On the origin of appetite: GLWamide in jellyfish represents an ancestral satiety neuropeptide.

Food intake is regulated by internal state. This function is mediated by hormones and neuropeptides, which are best characterized in popular model species. However, the evolutionary origins of such feeding-regulating neuropeptides are poorly understood. We used the jellyfish Cladonema to address this question. Our combined transcriptomic, behavioral, and anatomical approaches identified GLWamide as a feeding-suppressing peptide that selectively inhibits tentacle contraction in this jellyfish. In the fruit fly Drosophila, myoinhibitory peptide (MIP) is a related satiety peptide. Surprisingly, we found that GLWamide and MIP were fully interchangeable in these evolutionarily distant species for feeding suppression. Our results suggest that the satiety signaling systems of diverse animals share an ancient origin.

Tick CAPA propeptide cDNAs and receptor activity of endogenous tick pyrokinins and analogs: Towards discovering pyrokinin function in ticks.

Pyrokinins (PKs) are pleiotropic neuropeptides with significant roles in invertebrate physiology. Although functions of PKs are known in insects, there is a lack of knowledge of PK-encoding genes and PKs functions in ticks. Herein the first tick cDNAs of the capability (capa) gene were cloned from the southern cattle tick, Rhipicephalus microplus (Acari: Ixodidae), and the blacklegged tick, Ixodes scapularis. Each cDNA encoded one periviscerokinin and five different pyrokinins. Two PKs were identical in sequence in the two species. The three PKs unique to R. microplus (Rhimi-CAPA-PK1, -PK2, and -PK5) were tested on the recombinant R. microplus pyrokinin receptor using a calcium bioluminescence assay. The Rhimi-CAPA-PKs acted as agonists with EC50s ranging from 101-188 nM. Twenty PK analogs designed for enhanced bioavailability and biostability were tested on the receptor. Five of these were designed based on the sequences of the three unique Rhimi-CAPA-PKs. Eight PK analogs were also agonists; four of them were full agonists that exhibited comparable efficacy to the native Rhimi-CAPA-PKs, with EC50 ranging from 401 nM-1.9 µM. The structure-activity relationships (SAR) of all analogs were analyzed. Our results suggested that a positively charged, basic lysine at the variable position X of the PK active core (FXPRLamide) conferred enhanced affinity to the analogs in their interaction with the tick receptor. These analogs are promising tools to elucidate the pyrokinin function in ticks in vivo as these analogs are expected to have prolonged hemolymph residence time in comparison to the native peptides.

Sex-specific epigenetic alterations of the hypothalamic Agrp-Pomc system do not explain 'diabesity' in the offspring of high-fat diet (HFD) overfed maternal rats.

Maternal high-fat diet (HFD) overfeeding pre- and during pregnancy and lactation may 'program' a 'diabesity' predisposition in the offspring, for inconclusive reasons. Acquired alterations of the hypothalamic promoter methylation and mRNA expression of the satiety neurohormone Pomc are possibly of critical importance here. We investigated within one developmental approach, including male and female rats, the sex-specific DNA methylation pattern and corresponding mRNA expression of both Pomc and its endogenous functional antagonist Agrp in the hypothalamus of adult HFD offspring. Obesity and diabetic disturbances occurred in both male and female HFD offspring, accompanied by altered Pomc promoter methylation pattern. However, this was not related to significant Pomc mRNA expression alterations. In contrast, male-specific alterations of Agrp promoter methylation were found, even associated with reduced mRNA expression of this orexigenic/anabolic Pomc antagonist. In conclusion, acquired epigenetic alterations of the hypothalamic Agrp-Pomc system hardly explain the 'diabesity' phenotype in HFD offspring, while distinct vulnerability and functionality of Agrp promoter and related genomic regions methylation should be further investigated.

AMGSEFLamide, a member of a broadly conserved peptide family, modulates multiple neural networks in Homarus americanus.

