Live cell imaging of ß-tubulin mRNA reveals spatiotemporal expression dynamics in the filamentous fungus Aspergillus oryzae.

In filamentous fungi, microtubules are important for polar growth and morphological maintenance and serve as rails for intracellular trafficking. The molecular mechanisms associated with microtubules have been analyzed. However, little is known about when and where tubulin, a component of microtubules, is biosynthesized in multinuclear and multicellular filamentous fungi. In this study, we visualized microtubules based on the enhanced green fluorescence protein (EGFP)-labeled α-tubulin and ß-tubulin mRNA tagged by the EGFP-mediated MS2 system in living yellow Koji mold Aspergillus oryzae cells in order to understand the spatiotemporal production mechanism of tubulin. We found that mRNA of btuA, encoding for ß-tubulin, localized at dot-like structures through the apical, middle and basal regions of the hyphal cells. In addition, some btuA mRNA dots showed microtubule-dependent motor protein-like dynamics in the cells. Furthermore, it was found that btuA mRNA dots were decreased in the cytoplasm just before mitosis but increased immediately after mitosis, followed by a gradual decrease. In summary, the localization and abundance of ß-tubulin mRNA is spatiotemporally regulated in living A. oryzae hyphal cells.

Effect of Hita Tenryo Water™, a natural mineral water, on allergic symptoms induced by cedar in mice.

The number of patients with allergies to pollen and food is increasing worldwide. In Japan, the prevalence of cedar pollinosis, a type I allergy, is nearly 30% and accounts of hay fever are rising. A potential natural remedy for these allergic diseases may be Hita Tenryo Water™ (referred to simply as Hita Tenryo water), water that is pumped from deep underground in the Hita region of Oita, Japan, which has been the subject of various research reports. Here, we investigated the potential of using Hita Tenryo water to suppress the onset of cedar pollinosis in a mouse model and explored the immunological mechanism of the suppression. Test model mice were given Hita Tenryo water ad libitum to drink and received intraperitoneal administration of (i) tap water (Hw1), (ii) 25% Hita Tenryo water (Hw2) or (iii) 100% Hita Tenryo (Hw3). There were no significant differences in body weight change, feed intake, or water intake among the groups during the experimental period. We examined nose rubbing and sneezing as allergic symptoms. The frequency of rubbing and sneezing tended to decrease in the Hw1 and Hw2 group, and significantly decreased in the Hw3 group compared to control. Total IgE levels in serum were also significantly reduced in Hita Tenryo water intraperitoneal administration groups. In vitro examination of the rate of release of ß-hexosaminidase from BL-2H3 cells showed that there were no significantly differences between Hita Tenryo water-treated and control cells. In addition, measurement of Th2-related cytokine levels in concanavalin A-stimulated peripheral blood mononuclear cells revealed a significant decrease in IL-4, IL-6, and IL-10 levels in medium (p < 0.01). In contrast, production of IFN-γ significantly increased (p < 0.01). These results indicate that Hita Tenryo water may alleviate and/or suppress allergic symptoms.

Fructobacillus fructosus OS-1010 strain stimulates intestinal cells to secrete exosomes that activate muscle cells.

Fructobacillus is a lactic-acid bacterium recently identified in fructose-rich environments. Fructobacillus is also known to exhibit unusual growth characteristics due to an incomplete gene encoding alcohol/acetaldehyde hydrogenase, which results in an imbalance in the nicotinamide adenine mononucleotide (NAD+)/NADN levels. Recently, the addition of d-fructose to the culture medium of Fructobacillus strains increased the intracellular nicotinamide mononucleotide (NMN) content. In the present study, we evaluated the functionality of Fructobacillus that produces high levels of NMN, using one substrain (Fructobacillus fructosus OS-1010). Therefore, in this study, we examined its functionality in the interaction between intestinal cells and muscle cells. The results showed that supernatant derived from intestinal epithelial cells (Caco-2 cells) treated with F. fructosus OS-1010 activated muscle cells (C2C12 cells). Further analysis revealed that Caco-2 cells treated with F. fructosus OS-1010 secreted exosomes known as extracellular vesicles, which activated the muscle cells. Furthermore, pathway analysis of the target genes of miRNA in exosomes revealed that pathways involved in muscle cell activation, including insulin signaling and cardiac muscle regulation, neurotrophic factors, longevity, and anti-aging, can be activated by exosomes. In other words, F. fructosus OS-1010 could activate various cells such as the skin and muscle cells, by secreting functional exosomes from the intestinal tract.

