Human enhancer of invasion-cluster, a coiled-coil protein required for passage through mitosis

In a cross-species overexpression approach, we used the pseudohyphal transition of Saccharomyces cerevisiae as a model screening system to identify human genes that regulate cell morphology and the cell cycle. Human enhancer of invasion-cluster (HEI-C), encoding a novel evolutionarily conserved coiled-coil protein, was isolated in a screen for human genes that induce agar invasion in S. cerevisiae. In human cells, HEI-C is primarily localized to the spindle during mitosis. Depletion of HEI-C in vivo with short interfering RNAs results in severe mitotic defects. Analysis by immunofluorescence, flow cytometry analysis, and videomicroscopy indicates that HEI-C-depleted cells form metaphase plates with normal timing after G(2)/M transition, although in many cases cells have disorganized mitotic spindles. Subsequently, severe defects occur at the metaphase-anaphase transition, characterized by a significant delay at this stage or, more commonly, cellular disintegration accompanied by the display of classic biochemical markers of apoptosis. These mitotic defects occur in spite of the fact that HEI-C-depleted cells retain functional cell cycle checkpoints, as these cells arrest normally following nocodazole or hydroxyurea treatment. These results place HEI-C as a novel regulator of spindle function and integrity during the metaphase-anaphase transition.     

A polymorphic protease-activated receptor 2 (PAR2) displaying reduced sensitivity to trypsin and differential responses to PAR agonists

Protease-activated receptor 2 (PAR2) is a trypsin-activated member of a family of G-protein-coupled PARs. We have identified a polymorphic form of human PAR2 (PAR(2)F240S) characterized by a phenylalanine to serine mutation at residue 240 within extracellular loop 2, with allelic frequencies of 0.916 (Phe(240)) and 0.084 (Ser(240)) for the wild-type and mutant alleles, respectively. Elevations in intracellular calcium were measured in permanently transfected cell lines expressing the receptors. PAR(2)F240S displayed a significant reduction in sensitivity toward trypsin ( approximately 3.7-fold) and the PAR2-activating peptides, SLIGKV-NH(2) ( approximately 2.5-fold) and SLIGRL-NH(2) ( approximately 2.8-fold), but an increased sensitivity toward the selective PAR2 agonist, trans-cinnamoyl-LIGRLO-NH(2) ( approximately 4-fold). Increased sensitivity was also observed toward the selective PAR-1 agonist, TFLLR-NH(2) ( approximately 7-fold), but not to other PAR-1 agonists tested. Furthermore, we found that TLIGRL-NH(2) and a PAR4-derived peptide, trans-cinnamoyl-YPGKF-NH(2), were selective PAR(2)F240S agonists. By introducing the F240S mutation into rat PAR2, we observed shifts in agonist potencies that mirrored the human PAR(2)F240S, suggesting that Phe(240) is involved in determining agonist specificity of PAR2. Finally, differences in receptor signaling were paralleled in a cell growth assay. We suggest that the distinct pharmacological profile induced by this polymorphism will have important implications for the design of PAR-targeted agonists/antagonists and may contribute to, or be predictive of, an inflammatory disease.     

Transient gene expression from yeast artificial chromosome DNA in mammalian cells is enhanced by adenovirus.

The introduction of high molecular weight DNA into mammalian cells is useful for gene expression studies. However, current transfection strategies are inefficient, necessitating propagation of stable DNA transformants prior to analysis of gene expression. Here we demonstrate that transient lipid-mediated DNA transfection can be used to assess gene expression from yeast artificial chromosomes (YACs) containing the 230 kb cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and Escherichia coli lacZ . We also show that psoralen-UV inactivated adenovirus significantly enhances transfection efficiency. The ability to deliver high molecular weight DNA using lipid-mediated transfection should expedite the analysis of large human genes contained within artificial chromosome vectors.     

