Peptides (v.57, #C)

Ghrelin receptors in human gastrointestinal tract during prenatal and early postnatal development by Olivera Mitrović; Vladan Čokić; Dragoslava Đikić; Mirela Budeč; Sanja Vignjević; Tijana Subotički; Miloš Diklić; Rastko Ajtić (1-11).
The aim of our study was to investigate the appearance, density and distribution of ghrelin cells and GHS-R1a and GHS-R1b in the human stomach and duodenum during prenatal and early postnatal development. We examined chromogranin-A and ghrelin cells in duodenum, and GHS-R1a and GHS-R1b expression in stomach and duodenum by immunohistochemistry in embryos, fetuses, and infants. Chromogranin-A and ghrelin cells were identified in the duodenum at weeks 10 and 11 of gestation. Ghrelin cells were detected individually or clustered within the base of duodenal crypts and villi during the first trimester, while they were presented separately within the basal and apical parts of crypts and villi during the second and third trimesters. Ghrelin cells were the most numerous during the first (∼11%) and third (∼10%) trimesters of gestation development. GHS-R1a and GHS-R1b were detected at 11 and 16 weeks of gestation, showed the highest level of expression in Brunner's gland and in lower parts of duodenal crypts and villi during the second trimester in antrum, and during the third trimester in corpus and duodenum. Our findings demonstrated for the first time abundant duodenal expression of ghrelin cells and ghrelin receptors during human prenatal development indicating a role of ghrelin in the regulation of growth and differentiation of human gastrointestinal tract.
Keywords: Human fetal duodenum; GHS-R1a and GHS-R1b; Appearance; Distribution and density of ghrelin cells;

Serum levels of novel adipokines in patients with acute ischemic stroke: Potential contribution to diagnosis and prognosis by Nikolaos P.E. Kadoglou; Grigorios Fotiadis; Vaia Lambadiari; Eirini Maratou; George Dimitriadis; Christos D. Liapis (12-16).
This study evaluated serum levels of novel adipokines in acute ischemic stroke (AIS) and their association with prognosis. We enrolled 168 patients with AIS and 58 stroke-free age- and sex-matched individuals (controls). Clinical parameters, carotid ultrasound, metabolic profile, vaspin, apelin, visfatin, and ghrelin were assayed. Stroke-patients were sampled at hospital admission and were prospectively followed-up (median 16 months) for the cardiovascular endpoint (cardiovascular death/stroke/myocardial infarction). At admission, stroke-patients appeared with higher levels of systolic blood pressure, hsCRP and worse metabolic profile (p  < 0.05), (p  > 0.05). Compared to controls, AIS group had significantly higher serum concentrations of visfatin (22.92 ± 9.72 ng/ml vs 16.56 ± 7.82 ng/ml, p  = 0.006) and lower of vaspin (0.94 ± 0.43 ng/ml vs 1.84 ± 0.82 ng/ml, p  = 0.019) and ghrelin (3.47 ± 1.44 ng/ml vs 5.93 ± 2.78 ng/ml, p  < 0.001), while apelin did not differ between groups. Similar differences in adipokines were found between stroke subgroups with and without significant ipsilateral carotid stenosis (>50%) (p  < 0.05). In stepwise logistic regression analysis adjusted for diabetes, hypertension, dyslipidemia and age, visfatin (p  = 0.026) and ghrelin (p  = 0.012) proved to be independent predictors of AIS. During follow-up, 27 patients achieved cardiovascular endpoint. In addition to coronary artery disease and NIHSS score, visfatin serum levels was associated with cardiovascular endpoint (HR: 1.255, 95% CI: 1.025–1.576). Our results suggested the association of AIS with higher visfatin and lower vaspin and ghrelin serum levels. Visfatin levels can be a predictor of cardiovascular mortality and morbidity in AIS.
Keywords: Acute ischemic stroke; Apelin; Vaspin; Visfatin; Ghrelin; Adipokines;

