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Epidemiological and animal studies have demonstrated that ingestion of green tea enhances healthy life. However, caffeine in green tea can interfere with sleep. In this report, we examined the effect of green tea with lowered caffeine, low-caffeine green tea, on stress and sleep of the elderly. The participants (n = 10, mean age 89.3 ± 4.2 years) drank five cups/day of standard green tea for 1 week. Subsequently, they drank five cups/day of low-caffeine green tea for 2 weeks. Salivary α-amylase activity (sAA) was measured as a stress marker. Sleep stages were measured using a portable electroencephalography (n = 7, 6 female and 1 male). The level of sAA in the morning (sAAm) was significantly lower when the participants drank low-caffeine green tea than standard green tea. While the levels of sAAm were different among individuals, lower sAAm correlated with a higher quality of sleep. In those participants whose sAAm was lowered by the ingestion of low-caffeine green tea, some sleep parameters improved. Daily ingestion of low-caffeine green tea may be a beneficial tool for improving the quality of sleep of the elderly via the suppression of stress, although further research is required to fortify this hypothesis.

Chronic stress can impair the health of human brains. An important strategy that may prevent the accumulation of stress may be the consumption of functional foods. When senescence-accelerated mice prone 10 (SAMP10), a stress-sensitive strain, were loaded with stress using imposed male mouse territoriality, brain volume decreased. However, in mice that ingested theanine (6 mg/kg), the main amino acid in tea leaves, brain atrophy was suppressed, even under stress. On the other hand, brain atrophy was not clearly observed in a mouse strain that aged normally (Slc:ddY). The expression level of the transcription factor Npas4 (neuronal PAS domain protein 4), which regulates the formation and maintenance of inhibitory synapses in response to excitatory synaptic activity, decreased in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but increased in mice that ingested theanine. Lipocalin 2 (Lcn2), the expression of which increased in response to stress, was significantly high in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but not in mice that ingested theanine. These data suggest that Npas4 and Lcn2 are involved in the brain atrophy and stress vulnerability of SAMP10 mice, which are prevented by the consumption of theanine, causing changes in the expression of these genes.

Beta-cryptoxanthin (β-CRX, (3R)-β, β-caroten-3-ol) is an oxygenated carotenoid and a potent antioxidant that is abundant in Satsuma mandarin orange (Citrus unshiu MARC.), which is the most popular fruit in Japan. Since our preliminary data suggested that the ingestion of β-CRX had an anti-stress effect in female participants, the effect was evaluated in another set of female participants. The study design was a double-blind group comparison and participants (n = 23) were randomly assigned to β-CRX-rich orange juice or placebo (β-CRX was removed from orange juice) groups. β-CRX or placebo juice (125 mL, after breakfast) were consumed from 1 week prior to pharmacy practice and continued for 5 d into the practice period. Salivary α-amylase activity (sAA), a marker of sympathetic nervous system activity, was significantly higher in the evening than in the morning in the placebo-group during pharmacy practice, but not in the β-CRX-group. This result supports the anti-stress effect of β-CRX. The dose-dependency of β-CRX was observed in male mice that were loaded with stress. These results indicate that the ingestion of β-CRX is helpful to reduce stress.

The young leaves of green tea become lighter in color than usual when protected from sunlight by a shading net for about two weeks while growing. These leaves are called &ldquo, shaded white leaf tea&rdquo, or SWLT. In the eluate of SWLT, the amount of amino acids (361 mg/L) was significantly higher than that in regular tea (53.5 mg/L). Since theanine and arginine, the first and second most abundant amino acids in SWLT, have significant antistress effects, we examined the antistress effect of SWLT on humans. SWLT or placebo green tea (3 g) was eluted with room-temperature water (500 mL). Participants consumed the tea for one week prior to pharmacy practice and continued for 10 days in the practice period. The state-trait anxiety inventory, an anxiety questionnaire, tended to be scored lower in the SWLT group than the placebo, but other stress markers showed no differences. The effect of the difference in SWLT components examined with mice showed that aspartic acid and asparagine, which are abundant in SWLT, counteracted the antistress effects of theanine and arginine. Large amounts of caffeine also interfered with SWLT&rsquo, s antistress effect. Thus, SWLT, which is high in caffeine and amino acids, suppressed depressant behavior in mice.

Beta-cryptoxanthin [β-CRX, (3R)-β, β-caroten-3-ol] is a provitamin A and a potent antioxidant that is abundant in Satsuma mandarin orange (Citrus unshiu MARC.), which is the most popular fruit in Japan. The anti-stress effect of β-CRX on humans was evaluated in fifth-year university students during both routine daily life at the university and at pharmacy practice. The study design was a double-blind group comparison and participants (n=20; female 12, male 8) were randomly assigned to β-CRX-rich orange juice or placebo (β-CRX was removed from orange juice) groups. β-CRX or placebo juice (125 mL, once a day, after breakfast) were consumed from 10 d prior to the pharmacy practice and continued for 10 d into the practice period. To assess participants' anxiety, the state-trait anxiety inventory test was carried out before the pharmacy practice. Salivary α-amylase activity (sAA) was measured as a marker of sympathetic nervous system activity. In the placebo-group, sAA in the evening (post-practice sAA) tended to be higher than sAA in the morning (pre-practice sAA) during both routine daily life at the university and during pharmacy practice. In the β-CRX-group, the increase of post-practice sAA was suppressed in females. These results suggested that β-CRX has an anti-stress effect, at least, in females.