Rational development of beta-peptide inhibitors of human cytomegalovirus entry

Human cytomegalovirus (HCMV) is a pervasive and significant pathogen. At present, there is no HCMV vaccine, and the available drugs target only replication events. Thus, new therapeutic strategies are needed. HCMV fusion appears to require interactions of alpha-helical regions in viral surface glycoproteins gB and gH. Oligomers of beta-amino acids ("beta-peptides") are attractive unnatural scaffolds for mimicry of specific protein surfaces, because beta-peptides adopt predictable helical conformations and resist proteolysis. Here, we report the development of beta-peptides designed to mimic the gB heptad repeat and block HCMV entry. The most potent beta-peptide inhibits HCMV infection in a cell based-assay with an IC50 of approximately 30 microm. Consistent with our structure-based design strategy, inhibition is highly specific for HCMV relative to other related viruses. Mechanistic studies indicate that inhibitory beta-peptides act by disrupting membrane fusion. Our findings raise the possibility that beta-peptides may provide a general platform for development of a new class of antiviral agents and that inhibitory beta-peptides will constitute new tools for elucidating viral entry mechanisms.     

Variation in palaeo-shorelines explains contemporary population genetic patterns of rocky shore species

Processes driving and maintaining disjunct genetic populations in marine systems are poorly understood, owing to a lack of evidence of hard barriers that could have shaped patterns of extant population structure. Here, we map two genetically divergent lineages of an obligate rocky shore fish, Clinus cottoides, and model sea-level change during the last 110 000 years to provide the first evidence of a vicariant event along the southern coastline of Africa. Results reveal that lowered sea levels during glacial periods drastically reduced rocky intertidal habitat, which may have isolated populations in two refugia for at least 40 000 years. Contemporary coastal dynamics and oceanography explain secondary contact between lineages. This scenario provides an explanation for the origin of population genetic breaks despite a lack of obvious present-day geographical barriers and highlights the need for including palaeo-oceanography in unravelling extant population patterns.     

Alpha-chymotrypsin catalysis in imidazolium-based ionic liquids

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by alpha-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF(6)]), and in combination with supercritical carbon dioxide (SC-CO(2)). The activity of alpha-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. alpha-Chymotrypsin freeze-dried with K(2)HPO(4), KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF(6)] or [omim][PF(6)] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF(6)] than [bmim][PF(6)]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO(2). These results indicate that [bmim][PF(6)] and [omim][PF(6)] provide a relatively polar environment, which can be modified with nonpolar SC-CO(2) to optimize enzyme activity.     

Plant–herbivore–parasitoid interactions in an experimental freshwater tritrophic system: higher trophic levels modify competitive interactions between invasive macrophytes

Hydrobiologia - Natural enemies are known to modify competitive hierarchies among terrestrial plants. Here we examine whether the same applies to freshwater systems. Lagarosiphon major...     

Nest site choice by the intertidal spider Desis formidabilis (Araneae: Desidae) and nest utilisation by its hymenopteran egg parasitoid

1. Echthrodesis lamorali Masner, 1968 is the only known parasitoid of the eggs of the intertidal rocky shore spider Desis formidabilis O.P. Cambridge 1890 and is endemic to a small area of South Africa. 2. The abundance of spider nests and parasitoid presence were assessed in relation to their in‐ and between‐shore location at multiple sites within the distribution of E. lamorali along the Cape Peninsula (Western Cape, South Africa). 3. Desis formidabilis nests were more abundant in the mid‐shore zone than higher up or lower down the shore. Spider population sizes also differed between collection sites, with higher numbers recorded on the cooler western coast of the peninsula. 4. Evidence of parasitoid activity was recorded in 43.31% of the 127 nests and 13.85% of the 592 egg sacs they contained. 5. Where parasitoids gained entry to a spider egg sac, oviposition took place into all of the eggs present. 6. Incidence of wasp activity was positively correlated with spider nest concentration, not with height up the shore, suggesting that both the host and parasitoid are tolerant of salt‐water inundation. 7. These results should assist managers of the Table Mountain National Park, in which the full distribution of E. lamorali falls, to better understand this component of rocky shore community dynamics.