Serum antimicrobial peptides in patients with familial Mediterranean fever by Abdurrahman Tufan; Rıdvan Mercan; Ozge Tugce Pasaoglu; Hatice Pasaoglu; Mehmet Akif Ozturk; Berna Goker; Seminur Haznedaroglu (17-19).
Familial Mediterranean fever (FMF) is characterized by recurrent inflammation of serosal and synovial membranes. Despite the fact that it is a genetic disease, environmental factors, including infections, are shown to be triggering factors associated with the precipitation of attacks in FMF. Antimicrobial peptides (AMPs) are components of innate immunity which exert antimicrobial activity against many microorganisms. Human AMPs; cathelicidin (LL37) and defensins have immunomodulatory properties and are involved in the pathogenesis of many inflammatory disorders. Hence, we investigated serum AMPs in 23 newly diagnosed FMF patients. Blood samples were obtained at baseline, 6 months after initiation of colchicine and during an attack. Twenty-four healthy individuals constituted the control group. The concentrations of LL37, alpha-1, beta-1 and beta-2 defensins were determined by ELISA. Serum AMPs did not change during attacks and did not correlate with acute phase reactants. However, serum LL37 and defensins were found to be remarkably higher in FMF patients compared to healthy individuals both at baseline and 6 months after initiation of colchicine therapy which suggest that AMPs might have a role in the pathogenesis of FMF.
Keywords: Antimicrobial peptide; Cathelicidin; Defensin; Familial Mediterranean fever; Inflammation; Pathogenesis;

How does stress possibly affect cardiac remodeling? by Dejana Popovic; Bosiljka Plecas-Solarovic; Vesna Pesic; Milan Petrovic; Bosiljka Vujisic-Tesic; Bojana Popovic; Svetlana Ignjatovic; Arsen Ristic; Svetozar S. Damjanovic (20-30).
The aim of this study was to evaluate the predictive value of adrenocorticotropic hormone (ACTH), cortisol and ACTH receptor polymorphism (ACTHRP) for left ventricular (LV) remodeling. Thirty-six elite male athletes, as chronic stress adaptation models, and twenty sedentary age and sex-mached subjects emabarked on standard and tissue Doppler echocardiography to assess cardiac parameters at rest. They performed maximal cardiopulmonary test, which was used as an acute stress model. ACTH and cortisol were measured at rest (10 min before test), at beginning, at maximal effort, at 3rd min of recovery, using radioimmunometric and radioimmunoassey techniques, respectively. Promoter region of ACTHR gene (18p11.2) was analysed from blood samples using reverse polymerization reaction with the analysis of restriction fragment length polimorphisam by SacI restriction enzyme. Normal genotype was CTC/CTC, heterozygot for ACTHRP CTC/CCC and homozygot CCC/CCC. In all participants, ACTH and cortisol increased during acute stress, whereas in recovery ACTH increased and cortisol remained unchanged. 49/56 examiners manifested CTC/CTC, 7/56 CTC/CCC and 0/56 CCC/CCC. There was no difference in ACTHRP frequency between groups ( χ ( 1 ) 2 = 0.178 , p  = 0.67). LV mass (LVM) and LV end-diastolic volume (LVVd) were higher in athletes than in controls (p  < 0.01) and lower in CTC/CTC than in CTC/CCC genotype (219.43 ± 46.59(SD)g vs. 276.34 ± 48.86(SD)g, p  = 0.004; 141.24 ± 24.46(SD)ml vs. 175.29 ± 37.07(SD)ml, p  = 0.002; respectively). In all participants, predictors of LVM and LVVd were ACTH at rest (B  = −1.00,−0.44; β  = −0.30,−0.31; p  = 0.026,0.012, respectively) and ACTHRP (B  = 56.63,34; β  = 0.37,0.40; p  = 0.003,0.001, respectively). These results demonstrate that ACTH and ACTHRP strongly predict cardiac morphology suggesting possible regulatory role of stress system activity and sensitivity in cardiac remodeling.
Keywords: Stress; Adrenocorticotropic hormone; Cortisol; Adrenocorticotropic hormone receptor polymorphism; Left ventricular remodeling;

