Thermostable extracellular α-amylase and α-glucosidase ofLipomyces starkeyi

Summary Thermostable, extracellular -amylase and -glucosidase were produced byLipomyces starkeyi CBS 1809 in a medium containing maize starch and soya bean meal. Contrary to published findings which suggested a single cell-bound amylolytic system for another strain ofL. starkeyi, this study revealed the presence of two enzymes — an -amylase and an -glucosidase inL. starkeyi CBS 1809. The enzymes were separated by solvent and salt precipitation and ion-exchange chromatography on DEAE-Biogel-A. The -amylase and -glucosidase had pH optima at 4.0 and 4.5 and temperature optima at 70°C and 60°C, respectively. While the low pH optima are not unique the enzymes are very distinctive in yeasts in having very high temperature optima. The -glucosidase had highest activities on maltose and isomaltose (100) with relative rates of activity on maltotriose, isomaltotriose and p-nitrophenyl--d-glucoside of 59, 48 and 22, respectively. It was inactive towards sucrose. Both the -amylase and -glucosidase ofL. starkeyi were located extracellularly and had molecular weights of 76,000 and 35,000, respectively.     

Chemical Synthesis and Characterization of α-N-Octanoyl and Other α-N-Acyl Colistin Nonapeptide Derivatives

Eight α-N-acyl colistin nonapeptide derivatives including three aliphatic, four aromatic and one alicyclic derivatives were synthesized by the reaction of colistin nonapeptide with corresponding acid chlorides. This acylation reaction was carried out under the condition kept restrictedly at pH 5,0 in order to introduce an acyl group only to α-amino group but not to γ-amino group existing in a colistin nonapeptide molecule. Synthetic method and several physico-chemical natures of these acyl colistin nonapeptide derivatives are given in this paper.

All of the acylated derivatives thus synthesized exhibited characteristic antimicrobial activities. Antimicrobial spectra were substantially based upon a gram-negative type and not so much altered by chemical structures of acyl groups which were considerably differentiated from each other such as cyclic or chain form. Thus, more possible response of carbon size than its structure to the antimicrobial effectiveness was inferred. In spite of almost no toxicity and feeble antimicrobial activity of colistin nonapeptide itself, these acylated colistin nonapeptide derivatives showed a toxicity against mice at a dose of 16.9~70 mg/kg in LD₅₀, which, however, was inferior to the toxicity of colistin sulfate, possibly correspondent to their much weaker antimicrobial activities, as a whole. Hence, it seems likely that acyl part of these acylated colistin nonapeptide derivatives including that of colistin is seriously responsible for the biological activities.     

A Specific and Essential Role for Na,K-ATPase α3 in Neurons Co-expressing α1 and α3

Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na⁺ imaging techniques to study the role of α3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na⁺ concentration ([Na⁺]_i) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na⁺]_i, similar to what could be expected following suprathreshold neuronal activity, selective inhibition of α3 almost completely abolished the capacity to restore [Na⁺]_i in soma and dendrite. Recordings of Na,K-ATPase specific current supported the notion that when [Na⁺]_i is elevated in the neuron, α3 is the predominant isoform responsible for rapid extrusion of Na⁺. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na⁺ binding site. Following activation of protein kinase A, both the α3-dependent current and restoration of dendritic [Na⁺]_i were significantly attenuated, indicating that α3 is a target for phosphorylation and may participate in short term regulation of neuronal function.     

Isolation and Identification of α-Spinasterol and α-Spinasteryl D-Glucoside from Sugar Beet Pulp

A sterol and a steryl glucoside were isolated from dried beet pulp. The sterol was identified with α-spinasterol, the glucoside possessed chemical and physical properties such as follows: The molecular formula C₃₅H₅₈O₆, m.p. 292°, [ α]¹⁹_D-34.1°, acetate; m.p. 168°, benzoate; m.p. 175-177°, and positive for Molish and Lieber-mann-Burchard reactions. When it was hydrolyzed with 1% sulfuric acid, the crystal of α-spinasterol and D-glucose detectable by paper chromatography were obtained. These results gave evidence that the glucoside was in question to be α-spinasteryl D-glucoside.     

