A novel approach for whey recycling in production of bacterial insecticides

A simplified approach was devised to recycle sweet whey in production of spore-δ-endotoxin complex from certain entomopathogenic varieties ofBacillus thuringiensis Berliner. The process suggested aimed at the protection of the environment through dual channels namely biological oxygen demand (BOD) reduction of the byproduct under investigation and its incorporation in a microbial fermentation for production of pollution-free biological insecticides. The sweet whey could be used successfully for endotoxin production as complete fermentation media both as such and with simple treatments. Supplementation of whey media with ground leguminous seeds and fodder yeast resulted in marked increase in the yields of endotoxin produced but the toxicity was not increased proportionnally. Standard biological assays revealed high efficiency of certain strains ofB.t. var.entomocidus, kurstaki andgalleriae in producing endotoxins highly active against 3rd instar larvae ofSpodoptera littoralis Boisduval,Spodoptera exigua Hübner andHeliothis armigera Hübner. The suggested approach and the findings obtained are discussed in view of their application feasibilities.

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Résumé Une méthode simplifiée a été mise au point pour recycler le petit lait dans la production du complexe spore-δ-endotoxine de certaines variétés entomopathogènes deBacillus thuringiensis Berliner. Le procédé proposé a pour but la protection de l'environnement par un double canal chimique, c'est-à-dire la réduction de la demande biologique en oxygène du sous produit étudié et son incorporation dans une fermentation microbienne pour la fabrication d'insecticides biologiques non polluants. Le petit lait peut être employé avec succès pour la production d'endotoxine à la fois en tant que milieux complets de fermentation et avec des traitements simples. L'addition aux milieux à base de petit lait de graines de légumineuses et de levure alimentaire aboutit à une augmentation sensible des rendements en endotoxine mais la toxicité n'est pas accrue en proportion. Des essais biologiques normalisés montrent une activité élevée de certaines souches deB.t. var.entomocidus, kurstaki etgalleriae qui produisent des endotoxines très actives à l'égard des larves de 3e stage deSpodoptera littoralis Boisduval,Spodoptera exigua Hübner etHeliothis armigera Hübner. La méthode proposée et les résultats obtenus sont discutés en vue de leur faisabilité d'application.

     

Chemical changes in the haemolymph ofSpodoptera littoralis [Lep.: Noctuidae] as affected byBacillus thuringiensis

Analyses of the haemolymph of the larvae ofSpodoptera littoralis Boisduval with an amino acid analyser indicated the presence of a group of amino acids, some of which showed an obvious quantitative decrease as a result of treatment withBacillus thuringiensis Berliner such as threonine, serine, asparagine, alanine, valine, methionine, isoleucine, leucine, phenyl alanine and ornithine. Glutamic acid, cystine, β-alanine, γ-butyric acid and histidine contents of the haemolymph of diseased larvae were higher than those in the haemolymph of normal individuals. This increase in the content of some amino acids in infested larvae may be attributed to the possible dissolution of protein crystals ofBacillus thuringiensis. The activity of proteolytic enzymes in the gut may be partly involved in the reaction. B. thuringiensis was found to affect the concentration of some ions of the haemolymph ofS. littoralis. So, a marked decrease in sodium and potassium concentration was observed 4–5 days after treatment withB. thuringiensis. The change in Na⁺/K⁺ ratio in healthy and treated larvae possibly indicate their interference, in the larval toxicity withB. thuringiensis. An obvious decrease in the concentration of magnesium and zinc was also observed in the larval haemolymph after feeding on a diet containing theB. thuringiensis preparations. The pH value of the haemolymph however showed no change after treatment withB. thuringiensis.

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Résumé L'analyse de l'hémolymphe des chenilles deSpodoptera littoralis Boisduval à l'aide d'un analyseur d'acides aminés montre une diminution significative de la quantité de certains acides aminés à la suite d'un traitement parBacillus thuringiensis Berliner, tels que: thréonine, sérine, asparagine, alanine, valine, methionine, isoleucine, leucine, phenylalanine et ornithine. La teneur en acide glutamique, cystine, β alanine, γ acide butyrique et histidine de l'hémolymphe des larves traitées est plus élevée que celle des chenilles témoins. Cette augmentation est peut-être liée à la dissolution des protéines du cristal deB. thuringiensis. B. thuringiensis affecte également la concentration de certains ions dans l'hémolymphe deS. littoralis. On observe une nette réduction du taux de sodium et de potassium 4 à 5 jours après le traitement. Il est possible que cette modification du rapport Na⁺/K⁺ chez les larves malades traduise l'interférence de ces deux ions dans les phénomènes de toxicité deB. thuringiensis. Une diminution nette de la concentration en magnésium et en zinc est également notée dans l'hémolymphe des chenilles après alimentation sur un milieu traité parB. thuringiensis. Le pH de l'hémolymphe n'est pas affecté.

