Neuronal responses of melanin-concentrating hormone and corticotropin-releasing hormone to background color in the self-fertilizing fish, Kryptolebias marmoratus.

We examined neuronal responses of hypothalamic melanin-concentrating hormone (MCH) and corticotropin-releasing hormone (CRH) to background color in the self-fertilizing fish, Kryptolebias marmoratus. Fish were individually reared in lidless white or black cylindrical plastic containers for 15 days. The number of MCH-immunoreactive (ir) cell bodies in the nucleus lateralis tuberis (NLT) of the hypothalamus was significantly greater in the white-acclimated fish, while no significant differences were observed in the nucleus anterior tuberis (NAT) of the hypothalamus. Significant differences were not seen in the number of CRH-ir cell bodies in the NLT between the groups. The body of the white- and black-acclimated fish appeared lighter and darker, respectively, compared with the baseline color. In the black-acclimated fish, feeding activity was significantly greater with a tendency toward higher specific growth rate compared with the observations in white-acclimated fish. No significant inter-group cortisol level differences were observed. These results indicate that background color affects MCH neuronal activity in the NLT as well as body color adaptation but does not affect CRH neuronal activity in K. marmoratus.

Seasonal changes in NRF2 antioxidant pathway regulates winter depression-like behavior.

Seasonal changes in the environment lead to depression-like behaviors in humans and animals. The underlying mechanisms, however, are unknown. We observed decreased sociability and increased anxiety-like behavior in medaka fish exposed to winter-like conditions. Whole brain metabolomic analysis revealed seasonal changes in 68 metabolites, including neurotransmitters and antioxidants associated with depression. Transcriptome analysis identified 3,306 differentially expressed transcripts, including inflammatory markers, melanopsins, and circadian clock genes. Further analyses revealed seasonal changes in multiple signaling pathways implicated in depression, including the nuclear factor erythroid-derived 2-like 2 (NRF2) antioxidant pathway. A broad-spectrum chemical screen revealed that celastrol (a traditional Chinese medicine) uniquely reversed winter behavior. NRF2 is a celastrol target expressed in the habenula (HB), known to play a critical role in the pathophysiology of depression. Another NRF2 chemical activator phenocopied these effects, and an NRF2 mutant showed decreased sociability. Our study provides important insights into winter depression and offers potential therapeutic targets involving NRF2.

Immunohistochemical detection of prolactin-releasing peptide2 in the brain of the inshore hagfish Eptatretus burgeri.

Prolactin-releasing peptide2 (PrRP2) belongs to the RFamide peptide group and is a paralog of prolactin-releasing peptide (PrRP). Recent studies demonstrated that PrRP2, but not PrRP, regulates prolactin release in teleosts. The evolutionary origin of PrRP and PrRP2 dates back to at least early vertebrates because homologs of PrRP/PrRP2 were identified in lampreys, one of the earliest branch of vertebrates class Agnatha. However, PrRP/PrRP2 remains to be identified in hagfish, another representative species of class Agnatha. Here, we examined the distribution of PrRP2 in the brain and pituitary of the inshore hagfish Eptatretus burgeri to obtain further understanding of the neuroendocrine system of PrRP2. PrRP2-immunoreactive (ir) cell bodies were detected in the infundibular nucleus of hypothalamus (HYinf). PrRP2-ir fibers were restricted around PrRP2-ir cell bodies and were not detected in the dorsal wall of the neurohypophysis compared to the abundant PrRP2-ir fiber distribution in the brain and innervation to the pituitary in other vertebrates. To examine possible reciprocal connections of PrRP2 and other neuropeptides, we further conducted dual-label immunohistochemistry of PrRP2 and the PQRFamide (PQRFa) peptide or corticotropin-releasing hormone (CRH). Reciprocal connections are suggested between PrRP2 and PQRFa neurons as well as between PrRP2 and CRH neurons. The present study demonstrates, for the first time, that PrRP2 is expressed in the brain of inshore hagfish. The restricted distribution of PrRP2-ir fibers in the HYinf suggests that PrRP2 does not directly regulate the pituitary gland, but regulates the function of the HYinf where PQRFa and CRH are expressed.

Milk-derived peptides, Val-Pro-Pro and Ile-Pro-Pro, attenuate atherosclerosis development in apolipoprotein e-deficient mice: a preliminary study.

