Conversions of Microbial Products in Relation to Oxygen Supply in Inosine Fermentation

On the basis of the ratio (cell’s respiration rate/maximum oxygen demand of cells, r_{a b}/K_rM) as a new criterion of oxygen supply, symptoms of oxygen deficiency was described in inosine fermentation. Conversions of the products were observed to occur in relation to the extent of oxygen deficiency. When oxygen demand of the cells was satisfied (r_{a b}/K_rM = 1.0), the cells accumulated exclusively inosine. Under limited oxygen supply at the value of r_{a b}/K_rM 0.5~0.9, on the other hand, inosine formation was inhibited and acetoin was the predominant product. When oxygen supply was limited more strictly at the value of r_{a b}/K_rM smaller than 0.3, the cells excreted 2,3-butyleneglycol as the main product.     

Generation of the antioxidant yellow pigment derived from 4-methylthio-3-butenyl isothiocyanate in salted radish roots (takuan-zuke)

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) is a yellow pigment of salted radish roots (takuan-zuke) derived from 4-methylthio-3-butenyl isothiocyanate (MTBITC), the pungent component of radish roots. Here, we prepared salted radish and analyzed the behavior of the yellow pigment and related substances in the dehydration process and long-term salting process. All salted radish samples turned yellow, and their b^* values increased with time and temperature. The salted radish that was sun-dried and pickled at room temperature turned the brightest yellow, and the generation of TPMT was clearly confirmed. These results indicate that tissue shrinkage due to dehydration, salting temperature, and pH play important roles in the yellowing of takuan-zuke.     

Ameloblastin in Hertwig’s Epithelial Root Sheath Regulates Tooth Root Formation and Development

Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig’s epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2′-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21^Cip1 and p27^Kip1 was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.     

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose

The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.

Methods

Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.

Results

Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.

Conclusion

The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.     

Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D

Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta^+/+ and Ig-Hepta^−/− mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space.     

Intensive determination of storage condition effects on human plasma metabolomics

Introduction

Human plasma metabolomics offer powerful tools for understanding disease mechanisms and identifying clinical biomarkers for diagnosis, efficacy prediction and patient stratification. Although storage conditions can affect the reliability of data from metabolites, strict control of these conditions remains challenging, particularly when clinical samples are included from multiple centers. Therefore, it is necessary to consider stability profiles of each analyte.

Objectives

The purpose of this study was to extract unstable metabolites from vast metabolome data and identify factors that cause instability.

Method

Plasma samples were obtained from five healthy volunteers, were stored under ten different conditions of time and temperature and were quantified using leading-edge metabolomics. Instability was evaluated by comparing quantitation values under each storage condition with those obtained after ?80 °C storage.

Result

Stability profiling of the 992 metabolites showed time- and temperature-dependent increases in numbers of significantly changed metabolites. This large volume of data enabled comparisons of unstable metabolites with their related molecules and allowed identification of causative factors, including compound-specific enzymatic activity in plasma and chemical reactivity. Furthermore, these analyses indicated extreme instability of 1-docosahexaenoylglycerol, 1-arachidonoylglycerophosphate, cystine, cysteine and N⁶-methyladenosine.

Conclusion

A large volume of data regarding storage stability was obtained. These data are a contribution to the discovery of biomarker candidates without misselection based on unreliable values and to the establishment of suitable handling procedures for targeted biomarker quantification.

     

Replication Study in a Japanese Population of Six Susceptibility Loci for Type 2 Diabetes Originally Identified by a Transethnic Meta-Analysis of Genome-Wide Association Studies

AimWe performed a replication study in a Japanese population to evaluate the association between type 2 diabetes and six susceptibility loci (TMEM154, SSR1, FAF1, POU5F1, ARL15, and MPHOSPH9) originally identified by a transethnic meta-analysis of genome-wide association studies (GWAS) in 2014.MethodsWe genotyped 7,620 Japanese participants (5,817 type 2 diabetes patients and 1,803 controls) for each of the single nucleotide polymorphisms (SNPs) using a multiplex polymerase chain reaction invader assay. The association of each SNP locus with the disease was evaluated using logistic regression analysis.ResultsOf the six SNPs examined in this study, four (rs6813195 near TMEM154, rs17106184 in FAF1, rs3130501 in POU5F1 and rs4275659 near MPHOSPH9) had the same direction of effect as in the original reports, but two (rs9505118 in SSR1 and rs702634 in ARL15) had the opposite direction of effect. Among these loci, rs3130501 and rs4275659 were nominally associated with type 2 diabetes (rs3130501; p = 0.017, odds ratio [OR] = 1.113, 95% confidence interval [CI] 1.019–1.215, rs4275659; p = 0.012, OR = 1.127, 95% CI 1.026–1.238, adjusted for sex, age and body mass index), but we did not observe a significant association with type 2 diabetes for any of the six evaluated SNP loci in our Japanese population.ConclusionsOur results indicate that effects of the six SNP loci identified in the transethnic GWAS meta-analysis are not major among the Japanese, although SNPs in POU5F1 and MPHOSPH9 loci may have some effect on susceptibility to type 2 diabetes in this population.     

Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen–specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54^high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54^high or B2^high leukemia cells preferentially survived in vivo in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis in vitro and in vivo. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy.     

A high-molecular-weight,alkaline, and thermostable β-1,4-xylanase of a subseafloor <Emphasis Type="Italic">Microcella alkaliphila</Emphasis>

An endo β-1,4-xylanase (XynE15) from a culture broth of a deep subseafloor microorganism, Microcella alkaliphila JAM-AC0309, was purified to homogeneity. The molecular mass of XynE15 was approximately 150 kDa as judged by SDS-PAGE. The optimal pH and temperature for hydrolysis of xylan were pH 8 and 65 °C. The enzyme was stable to incubation for 30 min at up to 75 °C, and the half-life at 50 °C was 48 h. XynE15 hydrolyzed arabinoxylan, oat spelt xylan, and birchwood xylan well, but not avicel, carboxymethylcellulose, or arabinan. Xylooligosaccharides were hydrolyzed to mainly xylobiose from higher than xylotetraose. The genome sequencing analysis of strain JAM-AC03039 revealed that XynE15 was composed of 1,319 amino acids with one catalytic domain and three carbohydrate-binding domains belonging to glycoside hydrolase (GH) family 10 and carbohydrate-binding module (CBM) family 4, respectively.