Peptides (v.30, #2)

Enterostatin, a gut–brain peptide, inhibits dietary fat intake in rats. The purpose of this study was to identify the intracellular signaling pathways that are responsive to enterostatin and that modulate the effects of enterostatin on the expression of Agouti-related protein (AgRP). We used the hypothalamic GT1-7 neuronal cell line to identify the effects of enterostatin on cyclic AMP and ERK signaling using conventional immunoassays or Western blots to assay the activity of these pathways. Enterostatin enhanced the level of cyclic AMP, PKARIIβ and phospho-CREB and increased pERK levels in GT 1-7 cells. The effects on pERK were rapid (7.5 min) and dose-dependent. These signaling responses were blocked by an antibody to the enterostatin receptor (beta subunit of F1-ATPase), by the pERK inhibitor U0126 and by the P2Y receptor antagonist Suramin. Enterostatin showed a biphasic effect on AgRP mRNA, initially increasing but subsequently decreasing the levels. The cyclic AMP activator Sp-cAMP increased AgRP mRNA expression. Transfection of a wild type ERK construct reduced AgRP mRNA levels. Enterostatin inhibited expression of Krüppel-like factor 4 (KLF4), a transcriptional regulator of AgRP. KLF4 gene expression was increased by Sp-cAMP but decreased by wild-type ERK expression. U0126 blocked the effect of enterostatin on KLF4 expression. We conclude that enterostatin binding to its receptor activates the pERK pathway to inhibit AgRP gene expression but may enhance AgRP expression through activation of the cyclic AMP pathway. These pathways probably mediate the enterostatin inhibition of dietary fat intake.
Keywords: Enterostatin; AgRP; Krüppel-like factor 4; Amygdala; GT1-7 cells;

Electrophysiological effects of orexins/hypocretins on pedunculopontine tegmental neurons in rats: An in vitro study by Juhyon Kim; Kazuki Nakajima; Yutaka Oomura; Matthew J. Wayner; Kazuo Sasaki (191-209).
Orexin-A (ORX-A) and orexin-B (ORX-B) play critical roles in the regulation of sleep–wakefulness and feeding. ORX neurons project to the pedunculopontine tegmental nucleus (PPT), which regulates waking and rapid eye movement (REM) sleep. Thus, we examined electrophysiological effects of ORXs on rat PPT neurons with a soma size of more than 30 μm. Whole cell patch clamp recording in vitro revealed that ORX-A and ORX-B depolarized PPT neurons dose-dependently in normal and/or tetrodotoxin containing artificial cerebrospinal fluids (ACSFs), and the EC50 values for ORX-A and ORX-B were 66 nM and 536 nM, respectively. SB-334867, a selective inhibitor for ORX 1 (OX1) receptors, significantly suppressed the ORX-A-induced depolarization. The ORX-A-induced depolarization was reduced in high K+ ACSF with extracellular K+ concentration of 13.25 mM or N-methyl-d-glucamine (NMDG+)-containing ACSF in which NaCl was replaced with NMDG-Cl, and abolished in high K+-NMDG+ ACSF or in a combination of NMDG+ ACSF and recordings with Cs+-containing pipettes. An inhibitor of Na+/Ca2+ exchanger and chelating intracellular Ca2+ had no effect on the depolarization. Most of PPT neurons studied were characterized by an A-current or both A-current and a low threshold Ca2+ spike, and predominantly cholinergic. These results suggest that ORXs directly depolarize PPT neurons via OX1 receptors and via a dual ionic mechanism including a decrease of K+ conductances and an increase of non-selective cationic conductances, and support the notion that ORX neurons affect the activity of PPT neurons directly and/or indirectly to control sleep–wakefulness, especially REM sleep.
Keywords: Orexin/hypocretin; Pedunculopontine tegmental nucleus; Ascending arousal system; Cholinergic neurons; Sleep/wakefulness; REM sleep; SB-334867;

Inflammation-associated cachexia is associated with multiple chronic diseases and involves activation of appetite regulating centers in the arcuate nucleus of the hypothalamus (ARH). The nucleus of the solitary tract (NTS) in the brainstem has also been implicated as an important nucleus involved in appetite regulation. We set out to determine whether the NTS may be involved in inflammation-associated anorexia by injecting IL-1β into the 4th ventricle and assessing food intake and NTS neuronal activation. Injection of IL-1β produced a decrease in food intake at 3 and 12 h after injection which was ameliorated at the 12 h time point by a sub-threshold dose of agouti-related peptide (AgRP). Investigation into neuron types in the NTS revealed that IL-1β injection was associated with an increase in c-Fos activity in NTS neurons expressing tyrosine hydroxylase (TH). Additionally, injection of IL-1β into the 4th ventricle did not produce c-Fos activation of neurons expressing pro-opiomelanocortin (POMC) in the ARH, cells known to be involved in producing anorexia in response to systemic inflammation. Double-label in situ hybridization revealed that TH neurons did not express IL-1 receptor I (IL1-RI) transcript, demonstrating that c-Fos activation of TH neurons in this setting was not via direct stimulation of IL-1β on TH neurons themselves. We conclude that IL-1β injection into the 4th ventricle produces anorexia and is accompanied by an increase in activation in TH neurons in the NTS. This provides evidence that the brainstem may be an important mediator of anorexia in the setting of inflammation.
Keywords: Tyrosine hydroxylase; Brainstem; Appetite regulation; IL-1β; Cytokines; AgRP;

In older populations there is significant increase in incidence of impaired glucose tolerance and diabetes. Glucose-dependent insulinotropic polypeptide (GIP) improves glycemic control but its use as a therapeutic is hindered by short biological half-life. The present study examined effects of a longer-acting form of GIP, GIP[mPEG], on glucose homeostasis and beta-cell function in mice with age-related glucose intolerance. GIP[mPEG] decreased glucose and increased insulin concentrations when administered prior to a glucose challenge. Daily administration of GIP[mPEG] for 20 days had no effect on body weight and food intake. However, non-fasting glucose concentrations were decreased and insulin concentrations increased. Glycemic response to intraperitoneal glucose was improved and glucose-mediated insulin secretion enhanced. Insulin sensitivity, circulating triglycerides and resistin levels were unchanged by the treatment regimen, but plasma adiponectin levels increased. These data indicate that prolonged activation of the GIP receptor with GIP[mPEG] counters aspects of impaired beta-cell function and age-related glucose intolerance.
Keywords: Aging; GIP; GIP[mPEG]; Glucose tolerance; Insulin secretion;

