Stereochemistry of the hydrolysis of α,α-trehalose by trehalase,determined by using a labelled substrate

(1,1′-¹³C)α,α-Trehalose was obtained in 37% yield from the Pavia condensation of 2,3,4,6-tetra-O-benzyl-d-(1-¹³C)glucopyranose, in dichloromethane in the presence of trifluoromethanesulfonic anhydride, followed by the usual deprotection techniques. The hydrolysis of this substrate by cockchafer trehalase was monitored at 37° by using ¹³C-n.m.r. spectroscopy with short recording times. Equimolecular amounts of α- and β-d-glucopyranose are released simultaneously by the action of the enzyme. This result is consistent with a bimolecular substitution mechanism, taking into account previous results involving C-2 asymmetric participation in the catalytic step of hydrolysis of α,α-trehalose. For comparative evaluation of its accuracy, the usual polarimetric technique was also used for the determination of the anomeric configuration of the d-glucose released by the action of the enzyme on α,α-trehalose.     

Characterization of the extracellular,water-insoluble α-D-glucans of oral streptococci by methylation analysis,and by enzymic synthesis and degradation

Methylation analysis of water-insoluble α-D-glucans synthesized from sucrose by culture filtrates from several strains of Streptococcus spp. has proved that all of the glucans were highly branched and that the chains contained (1→6)- and (1→3)-linked D-glucose residues not involved in branch points. Hydrolysis of the glucans with a specific endo-(1→3)-α-D-glucanase demonstrated that the majority of the (1→3)-linked glucose residues were arranged in sequences. D-Glucose was the major product of the hydrolysis, and a small proportion of nigerose was also released. The use of a specific endo-(1→6)-α-D-glucanase similarly indicated that the glucans also contained sequences of (1→6)-linked α-D-glucose residues, and that those chains were branched. Two D-glucosyltransferases (GTF-S and GTF-I), which reacted with sucrose to synthesize a soluble glucan and a water-insoluble glucan, respectively, were separated from culture filtrates of S. mutans OMZ176. The soluble glucan was characterized as a branched (1→6)-α-D-glucan, whereas the insoluble one was a relatively linear (1→3)-α-D-glucan. The hypothesis is advanced that the glucosyltransferases can transfer glucan sequences by means of acceptor reactions similar to those proposed by Robyt for dextransucrase, leading to the synthesis of a highly branched glucan containing both types of chain. The resulting structure is consistent with the evidence obtained from methylation analysis and enzymic degradations, and explains the synergy displayed when the two D-glucosyltransferases interact with sucrose. Variations in one basic structure can account for the characteristics of water-insoluble glucans from S. sanguis and S. salivarius, and for the strain-dependent diversity of S. mutans glucans.     

Biocontrol of Fusarium sambucinum,dry rot of potato,by Serratia plymuthica 5–6

Serratia grimesii 4–9 and Serratia plymuthica 5–6, isolated from the rhizosphere of pea, Pisum sativum (L), were evaluated for their potential to suppress growth of Fusarium sambucinum in vitro and to reduce Fusarium dry rot in stored potatoes (Solanum tuberosum L). In vitro studies indicated that these bacterial isolates suppressed growth of F. sambucinum by 60% or more at both 15 and 25°C. In a potato tuber slice assay the number of infection sites in potato slices exposed to F. sambucinum and treated with S. grimesii 4–9 and S. plymuthica 5–6 was reduced by 96 and 97%, respectively, at 15°C. The diameter (mm) of the infection sites was reduced 91 and 96%, respectively, when compared to slices treated with F. sambucinum alone. Studies with Fusarium-infected whole potato tubers also showed significant reduction in dry rot formation following treatment with the bacterial isolates or the fungicide thiabendazole. When applied simultaneously with the pathogen, S. grimesii 4–9 and S. plymuthica 5–6 suppressed development of Fusarium dry rot by 60 and 77%, respectively, at 15°C and by 63 and 84%, respectively, at 25°C compared to tubers inoculated with the pathogen alone. Thiabendazole suppressed development of Fusarium dry rot by 66 and 81% at 15 and 25°C, respectively, compared to tubers inoculated with the pathogen alone. These studies demonstrate the potential of soil bacteria as biofungicides for managing post-harvest crop diseases. Due to the potential risks to human health associated with S. grimesii 4–9, S. plymuthica 5–6 is recommended for further study for biofungicide development.     

