Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine

In order to understand the structural basis for the high thermostability of phytase from Aspergillus fumigatus, its crystal structure was determined at 1.5 A resolution. The overall fold resembles the structure of other phytase enzymes. Aspergillus niger phytase shares 66% sequence identity, however, it is much less heat-resistant. A superimposition of these two structures reveals some significant differences. In particular, substitutions with polar residues appear to remove repulsive ion pair interactions and instead form hydrogen bond interactions, which stabilize the enzyme; the formation of a C-terminal helical capping, induced by arginine residue substitutions also appears to be critical for the enzyme's ability to refold to its active form after denaturation at high temperature. The heat-resilient property of A.fumigatus phytase could be due to the improved stability of regions that are critical for the refolding of the protein; and a heat-resistant A.niger phytase may be achieved by mutating certain critical residues with the equivalent residues in A.fumigatus phytase. Six predicted N-glycosylation sites were observed to be glycosylated from the experimental electron density. Furthermore, the enzyme's catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family. The trap of this catalytic intermediate confirms the two-step catalytic mechanism of the acid histidine phosphatase family.     

Systemic serotonin sulfate in opisthobranch mollusks

Serotonin (5-hydroxytryptamine, 5-HT) is a ubiquitous modulatory neurotransmitter with roles as a neurohormone and neurotransmitter. However, few studies have been performed characterizing this molecule and its related metabolites in circulating fluids. Here, we demonstrate native 5-HT sulfate, but much lower levels of 5-HT, in hemolymph of the marine mollusk Pleurobranchaea californica. The metabolite 5-HT sulfate forms from 5-HT uptake and metabolism in central ganglia of Aplysia californica and in the visceral nerve and eye of Pleurobranchaea, but not in hemolymph itself. In addition, 5-hydroxyindole acetic acid (5-HIAA), while not detected in hemolymph, forms in higher quantities than does 5-HT sulfate in the eye and visceral nerve, and gamma-glu-5-HT is also observed in this area but never in hemolymph. As systemic 5-HT sulfate appears not to originate from the optic region or from systemic 5-HT, 5-HT sulfate likely derives from the nervous system. Circulating 5-HT sulfate is at least 10-fold higher during the light portion of a 12 : 12-h light/dark cycle than during the dark portion (p < 0.0007), but there is no obvious trend for free systemic tryptophan (Trp) (p > 0.3) in Pleurobranchaea. 5-HT in mollusks is associated with general arousal state; thus, diurnal systemic changes in a 5-HT catabolite may reflect a regulatory role for indole catabolism in behavioral rhythms.     

Conformational changes in the nuclear lamina induced by herpes simplex virus type 1 require genes U(L)31 and U(L)34

The herpes simplex virus type 1 (HSV-1) U(L)31 and U(L)34 proteins are dependent on each other for proper targeting to the nuclear membrane and are required for efficient envelopment of nucleocapsids at the inner nuclear membrane. In this work, we show that whereas the solubility of lamins A and C (lamin A/C) was not markedly increased, HSV induced conformational changes in the nuclear lamina of infected cells, as viewed after staining with three different lamin A/C-specific antibodies. In one case, reactivity with a monoclonal antibody that recognizes an epitope in the lamin tail domain was greatly reduced in HSV-infected cells. This apparent HSV-induced epitope masking required both U(L)31 and U(L)34, but these proteins were not sufficient to mask the epitope in uninfected cells, indicating that other HSV proteins are also required. In the second case, staining with a rabbit polyclonal antibody that primarily recognizes epitopes in the lamin A/C rod domain revealed that U(L)34 is required for HSV-induced decreased availability of epitopes for reaction with the antibody, whereas U(L)31 protein was dispensable for this effect. Still another polyclonal antibody indicated virtually no difference in lamin A/C staining in infected versus uninfected cells, indicating that the HSV-induced changes are more conformational than the result of lamin depletion at the nuclear rim. Further evidence supporting an interaction between the nuclear lamina and the U(L)31/U(L)34 protein complex includes the observations that (i) overexpression of the U(L)31 protein in uninfected cells was sufficient to relocalize lamin A/C from the nuclear rim into nucleoplasmic aggregates, (ii) overexpression of U(L)34 was sufficient to relocalize some lamin A/C into the cytoplasm, and (iii) both U(L)31 and U(L)34 could directly bind lamin A/C in vitro. These studies suggest that the U(L)31 and U(L)34 proteins modify the conformation of the nuclear lamina in infected cells, possibly by direct interaction with lamin A/C, and that other proteins are also likely involved. Given that the nuclear lamina potentially excludes nucleocapsids from envelopment sites at the inner nuclear membrane, the lamina alteration may reflect a role of the U(L)31/U(L)34 protein complex in perturbing the lamina to promote nucleocapsid egress from the nucleus. Alternatively, the data are compatible with a role of the lamina in targeting the U(L)31/U(L)34 protein complex to the nuclear membrane.     

