Linalyl acetate exerts analgesic effects by inhibiting nociceptive TRPA1 in mice.

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.

Acyl modifications in bovine, porcine, and equine ghrelins.

Ghrelin is a peptide hormone with various important physiological functions. The unique feature of ghrelin is its serine 3 acyl-modification, which is essential for ghrelin activity. The major form of ghrelin is modified with n-octanoic acid (C8:0) by ghrelin O-acyltransferase. Various acyl modifications have been reported in different species. However, the underlying mechanism by which ghrelin is modified with various fatty acids remains to be elucidated. Herein, we report the purification of bovine, porcine, and equine ghrelins. The major active form of bovine ghrelin was a 27-amino acid peptide with an n-octanoyl (C8:0) modification at Ser3. The major active form of porcine and equine ghrelin was a 28-amino acid peptide. However, porcine ghrelin was modified with n-octanol (C8:0), whereas equine ghrelin was modified with n-butanol (C4:0) at Ser3. This study indicates the existence of structural divergence in ghrelin and suggests that it is necessary to measure the minor and major forms of ghrelin to fully understand its physiology.

Effect of a single warm compress prior to ophthalmic surgery on ocular surface and intraoperative visibility: a randomised controlled study.

PURPOSE: We investigated the effects of a warm compress immediately before surgery on the ocular surface and intraoperative visibility during surgery. METHODS: A randomised controlled quasi-experiment at Saitama Medical University Hospital. From November 2020 to September 2021, 200 patients scheduled for endophthalmic surgery were randomly assigned to a group that received a hot compress with a spontaneously heating eye mask (HM group) or a group that received only an eye mask (control group). The eye masks were applied for 20 min from 2 hours before surgery, and before and after mask application in the non-invasive tear break-up time (NIBUT), tear meniscus height (TMH) and obstruction score of the meibomian gland (meiboscore) were evaluated. The time from wetting to dry blurring of the corneal surface (corneal blurring time, CBT) was also compared before and after the warm compress. RESULTS: We enrolled 100 patients in the HM group (mean age 69.0±13.3 years) and 99 patients in the control group (mean age 69.5±16.2 years). In the control group, there were no significant changes in the NIBUT, meiboscore or TMH before and after eye mask use, whereas in the HM group, the NIBUT increased from 6.7±5.1 to 9.5±5.6 s (p<0.001), the meiboscore improved from 0.71±0.93 to 0.63±0.96 (p=0.03) and the TMH significantly improved from 0.22±0.08 to 0.24±0.08 mm (p<0.001). The CBT was longer the HM group than control group (33.5±13.4 s, 25.7±14.9 s, respectively, p=0.01). CONCLUSIONS: The condition of the ocular surface and intraoperative visibility improved after a single warm compress. TRIAL REGISTRATION NUMBER: UMIN R000047286.

Inhibitory effects of linalool, an essential oil component of lavender, on nociceptive TRPA1 and voltage-gated Ca2+ channels in mouse sensory neurons.

Linalool, an essential oil component of lavender is commonly used in fragrances. It is known that linalool has anxiolytic, sedative, and analgesic actions. However, the mechanism of its analgesic action has not yet been fully clarified. Pain signals elicited by the activation of nociceptors on peripheral neurons are transmitted to the central nervous system. In the present study, we investigated the effects of linalool on transient receptor potential (TRP) channels and voltage-gated channels, both of which are important for pain signaling via nociceptors in somatosensory neurons. For detection of channel activity, the intracellular Ca2+ concentration ([Ca2+]i) was measured using a Ca2+-imaging system, and membrane currents were recorded using the whole-cell patch-clamp technique. Analgesic actions were also examined in vivo. In mouse sensory neurons linalool at concentrations that did not induce [Ca2+]i increases did not affect [Ca2+]i responses to capsaicin and acids, TRPV1 agonists, but suppressed those induced by allyl isothiocyanate (AITC) and carvacrol, TRPA1 agonists. Similar inhibitory effects of linalool were observed in cells that heterologously expressed TRPA1. Linalool attenuated the [Ca2+]i increases induced by KCl and voltage-gated Ca2+ currents but only slightly suppressed voltage-gated Na＋currents in mouse sensory neurons. Linalool diminished TRPA1-mediated nociceptive behaviors. The present data suggest that linalool exerts an analgesic action via the suppression of nociceptive TRPA1 and voltage-gated Ca2+ channels.

