Stigmatellin Probes the Electrostatic Potential in the QB Site of the Photosynthetic Reaction Center

The electrostatic potential in the secondary quinone (Q_B) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to Q_B. It is controlled by the ionization states of residues in a strongly interacting cluster around the Q_B site. Reduction of the Q_B induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the Q_B binding pocket. It binds to the Q_B site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the Q_B pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the Q_B site was modulated by a single mutation, L213Asp→Ala, or double mutations, L213Asp-L212Glu→Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn→AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the Q_B site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.     

Impact of food processing and detoxification treatments on mycotoxin contamination

Mycotoxins are fungal metabolites commonly occurring in food, which pose a health risk to the consumer. Maximum levels for major mycotoxins allowed in food have been established worldwide. Good agricultural practices, plant disease management, and adequate storage conditions limit mycotoxin levels in the food chain yet do not eliminate mycotoxins completely. Food processing can further reduce mycotoxin levels by physical removal and decontamination by chemical or enzymatic transformation of mycotoxins into less toxic products. Physical removal of mycotoxins is very efficient: manual sorting of grains, nuts, and fruits by farmers as well as automatic sorting by the industry significantly lowers the mean mycotoxin content. Further processing such as milling, steeping, and extrusion can also reduce mycotoxin content. Mycotoxins can be detoxified chemically by reacting with food components and technical aids; these reactions are facilitated by high temperature and alkaline or acidic conditions. Detoxification of mycotoxins can also be achieved enzymatically. Some enzymes able to transform mycotoxins naturally occur in food commodities or are produced during fermentation but more efficient detoxification can be achieved by deliberate introduction of purified enzymes. We recommend integrating evaluation of processing technologies for their impact on mycotoxins into risk management. Processing steps proven to mitigate mycotoxin contamination should be used whenever necessary. Development of detoxification technologies for high-risk commodities should be a priority for research. While physical techniques currently offer the most efficient post-harvest reduction of mycotoxin content in food, biotechnology possesses the largest potential for future developments.     

Cortical localization of the Galpha protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the Galpha proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using co-immunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the Gbeta protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two Galpha proteins can activate the same pathway and that their dual presence is normally needed to counter Gbetagamma. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA-16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between Galpha proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.     

Multiple origin of metallicolous populations of the pseudometallophyte Arabidopsis halleri (Brassicaceae) in central Europe: the cpDNA testimony

The population structure of the pseudo-metallophyte herb, Arabidopsis halleri, was studied using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) on chloroplast DNA (cpDNA). The history of metallicolous (M) populations showing increased zinc tolerance was investigated. Eight primer-enzyme combinations out of 72 tested were applied to a total of 625 individuals from 28 widespread populations, 14 of them being M. Eleven distinct chlorotypes were found: five were common to nonmetallicolous (NM) and M populations, whereas six were only observed in one edaphic type (five in NM and one in M). No difference in chlorotype diversity between edaphic types was detected. Computed on the basis of chlorotype frequencies, the level of population differentiation was high but remained the same when taking into account levels of molecular divergence between chlorotypes. Isolation by distance was largely responsible for population differentiation. Geographically isolated groups of M populations were more genetically related to their closest NM populations than to each other. Our results suggest that M populations have been founded separately from distinct NM populations without suffering founding events and that the evolution towards increased tolerance observed in the distinct M population groups occurred independently.     

Comparison of haplotype frequencies differentiate fall armyworm (Lepidoptera: Noctuidae) corn-strain populations from Florida and Brazil

Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a major economic pest throughout the Western Hemisphere. Populations can be subdivided into two morphologically identical but genetically distinct strains (corn-strain and rice-strain) that differ in their host plant preferences. These strains can be distinguished by using polymorphisms in the mitochondrial cytochrome oxidase 1 gene. Additional sequence analysis of this locus identified two sites that were highly polymorphic in the corn-strain population and that produced four different haplotype subgroups. Comparisons of the frequency distribution of these haplotypes found no seasonal or plant host specificities, but they did demonstrate that the Brazil corn-strain population is different from corn-strain fall armyworm found in Florida. The development of a rapid means of distinguishing fall armyworm populations originating from Brazil versus Florida provides an opportunity for investigating and comparing the genetic complexity and long-range movements of this important agricultural pest.     

The paradox high zooplankton biomass-low vegetal particulate organic matter in high turbidity zones: What way for energy transfer?

Estuarine ecosystems are characterized by high zooplanktonic biomasses, essentially constituted by copepods and mysids whose nutritional requirements are mainly provided by phytoplankton, an easily available carbon form. The Gironde estuary is characterized by high turbidities which limit light penetration in the water column and therefore primary production. Consequently, primary production is low and its availability for higher trophic level is very limited. The main goal of this study was to characterize the total vegetal particulate organic matter (POM) in high turbidity zones of the Gironde estuary during summer (a critical period characterized by high heterotrophic bacterial degradation and high zooplanktonic biomasses) and to analyse its utilization by zooplankton, using prey/predator experiments and trophic biomarkers (fatty acids). The specific goals were to define (i) how vegetal POM was exploited by the different zooplanktonic groups (protozoa, copepods and mysids) and (ii) which alternative preys could be used when vegetal POM was not sufficient to ensure their nutritional requirements.Chlorophyll biomass was very low in the MTZ during summer 2002 (0.48 ± 0.03 mg m^− 3). Total zooplankton grazing was low (19% d^− 1) probably due to a large contribution of detritus originating from terrestrial plants in vegetal POM compared to phytoplankton. The highest grazing pressure was exercised by the mysid Mesopodopsis slabberi due to its high abundances and by its almost entirely herbivorous diet (phytoplankton and small terrestrial detritus). Grazing rates (19.7 ± 4.2 and 9.6 μgC cop^− 1 d^− 1 for juveniles and adults, respectively) seemed to be sufficient to satisfy their daily carbon requirement. Grazing rate of the copepod Eurytemora affinis (139 ngC cop^− 1 d^− 1) seemed to be insufficient to cover its nutritional requirements and the copepods probably needed to complete a great part of their diet from protozoa. Grazing rates of the mysid Neomysis integer (24.7 ± 0.01 and 20.89 ± 8.45 μgC cop^− 1 d^− 1 for juveniles and adults, respectively) were higher than those of M. slabberi when feeding only on phytoplankton. However, when other preys were introduced in its environment, N. integer only fed on the copepod E. affinis with a preference for nauplii. The study revealed the great importance of protozoa and bacteria in the trophic transfers between vegetal POM and zooplankton in the MTZ during summer, despite the low protozoa grazing pressure on vegetal POM (3.1%). The detritic food chain probably implies various trophic transfers with little direct relationships between vegetal POM and zooplankton.     

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.