Diversity and similarity of microbial communities in petroleum crude oils produced in Asia

To understand microbial communities in petroleum crude oils, we precipitated DNA using high concentrations of 2,2,4-trimethylpentane (isooctane) and purified. Samples of DNA from five crude oils, (Middle East, 3; China, 1; and Japan, 1) were characterized based upon their 16S rRNA gene sequences after PCR amplification and the construction of clone libraries. We detected 48 eubacterial species, one cyanobacterium, and one archaeon in total. The microbial constituents were diverse in the DNA samples. Most of the bacteria affiliated with the sequences of the three oils from the Middle East comprised similar mesophilic species. Acinetobacter, Propionibacterium, Sphingobium and a Bacillales were common. In contrast, the bacterial communities in Japanese and Chinese samples were unique. Thermophilic Petrotoga-like bacteria (11%) and several anaerobic-thermophilic Clostridia- and Synergistetes-like bacteria (20%) were detected in the Chinese sample. Different thermophiles (12%) and Clostridia (2%) were detected in the Japanese sample.     

Overproduction of Escherichia coli DNA polymerase DinB (Pol IV) inhibits replication fork progression and is lethal

Escherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We suggest that DinB targets Pol III, thereby acting as a brake on replication fork progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication to safeguard genome stability.     

Characterization of two different endo-alpha-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens

Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between alpha-GalNAc at the reducing end of mucin-type sugar chains and serine/threonine of proteins to release oligosaccharides. Previously, we identified the gene engBF encoding endo-alpha-GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Gal beta 1-3GalNAc (Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. 2005. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 280:37415-37422). Here we cloned a similar gene named engCP from Clostridium perfringens, a pathogenic enterobacterium, and characterized the gene product EngCP. Detailed analyses on substrate specificities of EngCP and EngBF using a series of p-nitrophenyl-alpha-glycosides chemically synthesized by the di-tert-butylsilylene-directed method revealed that both enzymes released Hex/HexNAc beta 1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Gal beta 1-3(GlcNAc beta 1-6)GalNAc, core 8 disaccharide Gal alpha 1-3GalNAc, and monosaccharide GalNAc. Our results suggest that EngCP possesses broader substrate specificity than EngBF. Actions of the two enzymes on native glycoproteins and cell surface glycoproteins were also investigated.     

Adsorption of gold(III), platinum(IV) and palladium(II) onto glycine modified crosslinked chitosan resin

The adsorption of Au(III), Pt(IV) and Pd(II) onto glycine modified crosslinked chitosan resin (GMCCR) has been investigated. The parameters studied include the effects of pH, contact time, ionic strength and the initial metal ion concentrations by batch method. The optimal pH for the adsorption of Au(III), Pt(IV) and Pd(II) was found to range from 1.0 to 4.0 and the maximum uptake was obtained at pH 2.0 for Au(III), Pt(IV) and Pd(II). The results obtained from equilibrium adsorption studies are fitted in various adsorption models such as Langmuir and Freundlich and the model parameters have been evaluated. The maximum adsorption capacity of GMCCR for Au(III), Pt(IV) and Pd(II) was found to be 169.98, 122.47 and 120.39mg/g, respectively. The kinetic data was tested using pseudo-first-order and pseudo-second-order kinetic models and an intraparticle diffusion model. The correlation results suggested that the pseudo-second-order model was the best choice among all the kinetic models to describe the adsorption behavior of Au(III), Pt(IV) and Pd(II) onto GMCCR. Various concentrations of HCl, thiourea and thiourea-HCl solutions were used to desorb the adsorbed precious metal ions from GMCCR. It was found that 0.7M thiourea-2M HCl solution provided effectiveness of the desorption of Au(III), Pt(IV) and Pd(II) from GMCCR. The modification of glycine on crosslinked chitosan resin (CCR) was studied by Fourier transform infrared spectrometry (FTIR) and scanning electron microscopy (SEM).     

Novel echinocandin antifungals. Part 1: novel side-chain analogs of the natural product FR901379

A series of novel acylated analogs of the novel water-soluble echinocandin FR901379 have been prepared and evaluated for antifungal and hemolytic activity. A relationship between antifungal activity and lipophilicity of the acyl side chain, expressed as ClogP was demonstrated, and an analog (3c) with 5.5- to 8-fold superior in vivo activity relative to the previously disclosed 4-(n-octyloxy)benzoyl side chain analog, FR131535 obtained.     

