Human Milk Exosomes Induce ZO-1 Expression via Inhibition of REDD1 Expression in Human Intestinal Epithelial Cells.

Human milk exosomes (HMEs) enhance intestinal barrier function and contribute to an improvement in inflammation and mucosal injury, such as necrotizing enteritis (NEC), in infants. Here, we aimed to elucidate the intracellular factors involved in HME-induced expression of zonula occludens-1 (ZO-1), a tight junction protein, in Caco-2 human intestinal epithelial cells. HME treatment for 72 h significantly increased transepithelial electrical resistance in these cells. The mean ZO-1 protein levels in cells treated with HME for 72 h were significantly higher than those in the control cells. The mRNA and protein levels of regulated in development and DNA damage response 1 (REDD1) in HME-treated cells were significantly lower than those in the control cells. Although HME treatment did not increase the mechanistic target of rapamycin (mTOR) level in Caco-2 cells, it significantly increased the phosphorylated mTOR (p-mTOR) level and p-mTOR/mTOR ratio. The ZO-1 protein levels in cells treated with an inducer of REDD1, cobalt chloride (CoCl2) alone were significantly lower than those in the control cells. However, ZO-1 protein levels in cells co-treated with HME and CoCl2 were significantly higher than those in cells treated with CoCl2 alone. Additionally, REDD1 protein levels in cells treated with CoCl2 alone were significantly higher than those in the control cells. However, REDD1 protein levels in cells co-treated with HME and CoCl2 were significantly lower than those in cells treated with CoCl2 alone. This HME-mediated effect may contribute to the development of barrier function in the infant intestine and protect infants from diseases.

Suppression of milk-derived miR-148a caused by stress plays a role in the decrease in intestinal ZO-1 expression in infants.

BACKGROUND & AIMS: Milk-derived miR-148a-3p (miR-148a), which is abundant in breast milk, has been shown to be associated with the development of infants' intestines. Although it is well known that stress during lactation changes milk constituents in terms of lipid and protein, no studies have examined the influence of stress on miR-148a expression in breast milk. The objective of this study is to investigate the relationship between stress and miR-148a expression in milk, and to evaluate whether the changes in milk-derived miR-148a expression-caused by the mother's exposure to stress-influence intestinal ZO-1 expression in infants. METHODS: The participants of this study were healthy Japanese women who were nursing. Psychological stress evaluation of the subjects was conducted using a short form of the Profile of Mood State Second Edition-Adult (POMS-2). Additionally, miR-148a expressions in restraint stressed nursing mice were investigated using quantitative real-time PCR. The levels of a tight junction protein zonula occludens-1 (ZO-1) and DNA methyltransferase 1 (DNMT1), which is a direct target of miR-148a, in ileum in neonatal mice breastfed by stressed nursing mice were investigated using Western blot. Furthermore, to investigate the influence of miR-148a on ZO-1 expression within the intestine, the levels of ZO-1 and DNMT1 in human intestinal epithelial Caco-2 cells with lentivirus-mediated miR-148a overexpression were evaluated. RESULTS: A significantly negative correlation was observed between relative miR-148a expression in breast milk and the total mood disturbance T-score. Each T-score on negative mood subscales of anger-hostility, confusion-bewilderment, depression-dejection, fatigue-inertia, and tension-anxiety was significantly negatively correlated with relative miR-148a expression in breast milk: a positive mood subscale vigor-activity T-score was significantly positively correlated with relative miR-148a expression in breast milk. A positive mood friendliness T-score, estimated separately from other scores, was significantly positively correlated with relative miR-148a expression in breast milk. Additionally, the relative expression of miR-148a in the milk obtained from stressed mice was significantly lower than that of control mice. The relative level of ZO-1 in ileum of neonatal mice nursed by stressed mice was significantly lower than that of neonatal mice nursed by control mice. Additionally, the relative level of DNMT1 in ileum of neonatal mice nursed by stressed mice was significantly higher than that of neonatal mice nursed by control mice. Furthermore, the relative level of ZO-1 in miR-148a-overexpressed Caco-2 cells was significantly higher than that in control cells. The relative level of DNMT1 in miR-148a-overexpressed Caco-2 cells was significantly lower than that in control cells. CONCLUSIONS: Mothers' exposure to stress during lactation may cause miR-148a expression in breast milk. Additionally, stressed-induced suppression of miR-148a expression in breast milk may cause a decrease in intestinal ZO-1 level via the increase in DNMT1 in infants' intestines. These observations are beneficial information for breastfeeding mothers and their families and perinatal medical professionals. Our findings encourage monitoring maternal psychological stress during lactation to promote breastfeeding and adequate infant nutrition.

