Pilot-scale production of mosquitocidal toxins by Bacillus thuringiensis and Lysinibacillus sphaericus under solid-state fermentation

Mosquitocidal toxins of Bacillus thuringiensis israelensis (Bti) and Lysinibacillus sphaericus 14N1 (Ls14N1) were produced under solid-state fermentation using agro-industrial wastes. Sugar beet pulp–sesame meal (1:1) and wheat germ meal–linen meal (1:1) at 9% were the efficient substrate mixtures for the growth and toxin production of Bti and Ls14N1, respectively. Bti was more active after the addition of beef extract (0.2%) or yeast extract (0.5%) to the medium. On the other hand, the addition of yeast extract (0.2%) or NYSM salts (2%) significantly enhanced the toxicity produced by Ls14N1. The optimum conditions for the maximum toxicity of Bti were at pH 7–8, 20–30% moisture, 4–10% inoculum and 7 days incubation. For Ls14N1, the best conditions were pH 6.5–7.5, 20–30% moisture, 4–10% inoculum and 5 days incubation. It was found that the best thickness of carrier-substrates in the plate (15?cm in diameter) for the maximum mosquitocidal activity was about 0.5?cm for Bti and 0.5–1?cm for Ls14N1. Pilot-scale production in aluminium trays applying the above conditions showed a decrement of toxicity of fermented cultures and some plates were contaminated. These problems were dissolved by reducing the moisture content to 15%, increasing inoculum to 10% and manual agitation of trays every-day.     

Lead acetate and arsenic trioxide induce instability of microsatellites at three different fragile sites (6q21, 9q32-9q33 and 15p14) within the genome of the rat

Human exposure to metals is of increasing concern due to the well-documented toxic and carcinogenic effects of metals and metal compounds, and the rising environmental levels due to industrial processes and pollution. It has been reported that metals can be genotoxic by several modes of action including generation of reactive oxygen species and inhibition of DNA repair. The aim of this study was to evaluate microsatellite instability (MSI) in three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located within three common fragile sites in the genome of the laboratory rat (6q21, 9q32-9q33 and 15p14) exposed to acute and chronic doses of a metal salt (lead acetate trihydrate) and a metalloid oxide (arsenic trioxide). In the acute and sub-chronic studies with the two compounds, MSI was observed in the three loci as deletions or additions of microsatellite repeats. The percentages of MSI were 36.4% and 42.1% for lead acetate and arsenic trioxide, respectively. Results of the present work indicate that the microsatellites located within fragile sites provide a convenient assay system to detect changes in DNA sequences resulting from exposure to genotoxic agents.     

A reversible decrease in ribulose 1,5‐bisphosphate carboxylase/oxygenase carboxylation activity caused by the aggregation of the enzyme's large subunit is triggered in response to the exposure of moderate irradiance‐grown plants to low irradiance

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) is highly regulated in response to fluctuations in the environment, including changes in irradiance. However, no complex data are available on Rubisco regulatory mechanisms triggered in plants which are submitted to moderate–low irradiance shift. Therefore, we investigated in a comprehensive way the changes at the level of amount of Rubisco protein, its structural organization and carboxylase activity of the holoenzyme as triggered by exposure of moderate irradiance‐grown Arabidopsis thaliana plants to low irradiance conditions. An exposure of moderate irradiance‐grown plants to low irradiance for a single photoperiod caused the exclusion of a certain pool of Rubisco under altered conditions owing to oxidative modifications resulting in the formation of protein aggregates involving Rubisco large subunit (LS). As a result, both initial and total Rubisco carboxylase activities were reduced, whereas Rubisco activation state remained largely unchanged. The results of the determination of reactive oxygen species indicated that a moderate/low irradiance transition had stimulated ¹O₂ accumulation and we strongly suggest that Rubisco oxidative modifications leading to formation of aggregates encompassing Rubisco‐LS were triggered by ¹O₂. When moderate irradiance regime was resumed, the majority of Rubisco‐LS containing aggregates tended to be resolubilized, and this allowed Rubisco carboxylation activities to be largely recovered, without changes in the activation state of the enzyme. In the longer term, these results allow us to better understand a complexity of Rubisco regulatory mechanisms activated in response to abiotic stresses and during recovery from the stresses.     

