Biomedical Research-tokyo最新文献

Kidney stone disease is a serious disease due to the severe pain it causes, high morbidity, and high recurrence rate. Notably, calcium oxalate stones are the most common type of kidney stone. Calcium oxalate appears in two forms in kidney stones: the stable phase, monohydrate (COM), and the metastable phase, dihydrate (COD). Particularly, COM stones with concentric structures are hard and difficult to treat. However, the factor determining the growth of either COM or COD crystals in the urine, which is supersaturated for both phases, remains unclear. This study shows that calcium phosphate ingredients preferentially induce COM crystal nucleation and growth, by observing and analyzing kidney stones containing both COM and COD crystals. The forms of calcium phosphate are not limited to Randall's plaques (1-2 mm size aggregates, which contain calcium phosphate nanoparticles and proteins, and form in the renal papilla). For example, aggregates of strip-shaped calcium phosphate crystals and fields of dispersed calcium phosphate microcrystals (nano to micrometer order) also promote the growth of concentric COM structures. This suggests that patients who excrete urine with a higher quantity of calcium phosphate crystals may be more prone to forming hard and troublesome COM stones.

Electromagnetic fields (EMFs) noninvasively promote fracture healing, prevent osteoporosis, promote diaphyseal growth, enhance differentiation, and stimulate cell division. However, no good model systems for analyzing bone regeneration have been reported. In this study, we examined the in vivo regeneration of scales having osteoblasts and osteoclasts using a new magnetic field generator for exposing aquatic animals to EMFs at a sine-wave frequency of 60 Hz. Goldfish scales were put into a fish-breeding space with the developed magnetic field generator and exposed to extremely low-frequency electromagnetic fields (ELF-EMFs) of 60 Hz at an intensity of 1, 3, and 5 mT for 10 days while being regenerated the scales. After exposure, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities in the goldfish scales were measured as markers of osteoblasts and osteoclasts, respectively. As a result, both ALP and TRAP activities in regenerating scales exposed to 3 mT ELF-EMFs were higher than those in regenerating scales exposed to 1 and 5 mT ELF-EMFs. Exposure of scales to 3 mT ELF-EMFs significantly enhanced the scale regeneration rate. Exposure of rat calvaria to 3 mT ELF-EMFs also increased both ALP and TRAP activities like in goldfish scales. Thus, we concluded that 3 mT ELF-EMFs contribute to the medical treatment of bone diseases.

In drug discovery research, it is important to identify target proteins of bioactive small-molecule compounds and analyse their functions. In this study, we examined whether target membrane proteins could be captured by compounds that bind to membrane proteins on the cell surface. For this purpose, we performed affinity purification using the compound-immobilized nanomagnetic beads. Affinity purification with nanomagnetic beads is known to be effective for determining the protein binding partners of small molecules. However, most previous studies have targeted proteins in the cytoplasm. As a model compound, we chose BMS-1166 (a representative small-molecule compound from Bristol Myers Squibb), a PD-1/PD-L1 immune checkpoint inhibitor that binds to PD- L1 and promotes PD-L1 dimerization. BMS-1166-immobilized beads were manufactured and incubated with extracts of cells with high PD-L1 protein expression. The bound protein was confirmed by western blotting and proteomic analysis to be PD-L1. BMS-1166-immobilized nano-magnetic beads were able to specifically bind and capture the membrane protein PD-L1. In addition, high-purity protein could be obtained from cell extracts in a single step. This is the first report of the purification of a membrane protein to high purity with nanobeads. Nanomagnetic beads with immobilized compounds are an effective tool for identifying the protein binding partners of small molecules, especially when the targets are membrane proteins.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

Copper (Cu) is known to induce oxidative stress and apoptosis in the liver, kidney, and brain. We previously demonstrated the molecular mechanism underlying the Cu-induced hepatic diurnal variation. However, the cellular molecule(s) involved in Cu-induced renal chronotoxicity remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying Cu-induced diurnal toxicity in the kidneys. We evaluated cell viability and clock gene expression levels in mouse renal cortex tubular cells (MuRTE61 cells) after Cu treatment. We also examined the Cu homeostasis- and apoptosis-related gene levels after period 1 (Per1) overexpression in MuRTE61 cells. Cu treatment decreased MuRTE61 cell viability in a dose-dependent manner. It increased the Per1 expression levels after 24 h. Notably, Per1 overexpression alleviated the Cu-induced inhibition of MuRTE61 cell viability. Moreover, Per1 overexpression downregulated the cleaved caspase-3 and reduced Cu levels by upregulating the antioxidant 1 copper chaperone (Atox1) levels. These results suggest that Cu-induced renal toxicity is associated with Per1 expression via the regulation of the copper chaperone, Atox1.

Linalool and linalyl acetate are major components of lavender essential oil. These substances possess many biological activities, such as anti-inflammatory activity, analgesic and anxiolytic effects, and anticonvulsant properties, and they also induce modulation of neuronal activity in the autonomic nervous system. However, there are no reports of the direct effects of linalool on respiratory activity. In the present study, we analyzed the effects of linalool and linalyl acetate on central respiratory activity in the brainstem-spinal cord preparation isolated from newborn rats. Linalool dose-dependently decreased the rate of respiratory activity. This effect was reversed by bicuculline, suggesting that linalool enhanced inhibitory synaptic connections via GABAA receptors. In addition, linalool reduced the coefficient of variation of inspiratory burst intervals and thus could work to stabilize the respiratory rhythm. Linalyl acetate did not cause inhibitory effects as observed in linalool treatment. Linalool depressed burst activity of pre-inspiratory neurons in the medullary respiratory networks and increased the amplitude of inspiratory inhibitory postsynaptic potentials of pre-inspiratory neurons. We concluded that linalool caused inhibitory effects on respiratory rhythm generation mainly through activation of presynaptic GABAA receptors of pre-inspiratory neurons.