The effects of soybean extracts were investigated in senescence-accelerated (SAMP10) mice, a mouse model of brain senescence with cognitive dysfunction. Mature soybeans are usually yellow. However, the green soybean retains green color after being ripened. Cognitive functions were significantly better-preserved in aged mice fed green soybean than age-matched control mice with or without yellow soybean feeding. Molecular mechanisms of the beneficial effect of green soybean on brain functions were examined through transcriptome analysis of SAMP10 hippocampus. The high expression of Ptgds was significantly associated with green soybean diet, which encodes lipocalin-type prostaglandin D2 synthase, a putative endogenous amyloid β(Αβ)-chaperone. In consonance, Aplp1 expression was significantly reduced, a member of amyloid precursor proteins. Furthermore, the amount of Aβ 40 and 42 was reduced in the insoluble fraction of cerebral cortex. These results suggest that the intake of green soybean ameliorates cognitive dysfunction of aged mice through the reduction of Aβ accumulation.

Caffeine, one of the main components in green tea, can interfere with sleep and block the effect of theanine. Since theanine, the main amino acid in tea leaves, has significant anti-stress effects in animals and humans, we examined the effects of green tea with lowered caffeine content, i.e., low-caffeine green tea (LCGT), on stress and quality of sleep of middle–aged individuals (n = 20, mean age 51.3 ± 6.7 years) in a double-blind crossover design. Standard green tea (SGT) was used as the control. These teas (≥300 mL/day), which were eluted with room temperature water, were consumed over a period of seven days after a single washout term. The level of salivary α-amylase activity (sAA), a stress marker, was significantly lower in participants that consumed LCGT (64.7 U/mL) than in those that consumed SGT (73.9 U/mL). Sleep quality was higher in participants that consumed a larger quantity of LCGT. In addition, a self-diagnostic check for accumulated fatigue was significantly lower in those participants that consumed LCGT than SGT. These results indicate that LCGT intake can reduce stress in middle-aged individuals and improve their quality of sleep. The reduction in caffeine is suggested to be a valid reason for enhancing the anti-stress effect of green tea.

Theanine, an amino acid in tea, has significant anti-stress effects on animals and humans. However, the effect of theanine was blocked by caffeine and gallate-type catechins, which are the main components in tea. We examined the anti-stress effect of green tea with lowered caffeine, low-caffeine green tea, on humans. The study design was a single-blind group comparison and participants (n=20) were randomly assigned to low-caffeine or placebo tea groups. These teas (≥500 mL/d), which were eluted with room temperature water, were taken from 1 week prior to pharmacy practice and continued for 10 d in the practice period. The participants ingested theanine (ca. 15 mg/d) in low-caffeine green tea. To assess the anxiety of participants, the state-trait anxiety inventory test was used before pharmacy practice. The subjective stress of students was significantly lower in the low-caffeine-group than in the placebo-group during pharmacy practice. The level of salivary α-amylase activity, a stress marker, increased significantly after daily pharmacy practice in the placebo-group but not in the low-caffeine-group. These results suggested that the ingestion of low-caffeine green tea suppressed the excessive stress response of students. This study was registered at the University Hospital Medical Information Network (ID No. UMIN14942).

Small-cell lung carcinoma releases progalanin. The released progalanin is activated via a nonclassical processing pathway, being processed into an active form of galanin (1-20) by plasmin in extracellular components. Plasmin is produced from plasminogen activators. To clarify the regulation of progalanin via plasminogen activation by urokinase and tissue-plasminogen activator (t-PA), we investigated the regulation mechanism for urokinase and t-PA expression and their effect on galanin activation. Additionally, we studied the effect of activated galanin on angiogenesis. To determine the effect of cell density, we measured the expression levels of urokinase and t-PA using real-time PCR and plasminogen/gelatin zymography in a cell culture. The urokinase expression increased under both high cell density and presence of cell membrane fractions. However, urokinase increments induced by conditioned medium were low. These results indicate that expression of plasminogen activators is regulated by cell membrane factors. We used tumor-bearing mice to clarify the expression of plasminogen activators and galanin activation. Real-time PCR showed that urokinase was substantially higher in the central parts of tumors compared to the periphery, and this was confirmed by plasminogen/gelatin zymography. To evaluate the biological effect of plasminogen activators on tumor growth, we used tranexamic acid as a plasminogen inhibitor. Tranexamic acid decreased galanin (1-20) and the hemoglobin content of tumors and suppressed tumor growth. Additionally, galanin had no effect on the hemoglobin content of tumors derived from cells lacking GALR2. These results demonstrate the regulation of urokinase expression in tumors through progalanin activation in extracellular compartments, and confirm that galanin plays a role in angiogenesis.

