Effect of fluid replacement with green tea on body fluid balance and renal responses under mild thermal hypohydration: a randomized crossover study.

PURPOSE: Maintaining an appropriate hydration level by ingesting fluid in a hot environment is a measure to prevent heat-related illness. Caffeine-containing beverages, including green tea (GT), have been avoided as inappropriate rehydration beverages to prevent heat-related illness because caffeine has been assumed to exert diuretic/natriuretic action. However, the influence of caffeine intake on urine output in dehydrated individuals is not well documented. The aim of the present study was to examine the effect of fluid replacement with GT on body fluid balance and renal water and electrolyte handling in mildly dehydrated individuals. METHODS: Subjects were dehydrated by performing three bouts of stepping exercise for 20 min separated by 10 min of rest. They were asked to ingest an amount of water (H2O), GT, or caffeinated H2O (20 mg/100 ml; Caf-H2O) that was equal to the volume of fluid loss during the dehydration protocol; fluid balance was measured for 2 h after fluid ingestion. RESULTS: The dehydration protocol induced hypohydration by ~ 10 g/kg body weight (~ 1% of body weight). Fluid balance 2 h after fluid ingestion was significantly less negative in all trials, and the fluid retention ratio was 52.2 ± 4.2% with H2O, 51.0 ± 5.0% with GT, and 47.9 ± 6.2% with Caf-H2O; those values did not differ among the trials. After rehydration, urine output, urine osmolality, and urinary excretions of osmotically active substances, sodium, potassium and chloride were not different among the trials. CONCLUSION: The data indicate that ingestion of GT or an equivalent caffeine amount does not worsen the hydration level 2 h after ingestion and can be effective in reducing the negative fluid balance for acute recovery from mild hypohydration. TRIAL REGISTRATION: ISRCTN53057185; retrospectively registered.

Endogenous Androgens Diminish Food Intake and Activation of Orexin A Neurons in Response to Reduced Glucose Availability in Male Rats.

Sex steroids modify feeding behavior and body weight regulation, and androgen reportedly augments food intake and body weight gain. To elucidate the role of endogenous androgens in the feeding regulation induced by reduced glucose availability, we examined the effect of gonadectomy (orchiectomy) on food intake and orexin A neuron's activity in the lateral hypothalamic/perifornical area (LH/PFA) in response to reduced glucose availability (glucoprivation) induced by 2-deoxy-d-glucose (2DG) administration in male rats. Rats (7W) were bilaterally orchiectomized (ORX group) or sham operated (Sham group). Seventeen days after the surgery, food intake response to 2DG (400 mg/kg, i.v.) was measured for 4 h after the infusion. The same experiment was performed for the immunohistochemical examination of c-Fos-expressing orexin A neurons in the LH/PFA and c-Fos expression in the arcuate nucleus (Arc). Food intake induced by glucoprivation was greater in the ORX group than the Sham group, and the glucoprivation-induced food intake was inversely correlated with plasma testosterone concentration. Glucoprivation stimulated c-Fos expression of the orexin A neurons at the LH/PFA and c-Fos expression in the dorsomedial Arc. The number and percentage of c-Fos-expressing orexin A neurons in the LH/PFA and c-Fos expression in the dorsomedial Arc were significantly higher in the ORX group than the Sham group. This indicates that endogenous androgen, possibly testosterone, diminishes the food intake induced by reduced glucose availability, possibly via the attenuated activity of orexin A neuron in the LH/PFA and neurons in the dorsomedial Arc.

Estradiol replacement improves high-fat diet-induced insulin resistance in ovariectomized rats.

The role of 17ß-estradiol (E2) in high-fat diet (HFD)-induced alteration of the protein kinase B (Akt) signaling pathway in ovariectomized (OVX) rats is unclear. Therefore, we examined whether chronic estrogen replacement restores HFD-induced impairment in insulin sensitivity by its effects concomitant with alterations in the Akt isoform 2 (Akt2) and Akt substrate of 160 kDa (AS160) phosphorylation in muscles of OVX rats. Nine-week-old female Wistar rats underwent ovariectomy under anesthesia; after 4 weeks, subcutaneous implantation of either E2 or placebo (PL) pellets was performed, and HFD feeding was initiated. Intravenous glucose tolerance tests were performed to assess insulin sensitivity. Following insulin injection into rats' portal vein, the liver and gastrocnemius muscle were dissected for insulin signaling analysis. We observed that HFD increased energy intake and body weight in the PL group; however, it was temporarily decreased in the E2 group. Adipose tissue accumulation was larger in HFD-fed rats than in normal chow diet (NCD)-fed rats in the PL group; however, this difference was not observed in the E2 group. HFD reduced insulin sensitivity in the PL group only. In vivo insulin stimulation increased Akt2 phosphorylation in the muscles of NCD-fed rats in both groups. In contrast, HFD affected insulin-stimulated phosphorylation of Akt2 and AS160 in the muscles of rats in the PL group but not in the E2 group. Our data suggest that E2 replacement improves HFD-induced insulin resistance, and this effect is accompanied by the alterations in the Akt2 and AS160 phosphorylation in insulin-stimulated muscles of OVX rats.

Estradiol Replacement Improves High-Fat Diet-Induced Obesity by Suppressing the Action of Ghrelin in Ovariectomized Rats.

This study aims to investigate the effects of estradiol replacement on the orexigenic action of ghrelin in ovariectomized (OVX) obese rats fed with a high-fat diet (HFD). Four weeks after OVX at 9 weeks of age, Wistar rats were subcutaneously implanted with either 17ß-estradiol (E2) or placebo (Pla) pellets and started on HFD feeding. After 4 weeks, growth hormone-releasing peptide (GHRP)-6, a growth hormone secretagogue receptor (GHSR) agonist injected intraperitoneally, induced changes in HFD intake, and c-Fos-positive neurons in the hypothalamic arcuate nucleus (ARC) were measured in both groups. The ghrelin protein and mRNA levels, as well as GHSR protein in stomach, were analyzed by Western blotting and real-time PCR. HFD increased energy intake and body weight in the Pla group, while it temporarily reduced these in the E2 group. GHRP-6 enhanced HFD intake and activated neurons in the ARC only in the Pla group. Furthermore, gastric ghrelin and GHSR protein levels were lower in the E2 group than in the Pla group, but plasma acyl ghrelin levels were similar in both groups. Our results suggest that E2 replacement improves obesity by inhibiting the orexigenic action of ghrelin via downregulation of ghrelin and its receptor in stomach in HFD-fed OVX rats.

Fluoxetine Mimics the Anorectic Action of Estrogen and Its Regulation of Circadian Feeding in Ovariectomized Female Rats.

Our previous study demonstrated that chronic estrogen replacement in ovariectomized rats reduces food intake and augments c-Fos expression in the suprachiasmatic nucleus (SCN), specifically during the light phase. Here, we hypothesized that serotonergic neurons in the central nervous system (CNS), which have anorectic action and play a role in regulating circadian rhythm, mediate the light phase-specific anorectic action of estrogen, and that selective serotonin reuptake inhibitors (SSRIs) mimic the hypophagic action of estrogen. Female Wistar rats were ovariectomized and treated with estradiol (E2) or cholesterol by subcutaneously implanting a silicon capsule containing E2 or cholesterol. Then, half of the cholesterol-treated rats were injected with the SSRI fluoxetine (5 mg/kg) (FLX group), while the remaining rats in the cholesterol-treated group (CON group) and all those in the E2 group were injected with saline subcutaneously twice daily at the onsets of the light and dark phases. Both E2 and FLX reduced food intake during the light phase but not the dark phase, and reduced body weight gain. In addition, both E2 and FLX augmented the c-Fos expression in the SCN, specifically during the light phase. These data indicate that FLX exerts estrogen-like antiobesity and hypophagic actions by modifying circadian feeding patterns, and suggest that estrogen regulates circadian feeding rhythm via serotonergic neurons in the CNS.

Endurance running exercise is an effective alternative to estradiol replacement for restoring hyperglycemia through TBC1D1/GLUT4 pathway in skeletal muscle of ovariectomized rats.

Menopause is a risk factor for impaired glucose metabolism. Alternative treatment of estrogen for postmenopausal women is required. The present study was designed to investigate the effects of 5-week endurance running exercise (Ex) by treadmill on hyperglycemia and signal pathway components mediating glucose transport in ovariectomized (OVX) placebo-treated rats, compared with 4-week 17ß-estradiol (E2) replacement or pair-feeding (PF) to the E2 group. Ex improved the hyperglycemia and insulin resistance index in OVX rats as much as E2 or PF did. However, Ex had no effect on body weight gain in the OVX rats. Moreover, Ex enhanced the levels of GLUT4 and phospho-TBC1D1 proteins in the gastrocnemius of the OVX rats, but E2 or PF did not. Instead, the E2 increased the Akt2/AS160 expression and activation in the OVX rats. This study suggests that endurance Ex training restored hyperglycemia through the TBC1D1/GLUT4 pathway in muscle by an alternative mechanism to E2 replacement.

Correlations between "hie-sho" interview score and progesterone, fat intake, and Kupperman index in pre- and post-menopausal women: a pilot study.

Japanese menopausal women who feel cold, even in a warm room, are said to be experiencing "hie-sho." We assessed the magnitude of coldness by a "hie-sho" interview score. The association between the magnitude of coldness and female hormones, fat intake, and menopausal symptoms is unknown. The aim of the present study was to elucidate the relationship between the hie-sho interview scores and female hormones, fat intake, Kupperman index in pre- (pre group) and post- (post group) menopausal women. The hie-sho interview scores, Kupperman index questionnaire results, dietary survey to analyze fat intake, and body weight were analyzed, and plasma estradiol, progesterone, and lipid levels were measured in the subjects in the pre (n = 9) and post (n = 11) groups. Plasma female hormones and fat intake were different, but the total Kupperman index was not different between pre and post groups. Plasma progesterone was positively correlated with the hie-sho score only in the post group. Plasma triglyceride was positively correlated with the hie-sho score only in the pre group. Intake of cholesterol, arachidonic acid, and docosapentaenoic acid was negatively correlated with the hie-sho score only in the pre group. The positive correlation between total Kupperman index and hie-sho score was observed only in the pre group. These results indicated that progesterone level was related to coldness in post-menopausal women. Fat intake, plasma triglyceride, and menopausal symptoms may be related to coldness in pre-menopausal women.

Estrogen replacement enhances insulin-induced AS160 activation and improves insulin sensitivity in ovariectomized rats.

Menopause predisposes women to impaired glucose metabolism, but the role of estrogen remains unclear. In this study, we examined the effects of chronic estrogen replacement on whole body insulin sensitivity and insulin signaling in ovariectomized rats. Female Wistar rats aged 9 wk were ovariectomized under anesthesia. After 4 wk, pellets containing either 17ß-estradiol (E2) or placebo (Pla) were subcutaneously implanted in the rats. After 4 wk of treatment, the intra-abdominal fat accumulation was greater in the Pla group than that in the E2 group. Hyperinsulinemic-euglycemic clamp analysis and intravenous glucose tolerance test revealed that insulin sensitivity was significantly lower in the Pla group than in the E2 group. In addition, Western blotting showed that in vivo insulin stimulation increased protein kinase B (Akt) phosphorylation to a similar degree in the gastrocnemius and liver of both groups, but phosphorylated Akt2 Ser474 was enhanced in the muscle of the E2 group compared with the Pla group. Moreover, insulin-stimulated phosphorylation of Akt substrate of 160 kDa (AS160) Thr642 was observed only in the E2 group, resulting in the difference between the two groups. Additionally, AS160 protein and mRNA levels were higher in muscle of the E2 group than the Pla group. In contrast, E2 replacement had no effect on glucose transporter 4 protein levels in muscle and glycogen synthase kinase-3ß in muscle and liver. These results suggest that estrogen replacement improves insulin sensitivity by activating the Akt2/AS160 pathway in the insulin-stimulated muscle of ovariectomized rats.

Are Wireless Electronic Stethoscopes Useful for Respiratory Rate Monitoring During Intravenous Sedation?

PURPOSE: Wireless stethoscopes can measure respiratory rate noninvasively and continuously, but there are no reports of their use during dental treatment. This study evaluated the usefulness of wireless stethoscopes during dental procedures requiring intravenous sedation (IVS). MATERIALS AND METHODS: This study was a prospective cohort study. The study sample consisted of dental patients who received IVS by propofol or midazolam administration at the Nippon Dental University Hospital (Tokyo, Japan). The predictor was respiratory rate measured using the wireless stethoscope (BrRR), and the outcome variable was respiratory rate measured during capnography (RR). Pearson correlation coefficients and paired-samples t tests were used for data analysis. A P value less than .05 was considered statistically significant. RESULTS: The study sample consisted of 12 patients. BrRR and RR were significantly and positively correlated (r = 0.93, P < .01). The mean ± standard deviation of BrRR was 14.16 ± 2.67 and that of RR was 14.32 ± 2.77. There was no significant difference between the 2 groups (P = .27). CONCLUSIONS: The results of this study suggest that wireless stethoscopes are suitable for monitoring respiratory rate during dental procedures requiring IVS because their use is as accurate as capnography.

Estrogen replacement attenuates stress-induced pressor responses through vasorelaxation via ß2-adrenoceptors in peripheral arteries of ovariectomized rats.

We examined whether chronic estrogen replacement has an inhibitory effect on stress-induced pressor responses via activation of ß2-adrenoceptor (AR) in peripheral arteries of ovariectomized rats. Female Wistar rats aged 9 wk were ovariectomized. After 4 wk, pellets containing either 17ß-estradiol (E2) or placebo (Pla) were subcutaneously implanted into the rats. After 4 wk of treatment, rats underwent cage-switch stress, and, in a separate experiment, a subset received an infusion of isoproterenol (ISO) with or without pretreatment with the ß1-AR blocker atenolol or the ß2-AR blocker butoxamine. In addition, the isolated mesenteric artery was used to assess the concentration-related relaxing responses to ISO and the ß1- or ß2-AR mRNA level. The cage-switch stress-induced pressor response was significantly attenuated in the E2-treated group compared with the Pla-treated group. Pretreatment with atenolol reduced blood pressure responses in both groups. However, butoxamine enhanced the pressor response only in the E2-treated group, resulting in no difference between the two groups. In addition, the intravenous ISO-induced depressor response was significantly enhanced in the E2-treated group compared with the Pla-treated group. Furthermore, the difference in the depressor response was abolished by pretreatment with butoxamine but not by atenolol. In the isolated mesenteric artery, butoxamine caused a rightward shift in ISO-induced concentration-related relaxation in the E2-treated group. The ß2-AR mRNA level in the mesenteric artery was higher in the E2-treated group than in the Pla-treated group. These results suggest that estrogen replacement attenuated the stress-induced pressor response probably by suppressing vasoconstriction via activation of ß2-ARs in peripheral arteries of ovariectomized rats. NEW & NOTEWORTHY In this study, we show, for the first time, that estrogen replacement has an inhibitory effect on the psychological stress-induced pressor response through vasorelaxation via ß2-adrenoceptors, probably due to overexpression of ß2-adrenoceptor mRNA, in peripheral arteries of ovariectomized rats.

S-equol Exerts Estradiol-Like Anorectic Action with Minimal Stimulation of Estrogen Receptor-α in Ovariectomized Rats.

Chronic estrogen replacement in ovariectomized rats attenuates food intake and enhances c-Fos expression in the suprachiasmatic nucleus (SCN), specifically during the light phase. S-equol, a metabolite of daidzein, has a strong affinity for estrogen receptor (ER)-ß and exerts estrogenic activity. The purpose of the present study was to elucidate whether S-equol exerts an estrogen-like anorectic effect by modifying the regulation of the circadian feeding rhythm in ovariectomized rats. Ovariectomized female Wistar rats were divided into an estradiol (E2)-replaced group and cholesterol (vehicle; Veh)-treated group. These animals were fed either a standard diet or an S-equol-containing diet for 13 days. Then, the brain, uterus, and pituitary gland were collected along with blood samples. In the rats fed the standard diet, E2 replacement attenuated food intake (P < 0.001) and enhanced c-Fos expression in the SCN (P < 0.01) during the light phase. Dietary S-equol supplementation reduced food intake (P < 0.01) and increased c-Fos expression in the SCN (P < 0.01) in the Veh-treated rats but not in the E2-replaced rats during the light phase. Dietary S-equol did not alter ER-α expression in the medial preoptic area or the arcuate nucleus, nor did dietary S-equol affect pituitary gland weight or endometrial epithelial layer thickness. By contrast, E2 replacement not only markedly decreased ER-α expression in these brain areas (P < 0.001) but also increased both the pituitary gland weight (P < 0.001) and the endometrial epithelial layer thickness (P < 0.001). Thus, dietary S-equol acts as an anorectic by modifying the diurnal feeding pattern in a manner similar to E2 in ovariectomized rats; however, the mechanism of action is not likely to be mediated by ER-α. The data suggest a possibility that dietary S-equol could be an alternative to hormone replacement therapy for the prevention of hyperphagia and obesity with a lower risk of adverse effects induced by ER-α stimulation.

Semaphorin 4D inhibits neutrophil activation and is involved in the pathogenesis of neutrophil-mediated autoimmune vasculitis.

OBJECTIVES: Inappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis. METHODS: Serum levels of soluble SEMA4D were evaluated by ELISA. Blood cell-surface expression of membrane SEMA4D was evaluated by flow cytometry. To determine the functional interactions between neutrophil membrane SEMA4D and endothelial plexin B2, wild-type and SEMA4D-/- mice neutrophils were cultured with an endothelial cell line (MS1) stained with SYTOX green, and subjected to neutrophil extracellular trap (NET) formation assays. The efficacy of treating human neutrophils with recombinant plexin B2 was assessed by measuring the kinetic oxidative burst and NET formation assays. RESULTS: Serum levels of soluble SEMA4D were elevated in patients with AAV and correlated with disease activity scores. Cell-surface expression of SEMA4D was downregulated in neutrophils from patients with AAV, a consequence of proteolytic cleavage of membrane SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects on endothelial cells. Membranous SEMA4D on neutrophils bound to plexin B2 on endothelial cells, and this interaction decreased NET formation. Recombinant plexin B2 suppressed neutrophil Rac1 activation through SEMA4D's intracellular domain, and inhibited pathogen-induced or ANCA-induced oxidative burst and NET formation. CONCLUSIONS: Neutrophil surface SEMA4D functions as a negative regulator of neutrophil activation. Proteolytic cleavage of SEMA4D as observed in patients with AAV may amplify neutrophil-mediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.

Polarization of M2 macrophages requires Lamtor1 that integrates cytokine and amino-acid signals.

Macrophages play crucial roles in host defence and tissue homoeostasis, processes in which both environmental stimuli and intracellularly generated metabolites influence activation of macrophages. Activated macrophages are classified into M1 and M2 macrophages. It remains unclear how intracellular nutrition sufficiency, especially for amino acid, influences on macrophage activation. Here we show that a lysosomal adaptor protein Lamtor1, which forms an amino-acid sensing complex with lysosomal vacuolar-type H+-ATPase (v-ATPase), and is the scaffold for amino acid-activated mTORC1 (mechanistic target of rapamycin complex 1), is critically required for M2 polarization. Lamtor1 deficiency, amino-acid starvation, or inhibition of v-ATPase and mTOR result in defective M2 polarization and enhanced M1 polarization. Furthermore, we identified liver X receptor (LXR) as the downstream target of Lamtor1 and mTORC1. Production of 25-hydroxycholesterol is dependent on Lamtor1 and mTORC1. Our findings demonstrate that Lamtor1 plays an essential role in M2 polarization, coupling immunity and metabolism.

Effects of estrogen replacement on stress-induced cardiovascular responses via renin-angiotensin system in ovariectomized rats.

