A systemic resistance inducing antiviral protein with N-glycosidase activity from Bougainvillea xbuttiana leaves

An antiviral protein from Bougainvillea xbuttiana leaves induced systemic resistance in host plants N. glutinosa and Cyamopsis tetragonoloba against TMV and SRV, respectively which was reversed by actinomycin D, when applied immediately or shortly after antiviral protein treatment. When the inhibitor was applied to the host plant leaves post inoculation, it was effective if applied upto 4 h after virus infection. It also delayed the expression of symptoms in systemic hosts of TMV. The inhibitor showed characteristic N-glycosidase activity on 25S rRNA of tobacco ribosomes, suggesting that it could also be interfering with virus multiplication through ribosome-inactivation process.     

Improved growth and essential oil yield and quality in Foeniculum vulgare mill on mycorrhizal inoculation supplemented with P-fertilizer

Two arbuscular mycorrhizal (AM) fungi Glomus macrocarpum and Glomus fasciculatum significantly improved growth and essential oil concentration of Foeniculum vulgare Mill. However, AM inoculation of plants along with phosphorus fertilization significantly enhanced growth, P-uptake and essential oil content of plants compared to either of the components applied separately. Among the two fungal inoculants, G. fasciculatum registered the highest growth at both levels of phosphorus used with up to 78% increase in essential oil concentration of fennel seeds over non-mycorrhizal control. The essential oil characterization by gas liquid chromatography revealed that the level of anethol was significantly enhanced on mycorrhization.     

Correcting improper chromosome-spindle attachments during cell division

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.     

Genetic association analysis of KCNQ3 and juvenile myoclonic epilepsy in a South Indian population

Juvenile myoclonic epilepsy (JME) is a common subtype of idiopathic generalized epilepsy that shows a complex pattern of inheritance. We have tested the association between JME phenotype and an intragenic marker in KCNQ3 by using the transmission disequilibrium test in 119 probands and their parents. Mutations in KCNQ3 are known to cause benign familial neonatal convulsions and are involved in the physiologically important M current in neurons. Our results provide suggestive evidence of allelic association between JME and KCNQ3 (P-value=0.008) and raise an interesting possibility of a genetic contribution to JME, viz., of a gene that causes a monogenic form of human epilepsy.     

EBNA1 partitions Epstein-Barr virus plasmids in yeast cells by attaching to human EBNA1-binding protein 2 on mitotic chromosomes

Epstein-Barr virus (EBV) episomal genomes are stably maintained in human cells and are partitioned during cell division by mitotic chromosome attachment. Partitioning is mediated by the viral EBNA1 protein, which binds both the EBV segregation element (FR) and a mitotic chromosomal component. We previously showed that the segregation of EBV-based plasmids can be reconstituted in Saccharomyces cerevisiae and is absolutely dependent on EBNA1, the EBV FR sequence, and the human EBNA1-binding protein 2 (EBP2). We have now used this yeast system to elucidate the functional contribution of human EBP2 to EBNA1-mediated plasmid partitioning. Human EBP2 was found to attach to yeast mitotic chromosomes in a cell cycle-dependent manner and cause EBNA1 to associate with the mitotic chromosomes. The domain of human EBP2 that binds both yeast and human chromosomes was mapped and shown to be functionally distinct from the EBNA1-binding domain. The functionality and localization of human EBP2 mutants and fusion proteins indicated that the attachment of EBNA1 to mitotic chromosomes is crucial for EBV plasmid segregation in S. cerevisiae, as it is in humans, and that this is the contribution of human EBP2. The results also indicate that plasmid segregation in S. cerevisiae can occur through chromosome attachment.     

Interactions of substrate with calreticulin,an endoplasmic reticulum chaperone

Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc(1)Man(7-9)GlcNAc(2) present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc(1)Man(7-9)GlcNAc(2), determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K(b)) and changes in enthalpy of binding (Delta H(b) degrees ) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 x 10(4) m(-1) at 279 K is obtained when glucose occurs in alpha-1,3 linkage to Man alpha Me as in Glc alpha 1-3Man alpha Me. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed.     

Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.     

Composition,acquisition, and distribution of the Vi exopolysaccharide-encoding Salmonella enterica pathogenicity island SPI-7

Vi capsular polysaccharide production is encoded by the viaB locus, which has a limited distribution in Salmonella enterica serovars. In S. enterica serovar Typhi, viaB is encoded on a 134-kb pathogenicity island known as SPI-7 that is located between partially duplicated tRNA(pheU) sites. Functional and bioinformatic analysis suggests that SPI-7 has a mosaic structure and may have evolved as a consequence of several independent insertion events. Analysis of viaB-associated DNA in Vi-positive S. enterica serovar Paratyphi C and S. enterica serovar Dublin isolates revealed the presence of similar SPI-7 islands. In S. enterica serovars Paratyphi C and Dublin, the SopE bacteriophage and a 15-kb fragment adjacent to the intact tRNA(pheU) site were absent. In S. enterica serovar Paratyphi C only, a region encoding a type IV pilus involved in the adherence of S. enterica serovar Typhi to host cells was missing. The remainder of the SPI-7 islands investigated exhibited over 99% DNA sequence identity in the three serovars. Of 30 other Salmonella serovars examined, 24 contained no insertions at the equivalent tRNA(pheU) site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNA(pheU)-phoN junction, which was not analyzed further. Sequence analysis of the SPI-7 region from S. enterica serovar Typhi strain CT18 revealed significant synteny with clusters of genes from a variety of saprophytic bacteria and phytobacteria, including Pseudomonas aeruginosa and Xanthomonas axonopodis pv. citri. This analysis suggested that SPI-7 may be a mobile element, such as a conjugative transposon or an integrated plasmid remnant.     

Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model

Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties. LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations. MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1. In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability. Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.