Molecular rearrangements of the extracellular vestibule in NMDAR channels during gating.

Many N-methyl-D-aspartate receptor (NMDAR) channel blockers that have therapeutic potential can be trapped in the closed state. Using a combination of the substituted cysteine accessibility method and open channel blockers, we found that the M3 segment forms the core of the extracellular vestibule, including a deep site for trapping blockers. The M3 segment, as well as more superficial parts of the extracellular vestibule, undergo extensive remodeling during channel closure, but do not define the activation gate, which is located deeper in the pore. Rather, the pore walls lining the extracellular vestibule constrict during channel closure. This movement is essential for coupling ligand binding to activation gate opening and accounts for the different mechanisms of open channel block, including trapping.     

Laboratory bioassays and field-cage trials of <Emphasis Type="Italic">Metarhizium</Emphasis> spp. isolates with field-collected Mormon crickets (<Emphasis Type="Italic">Anabrus simplex</Emphasis>)

The Mormon cricket (MC) is an important pest in the western United States. This study evaluated the virulence in the laboratory of 32 isolates of Metarhizium spp. towards field-collected MCs. Additionally, four isolates were tested in outdoor field-cage studies. All 32 Metarhizium isolates were pathogenic towards the MC (could induce some mortality in the laboratory), including four isolates of the grasshopper-specialist species M. acridum. Virulence varied considerably among the isolates. Field studies, conducted in 2008–2009, showed a statistically significant effect of fungal treatments in both years. Pairwise comparison of the survival curves, however, revealed that, in 2008, three of the isolates did not differ statistically from the non-fungus control treatment. In 2009, all three of the isolates tested had significantly lower survival rates than the control treatment: MCs exposed to M. robertsii isolate DWR 346 had the lowest survival with a LT₅₀ of 16 days. We hypothesize that the poor field performance resulted from a combination of negative environmental effects and isolate selection, and propose that further field studies with additional isolates are needed to identify an efficacious fungal agent for MC biocontrol.     

More plant biomass results in more offspring production in annuals,or does it?

Competitive ability in plants has been previously measured almost exclusively in terms of traits related to growth (biomass) or plant size. In this study, however, we used a multi‐species competition experiment with six annuals to measure relative competitive ability in terms of reproductive output, i.e. the number of offspring produced for the next generation. Under greenhouse conditions, plants of each species were started in pots from germinating seeds and were grown singly (free of competition) and at high density in both monocultures and in mixtures with all study species. Several traits traditionally regarded as determinants of competitive ability in plants were recorded for each species grown singly, including: seed mass, germination time, early growth rate and potential plant size (biomass and height). Under competition, several traits were recorded as indicators of relative performance in both monocultures and mixtures, including: biomass of survivors, total number of survivors, number of reproductive survivors, and reproductive output (total seed production) of the survivors. As expected, species that grew to a larger biomass in isolation had higher seed production in isolation. However, none of the traditional plant growth/size‐related traits, measured either in isolation or under competition, could predict between species variation in reproductive output under competition in either monocultures or mixtures. In mixtures, 97% of this variation in reproductive output could be explained by between‐species variation in the number of reproductive survivors. The results indicate that traits measured on plants grown singly may be poor predictors of reproductive output under competition, and that species’ rank order of competitive ability in terms of the biomass of survivors may bear no relationship to their rank order in terms of the number of offspring produced by these survivors. This has important implications for the interpretation of mechanisms of species coexistence and community assembly within vegetation.     

THE EFFECT OF GENETICALLY BASED DIFFERENCES IN SEED SIZE ON SEEDLING SURVIVAL IN ARABIDOPSIS THALIANA (BRASSICACEAE)

The relationship between genetically different seed sizes and seedling survival under severe nutrient deprivation was determined by comparing ten inbred lines of Arabidopsis thaliana. Seedlings were supplied with only sterile distilled water, and the number of days between germination and death (complete chlorosis) was recorded. Seedlings from genotypes with larger seeds survived longer than seedlings from genotypes with smaller seeds. These results suggest a genetically based adaptive significance of larger seed size resulting from a greater seedling tolerance of nutrient deprivation. This may confer a potentially important selective advantage when nutrient deprivation is the result of a low resource supply in the environment, or the result of nutrient depletion by neighbors.     

Functional roles of TAP and tapasin in the assembly of M3-N-formylated peptide complexes

H2-M3 is a MHC class Ib molecule with a high propensity to bind N-formylated peptides. Due to the paucity of endogenous Ag, the majority of M3 is retained in the endoplasmic reticulum (ER). Upon addition of exogenous N-formylated peptides, M3 trafficks rapidly to the cell surface. To understand the mechanism underlying Ag presentation by M3, we examined the role of molecular chaperones in M3 assembly, particularly TAP and tapasin. M3-specific CTLs fail to recognize cells isolated from both TAP-deficient (TAP(o)) and tapasin-deficient mice, suggesting that TAP and tapasin are required for M3-restricted Ag presentation. Impaired M3 expression in TAP(o) mice is due to instability of the intracellular pool of M3. Addition of N-formylated peptides to TAP(o) cells stabilizes M3 in the ER and partially restores surface expression. Surprisingly, significant amounts of M3 are retained in the ER in tapasin-deficient mice, even in the presence of N-formylated peptides. Our results define the role of TAP and tapasin in the assembly of M3-peptide complexes. TAP is essential for stabilization of M3 in the ER, whereas tapasin is critical for loading of N-formylated peptides onto the intracellular pool of M3. However, neither TAP nor tapasin is required for ER retention of empty M3.     