Arabidopsis VPS35, a retromer component, is required for vacuolar protein sorting and involved in plant growth and leaf senescence

The retromer complex is responsible for retrograde transport,which is coordinated with anterograde transport in the secretorypathway including vacuolar protein sorting. Yeast VPS35 is acomponent of the retromer complex that is essential for recognitionof specific cargo molecules. The physiological function of VPS35has not been determined in vacuolar protein sorting in higherorganisms. Arabidopsis thaliana has three VPS35 homologs designatedVPS35a, VPS35b and VPS35c. We isolated four vps35 mutants (vps35a-1,vps35b-1, vps35b-2 and vps35c-1) and then generated four doublemutants and one triple mutant. vps35a-1 vps35c-1 exhibited nounusual phenotypes. On the other hand, vps35b-1 vps35c-1 andthe triple mutant (vps35a-1 vps35b-2 vps35c-1) exhibited severephenotypes: dwarfism, early leaf senescence and fragmentationof protein storage vacuoles (PSVs). In addition, these mutantsmis-sorted storage proteins by secreting them out of the cellsand accumulated a higher level of vacuolar sorting receptor(VSR) than the wild type. VPS35 was localized in pre-vacuolarcompartments (PVCs), some of which contained VSR. VPS35 wasimmunoprecipitated with VPS29/MAG1, another component of theretromer complex. Our findings suggest that VPS35, mainly VPS35b,is involved in sorting proteins to PSVs in seeds, possibly byrecycling VSR from PVCs to the Golgi complex, and is also involvedin plant growth and senescence in vegetative organs.     

The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins

Double-stranded RNA induces RNA silencing and is cleaved into21–24 nt small RNA duplexes by Dicer enzyme. A strandof Dicer-generated small RNA duplex (called the guide strand)is then selected by a thermodynamic mechanism to associate withArgonaute (AGO) protein. This AGO–small RNA complex functionsto cleave mRNA, repress translation or modify chromatin structurein a sequence-specific manner. Although a model plant, Arabidopsisthaliana, contains 10 AGO genes, their roles and molecular mechanismsremain obscure. In this study, we analyzed the roles of ArabidopsisAGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAsthat associated with AGO2 was mainly adenine (85.7%) and thatwith AGO5 was mainly cytosine (83.5%). Small RNAs that wereabundantly cloned from the AGO2 immunoprecipitation fraction(miR163-LL, which is derived from the Lower Left of mature miR163in pre-miR163, and miR390) and from the AGO5 immunoprecipitationfraction (miR163-UL, which is derived from the Upper Left ofmature miR163 in pre-miR163, and miR390^*) are derived from thesingle small RNA duplexes, miR163-LL/miR163-UL and miR390/miR390^*.Each strand of the miR163-LL/miR163-UL duplex is selectivelysorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependentmanner rather than in a thermodynamic stability-dependent manner.Furthermore, we showed that both AGO2 and AGO5 have the abilityto bind cucumber mosaic virus-derived small RNAs. These resultsclearly indicate that the mechanism selecting the guide strandis different among AGO proteins and that multiple AGO genesare involved in anti-virus defense in plants.     

Temperature-dependent changes of cell shape during heterophyllous leaf formation in <Emphasis Type="Italic">Ludwigia </Emphasis><Emphasis Type="Italic">arcuata</Emphasis> (Onagraceae)

Although elongation of epidermal cells in submerged leaves is thought to be a common feature of heterophyllous aquatic plants, such elongation has not been observed in Ludwigia arcuata Walt. (Onagraceae). In this study we found that reduced culture temperature induced the elongation of epidermal cells of submerged leaves in L. arcuata. Since such submerged leaves also showed a reduction in the number of epidermal cells aligned across the leaf transverse axis, these data indicate that heterophyllous leaf formation in L. arcuata is partially temperature sensitive, i.e., the elongation of epidermal cells was temperature sensitive while the reduction in the number of epidermal cells did not show such temperature sensitivity. To clarify the mechanisms that cause such temperature sensitivity, we examined the effects of ethylene, which induced the formation of submerged-type leaves on aerial shoots at the relatively high culture-temperature of 28 degrees C. At 23 degrees C, ethylene induced both cell elongation and reduction in the number of epidermal cells across the leaf transverse axis, while cell elongation was not observed at 28 degrees C. Moreover, both submergence and ethylene treatment induced a change in the arrangement of cortical microtubules (MTs) in epidermal cells of developing leaves at 23 degrees C. Such changes in the arrangement of MTs was not induced at 28 degrees C. Factors involved in the temperature-sensitive response to ethylene would be critical for temperature-sensitive heterophyllous leaf formation in L. arcuata.