Three new species of bipolar budding yeasts of the genus Hanseniaspora and its anamorph Kloeckera isolated in Thailand

In the course of a survey of yeast biodiversity in the natural substrates in Thailand, eight strains were found to represent three hitherto undescribed species of Hanseniaspora/Kloeckera . They were isolated from insect frass, flower, lichen, rotted fruit and rotted wood. Based on the morphological and physiological characteristics, and sequences of D1/D2 domain, six strains represent a single species of the genus Hanseniaspora , described as Hanseniaspora thailandica sp. nov. (type BCC 14938^T=NBRC 104216^T=CBS 10841^T), and another strain as Hanseniaspora singularis sp. nov. (type BCC 15001^T=NBRC 104214^T=CBS 10840^T). A further strain, which belongs to Kloeckera and does not produce ascospores, is described as Kloeckera hatyaiensis sp. nov. (type BCC 14939^T=NBRC 104215^T=CBS 10842^T). Strains belonging to H. thailandica sp. nov. differed by 17–19 nucleotide substitutions from Hanseniaspora meyeri , the closest species. DNA reassociation between the two taxa showed 30–48% relatedness. Kloeckera hatyaiensis sp. nov. and H. singularis sp. nov. differed by eight and 16 nucleotide substitutions with one gap from the nearest species, Hanseniaspora clermontiae and Hanseniaspora valbyensis , respectively.     

Optimized synthesis and antiproliferative activity of desTHPdactylolides

Dactylolide and certain analogues are attractive targets for study due to their structural resemblance to zampanolide, a very promising anticancer lead compound and a unique covalent-binding microtubule stabilizing agent. The primary goal of this project is identification and synthesis of simplified analogues of dactylolide that would be easier to prepare and could be investigated for antiproliferative activity in comparison with zampanolide. Extension of Almann’s concept of a simplified zampanolide analogue to dactylolide in the form of desTHPdactylolide was attractive not only for reasons of synthetic simplification but also for the prospect that analogues of dactylolide could be prepared in both (17S) and (17R) configurations. Since Altmann’s overall yield for the six-step procedure leading to the C9–C18 fragment of desTHPdactylolide was only 8.7%, a study focused on optimized synthesis and antiproliferative evaluation of each enantiomer of desTHPdactylolide was initiated using Altmann’s route as a framework. To this end, two optimized approaches to this fragment C9–C18 were successfully developed by us using allyl iodide or allyl tosylate as the starting material for a critical Williamson ether synthesis. Both (17S) and (17R) desTHPdactylolides were readily synthesized in our laboratory using optimized methods in yields of 37–43%. Antiproliferative activity of the pair of enantiomeric desTHPdactylolides, together with their analogues, was evaluated in three docetaxel-sensitive and two docetaxel-resistant prostate cancer cell models using a WST-1 cell proliferation assay. Surprisingly, (17R) desTHPdactylolide was identified as the eutomer in the prostate cancer cell models. It was found that (17S) and (17R) desTHPdactylolide exhibit equivalent antiproliferative potency towards both docetaxel-sensitive (PC-3 and DU145) and docetaxel-resistant prostate cancer cell lines (PC-3/DTX and DU145/DTX).     

Wax-incorporated Emulsion Gel Beads of Calcium Pectinate for Intragastric Floating Drug Delivery

The purpose of this study was to prepare wax-incorporated pectin-based emulsion gel beads using a modified emulsion-gelation method. The waxes in pectin–olive oil mixtures containing a model drug, metronidazole, were hot-melted, homogenized and then extruded into calcium chloride solution. The beads formed were separated, washed with distilled water and dried for 12 h. The influence of various types and amounts of wax on floating and drug release behavior of emulsion gel beads of calcium pectinate was investigated. The drug-loaded gel beads were found to float on simulated gastric fluid if the sufficient amount of oil was used. Incorporation of wax into the emulsion gel beads affected the drug release. Water-soluble wax (i.e. polyethylene glycol) increased the drug release while other water-insoluble waxes (i.e. glyceryl monostearate, stearyl alcohol, carnauba wax, spermaceti wax and white wax) significantly retarded the drug release. Different waxes had a slight effect on the drug release. However, the increased amount of incorporated wax in the formulations significantly sustained the drug release while the beads remained floating. The results suggest that wax-incorporated emulsion gel beads could be used as a carrier for intragastric floating drug delivery.     

