Incidence and severity of bean common mosaic disease and resistance of popular bean cultivars to the disease in western Kenya

The common bean (Phaseolus vulgaris) is a high protein crop and the main legume in the cropping system of western Kenya. Despite its importance, common bean yields are low (<1.0 t/ha) and declining. Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) are the most common and most destructive viruses and can cause a yield loss as high as 100%. In Kenya, a limited number of cultivars and exotic genotypes with resistance to BCMV and BCMNV strains have been reported. This study sought to determine the distribution and screen popular cultivars for resistance to the viruses. In October 2016 and May 2017, two diagnostic surveys for bean common mosaic disease (BCMD) were conducted in seven counties of western Kenya namely Bungoma, Busia, Homa Bay, Nandi, Vihiga, Kakamega and Siaya. Leaf samples showing virus-like symptoms were collected and analysed by ELISA. Sixteen popularly grown bean cultivars together with cowpea (Vigna unguiculata), soybean (Glycine max), green grams (Vigna radiata) and groundnut (Arachis hypogaea) were planted in a greenhouse in a completely randomized block design with three replicates. The plants were inoculated with BCMNV isolate at 3-leaf stage. Data were taken weekly for 3 weeks on type of symptoms expressed and number of plants infected. In total, 270 bean farms were visited. Symptoms of mosaic, downward curling, local lesions, stunting or a combination of these were observed during both surveys. Mean virus incidence was higher in the short rain season (50.2%) than in the long rain season (35.6%). The mean BCMD severity on a scale of 0–3 was highest (2.3) in Kakamega County and lowest (0.5) in Siaya. On variety resistance tests to BCMNV isolate, 10 bean cultivars were susceptible, four tolerant and two resistant. BCMNV is widely distributed across counties probably because of use of uncertified seeds by farmers and inoculum pressure from seed and aphid vector. For improved yields of common bean, farmers should be advised to plant certified seeds for all legumes in the cropping system.     

Replication timing of 10 developmentally regulated genes in Physarum polycephalum.

We have tested the hypothesis which stipulates that only early-replicating genes are capable of expression. Within one cell type of Physarum - the plasmodium - we defined the temporal order of replication of 10 genes which were known to be variably expressed in 4 different developmental stages of the Physarum life cycle. Southern analysis of density-labeled, bromodesoxyuridine-substituted DNA reveals that 4 genes presumably inactive within the plasmodium, were not restricted to any temporal compartment of S-phase: 1 is replicated in early S-phase, 2 in mid S-phase and 1 in late S-phase. On the other hand, 4 out of 6 active genes analysed are duplicated early, with the first 30% of the genome. Surprisingly, the two others active genes are replicated late in S-phase. By gene-dosage analysis, based on quantitation of hybridization signals from early and late replicating genes throughout S-phase, we could pinpoint the replication of one of these two genes at a stage where 80-85% of the genome has duplicated. Our results demonstrate that late replication during S-phase does not preclude gene activity.     

Antiproliferative activity of levobupivacaine and aminoimidazole carboxamide ribonucleotide on human cancer cells of variable bioenergetic profile

We assessed the impact of ten mitoactive drugs on the viability and the proliferation of human cancer cells of variable origin and bioenergetics. A validated chemotherapeutic drug, doxorubicin, was used as a gold-standard for comparison. We also looked at the effect of these drugs on Rho(0) cells and on embryonic fibroblasts, both of which rely mainly on glycolysis to generate the vital ATP. The statistical analysis of the area under the curves revealed a cell-type specific response to mitodopant and mitotoxic compounds, in correlation with the contribution of glycolysis to cellular ATP synthesis. These findings indicate that the bioenergetic state of the cell determines in part the impact of mitodopants and mitotoxics on cancer cells viability.     

Chromogranin A Promotes Peptide Hormone Sorting to Mobile Granules in Constitutively and Regulated Secreting Cells: ROLE OF CONSERVED N- AND C-TERMINAL PEPTIDES*S?

Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N- and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N- and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.Eukaryotic cells share the capacity to rapidly secrete proteins through the constitutive secretory pathway. The fundamental feature of neuroendocrine and endocrine cells is the occurrence of dense-core secretory granules (DCGs),³ which are key cytoplasmic organelles responsible for secretion of hormones, neuropeptides, and neurotransmitters through the regulated secretory pathway (RSP). Storage at high concentrations of these secretory products is required for their finely tuned release in response to extracellular stimulation (1, 2). DCG biogenesis starts with the budding of immature secretory granules (ISGs) from the trans-Golgi network (TGN) through interactions between lipid rafts and protein components, in a similar manner to constitutive vesicle budding (2, 3). The ISG budding is followed by a multistep maturation process to form the mature secretory granules, including removal of the constitutive secretory proteins and lysosomal enzymes inadvertently packaged into ISGs (4).Despite increasing knowledge of the various steps of DCG formation, the nature of the sorting signals for entry of proteins into the DCGs and the molecular machinery required to generate secretory granules are not fully elucidated (5, 6). Several recent studies highlighted the role of members of the granin family, which may represent the driving force for granulogenesis in the TGN (2), although this notion has been a matter of debate (7). Granins are soluble acidic proteins widely distributed in endocrine and neuroendocrine cells, which are characterized by the ability to aggregate at acidic pH and a high Ca²⁺ environment (8, 9). These conditions are found in the lumen of the TGN allowing granins to aggregate in this compartment and to be segregated from constitutively secreted proteins (10, 11). The granin aggregates are believed to associate directly or indirectly with lipid rafts at the TGN to induce budding and formation of the ISGs. A prominent role of chromogranin A (CgA) in the regulation of DCG formation in endocrine and neuroendocrine cells has been proposed. Thus, depletion of CgA in PC12 cells led to a dramatic decrease in the number of DCGs (12), and exogenously expressed CgA in these depleted PC12 cells, as in DCG-deficient endocrine A35C and 6T3 cells, restored DCG biogenesis (12, 13). Besides, expression of granins in non-endocrine, constitutively secreting cells such as CV-1, NIH3T3, or COS-7 cells provoked the formation of DCG-like structures that release their content in response to Ca²⁺ influx (12, 14, 15). Further investigations performed in CgA null mice and transgenic mice expressing antisense RNA against CgA also revealed a reduction in the number of DCGs in chromaffin cells that was associated with an impairment of catecholamine storage, thus demonstrating the crucial role of CgA in normal DCG biogenesis (16, 17). In CgA knockout mice, the introduction of the gene expressing human CgA restored the regulated secretory phenotype (16). A different CgA null mice strain exhibited no discernable effect on DCG formation, but elevated catecholamine secretion (18), proving that CgA deficiency is associated with hormone storage impairment in neuroendocrine cells in vivo, a finding that was confirmed in vitro (19). The CgA^-/- mice strain generated by Hendy et al. (18) exhibited a compensatory overexpression of other granins, pointing to a possible overlap in granin function in secretory granule biogenesis.We reported previously that the frog CgA (fCgA) gene is coordinately regulated with the pro-opiomelanocortin (POMC) gene in the pituitary pars intermedia during the neuroendocrine reflex of skin color change, which allows amphibia to adapt to their environment through the release of POMC-derived melanotropic peptides (20, 21). Sequence comparison of fCgA with its mammalian orthologs revealed a high conservation of the N- and C-terminal domains, and far less conservation of the central part of the protein (Fig. 1A), suggesting that these domains may play a role in DCG formation and hormone release in various species (9, 20, 21). To assess the role of fCgA and its conserved N- and C-terminal regions in hormone sorting, storage, and secretion, we engineered different constructs that produce the native unmodified (no tag added) protein and truncated forms lacking the conserved N- and C-terminal domains, and we developed an antibody that specifically recognizes the central region of fCgA. Using the constitutively secreting COS-7 cells, which are devoid of DCGs, we could demonstrate for the first time that CgA is essential for targeting peptide hormones to newly formed mobile DCG-like structures. In the CgA-expressing AtT20 cells, which exhibit an only moderate capacity to sort secretory proteins to the regulated pathway (22), the granin plays a pivotal role in the sorting and release of POMC. The conserved terminal peptides of CgA are instrumental for these activities.Open in a separate windowFIGURE 1.Specificity of the antibody directed against frog CgA. A, scheme depicting the structure of fCgA and showing the high conservation of the terminal regions and the percentages of amino acid identity between frog and human CgA sequences. The highly conserved peptide WE14 and dibasic cleavage sites are also indicated. B, Western blot showing that the antibody developed against fCgA recognized the protein and several processing intermediates in frog but not rat pituitary extracts, whereas an antibody, directed against the WE14 conserved peptide, detected CgA and its processing products in both rat and frog pituitary extracts. C, immunofluorescence analysis of frog pituitary and adrenal glands, and rat adrenal gland using the antibodies against fCgA and WE14. cx, cortex; DL, distal lobe; IL, intermediate lobe; and m, medulla. Scale bars equal 10 μm.     

Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC–EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.     

Development of a fluorescence polarization assay to screen for inhibitors of the FtsZ/ZipA interaction

A fluorescence polarization competition assay has been developed to screen for inhibitors of the Escherichia coli FtsZ/ZipA protein-protein interaction. A previously published X-ray costructure demonstrated that a 17-amino-acid peptide, corresponding to FtsZ C-terminal residues 367-383 (FtsZ(367-383)), interacts with the C-terminal FtsZ binding domain of ZipA (ZipA(185-328)). Phage display was employed to identify a unique but related peptide which when further modified and labeled was shown to have a higher affinity to ZipA(185-328) than the FtsZ(367-383) peptide and binds to the same site. This peptide had a six fold increase in fluorescence polarization upon binding to ZipA(185-328) compared to a two fold increase for the FtsZ(367-383) fluorophore. As a result, assay parameters using the phage display peptide were further optimized and adapted for the high-throughput screen. A high-throughput screen of 250,000 compounds identified 29 hits with inhibition equal to or greater than 30% at 50 microg/ml. An X-ray costructure of a promising small molecule in this library complexed with ZipA(185-328) (KI=12 microM) revealed that the compound binds to the same hydrophobic pocket as the FtsZ(367-383) peptide.     