The senescence-accelerated mouse prone10 (SAMP10) strain, a model of aging, exhibits cognitive impairments and cerebral atrophy. We noticed that SAMP10/TaSlc mice, a SAMP10 substrain, have developed persistent glucosuria over the past few years. In the present study, we characterized SAMP10/TaSlc mice and further identified a spontaneous mutation in the Slc5a2 gene encoding sodium-glucose co-transporter (SGLT) 2. The mean concentration of urine glucose was high in SAMP10/TaSlc mice and increased further with advancing age, whereas other strains of senescence-accelerated mice, including SAMP1/SkuSlc, SAMP6/TaSlc and SAMP8/TaSlc or normal aging control SAMR1/TaSlc mice, exhibited no detectable glucose in urine. SAMP10/TaSlc mice consumed increasing amounts of food and water compared to SAMR1/TaSlc mice, suggesting the compensation of polyuria and the loss of glucose. Oral glucose tolerance tests showed decreased glucose reabsorption in the kidney of SAMP10/TaSlc mice. In addition, blood glucose levels decreased in an age-dependent fashion. The kidney was innately larger than that of control mice with no histological alterations. We examined the expression levels of glucose transporters in the kidney. Among SGLT1, SGLT2, glucose transporter (GLUT) 1 and GLUT2, we found a significant decrease only in the level of SGLT2. DNA sequencing of SGLT2 in SAMP10/TaSlc mice revealed a single nucleotide deletion of guanine at 1236, which resulted in a frameshift mutation that produced a truncated protein. We designate this strain as SAMP10/TaSlc- Slc5a2 slc (SAMP10-ΔSglt2). Recently, SGLT2 inhibitors have been demonstrated to be effective for the treatment of patients with type 2 diabetes (T2D). SAMP10-ΔSglt2 mice may serve as a unique preclinical model to study the link between aging-related neurodegenerative disorders and T2D.

Purpose Theanine, an amino acid in tea, has significant anti-stress effect on experimental animals under psychosocial stress. Anti-stress effect of theanine on humans was evaluated in 5th-year university students during pharmacy practice. Method The study design was a single-blind group comparison and participants (n = 20) were randomly assigned to theanine or placebo groups. Theanine or placebo (lactose) tablets (200 mg, twice a day, after breakfast and lunch) were taken from 1 week prior to the pharmacy practice and continued for 10 days in the practice period. To assess the anxiety of the participants, the state–trait anxiety inventory test was carried out before the pharmacy practice. Salivary α-amylase activity (sAA) was measured as a marker of sympathetic nervous system activity. Results In the placebo-group, sAA in the morning (pre-practice sAA) was higher than in theanine-group during the pharmacy practice ( p = 0.032). Subjective stress was significantly lower in the theanine-group than in the placebo-group ( p = 0.020). These results suggest that theanine intake had anti-stress effect on students. Furthermore, students with higher pre-practice sAA showed significantly higher trait anxiety in both groups ( p = 0.015). Similarly, higher pre-practice sAA was correlated to shorter sleeping time in both groups ( p = 0.41 × 10 − 3 ). Conclusion Stressful condition increased the level of sAA that was essentially affected by individual trait anxiety. The low levels of pre-practice sAA and subjective stress in the theanine-group suggest that theanine intake suppressed initial stress response of students assigned for a long-term commitment of pharmacy practice.

Green soybean (Glycine max L.), which was used in this study, retains the green color of the seed coat cotyledon even after ripening, even though many soybean cultivars become yellow during maturation. We have reported that ingestion of green soybean extract (GSE) suppressed cognitive dysfunction and reduced amyloid β accumulation more than yellow soybean extract (YSE) in aged senescence-accelerated (SAMP10) mice, a mouse model of brain senescence. To clarify the mechanism by which GSE suppresses cognitive dysfunction, we examined the effect of GSE and YSE on neurite outgrowth in human neuroblastoma SHSY-5Y cells. GSE significantly increased cell number and neurite outgrowth at 5 ng/ml (as isoflavones, 30 pg/ml), but the effect of YSE was lower than that of GSE. Although isoflavone aglycones, genistein and daidzein increased the number of SH-SY5Y cells, the effect was only observed at a high concentration [0.05 μM (13 ng/ml)]. ICR mice were fed a diet containing 3% soybean extract for 3 weeks (3-4 g/kg; as isoflavones, ca. 20 mg/kg) to examine cognitive function. Learning and memory abilities, as evaluated by a step-through passive avoidance task, a Y-maze and a novel object recognition test, were significantly higher in mice that ingested GSE than control mice that were fed a normal diet. The expression of brain-derived neurotrophic factor (BDNF) in the hippocampus, as detected by western blot analysis, was higher in mice that ingested GSE than in control mice. This study suggests that the ingestion of GSE enhances learning and memory abilities in mice. The effect by which GSE improved cognitive function appears to be caused, in part, by increased neuritogenesis and BDNF expression.