Effects of an ω3 fatty acid-biased diet on luteolysis, parturition, and uterine prostanoid synthesis in pregnant mice.

The ω3 polyunsaturated fatty acids (PUFAs) are known to have beneficial effects on health and diseases, and hence their intake is encouraged. However, it remains unknown as to how ω3 PUFAs affect female reproduction processes, in which ω6 PUFA-derived prostaglandin (PG) E2 and PGF2α play crucial roles. We therefore compared female reproductive performance between ω3 PUFA-biased linseed oil diet-fed (Lin) mice and ω6 PUFA-biased soybean oil diet-fed (Soy) mice. In Lin mice, the uterine levels of arachidonic acid (AA) and eicosapentaenoic acid (EPA) were 0.42 fold and 16 fold of those in Soy mice, respectively, with the EPA/AA ratio being 0.7 (vs 0.02 in Soy mice). Lin mice showed no alterations in any of the fertility indexes, including luteolysis and parturition. The uterine PG synthesis profiles of Lin mice were similar to those of Soy mice, but the levels of PGF2α and PGE2 were 50% of those in Soy mice, as a result of the increased EPA/AA ratio. PGF3α and PGE3 were undetectable in the uterine tissues of Soy and Lin mice. Interestingly, in Lin mice, 'luteolytic' PGF2α synthesis was considerably maintained even in the ω6 PUFA-reduced condition. These results suggest the existence of an elaborate mechanism securing PGF2α synthesis to a level that is sufficient for triggering luteolysis and parturition, even under ω6 PUFA-reduced conditions.

Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter.

A characteristic subset of microglia expressing CD11c appears in response to brain damage. However, the functional role of CD11c+ microglia, as well as the mechanism of its induction, are poorly understood. Here we report that the genetic ablation of signal regulatory protein α (SIRPα), a membrane protein, induced the emergence of CD11c+ microglia in the brain white matter. Mice lacking CD47, a physiological ligand of SIRPα, and microglia-specific SIRPα-knockout mice exhibited the same phenotype, suggesting that an interaction between microglial SIRPα and CD47 on neighbouring cells suppressed the emergence of CD11c+ microglia. A lack of SIRPα did not cause detectable damage to the white matter, but resulted in the increased expression of genes whose expression is characteristic of the repair phase after demyelination. In addition, cuprizone-induced demyelination was alleviated by the microglia-specific ablation of SIRPα. Thus, microglial SIRPα suppresses the induction of CD11c+ microglia that have the potential to accelerate the repair of damaged white matter.

The flavonoid Baicalein attenuates cuprizone-induced demyelination via suppression of neuroinflammation.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system characterized by recurrent and progressive demyelination/remyelination cycles, neuroinflammation, oligodendrocyte loss, and axonal pathology. Baicalein isolated from the roots of Scutellaria baicalensis has been shown to exert anti-inflammatory and antioxidant effects. The cuprizone model is an established mouse model of MS and causes demyelination and motor dysfunction and induces neuroinflammation, such as glial activation and pro-inflammatory cytokine production. To determine whether Baicalein attenuates cuprizone-induced demyelination, we administrated Baicalein to cuprizone-exposed mice. Baicalein attenuated weight loss (P<0.05) and motor dysfunction (P<0.05) in the cuprizone model mice. Baicalein treatment effectively suppressed the demyelination (P<0.01) and gene expressions of CNP (P<0.05) and MBP (P<0.05). Baicalein treatment also inhibited the cuprizone-induced increase in Iba1-positive microglia (P<0.001), GFAP-positive astrocytes (P<0.001), and the gene expressions of CD11b (P<0.01), GFAP (P<0.05), TNFα (P<0.05), IL-1ß (P<0.05), and iNOS (p<0.01). We found that Baicalein treatment attenuated cuprizone-induced demyelination, glial activation, pro-inflammatory cytokine expression, and motor dysfunction. Our results suggest that Baicalein may be a useful therapeutic agent in demyelinating diseases to suppress neuroinflammation.

Shp2 in forebrain neurons regulates synaptic plasticity, locomotion, and memory formation in mice.

Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K(+)-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation.

