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Background: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there., Methodology/principal Findings: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)gamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5)P(2), and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice., Conclusions/significance: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.

We have developed a new approach for monitoring the metabolic dynamics in microbial ecosystems using a combination of DNA fingerprinting and metabolome analysis based on stable-isotope-labeling technologies. Stable-isotope probing of DNA (DNA-SIP) has been used previously for the evaluation of cross-feeding in microbial communities. For the development and validation of our monitoring approach, fecal microbiota were analyzed with stable-isotope-labeled glucose used as the sole carbon source. In order to link the metabolic information and the microbial variability, we performed metabolic-microbial correlation analysis based on nuclear magnetic resonance (NMR) profiles and denaturing gradient gel electrophoresis (DGGE) fingerprints, which successfully identified the glucose-utilizing bacteria and their related extracellular metabolites. Moreover, our approach revealed information regarding the carbon flux, in that the "first" wave of extracellular metabolites secreted by the glucose-utilizing bacteria were incorporated into the "secondary" group of substrate-utilizing bacteria, and that this "secondary" group further produced their own secondary metabolized substrates. Thus, this approach is a powerful tool for monitoring the metabolic dynamics in microbial ecosystems and allows for the tracking of the carbon flux within a microbial community., (2010. Published by Elsevier B.V.)

Neuropeptide W (NPW), recently isolated from porcine hypothalamus, has been identified as the endogenous ligand for both NPBWR1 (GPR7) and NPBWR2 (GPR8), which belong to the orphan G protein-coupled receptor family. NPW is thought to play an important role in the regulation of feeding and drinking behavior, and to be related to the stress response. NPW-containing neurons are localized in several regions of the brain, including the hypothalamus, hippocampus, limbic system, midbrain, and brain stem. Accumulated evidence suggests that hypothalamic neuropeptides, such as neuropeptide Y (NPY), orexin, melanin-concentrating hormone (MCH), and proopiomelanocortin (POMC), are involved in the regulation of feeding behavior and energy homeostasis via neuronal circuits in the hypothalamus. NPW also forms part of the feeding-regulating neuronal circuitry in conjunction with other feeding-regulating peptide-containing neurons within the hypothalamus. We summarize our current understanding of the distribution of NPW and of the neuronal interactions between NPW and the different feeding-regulating peptide-containing neurons. This review also discusses evidence for the dichotomous actions of NPW on energy balance and the potential mechanisms involved.

It has been reported that melanocytes play important roles in skin and hair pigmentation and are differentiated from melanocyte stem cells (MSCs) residing in the bulge area of hair follicles. Recently, interest has been growing in MSCs because regulation of the upstream of differentiated melanocytes is essential for the determination of skin and hair pigmentation; however, their precise characteristics remain to be elucidated. The aim of this study is to explore cell-surface markers expressed on MSCs in order to understand their characteristics. To explore genes specifically expressed in the bulge region, we classified a hair follicle into four areas, hair bulb, hair bulb to bulge (lower bulge), bulge, and epidermis to bulge (upper bulge), and collected these areas from back skin sections of C57BL/6 mice by laser microdissection. Real-time RT-PCR performed on these areas revealed that Frizzled (Fzd)-4, Fzd7, low density lipoprotein receptor-related protein 5 (Lrp5), and Lrp6, receptors for Wnt molecules, were expressed higher in the bulge area than other areas. Furthermore, FACS analysis showed that populations of Fzd4(+) cells and Fzd7(+) cells were different from those of Kit(+) cells (precursor of melanocytes: melanoblasts). Fzd4(+) and Fzd7(+) cells isolated by FACS required a longer culture period to differentiate into mature melanocytes than Kit(+) cells. Up-regulation of mRNA expressions of melanocyte markers (dopa chrometautomerase: Dct, tyrosinase: Tyr, tyrosinase-related protein 1: Tyrp1) was observed in Fzd4(+) and Fzd7(+) cells following Kit(+) cells during differentiation. These results suggested that Fzd4(+) and Fzd7(+) cells were more immature than melanoblasts, therefore raising the possibility that Fzd4(+) and Fzd7(+) cells are MSCs., ((c) 2010 Elsevier Inc. All rights reserved.)

Neuropeptide W (NPW) is an anorectic peptide produced in the brain. Here, we showed that NPW was present in several hypothalamic nuclei, including the paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, lateral hypothalamus, and hypothalamic arcuate nucleus. NPW expression was significantly up-regulated in leptin-deficient ob/ob and leptin receptor-deficient db/db mice. The increase in NPW expression in ob/ob mice was abrogated to control levels after leptin replacement. Leptin induced suppressors of cytokine signaling-3 after phosphorylation of signal transducer and activator of transcription-3 in NPW-expressing neurons. In addition, we demonstrated that NPW reduces feeding via the melanocortin-4-receptor signaling pathway. We also showed that NPW activates proopiomelanocortin and inhibits neuropeptide Y neurons using loose-patch extracellular recording of these neurons identified by promoter-driven green fluorescent protein expression. This study indicates that NPW may play an important role in the regulation of feeding and energy metabolism under the conditions of leptin insufficiency.

Neuropeptide W (NPW), which was recently isolated from the porcine hypothalamus, has been identified as the endogenous ligand of the orphan G protein-coupled receptors GPR7 (NPBWR1) and GPR8 (NPBWR2). Infusion of NPW increases food intake in the light phase, whereas in the dark phase, it has the opposite effect. In this study, we used RT-PCR analysis to examine the gene expression of NPW mRNA in the rat brain, and performed a detailed analysis of the distribution of NPW-positive neurons by use of immunohistochemistry at both the light and electron microscopic levels. NPW mRNA expression was demonstrated in the hypothalamic paraventricular nucleus (PVN), arcuate nucleus (ARC), ventromedial nucleus (VMH) and lateral hypothalamus (LH). At the light microscopic level, NPW-like immunoreactive (NPW-LI) cell bodies were found in the preoptic area (POA), PVN, ARC, VMH, LH, PMD (dorsal premammillary nucleus), periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), and prepositus nucleus (Pr). NPW-LI axon terminals were shown in the POA, bed nucleus of the stria terminalis (BST), amygdala, PVN, ARC, VMH, LH, and PAG, LPB. In addition, at the electron microscopic level, NPW-LI cell bodies and dendritic processes were often seen to receive inputs from other unknown neurons in the ARC, PVN, VMH and amygdala. Our observations indicate that NPW-LI neurons widely distributed in the rat brain region. These finding suggest that NPW may have important roles in feeding behavior, energy homeostasis, emotional response and regulation of saliva secretion., (Copyright 2009 Elsevier Ltd. All rights reserved.)

