Increase of β-Fructofuranosidase Content in Tomato Fruit during the Ripening Process

β-Chloro-l-alanine was catalytically converted to pyruvate, ammonia and chloride by α-aminoisobutyrate (AIB) decomposing enzyme (α, β elimination), which was synchronously inactivated. There was a linear relationship between α, β elimination and inactivation. With apoenzyme, neither α, β elimination nor inactivation occurred. These facts suggest that α, β elimination is dependent on pyridoxal 5′-phosphate, and inactivation cooperates with α, β elimination (syncatalytic inactivation). But it seemed that d-form of β-chloroalanine was not a substrate for AIB decomposing enzyme, because just half amount of β-chloro-dl-alanine was decomposed to pyruvate by the enzyme.

An identical active site for each of following three reactions were shown by the fact that AIB decomposing activity, transamination activity and α, β elimination activity were lost in parallel. From a kinetic study, the affinity of the enzyme toward β-chloro-l-alanine was shown to be higher than that toward AIB or l-alanine. The turnover number, about 8,000, of α, β elimination during the inactivation of one mol of the enzyme was much larger than that of d-amino acid transaminase or alanine racemase.     

Chemical Structure of Mating Pheromone Peptides, αsk1 and αsk3 Pheromones,Produced by Mating Type α Cells of Saccharomyces kluyveri

Three peptides, α^sk1, α^sk2 and α^sk3 pheromones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, α^sk2 pheromone, having already been determined. The unknown N-terminus of α^sk1 pheromone was elucidated to be 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from α^sk1 pheromone, and the primary structure of α^sk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of α^sk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of α^sk2 pheromone.     

Influence of the solvent in low temperature glycosylations with O-(2,3,5,6-tetra-O-benzyl-β-d-galactofuranosyl) trichloroacetimidate for 1,2-cis α-d-galactofuranosylation

Glycosylation studies for the construction of 1,2-cis α-linkages with O-(2,3,5,6-tetra-O-benzyl-β-d-galactofuranosyl) trichloroacetimidate (1) and several acceptors, including d-mannosyl and l-rhamnosyl derivatives were performed. The reactions were conducted at low temperatures using CH₂Cl₂, Et₂O, and acetonitrile as solvents. A non-participating solvent such as CH₂Cl₂ at −78 °C, favored the α-d-configuration. In contrast, acetonitrile strongly favored the β-d-configuration, whereas no selectivities were observed with Et₂O. The use of thiophene as an additive did not enhance the α-d-selectivity as in the pyranose counterpart. Although selectivities strongly depended on the acceptor, trichloroacetimidate 1 constitutes a valuable donor for the synthesis of α-d-Galf-(1→2)-l-Rha and α-d-Galf-(1→6)-d-Man. As these motifs are present in pathogenic microorganisms, these procedures described here are useful for the straightforward synthesis of natural oligosaccharides.     

2,2′-Anhydro-4′-Thionucleosides: Precursors for 2′-Azido- and 2′-Chloro-4′-thionucleosides and for a Novel Thiolane to Thietane Rearrangement

Abstract

2,2′-Anhydro-4′-thio-β-and α-nucleosides 9 and 10 have been prepared by an in situ 4-thio-1,2-glycal addition route. They undergo ring-opening by azide or chloride ion to give, after deprotection, the 2′-substituted-4′-thionucleosides 13 and 14, whereas reactions with cyanide or fluoride sources lead to the unsaturated nucleosides 17 or 18, depending upon conditions. An unexpected and clean rearrangement to the thietane 23 occurs on treatment of uracil derivative 20 with DAST.     

Quinoxalines Formed from α-1, 4 Glucans by Reaction with o-Phenylenediamine in Aqueous Alkaline Solution

The alkylation of α, β-unsaturated esters with n-butylbromide gave stereoselectively α-butyl-β, γ-cis-unsaturated esters in the presence of alkali amide in liquid ammonia. On the other hand, the alkylation of β, γ-cis or trans-unsaturated esters afforded α-butyl-β, cis or trans-unsaturated esters with retention of configuration. These alkylations provide a new synthetic method of preparing α-alkyl β, γ-cis or trans-unsaturated esters.     

