Molecular Characterization of Cryptosporidium Isolates from Humans and Animals in Iran

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.     

Cell death in seedlings of the interspecific hybrid of <Emphasis Type="Italic">Nicotiana gossei</Emphasis> and <Emphasis Type="Italic">N. tabacum</Emphasis>; possible role of knob-like bodies formed on tonoplast in vacuolar-collapse-mediated cell death

Vacuolar collapse plays a direct role in the cell death of the interspecific hybrid of Nicotiana gossei Domin ×N. tabacum L. which exhibits hybrid lethality at the seedling stage. We have previously reported that cell death in these seedlings began at the base of hypocotyls and spread throughout the plant (Mino et al. 2002). A light microscopic analysis revealed that the process involved disruption of the intra-cellular membranes, plasmolysis, and retraction of the wall of the cell in hypocotyls. A transmission electron microscopic analysis showed that there were several abnormal structures, i.e. knob-like bodies on the tonoplast and small vesicles in the cytoplasm, and the disintegration of the tonoplast, in the cells of seedlings grown at 26°C. However, no such cytological defects were observed in the seedlings grown at 37°C, at which temperature the expression of lethality was suppressed. The activity levels of vacuolar processing enzyme (VPE), which might be involved in the vacuolar collapse of plant cells, temporarily increased in the seedlings grown at 26°C before apparent cell death proceeded, but it remained unchanged in the seedlings grown at 37°C. Applications of acetyl-l-tyrosyl-l-valyl-l-alanyl-l-aspart-1-aldehyde, an inhibitor for VPE, and cycloheximide to the seedlings suppressed VPE's activities, the formation of knob-like bodies on the tonoplast, and cell death. VPE might be involved in the structural anomalies on the tonoplast which lead to cell death triggered by vacuolar collapse in hybrid seedlings.     

Gene- and Protein-Delivered Zinc Finger–Staphylococcal Nuclease Hybrid for Inhibition of DNA Replication of Human Papillomavirus

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP–SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP–SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP–SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP–SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.     

Kinetochore-dependent microtubule rescue ensures their efficient and sustained interactions in early mitosis

1. Download : Download high-res image (303KB)
2. Download : Download full-size image

Highlights? Microtubule (MT) rescue is promoted by a laterally associated kinetochore (KT) ? MT rescue at and distal to the KT is promoted by Stu2 transfer and transport from the KT ? MT rescue prevents KT detachment from the MT end when KT end-on tethering fails ? MT extension following rescue facilitates collection of other widely scattered KTs     

Identification of a plant aminopeptidase with preference for aromatic amino acid residues as a novel member of the prolyl oligopeptidase family of serine proteases

Genome analysis has indicated that plants, like animals, possess a variety of protease genes. However, bulk of putative proteases has not been characterized at the enzyme level. In this article, a novel enzyme that hydrolyses phenylalanyl-4-methylcoumaryl 7-amide (phenylalanyl-MCA) was purified from cotyledons of daikon radish by ammonium sulphate fractionation and successive chromatography with DEAE-cellulose, phenyl-Sepharose, Sephacryl S-200 and Mini-Q. The molecular mass of the enzyme was estimated to be 78 kDa by SDS-PAGE under reducing conditions and 74 kDa by gel filtration, indicating that the enzyme is a monomer. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that the enzyme is an orthologue of Arabidopsis unidentified protein, acylpeptide hydrolase-like protein (AHLP; UniProt ID: Q9FG66) belonging to the prolyl oligopeptidase (POP) family of a serine-type peptidase predicted from genetic information. Good substrates identified for the enzyme include phenylalanyl-MCA, tyrosyl-MCA and enkephalin. Neither acylamino acid-releasing activity nor endopeptidase activity was detected. The enzyme cleaved enkephalin (YGGFM, YGGFL), whereas, BAM-12 P (YGGFMRRVGRPE) and dynorphin A (YGGFLRRIRPKLK) were not digested. These results suggested that the enzyme possesses strict size selectivity of substrate. We propose the name 'tyrosyl aminopeptidase' for the uncharacterized protein AHLP.     

