Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1

KAAT1 is a lepidopteran neutral amino acid transporter belonging to the NSS super family (SLC6), which has an unusual cation selectivity, being activated by K⁺ and Li⁺ in addition to Na⁺. We have previously demonstrated that Asp338 is essential for KAAT1 activation by K⁺ and for the coupling of amino acid and driver ion fluxes. By comparing sequences of NSS family members, site-directed mutagenesis, and expression in Xenopus laevis oocytes, we identified Lys102 as a residue likely to interact with Asp338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents in the presence of both Na⁺ and K⁺. However, in K102V/D338E double mutant, the K102V mutation reversed both the inhibition of Na⁺-dependent transport and the block in K⁺-dependent transport that characterize the D338E mutant. K⁺-dependent leucine currents were not observed in any mutants with D338E. In the presence of the oxidant Cu(II) (1,10-phenanthroline)₃, we observed specific and reversible inhibition of K102C/D338C mutant, but not of the corresponding single cysteine mutants, suggesting that these residues are sufficiently close to form a disulfide bond. Thus both structural and functional evidence suggests that these two residues interact. Similar results have been obtained mutating the bacterial transporter homolog TnaT. Asp338 corresponds to Asn286, a residue located in the Na⁺ binding site in the recently solved crystal structure of the NSS transporter LeuT_Aa (41). Our results suggest that Lys102, interacting with Asp338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway. residue interaction; oxidants; tertiary structure     

Depth-related changes to density,diversity and structure of benthic megafaunal assemblages in the Fimbul ice shelf region,Weddell Sea,Antarctica

The depth-related patterns in the benthic megafauna of the NE Weddell Sea shelf at the edge of the Fimbul Ice Shelf were investigated at seven sites using towed camera platform photographs. Megafaunal density decreased with depth from 77,939 ha⁻¹ at 245 m to 8,895 ha⁻¹ at 510 m. While diversity was variable, with H′ ranging between 1.34 and 2.28, there were no depth related patterns. Multivariate analyses revealed two distinct assemblages; a shallow assemblage with dense patches of suspension feeders in undisturbed areas and a deep assemblage where these were not present. Disturbance from icebergs explained many observed patterns in faunal distribution. In shallow waters probable effects of disturbance were observed as changes in successional stages; in deeper waters changes in habitat as a result of past disturbance explained faunal distributions. In deeper areas ice ploughing created a mosaic landscape of fine and coarse sediments. Total megafaunal density was highest in areas of coarse sediment (up to 2.9 higher than in finer sediment areas) but diversity was highest in intermediate areas (H′ = 2.35).     

Activation and Inactivation of Mechanosensitive Currents in the Chick Heart

The behavior of MS channels in embryonic chick ventricular myocytes activated by direct mechanical stimulation is strongly affected by inactivation. The amplitude of the current is dependent not only on the amplitude of the stimulus, but also the history of stimulation. The MS current inactivation appears to be composed of at least two contributions: (i) rearrangement of the cortical tension transducing elements and (ii) blocking action of an autocrine agent released from the cell. With discrete mechanical stimuli, the MS current amplitude in the second press of a double press protocol was always smaller than the amplitude of the first MS current. Occasionally, a large MS current occurred when the cell was first stimulated, but subsequently the cell became unresponsive. For a series of stimuli of varying amplitudes, the order in which they were applied to the cell affected the size of the observed MS current for a given stimulus magnitude. When continuous sinusoidal stimulation was applied to the cells, the MS current envelope either reached a steady state, or inactivated. With sinusoidal stimulation, the MS response could be enhanced or restored by simple perfusion of fluid across the cell. This suggests that mechanical stimulation of the cells produces an autocrine inhibitor of MS channels as well as resulting in cortical rearrangement. Received: 7 July 1999/Revised: 26 October 1999     

Packaging capacity and stability of human adenovirus type 5 vectors.

Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population.     

Genomic upregulation of cardiac Cav1.2α and NCX1 by estrogen in women

Background

Women have a higher risk of lethal arrhythmias than men in long QT syndrome type 2 (LQTS2), but the mechanisms remain uncertain due to the limited availability of healthy control human tissue. We have previously reported that in female rabbits, estrogen increases arrhythmia risk in drug-induced LQTS2 by upregulating L-type Ca²⁺ (I_Ca,L) and sodium-calcium exchange (I_NCX) currents at the base of the epicardium by a genomic mechanism. This study investigates if the effects of estrogen on rabbit I_Ca,L and I_NCX apply to human hearts.

Methods

Postmortem human left ventricular tissue samples were probed with selective antibodies for regional heterogeneities of ion channel protein expression and compared to rabbit myocardium. Functionally, I_Ca,L and I_NCX were measured from female and male cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) with the voltage-clamp technique from control and estrogen-treated iPS-CMs.

Results

In women (n = 12), Cav1.2α (primary subunit of the L-type calcium channel protein 1) and NCX1 (sodium-calcium exchange protein) levels were higher at the base than apex of the epicardium (40 ± 14 and 81 ± 30%, respectively, P < 0.05), but not in men (n = 6) or postmenopausal women (n = 6). Similarly, in cardiomyocytes derived from female human iPS-CMs, estrogen (1 nM, 1–2 days) increased I_Ca,L (31%, P < 0.05) and I_NCX (7.5-fold, ??90 mV, P < 0.01) and their mRNA levels (P < 0.05). Moreover, in male human iPS-CMs, estrogen failed to alter I_Ca,L and I_NCX.