Milk-derived peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), have angiotensin I-converting enzyme inhibitory activities and blood pressure-lowering effects. We examined the effects of these peptides on the development of atherosclerosis in apolipoprotein E-deficient [apoE(-/-)] mice. For 31 weeks, six-week-old male apoE(-/-) mice received a diet that included one of the following: fermented milk containing both VPP and IPP; casein hydrolysate containing both of these peptides; synthesized VPP; synthesized IPP; enalapril; captopril; or control diet. At the end of feeding, blood biochemistry, aortic atherogenesis, and gene expression by DNA microarray analysis were evaluated. There were no significant changes in the plasma lipid levels and 8-isoprostane, a marker of oxidative stress. The area ratio of intima to media in the aortic arch was significantly lower in the fermented milk, casein hydrolysate, synthesized VPP, enalapril, and captopril groups than in the control group. As is common with diets containing VPP and/or IPP, we observed reductions in mRNA expression of inflammatory cytokines, such as interleukin (IL)-6 and IL-1ß, oxidized low-density lipoprotein receptor, and transcription regulators. These results suggest that a continuous intake of VPP and IPP might be beneficial for preventing atherosclerosis caused by hypercholesterolemia.

Exposure to probiotic Lactobacillus acidophilus L-92 modulates gene expression profiles of epithelial Caco-2 cells.

To understand host gastrointestinal response after exposure to probiotic Lactobacillus acidophilus L-92, microarray analysis of cultured epithelial Caco-2 cells was performed. Of the 187 genes down-regulated after 4 h treatment with L-92, 25 were involved in RNA splicing; 12, in cell cycle; 8 were transcriptional regulators; 2 were involved in ubiquitin proteolysis; 2, in adhesion; 2, in meiosis; 2, in splicing; and 2 encoding cytokines. In the RNA splicing group, genes encoding small nuclear RNAs, nuclear pore complex interacting proteins, RNA binding motif proteins, and SMG1 homologs (phosphatidylinositol 3-kinase-related kinase) were identified. Among the only 13 genes up-regulated by the treatment, 5 were involved in histone structure, and 2 were involved in metabolism. Genes belonging to cell adhesion, transmembrane proteins, mitogen-activated protein kinase, immune response, DNA binding, inflammation, and protein synthesis groups were mainly up-regulated after 20 h of treatment, whereas no significantly down-regulated genes were observed. In the present transcriptome analysis, during the early stage of treatment (four hours of treatment) with L-92, genes involved in cell growth and cell meiosis were mainly repressed. During the late phase of treatment (20 h of treatment), the expression of the genes linked to cell adhesion activity and metabolism for cell growth was enhanced. From the present transcriptome analysis, we suggest that Caco-2 cells slow down cell death and turnover of RNA synthesis as an early response to L-92 treatment; at the late stage of treatment, the genes involved in cell proliferation, transcriptional activity, and apoptosis are activated.

Central distribution of kiss2 neurons and peri-pubertal changes in their expression in the brain of male and female red seabream Pagrus major.

kisspeptins that are encoded by kiss1 gene are now considered the key regulator of reproduction from a number of studies in mammals. In most vertebrates, a paralogue of kiss1, called kiss2, is also present, and the functional significance of kisspeptins is not known precisely. In the present study, we have cloned kiss2 from a perciform teleost, the red seabream Pagrus major. The amino acid sequence deduced from the red seabream kiss2 contained a highly conserved 10-amino-acid residue, Kiss2(10) or kp-10. A kiss1-like transcript was also identified, but it appears to be non-functional due to the presence of a "premature" stop codon. Neurons that express kiss2 mRNA were distributed in the dorsal (NRLd) and ventral (NRLv) parts of nucleus recessi lateralis in the hypothalamus. In some fish a few kiss2-expressing neurons were detected in the preoptic area and nucleus ventralis tuberis. The number of kiss2-expressing neurons in the NRLd was larger during the first spawning season in both males and females compared with fish in the post-spawning periods. In males the number of kiss2 neurons in the NRLd of maturing fish was also larger than those in the post-spawning periods. In males the number of kiss2 neurons in the NRLv showed a similar pattern of changes to that of NRLd, while significant changes were not detected for females. The numbers of gonadotropin-releasing hormone 1 (GnRH1)-immunoreactive neurons in the preoptic area showed a similar pattern of change as those of kiss2 cells of the NRLd in both males and females (and also the NRLv in males). These results are in good agreement with the hypothesis that kiss2 neurons are involved in pubertal processes via regulatory influences on GnRH1 neurons in red seabream.

Effects of Val-Pro-Pro and Ile-Pro-Pro on nondipper patients: a preliminary study.

