miRNA-based rapid differentiation of purified neurons from hPSCs advancestowards quick screening for neuronal disease phenotypes in vitro

Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene

Exogenous and endogenous ghrelin counteracts GLP-1 action to stimulate cAMP signaling and insulin secretion in islet β-cells

AbstractWe studied interactive effects of insulinotropic GLP-1 and insulinostatic ghrelin on rat pancreatic islets. GLP-1 potentiated glucose-induced insulin release and cAMP production in isolated islets and [Ca2+]i increases in single β-cells, and these potentiations were attenuated by ghrelin. Ghrelin suppressed [Ca2+]i responses to an adenylate cyclase activator forskolin. Moreover, GLP-1-induced insulin release and cAMP production were markedly enhanced by [d-lys3]-GHRP-6, a ghrelin receptor antagonist, in isolated islets. These results indicate that both exogenous and endogenous islet-derived ghrelin counteracts glucose-dependent GLP-1 action to increase cAMP production, [Ca2+]i and insulin release in islet β-cells, positioning ghrelin as a modulator of insulinotropic GLP-1

Burkholderia insecticola sp. nov., a gut symbiotic bacterium of the bean bug Riptortus pedestris

A Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacterium, designated strain RPE64(T), was isolated from the gut symbiotic organ of the bean bug Riptortus pedestris, collected in Tsukuba, Japan, in 2007. 16S rRNA gene sequencing showed that this strain belongs to the Burkholderia glathei Glade, exhibiting the highest sequence similarity to Burkholderia peredens LMG 29314(T) (100%), Burkholderia turbans LMG 29316(T) (99.52%) and Burkholderia ptereochthonis LMG 29326(T) (99.04 %). Phylogenomic analyses based on 107 single-copy core genes and Genome BLAST Distance Phylogeny confirmed B. peredens LMG 29314(T), B. ptereochthonis LMG 29326(T) and several uncultivated, endophytic Burkholderia species as its nearest phylogenetic neighbours. Digital DNA-DNA hybridization experiments unambiguously demonstrated that strain RPE64(T) represents a novel species in this lineage. The G+C content of its genome was 63.2 mol%. The isoprenoid quinone was ubiquinone 8 and the predominant fatty acid components were C-16:0, C-18:1 omega 7c and C-17:0 cyclo. The absence of nitrate reduction and the capacity to grow at pH 8 clearly differentiated strain RPE64(T) from related Burkholderia species. Based on these genotypic and phenotypic characteristics, strain RPE64(T) is classified as representing a novel species of the genus Burkholderia, for which the name Burkholderia insecticola sp. nov. is proposed. The type strain is RPE64(T) (=NCIMB 15023(T)=JCM 31142(T))

