Thermophilic and halophilic β-agarase from a halophilic archaeon Halococcus sp. 197A

An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a β-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl₂ or MgCl₂. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65–80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl₂, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.     

Matrix-Embedded Osteocytes Regulate Mobilization of Hematopoietic Stem/Progenitor Cells

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A Combination of a Dairy Product Fermented by Lactobacilli and Galactooligosaccharides Shows Additive Effects on Mineral Balances in Growing Rats with Hypochlorhydria Induced by a Proton Pump Inhibitor

This study aimed to investigate the effects of a combination of a dairy product fermented by lactobacilli (DFL) and galactooligosaccharides (GOS) on mineral balances in growing rats with hypochlorhydria induced by a proton pump inhibitor (PPI). Three-week-old male rats were assigned to receive one of six diets: a control diet, control diets containing 1.6 or 5.0 % GOS, a DFL diet and DFL diets containing 1.6 or 5.0 % GOS for 9 days. From day 5 of the feeding period, half of the rats fed with control diets were subcutaneously administered with saline, whereas the remaining rats were administered with PPI for 5 days. Calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe) and zinc (Zn) balances were determined from days 6 to 9. PPI administration significantly decreased the apparent absorption of Ca and Fe and increased urinary P excretion, resulting in decreased Ca, Fe and P retention. GOS dose-dependently increased the apparent absorption of Ca, Mg and Fe and urinary Mg excretion and decreased urinary P excretion. DFL significantly increased the apparent absorption of Ca and Mg and urinary Mg excretion. The combination of DFL and GOS additively affected these parameters, resulting in increased Ca, P and Fe retention, and it further increased the apparent absorption and retention of Zn at 5.0 % GOS. In conclusion, the combination of DFL and GOS improves Ca, P and Fe retention in an additive manner and increases the Zn retention in growing rats with hypochlorhydria induced by PPI.     

The microalga Parachlorella kessleri––A novel highly efficient lipid producer

The alga Parachlorella kessleri, strain CCALA 255, grown under optimal conditions, is characterized by storage of energy in the form of starch rather than lipids. If grown in the complete medium, the cultures grew rapidly, producing large amounts of biomass in a relatively short time. The cells, however, contained negligible lipid reserves (1–10% of DW). Treatments inducing hyperproduction of storage lipids in P. kessleri biomass were described. The cultures were grown in the absence or fivefold decreased concentration of either nitrogen or phosphorus or sulfur. Limitation by all elements using fivefold or 10‐fold diluted mineral medium was also tested. Limitation with any macroelement (nitrogen, sulfur, or phosphorus) led to an increase in the amount of lipids; nitrogen limitation was the most effective. Diluted nutrient media (5‐ or 10‐fold) were identified as the best method to stimulate lipid overproduction (60% of DW). The strategy for lipid overproduction consists of the fast growth of P. kessleri culture grown in the complete medium to produce sufficient biomass (DW more than 10 g/L) followed by the dilution of nutrient medium to stop growth and cell division by limitation of all elements, leading to induction of lipid production and accumulation up to 60% DW. Cultivation conditions necessary for maximizing lipid content in P. kessleri biomass generated in a scale‐up solar open thin‐layer photobioreactor were described. Biotechnol. Bioeng. 2013; 110: 97–107. © 2012 Wiley Periodicals, Inc.     

C-terminal truncation of Noxa1 greatly enhances its ability to activate Nox2 in a pure reconstitution system

Noxa1 activates Nox2 together with Noxo1 and Rac in a pure reconstitution system, but the resulting activity is considerably lower than that induced by p67^phox and p47^phox. In this study, we found that C-terminal-truncated forms of Noxa1 exhibited higher activities than full-length Noxa1. Of the truncations examined, Noxa1(1-225) showed the highest ability for activation. Kinetic studies revealed that Noxa1(1-225) had a threefold higher V_max value than full-length Noxa1 with a similar EC₅₀ value. The affinities of Noxo1 and RacQ61L were not much altered by the truncation. Conversely, the affinity of FAD for the Nox2 complex was enhanced after the truncation. In the absence of Noxo1, Noxa1(1-225) showed much higher activity with a lower EC₅₀ than full-length Noxa1. Noxa1(1-225) showed comparable activity to that of p67^phox with either Noxo1 or p47^phox, although the stability was lower than that with p67^phox and p47^phox. These findings indicate that the role of the C-terminal half of Noxa1 is autoinhibition. The data suggest a two-step autoinhibition mechanism, comprising self-masking to interrupt the binding to the oxidase, and holding of the activation domain in a suboptimal position to the oxidase. This study reveals that when both types of inhibition are released, Noxa1 achieves high-level superoxide production.     

Identification of a novel benzimidazole derivative as a highly potent NPY Y5 receptor antagonist with an anti-obesity profile

Optimization of HTS hit 1 for NPY Y5 receptor binding affinity, CYP450 inhibition, solubility and metabolic stability led to the identification of some orally available oxygen-linker derivatives for in vivo study. Among them, derivative 4i inhibited food intake induced by the NPY Y5 selective agonist, and chronic oral administration of 4i in DIO mice caused a dose-dependent reduction of body weight gain.     

Distinct expression patterns and roles of aldehyde dehydrogenases in normal oral mucosa keratinocytes: differential inhibitory effects of a pharmacological inhibitor and RNAi-mediated knockdown on cellular phenotype and epithelial morphology

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.