Contraceptive steroids from pharmaceutical waste perturbate junctional communication in Sertoli cells

The potential health impact of pharmaceutical waste is now a growing concern. Contraceptive steroids are prominent environmental contaminants and thus may act as endocrine disruptors. Numerous xenobiotics hamper Sertoli cells junctional communication which is known to participate in spermatogenesis control. This has been associated with male subfertility and testicular cancer. We investigated three contraceptive molecules found in the environment for their potential impact on Sertoli cells gap junction functionality: 17a-ethynylestradiol, medroxyprogesterone acetate and levonorgestrel. Four other non-steroid drugs also found in the environment were included in the study. Communication disruption was analyzed in vitro in murine seminiferous tubules and the 42GPA9 Sertoli cell line. Steroids modulated connexin43 trafficking and impaired junctional communication through rapid effects apparently acting on the cell membrane but not on Cx43 expression. The 4 non-steroid compounds showed no effect. Longer exposure to steroids increased gap junction impairment, which was associated in part with Na/K ATPase internalization. Estrogen receptors (ER) did not appear to be involved in gap junction disruption: Sertoli cells are devoid of ERα and only express the cytoplasmic β isoform. ERβ localization was not modified by either steroid. The threshold level was surprisingly low, around 10^?16 M. We conclude that steroidal pollutants disrupt Sertoli cells junctional communication in vitro at concentrations that can be found in the environment.     

Callose and cellulose synthase gene expression analysis from the tight cluster to the full bloom stage and during early fruit development in Malus × domestica

Apple (Malus × domestica) is an economically important temperate fruit-bearing crop which belongs to the family of Rosaceae and its pomaceous fruit is one of the most commonly cultivated. Several studies have demonstrated that the cell wall plays a pivotal role during flower and fruit development. It takes active part in pollen tube growth and contributes to determine the fruit firmness trait through the action of cell wall-related enzymes (i.e. polygalacturonase and pectinmethylesterase). We have investigated the expression of callose and cellulose synthase genes during flowering from tight cluster to anthesis and during early fruit development in domesticated apple. We also link the changes observed in gene expression to the profile of soluble non-structural carbohydrates at different developmental stages of flowers/fruitlets and to the qualitative results linked to wall polysaccharides’ composition obtained through near-infrared spectroscopy. This work represents an important addition to the study of tree physiology with respect to the analysis of the expression of callose and cellulose synthase genes during flower and early fruit development in domesticated apple.     

Binding of MgtR,a Salmonella Transmembrane Regulatory Peptide,to MgtC,a Mycobacterium tuberculosis Virulence Factor: A Structural Study

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.     

A General Approach for Haplotype Phasing across the Full Spectrum of Relatedness

Many existing cohorts contain a range of relatedness between genotyped individuals, either by design or by chance. Haplotype estimation in such cohorts is a central step in many downstream analyses. Using genotypes from six cohorts from isolated populations and two cohorts from non-isolated populations, we have investigated the performance of different phasing methods designed for nominally ‘unrelated’ individuals. We find that SHAPEIT2 produces much lower switch error rates in all cohorts compared to other methods, including those designed specifically for isolated populations. In particular, when large amounts of IBD sharing is present, SHAPEIT2 infers close to perfect haplotypes. Based on these results we have developed a general strategy for phasing cohorts with any level of implicit or explicit relatedness between individuals. First SHAPEIT2 is run ignoring all explicit family information. We then apply a novel HMM method (duoHMM) to combine the SHAPEIT2 haplotypes with any family information to infer the inheritance pattern of each meiosis at all sites across each chromosome. This allows the correction of switch errors, detection of recombination events and genotyping errors. We show that the method detects numbers of recombination events that align very well with expectations based on genetic maps, and that it infers far fewer spurious recombination events than Merlin. The method can also detect genotyping errors and infer recombination events in otherwise uninformative families, such as trios and duos. The detected recombination events can be used in association scans for recombination phenotypes. The method provides a simple and unified approach to haplotype estimation, that will be of interest to researchers in the fields of human, animal and plant genetics.     

Metallic Nanowire Array–Polymer Hybrid Film for Surface Plasmon Resonance Sensitivity Enhancement and Spectral Range Enlargement

In this paper, the coupling interaction is investigated between a metallic nanowire array and a metal film under the Kretschmann condition. The plasmonic multilayer is composed of a metallic nanowire array embedded in a polymer layer positioned above a metal film, exploiting the classical surface plasmon resonance (SPR) configuration. We analyze the influence of various structural parameters of the metallic nanowire array on the SPR spectrum of thin metal film. The results show that the coupling interactions of nanowires with the metal film can greatly affect SPR resonance wavelength and increase SPR sensitivity. The coupling strength of metallic nanowire array and metal film also impacts resonance wavelength, which can be used to adjust SPR range but have little effect on its sensitivity. The results are confirmed using a dipole coupling resonance model of metallic nanowire. We demonstrated that this nanostructured hybrid structure can be used for high sensitivity SPR monitoring in a large spectral range, which is important for advanced SPR measurement including fiber-optic SPR sensing technology.     

