たまご醤油の調製に関する研究 第2報 : ピザ台への麹菌培養と卵白液の醤油化

This research was aimed to produce a fermented egg white sauce that taste strong umami by use of pizzacrusts as a solid medium for koji cultivation. Pizza crusts were made from dough of bread flour and glutenpowder with yeast, and cut into small pieces about 5mm square sections. After adjusting moisture content at45%, Aspergillus oryzae was cultured on the pizza crusts. A. oryzae favorably grew into the crusts, because ofporous structure of the crusts. Enzyme activities of protease, acidic carboxypeptidase and amylase extractedfrom the crust koji were 2-3 times higher than those of usual Koji cultivated on delipidated soybean and wheat.The crusts koji were mixed with liquid egg white and salt to prepare egg white moromi (16% NaCl) for fermentation. Formol nitrogen and total amino acid content of the crusts moromi were gradually increased from0.12% and 0.37% at start to 0.93% and 8.8% at 6 months, respectively, resulting in much amino acid producedby degradation of proteins by enzymes in koji. The moromi at 6 months was filtered and sterilized to obtain afermented egg white sauce. The glutamic acid content of the egg white sauce was about 1.6%. This value wasabout 2 times higher comparing to that of soy sauce commercially available.It was revealed by sensory evaluation that pizza crusts koji with 16% salted liquid egg white produced fermented egg white sauce with unique egg flavor, much umami taste, and lighter color comparing to that ofusual soy sauce. Moreover, egg proteins were completely decomposed to peptides or amino acid, which couldnot be detected by an egg allergy detection ELISA kit in the fermented egg white sauce at 6 months. It might bethe first to utilize pizza crust to ferment A. oryzae. It also might be the first to use the crust koji with liquid eggwhite and salt during fermentation of egg white sauce. The egg white sauce will be a new fermented seasoningin our dietary life as well as in food industry

Expression of 20-hydroxyecdysone-induced genes in the silkworm brain and their functional analysis in post-embryonic development

金沢大学理工研究域自然システム学系The insect brain is the center of developmental control, from which ecdysone governs brain morphogenesis and regulates gene expression cascades associated with molting and metamorphosis. In order to identify the 20-hydroxyecdysone (20E)-inducible genes responsible for molting and metamorphosis, we constructed a 20E-induced subtraction complementary DNA library from the fifth instar larval brain of the silkworm Bombyx mori. We isolated 10 genes, designated as bombeil-1 to bombeil-10, three of which did not show any sequence similarity to previously identified Bombyx genes. Whole-mount in situ hybridization revealed that all of these bombeil messenger RNAs were exclusively located in two pairs of lateral neurosecretory cells in the larval brain, known as prothoracicotropic hormone (PTTH)-producing cells. RNA-interference knockdown targeting bombeil-2 resulted in larval-pupal molt defects, and adult wing and leg malformations. These results, together with the cell-specific co-localization of bombeil transcripts with PTTH, suggest that bombeil genes play important roles during larval-pupal-adult development. © 2008 Elsevier Ltd. All rights reserve

たまご醤油の調製に関する研究

It has been estimated about 500,000 tons of hen's eggs were utilized in food processing annually in Japan. Because of the use of egg yolk has been much higher than that of egg white in the food industry, a large quantity of surplus egg white remained as frozen egg white. Hence, it is worth to develop the use of egg white to normalize the balance for the utilization of egg components. In this report, we demonstrated the preparation of a fermented seasoning sauce in which egg white, whole egg or egg yolk liquid was added to soy sauce koji with large amount of salt. The soy sauce koji is a material that Aspergillus orizae was grown on mixtures of delipidated soy and roasted wheat grain. The salt and protein concentration in each egg component with koji mixture was adjusted to 15.8-16.1% and 12.5-13.0%, respectively, at the start of fermentation. During the fermentation for 6 months, total amino acids % in the supernatant of each mixture was increased to around 10%, with an exception that the value for egg yolk sauce was only 3.6%. The umami taste and flavor of the egg sauce are comparable and characteristic to that of the soy sauce. Moreover, color of the egg sauce is much lighter than that of the soy sauce卵白液や全卵液を用いた醤油は,従来の醤油麹のみを使用した対照醤油に対し,もろみの量を約2倍に増量しても同等の旨味をもつ醤油が得られた。　卵白液を含む醤油のホルモール態窒素量は約1.0%まで増加した。また,ペプチド鎖平均鎖長は,熟成前は約10で,熟成とともに約2まで分解された。　醗酵熟成中,鶏卵のタンパク質が醤油麹のプロテアーゼにより分解された結果,熟成期間6ヶ月目の卵白を含む醤油のアミノ酸総量は,約10%と対照醤油と同等の値を示した。卵白醤油はアミノ酸総量が高く,旨味や甘味の強い醤油となった。また,熟成期間6ヶ月目の波長550nmにおける吸光度は卵白醤油の色調が顕著に薄かった。以上の結果,卵白を用いたたまご醤油は経済的にも優れ,独特の風味と薄い色調を持ち合わせた,新規の醗酵調味料としての利用が期待できる

DGKγ Knock-Out Mice Show Impairments in Cerebellar Motor Coordination, LTD, and the Dendritic Development of Purkinje Cells through the Activation of PKCγ