Recent genomic/transcriptomic studies have identified a novel peptide family whose members share the carboxyl terminal sequence -GSEFLamide. However, the presence/identity of the predicted isoforms of this peptide group have yet to be confirmed biochemically, and no physiological function has yet been ascribed to any member of this peptide family. To determine the extent to which GSEFLamides are conserved within the Arthropoda, we searched publicly accessible databases for genomic/transcriptomic evidence of their presence. GSEFLamides appear to be highly conserved within the Arthropoda, with the possible exception of the Insecta, in which sequence evidence was limited to the more basal orders. One crustacean in which GSEFLamides have been predicted using transcriptomics is the lobster, Homarus americanus Expression of the previously published transcriptome-derived sequences was confirmed by reverse transcription (RT)-PCR of brain and eyestalk ganglia cDNAs; mass spectral analyses confirmed the presence of all six of the predicted GSEFLamide isoforms - IGSEFLamide, MGSEFLamide, AMGSEFLamide, VMGSEFLamide, ALGSEFLamide and AVGSEFLamide - in H. americanus brain extracts. AMGSEFLamide, of which there are multiple copies in the cloned transcripts, was the most abundant isoform detected in the brain. Because the GSEFLamides are present in the lobster nervous system, we hypothesized that they might function as neuromodulators, as is common for neuropeptides. We thus asked whether AMGSEFLamide modulates the rhythmic outputs of the cardiac ganglion and the stomatogastric ganglion. Physiological recordings showed that AMGSEFLamide potently modulates the motor patterns produced by both ganglia, suggesting that the GSEFLamides may serve as important and conserved modulators of rhythmic motor activity in arthropods.

Isolation and identification of endogenous RFamide-related peptides 1 and 3 in the mouse hypothalamus.

Although the RFamide-related peptide (RFRP) preproprotein sequence is known in mice, until now, the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study, we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using an immunoaffinity column conjugated with specific antibodies against the mouse C-terminus of RFRP-1 and RFRP-3. Employing liquid chromatography coupled with mass spectrometry, we demonstrated that RFRP1 consists of 15 amino acid residues and RFRP3 consists of 10 amino acid residues (ANKVPHSAANLPLRF-NH2 and SHFPSLPQRF-NH2, respectively). To investigate the distribution of RFRPs in the mouse central nervous system, we performed immunohistochemical staining of the brain sections collected from wild-type and Rfrp knockout animals. These data, together with gene expression in multiple tissues, provide strong confidence that RFRP-immunoreactive neuronal cells are localised in the dorsomedial hypothalamic nucleus (DMH) and between the DMH and the ventromedial hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and immunohistochemical visualisation of targeting RFRPs neurones in the mice brain provide the basis for further investigations of the functional biology of RFRPs.

Screening of 109 neuropeptides on ASICs reveals no direct agonists and dynorphin A, YFMRFamide and endomorphin-1 as modulators.

Acid-sensing ion channels (ASICs) belong to the DEG/ENaC gene family. While ASIC1a, ASIC1b and ASIC3 are activated by extracellular protons, ASIC4 and the closely related bile acid-sensitive ion channel (BASIC or ASIC5) are orphan receptors. Neuropeptides are important modulators of ASICs. Moreover, related DEG/ENaCs are directly activated by neuropeptides, rendering neuropeptides interesting ligands of ASICs. Here, we performed an unbiased screen of 109 short neuropeptides (<20 amino acids) on five homomeric ASICs: ASIC1a, ASIC1b, ASIC3, ASIC4 and BASIC. This screen revealed no direct agonist of any ASIC but three modulators. First, dynorphin A as a modulator of ASIC1a, which increased currents of partially desensitized channels; second, YFMRFamide as a modulator of ASIC1b and ASIC3, which decreased currents of ASIC1b and slowed desensitization of ASIC1b and ASIC3; and, third, endomorphin-1 as a modulator of ASIC3, which also slowed desensitization. With the exception of YFMRFamide, which, however, is not a mammalian neuropeptide, we identified no new modulator of ASICs. In summary, our screen confirmed some known peptide modulators of ASICs but identified no new peptide ligands of ASICs, suggesting that most short peptides acting as ligands of ASICs are already known.

Effects of Kisspeptin-10 on Hypothalamic Neuropeptides and Neurotransmitters Involved in Appetite Control.