The antistress effect of theanine (γ-glutamylethylamide), an amino acid in tea, was investigated using mice that were psychosocially stressed from a conflict among male mice in conditions of confrontational housing. Two male mice were housed in the same cage separated by a partition to establish a territorial imperative. When the partition was removed, the mice were co-housed confrontationally. As a marker for the stress response, changes in the adrenal gland were studied in comparison to group-housed control mice (six mice in a cage). Significant adrenal hypertrophy was observed in mice during confrontational housing, which was developed within 24 h and persisted for at least 1 week. The size of cells in the zona fasciculata of the adrenal gland, from which glucocorticoid is mainly secreted, increased (∼1.11-fold) in mice during confrontational housing, which was accompanied by a flattened diurnal rhythm of corticosterone and ACTH in blood. The ingestion of theanine (>5 μg ml(-1)) prior to confrontational housing significantly suppressed adrenal hypertrophy. An antidepressant, paroxetin, suppressed adrenal hypertrophy in a similar manner in mice during confrontational housing. In mice that ingested theanine, behavioural depression was also suppressed, and a diurnal rhythm of corticosterone and ACTH was observed, even in mice that were undergoing confrontational housing. Furthermore, the daily dose of theanine (40 μg ml(-1)) blocked the counteracting effects of caffeine (30 μg ml(-1)) and catechin (200 μg ml(-1)). The present study demonstrated that theanine prevents and relieves psychosocial stress through the modulation of hypothalamic-pituitary-adrenal axis activity.

Galanin is a neuropeptide that is widely distributed in the central and peripheral nervous systems. Some small cell lung carcinoma (SCLC) cell lines such as SBC-3A release only the high-molecular-mass form, with lower molecular mass forms being undetectable. To investigate the mechanism of processing of progalanin to active peptide, we studied galanin-LI in both the culture media of SBC-3A cells and in extracts from in vivo mouse SBC-3A tumors. SBC-3A cells were found to release high molecular mass galanin, but did not release active peptides. In contrast, tumor extract contained both high-molecular-mass galanin, and a cleaved lower-molecular-mass form of the peptide (8, 5 and 2 kDa). The lower-molecular-mass peptide was identified as galanin(1-20) by MALDI-TOF mass spectrometry. We then looked at MMP-2 and MMP-9 release from SBC-3A cells and tumor tissue treated with galanin and progalanin, as revealed by gelatin zymography. Galanin elicited pro-MMP-2 and pro-MMP-9 release from SBC-3A cells and tumor tissue; however, recombinant progalanin induced pro-MMP-2 and pro-MMP-9 release from tumor tissue only. This study has shown that the galanin-LI released from SCLC SBC-3A cells consisted of the high-molecular-mass peptide form, and was processed extracellularly to galanin(1-20). Furthermore, galanin was seen to induce pro-MMP-2 and pro-MMP-9 release from SBC-3A cells.

Relaxin-3 is a recently discovered member of the insulin superfamily and is a ligand for three orphan G-protein-coupled receptors: GPCR135, GPCR142 and LGR7 (leucine-rich repeat-containing G-protein-coupled receptor 7). GPCR135 mRNA is expressed in the pancreas, however, it is not known, whether the peptide affects pancreatic islet function. Reverse transcriptase (RT)-PCR and radioreceptor assay have shown that the relaxin-3 receptor (GPCR135) is expressed in pancreatic islets and rat insulinoma, but LGR7 is not expressed. Moreover, relaxin-3 has been revealed to inhibit the secretion of insulin from pancreatic islets. However, we can not detect relaxin-3 in small intestine and pancreas. These results suggest a novel role of relaxin-3 in the regulation of insulin release.

The production of melanin is regulated by α-melanocyte-stimulating hormone (α-MSH), which is produced from proopiomelanocortin (POMC). Keratinocytes release POMC along with lower levels of α-MSH and ACTH. To clarify the mechanism of melanogenesis after ultraviolet (UV)-irradiation, this study focused on the expression of POMC and POMC-derived peptides after UV-irradiation. Western blot analysis and immunoassays indicated that both POMC and α-MSH-like immunoreactivity (α-MSH-LI) increased after UV-irradiation. However, other POMC-derived products were very low. In hypophysectomized mice, α-MSH-LI increased to the same level as in control mice after UV-irradiation. Structural analysis revealed that the major α-MSH-LI product was ACTH(1–8). Furthermore, ACTH(1–8) competed with [125I]-α-MSH for receptor binding and increased melanin production via a melanocortin-1 receptor. These results suggested that melanin was produced through ACTH(1–8) after UV-irradiation. Trypsin-like enzymatic activity, which is responsible for POMC activation, increased after UV-irradiation and was identified as tryptase. In mast cell-deficient mice, which do not produce tryptase, α-MSH-LI levels were unchanged after UV-irradiation. The present study demonstrates the production of ACTH(1–8) from POMC by tryptase, which is a novel peptide-processing mechanism in the extracellular compartment of the skin.