Complexity of neural mechanisms underlying overconsumption of sugar in scheduled feeding: Involvement of opioids, orexin, oxytocin and NPY by Pawel K. Olszewski; Timothy J. Shaw; Martha K. Grace; Catherine E. Höglund; Robert Fredriksson; Helgi B. Schiöth; Allen S. Levine (226-233).
A regular daily meal regimen, as opposed to ad libitum consumption, enforces eating at a predefined time and within a short timeframe. Hence, it is important to study food intake regulation in animal feeding models that somewhat reflect this pattern. We investigated the effect of scheduled feeding on the intake of a palatable, high-sugar diet in rats and attempted to define central mechanisms – especially those related to opioid signaling – responsible for overeating sweet foods under such conditions. We found that scheduled access to food, even as challenging as 20 min per day, does not prevent overconsumption of a high-sucrose diet compared to a standard one. An opioid receptor antagonist, naloxone, at 0.3–1 mg/kg b. wt., decreased the intake of the sweet diet, whereas higher doses were required to reduce bland food consumption. Real-time PCR analysis revealed that expression of hypothalamic and brainstem genes encoding opioid peptides and receptors did not differ in sucrose versus regular diet-fed rats, which suggests that scheduled intake of sweet food produces only a transient change in the opioid tone. Intake of sugar was also associated with upregulation of orexin and oxytocin genes in the hypothalamus and NPY in the brainstem. We conclude that scheduled consumption of sugar diets is associated with activity of a complex network of neuroregulators involving opioids, orexin, oxytocin and NPY.
Keywords: Restriction; Diet; Rats; Deprivation; Peptides; Reward; Obesity;

Neuropeptide S inhibits the acquisition and the expression of conditioned place preference to morphine in mice by Wei Li; Ya-Hu Gao; Min Chang; Ya-Li Peng; Jia Yao; Ren-Wen Han; Rui Wang (234-240).
Neuropeptide S (NPS), a recently identified bioactive peptide, was reported to regulate arousal, anxiety, motoring and feeding behaviors. NPS precursor and NPS receptor mRNA were found in the amygdala, the ventral tegmental area (VTA) and the substantia nigra, the area thought to modulate rewarding properties of drugs. In the present study, we examined the influence of NPS on the rewarding action of morphine, using the unbiased conditioned place preference (CPP) paradigm. Morphine (1, 3 and 6 nmol, i.c.v.) induced a significant place preference. For testing the effect of NPS on the acquisition of morphine CPP, mice were given the combination of NPS and morphine on the conditioning days, and without drug treatment on the followed test day. To study the effect of NPS on the expression of morphine CPP, mice received the treatment of saline/morphine on the conditioning days, and NPS on the test day, 15 min before the placement in the CPP apparatus. Our results showed that NPS (0.3–10 nmol) alone neither induced place preference nor aversion, however, NPS (1 and 3 nmol) blocked the acquisition of CPP induced by 3 nmol morphine, and acquisition of 6 nmol morphine-induced CPP was also reduced by NPS (6 and 10 nmol). Moreover, the expression of CPP induced by 6 nmol morphine was also inhibited by NPS (0.1, 1 and 10 nmol). These results revealed the involvement of NPS in rewarding activities of morphine, and demonstrated the interaction between NPS system and opioid system for the first time.
Keywords: Neuropeptide S; Conditioned place preference; Acquisition; Expression; Morphine; Reward;

Effect of arginine vasopressin on acupuncture analgesia in the rat by Jun Yang; Yu Yang; Cheng-Hai Wang; Gen Wang; Hongtao Xu; Wen-Yan Liu; Bao-Cheng Lin (241-247).
Arginine vasopressin (AVP) has been proven to be involved in the process of pain regulation. This communication was designed to investigate the effect of AVP on acupuncture analgesia in the rat model. The results showed that intraventricular injection (icv) of AVP could enhance acupuncture analgesia in a dose-dependent manner, whereas icv of anti-AVP serum decreased acupuncture analgesia. However, neither intrathecal (ith) nor intravenous injection (iv) of AVP or anti-AVP serum could influence acupuncture analgesia. Electrical acupuncture of “Zusanli” points (St. 36) decreased AVP concentration in the hypothalamic paraventricular nucleus (PVN), and increased AVP concentration in the hypothalamic supraoptic nucleus (SON), periaqueductial gray (PAG), caudate nucleus (CdN) and raphe magnus nucleus (RMN), but did not change AVP concentration in the pituitary, spinal cord and plasma. The effect of AVP on acupuncture analgesia was partly reversed by pretreatment with naloxone, an opiate receptor antagonist. These data suggested that AVP in the brain played a role in the process of acupuncture analgesia in combination with the endogenous opiate peptide system.
Keywords: Arginine vasopressin; Acupuncture analgesia; Brain; Endogenous opiate peptide;

