Transglutaminase-like activity participates in cell wall biogenesis in <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

Transglutaminases (TGases) catalyze the cross-linking between protein molecules by formation of an amide bond between γ-carboxyamide group of glutamine and the ε-amine group of lysine under deamination of glutamine. We have demonstrated the participation of transglutaminase-like activity in the isolated cell walls and in the process of cell wall regeneration in protoplasts of the yeast Saccharomyces cerevisiae. A radioactive TGase substrate [³H]putrescine was incorporated into the isolated cell walls and into the TCA-insoluble fraction in regenerating protoplasts. The incorporation was increased by adding exogenous artificial substrate of TGase N,N’-dimethylcasein and was inhibited by TGase inhibitor cystamine and/or EDTA. These results suggest the existence of a TGase-type reaction involved in the formation of covalent cross-links between glycoprotein molecules during cell wall construction in S. cerevisiae.     

Transposon display supports transpositional activity of <Emphasis Type=Italic>P</Emphasis> elements in species of the <Emphasis Type=Italic>saltans</Emphasis> group of <Emphasis Type=Italic>Drosophila</Emphasis>

Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances between strains regarding the insertion polymorphism profile were estimated. Amplification of the P element based on copy number estimates was highly variable among the strains (D. sturtevanti, canonical 20.11, O-type 9.00; D. saltans, canonical 16.4, O-type 12.60 insertions, on average). The larger values obtained by TD compared to our previous data by Southern blotting support the higher sensitivity of TD over Southern analysis for estimating transposable element copy numbers. The higher numbers of the canonical P element and the greater divergence in its distribution within the genome of D. sturtevanti (24.8%) compared to the O-type (16.7%), as well as the greater divergence in the distribution of the canonical P element, between the D. sturtevanti (24.8%) and the D. saltans (18.3%) strains, suggest that the canonical element occupies more sites within the D. sturtevanti genome, most probably due to recent transposition activity. These data corroborate the hypothesis that the O-type is the oldest subfamily of P elements in the saltans group and suggest that the canonical P element is or has been transpositionally active until more recently in D. sturtevanti.     

Attenuation of fibrosis <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> with <Emphasis Type=Italic>SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Cryopreservation of <Emphasis Type=Italic>Robinia</Emphasis><Emphasis Type=Italic>pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Stable coexistence of two <Emphasis Type=Italic>Caldicellulosiruptor</Emphasis> species in a <Emphasis Type=Italic>de novo</Emphasis> constructed hydrogen-producing co-culture

Background  

Mixed culture enrichments have been used frequently for biohydrogen production from different feedstock. In spite of the several advantages offered by those cultures, they suffer poor H₂ yield. Constructing defined co-cultures of known H₂ producers may offer a better performance than mixed-population enrichments, while overcoming some of the limitations of pure cultures based on synergies among the microorganisms involved.     

Probiotic <Emphasis Type=Italic>Lactobacillus</Emphasis> strains: <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type=Italic>Arabidopsis </Emphasis><Emphasis Type=Italic>thaliana</Emphasis> L. and <Emphasis Type=Italic>Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

Construction,expression and characterization of fusion enzyme from <Emphasis Type=Italic>Arthrobacter oxydans</Emphasis> dextranase and <Emphasis Type=Italic>Klebsiella pneumoniae</Emphasis> amylase

An artificial fusion protein of Arthrobacter oxydans dextranase and Klebsiella pneumoniae α-amylase was constructed and expressed in Escherichia coli. Most of the expressed protein existed as an insoluble fraction, which was solubilized with urea. The purified fusion enzyme electrophoretically migrated as a single protein band; M = 137 kDa, and exhibited activities of both dextranase (10.8 U mg⁻¹) and amylase (7.1 U mg⁻¹), which were lower than that of reference dextranase (13.3 U mg⁻¹) and α-amylase (103 U mg⁻¹). The fusion enzyme displayed bifunctional enzyme activity at pH 5–7 at 37°C. These attributes potentially make the fusion enzyme more convenient for use in sugar processing than a two-enzyme system.     

The isolation and analysis of a soybean <Emphasis Type=Italic>CO</Emphasis> Homologue <Emphasis Type=Italic>GmCOL10</Emphasis>

The CONSTANS (CO) gene is a key regulator of the response to photoperiod in the model plant Arabidopsis thaliana, and its homologues are present in many plant species. We describe here the isolation of the CO homologue for zinc finger protein gene GmCOL10 (Glycine max CONSTANS-Like 10) from the soybean cultivar Kennong18. Sequence comparisons showed that the closest A. thaliana gene to GmCOL10 is COL5. The expression of GmCOL10 was regulated in a circadian manner, especially under short-day conditions. The expression of GmCOL10 was concentrated in vegetative organs, and in particular in the unifoliolates and cotyledons. An analysis of subcellular localization found GmCOL10 in the nucleus. Our data suggested that GmCOL10 was not related to the photoperiodic pathway of floral transition as Arabidopsis CO does.     

