Applying heat and mass balance theory to determine the flow rate for the measurement of benthic material flux in a flow-through system

Movement of water overlying the sediments has not been taken into consideration in most of the experiments conducted to estimate the dissolved material flux from the sediment. Even in recent experiments that incorporated the stirring motion, interpretation of the data is difficult, because the mixing rate used may be different from actual mixing rate in the field. We propose a method to estimate the in situ mixing rate that should be used to set the flow rate in a flow-through core incubation system. The flow rate is calculated from the vertical mixing rate of the water that is deduced from the heat diffusivity. Release rates of NO₃+NO₂–N obtained from our flow-through incubation system were higher by 1–3 orders of magnitude than those from the conventional diffusion calculation method that estimates the flux from the gradient of nutrient concentration across the sediment-water interface. Increase in NO₃+NO₂–N flux is considered to be due to intensification of the nitrification process as a result of an increase in dissolved oxygen (DO) supply with the motion of water. DO supply is also considered to be an important factor controlling macrofaunal abundance and consequently their excretory contributions to the fluxes of dissolved organic nitrogen as well as NH₄–N. From this point of view, we strongly recommend the application of heat and mass balance theory to estimate nitrogen flux using a flow-through experimental system.     

Structural switching of Cu,Zn-superoxide dismutases at loop VI: insights from the crystal structure of 2-mercaptoethanol-modified enzyme

Cu,Zn SOD1 (superoxide dismutase 1) is implicated in FALS (familial amyotrophic lateral sclerosis) through the accumulation of misfolded proteins that are toxic to neuronal cells. Loop VI (residues 102–115) of the protein is at the dimer interface and could play a critical role in stability. The free cysteine residue, Cys¹¹¹ in the loop, is readily oxidized and alkylated. We have found that modification of this Cys¹¹¹ with 2-ME (2-mercaptoethanol; 2-ME-SOD1) stabilizes the protein and the mechanism may provide insights into destabilization and the formation of aggregated proteins. Here, we determined the crystal structure of 2-ME-SOD1 and find that the 2-ME moieties in both subunits interact asymmetrically at the dimer interface and that there is an asymmetric configuration of segment Gly¹⁰⁸ to Cys¹¹¹ in loop VI. One loop VI of the dimer forms a 3₁₀-helix (Gly¹⁰⁸ to His¹¹⁰) within a unique β-bridge stabilized by a hydrogen bond between Ser¹⁰⁵-NH and His¹¹⁰-CO, while the other forms a β-turn without the H-bond. The H-bond (H-type) and H-bond free (F-type) configurations are also seen in some wild-type and mutant human SOD1s in the Protein Data Bank suggesting that they are interconvertible and an intrinsic property of SOD1s. The two structures serve as a basis for classification of these proteins and hopefully a guide to their stability and role in pathophysiology.     

Adipose-derived mesenchymal stromal cells from genetically modified pigs: immunogenicity and immune modulatory properties

Background aimsThe immunomodulatory and anti-inflammatory effects of mesenchymal stromal cells (MSC) could prove to be a potential therapeutic approach for prolongation of survival of cell xenotransplantation. Adipose (Ad) MSC from genetically modified pigs could be an abundant source of pig donor-specific MSC.MethodsPig (p) MSC were isolated from adipose tissue of α1,3-galactosyltransferase gene knock-out pigs transgenic for human (h) CD46 (GTKO/hCD46), a potential source of islets. After characterization with differentiation and flow cytometry (FCM), AdMSC were compared with bone marrow (BM) MSC of the same pig and human adipose-derived (hAd) MSC. The modulation of human peripheral blood mononuclear cell (hPBMC) responses to GTKO pig aortic endothelial cells (pAEC) by different MSC was compared by measuring 3H-thymidine uptake. The supernatants from the AdMSC cultures were used to determine the role of soluble factors.ResultsGTKO/hCD46 pAdMSC (i) did not express galactose-α1,3-galactose (Gal) but expressed hCD46, (ii) differentiated into chondroblasts, osteocytes and adipocytes, (iii) expressed stem cell markers, (iv) expressed lower levels of Swine Leucocyte Antigen I (SLAI), Swine Leucocyte Antigen II DR (SLAIIDR) and CD80 than pAEC before and after pig interferon (IFN)-γ stimulation. The proliferative responses of hPBMC to GTKO/hCD46 pAdMSC and hAdMSC stimulators were similar, and both were significantly lower than to GTKO pAEC (P < 0.05). The proliferation of hPBMC to GTKO pAEC was equally suppressed by GTKO/hCD46 pAdMSC and hAdMSC (P > 0.05). The supernatant from GTKO/hCD46 pAdMSC did not suppress the human xenoresponse to GTKO pAEC, which was cell–cell contact-dependent.ConclusionsInitial evidence suggests that genetically modified pAdMSC function across the xenogeneic barrier and may have a role in cellular xenotransplantation.     

MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.     

Synthesis and structure-activity relationships of potent 4-fluoro-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors

Dipeptidyl peptidase IV (DPP-IV) inhibitors are promising antidiabetic drugs, and several drugs are in the developmental stage. We previously reported that the introduction of fluorine to the 4-position of 2-cyanopyrrolidine enhanced the DPP-IV inhibitory effect. In the present report, we examined the structure-activity relationship (SAR) of 2-cyano-4-fluoropyrrolidine with N-substituted glycine at the 1-position. We report the identification of a potent and stable DPP-IV inhibitor (TS-021) with a long-term persistent plasma drug concentration and a potent antihyperglycemic activity.     

A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.     

Reducing effect of feeding powdered nacre of Pinctada maxima on the visceral fat of rats

An abdominal fat accumulation complicated by high blood triglycerides is regarded as a risk factor of metabolic syndrome. Feeding powdered nacre, mother of pearl, from Pinctada maxima, resulted in reduced body weight, visceral fat amount, and blood triglyceride level without influencing the food intake, body length, or amount of muscular tissue, suggesting that nacre powder specifically could decrease visceral fat.     

Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus

Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2^T was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into l-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.

     

Germination of the fully myco-heterotrophic orchid Cyrtosia septentrionalis is characterized by low fungal specificity and does not require direct seed-mycobiont contact

We examined the level of specificity exhibited by seeds of Cyrtosia septentrionalis for germination-inducing fungi. Three wood-decomposing Armillaria species (A. gallica, A. mellea subsp. nipponica and A. tabescens) known to colonize adult plants and an unidentified species of Polyporales isolated from naturally growing protocorms were tested. Xylobolus annosus, a free-living decomposer of Russulales, was included as a control. Seed germination occurred in a sawdust-based medium in sealed and unsealed containers in the presence of all fungi, indicating low mycorrhizal specificity in germination. Moreover, germination occurred even in modified containers in which the fungus was physically isolated from the orchid seeds, indicating that direct seed-fungus contact is not required. Higher germination percentages were observed in sealed containers in which a modified atmosphere, consisting of a lower O₂ and a higher CO₂ concentration, had been established in the air above the inoculated medium as a result of the saprophytic activity of the fungus. In nature, atmospheric conditions more effective for seed germination might be established by the action of mycobionts in decomposing wood. Seeds germinated in the presence of the unknown Polyporales species underwent further growth and development, as compared with the other fungi tested, when covered with soil.