Apicomplexan cytoskeleton and motors: Key regulators in morphogenesis, cell division, transport and motility

Protozoan parasites of the phylum Apicomplexa undergo a lytic cycle whereby a single zoite produced by the previous cycle has to encounter a host cell, invade it, multiply to differentiate into a new zoite generation and escape to resume a new cycle. At every step of this lytic cycle, the cytoskeleton and/or the gliding motility apparatus play a crucial role and recent results have elucidated aspects of these processes, especially in terms of the molecular characterization and interaction of the increasing number of partners involved, and the signalling mechanisms implicated. The present review aims to summarize the most recent findings in the field.     

Characterization of CamH from Methanosarcina thermophila,Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

The homotrimeric enzyme Mt-Cam from Methanosarcina thermophila is the archetype of the γ class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from M. thermophila (Mt-CamH) was overproduced in Escherichia coli and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from E. coli cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had k_cat and k_cat/K_m values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from E. coli cultured with supplemental iron (Fe-Mt-CamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective k_cat (k_cat^eff) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the k_cat/K_m^eff was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the k_cat^eff of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H₂O₂, which supports the hypothesis that Fe²⁺ has a role in the active site. The k_cat for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a “ping-pong” mechanism accepting a proton. The k_cat/K_m was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO₂ and HCO₃⁻ is separate from intermolecular proton transfer.Carbonic anhydrases (CAs) are metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate (CO₂ + H₂O ⇆ HCO₃⁻ + H⁺) and are distributed among metabolically diverse species belonging to all three domains of life, reflecting the importance of these enzymes in biology (10, 26). Five classes (α, β, γ, δ, and ζ classes) of CAs that evolved independently and have no significant sequence identity have been identified. Our current understanding of the biochemistry and biological roles of CAs is based largely on several α and β class enzymes from mammals and plants. Two other CAs from members of the Eucarya domain, which belong to the δ and ζ classes and were isolated from a marine diatom, have been characterized (21). Although CAs are widely distributed in diverse prokaryotes (25), relatively few CAs belonging to the α and β classes from members of the Bacteria domain have been characterized. Remarkably, only two CAs from members of the Archaea domain, one each from the β and γ classes, have been characterized biochemically (35). Although the γ class is widely distributed in diverse species belonging to all three domains (26, 31), only the archetype γ class enzyme (Mt-Cam) from the anaerobic acetate-utilizing methane-producing species Methanosarcina thermophila in the Archaea domain has been characterized biochemically and shown to have CA activity (36). Mt-Cam belongs to a superfamily of proteins, comprised mainly of acyltransferases, that share a distinctive left-handed parallel β-helix fold predicted by a unique sequence motif (17, 19). Mt-Cam overproduced in Escherichia coli and purified aerobically contains zinc in the active site (2). However, overproduction in the closely related species Methanosarcina acetivorans yields an enzyme with 3-fold-greater CA activity and iron in the active site, establishing iron as the physiologically relevant metal (18).The kinetic mechanism of Mt-Cam (1, 36) resembles that of all classes of CAs studied despite structural differences. The overall reaction occurs in two mechanistically distinct half-reactions shown in equations 1 and 2 and in equations 3 and 4, where M is a metal, E is the enzyme, and B is buffer. (1) (2) (3) (4) The first half-reaction is a nucleophilic attack of metal-bound hydroxide on carbon dioxide, yielding bicarbonate, which is reflected in the steady-state parameter k_cat/K_m. The second half-reaction is the rate-determining proton transfer from metal-bound water to buffer, which is reflected in the steady-state parameter k_cat. The proton extracted from the metal-bound water is transferred to the buffer, which acts as the second substrate in a ping-pong mechanism.Kinetic analyses of single-amino-acid variants of Mt-Cam have identified Gln75, Asn73, and Asn202 as residues involved in a hydrogen bond network required for the first half-reaction and Glu62 and Glu84 as residues required for the second half-reaction (36). Also, analyses of single-amino-acid variants have identified Arg59, Asp61, and Asp76 as residues important for the integrity of the active site (31). The crystal structure identified a histidine motif (His81, His117, and His122) ligating the active site metal (17). Although Mt-Cam is the only γ class CA that has been characterized biochemically, homologs of Mt-Cam from Arabadopsis thaliana have also been investigated (20). Structural modeling and sequence analysis of the homologs have shown that there is conservation in Mt-Cam of the overall fold, metal ligand (Mt-Cam His81, His117, and His122), and residues important for catalysis (Mt-Cam Gln75 and Gln73) and active site structure (Mt-Cam Arg59, Asp61, and Asp76). However, Glu62 and Glu84 in Mt-Cam, which are required for the second half-reaction, are not conserved in the A. thaliana homologs, and CA activity was not detected in the proteins overproduced in E. coli. Furthermore, the proton shuttle residue (PSR) Glu84 in Cam is located on an acidic loop (30) that is not present in the A. thaliana homologs. A 2004 database search queried with one of the A. thaliana homologs retrieved sequences of putative γ class homologs from cyanobacteria, alpha- and gammaproteobacteria, plants, and green algae (20). With only one exception, all of the sequences showed conservation with the Mt-Cam residues essential for metal binding and catalysis except Glu62 and acidic loop residues that include Glu84. The crystal structure of an Mt-Cam homolog from the anaerobic archaeon Pyrococcus horikoshii that was overproduced in E. coli (14) has left-handed parallel β-helix fold and active site architecture similar to that of Mt-Cam (13) with bicarbonate bound to the active site zinc. However, the acidic loop and the PSR Glu84 of Mt-Cam are not conserved in the P. horikoshii enzyme, and CA activity was not examined. Interestingly, calcium was also shown to be present in the P. horikoshii structure. An Mt-Cam homolog from the marine alga Emiliania huxleyi was overproduced in E. coli and purified to homogeneity (27). The purified enzyme was not characterized, and CA activity was not examined, although 2-fold-greater CA activity was detected in extracts of E. coli expressing the gene. An alignment of the E. huxleyi homolog with Mt-Cam revealed the absence of residues essential for ligation of the active site metal (Mt-Cam His81, His117, and His122), residues important for activity (Mt-Cam Gln75, Gln73, and Glu62), and the acidic loop containing the PSR Glu84.Since only the archetype of the γ class CAs has been characterized biochemically, our overall understanding of the γ class is vague. Sequence analyses of putative homologs have suggested that there is substantial diversity similar to that in the α class (28). In particular, the sequence analyses indicated that many homologs lack the acidic loop and the essential PSR Glu84 of Mt-Cam, suggesting that these homologs belong to a subclass of the γ class. However, the absence of CA activity reported for the two purified proteins belonging to the putative subclass (14, 27) calls into question the catalytic activity, metal content, and other properties. Here we describe a more recent sequence analysis of the γ class and biochemical characterization of a representative of the putative subclass, Mt-CamH from M. thermophila, that was overproduced in E. coli and had substantial CA activity, although it has properties that distinguish it from Mt-Cam.     

