Presence and Germination of the Probiotic Bacillus subtilis DE111® in the Human Small Intestinal Tract: A Randomized, Crossover, Double-Blind, and Placebo-Controlled Study.

Spore-based probiotics offer important advantages over other probiotics as they can survive the harsh gastric conditions of the stomach and bile salts in the small intestine, ultimately germinating in the digestive tract. A novel clinical trial in 11 ileostomy participants was conducted to directly investigate the presence and germination of the probiotic strain Bacillus subtilis DE111® in the small intestine. Three hours following ingestion of DE111®, B. subtilis spores (6.4 × 104 ± 1.3 × 105 CFU/g effluent dry weight) and vegetative cells (4.7 × 104 ± 1.1 × 105 CFU/g effluent dry weight) began to appear in the ileum effluent. Six hours after ingestion, spore concentration increased to 9.7 × 107 ± 8.1 × 107 CFU/g and remained constant to the final time point of 8 h. Vegetative cells reached a concentration of 7.3 × 107 ± 1.4 × 108 CFU/g at 7 h following ingestion. These results reveal orally ingested B. subtilis DE111® spores are able to remain viable during transit through the stomach and germinate in the small intestine of humans within 3 h of ingestion.

In the absence of thioredoxins, what are the reductants for peroxiredoxins in Thermotoga maritima?

Three peroxiredoxins (Prxs) were identified in Thermotoga maritima, which possesses neither glutathione nor typical thioredoxins: one of the Prx6 class; one 2-Cys PrxBCP; and a unique hybrid protein containing an N-terminal 1-Cys PrxBCP domain fused to a flavin mononucleotide-containing nitroreductase (Ntr) domain. No peroxidase activity was detected for Prx6, whereas both bacterioferritin comigratory proteins (BCPs) were regenerated by a NADH/thioredoxin reductase/glutaredoxin (Grx)-like system, constituting a unique peroxide removal system. Only two of the three Grx-like proteins were able to support peroxidase activity. The inability of TmGrx1 to regenerate oxidized 2-Cys PrxBCP probably results from the thermodynamically unfavorable difference in their disulfide/dithiol E(m) values, -150 and -315 mV, respectively. Mutagenesis of the Prx-Ntr fusion, combined with kinetic and structural analyses, indicated that electrons are not transferred between its two domains. However, their separate activities could function in a complementary manner, with peroxide originating from the chromate reductase activity of the Ntr domain reduced by the Prx domain.

Structure-function relationship of the chloroplastic glutaredoxin S12 with an atypical WCSYS active site.

Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol (28)WCSYS(32) active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys(87)) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys(87) exhibits a certain reactivity, since its pK(a) is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys(29)) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation.

Identification of intra- and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis.

Redox active proteins in plant mitochondria were examined using 2-D oxidant/reductant diagonal-SDS-PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in-gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O-acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox-functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins.

The Cytotoxic lipid peroxidation product 4-hydroxy-2-nonenal covalently modifies a selective range of proteins linked to respiratory function in plant mitochondria.

Plants encounter a variety of environmental stresses that affect their cellular machinery and that they adapt to on a daily basis. Lipid peroxidation is one consequence, at the cellular level, of such stresses and yields cytotoxic lipid aldehydes, including 4-hydroxy-2-nonenal (HNE), that react with specific sites in proteins, leading to diverse changes in protein function and/or stability. We have assessed the sensitivity of plant mitochondrial proteins to HNE modification, using one-dimensional and two-dimensional denaturing PAGE and blue native-PAGE coupled to immunological detection and tandem mass spectrometry identification. A select range of proteins was modified by exogenous application of HNE to mitochondria isolated from Arabidopsis cell cultures. These included a number of proteins that directly interact with the ubiquinone pool, as well as a number of soluble matrix proteins. Mitochondria isolated from cell cultures following hydrogen peroxide, antimycin A, or menadione treatment had significantly reduced respiratory capacity and elevated levels of HNE adduction to specific subsets of proteins. Targets identified included the proteins affected by direct application of HNE but also some new proteins, including a number of matrix dehydrogenases, the inner membrane adenine nucleotide translocator, and the outer membrane voltage-dependent anion channel. Degradation products of some proteins were also found to be HNE adducted, suggesting a link between HNE adduction and protein turnover. Some of the major enzyme complexes that were HNE adducted did not show demonstrable changes in their maximal activity measured with artificial acceptors, but changes did occur in associations between respiratory chain complexes following stress treatments.

Sensitivity of plant mitochondrial terminal oxidases to the lipid peroxidation product 4-hydroxy-2-nonenal (HNE).

We have investigated the effect of the lipid peroxidation product, HNE (4-hydroxy-2-nonenal), on plant mitochondrial electron transport. In mitochondria isolated from Arabidopsis thaliana cell cultures, HNE inhibited succinate-dependent oxygen consumption via the Aox (alternative oxidase), but had minimal effect on respiration via Cox (cytochrome c oxidase). Maximal Cox activity, measured with reduced cytochrome c as substrate, was only slightly inhibited by high concentrations of HNE, at which Aox was completely inhibited. Incubation with HNE prevented dimerization of the Aox protein, suggesting that one site of modification was the conserved cysteine residue involved in dimerization and activation of this enzyme (Cys(I)). However, a naturally occurring isoform of Aox lacking Cys(I) and unable to be dimerized, LeAox1b from tomato (Lycopersicon esculentum), was equally sensitive to HNE inhibition, showing that other amino acid residues in Aox also interact with HNE. The presence of HNE in vivo in Arabidopsis cell cultures was also investigated. Induction of oxidative stress in the cell cultures by the addition of hydrogen peroxide, antimycin A or menadione, caused a significant increase in hydroxyalkenals (of which HNE is the most prominent). Western blotting of mitochondrial proteins with antibodies against HNE adducts, demonstrated significant modification of proteins during these treatments. The implications of these results for the response of plants to reactive oxygen species are discussed.