Features of the kinetics of accumulating entomocidal exotoxins of Bacillus thuringiensis subsp. thuringiensis during fermentation stage of bitoxibacillin production

Accumulation of Bacillus thuringiensis subsp. thuringiensis beta-exotoxin (BET) in the course of industrial fermentation (a stage in the production of the entomocidal biopreparation bitoxibacillin) has been studied. It has been demonstrated in model experiments that the decrease in the content of BET in the culture fluid is accounted for by the toxin interaction with an attendant product, the exogenous metabolite (EM). EM has been isolated from the culture fluid and characterized. EM causes alkalization of the medium, exerts entomocidal effects (in Musca domestica) and fails to form salts on treatment with BaCl2. The absorption spectrum of EM is similar to that of BET, showing a maximum at lambda = 259 nm. The light-absorbing chromophore is a pyrimidine or purine base. A method for quantitative determination of both exotoxins (BET and EM) in bacterial preparations has been developed.     

Piracetam correction of deficient GABA-ergic inhibition due to early postnatal exposure to cycloheximide

Cycloheximide, administered to 7-day-old rats, caused the delay of CNS activity changes in grown-up rats. The animals showed impaired memory function, expressed in the decrease of habituation in the "open field" test, and the alteration of passive avoidance reflex. The increased number of intersignal reactions due to hypermotility was found during elaboration of avoidance reflex. The analysis of evoked potential recovery revealed the deficiency of GABA-ergic inhibition in the neocortex. Piracetam was shown to prevent completely behavioural disturbances and deficiency of GABA-ergic processes in grown-up animals.     

Chromosomal <Emphasis Type="Italic">flhB1</Emphasis> gene of the alphaproteobacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 is essential for correct assembly of both constitutive polar flagellum and inducible lateral flagella

Azospirillum brasilense has the ability of swimming and swarming motility owing to the work of a constitutive polar flagellum and inducible lateral flagella, respectively. The interplay between these flagellar systems is poorly understood. One of the key elements of the flagellar export apparatus is the protein FlhB. Two predicted flhB genes are present in the genome of A. brasilense Sp245 (accession nos. HE577327–HE577333). Experimental evidence obtained here indicates that the chromosomal coding sequence (CDS) AZOBR_150177 (flhB1) of Sp245 is essential for the production of both types of flagella. In an flhB1::?Omegon-Km mutant, Sp245.1063, defects in polar and lateral flagellar assembly and motility were complemented by expressing the wild-type flhB1 gene from plasmid pRK415. It was found that Sp245.1063 lost the capacity for slight but statistically significant decrease in mean cell length in response to transfer from solid to liquid media, and vice versa; in the complemented mutant, this capacity was restored. It was also shown that after the acquisition of the pRK415-harbored downstream CDS AZOBR_150176, cells of Sp245 and Sp245.1063 ceased to elongate on solid media. These initial data suggest that the AZOBR_150176-encoded putative multisensory hybrid sensor histidine kinase–response regulator, in concert with FlhB1, plays a role in morphological response of azospirilla to changes in the hardness of a milieu.     

3D in vitro co-culture models based on normal cells and tumor spheroids formed by cyclic RGD-peptide induced cell self-assembly

Objectives

To design novel 3D in vitro co-culture models based on the RGD-peptide-induced cell self-assembly technique.

Results

Multicellular spheroids from M-3 murine melanoma cells and L-929 murine fibroblasts were obtained directly from monolayer culture by addition of culture medium containing cyclic RGD-peptide. To reach reproducible architecture of co-culture spheroids, two novel 3D in vitro models with well pronounced core–shell structure from tumor spheroids and single mouse fibroblasts were developed based on this approach. The first was a combination of a RGD-peptide platform with the liquid overlay technique with further co-cultivation for 1–2 days. The second allowed co-culture spheroids to generate within polyelectrolyte microcapsules by cultivation for 2 weeks. M-3 cells (a core) and L-929 fibroblasts (a shell) were easily distinguished by confocal microscopy due to cell staining with DiO and DiI dyes, respectively.

Conclusions

The 3D co-culture spheroids are proposed as a tool in tumor biology to study cell–cell interactions as well as for testing novel anticancer drugs and drug delivery vehicles.

     

Immunoelectron microscopy investigation of the cell surface of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains

The genus-specific surface protein antigens of Azospirillum brasilense strains were visualized immunochemically. The procedure used for cell sample preparation was optimized to ensure that the surface protein structures were detected on cells in situ. Gold and gold-silver nanoparticles were conjugated to antibodies raised against the flagellin of A. brasilense type strain Sp7, against the lipopolysaccharide of A. brasilense Sp245, and against the genus-specific protein determinants of A. brasilense Sp7. Electron microscopic analysis using nanoparticle-labeled antibodies revealed antigenic determinants of the polar flagellum on the A. brasilense Sp245 cell surface, which in these bacteria are normally screened from the surroundings by a lipopolysaccharide sheath. Pili-like structures were detected on the Sp245 wild-type strain and on its Fla^– Swa^– Omegon-Km mutant SK048, which are presumably involved in microcolonial spreading in these bacteria.     

Effect of isolation stress on ethanol pharmacodynamics in the blood of rats

Head space chromatography was used to study the rate of ethanol excretion as criterion of the activity of the ethanol-oxidizing enzymatic system. Isolational stress was shown to be one of the factors determining induction of the ethanol-oxidizing erzymatic systems. The isolation-induced activity of these systems was reduced by diazepam administration. This fact may evidence the existence of central regulation of the activity of the enzymatic systems that metabolize ethanol.     

Microvascular permeability in rats susceptible and not susceptible to the development of experimental alcoholism

Using the fluorescence technique it has been shown that rats predisposed to the development of experimental alcoholism have a higher permeability of mesenterial microvessels as compared to non-predisposed animals. The role of enzymes and the endothelium in the mechanisms of permeability is discussed.