Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of genome fragments

Within a phylum Crenarchaeota, only some members of the hyperthermophilic class Thermoprotei, have been cultivated and characterized. In this study, we have constructed a metagenomic library from a microbial mat formation in a subsurface hot water stream of the Hishikari gold mine, Japan, and sequenced genome fragments of two different phylogroups of uncultivated thermophilic Crenarchaeota: (i) hot water crenarchaeotic group (HWCG) I (41.2 kb), and (ii) HWCG III (49.3 kb). The genome fragment of HWCG I contained a 16S rRNA gene, two tRNA genes and 35 genes encoding proteins but no 23S rRNA gene. Among the genes encoding proteins, several genes for putative aerobic-type carbon monoxide dehydrogenase represented a potential clue with regard to the yet unknown metabolism of HWCG I Archaea. The genome fragment of HWCG III contained a 16S/23S rRNA operon and 44 genes encoding proteins. In the 23S rRNA gene, we detected a homing-endonuclease encoding a group I intron similar to those detected in hyperthermophilic Crenarchaeota and Bacteria, as well as eukaryotic organelles. The reconstructed phylogenetic tree based on the 23S rRNA gene sequence reinforced the intermediate phylogenetic affiliation of HWCG III bridging the hyperthermophilic and non-thermophilic uncultivated Crenarchaeota.     

Anti-inflammatory cyathane diterpenoids from Sarcodon scabrosus

Four novel diterpenoids were isolated from the fruiting bodies of Sarcodon scabrosus (Fr.) Karst. (Boraginaceae) together with neosarcodonin A. One of the novel compounds was elucidated to be a cyathane diterpenoid, namely neosarcodonin O, by its spectral data. The others were characterized as 19-O-linoleoyl, 19-O-oleoyl and 19-O-stearoyl derivatives of sarcodonin A, after comparison with the authentic samples synthetically prepared from sarcodonin A. These compounds, together with the five 19-O-acyl derivatives synthesized from sarcodonin A, each exhibited inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation on mouse ears by topical application.     

HST-1/FGF-4 protects male germ cells from apoptosis under heat-stress condition

Apoptosis plays an important role in controlling the number of male germ cells and eliminating defective germ cells during testicular development and spermatogenesis. We show here that fibroblast growth factor-4 (HST-1/FGF-4) may play a critical role as a survival factor for germ cells, protecting them from apoptosis. Testes of adult male mice that received an adenovirus carrying human HST-1/FGF-4 (AxHST-1) or a control adenovirus (AxCAwt) were exposed to mild hyperthermia, which causes germ cell apoptosis. An in situ terminal-deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling (TUNEL) assay characterized germ cell apoptosis. The results indicated that HST-1/FGF-4 significantly reduced the apoptotic death of germ cells and prevented testicular weight loss and sperm count reduction. We also found that Hst-1/Fgf-4 present in testes is up-regulated in vivo when the testes are exposed to mild hyperthermia, and that endogenous Hst-1/Fgf-4 mRNA expression in Sertoli cells are also induced when the cells are exposed to mild hyperthermia in vitro. In addition, the MAPK cascade, which could increase an FGF-dependent survival signal, is activated by HST-1/FGF-4 stimuli in germ cells. On the other hand, upon HST-1/FGF-4 stimulation, lactate production from Sertoli cells were induced, which is indispensable nutrient for germ cell survival. These results suggest that HST-1/FGF-4 can act as an important physiological anti-apoptotic factor for male germ cells in stimulating lactate production of Sertoli cells upon heat stress, thereby promoting germ cell survival.     

Phosphatidylglycerol requirement for the function of electron acceptor plastoquinone Q(B) in the photosystem II reaction center

Phosphatidylglycerol (PG), a ubiquitous constituent of thylakoid membranes of chloroplasts and cyanobacteria, is demonstrated to be essential for the functionality of plastoquinone electron acceptor Q(B) in the photosystem II reaction center of oxygenic photosynthesis. Growth of the pgsA mutant cells of Synechocystis sp. PCC6803 that are defective in phosphatidylglycerolphosphate synthase and are incapable of synthesizing PG, in a medium without PG, resulted in a 90% decrease in PG content and a 50% loss of photosynthetic oxygen-evolving activity as reported [Hagio, M., Gombos, Z., Várkonyi, Z., Masamoto, K., Sato, N., Tsuzuki, M., and Wada, H. (2000) Plant Physiol. 124, 795-804]. We have studied each step of the electron transport in photosystem II of the pgsA mutant to clarify the functional site of PG. Accumulation of Q(A)(-) was indicated by the fast rise of chlorophyll fluorescence yield under continuous and flash illumination. Oxidation of Q(A)(-) by Q(B) plastoquinone was shown to become slow, and Q(A)(-) reoxidation required a few seconds when measured by double flash fluorescence measurements. Thermoluminescence measurements further indicated the accumulation of the S(2)Q(A)(-) state but not of the S(2)Q(B)(-) state following the PG deprivation. These results suggest that the function of Q(B) plastoquinone was inactivated by the PG deprivation. We assume that PG is an indispensable component of the photosystem II reaction center complex to maintain the structural integrity of the Q(B)-binding site. These findings provide the first clear identification of a specific functional site of PG in the photosynthetic reaction center.     