Further studies on the pharmacological features of the nociceptin/orphanin FQ receptor ligand ZP120 by Carmela Fischetti; Anna Rizzi; Elaine C. Gavioli; Giuliano Marzola; Claudio Trapella; Remo Guerrini; Jorgen S. Petersen; Girolamo Calo (248-255).
ZP120 is a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In previous studies, the effects of ZP120 were found to be sensitive to J-113397 in mouse tissues while resistant to UFP-101 in rat tissues. The aim of this study was to further investigate the ZP120 pharmacological profile using mouse and rat preparations, J-113397 and UFP-101, as well as NOP receptor knockout (NOP−/−) mice. Electrically stimulated mouse and rat vas deferens were used to characterize the pharmacology of ZP120 in vitro. For in vivo studies the tail-withdrawal assay was performed in wild type (NOP+/+) and NOP knockout (NOP−/−) mice. In the mouse and rat vas deferens ZP120 mimicked the effects of N/OFQ showing higher potency but lower maximal effects. In both preparations, J-113397 antagonized N/OFQ and ZP120 effects showing similar pK B values (≈7.8). UFP-101 antagonized the actions of N/OFQ (pK B values ≈7.3) but did not modify the effects of ZP120. The inhibitory effects of N/OFQ and ZP120 were no longer evident in vas deferens tissues taken from NOP−/− mice. In NOP+/+ mice subjected to the tail-withdrawal assay, ZP120 (1 nmol) mimicked the pronociceptive action of N/OFQ (10 nmol), producing longer lasting effects. The effects of both peptides were absent in NOP−/− animals. The NOP receptor ligand ZP120 is a high potency NOP selective partial agonist able to evoke long-lasting effects; its diverse antagonist sensitivity in comparison with N/OFQ may derive from different modality of binding to the NOP receptor.
Keywords: Nociceptin/orphanin FQ; NOP receptor; ZP120; Mouse and rat vas deferens; Tail-withdrawal assay;

Low circulating VVH7-like immunoreactivity (VVH7 i.r) level was amazingly observed in human diabetic sera. Here, we examined the impact of diabetes type, clinico-biological features and metabolic control on circulating VVH7 i.r level in this disease. ELISA test was used to measure VVH7 i.r in sera of 120 diabetic patients (type 1 diabetes in 64, type 2 diabetes in 56). Three enzymatic tests were also applied to determine serum cathepsin D (CD), dipeptidyl peptidase IV (DPP-IV) and angiotensin-converting enzyme (ACE) activities. A subgroup of 24 type 1 diabetic patients negative for microalbuminuria and hypertension were submitted to an ambulatory blood pressure monitoring to evaluate the relationship between VVH7 i.r level and blood pressure parameters. The mean serum concentration of VVH7 i.r was drastically reduced in diabetic patients (0.91 ± 0.93 μmol/l versus 5.63 ± 1.11 μmol/l in controls) (p  < 0.001). A negative correlation between VVH7 i.r level and daytime diastolic blood pressure existed in type 1 diabetic patients. There was no association of low VVH7 i.r with either type of diabetes or HbA1c level. An increase of cathepsin D activity was found in serum of diabetic patients compared to controls (0.47 U/ml versus 0.15 U/ml, respectively) whereas DPPIV activity was significantly decreased in diabetic sera (50.81 U/ml versus 282.10 U/l respectively). Diminution of VVH7 i.r in sera of diabetic patients was confirmed but still remained unexplained. Relationships between higher systolic blood pressure and decrease of VVH7 i.r reinforce the need to investigate this pathway in this disease to elucidate its role in macro- and micro-angiopathy.
Keywords: VVH7 i.r; diabetes; Serum angiotensin-converting-enzyme; Serum cathepsin D; Serum dipeptidyl peptidase IV;

Hepcidin, an antimicrobial peptide is downregulated in ceruloplasmin-deficient mice by Pei Guo; Rui Cui; Yan-Zhong Chang; Wen-Shuang Wu; Zhong-Ming Qian; Kunihiro Yoshida; Ya-Tiao Qiao; Shin’ichi Takeda; Xiang-Lin Duan (262-266).
Hepcidin, a principle regulator of iron metabolism, is synthesized by the liver. Contradictory results have been reported on the regulation of hepcidin expression in response to serum transferrin saturation and liver iron content. In the present study, we explore the expression of murine hepcidin mRNA and further analyze the relationship between liver hepcidin mRNA expression, liver iron stores, and serum iron level utilizing ceruloplasmin gene knockout mice. We find that hepcidin expression correlates significantly with serum transferrin saturation, whereas there is a negative correlation of hepcidin expression with liver tissue iron level.
Keywords: Iron; Hepcidin; Liver iron store; Serum iron; Transferrin saturation;

Antimicrobial human beta-defensin-2 stimulates migration, proliferation and tube formation of human umbilical vein endothelial cells by Adone Baroni; Giovanna Donnarumma; Iole Paoletti; Immacolata Longanesi-Cattani; Katia Bifulco; Maria Antonietta Tufano; Maria Vincenza Carriero (267-272).
Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which is released upon microbial invasion and contributes to mucosal and epithelial defense modulating both innate and adaptive immunity. We found that hBD-2 stimulates chemotaxis of human endothelial cells with an extent similar to that exerted by the vascular endothelial growth factor (VEGF). The hBD-2-dependent chemotaxis is dose-dependent, maximal effect being reached at 500 ng/ml concentration. In the absence of any growth factor, hBD-2 favors wound healing of endothelial cells, causing an about 2-fold increase in the speed of wound closure with respect to the control. Furthermore, hBD-2 promotes endothelial cell proliferation, although at a minor extent as compared to VEGF. When plated on matrigel enriched with angiogenic factors, endothelial cells form a three-dimensional network of tubes that gives rise to capillary-like structures. Similarly to VEGF, hBD-2 promotes capillary-like tube formation of human endothelial cells. Pro-angiogenic effect promoted by hBD-2 is dose-dependent, peaks at a 500 ng/ml hBD-2 concentration and is prevented by blocking anti-αvβ3 monoclonal antibody. However, hBD-2-induced pro-angiogenic activity is not due to endogenously produced VEGF because it is not prevented by neutralizing anti-VEGF antibodies. Overall, our findings suggest that hBD-2 could link inflammation and the host defense through its pro-angiogenic activity.
Keywords: Beta-defensins; Endothelial cells; Chemotaxis; Angiogenesis;