Many intestinal hormones are also distributed throughout the central nervous system (CNS). However, the existence of secretin, a typical intestinal hormone, has not been clearly shown in the brain. There have been conflicting data concerning the expression of secretin in the CNS. The aim of this study was to confirm the existence of secretin in the CNS immunochemically and histochemically. Proforms of secretin were detected in porcine cortex extracts by gel filtration chromatography and reverse-phase HPLC with a radioimmunoassay using different specific antibodies raised against secretin and prosecretin (1-41), indicating the production and processing of secretin precursor in the CNS. An immunocytochemical search in several regions of the rat CNS with the two antibodies revealed that secretin-positive neurons were concentrated in a limited area ranging from the corner of the dentate gyrus to the molecular layer in the hippocampus. In situ hybridization also supported this finding. The limited location of secretin-positive cells shown here caused difficulty in detecting secretin in the CNS. The hippocampus is thought to contribute to aging, and secretinergic neurons in this region might play an important role. This study suggests that secretin is produced in the CNS and may act as a neurotransmitter or neuromodulator in the hippocampus.

cope To understand the mechanism by which green tea lowers the risk of dementia, focus was placed on the metabolites of epigallocatechin gallate (EGCG), the most abundant catechin in green tea. Much of orally ingested EGCG is hydrolyzed to epigallocatechin (EGC) and gallic acid. In rats, EGC is then metabolized mainly to 5-(3’, 5’-dihydroxyphenyl)-γ-valerolactone (EGC-M5) and its conjugated forms, which are distributed to various tissues. Therefore, we examined the permeability of these metabolites into the blood-brain barrier (BBB), and nerve cell proliferation/differentiation in vitro. Methods and results The permeability of EGC-M5, glucuronide and the sulfate of EGC-M5, pyrogallol, as well as its glucuronide into the BBB were examined using a BBB model kit. Each brain- and blood-side sample was subjected to liquid chromatography tandem-mass spectrometry analysis. BBB permeability (%, in 0.5 h) was 1.9-3.7 %. In human neuroblastoma SH-SY5Y cells, neurite length was significantly prolonged by EGC-M5, and the number of neurites was increased significantly by all metabolites examined. Conclusion The permeability of EGC-M5 and its conjugated forms into the BBB suggests that they reached the brain parenchyma. In addition, the ability of EGC-M5 to affect nerve cell proliferation and neuritogenesis suggests that EGC-M5 may promote neurogenesis in the brain. This article is protected by copyright. All rights reserved

Galanin is a neuropeptide expressed in the central and peripheral nervous systems. Galanin is known to be biosynthesized in neural and endocrine cells, but little evidence exists for its synthesis in other cells. In this study, we explored galanin-releasing nonneural cells using radioimmunoassay, finding that some fibroblasts produced and released the galanin-like immunoreactive component (galanin-LI). The molecular weight of the galanin-LI obtained from the fibroblasts, as measured by gel filtration chromatography and Western blotting, was 14 kDa and suggested that the compound was progalanin. Peptide mass fingerprinting analysis identified the large form of galanin-LI as progalanin without its signal sequence. In addition, galanin-LI was located in the Golgi bodies and vesicle-like structures of the fibroblasts. Furthermore, the addition of brefeldin A, an inhibitor of transport from the ER, decreased the release of galanin-LI. In this study, we showed that the fibroblast, a nonneural and nonendocrine cell type, produced and released a galanin precursor, progalanin, without processing via Golgi bodies or secretory vesicles.

Chromogranin A (CgA) is a member of a family of highly acidic proteins, chromogranins, which are co-stored in the adrenergic neurons and paraneurons and co-released with adrenaline and noradrenaline (NAd) in response to adequate stimulation. The present study provides novel evidence that CgA-like immunoreactivity (IR) is stored in the exocrine cells in the granular convoluted tubule, and is secreted into saliva by stimulation with NAd and acetylcholine (ACh) in the isolated and perfused rat submandibular gland. NAd at 1 microM produced maximum secretion of CgA-like IR (<< 0.9 mM) and a marked increase in salivary flow. Further increases in NAd concentration (10 or 100 microM) yielded concentration-dependent decreases in both responses. ACh at 1 microM produced maximum salivary flow and a slight elevation of CgA-like IR secretion (6 microM); 100 microM ACh decreased the salivary flow but increased the CgA-like IR secretion (0.6 mM). Electron microscopic examination showed vigorous compound exocytosis of secretory granules in the cells of the granular convoluted tubule when the submandibular gland was stimulated with 1 microM NAd. These results provide an experimental basis for the view that the salivary CgA-like IR secretion may be a sensitive and quantitative index of the activity of the sympathetic nervous system innervating the gland.

Preeclampsia is a multisystemic disorder specific to human pregnancy. It is common and major complication, characterized by hypertension, proteinuria and oedema. This clinical condition adversely affects the mother and the foetus. The causative factors remain poorly understood. Oxidative stress is implicated in preeclampsia which could provide the linkage between decreased placental perfusion and the maternal syndrome. Pregnancy has also been associated with depressed cell mediated immunity. The increase in activity of adenosine deaminase (ADA) in preeclamptic women compared to normotensive pregnant women is reported in earlier studies. The main objective of the present study was to correlate the adenosine deaminase activity with the antioxidant status in preeclamptic pa-tients. The study groups aged between 20 – 35 years included 20 normotensive primigravida wom-en and 20 primigravida diagnosed as preeclampsia in their last trimester. The control group included 20 healthy nonpregnant women volunteers of the same age group. The diagnosis of preeclampsia was made by a gynecologist at Lady Goshen Hospital. The adenosine deaminase activity and antioxidant status in the form of superoxide dismutase (SOD), glu-tathione (GSH) and total antioxidant activity (TAA) were measured. The extent of oxidative stress was estimated by measuring plasma malondialdehyde (MDA) levels. The comparison of MDA between controls and preeclamptic patients showed significance (p 0.000). The total antioxidant activity (TAA) between the controls and preeclamptic pa-tients showed significance (p 0.000). However, ADA activity did not show statistical signifi-cance among the different study groups. The ADA activity may not be significant due to other systemic causes such as anemia and malnutrition.