Preoperative plasma leptin levels predict delirium in elderly patients after hip fracture surgery by Xue-Wu Chen; Jun-Wu Shi; Ping-Shan Yang; Zhu-Qi Wu (31-35).
Leptin is considered to be a modulator of the immune response. Hypoleptinemia increases the risk for Alzheimer's disease and vascular dementia. The present study aimed to investigate the ability of plasma leptin level to predict delirium in elderly patients after hip fracture surgery. Postoperative delirium (pod) was evaluated using the Confusion Assessment Method. Prolonged postoperative delirium (ppod) was defined as delirium lasting more than 4 weeks. Plasma leptin levels of 186 elderly patients and 186 elderly controls were measured by an enzyme-linked immunosorbent assay. Plasma leptin level was substantially lower in patients than in controls (4.6 ± 2.2 ng/ml vs. 7.5 ± 1.8 ng/ml, P  < 0.001). It was identified as an independent predictor for pod [odds ratio, 0.385; 95% confidence interval (CI), 0.286–0.517; P  < 0.001] and ppod (odds ratio, 0.283; 95% CI, 0.152–0.527; P  < 0.001) using a multivariate analysis, and had high area under receiver operating characteristic curve for pod [area under curve (AUC), 0.850; 95% CI, 0.790–0.898] and ppod (AUC, 0.890; 95% CI, 0.836–0.931). The predictive value of leptin was markedly bigger than that of age for pod (AUC, 0.705; 95% CI, 0.634–0.770; P  = 0.002) and ppod (AUC, 0.713; 95% CI, 0.642–0.777; P  = 0.019). In a combined logistic-regression model, leptin improved the AUC of age to 0.890 (95% CI, 0.836–0.931) (P  < 0.001) for pod and 0.910 (95% CI, 0.860–0.947) (P  = 0.005) for ppod. Thus, preoperative plasma leptin level may be a useful, complementary tool to predict delirium and also prolonged delirium in elderly patients after hip fracture surgery.
Keywords: Leptin; Elderly; Delirium; Hip fracture; Postoperative;

Hepcidin is an antimicrobial peptide and iron-regulatory molecule with highly conserved disulfide bridges among vertebrates, but structural insights into the function in fish remains largely missing. We demonstrate here that recombinant hepcidin-2 from zebrafish is capable of inhibiting the growth of the Gram-negative bacteria Escherichia coli and Vibrio anguillarum, and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentrations (MICs) of 18, 15, 13 and 9 μM, respectively. We also show by TEM examination that recombinant hepcidin-2 is directly cidal to the cells of E. coli and S. aureus. Moreover, we find that hepcidin-2 displays affinity to LPS, LTA and PGN. All these data indicate that hepcidin-2 is both a pattern recognition molecule, capable of identifying LPS, LTA and PGN, and an antibacterial effector, capable of inhibiting the growth of bacteria. The data also show that the antibacterial activity of hepcidin-2 depends upon the disulfide bridges.
Keywords: Zebrafish; Danio rerio; Hepcidin-2; Bactericidal activity;

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors by Alice B. Nongonierma; Catherine Mooney; Denis C. Shields; Richard J. FitzGerald (43-51).
Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC50 216 μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC50 values of 3.5 and 44.2 μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure–activity relationship modeling, rather than on docking, in computationally selecting peptides for screening.
Keywords: Dipeptidyl peptidase IV inhibitors; Molecular docking; Bioactive peptides; Milk; Predictive modeling; Hydrophobicity;

In insects, posttranslational modifications of neuropeptides are largely restricted to C- and N-terminal amino acids. The most common modifications, N-terminal pyroglutamate formation and C-terminal α-amidation, may prevent a fast degradation of these messenger molecules. This is particularly important for peptide hormones. Other common posttranslational modifications of proteins such as glycosylation and phosphorylation seem to be very rare in insect neuropeptides. To check this assumption, we used a computer algorithm to search an extensive data set of MALDI-TOF mass spectra from cockroach tissues for ion signal patterns indicating peptide phosphorylation. The results verify that phosphorylation is indeed very rare. However, a candidate was found and experimentally verified as phosphorylated CAPA pyrokinin (GGGGpSGETSGMWFGPRL-NH2) in the cockroach Lamproblatta albipalpus (Blattidae, Lamproblattinae). Tandem mass spectrometry revealed the phosphorylation site as Ser5. Phosphorylated CAPA pyrokinin was then also detected in most other cockroach lineages (e.g. Blaberidae, Polyphagidae) but not in closely related blattid species such as Periplaneta americana. This is remarkable since the sequence of CAPA pyrokinin is identical in Lamproblatta and Periplaneta. A consensus sequence of CAPA pyrokinins of cockroaches revealed a conserved motif that suggests phosphorylation by a Four-jointed/FAM20C related kinase.
Keywords: Insect neuropeptides; Posttranslational modification; Phosphorylation; CAPA pyrokinin; MALDI-TOF mass spectrometry;

