Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.     

Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts: CHANNELING OF ACETYL-CoA FROM PYRUVATE DEHYDROGENASE TO CARNITINE ACETYLTRANSFERASE*

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [¹³C₂,²H₃]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [¹³C₆]glucose or [1,2-¹³C₂]palmitate) or/and M1 acetyl-CoA (e.g. [1-¹³C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.     

Microbial community structure and dynamics in a mixotrophic nitrogen removal process using recycled spent caustic under different loading conditions

A laboratory-scale Bardenpho process was established to investigate the proper nitrogen loading rate (NLR) when modified spent caustic (MSC) is applied as electron donor and alkalinity source for denitrification. MSC injection induced autotrophic nitrogen removal with sulfur as electron donor and heterotrophic denitrification. The nitrogen removal rate (NRR) did not increase proportionally to NLR. Based on the total nitrogen concentration in the effluent observed in the trials with MSC, the NLR in the influent should not exceed 0.15 kg N/m³ d in order to satisfy water quality regulations. Microbial communities in the anoxic reactors were characterized by pyrosequencing of 16S rRNA gene sequences amplified by the polymerase chain reaction of DNA extracted from sludge samples. Microbial diversity was lower as MSC dosage was increased, and the injection of MSC caused an increase in SOB belonging to the genus Thiobacillus which is responsible for denitrification using sulfur.     

Statistical modelling and optimization of substrate composition for bacterial growth and cadmium removal using response surface methodology

In this study, substrate composition was optimized for the growth of Achromobacter xyloxidans and biosorption of Cd(II) from aqueous solution. Response surface methodology (RSM) was used to investigate the function of three independent operating variables, namely, peptone (2.5-10 g/L), beef extract (2.5-5.0 g/L) and incubation time (24-96 h), on dependent variables, i.e. sorption of Cd(II) ions, protein content and biomass growth of A. xyloxidans. The maximum Cd(II) removal efficiency of 69.2%, protein content 1.9 mg/L and growth 0.354 optical density was found at optimal conditions of peptone 10 g/L, incubation time 60 h and beef extract 2.5 g/L. The significance of independent variables and interactions between variables were tested by means of the analysis of variance (ANOVA) with 95% confidence limits and values of “Prob > F” less than 0.0500 indicate that model terms are significant. Fourier transfer infrared (FTIR) analysis was used to investigate sorption mechanism and involved functional groups in Cd(II) binding.     

Conversion for Avicel and AFEX pretreated corn stover by Clostridium thermocellum and simultaneous saccharification and fermentation: insights into microbial conversion of pretreated cellulosic biomass

In this study, efforts were taken to compare solubilization of Avicel and AFEX pretreated corn stover (AFEX CS) by SSF and Clostridium thermocellum fermentation, with an aim to gain insights into microbial conversion of pretreated cellulosic biomass. Solubilization rates for AFEX CS are comparable for the two systems while solubilization of Avicel is much faster by C. thermocellum. Initial catalyst loading impacts final cellulose conversion for SSF but not for C. thermocellum. Hydrolysis of the two substrates using cell-free C. thermocellum fermentation broth revealed much smaller difference in cellulose conversion than the difference observed for growing cultures. Tests on hemicellulose removal and particle size reduction for AFEX CS indicated that substrate accessibility is very important for enhanced solubilization by C. thermocellum.     

Substrate stiffness regulates apoptosis and the mRNA expression of extracellular matrix regulatory genes in the rat annular cells

Cells are subjected to static tension of different magnitudes when cultured on substrates with different stiffnesses. It has long been recognized that mechanical stress is an important modulator of the intervertebral disc degeneration. Here we studied the influence of substrate stiffness on cell morphology, apoptosis and extracellular matrix (ECM) metabolism of the rat annulus fibrosus (AF) cells which are known to be mechanosensitive cells. Polyacrylamide gel substrates with three different stiffnesses were prepared by varying the concentration of acrylamide and bisacrylamide, and the elastic modulus of the different gel substrates were measured with atomic force microscopy (AFM). First-passage rat annular cells were cultured on soft, intermediate, rigid substrates or plastics for 24 or 48 h. The percentages of apoptotic cells were detected by flow cytometry and caspase-3 activity, and morphologic changes were visualized by Hoechst 33258 staining and F-actin staining. In addition, the expression of ECM genes (Col1α1, Col2α1, aggrecan, MMP-3, MMP-13 and ADAMTS-5) were analyzed by RT-PCR. The three different substrates had elastic moduli varying between 1 ± 0.23 kPa (soft, 5% gel with 0.06% bis), 32 ± 2.89 kPa (intermediate, 10% gel with 0.13% bis) and 63 ± 3.45 kPa (rigid, 10% gel with 0.26% bis) with a thickness about 60-70 μm. Most of the rat AF cells appeared small and rounded, and lost most of their stress fibers when cultured on soft substrate. There was a significant increase in the percentage of apoptotic cells in the rat AF cells cultured on soft and intermediate substrates relative to those on plastic surface, with a parallel decrease in the area of cell spreading and nucleus. The AF cells grown on intermediate or rigid substrate had reduced expression of Col1α1, Col2α1 and aggrecan and enhanced expression of MMP-3, MMP-13, and ADAMTS-5 at 24 h or 48 h, respectively, relative to those cultured on plastic surface. Conversely, we observed an up-regulation of Col2α1 and aggrecan and no change in the gene expression of MMP-3, MMP-13, and ADAMTS-5 in AF cells on soft substrates. Rat AF cells are sensitive to substrate stiffness which can regulate the morphology, growth, apoptosis and ECM metabolism of rat AF cells, thus indicating the importance of substrate choice for cell transplantation and regeneration for the treatment of disc degeneration using tissue-engineering technique.     