Scalp bacterial species influence SIRT1 and TERT expression in keratinocytes.

Scalp bacteria on the human scalp and scalp hair comprise distinct community structures for sites and individuals. To evaluate their effect on human keratinocyte cellular activity, including that of the hair follicular keratinocytes, the expression of several longevity genes was examined using HaCaT cells. A screening system that uses enhanced green fluorescent protein (EGFP) fluorescence was established to identify scalp bacteria that enhance silent mating type information regulation 2 homolog-1 (SIRT1) promoter activity in transformed HaCaT cells (SIRT1p-EGFP). The results of quantitative polymerase chain reaction revealed that several predominant scalp bacteria enhanced (Cutibacterium acnes and Pseudomonas lini) and repressed (Staphylococcus epidermidis) the expressions of SIRT1 and telomerase reverse transcriptase (TERT) genes in HaCaT cells. These results suggest that the predominant scalp bacteria are related to the health of the scalp and hair, including repair of the damaged scalp and hair growth, by regulating gene expression in keratinocytes.

Deacylated Derivative of Hericenone C Treated by Lipase Shows Enhanced Neuroprotective Properties Compared to Its Parent Compound.

Hericium erinaceus, a mushroom species commonly known as Yamabushitake in Japan, is known to have a stimulatory effect on neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Hericenone C, a meroterpenoid with palmitic acid as the fatty acid side chain, is reported to be one such stimulant. However, according to the structure of the compound, the fatty acid side chain seems highly susceptible to lipase decomposition, under in vivo metabolic conditions. To study this phenomenon, hericenone C from the ethanol extract of the fruiting body was subjected to lipase enzyme treatment and observed for changes in the chemical structure. The compound formed after the lipase enzyme digestion was isolated and identified using LC-QTOF-MS combined with 1H-NMR analysis. It was found to be a derivative of hericenone C without its fatty acid side chain and was named deacylhericenone. Interestingly, a comparative investigation of the neuroprotective properties of hericenone C and deacylhericenone showed that the BDNF mRNA expression in human astrocytoma cells (1321N1) and the protection against H2O2-induced oxidative stress was considerably higher in the case of deacylhericenone. These findings suggest that the stronger bioactive form of the hericenone C compound is in fact deacylhericenone.

Molecular Mechanisms for the Carnosine-Induced Activation of Muscle-Brain Interaction.

Carnosine is known to improve brain function. The molecular basis for the carnosine-mediated interaction between intestinal cells and neuronal cells is that carnosine acts on intestinal cells and stimulates exosome secretion, which can induce neurite outgrowth in neuronal cells. This study aimed to infer the carnosine-mediated interaction between muscle cells and neuronal cells. The results revealed that carnosine induces muscle cell differentiation, as well as the secretion of exosomes and myokines that can act on neuronal cells. Carnosine acts not only on intestinal cells but also on muscle cells, stimulating the secretion of secretory factors including exosomes that induce neurite outgrowth in neuronal cells, as well as myokines known to be involved in neuronal cell activation. As the miRNAs in exosomes secreted from intestinal cells and muscle cells upon carnosine treatment are different, it could be assumed that carnosine acts on each cell to interact with neuronal cell through separate factors and mechanisms.

Identification and Functional Evaluation of Polyphenols That Induce Regulatory T Cells.

