Comparison of the apoptotic processes induced by the oxysterols 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7-hydroxycholesterol (7-OH) and cholesterol-5,6-epoxide (-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochromec release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7-OH; however, in -epoxide-treated cells, the inhibitor of cytochromec release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7-OH-treated cells but not in cells incubated with -epoxide. Trolox, a water-soluble synthetic analogue of -tocopherol, prevented 7-OH-induced apoptosis but did not protect against cell death induced by -epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7-OH or -epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7-OH and -epoxide in U937 cells.     

Distribution of Fucoidan Hydrolases and Some Glycosidases among Marine Invertebrates

Thirty-three species of marine invertebrates from the Sea of Japan were analyzed for contents of fucoidan hydrolases and some glycosidases. Fucoidan hydrolase activity was assessed by examining the effect of animal tissue extracts on fucoidans from the two brown seaweeds Laminaria cichorioides and Fucus evanescens, which have different structural characteristics. The activity of glycosidases (-glucosidase, -galactosidase, -fucosidase, and -mannosidase) was determined using p-nitrophenyl derivatives of sugars as substrates. It was found that glycosidases and fucoidan hydrolases of different specificities are fairly widely distributed among marine invertebrates. Mollusks and some species of echinoderms and arthropods showed the highest enzymatic activity. This research will enable us to choose organisms for the separation and study of fucoidan hydrolases and glycosidases, which may be useful in determining the structure of fucoidans.     

Linkage mapping of ‘25-kDa globulin’ genes on homoeologous group-1 chromosomes of bread and durum wheat

Acid polyacrylamide-gel electrophoresis (A-PAGE) of ethanol-soluble proteins from the endosperm of bread and durum wheats reveals some bands encoded by genes on the homoeologous group-1 chromosomes with higher mobility than the -gliadins. The isolation of these proteins showed that they were the previously described 25-kDa globulins encoded by genes at the Glo-A1, Glo-B1, and Glo-D1 loci. The variability found among a collection of 51 bread and 81 durum wheats was very low: two allelic variants at Glo-A1 and no variants at Glo-B1 in each of the two species, and two allelic variants at Glo-D1 in bread wheat. Inheritance studies of 25-kDa globulin genes on group-1 chromosomes of bread and durum wheat were carried out on the F₂ progeny from four crosses, two in bread wheat and two in durum wheat. The linkage mapping of the 1A 25-kDa globulin genes of bread wheat was done based on four prolamin loci: Glu-A1, Glu-A3, Gli-A1 and Gli -A3. The percentages of recombination and the distances found allowed a re-evaluation of the linkage map of endosperm protein loci on this chromosome. The Glo-A1 locus was found to be located at the distal end of the short arm of 1A chromosome, at a distance of 5.23±1.99 cM from Gli-A1, 6.85±2.22 cM from Glu-A3, 22.64±3.62 cM from Gli-A1, and at a recombination percentage of 49.30±4.40 from Glu-A1. A similar distance between Gli-A1 and Glo-A1 (4.82±1.75 and 6.66±2.26 cM) was found in durum wheat. The distance between Gli-D1 and Glo-D1 on chromosome 1D was 2.86±1.39 cM.     

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.     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Enzymatic modification of β-lactoglobulin with N-fatty-acyl-dipeptide by transglutaminase from Streptomyces mobaraense

-Lactoglobulin was enzymatically acylated with N-lauroyl-l-glutaminyl-glycine and N-lauroyl-l-glutamyl-l-lysine using transglutaminase from Streptomyces mobaraense. The modification of the protein with N-fatty-acyl-dipeptide was confirmed by SDS-PAGE, gel chromatography, HPLC, amino acid analysis, and TOF-MS. The degrees of the protein modification with N-lauroyl-l-glutaminyl-glycine and N-lauroyl-l-glutamyl-l-lysine were estimated to be 2–4 and 1.5 residues per molecule, respectively.     

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.     