The novel antimicrobial peptides from skin of Chinese broad-folded frog, Hylarana latouchii (Anura:Ranidae) by Hui Wang; Yi Lu; Xiuqing Zhang; Yuhong Hu; Haining Yu; Jingze Liu; Junshe Sun (273-282).
Broad-folded frogs (Hylarana latouchii), one member of 12 species of the genus Hylarana in the Chinese frog fauna, are widely distributed in the South of China. In this study, we purified and characterized three antimicrobial peptides from the skin secretion of H. latouchii. Five different cDNA fragments encoding the precursors of these antimicrobial peptides were cloned, and five mature antimicrobial peptides belonging to two different families were deduced from the five cDNAs. Structural characterization of the mature peptides had identified them as members of the brevinin-1 and temporin families. They were named brevinin-1LTa (FFGTALKIAANVLPTAICKILKKC), brevinin-1LTb (FFGTALKIAANILPTAICKILKKC), temporin-LTa (FFPLVLGALGSILPKIF-NH2), temporin-LTb (FIITGLVRGLTKLF-NH2) and temorin-LTc (SLSRFLSFLKIVYPPAF-NH2). Brevinin-1LTa, temporin-LTa, temporin-LTb and temporin-LTc with different antimicrobial activities induced significant morphological alterations of the tested microbial surfaces as shown by scanning electron microscopy, which indicated strong membrane disruption.
Keywords: Amphibian; Antimicrobial peptides; Hylarana latouchii; Temporin; Brevinin-1;

Epinecidin-1, an antimicrobial peptide from fish (Epinephelus coioides) which has an antitumor effect like lytic peptides in human fibrosarcoma cells by Wei-Ju Lin; Yi-Lun Chien; Chia-Yu Pan; Tai-Lang Lin; Jyh-Yih Chen; Shu-Jun Chiu; Cho-Fat Hui (283-290).
Epinecidin-1, a synthetic 21-mer antimicrobial peptide originally identified from grouper (Epinephelus coioides), specifically exhibited high antimicrobial activities against both Gram-negative and Gram-positive bacteria. In the current study we report on the in vitro cytotoxicity of the peptide, an important factor before it can be considered for further applications in cancer therapy. The cytotoxicity of epinecidin-1 was investigated against several cancer cells (A549, HA59T/VGH, HeLa, HepG2, HT1080, RAW264.7, and U937) and normal cells (AML-12, NIH3T3, and WS-1) with the MTT assay, and the inhibition of cancer cell growth was confirmed by a soft agar assay and scanning electron microscopy. However, cell variations were detected with AO/EtBr staining, while apoptosis and necrosis gene expressions in HT1080 cells after treatment with the epinecidin-1 peptide and Nec-1 showed that epinecidin-1 had an anti-necrosis function in HT1080 cells. The data presented here indicate that epinecidin-1 has in vitro antitumor activity against the HT1080 cell line, and functions like lytic peptides. In addition, our results suggest that epinecidin-1 may prove to be an effective chemotherapeutic agent for human fibrosarcoma cells in the future.
Keywords: Epinecidin-1; Tumor cell inhibition; Lytic peptide;

Hylin a1, the first cytolytic peptide isolated from the arboreal South American frog Hypsiboas albopunctatus (“spotted treefrog”) by Mariana S. Castro; Tânia Cristina G. Ferreira; Eduardo M. Cilli; Edson Crusca; Maria José Soares Mendes-Giannini; Antonio Sebben; Carlos André O. Ricart; Marcelo V. Sousa; Wagner Fontes (291-296).
RP-HPLC fractionation of the electrically stimulated skin secretion of the arboreal South American frog Hypsiboas albopunctatus (“spotted treefrog”) led to the isolation of a cytolytic C-terminally amidated peptide. This novel peptide, named hylin a1 (Hy-a1), consists of 18 amino acid residues (IFGAILPLALGALKNLIK-NH2). The α-helical structure of the synthetic hylin a1 peptide was confirmed by CD spectroscopy in the presence of 60% (v/v) TFE. The synthetic peptide displayed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa and also against fungi (Candida albicans, C. krusei, C. parapsilosis and Cryptococcus neoformans). Hylin a1 was also able to disrupt human erytrocytes (HC50  = 18 μM). Similarity analysis using PSI-BLAST revealed 50–44% of identity to maximins Hv, H16, H15 and H10 from Bombina maxima and also to hylins b1 and b2 (Hy-b1 and Hy-b2) from Hypsiboas lundii (synonym: Hyla biobeba).
Keywords: Amphibian; Hypsiboas; Skin secretion; Hemolysis; Antimicrobial peptide;

Molecular, mass spectral, and physiological analyses of orcokinins and orcokinin precursor-related peptides in the lobster Homarus americanus and the crayfish Procambarus clarkii by Patsy S. Dickinson; Elizabeth A. Stemmler; Elizabeth E. Barton; Christopher R. Cashman; Noah P. Gardner; Szymon Rus; Henry R. Brennan; Timothy S. McClintock; Andrew E. Christie (297-317).
Recently, cDNAs encoding prepro-orcokinins were cloned from the crayfish Procambarus clarkii; these cDNAs encode multiple copies of four orcokinin isoforms as well as several other peptides. Using the translated open reading frames of the P. clarkii transcripts as queries, five ESTs encoding American lobster Homarus americanus orthologs were identified via BLAST analysis. From these clones, three cDNAs, each encoding one of two distinct prepro-hormones, were characterized. Predicted processing of the deduced prepro-hormones would generate 13 peptides, 12 of which are conserved between the 2 precursors: the orcokinins NFDEIDRSGFGFN (3 copies), NFDEIDRSGFGFH (2 copies) and NFDEIDRSGFGFV (2 copies), FDAFTTGFGHN (an orcomyotropin-related peptide), SSEDMDRLGFGFN, GDY(SO3)DVYPE, VYGPRDIANLY and SAE. Additionally, one of two longer peptides (GPIKVRFLSAIFIPIAAPARSSPQQDAAAGYTDGAPV or APARSSPQQDAAAGYTDGAPV) is predicted from each prepro-hormone. MALDI-FTMS analyses confirmed the presence of all predicted orcokinins, the orcomyotropin-related peptide, and three precursor-related peptides, SSEDMDRLGFGFN, GDYDVYPE (unsulfated) and VYGPRDIANLY, in H. americanus neural tissues. SAE and the longer, unshared peptides were not detected. Similar complements of peptides are predicted from P. clarkii transcripts; the majority of these were detected in its neural tissues with mass spectrometry. Truncated orcokinins not predicted from any precursor were also detected in both species. Consistent with previous studies in the crayfish Orconectes limosus, NFDEIDRSGFGFN increased mid-/hindgut motility in P. clarkii. Surprisingly, the same peptide, although native to H. americanus, did not affect gut motility in this species. Together, our results provide the framework for future investigations of the regulation and physiological function of orcokinins/orcokinin precursor-related peptides in astacideans.
Keywords: Homarus americanus; Procambarus clarkii; Orcokinin; Orcomyotropin; Expressed sequence tag (EST); cDNA; Sinus gland; Commissural ganglion; Stomatogastric ganglion; Supraoesophageal ganglion; Matrix assisted laser desorption/ionization Fourier transform mass spectrometry;