Stablizing crude and ultrafiltrated α-amylase and α-glucosidase from Aspergillus oryzae by trehalose

Thermal resistance of freeze-dried -amylase and -glucosidase in trehalose matrices (1 to 20 % w/v) stored at 90 °C and relative humidities (RH) between 0 and 44 % was studied. At RH values up to 33 %, 10 % (w/v) trehalose was necessary to retain at least 50 % of -amylase activity. For -glucosidase, 10 % (w/v) trehalose was effective only at 0 % RH. Ultrafiltration of the crude enzymatic fermentation extracts enhanced enzyme stability per se. However, ultrafiltration in combination with 1 % (w/v) trehalose retained 74 % of -glucosidase and 95 % of -amylase activities. © Rapid Science Ltd. 1998     

Difference between light- and wound-induced biosynthesis of α-solanine and α-chaconine in potato tubers

Potato tuber tissues can incorporate mevalonic acid-2-14C into glycoalkaloids, namely α-chaconine and α-solanine. The percent incorporation of this labeled precursor into α-chaconine in light exposed tubers is more than that of mechanically injured tubers.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Identification and biology of α-secretase

Our knowledge of the etiology of Alzheimer's disease (AD) has advanced tremendously since the discovery of amyloid beta (Aβ) aggregation in diseased brains. Accumulating evidence suggests that Aβ plays a causative role in AD. The β-secretase enzyme, beta-site APP cleaving enzyme-1 (BACE1), is also implicated in AD pathogenesis, given that BACE1 cleavage of amyloid precursor protein is the initiating step in the formation of Aβ. As a result, BACE1 inhibition has been branded as a potential AD therapy. In this study, we review the identification and basic characteristics of BACE1, as well as the progress in our understanding of BACE1 cell biology, substrates, and phenotypes of BACE1 knockout mice that are informative about the physiological functions of BACE1 beyond amyloid precursor protein cleavage. These data are crucial for predicting potential mechanism-based toxicity that would arise from inhibiting BACE1 for the treatment or prevention of AD.     

Specificity and stereospecificity of α-chymotrypsin

1. The optically pure p-nitrophenyl esters of the d and l enantiomers of N-acetyl-tryptophan, N-acetylphenylalanine and N-acetyl-leucine, and the p-nitrophenyl ester of N-acetylglycine, have been prepared. 2. These materials are all substrates of α-chymotrypsin, and the rates of deacylation of the corresponding acyl-α-chymotrypsins have been determined. 3. As the size of the amino acid side chain increases, the l series deacylate progressively faster than the N-acetylglycyl-enzyme, and the d series progressively more slowly. 4. The results are interpreted in terms of a three-locus model of the enzyme's active site, which accounts for the interrelationship between substrate specificity and stereospecificity observed. 5. The concepts of negative specificity and of specificity saturation are introduced.     

The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein

α_S-Casein, the major milk protein, comprises α_S1- and α_S2-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α_S-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α_S-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α_S2-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α_S1-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α_S1-, α- and α_S-casein were all significantly less effective. We conclude that α_S1- and α-casein stabilise each other to facilitate optimal chaperone activity of α_S-casein. This work highlights the interdependency of casein proteins for their structural stability.     

Effects of α-ketoglutarate on neutrophil intracellular amino and α-keto acid profiles and ROS production

The aim of this study was to determine the effects of α-ketoglutarate on neutrophil (PMN), free α-keto and amino-acid profiles as well as important reactive oxygen species (ROS) produced [superoxide anion (O₂ ^?), hydrogen peroxide (H₂O₂)] and released myeloperoxidase (MPO) acitivity. Exogenous α-ketoglutarate significantly increased PMN α-ketoglutarate, pyruvate, asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Additionally, in parallel with intracellular α-ketoglutarate changes, increases in O₂ ^– formation, H₂O₂-generation and MPO acitivity have also been observed. We therefore believe that α-ketoglutarate is important for affecting PMN “susceptible free amino- and α-keto acid pools” although important mechanisms and backgrounds are not yet completely explored. Moreover, our results also show very clearly that changes in intragranulocytic α-ketoglutarate levels are relevant metabolic determinants in PMN nutrition considerably influencing and modulating the magnitude and quality of the granulocytic host defense capability as well as production of ROS.     

Differences between α- and β-Chain Mutants of Human Haemoglobin and between α- and β-Thalassaemia. Possible Duplication of the α-Chain Gene

Human adult haemoglobin consists of two unlike pairs of polypeptide chains, and can be described as α₂β₂. Amino-acid substitutions in either of the two types of chain result in α- and β-chain variants. In thalassaemia, which causes a lowered production of haemoglobin, the α or the β chain can be affected, the result being α- or β-thalassaemia. There is a quantitative difference in the proportion of α- and β-chain variants to normal haemoglobin in the respective heterozygotes, and there is also a difference in the pattern of inheritance of α- and β-thalassaemia: these could possibly be explained by assuming that man has one gene for the β- and two for the α-chain.     

