Preparative route to N-glycolylneuraminic acid phenyl 2-thioglycoside donor and synthesis of Neu5Gc-alpha-(2-->3')-lactosamine 3-aminopropyl glycoside

The spacer-armed trisaccharide, Neu5Gc-alpha-(2-->3')-lactosamine 3-aminopropyl glycoside, was synthesized by regio- and stereoselective sialylation of the suitably protected triol acceptor, 3-trifluoroacetamidopropyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-(6-O-benzyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside, with the donor methyl [phenyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha,beta-D-galacto-2-nonulopyranosid]onate. The donor was obtained, in turn, from methyl [phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha,beta-D-galacto-2-nonulopyranosid]onate by N-tert-butoxycarbonylation of the acetamido group followed by total N- and O-deacetylation, per-O-acetylation, subsequent Boc group removal, and N-acetoxyacetylation.     

Structural insight into the molecular basis of polyextremophilicity of short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus

Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme – half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous “resilience” of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.     

Temporal Constraints of Behavioral Inhibition: Relevance of Inter-stimulus Interval in a Go-Nogo Task

The capacity to inhibit prepotent and automatic responses is crucial for proper cognitive and social development, and inhibitory impairments have been considered to be key for some neuropsychiatric conditions. One of the most used paradigms to analyze inhibitory processes is the Go-Nogo task (GNG). This task has been widely used in psychophysical and cognitive EEG studies, and more recently in paradigms using fMRI. However, a technical limitation is that the time resolution of fMRI is poorer than that of the EEG technique. In order to compensate for these temporal constraints, it has become common practice in the fMRI field to use longer inter-stimulus intervals (ISI) than those used in EEG protocols. Despite the noticeable temporal differences between these two techniques, it is currently assumed that both approaches assess similar inhibitory processes. We performed an EEG study using a GNG task with both short ISI (fast-condition, FC, as in EEG protocols) and long ISI (slow-condition, SC, as in fMRI protocols). We found that in the FC there was a stronger Nogo-N2 effect than in the SC. Moreover, in the FC, but not in the SC, the number of preceding Go trials correlated positively with the Nogo-P3 amplitude and with the Go trial reaction time; and negatively with commission errors. In addition, we found significant topographical differences for the Go-P3 elicited in FC and SC, which is interpreted in terms of different neurotransmitter dynamics. Taken together, our results provide evidence that frequency of stimulus presentation in the GNG task strongly modulates the behavioral response and the evoked EEG activity. Therefore, it is likely that short-ISI EEG protocols and long-ISI fMRI protocols do not assess equivalent inhibitory processes.     

The influence of the electric diffusion potential on delayed fluorescence light curves of chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea

The potassium salt-induced transient increase of delayed fluorescence yield was studied in pea chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea.A simple kinetic model is proposed to account for the actinic light intensity dependence of the delayed fluorescence enhancement by the transmembrane diffusion potential induced by sudden salt addition. The electric field dependence of the rate constants for the recombination of primary separated charges with and without subsequent electronic excitation of reaction center chlorophyll was obtained.From the value of enhancement of delayed fluorescence by salt concentration gradients at saturating actinic light intensity, it is concluded that the distance, normal to thylakoid membrane surface, between the primary acceptor and the donor of Photosystem II is smaller than the membrane thickness.     

Improved linkage map and thirty new microsatellite markers for rat Chromosome 10

An improved linkage map for rat Chromosome (Chr) 10 with two F₂ populations was constructed. Thirty new microsatellite markers were generated from a Chr 10-specific, small-insert genomic library and mapped to rat Chr 10. Among them were the rat homologs for the mouse gene for light and heavy chains of myeloperoxidase and human neurofibromatosis 1. Eight newly generated markers (D10Mco62, D10Mco63, D10Mco64, D10Mco65, D10Mco67, D10Mco68, D10Mco70, and D10Mco74) were mapped to the region of the rat Chr 10 blood pressure QTL. The availability of such markers may be instrumental in the search for genes responsible for the hypertension. Received: 13 July 1998 / Accepted: 9 September 1998     

A halotolerant and thermotolerant <Emphasis Type="Italic">Bacillus</Emphasis> sp. degrades hydrocarbons and produces tensio-active emulsifying agent

A Bacillus sp. strain DHT, isolated from oil-contaminated soil, grew and produced biosurfactant when cultured in variety of substrate at salinities of up to 100 g l⁻¹ and temperatures up to 45°C. It was capable of utilizing crude oil, fuels, various pure alkanes and PAHs as a sole carbon and energy source across a wide range of temperature and salinity. Over the range evaluated, the degradation of hydrocarbon and biosurfactant production was not influenced by salinity (0–10% wv⁻¹) and temperature (30–45°C). The biosurfactant produced by the organism emulsified a range of hydrocarbons with hexadecane as the best substrate and toluene as the poorest. From 16S rDNA analysis, strain DHT was related to Bacillus licheniformis.     

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.     

Cutting Edge: Differential inhibition of TLR signaling pathways by cell-permeable peptides representing BB loops of TLRs

We designed cell-penetrating peptides comprised of the translocating segment of Drosophila antennapedia homeodomain fused with BB loop sequences of TLR2, TLR4, and TLR1/6. TLR2- and TLR4-BB peptides (BBPs) inhibited NF-kappaB translocation and early IL-1beta mRNA expression induced by LPS, and the lipopeptides S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys(4)-OH (P3C) and S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-Cys-Ser-Lys(4)-OH (P2C). TLR4- and TLR2-BBPs also strongly inhibited LPS-induced activation of ERK. Only TLR2-BBP significantly inhibited ERK activation induced by P3C, which acts via TLR2/1 heterodimers. BBPs did not inhibit activation of ERK induced by P2C, a TLR2/6 agonist. The TLR2-BBP induced weak activation of p38, but not ERK or cytokine mRNA. The TLR1/6-BBP failed to inhibit NF-kappaB or MAPK activation induced by any agonist. Our results suggest that the receptor BBPs selectively affect different TLR signaling pathways, and that the BB loops of TLR1/6 and TLR2 play distinct roles in formation of receptor heterodimers and recruitment of adaptor proteins.