     

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue,cell, and model membrane preparations

The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.     

Ryanodine-affinity chromatography purifies 106 kD Ca release channels from skeletal and cardiac sarcoplasmic reticulum

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the 'physiological' Ca2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca2+ release channels which are a minor component (0.1-0.3%) of SR proteins.     

New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n?=?16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n?=?16) after operation were cooled to 5 °C for 24 h, then frozen after 24 h pre-cooling to 5 °C, thawed and just after thawing their quality was analyzed. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.

     

Influence of Magnetic Field on Brain Activity During Administration of Caffeine

The aim of the present work is to evaluate the effect of caffeine, the world’s most popular psychoactive drug, on the electric activity of the rat’s brain that exposed to extremely low-frequency magnetic field (ELF-MF), during 15 days. The obtained results showed that administration of caffeine in a group of rats by dose of 10 mg/kg (equivalent to human daily consumption) caused a reduction in the mean power amplitude of electroencephalogram (EEG) trace for almost all frequency bands especially α (8–12 Hz). It was observed that the influence of caffeine was more evident in motor cortex than in visual cortex. While the exposure of another group to ELF-MF of intensity 0.2 mT during the same period caused an enhancement in the mean power amplitude of most EEG frequency bands; this was more observed in the right hemisphere of the brain than that of the left hemisphere. The administration of caffeine while rats were exposed to ELF-MF, led, after 5 days of exposure, to a great increase in the mean power amplitude of α band at all places of recording electrodes. It may be concluded that caffeine administration was more effective in reducing the hazardous of ELF-MF in motor cortex than in visual cortex.     

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.     

Enhancement of Bioavailability and Pharmacodynamic Effects of Thymoquinone Via Nanostructured Lipid Carrier (NLC) Formulation

Thymoquinone (TQ), obtained from black cumin (Nigella sativa), is a natural product with anti-oxidant, anti-inflammatory, and hepatoprotective effects but unfortunately with poor bioavailability. Aiming to improve its poor oral bioavailability, TQ-loaded nanostructured lipid carriers (NLCs) were prepared by high-speed homogenization followed by ultrasonication and evaluated in vitro. Bioavailability and pharmacodynamic studies were also performed. The resultant NLCs showed poor physical homogeneity in Compritol 888 ATO Pluronic F127 system which consequently produced larger particle size and polydispersity index, smaller zeta potential values, and lower short-term (30 days) physical stability than other systems. Encapsulation efficiency percentage (EE%) lied between 84.6?±?5% and 96.2?±?1.6%. TQ AUC_0–t values were higher in animals treated with NLCs, with a relative bioavailability of 2.03- and 3.97-fold (for F9 and F12, respectively) higher than TQ suspension, indicating bioavailability enhancement by NLC formulation. Hepatoprotective effects of F12 showed significant (P?<?0.05) decrease in both serum alanine amino transferase and aspartate amino transferase to reach 305.0?±?24.88 and 304.7?±?23.55 U/ml, respectively, when compared with untreated toxic group. Anti-oxidant efficacy of F12 showed significant (P?<?0.05) decline of malondialdehyde and elevation of reduced glutatione. This improvement was also confirmed histopathologically.     

Speeding up Monte Carlo molecular simulation by a non-conservative early rejection scheme

Monte Carlo (MC) molecular simulation describes fluid systems with rich information, and it is capable of predicting many fluid properties of engineering interest. In general, it is more accurate and representative than equations of state. On the other hand, it requires much more computational effort and simulation time. For that purpose, several techniques have been developed in order to speed up MC molecular simulations while preserving their precision. In particular, early rejection schemes are capable of reducing computational cost by reaching the rejection decision for the undesired MC trials at an earlier stage in comparison to the conventional scheme. In a recent work, we have introduced a ‘conservative’ early rejection scheme as a method to accelerate MC simulations while producing exactly the same results as the conventional algorithm. In this paper, we introduce a ‘non-conservative’ early rejection scheme, which is much faster than the conservative scheme, yet it preserves the precision of the method. The proposed scheme is tested for systems of structureless Lennard-Jones particles in both canonical and NVT-Gibbs ensembles. Numerical experiments were conducted at several thermodynamic conditions for different number of particles. Results show that at certain thermodynamic conditions, the non-conservative method is capable of doubling the speed of the MC molecular simulations in both canonical and NVT-Gibbs ensembles.