Psathyrella velutina Mushroom Lectin Exhibits High Affinity toward Sialoglycoproteins Possessing Terminal N-Acetylneuraminic Acid alpha 2,3-Linked to Penultimate Galactose Residues of Trisialyl N-Glycans. Comparison with other sialic acid-specific lectins

A lectin from the fruiting body of the Psathyrella velutina mushroom (PVL) was found to bind specifically to N-acetylneuraminic acid, as well as to GlcNAc (Ueda, H., Kojima, K., Saitoh, T., and Ogawa, H. (1999) FEBS Lett. 448, 75-80). In this study, the glycan sequences that PVL recognizes with high affinity on sialoglycoproteins were revealed. Among sialic acid-specific lectins only PVL could reveal the sialylated N-acetyllactosamine structure of glycoproteins in blotting studies, based on the dual specificity. The affinity of PVL to fetuin was measured by surface plasmon resonance to be 10(7) m(-1), which is an order of magnitude higher than those of Sambucus nigra agglutinin and Maackia amurensis mitogen, whereas affinity to asialofetuin was approximately 0 and to asialo-agalactofetuin was 10(8) m(-1), suggesting that PVL exhibits remarkably high affinities toward glycoproteins possessing trisialo- or GlcNAc-exposed glycans. Transferrin was separated into fractions that correspond to the sialylation states on an immobilized PVL column. Transferrin-possessing trisialoglycans containing alpha2,3-linked N-acetylneuraminic acid on the beta1,4-linked GlcNAc branch bound to the PVL column and eluted with GlcNAc; those containing only alpha2,6-linked sialic acids were retarded, whereas other transferrin fractions passed through the column. These results indicate that PVL is a lectin with potential for separation and detection of sialoglycoproteins because of its dual specificity toward sialoglycans and GlcNAc exposed glycans.     

Production of fertile transgenic barley (Hordeum vulgare L.) plant using the hygromycin-resistance marker

Fertile transgenic barley (Hordeum vulgare L.) plants were obtained by high velocity particle bombardment. The plasmid pBCl was used to deliver the selectable hph gene and reporter Gus gene into immature embryo. After the selection culture 18 hygromycin resistant plants were obtained. Samples for Southern hybridization and enzymatic Gus assay were obtained from 11 plants. Southern hybridization confirmed the presence of the hph gene in the 11 hygromycin resistant plants(T₀). Enzymatic assay indicated that all the t₀ plants that showed hph positive in Southern analysis possessed detectable amount of Gus activity. To date all the 11 t₀ plants reached maturity and mature seeds were obtained Transmission of the hph gene to progeny(T₁) of two independent t₀ plants was confirmed by Southern hybridization.Abbreviations Adh Alcohol Dehydrogenase - BA 6-Benzylaminopurine - cv cultivar - 2,4-D 2,4-Dichlorophenoxyacetic Acid - Gus -Glucuronidase - hph Hygromycin Phosphotransferase - 4MU 4-Methyl-umbelliferone     

Determination of oral mucosal Poisson’s ratio and coefficient of friction from in-vivo contact pressure measurements

Despite their considerable importance to biomechanics, there are no existing methods available to directly measure apparent Poisson’s ratio and friction coefficient of oral mucosa. This study aimed to develop an inverse procedure to determine these two biomechanical parameters by utilizing in vivo experiment of contact pressure between partial denture and beneath mucosa through nonlinear finite element (FE) analysis and surrogate response surface (RS) modelling technique. First, the in vivo denture–mucosa contact pressure was measured by a tactile electronic sensing sheet. Second, a 3D FE model was constructed based on the patient CT images. Third, a range of apparent Poisson’s ratios and the coefficients of friction from literature was considered as the design variables in a series of FE runs for constructing a RS surrogate model. Finally, the discrepancy between computed in silico and measured in vivo results was minimized to identify the best matching Poisson’s ratio and coefficient of friction. The established non-invasive methodology was demonstrated effective to identify such biomechanical parameters of oral mucosa and can be potentially used for determining the biomaterial properties of other soft biological tissues.     

Molecular Cloning of PbSTKL1 Gene from Plasmodiophora brassicae Expressed during Clubroot Development

Infection of crucifers by the obligate plant pathogen Plasmodiophora brassicae Woron. results in the formation of clubroot disease in these plants. Plasmodiophora brassicae gene expression during disease development was studied by differential display analysis of total RNA extracted from the roots of Chinese cabbage inoculated with the pathogen. In a series of experiments, 30 differentially expressed bands of cDNA were detected, and the expression of clone no. 17 was confirmed in clubbed roots. Southern blot analysis showed that this clone was a single‐copy gene in the P. brassicae genome. Putative amino acid sequence analysis of the full‐length cDNA of clone no. 17 (4.6 kb, designated PbSTKL1) revealed a serine/threonine kinase‐like domain at the C‐terminal region and a coiled‐coil structure in the middle region of the putative protein. PbSTKL1 expression increased strongly beginning 30 days after inoculation and was coincident with resting spore formation.