小林 謙 (コバヤシ ケン)
| 農学研究院 基盤研究部門 畜産科学分野 | 准教授 |
Last Updated :2025/12/04
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- 2024年09月, 日本畜産学会, 優秀論文賞
Establishment of an in vitro culture model to study milk production and the blood–milk barrier with bovine mammary epithelial cells
Yusaku Tsugami;Norihiro Suzuki;Manabu Kawahara;Takahiro Suzuki;Takanori Nishimura;Ken Kobayashi, 32344845 - 2017年10月, 日本乳房炎研究会, 髙居百合子学術賞
小林謙
論文
- Differential effects of type I, III, and V collagens on mammary epithelial development in vitro
Kazuki Ninomiya, Yuki Yasui, Liang Han, Tamaki Uehara, Takanori Nishimura, Ken Kobayashi
Tissue and Cell, 103165, 103165, Elsevier BV, 2025年09月
英語, 研究論文（学術雑誌） - Royal Jelly Induces Thin Hair Shaft Formation by Suppressing Proliferation of Hair Follicle Stem Cells in Mice.
Takumi Hamanishi, Haruta Koga, Takanori Nishimura, Ken Kobayashi
ACS omega, 10, 17, 17228, 17236, 2025年05月06日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Royal jelly (RJ), a honeybee product, is used as a cosmetic and food ingredient to improve skin condition. However, the influences of RJ on hair growth remain unclear. In this study, we investigated whether RJ regulates hair follicle development, hair shaft formation, and proliferation of hair follicle stem cells (HFSCs) using a gentle anagen induction model by shaving the back skin and a forced anagen induction model by depilating the back skin in mice. The results showed that topical application of RJ on depilated skin induced thinning of the hair shaft and smaller hair bulb formation during the anagen phase. In addition, RJ suppressed the proliferation of CK15-positive HFSCs in hair follicles at the early and middle anagen stages of shaved back skin. RJ suppressed the proliferation of cultured HFSCs in vitro. These findings suggested that RJ induces the formation of thin hair shafts by suppressing the HFSC proliferation. - Nicotinic acetylcholine receptors modulate casein secretion and claudin expression in mammary epithelial cells by regulating STAT5 and Akt pathways
Ken Kobayashi, Yuki Yasui, Tamaki Uehara, Kazuki Ninomiya, Hideki Miwa, Liang Han
Biochemical and Biophysical Research Communications, 152036, 152036, Elsevier BV, 2025年05月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Leptin on the apical surface inhibits casein production and STAT5 phosphorylation in mammary epithelial cells.
Lu Shan-Ni, Han Liang, Yuki Yasui, Kazuki Ninomiya, Tamaki Uehara, Takanori Nishimura, Ken Kobayashi
Experimental cell research, 443, 1, 114330, 114330, 2024年11月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Leptin is a peptide hormone present in both the blood and milk. A close relationship between leptin and milk production in lactating mammary glands has been previously reported. However, how leptin influences milk production in lactating mammary glands remains unclear. Also, whether leptin in milk or blood influences mammary epithelial cells (MECs) during lactation needs further investigation. This study investigated the effects of leptin on mouse MECs using a culture model in which MECs produced milk components and formed less permeable tight junctions. Our results showed that β-casein production in MEC was inhibited by leptin in a concentration-dependent manner. Leptin also inactivated the signal transducer and activator of transcription 5 (STAT5), a transcription factor that facilitates milk production in MECs. Leptin treatment induced the activation of p38 and c-Jun N-terminal kinase (JNK) in MEC before STAT5 inactivation, and anisomycin, an activator of p38 and JNK, induced the inactivation of STAT5. Furthermore, leptin exposure on the apical surface of MECs inhibited β-casein production and inactivated STAT5. However, leptin exposure on the basolateral surface hardly caused these effects. These findings suggested that milk leptin, but not plasma leptin, inhibited milk production in MECs. - Regulatory roles of dopamine D2 receptor in milk protein production and apoptosis in mammary epithelial cells.
Liang Han, Shan-Ni Lu, Takanori Nishimura, Ken Kobayashi
Experimental cell research, 439, 1, 114090, 114090, 2024年06月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Dopamine D2 receptors (D2Rs) play crucial roles in regulating diverse physiological functions of the central nervous system and peripheral organs. D2Rs are also expressed in mammary glands. However, which cell types express D2Rs and whether they are involved in milk production remains unclear. The present findings revealed that D2Rs are expressed in the apical regions of the lateral membranes of mammary epithelial cells (MECs) in lactating mice. We also investigated the effects of the D2R agonist bromocriptine and/or antagonist domperidone on intracellular cAMP levels, milk protein production, and apoptosis in a lactation culture model of MECs that produce major milk components like lactating MECs in vivo. We found that bromocriptine decreased intracellular cAMP levels, whereas domperidone dose-dependently neutralized this effect. Bromocriptine also inhibited casein and lactoferrin production and suppressed activities of STAT5 and glucocorticoid receptors (GRs). Domperidone neutralized the inhibition of casein production as well as STAT5 and GR inactivation induced by bromocriptine. Furthermore, D2R activation by bromocriptine induced apoptosis and inactivated ERK, a signaling molecule responsible for promoting cell proliferation and survival. Domperidone attenuated ERK inactivation and apoptosis induced by bromocriptine. These findings suggest that D2Rs play regulatory roles in milk protein production and apoptosis in MECs. - Murf1 alters myosin replacement rates in cultured myotubes in a myosin isoform-dependent manner.
Emi Uenaka, Koichi Ojima, Takahiro Suzuki, Ken Kobayashi, Susumu Muroya, Takanori Nishimura
In vitro cellular & developmental biology. Animal, 2024年05月17日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin-proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 replacement. In contrast to Murf2 or Murf3 overexpression, Murf1 overexpression selectively facilitated the GFP-Myh3 myosin replacement. Next, to examine the effects of Murf1 overexpression on the replacement of myosin isoforms, Cherry-Murf1 was coexpressed with GFP-Myh1, GFP-Myh4, or GFP-Myh7 in myotubes. Intriguingly, Murf1 overexpression enhanced the myosin replacement of GFP-Myh4 but did not affect those of GFP-Myh1 or GFP-Myh7. Surprisingly, overexpression of Murf1 did not enhance the ubiquitination of proteins. These results indicate that Murf1 selectively regulated myosin replacement in a Myh isoform-dependent fashion, independent of enhanced ubiquitination. This suggests that Murf1 may have a role beyond functioning as a ubiquitin ligase E3 in thick filament myosin replacement. - Culture models to investigate lactation and mastitis in bovine mammary epithelial cells
Tsugami Y, Nagasawa Y, Kobayashi K
Japan Agricultural Research Quarterly, 2024年, ［査読有り］, ［最終著者］ - Structural Features of Connective Tissue Formed around Resin Implants Subcutaneously Embedded in Dairy Cows.
Yuka Katayama, Osamu Ichii, Teppei Nakamura, Keita Yanase, Masaya Hiraishi, Takashi Namba, Yuki Otani, Teppei Ikeda, Erika Tsuji, Natsuko Tsuzuki, Ken Kobayashi, Yasuhiro Kon, Takanori Nishimura
Animals : an open access journal from MDPI, 13, 23, 2023年11月29日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Foreign body reactions (FBRs) are inadvertently observed in invading or artificially embedded materials, triggering inflammation and subsequent fibrotic processes to occur in situ. Here, we assessed the spatiotemporal formation of connective tissue around implanted materials to establish a technique using connective tissue formed by FBRs as xenografts. An acrylic resin implant, comprising a columnar inner rod and a tubular outer cylinder (OC) with several slits, was embedded in adult dairy cows. Tissues formed in the inner rod and OC groups were histologically analyzed at weeks 2, 4, 8, and 12. Edematous tissues with non-collagenous fibers formed for 2 weeks and showed increased cellularity after 4 weeks. The weight, thickness, amounts of total protein, collagen, DNA, and quantitative scores of α-smooth muscle actin-positive myofibroblasts or elastic fibers notably increased after 8 weeks, with condensed collagen fibers showing orientation. Inflammatory cells were primarily localized in tissues close to the OC, and their numbers increased, with the count of CD204+ cells peaking at 8 weeks and declining at 12 weeks. The count of Ki67+ proliferating cells slightly increased in tissues close to the OC; however, the number and lumen of CD31+ vessels increased. These results may help understand FBR-related tissue remodeling. - Effects of hydrostatic compression on milk production-related signaling pathways in mouse mammary epithelial cells
Ken Kobayashi, Liang Han, Shan-Ni Lu, Kazuki Ninomiya, Naoki Isobe, Takanori Nishimura
Experimental Cell Research, 431, 1, 113762, 113762, Elsevier BV, 2023年10月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Netrin-4 synthesized in satellite cell-derived myoblasts stimulates autonomous fusion
Takahiro Maeno, Rio Arimatsu, Koichi Ojima, Yuki Yamaya, Hikaru Imakyure, Naruha Watanabe, Yusuke Komiya, Ken Kobayashi, Mako Nakamura, Takanori Nishimura, Ryuichi Tatsumi, Takahiro Suzuki
Experimental Cell Research, 430, 1, 113698, 113698, Elsevier BV, 2023年09月, ［査読有り］
研究論文（学術雑誌） - Potential effects of gingerol topical application on components of the innate immunity in lactating goat mammary glands
Yusaku Tsugami, Takahiro Nii, Ken Kobayashi, Naoki Isobe
Innate Immunity, SAGE Publications, 2023年08月22日, ［査読有り］
英語, 研究論文（学術雑誌）, In the mammary glands, production of antimicrobial components and formation of less-permeable tight junctions (TJs) are important for safe milk production. Previously, we reported that local heat treatment of udders using disposable heating pad enhances the components of innate immunity in lactating goat mammary glands. Gingerol is a polyphenol present in ginger that can induce heat-like effects. However, oral administration of polyphenols causes a decrease in biological activity through conjugation and metabolic conversion. Here, we investigated the effects of gingerol on antimicrobial components and TJs by topically applying it to lactating goat udders. Gingerol application increased the somatic cell count, cathelicidin-2 concentration, and proportion of polymorphonuclear cells in the milk and interleukin-8 production. Moreover, gingerol treatment enhanced β-defensin-1 production in milk, cultured mammary epithelial cells, and cultured somatic cells. Contrastingly, gingerol treatment did not affect the concentrations of blood-derived components (Na+, albumin, and IgG) in the milk or the TJ barrier function of cultured mammary epithelial cells. These findings suggest that the topical application of gingerol, similar to local heat treatment, to udders enhances the components of innate immunity in mammary glands. These findings may be useful for the prevention of mastitis in milk-producing animals and, hence, safe and stable dairy production. - Culture Models to Investigate Mechanisms of Milk Production and Blood-Milk Barrier in Mammary Epithelial Cells: a Review and a Protocol
Ken Kobayashi
Journal of Mammary Gland Biology and Neoplasia, 28, 1, Springer Science and Business Media LLC, 2023年05月01日, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）, Abstract
Mammary epithelial cells (MECs) are the only cell type that produces milk during lactation. MECs also form less-permeable tight junctions (TJs) to prevent the leakage of milk and blood components through the paracellular pathway (blood-milk barrier). Multiple factors that include hormones, cytokines, nutrition, and temperature regulate milk production and TJ formation in MECs. Multiple intracellular signaling pathways that positively and negatively regulate milk production and TJ formation have been reported. However, their regulatory mechanisms have not been fully elucidated. In addition, unidentified components that regulate milk production in MECs likely exist in foods, for example plants. Culture models of functional MECs that recapitulate milk production and TJs are useful tools for their study. Such models enable the elimination of indirect effects via cells other than MECs and allows for more detailed experimental conditions. However, culture models of MECs with inappropriate functionality may result in unphysiological reactions that never occur in lactating mammary glands in vivo. Here, I briefly review the physiological functions of alveolar MECs during lactation in vivo and culture models of MECs that feature milk production and less-permeable TJs, together with a protocol for establishment of MEC culture with functional TJ barrier and milk production capability using cell culture inserts. - Sweet taste receptor subunit T1R3 regulates casein secretion and phosphorylation of STAT5 in mammary epithelial cells.
