Solution structure of the calmodulin‐like C‐terminal domain of Entamoeba α‐actinin2

Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross‐links, or caps the filament ends have been identified and the actin cross‐linker α‐actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α‐actinin is believed to be required for infection. To better understand the role of α‐actinin in the infectious process we have determined the solution structure of the C‐terminal calmodulin‐like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium‐binding EF‐hand motifs, connected with a mobile linker. Proteins 2016; 84:461–466. © 2016 Wiley Periodicals, Inc.     

A nondestructive method for extracting maternally derived egg yolk carotenoids

ABSTRACT.   Maternally deposited carotenoids are a prominent component of egg yolk and are vital for the development and growth of the embryo. In most studies of avian yolk carotenoids, eggs are destructively sampled and this may limit both the number of clutches studied and the research questions addressed. We describe an empirical field trial for a nondestructive biopsy method to extract small samples (0.05 ml) of egg yolk for high-performance liquid chromatography (HPLC) analysis of yolk carotenoid concentrations. We sampled 180 clutches ( N = 44 biopsies) of two species of introduced thrushes (genus Turdus ) from agricultural habitats in central North Island, New Zealand. Once the protocol was established, all biopsied eggs from clutches that were not depredated or deserted before candling were found to be developing normally after 3–5 d of incubation ( N = 28) and all hatched. Biopsy samples (>0.02 g) produced concentrations of yolk carotenoids (and variances) that were statistically indistinguishable from whole yolk destructive samples. In addition, our samples (>0.02 g) confirmed previously reported differences in yolk carotenoid concentrations between the two thrush species and revealed a significant decline in yolk carotenoid concentration with laying order. Further examination of how variability in yolk carotenoid concentration and identity influences offspring sex, success, and survival or, later in life, reproductive success and ability to efficiently incorporate dietary carotenoids into both integument and immune tissues will require larger sample sizes. Studies to date have been restricted by the number of destructive samples that investigators are willing (or permitted) to obtain from wild species. Thus, we hope that our nondestructive method of sampling yolk will promote further examination of the links between carotenoid uptake from the environment and maternal investment in the avian yolk.     

Hmi1p from Saccharomyces cerevisiae mitochondria is a structure-specific DNA helicase

Hmi1p is a Saccharomyces cerevisiae mitochondrial DNA helicase that is essential for the maintenance of functional mitochondrial DNA. Hmi1p belongs to the superfamily 1 of helicases and is a close homologue of bacterial PcrA and Rep helicases. We have overexpressed and purified recombinant Hmi1p from Escherichia coli and describe here the biochemical characteristics of its DNA helicase activities. Among nucleotide cofactors, the DNA unwinding by Hmi1p was found to occur efficiently only in the presence of ATP and dATP. Hmi1p could unwind only the DNA substrates with a 3'-single-stranded overhang. The length of the 3'-overhang needed for efficient targeting of the helicase to the substrate depended on the substrate structure. For substrates consisting of duplex DNA with a 3'-single-stranded DNA overhang, at least a 19-nt 3'-overhang was needed. In the case of forked substrates with both 3'- and 5'-overhangs, a 9-nt 3'-overhang was sufficient provided that the 5'-overhang was also 9 nt in length. In flap-structured substrates mimicking the chain displacement structures in DNA recombination process, only a 5-nt 3'-single-stranded DNA tail was required for efficient unwinding by Hmi1p. These data indicate that Hmi1p may be targeted to a specific 3'-flap structure, suggesting its possible role in DNA recombination.     

Size and temperature dependent foraging capacities and metabolism: consequences for winter starvation mortality in fish

The foraging related capacities, energy requirements and the ability of individuals to withstand starvation are strongly dependent on body size and temperature. In this study, we estimated size-dependent foraging rates and critical resource density (CRD) in small Arctic char ( Salvelinus alpinus ) under winter conditions and compared these with previous observations under summer conditions. We investigated if starvation mortality is size-dependent in the laboratory, and we assessed the potential for winter growth and the occurrence and size dependency of winter mortality both in a large scale pond experiment and in natural lakes. The efficiency of foraging on macroinvertebrates increased with size but was lower at 4 than 12°C, still CRD was lower at 4 than 12°C as metabolic rates decreased faster than foraging efficiency with temperature. When starved, small char died before large and at rates which suggest that YOY char need to feed during winter to avoid starvation. Results from both our pond experiment and field study indicate that survival of YOY char over winter is high, despite severe winter conditions, because YOY char are able to feed and grow during winter. In seasonal environments with declining resources, the size scaling and temperature dependency of foraging and metabolic demands may provide conditions which can favour either small or large individuals. This size advantage dichotomy relates to that larger individual's by having a higher CRD are more likely to start starving, but once resource levels are below CRD for all size classes, small individuals starve to death at a higher rate. Negative size-dependent winter mortality from starvation is suggested to be more pronounced in species that are not adapted to feed at low temperatures and in species feeding on zooplankton, since zooplankton abundance, in contrast to macroinvertebrate abundance, is generally low during winter.     