Regulatory T cells (Tregs) and CD4+/CD25+ T cells play an important role in the suppression of excessive immune responses, homeostasis of immune function, and oral tolerance. In this study, we screened for food-derived polyphenols that induce Tregs in response to retinaldehyde dehydrogenase (RALDH2) activation using macrophage-like THP-1 cells. THP-1 cells were transfected with an EGFP reporter vector whose expression is regulated under the control of mouse Raldh2 promoter and named THP-1 (Raldh2p-EGFP) cells. The THP-1 (Raldh2p-EGFP) cells were treated with 33 polyphenols after inducing their differentiation into macrophage-like cells using phorbol 12-myristate 13-acetate. Of the 33 polyphenols, five (kaempferol, quercetin, morin, luteolin and fisetin) activated Raldh2 promoter activity, and both quercetin and luteolin activated the endogenous Raldh2 mRNA expression and enzymatic activity. Furthermore, these two polyphenols increased transforming growth factor beta 1 and forkhead box P3 mRNA expression, suggesting that they have Treg-inducing ability. Finally, we verified that these polyphenols could induce Tregs in vivo and consequently induce IgA production. Oral administration of quercetin and luteolin increased IgA production in feces of mice. Therefore, quercetin and luteolin can induce Tregs via RALDH2 activation and consequently increase IgA production, suggesting that they can enhance intestinal barrier function.

Association of serum s-adenosylmethionine, s-adenosylhomocysteine, and their ratio with the risk of dementia and death in a community.

We examined the association of serum s-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH) (methionine metabolites), and their ratio on the risk of dementia and death in a community-dwelling population of older Japanese individuals. 1371 residents of Hisayama, Japan, aged 65 years or older and without dementia, were followed for a median of 10.2 years (2007-2017). We divided serum SAM, SAH, and SAM/SAH ratio into quartiles. Cox proportional hazards models were used to estimate the hazard ratios (HRs) and their 95% confidence intervals (CIs) of serum SAM, SAH, and SAM/SAH ratio levels on the risk of a composite outcome of all-cause dementia or death, and each outcome. During the follow-up, 635 participants developed all-cause dementia and/or died, of which 379 participants developed dementia and 394 deaths occurred. The multivariable-adjusted HRs of the composite outcome decreased significantly with increasing serum SAM levels (P for trend = 0.01), while they increased significantly with higher serum SAH levels (P for trend = 0.03). Higher serum SAM/SAH ratio levels were significantly associated with a lower risk of the composite outcome (P for trend = 0.002), as well as with lower risk of each outcome. Our findings suggest that the balance of methionine metabolites may closely associate with the risk of dementia and death.

Age-related functional decline of human B cells.

This study aimed to investigate the changes in B cell functional decline and antigen sensitization with aging using two Epstein Barr virus (EBV)-immortalized human B cell lines, one from a 22-year-old man (EBV-B young) and the other from a 65-year-old man (EBV-B old). The activity of senescence-associated ß-galactosidase, a marker of cellular senescence, was enhanced in the EBV-B old cells compared with EBV-B young cells. Moreover, the levels of p16, p21, IL-6, TNF-α, and TGF-ß1, which are senescence-associated secretary phenotypes, were also increased in EBV-B old cells. In vitro immunization of EBV-B cells with ß-lactoglobulin further showed that EBV-B old cells had a reduced cell population of naïve B cells than that of EBV-B young cells. Furthermore, HLA-DR expression, which is important for antigen presentation, was decreased in the EBV-B old cells. Comparative microarray analysis between EBV-B young and old cells also showed decreased expression of antibody genes, such as those of the heavy chain and light chain (κ chain). These results suggest that cellular senescence and decreased gene expression are responsible, at least in part, for the decline in B cell function and antigen sensitization capacity with aging, which ultimately impairs the function of the acquired immune system.

Essential Oils Derived from Cistus Species Activate Mitochondria by Inducing SIRT1 Expression in Human Keratinocytes, Leading to Senescence Inhibition.

