Supervillin binding to myosin II and synergism with anillin are required for cytokinesis

Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin''s role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.     

Stable germ line transformation of a leafy vegetable crop amaranth (Amaranthus tricolor L.) mediated by Agrobacterium tumefaciens

We have optimized a procedure for genetic transformation of a major leafy vegetable crop, Amaranthus tricolor L., using epicotyl explant co-cultivation with Agrobacterium tumefaciens. Two disarmed A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (nptII) and the β-glucuronidase gene (gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated Agrobacterium culture corresponding to OD₆₀₀???0.6 and diluted to 10⁹ cells ml^?1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg?ml^?1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the nptII and gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T₀). Among T₁ seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (χ ²) test. Southern hybridization of genomic DNA from kanamycin-resistant T₁ transgenic segregants to an nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T₁ transgenic plants, thereby confirming stable expression of the nptII gene.     

Role of Pore-Forming Toxins in Bacterial Infectious Diseases

SUMMARY

Pore-forming toxins (PFTs) are the most common bacterial cytotoxic proteins and are required for virulence in a large number of important pathogens, including Streptococcus pneumoniae, group A and B streptococci, Staphylococcus aureus, Escherichia coli, and Mycobacterium tuberculosis. PFTs generally disrupt host cell membranes, but they can have additional effects independent of pore formation. Substantial effort has been devoted to understanding the molecular mechanisms underlying the functions of certain model PFTs. Likewise, specific host pathways mediating survival and immune responses in the face of toxin-mediated cellular damage have been delineated. However, less is known about the overall functions of PFTs during infection in vivo. This review focuses on common themes in the area of PFT biology, with an emphasis on studies addressing the roles of PFTs in in vivo and ex vivo models of colonization or infection. Common functions of PFTs include disruption of epithelial barrier function and evasion of host immune responses, which contribute to bacterial growth and spreading. The widespread nature of PFTs make this group of toxins an attractive target for the development of new virulence-targeted therapies that may have broad activity against human pathogens.     

Artificial bare patches increase habitat for the endangered Ohlone tiger beetle (Cicindela ohlone)

The endangered Ohlone tiger beetle (Cicindela ohlone) depends on bare ground areas in California coastal grasslands to encounter mates, oviposit, and find prey. We tested habitat creation as a potential management strategy to increase the availability of oviposition sites for C. ohlone. We compared three different bare ground treatments by scraping off surface vegetation, ripping, and tamping the plots. We also tested whether bare ground creation expands C. ohlone range within a habitat patch by scraping plots at increasing distances from the core habitat and monitoring C. ohlone colonization. C. ohlone oviposited significantly more in artificial bare ground plots compared to controls both one and 2 years after the scrapes were created. Distance from the core habitat did not affect colonization nor did decompaction of scraped plots. Percent bare ground significantly predicted incidence of colonization. For the conservation of the endangered Ohlone tiger beetle, we recommend continued creation of scraped plots every 2 years in order to maintain bare ground and to ensure maximum usage by female C. ohlone as oviposition sites.     

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg^?1. The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5–11.5) and temperature (40–90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K _m and V _max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min^?1 mg^?1 protein by analyzing Michaelis–Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.     

Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae

Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo–devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion–deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pi_non/syn was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes.