MSP dynamics drives nematode sperm locomotion

Most eukaryotic cells can crawl over surfaces. In general, this motility requires three sequential actions: polymerization at the leading edge, adhesion to the substrate, and retraction at the rear. Recent in vitro experiments with extracts from spermatozoa from the nematode Ascaris suum suggest that retraction forces are generated by depolymerization of the major sperm protein cytoskeleton. Combining polymer entropy with a simple kinetic model for disassembly we propose a model for disassembly-induced retraction that fits the in vitro experimental data. This model explains the mechanism by which disassembly of the cytoskeleton generates the force necessary to pull the cell body forward and suggests further experiments that can test the validity of the models.     

Follicle-stimulating hormone affects metaphase I chromosome alignment and increases aneuploidy in mouse oocytes matured in vitro

Follicle-Stimulating Hormone (FSH) at a wide range of doses is routinely added to culture media during in vitro maturation (IVM) of oocytes, but the effects on oocyte health are unclear. The suggestion that superovulation may cause aneuploidy and fetal abnormalities prompted us to study the potential role of FSH in the genesis of chromosomal abnormalities during meiosis I. Mouse cumulus-oocyte complexes (COCs) isolated from the antral follicles of unprimed, sexually immature B6CBF1 mice were cultured in increasing concentrations of FSH. Following culture, matured oocytes were isolated, spread, stained with DAPI, and the numbers of chromosomes counted. Significantly increased aneuploidy, arising during the first meiotic division, was observed in metaphase II oocytes matured in higher concentrations of FSH (> or =20 ng/ml). The effect of FSH on spindle morphology and chromosome alignment during metaphase I was then explored using immunocytochemistry and three-dimensional reconstruction of confocal sections. High FSH had no effect on gross spindle morphology but did alter chromosome congression during prometaphase and metaphase, with the spread of chromosomes across the spindle at this time being significantly greater in oocytes cultured in 2000 ng/ml compared with 2 ng/ml FSH. Analysis of three-dimensional reconstructions of spindles in oocytes matured in 2000 ng/ml FSH shows that chromosomes are more scattered and farther apart than they are following maturation in 2 ng/ml FSH. These results demonstrate that exposure to high levels of FSH during IVM can accelerate nuclear maturation and induce chromosomal abnormalities and highlights the importance of the judicious use of FSH during IVM.     

IL-4 increases type 2, but not type 1, cytokine production in CD8+ T cells from mild atopic asthmatics

Background

Virus infections are the major cause of asthma exacerbations. CD8⁺ T cells have an important role in antiviral immune responses and animal studies suggest a role for CD8⁺ T cells in the pathogenesis of virus-induced asthma exacerbations. We have previously shown that the presence of IL-4 during stimulation increases the frequency of IL-5-positive cells and CD30 surface staining in CD8⁺ T cells from healthy, normal subjects. In this study, we investigated whether excess IL-4 during repeated TCR/CD3 stimulation of CD8⁺ T cells from atopic asthmatic subjects alters the balance of type 1/type 2 cytokine production in favour of the latter.

Methods

Peripheral blood CD8⁺ T cells from mild atopic asthmatic subjects were stimulated in vitro with anti-CD3 and IL-2 ± excess IL-4 and the expression of activation and adhesion molecules and type 1 and type 2 cytokine production were assessed.

Results

Surface expression of very late antigen-4 [VLA-4] and LFA-1 was decreased and the production of the type 2 cytokines IL-5 and IL-13 was augmented by the presence of IL-4 during stimulation of CD8⁺ T cells from mild atopic asthmatics.

Conclusion

These data suggest that during a respiratory virus infection activated CD8⁺ T cells from asthmatic subjects may produce excess type 2 cytokines and may contribute to asthma exacerbation by augmenting allergic inflammation.     

The role of prey nutritional status in governing protozoan nitrogen regeneration efficiency

Laboratory experiments were conducted to study nitrogen (N) regeneration by the heterotrophic marine dinoflagellate Oxyrrhis marina when ingesting phytoplankton prey of two different species and of two alternative carbon:nitrogen (C:N) ratios. Experiments were conducted in the presence of L-methionine sulfoximine (MSX) which acts as a glutamine synthetase inhibitor. Utilisation by phytoplankton of N regenerated by protozoans and other organisms drives secondary production in marine food webs. However, the rapid utilisation of this N by phytoplankton has previously hampered accurate assessment of the efficiency of protozoan N regeneration. This phenomenon is particularly problematic when the phytoplankton are nutrient stressed and most likely to rapidly utilise N. The use of MSX prevented significant utilisation by phytoplankton of protozoan regenerated N. Hence, by removing the normal pathway of N cycling, we were able to determine the N regeneration efficiency (NRE) of the protozoan. The results suggested that predator NRE could be explained in terms of the relative CN stoichiometry of prey and predator. Using a mathematical model we demonstrated that changing the method used to simulate the NRE of the protozoan trophic level has the potential to markedly modify the predicted dynamics of the simulated microbial food web.     

