Location of O-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated Eucalyptus wood

A combination of techniques was used to localise the O-acetyl substituents in xylo-oligosaccharides, which are present in hydrolysates of hydrothermally treated Eucalyptus wood. Reversed-phase (RP)-high performance liquid chromatography (HPLC) coupled on-line to both a mass spectrometer and an evaporating light scattering (ELS) detector provided data about the order of elution of the various O-acetylated oligomers. The retention of the oligomers on the column depended on the number and position of the O-acetyl substituents within the xylo-oligosaccharides. One dimensional (1D)- and two dimensional (2D)-(1)H NMR spectroscopy was used to study the structural features of several xylotetramers separated by RP-HPLC, each having one O-acetyl substituent. O-Acetyl migration was proven to have occurred in these xylo-oligosaccharides. Mainly O-acetyl migration within the same xylosyl residue was observed. RP-HPLC-NMR was performed in order to study the structural features of the acetylated oligomers 'on-line' avoiding O-acetyl migration. Finally, the precise location of the 2-O- or 3-O-acetyl substituent in 6 xylotetramers and 4 xylotrimers separated by RP-HPLC was determined.     

Porous scaffolds from high molecular weight polyesters synthesized via enzyme-catalyzed ring-opening polymerization

Several aliphatic polyesters have been synthesized until now using enzyme-catalyzed ring-opening polymerization (ROP) of different lactones, although their molecular weight, hence mechanical strength, was not sufficient enough to fabricate porous scaffolds from them. To achieve this target, 1,5-dioxepan-2-one (DXO) and epsilon-caprolactone (CL) were polymerized in bulk with Lipase CA as catalyst at 60 degrees C, and porous scaffolds were prepared from the polymers obtained thereof using a salt leaching technique. The CL/DXO molar feed ratio was varied from 1.5 to 10, and the reactivity ratios of CL and DXO were determined using the Kelen-Tudos method under such conditions of polymerization. NMR results showed a slightly lower CL/DXO molar ratio in the copolymers than in the feed due to high reactivity of DXO toward Lipase CA catalysis. The crystallinity of the PCL segment of the copolymers was affected by the presence of soft and amorphous DXO domains. The copolymers having high CL content were thermally more stable. The porosity of the scaffolds was in the range 82-88%, and the SEM analysis showed interconnected pores in the scaffolds. Of the two parameters which could affect the mechanical properties, viz., the copolymer composition and the scaffold pore size, the pore size showed a significant effect on the mechanical properties of the scaffolds. The porous scaffolds developed in this way for tissue engineering are free from toxic organometallic catalyst residues, and they are highly suitable for biomedical applications.     

A simple method for the synthesis of benzyl 4-O-benzylhexopyranosides

The kinetics of hydrolysis in dilute sulfuric acid of xylo-oligosaccharides ranging between the di- and penta-oligosaccharides has been studied. One of the two terminal bonds and each internal bond of all xylo-oligosaccharides tested were hydrolysed at the same rate. The hydrolytic rate of the other terminal bond was the same as that of xylobiose, which was 1.8 times greater than that of an internal bond. The rates of hydrolysis of xylo-oligosaccharides have been described as functions of the reaction temperature and concentration of sulfuric acid. It has been shown that the yield of xylose in hydrolysis of xylo-oligosaccharides by sulfuric acid may be calculated from the ratio ( 1.8) of the rate for xylobiose to that of an internal bond and the empirical equation that describes the rate-constant for xylobiose.     

An alternative approach for the synthesis of fluorogenic substrates of endo-beta-(1-->4)-xylanases and some applications

Fluorogenic substrates of endo-beta-(1-->4)-xylanases (EXs), 4-methylumbelliferyl beta-glycosides of xylobiose and xylotriose were synthesized from fully acetylated oligosaccharides using the alpha-trichloroacetimidate procedure. A commercially available syrup containing xylose and xylo-oligosaccharides was used as the starting material. Both fluorogenic glycosides were found to be suitable substrates for EXs, particularly for sensitive detection of the enzymes in electrophoretic gels and their in situ localization on sections of fruiting bodies of some plants, such as tomato, potato and eggplant, all of the family Solanaceae.     