Cross-Reactive Neutralizing Antibodies Directed against Pandemic H1N1 2009 Virus Are Protective in a Highly Sensitive DBA/2 Mouse Influenza Model

Our ability to rapidly respond to an emerging influenza pandemic is hampered somewhat by the lack of a susceptible small-animal model. To develop a more sensitive model, we pathotyped 18 low-pathogenic non-mouse-adapted influenza A viruses of human and avian origin in DBA/2 and C57BL/6 mice. The majority of the isolates (13/18) induced severe morbidity and mortality in DBA/2 mice upon intranasal challenge with 1 million infectious doses. Also, at a 100-fold-lower dose, more than 50% of the viruses induced severe weight loss, and mice succumbed to the infection. In contrast, only two virus strains were pathogenic for C57BL/6 mice upon high-dose inoculation. Therefore, DBA/2 mice are a suitable model to validate influenza A virus vaccines and antiviral therapies without the need for extensive viral adaptation. Correspondingly, we used the DBA/2 model to assess the level of protection afforded by preexisting pandemic H1N1 2009 virus (H1N1pdm) cross-reactive human antibodies detected by a hemagglutination inhibition assay. Passive transfer of these antibodies prior to infection protected mice from H1N1pdm-induced pathogenicity, demonstrating the effectiveness of these cross-reactive neutralizing antibodies in vivo.Respiratory tract infections are the third leading cause of mortality in the world (27). Influenza, a disease of the airways caused by influenza viruses, is responsible for approximately half a million deaths and 3 to 5 million hospitalizations per year (28). In addition to the annual disease burden, influenza A virus is more notoriously known for its ability to cause pandemics. Three pandemics have been reported in the twentieth century: the first that occurred in 1918 (Spanish influenza) killed 20 to 50 million individuals (15); the other two in 1957 and 1968, although less lethal, killed millions due to the lack of preexisting immunity. In April 2009, two cases of febrile illness were confirmed to be caused by swine-origin influenza A virus (H1N1) (4, 8). Continuous spread within North America and other parts of the world has signaled the first influenza pandemic of this century.To study the pathogenicity of influenza A viruses, including the current pandemic A (H1N1) 2009 virus (H1N1pdm), in mammalian hosts and to determine the effectiveness of pharmaceutical interventions, it is essential to have a sensitive animal model. Although influenza has some important differences in mice and humans, a murine model is the only animal model thus far described that allows for relatively high group numbers and any relatively high throughput. Unfortunately, only a few strains of influenza A virus—almost exclusively belonging to the highly pathogenic avian influenza virus isolates of the H5 and H7 subtypes—are pathogenic in most commonly used mouse strains without adaptation through serial passaging. The hemagglutinin (HA) proteins of these H5 and H7 viruses contain a basic amino acid cleavage site, allowing them to spread systemically (12, 19, 26). Most other subtypes of influenza virus, including H1N1 and H3N2, either do not infect or cause very mild disease in mice. The requirement for adaptation of a pandemic virus to commonly used mouse strains can lead to a delay in the gathering of important data to help guide public health control strategies. As such, the lack of a sensitive small-animal model to study the infection dynamics of various subtypes of avian influenza viruses severely hampers the rapid and effective response required during a pandemic or prepandemic situation.This study was designed to demonstrate the utility of DBA/2 mice, previously reported to be susceptible to highly pathogenic influenza viruses (1), to study infections caused by several influenza A virus subtypes isolated from birds or humans without the need for prior adaptation. To assess the utility of the model to respond to emerging strains, we used DBA/2 mice to examine the functional activity of sera from individuals previously shown to have preexisting cross-reactive H1N1pdm antibodies. It is hypothesized that these individuals may be partially protected from infection because of the presence of cross-reactive neutralizing antibodies produced after infection with a different but related H1N1 virus. This hypothesis is supported by in vitro microneutralization and hemagglutination inhibition (HI) assays (2, 10); however, it is not yet known whether these antibodies are also functional in vivo.     