<Emphasis Type="Italic">C. elegans</Emphasis> serine-threonine kinase KIN-29 modulates TGFβ signaling and regulates body size formation

Background  

In C. elegans there are two well-defined TGFβ-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFβ signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29.     

Parasite regulation by host hormones: an old mechanism of host exploitation?

Recent experimental evidence suggests that parasites can not only evade immune responses actively but also exploit the hormonal microenvironment within the host to favor their establishment, growth and reproduction. The benefit for parasites of hormonal exploitation is so great that they have evolved structures similar to the steroid and protein hormone receptors expressed in upper vertebrates that can bind to the hormonal metabolites synthesized by the host. This strategy is exemplified by two parasites that respond to adrenal steroids and sexual steroids, respectively: Schistosoma mansoni and Taenia crassiceps. Understanding how the host endocrine system can, under certain circumstances, favor the establishment of a parasite, and characterizing the parasite hormone receptors that are involved might aid the design of hormonal analogs and drugs that affect the parasite exclusively.     

Molecular evidence for the existence of natural hybrids in the genus Zygosaccharomyces

26S rDNA D1/D2 sequencing was used to characterise a number of food-associated Zygosaccharomyces rouxii strains held at the National Collection of Yeast Cultures. In the course of this study, four strains (NCYC 1682, NCYC 3042, NCYC 3060 and NCYC 3061) were identified which appeared, based on their D1/D2 sequences, to belong to a novel Zygosaccharomyces species. However, subsequent sequence analysis showed that NCYC 1682, NCYC 3060 and NCYC 3061 possess two highly divergent copies of the nuclear-encoded ADE2, HIS3 and SOD2 genes, indicating these three strains are in fact hybrids. NCYC 3042, however, does appear to represent a novel species which may be hypothesized to have crossed with Z. rouxii and given rise to hybrid strains. Additional approaches to define precise taxonomic status and mechanisms of hybrid genome formation amongst yeast species are discussed.     

Effect of Screening Out Implausible Energy Intake Reports on Relationships between Diet and BMI

Objective: We present an updated method for identifying physiologically implausible dietary reports by comparing reported energy intake (rEI) with predicted energy requirements (pER), and we examine the impact of excluding these reports. Research Methods and Procedures: Adult data from the Continuing Survey of Food Intakes by Individuals 1994 to 1996 were used. pER was calculated from the dietary reference intake equations. Within‐subject variations and errors in rEI [coefficient of variation (CV) ～ 23%] over 2 days (d), pER (CV ～ 11%), and measured total energy expenditure (mTEE; doubly labeled water, CV ～ 8.2%) were propagated, where ±1 SD = . Thus, a report was identified as implausible if rEI was not within 78% to 122% of pER. Multiple cut‐offs between ±1 and ±2 SD were tested. Results: %rEI/pER = 81% in the total sample (n = 6499) and progressively increased to 95% in the ±1 SD sample (n = 2685). The ±1 to 1.4 SD samples yielded rEI‐weight associations closest to the theoretical relationship (mTEE to weight). Weak or spurious diet—BMI associations were present in the total sample; ±1 to 1.4 SD samples showed the strongest set of associations and provided the maximum n while maintaining biological plausibility. Discussion: Our methodology can be applied to different data sets to evaluate the impact of implausible rEIs on health outcomes. Implausible rEIs reduce the overall validity of a sample, and not excluding them may lead to inappropriate conclusions about potential dietary causes of health outcomes such as obesity.     

Probing the mechanism of the Archaeoglobus fulgidus inositol-1-phosphate synthase

myo-Inositol-1-phosphate synthase (mIPS) catalyzes the conversion of glucose-6-phosphate (G-6-P) to inositol-1-phosphate. In the sulfate-reducing archaeon Archaeoglobus fulgidus it is a metal-dependent thermozyme that catalyzes the first step in the biosynthetic pathway of the unusual osmolyte di-myo-inositol-1,1'-phosphate. Several site-specific mutants of the archaeal mIPS were prepared and characterized to probe the details of the catalytic mechanism that was suggested by the recently solved crystal structure and by the comparison to the yeast mIPS. Six charged residues in the active site (Asp225, Lys274, Lys278, Lys306, Asp332, and Lys367) and two noncharged residues (Asn255 and Leu257) have been changed to alanine. The charged residues are located at the active site and were proposed to play binding and/or direct catalytic roles, whereas noncharged residues are likely to be involved in proper binding of the substrate. Kinetic studies showed that only N255A retains any measurable activity, whereas two other mutants, K306A and D332A, can carry out the initial oxidation of G-6-P and reduction of NAD+ to NADH. The rest of the mutant enzymes show major changes in binding of G-6-P (monitored by the 31P line width of inorganic phosphate when G-6-P is added in the presence of EDTA) or NAD+ (detected via changes in the protein intrinsic fluorescence). Characterization of these mutants provides new twists on the catalytic mechanism previously proposed for this enzyme.     

15S-Lipoxygenase-2 mediates arachidonic acid-stimulated adhesion of human breast carcinoma cells through the activation of TAK1, MKK6, and p38 MAPK

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.