[Efficacy of the Extract from Fermentation of Soybean and Rice Bran on Hyperglycemia].

In recent years, lifestyle-related diseases such as hypertension and diabetes have been on the rise. These conditions can cause serious conditions such as myocardial and cerebral infarctions. Therefore, proper control of blood pressure and blood glucose levels is important issues in preventive medicine. Traditional fermented foods have been shown to have various functions, and their effects on lifestyle-related diseases have attracted particular attention. In this study, we investigated the effects of fermented soybeans and rice bran (OE-1) and supplements containing OE-1 on blood glucose levels and weight changes. We identified an inhibitory effect on elevated blood glucose levels upon administration of OE-1, and this effect was thought to be due to digestive enzyme inhibition. These effects of foods containing OE-1 are expected to have a positive effect on the prevention and improvement of lifestyle-related diseases as health foods.

Cre/LoxP Genetic Recombination Sustains Cartilage Anabolic Factor Expression in Hyaluronan Encapsulated MSCs Alleviates Intervertebral Disc Degeneration.

(1) Background: Inexplicable low back and neck pain frequently results from spinal disc degeneration with an imbalanced intervertebral disc (IVD) cell homeostasis. We hypothesize that introducing MSC expressing a sustained cartilage-anabolic factor in the IVD may stimulate the mucoid materials secreted from the IVD cells, promote the MSC's chondrogenesis and maintain the hydration content providing mechanical strength to decelerate the disc degeneration progression; (2) Methods: This study expressed a cartilage-anabolic factor runx1 by a baculoviral vector (BV) transduced MSCs through a Cre/LoxP gene editing and recombination system for sustained recombinant runx1 transcription factor production. The Cre/LoxP BV modified MSCs were encapsulated by hyaluronan hydrogel, due to its' vital composition in ECM of a healthy disc and transplanted to a punctured coccygeal disc in rats through micro-injection, followed by X-ray radiography and histological analysis at the 4- and 12-weeks post-transplantation; (3) Results: Data reveals the Cre/LoxP BV system-mediated long-termed runx1 gene expression, possessing good biosafety characteristics in the in vitro cell transduction and in vivo MSCs transplantation, and maintained superior hydration content in the disc than that of mock transduced MSCs; (4) Conclusions: This proof-of-concept study fulfills the need of implanting therapeutic cells accompanied with microinjection in the disc, such as a discography and paves a road to manufacture composite hyaluronan, such as peptide modified hyaluronan as an MSC carrier for IVD regeneration in the future study.

Runx1 Messenger RNA Delivered by Polyplex Nanomicelles Alleviate Spinal Disc Hydration Loss in a Rat Disc Degeneration Model.

Vertebral disc degenerative disease (DDD) affects millions of people worldwide and is a critical factor leading to low back and neck pain and consequent disability. Currently, no strategy has addressed curing DDD from fundamental aspects, because the pathological mechanism leading to DDD is still controversial. One possible mechanism points to the homeostatic status of extracellular matrix (ECM) anabolism, and catabolism in the disc may play a vital role in the disease's progression. If the damaged disc receives an abundant amount of cartilage, anabolic factors may stimulate the residual cells in the damaged disc to secrete the ECM and mitigate the degeneration process. To examine this hypothesis, a cartilage anabolic factor, Runx1, was expressed by mRNA through a sophisticated polyamine-based PEG-polyplex nanomicelle delivery system in the damaged disc in a rat model. The mRNA medicine and polyamine carrier have favorable safety characteristics and biocompatibility for regenerative medicine. The endocytosis of mRNA-loaded polyplex nanomicelles in vitro, mRNA delivery efficacy, hydration content, disc shrinkage, and ECM in the disc in vivo were also examined. The data revealed that the mRNA-loaded polyplex nanomicelle was promptly engulfed by cellular late endosome, then spread into the cytosol homogeneously at a rate of less than 20 min post-administration of the mRNA medicine. The mRNA expression persisted for at least 6-days post-injection in vivo. Furthermore, the Runx1 mRNA delivered by polyplex nanomicelles increased hydration content by ≈43% in the punctured disc at 4-weeks post-injection (wpi) compared with naked Runx1 mRNA administration. Meanwhile, the disc space and ECM production were also significantly ameliorated in the polyplex nanomicelle group. This study demonstrated that anabolic factor administration by polyplex nanomicelle-protected mRNA medicine, such as Runx1, plays a key role in alleviating the progress of DDD, which is an imbalance scenario of disc metabolism. This platform could be further developed as a promising strategy applied to regenerative medicine.