Much clinical evidence on the antihypertensive effects of the milk-derived antihypertensive peptides Val-Pro-Pro and Ile-Pro-Pro (lactotripeptides) has been reported. However, circadian rhythm effects determined by ambulatory blood pressure monitoring (ABPM) to eliminate the confounding influence of the white-coat effect have not been fully studied. Twelve hypertensive patients not receiving antihypertensive medication (2 men, 10 women; mean age±standard deviation, 63.5±8.3 years) who had been visiting our clinic for more than 1 year participated in this study. Mean (±standard deviation) systolic blood pressure (SBP) and diastolic blood pressure (DBP) were 142.4±2.6 and 83.5±6.4 mm Hg, respectively, at the first office visit. After patients ingested a fermented milk product containing antihypertensive peptides (2.53 mg Val-Pro-Pro; 1.52 mg Ile-Pro-Pro) for more than 4 weeks, both office SBP and DBP were significantly reduced to a mean (±standard deviation) of 133.3±7.0 mm Hg and 76.5±8.4 mm Hg (P<.001 and P<.005 by paired t-test), respectively. The 24-hour SBP and DBP determined by ABPM were reduced from 127.3±2.4 and 78.7±2.3 mm Hg to 120.2±2.4 and 75.0±2.2 mm Hg (P<.001 and P<.05), respectively. Awake-time SBP (08:00-21:00), night-time SBP (0:00-05:00), and early-morning SBP (06:00-07:00) were reduced from 130.9±2.4 to 123.3±2.3 mm Hg, 118.7±2.9 to 113.2±3.4 mm Hg, and 132.8±4.3 to 122.4±3.9 mm Hg (by paired t-test: P<.001, P<.05, and P<.05), respectively. As seen with DBP measured by ABPM, 24-hour DBP and awake-time DBP were significantly reduced from 78.7±2.3 to 75.0±2.2 mm Hg and 82.1±2.5 to 77.3±2.2 mm Hg (P<.05 and P<.01), respectively. Office BP and 24-hour blood pressure did not significantly differ between the dipper and nondipper groups at baseline. However, after treatment, night-time and early-morning blood pressure were significantly reduced from baseline in the nondipper group (-8.5±2.5 and -15.6±3.7 mm Hg; P<.05 and P<.01, respectively) but not in the dipper group (-2.5±3.6 and -1.2±4.7 mm Hg; P not significant), and the reduction in early-morning blood pressure significantly differed between the groups (P<.05). These results suggest that Val-Pro-Pro and Ile-Pro-Pro decrease blood pressure in patients with stage I hypertension and result not only in lower blood pressure at night-time but also in lower early-morning SBP in nondipper patients.

Beneficial potential of casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro on central blood pressure and hemodynamic index: a preliminary study.

Pharmaceutical angiotensin-converting enzyme (ACE) inhibitors have been shown to reduce arterial stiffness; the possible effect of food-derived putative ACE inhibitory peptides on this degenerative process, however, has not been reported. In the present study, casein hydrolysate containing the lactotripeptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), which has been found to have an antihypertensive effect in a number of clinical studies, was investigated for its ability to improve hemodynamic parameters, including central systolic blood pressure (cSBP), in hypertensive subjects. Twelve hypertensive subjects who were not on prescribed medication were monitored for various hemodynamic parameters, including brachial blood pressure (peripheral blood pressure), cSBP, and augmentation index (AI), at the start and then after 3, 6, and 9 weeks of a daily treatment comprising four tablets containing VPP and IPP. Compared with basal levels, treatment with casein hydrolysate for 6 and/or 9 weeks showed a significant reduction in peripheral systolic and diastolic blood pressure, AI, and cSBP, but not in heart rate or pulse pressure. cSBP showed a reduction sooner and greater (-21.8 mm Hg) than did brachial systolic blood pressure (-16.4 mm Hg) during the 9-week treatment. Although small and not placebo-controlled, this study suggests that continuous intake of VPP and IPP might have the potential to improve arterial stiffness as well as cSBP and peripheral brachial blood pressure.

Effect of milk fermented with Lactobacillus acidophilus strain L-92 on symptoms of Japanese cedar pollen allergy: a randomized placebo-controlled trial.

A placebo-controlled, single-blind study was conducted to evaluate the effects of Lactobacillus acidophilus strain L-92 (L-92) on the symptoms of Japanese cedar-pollen allergy. This study was carried out during the 2002 and 2003 seasons of Japanese cedar pollination. Twenty-three in-house volunteers were asked to drink 100 ml of heat-treated milk fermented with L-92 containing 5 x 10(10) of the bacteria, twice a day, for 6 consecutive weeks. A similar study was carried out during the 2003 season for 10 weeks, but the daily dose of bacteria was 2 x 10(10). A significant improvement of the ocular symptom-medication score (SMS) was observed in 2002 and of the score of distress of life in 2003. These data show that a daily oral intake of not less than 2 x 10(10) heat-treated L-92 cells improved the symptoms of Japanese cedar pollinosis, thereby contributing to reduce the dose of concomitant medications. However, no blood parameter was significantly affected in these trials.

