Pichia koratensis sp. nov., a new ascomycetous yeast related to Pichia acaciae isolated from insect frass in Thailand

Two yeast strains (ST-235 and ST-237) isolated from insect frass collected in northeastern Thailand, were identified as Pichia acaciae and a hitherto undescribed species based on the sequence analysis of the D1/D2 domain of 26S rDNA, ITS regions, and DNA-DNA reassociation experiments. The latter strain, ST-237 (=BCC 11769=NBRC 103638=JCM 12576), was described as Pichia koratensis sp. nov. Pichia koratensis is closely related to Pichia acaciae in the D1/D2 domain sequence of 26S rDNA and phenotypical characteristics but differs by 5 nucleotides (0.9%) from the type strain of P. acaciae and is clearly distinguished by the assimilation and fermentation of sucrose.     

Revealing differentially expressed proteins in two morphological forms of <Emphasis Type="Italic">Spirulina platensis</Emphasis> by proteomic analysis

Spirulina is distinguished from other cyanobacteria by its spiral morphology; however, this cyanobacterium has frequently been observed with a linear morphology in laboratory and industrial conditions. In our laboratory conditions, the simultaneously presence of the linear and spiral forms has also been observed. In the present study, the two forms of S. platensis C1 were separated and grown as axenic cultures in order to study the proteins that were differentially expressed in the soluble and insoluble protein fractions of the spiral and the linear forms. Two dimensional-differential gel electrophoresis (2D-DIGE) was performed to separate differentially expressed proteins that were subsequently identified by mass spectrometry. The differentially expressed proteins suggested two points. First, the morphological change is possibly induced by various environmental stresses such as oxygen level, carbon dioxide level, nutrient availability, and light. Second, the change of cell-shape might be a result of the change in a cell shape determination mechanism. Thus, this study is the first to show evidence at the protein level that may explain this morphological transformation in Spirulina.     

Revealing the complementation of ferredoxin by cytochrome b 5 in the Spirulina-∆6-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b 5 domain and Spirulina-∆6-acyl-lipid desaturase

Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b ₅. The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-∆⁶ desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-∆⁶ desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b ₅ domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-∆⁶ desaturase fused or co-expressed with cytochrome b ₅ in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b ₅. Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b ₅ domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b ₅ domain in the desaturation process of Spirulina-∆⁶ desaturase. Furthermore, the free form of cytochrome b ₅ domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.     

Production of red pigments by the insect pathogenic fungus Cordyceps unilateralis BCC 1869

Production of red pigments (naphthoquinones) by the insect pathogenic fungus Cordyceps unilateralis BCC 1869 was investigated in this study. Cultivation conditions, including temperature, intitial pH of medium, and aeration, were optimised to improve the yield of total naphthoquinones in shake-flask culture of C. unilateralis. The highest yield of total naphthoquinones (3 g L^–1) was obtained from a 28-day culture grown in potato dextrose broth with an initial pH of 7.0, at 28°C with shaking-induced aeration at 200 rpm. An extraction process for isolation of the targeted naphthoquinone, 3,5,8-trihydroxy-6-methoxy-2-(5-oxohexa-1,3-dienyl)-1,4-naphthoquinone (3,5,8-TMON), from a culture of C. unilateralis, was also developed. The yield of 3,5,8-TMON obtained was about 1.2 g L^–1 or 40% of total naphthoquinones. The stability of 3,5,8-TMON was very high, even upon exposure to strong sunlight (70,000 lx), high temperature up to 200°C, and acid and alkali solutions at concentrations of 0.1 M     

A mixture theory analysis for passive transport in osmotic loading of cells

The theory of mixtures is applied to the analysis of the passive response of cells to osmotic loading with neutrally charged solutes. The formulation, which is derived for multiple solute species, incorporates partition coefficients for the solutes in the cytoplasm relative to the external solution, and accounts for cell membrane tension. The mixture formulation provides an explicit dependence of the hydraulic conductivity of the cell membrane on the concentration of permeating solutes. The resulting equations are shown to reduce to the classical equations of Kedem and Katchalsky in the limit when the membrane tension is equal to zero and the solute partition coefficient in the cytoplasm is equal to unity. Numerical simulations demonstrate that the concentration-dependence of the hydraulic conductivity is not negligible; the volume response to osmotic loading is very sensitive to the partition coefficient of the solute in the cytoplasm, which controls the magnitude of cell volume recovery; and the volume response is sensitive to the magnitude of cell membrane tension. Deviations of the Boyle-van't Hoff response from a straight line under hypo-osmotic loading may be indicative of cell membrane tension.     