Cistus L. is a genus of dicotyledonous perennial herbaceous plants. Cistus species have been commonly used in folk medicine in the Mediterranean region. In the present study, the biological activities of essential oils derived from Cistus species (Cistus laurifolius, C. monspeliensis, C. creticus, and C. salviifolius) were evaluated. Essential oils derived from C. laurifolius and C. monspeliensis were found to augment the expression of SIRT1, an anti-aging gene, in the normal culture of HaCaT cells. Furthermore, these essential oils increased the number and size of mitochondria and augmented their activity. These effects were thought to be caused by the up- and downregulated expression of MITOL and Drp1 in HaCaT cells, respectively, in response to the essential oil treatment. In addition, these essential oils were found to attenuate ultraviolet-B-induced mitochondrial damage and cellular senescence in HaCaT cells. These findings indicate that essential oils derived from C. laurifolius and C. monspeliensis may inhibit skin aging through mitochondrial regulation via SIRT1 activation.

Paramylon extracted from Euglena gracilis EOD-1 augmented the expression of SIRT1.

Euglena gracilis, a type of microalgae, contains several nutrients and accumulates paramylon, a ß-1,3-glucan. In recent studies, paramylon has shown to exhibit various activities including immunomoduratory and hepatoprotective effects. In the present study, using an in vitro cell culture system, we aimed to determine whether paramylon derived from the E. gracilis EOD-1 strain, which produces large amounts of paramylon, can augment SIRT1 expression in epidermal cells via activating gut-skin interactions. Results showed that paramylon augmented the expression of SIRT1 in Caco-2 cells, a human intestinal cell line. Furthermore, microarray analysis of Caco-2 cells treated with paramylon showed that paramylon activates epidermal cells through inducing the secretion of factors from intestinal cells. Then, we focused on skin cells as target cells of paramylon-activated intestinal cells. Results showed that secretory factors from Caco-2 cells treated with paramylon augmented the expression of SIRT1 in HaCaT cells, a human keratinocyte cell line, and that expression level of genes related to the growth and maintenance of epidermal cells were significantly changed in Caco-2 cells treated with paramylon as evidenced by microarray analysis. All these results suggest that paramylon can activate epidermal cells by inducing the production of secretory factors from intestinal cells.

Exosome-Mediated Activation of Neuronal Cells Triggered by γ-Aminobutyric Acid (GABA).

γ-Aminobutyric acid (GABA) is a potent bioactive amino acid, and several studies have shown that oral administration of GABA induces relaxation, improves sleep, and reduces psychological stress and fatigue. In a recent study, we reported that exosomes derived from GABA-treated intestinal cells serve as signal transducers that mediate brain-gut interactions. Therefore, the purpose of this study was to verify the functionality of GABA-derived exosomes and to examine the possibility of improving memory function following GABA administration. The results showed that exosomes derived from GABA-treated intestinal cells (Caco-2) activated neuronal cells (SH-SY5Y) by regulating genes related to neuronal cell functions. Furthermore, we found that exosomes derived from the serum of GABA-treated mice also activated SH-SY5Y cells, indicating that exosomes, which are capable of activating neuronal cells, circulate in the blood of mice orally administered GABA. Finally, we performed a microarray analysis of mRNA isolated from the hippocampus of mice that were orally administered GABA. The results revealed changes in the expression of genes related to brain function. Gene Set Enrichment Analysis (GSEA) showed that oral administration of GABA affected the expression of genes related to memory function in the hippocampus.

Exosomes Derived from Fisetin-Treated Keratinocytes Mediate Hair Growth Promotion.