Structural and Immunochemical Studies of Two Cross-Reactive Proteus mirabilis O-Antigens,O6 and O23, Containing β1→3-Linked 2-Acetamido-2-Deoxy-d-Glucopyranose Residues

A marked serological cross-reactivity was observed by ELISA and a precipitation test between anti-Proteus mirabilis O23 serum and the lipopolysaccharide as well as the O-specific polysaccharide from the Proteus mirabilis strain belonging to serogroup O6. The structures of the O-specific polysaccharides were elucidated using chemical and NMR spectroscopic analyses, and the only common component, 2-acetamido-2-deoxy-β-d -glucopyranose (β-d -GlcNAc), was revealed, which was suggested to be responsible for the cross-reactivity observed. Both anti-O23 and anti-O6 sera were shown to react with 1, 3-linked β-d -GlcNAc-containing O-antigen from Salmonella enterica ssp. arizonae O59 also. The lack of reactivity of Smith-degraded P. mirabilis O6 O-specific polysaccharide with homologous antiserum indicated the crucial role of α-d -glucuronic acid in specific antibody binding.     

Reactions of O-Substituted L-Homoserines Catalyzed by L-Methionine γ-Lyase and Their Mechanism

L-Methionine γ-lyase (EC 4.4.1.11) catalyzes α,γ-elimination of O-substituted L-homoserines (i.e., ROCH₂CH₂CH(NH₂)COOH; R = acetyl, succinyl, or ethyl) to produce α-ketobutyrate, ammonia, and the corresponding carboxylate or alcohol, and also their γ-replacement reactions with various thiols to produce the corresponding S-substituted L-homocysteines. The reactivities of O-substituted L-homoserines in α,γ-elimination relative to that of L-methionine were as follows: O-acetyl, 140%; O-succinyl, 17%; and O-ethyl-L-homoserine, 99%. However, the enzyme does not catalyze the synthesis of O-substituted L-homoserines from alcohol or carboxylic acids in a γ-replacement reaction. We have analyzed the α,γ-elimination of O-acetyl-L-homoserine in deuterium oxide by ¹H-NMR. The [β-²H, γ-²H]-species of α-ketobutyrate was exclusively formed from O-acetyl-L-homoserine. The enzyme catalyzes deamination of L-vinylglycine to give the identically labeled α-ketobutyrate species. Incubation of the enzyme with O-acetyl-L-homoserine resulted in the appearance of a new absorption band at 480 nm, which was observed also with L-vinylglycine. These results strongly suggest that the α,γ-elimination and γ-replacement reactions of O-acetyl-L-homoserine proceed through the stabilized α-carbanion of a Schiff base between pyridoxal 5'-phosphate and vinylglycine, which has been suggested as the key intermediate of L-methionine γ-lyase-caralyzed reactions of S-substituted L-homocysteines [N. Esaki, T. Suzuki, H. Tanaka, K. Soda and R. R. Rando, FEBS Lett., 84, 309 (1977).     

Cell adhesion and integrin expression are modulated by oxidative stress in EA.hy 926 cells

The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125 μM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin αv and α5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that αvβ1 and αvβ3 integrins were involved in adhesion of cells to Vn, and αvβ3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, α5β1 and αvβ1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM.     

Glomerular Extracellular Matrix Components and Integrins

It has become apparent that extracellular matrix components and their cellular receptors, the integrins, are important regulators of glomerular development and function. In this rapidly evolving field we studied the production of extracellular matrix components and integrins by rat glomerular visceral epithelial and mesangial cells, using molecular probes and antibodies that have recently become available. Special attention was paid to laminin isoforms and to splice variants of the integrin subunits α3 and α6. Results were compared to the in vivo expression in human fetal, newborn and adult kidneys.

The mesangial cells were found to produce laminin-1, nidogen and two as yet unidentified laminin isoforms with putative α chains of about 395 (m) and of 375 kDa (cry), tentatively described before as bovine kidney laminin. Furthermore, they expressed the integrins α1β1, α2β, α3Aβ1, α5β1, αvβ3, αvβ5, and small amounts of α6Aβ1 and α6Bβ1. The glomerular visceral epithelial cells produced the two new laminin isoforms mentioned above, laminin-5, but no laminin-1 or nidogen. The integrins α2β1, αAβ1, α6Aβ4, αBβ4 and the integrin subunit av were found to be expressed.