Surface-tethered DNA complexes for enhanced gene delivery

Overcoming the barriers to efficient gene transfer is a fundamental goal of biotechnology. A versatile approach to enhance the delivery of nonviral DNA involves complexation with cationic polymers, which can be designed to overcome the barriers to effective gene transfer. More recently, DNA release from a polymer substrate or scaffold has been shown to enhance gene transfer, likely by increasing DNA concentrations in the cell microenvironment. We propose a novel approach that combines these two strategies in which cationic polymer/DNA complexes are tethered to a substrate that supports cell adhesion. The cationic polymers package the DNA for efficient internalization and the surface tethering functions to maintain elevated concentrations in the cell microenvironment for cells adhered to the substrate. The cationic polymer polylysine (degree of polymerization equal to 19 or 150) was modified with biotin groups, which was confirmed by mass spectrometry and biochemical analysis. Complex formation of DNA with biotinylated-polylysine, or mixtures of biotinylated and nonbiotinylated polylysines, was confirmed by gel electrophoresis. Plasmid DNA encoding for the reporter gene beta-galactosidase was complexed with different mixtures of biotinylated and nonbiotinylated polylysine and incubated on neutravidin (nonglycosylated avidin)-coated surfaces. DNA surface densities ranging from 0.1 to 4.3 microg/cm2 were observed and found to be a function of the number of biotin groups, the molecular weight of the polylysine, and the amount of DNA. HEK293T or NIH/3T3 cells were then seeded onto the DNA-modified surfaces, and transfection was quantified at 48 and 96 h. Transfection by the DNA surfaces was observed with both cell lines, and expression levels up to 100 fold greater than bulk delivery of the complexes was obtained. Transfection was found to be a function of the surface DNA quantities and the number of tethers on the complex. Transfected cells were observed only in the region in which DNA complexes were tethered, suggesting that the location of transfected cells can be specifically controlled. Surface tethering of DNA represents a promising approach to enhancing gene transfer and spatially controlling gene delivery, which may have applications to a multitude of fields ranging from tissue engineering to functional genomics.     

Mutation of a novel gene results in abnormal development of spermatid flagella,loss of intermale aggression and reduced body fat in mice

ROSA22 male mice are sterile due to a recessive gene-trap mutation that affects development of the spermatid flagellum. The defect involves the flagellar axoneme, which becomes unstable around the time of its assembly. Despite a subsequent complete failure in flagellar assembly, development of the spermatid head appears normal and the spermatid head is released at the correct stage in spermatogenesis. The mutation is pleiotropic. Although ROSA22 homozygote males have normal levels of circulating testosterone and display normal mating behavior, they do not exhibit intermale aggressive behavior and have reduced body fat. The mutated gene (Gtrgeo22) maps to mouse chromosome 10 and is closely flanked by two known genes, Madcam1 and Cdc34. Ribonuclease protection analysis indicates that expression of the flanking genes is unaffected by the mutation. Gtrgeo22 is expressed at low levels in epithelial cells in several tissues, as well as in testis and brain. Analysis of the peptide coding sequence suggests that Gtrgeo22 encodes a novel transmembrane protein, which contains dileucine and tyrosine-based motifs involved in intracellular sorting of transmembrane proteins. Analysis of the Gtrgeo22 gene product should provide novel insight into the molecular basis for intermale aggression and sperm flagellar development.     

Bacterial recovery from ancient glacial ice

Ice that forms the bottom 18 m of a 308 m ice core drilled from the Guliya ice cap on the Qinghan-Tibetan plateau in Western China is over 750000 years old and is the oldest glacial ice known to date. Fourteen bacterial isolates have been recovered from samples of this ice from approximately 296 m below the surface (mbs). Based on 16S rDNA sequences, these are members of the alpha- and beta-proteobacterial, actinobacterial and low-G + C Gram-positive bacterial lineages. 16S rDNA molecules have also been amplified directly, cloned and sequenced from the ice-core melt water. These originated from Pseudomonas and Acinetobacter gamma-proteobacterial species. These results demonstrate that bacteria can be recovered from water ice that has frozen for time periods relevant to biological survival through terrestrial ice ages or during interplanetary transport.     

Long‐term sound and movement recording tags to study natural behavior and reaction to ship noise of seals

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Improved fertilization and embryo development resulting in birth of live piglets after intracytoplasmic sperm injection and in vitro culture in a cysteine-supplemented medium

The effects of cysteine treatment on fertilization rate, intracellular concentration of glutathione, and embryo development in vitro and after embryo transfer were examined following intracytoplasmic sperm injection (ICSI) of in vitro-matured porcine oocytes using a piezo drive unit. Culture of presumed zygotes after ICSI with 1.71-3.71 mM cysteine for 3-12h improved (P<0.05) fertilization rates as compared to treatment with 0.57 mM cysteine or to controls (0mM) (56 to 68%, 48%, 35%, respectively). Extension of treatment time with cysteine beyond 3h did not further increase fertilization rates, suggesting that cysteine promoted early developmental events after ICSI (e.g. decondensation of sperm chromatin). There was no effect of cysteine supplementation on oocyte glutathione levels after ICSI. Pretreatment of spermatozoa for 3h with 1.71 mM cysteine did not improve fertilization rates. The incidence of blastocysts formation when cultured in 1.71 mM cysteine for 3h after ICSI was 31%, which was higher (P<0.05) than controls (18%). Transfer of 20-38 embryos cultured with 1.71 mM cysteine for 3h after ICSI to each of seven recipients yielded three deliveries with an average litter size of 4.0. We concluded that cysteine supplementation for the first 3h after ICSI improved fertilization and embryo development rates, with no influence on glutathione levels in oocytes, and that the cysteine-treated ICSI embryos developed to full term. The study also showed that porcine oocytes matured in a chemically defined medium had the ability for full-term development after piezo-ICSI without additional treatments for oocyte activation.