Hypothermia-dependent and -independent effects of forced swim on the phosphorylation states of signaling molecules in mouse hippocampus.

Forced swim (FS) stress induces diverse biochemical responses in the brain of rodents. Here, we examined the effect of hypothermia induced by FS in cold water on the phosphorylation of FS-sensitive signaling molecules in the mouse brain. As we have shown previously, FS in cold water induced a significant increase in the level of tyrosine phosphorylation of SIRPα, a neuronal membrane protein, in mouse hippocampus, while such effect of FS was markedly reduced in mice subjected to FS in warm water. FS in cold water also induced phosphorylation of mitogen-activated protein kinase kinase (MEK) as well as of cAMP response element-binding protein (CREB), or dephosphorylation of α isoform of Ca(2+)/calmodulin-dependent protein kinase II (αCaMKII) in the hippocampus. These effects of FS on the phosphorylation of these molecules were also lost in mice subjected to FS in warm water. Genetic ablation of SIRPα did not change the phosphorylation states of these molecules in the brain. Forced cooling of anesthetized mice, which induced a marked increase in the phosphorylation of SIRPα, induced dephosphorylation of αCaMKII in the brain, while the same treatment did not affect the phosphorylation level of MEK and CREB. Hibernation also induced an increase and a decrease of the phosphorylation of SIRPα and αCaMKII, respectively, in the brain of chipmunk. These results suggest that hypothermia is a major element that determines the levels of phosphorylation of αCaMKII and SIRPα during the FS in cold water, while it is not for the phosphorylation levels of MEK and CREB.

Dendritic cell-specific ablation of the protein tyrosine phosphatase Shp1 promotes Th1 cell differentiation and induces autoimmunity.

Dendritic cells (DCs) promote immune responses to foreign Ags and immune tolerance to self-Ags. Deregulation of DCs is implicated in autoimmunity, but the molecules that regulate DCs to protect against autoimmunity have remained unknown. In this study, we show that mice lacking the protein tyrosine phosphatase Shp1 specifically in DCs develop splenomegaly associated with more CD11c(+) DCs. Splenic DCs from the mutant mice showed upregulation of CD86 and CCR7 expression and of LPS-induced production of proinflammatory cytokines. The mice manifested more splenic Th1 cells, consistent with the increased ability of their DCs to induce production of IFN-γ by Ag-specific T cells in vitro. The number of splenic CD5(+)CD19(+) B-1a cells and the serum concentrations of Igs M and G2a were also increased in the mutant mice. Moreover, aged mutant mice developed glomerulonephritis and interstitial pneumonitis together with increased serum concentrations of autoantibodies. Shp1 is thus a key regulator of DC functions that protects against autoimmunity.

Hypothermia-induced tyrosine phosphorylation of SIRPα in the brain.

Signal regulatory protein α (SIRPα) is a neuronal membrane protein that undergoes tyrosine phosphorylation in the brain of mice in response to forced swim (FS) stress in cold water, and this response is implicated in regulation of depression-like behavior in the FS test. We now show that subjection of mice to the FS in warm (37 °C) water does not induce the tyrosine phosphorylation of SIRPα in the brain. The rectal temperature (T(rec) ) of mice was reduced to 27° to 30 °C by performance of the FS for 10 min in cold water, whereas it was not affected by the same treatment in warm water. The level of tyrosine phosphorylation of SIRPα in the brain was increased by administration of ethanol or picrotoxin, starvation, or cooling after anesthesia, all of which also induced hypothermia. Furthermore, the tyrosine phosphorylation of SIRPα in cultured hippocampal neurons was induced by lowering the temperature of the culture medium. CD47, a ligand of SIRPα, as well as Src family kinases or SH2 domain-containing protein phosphatase 2 (Shp2), might be important for the basal and the hypothermia-induced tyrosine phosphorylation of SIRPα. Hypothermia is therefore likely an important determinant of both the behavioral immobility and tyrosine phosphorylation of SIRPα observed in the FS test.

Physiological characterization of Campylobacter jejuni under cold stresses conditions: its potential for public threat.