Ken Kobayashi, Liang Han, Taku Koyama, Shan-Ni Lu, Takanori Nishimura
Biochimica et biophysica acta. Molecular cell research, 1870, 4, 119448, 119448, 2023年04月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, During lactation, mammary epithelial cells (MECs) on the apical membrane are in contact with lactose in milk, while MECs on the basolateral membrane are in contact with glucose in blood. Both glucose and lactose are sweeteners that are sensed by a sweet taste receptor. Previously, we have shown that lactose exposure on the basolateral membrane, but not the apical membrane, inhibits casein production and phosphorylation of STAT5 in MECs. However, it remains unclear whether MECs have a sweet taste receptor. In this study, we confirmed that the sweet taste receptor subunit T1R3 existed in both the apical and basolateral membranes of MECs. Subsequently, we investigated the influence of apical and basolateral sucralose as a ligand for the sweet taste receptor using a cell culture model. In this model, upper and lower media were separated by the MEC layer with less-permeable tight junctions. The results showed in the absence of glucose, both apical and basolateral sucralose induced phosphorylation of STAT5, which is a positive transcriptional factor for milk production. In contrast, the T1R3 inhibitor basolateral lactisole reducing phosphorylated STAT5 and secreted caseins in the presence of glucose. Furthermore, exposure of the apical membrane to sucralose in the presence of glucose inhibited the phosphorylation of STAT5. Simultaneously, GLUT1 was partially translocated from the basolateral membrane to the cytoplasm in MECs. These results suggest that T1R3 functions as a sweet receptor and is closely involved in casein production in MECs. - Menthol application on healthy and inflamed goat udders changes antimicrobial components in milk
Ruri Ohno, Naoki Suzuki, Yusaku Tsugami, Takahiro Nii, Ken Kobayashi, Naoki Isobe
Animal Science Journal, 94, 1, e13832, Wiley, 2023年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mammary glands with mastitis are usually treated with antibiotics in combination with anti-inflammatory drug application on the udder skin. Menthol is an anti-inflammatory drug. The aim of the present study was to investigate the effect of surface application of menthol on goat udders on the production of antimicrobial components in milk. Goats (5 Shiba and 11 Tokara goats) were subjected to menthol application to the udder under both healthy and inflammatory conditions. An intramammary infusion of lipopolysaccharides was carried out to induce inflammatory conditions in the udder. Milk samples were collected to determine somatic cell count (SCC) and sodium ion (Na+ ), antimicrobial component and cytokine concentrations. In healthy udders, menthol application increased the concentration of antimicrobial components (S100A7 and S100A8), but not in the control. In the inflamed udder, antimicrobial component (lactoferrin, S100A7, and S100A8) and inflammatory cytokine (IL-1β) concentrations were higher in the menthol group than in the control group. These results suggest that menthol application on udders augments the antimicrobial component concentration in the mammary gland under both healthy and inflammatory conditions. - Modulation of Milk and Lipid Synthesis and Secretion in a3-Dimensional Mouse Mammary Epithelial Cell Culture Model: Effects of Palmitate and Orlistat.
Michael G Ross, Ken Kobayashi, Guang Han, Mina Desai
Nutrients, 14, 23, 2022年11月22日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Human milk synthesis is impacted by maternal diet, serum composition, and substrate uptake and synthesis by mammary epithelial cells (MECs). The milk of obese/high-fat-diet women has an increased fat content, which promote excess infant weight gain and the risk of childhood/adult obesity. Yet, the knowledge of milk synthesis regulation is limited, and there are no established approaches to modulate human milk composition. We established a 3-dimensional mouse MEC primary culture that recreates the milk production pathway and tested the effects of the major saturated fatty acid in human milk (palmitate) and a lipoprotein lipase inhibitor (orlistat) on triglyceride production. Positive immunostaining confirmed the presence of milk protein and intracellular lipid including milk globules in the cytoplasm and extracellular space. The treatment with palmitate activated "milk" production by MECs (β-casein) and the lipid pathway (as evident by increased protein and mRNA expression). Consistent with these cellular changes, there was increased secretion of milk protein and triglyceride in MEC "milk". The treatment with orlistat suppressed milk triglyceride production. Palmitate increased milk and lipid synthesis, partly via lipoprotein lipase activation. These findings demonstrate the ability to examine MEC pathways of milk production via both protein and mRNA and to modulate select pathways regulating milk composition in MEC culture. - Early effects of lipoteichoic acid from Staphylococcus aureus on milk production-related signaling pathways in mouse mammary epithelial cells.
Ken Kobayashi, Naoki Omatsu, Liang Han, Lu Shan-Ni, Takanori Nishimura
Experimental cell research, 420, 1, 113352, 113352, 2022年11月01日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Staphylococcus aureus causes subclinical mastitis; lipoteichoic acid (LTA) from S. aureus causes mastitis-like adverse effects on milk production by mammary epithelial cells (MECs). Here, we investigated the early effects of LTA from S. aureus on mouse MECs using a culture model, in which MECs produced milk components and formed less permeable tight junctions (TJs). In MECs of this model, Toll-like receptor 2 (receptor for LTA), was localized on the apical membrane, similar to MECs in lactating mammary glands. LTA weakened the TJ barrier within 1 h, concurrently with localization changes of claudin 4. LTA treatment for 24 h increased αS1-casein and decreased β-casein levels. In MECs exposed to LTA, the activation level of signal transducer and activator of transcription 5 (major transcriptional factor for milk production) was low. LTA activated signaling pathways related to cell survival (extracellular signal-regulated kinase, heat shock protein 27, and Akt) and inflammation (p38, c-Jun N-terminal kinase, and nuclear factor κB). Thus, LTA caused abnormalities in casein production and weakened the TJs by affecting multiple signaling pathways in MECs. LTA-induced changes in signaling pathways were not uniform in all MECs. Such complex and semi-negative actions of LTA may contribute to subclinical mastitis caused by S. aureus. - Endogenous slow and fast myosin dynamics in myofibers isolated from mice expressing GFP-Myh7 and Kusabira Orange-Myh1.
Koichi Ojima, Masahiro Kigaki, Emi Ichimura, Takahiro Suzuki, Ken Kobayashi, Susumu Muroya, Takanori Nishimura
American journal of physiology. Cell physiology, 323, 2, C520-C535, 2022年06月27日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Skeletal muscle consists of slow and fast myofibers in which different myosin isoforms are expressed. Approximately 300 myosins form a single thick filament in the myofibrils, where myosin is continuously exchanged. However, endogenous slow and fast myosin dynamics have not been fully understood. To elucidate those dynamics, here we generated mice expressing green fluorescence protein-tagged slow myosin heavy chain (GFP-Myh7) and Kusabira Orange fluorescence protein-tagged fast myosin heavy chain (KuO-Myh1). First, these mice enabled us to distinguish between GFP- and KuO-myofibers under fluorescence microscopy: GFP-Myh7 and KuO-Myh1 were exclusively expressed in slow myofibers and fast myofibers, respectively. Next, to monitor endogenous myosin dynamics, fluorescence recovery after photobleaching (FRAP) was conducted. The mobile fraction (Mf) of GFP-Myh7 and that of KuO-Myh1 were almost constant values independent of the regions of the myofibers and the muscle portions where the myofibers were isolated. Intriguingly, proteasome inhibitor treatment significantly decreased the Mf in GFP-Myh7 but not in KuO-Myh1 myofibers, indicating that the response to a disturbance in protein turnover depended on muscle fiber type. Taken together, the present results indicated that the mice we generated are promising tools not only for distinguishing between GFP- and KuO-myofibers but also for studying the dynamics of endogenous myosin isoforms by live-cell fluorescence imaging. - Lactose on the basolateral side of mammary epithelial cells inhibits milk production concomitantly with signal transducer and activator of transcription 5 inactivation.
Ken Kobayashi, Haruka Wakasa, Liang Han, Taku Koyama, Yusaku Tsugami, Takanori Nishimura
Cell and tissue research, 389, 3, 501, 515, 2022年06月24日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Mammary epithelial cells (MECs) are the only cells capable of synthesizing lactose. During lactation, alveolar MECs secrete lactose through the apical membrane into the alveolar lumen, whereas alveolar tight junctions (TJs) block the leakage of lactose into the basolateral sides of the MECs. However, lactose leaks from the alveolar lumen into the blood plasma in the mastitis and after weaning. This exposes the basolateral membrane of MECs to lactose. The relationship between lactose in blood plasma and milk production has been suggested. The present study determined whether lactose exposure on the basolateral membrane of mouse MECs adversely affects milk production in vitro. Restricted exposure to lactose on the basolateral side of the MECs was performed using a culture model, in which MECs on the cell culture insert exhibit milk production and less-permeable TJs. The results indicated that lactose exposure on the basolateral side inhibited casein and lipid production in the MECs. Interestingly, lactose exposure on the apical side did not show detectable effects on milk production in the MECs. Basolateral lactose exposure also caused the inactivation of STAT5, a primary transcriptional factor for milk production. Furthermore, p38 and JNK were activated by basolateral lactose exposure. The activation of p38 and JNK following anisomycin treatment reduced phosphorylated STAT5, and inhibitors of p38 blocked the reduction of phosphorylated STAT5 by basolateral lactose exposure. These findings suggest that lactose functions as a partial inhibitor for milk production but only when it directly makes contact with the basolateral membrane of MECs. - Adverse Effects of High Temperature On Mammary Alveolar Development In Vitro.