Synthesis and initial evaluation of novel,non-peptidic antagonists of the αv-integrins αvβ3 and αvβ5

The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the α_v-integrins α_vβ₃ and α_vβ₅ is described. High-throughput screening of an extensive series of ECLiPS? compound libraries led to the identification of compound 1 as a dual inhibitor of the α_v-integrins α_vβ₃ and α_vβ₅. Optimization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of compounds 15a and 15b with reduced PSA and much improved potency for both the α_vβ₃ and α_vβ₅ integrins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and compound 15a also exhibited a promising Caco-2 permeability profile.     

Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Characterization of α2,3- and α2,6-sialyltransferases from Helicobacter acinonychis

Genome sequence data were used to clone and express two sialyltransferase enzymes of the GT-42 family from Helicobacter acinonychis ATCC 51104, a gastric disease isolate from Cheetahs. The deposited genome sequence for these genes contains a large number of tandem repeat sequences in each of them: HAC1267 (RQKELE)(15) and HAC1268 (EEKLLEFKNI)(13). We obtained two clones with different numbers of repeat sequences for the HAC1267 gene homolog and a single clone for the HAC1268 gene homolog. Both genes could be expressed in Escherichia coli and sialyltransferase activity was measured using synthetic acceptor substrates containing a variety of terminal sugars. Both enzymes were shown to have a preference for N-acetyllactosamine, and they each made a product with a different linkage to the terminal galactose. HAC1267 is a mono-functional α2,3-sialyltransferase, whereas HAC1268 is a mono-functional α2,6-sialyltransferase and is the first member of GT-42 to show α2,6-sialyltransferase activity.     

Inhibitors of α-glucosidase, α-amylase and lipase from Chrysanthemum morifolium

A new endoperoxysesquiterpene lactone, 10α-hydroxy-1α,4α-endoperoxy-guaia-2-en-12,6α-olide (1), together with a flavanone, eriodictyol (2), and two flavone glycosides, acacetin-7-O-β-d-glucopyranoside (3) and acacetin-7-O-α-l-rhamopyranoside (4), were isolated from the methanol extract of Chrysanthemum morifolium flowers by a bioassay-guided fractionation. Compound 1 showed strong inhibitory effects against α-glucosidase and lipase activities, with IC₅₀ values of 229.3 and 161.0 μM, respectively. The flavone glycosides 3 and 4 inhibited both α-glucosidase and α-amylase, while flavanone 2 was only effective against α-amylase.     

Syntheses and spectroscopic properties of α- and β-phosphatidylcholines and phosphatidylethanolamines

The widely used partial synthesis of phospholipids via deacylation of naturally occurring phospholipids, followed by reacylation with fatty acid anhydrides, is accompanied by phosphoryl migration. The resulting mixture of α- and β-phospholipids was separated by short-column chromatography. Milder acylation procedures in which no phosphoryl migration occurs, were developed. 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine was prepared in 50% yield by acylation of sn-glycero-3-phosphocholine (GPC) with N-linoleoylimidazole. Detailed NMR and infrared spectra of α- and β-phosphatidylcholines (PCs) and -ethanolamines (PEs) are reported and the differences between isomers discussed.     

Serum α-tocopherol and selenium in belgian infants and children

Previous studies showed low selenium (Se) concentrations in Belgian children. Serum α-tocopherol, retinol, total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol, selenium (Se), and thiobarbituric acid-reactive substances were examined. In order to obtain further information on the Se status in Belgian children, Se, α-tocopherol, retinol, and lipid concentrations were examined and signs of peroxidative lipid damage were evaluated in a subgroup. The study was performed in 524 children (0–14 yr old) during vaccination campaigns. Three age groups were analyzed: 0–1, 1–4, and 4–14 yr. In 87 of them, where sufficient amounts of serum were available, analysis of thiobarbituric acid-reactive substances was done. Infants have high serum α-tocopherol concentrations: (23.2 μmol/L [median and interquartile range: 18.6–30.2]) and low Se concentrations (0.37 mol/L [0.27–0.47]). Se concentrations rise significantly during the first 4 yr (p < 0.0001) (Mann-Whitney U-test, tied p-values): 0.70 μmol/L (0.59–0.82); in the 4–14 yr olds, it was 0.75 μmol/L (0.67–0.86). These values remain low compared to results coming from other parts of the world. α-Tocopherol concentrations decrease significantly after infancy (p < 0.0001). The ratio α-tocopherol/total cholesterol is higher in infants. This is induced by the high vitamin E content of infant formulas. Signs of serum lipid peroxidation could not be detected by analysis of serum malondialdehyde concentrations. High α-tocopherol concentrations, as those observed in infant serum lipids, could be one of the protective mechanisms from the peroxidative lipid damages, sometimes observed in a low-Se status.