Identification of the ghrelin-GHSR 1 system and its influence in the modulation of induced ocular hypertension in rabbit and rat eyes by A. Rocha-Sousa; P. Pereira-Silva; M. Tavares-Silva; S. Azevedo-Pinto; J. Rodrigues-Araújo; S. Pinho; A. Avelino; F. Falcão-Reis; A. Leite-Moreira (59-66).
Recent studies evidenced a decrease in ghrelin's aqueous humor levels in patients with glaucoma. The goal of our investigation was to study the effect of the ghrelin-GHSR-1a system in the modulation of intraocular pressure in acute ocular hypertension models and its expression and distribution in ocular tissues. Two animal models of acute ocular hypertension were used to study the effect of the ghrelin-GHSR-1a system in the modulation of intraocular pressure: the rabbit and the rat. Ocular hypertension was induced by an intravitreal injection of 20% NaCl. Ghrelin or des-acyl ghrelin were delivered subconjunctivally and the intraocular pressure was assessed by a rebound tonometer that was calibrated for each species. In addition, we have studied the influence of nitric oxide and prostaglandins on ghrelin's effect in the rabbit animal model. Finally, we determined by immunofluorescence the expression of ghrelin and GHSR-1 in the rat's ocular tissue. Ghrelin decreased the intraocular pressure in both animal models (maximum decrease: 43.8 ± 12.0% in the rabbit and 29.0 ± 7.46% in the rat). In the rabbit, this effect was blunted in the presence of l-NAME and ketorolac. Des-acyl ghrelin only decreased the intraocular pressure in the rat (maximum decrease: 34.9 ± 8.15%). Ghrelin expression was detected in the ciliary processes and GHSR-1 expression was detected in the trabecular meshwork and ciliary body. The ghrelin-GHSR-1 system is expressed in the anterior segment of the eye. Ghrelin and des-acyl ghrelin are responsible for a hypotensive effect in acute ocular hypertension animal models.
Keywords: Ghrelin; Des-acyl ghrelin; GHSR-1a; Intraocular pressure;

Frog skin constitutes a rich source of peptides with a wide range of biological properties. These include host-defense peptides with cytotoxic activities against bacteria, fungi, protozoa, viruses, and mammalian cells. Several hundred such peptides from diverse species have been described. Although attention has been focused mainly on antimicrobial activity, the therapeutic potential of frog skin peptides as anti-infective agents remains to be realized and no compound based upon their structures has yet been adopted in clinical practice. Consequently, alternative applications are being explored. Certain naturally occurring frog skin peptides, and analogs with improved therapeutic properties, show selective cytotoxicity against tumor cells and viruses and so have potential for development into anti-cancer and anti-viral agents. Some peptides display complex cytokine-mediated immunomodulatory properties. Effects on the production of both pro-inflammatory and anti-inflammatory cytokines by peritoneal macrophages and peripheral blood mononuclear cells have been observed so that clinical applications as anti-inflammatory, immunosuppressive, and immunostimulatory agents are possible. Several frog skin peptides, first identified on the basis of antimicrobial activity, have been shown to stimulate insulin release both in vitro and in vivo and so show potential as incretin-based therapies for treatment of patients with Type 2 diabetes mellitus. This review assesses the therapeutic possibilities of peptides from frogs belonging to the Ascaphidae, Alytidae, Pipidae, Dicroglossidae, Leptodactylidae, Hylidae, and Ranidae families that complement their potential role as anti-infectives for use against multidrug-resistant microorganisms.
Keywords: Host-defense peptide; Frog skin; Anti-cancer; Anti-viral; Immunomodulatory; Type 2 diabetes;