Campylobacter jejuni is the major cause of human gastroenteritis worldwide. Under stress conditions, C. jejuni can enter a viable but non-culturable (VBNC) state. We found that the C. jejuni was able to enter a VBNC state by prolonged incubation at 4°C. The standard isolation methods using pre-enrichment steps in Bolton broth or Preston broth could not detect the VBNC cells in spiked chicken meat. The transcription levels of virulence-associated genes (flaA, flaB, cadF, ciaB, cdtA, cdtB and cdtC) were expressed in VBNC cells but in low levels. The VBNC cells retained the ability to invade Caco-2 human intestinal epithelial cells in vitro. In most cases, VBNC cells failed to resuscitate in Caco-2 cells, but in some experiments, they formed colonies after co-incubation with host cells. Collectively, C. jejuni enters into a VBNC state at 4°C and the VBNC C. jejuni remains virulent which may possibly lead to disease in humans. C. jejuni in VBNC state is a potential concern for food safety.

Signal regulatory protein α regulates the homeostasis of T lymphocytes in the spleen.

The molecular basis for formation of lymphoid follicle and its homeostasis in the secondary lymphoid organs remains unclear. Signal regulatory protein α (SIRPα), an Ig superfamily protein that is predominantly expressed in dendritic cells or macrophages, mediates cell-cell signaling by interacting with CD47, another Ig superfamily protein. In this study, we show that the size of the T cell zone as well as the number of CD4(+) T cells were markedly reduced in the spleen of mice bearing a mutant (MT) SIRPα that lacks the cytoplasmic region compared with those of wild-type mice. In addition, the expression of CCL19 and CCL21, as well as of IL-7, which are thought to be important for development or homeostasis of the T cell zone, was markedly decreased in the spleen of SIRPα MT mice. By the use of bone marrow chimera, we found that hematopoietic SIRPα is important for development of the T cell zone as well as the expression of CCL19 and CCL21 in the spleen. The expression of lymphotoxin and its receptor, lymphotoxin ß receptor, as well as the in vivo response to lymphotoxin ß receptor stimulation were also decreased in the spleen of SIRPα MT mice. CD47-deficient mice also manifested phenotypes similar to SIRPα MT mice. These data suggest that SIRPα as well as its ligand CD47 are thus essential for steady-state homeostasis of T cells in the spleen.

Essential roles of SIRPα in homeostatic regulation of skin dendritic cells.

Signal regulatory protein α (SIRPα) is an immunoglobulin superfamily protein that is predominantly expressed in dendritic cells (DCs). Its cytoplasmic region binds SHP-1 or SHP-2 protein tyrosine phosphatases, while its extracellular region interacts with CD47, another immunoglobulin superfamily protein, constituting cell-cell signaling. SIRPα was previously shown to be important for development of contact hypersensitivity, likely as a result of its positive regulation of the priming by DCs of CD4(+) T cells. However, the mechanism by which SIRPα regulates DC functions remains unknown. Here we found that the number of I-A(+) cells, which represent migratory DCs such as Langerhans cells (LCs) or dermal DCs from the skin, in the peripheral lymph nodes (LNs) was markedly decreased in mice expressing a mutant form of SIRPα that lacks the cytoplasmic region compared with that of wild-type (WT) mice. In addition, an increase of fluorescein isothiocyanate (FITC)-bearing I-A(+) cells in the draining lymph nodes (LNs) after skin-painting with FITC was markedly blunted in SIRPα mutant mice. However, migratory ability, as well as expression of CCR7, of bone marrow-derived DCs prepared from SIRPα mutant mice were not impaired. By contrast, the number of I-A(+) LCs in the epidermis of SIRPα mutant mice was markedly decreased compared with that of WT mice. In addition, the mRNA expression of transforming growth factor-ß receptor II in LCs of SIRPα mutant mice was markedly decreased compared with that of WT mice. These results suggest that SIRPα is important for homeostasis of LCs in the skin, as well as of migratory DCs in the LNs, but unlikely for migration of these cells from the skin to draining LNs.

Regulation by SIRPα of dendritic cell homeostasis in lymphoid tissues.

