Sterilization of boll weevil pupae with fractionated doses of gamma irradiation

Fractionated doses of 6,250–8,000 rads of gamma irradiation when administered to pupae of the boll weevil, Anthonomus grandis Boh., sexually sterilized both sexes. Mortality of males thus treated with 6,250 and 8,000 rads via fractionation was 14% and 27% respectively, by 5 days posttreatment compared with 46% mortality when an equivalent acute dose was administered to newly emerged adults. Pheromone production of males irradiated at 6,250 rads was one-third that of the control for the first 4 days, but equal that of the control during 5–11 days posttreatment. This procedure lends itself to the large-scale sterilization of weevils needed in an eradication program. This technique is applicable to other insects that are highly susceptible to acute doses.

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Zusammenfassung Durch fraktionierte Dosen von insgesamt 6250 und 8000 rads einer Gammabestrahlung (25 bzw. 32 Teilbehandlungen zu 250 rads mit 4 h-Intervallen) wurden Puppen des Baumwollkapselkäfers, Anthonomus grandis Boheman, in beiden Geschlechtern sexuell sterilisiert. Die Sterblichkeit, 5 d nach der Behandlung, betrug bei den so behandelten Männchen bei 6250 rads 14%, bei 8000 rads 27% im Vergleich zu 46%, wenn eine äquivalente akute Dosis bei frisch geschlüpften Käfern angewendet wurde. Die Pheromonproduktion der Männchen in den ersten 4 d erreichte bei 6250 rads-Bestrahlung 1/3 derjenigen der Kontrolltiere; 5–11 d nach der Behandlung war kein Unterschied. Dieses Verfahren eignet sich für die Massensterilisation von Kapselkäfern, wie sie bei einem Ausrottungsprogramm nötig ist. Diese Technik ist anwendbar auf andere Insekten, die gegenüber akuten Dosen hochempfindlich sind.

     

Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re-initiation of DNA replication

Initiation and re-initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single-stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis, these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature-sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSB-coated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo. Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re-initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.     

Range contraction and increasing isolation of a polar bear subpopulation in an era of sea‐ice loss

Climate change is expected to result in range shifts and habitat fragmentation for many species. In the Arctic, loss of sea ice will reduce barriers to dispersal or eliminate movement corridors, resulting in increased connectivity or geographic isolation with sweeping implications for conservation. We used satellite telemetry, data from individually marked animals (research and harvest), and microsatellite genetic data to examine changes in geographic range, emigration, and interpopulation connectivity of the Baffin Bay (BB) polar bear (Ursus maritimus) subpopulation over a 25‐year period of sea‐ice loss. Satellite telemetry collected from n = 43 (1991–1995) and 38 (2009–2015) adult females revealed a significant contraction in subpopulation range size (95% bivariate normal kernel range) in most months and seasons, with the most marked reduction being a 70% decline in summer from 716,000 km² (SE 58,000) to 211,000 km² (SE 23,000) (p < .001). Between the 1990s and 2000s, there was a significant shift northward during the on‐ice seasons (2.6^° shift in winter median latitude, 1.1^° shift in spring median latitude) and a significant range contraction in the ice‐free summers. Bears in the 2000s were less likely to leave BB, with significant reductions in the numbers of bears moving into Davis Strait (DS) in winter and Lancaster Sound (LS) in summer. Harvest recoveries suggested both short and long‐term fidelity to BB remained high over both periods (83–99% of marked bears remained in BB). Genetic analyses using eight polymorphic microsatellites confirmed a previously documented differentiation between BB, DS, and LS; yet weakly differentiated BB from Kane Basin (KB) for the first time. Our results provide the first multiple lines of evidence for an increasingly geographically and functionally isolated subpopulation of polar bears in the context of long‐term sea‐ice loss. This may be indicative of future patterns for other polar bear subpopulations under climate change.     

Na+-K+-2Cl- cotransporters and Cl- channels regulate citric acid cough in guinea pigs.

Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and it was present on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane-anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1 mM to 2 M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of Cl- from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor furosemide (10-100 microM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing (P < 0.05). Localized tracheal pretreatment with the Cl- channel inhibitors DIDS or niflumic acid (100 microM) also significantly reduced cough, whereas the GABAA receptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate Cl- due to the expression of the furosemide-sensitive Cl- transporter, NKCC1. Efflux of intracellular Cl-, in part through calcium-activated Cl- channels, may play an important role in regulating airway afferent neuron activity.     

Membrane Toxicity of Abnormal Prion Protein in Adrenal Chromaffin Cells of Scrapie Infected Sheep

Transmissible spongiform encephalopathies (TSEs) or prion diseases are associated with accumulations of disease specific PrP (PrP^d) in the central nervous system (CNS) and often the lymphoreticular system (LRS). Accumulations have additionally been recorded in other tissues including the peripheral nervous system and adrenal gland. Here we investigate the effect of sheep scrapie on the morphology and the accumulation of PrP^d in the adrenal medulla of scrapie affected sheep using light and electron microscopy. Using immunogold electron microscopy, non-fibrillar forms of PrP^d were shown to accumulate mainly in association with chromaffin cells, occasional nerve endings and macrophages. PrP^d accumulation was associated with distinctive membrane changes of chromaffin cells including increased electron density, abnormal linearity and invaginations. Internalisation of PrP^d from the chromaffin cell plasma membrane occurred in association with granule recycling following hormone exocytosis. PrP^d accumulation and internalisation from membranes is similarly associated with perturbations of membrane structure and trafficking in CNS neurons and tingible body macrophages of the LRS. These data suggest that a major toxic effect of PrP^d is at the level of plasma membranes. However, the precise nature of PrP^d-membrane toxicity is tissue and cell specific suggesting that the normal protein may act as a multi-functional scaffolding molecule. We further suggest that the co-localisation of PrP^d with exocytic granules of the hormone trafficking system may provide an additional source of infectivity in blood.     

Replication terminus for DNA polymerase I during initiation of pAMβ1 replication: role of the plasmid-encoded resolution system

Replication of plasmid pAMβ1 is initiated by DNA polymerase I (Pol I) and completed by DNA polymerase III holoenzyme contained in the replisome machinery. In this study we report that initiation of DNA replication generates D-loop structures containing the nascent leading strand paired to its template, and that D-loop extension is arrested ≈230 bp from the initiation site of DNA synthesis in the presence of the plasmid-encoded resolvase. In vitro and in vivo data suggest that this arrest is caused by a collision between Pol I and the resolvase bound to its target. As the arrested D-loop replication intermediates carry a single-stranded primosome-assembly site, we hypothesize that the biological role of the replication arrest is to limit the region replicated by Pol I and to promote the replacement of Pol I by the replisome in order to initiate concerted synthesis of the leading and lagging strands.     

Lead discovery using molecular docking

As the structures of more and more proteins and nucleic acids become available, molecular docking is increasingly considered for lead discovery. Recent studies consider the hit-rate enhancement of docking screens and the accuracy of docking structure predictions. As more structures are determined experimentally, docking against homology-modeled targets also becomes possible for more proteins. With more docking studies being undertaken, the 'drug-likeness' and specificity of docking hits is also being examined.     

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.Fibulins are a family of extracellular glycoproteins containing contiguous calcium-binding epidermal growth factor-like domains (cbEGFs)³ and a characteristic C-terminal fibulin (FC) domain (1–3). Recent studies have revealed that fibulin-4 and -5 are both essential for elastic fiber formation (4–7). Fibulin-4 is widely expressed from early embryogenesis and is necessary for normal vascular, lung, and skin development, since mice that lack fibulin-4 do not form elastic fibers and die perinatally (5). Furthermore, mice with reduced fibulin-4 expression develop aneurysms (8). Fibulin-5 is abundant in the aorta and large arteries during embryogenesis and following vascular injury (9, 10). Lack of fibulin-5 causes a less severe phenotype, with viable homozygous mice, but the elastic fibers in skin, lungs, and aorta are irregular and fragmented (6, 7), and there is altered vascular remodeling (11). These mice models also highlight that fibulin-4 and -5 have non-compensatory roles in elastic fiber formation. Mutations in both molecules can cause cutis laxa, a heritable disorder associated with elastic fiber degeneration leading to sagging skin, vascular tortuosity, and emphysematous lungs (12–15). A third isoform, fibulin-3, may play a minor role in elastic fiber formation, since its deficiency disrupts elastic fibers in Bruch''s membrane of the eye (16) and vaginal tissues (17).Elastic fiber formation is a complex multistep process (18–20). Initial pericellular microassembly of tropoelastin, which may involve the 67-kDa elastin-binding protein receptor, generates elastin globules that are stabilized by desmosine cross-links catalyzed mainly by lysyl oxidase (LOX) but also by LOXL1 (LOX-like 1). These globules are deposited on a fibrillin microfibril template, where they coalesce and undergo further cross-linking to form the elastin core of mature fibers. The ability of fibulin-4 and -5 to bind tropoelastin and fibrillin-1, the major structural component of microfibrils, supports a model in which these fibulins direct elastin deposition on microfibrils (4–7, 21–25). This model does not delineate the unique molecular contributions of fibulin-4 and -5 to elastic fiber formation, but some molecular differences have emerged. Tropoelastin was bound more strongly by fibulin-5 than by fibulin-4, whereas fibulin-5 was at the microfibril-elastin interface, but perichondrial fibulin-4 localized mainly to microfibrils (4).Fibulin-4 null mice offer tantalizing clues to how fibulin-4 contributes to elastic fiber formation (5). They had dramatically reduced (94%) desmosine cross-links despite no change in elastin or LOX expression levels, and electron-dense rodlike structures were prominent within elastin aggregates. Morphologically similar structures seen after chemically inhibiting LOX were previously identified as glycosaminoglycans, which can bind charged free ϵ-amino groups on lysines in tropoelastin (26). However, fibulin-4^+/− mice showed ∼20% increase in desmosine (5). LOX-null mice have phenotypic features similar to those of fibulin-4 null mice, dying perinatally with 60% reduced desmosine cross-links and major abnormalities in vascular and other elastic tissues (27, 28). In contrast, LOXL1-null mice are viable but have reduced desmosine (29), whereas fibulin-5 null mice have a 16% reduction in desmosine cross-links and survive well into adulthood (7). Detection of the LOXL1 pro-domain in fibulin-5 null mice skin but not wild-type skin implicates fibulin-5 in activation of LOXL1 (30).We and others have shown that fibrillin-1 and the microfibrillar protein MAGP-1 can both directly bind tropoelastin (31–34). However, the fibulin-null mice show that the fibrillin-1 interaction with tropoelastin is insufficient to support elastic fiber formation in vivo. Fibulin-5 has been reported to facilitate tropoelastin binding to the N-terminal half of fibrillin-1 (21). A study of elastin polypeptide self-assembly through coacervation and maturation phases showed that, although the N-terminal half of fibrillin-1 increased maturation velocity and droplet clustering, fibulin-4 and -5 both slowed maturation and limited globule growth (35). These studies imply that fibulins and fibrillin-1 act together to regulate elastin accretion on microfibrils.To gain further insights into the contributions of fibulin-4 and -5 to elastic fiber formation, we have delineated how they interact with tropoelastin, LOX, and fibrillin-1. Novel findings are that fibulin-4 directly binds LOX, and this interaction enhances fibulin-4 binding to tropoelastin, thus forming a ternary complex that may be critical for elastin cross-linking. Fibulin-5 can concurrently bind fibulin-4 and tropoelastin, but the interaction of both fibulins with fibrillin-1 strongly inhibits their binding to tropoelastin. These interactions indicate the molecular basis of how fibulins act as chaperones for deposition of elastin onto microfibrils. Our study thus provides a molecular account of the differential roles of fibulins-4 and -5 in elastic fiber formation.     

Plasmodium research in the postgenomic era

The complete genomic sequence of Plasmodium falciparum strain 3D7 was published in October 2002. At the Next Steps in Malaria Research meeting in April 2005, the next practical steps were considered and the priorities ranked for postgenomic research in Plasmodium. The high-throughput approaches that will help to answer the major biological questions regarding Plasmodium should, like the genome project itself, build community-shared resources, and efforts must be made to help researchers ready themselves to use the tools that will become available.