Twig-Nesting Ants: The Hidden Predators of the Coffee Berry Borer in Chiapas,Mexico

Coffee is a globally important crop that is subject to numerous pest problems, many of which are partially controlled by predatory ants. Yet several studies have proposed that these ecosystem services may be reduced where agricultural systems are more intensively managed. Here we investigate the predatory ability of twig-nesting ants on the main pest of coffee, the coffee berry borer (Hypothenemus hampei) under different management systems in southwest Chiapas, Mexico. We conducted both laboratory and field experiments to examine which twig-nesting ant species, if any, can prey on free-living borers or can remove borers embedded in coffee fruits and whether the effects of the twig-nesting ant community differ with habitat type. Results indicate that several species of twig-nesting ants are effective predators of both free-living borers and those embedded in coffee fruits. In the lab, Pseudomyrmex ejectus, Pseudomyrmex simplex, and Pseudomyrmex PSW-53 effectively removed free-living and embedded borers. In the field, abundance, but not diversity, of twig-nesting ant colonies was influenced by shade management techniques, with the highest colony abundance present in the sites where shade trees were recently pruned. However, borer removal rates in the field were significant only in the shadiest site, but not in more intensively managed sites. This study provides evidence that twig-nesting ants can act as predators of the coffee berry borer and that the presence of twig-nesting ants may not be strongly linked to shade management intensity, as has been suggested for other arthropod predators of the borer.     

Toxicity of three acaricides to Tetranychus urticae (Tetranychidae: Acari) and Orius insidiosus (Anthocoridae: Hemiptera)

Management for twospotted spider mite, Tetranychus urticae Koch, populations in peanut, Arachis hypogaea L., relies on acaricides. The outcomes of acaricide applications are most predictable when complete information on their toxicity and specificity is available. Specifically, the degrees to which acaricides impact different stages of T. urticae and natural enemies combined determine the overall efficacy of an acaricide application. The objectives of this study were to determine stage-specific direct and residual efficacies of three acaricides (fenpropathrin, etoxazole, and propargite) against T. urticae, and the direct and residual toxicity of the acaricides to Orius insidiosus (Say) adults. Direct toxicity of acaricides to T. urticae was measured on peanut cuttings. All acaricide treatments caused significant mortality to a mixed stage population of T. urticae, and mortality did not differ among the acaricides 7 d after treatment. When toxicity to eggs was tested, the proportion of eggs that hatched for all acaricide treatments was significantly lower than the control, with etoxazole and propargite causing 100% mortality. Exposure to acaricide residues caused < 30% mortality of T. urticae adults 1 and 2 d after treatment and was not significantly different from the control. Fenpropathrin and propargite caused 100% mortality and etoxazole caused > 50% mortality of O. insidious adults after direct exposure to the acaricides. Residual toxicity of acaricides to O. insidiosus adults varied but remained toxic to O. insidiosus longer than to T. urticae. Fenpropathrin had the longest residual effect on O. insidiosus adults, causing > 95% mortality after 14 d; etoxazole and propargite caused < 30% mortality after 14 d.     

Plant responses to potassium deficiencies: a role for potassium transport proteins

The availability of potassium to the plant is highly variable, due to complex soil dynamics, which are strongly influenced by root-soil interactions. A low plant potassium status triggers expression of high affinity K+ transporters, up-regulates some K+ channels, and activates signalling cascades, some of which are similar to those involved in wounding and other stress responses. The molecules that signal low K+ status in plants include reactive oxygen species and phytohormones, such as auxin, ethylene and jasmonic acid. Apart from up-regulation of transport proteins and adjustment of metabolic processes, potassium deprivation triggers developmental responses in roots. All these acclimation strategies enable plants to survive and compete for nutrients in a dynamic environment with a variable availability of potassium.