Fed-batch fermentation of xylose by a fast-growing mutant of xylose-assimilating recombinant Saccharomyces cerevisiae

Mutants of xylose-assimilating recombinant Saccharomyces cerevisiae carrying the xylose reductase and xylitol dehydrogenase genes on plasmid pEXGD8 were selected, after ethyl methanesulfonate treatment, for their rapid growth on xylose medium. The fastest growing strain (strain IM2) showed a lower activity of xylose reductase but a higher ratio of xylitol dehydrogenase to xylose reductase activities than the parent strain, as well as high xylulokinase activity. Southern hybridization of the chromosomal DNA indicated that plasmid pEXGD8 was integrated into the chromosome of mutant IM2, resulting in an increase in the stability of the cloned genes. In batch fermentation under O₂ limitation, the yield and production rate of ethanol were improved 1.6 and 2.7 times, respectively, compared to the parent strain. In fed-batch culture with slow feeding of xylose and appropriate O₂ supply at a low level, xylitol excreted from the cells was limited and the ethanol yield increased 1.5 times over that in the batch culture, with a high initial concentration of xylose, although the production rate was reduced. The results suggested that slow conversion of xylose to xylitol led to a lower level of intracellular xylitol, resulting in less excretion of xylitol, and an increase in the ethanol yield. It was also observed that the oxidation of xylitol was strongly affected by the O₂ supply.Correspondence to: T. Yoshida     

Polyethylene Glycol on Stability of Chitosan Microparticulate Carrier for Protein

Stability enhancement of protein-loaded chitosan microparticles under storage was investigated. Chitosan glutamate at 35 kDa and bovine serum albumin as model protein drug were used in this study. The chitosan microparticles were prepared by ionotropic gelation, and polyethylene glycol 200 (PEG 200) was applied after the formation of the particles. All chitosan microparticles were kept at 25°C for 28 days. A comparison was made between those preparations with PEG 200 and without PEG 200. The changes in the physicochemical properties of the microparticles such as size, zeta potential, pH, and percent loading capacity were investigated after 0, 3, 7, 14, and 28 days of storage. It was found that the stability decreased upon storage and the aggregation of microparticles could be observed for both preparations. The reduction in the zeta potential and the increase in the pH, size, and loading capacity were observed when they were kept at a longer period. The significant change of those preparations without PEG 200 was evident after 7 days of storage whereas those with PEG 200 underwent smaller changes with enhanced stability after 28 days of storage. Therefore, this investigation gave valuable information on the stability enhancement of the microparticles. Hence, enhanced stability of chitosan glutamate microparticles for the delivery of protein could be achieved by the application of PEG 200.     

Antivibrio compounds produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. W3: characterisation and assessment of their safety to shrimps

In order to explore compounds naturallly inhibitory to shrimp pathogenic vibrios, a culture filtrate of Pseudomonas sp. W3 at a pH of 2 was extracted with ethyl acetate (EtOAc) to produce 82.15 mg/l of a yellow–brown extract (EtOAc-W3) that had MIC values of 225-450 μg/ml against the growth of 18 shrimp pathogenic Vibrio harveyi strains. The MIC of EtOAc-W3 against the most pathogenic strain PSU 2015 was 450 μg/ml and this strain had the lowest LD₅₀ (50% lethal dose) to pacific white shrimp (Litopenaeus vannamei, PL 21). At this MIC value, EtOAc-W3 in artificial sea water (ASW) killed strain PSU 2015; however in natural sea water, only a partial growth inhibition was observed. The toxicity to pacific white shrimp and antivibrio activity of the EtOAc-W3 were investigated by conducting an experiment with 4 sets; native control (commercial ASW), EtOAc-W3 control (MIC/10, 45 μg/ml), challenge (inoculation 6.0 × 10⁶ c.f.u./ml PSU 2015) and treatment (6.0 × 10⁶ c.f.u./ml PSU 2015 + 45 μg/ml EtOAc-W3). The same experiment was repeated by increasing the dose of EtOAc-W3 to 90 μg/ml (MIC/5). Both concentrations of EtOAc-W3 tested had no toxicity to postlarval shrimps. A significant decrease in shrimp mortality was observed over a 72 h period as approximately 80% of the shrimps died in each challenge set but only 63 and 23% died in the presence of 45 and 90 μg/ml EtOAc-W3. The major component of EtOAc-W3 was supposed to be 2-heptyl-4-quinolone (HHQ) by FAB-MS and ¹H-NMR analyses of the purified fraction.     