We show that during nephrogenesis, the laminin α1 chain disappears and is replaced by another a chain, possibly one of the two as yet unidentified α chains mentioned above. The laminin β1 chain is replaced by the β2 chain somewhat later in glomerular development. In general, the integrins found to be expressed in glomeruli of adult kidney were consistent with those found in cultured glomerular visceral epithelial and mesangial cells. No splice variant switch of the integrin α3 or α6 subunits could be demonstrated during nephrogenesis.

Our results suggest an important role for the mesangial cell in providing nidogen as a crucial component of the supramolecular stucture of the glomerular basement membrane. Furthermore our results indicate that laminin αxβ2γ1 and αβ2γ1 isoforms are important in the glomerulus of adult kidney and that the integrin α3Aβ1 is the main integrin receptor for laminin isoforms on glomerular visceral epithelial and mesangial cells, both in vitro and in vivo.     

Studies on the Organophosphorus Insecticides

The reaction of O,O-dialkyl phosphorochloridates or phosphorochloridothioates with sodium salts of benzoylacetone and the reaction of trialkyl phosphites with α-chloro-benzoylacetone or acetophenone were attempted with a view to prepare the low toxic phosphorus insecticides containing acyl-vinyl group. It was found that the products did not have a phosphonate-form but a phosphorate-form and the phenyl-acyl-vinyl group had linkage with phosphorus atom by the enol-form of next carbonyl group of the phenyl-group.     

Quantitative Study of Anomeric Forms of Maltose Produced by α- and βAmylases

Anomeric forms of glucose and maltose produced from phenyl, p-nitrophenyl, p-tert-butylphenyl, p-ethylphenyl and p-chlorophenyl α-maltosides and maltopentaose by α- and β-amylases were determined quantitatively by a gas-liquid chromatographic method. All of the three kinds of α-amylases tested, B. subtilis saccharifying α-amylase, Taka-amylase A, and porcine pancreas α-amylase, were found to produce only α-maltose from the maltosides. Sweet potato and barley β-amylases produced β-maltose from maltopentaose.

Saccharifying α-amylase from B. subtilis also released α-maltose from all the maltosides mentioned above, contrary to the report by Shibaoka et al. that the enzyme released β-maltose from maltosides other than phenyl α-maltoside: FEBS Lett., 16, 33 (1971); J. Biochem., 77, 1215 (1975). It appears unlikely that the α-amylase releases β-maltose, depending on the kind of substrate.     

Comparison by 1H-nmr spectroscopy of the conformation of the 2600 dalton antifreeze glycopeptide of polar cod with that of the high molecular weight antifreeze glycoprotein

The antifreeze glycopeptide (AFGP-8) from polar cod, B. saida, is a 14-amino acid polypeptide having alternating glycotripeptide sequences of Ala-[Gal(β1 → 3)GalNAc(β1 → O)]-Thr-Pro and Ala-[Gal(β1 → 3)GalNAc(β1 → O)]-Thr-Ala, with alanyl residues at amino and carboxy terminals. Conformational studies of AFGP-8 have been carried out by ¹H-nmr and empirical energy calculations to investigate the difference in its antifreeze behavior from that of the more active high-molecular weight AFGP 1-4 of P. borchgrevinki. The ¹H-nmr spectra, including the resonances of the exchangeable amide protons, were assigned by two-dimensional correlated spectroscopy (COSY), one-dimensional difference decoupling, and nuclear Overhauser effect (NOE) measurements. For the four threonyl residues, the amide proton coupling constants and the small coupling constants between H^α and H^β indicate similar conformations, despite significant chemical shift differences. The strong NOE between the α protons and the amide protons of the residue following together with large temperature coefficients of chemical shifts, indicate an extended conformation not consisting of α-helix, turns or bends. Energy computations indicate several low-energy conformations consistent with the observed coupling constants for ?. Among these, a left-handed helical conformation with three repeating residues per turn has been proposed, which is in accordance with the observed NOE between the methyl group of the α-GalNAc and Ala H^βs. While the observed Overhauser effects in the threonyl side chain suggest a certain amount of conformational averaging, the effect involving the acetmido methyl of α-GalNAc and H^βs of Ala indicate that it as is a major conformer. In view of the close similarity between the conformations of AFGP-8 and the more active antifreeze polymer, AFGP 1-4, we propose that the difference in their activities is due to the length of the regular repeating structure with glycosylation at every third amino acid residue, and not due to any fundamental difference in their conformations.     