A simple and highly sensitive high-performance liquid chromatography procedure was developed for the determination of carnosine in urine. Carnosine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 15 min in borate buffer (20 mmol l(-1), pH 9.0) to produce fluorescent sulfonamides. After hydrolysis of the reaction mixture with formic acid at 100 degrees C for 15 min, the fluorescent derivative of carnosine was separated on a reversed-phase column with a linear gradient elution using solvents of (A) acetate buffer (0.1 mmol l(-1), pH 7.0) and (B) acetonitrile at a flow-rate of 1.0 ml/min and was detected at excitation and emission wavelengths of 318 and 400 nm, respectively. The detection limit of carnosine was 4 fmol at a signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 2.7-4.6% and 0.4-5.2%, respectively. The concentration of carnosine in normal human urine was found to be 4.6-125 nmol (mg creatinine)(-1) (mean+/-SD: 21.6+/-26.6 nmol (mg creatinine)(-1), n=20)., (2009 Elsevier B.V. All rights reserved.)

The number of foreign workers in Japan, especially temporary workers, has been increasing recently. However, little is known about the mental health status of the foreign workers working temporarily in Japan. We examined the depressive symptoms in 81 Chinese factory workers and attempted to identify the determining factors. The subjects were requested to complete individual questionnaires on sociodemographic variables (sex, age group, and residence period in Japan), working condition variables (number of working days per week and working hours per day), health administration variables (health checkups and health education), a social support variable (interpreters at workplace), and health behavior variables (alcohol consumption, current smoking, and regular exercise). The 20-item Center for Epidemiologic Studies Depression scale (CES-D) was used to measure the depressive symptoms. Of all the subjects, 95% and 84% received health checkups and health education, respectively, at their workplaces. The results of multiple linear regression analysis showed that long working hours per day were significantly associated with high CES-D score. Further, we found that older age (30-49 yr) was marginally associated with high CES-D score. Health administration considering working time and age would be important for decreasing depressive symptoms among foreign workers.

Enrichment of anammox bacteria from three types of seed sludge, sewage, digester, and nitrification sludges, was conducted using a nonwoven fabric carrier for immobilizing the anammox bacteria, and the microbial diversity of the enriched anammox culture was investigated. About four months later, simultaneous removals of ammonium and nitrite, and production of a small amount of nitrate, which is unique to the anammox reaction, were observed in all 3 sludge reactors. Results of 16S rRNA gene analysis indicated that anammox bacteria were cultivated and diversified in each sludge type. Moreover, the microbial diversity of anammox bacteria was higher in the enriched culture from sewage sludge compared to the other two types of seed sludge. Bacillus sp. coexisted in the anammox culture cultivated from sewage sludge. These results suggest that differences in the anammox community in the enriched culture were caused by differences in the type of seed sludge.

Herein is reported a rare case of carcinoma arising from papilloma of the breast. A 63-year-old postmenopausal woman noticed a nodule approximately 1 cm in diameter in her left breast. Ultrasonography indicated a mass with a solid pattern within an intracystic tumor measuring 1.5 x 1.5 x 1.4 cm in diameter located near the left nipple. On total image analysis malignancy could not be denied, therefore lumpectomy with resection of the surrounding tissue was performed. Histologically the tumor consisted of cancerous and papilloma components. The cancer cells had high-grade nuclear atypia, were irregular, and contained abundant eosinophilic cytoplasm with a thin vascular stalk. In contrast, the tumor cells had no atypia, and had a thick stroma in the papilloma components. Both lesions could be distinguished clearly from each other. In addition, a transition from papillary to cancerous elements in some areas was seen. An additional partial mastectomy was performed after the lumpectomy but no carcinoma foci were noted in the excised tissue. Possible occurrence of cancerous change in solitary intraductal papilloma of the breast was suspected.

The correlations between mRNA expressions of 5-fluorouracil(5-FU)-related enzymes; thymidylate synthase(TS), dihydropyrimidine dehydrogenase(DPD), thymidine phosphorylase(TP), and orotate phosphoribosyltransferase (OPRT)in breast cancers, along with disease-free survival(DFS), were investigated in 35 patients treated with postoperative adjuvant chemotherapy using cyclophosphamide, methotrexate and 5-fluorouracil(CMF). The patients treated with CMF were divided into two groups, a lower group(L group)and a higher group(H group), according to the median value of the mRNA expression for each enzyme in 220 breast cancer specimens, which were resected between 1996 and 1998 in our institute. 5-year DFS was not significantly different between TS-L and H group(60% and 80%, p=0.38), DPD-L and H group(57.9% and 86.7%, p=0.088), and TP-L and H group(70% and 73.3%, p=0.89), respectively. 5-year DFS in the OPRT-H group(88.9%)was significantly better than that in the OPRT-L group(50%) (p=0.024). In the OPRT-H group, despite the fact that the proportion of patients with lymph node involvement in the CMF group was significantly higher than that in the postoperative adjuvant hormone therapy group, 5-year DFS was not significantly different between the two groups(p=0.10). Our results suggest that OPRT level was the significant predictive marker for DFS in the breast cancer patients treated with postoperative adjuvant chemotherapy using CMF.

An anaerobic ammonium oxidation (anammox) process for ammonia-rich wastewater treatment has not been reported at temperatures below 15 degrees C. This study used a gel carrier with entrapped anammox bacteria to obtain a stable nitrogen removal performance at low temperatures. In a continuous feeding test, a high nitrogen conversion rate (6.2 kg N m(-3) day(-1)) was confirmed at 32 degrees C. Nitrogen removal activity decreased gradually with decreasing operation temperature; however, it still occurred at 6 degrees C. Nitrogen conversion rates at 22 and 6.3 degrees C were 2.8 and 0.36 kg N m(-3) day(-1), respectively. Moreover, the stability of anammox activity below 20 degrees C was confirmed for more than 130 days. In batch experiments, anammox gel carriers were characterized with respect to temperature. The optimum temperature for anammox bacteria was found to be 37 degrees C. Furthermore, it was clear that the temperature dependence changed at about 28 degrees C. The apparent activation energy in the temperature range from 22 to 28 degrees C was calculated as 93 kJ mol(-1), and that in the range from 28 to 37 degrees C was 33 kJ mol(-1). This value agrees with the result of a continuous feeding test (94 kJ mol(-1), between 6 and 22 degrees C). The nitrogen removal performance demonstrated at the low temperatures used in this study will open the door for the application of anammox processes to many types of industrial wastewater treatment.

