Structural characterization of heterodimeric laccases from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

The subfamily of POXA3 laccase isoenzymes produced by the fungus Pleurotus ostreatus has been characterized as an example of the complexity and heterogeneity of fungal isoenzyme patterns. Two isoenzymes, POXA3a and POXA3b, were previously purified, exhibiting an unusual heterodimeric structure composed of a large (67 kDa) and a small (18 or 16 kDa) subunit. A unique gene encodes the large subunit of both POXA3a and POXA3b, but alternative splicing produces two variants—differing for an insertion of four amino acids—for each isoenzyme. Two genes encoding POXA3a and POXA3b small subunits have been identified, and the corresponding amino acid sequences show only two amino acid substitutions. The 18- and 16-kDa subunits of both POXA3a and POXA3b differ for N-glycosylation at Asn150 of the 16-kDa subunit. The POXA3 large subunit 3D model allows us to highlight peculiarities of this molecule with respect to the laccases whose 3D structures are known.     

Knocking down the diencephalic thyrotropin-releasing hormone precursor gene normalizes obesity-induced hypertension in the rat

We recently showed that diencephalic TRH may mediate the central leptin-induced pressor effect. Here, to study the role of TRH in obesity-induced hypertension (OIH), we used a model of OIH produced by a high-fat diet (HFD, 45 days) in male Wistar rats. After 4 wk, body weight and systolic arterial blood pressure (SABP) increased in HFD animals. Plasma leptin was correlated with peritoneal adipose tissue. Then, we treated OIH animals with an antisense oligodeoxynucleotide and small interfering (si)RNA against the prepro-TRH. Antisense significantly decreased diencephalic TRH content and SABP at 24 and 48 h posttreatment. Similar effects were observed with siRNA against prepro-TRH but for up to 4 wk. Conversely, vehicle, an inverted antisense sequence and siRNA against green fluorescence protein, produced no changes. SABP decrease seems to be owing to an inhibition of the obesity-enhanced sympathetic outflow but not to an alteration in thyroid status. Using a simple OIH model we demonstrated, for the first time, that central TRH participates in the hypertension induced by body weight gain probably through its well-known action on sympathetic activity. Thus the TRH-leptin interaction may contribute to the strong association between hypertension and obesity.     

Neutrophils activate macrophages for intracellular killing of Leishmania major through recruitment of TLR4 by neutrophil elastase

We investigated the role of neutrophil elastase (NE) in interactions between murine inflammatory neutrophils and macrophages infected with the parasite Leishmania major. A blocker peptide specific for NE prevented the neutrophils from inducing microbicidal activity in macrophages. Inflammatory neutrophils from mutant pallid mice were defective in the spontaneous release of NE, failed to induce microbicidal activity in wild-type macrophages, and failed to reduce parasite loads upon transfer in vivo. Conversely, purified NE activated macrophages and induced microbicidal activity dependent on secretion of TNF-alpha. Induction of macrophage microbicidal activity by either neutrophils or purified NE required TLR4 expression by macrophages. Injection of purified NE shortly after infection in vivo reduced the burden of L. major in draining lymph nodes of TLR4-sufficient, but not TLR4-deficient mice. These results indicate that NE plays a previously unrecognized protective role in host responses to L. major infection.     

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.     

Response to novel food and the role of social influences in common marmosets (Callithrix jacchus) and Goeldi's monkeys (Callimico goeldii)

Neophobia, defined as showing caution toward novel features of the environment, is widespread in birds and mammals; it can be affected by ecology, early experience, and social context. In this study, we aimed to (i) investigate the response to novel food in adult common marmosets and Goeldi's monkeys and (ii) assess the role of social influences. We used an experimental paradigm employed previously with capuchin monkeys and children, in which a subject (observer) was presented with a novel food under three conditions: (i) Presence: group members did not have food; (ii) Different color: group members received familiar food whose color differed from that of the observer's novel food; (iii) Same color: group members received familiar food of the same color as the observer's novel food. Although most common marmosets tasted and/or ate the novel food, none of the Goeldi's monkeys ate it and only two sampled it. Differences in home range size and early social experience might explain the divergent behavior of the two species. Observers of both species similarly attended to group members and their visual attention increased with the number of group members eating, especially when the observer's and group members' foods were perceptually similar. However, we observed social influences on explorative behavior in Goeldi's monkeys but not on explorative or eating behavior in common marmosets. This result might be explained by the different pattern of response to novel food observed in the two species. Moreover, social influences on Goeldi's monkeys' behavior were nonspecific, i.e. they were not based on an appreciation that the food is safe because eaten by group members.     

Absence of Classical Heat Shock Response in the Citrus Pathogen <Emphasis Type="Italic">Xylella fastidiosa</Emphasis>

The fastidious bacterium Xylella fastidiosa is associated with important crop diseases worldwide. We have recently shown that X. fastidiosa is a peculiar organism having unusually low values of gene codon bias throughout its genome and, unexpectedly, in the group of the most abundant proteins. Here, we hypothesized that the lack of codon usage optimization in X. fastidiosa would incapacitate this organism to undergo quick and massive changes in protein expression as occurs in a classical stress response. Proteomic analysis of the response to heat stress in X. fastidiosa revealed that no changes in protein expression can be detected. Moreover, stress-inducible proteins identified in the closely related citrus pathogen Xanthomonas axonopodis pv citri were found to be constitutively expressed in X. fastidiosa. These proteins have extremely high codon bias values in the X. citri and other well-studied organisms, but low values in X. fastidiosa. Because biased codon usage is well known to correlate to the rate of protein synthesis, we speculate that the peculiar codon bias distribution in X. fastidiosa is related to the absence of a classical stress response, and, probably, alternative strategies for survival of X. fastidiosa under stressfull conditions.     