Haruka Wakasa, Yusaku Tsugami, Taku Koyama, Liang Han, Takanori Nishimura, Naoki Isobe, Ken Kobayashi
Journal of mammary gland biology and neoplasia, 27, 2, 155, 170, 2022年05月17日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, In the mammary glands during pregnancy, the alveolar buds are first branched from the mammary ducts after which they form the alveolar luminal structure for milk production postparturition. Body temperature could increase for several reasons, such as infectious disease and heat stress. We have previously reported that high temperature adversely effects on the lactation capacity of mouse mammary epithelial cells (MECs). However, it remains unclear how high temperature influences mammary morophogenesis during pregnancy. In this study, we investigated the effects of high temperature on this mammary alveolar development process using two types of culture models including embedded organoids of MECs in Matrigel; these models reproduced mammary alveolar bud induction and alveolar luminal formation. Results showed that a culture temperature of 41 °C repressed alveolar bud induction and inhibited alveolar luminal formation. In addition, the treatment at 41 °C decreased the number of proliferating mammary epithelial cells but did not affect cell migration. Levels of phosphorylated Akt, -ERK1/2, -HSP90, and -HSP27 were increased in organoids cultured at 41 °C. The specific inhibitors of HSP90 and HSP27 exacerbated the disruption of organoids at 41 °C but not at 37 °C. Furthermore, the organoids precultured at 37 and 41 °C in the alveolar luminal formation model showed differences in the expression levels of caseins and tight junction proteins, which express in MECs in lactating mammary glands, after induction of MEC differentiation by prolactin and dexamethasone treatment in vitro. These results suggest that elevated temperature directly hinders mammary alveolar development; however, heat shock proteins may mitigate the adverse effects of high temperatures. - Thick filament-associated myosin undergoes frequent replacement at the tip of the thick filament.
Emi Ichimura, Koichi Ojima, Susumu Muroya, Ken Kobayashi, Takanori Nishimura
FEBS open bio, 12, 4, 852, 863, 2022年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Myosin plays a fundamental role in muscle contraction. Approximately 300 myosins form a bipolar thick filament, in which myosin is continuously replaced by protein turnover. However, it is unclear how rapidly this process occurs and whether the myosin exchange rate differs depending on the region of the thick filament. To answer this question, we first measured myosin release and insertion rates over a short period and monitored myotubes expressing a photoconvertible fluorescence protein-tagged myosin, which enabled us to monitor myosin release and insertion simultaneously. About 20% of myosins were replaced within 10 min, while 70% of myosins were exchanged over 10 h with symmetrical and biphasic alteration of myosin release and insertion rates. Next, a fluorescence pulse-chase assay was conducted to investigate whether myosin is incorporated into specific regions in the thick filament. Newly synthesized myosin was located at the tip of the thick filament rather than the center in the first 7 min of pulse-chase labeling and was observed in the remainder of the thick filament by 30 min. These results suggest that the myosin replacement rate differs depending on the regions of the thick filament. We concluded that myosin release and insertion occur concurrently and that myosin is more frequently exchanged at the tip of the thick filament. - Isoflavones and their metabolites influence the milk production ability of bovine mammary epithelial cells in a type‐specific manner
Yusaku Tsugami, Haruka Wakasa, Manabu Kawahara, Takanori Nishimura, Ken Kobayashi
Animal Science Journal, 93, 1, e13720, Wiley, 2022年01月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Dairy cows feed on isoflavones as physiologically active substances present in legumes. However, the influences of isoflavones (biochanin A, genistein, formononetin, and daidzein) and their metabolites (p-ethylphenol and equol) on milk components production, tight junctions (TJs), and their regulatory pathways are unclear in bovine mammary epithelial cells (BMECs). In this study, we investigated the influences of isoflavones and their metabolites in BMECs using an in vitro culture model. The influences of isoflavones on milk components production, TJ proteins, and STAT5/STAT3 signaling pathways were different in a type-specific manner. Biochanin A decreased the mRNA expression and secretion of both β-casein and lactoferrin while a decrease in activated STAT5 and an increase in activated STAT3. In contrast, equol increased claudin-3, which is the main components for less-permeable TJs in lactation, while an increase in activated STAT5. In addition, a mixture of multiple isoflavones based on the intake of red clover increased secretion of lactoferrin, mRNA expression of β-casein, and amount of claudin-3, but a mixture based on soy did not affect the BMECs. Thus, these results indicate that isoflavones in legumes and the metabolic activity of isoflavones in dairy cows when feeding legumes may affect the milk production ability in BMECs. - IL-1β is a key inflammatory cytokine that weakens lactation-specific tight junctions of mammary epithelial cells.
Ken Kobayashi, Kota Matsunaga, Yusaku Tsugami, Haruka Wakasa, Takanori Nishimura
Experimental cell research, 409, 2, 112938, 112938, 2021年12月15日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, In lactating mammary glands, alveolar mammary epithelial cells (MECs) produce milk and form less-permeable tight junctions (TJs). However, alveolar TJs are weakened with a reduction in milk production in mammary glands due to mastitis or weaning in the presence of high levels of IL-1β, IL-6, or TNF-α. In this study, using in vitro cultured model of MECs with milk-producing ability and lactation-specific TJs, we investigated whether the aforementioned cytokines affect MEC TJs. The results showed that TNF-α, IL-1β, and IL-6 affected lactation-specific TJs in different ways. In particular, upon activation of p38 and JNK signalling, IL-1β caused rapid disruption of TJs at tricellular contact points. IL-1β treatment led to decreased CLDN3, CLDN4, and OCLN levels and a weakened TJ barrier. The adverse effects of IL-1β on TJs were mimicked by anisomycin, which is an activator of p38 and JNK signalling, and were blocked by MEC pretreatment with a p38 inhibitor but not a JNK inhibitor. The mislocalization of tricellulin at tricellular contact areas was confirmed in MECs treated with IL-1β or anisomycin. These results indicate that IL-1β is a key cytokine that adversely affects the TJs between MECs by activating p38. - Local Heat Treatment of Goat Udders Influences Innate Immune Functions in Mammary Glands.
Yusaku Tsugami, Yuki Ishiba, Naoki Suzuki, Takahiro Nii, Ken Kobayashi, Naoki Isobe
Journal of mammary gland biology and neoplasia, 26, 4, 387, 397, 2021年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Heat stress and mastitis adversely affect milk production in dairy ruminants. Although the udder temperature is elevated in both conditions, the influence of this local temperature rise on milk production and immune function of ruminant mammary glands remains unclear. To address this question, we heated the mammary glands of goats by covering one half of the udder with a disposable heating pad for 24 h, the other uncovered half served as a control. Sixteen Tokara goats (1-5 parity) and three Shiba goats (1-2 parity) at the mid-lactation stage were individually housed, fed 0.6 kg of hay cubes and 0.2 kg of barley per day, and had free access to water and trace-mineralized salt blocks. Milk samples were collected every 6 h for 24 h after covering (n = 16), and deep mammary gland tissue areas were collected after 24 h (n = 5). The concentrations of antimicrobial components [lactoferrin, β-defensin-1, cathelicidin-2, cathelicidin-7, and immunoglobulin A (IgA)] in milk were measured by the enzyme-linked immunosorbent assay (ELISA). The localization of IgA was examined by immunohistochemistry. The mRNA expression and protein concentrations of C-C motif chemokine ligand-28 (CCL-28) and interleukin (IL)-8 in the mammary gland tissue were measured using quantitative polymerase chain reaction and ELISA, respectively. The somatic cell count in milk was significantly higher in the local heat-treatment group than in the control group after 12 h of treatment. The treatment group had significantly higher concentrations of cathelicidin-2 and IgA than the control group after 24 h of treatment. In addition, the number of IgA-positive cells in the mammary stromal region and the concentration of CCL-28 in the mammary glands were increased by local heat treatment. In conclusion, a local rise in udder temperature enhanced the innate immune function in mammary glands by increasing antimicrobial components. - GAD67-mediated GABA Synthesis and Signaling Impinges on Directing Basket Cell Axonal Projections Toward Purkinje Cells in the Cerebellum
Hideki Miwa, Ken Kobayashi, Shinobu Hirai, Mitsuhiko Yamada, Masahiko Watanabe, Haruo Okado, Yuchio Yanagawa
The Cerebellum, 21, 6, 905, 919, Springer Science and Business Media LLC, 2021年10月21日, ［査読有り］
研究論文（学術雑誌） - Genistein Directly Represses the Phosphorylation of STAT5 in Lactating Mammary Epithelial Cells.
Yusaku Tsugami, Haruka Wakasa, Takanori Nishimura, Ken Kobayashi
ACS omega, 6, 35, 22765, 22772, 2021年09月07日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Genistein is a soy isoflavone and shows various physiological activities, such as affinities for estrogen receptors (ERs) and inhibitory effects on the epidermal growth factor receptor (EGFR) pathway. A previous study reported that genistein downregulates milk production ability in mammary epithelial cells (MECs) while decreasing the phosphorylation of STAT5. The ER and EGFR pathways indirectly regulate STAT5. In this study, the repressing mechanism of genistein against the phosphorylation of STAT5 was investigated using a culture model of mouse MECs with milk production ability. The results revealed that genistein did not influence the behavior of ERα and ERβ, whereas genistein immediately repressed the phosphorylation of ERK1/2. However, the decrease in phosphorylated STAT5 occurred independent of the phosphorylation of EGFR. Genistein repressed new phosphorylation of STAT5 by prolactin without influencing the phosphorylation of JAK2. In conclusion, this study indicates that genistein directly inhibits the phosphorylation of STAT5 in lactating MECs. - The ubiquitin ligase Ozz decreases the replacement rate of embryonic myosin in myofibrils.
Emi Ichimura, Koichi Ojima, Susumu Muroya, Takahiro Suzuki, Ken Kobayashi, Takanori Nishimura
Physiological reports, 9, 17, e15003, 2021年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle-specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP-Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP-Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz. - Abundant Synthesis of Netrin-1 in Satellite Cell-Derived Myoblasts Isolated from EDL Rather Than Soleus Muscle Regulates Fast-Type Myotube Formation.
