The Drosophila kinesin-like protein KLP67A is essential for mitotic and male meiotic spindle assembly

We have performed a mutational analysis together with RNA interference to determine the role of the kinesin-like protein KLP67A in Drosophila cell division. During both mitosis and male meiosis, Klp67A mutations cause an increase in MT length and disrupt discrete aspects of spindle assembly, as well as cytokinesis. Mutant cells exhibit greatly enlarged metaphase spindle as a result of excessive MT polymerization. The analysis of both living and fixed cells also shows perturbations in centrosome separation, chromosome segregation, and central spindle assembly. These data demonstrate that the MT plus end-directed motor KLP67A is essential for spindle assembly during mitosis and male meiosis and suggest that the regulation of MT plus-end polymerization is a key determinant of spindle architecture throughout cell division.     

Identification and characterization of PiORP1, a Petunia oxysterol-binding-protein related protein involved in receptor-kinase mediated signaling in pollen, and analysis of the ORP gene family in Arabidopsis

Oxysterol-binding proteins (OSBPs) and oxysterol-binding-protein related proteins (ORPs) are encoded by most eukaryotic genomes examined to date; however, they have not yet been characterized in plants. Here we report the identification and characterization of PiORP1, an ORP of Petunia inflata that interacts with the cytoplasmic kinase domain of a receptor-like kinase, named PRK1, of P. inflata. PiORP1 is phosphorylated by PRK1 in vitro and therefore may be involved in PRK1 signaling during pollen development and growth. RNA gel blot analysis showed that PiORP1 and PRK1 had very similar expression patterns in developing pollen, mature pollen and pollen tubes. GFP fusion proteins of PiORP1 localized in the plasma membrane of pollen tubes at distinct foci and its PH domain alone was sufficient to mediate this localization. The sequence for the oxysterol-binding domain of PiORP1 was used to search the genome of Arabidopsis; 12 ORPs were identified and phylogenetic analysis revealed that they fell into two distinct clades, consistent with the ORPs of other eukaryotes. RT-PCR analysis showed that all 12 Arabidopsis ORPs were expressed; 10 were expressed in most of the tissues examined under normal growth conditions, but only three were expressed in pollen. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. GenBank accession number for PiORP1: DQ241801     

Evidence of a Male Sex Pheromone in the Round Goby (Neogobius melanostomus)

The reproductive success of the round goby (Neogobius melanostomus), an invasive fish, may be mediated by the use of pheromones. We hypothesized that reproductive male (RM) round gobies release sex pheromone to which reproductive females (RF) respond. In this study, we compared behavioural and electrophysiological responses of reproductive and non-reproductive female round gobies to conspeci fic males. Results of behavioural experiments in the laboratory showed that RF spent significantly more time near the source of the male odour compared with odours from control water. However, RF did not distinguish between odours from non-reproductive male (non-RM) water and control water. Non-reproductive females (non-RF) were not attracted to odours released from RM or non-RM water. Results of electro-olfactogram (EOG) responses showed that both RF and non-RF discriminated between HPLC fractionated RM and non-RM odours. However, the EOG responses of RF were about eight-fold higher than non-RF exposed to RM odours. These findings confirm that RM round gobies release a pheromone signal that attracts RF. The results of this research may be useful in developing control strategy using natural pheromones to disrupt the reproductive behaviour of the invasive round goby and to curtail its effects on native species. An erratum to this article is available at .     

The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography

Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.     

Nef alleles from human immunodeficiency virus type 1-infected long-term-nonprogressor hemophiliacs with or without late disease progression are defective in enhancing virus replication and CD4 down-regulation

Infection with human immunodeficiency virus (HIV)-encoding defective nef variants may contribute to a relatively benign course of disease in a minority of long-term nonprogressors (LTNP). We have examined the functions of nef alleles from six individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Three out of six individuals have progressed to HIV disease (late progressors [LP]), whereas the three remainders have maintained their LTNP status at least up to 2003. The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells of all six individuals in 1995 and 1998. The proportion of sequences containing mutations not yielding Nef expression significantly diminished in 1998 versus that in 1995. Several previously defined functional regions of intact nef alleles were highly conserved. However, the major variant obtained in 1998 from plasma RNA of five out of six individuals significantly reduced HIV infectivity/replication and impaired Nef-mediated CD4 but not major histocompatibility complex class I antigen down-modulation from the cell surface. Thus, functional alterations of the nef gene are present in both LP and LTNP, suggesting that Nef defectiveness in vitro is not necessarily associated with the long-term maintenance of LTNP status. Of interest is the fact that isolates from three out of three LP showed a dual CCR5/CXCR4 coreceptor use (R5X4), in contrast to those from LTNP, which were exclusively R5. Thus, in vivo evolution of gp120 Env to CXCR4 use appears to be associated with HIV disease progression in individuals infected with nef-defective viruses.     

