Identification of the benzodiazepines as a new class of antileishmanial agent

The continual increase in drug resistance; the lack of new chemotherapeutic agents; the toxicity of existing agents and the increasing morbidity with HIV co-infection mean the search for new antileishmanial agents has never been more urgent. We have identified the benzodiazepines as a structural class for antileishmanial hit optimisation, and demonstrated that their in vitro activity is comparable with the clinically used drug, sodium stibogluconate, and that the compounds are not toxic to macrophages.     

Efficient surface patterning of oligonucleotides inside a glass capillary through oxime bond formation

The efficient surface patterning of oligonucleotides was accomplished onto the inner wall of fused-silica capillary tubes as well as on the surface of glass slides through oxime bond formation. The robustness of the method was demonstrated by achieving the surface immobilization of up to three different oligonucleotide sequences inside the same capillary tube. The method involves the preparation of surfaces grafted with reactive aminooxy functionalities masked with the photocleavable protecting group, 2-(2-nitrophenyl) propyloxycarbonyl group (NPPOC). Briefly, NPPOC-aminooxy silane 1 was prepared and used to silanize the glass surfaces. The NPPOC group was cleaved under brief irradiation to unmask the reactive aminooxy group on surfaces. These reactive aminooxy groups were allowed to react with aldehyde-containing oligonucleotides to achieve an efficient surface immobilization. The advantage associated with the present approach is that it combines the high-coupling efficiency of oxime bond formation with the convenience associated with the use of photolabile groups. The present strategy thus offers an alternative approach for the immobilization of biomolecules in the microchannels of "labs on a chip" devices.     

A novel cold-inducible expression system for Bacillus subtilis

Production of recombinant proteins at low temperatures is one strategy to prevent formation of protein aggregates and the use of an expensive inducer such as IPTG. We report on the construction of two expression vectors both containing the cold-inducible des promoter of Bacillus subtilis, where one allows intra- and the other extracellular synthesis of recombinant proteins. Production of recombinant proteins started within the first 30min after temperature downshock to 25 degrees C and continued for about 5h.     

One step engineering of T7-expression strains for protein production: increasing the host-range of the T7-expression system

The T7-expression system has been very useful for protein expression in Escherichia coli. However, it is often desirable to over-express proteins in species other than E. coli. Here, we constructed an inducible broad-host-range T7-expression transposon, which allows simple one-step construction of T7-expression strains in various species, providing the option to over-express proteins of interest in a broader host-range. This transposon contains the T7 RNA polymerase driven by the lacUV5 promoter, which is repressed by the lac-repressor. Leaky expression is prevented by the presence of T7-lysozyme on this construct. The complete T7-expression system is flanked by mariner transposon repeats of the suicidal R6Kgammaori plasmid, pBT20-Deltabla. Stable integration of the whole system is possible by a one-step selection for a Flp-excisable Gm(R)-marker. We showed the engineering of E. coli, Pseudomonas aeruginosa, Erwinia carotovora, Salmonella choleraesuis, Agrobacterium tumefaciens, and Chromobacterium violaceum strains with this construct and demonstrated the expression of the Burkholderia pseudomallei Asd protein in these hosts, by induction with isopropyl-beta-d-thiogalactopyranoside (IPTG).     

A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization

A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36G37, a conserved pentapeptide G103HSLG107 and a conserved catalytic His265 and Asp237. The EstR sequence shared 64-70 and 44-48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni-NTA-purified esterase EstR showed an optimal activity in the temperature range 60-65 degrees C and pH range 7.5-9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl fluoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.     

Association of the FADS2 gene with omega-6 and omega-3 PUFA Concentration in the egg yolk of Japanese quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low omega-6:omega-3 PUFA ratio (h2 = 0.36-0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the omega-6 and omega-3 PUFAs (P < 0.05). No effects of the other SNPs were found - indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to omega-6 and omega-3 PUFA concentration in the egg yolk.     

Amino acid and nucleotide sequence, adjacent genes, and heterologous expression of hiracin JM79, a sec-dependent bacteriocin produced by Enterococcus hirae DCH5, isolated from Mallard ducks (Anas platyrhynchos)

The primary structure of a bacteriocin produced by Enterococcus hirae DCH5 was determined by combined amino acid and DNA sequencing. Nucleotide analysis of a 2838-bp DNA fragment of E. hirae DCH5 revealed five putative ORFs. The first orf (hirJM79) encodes a 74-amino-acid peptide containing an N-terminal signal peptide of 30 amino acids, followed by the amino acid sequence of the mature bacteriocin, hiracin JM79 (HirJM79), of 44 amino acids. The second orf (hiriJM79) encodes the putative immunity protein of HirJM79. Contiguous ORFs encode a putative mobilization protein (orfC), a relaxase/mobilization nuclease domain (orfD), and a hypothetical protein (orfE). The production and functional expression of HirJM79 by heterologous hosts suggest that hirJM79 is the minimum requirement for production of biologically active HirJM79, that HirJM79 is most likely externalized by the general secretory pathway or sec-dependent pathway, and that HiriJM79 is the immunity protein for HirJM79.     

Trapping choline oxidase in a nonfunctional conformation by freezing at low pH

Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.     

Degradation of polyethylene succinate (PES) by a new thermophilic <Emphasis Type="Italic">Microbispora</Emphasis> strain

Thermophilic actinomycetes were isolated from sediment of the Chingshuei hot spring in north Taiwan, and the strain HS 45-1 was selected from colonies which formed distinct clear zones on agar plate with emulsified polyethylene succinate (PES). The film of PES disappeared within 6 days in liquid cultures at 50°C. The strain HS 45-1 was also able to degrade poly (ε-carpolactone) (PCL) and poly (3-hydroxybutyrate) (PHB) films completely within 6 days in liquid cultures. Basing on the results of phynotypic characteristics, phylogenetic studies and DNA-DNA hybridization, strain HS 45-1 should be assigned to Micorbispora rosea subsp. taiwanensis.     

Physiological versatility of the genus Rhodocista

A new purple bacterium (strain T4), capable of heterotrophic aerobic and phototrophic anaerobic growth, was isolated from waste water of a noodle factory near Hanoi, Vietnam. A comparison of 16S rDNA sequences revealed its association with the genus Rhodocista. The isolate, tentatively named "Rhodocista hanoiensis", forms cysts after growth on butyrate-containing plates at 42 degrees C. The vegetative cells form short (under aerobic conditions) or long curve-shaped rods. In contrast to other species of this genus T4 does not need any supplines (growth factors, not synthesized by the organisms). Comparative studies of T4 with Rhodocista centenaria (Rhodospirillum centenum) and Rhodocista pekingensis revealed a remarkable physiological versatility regarding nutrient spectra and survival properties of this genus.