Takahiro Suzuki, Aika Mori, Takahiro Maeno, Rio Arimatsu, Emi Ichimura, Yuriko Nishi, Kouga Hisaeda, Yuki Yamaya, Ken Kobayashi, Mako Nakamura, Ryuichi Tatsumi, Koichi Ojima, Takanori Nishimura
International journal of molecular sciences, 22, 9, 2021年04月26日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Resident myogenic stem cells (satellite cells) are attracting attention for their novel roles in myofiber type regulation. In the myogenic differentiation phase, satellite cells from soleus muscle (slow fiber-abundant) synthesize and secrete higher levels of semaphorin 3A (Sema3A, a multifunctional modulator) than those derived from extensor digitorum longus (EDL; fast fiber-abundant), suggesting the role of Sema3A in forming slow-twitch myofibers. However, the regulatory mechanisms underlying fast-twitch myotube commitment remain unclear. Herein, we focused on netrin family members (netrin-1, -3, and -4) that compete with Sema3A in neurogenesis and osteogenesis. We examined whether netrins affect fast-twitch myotube generation by evaluating their expression in primary satellite cell cultures. Initially, netrins are upregulated during myogenic differentiation. Next, we compared the expression levels of netrins and their cell membrane receptors between soleus- and EDL-derived satellite cells; only netrin-1 showed higher expression in EDL-derived satellite cells than in soleus-derived satellite cells. We also performed netrin-1 knockdown experiments and additional experiments with recombinant netrin-1 in differentiated satellite cell-derived myoblasts. Netrin-1 knockdown in myoblasts substantially reduced fast-type myosin heavy chain (MyHC) expression; exogenous netrin-1 upregulated fast-type MyHC in satellite cells. Thus, netrin-1 synthesized in EDL-derived satellite cells may promote myofiber type commitment of fast muscles. - Lipopolysaccharide and lipoteichoic acid influence milk production ability via different early responses in bovine mammary epithelial cells.
Yusaku Tsugami, Haruka Wakasa, Manabu Kawahara, Takanori Nishimura, Ken Kobayashi
Experimental cell research, 400, 2, 112472, 112472, 2021年03月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are cell wall components of Escherichia coli and Staphylococcus aureus, which cause clinical and subclinical mastitis, respectively. However, the reason of the difference in symptoms by pathogen type remains unclear. In this study, the influence of LPS and LTA on early response and milk production in lactating bovine mammary epithelial cells (BMECs) was comparatively investigated. The results showed that LPS decreased the secretion of β-casein, lactose, and triglycerides, whereas LTA decreased the secretion of lactose and triglycerides but increased lactoferrin production without any influence on β-casein secretion. In addition, the influence of milk lipid droplet size in BMECs and gene expression related to milk fat synthesis was different between LPS and LTA. LPS increased the gene expression of interleukin (IL)-1β, tumor necrosis factor-α, and IL-8 through the activation of the nuclear factor-κB (NF-κB), p38, and c-Jun N-terminal kinase pathways, whereas LTA increased IL-1β and CC chemokine ligand 5 expression through the activation of the NF-κB pathway. Moreover, these cytokines and chemokines differently affected the milk production ability of BMECs. These results suggested that the pathogen-specific symptoms may be related to the differences in the early response of BMECs to bacterial toxins. - Adverse effects of LPS on membrane proteins in lactating bovine mammary epithelial cells.
Yusaku Tsugami, Haruka Wakasa, Manabu Kawahara, Atsushi Watanabe, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
Cell and tissue research, 384, 2, 435, 448, 2021年01月12日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Mastitis causes a decrease in milk yield and abnormalities in milk components from dairy cows. Escherichia coli and the E. coli lipopolysaccharide (LPS) cell wall component directly downregulate milk production in bovine mammary epithelial cells (BMECs). However, the detailed mechanism by which this occurs in BMECs remains unclear. Various membrane proteins, such as immune sensors (Toll-like receptors, TLR), nutrient transporters (glucose transporter and aquaporin), and tight junction proteins (claudin and occludin) are involved in the onset of mastitis or milk production in BMECs. In this study, we investigated the influence of LPS on membrane proteins using an in vitro culture model. This mastitis model demonstrated a loss of glucose transporter-1 and aquaporin-3 at lateral membranes and a decrease in milk production in response to LPS treatment. LPS disrupted the tight junction barrier and caused compositional changes in localization of claudin-3 and claudin-4, although tight junctions were maintained to separate the apical membrane domains and the basolateral membrane domains. LPS did not significantly affect the expression level and subcellular localization of epidermal growth factor receptor in lactating BMECs with no detectable changes in MEK1/2-ERK1/2 signaling. In contrast, NFκB was concurrently activated with temporal translocation of TLR-4 in the apical membranes, whereas TLR-2 was not significantly influenced by LPS treatment. These findings indicate the importance of investigating the subcellular localization of membrane proteins to understand the molecular mechanism of LPS in milk production in mastitis. - Suppressive effects of curcumin on milk production without inflammatory responses in lactating mammary epithelial cells.
Ken Kobayashi, Yusaku Tsugami, Norihiro Suzuki, Takahiro Suzuki, Takanori Nishimura
Phytomedicine : international journal of phytotherapy and phytopharmacology, 80, 153360, 153360, 2021年01月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Curcumin is a naturally occurring polyphenol found in Curcuma longa with multiple therapeutic properties, such as anti-inflammatory, wound healing and anti-cancer effects. Curcuma longa is also used as a galactagogue to improve milk production during lactation. PURPOSE: To assess curcumin could have therapeutic potential for breastfeeding mothers, we investigated whether and how curcumin influences milk production in lactating mammary epithelial cells (MECs) at the cellular and molecular levels. METHODS: We prepared a lactating MEC culture model that produced milk components and formed less-permeable tight junctions (TJs) to investigate the molecular mechanism of curcumin on milk production, TJs, and inflammation in vitro. RESULTS: Curcumin downregulated milk production in lactation MECs concurrently with inactivation of lactogenesis-relating signaling (STAT5 and glucocorticoid receptor). The maintenance of a less-permeable TJ barrier was also confirmed, although the TJ protein claudin-4 increased. Curcumin inactivated NFκB and STAT3 signaling, which are closely involved in inflammatory responses in weaning and mastitis mammary glands. The expression levels of IL-1β and TNF-α were also decreased by curcumin treatment. Furthermore, curcumin blocked activation of inflammatory signaling by lipopolysaccharide treatment in MECs, similar to those in MECs that were treated with diclofenac sodium. The drastic phosphorylation of ERK was induced by curcumin treatment in the absence of EGF. U0126, an inhibitor of ERK phosphorylation, attenuated the adverse effects of curcumin on lactating MECs. CONCLUSION: The results of the present study suggests that curcumin downregulates milk production via inactivation of STAT5 and GR signaling with concurrent suppression of inflammatory responses via STAT3 and NFκB signaling in MECs. These findings provide new insights into the role of curcumin as a mild suppressor of milk production without inflammatory damages in breastfeeding mothers. - Yes-associated protein 1 translocation through actin cytoskeleton organization in trophectoderm cells.
Shota Yamamura, Nanami Goda, Hiroki Akizawa, Nanami Kohri, Ahmed Z Balboula, Ken Kobayashi, Hanako Bai, Masashi Takahashi, Manabu Kawahara
Developmental biology, 468, 1-2, 14, 25, 2020年12月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells. - Menthol from Mentha piperita Suppresses the Milk Production of Lactating Mammary Epithelial Cells In Vivo and In Vitro.
Norihiro Suzuki, Yusaku Tsugami, Haruka Wakasa, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
Molecular nutrition & food research, e2000853, 2020年11月14日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, SCOPE: Peppermint is traditionally used as an antigalactagogue in breastfeeding women. However, the suppressive mechanism remains unclear. The authors investigate whether and how peppermint influences milk production at the cellular and molecular levels. METHODS AND RESULTS: A lactating mammary epithelial cell (MEC) culture model that produces major milk components is prepared. Peppermint oil (PMO) suppresses β-casein production in conjunction with the induced enlargement of cytoplasmic lipid droplets (CLDs). PMO also significantly inactivates STAT5 and mTOR in the lactogenic signaling pathway. Menthol treatment, which is a primary PMO component, leads to decreases in β-casein production, enlarged CLDs, the inactivated STAT5 and mTOR. Eucalyptol and menthyl acetate, other components of peppermint, does not show suppressive effects on lactating MECs. The inactivation of STAT5 and mTOR upon menthol administration is also evident in alveolar MECs of lactating mice. Furthermore, lactating MECs expresses TRPM8 and TRPA1, which are menthol receptors known as cold receptors. Agonists of TRPM8 and TRPA1 suppresses β-casein production and inactivation of STAT5 and mTOR in the lactating MECs. CONCLUSION: These findings indicate that peppermint has potential as an antigalactagogue. Menthol is suggested to be an active compound in peppermint. TRPM8 and TRPA1 may function as receptors for menthol. - Distinct Expression Patterns of Fibrillar Collagen Types I, III, and V in Association with Mammary Gland Remodeling during Pregnancy, Lactation and Weaning.
Shiori Tsutsui, Haruka Wakasa, Yusaku Tsugami, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
Journal of mammary gland biology and neoplasia, 25, 3, 219, 232, 2020年09月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The mammary gland structurally and functionally remodels during pregnancy, during lactation and after weaning. There are three types of fibrillar collagens, types I, III, and V, in mammary stromal tissue. While the importance of the fibrillar structure of collagens for mammary morphogenesis has been suggested, the expression patterns of each type of fibrillar collagen in conjunction with mammary remodeling remain unclear. In this study, we investigated their expression patterns during pregnancy, parturition, lactation and involution. Type I collagen showed a well-developed fibril structure during pregnancy, but the fibrillar structure of type I collagen then became sparse at parturition and during lactation, which was concurrent with the downregulation of its mRNA and protein levels. The well-developed fibrillar structure of type I collagen reappeared after weaning. On the other hand, type V collagen showed a well-developed fibrillar structure and upregulation in the lactation period but not in the periods of pregnancy and involution. Type III collagen transiently developed a dense fibrillar network at the time of parturition and exhibited drastic increases in mRNA expression. These results indicate that each type of fibrillar collagen is distinctly involved in structural and functional remodeling in mammary glands during pregnancy, parturition, lactation, and involution after weaning. Furthermore, in vitro studies of mammary epithelial cells showed regulatory effects of type I collagen on cell adhesion, cell proliferation, ductal branching, and β-casein secretion. Each type of fibrillar collagen may have different roles in defining the cellular microenvironment in conjunction with structural and functional mammary gland remodeling. - T cells are the main population in mouse breast milk and express similar profiles of tight junction proteins as those in mammary alveolar epithelial cells.
Ryoyo Ikebuchi, Maika Fujimoto, Taiki Moriya, Yutaka Kusumoto, Ken Kobayashi, Michio Tomura
Journal of reproductive immunology, 140, 103137, 103137, Elsevier BV, 2020年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Immune cells are present in the breast milk of several mammalian species; however, their immunological function and transmigration mechanisms to milk remain unknown. Some researchers hypothesize that milk leukocytes have a mammary gland (MG) origin and transmigrate thorough the paracellular pathway, but mammary alveolar epithelial cells strictly regulate the paracellular movement of milk components during lactation via barrier structures, such as tight junctions (TJs). To investigate this discrepancy, we compared leukocyte populations in mouse MG and milk and explored TJ protein expression profiles in MG leukocytes. The main subsets of milk leukocytes were CD8+ and CD4+ T cells displaying the memory phenotype. The proportions of myeloid, B, and dendritic cells were significantly lower in milk than in the MG. CD8+ T cells expressed genes encoding the TJ proteins claudin-3, -7, -12, and ZO-1 at higher levels when compared with myeloid and B cells in the MG among lactating mice. Alveolar epithelial cells in the MG expressed claudin-3, -4, and -7. Administration of FTY720, an inhibitory agonist of sphingosine 1-phosphate receptor 1 that stabilizes TJ permeability, increased the myeloid cell proportion in milk. Different leukocyte populations in the MG and milk suggest active and selective mechanisms of cell transmigration to milk. Both TJ-forming components in alveolar epithelial cells from the MG and TJ protein expression profiles in leukocytes from the MG appear to regulate milk leukocyte populations. T cells are the main population in mouse breast milk and express similar profiles of TJ proteins as those in mammary alveolar epithelial cells. - Distinct expression patterns of aquaporin 3 and 5 in ductal and alveolar epithelial cells in mouse mammary glands before and after parturition.