House musk shrew (Suncus murinus, order: Insectivora) as a new model animal for motilin study by Chihiro Tsutsui; Kie Kajihara; Takatsugu Yanaka; Ichiro Sakata; Zen Itoh; Sen-ichi Oda; Takafumi Sakai (318-329).
Although many studies have demonstrated the action of motilin on migrating motor complex by using human subjects and relatively large animals, the precise physiological mechanisms of motilin remain obscure. One reason for the lack of progress in this research field is that large animals are generally not suitable for molecular-level study. To overcome this problem, in this study, we focused on the house musk shrew (Suncus murinus, order: Insectivora, suncus named as laboratory strain) as a small model animal, and we present here the results of motilin gene cloning and its availability for motilin study. The motilin gene has a high homology sequence with that of other mammals, including humans. Suncus motilin is predicted to exist as a 117-residue prepropeptide that undergoes proteolytic cleavage to form a 22-amino-acid mature peptide. The results of RT-PCR showed that motilin mRNA is highly expressed in the upper small intestine, and low levels of expression were found in many tissues. Morphological analysis revealed that suncus motilin-producing cells were present in the upper small intestinal mucosal layer but not in the myenteric plexus. Administration of suncus motilin to prepared muscle strips of rabbit duodenum showed almost the same contractile effect as that of human motilin. Moreover, suncus stomach preparations clearly responded to suncus or human motilin stimulation. To our knowledge, this is the first report that physiological active motilin was determined in small laboratory animals, and the results of this study suggest that suncus is a suitable model animal for studying the motilin-ghrelin family.
Keywords: cDNA cloning; Tissue distribution; Gastrointestinal tract; Smooth muscle contraction;

Passive transfer of Plasmodium falciparum MSP-2 pseudopeptide-induced antibodies efficiently controlled parasitemia in Plasmodium berghei-infected mice by Paola A. Martínez; Nubia Yandar; Liliana P. Lesmes; Martha Forero; Oscar Pérez-Leal; Manuel Elkin Patarroyo; José Manuel Lozano (330-342).
We have developed monoclonal antibodies directed against the pseudopeptide ψ-130, derived from the highly conserved malarial antigen Plasmodium falciparum merozoite surface protein 2 (MSP-2), for obtaining novel molecular tools with potential applications in the control of malaria. Following isotype switching, these antibodies were tested for their ability to suppress blood-stage parasitemia through passive immunization in malaria-infected mice. Some proved totally effective in suppressing a lethal blood-stage challenge infection and others reduced malarial parasitemia. Protection against P. berghei malaria following Ig passive immunization can be associated with specific immunoglobulins induced by a site-directed designed MSP-2 reduced amide pseudopeptide.
Keywords: Pseudopeptide; Ig isotype; Murine malarial infection; In vivo neutralizing activity; Anti-malarial vaccine;

Identification of nose-to-brain homing peptide through phage display by Xiao-Mei Wan; Yong-Ping Chen; Wen-Rui Xu; Wen-jun Yang; Long-Ping Wen (343-350).
Brain delivery of drug molecules through the nasal passage represents a viable approach for bypassing the blood-brain barrier (BBB) but remains a major challenge due to lack of efficient homing carriers. To screen for potential peptides with the ability to transport into the brain via the nasal passage, we applied a C7C phage peptide display library (Ph.D.-C7C) intra-nasally to anesthetized rats and recovered phage from the brain tissue 45 min after phage administration. After three rounds of panning, 10 positive phage clones were selected and sequenced. Clone7, which exhibited highest translocation efficiency, was chosen for further studies. After nasal administration, Clone7 entered the brain within 30 min and exhibited translocation efficiency about 50-fold higher than a random phage. A 11-amino acid synthetic peptide derived from the displayed sequence of Clone7 (ACTTPHAWLCG) efficiently inhibited the nasal-brain translocation of Clone7. Both phage recovery results and fluorescent microscopy images revealed the presence of many more Clone7 phage in the brain than in the liver, kidney and other internal organs after the nasal administration, suggesting that Clone7 bypassed the BBB and entered brain directly. Furthermore, both Clone7 and the ACTTPHAWLCG peptide were found to be heavily distributed along the olfactory nerve after the nasal administration, further suggesting a direct passage route into the brain via the olfactory region. These results demonstrated the feasibility of using the in vivo phage display approach for selecting peptides with the nose-to-brain homing capability and may have implications for the development of novel targeting carriers useful for brain delivery.
Keywords: Blood-brain barrier; Brain; Drug delivery; Intranasal; Phage display;