Energy homeostasis is regulated by endocrine factors. The concentration of relaxin-3 in serum is related to body mass index. However, relaxin-3 is found only in the brain and testis. In this study, we examined the expression of relaxin- 3 in adipose tissue and its effects on adipogenesis. The expression of relaxin-3 was determined using RT-PCR, a relaxin- 3 C-peptide-specific radioimmunoassay, specifically in the stromal-vascular fraction (SVF) cells rather than adipocytes. The release of C-peptide was regulated by glucose concentration in the SVF cells. However, the differentiated adipocytes did not express relaxin-3. In glucose perfusion experiments, C-peptide was released in response to high glucose concentrations in the mesenteric perfusate, opposite to insulin release. Additionally, GPCR135 mRNA was expressed in adipocytes. Relaxin-3 increased triglycerides in adipocytes and decreased lipase activity. The present study showed that relaxin-3 is secreted from SVF cells and that it regulates lipid accumulation in adipocytes.

Progalanin is released from the small cell lung carcinoma line SBC-3A and converted to its active form by plasmin. To elucidate the role of progalanin activation in the extracellular compartment, matrix metalloproteinase (MMP) activity was studied in SBC-3A cells treated with progalanin siRNA, and angiogenesis was measured in tumor tissue originating from SBC-3A cell transplantation into mice. Progalanin siRNA caused downregulation of progalanin expression for approximately 8 days. MMP activity and angiogenesis were reduced in tumors induced by transplantation of progalanin siRNA-treated SBC-3A cells. In contrast, MMP-9 and MMP-2 activity and angiogenesis increased in tumors originating from progalanin siRNA-treated SBC-3A cells in the presence of galanin and progalanin. Furthermore, injection of tranexamic acid, a plasmin inhibitor, more markedly reduced MMP-9 and MMP-2 activity and angiogenesis in tumors originating from progalanin siRNA-treated SBC-3A cells and in tumor tissue originating from progalanin siRNA-treated SBC-3A cells in the presence of progalanin. The reduction of MMP-9 and MMP-2 activity with tranexamic acid was restored by galanin, but not by progalanin. Moreover, tranexamic acid reduced angiogenesis in control siRNA-treated SBC-3A cells. These results suggest that the activation of progalanin by plasmin in the extracellular compartment was involved in MMP-9 and MMP-2 activation and in angiogenesis in tumor tissue.

Galanin is a neuropeptide widely distributed in the central and peripheral nervous systems. Although its role in non-neural cells is poorly understood, it is known that during inflammation, the dermis layer of the skin produces and releases galanin. The aim of this report is to study the expression of galanin in granulation tissue. After inducing inflammation by cotton thread implantation, galanin-like immunoreactivity (galanin-LI) in plasma reached a maximum on the third day. Galanin-LI was observed in fibroblast-like cells occurring close to collagen fibers in developing granulation tissue. Furthermore, galanin receptor subtypes 1 and 2 (GALR1 and GALR2)-expressing cells were observed around microvessels and were found to produce desmin. Galanin was injected along the cotton threads immediately after implantation, resulting in rapid formation of granulation tissue, and an increase in the contents of microvessels, indicating a stimulatory effect of galanin on the process of angiogenesis in granulation tissue. The results demonstrate that some galanin was released from fibroblast-like cells during the formation of granulation tissue, and that it stimulated angiogenesis.

Galanin is a neuropeptide that is widely distributed in the central and peripheral nervous systems. In a previous study, we showed that a small cell lung carcinoma (SCLC) cell line, SBC-3A, released progalanin but not galanin, and that progalanin was then converted to galanin(1-20), the active form. Because the galanin(1-20) had undergone hydrolysis at Arg and Lys residues, the protease concerned was surmised to have a trypsin-like activity. The present study was performed to identify the trypsin-like protease which had previously been found to activate progalanin in this tumor tissue. The protease was isolated using chromatography and electrophoresis, and identified in tumor extracts from SBC-3A tumor- bearing mice; the major protease was found to be plasmin. We next confirmed that extracellular processing of progalanin occurs in SCLC tumor tissue (tumors produced by the implantation of SBC-3A cells into mice), and in two types of breast tumor tissue (obtained by implantation into mice of BT-549 and MDA-MB-436 cells). In cell culture, processed forms of progalanin were undetectable in SBC-3A, BT-549 or MDA-MB-436 cells. Conversely, gel filtration chromatography analysis of tumor extracts from SBC-3A, BT-549 and MDA-MB-436-bearing mice, revealed that galanin-like immunoreactivity (galanin-LI) in these tumor extracts was due to the presence of progalanin (14 kDa) and galanin(1-20) (2 kDa). Moreover, trypsin-like protease activity was elevated, and plasmin was expressed abundantly in SBC-3A, BT-549 and MDA-MB-436 tumors in mice. In addition, tranexamic acid, a plasmin inhibitor, inhibited progalanin conversion to galanin(1-20). The present study revealed that plasmin was present in tumor tissue, and that it was responsible for processing progalanin to galanin(1-20) in the extracellular environment.