Human telomeres have different overhang sizes at leading versus lagging strands

G-rich 3' telomeric overhangs are required both for forming the distinct telomere structures to protect chromosome ends and for extending telomeres by telomerase. However, little is known about the molecular mechanisms generating telomere overhangs in human cells. We show here that cultured normal human diploid cells have longer G overhangs at telomeres generated by lagging-strand synthesis than by leading-strand synthesis. We also demonstrate that telomerase expression results in elongated overhangs at the leading daughter telomeres. Thus, the overhangs at the leading and lagging daughter telomeres are generated differently in human cells, and telomerase may preferentially affect overhangs generated at the telomeres produced by leading-strand synthesis.     

GTP drives myosin light chain 1 interaction with the class V myosin Myo2 IQ motifs via a Sec2 RabGEF-mediated pathway

The yeast myosin light chain 1 (Mlc1p) belongs to a branch of the calmodulin superfamily and is essential for vesicle delivery at the mother-bud neck during cytokinesis due to is ability to bind to the IQ motifs of the class V myosin Myo2p. While calcium binding to calmodulin promotes binding/release from the MyoV IQ motifs, Mlc1p is unable to bind calcium and the mechanism of its interaction with target motifs has not been clarified. The presence of Mlc1p in a complex with the Rab/Ypt Sec4p and with Myo2p suggests a role for Mlc1p in regulating Myo2p cargo binding/release by responding to the activation of Rab/Ypt proteins. Here we show that GTP or GTPgammaS potently stimulate Mlc1p interaction with Myo2p IQ motifs. The C-terminus of the Rab/Ypt GEF Sec2p, but not Sec4p activation, is essential for this interaction. Interestingly, overexpression of constitutively activated Ypt32p, a Rab/Ypt protein that acts upstream of Sec4p, stimulates Mlc1p/Myo2p interaction similarly to GTP although a block of Ypt32 GTP binding does not completely abolish the GTP-mediated Mlc1p/Myo2p interaction. We propose that Mlc1p/Myo2p interaction is stimulated by a signal that requires Sec2p and activation of Ypt32p.     

Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. We have engineered a vector that expresses the fusion protein containing the following: (a) green fluorescent protein as a reporter of localization, (b) bacterial maltose-binding protein to increase the size of the reporter polypeptide, and (c) galectin-3, whose sequence we wished to dissect in search of amino acid residues vital for nuclear localization. In mouse 3T3 fibroblasts transfected with this expression construct, the full-length galectin-3 (residues 1-263) fusion protein was localized predominantly in the nucleus. Mutants of this construct, containing truncations of the galectin-3 polypeptide from the amino terminus, retained nuclear localization through residue 128; thus, the amino-terminal half was dispensable for nuclear import. Mutants of the same construct, containing truncations from the carboxyl terminus, showed loss of nuclear localization. This effect was observed beginning with truncation at residue 259, and the full effect was seen with truncation at residue 253. Site-directed mutagenesis of the sequence ITLT (residues 253-256) suggested that nuclear import was dependent on the IXLT type of nuclear localization sequence, first discovered in the Drosophila protein Dsh (dishevelled). In the galectin-3 polypeptide, the activity of this nuclear localization sequence is modulated by a neighboring leucine-rich nuclear export signal.     

Ethnobotany of the Balti community,Tormik valley,Karakorum range,Baltistan, Pakistan

Background

Limited health facilities and malnutrition are major problems in the Karakorum Range of Northern Pakistan, often resulting in various human disorders. Since centuries, however, local communities in these areas have developed traditional methods for treating various ailments and local foods capes that can be significant for devising public health and nutritional policies. This study was intended to document the ethnobotanical knowledge of the local peoples in the Tormik Valley, especially in the medical and food domains.

Methods

Field trips were undertaken in 14 different villages of the study area from 2010 to 2012. Ethnobotanical data were gathered using semi-structured interviews and group conversation with 69 informants. Details about local uses of plant species were recorded along with demographic characteristics of the visited communities. Relative frequency citation index (RFCi) and preference ranking index (PRi) tools were applied to determine the cultural significance of the reported species.

Results

Sixty-three plant species, with a predominance of Asteraceae and Fabaceae family members, as well as their detailed folk uses were documented. Forty-three percent of the species were used to treat various diseases, 21 % were consumed as wild fruits and vegetables and 53 % of the species had multipurpose applications. Thymus linearis Benth, Hippophae rhamnoides ssp. turkestanica L. and Convolvulus arvensis L. were found to be the most utilized medicinal plant species, i.e. those with significant RFCi values (0.54, 0.51 and 0.48, respectively). Betula utilis D. Don was the most versatile taxon (seven different ways of utilization); being this species a common and easily accessible subalpine tree and then under anthropogenic pressure, the implementation of concrete strategies aimed at its in-situ and ex-situ conservation is strongly recommended.

Conclusion

The valleys in the Karakorum Mountains in the Northern Pakistan host significant Traditional Knowledge on local food and medicinal plant species, which need to be reconsidered and cautiously re-evaluated by ethnopharmacologists, and public health/nutrition actors. Furthermore, germane trans-disciplinary investigations are suggested to ensure the dynamic conservation of precious local knowledge systems, as well as plant diversity in Pakistani mountain regions.