Ethnobotanical Survey on Skin Whitening Prescriptions of Traditional Chinese Medicine in Taiwan.

The increasing interest and demand for skin whitening products globally, particularly in Asia, have necessitated rapid advances in research on skin whitening products used in traditional Chinese medicine (TCM). Herein, we investigated 74 skin whitening prescriptions sold in TCM pharmacies in Taiwan. Commonly used medicinal materials were defined as those with a relative frequency of citation (RFC) > 0.2 and their characteristics were evaluated. Correlation analysis of commonly used medicinal materials was carried out to identify the core component of the medicinal materials. Of the purchased 74 skin whitening prescriptions, 36 were oral prescriptions, 37 were external prescriptions, and one prescription could be used as an oral or external prescription. After analysis, 90 traditional Chinese medicinal materials were obtained. The Apiaceae (10%; 13%) and Leguminosae (9%; 11%) were the main sources of oral and external medicinal materials, respectively. Oral skin whitening prescriptions were found to be mostly warm (46%) and sweet (53%), while external skin whitening prescriptions included cold (43%) and bitter (29%) medicinal materials. Additionally, mainly tonifying and replenishing effects of the materials were noted. Pharmacological analysis indicated that these medicinal materials may promote wound healing, treat inflammatory skin diseases, or anti-hyperpigmentation. According to the Spearman correlation analysis on interactions among medicinal materials with an RFC > 0.2 in the oral skin whitening prescriptions, Paeonia lactiflora Pall. (white) and Atractylodes macrocephala Koidz. showed the highest correlation (confidence score = 0.93), followed by Ziziphus jujuba Mill. (red) and Astragalus propinquus Schischkin (confidence score = 0.91). Seven medicinal materials in external skin whitening prescriptions with an RFC > 0.2, were classified as Taiwan qi bái sàn (an herbal preparation), including Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav., Wolfiporia extensa (Peck) Ginns, Bletilla striata (Thunb.) Rchb. f., Atractylodes macrocephala Koidz., Ampelopsis japonica (Thunb.) Makino, Paeonia lactiflora Pall. (white), and Bombyx mori Linnaeus. Skin whitening prescriptions included multiple traditional Chinese medicinal materials. Despite the long history of use, there is a lack of studies concerning skin whitening products, possibly due to the complex composition of traditional Chinese medicine. Further studies are required to assess the efficacy and safety of these traditional Chinese medicinal materials for inclusion in effective, safe, and functional pharmacological products.

Population Pharmacokinetics of Teicoplanin and Its Dosing Recommendations for Neutropenic Patients With Augmented Renal Clearance for Hematological Malignancies.