The purpose of this study was to determine whether chronic estrogen replacement in ovariectomized rats inhibits the pressor response to psychological stress by attenuating the activation of the renin-angiotensin system. Female Wistar rats aged 9 wk were ovariectomized. After 4 wk, the rats were randomly assigned to be implanted subcutaneously with pellets containing either 17ß-estradiol (E2) or placebo (Pla). After 4 wk of treatment, the rats underwent cage-switch stress and, in a separate experiment, a subset received an infusion of angiotensin II. The cage-switch stress rapidly elevated blood pressure (BP) and heart rate (HR) as measured by radiotelemetry in both groups. However, the BP and HR responses to the stress were significantly attenuated in the E2 group compared with the Pla group. An angiotensin II type 1 receptor blocker, losartan, given in drinking water, abolished the difference in the pressor response to stress between the two groups. Moreover, the stress-induced elevation in plasma renin activity and angiotensin II concentration was significant in the Pla group, but not in the E2 group. In addition, the expression of renin mRNA in the kidney was lower in the E2 group relative to the Pla group. Finally, we found that intravenous angiotensin II infusion increased BP and decreased HR to a similar degree in both groups. These results suggest that the inhibitory effects of estrogen on psychological stress-induced activation of the renin-angiotensin system could be at least partially responsible for the suppression of the pressor responses to psychological stress seen in estrogen-replaced ovariectomized rats.

mTOR Complex Signaling through the SEMA4A-Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells.

Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4(+) T cell responses, the involvement of mTOR in CD8(+) T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8(+) T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A(-/-) CD8(+) T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8(+) T cell responses were significantly impaired in SEMA4A(-/-) mice. Furthermore, SEMA4A(-/-) CD8(+) T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8(+) T cells. IFN-γ production and mTORC1 activity in SEMA4A(-/-) CD8(+) T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8(+) T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8(+) T cells, but also reveal a novel link between a semaphorin and mTOR signaling.

Semaphorin 4D Contributes to Rheumatoid Arthritis by Inducing Inflammatory Cytokine Production: Pathogenic and Therapeutic Implications.

OBJECTIVE: Semaphorin 4D (Sema4D)/CD100 has pleiotropic roles in immune activation, angiogenesis, bone metabolism, and neural development. We undertook this study to investigate the role of Sema4D in rheumatoid arthritis (RA). METHODS: Soluble Sema4D (sSema4D) levels in serum and synovial fluid were analyzed by enzyme-linked immunosorbent assay. Cell surface expression and transcripts of Sema4D were analyzed in peripheral blood cells from RA patients, and immunohistochemical staining of Sema4D was performed in RA synovium. Generation of sSema4D was evaluated in an ADAMTS-4-treated monocytic cell line (THP-1 cells). The efficacy of anti-Sema4D antibody was evaluated in mice with collagen-induced arthritis (CIA). RESULTS: Levels of sSema4D were elevated in both serum and synovial fluid from RA patients, and disease activity markers were correlated with serum sSema4D levels. Sema4D-expressing cells also accumulated in RA synovium. Cell surface levels of Sema4D on CD3+ and CD14+ cells from RA patients were reduced, although levels of Sema4D transcripts were unchanged. In addition, ADAMTS-4 cleaved cell surface Sema4D to generate sSema4D in THP-1 cells. Soluble Sema4D induced tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) production from CD14+ monocytes. IL-6 and TNFα induced ADAMTS-4 expression in synovial cells. Treatment with an anti-Sema4D antibody suppressed arthritis and reduced proinflammatory cytokine production in CIA. CONCLUSION: A positive feedback loop involving sSema4D/IL-6 and TNFα/ADAMTS-4 may contribute to the pathogenesis of RA. The inhibition of arthritis by anti-Sema4D antibody suggests that Sema4D represents a potential therapeutic target for RA.

A point mutation in Semaphorin 4A associates with defective endosomal sorting and causes retinal degeneration.