Diacylglycerol kinase γ (DGKγ) regulates protein kinase C (PKC) activity by converting DG to phosphatidic acid (PA). DGKγ directly interacts with PKCγ and is phosphorylated by PKCγ, resulting in the upregulation of lipid kinase activity. PKC dysfunction impairs motor coordination, indicating that the regulation of PKC activity is important for motor coordination. DGKγ and PKC are abundantly expressed in cerebellar Purkinje cells. However, the physiological role of DGKγ has not been elucidated. Therefore, we developed DGKγ knock-out (KO) mice and tested their cerebellar motor coordination. In DGKγ KO mice, cerebellar motor coordination and long-term depression (LTD) were impaired, and the dendrites of Purkinje cells from DGKγ KO mice were significantly retracted. Interestingly, treatment with the cPKC inhibitor Gö6976 (Gö) rescued the dendritic retraction of primary cultured Purkinje cells from DGKγ KO mice. In contrast, treatment with the PKC activator 12-o-tetradecanoylphorbol 13-acetate (TPA) reduced morphologic alterations in the dendrites of Purkinje cells from wild-type (WT) mice. In addition, we confirmed the upregulation of PKCγ activity in the cerebellum of DGKγ KO mice and rescued impaired LTD in DGKγ KO mice with a PKCγ-specific inhibitor. Furthermore, impairment of motor coordination observed in DGKγ KO mice was rescued in tm1c mice with DGKγ reexpression induced by the FLP-flippase recognition target (FRT) recombination system. These results indicate that DGKγ is involved in cerebellar LTD and the dendritic development of Purkinje cells through the regulation of PKCγ activity, and thus contributes to cerebellar motor coordination

Intravenous Administration of Mesenchymal Stem Cell-Derived Exosome Alleviates Spinal Cord Injury by Regulating Neutrophil Extracellular Trap Formation through Exosomal miR-125a-3p

Spinal cord injury (SCI) leads to devastating sequelae, demanding effective treatments. Recent advancements have unveiled the role of neutrophil extracellular traps (NETs) produced by infiltrated neutrophils in exacerbating secondary inflammation after SCI, making it a potential target for treatment intervention. Previous research has established that intravenous administration of stem cell-derived exosomes can mitigate injuries. While stem cell-derived exosomes have demonstrated the ability to modulate microglial reactions and enhance blood–brain barrier integrity, their impact on neutrophil deactivation, especially in the context of NETs, remains poorly understood. This study aims to investigate the effects of intravenous administration of MSC-derived exosomes, with a specific focus on NET formation, and to elucidate the associated molecular mechanisms. Exosomes were isolated from the cell supernatants of amnion-derived mesenchymal stem cells using the ultracentrifugation method. Spinal cord injuries were induced in Sprague-Dawley rats (9 weeks old) using a clip injury model, and 100 μg of exosomes in 1 mL of PBS or PBS alone were intravenously administered 24 h post-injury. Motor function was assessed serially for up to 28 days following the injury. On Day 3 and Day 28, spinal cord specimens were analyzed to evaluate the extent of injury and the formation of NETs. Flow cytometry was employed to examine the formation of circulating neutrophil NETs. Exogenous miRNA was electroporated into neutrophil to evaluate the effect of inflammatory NET formation. Finally, the biodistribution of exosomes was assessed using 64Cu-labeled exosomes in animal positron emission tomography (PET). Rats treated with exosomes exhibited a substantial improvement in motor function recovery and a reduction in injury size. Notably, there was a significant decrease in neutrophil infiltration and NET formation within the spinal cord, as well as a reduction in neutrophils forming NETs in the circulation. In vitro investigations indicated that exosomes accumulated in the vicinity of the nuclei of activated neutrophils, and neutrophils electroporated with the miR-125a-3p mimic exhibited a significantly diminished NET formation, while miR-125a-3p inhibitor reversed the effect. PET studies revealed that, although the majority of the transplanted exosomes were sequestered in the liver and spleen, a notably high quantity of exosomes was detected in the damaged spinal cord when compared to normal rats. MSC-derived exosomes play a pivotal role in alleviating spinal cord injury, in part through the deactivation of NET formation via miR-125a-3p

Sequential Sensing by TLR2 and Mincle Directs Immature Myeloid Cells to Protect against Invasive Group A Streptococcal Infection in Mice

Summary: Severe invasive group A Streptococcus (GAS) infection evades anti-bacterial immunity by attenuating the cellular components of innate immune responses. However, this loss of protection is compensated for by interferon (IFN)-γ-producing immature myeloid cells (γIMCs), which are selectively recruited upon severe invasive GAS infection in mice. Here, we demonstrate that γIMCs provide this IFN-γ-mediated protection by sequentially sensing GAS through two distinct pattern recognition receptors. In a mouse model, GAS is initially recognized by Toll-like receptor 2 (TLR2), which promptly induces interleukin (IL)-6 production in γIMCs. γIMC-derived IL-6 promotes the upregulation of a recently identified GAS-sensing receptor, macrophage-inducible C-type lectin (Mincle), in an autocrine or paracrine manner. Notably, blockade of γIMC-derived IL-6 abrogates Mincle expression, downstream IFN-γ production, and γIMC-mediated protection against severe invasive GAS infection. Thus, γIMCs regulate host protective immunity against severe invasive GAS infection via a TLR2–IL-6–Mincle axis. : Matsumura et al. show that γIMCs sequentially sense group A Streptococcus (GAS) through TLR2 and Mincle. Specifically, TLR2-triggered production of IL-6 functions as an intermediate that amplifies Mincle expression to maximize host protection through IFN-γ production. The sequential sensing is a distinct feature in γIMCs following severe invasive GAS infection. Keywords: group A Streptococcus infections, immature myeloid cells, Toll-like receptors, C-type lectin receptors, cytokine