Microwave‐assisted solid‐phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues

We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C‐terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80‐amino acid peptide sequence of rat NPGL by microwave‐assisted solid‐phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1‐[Bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium‐3‐oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione‐containing redox buffer and 50% acetonitrile. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Genetic isolation of transport signals directing cell surface expression

Membrane proteins represent approximately 30% of the proteome in both prokaryotes and eukaryotes. The spatial localization of membrane-bound proteins is often determined by specific sequence motifs that may be regulated in response to physiological changes, such as protein interactions and receptor signalling. Identification of signalling motifs is therefore important for understanding membrane protein expression, function and transport mechanisms. We report a genetic isolation of novel motifs that confer surface expression. Further characterization showed that SWTY, one class of these isolated motifs with homology to previously reported forward transport motifs, has the ability to both override the RKR endoplasmic reticulum localization signal and potentiate steady-state surface expression. The genetically isolated SWTY motif is functionally interchangeable with a known motif in cardiac potassium channels and an identified motif in an HIV coreceptor, and operates by recruiting 14-3-3 proteins. This study expands the repertoire of and enables a screening method for membrane trafficking signals.     

A universal and reliable assay for molecular sex identification of three‐spined sticklebacks (Gasterosteus aculeatus)

In heterogametic species, biological differences between the two sexes are ubiquitous, and hence, errors in sex identification can be a significant source of noise and bias in studies where sex‐related sources of variation are of interest or need to be controlled for. We developed and validated a universal multimarker assay for reliable sex identification of three‐spined sticklebacks (Gasterosteus aculeatus). The assay makes use of genotype scores from three sex‐linked loci and utilizes Bayesian probabilistic inference to identify sex of the genotyped individuals. The results, validated with 286 phenotypically sexed individuals from six populations of sticklebacks representing all major genetic lineages (cf. Pacific, Atlantic and Japan Sea), indicate that in contrast to commonly used single‐marker‐based sex identification assays, the developed multimarker assay should be 100% accurate. As the markers in the assay can be scored from agarose gels, it provides a quick and cost‐efficient tool for universal sex identification of three‐spined sticklebacks. The general principle of combining information from multiple markers to improve the reliability of sex identification is transferable and can be utilized to develop and validate similar assays for other species.     

Spatial changes in carbon and nitrogen stable isotopes of the plankton food web in a saline lake ecosystem

We investigated spatial changes in the isotope ratios of the plankton food web in Lake Chany, Siberia, Russia, especially at an estuarine transition zone of the lake. The δ¹³C values of particulate organic matter (POM) varied among the sampling sites, and increased with increasing pH of the lake water. This may reflect a shift by phytoplankton from using CO₂ to using bicarbonate for photosynthesis with increasing pH. The δ¹³C values of zooplankton community also changed at each site along with those of the POM. This was indicative of carbon isotope changes of plankton food webs between the stations along an environmental gradient.     

Quantitative genetics of body size and timing of maturation in two nine-spined stickleback (Pungitius pungitius) populations

Due to its influence on body size, timing of maturation is an important life-history trait in ectotherms with indeterminate growth. Comparison of patterns of growth and maturation within and between two populations (giant vs. normal sized) of nine-spined sticklebacks (Pungitius pungitius) in a breeding experiment revealed that the difference in mean adult body size between the populations is caused by differences in timing of maturation, and not by differential growth rates. The fish in small-sized population matured earlier than those from large-sized population, and maturation was accompanied by a reduction in growth rate in the small-sized population. Males matured earlier and at smaller size than females, and the fish that were immature at the end of the experiment were larger than those that had already matured. Throughout the experimental period, body size in both populations was heritable (h² = 0.10–0.64), as was the timing of maturation in the small-sized population (h² = 0.13–0.16). There was a significant positive genetic correlation between body size and timing of maturation at 140 DAH, but not earlier (at 80 or 110 DAH). Comparison of observed body size divergence between the populations revealed that Q _ST exceeded F _ST at older ages, indicating adaptive basis for the observed divergence. Hence, the results suggest that the body size differences within and between populations reflect heritable genetic differences in the timing of maturation, and that the observed body size divergence is adaptive.     