Although patients with chronic kidney disease (CKD) have a higher risk of colorectal cancer (CRC) aggravation, the connection between these two diseases is not well understood. Recent studies have shown that both CKD and CRC aggravation are closely related to an increased abundance of indole-producing Fusobacterium nucleatum in the gut. The indole absorbed from the gut is eventually metabolized to indoxyl sulfate in the liver. Since indoxyl sulfate is involved not only in accelerating CKD progression but also in the initiation and development of its associated complications, the present study aimed to clarify whether indoxyl sulfate induces the proliferation of CRC cells. This study found that indoxyl sulfate induced the proliferation of CRC-derived HCT-116 cells by activating the aryl hydrocarbon receptor (AhR) and the proto-oncogene Akt. The AhR antagonist CH223191 and Akt inhibitor MK2206 suppressed indoxyl sulfate-induced proliferation of HCT-116 cells. We also found that indoxyl sulfate upregulated epidermal growth factor receptor (EGFR) expression, which is associated with poor prognosis of CRC, whereas CH223191 and MK2206 repressed EGFR expression. Furthermore, indoxyl sulfate increased the sensitivity of CRC cells to EGF by upregulating EGFR expression. These findings suggest that indoxyl sulfate may be an important link between CKD and CRC aggravation.

Transient receptor potential vanilloid 1 (TRPV1) is primarily expressed in sensory neurons and functions as a nociceptive channel. TRPV1 is activated by capsaicin, acidic pH, and noxious heat. Compounds inhibiting TRPV1 have been explored to develop analgesic drugs. In this study, the effect of linalyl acetate (LA), a lavender essential oil component that exerts analgesic effects, on TRPV1 was investigated by measuring intracellular Ca2+ concentration ([Ca2+]i) and whole-cell membrane currents. The analgesic effects of LA on TRPV1-mediated pain were also examined. LA inhibited [Ca2+]i responses to capsaicin, acidic pH, and heat in mouse sensory neurons. Unlike the transient LA-inhibition on capsaicin- and heat-responses, its inhibition on acid-responses persisted even after the LA removal. In TRPV1-expressing HEK293 cells, LA reversibly suppressed [Ca2+]i responses to capsaicin and heat, but persistently inhibited those to acids. Similarly, LA reversibly attenuated current responses to capsaicin but durably suppressed those to acids. LA sustainingly inhibited the responses to spermine, an endogenous TRPV1 agonist, and reduced pain-related behaviors induced by spermine and noxious heat. These results indicate that LA inhibits TRPV1 in a mode-independent manner, with long-lasting inhibition of acid-induced TRPV1 activation. These inhibitory actions of LA on TRPV1 may be related to its analgesic effects.

Tramadol and duloxetine, reuptake inhibitors of serotonin and noradrenaline, are widely used analgesics. Cytoplasmic serotonin in human platelets reportedly regulates the activity of low-molecular-weight GTP-binding proteins via serotonylation, leading to the modulation of platelet functions. We recently showed that the combination of thrombopoietin and collagen in the low doses synergistically induces human platelet activation via Rac and Rho/Rho-kinase. In the present study, we investigated the effects of tramadol and duloxetine on the synergistic effect, and the mechanism. Tramadol reduced the platelet aggregation and the release of PDGF-AB by the combination of thrombopoietin and collagen in the low doses. The aggregation and the release were also inhibited by duloxetine. Not reboxetine, a specific inhibitor of noradrenaline transporter, but fluvoxamine and sertraline, specific inhibitors of serotonin transporter suppressed the aggregation and the release. Tramadol, duloxetine, fluvoxamine and sertraline but not reboxetine attenuated the levels of GTP-Rac and GTP-Rho, and phospho-cofilin induced by the combination. Taken together, our results strongly suggest that tramadol and duloxetine, not as noradrenaline reuptake inhibitor but as serotonin reuptake inhibitor, suppress the activation of Rac and Rho/Rho-kinase elicited by the combination of subthreshold thrombopoietin and collagen, leading to the attenuation of human platelet activation.

Clary sage essential oil (CSEO) is utilized in perfumery, aromatherapy, and skincare. Linalyl acetate (LA), a primary component of CSEO, possesses sedative, anxiolytic, and analgesic properties. However, the mechanism of its analgesic action is not clearly understood. Transient receptor potential ankyrin 1 (TRPA1) channel, a non-selective cation channel, is mainly expressed in sensory neurons and serves as a sensor of various irritants. In this study, we investigated the effects of LA on TRPA1 channel using heterologous expression system and isolated sensory neurons. To detect channel activity, we employed Ca2+ imaging and the whole-cell patch-clamp technique. The analgesic action of LA was measured in a pain-related behavioral mouse model. In cells that heterologously expressed TRPA1, LA diminished [Ca2+]i and current responses to allylisothiocyanate (AITC) and carvacrol: exogenous TRPA1 agonists, and the inhibitory effects were more pronounced for the former than for the latter. Moreover, LA suppressed [Ca2+] i and current responses to PGJ2: an endogenous TRPA1 agonist. Similar inhibitory actions were observed in native TRPA1 channels expressed in mouse sensory neurons. Furthermore, LA diminished PGJ2-induced nociceptive behaviors in mice. These findings suggest that analgesic effects of LA exert through inhibition of nociceptive TRPA1, making it a potential candidate for novel analgesic development.