The molecular basis for regulation of dendritic cell (DC) development and homeostasis remains unclear. Signal regulatory protein α (SIRPα), an immunoglobulin superfamily protein that is predominantly expressed in DCs, mediates cell-cell signaling by interacting with CD47, another immunoglobulin superfamily protein. We now show that the number of CD11c(high) DCs (conventional DCs, or cDCs), in particular, that of CD8-CD4+ (CD4+) cDCs, is selectively reduced in secondary lymphoid tissues of mice expressing a mutant form of SIRPα that lacks the cytoplasmic region. We also found that SIRPα is required intrinsically within cDCs or DC precursors for the homeostasis of splenic CD4+ cDCs. Differentiation of bone marrow cells from SIRPα mutant mice into DCs induced by either macrophage-granulocyte colony-stimulating factor or Flt3 ligand in vitro was not impaired. Although the accumulation of the immediate precursors of cDCs in the spleen was also not impaired, the half-life of newly generated splenic CD4+ cDCs was markedly reduced in SIRPα mutant mice. Both hematopoietic and nonhematopoietic CD47 was found to be required for the homeostasis of CD4+ cDCs and CD8-CD4- (double negative) cDCs in the spleen. SIRPα as well as its ligand, CD47, are thus important for the homeostasis of CD4+ cDCs or double negative cDCs in lymphoid tissues.

DNA markers indicate low genetic diversity and high genetic divergence in the landlocked freshwater goby, Rhinogobius sp. YB, in the Ryukyu Archipelago, Japan.

Genetic diversity and genetic divergence were investigated in the landlocked goby Rhinogobius sp. YB by analysis of seven microsatellite DNA loci and the mtDNA control region sequence, and were compared with those of the closely related amphidromous species Rhinogobius sp. DA. Samples of Rhinogobius sp. YB and Rhinogobius sp. DA were collected from seven and four rivers, respectively. All pairwise Fst tests based on microsatellite DNA showed significant genetic differences, except for one pair of populations of Rhinogobius sp. DA (P<0.00064, alpha=78). The average Nei's genetic distance was 0.616 in Rhinogobius sp. YB and 0.394 in Rhinogobius sp. DA. Forty-two haplotypes were detected in both species, and almost all Rhinogobius sp. YB populations included different haplotypes. The means of allelic richness, Ho, and He in Rhinogobius sp. YB (2.057, 0.149, and 0.156, respectively) were significantly lower than in Rhinogobius sp. DA (4.868, 0.366, and 0.403, respectively; P<0.05). The high genetic divergence and low genetic diversity in Rhinogobius sp. YB may have resulted from repeated colonizations of rivers by different founders. Efforts to conserve genetic resources should take these evolutionarily significant units (ESU) of Rhinogobius sp. YB into account. The genetic markers used in this study provide simple and highly informative indicators for Rhinogobius sp. YB population management.

1H NMR-based metabonomic analysis of urine from young spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) and their substrains are a useful model for studying essential hypertension which is a complex, polygenic, and multifactorial disorder. Their genetic and metabolic features are of great interest because they may provide insights into the mechanism of blood pressure regulation. We have compared urinary metabolic profiles of young SHR with those of their age-matched normotensive controls, Wistar Kyoto rats, using (1)H NMR-based metabonomics. Principal components analysis was applied to the NMR spectral data after data-reduced and normalized by the total integral or the creatinine integral. Consequently, a clear separation of urine samples between the two strains was observed in the principal components scores plot. The loadings plot from the data normalized by the creatinine integral showed that many metabolites such as citrate, alpha-ketoglutarate, and hippurate contributed to the separation, and the urinary levels of most metabolites used in this study, including these three, were lower in SHR than in Wistar Kyoto rats. These metabolic changes may be concerned with blood pressure regulation in SHR, although a relation to other strain differences cannot be ruled out. The present study suggests the usefulness of a (1)H NMR-based metabonomic approach using SHR in the field of hypertension research.

The proposal of measurement of RNs at acute care setting cognitive process when they make cognitive error.

This paper presents the proposal for the design of a measurement of cognitive process of nurses at acute care setting when they make cognitive error. nurses especially at acute care setting perform their numerous nursing tasks simultaneously in time-stressed, information-rich situation. To cope with these severe cognitive demands expertise nurses develop simplification strategies not to consume their mental resources so much. However medical errors happen, regardless of nurses' experience. The resource allocation theory of Norman and Bobrow suggests that inappropriate selection of simplification strategy when the task is needed more attention induce the cognitive error. In this paper too much mental workload is presumed one of the reasons to make inappropriate selection. And the study design to measure cognitive process under the different mental workload situation by talk aloud protocol and psycho-physiological response is proposed.