Double-sides sticking mechanism of vinblastine interacting with α,β-tubulin to get activity against cancer cells

Abstract

Vinblastine (VLB) and its derivatives have been used for clinical first-line drugs to treat various cancers. Due to the resistance and serious side effects from using VLB and its derivatives, there is a need to discover and develop novel VLB derivatives with high activity against cancer cells. In order to better discover and develop new VLB derivatives, we need to study the structural basis of VLB's anti-cancer cytotoxicity and the mechanism of its interaction with α,β-tubulins. Based on the crystal structure of α,β-microtubule complex protein, the molecular dynamics method including the sampling PMF method was used to study the variation of dissociation free energy (ΔG) of α,β-tubulins under different system conditions, and then from which to study the mechanism of the interaction between VLB and α,β-tubulins. The obtained results show that the dissociation of pure α,β-tubulins requires 197.8?kJ·mol^?1 for ΔG. When the VLB molecule exists between the interface of α,β-tubulins, the dissociation ΔG of α,β-tubulins reaches 220.5?kJ·mol^?1, which is greater than that of pure α,β-tubulin. The VLB molecule is formed by connecting a vindoline moiety (VM) molecule with a catharanthine moiety (CM) molecule through a carbon-carbon bond, which is a larger molecule. When the CM molecule exists in the middle of α,β-tubulin interface, the dissociation ΔG of α,β-tubulins is 46.2?kJ·mol^?1, during which the CM moves with β-tubulin. When the VM molecule exists between the middle of α,β-tubulin interface, the dissociation ΔG of α,β-tubulins is 86.7?kJ·mol^?1, during which it moves with α-tubulin. Therefore, the VLB molecule is like a double-sides tape to stick α-tubulin and β-tubulin together. The VLB molecule intervenes the dynamic equilibrium between dissociation and aggregation of α-tubulin and β-tubulin by a double-sides sticking mechanism to exert high activity with toxicity against cancer cell. Besides, our results demonstrate that VLB has its structural basis for anticancer cytotoxicity due to its two compositions composed of a CM molecule and a VM molecule although they have little toxicity against cancer cell alone.     

Bacterial species selective toxicity of two isomeric α/β-peptides: Role of membrane lipids

We have studied how membrane interactions of two synthetic cationic antimicrobial peptides with alternating α- and β-amino acid residues (“α/β-peptides”) impact toxicity to different prokaryotes. Electron microscopic examination of thin sections of Escherichia coli and of Bacillus subtilis exposed to these two α/β-peptides reveals different structural changes in the membranes of these bacteria. These two peptides also have very different effects on the morphology of liposomes composed of phosphatidylethanolamine and phosphatidylglycerol in a 2:1 molar ratio. Freeze fracture electron microscopy indicates that with this lipid mixture, α/β-peptide I induces the formation of a sponge phase. ³¹P NMR and X-ray diffraction are consistent with this conclusion. In contrast, with α/β-peptide II and this same lipid mixture, a lamellar phase is maintained, but with a drastically reduced d-spacing. α/β-Peptide II is more lytic to liposomes composed of these lipids than is I. These findings are consistent with the greater toxicity of α/β-peptide II, relative to α/β-peptide I, to E. coli, a bacterium having a high content of phosphatidylethanolamine. In contrast, both α/β-peptides display similar toxicity toward B. subtilis, in accord with the greater anionic lipid composition in its membrane. This work shows that variations in the selectivity of these peptidic antimicrobial peptides toward different strains of bacteria can be partly determined by the lipid composition of the bacterial cell membrane.     

Cystine-knot peptides engineered with specificities for α(IIb)β(3) or α(IIb)β(3) and α(v)β(3) integrins are potent inhibitors of platelet aggregation

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ～20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α_IIbβ₃, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K_D) constants for α_IIbβ₃ integrin ranging from 60 to 90 nM, and did not bind to α_vβ₃, α_vβ₅, or α₅β₁ integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α_IIbβ₃ and α_vβ₃ integrins with K_D values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α_vβ₅ or α₅β₁ integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.     