Orexin-A and -B (identical to hypocretin-1 and -2) are hypothalamic neuropeptides that regulate appetite and arousal. Orexins-producing neurons project their axons to various brain regions, including the olfactory bulb. In the present study, to understand the relationship between orexins and olfaction, we investigated the distribution of the orexin-A- and -B-immunoreactive (ir) fibers in the rat olfactory bulb and the contents of orexin-A and -B in the rat olfactory bulb after food deprivation for 48 h by using immunohistochemistry and radioimmunoassay, respectively. Both orexin-A- and -B-ir fibers are similarly wide spread from the glomerular layer of the olfactory bulb where the terminals of the peripheral olfactory nerves make synapses with the mitral cells or the tufted cells, to the piriform cortex. Dense orexin-A- and -B-ir fibers were observed mainly in the granular cell layer and anterior olfactory nucleus. The contents of orexin-A and -B (pg/10 mg wet weight tissue) in fed rats (mean+/-S.E.M., n=6) were 2.72+/-0.24 and 6.31+/-0.63, respectively. Fasting for 48 h significantly reduced the contents of orexin-B, but not orexin-A. Orexins in the rat olfactory bulb may be involved in not only olfactory system but also energy balance.

Neuropeptide W (NPW) is an endogenous ligand for GPR7, a member of the G-protein-coupled receptor family. NPW plays an important role in the regulation of both feeding and energy metabolism, and is also implicated in modulating responses to an acute inflammatory pain through activation of the hypothalamus-pituitary-adrenal axis. GPR7 mRNA has been shown to be expressed in the hypothalamus, pituitary gland and adrenal cortex. Similarly, NPW expression has been demonstrated in the brain and pituitary gland. However, the precise distribution of NPW-producing cells in the adrenal gland remains unknown. The aim of this study was to explore the distribution and localization of NPW immunoreactivity in the rat adrenal gland. Total RNA was prepared from the hypothalamus, pituitary gland and adrenal gland. RT-PCR revealed the expression of NPW mRNA in these tissues, while in situ hybridization demonstrated the presence of NPW mRNA in the adrenal medulla. When immunohistochemistry was performed on sections of adrenal gland, NPW-like immunoreactivity (NPW-LI) was observed in the medulla but not in the cortex. Moreover, NPW-LI was found to be co-localized in cells which expressed dopamine beta hydroxylase but not phenylethanolamine-N-methyltransferase. The finding that NPW is expressed in noradrenalin-containing cells in the adrenal medulla suggests that it may play an important role in endocrine function in the adrenal gland.

Neuropeptide W (NPW), a novel endogenous peptide for G protein-coupled receptors GPR7 and GPR8, is expressed in the gastric antral mucosa of rat, mouse, and human stomachs. Here, we studied the ontogeny of NPW in the developing rat stomach. Real-time RT-PCR showed that NPW gene expression was initially detectable in embryonic day 14 (E14) stomach and gradually increased during the progress of age until birth, postnatal day 1 (P1). NPW mRNA level in the stomach increased again from the weaning period (P21) until reaching adulthood. Immunohistochemistry using polyclonal antibodies raised against rat NPW revealed that NPW-positive cells were detected in the P1 antral stomach and gradually increased during the development of age. Furthermore, double immunohistochemistry demonstrated that NPW colocalized with gastrin in P1 rat stomach. These data will provide clues to physiological functions of NPW in the development of rat stomach.

Neuropeptide W (NPW) was recently discovered as the endogenous ligand for GPR7 and GPR8, which are orphan G protein-coupled receptors isolated from the porcine brain. These receptors are assumed to be involved in feeding regulation and/or energy homeostasis. Recent anatomical studies have revealed that high levels of GPR7 mRNA are distributed in the brain, including the hypothalamus and amygdala. However immunohistochemical studies on the distribution and localization of NPW have revealed differing results concerning whether or not NPW-containing cell bodies and their processes are present in the hypothalamus. Only a few immunohistochemical reports have been published concerning the presence of NPW-containing neurons in the brains of rodents, while there have been no anatomical studies of the co-localization of this neuropeptide with other transmitters. On this basis, we used a specific antiserum against NPW to determine immunohistochemically the presence of NPW-containing neurons in the rat hypothalamus. Many NPW-like immunoreactive cell bodies and their processes could be detected in the caudal region of the lateral hypothalamus but not in its anterior or middle regions. Given this positive identification of NPW-containing neurons in the lateral hypothalamus, we further studied the nature of interaction between NPW-containing neurons and neurons containing feeding regulating peptides such as orexin- and melanin-concentrating hormone (MCH). Very close interactions between NPW-containing nerve processes and orexin- and MCH-containing neuronal cell bodies and processes could be observed. These morphological findings strongly suggest that NPW is involved in the regulation of feeding and/or sleep/arousal behavior through orexin- and/or MCH-mediated neuronal pathways.

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway in the biological nitrogen cycle and a new cost-effective way to remove ammonium from wastewater. We have so far developed new immobilization technique that anammox bacteria entrapped in polyethylene glycol (PEG) gel carrier. However, fate and behavior of anammox bacteria in a gel carrier is not well understood. In the present study, we focused on the population changes of anammox bacteria in a gel carrier. Three specific primer sets were designed for real-time PCR. For quantification of anammox bacteria in a gel carrier, real-time PCR was performed. The anammox bacteria related to HPT-WU-N03 clone were increased the rate in anammox population, and found to be a major population of anammox bacteria in a gel carrier. Furthermore, from the results of nitrogen removal performance and quantification of anammox bacteria, the correlation coefficient between copy numbers of anammox bacteria and nitrogen conversion rate was calculated as 0.947 in total anammox population. This is the first report that population changes of anammox bacteria immobilized in a gel carrier were evaluated., (Copyright IWA Publishing 2008.)

Anaerobic ammonium-oxidizing (anammox) bacteria were immobilized in polyethylene glycol gel carriers. A small amount of seed sludge [0.24% (w/v)] was entrapped in the carriers, and continuous feeding tests were performed. Nitrogen removal activity increased gradually, reaching 3.7 kg N/m(3) reactor per day on day 67. The average of nitrogen conversion rate was calculated as 3.4 kg N/m(3) reactor per day. Microscopic examination clearly showed that small red clusters formed in the gel carrier. Moreover, fluorescence in situ hybridization analysis revealed that these clusters consisted of anammox bacteria. From real-time polymerase chain reaction analysis, the growth of anammox bacteria in the gel carriers was clearly shown by increased concentration of 16S rRNA gene of planctomycete from 4.3 x 10(8) to 4.2 x 10(9) copies/ml between days 41 and 55. To determine the effects of inoculation on the start-up of the reactor, the amount of seed sludge in the gel carrier was varied and it was found that the start-up period could be reduced to as little as 25 days when a sludge concentration of 1.4% (w/v) was used. This is the first report of successful immobilization and cultivation of anammox bacteria in a gel carrier.