Deslorelin, a luteinizing hormone releasing hormone (LHRH) agonist, is transported via the LHRH-receptor (LHRH-R) and exhibits regional variation as follows: inferior turbinate posterior (ITP) > medium turbinate posterior (MTP) > medium turbinate anterior (MTA) of the bovine nasal mucosa. Differential LHRH-R expression in various regions of the nose is a potential explanation for regional variation in deslorelin transport. Thus, the objective was to determine whether LHRH-R expression exhibits regional variation in bovine nasal mucosa. LHRH-R density (B max) and affinity constant (K d) were determined by saturation experiments using 0.5 mg tissue in the presence of increasing amounts of I125-deslorelin (100–100,000 cpm) at 4 °C for 4 h. The 50% inhibitory concentration (IC50) was determined by competition experiments using various amounts of unlabelled deslorelin (0.01–3000 ng) at 4 °C for 4 h. LHRH-R mRNA and protein expressions were determined using real-time PCR and Western blot analysis, respectively. LHRH-R B max and K d varied between the regions of excised bovine nasal mucosa: ITP > MTP > MTA. The inhibition experiments yielded two IC50 concentrations which exhibited trends similar to B max and K d. Real-time PCR and Western blot analysis indicated that LHRH-R expression exhibits similar trends: ITP > MTP > MTA. We identified two deslorelin binding sites in the nasal tissues, with high affinity sites representing approximately 60–70% of the total sites available. In summary, regional differences in nasal deslorelin transport correlate with regional differences in LHRH-R expression, with LHRH-R expression, peptide binding, and transport being the highest in the inferior turbinate posterior region of the nose.
Keywords: Deslorelin; Nasal turbinates; Luteinizing hormone releasing hormone (LHRH); LHRH-receptor (LHRH-R);

GalNAc-transferase specificity prediction based on feature selection method by Lin Lu; Bing Niu; Jun Zhao; Liang Liu; Wen-Cong Lu; Xiao-Jun Liu; Yi-Xue Li; Yu-Dong Cai (359-364).
Keywords: GalNAc-transferase; Maximum Relevance Minimum Redundancy method; Nearest Neighbor Algorithm; Amino Acid Index database; O-glycosylation;

Possible involvement of corticotropin-releasing factor receptor signaling on vascular inflammation by Yuri Inada; Keiichi Ikeda; Katsuyoshi Tojo; Masaya Sakamoto; Yuko Takada; Naoko Tajima (365-372).
Based on the reported anti-inflammatory and anti-stress responses by corticotropin-releasing factor (CRF) receptor signaling, endogenous CRF receptor agonists, CRF, urocortin (UCN) I and its related peptides, may play protective roles against cardiovascular stresses via the CRF receptor signaling. Therefore, the present study was designed to evaluate the involvement of CRF receptor signaling against vascular inflammatory stress using human aortic endothelial cells (HAECs). In addition, due to the possible involvement of CRF receptor signaling in the effects of statin on endothelial cells, the effects of pitavastatin on the expression of UCN-related peptides in HAECs were also evaluated. HAECs expressed all UCNs, CRF type 1 receptor (CRF-R1), and CRF type 2 (CRF-R2)α and CRF-R2β mRNAs. Real time PCR analysis revealed that UCN I mRNA was down-regulated, whereas UCN II mRNA was up-regulated by tumor necrosis factor (TNF)-α. Selective blockade of CRF-R1 resulted in significant increase in TNF-α-induced expression of vascular adhesion molecule-1 at mRNA level and E-selectin at mRNA and protein levels. Pitavastatin up-regulated UCN I mRNA without TNF-α, but co-incubation with pitavastatin and TNF-α resulted in decrease in UCN I mRNA. On the contrary, UCN II, CRF-R1, and CRF-R2 mRNAs were markedly increased by co-incubation of pitavastatin and TNF-α. These facts indicate that CRF-R1 signaling may have protective role against TNF-α-induced vascular inflammation. In addition, because of up-regulation of CRF-R1 mRNA by pitavastatin with or without TNF-α, CRF-R1 may be involved in the vasoprotective effects of pitavastatin.
Keywords: Urocortin; CRH receptors; Human aortic endothelial cells; TNF-α; Pitavastatin;

Periodic acceleration (pGz) acutely increases endothelial and neuronal nitric oxide synthase expression in endomyocardium of normal swine by Jose A. Adams; Heng Wu; Jorge A. Bassuk; Jaqueline Arias; Arkady Uryash; Paul Kurlansky (373-377).
Introduction: Periodic acceleration (pGz) is a non-invasive method of increasing pulsatile shear stress to the endothelium. pGz is achieved by the sinusoidal head to foot motion to the supine body. pGz increases endogenous production of nitric oxide in whole animal models and isolated perfused vessel preparations, and is cardioprotective when applied prior to, during and after ischemia reperfusion. In part, the protective effects of pGz are attributable to nitric oxide (NO). The purpose of this investigation was to determine whether pGz up-regulates NOS isoforms in the endomyocardium. Methods and results: Fifteen swine weight 15–20 kg, were anesthetized, instrumented to measure hemodynamics and randomized. Ten animals received 1 h of pGz at 180 cycles/min and Gz ± 3.9 m/s2 [pGz] in addition to conventional ventilatory support and five served as time controls. Results: pGz produced a 2.3 ± 0.4 and a 6.6 ± 0.1 fold significant increase in eNOS and phosphorylated eNOS, 3.6 ± 1.1 fold increase in nNOS, and no significant change in iNOS. pGz also produced a 2.4 ± 0.3 and 3.9 ± 0.2 folds significant increase in both total(t-Akt) and phosphorylated (p-Akt) Akt. Conclusions: pGz is associated with an increase in both total and phosphorylated eNOS and nNOS protein expression in endomyocardium, and induced significant increase in total and phosphorylated-Akt. The data indicates that pGz is a novel method to induce eNOS and nNOS production in the endomyocardium. Therefore, pGz may serve as a powerful non-invasive intervention to activate the beneficial cardiac effects of endothelial and neuronal NOS.
Keywords: Periodic acceleration; Nitric oxide; Endothelial nitric oxide synthase; Neuronal nitric oxide synthase; Inducible nitric oxide synthase; Akt; Pulsatile shear stress;