Basic studies and applications on bioremediation of DDT: A review

The persistent insecticide DDT (1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane) has been widely used for pest control in the management of mosquito-borne malaria and is still used for that purpose in some tropical countries. Considering the potential for negative effects due to DDT contamination, it is necessary to determine effective methods of remediation. Several methods have been used to degrade or transform DDT into less toxic compounds. Bacteria and white-rot fungi (WRF) have been shown to enhance the degradation process in soil using both pure and mixed cultures. Recently, a biological approach has been used as an environmentally-friendly treatment, using new biological sources to degrade DDT, e.g. brown-rot fungi (BRF), cattle manure compost (CMC) and spent mushroom waste (SMW). In this review, the abilities of BRF, CMC and SMW to degrade DDT are discussed, including the mechanisms and degradation pathways. Furthermore, application of these sources to contaminated soil is also described. The review discusses which is the best source for bioremediation of DDT.     

Validation of thermal composting process using olive mill solid waste for industrial scale cultivation of Agaricus bisporus

The production of a substrate containing destoned olive mill solid waste for the cultivation of Agaricus bisporus (Lange) Imbach on an industrial scale was studied. A standard mushroom compost (C) mainly made from straw and poultry manure was compared with the experimental compost (EC) containing the same ingredients as (C) but with added olive mill solid waste (10.6% w/w). Microbial indicators such as counts of heterotrophic microbes and actinomycetes were higher in EC than in C. In addition, compost selectivity as indicated by higher mushroom yield and biological efficiency of EC was higher than that of C. Market quality of the mushrooms produced in both C and EC were comparable. These findings support our work that olive mill solid waste can be used safely in thermal composting process to produce a selective substrate for industrial-scale cultivation of A. bisporus. This study also demonstrates an environmentally sustainable system to manage solid waste from olive oil extraction processes thus overcoming environmental pollution brought about by irrational disposal of the waste on farm lands.     

Substrate profiling of IGF-1R and InsR: identification of a potent pentamer substrate

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. As part of a study on the substrate requirements of Insulin-like Growth Factor 1 Receptor (IGF-1R) and Insulin Receptor (InsR), we evaluated and applied a universal assay system able to monitor the phosphorylation of unlabelled peptides of any length in real time. In contrast to already reported profiling methodologies, we were able to assess the k(cat)/K(M) ratio of peptides as short as tetramers. Notably, we were able to identify an efficient pentamer substrate that exhibited kinetic properties close to those of a 250-amino acid protein derived from IRS-1, a natural substrate of IGF-1R and InsR.     

Waste biomass of Nostoc linckia as adsorbent of crystal violet dye: Optimization based on statistical model

The potential of spent biomass of a hydrogen producing cyanobacterial strain Nostoc linckia from a hydrogen fermentor was studied for decolorization of a tri-phenylmethane dye, crystal violet. The waste cyanobacterial biomass immobilized in calcium alginate was used as a biosorbent and the process variables were optimized for maximum dye removal using the statistical response surface methodology (RSM). Batch mode experiments were performed to determine the kinetic behavior of the dye in aqueous solution allowing the computation of kinetic parameters. Influence of interacting parameters like temperature (25-35 °C), pH (4-8), initial dye concentration (100-200 mg/L) and cyanobacterial dose (0.2-0.4 g) on dye removal were examined using central composite design (CCD) which included two additional levels for each parameter. Second-order polynomial regression model, was applied which was statistically validated using analysis of variance. Ability of the immobilized biomass to decolorize the dye was maximum (72%) at pH 8.0, temperature 35 °C, 200 mg/L initial dye concentration and 0.2 g cyanobacterial dose. Adsorption of the dye on cell surface was further confirmed by scanning electron micrographs of the biomass before and after dye loading. FT-IR studies revealed that decolorization was due to biosorption mediated mainly by functional groups like hydroxyl, amide, carboxylate, methyl and methylene groups present on the cell surface.