Fiber connections of the anterior preglomerular nucleus in cyprinids with notes on telencephalic connections of the preglomerular complex.

Fiber connections of the anterior preglomerular nucleus (PGa) were studied in carp and goldfish by tracer injection experiments to the nucleus and telencephalon. The PGa received fibers from the central nucleus of semicircular torus, perilemniscular nucleus, anterior tuberal nucleus, and medial pretoral nucleus, all of which are presumed auditory structures. The PGa projected to the dorsal (dDm) and ventral (vDm) regions of medial part of dorsal telencephalic area. The caudomedial region of lateral preglomerular nucleus (PGl) and medial zone of medial preglomerular nucleus (PGm), which also receive auditory toral fibers, projected to the same telencephalic regions as did PGa. These preglomerular structures and the PGa also received in common descending fibers from a rostromedial portion of dDm. The PGa also received fibers from the parvocellular and magnocellular preoptic nuclei, and suprachiasmatic nucleus and projected to the anterior tuberal nucleus and medial inferior lobe, suggesting neurohormonal and circadian control on the PGa and auditory influences on hypothalamic functions. Of other diencephalic nuclei that receive auditory toral fibers, only small numbers of neurons were labeled in the central posterior thalamic nucleus and anterior tuberal nucleus even after large injections to the dorsal telencephalic area. Thus, the PGa and closely related preglomerular regions, not the dorsal thalamus, appear to constitute the major auditory relay station to the dorsal telencephalic area. The rostrolateral region of PGl, rostral and lateral zones of PGm, commissural preglomerular nucleus, and preglomerular tertiary gustatory nucleus, which do not receive auditory toral fibers, also projected to the dorsal telencephalic area.

Antihypertensive effect of casein hydrolysate in a placebo-controlled study in subjects with high-normal blood pressure and mild hypertension.

We describe a clinical trial to study the efficacy of a casein hydrolysate, prepared using an Aspergillus oryzae protease, containing the major angiotensin-I-converting enzyme inhibitory peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) in a single-blind, placebo-controlled study. A total of 131 volunteers with high-normal blood pressure and mild hypertension were randomly divided into four groups (n 32 or 33 in each group). Each volunteer was given two tablets containing four different dosages of VPP and IPP (VPP+IPP: 0, 1.8, 2.5 and 3.6 mg), daily for 6 weeks. A significant decrease in systolic blood pressure was observed at 6 weeks in the active group receiving 1.8 mg (P<0.01) VPP and IPP; in the active groups receiving either 2.5 mg or 3.6 mg, systolic blood pressure was decreased at both 3 weeks (P<0.05 and P<0.05) and 6 weeks (P<0.001 and P<0.0001) compared with systolic blood pressure measured before treatment. Changes in the systolic blood pressure after 6 weeks of treatment in the four groups were --1.7, --6.3, --6.7 and --10.1 mmHg, and these effects were dose dependent. In addition, a significant difference in systolic blood pressure between the placebo group and the VPP and IPP group receiving 3.6 mg was observed (P<0.001) by two-way ANOVA. The antihypertensive effect was greater in mildly hypertensive subjects (n 20 or 21 in each group) than in any of the other subjects. No significant change of diastolic blood pressure was observed for all the test groups, and no differences in diastolic blood pressure in the test sample groups compared with the placebo group were observed during the test period.

Biogenic peptides and their potential use.

This paper reviews bioactive peptides, biogenic peptides, opioid peptides, immunostimulating peptides, mineral soluble peptides, antihypertensive peptides and antimicrobial peptides originating from food materials and enzymatic hydrolysis of proteins. Antihypertensive peptides are extensively reviewed and have been divided into angiotensin I-converting enzyme inhibitory peptides and others. These peptides are produced in the enzymatic hydrolysate of treated food materials such as milk, animal and fish meat, maize, wheat, soybeans and egg, and also from microbe-fermented products. Peptides with strong antihypertensive effects on spontaneously hypertensive rats are discussed and are divided into high and low angiotensin I-converting enzyme inhibitory activities. In addition, new topics from our studies on antihypertensive peptides are introduced. Efficacies of these peptides in clinical studies and differences with medicinal substances are summarized. Recent studies in this area shown the possibility of using biogenic peptides for improvements in treatment or prevention of hypertension.