Plasma lipase, C-peptide reactivity (CPR) and human pancreatic polypeptide (HPP) responses after ingestion of elemental diet were studied in 27 patients with chronic pancreatitis. These subjects were classified into 3 groups according to ERP findings; minimum or mild (MIP, n = 17), moderate (MOP, n = 6) and advanced (ADP, n = 4). Basal plasma lipase levels in the MIP and MOP patients were significantly higher than that in the controls (P0.05). Plasma CPR response (sigma delta CPR) in MIP cases were significantly higher than that in controls (P0.05). Also, plasma HPP (response (sigma delta HPP) in MIP cases were significantly higher than that in controls (P0.05). Plasma CPR and HPP responses correlated with the severity of chronic pancreatitis. Fourteen of the 17 MIP patients (82%) showed higher levels of basal lipase or sigma delta HPP in comparison to the respective normal ranges. This study suggested that the ED test may be more sensitive for detection of mild chronic pancreatitis and that it may be useful for evaluating exocrine and endocrine pancreatic functions in various stages of chronic pancreatitis.

The C-terminal [19-29] fragment of glucagon is a modulator of the action of glucagon, the mothermolecule. This was measured directly on cultured cardiac myocytes (inotropic effect) and on the insulin-secreting βTC cell line (insulin release). In both models, the [19-29] fragment clone behaves, at very low concentrations (1-100 pM), as an antagonist (negative inotropic effect, inhibition of insulin release) of the glucagon action. When combined with glucagon, it still antagonized the glucagon action (by 70% at 100 pM in WTC cells) or potentiates the glucabon action (at nM concentrations in the cardiac myocytes)

Alterations in the levels of substance P (SP)-like and vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the respiratory tract of aging guinea pigs were measured using specific radioimmunoassays (RIAs). Normal Hartley male guinea pigs were classified into four groups by age. The trachea, major bronchus and inferior lung were collected from anesthetized guinea pigs. RIAs were done using synthetic SP and guinea-pig VIP (gpVIP) as standard antigens. The age of guinea pigs should be considered when measuring the content of neuropeptides in the airway. Changes in bronchial hyper-reactivity due to aging or to out-grow phenomenon in asthmatic children are possibly related to neuropeptides

Helodermin-like and salmon calcitonin (sCT)-like immunoreactivities co-existed in a subset of human calcitonin (hCT)-containing cells in normal human thyroid tissue and medullary thyroid carcinomas. Helodermin/sCT-immunoreactive cells were mostly different from calcitonin gene-related peptide (CGRP)-positive cells. Helodermin and sCT immunoreactivities were not identified in pulmonary and pancreatic hCT-positive neuroendocrine tumors, except for a few lung tumor cells showing positive staining with one of two sCT antisera used. Helodermin immunoreactivity demonstrated by rabbit antiserum R0086 was completely abolished in the presence of synthetic sCT, while sCT immunoreactivity was not absorbed by synthetic helodermin. The carboxyl terminal Arg30-Thr31 sequence (and Pro35 amide structure) of helodermin would be the epitopic site recognized by this antiserum, since a similar amino acid sequence is present in sCT molecules but absent from hCT and CGRP.

To clarify the dopaminergic regulation of neuropeptide Y (NPY) neurons, the effect of haloperidol on NPY in basal ganglia and the cerebral cortex of the rat brain was investigated by sensitive radioimmunoassay and immunocytochemistry using antiserum against rat NPY. After repeated intraperitoneal injections of haloperidol (5 mg/kg) for 6 days, the content of immunoreactive NPY was significantly decreased in the caudate-putamen, but significantly increased in the lateral prefrontal cortex. After treatment for 21 days, the content of immunoreactive NPY in the caudate-putamen remained significantly low, but the extent of change in the lateral prefrontal cortex diminished. In the medial prefrontal cortex, piriform cortex, parietal cortex and nucleus accumbens, no significant changes were found after treatment for either 6 or 21 days. These findings were compatible with those obtained by immunocytochemistry using the same antiserum: an increase of immunoreactive fibers and terminals in the lateral prefrontal cortex and their decrease in the caudate-putamen. However, in the nucleus accumbens the density of immunoreactive fibers and terminals was decreased in the rostral portion, but not in the caudal portion after haloperidol treatment for 6 and 21 days. These findings suggest that dopaminergic afferents region-specifically regulate dopamine-sensitive NPY neurons in the rat brain.

