Guidelines for safety tests and registration of bacterial pesticides

Guidelines have been formulated for safety tests and registration of new microbial pesticides containing pathogenic or inhibitory bacteria. These guidelines are intended for the advice of interested firms and governmental agencies. They are based on over a decade's experience and intense recent activity in this field. For the registration of a new microbial product, information is required on the identity of the new bacterium, its biological properties, production, formulation, quality control, application and efficacy. For safety assessment a series of tests on the infectivity, toxicity and allergenicity etc. in laboratory mammals is arranged in 3 tiers. Entirely negative results in tier 1 indicate acceptance of the product as safe. Positive results may lead to its rejection or to further lengthier tests in tiers 2 and 3, culminating in rejection or acceptance after a risk-benefit analysis, possibly with restrictions concerning its use required on the label. Data on residues, inactivation and degradability may be necessary. Information on exposure of humans during production and use are particularly valuable. The intended use of the product greatly influences both safety test requirements and tests on non-target organisms and wildlife, e.g. bees, important predators and parasites of the target species, earthworms, birds and fish. Also phytotoxicity and phytopathogenicity must be considered. Aspects of current interest are critically discussed. Any fundamental studies not justified as mandatory requirements for testing a new product should be funded by non-industrial sources.     

Birbeck Granule-Like “Organized Smooth Endoplasmic Reticulum” Resulting from the Expression of a Cytoplasmic YFP-Tagged Langerin

Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of “Organized Smooth Endoplasmic Reticulum” (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a “double-lock” mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.     

A blue variant in the rainbow trout, Oncorhynchus mykiss Walbaum

A blue variant of the rainbow trout, which appeared in a French fish farm, displayed an iridescent body color that was cobalt blue on the back, lighter on the undersides, and silvery on the belly and which held up to adult stage. This color was supposed to result from a Tyndall effect involving a structural arrangement of melanin pigments because it disappeared when it was associated with a depigmenting gene. This blue variant appeared to be governed by an autosomal recessive gene. Blue fry survival and body weight were about 25% less than those of wild-type sibs, but no major problem was observed in further breeding performances, including reproduction. These features do not correspond with those of the blue variants previously described in the rainbow trout.     

Activation of Store-Operated Calcium Entry in Airway Smooth Muscle Cells: Insight from a Mathematical Model

Intracellular dynamics of airway smooth muscle cells (ASMC) mediate ASMC contraction and proliferation, and thus play a key role in airway hyper-responsiveness (AHR) and remodelling in asthma. We evaluate the importance of store-operated entry (SOCE) in these dynamics by constructing a mathematical model of ASMC signaling based on experimental data from lung slices. The model confirms that SOCE is elicited upon sufficient depletion of the sarcoplasmic reticulum (SR), while receptor-operated entry (ROCE) is inhibited in such conditions. It also shows that SOCE can sustain agonist-induced oscillations in the absence of other influx. SOCE up-regulation may thus contribute to AHR by increasing the oscillation frequency that in turn regulates ASMC contraction. The model also provides an explanation for the failure of the SERCA pump blocker CPA to clamp the cytosolic of ASMC in lung slices, by showing that CPA is unable to maintain the SR empty of . This prediction is confirmed by experimental data from mouse lung slices, and strongly suggests that CPA only partially inhibits SERCA in ASMC.     

ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium

To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the ‘fitness’ of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation.     

Endophytic Neotyphodium lolii induced tolerance to Zn stress in Lolium perenne

The effect of Zn on growth, chlorophyll a fluorescence, net photosynthetic rate, gas exchange, water content and mineral concentrations (Zn, Mn and Mg) in ryegrass infected with or free from Neotyphodium lolii was studied by addition of ZnSO₄ (0–20 m M ) to the nutrient solution. Zn induced a decrease in growth of plants at 1, 5 and 10 m M and cessation of growth at 20 m M ZnSO₄. From 1 to 10 m M , the decrease was less pronounced in the presence of the endophytic fungus than in its absence. The growth limitation was due to an accumulation of Zn in leaves. From 8 to 15 days, the presence of the fungus in the plant led to a limitation of the Zn concentration in the leaves (24–32% lower with N. lolii than without). This restriction of Zn concentrations in leaves also had a beneficial effect on photosystem II (PSII) activities, net photosynthetic rate and internal CO₂ concentration. Particularly at 1 and 5 m M , the quantum yield of electron flow throughout PSII was greater in the presence of the fungus than in its absence and at 5 and 10 m M , the internal CO₂ concentration was maintained at a normal level. Compared with the endophyte-free ryegrass, the symbiotic plants showed higher values of total dry weight and tiller number, indicating a tolerance to environmental Zn stress.     

Rab11A controls the biogenesis of Birbeck granules by regulating Langerin recycling and stability

The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A-RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system.