An alpha-L-fucosidase from Thermus sp. with unusually broad specificity

An -L-fucosidase (E.C. 3.2.1.51) exhibiting a wide aglycon specificity expressed in ability of cleaving 1 6-, 1 3-, 1 4-, and 1 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The -L-fucosidase hydrolyzes p-nitrophenyl -L-fucopyranoside with V _max of 12.0 ± 0.1 M/min/mg and K _m = 0.20 ± 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 ± 10 kDa consisting of four identical subunits with a molecular mass of 61.0 ± 0.5 kDa. The N-terminal sequence showed homology to some -L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl -L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the -L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl -L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.     

Investigation of the Relationship between the Lectin Activity of Azospirilla and Their α-Glucosidase, β-Glucosidase,and β-Galactosidase

The activities of -glucosidase, -glucosidase, and -galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilenseSp7 and Azospirillum lipoferum59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol–acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied for the azospirilla studied. The cofunction of the A. brasilenseSp7 lectin and -galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum59b lectin and glucosidases was revealed. The lectin from A. lipoferum59b may possess saccharolytic activity.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Evidence suggesting energy-dependent formaldehyde transport in an RuMP-type methylotroph (T15)

Formaldehyde accumulation ratios ([¹⁴CH₂O]_i/[¹⁴CH₂O]_o) as high as 12-fold were measured in anaerobic, CH₃OH-energized, whole cell suspensions of the ribulose monophosphate (RuMP)-type methylotrophic strain T15. Uptake kinetics were extremely rapid, enabling the attainment of equilibrium in only 10–30 s. Transport appears to be energy-dependent and associated with the protonmotive force (pmf). Anaerobic incubation with 5 M carbonyl p-(trifluoromethoxy)-phenylhydrazone (FCCP) led to 70%–90% reduction of the accumulation ratio. Though not as pronounced, diminished uptake was also observed in the presence of 140 M nigericin, 161 M valinomycin and 90 mM KSCN, commensurate with their effects on pmf. Accumulation of CH₂O as a function of external pH followed a trend more similar to that of pmf than either pH or . Preventing energization by incubation with 100 M N,N-dicyclohexylcarbodiimide (DCCD) led to nearly 80% inhibition of CH₂O transport. Over short time periods it was possible to chase accumulated ¹⁴CH₂O from previously loaded cells by collapsing pmf; however, this technique also indicated that significant ¹⁴CH₂O incorporation began to occur within 3 min.Abbreviations FCCP Carbonyl cyanide p-(trifluoromethyoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - RuMP ribulose monophosphate - TPP⁺ tetra[U-¹⁴C]phenylphosphonium - pmf protonmotive force     

Control of nitrogenase inAzospirillum sp.

Many N₂-fixing organisms can turn off nitrogenase activity in the presence of NH₄ ⁺ and turn it on again when the NH₄ ⁺ is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH₄ ⁺ is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genusAzospirillum possess the DRAT and DRAG systems (A. lipoferum andA. brasilense), whereasA. amazonense in the same genus lacks DRAT and DRAG.A. amazonense responds to NH₄ ⁺ but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them intoA. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function inAcetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N₂ at a pH as low as 3.0.A. diazotrophicus does show a rather sluggish response to NH₄ ⁺. A level of about 10 M NH₄ ⁺ is required to switch off the system. The response to NH₄ ⁺ is influenced by the dissolved oxygen concentration (DOC) as has been reported forAzospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O₂ seems optimal for the response inA. diazotrophicus.     

Efficient catalysis by pyridinium sulfonate in glycosylation involving an oxazoline intermediate derived from per-O-acetyl-N-acetyllactosamine andN,N′-diacetylchitobiose

New and facileN-acetyllactosaminidation andN,N-diacetylchitobiosylation with various alcohols, thiols, and sugar derivatives through the 1,2-oxazoline intermediates catalyzed by pyridiniump-toluenesulfonate were achieved.     