Yoshiki Kaihoko, Yusaku Tsugami, Norihiro Suzuki, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
Cell and tissue research, 380, 3, 513, 526, Springer Science and Business Media LLC, 2020年06月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Milk osmolarity maintains an isotonic status for suckling infants during lactation. However, it remains unclear how the water content in milk is regulated in lactating mammary glands. In lactating mammary alveoli and ducts, mammary epithelial cells (MECs) are in direct contact with milk. In this study, we focus on two types of water channels, aquaporin 3 (AQP3) and AQP5, in alveolar and ductal MECs before and after parturition. AQP3 showed diffuse localization in the cytoplasm of ductal MECs but concentrated localization in the basolateral membrane of alveolar MECs during the late pregnancy and lactation periods. Translocation of AQP5 from the cytoplasm toward the apical membrane occurred in ductal MECs immediately before parturition. Subsequently, we examined the hormonal influences on the expression of AQP3 and AQP5 in cultured MECs in vitro. Progesterone and estrogen distinctly increased AQP3 and AQP5 in cultured MECs, respectively. Cotreatment with prolactin and dexamethasone significantly decreased both AQP3 and AQP5. Prolactin also facilitated the translocation of AQP5 into the apical membrane of MECs. In cultured MECs, AQP3 was homogeneously expressed in MECs, whereas AQP5 showed different expression levels between MECs regardless of the hormonal treatment. Different activation states of the prolactin/STAT5 pathway were also observed between ductal and alveolar MECs. These findings suggest that the expression pattern of AQP3 and AQP5 is distinctly regulated by lactogenic hormones in alveolar and ductal MECs before and after parturition. AQP5 expressed in ductal MECs may function as a water channel to regulate milk osmolarity in mice. - Regulatory Effects of Soy Isoflavones and Their Metabolites in Milk Production via Different Ways in Mice.
Yusaku Tsugami, Norihiro Suzuki, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
Journal of agricultural and food chemistry, 68, 21, 5847, 5853, American Chemical Society (ACS), 2020年05月27日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Soy products contain abundant genistein and daidzein isoflavones. Orally ingested soy isoflavones are partially metabolized to isoflavan by enteric bacteria. Their levels in the blood increase after soy products are eaten. In this study, we investigated the influence of genistein, daidzein, and equol by intraperitoneal administration in lactating mice. Genistein decreased the secretion of α- and β-casein and downregulated the gene expression of Csn1, Csn2, Csn3, and Wap while inactivating the signal transducer and activator of transcription 5 (STAT5) and ERK1/2. In contrast, equol increased Csn1-3 expression while inactivating STAT3. Daidzein did not show inhibitory effects on milk production. The effects of genistein and equol were also confirmed in lactating mammary epithelial cells (MECs), which were cultured in the presence of soy isoflavones and equol at physiological concentrations for 7 days. These findings indicate that genistein, daidzein, and equol influence milk production in MECs in vivo and in vitro in distinctly different ways. - Adverse Effects of Coumestrol and Genistein on Mammary Morphogenesis and Future Milk Production Ability of Mammary Epithelial Cells.
Aogu Kumai, Yusaku Tsugami, Haruka Wakasa, Norihiro Suzuki, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
Advanced biosystems, 4, 4, e1900187, 1900187, Wiley, 2020年04月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Isoflavones are a class of flavonoids present in legumes and are called phytoestrogens because of their estrogen-like activity. Endogenous estrogen is well known to regulate mammary gland morphogenesis during pregnancy. Each isoflavone also has different physiological activities. However, it is difficult to investigate the direct effect of each isoflavone in mammary morphogenesis in vivo because isoflavones are metabolized into different isoflavones by enteric bacteria. In this study, investigated are the direct influences of coumestrol, daidzein, and genistein on mammary structure development and future milk production ability of mammary epithelial cells (MECs) using in vitro culture models. Mouse MECs are cultured in Matrigel with basic fibroblast growth factor and epidermal growth factor to induce ductal branching and alveolar formation, respectively. Coumestrol and genistein inhibit ductal branching and alveolar formation by affecting the proliferation and migration of MECs with the induction of apoptosis. Daidzein hardly influences mammary structure development. Furthermore, pretreatment with coumestrol adversely affects the induction of milk production ability of MECs. These results suggest that each isoflavone differentially influences mammary morphogenesis and future milk production by affecting MEC behaviors. These results also suggest that the culture models are effective to study mammary epithelial morphogenesis in vitro. - Nicotine directly affects milk production in lactating mammary epithelial cells concurrently with inactivation of STAT5 and glucocorticoid receptor in vitro.
Kobayashi, K., Tsugami, Y., Suzuki, N., Suzuki, T., and Nishimura, T.
Toxicology in Vitro, 63, 104741, 104741, 2020年03月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Nicotine from tobacco smoke is absorbed into the bloodstream and transferred into breast milk in breastfeeding mothers. Smoking causes a decrease in breast milk volume, adverse changes to the milk composition, and a shortened lactation period. Breast milk is produced by mammary epithelial cells (MECs) in mammary glands during lactation. However, it remains unclear whether nicotine directly affects milk production in lactating MECs. To address this issue, we prepared a culture model with high milk production ability and less-permeable tight junctions (TJs) by seeding mouse MECs on a cell culture insert. Lactating MECs showed expression of α2, α3, β2, and β4 of nicotinic acetylcholine receptors. The high concentration of nicotine at 10-100 μM inhibited β-casein secretion and caused abnormal localization of TJ proteins. We subsequently investigated whether nicotine at a physiological concentration could affect lactating MECs. Nicotine at 1.0 μM directly inhibited α- and β-casein secretion in lactating MECs concurrently with inactivation of STAT5 and glucocorticoid receptor without affecting the TJ barrier. Nicotine treatment also induced MEC apoptosis concurrently with inactivation of Akt. These results support the adverse effects of nicotine on breastfeeding in smoking mothers. - Establishment of an in vitro culture model to study milk production and the blood–milk barrier with bovine mammary epithelial cells
Yusaku Tsugami, Norihiro Suzuki, Manabu Kawahara, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
Animal Science Journal, 91, 1, Wiley, 2020年01月, ［査読有り］, ［最終著者, 責任著者］
研究論文（学術雑誌） - Adipocytes suppress differentiation of muscle cells in a co-culture system
Kangmin Seo, Takahiro Suzuki, Ken Kobayashi, Takanori Nishimura
Animal Science Journal, 90, 3, 423, 434, Wiley, 2019年03月, ［査読有り］
研究論文（学術雑誌） - Potential of Gouda cheese whey to improve epidermal conditions by regulating proliferation and differentiation of keratinocytes
Shinagawa F, Takata S, Toba Y, Ikuta M, Hioki S, Suzuki T, Nishimura T, Nakamura R, Kobayashi K
International Dairy Journal, 87, 100, 106, 2018年12月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌） - Application of red pigment producing edible fungi for development of a novel type of functional cheese
Kumura Haruto, Ohtsuyama Takeru, Matsusaki Yoh-hey, Taitoh Miho, Koyanagi Haruka, Kobayashi Ken, Hayakawa Toru, Wakamatsu Jun-ichi, Ishizuka Satoshi
JOURNAL OF FOOD PROCESSING AND PRESERVATION, 42, 10, 2018年10月, ［査読有り］
英語, 研究論文（学術雑誌） - IL-1β directly inhibits milk lipid production in lactating mammary epithelial cells concurrently with enlargement of cytoplasmic lipid droplets
Matsunaga K, Tsugami Y, Kumai A, Suzuki T, Nishimura T, Kobayashi K
Experimental Cell Research, 370, 2, 365, 372, 2018年09月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌） - Moderate High Temperature Condition Induces the Lactation Capacity of Mammary Epithelial Cells Through Control of STAT3 and STAT5 Signaling
Ken Kobayashi, Yusaku Tsugami, Kota Matsunaga, Takahiro Suzuki, Takahiro Nishimura
Journal of Mammary Gland Biology and Neoplasia, 23, 1-2, 75, 88, Springer Science and Business Media LLC, 2018年06月, ［査読有り］, ［最終著者, 責任著者］
研究論文（学術雑誌） - Reciprocal regulation of TEAD4 and CCN2 for the trophectoderm development of the bovine blastocyst
Hiroki Akizawa, Ken Kobayashi, Hanako Bai, Masashi Takahashi, Shinjiro Kagawa, Hiroaki Nagatomo, Manabu Kawahara
Reproduction, 155, 6, 563, 571, Bioscientifica, 2018年06月, ［査読有り］
研究論文（学術雑誌）, The first segregation at the blastocyst stage is the symmetry-breaking event to characterize two cell components; namely, inner cell mass (ICM) and trophectoderm (TE). TEA domain transcription factor 4 (TEAD4) is a well-known regulator to determine TE properties of blastomeres in rodent models. However, the roles of bovine TEAD4 in blastocyst development have been unclear. We here aimed to clarify the mechanisms underlining TE characterization by TEAD4 in bovine blastocysts. We first found that theTEAD4 mRNA expression level was greater in TE than in ICM, which was further supported by TEAD4 immunofluorescent staining. Subsequently, we examined the expression patterns of TE-expressed genes;CDX2 ,GATA2 andCCN2 , in theTEAD4 -knockdown (KD) blastocysts. These expression levels significantly decreased in theTEAD4 KD blastocysts compared with controls. Of these downregulated genes, theCCN2 expression level decreased the most. We further analyzed the expression levels of TE-expressed genes;CDX2 ,GATA2 andTEAD4 in theCCN2 KD blastocysts. Strikingly, theCCN2 KD blastocysts showed the downregulation ofCDX2 ,GATA2 andTEAD4 . Furthermore, the ratio of TE-to-ICM cell numbers in theCCN2 KD blastocysts significantly decreased compared to controls. To our knowledge, this is the first study showing the regulation ofCCN2 expression thoroughTEAD4 in mammalian embryos. Not only that, this study also provides evidence that reciprocal regulation ofTEAD4 andCCN2 is required for TE development with appropriate gene expression in bovine blastocysts. - Phytoestrogens Weaken the Blood-Milk Barrier in Lactating Mammary Epithelial Cells by Affecting Tight Junctions and Cell Viability
Yusaku Tsugami, Kota Matsunaga, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 65, 50, 11118, 11124, 2017年12月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌） - Isoflavones and their metabolites influence the milk component synthesis ability of mammary epithelial cells through prolactin/STAT5 signaling
Yusaku Tsugami, Kota Matsunaga, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
MOLECULAR NUTRITION & FOOD RESEARCH, 61, 10, 201700156, 2017年10月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌） - Developmental Expression of Claudins in the Mammary Gland
Heidi K. Baumgartner, Michael C. Rudolph, Palaniappian Ramanathan, Valerie Burns, Patricia Webb, Benjamin G. Bitler, Torsten Stein, Ken Kobayashi, Margaret C. Neville
JOURNAL OF MAMMARY GLAND BIOLOGY AND NEOPLASIA, 22, 2, 141, 157, 2017年06月, ［査読有り］
英語, 研究論文（学術雑誌） - Adjunctive Application of Solid-State Culture Products from Aspergillus Oryzae for Semi-Hard Cheese
Haruto Kumura, Chiharu Saito, Yuko Taniguchi, Taiki Machiya, Yutaroh Takahashi, Ken Kobayashi, Akira Kimura
Advances in Dairy Research, 05, 03, OMICS Publishing Group, 2017年, ［査読有り］
研究論文（学術雑誌） - Distinct roles of prolactin, epidermal growth factor, and glucocorticoids in β-casein secretion pathway in lactating mammary epithelial cells.
Kobayashi K, Oyama S, Kuki T, Tsugami Y, Matsunaga K, Suzuki T, Nishimura T
Molecular and Cellular Endocrinology, 440, C, 16, 24, 2017年01月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Prolactin and glucocorticoid signalling induces lactation-specific tight junctions concurrent with β-casein expression in mammary epithelial cells.
Kobayashi K, Tsugami Y, Matsunaga K, Oyama S, Kuki T, Kumura H
Biochimica et Biophysica Acta - Molecular and Cell Research, 1863, 8, 2006, 2016, 2016年08月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Characterization of Toll-Like Receptor 9 expression during Mouse Preimplantation Development
Keisuke Sasaki, Aiko Tanaka, Hiroaki Nagatomo, Hidehiko Ogawa, Ken Kobayashi, Masashi Takahashi, Manabu Kawahara
Journal of Genital System & Disorders, 05, 01, {OMICS} Publishing Group, 2016年, ［査読有り］
英語, 研究論文（学術雑誌） - Pro-inflammatory cytokine TNF-α is a key inhibitory factor for lactose synthesis pathway in lactating mammary epithelial cells
Ken Kobayashi, Chinatsu Kuki, Shoko Oyama, Haruto Kumura
Experimental Cell Research, 340, 2, 295, 304, 2016年01月, ［査読有り］, ［筆頭著者, 責任著者］
研究論文（学術雑誌） - Mitochondrial Function in Murine Skin Epithelium Is Crucial for Hair Follicle Morphogenesis and Epithelial-Mesenchymal Interactions
Jennifer E. Kloepper, Olivier R. Baris, Karen Reuter, Ken Kobayashi, Daniela Weiland, Silvia Vidali, Desmond J. Tobin, Catherin Niemann, Rudolf J. Wiesner, Ralf Paus
JOURNAL OF INVESTIGATIVE DERMATOLOGY, 135, 3, 679, 689, 2015年03月, ［査読有り］
英語, 研究論文（学術雑誌） - Early down-regulation of milk production after weaning by pup removal and prior to involution in mouse mammary glands
Takaaki Uejyo, Chinatsu Kuki, Shoko Oyama, Haruto Kumura, Ken Kobayashi
CELL AND TISSUE RESEARCH, 359, 2, 643, 653, 2015年02月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌） - Stimulatory Effect of Brazilian Propolis on Hair Growth through Proliferation of Keratinocytes in Mice
Shota Miyata, Yozo Oda, Chika Matsuo, Haruto Kumura, Ken Kobayashi
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 62, 49, 11854, 11861, 2014年12月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌） - Underlying mechanisms involved in the decrease of milk secretion during Escherichia coli endotoxin induced mastitis in lactating mice
Ken Kobayashi, Shoko Oyama, Takaaki Uejyo, Chinatsu Kuki, Md Morshedur Rahman, Haruto Kumura
VETERINARY RESEARCH, 44, 119, 2013年12月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌） - Histological analysis of mammary gland remodeling caused by lipopolysaccharide in lactating mice
Ken Kobayashi, Takaaki Uejyo, Shoko Oyama, Md. Morshedur Rahman, Haruto Kumura
Cell and Tissue Research, 354, 2, 495, 506, 2013年11月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - The immune system of mouse vibrissae follicles: cellular composition and indications of immune privilege
Marta Bertolini, Katja C. Meyer, Radomir Slominski, Ken Kobayashi, Ralf J. Ludwig, Ralf Paus
EXPERIMENTAL DERMATOLOGY, 22, 9, 593, 598, 2013年09月, ［査読有り］
英語, 研究論文（学術雑誌） - Lipopolysaccharide Disrupts the Milk-Blood Barrier by Modulating Claudins in Mammary Alveolar Tight Junctions
Ken Kobayashi, Shoko Oyama, Atsushi Numata, Md. Morshedur Rahman, Haruto Kumura
PLoS ONE, 8, 4, e62187, 2013年04月23日, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Potential of polar lipids from bovine milk to regulate the rodent dorsal hair cycle
H. Kumura, T. Sawada, Y. Oda, M. Konno, K. Kobayashi
JOURNAL OF DAIRY SCIENCE, 95, 7, 3629, 3633, 2012年07月, ［査読有り］, ［最終著者］
英語, 研究論文（学術雑誌） - Distinct behavior of claudin-3 and -4 around lactation period in mammary alveolus in mice
Ken Kobayashi, Haruto Kumura
HISTOCHEMISTRY AND CELL BIOLOGY, 136, 5, 587, 594, 2011年11月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Progesterone maintains amniotic tight junctions during midpregnancy in mice
Ken Kobayashi, Hideki Miwa, Masato Yasui
MOLECULAR AND CELLULAR ENDOCRINOLOGY, 337, 1-2, 36, 42, 2011年04月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Apoptosis in Mouse Amniotic Epithelium Is Induced by Activated Macrophages Through the TNF Receptor Type 1/TNF Pathway
Ken Kobayashi, Kazuo Umezawa, Masato Yasui
BIOLOGY OF REPRODUCTION, 84, 2, 248, 254, 2011年02月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Inflammatory mediators weaken the amniotic membrane barrier through disruption of tight junctions¬.
Kobayashi K, Miwa H, Yasui M
The Journal of Physiology, 588, 24, 4859, 4869, 2010年12月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Cellular and subcellular localization of aquaporins 1, 3, 8, and 9 in amniotic membranes during pregnancy in mice
Ken Kobayashi, Masato Yasui
CELL AND TISSUE RESEARCH, 342, 2, 307, 316, 2010年11月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - A tissue-engineered trachea derived from a framed collagen scaffold, gingival fibroblasts and adipose-derived stem cells
Ken Kobayashi, Teruhisa Suzuki, Yukio Nomoto, Yasuhiro Tada, Masao Miyake, Akihiro Hazama, Ikuo Wada, Tatsuo Nakamura, Koichi Omori
BIOMATERIALS, 31, 18, 4855, 4863, 2010年06月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌） - Expression and Distribution of Tight Junction Proteins in Human Amnion During Late Pregnancy
K. Kobayashi, I. Kadohira, M. Tanaka, Y. Yoshimura, K. Ikeda, M. Yasui
PLACENTA, 31, 2, 158, 162, 2010年02月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Bioengineered Trachea With Fibroblasts in a Rabbit Model
Wataru Okano, Yukio Nomoto, Ikuo Wada, Ken Kobayashi, Masao Miyake, Tatsuo Nakamura, Koichi Omori
ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY, 118, 11, 796, 804, 2009年11月, ［査読有り］
英語, 研究論文（学術雑誌） - Dynamic Changes in Amniotic Tight Junctions during Pregnancy
K. Kobayashi, T. Inai, Y. Shibata, M. Yasui
PLACENTA, 30, 10, 840, 847, 2009年10月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌） - Clinical application of in situ tissue engineering using a scaffolding technique for reconstruction of the larynx and trachea
Koichi Omori, Yasuhiro Tada, Teruhisa Suzuki, Yukio Nomoto, Takashi Matsuzuka, Ken Kobayashi, Tatsuo Nakamura, Shinichi Kanemaru, Masaru Yamashita, Ryo Asato
ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY, 117, 9, 673, 678, 2008年09月, ［査読有り］
英語, 研究論文（学術雑誌） - Regeneration of the trachea using a bioengineered scaffold with adipose-derived stem cells
Teruhisa Suzuki, Ken Kobayashi, Yasuhiro Tada, Yukie Suzuki, Ikuo Wada, Tatuo Nakamura, Koichi Omori
ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY, 117, 6, 453, 463, 2008年06月, ［査読有り］
英語, 研究論文（学術雑誌） - Regeneration of tracheal epithelium utilizing a novel bipotential collagen scaffold
Yasuhiro Tada, Teruhisa Suzuki, Toshiaki Takezawa, Yukio Nomoto, Ken Kobayashi, Tatuo Nakamura, Koichi Omori
ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY, 117, 5, 359, 365, 2008年05月, ［査読有り］
英語, 研究論文（学術雑誌） - Effect of fibroblasts on epithelial regeneration on the surface of a bioengineered trachea
Yukio Nomoto, Ken Kobayashi, Yasuhiro Tada, Ikuo Wada, Tatsuo Nakamura, Koichi Omori
ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY, 117, 1, 59, 64, 2008年01月, ［査読有り］
英語, 研究論文（学術雑誌） - Potential of heterotopic fibroblasts as autologous transplanted cells for tracheal epithelial regeneration
Ken Kobayashi, Teruhisa Suzuki, Yukio Nomoto, Yasuhiro Tada, Masao Miyake, Akihiro Hazama, Tatsuo Nakamura, Koichi Omori
TISSUE ENGINEERING, 13, 9, 2175, 2184, 2007年09月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌） - エゾシカ外皮の鞣しに伴う変化
福永重治, 杉浦智樹, 小林 謙, 山川育生, 竹ノ内一昭, 中村富美男
北海道畜産学会報, 49, 43, 50, 2007年, ［査読有り］
日本語, 研究論文（学術雑誌） - Effect of fibroblasts on tracheal epithelial regeneration in vitro
Ken Kobayashi, Yukio Nomoto, Teruhisa Suzuki, Yasuhiro Tada, Masao Miyake, Akihiro Hazama, Shinichi Kanemaru, Tatsuo Nakamura, Koichi Omori
TISSUE ENGINEERING, 12, 9, 2619, 2628, 2006年09月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌） - Tissue engineering for regeneration of the tracheal epithelium
Yukio Nomoto, Teruhisa Suzuki, Yasuhiro Tada, Ken Kobayashi, Masao Miyake, Akihiro Hazama, Ikuo Wada, Shinichi Kanemaru, Tatsuo Nakamura, Koichi Omori
ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY, 115, 7, 501, 506, 2006年07月, ［査読有り］
英語, 研究論文（学術雑誌） - コラーゲンの産生異常がニワトリ羽毛の初期発生に及ぼす影響
小林 謙, 福永重治, 竹之内一昭, 加藤(森)ゆうこ, 中村富美男
北海道畜産学会報, 48, 53, 58, 2006年, ［査読有り］, ［筆頭著者］
日本語, 研究論文（学術雑誌） - ニワトリ初生羽の発生に伴う真皮コラーゲンの形態変化
小林 謙, 福永重治, 竹之内一昭, 加藤(森)ゆうこ, 中村富美男
北海道畜産学会報, 47, 65, 71, 2005年, ［査読有り］, ［筆頭著者］
日本語, 研究論文（学術雑誌） - Functional role of type VI collagen during early feather development of the chicken embryo in vitro
Ken Kobayashi, Shigeharu Fukunaga, Kazuaki Takenouchi, Yuko Kato-Mori, Fumio Nakamura
Animal Science Journal, 76, 3, 273, 282, Blackwell Publishing, 2005年, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌） - Functional indispensability of fibronectin associated on the mesenchymal cell surface during the early period of feather development in the chick embryo in vitro
Ken Kobayashi, Shigeharu Fukunaga, Kazuaki Takenouchi, Fumio Nakamura
Animal Science Journal, 75, 5, 441, 449, 2004年10月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌） - ウシ脊髄における正常型細胞性プリオンタンパク質（PrPc）の局在
中村富美男, 田中誠一, 小林 謙, 關 巌, 李 昇遠, 福永重治
北海道畜産学会報, 45, 25, 31, 2003年, ［査読有り］
日本語, 研究論文（学術雑誌） - Immunohistochemical detection of cellular prion prorein (PrPc) in the rat central nervous system.
