Biomedical Research-tokyo最新文献

A live assay tool has been established to uncover the precise molecular mechanisms underlying complex cell fusion events in myoblasts. The novel cell-based assay, HiMy (HiBiT-based myoblast fusion), utilizes a recently developed split-luciferase technology. The assay successfully detected cell fusion in differentiating C2C12 myoblast cultures. This allowed us to measure mixing of the cytoplasm, which occurred several hours after the initiation of C2C12 differentiation. Unlike what was reported earlier, the fusion was detected a few hours after the initiation of differentiation. Thus, this assay is sensitive enough to monitor fusion events before they become detectable using conventional methods. Furthermore, a panel of laboratory compounds, including a variety of inhibitors of cellular enzymes or activities, were assayed using the HiMy assay. Lovastatin, a cholesterol biogenesis inhibitor, decreased HiMy activity by approximately 50%. In contrast, mevalonolactone, a precursor for cholesterol synthesis, increased fusion activity. These results confirmed the previous finding that the amount of cellular cholesterol positively correlates with the rate of myoblast fusion during myogenesis. These results indicate that the novel cell fusion assay is a quick, accurate, and robust method to monitor intercellular fusion events.

The inhibitory action of the secondary bile acid lithocholic acid (LCA) on neurally evoked Cl^-/HCO₃^- secretion was investigated using the Ussing-chambered mucosal-submucosal preparation from the rat distal colon. Electrical field stimulation (EFS) evoked cholinergic and noncholinergic secretory responses in the rat distal colon. The responses were almost completely blocked by TTX (10^-6 M) but not atropine (10^-5 M) or hexamethonium (10^-4 M). The selective antagonist for VIP receptor 1 (VPAC1) greatly reduced the EFS-evoked response. Thus, the rat distal colon may be predominantly innervated by noncholinergic VIP secretomotor neurons. Basolateral addition of 6 × 10^-5 M LCA inhibited the EFS-evoked response. The inhibitory action of LCA was partly rescued by the Y2R antagonist BIIE0246. The bile acid receptor TGR5 agonist INT-777 mimicked the LCA-induced inhibitory action. Immunohistochemical staining showed the colocalization of TGR5 and PYY on L cells. TGR5 immunoreactivity was also found in VIP-immunoreactive submucosal neurons which also expressed the PYY receptor, Y2R. These results suggest that LCA inhibits neurally evoked Cl^-/HCO₃^- secretion through the activation of TGR5 on L cells and cholinergic- and VIP-secretomotor neurons in the submucosal plexus. Furthermore, the inhibitory mechanism may involve TGR5-stimulated PYY release from L cells and Y2R activation in VIP-secretomotor neurons.

Selective estrogen receptor modulator (SERM) binds to estrogen receptors (ERs) and acts as both an agonist or an antagonist, depending on the target tissue. Raloxifene and bazedoxifene as SERMs are currently used hormone replacement medicines for postmenopausal osteoporosis. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts promotes osteoclastogenesis. We have previously demonstrated that transforming growth factor (TGF)-β induces the synthesis of M-CSF via SMAD2/3, p38 mitogen-activated protein kinase (MAPK), p44/p42 MAPK and c-Jun N-terminal kinase (JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether SERM affects the M-CSF synthesis by TGF-β in MC3T3-E1 cells. Raloxifene and bazedoxifene significantly suppressed the synthesis of M-CSF. PPT, an ERα agonist, but not ERB041, an ERβ agonist, inhibited the release of M-CSF. MPP, an ERα antagonist, reversed the suppression by raloxifene of the M-CSF release. Raloxifene attenuated the TGF-β-induced phosphorylation of JNK but not SMAD3, p42 MAPK and p38 MAPK. Bazedoxifene and PPT also inhibited the phosphorylation of JNK. Furthermore, MPP, an ERα antagonist, reversed the suppression by both raloxifene and bazedoxifene of the phosphorylation of JNK. Our results strongly indicate that raloxifene and bazedoxifene, SERMs, suppress the TGF-β-induced synthesis of M-CSF through ERα-mediated inhibition of JNK pathway in osteoblasts.

