The microtubules dance and the spindle poles swing

Using live imaging and computer simulation, Kozlowski et al. (2007) show that an interplay between spindle pole movements, microtubule dynamics, and microtubule bending contribute to asymmetric spindle placement in the C. elegans embryo.     

Stability of a receptor-binding active human immunodeficiency virus type 1 recombinant gp140 trimer conferred by intermonomer disulfide bonding of the V3 loop: differential effects of protein disulfide isomerase on CD4 and coreceptor binding

Stable trimeric forms of human immunodeficiency virus recombinant gp140 (rgp140) are important templates for determining the structure of the glycoprotein to assist in our understanding of HIV infection and host immune response. Such information will aid the design of therapeutic drugs and vaccines. Here, we report the production of a highly stable and trimeric rgp140 derived from a HIV type 1 (HIV-1) subtype D isolate that may be suitable for structural studies. The rgp140 is functional in terms of binding to CD4 and three human monoclonal antibodies (17b, b12, and 2G12) that have broad neutralizing activities against a range of HIV-1 isolates from different subtypes. Treatment of rgp140 with protein disulfide isomerase (PDI) severely restricted 17b binding capabilities. The stable nature of the rgp140 was due to the lack of processing at the gp120/41 boundary and the presence of an intermonomer disulfide bond formed by the cysteines of the V3 loop. Further characterization showed the intermonomer disulfide bond to be a target for PDI processing. The relevance of these findings to the roles of the V3 domain and the timing of PDI action during the HIV infection process are discussed.     

Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which beta(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis.     

Long-term effects of graduated compression stockings on cardiorespiratory performance

The use of graduated compression stockings (GCS) in sport has been increasing in the last years due to their potential positive effects for athletes. However, there is little evidence to support whether these types of garments actually improve cardiorespiratory performance. The aim of this study was to examine the cardiorespiratory responses of GCS during running after three weeks of regular use. Twenty recreational runners performed three tests on different days: test 1) – a 5-min maximal effort run in order to determine the participants’ maximal aerobic speed; and tests 2) and 3) – a fatigue running test of 30 minutes at 80% of their maximal aerobic speed with either GCS or PLACEBO stockings at random. Cardiorespiratory parameters (minute ventilation, heart rate, relative oxygen consumption, relative carbon dioxide production, ventilatory equivalents for oxygen and carbon dioxide, and oxygen pulse) were measured. Before each test in the laboratory, the participants trained with the randomly assigned stockings (GCS or PLACEBO) for three weeks. No significant differences between GCS and PLACEBO were found in any of the cardiorespiratory parameters. In conclusion, the present study provides evidence that running with GCS for three weeks does not influence cardiorespiratory parameters in recreational runners.     

p53-mediated transcriptional regulation and activation of the actin cytoskeleton regulatory RhoC to LIMK2 signaling pathway promotes cell survival

The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIM kinase 2 (LIMK2) are direct p53 target genes induced by genotoxic agents. Although RhoC and LIMK2 have well-established roles in actin cytoskeleton regulation, our results indicate that activation of LIMK2 also has a pro-survival function following DNA damage. LIMK inhibition by siRNA-mediated knockdown or selective pharmacological blockade sensitized cells to radio- or chemotherapy, such that treatments that were sub-lethal when administered singly resulted in cell death when combined with LIMK inhibition. Our findings suggest that combining LIMK inhibitors with genotoxic therapies could be more efficacious than single-agent administration, and highlight a novel connection between actin cytoskeleton regulators and DNA damage-induced cell survival mechanisms.     

The E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of active Rac1

Rac1 small GTPase controls essential aspects of cell biology and is a direct target of numerous bacterial virulence factors. The CNF1 toxin of pathogenic Escherichia coli addresses Rac1 to ubiquitin-proteasome system (UPS). We report the essential role of the tumor suppressor HACE1, a HECT-domain containing E3 ubiquitin-ligase, in the targeting of Rac1 to UPS. HACE1 binds preferentially GTP-bound Rac1 and catalyzes its polyubiquitylation. HACE1 expression increases the ubiquitylation of Rac1, when the GTPase is activated by point mutations or by the GEF-domain of Dbl. RNAi-mediated depletion of HACE1 blocks the ubiquitylation of active Rac1 and increases GTP-bound Rac1 cellular levels. HACE1 antagonizes cell isotropic spreading, a hallmark of Rac1 activation, and is required for endothelial cell monolayer invasion by bacteria. Together, these data establish the role of the HACE1 E3 ubiquitin-ligase in controlling Rac1 ubiquitylation and activity.     

Backpack load affects lower limb muscle activity patterns of female hikers during prolonged load carriage

This study investigated the effect of prolonged load carriage on lower limb muscle activity displayed by female recreational hikers. Electromyography (EMG) signals from vastus lateralis (VL), biceps femoris (BF), semitendinosus (ST), tibialis anterior (TA) and gastrocnemius (GM) were recorded for fifteen female hikers carrying four loads (0%, 20%, 30% and 40% body weight (BW)) over 8 km. Muscle burst duration, muscle burst onset relative to initial contact and integrated EMG signals (iEMG) were calculated to evaluate muscle activity, whereas the shift in mean power frequency (MPF) was used to evaluate muscle fatigue. Increased walking distance significantly decreased the MPF of TA; decreased the iEMG for VL, ST and GM; and shortened VL muscle burst duration. Furthermore, carrying 20–40% BW loads significantly increased VL and GM iEMG and increased BF muscle burst duration, whereas a 40% BW load caused a later VL muscle burst onset. The differences observed in muscle activity with increased load mass seem to be adjustments aimed at maintaining balance and attenuating the increased loads placed on the lower limbs during gait. Based on the changes in muscle activity, a backpack load limit of 30% BW may reduce the risk of lower limb injury for female hikers during prolonged walking.     

Evolution of the hymenopteran megaradiation

The Hymenoptera--ants, bees and wasps--represent one of the most successful but least understood insect radiations. We present the first comprehensive molecular study spanning the entire order Hymenoptera. It is based on approximately 7 kb of DNA sequence from 4 gene regions (18S, 28S, COI and EF-1α) for 116 species representing all superfamilies and 23 outgroup taxa from eight orders of Holometabola. Results are drawn from both parsimony and statistical (Bayesian and likelihood) analyses, and from both by-eye and secondary-structure alignments. Our analyses provide the first firm molecular evidence for monophyly of the Vespina (Orussoidea+Apocrita). Within Vespina, our results indicate a sister-group relationship between Ichneumonoidea and Proctotrupomorpha, while the stinging wasps (Aculeata) are monophyletic and nested inside Evaniomorpha. In Proctotrupomorpha, our results provide evidence for a novel core clade of proctotrupoids, and support for the recently proposed Diaprioidea. An unexpected result is the support for monophyly of a clade of wood-boring sawflies (Xiphydrioidea+Siricoidea). As in previous molecular studies, Orussidae remain difficult to place and are either sister group to a monophyletic Apocrita, or the sister group of Stephanidae within Apocrita. Both results support a single origin of parasitism, but the latter would propose a controversial reversal in the evolution of the wasp-waist. Generally our results support earlier hypotheses, primarily based on morphology, for a basal grade of phytophagous families giving rise to a single clade of parasitic Hymenoptera, the Vespina, from which predatory, pollen-feeding, gall-forming and eusocial forms evolved.