Two new anamorphic yeasts, Candida thailandica sp. nov. and Candida lignicola sp. nov., isolated from insect frass in Thailand

Two new yeast strains of the genus Candida were isolated from insect frass collected in Khao-Yai National Park, Nakhonrachasima, Thailand. Based on the morphological, physiological and chemotaxonomic characteristics, and sequence analysis of the D1/D2 domain of 26S rRNA gene, these two strains were found to represent two distinct undescribed species and were named Candida thailandica sp. nov. (ST-17 = BCC 7717(T) = NBRC 102562(T)=CBS 10 610) and Candida lignicola sp. nov. (ST-33 = BCC 7733(T) = NBRC 102564(T) = CBS 10612). In the D1/D2 domain of 26S rRNA gene, C. thailandica (GeneBank accession no. AY228491) differs from Candida tsuchiyae, the nearest species, in 66 nucleotide substitutions (10%) and C. lignicola (GeneBank accession no. AY845350) differs from Candida coipomoensis, the nearest species, in nine nucleotides (1.6%). These two new species are clearly distinguished from their closest species by the assimilation of several carbon compounds.     

Biodegradation of crude oil by soil microorganisms in the tropic

Five microorganisms, three bacteria and two yeasts, capable of degrading Tapis light crude oil were isolated from oil-contaminated soil in Bangkok, Thailand. Soil enrichment culture was done by inoculating the soil in mineral salt medium with 0.5% v/v Tapis crude oil as the sole carbon source. Crude oil biodegradation was measured by gas chromatography method. Five strains of pure microorganisms with petroleum degrading ability were isolated: three were bacteria and the other two were yeasts. Candida tropicalis strains 7Y and 15Y were identified as efficient oil degraders. Strain 15Y was more efficient, it was able to reduce 87.3% of the total petroleum or 99.6% of n-alkanes within the 7-day incubation period at room temperature of 25 ± 2 °C.     

Control of aggression and dominance in white-throated sparrows by testosterone and its metabolites

In three experiments, we investigated whether testosterone itself or its metabolites activate aggression and dominance in white-throated sparrows Zonotrichia albicollis. Groups of five to six sparrows, each treated with a different steroid implanted subcutaneously, were observed in outdoor aviaries during late winter to determine the birds' rates of aggression (supplantations and attacks scaled to the number of available subordinates) and dominance rankings with opponents not previously encountered. In Experiment 1, testosterone (T) had a greater effect on aggression and dominance than did androstenedione, 5α-dihydrotestosterone (D), androsterone, or estradiol (E). In Experiment 2, birds with T or D + E had higher aggression scores and dominance ranks than birds with either D or E alone. Birds with T and D + E did not differ. The testosterone metabolites, D and E, thus acted synergistically to determine rates of aggression and dominance ranks. To corroborate these results, in Experiment 3 we treated T-implanted birds with the following blocking agents: ATD, expected to reduce conversion of T to E (AT birds); progesterone, expected to reduce conversion of T to D (PT birds); or both (APT birds). The APT birds had lower aggression scores and dominance ranks than did AT or PT birds, despite having higher mean levels of circulating T than AT or PT birds or birds implanted with T alone. Cyproterone acetate also reduced aggression scores and dominance in T-implanted birds. We conclude that the hormonal control of aggression and dominance in these birds requires conversion of testosterone to both androgenic and estrogenic metabolites.