High levels of fluctuating asymmetry in isolated stickleback populations

ABSTRACT: BACKGROUND: Fluctuating asymmetry (FA), defined as small random deviations from the ideal bilateral symmetry, has been hypothesized to increase in response to both genetic and environmental stress experienced by a population. We compared levels of FA in 12 bilateral meristic traits (viz. lateral-line system neuromasts and lateral plates), and heterozygosity in 23 microsatellite loci, among four marine (high piscine predation risk) and four pond (zero piscine predation risk) populations of nine-spined sticklebacks (Pungitius pungitius). RESULTS: Pond sticklebacks had on average three times higher levels of FA than marine fish and this difference was highly significant. Heterozygosity in microsatellite markers was on average two times lower in pond (HE [almost equal to] 0.3) than in marine (HE [almost equal to] 0.6) populations, and levels of FA and heterozygosity were negatively correlated across populations. However, after controlling for habitat effect on heterozygosity, levels of FA and heterozygosity were uncorrelated. CONCLUSIONS: The fact that levels of FA in traits likely to be important in the context of predator evasion were elevated in ponds compared to marine populations suggests that relaxed selection for homeostasis in ponds lacking predatory fish may be responsible for the observed habitat difference in levels of FA. This inference also aligns with the observation that the levels of genetic variability across the populations did not explain population differences in levels of FA after correcting for habitat effect. Hence, while differences in strength of selection, rather than in the degree of genetic stress could be argued to explain habitat differences in levels of FA, the hypothesis that increased FA in ponds is caused by genetic stress cannot be rejected.     

Topical application of a plant extract to different life stages of Trichoplusia ni fails to influence feeding or oviposition behaviour

We have previously determined that larval feeding experience with a feeding/oviposition deterrent modified the feeding responses of larvae and oviposition responses of subsequent moths. These behavioural changes were attributed to learning, but the possibility of chemical legacy could not be ruled out. In the present study, we have topically applied a feeding/oviposition deterrent plant extract from Hoodia gordonii (Masson) Sweet ex Decne (Asclepiadaceae) to larvae, pupae, and adults of Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) to determine whether the feeding response of larvae and oviposition response of subsequent female moths is similarly modified by chemicals applied to the external surface of the insect. Our results indicate that traces of the extract that may be present internally or externally on the larvae do not reduce the feeding deterrent response of larvae. Furthermore, traces of the extract in or on larvae, pupae, or adult moths did not alter oviposition choice of female moths, leading us to discount the role of experience through topical application in this study. The fact that feeding/oviposition choice was only influenced by prior feeding experience of the larvae and not by topical administration suggests that habituation via sensory stimulation through mouthpart chemosensilla is likely a central phenomenon. Continuous exposure of adult moths to the extract over a period of 7 days did not affect the oviposition response of the female moths, ruling out the role of adult experience on host-plant selection in T. ni . To the best of our knowledge, this is the first study to examine the role of experience via topical application of chemicals onto all life stages of the insect except the egg. Chemical legacy may not be playing a role in influencing the oviposition choices of female T. ni moths.     

Advances in soil microbial ecology and the biodiversity

Recent studies on the colony formation of soil bacteria opened the way to categorize soil bacteria into colony forming curve (CFC) groups of different growth rates. A bacterial culture collection comprising organisms from every CFC group is called an ecocollection. Outlines of ECs of paddy soil 1992 and grassland soil 1987 and 1992 were described. Phylogenetic studies by 16S rDNA sequencing showed a great diversity of culture strains of the ecocollections (EC). A set of alternative concepts was proposed; the active and the quiescent forms of bacterial cells in soil. The former is able to be cultivated and thus counted by the plate method, while the latter is not unless it transforms into the former. Based on the results several points required for extensive cataloguing of soil bacteria were noted.