Nakamura F, Seki I, Kobayashi K, Tanaka M, Fukunaga S
Animal Science Journal, 73, 6, 553, 556, 2002年12月, ［査読有り］
英語, 研究論文（学術雑誌）
その他活動・業績
- 培養ミルクと乳腺上皮細胞の培養モデル
小林謙, 乳業技術, 73, 2024年 - 泌乳誘導下におけるMycoplasma bovis感染がウシ乳腺上皮細胞の乳汁合成機能および炎症応答に与える影響
永光理子, 今泉法子, 小林謙, 盧尚建, 楜澤共生, 河合一洋, 権平智, 樋口豪紀, 家畜衛生学雑誌, 50, 3, 2024年 - 乳腺上皮細胞における泌乳の調節機構
小林謙, 日本小児科学会雑誌, 127, 2, 2023年 - 乳分泌のメカニズムと乳分泌培養モデルの作製法について
小林謙, 科学と工業, 97, 12, 2023年 - 乳房炎について乳腺上皮細胞の視点から考える
小林謙, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM), 2021年 - 脂肪細胞が筋細胞の筋線維型決定に及ぼす影響
趙暁琳, 徐康びん, 鈴木貴弘, 小林謙, 西邑隆徳, 日本畜産学会大会講演要旨, 125th, 2019年 - ペパーミントが乳腺上皮細胞の乳産生能を低下させる機序に関する研究
鈴木徳宏, 隈井仰, 津上優作, 鈴木貴弘, 西邑隆徳, 小林謙, 日本畜産学会大会講演要旨, 125th, 2019年 - 筋幹細胞が合成するNetrin-1は速筋型筋線維の形成誘導に関与する
鈴木貴弘, 森愛華, 久枝皓雅, 西百合子, 有松里央, 小林謙, 辰巳隆一, 尾嶋孝一, 西邑隆徳, 日本筋学会学術集会プログラム・抄録集, 5th, 2019年 - 筋原線維内のミオシン分子置換様相は筋線維型によって異なる
城垣誠大, 尾嶋孝一, 鈴木貴弘, 小林謙, 西邑隆徳, 日本畜産学会大会講演要旨, 125th, 2019年 - 培養基質の弾性率が筋芽細胞による自律的な筋線維型制御に与える影響
山谷有希, 有松里央, 前野岳大, 久江未記, 小林謙, 辰巳隆一, 尾嶋孝一, 西邑隆徳, 鈴木貴弘, 日本筋学会学術集会プログラム・抄録集, 5th, 2019年 - 筋幹細胞の合成・分泌因子Netrin-1による速筋型筋線維の形成誘導
鈴木貴弘, 森愛華, 久枝皓雅, 西百合子, 有松里央, 小林謙, 辰巳隆一, 尾嶋孝一, 西邑隆徳, 日本畜産学会大会講演要旨, 125th, 2019年 - ウシ乳腺上皮細胞を用いたin vitro乳分泌モデルの作製とその有用性の検討
津上優作, 鈴木徳宏, 隈井仰, 鈴木貴弘, 西邑隆徳, 小林謙, 日本畜産学会大会講演要旨, 125th, 2019年 - イヌ肺動脈に異種移植したウシ由来バイオシートの組織構造
今山知佳, 鈴木貴弘, 小林謙, 三谷朋弘, 市居修, 中山泰秀, 古越真耶, 池田哲平, 小千田圭吾, 西邑隆徳, 日本畜産学会大会講演要旨, 124th, 115, 115, 2018年03月28日
(公社)日本畜産学会, 日本語 - 衛星細胞由来のSema3Aによる遅筋線維の形成誘導メカニズムの解析
橋本尚弥, 西百合子, 森愛華, 小林謙, 辰巳隆一, 西邑隆徳, 鈴木貴弘, 日本畜産学会大会講演要旨, 124th, 2018年 - マウス系統種の違いによる衛星細胞の分化能の比較
西百合子, 有松里央, 森愛華, 福地達貴, 久枝皓雅, 小林謙, 辰巳隆一, 西邑隆徳, 鈴木貴弘, 日本筋学会学術集会プログラム・抄録集, 4th, 2018年 - 衛星細胞におけるmyogeninの機能は局在部位で異なる
有松里央, 西百合子, 小林謙, 西邑隆徳, 鈴木貴弘, 日本筋学会学術集会プログラム・抄録集, 4th, 2018年 - 発酵乳製品用風味強化剤としてのAspergillus oryzaeの応用に関する研究
細野誠, 齋藤千春, 谷口悠子, 小林謙, 若松純一, 木村彰, 玖村朗人, 日本農芸化学会大会講演要旨集(Web), 2017, ROMBUNNO.3A05p09 (WEB ONLY), 2017年03月05日
日本語 - ウシ生体内で形成させた移植医療用組織の構造
今山 知佳, 鈴木 貴弘, 小林 謙, 三谷 朋弘, 市居 修, 小千田 圭吾, 池田 哲平, 寺澤 武, 中山 泰秀, 西邑 隆徳, 日本畜産学会大会講演要旨集, 122回, 175, 175, 2017年03月
(公社)日本畜産学会, 日本語 - 家畜生体内で再生医療移植用組織をつくる
西邑 隆徳, 今山 知佳, 鈴木 貴弘, 小林 謙, 三谷 朋弘, 市居 修, 小千田 圭吾, 池田 哲平, 寺澤 武, 中山 泰秀, 日本畜産学会大会講演要旨集, 122回, 175, 175, 2017年03月
(公社)日本畜産学会, 日本語 - 筋幹細胞分泌因子netrinの筋線維型制御作用を検証する
鈴木貴弘, 西百合子, 橋本尚弥, 小林謙, 辰巳隆一, 西邑隆徳, 日本畜産学会大会講演要旨, 122nd, 2017年 - イソフラボンが乳腺上皮細胞における乳産生へ及ぼす影響
津上優作, 松長康太, 鈴木貴弘, 西邑隆徳, 小林謙, 日本畜産学会大会講演要旨, 122nd, 2017年 - 炎症性サイトカインが乳腺上皮細胞の乳成分産生に及ぼす影響
松長康太, 津上優作, 鈴木貴弘, 西邑隆徳, 小林謙, 日本畜産学会大会講演要旨, 122nd, 2017年 - 温度変化が乳腺上皮細胞の乳成分産生能力に及ぼす影響
小林謙, 津上優作, 松長康太, 鈴木貴弘, 西邑隆徳, 日本畜産学会大会講演要旨, 122nd, 2017年 - 筋幹細胞由来分泌因子による筋線維型制御機構に関する研究-速筋化誘導因子の候補netrinに着目して-
鈴木貴弘, 森愛華, 西百合子, 橋本尚弥, 小林謙, 辰巳隆一, 西邑隆徳, 日本筋学会学術集会プログラム・抄録集, 3rd, 2017年 - 気道再建と再生医療
大森孝一, 中村達雄, 多田靖宏, 野本幸男, 鈴木輝久, 小林 謙, 金丸眞一, 安里 亮, 山下 勝, 耳鼻と臨床, 54, 5, 271, 279, 2008年
書籍等出版物
共同研究・競争的資金等の研究課題
- 物理的刺激を起点として乳腺上皮細胞の乳分泌を調節する機構の解明
基盤研究（B）
2024年04月 - 2028年03月
日本学術振興会, 北海道大学大学院, 研究代表者 - 血液中の白血球が乳腺上皮細胞の血液乳関門を通過して乳汁中に移行する機構の解明
科学研究費助成事業
2025年06月27日 - 2027年03月31日
小林 謙
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 25K22409 - 乳腺上皮細胞のアセチルコリン代謝が妊娠期の乳腺発達と泌乳期の乳産生を調節する機構
2025年04月 - 2026年03月
公益財団法人 喫煙科学研究財団, 研究代表者 - 血流と搾乳を模した2系統の灌流培養システムを基盤とする培養乳生産技術の開発
科学研究費助成事業
2023年06月30日 - 2025年03月31日
小林 謙
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 23K18070 - 乳腺上皮細胞に発現する機械刺激受容体Piezo2が乳産生を制御する機構の解明
研究助成(一般)
2024年07月 - 2025年03月
秋山記念生命科学振興財団, 研究代表者 - 血流と搾乳を模した2系統の灌流培養システムを用いて母乳分泌調節性食品の同定と作用機序解明を培養下で可能にする方法の確立
2024年
一般財団法人 糧食研究会, 研究代表者 - 黄色ブドウ球菌が乳腺上皮細胞に寄生して感染防御バリアを脆弱化させる機構の解明
科学研究費助成事業
2021年07月09日 - 2023年03月31日
小林 謙
黄色ブドウ球菌は伝染性の乳房炎を引き起こす。黄色ブドウ球菌による乳房炎は難治性であり、その治療のための抗生物質の投与は黄色ブドウ球菌の薬剤耐性化を誘発する。そのため、黄色ブドウ球菌の乳房炎を効果的に治療する方策が求められている。しかし、黄色ブドウ球菌の乳房炎がなぜ難治性なのか、その感染戦略の全容はわかっていない。そこで本研究では、“黄色ブドウ球菌は乳腺上皮細胞に寄生し、その感染防御バリアを細胞内部から崩壊させることで難治性乳房炎を引き起こす”と仮説を立て、仮説を実証するため、以下の実験を行った。
まず、マウスから乳腺上皮細胞を単離し、cell culture insert上にコンフルエントになるまで培養した後、生体同様に乳産生とタイトジャンクション形成を行うよう、プロラクチンやデキサメタゾンを含む培地を用いて分化誘導した。この乳腺上皮細胞の培地に蛍光色素で標識した黄色ブドウ球菌を添加すると、培地中や乳腺上皮細胞のみならず、乳腺上皮細胞内にも侵入する様子が観察された。また、感染した細胞では細胞の形態が変化しており、アクチン線維のネットワークの変化が起きていた。続いて、細胞外の黄色ブドウ球菌を抗生物質で除去し、その3日後に乳腺上皮細胞の乳産生能力とタイトジャンクションバリア機能を評価した。その結果、代表的な乳タンパク質であるカゼインの産生量減少とタイトジャンクション構成タンパク質の質的変化が確認された。また、黄色ブドウ球菌由来毒素であるリポタイコ酸が培地中に存在した場合においても乳腺上皮細胞の乳産生能力とタイトジャンクションの変化が起きていた。