Fungal Reduction of C6 α,β-Unsaturated Carboxylic Acids

The metabolism of sorbic acid (trans-2,trans-4-hexadienoic acid) and its related compounds by Mucor sp. A-73 was investigated. Sorbic acid was reduced by this fungus to trans-4-hexenol (more than 90% yield). In a series of hexamonoenoic acids, carboxyl groups and α,β-double bond were reduced, but β,γand γ,δ double bonds were hardly reduced. The reduction of cis-2-hexenoic acid was slower than that of the corresponding trans isomer. Sorbic alcohol, one of α,β-unsaturated alcohols, was converted well to trans-4-hexenol by the fungus. These results showed that this fungus could carry out two independent reductions: (i) carboxyl group→alcohol, (ii) α,β-unsaturated alcohol→αβ-saturated one. Furthermore, α,β-unsaturated alcohols were temporarily detected in the course of fungal reductions of some α,β-unsaturated acids. The fact suggested that the reduction of α,β-unsaturated acids to α,β-saturated alcohols was initiated by the reaction (i) and followed by (ii). The biological hydrogenation of α,β-unsaturated alcohols is a new reaction.     

Studies on Abscisic Acid

Methyl α-cyclocitrylideneacetate was successively oxidized with selenium dioxide and chromium trioxide-pyridine complex to give methyl 1′-hydroxy-α-cyclocitrylideneacetate and a mixture of methyl 3′-keto-β-cyclocitrylideneacetate and methyl 4′-keto-α-cyclocitrylideneacetate. Further, oxidation of methyl α-cyclocitrylideneacetate with tert-butyl chromate afforded methyl 4′-keto-α-cyclocitrylideneacetate and methyl 1′-hydroxy-4′-keto-α-cyclocitry-lineacetate. Similarly, methyl α-cyclogeranate was oxidized to methyl 3-keto-β-cyclogeranate and methyl 4-keto-α-cyclogeranate. Methyl l′-hydroxy-4′-keto-α-cyclocitrylideneacetate, methyl l-hydroxy-4-keto-α-cyclogeranate and their related compounds did not show growth inhibitory activities on rice seedlings.     

Stereochemistry of the β-cyanoalanine synthetase and S-alkylcysteine lyase reactions

The stereochemistry of the replacement of the SH-group of cysteine by CN catalyzed by β-cyanoalanine synthetase was studied using cysteine stereospecifically tritiated at C-3. Analysis of the resulting β-cyanoalanine by conversion into fumarate via aspartate and malate showed that the reaction had occurred with retention of configuration at C-3. Using cystine stereospecifically labeled at C-3 with tritium or with tritium and deuterium, it was found that the α,β-elimination reaction catalyzed by S-alkylcysteine lyase involves stereo-specific replacement of the β-substituent of the substrate by a hydrogen derived from the solvent, D₂O or H₂O, with retention of configuration to give pyruvate containing a chiral methyl group. The results are discussed, particularly in the light of mechanistic proposals by Braunstem and co-workers.     

Purification,Characterization, and Crystallization of Single Molecular Species of β-Conglycinin from Soybean Seeds

Four major molecular species of β-conglycinin, α₃, α_2β, αβ₂, and β₃, were isolated and purified from seeds of an α' subunit-deficient strain of soybeans (Glycine max). All components were found to be homogeneous by high pressure liquid chromatography, SDS-polyacrylamide gel electrophoresis, and amino acid and amino terminal sequence analyses. The amino acid compositions of the α₃ and β₃ components agreed fairly well with the compositions deduced from the cDNA sequences, and all of the components were highly glycosylated. The α₃ and β₃ components were compared regarding their secondary structures. The secondary structure of the α₃ component deduced from CD measurements showed a higher α-helix content than that of the β₃ component. The β₃ component was crystallized by decreasing the ionic strength from 0.5 to 0.14 in phosphate buffer, pH 7.3, and the crystals grew to a size (1.0 mm × 0.2 mm × 0.2 mm) suitable for X-ray crystallographic analysis. A preliminary X-ray analysis showed that the crystal belonged to an orthorhombic crystal system having the space group P2₁2₁2₁ and unit cell dimensions of a = 185.1 Å, b = 107.9 Å, and c = 97.6 Å.     

A recombinant Escherichia coli expressing an α-alkyl-β-ketoester reductase with unusual stereoselectivity

A recombinant Escherichia coli expressing an α-alkyl-β-ketoester reductase that yields the anti (2R,3R) α-alkyl-β-hydroxyester as the major product was constructed. The enzyme was identified from genomic libraries of Paucimonas lemoignei, cloned and expressed in E. coli providing a whole cell biocatalyst with novel stereoselectivity. This constitutes the first report on a whole cell biocatalyst for the synthesis of (2R,3R) α-alkyl-β-hydroxy-esters.