Besides its role as key regulator in gonadotropin releasing hormone secretion, reproductive function, and puberty onset, kisspeptin has been proposed to act as a bridge between energy homeostasis and reproduction. In the present study, to characterize the role of hypothalamic kisspeptin as metabolic regulator, we evaluated the effects of kisspeptin-10 on neuropeptide Y (NPY) and brain-derived neurotrophic factor (BDNF) gene expression and the extracellular dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytriptamine, 5-HT), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIIA) concentrations in rat hypothalamic (Hypo-E22) cells. Our study showed that kisspeptin-10 in the concentration range 1 nM⁻10 µM was well tolerated by the Hypo-E22 cell line. Moreover, kisspeptin-10 (100 nM⁻10 µM) concentration independently increased the gene expression of NPY while BDNF was inhibited only at the concentration of 10 µM. Finally, kisspeptin-10 decreased 5-HT and DA, leaving unaffected NE levels. The inhibitory effect on DA and 5-HT is consistent with the increased peptide-induced DOPAC/DA and 5-HIIA/5-HT ratios. In conclusion, our current findings suggesting the increased NPY together with decreased BDNF and 5-HT activity following kisspeptin-10 would be consistent with a possible orexigenic effect induced by the peptide.

Isolation and functional characterization of the pheromone biosynthesis activating neuropeptide receptor of Chinese oak silkworm, Antheraea pernyi.

Insect pheromone biosynthesis activating neuropeptide (PBAN) controls the synthesis and actuating of sex pheromones of female adult. In the current examination, the full-length cDNA encoding the PBAN receptor was cloned from the pheromone gland (PG) of Antheraea pernyi (AntpePBANR). The AntpePBANR displayed the characteristic seven transmembrane areas of the G protein-coupled receptor (GPCR) and was closely related to the PBANR from Bombyx mori and Manduca sexta in the phylogenetic tree. The AntpePBANR expressed in mammalian cell lines were enacted by AntpePBAN in a concentration-dependent manner. AntpePBANR activation resulted in the calcium mobilization but did not activate the cAMP elevation pathway. Cells expressing AntpePBANR were profoundly responsive to Antpe-γ-SGNP (suboesophageal ganglion neuropeptides) and Antpe-DH (diapause hormone), different individuals from FXPRLamide (X = T, S or V) family in A. pernyi. Deletion of residues in the C-terminal hexapeptide (FSPRLamide) proved that P, R and L played the key parts in initiating the AntpePBANR, the amination to the last C terminal residues which can also likewise impact the activation of AntpePBAN receptor altogether. The mRNA of the AntpePBANR gene demonstrated the most noteworthy transcript levels in pheromone gland followed by fat body.

The hypothalamic NPVF circuit modulates ventral raphe activity during nociception.

RFamide neuropeptide VF (NPVF) is expressed by neurons in the hypothalamus and has been implicated in nociception, but the circuit mechanisms remain unexplored. Here, we studied the structural and functional connections from NPVF neurons to downstream targets in the context of nociception, using novel transgenic lines, optogenetics, and calcium imaging in behaving larval zebrafish. We found a specific projection from NPVF neurons to serotonergic neurons in the ventral raphe nucleus (vRN). We showed NPVF neurons and vRN are suppressed and excited by noxious stimuli, respectively. We combined optogenetics with calcium imaging and pharmacology to demonstrate that stimulation of NPVF cells suppresses neuronal activity in vRN. During noxious stimuli, serotonergic neurons activation was due to a suppression of an inhibitory NPVF-ventral raphe peptidergic projection. This study reveals a novel NPVF-vRN functional circuit modulated by noxious stimuli in vertebrates.

Newly identified allatostatin Bs and their receptor in the two-spotted cricket, Gryllus bimaculatus.

