Finding the best antibody dilution by repeated immunostaining of the same tissue section

One can find the optimal antibody dilution for immunostaining by repeated staining on the same tissue section by using a less dilute antibody for each attempt. Using secondary antibody and horseradish peroxidase conjugated to a dextran polymer, a section stained repeatedly with several dilutions of antibody appears as good as a section stained with only the last dilution.     

Long-term H<Subscript>2</Subscript> photoproduction from starch by co-culture of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> and <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> in a repeated batch process

Objectives

To prove the possibility of efficient starch photofermentation in co-culture of heterotrophic and phototrophic bacteria over prolonged period.

Results

Repeated batch photofermentation of starch was demonstrated in co-culture Clostridium butyricum and Rhodobacter sphaeroides under microaerobic conditions. It continued 15 months without addition of new inoculum or pH regulation when using 4–5 g starch l^?1 and 0.04 g yeast extract l^?1. The complete degradation of starch without volatile fatty acids accumulation was shown in this co-culture. The average H₂ yield of 5.2 mol/mol glucose was much higher than that in Clostridium monoculture. The species composition of co-culture was studied by q-PCR assay. The concentration of Clostridium cells in prolonged co-culture was lower than in monoculture and even in a single batch co-culture. This means that Clostridia growth was significantly limited whereas starch hydrolysis still took place.

Conclusion

The prolonged repeated batch photofermentation of starch by co-culture C. butyricum and R. sphaeroides provided efficient H₂ production without accumulation of organic acids under conditions of Clostridia limitation.

     

Histochemical changes in neonatal liver caused by vaginal instillation of magnetic nanoparticles in pregnant mice

Drug delivery through the vagina is a novel and effective approach for treating embryonic diseases. Magnetic nanoparticles (MNPs) currently are used as drug delivery systems. The safety of MNPs for use with embryonic tissues remains unclear. We used pregnant mice to investigate the possible toxicity of MNPs toward neonatal liver at three embryonic ages using histochemical and immunohistochemical techniques. MNPs were instilled through the vaginas of pregnant mice at days 12 (E12), 15 (E15) and 17 (E17) after fertilization. We found MNPs in the neonatal liver parenchyma after delivery of the pups on day 20. We observed that MNPs caused mild apoptosis of hepatocytes, cytoplasmic vacuolation and lymphocytic infiltration in the neonatal liver after treatment at E15 compared to instillation at E12 and E17. We observed also that MNPs increased the production of caspase proteins and tumor necrosis factor receptor 2 proteins, which are indicators of apoptosis, in the neonatal liver after instillation of MNPs at E15 compared to instillation at E12 and E17. MNPs also increased the number of collagen fibers and the amounts of connective tissue growth factors in the neonatal liver parenchyma after instillation at E15 compared to instillation at E12 and E17. The general carbohydrates in the neonatal liver were decreased in a time-dependent manner after instillation at E17, E15 and E12 owing to the presence of MNPs in the parenchyma. Overall, we determined that MNPs were mildly toxic to neonatal liver.     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.     

Genome organization of the plasmid of IncQ/P4 group and its vector derivatives

The genome organization and functioning of IncQ/P4 plasmids are reviewed. Based on these plasmids, cloning vectors have been constructed for broad host range of gram-negative bacteria. Together with one- and two-replicon vectors for cloning via insertion inactivation of markers, specialized plasmid vectors are described: cosmids, promoter-probe vectors, vectors for direct selection of recombinant molecules. Examples of using broad host range vectors for gene cloning and expression in non-enteric gram-negative bacteria are presented.     

Development of gene transfer systems in Methylobacillus flagellatum KT: Isolation of auxotrophic mutants

A collection of polyauxotrophic mutants of the obligate methylotroph Methylobacillus flagellatum KT was obtained. On the first step two stable auxotrophic mutants with a high requirement for amino acids supplements were isolated by treatment with nitrosoguanidine and selection on complete medium. Spontaneous variants of these mutants with a low requirement for nutrient supplements were the base for obtaining polyauxotrophic strains. It was shown, that the growth of mutants of M. flagellatum KT is inhibited by complete medium. Some amino acids and nucleotides are the inhibitor components of complete media. An approach for selection of auxotrophic mutants of individual genes was worked out on minimal medium. The optimal conditions for nitrosoguanidine mutagenesis of M. flagellatum KT were developed. The possible mechanisms of action of some of the nutrient supplements on the growth of M. flagellatum KT are discussed.     

Characteristics of Methylobacillus flagellatum KT mutants, deficient for phosphoglucoisomerase, an enzyme of the ribulose monophosphate cycle of obligate methylotrophic bacteria

The activities of the key enzymes of ribulose monophosphate cycle for formaldehyde oxidation and assimilation were tested in crude extracts from temperature sensitive mutants of obligatemethylotroph M. flagellatum KT. Two mutants deficient in phosphoglucoisomerase activity were identified during this screening. Phosphoglucoisomerase of T525 pgi-1 mutant was active both at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. Complete inactivation of the enzyme at 42 degrees C occurred in 2 h in vitro, while in vivo incubation at nonpermissive temperature for more than 10 h was required for the enzyme inactivation. Phosphoglucoisomerase activity of T566 pgi-2 was 5-fold lower as compared with the one from the parent strain incubated at 30 degrees C. The enzyme was inactivated in 2 min. in crude extract at nonpermissive temperature.     

Nitrogen-fixing cyanobacteria: A review

The review sums up recent data on the key enzymes involved in hydrogen evolution. Hydrogen release by cyanobacteria is due to the effects of nitrogenases and hydrogenases. Cyanobacteria hold important practical implications as transformers of solar energy into molecular hydrogen viewed as a fuel. Avenues of further research in the field are discussed.