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.     

Tissue and serum α2-3- and α2-6-linkage specific sialylation changes in oral carcinogenesis

Increased sialylation of cell surface glycoconjugates is among the key molecular changes associated with malignant transformation and cancer progression. We investigated significance of linkage-specific sialylation changes in oral carcinogenesis. Tissue and serum levels of total sialic acid (TSA), linkage-specific sialyltransferases (ST) and sialoproteins were analyzed from patients with oral precancerous conditions (OPC) and oral cancer as well as the post-treatment follow-up blood samples of oral cancer patients. TSA levels were measured using a spectrophotometric method. The linkage-specific lectins, Sambusus nigra (SNA) and Maackia amurensis (MAM) detects α2-6- and α2-3-linked sialic acid, respectively, were used to analyze ST activity and sialoproteins. Malignant tissues showed significantly higher levels of TSA, reactivity of SNA and MAM, and α2,3-ST activity compared to the adjacent normal tissues. α2,6-ST was also higher in malignant tissues. Similarly, the marker levels were higher in precancerous tissues than their adjacent normal tissues. Serum levels of TSA, TSA/ total proteins, α2-6-sialoproteins and α2,6-ST were markedly increased in untreated oral cancer patients compared to the controls and OPC as well as responder (CR) patients. Serum levels of the markers were higher or comparable between untreated oral cancer patients and non-responders (NR). Serum levels of α2-3-sialylation were elevated in non-responders compared with the responders. Further, the observed sialylation changes in tissue and serum were found to be associated with various clinicopathological features and disease progression. Thus, the data suggest potential utility of sialylation markers in early detection, prognostication and treatment monitoring of oral cancer.     

Thermostable α-1,4- and α-1,6-glucosidase enzymes from Bacillus sp. Isolated from a marine environment

Penaeus vannamei (the shrimp) is an omnivorous species and it can be assumed that a high level of carbohydrates is necessary for its growth. -1,4- and 1,6-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates, principally starch. However, the shrimp's hepatopancreas produces only -1,4-glucosidases, which limits the growth rate in different sources of starch. In order to identify strains with -1,4- and 1,6-glucosidase enzymes with potential uses in shrimp feed production, Bacillus strains were isolated from marine environments. One strain produced large amounts of an extracellular thermostable -glucosidase that permitted good growth on starch. The organism was identified by polymorphism (restriction-fragment-length polymorphism, RFLP), sequenced, and named B. subtilis LMM-12.     

Formation of α-D-glucose-1-phosphate by thermophilic α-1,4-D-glucan phosphorylase

For the production of α-D-glucose-1-phosphate (G-1-P), α-1,4-D-glucan phosphorylase from Thermus caldophilus GK24 was partially purified to a specific activity of 13 U mg⁻¹ and an enzyme recovery of 15%. The amount of G-1-P reached maximum (18%) when soluble starch was used as substrate, and the smallest substrate for G-1-P formation was maltotriose. The structure of purified G-1-P was confirmed by comparison to ¹³C-NMR data for an authentic sample. In addition to G-1-P, glucose-6-phosphate (12%) was simultaneously produced when 10 mM maltoheptaose was used as substrate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 89–93. Received 12 May 1999/ Accepted in revised form 29 August 1999     

Enzymatic synthesis of α-2-deoxyglucosyl derivatives catalyzed by organic solvent-resistant α-glucosidase

Organic solvent-resistant Aspergillus niger α-glucosidase (ANGase) can synthesize α-2-deoxyglucosyl derivatives (2DDs) in water-organic solvent media by a trans-addition reaction from d-glucal to various acceptors. Herein, we studied the influence of four different solvents on ANGase stability and activity. ANGase exhibited 47 or 43% residual activity following incubation in 50% (v/v) or in 70% (v/v) acetone for 4 h, respectively. When various carbohydrates were used as acceptor molecules, ANGase catalyzed the addition reaction of four different sugar alcohols, glucose, sucrose, or trehalose to d-glucal. Among the acceptor molecules tested, xylitol was the best acceptor by producing the highest yield (87% addition). The concentration of acetone/acceptor influenced the formation of 2DDs and the yields. We confirmed the molecular weight of five kinds of products by mass spectrometry and enzymatic hydrolysis. Current method is useful for the production of carbohydrates containing 2-deoxyglucose moiety.     