High genetic diversity of Vibrio cholerae in the European lake Neusiedler See is associated with intensive recombination in the reed habitat and the long‐distance transfer of strains

Coastal marine Vibrio cholerae populations usually exhibit high genetic diversity. To assess the genetic diversity of abundant V. cholerae non‐O1/non‐O139 populations in the Central European lake Neusiedler See, we performed a phylogenetic analysis based on recA, toxR, gyrB and pyrH loci sequenced for 472 strains. The strains were isolated from three ecologically different habitats in a lake that is a hot‐spot of migrating birds and an important bathing water. We also analyzed 76 environmental and human V. cholerae non‐O1/non‐O139 isolates from Austria and other European countries and added sequences of seven genome‐sequenced strains. Phylogenetic analysis showed that the lake supports a unique endemic diversity of V. cholerae that is particularly rich in the reed stand. Phylogenetic trees revealed that many V. cholerae isolates from European countries were genetically related to the strains present in the lake belonging to statistically supported monophyletic clades. We hypothesize that the observed phenomena can be explained by the high degree of genetic recombination that is particularly intensive in the reed stand, acting along with the long distance transfer of strains most probably via birds and/or humans. Thus, the Neusiedler See may serve as a bioreactor for the appearance of new strains with new (pathogenic) properties.     

Reporter-linked monitoring of transgene expression in living cells using the ecdysone-inducible promoter system

Inducible promoter systems such as the ecdysone-inducible system or the tetracycline-regulated expression systems have proven to be powerful tools in studying gene function. In practice, such systems have met with the difficulty that either the vector expressing the transactivator gene or the vector carrying the response element are frequently silenced by flanking genomic sequences after stable integration. In order to identify those cells in a heterogeneous population in which a transgene is expressed from an ecdysone-inducible promoter, we have created the vector p2ER-EGFP/mcs that contains two ecdysone-inducible expression cassettes in tandem. Using two reporter genes, lacZ and green fluorescent protein (EGFP), we demonstrate that the expression of both genes can be co-induced from a very low baseline in CHO cells expressing the modified ecdysone receptor and the retinoid X receptor. The expression of EGFP and lacZ from vector p2ER-EGFP/lacZ follows the same Muristerone A concentration-dependence as that of EGFP from vector pER-EGFP, indicating that the juxtaposition of the two inducible promoters in vector p2ER-EGFP/mcs does not cause cross interference between them. We suggest that this modification of the ecdysone-inducible promoter system will allow for the visual control of the induced expression of other genes by Muristerone A.     

In Vivo Studies on the Extracellular, and Veratrine-Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion

The effects of corticostriatal deafferentation (decortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine-releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra- and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. gamma-Aminobutyric acid (GABA), taurine (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month post-lesion. The intracellular pool of Glu and Asp was also reduced in kainate-lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of noneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion-induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood-brain barrier transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular Origins of Endogenous Amino Acids Released into the Extracellular Fluid of the Rat Striatum During Severe Insulin-Induced Hypoglycemia

The effect of severe insulin-induced hypoglycemia on the extracellular levels of endogenous amino acids in the rat striatum was examined using the brain microdialysis technique. A characteristic pattern of alterations consisting of a 9-12-fold increase in aspartate (Asp), and more moderate increases in glutamate (Glu), taurine (Tau), and gamma-aminobutyric acid (GABA), was noted following cessation of electroencephalographic activity (isoelectricity). Glutamine (Gln) levels were reduced both during and after the isoelectric period and there was a delayed increase in extracellular phosphoethanolamine (PEA) content. The effects of decortication and excitotoxin lesions on the severe hypoglycemia-evoked efflux of endogenous amino acids in the striatum were also examined. Decortication reduced the release of Glu and Asp both 1 week and 1 month post-lesion. The efflux of other neuroactive amino acids was not affected significantly. In contrast, GABA, Tau, and PEA efflux was attenuated in kainate-lesioned striata. Glu and Asp release was also reduced under these conditions, and a smaller decrease in extracellular Gln was noted. These data suggest that GABA, Glu, and Asp are released primarily from their transmitter pools during severe hypoglycemia. The releasable pools of Tau and PEA appear to be located in kainate-sensitive striatal neurons. The significance of these results is discussed with regard to the excitotoxic theory of hypoglycemic cell death.     

Species composition and vegetation structure of an upper montane forest at the summit of Mt. Doi Inthanon, Thailand

Upper montane forest (UMF) within Doi Inthanon National Park, Northern Thailand, was investigated by means of fifty, 40 × 40 m stratified random plots situated between 2080 and 2565 m altitude. The aim was to address a number of community ecological questions concerning woody species composition and structural heterogeneity of the forest. A total of 7474 individuals of trees and woody climbers ≥ 15 cm gbh (girth at breast height) were included in the study and these were identified to 47 species, 39 genera and 26 families. The average density was 934 individuals/ha and the average stem basal area was 71.8 m²/ha. The most important species were: Quercus eumorpha, Sjzygium angkae, Litsea martabanica, Helicia nilagirica, Lindera caudata, Schima wallichii, Osmanthus fiagrans, Eurya acuminata, Myrsine semiserrata and Ilex umbellulata and the most important families were Fagaceae, Lauraceae, Theaceae and Myrtaceae. Altitude was the most important environmental variable explaining species composition and vegetation structure. Most of the calculated vegetation variables showed significant correlation with altitude: species richness, family richness, diversity, density and crown cover declined with altitude, average tree height was uncorrelated with altitude and basal area increased with altitude. An analysis of size class distributions indicated good forest conditions and reverse-J-shaped age class distribution of most species.