Long-term calcineurin inhibitor (CNI) administration causes irreversible nephrotoxicity. Therefore, early CNI-induced nephrotoxicity detection is necessary for patients who will need long-term CNI administration. There is no pathological indicator for early CNI-induced nephrotoxicity. Here, serial protocol kidney biopsy specimens from five kidney-transplant patients with severe CNI-induced nephrotoxicity were examined. We observed that the increase in CD44 expression in glomerular parietal epithelial cells (PECs) preceded the chronic pathological changes of CNI-induced nephrotoxicity such as tubular atrophy/interstitial fibrosis, arterial hyaline thickening, and focal segmental glomerulosclerosis (FSGS). This result suggests that CD44-positive PECs have pivotal roles in FSGS development in human CNI-induced nephrotoxicity as well as rodent models. CD44 could be useful as a pathological marker for early CNI-induced nephrotoxicity detection post kidney transplantation.

Gut epithelial cells are characterized by rapid, constant cell renewal. The disposal of aging epithelial cells around the villus tips of the small intestine occurs so regularly that it has been regarded as a consequence of well-controlled cell death, designated as apoptosis. However, the notion of live cell extrusion in the intestine has been intensively built among researchers, and the disposal processes of effete epithelial cells display species and regional differences. Chemical mediators and mechanical forces rising from surrounding cells contribute to the regulated cell replacement. Cytotoxic intraepithelial lymphocytes and lamina propria macrophages play a leading role in the selection of disposal cells and their extrusion to maintain fully the epithelial homeostasis in tandem with the dynamic reconstruction of junctional devices. Lymphocyte-mediated cell killing is predominant in the mouse and rat, while the disposal of epithelial cells in the guinea pig, monkey, and human is characterized by active phagocytosis by subepithelially gathering macrophages. The fenestrated basement membrane formed by immune cells supports their involvement and explains species differences in the disposal of epithelial cells. Via these fenestrations, macrophages and dendritic cells can engulf apoptotic epithelial cells and debris and convey substantial information to regional lymph nodes. In this review, we attempt to focus on morphological aspects concerning the apoptosis and disposal process of effete epithelial cells; in vitro or ex vivo analyses using cultured monolayer has become predominant in recent studies concerning the exfoliation of apoptotic enterocytes. Furthermore, we give attention to their species differences, which is controversial but crucial to our understanding.

Natriuretic peptides (NPs) are a family of peptide hormones produced in cardiac muscle cells and consist mainly of three types: atrial NP (ANP), B-type (or brain) NP (BNP), and C-type NP. We herein report the effects of ANP and BNP on central respiratory activity in brainstem-spinal cord preparation isolated from newborn rats. Bath application of these peptides (100 nM) induced a weak transient depression of the respiratory rhythm followed by recovery. Respiratory-related neurons in the rostral ventrolateral medulla showed a tendency for transient hyperpolarization followed by recovery during the application of ANP or BNP. The application of a membrane-permeable cGMP, 8-Br-cGMP (10 or 20 μM), did not induce significant effects on respiratory rhythm, suggesting no involvement of guanylyl cyclase in effects of ANP or BNP. We also examined effects of BNP on respiratory depression induced by the sedative dexmedetomidine, which exerts an inhibitory influence on respiratory rhythm. When pretreated with 50 nM BNP, the inhibitory effect of 100 nM dexmedetomidine was significantly reduced. Our findings suggest that ANP and BNP act as mild excitatory agents with sustained effects on respiratory rhythm after an initial transient depression.

Cell-free DNA (cfDNA) is a fragment of DNA circulating in the blood, and its concentration is often elevated in cancer patients. To investigate the relationships between serum cfDNA concentration and clinical characteristics, including prognosis, we measured serum cfDNA concentration in 114 newly diagnosed lymphoma patients. The cfDNA concentrations in diffuse large B cell lymphoma (DLBCL) (62.5 ng/mL) and follicular lymphoma patients (51.6 ng/mL) were significantly elevated compared to healthy individuals (7.5 ng/mL, P < 0.001). In DLBCL, patients with elevated serum cfDNA (> 38.9 ng/mL) at diagnosis had significantly shorter time-to-progression compared to those without (P = 0.033). The addition of cfDNA concentration to the international prognostic index showed improved predictive power for time-to-progression. Moreover, cfDNA added significant prognostic value to other inflammatory markers such as B symptoms and sIL2R. There was a trend towards shorter progression-free survival and overall survival in patients with elevated cfDNA. Furthermore, B symptoms (P = 0.038), bulky masses (P = 0.031), non-GCB subtype (P = 0.012), and serum sIL-2R levels > 2,000 U/mL (P = 0.012) were associated with higher cfDNA levels. Our study showed that serum cfDNA concentration at diagnosis was associated with certain clinicopathological characteristics, and may be predictive of survival outcomes in DLBCL patients.