Chemoenzymatic synthesis of C8-modified sialic acids and related α2-3- and α2-6-linked sialosides

Naturally occurring 8-O-methylated sialic acids, including 8-O-methyl-N-acetylneuraminic acid and 8-O-methyl-N-glycolylneuraminic acid, along with 8-O-methyl-2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (Kdn8Me) and 8-deoxy-Kdn were synthesized from corresponding 5-O-modified six-carbon monosaccharides and pyruvate using a sialic acid aldolase cloned from Pasteurella multocida strain P-1059 (PmNanA). In addition, α2-3- and α2-6-linked sialyltrisaccharides containing Neu5Ac8Me and Kdn8Deoxy were also synthesized using a one-pot multienzyme approach. The strategy reported here provides an efficient approach to produce glycans containing various C8-modified sialic acids for biological evaluations.     

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.     

Attempt at Affinity Labeling of α- and β- Amylases by α- and β-d-Glucopyranosides and α- and β-Maltooligosaccharides with 2,3-Epoxypropyl Residue as Aglycone: Specific Inactivation of β-Amylases

Among 2,3-epoxypropyl α-d-glucopyranoside and 2,3-epoxypropyl α-maltooligosaccharides and the β-anomers, 2,3-epoxypropyl α-d-glucopyranoside (α-EPG) strongly inactivated the β-amylases [EC 3.2.1.2] of sweet potato, barley, and Bacillus, cereus, in addition to soybean β amylase [J. Biochem., 99, 1631 (1986)]. However, none of the compounds used inactivated any α-amylases [EC 3.2.1.1] of porcine pancreas, Aspergillus oryzae, or Bacillus amyloliquefaciens. Irreversible incorporation of ¹⁴C-labeled α-EPG into β-amylases was stoichiometric, i.e., one α-EPG per active site of the enzyme was bound, and the inactivations were almost complete. The results suggest that α-EPG is an affinity labeling reagent selective for β-amylase. Slow inactivations by the other compounds were also observed, depending on the difference of source of β amylase.     

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.     

Surface α2-3- and α2-6-sialylation of human monocytes and derived dendritic cells and its influence on endocytosis

Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that α2-3-sialylated O-glycans and α2-6- and α2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented α2-6-sialylated N-glycans; ST3Gal1 contributes for the α2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased α2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.     

AHL-dependent quorum sensing inhibition: synthesis and biological evaluation of α-(N-alkyl-carboxamide)-γ-butyrolactones and α-(N-alkyl-sulfonamide)-γ-butyrolactones

New N-acylhomoserine lactone (AHL) analogues in which the amide function is replaced by a reverse-amide one have been studied as AHL QS modulators. The series of compounds consists of α-(N-alkyl-carboxamide)-γ-butyrolactones, α-(N-alkyl-sulfonamide)-γ-butyrolactones, and 2-(N-alkyl-carboxamide)-cyclopentanones and cyclopentanols. Most active compounds exhibited antagonist activities against LuxR reaching the 30 μM range.     

Structure-function relationships in human α- and γ-thrombins

Summary Human pro-coagulant -thrombin may be proteolyzed under controlled conditions to the non-coagulant - and -thrombin forms. These derivative forms nonetheless retain esterase and amidase activities with small substrates as well as several other thrombin functions. Structurally, human -thrombin consists of three non-covalently associated fragments which retain structural integrity as measured by several spectroscopic criteria as well as enzymatic function. The protein folding characteristics of three-chain -thrombin indicate that each fragment (domain) contains sufficient information to result in a correct renaturation of protein conformation. Those subtle structural differences which distinguish - from -thrombin are most likely the obstructions to fibrinogen binding which account for the loss of clotting activity.     

Separation of α- and β-Globin Messenger RNAs

THE 10S RNA fraction of reticulocytes from various species contains the haemoglobin messenger RNA^1–4. When this 10S RNA fraction is added to a cell-free system derived from reticulocytes or Krebs II ascites cells, it directs the synthesis of α and β chains of haemoglobin^5–8. The α and β messenger RNA molecules contained in this fraction, however, have not yet been separated and identified. When reticulocyte. RNA of mouse is subjected to electrophoresis on 6% polyacrylamide gels, the 10S fraction contains two major bands and three minor bands⁹, suggesting that the major lOS RNA bands contain the messenger RNAs for the α- and β-globin chains.     