A cDNA encoding allatostatin Bs (ASTBs) containing the W(X)6W motif was identified using a database generated by a next generation sequencer (NGS) in the two-spotted cricket, Gryllus bimaculatus. The contig sequence revealed the presence of five novel putative ASTBs (GbASTBs) in addition to GbASTBs previously identified in G. bimaculatus. MALDI-TOF MS analyses revealed the presence of these novel and previously identified GbASTBs with three missing GbASTBs. We also identified a cDNA encoding G. bimaculatus GbASTB receptor (GbASTBR) in the NGS data. Phylogenetic analysis demonstrated that this receptor was highly conserved with other insect ASTBRs, including the sex peptide receptor of Drosophila melanogaster. Calcium imaging analyses indicated that the GbASTBR heterologously expressed in HEK293 cells exhibited responses to all identified GbASTBs at a concentration range of 10(-10)-10(-5)M.

Forces generated during stretch in the heart of the lobster Homarus americanus are anisotropic and are altered by neuromodulators.

Mechanical and neurophysiological anisotropies mediate three-dimensional responses of the heart of ITALIC! Homarus americanus Although hearts ITALIC! in vivoare loaded multi-axially by pressure, studies of invertebrate cardiac function typically use uniaxial tests. To generate whole-heart length-tension curves, stretch pyramids at constant lengthening and shortening rates were imposed uniaxially and biaxially along longitudinal and transverse axes of the beating whole heart. To determine whether neuropeptides that are known to modulate cardiac activity in ITALIC! H. americanusaffect the active or passive components of these length-tension curves, we also performed these tests in the presence of SGRNFLRFamide (SGRN) and GYSNRNYLRFamide (GYS). In uniaxial and biaxial tests, both passive and active forces increased with stretch along both measurement axes. The increase in passive forces was anisotropic, with greater increases along the longitudinal axis. Passive forces showed hysteresis and active forces were higher during lengthening than shortening phases of the stretch pyramid. Active forces at a given length were increased by both neuropeptides. To exert these effects, neuropeptides might have acted indirectly on the muscle via their effects on the cardiac ganglion, directly on the neuromuscular junction, or directly on the muscles. Because increases in response to stretch were also seen in stimulated motor nerve-muscle preparations, at least some of the effects of the peptides are likely peripheral. Taken together, these findings suggest that flexibility in rhythmic cardiac contractions results from the amplified effects of neuropeptides interacting with the length-tension characteristics of the heart.

[des-Arg(1)]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom.

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

Effect and safety of Shengxuening (extract from excrement of bombyxin) for renal anemia: a systematic review.

OBJECTIVE: To assess the effect and safety of Shengxuening (SXN), extract from excrement of bombyxin, in the treatment of renal anemia, compared to ferrous succinate and ferrous sulfate. METHODS: According to the participant, intervention, comparison, outcomes, study design (PICOS) principles, we searched the Chinese Biomedical Literature Database, China National Knowledge Infrastructure Database, Chinese Evidence-Based Medicine Database, Wanfang Database (From establishment to December 2014). Two reviewers selected articles independently according to the inclusion and exclusion criteria. The quality of included studies was assessed by using the Cochrane Handbook. All statistical analyses were conducted by using Revman (vision 5.2) software. RESULTS: A total of 14 randomized controlled trials (RCTs) were enrolled in the review. The results revealed that, when compared with blank group, SXN significantly improved the hemoglobin (P >) levels [MD = 6.29, 95% CI (1.65-10.94), P < 0.0008] and albumin (ALB) [MD = 10.98, 95% CI (6.97-14.99), P < 0.00001]. In addition, SXN could significantly increase the P > levels [MD = 10.98, 95% CI (6.97, 14.99), P < 0.00001]. Compared with other oral medicine SXN could improve the P > levels effectively [MD = 8.49, 95% CI (2.40, 14.58), P = 0.006]. And the subgroups analysis shown that compared with ferrous-sulfate there were significant differences [MD = 17.4, 95% CI (15.06, 19.73), P < 0.000 01] and the result of ferrous-succinate had significant differences [MD = 5.34, 95% CI (2.12, 8.56), P = 0.001] too. Compared with Intravenous iron groups, there were statistical differences [MD = － 5.04, 95% CI (－ 9.59, － 0.50), P = 0.03]. In the safety analysis, the rate of adverse reactions in SXN groups and control groups were 19.3% and 3.7%, respectively (P < 0.000 01). Due to our studies were of poor methodological quality, and the sample size were small, the results were influenced by bias. CONCLUSION: Our findings suggest that the SXN had better effect and was safer in the treatment of RA than ferrous succinate and ferrous sulfate.