Isolation and partial characterization of a 30 kDa adhesin from Paracoccidioides brasiliensis

The virulence of Paracoccidioides brasiliensis can be attenuated or lost after long periods of repeated subculturing and reestablished after animal inoculation. Only one adhesin (gp43) has been described until now, among the various identified components of P. brasiliensis, and gp43 shows adhesion to laminin. Thus, the present study was designed to isolate and characterize factors putatively related to the capacity of this fungus to adhere to the host by comparing P. brasiliensis samples, taken before and after animal inoculation. The two samples differed in their pattern of adhesion and invasion. The sample recently isolated from animals (Pb18b) demonstrated a greater capacity to adhere and to invade the Vero cells than the one subcultured in vitro (Pb18a). Extract from Pb18b also showed higher levels of protein expression than that from Pb18a, when two-dimensional electrophoresis gels were compared. A protein species of 30 kDa, pI 4.9, was more evident in the Pb18b extract and had properties of adhesin. Laminin, but none of the other extracellular matrix (ECM) components, such as fibronectin, collagen I and IV, bound specifically to the P. brasiliensis 30 kDa protein. The roles of 30 kDa and gp43 in cellular interactions were investigated and the adhesion of P. brasiliensis yeast cells was intensively inhibited by pre-treatment of epithelial cells with 30 kDa protein and gp43. Thus, this study presents evidence that adhesion capacity could be related to virulence, and that a 30 kDa adhesin accumulated differentially in samples with different levels of pathogenicity. This protein and its adhesion characteristics are being published for the first time and may be related to the virulence of P. brasiliensis.     

Glutathione modulates lipid composition of human colon derived HT-29 cells

Glutathione (GSH) is important in maintaining intracellular thiol status. The present study looked at the effect of GSH depletion on lipid composition of colon-derived HT-29 cells. GSH was depleted in HT-29 cells by incubation either with buthionine-S, R-sulfoximine (BSO) or diethylmaleate (DEM). GSH was restored during early periods of cell growth by supplementation of growth medium with either GSH ester or N-acetyl cysteine (NAC). Lipids were analysed following GSH depletion and supplementation. Among the neutral lipids, an increase in free cholesterol and diacylglycerol and decrease in cholesteryl ester and triacylglycerol were seen in GSH-depleted cells as compared to control cells. There were no detectable free fatty acids either in control or GSH-depleted cells. Among the phospholipids, a decrease in phosphatidylcholine and phosphatidylinositol and an increase in phosphatidylethanolamine were observed. These changes were almost completely reversed by supplementation of BSO-treated cells with GSH ester and partially reversed by N-acetyl cysteine. These results suggest that the GSH status of the cell plays an important role in the lipid composition of the cells.     

Drying of mucilage causes water repellency in the rhizosphere of maize: measurements and modelling

Background and Aims

Although maize roots have been extensively studied, there is limited information on the effect of root exudates on the hydraulic properties of maize rhizosphere. Recent experiments suggested that the mucilaginous fraction of root exudates may cause water repellency of the rhizosphere. Our objectives were: 1) to investigate whether maize rhizosphere turns hydrophobic after drying and subsequent rewetting; 2) to test whether maize mucilage is hydrophobic; and 3) to find a quantitative relation between rhizosphere rewetting, particle size, soil matric potential and mucilage concentration.

Methods

Maize plants were grown in aluminum containers filled with a sandy soil. When the plants were 3-weeks-old, the soil was let dry and then it was irrigated. The soil water content during irrigation was imaged using neutron radiography. In a parallel experiment, ten maize plants were grown in sandy soil for 5 weeks. Mucilage was collected from young brace roots growing above the soil. Mucilage was placed on glass slides and let dry. The contact angle was measured with the sessile drop method for varying mucilage concentration. Additionally, capillary rise experiments were performed in soils of varying particle size mixed with maize mucilage. We then used a pore-network model in which mucilage was randomly distributed in a cubic lattice. The general idea was that rewetting of a pore is impeded when the concentration of mucilage on the pore surface (g cm^?2) is higher than a given threshold value. The threshold value depended on soil matric potential, pore radius and contract angle. Then, we randomly distributed mucilage in the pore network and we calculated the percolation of water across a cubic lattice for varying soil particle size, mucilage concentration and matric potential.

Results

Our results showed that: 1) the rhizosphere of maize stayed temporarily dry after irrigation; 2) mucilage became water repellent after drying. Mucilage contact angle increased with mucilage surface concentration (gram of dry mucilage per surface area); 3) Water could easily cross the rhizosphere when the mucilage concentration was below a given threshold. In contrast, above a critical mucilage concentration water could not flow through the rhizosphere. The critical mucilage concentration decreased with increasing particle size and decreasing matric potential.

Conclusions

These results show the importance of mucilage exudation for the water fluxes across the root-soil interface. Our percolation model predicts at what mucilage concentration the rhizosphere turns hydrophobic depending on soil texture and matric potential. Further studies are needed to extend these results to varying soil conditions and to upscale them to the entire root system.