The highly stable alcohol dehydrogenase of Thermomicrobium roseum: purification and molecular characterization

An alcohol dehydrogenase (ADH) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. The native enzyme was found to be a homo-dimer of 43-kDa subunits. The pI of the enzyme was determined to be 6.2, while its optimum pH is 10.0. The enzyme oxidized mainly primary aliphatic alcohols and exhibited high substrate specificity towards ethanol, n-propanol and crotyl alcohol. The highest reaction rate was observed when ethanol was used as substrate and the K(m) value of the enzyme for ethanol was 24.2 mM. Pyrazole notably inhibited the enzymatic activity. The enzyme had the optimal temperature of 70 degrees C and was highly stable against high temperature.     

Characterization of a serine proteinase mediating encystation of Acanthamoeba

Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.     

Phylogenetic Analysis of AA-genome Oryza Species (Poaceae) Based on Chloroplast, Mitochondrial, and Nuclear DNA Sequences

Species in the genus Oryza (Poaceae) contain 10 genomic types and are distributed in pan-tropics of the world. To explore phylogenetic relationships of Oryza species having the AA-genome, DNA sequences of the chloroplast trnL intron and trnL-trnF spacer, mitochondrial nad1 intron 2, and nuclear internal transcribed spacer were analyzed, based on materials from 6 cultivated (O. sativa and O. glaberrima) and 13 wild accessions, in addition to a CC-genome species (O. officinalis) that was used as an outgroup. Analyses of the combined sequence data set from different sources provide a much better resolution of the AA-genome species than the individual data set, indicating the limitation of a single gene in phylogenetic reconstruction. The phylogeny based on the combined data set demonstrated an apparent grouping of the AA-genome Oryza species that was well associated with their geographic origin, although the Australian O. meridionalis showed its affinity with the African species. The geographic pattern of the phylogenetic relationship was probably attributed to the frequent genetic exchange and introgression among the AA-genome species from the same continents. In addition, Asian cultivated rice O. sativa showed its close relation to O. rufipogon and O. nivara, whereas African cultivated rice O. glaberrima was closely linked to O. barthii and O. longistaminata, indicating the independent domestication of the two cultivated species in different geographic locations.     

MHC gene configuration variation in geographically disparate populations of California sea lions (Zalophus californianus)

Major histocompatibility complex (MHC) class II DRB genotypes were examined in two geographically isolated populations of California sea lions (Zalophus californianus) (Gulf of California and California coastal Pacific Ocean). Genomic DNA from 227 California sea lions was examined using eight sequence-specific primer (SSP) pairs flanking the putative peptide-binding site. A total of 40 different Zaca-DRB genotype configurations were identified among the 227 individuals. Using SSP-PCR, significant differences were found between coastal California and Gulf of California Zalophus populations in numbers of DRB sequences per individual and configuration of sequences within individuals. Additionally, unique local patterns of MHC diversity were identified among the Midriff Island animals. These population differences are consistent with either ecologically distinct patterns of selection pressures and/or geographical isolation. The consequences of these partitioned MHC configurations at the population level are as yet unknown; however, the worldwide increase in emerging marine diseases lends urgency to their examination.     

Microsatellite mapping of a Triticum urartu Tum. derived powdery mildew resistance gene transferred to common wheat (Triticum aestivum L.)

A powdery mildew resistance gene from Triticum urartu Tum. accession UR206 was successfully transferred into hexaploid wheat (Triticum aestivum L.) through crossing and backcrossing. The F₁ plants, which had 28 chromosomes and an average of 5.32 bivalents and 17.36 univalents in meiotic pollen mother cells (PMC), were obtained through embryos rescued owing to shriveling of endosperm in hybrid seed of cross Chinese Spring (CS) × UR206. Hybrid seeds were produced through backcrossing F₁ with common wheat parents. The derivative lines had normal chromosome numbers and powdery mildew resistance similar to the donor UR206, indicating that the powdery mildew resistance gene originating from T. urartu accession UR206 was successfully transferred and expressed in a hexaploid wheat background. Genetic analysis indicated that a single dominant gene controlled the powdery mildew resistance at the seedling stage. To map and tag the powdery mildew resistance gene, 143 F₂ individuals derived from a cross UR206 × UR203 were used to construct a linkage map. The resistant gene was mapped on the chromosome 7AL based on the mapped microsatellite makers. The map spanned 52.1 cM and the order of these microsatellite loci agreed well with the established microsatellite map of chromosome arm 7AL. The resistance gene was flanked by the microsatellite loci Xwmc273 and Xpsp3003, with the genetic distances of 2.2 cM and 3.8 cM, respectively. On the basis of the origin and chromosomal location of the gene, it was temporarily designated PmU.     

Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.