Neuropeptidergic Signaling in the American Lobster Homarus americanus: New Insights from High-Throughput Nucleotide Sequencing.

Peptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

Chronic Mild Cold Conditioning Modulates the Expression of Hypothalamic Neuropeptide and Intermediary Metabolic-Related Genes and Improves Growth Performances in Young Chicks.

BACKGROUND: Low environmental temperatures are among the most challenging stressors in poultry industries. Although landmark studies using acute severe cold exposure have been conducted, still the molecular mechanisms underlying cold-stress responses in birds are not completely defined. In the present study we determine the effect of chronic mild cold conditioning (CMCC) on growth performances and on the expression of key metabolic-related genes in three metabolically important tissues: brain (main site for feed intake control), liver (main site for lipogenesis) and muscle (main site for thermogenesis). METHODS: 80 one-day old male broiler chicks were divided into two weight-matched groups and maintained in two different temperature floor pen rooms (40 birds/room). The temperature of control room was 32°C, while the cold room temperature started at 26.7°C and gradually reduced every day (1°C/day) to reach 19.7°C at the seventh day of the experiment. At day 7, growth performances were recorded (from all birds) and blood samples and tissues were collected (n = 10). The rest of birds were maintained at the same standard environmental condition for two more weeks and growth performances were measured. RESULTS: Although feed intake remained unchanged, body weight gain was significantly increased in CMCC compared to the control chicks resulting in a significant low feed conversion ratio (FCR). Circulating cholesterol and creatine kinase levels were higher in CMCC chicks compared to the control group (P<0.05). CMCC significantly decreased the expression of both the hypothalamic orexigenic neuropeptide Y (NPY) and anorexigenic cocaine and amphetamine regulated transcript (CART) in chick brain which may explain the similar feed intake between the two groups. Compared to the control condition, CMCC increased the mRNA abundance of AMPKα1/α2 and decreased mTOR gene expression (P<0.05), the master energy and nutrient sensors, respectively. It also significantly decreased the expression of fatty acid synthase (FAS) gene in chick brain compared to the control. Although their roles are still unknown in avian species, adiponectin (Adpn) and its related receptors (AdipoR1 and 2) were down regulated in the brain of CMCC compared to control chicks (P<0.05). In the liver, CMCC significantly down regulated the expression of lipogenic genes namely FAS, acetyl-CoA carboxylase alpha (ACCα) and malic enzyme (ME) and their related transcription factors sterol regulatory element binding protein 1/2 (SREBP-1 and 2). Hepatic mTOR mRNA levels and phosphorylated mTOR at Ser2448 were down regulated (P<0.05), however phosphorylated ACCαSer79 (inactivation) was up regulated (P<0.05) in CMCC compared to control chicks, indicating that CMCC switch hepatic catabolism on and inhibits hepatic lipogenesis. In the muscle however, CMCC significantly up regulated the expression of carnitine palmitoyltransferase 1 (CPT-1) gene and the mRNA and phosphorylated protein levels of mTOR compared to the control chicks, indicating that CMCC enhanced muscle fatty acid ß-oxidation. CONCLUSIONS: In conclusion, this is the first report indicating that CMCC may regulate AMPK-mTOR expression in a tissue specific manner and identifying AMPK-mTOR as a potential molecular signature that controls cellular fatty acid utilization (inhibition of hepatic lipogenesis and induction of muscle fatty acid ß-oxidation) to enhance growth performance during mild cold acclimation.

Lipid-Conjugation of Endogenous Neuropeptides: Improved Biotherapy against Human Pancreatic Cancer.