Di-ddU-α-Hydroxyphosphonate and α-Hydroxy- α- METHYLPHOSPHONATES as Potential Prodrugs of Antiviral Nucleoside Analogues

Abstract

The synthesis of a potential new prodrug system for the antiviral nucleoside ddU 1 based on α-hydroxybenzylphosphonates 2 and 3 is described. In principle, 2, 3 are able to hydrolyze via different mechanisms yielding ddU H-phosphonate 4 or ddU monophosphate 5, respectively.     

Genetic polymorphism of αs1- and αs2-caseins in Hungarian Milking Goats

The aim of this study was to perform an initial characterization of milk quality and to determine genetic polymorphism at the CSN1S1 and CSN1S2 locus in two herds of local dairy goats (Hungarian Milking). The fat, protein and lactose level of milk samples in Hungarian Milking Goats were compared to other local goat breeds worldwide and it was concluded that the mean milk production of the Hungarian goats should be improved. The presence of the A, B, C + D, E, F and O alleles of CSN1S1 locus and A + B + C + E, D, F and O alleles of CSN1S2 locus were genotyped for by PCR-AS and PCR-RFLP methods in 103 goats. The strong B allele of CSN1S1 is more frequent in the local Hungarian Milking than in the imported Alpine and Saanen goats. The relatively high incidence of the O allele of CSN1S2 gene is also characteristic for the Hungarian Milking Goats and those special allele distribution patterns could be used to develop selection strategies to breed specialised lines of Hungarian local breeds.     

Hydrolysis of α-1,6-Glucosidic Linkages by an α-Amylase from Thermoactinomyces vulgaris R-47

We studied DNA breakage by phenyl compounds present in foodstuffs in vitro using γDNA and in cultured mammalian cells using RFL and HeLa cells. Strong in vitro activity was detected in o- and p-dihydroxyphenols, but the m-isomer had no activity. The same results were obtained with aminophenols and phenylenediamines. In flavonoids, the 3-OH group seemed to be active in the DNA breakage, in addition to the odiphenol group. Cellular DNA breakage by the compounds was different from their in vitro activity and varied with the cell lines. RFL cells were preferable to HeLa cells for screening for DNA breaking substances, because of their greater sensitivity.     

Characterization of the mRNAs for α-, β- and γ-actin

The mRNAs for α-, β- and γ-actin have been characterized with respect to molecular weight and poly(A) content. Polyacrylamide gel electrophoresis under denaturing conditions shows that the mRNA for α-actin (muscle-specific actin) is approximately 4.6 × 10⁵ daltons in size, and that the mRNAs for β- and γ-actin (nonmuscle actins) are much larger, approximately 6.6 × 10⁵ daltons in size. We therefore calculate that the noncoding regions of the β- and γ-actin mRNAs contain about 800 nucleotides. This is in marked contrast to the noncoding regions of α-actin mRNA which contain only about 180 nucleotides. During electrophoresis in high-resolution nondenaturing gels, the β-actin mRNA migrates slightly slower than the γ-actin mRNA. This indicates either that β-actin mRNA is about 100 nucleotides longer than γ-actin mRNA, or that these mRNAs differ in secondary structure. Fractionation of actin mRNA on the basis of poly(A) content shows that a substantial portion of the β-actin mRNA, but very little of the α- or γ-actin mRNAs, fails to bind to oligo(dT)-cellulose. Much of this poly(A)-deficient β-actin mRNA, however, does bind to poly(U)-Sepharose, a substrate with higher affinity for short poly(A) sequences. This indicates that many of these β-actin mRNA molecules are polyadenylated, but that they have unusually short poly(A) tails. The finding that β- and γ-actins are translated from mRNAs of different electrophoretic mobility and different poly(A) content strongly suggests that these two closely related proteins are products of different genes.     

Klotho is a substrate for α-, β- and γ-secretase

Klotho is an anti-aging protein with different functions of the full-length membrane protein and the secreted hormone-like form. Using overexpression and knock-down approaches as well as embryonic fibroblasts of knock-out mice we present evidence that Klotho is shedded by the α-secretases ADAM10 and 17 as well as by the β-secretase β-APP cleaving enzyme 1. The remaining membrane-bound fragment is a substrate for regulated intramembrane proteolysis by γ-secretase. Our data suggest that therapeutic approaches targeting these proteases should be carefully analyzed for potential side effects on Klotho-mediated physiological processes.