Recent advances in peptidomics have enabled the identification of previously uncharacterized peptides. However, sequence information alone does not allow us to identify candidates for bioactive peptides. To increase an opportunity to discover bioactive peptides, we have focused on C-terminal amidation, a post-translational modification shared by many bioactive peptides. We analyzed peptides secreted from human medullary thyroid carcinoma TT cells that produce amidated peptides, and we identified two novel amidated peptides, designated neuroendocrine regulatory peptide (NERP)-1 and NERP-2. NERPs are derived from distinct regions of the neurosecretory protein that was originally identified as a product of a nerve growth factor-responsive gene in PC12 cells. Mass spectrometric analysis of the immunoprecipitate using specific antibodies as well as reversed phase-high performance liquid chromatography coupled with radioimmunoassay analysis of brain extract demonstrated the endogenous presence of NERP-1 and NERP-2 in the rat. NERPs are abundant in the paraventricular and supraoptic nuclei of the rat hypothalamus and colocalized frequently with vasopressin but rarely with oxytocin. NERPs dose-dependently suppressed vasopressin release induced by intracerebroventricular injection of hypertonic NaCl or angiotensin II in vivo. NERPs also suppressed basal and angiotensin II-induced vasopressin secretion from hypothalamic explants in vitro. Bioactivity of NERPs required C-terminal amidation. Anti-NERP IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release. These findings suggest that NERPs are novel modulators in body fluid homeostasis.

Age-related decreases in energy expenditure have been associated with the loss of skeletal muscle and decline of food intake, possibly through a mechanism involving changes of growth hormone (GH) secretion and feeding behavior. Age-related declines of growth hormone secretion and food intake have been termed the somatopause and anorexia of ageing, respectively. Ghrelin, a 28-amino-acid peptide, was isolated from human and rat stomachs as an endogenous ligand of growth hormone secretagogue receptor. Ghrelin stimulates growth hormone release and food intake when peripherally administered to rodents and humans. Here, we investigate the relationship between age-related decline of growth hormone secretion and/or food intake and ghrelin function. Ghrelin (10 nmol/kg body weight) was administered intravenously to male 3-, 12-, 24-and 27-month-old Long-Evans rats, after which growth hormone concentrations and 2 h food intake were measured. An intravenous administration of ghrelin to rats increased food intake in all generations. In addition, to orexigenic effect by ghrelin, intravenous administration of ghrelin elicited a marked increase in plasma GH levels, with the peak occurring 15 min after administration. These findings suggest that the aged rats maintain the reactivity to administered exogenous ghrelin.

Pregnancy-specific glycoproteins (Psgs) secreted by the placenta regulate the immune system to ensure the survival of the fetal allograft by inducing IL-10, an anti-inflammatory cytokine. However, it is unknown whether Psgs are involved in more general aspects of immune response other than maternal immunity. Here, we report that Psg18 is highly expressed in the follicle-associated epithelium (FAE) overlaying Peyer's patches (PPs). Bioinformatics analysis with Reference Database for Immune Cells (RefDIC) as well as RT-PCR data demonstrated that Psg18 is exclusively expressed in FAE in adult mice, in contrast to other Psg family members that are either not expressed or only slightly expressed in FAE. Psg18 expression was observed in FAE of germ-free-conditioned mice, and was slightly upregulated after bacterial inoculation. In situ hybridization analysis revealed that Psg18 is widely expressed throughout FAE. Furthermore, Psg18 protein is deposited on the extracellular matrix in the subepithelial dome beneath FAE, where antigen-presenting cells accumulate. These results suggest that Psg18 is an FAE-specific marker protein that could promote interplay between FAE and immune cells in mucosa-associated lymphoid tissues.

Expressions of the growth hormone secretagogue receptor (GHS-R) mRNA and its protein were confirmed in rat fetal spinal cord tissues by RT-PCR and immunohistochemistry. In vitro, over 3 nM ghrelin and des-acyl ghrelin induced significant proliferation of primary cultured cells from the fetal spinal cord. The proliferating cells were then double-stained using antibodies against the neuronal precursor marker, nestin, and the cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), and the nestin-positive cells were also found to be co-stained with antibody against GHS-R. Furthermore, binding studies using [125I]des-acyl ghrelin indicated the presence of a specific binding site for des-acyl ghrelin, and confirmed that the binding was displaced with unlabeled des-acyl ghrelin or ghrelin. These results indicate that ghrelin and des-acyl ghrelin induce proliferation of neuronal precursor cells that is both dependent and independent of GHS-R, suggesting that both ghrelin and des-acyl ghrelin are involved in neurogenesis of the fetal spinal cord.

Background: Ghrelin is a body weight-regulating peptide produced and secreted primarily by the gastric mucosa. Helicobacter pylori infection impairs gastric ghrelin production, leading to a lower plasma ghrelin concentration. However, the effect of H. pylori eradication on plasma ghrelin levels and its relation to body weight change after H. pylori cure are still uncertain. We examined the association of plasma ghrelin levels with gastric ghrelin production and body weight change before and after H. pylori eradication., Methods: Plasma ghrelin concentrations, gastric ghrelin expression, and body weight were determined in a total of 134 consecutive individuals before and 12 weeks after successful H. pylori eradication. Gastric ghrelin expression was evaluated by determining mRNA expression levels and the number of ghrelin-producing cells in gastric mucosa biopsy specimens by real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively., Results: Plasma ghrelin concentration increased in 50 patients and decreased in 84 patients after H. pylori eradication. After H. pylori cure, however, gastric preproghrelin mRNA expression was increased nearly fourfold (P < 0.0001), and the number of ghrelin-positive cells was increased or unchanged. In contrast, plasma ghrelin changes after H. pylori cure were inversely correlated with both body weight change (P < 0.0001) and initial plasma ghrelin levels (P < 0.0001)., Conclusions: Changes in plasma ghrelin concentrations before and after H. pylori cure were inversely correlated with body weight change and initial plasma ghrelin levels but not with gastric ghrelin production in Japanese patients.

Ghrelin, a gastrointestinal peptide, stimulates feeding when administered peripherally. Blockade of the vagal afferent pathway abolishes ghrelin-induced feeding, indicating that the vagal afferent pathway may be a route conveying orexigenic ghrelin signals to the brain. Here, we demonstrate that peripheral ghrelin signaling, which travels to the nucleus tractus solitarius (NTS) at least in part via the vagus nerve, increases noradrenaline (NA) in the arcuate nucleus of the hypothalamus, thereby stimulating feeding at least partially through alpha-1 and beta-2 noradrenergic receptors. In addition, bilateral midbrain transections rostral to the NTS, or toxin-induced loss of neurons in the hindbrain that express dopamine beta hydroxylase (an NA synthetic enzyme), abolished ghrelin-induced feeding. These findings provide new evidence that the noradrenergic system is necessary in the central control of feeding behavior by peripherally administered ghrelin.