Neuropeptides are small neuronal signaling molecules that act as neuromodulators for a variety of neural functions including analgesia, reproduction, social behavior, learning, and memory. One of the endogenous neuropeptides-Met-Enkephalin (Met-Enk), has been shown to display an inhibitory effect on cell proliferation and differentiation. Here, a novel lipid-modification approach is shown to create a small library of neuropeptides that will allow increased bioavailability and plasma stability after systemic administration. It is demonstrated, on an experimental model of human pancreatic adenocarcinoma, that lipid conjugation of Met-Enk enhances its tumor suppression efficacy compared to its nonlipidated counterparts, both in vitro and in vivo. More strikingly, the in vivo studies show that a combination therapy with a reduced concentration of Gemcitabine has suppressed the tumor growth considerably even three weeks after the last treatment.

VGF changes during the estrous cycle: a novel endocrine role for TLQP peptides?

Although the VGF derived peptide TLQP-21 stimulates gonadotropin-releasing hormone (GnRH) and gonadotropin secretion, available data on VGF peptides and reproduction are limited. We used antibodies specific for the two ends of the VGF precursor, and for two VGF derived peptides namely TLQP and PGH, to be used in immunohistochemistry and enzyme-linked immunosorbent assay complemented with gel chromatography. In cycling female rats, VGF C-/N-terminus and PGH peptide antibodies selectively labelled neurones containing either GnRH, or kisspeptin (VGF N-terminus only), pituitary gonadotrophs and lactotrophs, or oocytes (PGH peptides only). Conversely, TLQP peptides were restricted to somatostatin neurones, gonadotrophs, and ovarian granulosa, interstitial and theca cells. TLQP levels were highest, especially in plasma and ovary, with several molecular forms shown in chromatography including one compatible with TLQP-21. Among the cycle phases, TLQP levels were higher during metestrus-diestrus in median eminence and pituitary, while increased in the ovary and decreased in plasma during proestrus. VGF N- and C-terminus peptides also showed modulations over the estrous cycle, in median eminence, pituitary and plasma, while PGH peptides did not. In ovariectomised rats, plasmatic TLQP peptide levels showed distinct reduction suggestive of a major origin from the ovary, while the estrogen-progesterone treatment modulated VGF C-terminus and TLQP peptides in the hypothalamus-pituitary complex. In in vitro hypothalamus, TLQP-21 stimulated release of growth hormone releasing hormone but not of somatostatin. In conclusion, various VGF peptides may regulate the hypothalamus-pituitary complex via specific neuroendocrine mechanisms while TLQP peptides may act at further, multiple levels via endocrine mechanisms involving the ovary.

Demonstration of binding of neuronal calcium sensor-1 to the cav2.1 p/q-type calcium channel.

In neurons, entry of extracellular calcium (Ca(2+)) into synaptic terminals through Cav2.1 (P/Q-type) Ca(2+) channels is the driving force for exocytosis of neurotransmitter-containing synaptic vesicles. This class of Ca(2+) channel is, therefore, pivotal during normal neurotransmission in higher organisms. In response to channel opening and Ca(2+) influx, specific Ca(2+)-binding proteins associate with cytoplasmic regulatory domains of the P/Q channel to modulate subsequent channel opening. Channel modulation in this way influences synaptic plasticity with consequences for higher-level processes such as learning and memory acquisition. The ubiquitous Ca(2+)-sensing protein calmodulin (CaM) regulates the activity of all types of mammalian voltage-gated Ca(2+) channels, including the P/Q class, by direct binding to specific regulatory motifs. More recently, experimental evidence has highlighted a role for additional Ca(2+)-binding proteins, particularly of the CaBP and NCS families in the regulation of P/Q channels. NCS-1 is a protein found from yeast to humans and that regulates a diverse number of cellular functions. Physiological and genetic evidence indicates that NCS-1 regulates P/Q channel activity, including calcium-dependent facilitation, although a direct physical association between the proteins has yet to be demonstrated. In this study, we aimed to determine if there is a direct interaction between NCS-1 and the C-terminal cytoplasmic tail of the Cav2.1 α-subunit. Using distinct but complementary approaches, including in vitro binding of bacterially expressed recombinant proteins, fluorescence spectrophotometry, isothermal titration calorimetry, nuclear magnetic resonance, and expression of fluorescently tagged proteins in mammalian cells, we show direct binding and demonstrate that CaM can compete for it. We speculate about how NCS-1/Cav2.1 association might add to the complexity of calcium channel regulation mediated by other known calcium-sensing proteins and how this might help to fine-tune neurotransmission in the mammalian central nervous system.