BACKGROUND: Plasma teicoplanin concentrations do not reach the therapeutic range in several patients with hematological malignancies. Nevertheless, the characteristics of the population pharmacokinetic (PPK) models have not been clarified for malignancy. The decrease in the teicoplanin concentration in patients with cancer has been attributed to augmented renal clearance (ARC). It is essential to identify the causative factors of ARC to construct a PPK model to optimize the administration method. The authors aimed to establish a PPK model and develop an appropriate dosing regimen for teicoplanin in patients with hematological malignancies. METHODS: PPK analysis was performed using therapeutic drug monitoring (TDM) data from 119 patients with hematological malignancies. The developed model was verified by predictive performance. RESULTS: The covariates affecting systemic clearance were serum creatinine, presence or absence of neutropenia (<500/µL), and body size descriptor. Patients with hematologic malignancies and neutropenia showed a 25% increase in clearance compared with those with a normal neutrophil count. The PPK model was constructed based on the presence or absence of neutropenia. This model allowed the selection of the most appropriate dosage regimen out of those recommended by the TDM guidelines for patients with eGFR of >60 mL/min/1.73 m2. The PPK model predicted a dosing regimen for achieving a 10% improvement in the coverage probability of the target concentration range during the loading and maintenance phases. CONCLUSIONS: The PPK model may help optimize dose regimens and evaluate dosing methods, using comparative simulations, in patients with hematological malignancies.

Norepinephrine transporter expressed on mammary epithelial cells incorporates norepinephrine in milk into the cells.

Mammary epithelial cells synthesize and secrete norepinephrine (NE) into breast milk to regulate ß-casein expression through the adrenergic ß2 receptor. We investigated the expression, localization, and roles of NE transporter (NET) in the mammary epithelium during lactation. mRNA and protein levels of NET were determined in primary normal human mammary epithelial cells (pHMECs) and non-malignant human mammary epithelial MCF-12A cells. In nursing CD1 mice, NET localized to the apical membranes of the mammary epithelium. The intracellular NE content of pHMECs incubated with NE increased. Although the ß-casein concentration in milk was slightly higher at day 10 than at day 2 of lactation, the NE concentration and lactation-related proteins were only slightly changed on days 2-10. Restraint stress increased the NE concentration in milk from nursing mice and NET protein levels were significantly higher than in non-stressed nursing mice. NET is expressed on the apical membrane of mammary epithelial cells and incorporates NE in milk into cells, potentially regulating the NE concentration in milk.

Dictamnine delivered by PLGA nanocarriers ameliorated inflammation in an oxazolone-induced dermatitis mouse model.

Dictamnine is an active pharmaceutical ingredient in Dictamnus dasycarpus, a Chinese herbal medicine widely used for the treatment of skin inflammations such as atopic dermatitis (AD). Oxazolone has been demonstrated to induce significant skin inflammation and produce inflammatory cytokine expression identical to that of AD. An in vitro HaCaT inflammation model treated with dictamnine, which efficiently scavenged the reactive oxygen species (ROS) and mitochondrial ROS (mROS), and it reduced interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) expression, NLRP3 inflammasome activation, and NF-κB expression. To explore the anti-inflammatory mechanism of dictamnine and enhance sustained drug release and penetration into epidermal structures in a dermatitis mouse model, we prepared PLGA-nanocarrier-encapsulated dictamnine (Dic-PLGA-NC) in a specifically designed bioreactor, namely an ultrasound composite streams-impinging mixer (U-SiM). Mouse dermatitis model was treated with Dic-PLGA-NC medication, spleens were collected to evaluate body weight ratio, and skin was retrieved for histological examination and two-photon microscopy. The data demonstrate that Dic-PLGA-NC efficiently penetrated the dermal layer, making it superior to naked dictamnine; moreover, it ameliorated the dermatitis symptoms and inflammatory cytokine expression in vivo. Dic-PLGA-NC produced using the U-SiM bioreactor could be used in new manufacturing processes for drugs to treat AD.

Physiologic changes in serotonin concentrations in breast milk during lactation.