Ligand Binding to the FA3-FA4 Cleft Inhibits the Esterase-Like Activity of Human Serum Albumin

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of K _s, k ₊₂, and k ₊₂/K _s obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k ₊₃ << k ₊₂). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pK _a-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>_a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.     

Physiological characteristics of citrus plants infested with citrus blackfly

Aleurocanthus woglumi (Ashby, 1915) is an important agricultural pest that causes yield losses of 20–80% in citrus plants by removing plant nutrients while feeding and allowing the formation of sooty mold. The objective of this study was to evaluate physiological changes in citrus plants in response to A. woglumi infestation under field conditions. The experiment was conducted in a citrus orchard in Paço do Lumiar, Maranhão, Brazil. Thirty-two citrus plants were used, including eight of each of the following varieties: Tahiti lime, Tanjaroa tangerine, Nissey tangerine, and Ponkan tangerine. Four random plants with A. woglumi infestation and four plants free from this pest were selected from each variety. The physiological parameters evaluated were photochemical efficiency and gas exchange. Regarding photochemical efficiency, infested plants presented photoinhibition damage, with a performance index of 4.22. The gas exchange parameters of infested plants changed, with reductions in photosynthetic CO₂ assimilation of 69.7% (Tahiti), 64% (Tanjaroa), 68.8% (Nissey) and 63.3% (Ponkan). Plants infested with A. woglumi also presented physiological changes; their photosynthetic CO₂ assimilation, stomatal conductance, instantaneous transpiration, and performance indexes were affected. The infested citrus plants showed photoinhibition of photosystem II. The photosynthetic CO₂ assimilation decreased approximately 70% in Tahiti lime, Tanjaroa tangerine, Nissey tangerine, and Ponkan tangerine plants infested with A. woglumi.     

Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.     

Phylogenetic diversity and genome sizes of Astragalus (Fabaceae) in the Lebanon biogeographical crossroad

The Lebanese mountain range is an important zone of plant species richness and endemism where the genus Astragalus constitutes a principal component of plant biodiversity. Most of endemic Astragalus taxa, living in mountains and arid zones of Mounts Lebanon and anti-Lebanon, are characterized by a cushion, spiny vegetative form, named “tragacanthic”, which is a remarkable example of vegetative convergence evolution. Because of determination difficulties, taxonomic uncertainties, and discrepancy in the number of taxa listed according to authors, new data are hardly needed to improve systematics of Astragalus and to investigate the role of the Lebanese mountain range as refugia of biodiversity. Before this study only two values on the genome size of Astragalus were reported in the literature and no previous molecular studies had been carried out on Astragalus genus in Lebanon. We examined the utility of rDNA ITS molecular markers to distinguish Astragalus species of Lebanese mountain range and the variation range of their genome size. The main results revealed a striking diversity in Lebanese Astragalus species with the emphasis of a huge variation of genome sizes, an important inter-specific chromosome polymorphism and the existence of a high phylogenetic diversity. The strict endemic species of the Lebanese mountains are positioned throughout the phylogeny. These results confirm that the Lebanon and anti-Lebanon mounts constitute a third diversity center for Astragalus and that high altitude areas are important refugia of plant biodiversity despite centuries of exploitation by humans.     

Increased p wave dispersion in elite athletes

Background

Few studies have been performed on P wave indices in athletes. The aim of this study was to determine the behaviour of maximum P wave duration (Pmax), minimum P wave duration (Pmin) and P wave dispersion (PWD) in young high performance athletes, as well as the relationship of PWD with training history, heart rate (HR) and echocardiographic parameters.

Methods

We performed a cross-sectional observational study in 38 athletes of high performance in sports: water polo, distance running and weight lifting compared with 34 sedentary controls.

Results

The average age in both groups was 20.6 years. Note that PWD was increased in athletes (57 ± 14 ms vs. 40 ± 12 ms, p <0.001) while Pmin was significantly lower (57 ± 13 ms vs. 72 ± 13 ms, p <0.001), and there was no difference when comparing Pmax (114 ± 9 ms vs. 117 ± 14 ms, p> 0.05). The correlation between the duration of training (r = 0.511) and resting HR (r = 0.461) with PWD was significant (p <0.01).

Conclusions

PWD is increased in young athletes of high performance and was positively correlated with duration of training and baseline HR. The increase in PWD was secondary to a significant decrease in Pmin.