Enhanced telomerase reverse transcriptase (TERT) levels in dermal keratinocytes can serve as a novel target for hair growth promotion. Previously, we identified fisetin using a system for screening food components that can activate the TERT promoter in HaCaT cells (keratinocytes). In the present study, we aimed to clarify the molecular basis of fisetin-induced hair growth promotion in mice. To this end, the dorsal skin of mice was treated with fisetin, and hair growth was evaluated 12 days after treatment. Histochemical analyses of fisetin-treated skin samples and HaCaT cells were performed to observe the effects of fisetin. The results showed that fisetin activated HaCaT cells by regulating the expression of various genes related to epidermogenesis, cell proliferation, hair follicle regulation, and hair cycle regulation. In addition, fisetin induced the secretion of exosomes from HaCaT cells, which activated ß-catenin and mitochondria in hair follicle stem cells (HFSCs) and induced their proliferation. Moreover, these results revealed the existence of exosomes as the molecular basis of keratinocyte-HFSC interaction and showed that fisetin, along with its effects on keratinocytes, caused exosome secretion, thereby activating HFSCs. This is the first study to show that keratinocyte-derived exosomes can activate HFSCs and consequently induce hair growth.

Effect of extracts and isolated compounds derived from Retama monosperma (L.) Boiss. on anti-aging gene expression in human keratinocytes and antioxidant activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Moroccan folk medicine treats skin cicatrization with Retama monosperma (L.) Boiss. locally named "Rtem", but the mechanism involved is still not well known. Traditional healers use the plant in small doses as an anthelmintic, disinfectant and an effective abortive. In addition, the cladodes powder mixed with honey is employed as purgative and vermifuge. Equally, the SIRT1 and SIRT3 genes activation and sirtuin proteins expression, which delay cellular senescence, participate in wound healing and skin regeneration especially, SIRT1 the most studied gene, leads to fast skin restoration and cicatrization. AIM OF THE STUDY: In this study, we evaluated the ability of the Retama monosperma (L.)Boiss. flowers and seeds extracts and the isolated compounds in augmenting the SIRT1 and SIRT3 gene expression in HaCaT cells and expressing the antioxidant activity. MATERIALS AND METHODS: We examined for quantitative expression levels of SIRT1 and SIRT3 in HaCaT cell by qRT-PCR and the antioxidant activity by four tests (conjugated diene, TBARS assay, DPPH scavenging activity and H2O2 radical scavenging assay) of diethyl ether extract of flowers (DEF extract) and ethyl acetate extract of seeds (EAS extract) of R. monosperma(L.) Boiss. and the isolated compounds (quercetin, 6-methoxykaempferol, kaempferol and genistein). RESULTS: The screening system by EGFP fluorescence revealed that all samples and resveratrol significantly increase SIRT1 and SIRT3 promoters activities in HaCaT cells with p< 0.05. Furthermore, EAS, quercetin, 6-methoxykaempferol and kaempferol increase significantly (p< 0.05) SIRT1 (3.43, 1.18, 2.62, and 1.72 expression quantity, respectively) and SIRT3 (16.27, 5.01, 3.01, and 6.18 expression quantity, respectively) in HaCaT cells. On the other hand, genistein has a moderate activity on SIRT1 and SIRT3 with 1.43 and 2.04 expression levels. For the antioxidant activity, the EAS and the pure compounds exhibited stronger antioxidant activity than BHT. While DEF and genistein have a moderate antioxidant activity when compared with BHT. CONCLUSIONS: In this study, the expression levels of SIRT1 and SIRT3 in HaCaT cells increase in the presence of extracts of R. monosperma (L.) Boiss. and the pure compounds.

Soyasaponin ameliorates obesity and reduces hepatic triacylglycerol accumulation by suppressing lipogenesis in high-fat diet-fed mice.