Organization, Structure, and Assembly of α-Carboxysomes Determined by Electron Cryotomography of Intact Cells

Carboxysomes are polyhedral inclusion bodies that play a key role in autotrophic metabolism in many bacteria. Using electron cryotomography, we examined carboxysomes in their native states within intact cells of three chemolithoautotrophic bacteria. We found that carboxysomes generally cluster into distinct groups within the cytoplasm, often in the immediate vicinity of polyphosphate granules, and a regular lattice of density frequently connects granules to nearby carboxysomes. Small granular bodies were also seen within carboxysomes. These observations suggest a functional relationship between carboxysomes and polyphosphate granules. Carboxysomes exhibited greater size, shape, and compositional variability in cells than in purified preparations. Finally, we observed carboxysomes in various stages of assembly, as well as filamentous structures that we attribute to misassembled shell protein. Surprisingly, no more than one partial carboxysome was ever observed per cell. Based on these observations, we propose a model for carboxysome assembly in which the shell and the internal RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) lattice form simultaneously, likely guided by specific interactions between shell proteins and RuBisCOs.     

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α

POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.     

Inhibition of Swarming of Proteus by Sodium Tetradecyl Sulfate, β-Phenethyl Alcohol,and p-Nitrophenylglycerine

The effects of sodium tetradecyl sulfate (STS), β-phenethyl alcohol (PEA), and p-nitrophenylglycerol (PNPG) on motility, swarming, flagellation, and growth of Proteus were examined. Growth-inhibitory concentrations (GIC) and swarming-inhibitory concentrations (SIC) were determined. A characterization of the swarming-inhibitory efficacy of these compounds was based on their GIC/SIC ratio and their concentration inhibition curves. Using the homologous series of sodium alkyl sulfates as a standard reference, we showed that PNPG was more effective than STS, which was the most effective of the homologous series. PEA was less effective than sodium decyl sulfate but more effective than sodium octyl sulfate. Motility tests in liquid medium and electron microscope investigations indicated that the modes of action of the three compounds, all of which effectively inhibit the swarming of Proteus, are different. Whereas STS and PEA inhibit swarming by inhibition of motility, PNPG seems to act on the swarming mechanism sensu strictori, without impairment of motility. STS immobilizes by inhibition of flagellum formation or by some lytic action on the flagella already synthesized. PEA acts by impairing flagellar function, but leaves the flagella morphologically intact.     

Oxidation of 2,2,7,8-tetramethyl-6-chromanol,the model compound of γ-tocopherol,by hypochlorous acid

Abstract

Due to its high concentration in soybean oil, γ-tocopherol is probably the most important form of vitamin E in Western diets, whereas, α-tocopherol is by far the most important form of vitamin E as a dietary supplement. Recent studies have shown that γ-tocopherol is an excellent scavenger of nitric oxide and possibly other dangerous electrophiles while α-tocopherol has little if any scavenger activity.¹ The ability of γ-tocopherol to act as a scavenger of electrophiles is due to an absence of substituents in the activated 5-position of the chromanol ring.     

Intestinal Absorbability of Wheat Allergens,Subunits of a Wheat α-Amylase Inhibitor,Expressed by Bacteria

Wheat CM2, CM3 and CM16 proteins are known as subunits of the tetrameric α-amylase inhibitor as well as major allergens to baker’s asthma. The purpose of this study is to produce these CM proteins by bacteria in a quantity adequate for studying thepenetration characteristics of the CM proteins through intestinal mcosa in rats and Caco-2 cells. cDNAs encoding the mature proteins were expressed in Escherichia coli and purified by an Ni²⁺-chelating column. The recombinant proteins were radioiodinated and admministrered orally to rats or applied to the apical site of the Caco-2 cell monolayer. The radioactivity in the trichloroacetic acid-insoluble fraction, which was mainly composed of peptides with molecular mass less than that of the intact CM proteins, in the serum and the basolateral medium was highest in recombinant CM3. Accordingly, the intestinal absorption of these three proteins in the form present in wheat should be evaluated.     