Change in Volatile Compounds in Miso-soup by Heating

わが国の伝統的な発酵調味料である味噌は、製法や産地によりその風味は異なり、また、種類は多く地域性の高い調味料である。さらに、原料の大豆や発酵・熟成に由来する多くの機能性を持つ優れた食品でもある。味噌は主として、味噌汁として食され、その味噌汁の調理にあたっては、味噌を溶かし短時間沸騰させる。そのことにより、香味が最大限引き出され、おいしさが感じられる。しかし、過度の加熱では、多くの香気成分が揮発、あるいは逆に増加することで風味が損なわれ、おいしさは減少することが知られている。本研究では、味噌汁中の香気成分が味噌の種類や加熱時間の長短によりどのように変化するか香気成分捕集方法として動的ヘッドスペース法を用いて検討した。実験には、越後味噌、信州味噌、八丁味噌、西京味噌の4種類の味噌を用いて、未加熱、加熱時間10～60分での香気成分の変化をガスクロマトグラフィー(以下、GC)により分析し、比較検討を行った。GC分析の結果検出された総ピーク数は、越後味噌111、信州味噌106、八丁味噌113、西京味噌65であり、越後味噌、信州味噌、八丁味噌の香気成分は西京味噌に比べ、多種類の成分より構成されていた。また、GCパターンを比較すると、越後味噌、信州味噌は類似し、八丁味噌、西京味噌はそれぞれ特有のGCパターンを示した。GCの総ピーク面積は、越後味噌が一番大きく、次いで西京味噌、八丁味噌で、信州味噌は一番少なく、他の味噌の約41～50%に相当した。標準化合物および文献をもとに、各味噌汁中の香気成分を解析したところ、味噌汁の香気成分として、アルコール13、アルデヒド7、有機酸4、エステル12、含硫化合物2、炭化水素5、ケトン類1の総計44種類の化合物を同定または推定した。これら化合物のうち、94～99%占めたのがアルコール類で、このうちethanolが一番多く、越後味噌で65.14%、信州味噌で67.05%、八丁味噌で82.82%、西京味噌で93.19%であった。八丁味噌と西京味噌はその発酵・熟成には酵母の関与が比較的少ないにもかかわらず、ethanol量の多いのは上述の「酒精」の添加によるものと考えられる。そして、ethanolのほか、2-methylbutanol、3-methylbutanol、methanol、2-methyl-1-propanol、n-butanol、n-propanolなどが主なアルコール類として検出された。越後味噌、八丁味噌、西京味噌中の多くのアルコール類は加熱により減少する傾向を示したが、信州味噌では増加またはほとんど変化しないという特徴が認められた。各味噌汁中のアルデヒド類としてethanal、hexanal、benzaldehydeなどが検出され、これらは一般に加熱により増加する傾向にあり、他の香気成分との違いが認められた。アルデヒド類の加熱による増加は一般的にも知られ、その原因は味噌中の遊離アミノ酸からストレッカー分解により生成されるためと考えられる。有機酸のacetic acidは越後味噌、butyric acidは八丁味噌、2-methylbutyric acidと3-methylbutyric acidは信州味噌中で最も多いことが認められた。そして、加熱によりacetic acidは減少、butyric acidは増加、2-methylbutyric acidと3-methylbutyric acidは変化がないという,それぞれの特徴を示した。各味噌汁中で検出された主なエステル類はethyl acetate、ethyl heptanoate、ethyl lactateおよび2-methylbutyl acetateと3-methylbutyl acetateなどであり、量的にはethyl acetateは八丁味噌、ethyl heptanoateは越後味噌、ethyl lactateは信州味噌に多いことが認められた。これらのうち量的に多かったethyl acetateも加熱により増加する傾向にあったが、八丁味噌では加熱10分で一旦急激に減少することが認められた。そして、エステル類はその種類や味噌の違いにより加熱による傾向が異なったが、信州味噌ではethyl dodecanoateのみ減少傾向を示し、その他多くのエステル類は増加する傾向があった。含硫化合物のdimethyl disulfideは加熱により減少し、3-methylthiopropanal (methional)は増加した。また、炭化水素類では八丁味噌においてtridecane、hexadecaneに増加がみられた。これらの結果から、味噌汁の香気成分の中には加熱により減少するもの、逆に、増加するもの、また、味噌の種類による違いなど解析することができた。また、前報では香気成分捕集法として静的ヘッドスペース法を用い、味噌汁の香気成分として15化合物を同定または推定したが、本研究での動的ヘッドスペース法では44化合物が同定または推定されたことから、後者の分析法の優位性が認められた

Reconstruction-Dependent Recovery from Anorexia and Time-Related Recovery of Regulatory Ghrelin System in Gastrectomized Rats

Gastrectomy reduces food intake and body weight (BW) hampering recovery of physical conditions. It also reduces plasma levels of stomach-derived orexigenic ghrelin. This study explored changes in orexigenic ghrelin system in rats receiving total gastrectomy with Billroth II (B-II) or Roux-en-Y (R-Y) method. Feeding and BW were reduced by gastrectomy and subsequently recovered to a greater extent with R-Y than B-II while plasma ghrelin decreased similarly. At postoperative 12th week, ghrelin contents increased in the duodenum and pancreas, plasma ghrelin levels increased upon fasting, and ghrelin injection promoted feeding but not in earlier periods. In summary, gastrectomized rats partially recover feeding and BW, in a reconstruction-dependent manner. At 12th week, ghrelin is upregulated in extra-stomach tissues, plasma ghrelin levels are physiologically regulated, and orexigenic effect of exogenous ghrelin is restored. This time-related recovery of ghrelin system may provide a strategy for promoting feeding, BW, and thereby physical conditions in gastrectomized patients