Ghrelin, an acylated peptide produced predominantly in the stomach, stimulates feeding and GH secretion via interactions with the GH secretagogue type 1a receptor (GHS-R1a), the functionally active form of the GHS-R. Ghrelin molecules exist in the stomach and hypothalamus as two major endogenous forms, a form acylated at serine 3 (ghrelin) and a des-acylated form (des-acyl ghrelin). Acylation is indispensable for the binding of ghrelin to the GHS-R1a. Ghrelin enhances feeding via the neuronal pathways of neuropeptide Y and orexin, which act as orexigenic peptides in the hypothalamus. We here studied the effect of des-acyl ghrelin on feeding behavior. Intracerebroventricular (icv) administration of rat des-acyl ghrelin to rats or mice fed ad libitum stimulated feeding during the light phase; neither ip nor icv administration of des-acyl ghrelin to fasting mice suppressed feeding. The icv administration of des-acyl ghrelin induced the expression of Fos, a marker of neuronal activation, in orexin-expressing neurons of the lateral hypothalamic area, but not neuropeptide Y-expressing neurons of the arcuate nucleus. Peripheral administration of des-acyl ghrelin to rats or mice did not affect feeding. Although icv administration of ghrelin did not induce food intake in GHS-R-deficient mice, it did in orexin-deficient mice. In contrast, icv administration of des-acyl ghrelin stimulated feeding in GHS-R-deficient mice, but not orexin-deficient mice. Des-acyl ghrelin increased the intracellular calcium concentrations in isolated orexin neurons. Central des-acyl ghrelin may activate orexin-expressing neurons, perhaps functioning in feeding regulation through interactions with a target protein distinct from the GHS-R.

The CT-R [calcitonin (CT) receptor] is expressed in the central nervous system and is involved in the regulation of food intake, thermogenesis, and behaviors. CT-R-stimulating peptide-1 (CRSP-1), a potent ligand for the CT-R, was recently isolated from the porcine brain. In this study, we first confirmed that porcine CRSP-1 (pCRSP-1) enhanced the cAMP production in COS-7 cells expressing recombinant rat CT-R, and then we examined the central effects of pCRSP-1 on feeding and energy homeostasis in rats. Intracerebroventricular (icv) administration of pCRSP-1 to free-feeding rats suppressed food intake in a dose-dependent manner. Chronic icv infusion of pCRSP-1 suppressed body weight gain over the infusion period. Furthermore, icv administration of pCRSP-1 increased body temperature and decreased locomotor activity. The central effects of pCRSP-1 were more potent than those of porcine CT in rats. In contrast, ip administration of pCRSP-1 did not elicit any anorectic or catabolic effects. Administration icv of pCRSP-1 also induced mild dyskinesia of the lower extremities and decreased gastric acid output. Fos expression induced by icv administration of pCRSP-1 was detected in the neurons of the paraventricular nucleus, dorsomedial hypothalamic nucleus, arcuate nucleus, locus coeruleus, and nucleus of solitary tract, areas that are known to regulate feeding and energy homeostasis. Administration icv of pCRSP-1 increased plasma concentrations of ACTH and corticosterone, implying that the hypothalamic-pituitary-adrenocortical axis might be involved in catabolic effects of pCRSP-1. These results suggest that CRSP-1 can function as a ligand for the CT-R and may act as a catabolic signaling molecule in the central nervous system.

The doubling time of anaerobic ammonium-oxidizing (anammox) bacteria in an anaerobic biological filtrated (ABF) reactor was determined. Fluorescence in situ hybridization analysis was used to detect and count anammox bacteria cells in anammox sludge. As a result, the populations of anammox bacteria at 14th and 21st days were 1.1 x 10(6) and 1.7 x 10(7) cells/ml reactor, respectively. From these results, the doubling time of anammox bacteria was calculated as 1.8 days, and the specific growth rate (mu) was 0.39 day(-1). This result indicated that the anammox bacteria have higher growth rate than the reported value (doubling time, 11 days). Furthermore, it was clearly demonstrated that nitrogen conversion rate was proportional to the population of anammox bacteria. Maintaining the ideal environment for the growth of anammox bacteria in the ABF reactor might lead to faster growth. This is the first report of the growth rate of anammox bacteria based on the direct counting of anammox bacteria.

Ghrelin, an acylated peptide serving as an endogenous ligand for GH secretagogue receptor (GHS-R), was originally isolated from rat and human stomach. In this study, we report the critical role of maternal ghrelin in fetal development. High levels of ghrelin receptor (GHS-R) mRNA were detected in various peripheral fetal tissues beginning at embryonic d 14 and lasting until birth. Fetal GHS-R expression was also confirmed in fetal tissues by immunohistochemistry. Autoradiography revealed that both des-acyl ghrelin and acyl ghrelin bind to fetal tissues. Chronic treatment of mothers with ghrelin resulted in a significant increase in birth weight in comparison to newborns from saline-treated mothers. Even when maternal food intake after ghrelin treatment was restricted through paired feeding, significant stimulation of fetal development still occurred. Conversely, active immunization of mothers against ghrelin decreased fetal body weight during pregnancy. A single ghrelin injection into the mother increased circulating ghrelin levels in the fetus within 5 min of injection, suggesting that maternal ghrelin transits easily to the fetal circulation. High levels of des-acyl ghrelin were detected in fetal blood and amniotic fluid. Both acylated and des-acyl ghrelin increased [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation of cultured fetal skin cells in a dose-dependent manner, and calcium-imaging analysis revealed that acyl and des-acyl ghrelin increased the Ca2+ influx in discrete cultured fetal skin cells, respectively. These results indicate that maternal ghrelin regulates fetal development during the late stages of pregnancy.

Human beta-defensin (hBD)-3, a 45 amino acid antimicrobial peptide, was originally isolated from human skin. hBD-3 mRNA has also been detected in the airways by RT-PCR. While hBD-3 may be involved in antimicrobial defences within the respiratory tract, the presence of hBD-3 peptide in the respiratory system has not yet been confirmed. The antimicrobial activity of the synthesised hBD-3 peptide was measured by a radial diffusion assay and a colony count assay. The present authors confirmed the presence of hBD-3 peptide in homogenates of human lung and serum using reverse-phase HPLC coupled with a highly sensitive RIA. The localisation of the hBD-3 peptide was investigated by immunohistochemistry. In addition, the serum concentrations of hBD-3 were measured by RIA. hBD-3 exhibited a strong antimicrobial activity, which was unaffected by increasing salt concentrations. Immunohistochemically, the current authors observed the expression of hBD-3 in bronchial and bronchiolar epithelial cells. The mean+/-sd serum concentration of hBD-3 in patients with bacterial pneumonia was 239.4+/-17.8 pg x mL(-1) in the acute phase and, decreased to 159.3+/-20.1 pg x mL(-1) after the completion of therapy. In conclusion, these findings will help elucidate the role of human beta-defensin-3 in host immune responses and identify the pathophysiological significance of this molecule in respiratory infections.

