Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.     

Barley microgreen incorporation in diet-controlled diabetes and counteracted aflatoxicosis in rats

Increased environmental pollution and unhealthy lifestyle are blamed for escalated chronic diseases. Exposure to aflatoxins was recently suggested to have a role in the increased incidence of type 2 diabetes mellitus. Diet modification and consumption of different functional food are now gaining attention, especially in diabetes management. This study investigates the effect of a diet containing barley microgreen against diabetes induced by streptozotocin with or without aflatoxin administration in rats. Barley microgreen was rich in 3′-Benzyloxy-5,6,7,4′-tetramethoxyflavone (48.8% of total) followed by 5β,7βH,10α-Eudesm-11-en-1α-ol (18.46%). Streptozotocin injection and/or aflatoxin administration significantly elevated glucose level, decreased insulin level, decreased β-cell function, deteriorated liver and kidney function parameters, and induced oxidative stress in the liver. Histopathology revealed irregular small-sized islets and decreased area % of insulin-positive beta cells in the pancreas, hepatic degeneration, nephropathy, and neuropathy in diabetic and/or aflatoxin administered rats compared to control. Barley microgreen diet fed to diabetic rats with or without aflatoxin alleviated all evaluated parameters. Barley microgreen diet also ameliorated the toxic effect of aflatoxin. In conclusion, exposure to aflatoxin aggravated diabetes and its complication. The incorporation of barley microgreen in the diet was able to control type 2 diabetes mellitus and the improved outcomes observed with barley microgreen treatments involved or occurred in conjunction with improved biomarkers of oxidative stress.     

The presence of ADP-ribosylated Fe protein of nitrogenase in Rhodobacter capsulatus is correlated with cellular nitrogen status

The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH4+. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH4+, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH4+, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions.     

Laboratory photobioreactors

Growing phototrophic microorganisms consume the light energy. These microorganisms most often suffer from light deficiency because of exponential decrease in the energy of light passing through an absorbing medium. Therefore, effective distribution of light within the cultures is needed for their intensive cultivation. This is possible in special devices called photobioreactors. The photobioreactors described in the literature are classified into several types according to their geometric features. Their advantages and drawbacks are analyzed. Criteria currently used for comparing various photobioreactors are specified.     

Hydrogen production by cyanobacteria in an automated outdoor photobioreactor under aerobic conditions

The possibility of hydrogen production by a hydrogenase impaired mutant strain of Anabaena variabilis in outdoor culture was studied. A computer-controlled rooftop (outdoor) tubular photobioreactor (4.35 L) was assembled. H(2) production rates by A. variabilis PK84 grown in CO(2) + air in the photobioreactor were measured together with other parameters such as temperature, irradiance, pH, dry biomass weight, and pO(2), and Chl a concentrations during summer months of 1998 and 1999. Efficiencies of light energy bioconversion to H(2) energy and energy accumulated in biomass were calculated. The influence of irradiance, temperature, and mode of cultivation on H(2) production and efficiency of light energy bioconversion were evaluated. The culture produced up to 1.1 L H(2) day(-1) PhBR(-1). The efficiency of light energy to H(2) energy bioconversion on some days was 0.094%. However, the conditions for maximum H(2) photoproduction and for maximum efficiency of light energy to H(2) energy bioconversion were not the same. A. variabilis PK84 could produce hydrogen for prolonged periods (up to 40 days) without injection of fresh inoculum. During this period photobioreactor produced 24.5 L of H(2). Possibilities for increasing the efficiency of light energy conversion are discussed.     

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.     

New Photosensitizers of Bacteriochlorin Series for Photodynamic Cancer Therapy

New derivatives of bacteriochlorophyll a bearing an extra glutarimide exocycle were synthesized, and their reactivity was studied. Acetyl group in 3-acetyl-2,7,12,18-tetramethyl-8-ethyl-13,15-dicarboxy-17-carboxyethyl-7,8,17,18-tetrahydroporphyrin (bacteriochlorin p) was chemically modified into -hydroxyethyl and vinyl groups. A simple method of preparation of vinylbacteriopurpurin esters under the catalysis by p-toluenesulfonic acid was proposed. The resulting compounds exhibit a high adsorption in the visible and near IR areas of electronic spectra, a reasonable stability, and amphiphilic properties and, therefore, may be regarded as promising photosensitizers for the photodynamic cancer therapy.     

T-cell ubiquitin ligand affects cell death through a functional interaction with apoptosis-inducing factor, a key factor of caspase-independent apoptosis

The lymphoid protein T-cell ubiquitin ligand (TULA)/suppressor of T-cell receptor signaling (Sts)-2 is associated with c-Cbl and ubiquitylated proteins and has been implicated in the regulation of signaling mediated by protein-tyrosine kinases. The results presented in this report indicate that TULA facilitates T-cell apoptosis independent of either T-cell receptor/CD3-mediated signaling or caspase activity. Mass spectrometry-based analysis of protein-protein interactions of TULA demonstrates that TULA binds to the apoptosis-inducing protein AIF, which has previously been shown to function as a key factor of caspase-independent apoptosis. Using RNA interference, we demonstrate that AIF is essential for the apoptotic effect of TULA. Analysis of the subcellular localization of TULA and AIF together with the functional analysis of TULA mutants is consistent with the idea that TULA enhances the apoptotic effect of AIF by facilitating the interactions of AIF with its apoptotic co-factors, which remain to be identified. Overall, our results shed new light on the biological functions of TULA, a recently discovered protein, describing its role as one of very few known functional interactors of AIF.     

Comparison of genotypic and biochemical characteristics of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> strains isolated from sour milk products

Overall, 72 strains of lactic acid thermophilic streptococci isolated from sour milk products manufactured in various regions of Russia and European countries were analyzed using classical microbiological and molecular biological methods. Physiological and biochemical properties and genetic diversity of these Streptococcus thermophilus strains were studied, and a comparative analysis of the nucleotide sequences of the 16S rRNA gene was conducted. It has been demonstrated that the homology of proximal parts of the 16S rRNA gene of all the strains studied towards one another and towards the reference strain ATCC19258 amounts to 100%. As for the sugar fermentation, some strains display the characteristics untypical of the S. thermophilus members. The data obtained suggest that it is preferable to use gene 16S rRNA sequencing data for identification of natural isolates of closely related lactic acid bacterial species; moreover, this method is recommended for a precise species identification of industrial bacterial strains used in the food industry.     

Synthesis of bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis> by the purple nonsulfur bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

The ability of purple nonsulfur bacteria Rhodobacter capsulatus B10 to synthesize bacteriochlorophyll under phototrophic and dark conditions was studied. The modes for cultivation in the dark with oxygen limitation in a continuous culture at D = 0.1 h^?1 were selected. The yield of biomass reached 20 g/l; the bacteriochlorophyll a output of the process amounted to 16.6 mg/l h^?1.