Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using X-RACE technology and named AtKP1 (for Arabidopsis kinesin protein 1). This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coli and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.

Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylori

Vibrational spectroscopy and crystal structure analysis of two polymorphs of the di-amino acid peptide cyclo(L-Glu-L-Glu)

Cyclo(L-Glu-L-Glu) has been crystallised in two different polymorphic forms. Both polymorphs are monoclinic, but form 1 is in space group P21 and form 2 is in space group C2. Raman scattering and FT-IR spectroscopic studies have been conducted for the N,O-protonated and deuterated derivatives. Raman spectra of orientated single crystals, solid-state and aqueous solution samples have also been recorded. The different hydrogen-bonding patterns for the two polymorphs have the greatest effect on vibrational modes with N&bond;H and C&dbond;O stretching character. DFT (B3-LYP/cc-pVDZ) calculations of the isolated cyclo(L-Glu-L-Glu) molecule predict that the minimum energy structure, assuming C2 symmetry, has a boat conformation for the diketopiperazine ring with the two L-Glu side chains being folded above the ring. The calculated geometry is in good agreement with the X-ray crystallographic structures for both polymorphs. Normal coordinate analysis has facilitated the band assignments for the experimental vibrational spectra. Copyright © 2009 John Wiley & Sons, Ltd.

Evolution of the Diatoms: VIII. Re-Examination of the SSU-Rrna Gene Using Multiple Outgroups and a Cladistic Analysis of Valve Features.

The resolution of the SSU rRNA gene for phylogenetic analysis in the diatoms has been evaluated by Theriot et al. who claimed that the SSU rRNA gene could not be used to resolve the monophyly of the three diatoms classes described by Medlin and Kaczmarska. Although they used both only bolidomonads and heterokonts as outgroups, they did not explore outgroups further away than the heterokonts. In this study, the use of the multiple outgroups inside and outside the heterokonts with the rRNA gene for recovering the three monophyletic clades at the class level is evaluated. Trees with multiple outgroups ranging from only bolidophytes to Bacteria and Archea were analyzed with Bayesian and Maximum Likelihood analyses and two data sets were recovered with the classes being monophyletic. Other data sets were analyzed with non-weighted and weighted maximum parsimony. The latter reduced the number of clades and lengthened branch lengths between the clades. One data set using a weighted analysis recovered the three classes as monophyletic. Taking only bolidophytes as the only outgroup never produced monophyletic clades. Multiple outgroups including many heterokonts and certain members of the crown group radiation recovered monophyletic clades. The three classes can be defined by clear morphological differences primarily based on auxospore ontogeny and envelope structure, the presence or absence of a structure (tube process or sternum) associated with the annulus and the location of the cribrum in those genera with loculate areolae. A cladistic analysis of some of these features is presented and recovers the three classes.

Authoritarianism, threat, and prejudice: An analysis of mediation and moderation

Right-wing authoritarianism is a central construct in individual differences approaches to prejudice. Its power to predict prejudice is often attributed to perceived threat. However, the exact moderating and mediating processes involved are little understood. In two studies (Ns=53, 84), exposure to threatening versus nonthreatening information about an ethnic out-group had reliable indirect effects on prejudice in authoritarians, but not in nonauthoritarians, largely because authoritarians were more likely to perceive actual threat when they interpreted the information received to represent a threatening argument. Additionally, in Study 2, authoritarians reacted more strongly with negative emotions when they perceived actual threat.

An Investigation of Compressor Map Width Enhancement and a Detailed Analysis of Inducer Flow Field

This paper describes an investigation of various shroud bleed slot configurations of a centrifugal compressor using CFD with a manual multi-block structured grid generation method. The compressor under investigation is used in a turbocharger application for a heavy duty diesel engine of approximately 400 hp. The baseline numerical model has been developed and validated against experimental performance measurements. The influence of the bleed slot flow field on a range of operating conditions between surge and choke has been analysed in detail.
The impact of the returning bleed flow on the incidence at the impeller blade leading edge due to its mixing with the main through-flow has also been studied. From the baseline geometry, a number of modifications to the bleed slot width have been proposed, and a detailed comparison of the flow characteristics performed. The impact of slot variations on the inlet incidence angle has been investigated, highlighting the improvement in surge and choked flow capability. Along with this, the influence of the bleed slot on stabilising the blade passage flow by the suction of the tip and over-tip vortex flow by the slot has been considered near surge.

Death and football: An analysis of men's talk about emotions

This study is concerned with men's talk about emotions and with how emotion discourses function in the construction and negotiation of masculine ways of doing emotions and of consonant masculine subject positions. A sample group of 16 men, who were recruited from two social contexts in England, participated in focus groups on 'men and emotions'. Group discussions were transcribed and analysed using discourse analysis. Participants drew upon a range of discursive resources in constructing masculine emotional behaviour and negotiating masculine subject positions. They constructed men as emotional beings, but only within specific, rule-governed contexts, and cited death, a football match and a nightclub scenario as prototypical contexts for the permissible/understandable expression of grief, joy and anger, respectively. However, in the nightclub scenario, the men distanced themselves from the expression of anger as violence, whilst maintaining a masculine subject position. These discursive practices are discussed in terms of the possibilities for effecting change in men's emotional lives.

Molecular dynamics and principal components analysis of human telomeric quadruplex multimers.

Guanine-rich DNA repeat sequences located at the terminal ends of chromosomal DNA can fold in a sequence-dependent manner into G-quadruplex structures, notably the terminal 150–200 nucleotides at the 3' end, which occur as a single-stranded DNA overhang. The crystal structures of quadruplexes with two and four human telomeric repeats show an all-parallel-stranded topology that is readily capable of forming extended stacks of such quadruplex structures, with external TTA loops positioned to potentially interact with other macromolecules. This study reports on possible arrangements for these quadruplex dimers and tetramers, which can be formed from 8 or 16 telomeric DNA repeats, and on a methodology for modeling their interactions with small molecules. A series of computational methods including molecular dynamics, free energy calculations, and principal components analysis have been used to characterize the properties of these higher-order G-quadruplex dimers and tetramers with parallel-stranded topology. The results confirm the stability of the central G-tetrads, the individual quadruplexes, and the resulting multimers. Principal components analysis has been carried out to highlight the dominant motions in these G-quadruplex dimer and multimer structures. The TTA loop is the most flexible part of the model and the overall multimer quadruplex becoming more stable with the addition of further G-tetrads. The addition of a ligand to the model confirms the hypothesis that flat planar chromophores stabilize G-quadruplex structures by making them less flexible.