OBJECTIVES: Serotonin (5-hydroxytryptamine; 5-HT) plays an important role in milk volume homeostasis in the mammary glands during lactation, and 5-HT in milk also may affect infant development. The aim of this study was to investigate changes in 5-HT concentration in breast milk according to the duration of lactation and evaluate whether the 5-HT concentration varied before and after nursing. METHODS: Healthy nursing Japanese women who had a natural delivery or underwent a cesarean delivery at Iwate Medical University Hospital were included in this study. RESULTS: The mean 5-HT concentration in milk was obtained from multiparous mothers 6 to 7 d after delivery (colostrum) and was significantly higher compared with primiparous mothers (24.3 ± 2.63 versus 18.5 ± 2.60 ng/mL). Additionally, mean 5-HT concentration increased with increasing lactation duration in primiparous women (colostrum: 18.5 ± 2.60; 1 mo postdelivery: 19.8 ± 2.46; 3 mo postdelivery: 22.7 ± 2.55 ng/mL); in particular, the mean 5-HT concentration in breast milk 3 mo after delivery was significantly higher than in colostrum. The mean 5-HT concentrations in breast milk in primiparous mothers immediately before nursing, 1 to 2 h after nursing, and immediately before the next nursing event were 23.6 ± 1.48, 22.82 ± 1.65, and 21.84 ± 1.31 ng/mL, respectively; mean 5-HT concentrations in multiparous women were 25.4 ± 1.65, 23.6 ± 2.20, or 22.4 ± 2.09 ng/mL, respectively. There was no significant difference in 5-HT concentrations at each time point between the groups. CONCLUSION: This information may be useful in determining the role of 5-HT in breast milk on infant development and growth.

Engulfment of Toxic Amyloid ß-protein in Neurons and Astrocytes Mediated by MEGF10.

Amyloid-ß proteins (A ß), including Aß42 and A ß 43, are known pathogenesis factors of Alzheimer's disease (AD). Unwanted substances in the brain, including A ß, are generally removed by microglia, astrocytes, or neurons via a phagocytosis receptor. We observed that neurons and astrocytes engulfed A ß 42 and A ß 43, which are more neurotoxic than A ß 40. We previously showed that multiple-EGF like domains 10 (MEGF10) plays an important role in apoptotic cell elimination and is expressed in mammalian neurons and astrocytes. Therefore, we assessed whether MEGF10 is involved in A ß42 and A ß43 engulfment in MEGF10-expressing neurons and astrocytes. We found that MEGF10-expressing astrocytes and neurons engulfed A ß42 and A ß43 but not A ß40. Furthermore, incubation of the neurons and astrocytes with A ß42 and A ß43a ugmented MEGF10 phosphorylation; however, incubation with A ß40 did not have this augmenting effect. Our findings suggest that MEGF10 plays a phagocytosis receptor function for A ß42 and A ß43 in neurons and astrocytes.

Interaction between Angiotensin Receptor and ß-Adrenergic Receptor Regulates the Production of Amyloid ß-Protein.

Alzheimer's disease (AD) is characterized by the formation of extracellular amyloid plaques containing the amyloid ß-protein (Aß) within the parenchyma of the brain. Aß is considered to be the key pathogenic factor of AD. Recently, we showed that Angiotensin II type 1 receptor (AT1R), which regulates blood pressure, is involved in Aß production, and that telmisartan (Telm), which is an angiotensin II receptor blocker (ARB), increased Aß production via AT1R. However, the precise mechanism underlying how AT1R is involved in Aß production is unknown. Interestingly, AT1R, a G protein-coupled receptor, was strongly suggested to be involved in signal transduction by heterodimerization with ß2-adrenergic receptor (ß2-AR), which is also shown to be involved in Aß generation. Therefore, in this study, we aimed to clarify whether the interaction between AT1R and ß2-AR is involved in the regulation of Aß production. To address this, we analyzed whether the increase in Aß production by Telm treatment is affected by ß-AR antagonist using fibroblasts overexpressing amyloid precursor protein (APP). We found that the increase in Aß production by Telm treatment was decreased by the treatment of ß2-AR selective antagonist ICI-118551 more strongly than the treatment of ß1-AR selective antagonists. Furthermore, deficiency of AT1R abolished the effect of ß2-AR antagonist on the stimulation of Aß production caused by Telm. Taken together, the interaction between AT1R and ß2-AR is likely to be involved in Aß production.

Stress-Induced Suppression of Milk Protein Is Involved in a Noradrenergic Mechanism in the Mammary Gland.

