness and interventricular septum thickness. Mean plasma globotriaosylceramide normalised within 6 months. This 10-year study documents the effectiveness of agalsidase beta (1 mg/kg/2 weeks) in patients with Fabry disease. Most patients remained alive and event-free. Patients who initiated treatment at a younger age and with less kidney involvement benefited the most from therapy. Patients who initiated treatment at older ages and/or had advanced renal disease experienced disease progression. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Ten-year outcome of enzyme replacement therapy with agalsidase beta in patients with Fabry disease

PubMed Central

Germain, Dominique P; Charrow, Joel; Desnick, Robert J; Guffon, Nathalie; Kempf, Judy; Lachmann, Robin H; Lemay, Roberta; Linthorst, Gabor E; Packman, Seymour; Scott, C Ronald; Waldek, Stephen; Warnock, David G; Weinreb, Neal J; Wilcox, William R

2015-01-01

Background Fabry disease results from deficient α-galactosidase A activity and globotriaosylceramide accumulation causing renal insufficiency, strokes, hypertrophic cardiomyopathy and early demise. We assessed the 10-year outcome of recombinant α-galactosidase A therapy. Methods The outcomes (severe clinical events, renal function, cardiac structure) of 52/58 patients with classic Fabry disease from the phase 3 clinical trial and extension study, and the Fabry Registry were evaluated. Disease progression rates for patients with low renal involvement (LRI, n=32) or high renal involvement (HRI, n=20) at baseline were assessed. Results 81% of patients (42/52) did not experience any severe clinical event during the treatment interval and 94% (49/52) were alive at the end of the study period. Ten patients reported a total of 16 events. Patients classified as LRI started therapy 13 years younger than HRI (mean 25 years vs 38 years). Mean slopes for estimated glomerular filtration rate for LRI and HRI were −1.89 mL/min/1.73 m2/year and −6.82 mL/min/1.73 m2/year, respectively. Overall, the mean left ventricular posterior wall thickness and interventricular septum thickness remained unchanged and normal. Patients who initiated treatment at age ≥40 years exhibited significant increase in left ventricular posterior wall thickness and interventricular septum thickness. Mean plasma globotriaosylceramide normalised within 6 months. Conclusions This 10-year study documents the effectiveness of agalsidase beta (1 mg/kg/2 weeks) in patients with Fabry disease. Most patients remained alive and event-free. Patients who initiated treatment at a younger age and with less kidney involvement benefited the most from therapy. Patients who initiated treatment at older ages and/or had advanced renal disease experienced disease progression. PMID:25795794