Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa

A reduction in plasma membrane cholesterol is one of the early events that either triggers or is closely associated with capacitation of mammalian spermatozoa. In this investigation, we have examined the effects of cholesterol efflux on tyrosine phosphorylation, lipid diffusion, and raft organization in boar spermatozoa. Results show that a low level of cholesterol efflux, mediated by 5 mM methyl-beta-cyclodextrin (MBCD), enhances capacitation and induces phosphorylation of two proteins at 26 and 15 kDa without affecting sperm viability. Lipid diffusion rates under these conditions are largely unaffected except when cholesterol efflux is excessive. Low-density Triton X100-insoluble complexes (lipid rafts) were isolated from spermatozoa and found to have a restricted profile of proteins. Capacitation-associated cholesterol efflux has no effect on raft composition, but cholesterol depletion destabilizes them completely and phosphorylation is suppressed. During MBCD-mediated capacitation, the distribution of GM1 gangliosides on spermatozoa changes in a sequential manner from overlying the sperm tail to clustering on the sperm head. It is concluded that there is a safe window for removal of plasma membrane cholesterol from spermatozoa within which protein phosphorylation and polarized migration of lipid rafts take place. A preferential loss of cholesterol from the nonraft pool may be the stimulus that promotes raft clustering over the anterior sperm head.     

A trophic pathway from biogenic methane supports fish biomass in a temperate lake ecosystem

Although some primary consumers such as chironomid larvae are known to exploit methane‐derived carbon via microbial consortia within aquatic food webs, few studies have traced the onward transfer of such carbon to their predators. The ruffe Gymnocephalus cernuus is a widespread benthivorous fish which feeds predominantly on chironomid larvae and is well adapted for foraging at lower depths than other percids. Therefore, any transfer of methanogenic carbon to higher trophic levels might be particularly evident in ruffe. We sampled ruffe and chironomid larvae from the littoral, sub‐littoral and profundal areas of Jyväsjärvi, Finland, a lake which has previously been shown to contain chironomid larvae exhibiting the very low stable carbon isotope ratios indicative of methane exploitation. A combination of fish gut content examination and stable isotope analysis was used to determine trophic linkages between fish and their putative prey. Irrespective of the depth from which the ruffe were caught, their diet was dominated by chironomids and pupae although the proportions of taxa changed. Zooplankton made a negligible contribution to ruffe diet. A progressive decrease in δ¹³C and δ¹⁵N values with increasing water column depth was observed for both chironomid larvae and ruffe, but not for other species of benthivorous fish. Furthermore, ruffe feeding at greater depths were significantly larger than those feeding in the littoral, suggesting an ontogenetic shift in habitat use, rather than diet, as chironomids remained the predominant prey item. The outputs from isotope mixing models suggested that the incorporation of methane‐derived carbon to larval chironomid biomass through feeding on methanotrophic bacteria increased at greater depth, varying from 0% in the littoral to 28% in the profundal. Using these outputs and the proportions of littoral, sub‐littoral or profundal chironomids contributing to ruffe biomass, we estimated that 17% of ruffe biomass in this lake was ultimately derived from chemoautotrophic sources. Methanogenic carbon thus supports considerable production of higher trophic levels in lakes.     

Effects of long‐term grazing management on sand dune vegetation of high conservation interest

Question: Can long‐term grazing management maintain and restore species‐rich sand dune plant communities within a sand dune site of high conservation interest? Location: Newborough Warren, North Wales, UK. Methods: Vegetation changes that occurred between 1987 and 2003, subsequent to grazing by domestic livestock being introduced to the site after decades with little or no stock grazing, were analysed using data collected from permanent monitoring quadrats over a 16‐year period. Results: At the plant community level, grazing brought about a shift from a tall‐grass dominated, species‐poor community to a more species‐rich community in the dry dunes, but did not change community type in dune slacks. However, at the species level, grazing enhanced the abundance of some desired perennial, annual and biennial species, graminoids and bryophytes in both habitat types. The increased frequency of positive indicator species for habitat condition suggests that grazing was beneficial for species of conservation interest. Ellenberg nitrogen (N) values decreased after grazing in dry habitats but showed no long‐term change independent of grazing, suggesting no increase in site fertility over the study period. Surprisingly, light (L) values also decreased in the dry dunes after grazing. Conclusions: Long‐term grazing management can play an important role for the conservation of dune communities and associated species. Because of its positive effects on species diversity, plant communities and habitat condition in sand dunes, livestock grazing is recommended for conservation management.     

Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.     

Homology between hydroxybutyryl and hydroxyacyl coenzyme A dehydrogenase enzymes from Clostridium acetobutylicum fermentation and vertebrate fatty acid beta-oxidation pathways.

The enzymes NAD-dependent beta-hydroxybutyryl coenzyme A dehydrogenase (BHBD) and 3-hydroxyacetyl coenzyme A (3-hydroxyacyl-CoA) dehydrogenase are part of the central fermentation pathways for butyrate and butanol production in the gram-positive anaerobic bacterium Clostridium acetobutylicum and for the beta oxidation of fatty acids in eucaryotes, respectively. The C. acetobutylicum hbd gene encoding a bacterial BHBD was cloned, expressed, and sequenced in Escherichia coli. The deduced primary amino acid sequence of the C. acetobutylicum BHBD showed 45.9% similarity with the equivalent mitochondrial fatty acid beta-oxidation enzyme and 38.4% similarity with the 3-hydroxyacyl-CoA dehydrogenase part of the bifunctional enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase from rat peroxisomes. The pig mitochondrial 3-hydroxyacyl-CoA dehydrogenase showed 31.7% similarity with the 3-hydroxyacyl-CoA dehydrogenase part of the bifunctional enzyme from rat peroxisomes. The phylogenetic relationship between these enzymes supports a common evolutionary origin for the fatty acid beta-oxidation pathways of vertebrate mitochondria and peroxisomes and the bacterial fermentation pathway.     

Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens

Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C. The enterotoxin was detected by serological and biological assays. Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage. The culture maintained enterotoxigenicity throughout cultivation in a continuous system. The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium. No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.     

Correlates of species richness in mammals: body size, life history, and ecology

We present the most extensive examination to date of proposed correlates of species richness. We use rigorous phylogenetic comparative techniques, data for 1,692 mammal species in four clades, and multivariate statistics to test four hypotheses about species richness and compare the evidence for each. Overall, we find strong support for the life-history model of diversification. Species richness is significantly correlated with shorter gestation period in the carnivores and large litter size in marsupials. These traits and short interbirth intervals are also associated with species richness in a pooled analysis of all four clades. Additionally, we find some support for the abundance hypotheses in different clades of mammals: abundance correlates positively with species richness in primates but negatively in microchiropterans. Our analyses provide no evidence that mammalian species richness is associated with body size or degree of sexual dimorphism.     

DNA abasic lesions in a different light: solution structure of an endogenous topoisomerase II poison

Topoisomerase II is the target for several anticancer drugs that "poison" the enzyme and convert it to a cellular toxin by increasing topoisomerase II-mediated DNA cleavage. In addition to these "exogenous topoisomerase II poisons," DNA lesions such as abasic sites act as "endogenous poisons" of the enzyme. Drugs and lesions are believed to stimulate DNA scission by altering the structure of the double helix within the cleavage site of the enzyme. However, the structural alterations that enhance cleavage are unknown. Since abasic sites are an intrinsic part of the genetic material, they represent an attractive model to assess DNA distortions that lead to altered topoisomerase II function. Therefore, the structure of a double-stranded dodecamer containing a tetrahydrofuran apurinic lesion at the +2 position of a topoisomerase II DNA cleavage site was determined by NMR spectroscopy. Three major features distinguished the apurinic structure ( = 0.095) from that of wild-type ( = 0.077). First, loss of base stacking at the lesion collapsed the major groove and reduced the distance between the two scissile phosphodiester bonds. Second, the apurinic lesion induced a bend that was centered about the topoisomerase II cleavage site. Third, the base immediately opposite the lesion was extrahelical and relocated to the minor groove. All of these structural alterations have the potential to influence interactions between topoisomerase II and its DNA substrate.     

Hemps, a novel EGF-like protein, plays a central role in ascidian metamorphosis

All chordates share several characteristic features including a dorsal hollow neural tube, a notochord, a pharynx and an endostyle. Unlike other chordate taxa, ascidians have a biphasic life-history with two distinct body plans. During metamorphosis, the larval nerve cord and notochord degenerate and the pharyngeal gill slits and endostyle form. While ascidians, like other marine invertebrates, metamorphose in response to specific environmental cues, it remains unclear how these cues trigger metamorphosis. We have identified a novel gene (Hemps) which encodes a protein with a putative secretion signal sequence and four epidermal growth factor (EGF)-like repeats which is a key regulator of metamorphosis in the ascidian Herdmania curvata. Expression of Hemps increases markedly when the swimming tadpole larva becomes competent to undergo metamorphosis and then during the first 24 hours of metamorphosis. The Hemps protein is localised to the larval papillae and anterior epidermis of the larva in the region known to be required for metamorphosis. When the larva contacts an inductive cue the protein is released, spreading posteriorly and into the tunic as metamorphosis progresses. Metamorphosis is blocked by incubating larvae in anti-Hemps antibodies prior to the addition of the cue. Addition of recombinant Hemps protein to competent larvae induces metamorphosis in a concentration-dependent manner. A subgroup of genes are specifically induced during this process. These results demonstrate that the Hemps protein is a key regulator of ascidian metamorphosis and is distinct from previously described inducers of this process in terrestrial arthropods and aquatic vertebrates.     

Rapid Sample Processing for Detection of Food-Borne Pathogens via Cross-Flow Microfiltration

This paper reports an approach to enable rapid concentration and recovery of bacterial cells from aqueous chicken homogenates as a preanalytical step of detection. This approach includes biochemical pretreatment and prefiltration of food samples and development of an automated cell concentration instrument based on cross-flow microfiltration. A polysulfone hollow-fiber membrane module having a nominal pore size of 0.2 μm constitutes the core of the cell concentration instrument. The aqueous chicken homogenate samples were circulated within the cross-flow system achieving 500- to 1,000-fold concentration of inoculated Salmonella enterica serovar Enteritidis and naturally occurring microbiota with 70% recovery of viable cells as determined by plate counting and quantitative PCR (qPCR) within 35 to 45 min. These steps enabled 10 CFU/ml microorganisms in chicken homogenates or 10² CFU/g chicken to be quantified. Cleaning and sterilizing the instrument and membrane module by stepwise hydraulic and chemical cleaning (sodium hydroxide and ethanol) enabled reuse of the membrane 15 times before replacement. This approach begins to address the critical need for the food industry for detecting food pathogens within 6 h or less.