Gonadotropin-releasing hormone(GnRH)-associated peptide (GAP) is a 56-amino acid peptide found on the C-terminal of the GnRH (also called luteinizing hormone-releasing hormone) precursor and is assumed to be co-produced with GnRH. The purpose of this report is to demonstrate the presence of GAP immunoreactivity in bovine colostrum. Radioimmunoassay of acidified methanolic extracts demonstrated a concentration of GAP immunoreactivity of approximately 1.5 +/- 0.1 pmol/g dry skim bovine colostrum. Gel filtration (Sephadex G-10) and high-performance liquid chromatography of extracts containing GAP immunoreactivity showed it to be of low molecular weight and a high hydrophobic character. The presence of GAP immunoreactivity in bovine colostrum suggests that the GnRH precursor is synthesized and processed in mammary tissue itself.

The accumulation of oxidative damage is believed to contribute to senescence. We have previously found that the consumption of green tea catechins (GT-catechin), which are potent antioxidants, decreases oxidative damage to DNA and improves brain function in aged mice with accelerated senescence (SAMP10 mice). To investigate the mechanisms underlying the beneficial effects of GT-catechin, we measured the activities of antioxidative enzymes in the brains of aged SAMP10 mice. The activity of glutathione peroxidase (GPx), an essential enzyme for reduction of hydrogen and lipid peroxides, was significantly lower in aged mice than in younger ones. However, the decline in activity was prevented in aged mice that had consumed GT-catechin. The increased level of carbonyl proteins, a marker of oxidative damage in proteins, was also significantly reduced in aged mice that had consumed GT-catechin. The activities of superoxide dismutase and catalase were not decreased in aged mice. These results suggest that decreased activity of GPx importantly contributes to brain dysfunction in ageing SAMP10 mice. Furthermore, the intake of GT-catechin protected the decline in GPx activity and age-related oxidative damage in the brain.

Proopiomelanocortin (POMC) is the precursor of melanocyte-stimulating hormone (MSH) and beta-endorphin, and is suggested to have evolved by the insertion and deletion of ancestral MSH segments. Here, the primary structure of POMC was determined with cDNA cloning of brown tree snakes of Squamata and American alligators of Crocodylia to show an overview of the molecular evolution of POMC in reptiles. Snake and alligator POMCs are composed of alpha-, beta-, and gamma-MSH segments and a single beta-END segment as in other tetrapods; however, the gamma-MSH segment in snake POMC has a mutation in the essential sequence from His-Phe-Arg-Trp to His-(d)-(d)-Arg, in which (d) means deletion. It is conceivable that the ancestry of snake gamma-MSH had weak functional constraint and lacked biological significance during evolution. Phylogenetic analyses using the neighbor-joining method show that snake prePOMC is most diverged, and alligator prePOMC is most conserved in reptilian POMCs while it shows the highest sequence identity with ostrich prePOMC. These relationships are comparable to those observed in mitochondrial DNA. On the other hand, analyses of the pituitary with mass spectrometry revealed several peptides by post-translational processing as predicted by the locations of processing sites consisting of basic amino acid residues in snake and alligator POMCs. Remarkably, the monobasic site at the N-terminal side of the snake beta-MSH is suggested to act as a processing site. Thus, the study shows the divergence of snake POMC such as the critical mutation of gamma-MSH and high conservation of hormone organization of alligator POMC.

Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1-15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a recently isolated active peptide of the VIP (vasoactive intestinal peptide) family. Two bioactive forms, PACAP38 and PACAP27, a shorter N-terminal amidated peptide of PACAP38, have been identified. In this study, we explored the action of PACAP27 in rat dispersed pancreatic acini and the characteristics of its binding sites. PACAP27 stimulated amylase secretion and intracellular cAMP production in a dose-dependent manner. Adding 0.5 mM IBMX increased the potency of PACAP27 on the amylase secretion, but did not change the efficacy. The biological action of PACAP27 was mediated via intracellular cAMP as is also the case with PACAP38 or VIP. The time course study, however, revealed that PACAP stimulated the initial amylase secretion greater than VIP, suggesting an involvement of mechanisms other than intracellular cAMP. Binding studies using 125I-PACAP27 and 125I-VIP indicated that the binding sites for PACAP27 interacted with PACAP27 and VIP with a similar affinity. These observations suggested the presence of type II PACAP-binding sites in the normal acini of the rat pancreas which may be functionally coupled with acinar enzyme secretion.

23 galanin-related peptides were synthesized by solid phase technology or conventional solution method. The purity of the products was carefully assessed by routine analytical criteria. Using these synthetic peptides, we have investigated the effects of galanins and structurally modified galanin peptides on glucose-stimulated insulin release using the isolated perfused rat pancreas, gastrin and somatostatin release using the isolated perfused rat stomach, the neurally-evoked muscle contractions in guinea pig ileum and the C-fiber response in the isolated spinal cord of the new born rat. The results suggest that the galanin amino-terminal 1–15 sequence is crucial for its activity in the above four systems. With the goal of developing a specific antagonist of galanin, synthetic glanin(1–15) analogues [ d -Thr6, d -Trp8,9]galanin(1–15)ol, and [ d -Trp8,9]galanin(1–15)ol were found to be a potent antagonist for inhibitory effect of galanin on glucose-induced insulin release.