Galanin-like peptide (GALP) is produced in neurones in the hypothalamic arcuate nucleus and is implicated in the neural control of feeding behaviour. Previously, we have reported that GALP immunoreactive fibres were in direct contact with orexin/hypocretin immunoreactive neurones in the rat lateral hypothalamus using double-immunofluorescence. Centrally administered GALP is known to stimulate feeding behaviour. However, the target neurones of this action have not been clarified. The present study aimed to determine features of the GALP-mediated neuronal feeding pathway in rat. Accordingly, at the ultrastructural level, GALP-immunoreactive axon terminals were found to make synapses on orexin/hypocretin immunoreactive cell bodies and dendritic processes in the lateral hypothalamus. c-Fos immunoreactivity was expressed in orexin/hypocretin-immunoreactive neurones but not in melanin concentrating hormone-immunoreactive neurones in the lateral hypothalamus at 90 min after the application of GALP by i.c.v. infusion. Furthermore, to determine whether GALP regulates feeding behaviour via orexin/hypocretin neurones, the feeding behaviour of rats was studied following GALP i.c.v. injection with or without anti-orexin A and B immunoglobulin (IgG) pretreatment. The anti-orexin IgGs markedly inhibited GALP-induced hyperphagia. These results suggest that orexin/hypocretin-containing neurones in the lateral hypothalamus are targeted by GALP, and that GALP-induced hyperphagia is mediated via orexin/hypocretin neurones in the rat hypothalamus.

Neuropeptide W (NPW) is a 30-amino-acid peptide initially isolated from the porcine hypothalamus as an endogenous ligand for the G protein-coupled receptors GPR7 and GPR8. An intracerebroventricular administration of NPW increased serum prolactin and corticosterone concentrations, decreased dark-phase feeding, raised energy expenditure, and lowered body weight. Peripherally, GPR7 receptors are abundantly expressed throughout the gastrointestinal tract; the presence of NPW in the gastrointestinal endocrine system, however, remains unstudied. Using monoclonal and polyclonal antibodies raised against rat NPW, we studied the localization of NPW in the rat, mouse, and human stomach by light and electron microscopy. NPW-immunoreactive cells were identified within the gastric antral glands in all three species. Double immunohistochemistry and electron-microscopic immunohistochemistry studies in rats demonstrated that NPW is present in antral gastrin (G) cells. NPW immunoreactivity localized to round, intermediate-to-high-density granules in G cells. NPW-immunoreactive cells accounted for 90% chromagranin A- and 85% gastrin-immunoreactive endocrine cells in the rat gastric antral glands. Using reversed-phase HPLC coupled with enzyme immunoassays specific for NPW, we detected NPW30 and its C-terminally truncated form, NPW23, in the gastric mucosa. Plasma NPW concentration of the gastric antrum was significantly higher than that of the systemic vein, suggesting that circulating NPW is derived from the stomach. Plasma NPW concentration of the gastric antrum decreased significantly after 15-h fast and increased after refeeding. This is the first report to clarify the presence of NPW peptide in the stomachs of rats, mice, and humans. In conclusion, NPW is produced in gastric antral G cells; our findings will provide clues to additional mechanisms of the regulation of gastric function by this novel brain/gut peptide.

Background: Human beta-defensin-4 (hBD-4), a new member of the beta-defensin family, was discovered by an analysis of the genomic sequence. The objective of this study was to clarify hBD-4 expression in human lung tissue, along with the inducible expression in response to infectious stimuli, localization, and antimicrobial activities of hBD-4 peptides. We also investigated the participation of hBD-4 in chronic lower respiratory tract infections (LRTI) by measuring the concentrations of hBD-4 peptides in human bronchial epithelial lining fluid (ELF)., Methods: The antimicrobial activity of synthetic hBD-4 peptides against E. coli and P. aeruginosa was measured by radial diffusion and colony count assays. We identified hBD-4 in homogenated human lung tissue by reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay (RIA). Localization of hBD-4 was studied through immunohistochemical analysis (IHC). We investigated the effects of lipopolysaccharide (LPS) on hBD-4 expression and its release from small airway epithelial cells (SAEC). We collected ELF from patients with chronic LRTI using bronchoscopic microsampling to measure hBD-4 concentrations by RIA., Results: hBD-4 exhibited salt-sensitive antimicrobial activity against P. aeruginosa. We detected the presence of hBD-4 peptides in human lung tissue. IHC demonstrated the localization of hBD-4-producing cells in bronchial and bronchiolar epithelium. The levels of hBD-4 peptides released from LPS-treated SAECs were higher than those of untreated control cells. ELF hBD-4 was detectable in 4 of 6 patients with chronic LRTI, while the amounts in controls were all below the detectable level., Conclusion: This study suggested that hBD-4 plays a significant role in the innate immunity of the lower respiratory tract.

Ghrelin and cholecystokinin (CCK) are gastrointestinal hormones regulating feeding. Both transmitted via the vagal afferent, ghrelin elicits starvation signals, whereas CCK induces satiety signals. We investigated the interaction between ghrelin and CCK functioning in short-term regulation of feeding in Otsuka Long-Evans Tokushima fatty (OLETF) rats, which have a disrupted CCK type A receptor (CCK-AR), and their lean littermates, Long-Evans Tokushima Otsuka (LETO) rats. Intravenous administration of ghrelin increased 2-h food intake in both OLETF and LETO rats. Because OLETF rats are CCK insensitive, iv-administered CCK decreased 2-h food intake in LETO, but not in OLETF, rats. Although preadministration of CCK to LETO rats blocked food intake induced by ghrelin, CCK preadministration to OLETF rats did not affect ghrelin-induced food intake. Conversely, preadministration of ghrelin to LETO rats blocked feeding reductions induced by CCK. In electrophysiological studies, once gastric vagal afferent discharges were altered by ghrelin or CCK administration, they could not be additionally affected by serial administrations of either CCK or ghrelin, respectively. The induction of Fos expression in the hypothalamic arcuate nucleus by ghrelin was also attenuated by CCK preadministration. Using immunohistochemistry, we also demonstrated the colocalization of GH secretagogue receptor (GHS-R), the cellular receptor for ghrelin, with CCK-AR in vagal afferent neurons. These results indicate that the vagus nerve plays a crucial role in determining peripheral energy balance. The efficiency of ghrelin and CCK signal transduction may depend on the balance of their respective plasma concentration and/or on interactions between GHS-R and CCK-AR.

Peptide YY (PYY), an anorectic peptide, is secreted postprandially from the distal gastrointestinal tract. PYY(3-36), the major form of circulating PYY, binds to the hypothalamic neuropeptide Y Y2 receptor (Y2-R) with a high-affinity, reducing food intake in rodents and humans. Additional gastrointestinal hormones involved in feeding, including cholecystokinin, glucagon-like peptide 1, and ghrelin, transmit satiety or hunger signals to the brain via the vagal afferent nerve and/or the blood stream. Here we determined the role of the afferent vagus nerve in PYY function. Abdominal vagotomy abolished the anorectic effect of PYY(3-36) in rats. Peripheral administration of PYY(3-36) induced Fos expression in the arcuate nucleus of sham-operated rats but not vagotomized rats. We showed that Y2-R is synthesized in the rat nodose ganglion and transported to the vagal afferent terminals. PYY(3-36) stimulated firing of the gastric vagal afferent nerve when administered iv. Considering that Y2-R is present in the vagal afferent fibers, PYY(3-36) could directly alter the firing rate of the vagal afferent nerve via Y2-R. We also investigated the effect of ascending fibers from the nucleus of the solitary tract on the transmission of PYY(3-36)-mediated satiety signals. In rats, bilateral midbrain transections rostral to the nucleus of the solitary tract also abolished PYY(3-36)-induced reductions in feeding. This study indicates that peripheral PYY(3-36) may transmit satiety signals to the brain in part via the vagal afferent pathway.

Ghrelin, an acylated peptide originally identified in rat stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), stimulates both food intake and growth hormone (GH) secretion. Ghrelin is predominantly synthesized by a subset of endocrine cells in the oxyntic gland of human and rat stomach. Previous studies using immunohistochemistry have shown that ghrelin is also present in the hypothalamic arcuate nucleus, a region critical for the control of feeding and GH secretion, but its expression pattern in this region and the details of its molecular form has yet to be clarified. In this report, we examined the presence of ghrelin in the arcuate nucleus using reverse-phase liquid chromatography combined with radioimmunoassay (RIA) and immunohistochemistry. Neurons in the arcuate nucleus were observed to react positively to ghrelin antibodies. In addition, we confirmed the existence of ghrelin mRNA expression using the reverse-transcription polymerase chain reaction (RT-PCR). We also observed the colocalization of GHS-R with neuropeptide Y (NPY) and growth-hormone-releasing hormone (GHRH) in the arcuate nucleus. The present study clearly indicates that ghrelin is synthesized in the arcuate nucleus, which will further our understanding of ghrelin's actions in the central nervous system, including feeding behavior and GH secretion.

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.

Objective: In recent years great advances have been made in our understanding of the peripheral signals produced within the gastrointestinal tract that regulate appetite, such as ghrelin and peptide YY (PYY). While ghrelin elicites hunger signals, PYY elicites satiety. Therefore, alterations in hormone physiology may play a role in the pathogenesis of bulimia nervosa (BN). In this study, we investigated the postprandial profile of ghrelin and PYY levels in patients with BN., Design and Patients: Postprandial plasma ghrelin and PYY levels and insulin and glucose responses were measured in 10 patients with BN and 12 control patients in response to a standard 400 kcal meal., Results: Basal ghrelin levels present in BN subjects (265.0 +/- 25.5 pmol/l) were significantly higher than those in healthy controls (199.3 +/- 18.4 pmol/l, P < 0.05), while basal PYY levels were equivalent in BN (14.6 +/- 1.3 pmol/l) and control (12.8 +/- 1.1 pmol/l, P = 0.30) subjects. Postprandial ghrelin suppression (decremental ghrelin area under the curve) was significantly attenuated in BN patients, compared to controls (-96.3 +/- 26.8 pmol/l x 3 h vs. -178.2 +/- 25.7 pmol/l x 3 h, P < 0.05). After a meal, the incremental PYY area under the curve in BN patients was significantly blunted from that observed in controls (9.2 +/- 2.6 pmol/l x 3 h vs. 26.8 +/- 3.2 pmol/l x 3 h, P < 0.01). Glucose and insulin responses to meals were similar between the two groups., Conclusions: BN patients exhibit elevated ghrelin levels before meals with reduced ghrelin suppression after eating. In bulimia nervosa subjects, the rise in PYY levels after meals is also blunted. A gut-hypothalamic pathway involving peripheral signals, such as ghrelin and PYY, may be involved in the pathophysiology of BN.

Ghrelin is primarily secreted from the stomach and has been implicated in the coordination of eating behavior and weight regulation. The effects of Helicobacter pylori infection on plasma ghrelin concentration and gastric ghrelin production still have not been well known. We determined plasma ghrelin concentration in a total of 160 consecutive individuals with normal body mass index including 110 H. pylori-infected and 50 H. pylori-negative subjects. The expression levels of ghrelin mRNA and ghrelin-producing cells in the gastric mucosa were quantified with real-time quantitative RT-PCR and immunohistochemistry, respectively. The severity of gastric atrophy was evaluated by serum pepsinogen concentrations. Plasma ghrelin concentration, gastric ghrelin mRNA, and ghrelin-positive cell numbers in gastric mucosa were significantly lower in H. pylori-infected subjects. The decrease in plasma ghrelin concentration in H. pylori-positive subjects was accompanied by an attenuation of ghrelin mRNA expression and a reduction of ghrelin-positive cell numbers in the gastric mucosa. Moreover, lower serum pepsinogen I concentrations and I/II ratio were significantly associated with lower plasma ghrelin concentrations in H. pylori-positive subjects. These findings suggest that impaired gastric ghrelin production in association with atrophic gastritis induced by H. pylori infection accounts for the decrease in plasma ghrelin concentration.

Ghrelin, a brain-gut peptide discovered from the stomach, stimulates growth hormone release, food intake, adiposity, and weight gain. Circulating ghrelin levels are modulated under conditions of positive and negative energy balance, however its effect on macronutrient selection is not known. The present experiment investigates the effect of ghrelin on single and two-diet feeding paradigms in high-carbohydrate (HC) and high-fat (HF) preferring rats. In the macronutrient selection test in which rats were given free access to either high-carbohydrate or high-fat diet, an intracerebroventricular (i.c.v.) administration of ghrelin potently enhanced fat intake over carbohydrate intake in both HC- and HF-preferring rats. In the diet preference test in which rats were given free access to both high-carbohydrate and high-fat diets simultaneously, an i.c.v. administration of ghrelin also preferentially enhanced fat consumption over carbohydrate in both HF- and HC-preferring rats. Intracerebroventricular administrations of galanin and neuropeptide Y enhanced fat and carbohydrate ingestion, respectively. Centrally administered ghrelin enhanced fat ingestion. These results provide further insights for the role of ghrelin in feeding behavior and the development of obesity.