Plant growth promotion by Azospirillum sp. in sugarcane is influenced by genotype and drought stress

Azospirillum influences growth and development of several crops by producing phytohormones such as auxins which have a major impact on root development. An improved root system leads to better water and nutrient uptake that in turn may influence yield positively. In this study, two agronomically contrasting sugarcane cvs R 570 and M 1176/77 adapted to different agroclimatic zones were inoculated with Azospirillum sp., with and without drought stress, to gauge how far they could benefit from this bacterial association. As early as 103 days after planting, cv M 1176/77 responded positively to inoculation with 15% improved growth (shoot height) and 75% more root dry mass when subjected to drought stress, whereas cv R 570 responded negatively particularly in the absence of drought stress. The significant interaction of cultivar x water regime x Azospirillum inoculation suggests a complex interplay of these factors, possibly involving the indigenous plant auxin pool. Therefore, plant genotype needs to be taken into account when recommending bacterial inoculation for direct plant growth promotion. Furthermore, enhanced growth under sub-optimal water conditions shows clearly the benefits that could be obtained in semi-arid conditions where water deficits frequently occur.     

Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon

As for other mRNA measurement methods, quantitative RT-PCR results need to be normalized relative to stably expressed genes. Widely used normalizing genes include beta-actin and glyceraldehyde-3-phosphate dehydrogenase. It has, however, become clear that these and other normalizing genes can display modulated patterns of expression across tissue types and during complex cellular processes such as cell differentiation and cancer progression. Our objective was to set the basis for identifying normalizing genes that displayed stable expression during enterocytic differentiation and between healthy tissue and adenocarcinomas of the human colon. We thus identified novel potential normalizing genes using previously generated cDNA microarray data and examined the alterations of expression of two of these genes as well as seven commonly used normalizing genes during the enterocytic differentiation process and between matched pairs of resection margins and primary carcinomas of the human colon using real-time RT-PCR. We found that ribosomal phosphoprotein P0 was particularly stable in all intestinal epithelial cell extracts, thereby representing a particularly robust housekeeping reference gene for the assessment of gene expression during the human enterocytic differentiation process. On the other hand, beta-2-microglobulin generated the best score as a normalizing gene for comparing human colon primary carcinomas with their corresponding normal mucosa of the resection margin, although others were found to represent acceptable alternatives. In conclusion, we identified and characterized specific normalizing genes that should significantly improve quantitative mRNA studies related to both the differentiation process of the human intestinal epithelium and adenocarcinomas of the human colon. This approach should also be useful to validate normalizing genes in other intestinal contexts.     

Retinoic acid signalling is required for specification of pronephric cell fate

The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.