Effect of olfaction on the sensitivity of bitter taste

味覚と嗅覚の受容は異なる経路で脳へと伝達される一方、嗅覚の存在によって味の感じ方が変わることが知られているが、香気成分が味成分の受容そのものに与える影響については明確にされていない。本研究では、ラットを用いた飲水選択嗜好実験を行い、香気成分の添加により苦味溶液の選択率に変化が見れるかどうかを確認することにより、香気成分が苦味の感受性に与える影響について検討した。濃度の異なる塩酸キニーネ水溶液を用いた４瓶選択嗜好実験では、通常水群、香気水群（苦味溶液に10ppmの酪酸ブチルを添加）ともに苦味濃度が増加するほど嗜好率が低下したが、香気水群では0.001mMの苦味溶液で嗜好率が有意に増加した（p＜0.01)。一方、不快臭を示すヘキサナールを用いた場合では、個体差が大きく有意差は認められなかった。ヒトを用いた官能検査では、10ppmの酪酸ブチル、ヘキサナール、イソバレルアルデヒドのいづれの添加でも、0.01mM塩酸キニーネ水溶液の苦味を増加させた。これらの結果から、酪酸ブチルのような香気成分は苦味の感受性を変化させることが示唆され、香気成分の添加により苦味を修飾できる可能性が示された。It is known that not only taste but also olfaction (aroma) largely contributes the flavor of food, however, there is insufficient information about the interaction of olfaction and sensitivity of taste. In the present study, we investigated the effect of olfaction on the sensitivity of bitter taste by four-bottle preference test using rats. Ten male Sprague Dawley rats (6-weeks age) were divided into two groups: control and odor groups, and the drinking water of the odor group were added 10 ppm of butyl acetate (fruit-like odor). Rats were provided four bottles of drinking water containing different concentrations of quinine hydrochloride (0, 0.001, 0.01, 0.1 mM), and the preference rates were measured. The positions of the 4 bottles were randomized and switched every 48 h to avoid positional preference. During 8 days of experimental period, the volume of the 0.001 mM quinine hydrochloride consumed by the rats in the odor group was higher than that in the control group (p<0.01). In contrast, the addition of 10 ppm of 1-hexanal (off-flavor odor) had no effect on the preference rate of the 0.001 mM quinine hydrochloride by rats. Further, any addition of 10 ppm of butyl acetate, 1-hexanal, or isovaleraldehyde increased the sensitivity of bitter taste of 0.01mM quinine hydrochloride by human sensory evaluation. The results indicate that some odors such as butyl acetate may modify sensitivity of bitter taste in human and rat

C-type natriuretic peptide (CNP)/guanylate cyclase B (GC-B) system and endothelin-1(ET-1)/ET receptor A and B system in human vasculature

To assess the physiological and clinical implications of the C-type natriuretic peptide (CNP)/guanylyl cyclase B (GC-B) system in the human vasculature, we have examined gene expressions of CNP and its receptor, GC-B, in human vascular endothelial cells (ECs) and smooth muscle cells (SMCs) and have also compared the endothelin-1(ET-1)/endothelin receptor-A (ETR-A) and endothelin receptor-B (ETR-B) system in human aortic ECs (HAECs) and vascular SMCs (HSMCs) in vitro. We also examined these gene expressions in human embryonic stem (ES)/induced pluripotent stem cell (iPS)-derived ECs and mural cells (MCs). A little but significant amount of mRNA encoding CNP was detected in both human ES-derived ECs and HAECs. A substantial amount of GC-B was expressed in both ECs (iPS-derived ECs and HAECs) and SMCs (iPS-derived MCs and HSMCs). ET-1 was expressed solely in ECs. ETR-A was expressed in SMCs, while ETR-B was expressed in ECs. These results indicate the existence of a vascular CNP/GC-B system in the human vascular wall, indicating the evidence for clinical implication of the CNP/GC-B system in concert with the ET-1/ETR-A and ETR-B system in the human vasculature.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Menaquinone-4 Amplified Glucose-Stimulated Insulin Secretion in Isolated Mouse Pancreatic Islets and INS-1 Rat Insulinoma Cells

Vitamin K2 is indispensable for blood coagulation and bone metabolism. Menaquinone-4 (MK-4) is the predominant homolog of vitamin K2, which is present in large amounts in the pancreas, although its function is unclear. Meanwhile, &#946;-cell dysfunction following insulin secretion has been found to decrease in patients with type 2 diabetes mellitus. To elucidate the physiological function of MK-4 in pancreatic &#946;-cells, we studied the effects of MK-4 treatment on isolated mouse pancreatic islets and rat INS-1 cells. Glucose-stimulated insulin secretion significantly increased in isolated islets and INS-1 cells treated with MK-4. It was further clarified that MK-4 enhanced cAMP levels, accompanied by the regulation of the exchange protein directly activated by the cAMP 2 (Epac2)-dependent pathway but not the protein kinase A (PKA)-dependent pathway. A novel function of MK-4 on glucose-stimulated insulin secretion was found, suggesting that MK-4 might act as a potent amplifier of the incretin effect. This study therefore presents a novel potential therapeutic approach for impaired insulinotropic effects