Terminal glycosylation and disease: Influence on cancer and cystic fibrosis

Terminal glycosylation has been a recurring theme of the laboratory. In cystic fibrosis (CF), decreased sialic acid and increased fucosyl residues in 1,3 position to antennary N -acetyl glucosamine is the CF glycosylation phenotype. The glycosylation phenotype is reversed by transfection of CF airway cells with wtCFTR. In neuronal cells, polymers of 2,8sialyl residues are prominent in oligodendrocytes and human neuroblastoma. These findings are discussed in relationship to early studies in our laboratories and those of other investigators. The potential extension of these concepts to future clinical therapeutics is presented.     

Synthesis ofα-triazolylα-amino acid derivatives

Summary We report the synthesis of-triazolyl-amino esters by 1,3 dipolar cycloaddition of acetylenic compounds and-azido-amino esters.     

Synthesis of Alkyl Glycosides Catalyzed by β-Glycosidases in a System of Reverse Micelles

A basic possibility of enzymic synthesis of alkyl glycosides in a system of the Aerosol-OT (AOT) reverse micelles was studied. Octyl -D-galactopyranoside and octyl -D-glucopyranoside were synthesized from the corresponding sugars (lactose or glucose) and octyl alcohol under catalysis with glycolytic enzymes, -galactosidase and -glucosidase, respectively. The transglycosylation/hydrolysis ratio was shifted toward transglycosylation by using octyl alcohol, one of the substrates, as an organic solvent. The alkyl glycosides were thus obtained in one step from a hydrophilic mono- or disaccharide and a hydrophobic aliphatic alcohol. The direction of the reaction was shown to depend on the pH of aqueous solution solubilized in reverse micelles. The maximum yields were 45% and 40% for octyl galactoside and octyl glucoside, respectively; they markedly exceeded the yields of enzymic syntheses in a two-phase system reported previously.     

Relative levels of guanosine 5′-diphosphate 3′-diphosphate (ppGpp) in some N2 fixing bacteria during derepression and repression of nitrogenase

Addition of ammonium to N₂ fixing cultures of Azotobacter vinelandii, Klebsiella pneumoniae and Clostridium pasteurianum rapidly reduced the intracellular levels of guanosine 5-diphosphate 3-diphosphate (ppGpp) by 70–90%. This change might reflect a regulatory role of ppGpp in nitrogen metabolism.Abbreviations ppGpp guanosine 5-diphosphate 3-diphosphate     

Afferent bursting activity of ruff lateral line induced by background noise stimulation

Summary Single unit resting activities were recorded from fibres innervating a neuromast of the supra-orbital canal of the lateral line system of the ruff (Acerina cernua). The interval histogram of 1 of the 4 types of resting activity had a bimodal distribution (bursting activity). The resting activity of these fibres was compared with the measured vibration of the experimental table. The conclusion that the bursting activity is not spontaneous but is caused by small background vibrations of the table was supported by recording of extracellular hair cell responses.Abbreviation ISI Interspike interval     

Chitovibrin: a chitin-binding lectin fromVibrio parahemolyticus

A novel 134 kDa, calcium-independent chitin-binding lectin, chitovibrin, is secreted by the marine bacteriumVibrio parahemolyticus, inducible with chitin or chitin-oligomers. Chitovibrin shows no apparent enzymatic activity but exhibits a strong affinity for chitin and chito-oligomers >dp9. The protein has an isoelectric pH of 3.6, shows thermal tolerance, binds chitin with an optimum at pH 6 and is active in 0–4m NaCl. Chitovibrin appears to be completely different from other reported Vibrio lectins and may function to bindV. parahemolyticus to chitin substrates, or to capture or sequester chito-oligomers. It may be a member of a large group of recently described proteins in Vibrios related to a complex chitinoclastic (chitinivorous) system.Abbreviations (GlcNAc)₂ N,N-diacetylchitobiose - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PTS phosphotransferase system