Semaphorin 4A (Sema4A) has an essential role in photoreceptor survival. In humans, mutations in Sema4A are thought to contribute to retinal degenerative diseases. Here we generate a series of knock-in mouse lines with corresponding mutations (D345H, F350C or R713Q) in the Sema4A gene and find that Sema4A(F350C) causes retinal degeneration phenotypes. The F350C mutation results in abnormal localization of the Sema4A protein, leading to impaired endosomal sorting of molecules indispensable for photoreceptor survival. Additionally, protein structural modelling reveals that the side chain of the 350th amino acid is critical to retain the proper protein conformation. Furthermore, Sema4A gene transfer successfully prevents photoreceptor degeneration in Sema4A(F350C/F350C) and Sema4A(-/-) mice. Thus, our findings not only indicate the importance of the Sema4A protein conformation in human and mouse retina homeostasis but also identify a novel therapeutic target for retinal degenerative diseases.

Syntheses, characterization, and antitumor activities of platinum(II) and palladium(II) complexes with sugar-conjugated triazole ligands.

Four platinum(II) and palladium(II) complexes with sugar-conjugated bipyridine-type triazole ligands, [Pt(II)Cl(2)(AcGlc-pyta)] (3), [Pd(II)Cl(2)(AcGlc-pyta)] (4), [Pt(II)Cl(2)(Glc-pyta)] (5), and [Pd(II)Cl(2)(Glc-pyta)] (6), were prepared and characterized by mass spectrometry, elemental analysis, (1)H- and (13)C-NMR, IR as well as UV/VIS spectroscopy, where AcGlc-pyta and Glc-pyta denote 2-[4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl]ethyl 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranoside (1) and 2-[4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl]ethyl ß-D-glucopyranoside (2), respectively. The solid-state structure of complex 6 was determined by single-crystal X-ray-diffraction analysis. These complexes exhibited in vitro cytotoxicity against human cervix tumor cells (HeLa) though weaker than that of cisplatin.

Chronic oestrogen replacement in ovariectomised rats attenuates food intake and augments c-Fos expression in the suprachiasmatic nucleus specifically during the light phase.

Oestrogen replacement in ovariectomised (OVX) rats has been reported to attenuate food intake, especially during the light phase. To gain better insight into the central mechanism of oestrogen-induced reduction of food intake, we examined the effect of chronic oestrogen replacement in OVX rats on c-Fos expression in the suprachiasmatic nucleus (SCN) and on food intake during the light and dark phases. Eight-week-old female rats were ovariectomised and implanted with either an oestradiol (E2) or a vehicle pellet (Veh) subcutaneously. The animals were housed in an environment with a 12 h light-12 h dark cycle with the lights on at 07.00 hours. The amount of spontaneous food intake relative to each animal's body weight was significantly less for the E2 group than for the Veh group during the light phase, but there were no differences shown between these groups during the dark phase. There were no differences shown in the number of c-Fos-immunoreactive cells in the SCN in the E2 group compared with the Veh group during the early dark phase (22.00 hours; Zeitgeber time 15.00 (ZT15)), but the number was significantly higher than in the Veh group during the early light phase (10.00 hours; ZT3). This finding suggests that chronic oestrogen replacement chronically enhances SCN activity, specifically during the light phase. The oestrogen-induced enhancement of SCN activity during the light phase is possibly involved in the light phase-specific attenuation of food intake by oestrogen replacement in OVX rats.

Effects of estrogen on stress-induced activation of peptide neurons in PVN of ovariectomized rats.

Estrogen has been implicated in brain function related to stress responses. We investigated whether estrogen affects psychological stress-induced activation of peptide-containing or nitric oxide-producing neurons in the paraventricular hypothalamic nucleus (PVN) in ovariectomized (OVX) rats, both placebo-treated (OVX + Pla) and estrogen-treated (OVX + E2) by comparison of c-Fos expression using immunohistochemistry. Cage-switch stress increased activation in oxytocinergic neurons in the parvocellular PVN (pPVN) in OVX + Pla, but not in that of OVX + E2, rats. Moreover, the stress-induced activation in NADPH-diaphorase-positive neurons in the pPVN was larger in the OVX + E2 than in the OVX + Pla group. These findings suggest that estrogen attenuates the activation of oxytocinergic neurons in the pPVN, at least in part via nitric oxide.