Effect of abscisic acid and proline on <Emphasis Type=Italic>in vitro</Emphasis> flowering in <Emphasis Type=Italic>Vigna aconitifolia</Emphasis>

An experiment was taken up to find out possibilities of manipulating the in vitro flowering in moth bean. Abscisic acid (ABA) and proline both alone and in combination influenced days to flower induction, number of flowers per plant, number of pods per plant and seeds per pod. Frequency of flowering plants approached 100 % at 1 and 3 μM ABA and 800 μM proline. The range of flowering period (3 to 23.6 d) has also been influenced by various treatments.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

<Emphasis Type=Italic>Agrobacterium tumefaciens</Emphasis>-mediated transformation of oleaginous yeast <Emphasis Type=Italic>Lipomyces</Emphasis> species

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.     

<Emphasis Type=Italic>Boletus kermesinus</Emphasis>, a new species of <Emphasis Type=Italic>Boletus</Emphasis> section <Emphasis Type=Italic>Luridi</Emphasis> from central Honshu,Japan

Boletus kermesinus, a new species of Boletus section Luridi, is fully described and illustrated based on the materials collected in subalpine coniferous forests of central Honshu, Japan. It has distinctive features of dark-red basidiomata having distinct viscidity in the pileus surface, usually unchanging flesh, discolorous red pores, and an entirely reticulate stipe becoming coarsely lacerate-rimose with age.     

Interaction of <Emphasis Type=Italic>Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type=Italic>Campylobacter jejuni</Emphasis> and <Emphasis Type=Italic>Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Chemical composition of the essential oil from two wormwood species <Emphasis Type=Italic>Artemisia frigida</Emphasis> and <Emphasis Type=Italic>Artemisia argyrophylla</Emphasis>

The composition of the essential oil from the wormwood sage (Artemisia frigida Willd., Asteraceae) of populations growing in the Altai Territory, the Altai Republic, the Khakass Republic, the Tuva Republic, and the East-Kazakhstan region of the Republic of Kazakhstan and the representative species of the silver-leaved wormwood Artemisia argyrophylla Ledeb. growing in the Republic Altai has been studied by chromato-mass spectrometry. An analysis of 15 samples of the essential oil from A. frigida obtained over a period from 1999 to 2007 indicates that samples from different populations have similar sets of the main components: α-pinene (0.2–7.8%), camphene (1.9–5.8%), 1,8-cineole (8.9–33.8%), camphor (6.7–40.0%), borneol (3.9–12.3%), terpine-4-ol (1.5–6.5%), bornyl acetate (1.4–22.0%), and germacrene D (1.4–14.6%). Some samples contain substantial amounts of α- and β-thujones (in total up to 19.1%), which are completely absent in other samples. Some samples contain santolina alcohol (up to 13.8%) and its acetate (up to 4.8%). As differentiated from A. frigida, the essential oil of A. argyrophylla contains yomogi alcohol (1.2%), artemisia ketone (12.9%), artemisia alcohol (3.1%), artemisia alcohol acetate (3.9%), and small amounts of camphor (3.2%), borneol (0.3%), and bornyl acetate (0.2%).     

Four new species of <Emphasis Type=Italic>Crinipellis </Emphasis>and <Emphasis Type=Italic>Marasmius </Emphasis>in eastern Honshu,Japan

 Four new species of Crinipellis and Marasmius (Agaricales, Basidiomycetes) in eastern Honshu, Japan, are described and illustrated: (1) Crinipellis conchata sp. nov. (section Excentricinae), forming a conchate pileus and a strongly excentric, short stipe, was found on a dead twig of Trachelospermum asiaticum in Mt. Takao, Tokyo; (2) Marasmius funalis sp. nov. (section Androsacei), forming a densely white-hispid, dark brown stipe bearing numerous setiform caulocystidia, was found on a dead twig of Cryptomeria japonica or on leaf litter in Tokyo and Kanagawa; (3) Marasmius maculosus sp. nov. (section Sicci), having a relatively large, reddish-brown pileus distinctly mottled with pale colored spots and Siccus-type cheilocystidia and pileipellis cells with relatively long setulae, was found on leaf litter in the lowland forest of Kanagawa and Chiba; and (4) Marasmius sasicola sp. nov. (section Marasmius), having a small, plicate-sulcate pileus, a filiform, wiry, blackish stipe, collariate lamellae, and Siccus-type cheilocystidia and pileipellis elements, was found on fallen dead leaves of grass bamboo in Kanagawa. Received: January 30, 2002 / Accepted: May 24, 2002