Elastomeric hydrolyzable porous scaffolds: copolymers of aliphatic polyesters and a polyether-ester

Porous scaffolds of 1,5-dioxepan-2-one (DXO), L-lactide (LLA), and epsilon-caprolactone (CL) were prepared by a solvent casting, salt particulate leaching technique in which the composites were detached from their mold using a novel methanol swelling procedure. By incorporating DXO segments into polymers containing LLA or CL, an increase in hydrophilicity is achieved, and incorporating soft amorphous domains in the crystalline sections enables tailoring of the mechanical properties. The porosities of the scaffolds ranged from 89.2% to 94.6%, and the pores were shown to be interconnected. The materials were synthesized by bulk copolymerization of 1,5-dioxepan-2-one (DXO), L-lactide (LLA), and epsilon-caprolactone (CL) using stannous 2-ethylhexanoate as catalyst. The copolymers formed varied in structure; poly(DXO-co-CL) is random in its arrangement, whereas poly(DXO-co-LLA) and poly(LLA-co-CL) are more blocky in their structures.     

Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans

Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated xylo-oligosaccharides and complex non-soluble acetylglucuronoxylan. Both enzymes performed optimally at pH 7.0 and 40 °C.Axe2 has a clear preference for acetylated xylo-oligosaccharides (AcXOS) with a high degree of substitution and Axe3 does not show such preference. Axe3 has a preference for large AcXOS (DP 9-12) when compared to smaller AcXOS (especially DP 4-7) while for Axe2 the size of the oligomer is irrelevant. Even though there is difference in substrate affinity towards acetylated xylooligosaccharides from Eucalyptus wood, the final hydrolysis products are the same for Axe2 and Axe3: xylo-oligosaccharides containing one acetyl group located at the non-reducing xylose residue remain as examined using MALDI-TOF MS, CE-LIF and the application of an endo-xylanase (GH 10).     

Resorbable and highly elastic block copolymers from 1,5-dioxepan-2-one and L-lactide with controlled tensile properties and hydrophilicity

New resorbable and elastomeric ABA tri- and multiblock copolymers have been successfully synthesized by combining ring-opening polymerization with ring-opening polycondensation. Five different poly(L-lactide-b-1,5-dioxepan-2-one-b-L-lactide) triblock copolymers and one new poly(L-lactide-b-1,5-dioxepan-2-one) multiblock copolymer have been synthesized. The triblock copolymers were obtained by ring-opening polymerization of 1,5-dioxepan-2-one (DXO) and L-lactide (LLA) with a cyclic tin initiator. The new multiblock copolymer was prepared by ring-opening polycondensation of a low molecular weight triblock copolymer with succinyl chloride. The molecular weight and the composition of the final copolymers were easily controlled by adjusting the monomer feed ratio, and all of the polymers obtained had a narrow molecular weight distribution. It was possible to tailor the hydrophilicity of the materials by changing the DXO content. Copolymers with a high DXO content had a more hydrophilic surface than those with a low DXO content. The receding contact angle varied from 27 to 44 degrees. The tensile properties of the copolymers were controlled by altering the PDXO block length. The tensile testing showed that all the polymers were very elastic and had very high elongations-at-break (epsilon(b)). The copolymers retained very good mechanical properties (epsilon(b) approximately 600-800% and sigma(b) approximately 8-20 MPa) throughout the in vitro degradation study (59 days).     

Theoretical investigation of hydrogen bonding effects on oxygen, nitrogen, and hydrogen chemical shielding and electric field gradient tensors of chitosan/HI salt

A density functional theory study has been carried out to calculate the (17)O, (15)N, (13)C, and (1)H chemical shielding as well as (17)O, (14)N, and (2)H electric field gradient tensors of chitosan/HI type I salt. These calculations were performed using the B3LYP functional and 6-311++G (d,p) and 6-31++G (d,p) basis sets. Calculated EFG and chemical shielding tensors were used to evaluate the (17)O, (14)N, and (2)H nuclear quadruple resonance, NQR, and (17)O, (15)N, (13)C, and (1)H nuclear magnetic resonance, NMR, parameters in the cluster model, which are in good agreement with the available experimental data. The difference in the isotropic shielding (sigma(iso)) and quadrupole coupling constant (C(Q)) between monomer and target molecule in the cluster was analyzed in detail. It was shown that both EFG and CS tensors are sensitive to hydrogen-bonding interactions, and calculating both tensors is an advantage. A different influence of various hydrogen bond types, N-Hcdots, three dots, centeredI, O-Hcdots, three dots, centeredI, and N-Hcdots, three dots, centeredO was observed on the calculated CS and EFG tensors. On the basis of this study, nitrogen and O-6 are the most important nuclei to confirm crystalline structure of chitosan/HI. These nuclei have large change in their CS and EFG tensors because of forming intermolecular hydrogen bonds. Moreover, the quantum chemical calculations indicated that the intermolecular hydrogen-bonding interactions play an essential role in determining the relative orientation of CS and EFG tensors of O-6 and nitrogen atoms in the molecular frame axes.     