Targeted mutagenesis of a fatty acid Delta6-desaturase from Mucor rouxii: role of amino acid residues adjacent to histidine-rich motif II

The amino acid residues serine at position 213 (S213) and lysine at position 218 (K218), which are present in close proximity to the histidine-rich motif II of Mucor rouxii fatty acid Delta(6)-desaturase isoform II, were targeted for studying structure-function relationships using site-directed mutagenesis. The mutants were functionally characterized in a heterologous host, Saccharomyces cerevisiae. Substrate specificity and preference studies revealed that S213 and K218 are involved in substrate recognition. K218 plays a role in substrate preference by involvement in the binding of substrates, particularly C15-C18 monoene fatty acids. Modification of the M. rouxii Delta(6)-desaturase therefore has potential in specifically altering substrate utilization for production of desired fatty acids.     

Design and synthesis of thiourea based receptor containing naphthalene as oxalate selective sensor

The thiourea based receptor containing naphthalene groups (1), has been successfully designed and synthesized for application as an oxalate receptor. A density functional theory at B3LYP/6-31G(d,p) level of theory has been applied to predict the binding ability between 1 and selected anions, i.e., oxalate, malonate, succinate, glutarate, dihydrogen phosphate, and hydrogen sulphate. Calculation results point out that receptor 1 shows the strongest interaction to oxalate ion with the binding free energy of 172.48 kcal mol⁻¹. The recognition ability of 1 to the selected anions has been also investigated by means of the absorption and emission techniques. Experimental results are in excellent agreement with the calculation data in which receptor 1 shows highly selective for oxalate ion over the other anions with logβ of 3.82 (0.02) M⁻¹ by means of the size of binding cavity.      

Visualization of neuropeptides in paraffin-embedded tissue sections of the central nervous system in the decapod crustacean, Penaeus monodon, by imaging mass spectrometry

The distributions of neuropeptides in paraffin-embedded tissue sections (PETS) of the eyestalk, brain, and thoracic ganglia of the shrimp Penaeus monodon were visualized by imaging mass spectrometry (IMS). Peptide signals were obtained from PETS without affecting morphological features. Twenty-nine neuropeptides comprising members of FMRFamide, SIFamides, crustacean hyperglycaemic hormone, orcokinin-related peptides, tachykinin-related peptides, and allatostatin A were detected and visualized. Among these findings we first identified tachykinin-related peptide as a novel neuropeptide in this shrimp species. We found that these neuropeptides were distributed at specific areas in the three neural organs. In addition, 28 peptide sequences derived from 4 types of constitutive proteins, including actin, histones, arginine kinase, and cyclophilin A were also detected. All peptide sequences were verified by liquid chromatography-tandem mass spectrometry. The use of IMS on acetic acid-treated PETS enabled us to identify peptides and obtain their specific localizations in correlation with the undisturbed histological structure of the tissue samples.     

Draft genome sequence of Arthrospira platensis C1 (PCC9438)

Arthrospira platensis is a cyanobacterium that is extensively cultivated outdoors on a large commercial scale for consumption as a food for humans and animals. It can be grown in monoculture under highly alkaline conditions, making it attractive for industrial production. Here we describe the complete genome sequence of A. platensis C1 strain and its annotation. The A. platensis C1 genome contains 6,089,210 bp including 6,108 protein-coding genes and 45 RNA genes, and no plasmids. The genome information has been used for further comparative analysis, particularly of metabolic pathways, photosynthetic efficiency and barriers to gene transfer.