Conclusions

The results show that estrogen upregulates cardiac I_Ca,L and I_NCX in women through genomic mechanisms that account for sex differences in Ca²⁺ handling and spatial heterogeneities of repolarization due to base-apex heterogeneities of Cav1.2α and NCX1. By analogy with rabbit studies, these effects account for human sex-difference in arrhythmia risk.

     

Olfactory navigation during spawning migrations: a review and introduction of the Hierarchical Navigation Hypothesis

Migrations are characterized by periods of movement that typically rely on orientation towards directional cues. Anadromous fish undergo several different forms of oriented movement during their spawning migration and provide some of the most well‐studied examples of migratory behaviour. During the freshwater phase of the migration, fish locate their spawning grounds via olfactory cues. In this review, we synthesize research that explores the role of olfaction during the spawning migration of anadromous fish, most of which focuses on two families: Salmonidae (salmonids) and Petromyzontidae (lampreys). We draw attention to limitations in this research, and highlight potential areas of investigation that will help fill in current knowledge gaps. We also use the information assembled from our review to formulate a new hypothesis for natal homing in salmonids. Our hypothesis posits that migrating adults rely on three types of cues in a hierarchical fashion: imprinted cues (primary), conspecific cues (secondary), and non‐olfactory environmental cues (tertiary). We provide evidence from previous studies that support this hypothesis. We also discuss future directions of research that can test the hypothesis and further our understanding of the spawning migration.     

Modeling the spatial distribution of anthrax in southern Kenya

BackgroundAnthrax is an important zoonotic disease in Kenya associated with high animal and public health burden and widespread socio-economic impacts. The disease occurs in sporadic outbreaks that involve livestock, wildlife, and humans, but knowledge on factors that affect the geographic distribution of these outbreaks is limited, challenging public health intervention planning.MethodsAnthrax surveillance data reported in southern Kenya from 2011 to 2017 were modeled using a boosted regression trees (BRT) framework. An ensemble of 100 BRT experiments was developed using a variable set of 18 environmental covariates and 69 unique anthrax locations. Model performance was evaluated using AUC (area under the curve) ROC (receiver operating characteristics) curves.ResultsCattle density, rainfall of wettest month, soil clay content, soil pH, soil organic carbon, length of longest dry season, vegetation index, temperature seasonality, in order, were identified as key variables for predicting environmental suitability for anthrax in the region. BRTs performed well with a mean AUC of 0.8. Areas highly suitable for anthrax were predicted predominantly in the southwestern region around the shared Kenya-Tanzania border and a belt through the regions and highlands in central Kenya. These suitable regions extend westwards to cover large areas in western highlands and the western regions around Lake Victoria and bordering Uganda. The entire eastern and lower-eastern regions towards the coastal region were predicted to have lower suitability for anthrax.ConclusionThese modeling efforts identified areas of anthrax suitability across southern Kenya, including high and medium agricultural potential regions and wildlife parks, important for tourism and foreign exchange. These predictions are useful for policy makers in designing targeted surveillance and/or control interventions in Kenya.We thank the staff of Directorate of Veterinary Services under the Ministry of Agriculture, Livestock and Fisheries, for collecting and providing the anthrax historical occurrence data.     

Avian species diversity in different habitat types in and around North Nandi Forest,Kenya

Forest fragmentation and degradation leads to formation of modified habitats whose ability to support existing avifaunal diversity is still largely unknown. Bird diversity in indigenous forest, disturbed forest, plantation forest and farmlands adjacent to North Nandi Forest reserve was studied between January 2015 and June 2015. The distribution of bird feeding guilds in these habitat patches was also evaluated. Birds were surveyed using point counts, timed species counts and line transects and classified into six feeding guilds. A total of 3,232 individual birds of 151 species were recorded in the four habitats. Significant difference on bird abundance across the four habitats (F = 15.141, P ≤ 0.05, df = 3, 1121) was noted. Shannon–Weiner diversity index H′ for bird community ranged from 3.06 for plantation forest to 4.05 for disturbed forest showing a relatively diverse bird community. Insectivores (F = 3.090, P ≤ 0.05, df = 3, 297) dominated the foraging species assemblage in all the habitats significantly. Linear regression analysis revealed a strong linear relationship on bird species richness and abundance with vegetation variables (P < 0.01 in all cases). The results indicate that disturbed forest and indigenous forest support high bird species richness than plantation forest and farmlands. However, high bird abundance was observed in farmlands and plantation forest as opposed to indigenous forest and disturbed forest as they provide dispersal routes over a short distance and are important for creating corridors between primary forests.     

Using an integrative research approach to improve fish migrations in regulated rivers: a case study on Pacific Salmon in the Seton River,Canada

Hydrobiologia - Many of the world’s rivers are dammed, altering the physiology, behaviour, ecology and survival of fish. Integrative research has the potential to improve our understanding of...