Cross-talk between apoptosis and cytokines in the regulation of parasitic infection

Parasitic diseases have worldwide medical and economical impact. Host T lymphocytes and the cytokines they produce determine the outcome of parasitic infections. Programmed cell death by apoptosis is induced in the course of parasitic infections, and affects cytokine production by removing activated effector T and B cells. In addition, engulfment of apoptotic cells promotes the secretion of cytokines that regulate intracellular replication of protozoan parasites. In this review, we discuss how the cross-talk between apoptosis and cytokines regulates parasitic infection.     

Antimicrobial isoflavonoids from Erythrina crista galli infected with Phomopsis sp

The isoflavonoids coumestrol, genistein and daidzein have been isolated and identified by bioassay-guided fractionation from the acetone extract of Erythrina crista galli young twigs infected with Phomopsis sp. These compounds showed antimicrobial activity against Bacillus brevis (MIC values 16.3, 64.8 and 137.8 microM, respectively). This is the first time that coumestrol, besides lutein and n-nonacosane, are reported in this species.     

Limits of life in MgCl2-containing environments: chaotropicity defines the window

The biosphere of planet Earth is delineated by physico-chemical conditions that are too harsh for, or inconsistent with, life processes and maintenance of the structure and function of biomolecules. To define the window of life on Earth (and perhaps gain insights into the limits that life could tolerate elsewhere), and hence understand some of the most unusual biological activities that operate at such extremes, it is necessary to understand the causes and cellular basis of systems failure beyond these windows. Because water plays such a central role in biomolecules and bioprocesses, its availability, properties and behaviour are among the key life-limiting parameters. Saline waters dominate the Earth, with the oceans holding 96.5% of the planet's water. Saline groundwater, inland seas or saltwater lakes hold another 1%, a quantity that exceeds the world's available freshwater. About one quarter of Earth's land mass is underlain by salt, often more than 100 m thick. Evaporite deposits contain hypersaline waters within and between their salt crystals, and even contain large subterranean salt lakes, and therefore represent significant microbial habitats. Salts have a major impact on the nature and extent of the biosphere, because solutes radically influence water's availability (water activity) and exert other activities that also affect biological systems (e.g. ionic, kosmotropic, chaotropic and those that affect cell turgor), and as a consequence can be major stressors of cellular systems. Despite the stressor effects of salts, hypersaline environments can be heavily populated with salt-tolerant or -dependent microbes, the halophiles. The most common salt in hypersaline environments is NaCl, but many evaporite deposits and brines are also rich in other salts, including MgCl(2) (several hundred million tonnes of bischofite, MgCl(2).6H(2)O, occur in one formation alone). Magnesium (Mg) is the third most abundant element dissolved in seawater and is ubiquitous in the Earth's crust, and throughout the Solar System, where it exists in association with a variety of anions. Magnesium chloride is exceptionally soluble in water, so can achieve high concentrations (> 5 M) in brines. However, while NaCl-dominated hypersaline environments are habitats for a rich variety of salt-adapted microbes, there are contradictory indications of life in MgCl(2)-rich environments. In this work, we have sought to obtain new insights into how MgCl(2) affects cellular systems, to assess whether MgCl(2) can determine the window of life, and, if so, to derive a value for this window. We have dissected two relevant cellular stress-related activities of MgCl(2) solutions, namely water activity reduction and chaotropicity, and analysed signatures of life at different concentrations of MgCl(2) in a natural environment, namely the 0.05-5.05 M MgCl(2) gradient of the seawater : hypersaline brine interface of Discovery Basin - a large, stable brine lake almost saturated with MgCl(2), located on the Mediterranean Sea floor. We document here the exceptional chaotropicity of MgCl(2), and show that this property, rather than water activity reduction, inhibits life by denaturing biological macromolecules. In vitro, a test enzyme was totally inhibited by MgCl(2) at concentrations below 1 M; and culture medium with MgCl(2) concentrations above 1.26 M inhibited the growth of microbes in samples taken from all parts of the Discovery interface. Although DNA and rRNA from key microbial groups (sulfate reducers and methanogens) were detected along the entire MgCl(2) gradient of the seawater : Discovery brine interface, mRNA, a highly labile indicator of active microbes, was recovered only from the upper part of the chemocline at MgCl(2) concentrations of less than 2.3 M. We also show that the extreme chaotropicity of MgCl(2) at high concentrations not only denatures macromolecules, but also preserves the more stable ones: such indicator molecules, hitherto regarded as evidence of life, may thus be misleading signatures in chaotropic environments. Thus, the chaotropicity of MgCl(2) would appear to be a window-of-life-determining parameter, and the results obtained here suggest that the upper MgCl(2) concentration for life, in the absence of compensating (e.g. kosmotropic) solutes, is about 2.3 M.