Hypertensive activity of synthesized PTH(25–34) and Ac-PTH(25–30)–NH2 in rats by Artur Lehmann; Konrad Boblewski; Arkadiusz Bonna; Zbigniew Maćkiewicz; Apolonia Rybczyńska (378-384).
Parathyroid hormone (PTH) is secreted by parathyroid glands and is the main known factor that control plasma calcium concentration. There are many indications that PTH or products of PTH degradation influence the mean arterial blood pressure (MAP). These observations might be important in diseases accompanied with the overproduction of PTH such as primary hyperparathyroidism (PHPT). It was shown that the six amino acids PTH precursor—PRO-PTH with reversed sequence (PRO-rs), which contains a rare tripeptide -Arg-Lys-Lys- fragment, induces significant hypertensive response in rats. This strong alkali tripeptide is also present in the position 25–27 of the PTH molecule. The aim of the present study was to synthesize, by the solid phase peptide synthesis method, PTH fragments including the -Arg-Lys-Lys- sequence and test their influence on blood pressure and calcium plasma concentration in rats. Our study demonstrated that PTH(25–34) and the acetylated amide analogue of PTH(25–30), (Ac-PTH(25-30)–NH2) were hypertensive in the physiological doses. The presence of strong alkali sequence -Arg-Lys-Lys- in PTH(25–30) fragment is not sufficient to induce hypertension either in physiological or pharmacological doses in rats. Therefore, both the proximity of the -Arg-Lys-Lys- sequence and length of the peptide might also play roles as pressure factors.
Keywords: PTH; Blood pressure; Plasma calcium concentration; Primary hyperparathyroidism;

Plasma brain natriuretic peptide at rest and after adenosine-induced myocardial ischemia in normotensive and essential hypertensive patients with suspected coronary artery disease by Fiorella Fontana; Pasquale Bernardi; Carmine Pizzi; Rosanna Di Toro; Santi Spampinato; Emilio Merlo Pich (385-390).
This study investigated plasma brain natriuretic peptide (BNP) levels in normotensive and hypertensive patients with suspected coronary artery disease during radionuclide pharmacological stress testing. Twenty-seven normotensive patients (15 males, aged 63.0 ± 4.5 years and 12 females, aged 63.0 ± 4.1 years) and 38 essential hypertensive patients (25 males, aged 63.3 ± 3.3 years and 13 females, aged 64.6 ± 2.6 years) with chest pain and exercise stress testing inconclusive for coronary artery disease underwent myocardial perfusion single-photon emission computed tomography (SPECT) using adenosine infusion. SPECT identified patients without (16 normotensive and 22 hypertensive) and patients with (11 normotensive and 16 hypertensive) transient perfusion defects. Basal BNP levels in normotensive patients without transient myocardial ischemia (3.1 ± 1.2 fmol/ml) were significantly (P  < 0.01) lower than those observed in normotensive patients with transient ischemia (8.2 ± 1.2 fmol/ml), whereas BNP levels in hypertensive patients without transient ischemia (8.2 ± 1.0 fmol/ml) did not significantly differ from those in hypertensive patients with transient ischemia (8.1 ± 2.0 fmol/ml). No significant difference was found in BNP levels between males or females either in normotensive or hypertensive patients without or with ischemia. Adenosine infusion did not significantly change BNP levels in any subject group without or with myocardial perfusion defects. Our findings show that increases in BNP allow early detection of myocardial ischemia in normotensive patients, but not in hypertensive patients with suspected coronary artery disease. Adenosine-induced myocardial ischemia does not affect BNP production already activated by coronary artery disease in normotensive patients and by hemodynamic changes in hypertensive patients.
Keywords: Brain natriuretic peptide; Adenosine; Myocardial scintigraphy; Myocardial ischemia; Coronary artery disease; Essential hypertension;

Upregulated expression of intermedin and its receptor in the myocardium and aorta in spontaneously hypertensive rats by Qiang Zeng; Ying Yuan; Xi Wang; Hong Mei Wu; Li Fan; Yong Fen Qi; Chao Shu Tang; Yan Cai; Chun Shui Pan (391-399).
Intermedin (IMD), also called adrenomedullin 2 (ADM2), is a 47-amino acid peptide belonging to the calcitonin/calcitonin gene-related peptide (CGRP) family. IMD has similar or more potent vasodilatory and hypotensive actions compared with adrenomedullin (ADM) and CGRP. This study was designed to explore the role of IMD and its receptor in the pathogenesis of spontaneous hypertension and cardiac hypertrophy. Radioimmunoassay was employed to determine plasma immunoreactive IMD concentration and tissue immunoreactive IMD levels in the myocardium and aorta as well as cAMP concentration in the cardiovascular tissues in 13-week-old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). The mRNA expression of IMD, its receptor, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP)) were determined by semi-quantitative RT-PCR. Protein levels of CRLR and RAMPs were assayed by Western blotting. Our results showed that immunoreactive IMD concentration was enhanced in the SHR myocardium, aortas and plasma. Both the mRNA and protein levels of IMD, as well as those of CRLR and RAMP 1–3 were upregulated in SHRs. IMD affected cAMP generation in the myocardium and aorta, which were not attenuated by prior addition of either CGRP8–37 or ADM22–52 alone. These results indicate that the elevation of IMD and its receptor in the cardiovascular tissue may play an important role in the pathogenesis of spontaneous hypertension.
Keywords: Spontaneously hypertensive rats; Intermedin/adrenomedullin 2; Calcitonin receptor-like receptor; Receptor activity-modifying proteins; cAMP;

Increased expression of urotensin II-related peptide and its receptor in kidney with hypertension or renal failure by Nobuyoshi Mori; Takuo Hirose; Takashi Nakayama; Osamu Ito; Masayuki Kanazawa; Yutaka Imai; Masahiro Kohzuki; Kazuhiro Takahashi; Kazuhito Totsune (400-408).
Urotensin II-related peptide (URP) is a novel vasoactive peptide that shares urotensin II receptor (UT) with urotensin II. In order to clarify possible changes of URP expression in hypertension and chronic renal failure (CRF), the expressions of URP and UT were studied by quantitative RT-PCR and immunohistochemistry in kidneys obtained from spontaneous hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and WKY with CRF due to 5/6 nephrectomy. Expression levels of URP mRNA and UT mRNA were significantly higher in the kidneys obtained from SHR compared with age-matched WKY (at 5–16 and 16 weeks old, respectively). A dissection study of the kidney into three portions (inner medulla, outer medulla and cortex) showed that the expression levels of URP mRNA and UT mRNA were highest in the inner medulla and the outer medulla, respectively, in both SHR and WKY. The expression levels of URP and UT mRNAs were greatly elevated in the remnant kidneys of CRF rats at day 56 after nephrectomy, compared with sham-operated rats (about 6.5- and 11.9-fold, respectively). Immunohistochemistry showed that URP immunostaining was found mainly in the renal tubules, vascular smooth muscle cells and vascular endothelial cells. UT immunoreactivity was localized in the renal tubules and vascular endothelial cells. These findings suggest that the expressions of URP and UT mRNAs in the kidney are enhanced in hypertension and CRF, and that URP and its receptor have important pathophysiological roles in these diseases.
Keywords: Urotensin II; Urotensin II-related peptide; Kidney; Polymerase chain reaction; Immunohistochemistry; Vasoactive peptide;

