A mechanistic molecular test of the plant-sanction hypothesis in legume–rhizobia mutualism

The origin and persistence of mutualism is difficult to explain because of the widespread occurrence of exploitative, ‘cheating’ partners. As a policing strategy stabilising intraspecific cooperation, host sanctions against non-N₂ fixing, cheating symbionts have been proposed to stabilise mutualism in legume-rhizobium symbiosis. Mechanism of penalisations would include decreased nodular rhizobial viability and/or early nodule senescence. We tested these potential mechanisms of penalisations in split-root experiments using two soybean varieties and two rhizobial strains, a cooperative, normal N₂-fixing strain and an isogenic non-fixing derivative. We found no differences in the number of viable rhizobia recovered from nodules and no differential expression of a nodular senescence molecular marker. Thus, our results do not support the hypothesis of plant sanctions acting against cheating rhizobia in our experimental conditions.     

Structure and Function Analysis of Therapeutic Monoclonal Antibodies against Dengue Virus Type 2

Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C′ loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.Dengue virus (DENV), a member of the Flaviviridae family of RNA viruses, is related to several other human pathogens of global concern, including yellow fever and tick-borne, West Nile, and Japanese encephalitis viruses. DENV infection in humans occurs after Aedes aegypti or Aedes albopictus mosquito inoculation and results in clinical disease, ranging from a febrile illness (dengue fever [DF]) to a life-threatening hemorrhagic and capillary leak syndrome (dengue hemorrhagic fever [DHF]/dengue shock syndrome [DSS]). Globally, there is significant diversity among DENV strains, including four distinct serotypes (DENV type 1 [DENV-1], DENV-2, DENV-3, and DENV-4) that differ at the amino acid level by 25 to 40%. Additional complexity occurs within each serotype, as genotypes vary from one another by up to 3% at the amino acid level (21, 49). No approved antiviral treatment is currently available, and several candidate tetravalent vaccines remain in clinical development (reviewed in reference 11). Because of the increased geographic range of its mosquito vectors, urbanization, and international travel, DENV continues to spread worldwide and now causes an estimated 50 to 100 million infections and 250,000 to 500,000 cases of DHF/DSS per year, with 2.5 billion people at risk (68).DENV is an enveloped icosahedral virus with a single-stranded, positive-polarity RNA genome. The 10.7-kb genome is translated as a single polyprotein, which is cleaved into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by host and viral proteases. The mature DENV virion is ∼500 Å in diameter, with a highly organized outer protein shell, a 50-Å lipid membrane bilayer, and a nucleocapsid core (26). Mature DENV virions are covered by 90 anti-parallel E protein homodimers, arranged flat along the surface with quasi-icosahedral symmetry. The immature virion, which lacks cleavage of the prM protein, has a rough surface with 60 spikes each composed of three prM-E heterodimers (7, 73). Exposure to mildly acidic conditions in the trans-Golgi network promotes virus maturation through a structural rearrangement of the flavivirus E proteins and cleavage of prM to M by a furin-like protease (29, 66, 69, 70). The ectodomain of DENV E protein is comprised of three discrete domains (34-36, 39). Domain I (DI) is a central, eight-stranded β-barrel, which contains a single N-linked glycan in most DENV strains. DII is a long, finger-like protrusion from DI, with the highly conserved fusion peptide at its distal end and a second N-linked glycan that recognizes DC-SIGN (37, 38, 46, 59). DIII, which adopts an immunoglobulin-like fold, has been suggested to contain cell surface receptor recognition sites (5, 64, 71). Several groups have recently defined contact residues for type-specific, subcomplex-specific, and cross-reactive monoclonal antibodies (MAbs) that recognize DIII of DENV-2 (16, 17, 31, 47, 57, 61). Type-specific MAbs with neutralizing activity against DENV-2 localized to the BC, DE, and FG loops on the lateral ridge of DIII, whereas subcomplex-specific MAbs recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310.To date, no study has compared the in vitro inhibitory activity of MAbs in cells against a genetically diverse range of DENV-2 strains and their protective capacity in animals. Here, we had the goal of generating strongly neutralizing MAbs that would recognize virtually all DENV-2 strains and function as a possible postexposure therapy. Twenty-four new anti-DENV-2 mouse MAbs were generated with moderate or strong neutralizing activity against the homologous virus in cell culture assays. Binding sites were mapped for the majority of these by yeast surface display, identifying distinct epitopes in regions in DI (lateral ridge), DII (dimer interface, lateral ridge, and fusion loop), and DIII (lateral ridge, C-C′ loop, and A strand). Several MAbs failed to neutralize efficiently at least one DENV-2 strain of a distinct genotype, suggesting that antibody recognition of neutralizing epitopes varies among DENV-2 genotypes.To begin to assess the utility of this new panel of inhibitory MAbs as possible therapeutics against DENV-2, we evaluated their protective capacity in a stringent intracranial challenge model in BALB/c mice. Among the 16 neutralizing MAbs tested in mice, most were protective when given as prophylaxis. Seven of these had postexposure therapeutic activity when administered as a single dose by intraperitoneal route even 3 days after intracranial infection. For the MAbs with the greatest therapeutic potential, protection was confirmed with an antibody-enhanced vascular leakage mouse model (2, 72) of DENV-2 infection.     

Characterization of self-association and heteroassociation of bacterial cell division proteins FtsZ and ZipA in solution by composition gradient-static light scattering

We have characterized the self-association of FtsZ in its GDP-bound state (GDP-FtsZ) and the heteroassociation of FtsZ and a soluble recombinant ZipA (sZipA) lacking the N-terminal transmembrane domain by means of composition gradient-static light scattering (CG-SLS) and by measurement of sedimentation equilibrium. CG-SLS experiments at high ionic strengths and in the presence of 5 mM Mg(2+) show that, while FtsZ self-associates in a noncooperative fashion, sZipA acts as a monomer. CG-SLS data obtained from mixtures of FtsZ (A) and sZipA (B) in the presence of Mg(2+) are quantitatively described by an equilibrium model that takes into account significant scattering contributions from B, A(1), A(2), A(3), A(4), A(5), A(6), A(1)B, A(2)B, A(3)B, and A(4)B. However, in the absence of Mg(2+) (with EDTA), the data are best explained by an equilibrium model in which only B, A(1), A(2), A(3), A(1)B, and A(2)B contribute significantly to scattering. The best-fit molecular weights of monomeric A and B are in good agreement with values calculated from amino acid composition and with values obtained from sedimentation equilibrium. The latter technique also confirmed the interaction between sZipA and GDP-FtsZ. Moreover, the association model that best describes the CG-SLS data is in qualitative agreement with the sedimentation data. From these results, it follows that the binding of sZipA to GDP-FtsZ is of moderate affinity and does not significantly affect the interactions between FtsZ monomers. Under the working conditions used, only one sZipA binds to FtsZ oligomers with a length of six at most. The observed behavior would be compatible with FtsZ fibrils being anchored in vivo to the bacterial inner plasma membrane by substoichiometric binding of membrane-bound ZipA.     

Comparative analysis of the outdoor culture of Haematococcus pluvialis in tubular and bubble column photobioreactors

The present paper makes a comparative analysis of the outdoor culture of H. pluvialis in a tubular photobioreactor and a bubble column. Both reactors had the same volume and were operated in the same way, thus allowing the influence of the reactor design to be analyzed. Due to the large changes in cell morphology and biochemical composition of H. pluvialis during outdoor culture, a new, faster methodology has been developed for their evaluation. Characterisation of the cultures is carried out on a macroscopic scale using a colorimetric method that allows the simultaneous determination of biomass concentration, and the chlorophyll, carotenoid and astaxanthin content of the biomass. On the microscopic scale, a method was developed based on the computer analysis of digital microscopic images. This method allows the quantification of cell population, average cell size and population homogeneity. The accuracy of the methods was verified during the operation of outdoor photobioreactors on a pilot plant scale. Data from the reactors showed tubular reactors to be more suitable for the production of H. pluvialis biomass and/or astaxanthin, due to their higher light availability. In the tubular photobioreactor biomass concentrations of 7.0 g/L (d.wt.) were reached after 16 days, with an overall biomass productivity of 0.41 g/L day. In the bubble column photobioreactor, on the other hand, the maximum biomass concentration reached was 1.4 g/L, with an overall biomass productivity of 0.06 g/L day. The maximum daily biomass productivity, 0.55 g/L day, was reached in the tubular photobioreactor for an average irradiance inside the culture of 130 microE/m2s. In addition, the carotenoid content of biomass from tubular photobioreactor increased up to 2.0%d.wt., whereas that of the biomass from the bubble column remained roughly constant at values of 0.5%d.wt. It should be noted that in the tubular photobioreactor under conditions of nitrate saturation, there was an accumulation of carotenoids due to the high irradiance in this reactor, their content in the biomass increasing from 0.5 to 1.0%d.wt. However, carotenoid accumulation mainly took place when nitrate concentration in the medium was below 5.0mM, conditions which were only observed in the tubular photobioreactor. A similar behaviour was observed for astaxanthin, with maximum values of 1.1 and 0.2%d.wt. measured in the tubular and bubble column photobioreactors, respectively. From these data astaxanthin productivities of 4.4 and 0.12 mg/L day were calculated for the tubular and the bubble column photobioreactors. Accumulation of carotenoids was also accompanied by an increase in cell size from 20 to 35 microm, which was only observed in the tubular photobioreactors. Thus it may be concluded that the methodology developed in the present study allows the monitoring of H. pluvialis cultures characterized by fast variations of cell morphology and biochemical composition, especially in outdoor conditions, and that tubular photobioreactors are preferable to bubble columns for the production of biomass and/or astaxanthin.     

17β-Estradiol treatment is unable to reproduce p85α redistribution associated with gestational insulin resistance in rats

Maternal metabolic adaptations are essential to ensure proper fetal development. According to changes in insulin sensitivity, pregnancy can be divided into two periods: early pregnancy, characterized by an increase in maternal insulin sensitivity, and late pregnancy, in which there is a significant increase in insulin resistance. The aims of the present work were two-fold: firstly, the molecular mechanisms associated with the development of pregnancy-related insulin resistance in peripheral tissues, mainly retroperitoneal adipose tissue and skeletal muscle, were studied in pregnant rats at 6, 11, and 16 days gestation. Secondly, the role of 17β-estradiol in this process was elucidated in an animal model consisting of ovariectomized rats treated with 17β-estradiol to mimic plasma gestational levels. The results support the conclusion that retroperitoneal adipose tissue plays a pivotal role in the decrease in insulin sensitivity during pregnancy, through a mechanism that involves p85α redistribution to the insulin receptor and impairment of Glut4 translocation to the plasma membrane. Treatment with 17β-estradiol did not reproduce the molecular adaptations that occur during pregnancy, suggesting that other hormonal factors presents in gestation but absent in our experimental model are responsible for p85α redistribution to the insulin receptor.     

Rapid biological colonization of a granitic building by lichens

The Galician Centre of Contemporary Art (Santiago de Compostela, Spain) is a large, modern building finished with a granitic cladding. Completed in September 1993, it is already showing signs of deterioration, including biological colonization and intense blackening of exterior surfaces, and the breaking off of cladding. In this work we investigated the causes of this premature deterioration. Biological colonization was most intense on the lower areas of shaded walls, the most abundant organisms being the lichens Trapelia coarctata and T. involuta. The blackening was chiefly due to the build up of microorganisms or their remains (no elements indicative of atmospheric pollution were detected). Both the cladding and granite from the source quarry showed signs of severe weathering (fissuration, high porosity, the presence of kaolinite) and had high capillarity, rapidly absorbing large amounts of water and releasing it only slowly. Because of this and the wet climate in Santiago the cladding was almost permanently damp, which, together with its weathered condition, made it highly bioreceptive and, at the same time, reduced its mechanical resistance, making it susceptible to breakage.     

Lipid polymorphism: a correction. The structure of the cubic phase of extinction symbol Fd-- consists of two types of disjointed reverse micelles embedded in a three-dimensional hydrocarbon matrix.

The X-ray scattering study of a cubic phase of extinction symbol Fd--, recently performed on a lipid extract (PFL) from Pseudomonas fluorescens [Mariani et al. (1990) Biochemistry 29, 6799-6810] has been extended to several other systems, all consisting of mixtures of water-miscible (MO, PC, PE, oleate) and of water-immiscible (FA, DG) lipids, plus water. In all of these systems the cubic phase was observed in the presence of excess water. Some inconsistencies observed between PFL and the other systems, the fact that in PFL one of the reflections of the cubic phase happened to coincide with the strongest reflection of the hexagonal phase, and the finding, in one of the original cubic samples of PFL kept in the cold for more than 3 years, that the intensity of one of the reflections had decreased dramatically all indicated that a nonnegligible amount of a hexagonal impurity was in fact present in the samples of PFL originally thought to contain a pure cubic phase. The intensities were corrected for that impurity and analyzed again using a pattern recognition approach based upon the axiom that the histogram of the electron density maps is invariant with respect to physical structure, when different phases are compared whose chemical composition is the same. The hexagonal phase provided the reference phase for the comparison. The moments mean value of (delta rho)n were used to compare the histograms.(ABSTRACT TRUNCATED AT 250 WORDS)     

ssp Genes and spore osmotolerance in Bacillus thuringiensis israelensis and Bacillus sphaericus

It was shown previously that spores and vegetative cells of Bacillus sphaericus (Bf) and Bacillus thuringiensis israelensis (Bti) are very sensitive to osmotic variations. Since spore osmotolerance has been associated with their SASP (small acid soluble spore proteins) content coded by ssp genes, hybridization assays were performed with sspE and sspA genes from B. subtilis as probes and showed that Bti and Bf strains could lack an sspE-like gene. The B. subtilis sspE gene was then introduced into Bti 4Q2 strain; spores were obtained and showed a 65 to 650 times higher level of osmotolerance to NaCl, without affecting other important properties: hypoosmotic resistance in vegetative cells, spore UV resistance, and larvicidal activity against diptera larvae.