Advanced Microbial Taxonomy Combined with Genome-Based-Approaches Reveals that Vibrio astriarenae sp. nov., an Agarolytic Marine Bacterium,Forms a New Clade in Vibrionaceae

Advances in genomic microbial taxonomy have opened the way to create a more universal and transparent concept of species but is still in a transitional stage towards becoming a defining robust criteria for describing new microbial species with minimum features obtained using both genome and classical polyphasic taxonomies. Here we performed advanced microbial taxonomies combined with both genome-based and classical approaches for new agarolytic vibrio isolates to describe not only a novel Vibrio species but also a member of a new Vibrio clade. Two novel vibrio strains (Vibrio astriarenae sp. nov. C7^T and C20) showing agarolytic, halophilic and fermentative metabolic activity were isolated from a seawater sample collected in a coral reef in Okinawa. Intraspecific similarities of the isolates were identical in both sequences on the 16S rRNA and pyrH genes, but the closest relatives on the molecular phylogenetic trees on the basis of 16S rRNA and pyrH gene sequences were V. hangzhouensis JCM 15146^T (97.8% similarity) and V. agarivorans CECT 5085^T (97.3% similarity), respectively. Further multilocus sequence analysis (MLSA) on the basis of 8 protein coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) obtained by the genome sequences clearly showed the V. astriarenae strain C7^T and C20 formed a distinct new clade protruded next to V. agarivorans CECT 5085^T. The singleton V. agarivorans has never been included in previous MLSA of Vibrionaceae due to the lack of some gene sequences. Now the gene sequences are completed and analysis of 100 taxa in total provided a clear picture describing the association of V. agarivorans into pre-existing concatenated network tree and concluded its relationship to our vibrio strains. Experimental DNA-DNA hybridization (DDH) data showed that the strains C7^T and C20 were conspecific but were separated from all of the other Vibrio species related on the basis of both 16S rRNA and pyrH gene phylogenies (e.g., V. agarivorans CECT 5085^T, V. hangzhouensis JCM 15146^T V. maritimus LMG 25439^T, and V. variabilis LMG 25438^T). In silico DDH data also supported the genomic relationship. The strains C7^T also had less than 95% average amino acid identity (AAI) and average nucleotide identity (ANI) towards V. maritimus C210, V. variabilis C206, and V. mediterranei AK1^T, V. brasiliensis LMG 20546^T, V. orientalis ATCC 33934^T, and V. sinaloensis DSM 21326. The name Vibrio astriarenae sp. nov. is proposed with C7 as the type strains. Both V. agarivorans CECT 5058^T and V. astriarenae C7^T are members of the newest clade of Vibrionaceae named Agarivorans.     

Formation of interacting spins on flavosemiquinone and tyrosine radical in photoreaction of a blue light sensor BLUF protein TePixD

Light-induced radicals were detected by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) in the BLUF-domain protein TePixD of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The illumination of TePixD at 5-200 K derived an EPR signal with a separation of 85 G between the main peaks around g = 2, showing a typical Pake's pattern of magnetic dipole-dipole interaction between two nearby radicals. Longer illumination induced an EPR signal at g = 2.0045, which was assigned as a neutral flavosemiquinone FADH(*). The FADH(*) formation occurred in parallel with a decrease in Pake's doublet. The Pake's doublet was not detected in a mutant TePixD protein in which a tyrosine residue was replaced with phenylalanine (Y8F protein). A pulsed ENDOR study suggested that the Pake's doublet had arisen from the interaction between a neutral flavosemiquinone radical and a neutral tyrosine radical, i.e., the FADH(*)-Y8(*) state. An EPR simulation of the Pake's doublet showed that the distance between FAD and Y8 is 2.2 A shorter than that calculated from the X-ray crystallography structure in the dark-adapted state, suggesting the modification of the protein conformation in the photoinduced FADH(*)-Y8(*) state. The Pake's doublet signal was detected by 10 K illumination in the sample which was immediately frozen after 273 K illumination, corresponding to the red-shifted state F(490). On the other hand, the signal was not detected in the sample which was incubated for 10 min at 273 K in the dark after 273 K illumination, corresponding to the dark-adapted state D(471). In the sample annealed at 160 K for 10 min after 160 K illumination, corresponding to the partially red-shifted state J(11), the Pake's doublet signal was detected by the 10 K illumination. On the basis of these observations, we concluded that the interaction with the FADH(*)-Y8(*) state occurred after the second photoexcitation of the photoinduced red-shifted states in the photocycle of TePixD.