Effect of RFRP-3 on reproduction is sex- and developmental status-dependent in the striped hamster (Cricetulus barabensis).

RFamide-related peptides (RFRPs) are orthologous to gonadotropin-inhibitory hormone (GnIH) inhibiting gonadotropin release. There are only two RFRP sequences (RFRP-1 and RFRP-3) encoded in rodents. RFRP-3, which was considered as a hypothetical inhibitor on GnRH, shows a stimulatory effect on the male Syrian and male Siberian hamster in short days. As a dominant rodent pest in northern China farmland, the striped hamster (Cricetulus barabensis) has higher reproductive activities and could act as a model to study the mechanism of reproduction. However, the effect of RFRP-3 on the reproductive activity for the striped hamster is less understood. In the study, we cloned 643 bp RFRP cDNA from the striped hamster hypothalamus, which contained an ORF of 570 bp encoding two RFamide-related peptide (RFRP) sequences: SPAPANKVPHSAANLPLRF-NH2 (C. barabensis RFRP-1) and TLSRVPSLPQRF-NH2 (C. barabensis RFRP-3). We also investigated the expression variation of RFRP mRNA and GnRH mRNA in the hypothalamus from hamsters with different developmental statuses (7-week-, 13-week- and 1.5-year-olds) using FQ-PCR, in which the 13-week-old female individuals were in estrous. The striped hamsters that are 7 weeks and 1.5 years old are non-breeding individuals, and those that are 13-week hamsters have breeding phenomena. The highest hypothalamus RFRP mRNA level was found in breeding males as compared to non-breeding males. Conversely, the lowest RFRP mRNA level in the hypothalamus was observed in breeding females, with no significant level when the breeding females were compared to the 7-week-old individuals. Additionally, the investigation of GnRH expression level showed a declining expression trend across the developmental stages (7-week-, 13-week- and 1.5-year-olds) in both sexes. Significant negative and positive relationships were detected in the 13-week estrous female (r=-0.997, P=0.035) and the 13-week male (r=0.998, P=0.029) striped hamsters respectively, which suggest that RFRP-3 has inhibitory and stimulatory effects on female and male adults respectively. Our results suggest that the effects of RFRP-3 on reproduction are sex- and developmental status-dependent in the striped hamster.

Neuropeptide B in Nile tilapia Oreochromis niloticus: molecular cloning and its effects on the regulation of food intake and mRNA expression of growth hormone and prolactin.

Neuropeptide B (NPB) regulates food intake, energy homeostasis and hormone secretion in mammals via two G-protein coupled receptors, termed as GPR 7 and GPR 8. However, there is no study that reports the function of NPB in teleosts. In this study, the full-length cDNA of prepro-NPB with the size of 663bp was cloned from the hypothalamus of Nile tilapia. The CDS of the prepro-NPB is 387bp which encodes a precursor protein with the size of 128a.a. This precursor contains a mature peptide with the size of 29a.a, and it was named as NPB29. Tissue distribution study showed that this gene was mainly expressed in different parts of brain, especially in the diencephalon as well as hypothalamus, and the spinal cord in Nile tilapia. Fasting significantly stimulated the mRNA expression of NPB in the brain area without hypothalamus, and refeeding after fasting for 3 and 14days also showed similar effects on NPB expression. While, only short-term fasting (3days) and refeeding after fasting for 7 and 14days induced mRNA expression of NPB in the hypothalamus. Intraperitoneal (i.p.) injection of NPB remarkably elevated the mRNA expression of hypothalamic neuropeptide Y (NPY), cholecystokinin 1 (CCK1) and pituitary prolactin (PRL), whereas significantly inhibited growth hormone (GH) expression in pituitary. These observations in the present study suggested that NPB may participate in the regulation of feeding and gene expression of pituitary GH and PRL in Nile tilapia.