Stress decreases milk components such as milk protein and milk yield. The objective of this study was to investigate whether noradrenaline (NA) in milk constituted a factor associated with stress-induced changes in milk proteins such as ß-casein. Breast milk obtained from eight healthy, nursing women contained NA at concentrations ranging from 12.7 to 115.5 nM. The expression of tyrosine hydroxylase (TH), a rate-limiting enzyme of NA synthesis, was observed in primary normal human mammary epithelial cells (HMECs), and in MCF-12A and MCF-10A cell lines. The mean NA concentration in culture medium used by MCF-12A transfected with TH small interfering RNA (siRNA) was significantly lower than that of cells transfected with control siRNA. NA concentration in milk in restraint-stressed nursing mice was significantly higher than that in nonstressed nursing mice, owing to elevated TH expression in the mammary epithelium. The mean ß-casein concentration in milk in restraint-stressed mice was significantly lower than that in nonstressed mice. NA treatment resulted in a concentration-dependent decrease in ß-casein expression in HMECs. ß2 adrenergic receptor (ADRB2) expression was observed in HMECs, MCF-12A, and MCF-10A, and immunohistochemical analysis of ADRB2 using mammary epithelium sections obtained from mice at day 10 of lactation showed that ADRB2 was expressed at the apical membrane of mammary epithelium. Treatment with salbutamol, an ADRB2 stimulant, decreased ß-casein expression in a concentration-dependent manner in MCF-12A. Our results showed that endogenous NA derived from mammary epithelial cells likely comprises one of the factors involved in stress-induced changes in milk proteins such as ß-casein.

A clinical dose of angiotensin-converting enzyme (ACE) inhibitor and heterozygous ACE deletion exacerbate Alzheimer's disease pathology in mice.

Inhibition of angiotensin-converting enzyme (ACE) is a strategy used worldwide for managing hypertension. In addition to converting angiotensin I to angiotensin II, ACE also converts neurotoxic ß-amyloid protein 42 (Aß42) to Aß40. Because of its neurotoxicity, Aß42 is believed to play a causative role in the development of Alzheimer's disease (AD), whereas Aß40 has neuroprotective effects against Aß42 aggregation and also against metal-induced oxidative damage. Whether ACE inhibition enhances Aß42 aggregation or impairs human cognitive ability are very important issues for preventing AD onset and for optimal hypertension management. In an 8-year longitudinal study, we found here that the mean intelligence quotient of male, but not female, hypertensive patients taking ACE inhibitors declined more rapidly than that of others taking no ACE inhibitors. Moreover, the sera of all AD patients exhibited a decrease in Aß42-to-Aß40-converting activity compared with sera from age-matched healthy individuals. Using human amyloid precursor protein transgenic mice, we found that a clinical dose of an ACE inhibitor was sufficient to increase brain amyloid deposition. We also generated human amyloid precursor protein/ACE+/- mice and found that a decrease in ACE levels promoted Aß42 deposition and increased the number of apoptotic neurons. These results suggest that inhibition of ACE activity is a risk factor for impaired human cognition and for triggering AD onset.

Iron treatment inhibits Aß42 deposition in vivo and reduces Aß42/Aß40 ratio.

Alzheimer's disease (AD) is characterized by the formation of extracellular amyloid plaques containing the amyloid ß-protein (Aß) within the parenchyma of the brain. Aß42, which is 42 amino acids in length, is considered to be the key pathogenic factor in AD. Iron deposition is found abundantly in the amyloid plaques of AD patients; however, whether iron intake exacerbates amyloid deposition in vivo is unknown. Here, we treated AD model mice with iron-containing water and found that Aß42 deposition in the brain was significantly inhibited, along with a decrease in iron deposition. Iron treatment did not change the overall levels of iron in the brain or serum. Interestingly, Aß40 generation was significantly increased by iron treatment in amyloid precursor protein (APP)-overexpressing fibroblasts, whereas Aß42 generation did not change, which led to a decreased Aß42/Aß40 ratio. Because Aß40 can inhibit Aß42 aggregation in vitro, and Aß40 inhibits amyloid formation in vivo, our results suggest that iron can selectively enhances Aß40 generation and inhibit amyloid deposition by reducing the Aß42/Aß40 ratio. Thus, iron may be used as a novel treatment for reducing the Aß42/Aß40 ratio and Aß42 deposition in AD.

[Endogenous Serotonin and Milk Production Regulation in the Mammary Gland].