Interleukin-6 signaling activates signal transducer and activator of transcription 3 (STAT3), resulting in matrix metalloproteinase-3 (MMP-3) production. The hip joints with rapidly destructive coxopathy (RDC) show rapid chondrolysis, probably by increased MMP-3. This study aimed to elucidate STAT3 activation in the synovial tissues with joint destruction in the early stage of RDC. Synovial tissues within 7 months from the disease onset were obtained from four RDC patients with femoral head destruction and high serum levels of MMP-3. RDC synovial tissues demonstrated the synovial lining hyperplasia with an increase of CD68-positive macrophages and CD3-positive T lymphocytes. STAT3 activation was found in the synovial tissues by immunohistochemistry using anti-phospho-STAT3 antibody. The majority of phospho-STAT3-positive cells were the synovial lining cells and exhibited negative expression of the macrophage or T cell marker. Treatment with CP690,550, a Janus Kinase inhibitor, resulted in a decrease in phospho-STAT3-positive cells, especially with high intensity, indicating effective suppression of STAT3 activation in RDC synovial tissues. Inhibitory effect of CP690,550 could work through the Janus Kinase/STAT3 axis in the synovial tissues in the early stage of RDC. Thus, STAT3 may be a potential therapeutic target for prevention of joint structural damage in RDC.

Mammals express a set of chitinase family proteins, comprising chitinases, which can hydrolyze chitin, and chitinase-like proteins without the chitinase activity but possessing chitin-binding properties. They act as endogenous lectins, regulating various physiological/pathological events. Ym1, originally identified as an eosinophil chemotactic factor or a macrophage-derived protein in parasite-infected mice, is a rodent-specific chitinase-like protein. Ym1 is also purified from eosinophilic crystals formed in the lung and urinary system in various disease models. We previously reported that major cellular sources of murine Ym1 are alveolar macrophages in the lung and neutrophils/monocytes lineage cells of the spleen and bone marrow under normal conditions. We here analyzed the detailed cellular expression of Ym1 in Mesocestoides corti (M. corti)-infected mice. Ym1 was significantly increased in the liver containing the larvae, lung, and peritoneal exudate cells in M. corti-infected mice, where activated macrophages expressed Ym1. Characteristic needle-shaped eosinophilic crystals appeared in the larvae-free lung, and Ym1 was localized to endoplasmic reticulum of activated alveolar macrophages. Moreover, swollen mesothelial cells covering the liver, spleen, and heart expressed Ym1 abundantly. Although the role of Ym1 in parasitic infection remains unclear, our findings focusing on an endogenous lectin may help in better understanding defense mechanism against parasites.

Arginine methylation is a common post-translational modification which functions as an epigenetic regulator of transcription and plays a key role in various cell signaling pathways. The methylation of arginine residues is catalyzed by protein arginine methyltransferase (PRMT). However, the expression pattern and underlying mechanism of PRMTs and protein methylation profile in lipopolysaccharide (LPS)-induced innate immune responses are poorly understood. Using a shotgun proteomic approach, we found that LPS stimulation increased arginine and proline metabolism and responses to inflammation and bacterial infections. In comparison, cysteine and methionine metabolism, the pentose phosphate pathway, purine metabolism, and protein methylation factors were also decreased in LPS stimulated murine macrophage cell lines. We revealed that LPS stimulation downregulated PRMT1, PRMT5, and protein arginine methylation profiles in RAW264.7 cells using western blot analysis. Additionally, this phenomenon occurred in parallel with nitric oxide accumulation in LPS-induced macrophages. Using inflammation models, we demonstrate for the first time that LPS stimulation decreases PRMTs, leading to the decreasing of arginine methylation in macrophages.