Mechanism and stereochemistry of α,β-elimination of l-tyrosine catalysed by tyrosine phenol-lyase

The decomposition of l-tyrosine and its α-deuterated analogue under the action of extracts from Escherichia intermedia A-21 with high tyrosine phenol-lyase [l-tyrosine phenol-lyase (deaminating), EC 4.1.99.2] activity has been studied. The mass spectrometric data for samples of phenol produced by decomposition of deutero-l-tyrosine in water and D₂O-water (10:1) mixture, and by decomposition of normal l-tyrosine in D₂O-water (10:1), show that the process is accompanied by the intramolecular transfer of D or H to the leaving phenol group. The degree of transfer is 7–10%. Thus, the abstraction of α-proton and the subsequent protonation of the aromatic ring are accomplished by the same functional group of the enzyme. This is indicative of the cis-orientation of α-proton and the phenol fragment in relation to the plane of Schiff's base of α-aminoacrylate with pyridoxal phosphate during α,β-elimination. The isotope effect of the studied enzymic reaction is ≈3, which allows us to consider the α-proton abstraction as the limiting stage of the process.     

Investigations of Biodeterioration by Fungi in Historic Wooden Churches of Chiloé, Chile

The use of wood in construction has had a long history and Chile has a rich cultural heritage of using native woods for building churches and other important structures. In 2000, UNESCO designated a number of the historic churches of Chiloé, built entirely of native woods, as World Heritage Sites. These unique churches were built in the late 1700 s and throughout the 1800 s, and because of their age and exposure to the environment, they have been found to have serious deterioration problems. Efforts are underway to better understand these decay processes and to carryout conservation efforts for the long-term preservation of these important structures. This study characterized the types of degradation taking place and identified the wood decay fungi obtained from eight historic churches in Chiloé, seven of them designated as UNESCO World Heritage sites. Micromorphological observations identified white, brown and soft rot in the structural woods and isolations provided pure cultures of fungi that were identified by sequencing of the internal transcribed region of rDNA. Twenty-nine Basidiomycota and 18 Ascomycota were found. These diverse groups of fungi represent several genera and species not previously reported from Chile and demonstrates a varied microflora is causing decay in these historic buildings.     

Steady-state kinetics, micellar effects, and the mechanism of peroxidase-catalyzed oxidation of n -alkylferrocenes by hydrogen peroxide

 Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C₅H₁₁) by H₂O₂ to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25  °C. The rate of oxidation of ferrocenes with longer alkyl radicals is too slow to be measured. The reaction obeying the [RFc]:[H₂O₂] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic second-order rate constants k _w are (1.9 ± 0.5)×10⁵, (2.7 ± 0.1)×10⁴, and (5.9 ± 0.6)×10³ M^–1 s^–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple in terms of the Marcus formalism. Received: 15 July 1996 / Accepted: 15 November 1996     

Characterisation of the thiol–disulphide chemistry of desmopressin by LC, μ-LC, LC-ESI-MS and Maldi-Tof

Summary. To date, the majority of therapeutic peptides and proteins have to be administered via parenteral routes, which are painful and inconvenient. In order to gain sufficient high blood concentrations after oral application, various barriers in the gastrointestinal tract have to be overcome. Apart from a poor membrane uptake and intense enzymatic degradation, this study will demonstrate that thiol–disulphide reactions are an underestimated essential part of the presystemic metabolism. Glutathione, integrative part of the antioxidant defence system in the gastrointestinal tract, may play an important role in the inactivation of orally given peptides and proteins. In order to verify this hypothesis, desmopressin which bears a single disulphide bond was used as model peptide drug. Desmopressin was incubated with GSH in various concentrations, and the extent of thiol/disulphide exchange reactions between the peptide drug and GSH was investigated in dependence on pH and ratio of reactants determined as a function of time via HPLC, LC-MS and Maldi-Tof-MS analyses. Results showed that desmopressin is degraded by 1% reduced glutathione at pH 4 and pH 5.5. In presence of 0.01%, 0.1% and 1% of reduced glutathione 6.1%, 19.4% and 52.1% of desmopressin, respectively, were degraded. The masses of the conjugates after deconvolution measured by liquid chromatography and electrospray ionisation mass spectrometric detection were m/z 1069.67, m/z 1376.50, m/z 1683.40 and m/z 2138. These molecular masses, confirmed by Maldi-Tof-MS analysis, correspond with the masses of conjugates expected in theory. Under defined conditions, these results reveal that thiol–disulphide exchange reactions have a considerable impact on the alteration of peptide drugs and proteins.     

Who, what, where? Recognition and localization of acoustic signals by insects

Insects, like all hearing animals, must analyze acoustic signals to determine both their content and their location. Neurophysiological experiments, together with behavioral tests, are beginning to reveal the mechanisms underlying these signal-analysis tasks. Work summarized here focusses on two issues: first, how insects analyze the temporal structure of a single signal in the presence of other competing signals; and second, how the signal's location is represented by the binaural difference in neural activity.     

Conformations of disaccharides by empirical,force-field calculations: Part III, β-gentiobiose

The conformational flexibility of β-gentiobiose has been studied by using convergent energy minimisation in a new force-field, with relaxation of all degrees of freedom. Twenty-four local minima are found in the φ,ψ,ω-space. The free-enthalpy differences are 1.7, 3.7, 5.1, 5.3, and from 5.9 to 29.4 kJ.mol^?1 above the lowest minimum, corresponding to a distribution of 40:20:9:5:5:21 at 298 K. Each minimum is surrounded by a manifold of minimum conformers that differ only in exocyclic torsions. The average conformation of β-gentiobiose is not fully extended, but to some degree coiled. Three conformers are shown in stereo.     

Characterization of Antimicrobial-bearing Liposomes by ζ-Potential, Vesicle Size, and Encapsulation Efficiency

Liposome entrapment may improve activity of protein or polypeptide antimicrobials against a variety of microorganisms. In this study, ability of liposomes to withstand exposure to environmental and chemical stresses typically encountered in foods and food processing operations were tested. Liposomes consisting of distearoylphosphatidylcholine (PC) and distearoylphosphatidylglycerol (PG), with 0, 5, or 10 μg/ml of the antimicrobial peptide nisin entrapped, were exposed to elevated temperatures (25–75 °C) and a range of pH (5.5–11.0). Ability of liposomes to maintain integrity was assessed by measuring the encapsulation efficiency (EE), ζ-potential, and particle size distribution of liposomes. Distearoylphosphatidylcholine, PC/PG 8:2, and PC/PG 6:4 (mole fraction) liposomes retained between ~70–90% EE despite exposure to elevated temperature and alkaline or acidic pH. Particle size of liposomes averaged between 100 and 240 nm depending on liposome preparation. Liposomal surface charge depended primarily on phospholipid composition and changed little with inclusion of nisin. Surface charge was not affected by temperature for PC and PC/PG 8:2 but decreased for PC/PG 6:4 liposomes. Our results suggest that liposomes containing nisin may be suitable for use as antimicrobial-active ingredients in low- or high-pH foods subjected to moderate heat treatments.     

Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration

Several esters of 4-methylumbelliferone and 2-naphthol were synthesized and examined as possible spectrofluorimetric titrants for bovine alpha-chymotrypsin, trypsin, thrombin, Factor Xa and for subtilisin Novo. 4-Methylumbelliferyl p-guanidinobenzoate hydrochloride (MUGB) is a satisfactory titrant for alpha- and beta-trypsin, thrombin and Factor Xa and 4-methylumbelliferyl p-(NNN-trimethylammonium)cinnamate (MUTMAC) is a good titrant for alpha-chymotrypsin. The amount of enzyme used for spectrofluorimetric titration is 0.02-3.00nmol and the amount of 4-methylumbelliferone liberated is independent of the concentration of titrant and stoicheiometrically equal to the amount of enzyme used. Results obtained with MUGB and MUTMAC have been checked by spectrophotometric titration with p'-nitrophenyl p-guanidinobenzoate hydrochloride and p-nitrophenyl N(2)-acetyl-N(1)-benzylcarbazate respectively. p-Nitrophenyl N(2)-acetyl-N(1)-(9-anthrylmethyl)carbazate has been synthesized; it did not react with alpha-chymotrypsin. A satisfactory spectrofluorimetric titrant for subtilisin Novo was not discovered.     

Degradation of arylglycerol-β-aryl ethers,lignin substructure models,by Fusarium solani

Degradation of arylglycerol--aryl ethers, the most important substructure in lignin, by Fusarium solani M-13-1 was investigated. The fungus was shake-cultured in mineral salts media which contained either guaiacylglycerol--vanillic acid ether (2), syringylglycerol--vanillin ether (4), veratrylglycerol-vanillin ether (17) or glycerol-2-vanillic acid ether (9) as sole carbon source. Culture filtrates from incubations with 4 contained syringylglycerol-vanillic acid ether (6), 9 and 2,6-dimethoxy-p-benzoquinone (16). Culture filtrates from incubations with 2 also contained 9. Veratrylglycerol--vanillic acid ether (18) derived from 17 was not metabolized further. These results inidicate that the alkyl-aryl C-C bond in both 2 and 5 was cleaved by phenol oxidizing enzymes with formation of 9 and methoxy-p-benzoquinone (15 and 16). Compound 9 was converted to glycerol-2-vanillic acid ether monoacetate (10), glyceric acid-2-vanillic acid ether (11) and ethylene glycol monovanillic acid ether (12).Non-Standard Abbreviations Ar aromatic - THF tetrahydrofuran - TLC thin layer chromatography     

Immunogenicity of Rabies Virus Inactivated by β-Propiolactone, Acetylethyleneimine, and Ionizing Irradiation

Ionizing radiation, beta-propiolactone, and acetylethyleneimine were compared for their ability as virus-inactivating agents for the preparation of rabies vaccine. Each agent reduced viral infectivity exponentially; ionizing radiation also destroyed viral hemagglutinin. The vaccine prepared by ionizing radiation was equal or superior to that prepared by beta-propiolactone in its ability to protect mice from rabies infection. The acetylethyleneimine-treated vaccine was a less potent immunogen.     

Dissociation of β-Sheet Stacking of Amyloid β Fibrils by Irradiation of Intense,Short-Pulsed Mid-infrared Laser

Structure of amyloid β (Aβ) fibrils is rigidly stacked by β-sheet conformation, and the fibril state of Aβ is profoundly related to pathogenesis of Alzheimer’s disease (AD). Although mid-infrared light has been used for various biological researches, it has not yet been known whether the infrared light changes the fibril structure of Aβ. In this study, we tested the effect of irradiation of intense mid-infrared light from a free-electron laser (FEL) targeting the amide bond on the reduction of β-sheet content in Aβ fibrils. The FEL reduced entire contents of proteins exhibiting β-sheet structure in brain sections from AD model mice, as shown by synchrotron-radiation infrared microscopy analysis. Since Aβ_1-42 fibril absorbed a considerable FEL energy at amide I band (6.17 μm), we irradiated the FEL at 6.17 μm and found that β-sheet content of naked Aβ_1-42 fibril was decreased using infrared microscopic analysis. Consistent with the decrease in the β-sheet content, Congo-red signal is decreased after the irradiation to Aβ_1-42 fibril. Furthermore, electron microscopy analysis revealed that morphologies of the fibril and proto-fibril were largely changed after the irradiation. Thus, mid-infrared light dissociates β-sheet structure of Aβ fibrils, which justifies exploration of possible laser-based therapy for AD.     

Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis

Random mutagenesis was performed on β-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K–T307I were approx. 140, 190 and 200%, respectively, of wild type β-agarase (661 U/mg) at 40°C. All three mutant enzymes were stable up to 50°C and E99K–T307I had the highest thermostability. The melting temperature (T _m) of E99K–T307I, determined by CD spectra, was increased by 5.2°C over that of the wild-type enzyme (54.6°C). Activities of both the wild-type and E99K–T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl₂. The E99K–T307I enzyme was stable at 55°C with 1 mM CaCl₂, reaching 260% of the activity the wild-type enzyme held at 40°C without CaCl₂.