Soyasaponins are triterpenoid glycosides found in soybean. We investigated whether soyasaponin ameliorates lipid metabolism and its possible mechanisms. In C57BL/6J mice fed a high-fat diet (HFD), soyasaponin (SAP) was orally administered for 9 weeks. Additionally, we evaluated the effect of soyasapogenols on 3T3-L1 adipocytes. In HFD-fed mice, the SAP significantly reduced body weight by 7% and relative adipose tissue weight by 35%. X-ray computed tomography demonstrated that the SAP reduced visceral and subcutaneous adipose tissue weights during week 3 of feeding. The SAP reduced sterol regulatory element-binding protein-1c (SREBP-1c) mRNA levels by 32% in the epididymal adipose tissue, significantly decreasing the triacylglycerol (TAG) content by 37% and SREBP-1c and fatty acid synthase mRNA levels by 52% and 61%, respectively, in the liver. In 3T3-L1 adipocytes, soyasapogenol B significantly decreased lipid droplets. The SAP containing soyasaponin A and B as conjugates demonstrate anti-obesity effects by suppressing adipocyte differentiation and lipogenesis, with a preventive effect on hepatic TAG accumulation by suppressing lipogenesis. PRACTICAL APPLICATION: Soyasaponin is one of the oleanane triterpenoids in soybeans. We have demonstrated that soyasaponin potently reduces body weight and white adipose tissue weight, and hepatic triacylglycerol accumulation in high-fat diet-fed mice. Thus, soyasaponin is a beneficial compound to prevent obesity and fatty liver.

NFAT5 promotes oral squamous cell carcinoma progression in a hyperosmotic environment.

Epidermal growth factor receptor (EGFR) is highly expressed in several types of cancer cells including oral squamous cell carcinoma (OSCC). EGF/EGFR signaling is recognized as an important molecular target in cancer therapy. However, cancer cells often become tolerant to EGF/EGFR signaling-targeted therapies. In the tumor microenvironment, the tumor incites inflammation and the inflammation-derived cytokines make a considerable impact on cancer development. In addition, hyperosmolarity is also induced, but the role of osmotic stress in cancer development has not been fully understood. This study demonstrates molecular insights into hyperosmolarity effect on OSCC development and shows that NFAT5 transcription factor plays an important functional role in enhancing the oral cancer cell proliferation by inducing the EGFR translocation from the endoplasmic reticulum to the plasma membrane through increase the expression of DPAGT1, an essential enzyme for catalyzing the first committed step of N-linked protein glycosylation. These results suggest that hyperosmolarity-induced intra-nuclear translocation of NFAT5 essential for DPAGT1 activation and EGFR subcellular translocation responsible for OSCC tumor progression.

Correlative Localization Analysis Between mRNA and Enhanced Green Fluorescence Protein-Fused Protein by a Single-Molecule Fluorescence in situ Hybridization Using an egfp Probe in Aspergillus oryzae.

Although subcellular localization analysis of proteins fused with enhanced green fluorescence protein (EGFP) has been widely conducted in filamentous fungi, little is known about the localization of messenger RNAs (mRNAs) encoding the EGFP-fused proteins. In this study, we performed single-molecule fluorescence in situ hybridization (smFISH) using an egfp probe to simultaneously visualize EGFP-fused proteins and their mRNAs in the hyphal cells of the filamentous fungus Aspergillus oryzae. We investigated the subcellular localization of mRNAs encoding cytoplasmic EGFP, an actin marker protein Lifeact tagged with EGFP, and several EGFP-fused proteins AoSec22, AoSnc1, AoVam3, and AoUapC that localize to the endoplasmic reticulum (ER), the apical vesicle cluster Spitzenkörper, vacuolar membrane, and plasma membrane, respectively. Visualization of these mRNAs by smFISH demonstrated that each mRNA exhibited distinct localization patterns likely depending on the mRNA sequence. In particular, we revealed that mRNAs encoding Lifeact-EGFP, EGFP-AoSec22, EGFP-AoVam3, and AoUapC-EGFP, but not cytoplasmic EGFP and EGFP-AoSnc1, were preferentially localized at the apical cell, suggesting certain mechanisms to regulate the existence of these transcripts among hyphal regions. Our findings provide the distinct localization information of each mRNA in the hyphal cells of A. oryzae.