Identification of novel synthetic peptide showing angiogenic activity in human endothelial cells by Chang Hee Lee; Mi-sook Lee; Sun-jin Kim; Young-tae Je; Sung Ho Ryu; Taehoon Lee (409-418).
A novel synthetic hexapeptide (SFKLRY-NH2) that displays angiogenic activity has been identified by positional scanning of a synthetic peptide combinatorial library (PS-SPCL). The peptide induced proliferation, migration, and capillary-like tube formation in primary cultured HUVECs, and augmented vessel sprouting ex vivo while attenuated by the treatment with pertussis toxin (PTX) or U73122 (PLC-inhibitor) suggesting the influence of PTX-sensitive G-proteins and PLC. In addition, SFKLRY-NH2 up-regulated the expression of VEGF-A in HUVECs and the neutralizing antibody against VEGF suppressed SFKLRY-NH2-induced tube formation activity. Taken together, these results suggest that SFKLRY-NH2 may induce blood vessel formation by PTX-sensitive G protein-coupled receptor-PLC-Ca2+ signaling cascade leading into VEGF-A expression in HUVECs.
Keywords: SFKLRY-NH2; GPCR; Calcium; VEGF; HUVECs; Angiogenesis; PS-SPCL;

Acute neurohumoral modulation of diastolic function by Ricardo Ladeiras-Lopes; João Ferreira-Martins; Adelino F. Leite-Moreira (419-425).
Diastole plays a central role in cardiovascular homeostasis. Its two main determinants, myocardial relaxation and passive properties of the ventricular wall, are nowadays regarded as physiological mechanisms susceptible of active modulation. Furthermore, diastolic dysfunction and heart failure with normal ejection fraction (previously called diastolic heart failure) are two subjects of major clinical relevance and an intense area of research. The role of several neurohumoral mediators like angiotensin-II and endothelin-1 on the modulation of diastolic function was systematically described as having only chronic deleterious effects such as cardiac hypertrophy and fibrosis. However, over the last years a growing body of evidence described a new role for several peptides on the acute modulation of diastolic function. In the acute setting, some of these mediators may have the potential to induce an adaptive cardiac response. In this review, we describe the role of angiotensin-II, endothelin-1, nitric oxide, urotensin-II and ghrelin on the acute modulation of diastolic function, emphasizing its pathophysiological relevance. Only a thorough understanding of diastolic physiology as well as its active modulation, both in the acute and chronic settings, will improve our knowledge on diastolic dysfunction and allow us to solve the enigmas of heart failure with normal ejection fraction.
Keywords: Diastole; Cardiovascular physiology; Heart failure; Angiotensin-II; Endothelin-1;

Lunasin, a novel seed peptide for cancer prevention by Blanca Hernández-Ledesma; Chia-Chien Hsieh; Ben O. de Lumen (426-430).
Carcinogenesis is a multistage process derived from a combination of multiple heritable and environmental factors. It has been reported that populations consuming high levels of soybean products have both lower cancer incidence and mortality rates in the western countries. Lunasin is a novel and promising peptide initially discovered in soy and now found in wheat, barley and other seeds. Its cancer-preventive efficacy has been shown in mammalian cells which were induced by chemical carcinogens and viral oncogenes. Moreover, this peptide has been found to prevent skin cancer in a mouse cancer model induced by chemical carcinogens. Its bioavailability after oral administration makes it a perfect candidate as a chemopreventive agent. The purpose of this article is to review the discovery of this seed peptide and the most recent evidence on its possible benefits as an anticancer agent.
Keywords: Lunasin; Chemopreventive seed peptide; Cancer prevention;

Is GPR39 the natural receptor of obestatin? by Xiao-Ying Dong; Jin-Ming He; Sheng-Qiu Tang; Hai-Yun Li; Qing-Yan Jiang; Xiao-Ting Zou (431-438).
GPR39, an orphan receptor belonging to the family of G protein-coupled receptors, was originally reported to be the receptor of obestatin. However recently, numerous reports have questioned this conclusion. In mammals, GPR39 was reported to be involved in the regulation of gastrointestinal and the metabolic functions. In this article, a latest and brief review on the receptor family, structure, distribution and physiological functions of GPR39 has been reported.
Keywords: Distribution; Function; GPR39; Receptor family; Structure; Obestatin;

Obestatin, obesity and diabetes by An-Jing Ren; Zhi-Fu Guo; Yang-Kai Wang; Li Lin; Xing Zheng; Wen-Jun Yuan (439-444).
The high prevalence of obesity and diabetes will lead to higher rates of morbidity and mortality. It is well known that ghrelin plays a potential role in obesity and diabetes. Obestatin, a novel 23 amino acid amidated peptide encoded by the same gene that encodes ghrelin, was initially reported to have opposite actions to ghrelin in the regulation of food intake, emptying of the stomach and body weight. Recent work suggests that obestatin also regulate β-cell survival and insulin secretion. The ghrelin–obestatin system is, therefore, a promising target for the developing of new drugs for the treatment of obesity and diabetes. This review summarizes the interrelationship between obestatin, obesity and diabetes.
Keywords: Obestatin; Ghrelin; Obesity; Diabetes;