以上の結果より、黄色ブドウ球菌は細胞内外から乳腺上皮細胞の性質を変化させることが示唆された。
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 21K19174 - 乳腺上皮細胞のうま味受容体が母乳中のグルタミン酸濃度を感知する機構の解明
研究助成金
2022年 - 2023年
日本うま味調味料協会技術部会, 研究代表者 - 泌乳期乳腺におけるアセチルコリンが乳腺上皮細胞の乳産生能力を調節する機構の解明
一般研究助成
2021年 - 2023年
公益財団法人 喫煙科学研究財団, 研究代表者 - 辛味成分が乳腺上皮細胞のTRPA1を介して母乳分泌を調節する機構の解明
研究助成
2023年
公益財団法人 浦上食品・食文化振興財団, 研究代表者 - 乳腺上皮細胞の灌流培養システムを用いた培養乳の生産に関する研究
研究助成
2022年
公益財団法人 寿原記念財団, 研究代表者 - 泌乳期における非糖質甘味料の摂取が血液乳関門に及ぼす影響
研究助成
2022年
公益財団法人 日本食品化学研究振興財団 - 培養乳の持続的生産を可能にする乳腺上皮細胞の培養モデルに関する基礎研究
学術研究助成
2022年
公益財団法人 アサヒグループ学術振興財団, 研究代表者 - 食品由来成分の母乳移行性を評価する血液乳関門モデルの作製
学術奨励金
2022年
一般財団法人 東洋水産財団, 研究代表者 - 乳腺上皮細胞の温度応答性分子群が乳腺組織の形態形成と機能発現を制御する機構の解明
基盤研究（B）
2018年04月 - 2021年03月
小林謙
日本学術振興会, 研究代表者, 競争的資金 - マメ科植物に含有する多様な植物エストロゲンが泌乳期乳腺の血液乳関門に及ぼす影響
研究助成
2021年
一般財団法人 旗影会, 研究代表者 - 甘味と旨味が乳腺上皮細胞の母乳分泌を調節する機構の解明
研究助成
2021年
公益財団法人 サッポロ生物科学振興財団, 研究代表者 - 暑熱ストレスが乳分泌細胞の乳産生能力を低下させる機序の解明
研究助成
2020年
公益財団法人 栗林育英学術財団, 研究代表者 - 乳腺上皮細胞に発現する甘味受容体TAS1R3が乳産生を制御する機構に関する研究
2020年
公益財団法人 秋山記念生命科学振興財団, 研究助成（一般）, 研究代表者 - 大豆イソフラボンとその代謝産物が母乳分泌に与える効果の検証
研究助成
2020年
公益財団法人 タカノ農芸化学研究助成財団, 研究代表者 - 温度受容体であるTRPチャネルの活性化が線維芽細胞の皮膚恒常性維持機能へ及ぼす影響
研究助成
2017年 - 2019年
一般財団法人 ホーユー科学財団, 研究代表者 - 食品由来成分が母乳分泌へ与える影響を数値化する培養モデルの開発
学術研究助成
2019年
公益財団法人 飯島藤十郎記念食品科学振興財団 - グルコースセンサーの不適切な活性化が授乳期の母乳トラブルを引き起こす機序の解明
公益財団法人 三島海雲記念財団
2019年
個人研究奨励金, 研究代表者 - 妊娠期における非糖質系甘味料の摂取が乳腺発達と分娩後の乳産生に及ぼす影響
研究助成
2019年
公益財団法人 日本食品化学研究振興財団, 研究代表者 - ニコチンが乳腺上皮細胞の乳汁分泌能に及ぼす影響
若手研究助成
2016年 - 2018年
公益財団法人 喫煙科学研究財団, 研究代表者 - 現代の食生活の中で母乳育児を改善するために母乳分泌調節性食品（Galactogogue）をスクリーニングする方法の確立とその実用性の検証
研究助成
2018年
一般財団法人 糧食研究会, 研究代表者 - クルクミンが母乳産生を改善する効果の検証
研究助成
2018年
公益財団法人 山崎香辛料振興財団, 研究代表者 - 母乳分泌を調節する食品成分の同定とその作用機構解明
研究助成金
2018年
公益財団法人 食生活研究会, 研究代表者 - 温度感受性TRPチャネルを介した乳汁分泌調節機序に関する研究
基盤研究 (C)
2014年04月 - 2017年03月
小林謙
日本学術振興会, 研究代表者, 競争的資金 - マメ科植物に含有する多様な植物エストロゲンが乳腺発達と乳産生に及ぼす影響
研究助成
2017年
一般社団法人 旗影会, 研究代表者 - 母乳分泌に影響を及ぼす生理活性物質の探索と作用メカニズムに関する研究
学術研究助成
2017年
公益社団法人 アサヒグループ学術振興財団, 研究代表者 - レモングラスとペパーミントに含まれる有効成分が母乳分泌を調節する効果
研究助成
2017年
公益社団法人 日本アロマ環境協会, 研究代表者 - 授乳期における非糖質系甘味料の摂取が乳腺上皮細胞の乳汁分泌能に及ぼす影響
研究助成
2016年
公益財団法人 日本食品化学研究振興財団, 研究代表者 - ローヤルゼリーとプロポリスが発毛・育毛に及ぼす相乗効果の検証
みつばち研究助成基金
2013年 - 2015年
山田養蜂場, 研究代表者 - 泌乳期乳腺の乳成分産生を調節するメントールに関する研究
研究助成
2015年
公益財団法人 戸部眞紀財団, 研究代表者 - マメ科牧草に含まれるイソフラボンが泌乳期乳腺の乳汁分泌に与える影響
研究助成
2015年
公益財団法人 栗林育英学術財団, 研究代表者 - タイトジャンクションが乳腺腺胞細胞の乳汁分泌能を調節する機序の解明
科研費若手研究 (B)
2012年04月 - 2014年03月
小林謙
乳汁とは泌乳期乳腺の腺胞上皮細胞から分泌される液体である。泌乳期乳腺では分泌した乳汁が体内に逆流することを防ぐため、乳腺胞上皮細胞間に逆流を防止するバリアを発達させる。本研究ではこのタイトジャンクション（TJ）が泌乳期乳腺の乳汁分泌能に果たす役割を解明する為、TJを構成するクローディンに着目して調べた。
その結果、クローディン-3が泌乳期乳腺のTJを構成すること、クローディン‐4の発現量増加がバリア脆弱化を誘導すること、クローディン-4の発現が乳汁分泌能を調節する多様な生理活性物質によって制御されることがわかり、TJと乳汁分泌能が分子レベルで高度に連動していることが明らかになった。
日本学術振興会, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 24780281 - 乳腺胞上皮細胞と脂肪細胞に着目した泌乳期乳腺におけるグルコース吸収経路の解明
研究助成（奨励）
2014年
公益財団法人 秋山記念生命科学振興財団, 研究代表者 - マメ科牧草含有の植物エストロゲンが泌乳期乳腺の乳分泌能に及ぼす影響
研究助成
2014年
一般財団法人旗影会, 研究代表者 - 飼料添加物に用いられる成分が乳腺上皮細胞に及ぼす影響
受託研究
2013年
日本ニュートリション株式会社 - ホエーの入浴剤としての効能に関する基礎研究
研究シーズ発掘補助金
2013年
公益財団法人ノーステック財団, 研究代表者 - 乳期乳腺におけるカゼインミセルサイズの調節因子に関する研究
一般公募研究助成
2013年
一般財団法人 糧食研究会, 研究代表者 - ハチミツが毛包発達に及ぼす作用に関する基礎的検証
みつばち研究助成基金
2011年 - 2012年
山田養蜂場, 研究代表者 - ヒト羊膜上皮層のクローディンによる傍細胞経路の調節機構
科研費若手研究 (B)
2008年04月 - 2011年03月
小林謙
羊膜上皮層は羊水と直に接する組織であり、羊膜上皮層の細胞間に存在する密着結合は羊水成分と胎盤側成分の傍細胞経路(上皮細胞間隙を通る経路)による物質移動を調節している。本研究では早産や絨毛膜羊膜炎に繋がる基礎的知見を得るため、密着結合およびその構成タンパク質であるクローディンの調節機構について調べた。その結果、密着結合の物質透過性はクローディンの挙動を介してホルモンや炎症性サイトカインなどによって調節されていることが明らかになった。
日本学術振興会, 若手研究(B), 慶應義塾大学, 研究代表者, 競争的資金, 20790177 - 手術時における脂肪組織含有幹細胞群の回収方法と移植法の開発
科学研究費助成事業
2009年 - 2010年
鈴木 輝久, 小林 謙, 岡野 渉, 鈴木 政博
SVFはASCと比べ、すぐに回収でき、また、細胞培養を介さない点で非常に有利である。本研究ではSVFの気管再生における促進効果を確認した。また、SVFは幹細胞のマーカーであるCD90,CD105,CD29を発現しASCと同様に多分化能を有することを確認した。本結果は、SVFが再生医療において臨床応用可能である移植細胞群であることを示した。
日本学術振興会, 若手研究(B), 福島県立医科大学, 21791631 - 三次元培養モデルを用いた腹膜透析調整機序に関する研究
バクスターPD研究基金
2007年 - 2008年
バクスター株式会社, 研究代表者