The effects of transmitters on the affinity of the insoluble fraction of Ox brain for Na+ and K+

The insoluble fraction of ox-brain, which had previously been shown to have a non-linear affinity for Na⁺ and K⁺, was prepared. Acetylcholine (1×10^–8 mol/l and 1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero, while at 1×10^–6 mol/l, the affinity for the cations was almost as high as in the absence of the transmitter; the affinities for Na⁺ and K⁺ were particularly high, when the supernatant concentrations of these ions exceeded 80–100 mM. Addition of eserine (3×10^–5 mol/l) considerably modified the response of the fraction to acetylcholine (1×10^–5 mol/l). Atropine (1×10^–8 mol/l) in the absence or presence of acetylcholine (1×10^–5, or 1×10^–4 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Epinephrine (3×10^–10 mol/l) lowered the affinity for Na⁺ and K⁺, while ergotamine itself (1×10^–5 mol/l) reduced it to zero. The addition of both epinephrine and ergotamine at the latter concentrations restored the affinity of the fractions for Na⁺ and K⁺ to what it had been in the absence of the transmitter or antagonist, previously reported. Norepinephrine (3×10^–10 mol/l), or ouabain (1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Thus, the transmitters and antagonists altered the affinity of the insoluble fraction for Na⁺ and K⁺ nonlinearity, dependent upon their concentrations, the concentrations of the cations, and the interaction of transmitter and antagonist.     

Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P. ruminicola subsp. brevis) B(1)4

Freshly harvested whole cells from cultures of P. bryantii B(1)4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantii B(1)4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation with P. bryantii showed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens gave little increase in overall pentose utilization suggesting that external P. bryantii xylanases are as effective as the cloned R. flavefaciens enzymes in releasing products that can be utilised by P. bryantii cells. The xylanase system of P. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

Methionine Aminopeptidase II: A Molecular Chaperone for Sarcoplasmic Reticulum Calcium ATPase

The monoclonal antibody to the β-subunit of H⁺/K⁺-ATPase (mAbHKβ) cross-reacts with a protein that acts as a molecular chaperone for the structural maturation of sarcoplasmic reticulum (SR) Ca²⁺-ATPase. We partially purified a mAbHKβ-reactive 65-kDa protein from Xenopus ovary. After in-gel digestion and peptide sequencing, the 65-kDa protein was identified as methionine aminopeptidase II (MetAP2). The effects of MetAP2 on SR Ca²⁺-ATPase expression were examined by injecting the cRNA for MetAP2 into Xenopus oocytes. Immunoprecipitation and pulse-chase experiments showed that MetAP2 was transiently associated with the nascent SR Ca²⁺-ATPase. Synthesis of functional SR Ca²⁺-ATPase was facilitated by MetAP2 and prevented by injecting an antibody specific for MetAP2. These results suggest that MetAP2 acts as a molecular chaperone for SR Ca²⁺-ATPase synthesis.     

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

Inactivation of BK channels by the NH2 terminus of the beta2 auxiliary subunit: an essential role of a terminal peptide segment of three hydrophobic residues

An auxiliary beta2 subunit, when coexpressed with Slo alpha subunits, produces inactivation of the resulting large-conductance, Ca(2+) and voltage-dependent K(+) (BK-type) channels. Inactivation is mediated by the cytosolic NH(2) terminus of the beta2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH(2) terminus in the inactivation process. The beta2 NH(2) terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2-4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH(2)-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process.     

Copolymers from unsaturated macrolactones: toward the design of cross-linked biodegradable polyesters

The enzymatic synthesis of a series of random copolyesters by ring-opening polymerization of unsaturated macrolactones like globalide and ambrettolide with 1,5-dioxepan-2-one (DXO) and 4-methyl caprolactone (4MeCL) was investigated. (13)C NMR diad analysis confirmed the randomness of all copolymers irrespective of the comonomer ratios. Thermal investigation showed that incorporating the comonomers lowered the melting points of the polymers as compared with the macrolactone homopolymers. The decrease was dependent on the comonomer ratio. The unsaturated copolymers were thermally cross-linked using dicumyl peroxide, which resulted in completely amorphous insoluble networks. It was found that 10% incorporation of the unsaturated macolactone was sufficient to obtain a gel content of 95 wt %. Preliminary degradation tests confirm that the cross-linked copolymers are enzymatically degradable and that the incorporation of hydrophilic comonomers like DXO enhances degradation.     

1,3-Dideoxynojirimycin-3-yl glycosides of beta-(1-->3)- and beta-(1-->6)-linked gluco-oligosaccharides

Standard chemical methods involving the use of O-acetylated glycosyl trichloroacetimidates as glycosylating agents were used to prepare the five 1,3-dideoxynojirimycin-3-yl beta-(1-->3)-linked oligo-glucosides (1-5) and also the beta-(1-->6)-bonded glucobiose (gentiobiose)-based analogue 6 as potential fungicides. In the course of the work, the beta-(1-->6), beta-(1-->6)-linked analogue 8 of 6 and 6-O- and 4-O-beta-glucopyranosyl-deoxynojirimycins 7 and 9, respectively, were also produced.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Fragmentation pattern of underivatised xylo-oligosaccharides and their alditol derivatives by electrospray tandem mass spectrometry

Maltopentaose and olive pulp xylo-oligosaccharides and the correspondent alditol derivatives were analysed by ESI-MS and ESI-MS/MS. The ESI-MS spectrum of maltopentaose and maltopentaose alditols showed [M+Na]⁺and [M+H]⁺ ions. ESI-MS spectrum of xylo-oligosaccharides and their alditols showed [M+Na]⁺of neutral (Xyl_3–6) and acidic (Xyl_2–3MeGlcA and Xyl_2–3GlcA) xylo-oligosaccharides. The ESI-MS/MS spectra of maltopentaose and underivatised xylo-oligosaccharides presented fragments of glycosidic cleavages attributed to B/Z and C/Y ions. On the other hand, MS/MS spectra of the correspondent alditols showed glycosidic cleavages unambiguously identified as B-type and Y-type ions. Y-type fragment ions showed higher abundance in the MS/MS spectra of the alditol derivatives when compared to the non-reduced samples. The study of the oligoxylosyl alditols fragmentation permits to distinguish fragmentation pathways that occur both from the reducing end and from the non-reducing end of the xylan chain, allowing to obtain more information about the localization of the acidic substituent along the glucuronoxylan backbone.     

Solute carrier family 26 member a2 (Slc26a2) protein functions as an electroneutral SOFormula/OH-/Cl- exchanger regulated by extracellular Cl-

Slc26a2 is a ubiquitously expressed SO(4)(2-) transporter with high expression levels in cartilage and several epithelia. Mutations in SLC26A2 are associated with diastrophic dysplasia. The mechanism by which Slc26a2 transports SO(4)(2-) and the ion gradients that mediate SO(4)(2-) uptake are poorly understood. We report here that Slc26a2 functions as an SO(4)(2-)/2OH(-), SO(4)(2-)/2Cl(-), and SO(4)(2-)/OH(-)/Cl(-) exchanger, depending on the Cl(-) and OH(-) gradients. At inward Cl(-) and outward pH gradients (high Cl(-)(o) and low pH(o)) Slc26a2 functions primarily as an SO(4)(2-)(o)/2OH(-)(i) exchanger. At low Cl(-)(o) and high pH(o) Slc26a2 functions increasingly as an SO(4)(2-)(o)/2Cl(-)(i) exchanger. The reverse is observed for SO(4)(2-)(i)/2OH(-)(o) and SO(4)(2-)(i)/2Cl(-)(o) exchange. Slc26a2 also exchanges Cl(-) for I(-), Br(-), and NO(3)(-) and Cl(-)(o) competes with SO(4)(2-) on the transport site. Interestingly, Slc26a2 is regulated by an extracellular anion site, required to activate SO(4)(2-)(i)/2OH(-)(o) exchange. Slc26a2 can transport oxalate in exchange for OH(-) and/or Cl(-) with properties similar to SO(4)(2-) transport. Modeling of the Slc26a2 transmembrane domain (TMD) structure identified a conserved extracellular sequence (367)GFXXP(371) between TMD7 and TMD8 close to the conserved Glu(417) in the permeation pathway. Mutation of Glu(417) eliminated transport by Slc26a2, whereas mutation of Phe(368) increased the affinity for SO(4)(2-)(o) 8-fold while reducing the affinity for Cl(-)(o) 2 fold, but without affecting regulation by Cl(-)(o). These findings clarify the mechanism of net SO(4)(2-) transport and describe a novel regulation of Slc26a2 by an extracellular anion binding site and should help in further understanding aberrant SLC26A2 function in diastrophic dysplasia.