Objectives: To report the clinical features of two Japanese brothers with familial leptomeningeal amyloidosis, showing a causative gene abnormality of a transthyretin (TTR) variant Asp18Gly, previously reported only in a Hungarian family., Methods: The authors reported on a 42 year old man (patient 1) and his 45 year old brother (patient 2), both suffering from subarachnoid haemorrhage (SAH) without and with hydrocephalus, respectively. DNA sequences of the TTR gene were determined in both patients and the patients' clinical features described. A surgical biopsy of the leptomeninges was performed on patient 1., Results: DNA sequence analyses demonstrated the glycine-for-aspartate substitution at position 18 of the TTR variant. Both patients revealed pyramidal tract signs and cerebellar ataxia. Audiometric studies showed bilateral, mild sensorineural hearing loss in the patients whose cerebrospinal fluid (CSF) protein levels increased. T1 weighted MRI after contrast administration showed diffuse leptomeningeal enhancement along the Sylvian fissures and over the surface of the brainstem, cerebellum, and spinal cord. Gradient echo T2* weighted MRI showed superficial siderosis mainly in the cerebellum. A biopsy of the leptomeninges was obtained from the spinal cord of patient 1. While performing the biopsy, the authors observed the varicose, elongating, and fragile veins on the dorsal surface of the spinal cord. Immunohistochemical study revealed marked deposits of TTR derived amyloid on his leptomeninges., Conclusions: This is the second report of familial leptomeningeal amyloidosis with an Asp18Gly TTR gene mutation, clinically causing only CNS symptoms. Repeated SAH from fragile veins on the dorsal surface of the spinal cord seemed to induce superficial siderosis of the CNS. So far, there have been two reliable hallmarks leading to the diagnosis of leptomeningeal amyloidosis: diffuse leptomeningeal enhancement on contrast MRI and greatly increased CSF protein content. This study has contributed a third hallmark: the presence of superficial siderosis is useful in diagnosing leptomeningeal amyloidosis.

Objective: Defensins (alpha- and beta-defensins) are endogenous antimicrobial peptides. Little is known about alpha-defensins during Helicobacter pylori infection., Methods: The concentrations of human neutrophil peptides (HNP-1, -2, and -3), the major components of neutrophils-derived alpha-defensins, were measured by radioimmunoassay (RIA) in plasma and gastric juice of 61 H. pylori-infected and 33 uninfected subjects, and before and after anti-H. pylori treatment in 12 patients with H. pylori-associated gastritis. Interleukin (IL)-8 concentrations in gastric juice were measured by enzyme-linked immunosorbent assay. Histological grades of gastritis and neutrophil counts (/mm(2)) infiltrating in the gastric mucosa were determined using two biopsy specimens taken from the antrum and corpus. Immunohistochemistry and reverse-phase high performance liquid chromatography (RP-HPLC) were used to identify HNPs 1-3., Results: HNP 1-3 concentrations in gastric juice were significantly higher in H. pylori-positive than in H. pylori-negative patients and significantly decreased after cure. HNP 1-3 concentrations in gastric juice correlated with IL-8 levels and neutrophil densities in the gastric mucosa and were associated with histological degree of gastritis, especially the grades of activity. Intense immunoreactivity for anti-HNPs 1-3 antiserum was noted in infiltrating neutrophils in H. pylori-infected mucosa. In RP-HPLC analysis, all of the HNP 1-3 molecules were identified as their mature forms. Plasma HNP 1-3 concentrations were similar in H. pylori-infected and non-infected groups and showed no correlations with other parameters., Conclusions: We demonstrated significantly elevated levels of HNPs 1-3 in gastric juice during H. pylori infection. The elevation of HNPs is presumably secondary to H.pylori-associated gastric inflammation involving neutrophil infiltration.

Background: Sarcoidosis is a systemic granulomatous disease of unknown aetiology. It has been suggested that T helper type 1 (Th1) polarisation is associated with the pathophysiology of sarcoidosis, but the mechanism of skewing towards Th1 has not been elucidated. Dendritic cells (DCs) are known to regulate immune responses. This study was performed to determine whether DCs are involved in the aetiology of sarcoidosis., Methods: The numbers of peripheral blood DCs in 24 patients with sarcoidosis were analysed and biopsy specimens from four patients were stained immunohistochemically using monoclonal antibodies., Results: The numbers of both myeloid and lymphoid DC subsets were significantly decreased in the blood and mature DCs were found in the granulomas of patients with sarcoidosis. A number of interferon-gamma (IFN-gamma) producing T cells were also detected in the sarcoid granuloma, as well as many interleukin (IL)-4 producing T cells. Double staining of the biopsy specimen using anti-fascin and anti-CD3 antibodies showed an anatomical interaction between DCs and T cells., Conclusions: These findings suggest that the blood DC subsets may migrate into the affected tissues, contributing to the formation of the granulomas in sarcoidosis. It is hypothesised that the migrating DCs may regulate the T cell response in sarcoidosis, at least in the granulomatous lesions.

Ghrelin is a gastrointestinal peptide that stimulates food intake and growth hormone (GH) secretion. We studied the biosynthesis and secretion of ghrelin in a cancer cachexia mouse model. G361, a human melanoma cell line, was inoculated into nude mice. The body weight was reduced and the plasma concentration of interleukin-1beta (IL-1beta) was markedly higher in tumor-inoculated mice compared with vehicle-treated mice. Furthermore, white adipose tissue (WAT) weight, blood sugar level, and plasma concentrations of leptin and nonesterified fatty acids (NEFA) were significantly lower in tumor-inoculated mice. The plasma concentration of ghrelin increased with the progression of cachexia. The levels of both ghrelin peptide and mRNA in the stomach were also upregulated in tumor-inoculated mice. This study demonstrates that both ghrelin biosynthesis and secretion are stimulated in the long-term negative energy balance of tumor-inoculated cachectic mice. These findings suggest the involvement of ghrelin in the regulation of energy homeostasis in cancer cachexia.

Neuromedin U (NMU), a hypothalamic peptide, has been known to be involved in feeding behavior as a catabolic signaling molecule. However, little is known about the participation of NMU in the neuronal network. One NMU receptor, NMU2R, is abundantly expressed in the hypothalamic paraventricular nucleus, where corticotrophin-releasing hormone (CRH) is synthesized. The functions of CRH, regulation of stress response and feeding behavior, are comparable with those of NMU. Here, we have investigated the functional relationships between NMU and CRH using CRH knockout (KO) mice. Intracerebroventricular administration of NMU suppressed dark-phase food intake and fasting-induced feeding in wild-type mice. In contrast, these suppressions were not observed in CRH KO mice. NMU-induced increases in oxygen consumption and body temperature were attenuated in CRH KO mice. These results suggest that NMU plays a role in feeding behavior and catabolic functions via CRH. This study demonstrates a novel hypothalamic pathway that links NMU and CRH in the regulation of feeding behavior and energy homeostasis.