Fisetin Promotes Hair Growth by Augmenting TERT Expression.

Although thinning hair and alopecia are not recognized as severe diseases, hair loss has implications for mental health and quality of life; therefore, a large number of studies have been carried out to develop novel hair growth agents. In the present study, we aimed to examine the potential of telomerase reverse transcriptase (TERT), because TERT overexpression in skin activates resting hair follicle bulge stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To this end, we screened polyphenols that activate TERT expression in keratinocytes, and identified resveratrol and fisetin as strong hTERT-augmenting compounds. These polyphenols also regulated the gene expression of cytokines such as IGF-1 and KGF, which activate the ß-catenin pathway, and TGF-ß1, which plays an important role in maintaining the niche of hair follicle stem cells, thus are thought to play roles in promoting hair growth. We additionally showed that these polyphenols, especially fisetin, promoted hair growth from the shaved dorsal skin of mice, which suggests that these polyphenols activate the transition from telogen to anagen phase. Histological studies indicated that the dorsal skin of mice treated with these polyphenols contained numerous hair follicles and was thickened compared with that in control mice. Furthermore, on the dorsal skin of mice treated with resveratrol and fisetin, a number of proliferating cells (Ki67+ cells) were observed around the hair papilla. These results suggest that resveratrol and fisetin induce a shift from telogen to anagen in the hair follicle by inducing proliferation of hair follicle bulge stem cells, thus promoting hair growth.

γ-Aminobutyric acid (GABA) activates neuronal cells by inducing the secretion of exosomes from intestinal cells.

The oral administration of γ-aminobutyric acid (GABA) has been shown to affect brain functions. However, the molecular mechanisms underlying GABA-induced gut-brain interactions have not yet been fully elucidated. As the blood-brain barrier is impermeable to GABA, we hypothesized that the gut-brain interaction might be stimulated by some secretory factors derived from the gut. Then we focused on exosomes as a secretory mediator. In the present study, we investigated whether exosomes derived from GABA-treated intestinal cells activate neuronal cells. Our results revealed that secretory factors derived from GABA-treated intestinal cells (Caco-2) activated neuronal cells (SH-SY5Y). Further investigation revealed that exosomes derived from GABA-treated Caco-2 cells were responsible for activating the SH-SY5Y cells. These results suggested that GABA-activated intestinal cells induce the secretion of exosomes that activate neuronal cells. MicroRNAs in the exosomes derived from GABA-treated intestinal cells may play a key role in the activation of neuronal cells.

Characterization and functional analysis of ERAD-related AAA+ ATPase Cdc48 in Aspergillus oryzae.

Aspergillus oryzae can secrete large amounts of enzymes. However, the production of abundant secretory proteins triggers the unfolded protein response (UPR) in the endoplasmic reticulum (ER), and it is not clear how ER-associated protein degradation (ERAD) contributes to bulk protein production in A. oryzae. Here we identified AoCdc48, the sole A. oryzae ortholog of Saccharomyces cerevisiae AAA+ ATPase Cdc48, a component of the ERAD machinery. We found that AoCdc48 localizes in both nuclei and cytoplasm. Generation of an Aocdc48 conditional mutant showed that Aocdc48 repression leads to reduced cell growth and aberrant hyphal morphology. When Aocdc48-repressed cells were cultured on starch-containing plates, the α-amylase-encoding gene amyB was about 1.3-fold higher expressed. Indeed, a halo produced by secreted amylase was seen on potato starch-containing plates even when there was almost no growth under Aocdc48 repression. Fluorescence microscopy revealed that although AmyB seemed to be secreted, various organelle distributions were aberrant in Aocdc48-repressed cells. We found that D1 AAA domain is crucial for cell viability. Finally, we show that Aocdc48-overexpression also causes defects of cell growth, colonial morphology and conidial formation. Collectively, our results suggest that AoCdc48 is essential for growth and organelle distribution but dispensable for amylase secretion.