　Intrinsic serotonin (5-hydroxytryptamine; 5-HT) synthesized within the mammary epithelium has an important physiological role in milk volume homeostasis in many species including mice, cows, and humans. During lactation, mammary epithelial cells activate 5-HT synthesis by tryptophan hydroxylase 1 (TPH1). TPH1 catalyzes the rate-limiting step in 5-HT biosynthesis within mammary glands. 5-HT synthesized in mammary glands is released into both the apical (milk) and basolateral spaces by a vesicular monoamine transporter. 5-HT released into milk is incorporated by the apical membrane-expressed serotonin reuptake transporter and degraded by the monoamine oxidase A enzyme. Suckling maintains 5-HT at low levels in milk. When the mammary gland becomes filled with milk, 5-HT provides a negative feedback signal that suppresses further milk synthesis in the mammary epithelium. Our research, using human mammary epithelial MCF-12A cells, shows that the expression of ß-casein, a differentiation marker, is suppressed via 5-HT-mediated inhibition of signal transducer and activator of transcription 5. Additionally, our results show that reduced ß-casein expression in MCF-12A cells is associated with 5-HT7 receptor expression. Furthermore, we show that 5-HT7 receptor-mediated suppression of ß-casein expression is involved in the activation of protein kinase A and protein-tyrosine phosphatase 1B. Thus, this mechanism might be associated with the feedback signals by 5-HT within the mammary epithelium. Hence, further research that builds on our findings should include the elucidation of the physiological roles of 5-HT present in milk synthesized by mammary epithelial cells in vivo and its effects on nursing infants.

An E3 Ubiquitin Ligase, Synoviolin, Is Involved in the Degradation of Homocysteine-Inducible Endoplasmic Reticulum Protein.

Homocysteine-inducible endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible membrane protein involved in ER-associated degradation. Herp expression is maintained at low levels through a strict regulatory mechanism, but the details of this mechanism and the reasons why Herp expression is restricted in this manner remain unclear. Here, we show that Herp degradation involves synoviolin, an ER-resident E3 ubiquitin ligase. Herp protein levels were found to be markedly elevated in synoviolin-null cells, and Herp expression decreased when synoviolin was overexpressed. However, the lysine residues of Herp, which are ubiquitinated by E3 ubiquitin ligase, were not sufficient for regulation of Herp degradation. These results suggest that Herp degradation is mediated via synoviolin and that Herp ubiquitination involves amino acids other than lysine.

Expression of MEGF10 in cholinergic and glutamatergic neurons.

Multiple-EGF like domains 10 (MEGF10) is the mammalian homologue of Draper, a Drosophila phagocytosis receptor that plays an important role in synapse elimination and cell type-specific recognition. However, the expression and function of MEGF10 in the brain remain to be elucidated. Therefore, we aimed to clarify the regions and types of neurons that express MEGF10 in the brain, and to determine whether cells expressing MEGF10 possess phagocytic abilities. Our results indicated that MEGF10 is expressed in cholinergic and glutamatergic neurons of the cortex, hippocampus, and substantia nigra. Furthermore, the ratio of neurons expressing MEGF10 was higher in the cortex and hippocampus of hAPP transgenic mice than in those of wild-type mice. Phagocytic activity was also observed in neuronal cells expressing MEGF10. Thus, our results indicate that MEGF10 may be responsible for phagocytic activity targeted toward unwanted substances such as amyloid in cholinergic and glutamatergic neurons.

Analysis of serotonin concentrations in human milk by high-performance liquid chromatography with fluorescence detection.

Serotonin (5-hydroxytryptamine, 5-HT) plays an important role in milk volume homeostasis in the mammary gland during lactation; 5-HT in milk may also affect infant development. However, there are few reports on 5-HT concentrations in human breast milk. To address this issue, we developed a simple method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for measuring 5-HT concentrations in human breast milk. Breast milk samples were provided by four healthy Japanese women. Calibration curves for 5-HT in each sample were prepared with the standard addition method between 5 and 1000 ng/ml, and all had correlation coefficients >0.999. The recovery of 5-HT was 96.1%-101.0%, with a coefficient of variation of 3.39%-8.62%. The range of 5-HT concentrations estimated from the calibration curves was 11.1-51.1 ng/ml. Thus, the HPLC-FD method described here can effectively extract 5-HT from human breast milk with high reproducibility.

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein.

Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin.