A Chimeric Arabinogalactan Protein Promotes Somatic Embryogenesis in Cotton Cell Culture

Arabinogalactan proteins (AGPs) are a family of extracellular plant proteoglycans implicated in many aspects of plant growth and development, including in vitro somatic embryogenesis (SE). We found that specific AGPs were produced by cotton (Gossypium hirsutum) calli undergoing SE and that when these AGPs were isolated and incorporated into tissue culture medium, cotton SE was promoted. When the AGPs were partly or fully deglycosylated, SE-promoting activity was not diminished. Testing of AGPs separated by reverse-phase high-performance liquid chromatography revealed that the SE-promoting activity resided in a hydrophobic fraction. We cloned a full-length complementary DNA (cotton PHYTOCYANIN-LIKE ARABINOGALACTAN-PROTEIN1 [GhPLA1]) that encoded the protein backbone of an AGP in the active fraction. It has a chimeric structure comprising an amino-terminal signal sequence, a phytocyanin-like domain, an AGP-like domain, and a hydrophobic carboxyl-terminal domain. Recombinant production of GhPLA1 in tobacco (Nicotiana tabacum) cells enabled us to purify and analyze a single glycosylated AGP and to demonstrate that this chimeric AGP promotes cotton SE. Furthermore, the nonglycosylated phytocyanin-like domain from GhPLA1, which was bacterially produced, also promoted SE, indicating that the glycosylated AGP domain was unnecessary for in vitro activity.Arabinogalactan proteins (AGPs) comprise a diverse group of plant proteoglycans (for review, see Fincher et al., 1993; Nothnagel, 1997; Seifert and Roberts, 2007; Ellis et al., 2010). They are structurally complex, generally consisting of a Pro-, Ala-, Ser-, and Thr-rich protein backbone that is extensively modified, principally by hydroxylation of Pro residues (to Hyp) and subsequent glycosylation through O-linkages with type II arabinogalactans (Tan et al., 2003; Shimizu et al., 2005). Many AGPs also have a C-terminal hydrophobic domain that is processed and replaced with a glycosylphosphatidylinositol (GPI) anchor, which acts to tether the molecule to the extracellular face of the plasma membrane (Schultz et al., 1998). AGPs are also defined by their ability to be bound and precipitated by the synthetic dye β-glucosyl Yariv reagent (β-GlcY) and related molecules (Yariv et al., 1967). These dyes have been useful in isolating, localizing, and quantifying AGPs.AGPs are grouped into three subclasses (Schultz et al., 2002): AGPs have an N-terminal signal sequence, an arabinogalactosylated domain, and a hydrophobic C-terminal domain; “chimeric AGPs” contain at least one arabinogalactosylated domain and a domain with an unrelated motif; while “hybrid AGPs” contain arabinogalactosylated as well as different Pro/Hyp-rich glycoprotein motifs.AGPs are implicated in many aspects of plant cell growth and development. Historically, it was not possible to assign roles to individual AGPs, as tests were conducted with unfractionated mixtures of AGPs. More recently, individual AGPs, mainly from Arabidopsis (Arabidopsis thaliana), have been studied using techniques such as mutant analysis and gene knockout/silencing, providing evidence for roles of individual AGPs in cell expansion, root and seed regeneration, the coordination of vascular development, both male and female gametogenesis, the development of cotton fibers, and as contributors to plant stem strength (Shi et al., 2003; van Hengel and Roberts, 2003; Acosta-García and Vielle-Calzada, 2004; Motose et al., 2004; Yang et al., 2007; Levitin et al., 2008; Coimbra et al., 2009; Li et al., 2010; MacMillan et al., 2010).Conditioned media from in vitro embryogenic cultures contain factors that can promote somatic embryogenesis (SE), implying the presence of secreted signaling molecules (de Vries et al., 1988). There is evidence that secreted AGPs, which are components of conditioned media, are involved in SE. For example, SE in carrot (Daucus carota) and spruce (Picea abies) cell cultures was promoted when AGPs from conditioned media were added exogenously (Kreuger and van Holst, 1993; Egertsdotter and von Arnold, 1995). Subsequent studies showed the association of particular AGP epitopes with SE-promoting activity and the involvement of AGPs in SE for several other species (Kreuger et al., 1995; McCabe et al., 1997; Toonen et al., 1997; Chapman et al., 2000; Saare-Surminski et al., 2000; Ben Amar et al., 2007). There is also evidence that SE-promoting AGPs may be cleaved by an endochitinase (Egertsdotter and von Arnold, 1988; Domon et al., 2000; van Hengel et al., 2001, 2002), but neither the identity of the individual AGP(s) involved in promoting SE nor the mechanism of action has been established.In this study, we focused on SE in cotton (Gossypium hirsutum ‘Coker 315’), which is a limiting step in cotton transformation, and the potential role of AGPs in this process. We show that cotton calli undergoing somatic embryogenesis secrete an AGP fraction that promotes SE when incorporated back into the growth medium. We report the cloning and sequencing of a complementary DNA (cDNA) encoding a chimeric AGP present in this fraction and show that this molecule promotes SE.     

Isolated regulatory domains of cGMP-dependent protein kinase Ialpha and Ibeta retain dimerization and native cGMP-binding properties and undergo isoform-specific conformational changes

Molecular mechanisms that provide for cGMP activation of cGMP-dependent protein kinase (PKG) are unknown. PKGs are dimeric; each monomer contains a regulatory (R) and catalytic (C) domain. In this study, isolated recombinant R domains of PKGIalpha-(Delta349-670) and PKGIbeta-(Delta364-685) containing the dimerization and autoinhibitory subdomains and two allosteric cGMP-binding sites were expressed in Sf9 cells. Both R domains were dimers with elongated conformations (Stokes radii of 44 and 51 A, respectively, and frictional coefficients of 1.6 and 1.8, respectively). Exchange dissociation kinetics and K(D) values for cGMP were similar for each holoenzyme and its isolated R domain, indicating that under these conditions the C domain does not appreciably alter cGMP-binding functions of the R domain. As determined by gel filtration chromatography, cGMP binding caused elongation of the PKGIalpha-isolated R domain and contraction of the PKGIbeta-isolated R domain. Cyclic GMP-bound forms of the isoforms have similar physical dimensions that may reflect a common conformation of active isoforms. Elongation of the PKGIbeta holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGIalpha R domain may partially account for the elongation of wild type PKGIalpha associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation.     

Differential modulation of Alzheimer's disease amyloid beta-peptide accumulation by diverse classes of metal ligands

Biometals have an important role in AD (Alzheimer's disease) and metal ligands have been investigated as potential therapeutic agents for treatment of AD. In recent studies the 8HQ (8-hydroxyquinoline) derivative CQ (clioquinol) has shown promising results in animal models and small clinical trials; however, the actual mode of action in vivo is still being investigated. We previously reported that CQ-metal complexes up-regulated MMP (matrix metalloprotease) activity in vitro by activating PI3K (phosphoinositide 3-kinase) and JNK (c-jun N-terminal kinase), and that the increased MMP activity resulted in enhanced degradation of secreted Abeta (amyloid beta) peptide. In the present study, we have further investigated the biochemical mechanisms by which metal ligands affect Abeta metabolism. To achieve this, we measured the effects of diverse metal ligands on cellular metal uptake and secreted Abeta levels in cell culture. We report that different classes of metal ligands including 8HQ and phenanthroline derivatives and the sulfur compound PDTC (pyrrolidine dithiocarbamate) elevated cellular metal levels (copper and zinc), and resulted in substantial loss of secreted Abeta. Generally, the ability to inhibit Abeta levels correlated with a higher lipid solubility of the ligands and their capacity to increase metal uptake. However, we also identified several ligands that potently inhibited Abeta levels while only inducing minimal change to cellular metal levels. Metal ligands that inhibited Abeta levels [e.g. CQ, 8HQ, NC (neocuproine), 1,10-phenanthroline and PDTC] induced metal-dependent activation of PI3K and JNK, resulting in JNK-mediated up-regulation of metalloprotease activity and subsequent loss of secreted Abeta. The findings in the present study show that diverse metal ligands with high lipid solubility can elevate cellular metal levels resulting in metalloprotease-dependent inhibition of Abeta. Given that a structurally diverse array of ligands was assessed, the results are consistent with the effects being due to metal transport rather than the chelating ligand interacting directly with a receptor.     

Trauma-Related Mortality among Adults in Rural Western Kenya: Characterising Deaths Using Data from a Health and Demographic Surveillance System

Background

Information on trauma-related deaths in low and middle income countries is limited but needed to target public health interventions. Data from a health and demographic surveillance system (HDSS) were examined to characterise such deaths in rural western Kenya.

Methods And Findings

Verbal autopsy data were analysed. Of 11,147 adult deaths between 2003 and 2008, 447 (4%) were attributed to trauma; 71% of these were in males. Trauma contributed 17% of all deaths in males 15 to 24 years; on a population basis mortality rates were greatest in persons over 65 years. Intentional causes accounted for a higher proportion of male than female deaths (RR 2.04, 1.37-3.04) and a higher proportion of deaths of those aged 15 to 65 than older people. Main causes in males were assaults (n=79, 25%) and road traffic injuries (n=47, 15%); and falls for females (n=17, 13%). A significantly greater proportion of deaths from poisoning (RR 5.0, 2.7-9.4) and assault (RR 1.8, 1.2-2.6) occurred among regular consumers of alcohol than among non-regular drinkers. In multivariate analysis, males had a 4-fold higher risk of death from trauma than females (Adjusted Relative Risk; ARR 4.0; 95% CI 1.7-9.4); risk of a trauma death rose with age, with the elderly at 7-fold higher risk (ARR 7.3, 1.1-49.2). Absence of care was the strongest predictor of trauma death (ARR 12.2, 9.4-15.8). Trauma-related deaths were higher among regular alcohol drinkers (ARR 1.5, 1.1-1.9) compared with non-regular drinkers.

Conclusions

While trauma accounts for a small proportion of deaths in this rural area with a high prevalence of HIV, TB and malaria, preventive interventions such as improved road safety, home safety strategies for the elderly, and curbing harmful use of alcohol, are available and could help diminish this burden. Improvements in systems to record underlying causes of death from trauma are required.     

Visual Impairment as a Function of Visual Acuity in Both Eyes and Its Impact on Patient Reported Preferences

Purpose

To assess the impact of VA loss on patient reported utilities taking both eyes into account compared to taking only the better or the worse eye into account.

Methods

In this cross-sectional study 1085 patients and 254 controls rated preferences with the generic health-related (EQ-5D; n = 868) and vision-specific (Vision and Quality of Life Index (VisQoL); n = 837) multi-attribute utility instruments (MAUIs). Utilities were calculated for three levels of VA in the better and worse eyes, as well as for 6 different vision states based on combinations of the better and worse eye VA.

Results

Using the VisQoL, utility scores decreased significantly with deteriorating vision in both the better and worse eyes when analysed separately. When stratified by the 6 vision states, VisQoL utilities decreased as VA declined in the worse eye despite stable VA in the better eye. Differences in VisQoL scores were statistically significant for cases where the better eye had no vision impairment and the worse seeing fellow eye had mild, moderate or severe vision impairment. In contrast, the EQ-5D failed to capture changes in better or worse eye VA, or any of the six vision states.

Conclusions

Calculating utilities based only on better eye VA or using a generic MAUI is likely to underestimate the impact of vision impairment, particularly when the better eye has no or little VA loss and the worse eye is moderately to severely visually impaired. These findings have considerable implications for the assessment of overall visual impairment as well as economic evaluations within eye health.     

Assessing the efficacy of spray-delivered ‘eco-friendly’ chemicals for the control and eradication of marine fouling pests

Despite its frequent use in terrestrial and freshwater systems, there is a lack of published experimental research examining the effectiveness of spray-delivered chemicals for the management of non-indigenous and/or unwanted pests in marine habitats. This study tested the efficacy of spraying acetic acid, hydrated lime and sodium hypochlorite for the control of marine fouling assemblages. The chemicals are considered relatively ‘eco-friendly’ due to their low toxicity and reduced environmental persistence compared to synthetic biocides, and they were effective in controlling a wide range of organisms. Pilot trials highlighted acetic acid as the most effective chemical at removing fouling cover, therefore it was selected for more comprehensive full-scale trials. A single spray of 5% acetic acid with an exposure time of 1 min effectively removed up to 55% of the invertebrate species present and 65% of the cover on fouled experimental plates, while one application of 10% acetic acid over 30 min removed up to 78% of species present and 95% of cover. Single-spray treatments of 5% acetic acid reduced cover of the tunicate pest species Didemnum vexillum by up to 100% depending on the exposure duration, while repeat-spraying ensured that even short exposure times (1 min) achieved ～99% mortality. Both 5 and 10% acetic acid solutions appeared equally effective at removing the majority of algal species. This technique could be used for controlling the introduction of unwanted species or preventing the spread of pests, and is applicable to use on a variety of natural and artificial substrata, or for the treatment of structures that can be removed from the water.     

Fighting by Kittens and Piglets during Suckling: What Does it Mean?

Newborn kittens and piglets show some notable similarities in suckling‐associated behaviours. Both develop a pattern of teat use in which each member of the litter predominantly occupies one or two particular nipples or teats, both show agonistic behaviour towards littermates during suckling, and both are born with ‘weapons’–kittens with sharp claws and piglets with sharp needle teeth. Traditionally, the differential use of nipples or teats has been thought to be due to differential quality of the mammary glands. However, neither our findings in kittens nor an examination of the literature on piglets support this view. As mammary glands usually are of equal quality and as there are normally more glands than young, there seems little reason for littermates to fight. Furthermore, there is also little evidence that individual performance in suckling contests or use of ‘weapons’ is related to weight gain or to dominance relations. Given the present lack of satisfying adaptive explanations for the seemingly aggressive behaviour and use of weapons by kittens and piglets, we suggest these to be epiphenomena of developmental processes anticipating the use of these behaviours and structures in later adaptive contexts.     

A phylogeographic study of the stoneplant Conophytum (Aizoaceae; Ruschioideae; Ruschieae) in the Bushmanland Inselberg Region (South Africa) suggests anemochory

The Bushmanland Inselberg Region (BIR) of South Africa provides an ideal system to study population interactions, as these inselbergs function as islands of Succulent Karoo surrounded by Nama Karoo vegetation. The population genetics of four Conophytum taxa endemic to the quartz-associated habitats of inselbergs in the BIR were investigated using amplified fragment length polymorphisms (AFLP), namely C. marginatum subsp. haramoepense, C. marginatum subsp. marginatum, C. maughanii, and C. ratum. Conophytum marginatum colonizes the quartz outcrops on the summits of the inselbergs, while C. maughanii and C. ratum occupy quartz patches at the summit and base of the inselbergs. A total of 24 populations were sampled to assess genetic differentiation between populations of each species, specifically between summit and base populations of C. ratum, eastern and western populations of C. maughanii and populations of the two subspecies of C. marginatum. Moderate levels of genetic differentiation were recovered between the summit and base populations of C. ratum, with an indication of some genetic connectivity between the populations. Slight differentiation between the eastern and western populations of C. maughanii was recovered, however, this was not reflected in the PCoA and STRUCTURE results. In C. marginatum, no significant genetic differentiation was recovered between populations of the subspecies. These results may reflect evidence of different dispersal mechanisms in the species, with the genetic connectivity between populations of C. ratum possibly indicating dispersal through hygrochastic capsules, while genetic connectivity between populations of C. maughanii and C. marginatum may, for the first time, suggest long-distance dispersal, i.e. anemochory. This study provides the first insights into population interactions across the BIR and highlights the importance of conservation in the region, particularly of the Gamsberg, in light of the recent mining activities.     

The genetic drivers for the successful invasive potential of a generalist bird,the House crow

Biological Invasions - What kind of genetic structure helps the rapid range expansion of the invasive species is fundamental to understand spread of invasion. The House crow (Corvus splendens), an...