Cell penetrating peptides: Efficient vectors for delivery of nanoparticles, nanocarriers, therapeutic and diagnostic molecules by Samad Mussa Farkhani; Alireza Valizadeh; Hadi Karami; Samane Mohammadi; Nasrin Sohrabi; Fariba Badrzadeh (78-94).
Efficient delivery of therapeutic and diagnostic molecules to the cells and tissues is a difficult challenge. The cellular membrane is very effective in its role as a selectively permeable barrier. While it is essential for cell survival and function, also presents a major barrier for intracellular delivery of cargo such as therapeutic and diagnostic agents. In recent years, cell-penetrating peptides (CPPs), that are relatively short cationic and/or amphipathic peptides, received great attention as efficient cellular delivery vectors due to their intrinsic ability to enter cells and mediate uptake of a wide range of macromolecular cargo such as plasmid DNA (pDNA), small interfering RNA (siRNAs), drugs, and nanoparticulate pharmaceutical carriers. This review discusses the various uptake mechanisms of these peptides. Furthermore, we discuss recent advances in the use of CPP for the efficient delivery of nanoparticles, nanocarriers, DNA, siRNA, and anticancer drugs to the cells. In addition, we have been highlighting new results for improving endosomal escape of CPP-cargo molecules. Finally, pH-responsive and activable CPPs for tumor-targeting therapy have been described.
Keywords: Cell penetrating peptides; Drug delivery; Endosomal escape; Nanocarrier; Nanoparticle; Gene delivery;

Biologically active leptin-related synthetic peptides activate STAT3 via phosphorylation of ERK1/2 and PI-3K by Hung-Yun Lin; Sheng-Huei Yang; Heng-Yuan Tang; Guei-Yun Cheng; Paul J. Davis; Patricia Grasso (95-100).
The effects of leptin-related synthetic peptides [d-Leu-4]-OB3 and OB3 on energy balance and glucose homeostasis in ob/ob and db/db mice have been confirmed. The molecular basis of these effects, however, remains unclear. In the present study, we examined the ability of these peptides to activate signal transduction pathways known to be involved in transduction of the leptin signal. In a specific and concentration-dependent manner, [d-Leu-4]-OB3 induced phosphorylation of ERK1/2, PI-3K, Ser-727 STAT3, and Tyr-705 of STAT3. OB3 also induced activation of STAT3 via phosphorylation of ERK1/2, STAT3 Ser-727, STAT3 Tyr-705 and PI-3K p85, but to a lesser degree. Using PD98059 and LY294002, specific inhibitors of MEK and PI-3K, respectively, we were able to identify the signal transduction pathways involved in peptide-induced STAT3 activation. [d-Leu-4]-OB3 induced serine phosphorylation of STAT3 primarily through activation of ERK1/2. Tyrosine phosphorylation of STAT3, however, was induced primarily through activation of PI-3K. Our data suggest that in db/db mice, [d-Leu-4]-OB3 binding to short isoforms of the leptin receptor induces intracellular signaling cascades which do not require OB-Rb activation. These signals may ultimately result in peptide effects on transcriptional and translational events associated with energy balance and glycemic regulation. In summary, we have shown for the first time that, similar to leptin, bioactive leptin-related synthetic peptide analogs activate STAT3 via phosphorylation of serine and tyrosine residues by multiple signal transduction pathways.
Keywords: Leptin; Mitogen-activated protein kinase (MAPK); Phosphatidylinositol 3-kinase (PI-3K); Signal transducer and activator of transduction-3 (STAT3);

Angiotensin II and 1-7 during aging in Metabolic Syndrome rats. Expression of AT1, AT2 and Mas receptors in abdominal white adipose tissue by M.E. Rubio-Ruíz; L. Del Valle-Mondragón; V. Castrejón-Tellez; E. Carreón-Torres; E. Díaz-Díaz; V. Guarner-Lans (101-108).
Renin-Angiotensin System (RAS) plays an important role in the development of Metabolic Syndrome (MS) and in aging. Angiotensin 1-7 (Ang 1-7) has opposite effects to Ang II. All of the components of RAS are expressed locally in adipose tissue and there is over-activation of adipose RAS in obesity and hypertension. We determined serum and abdominal adipose tissue Ang II and Ang 1-7 in control and MS rats during aging and the expression of AT1, AT2 and Mas in white adipose tissue. MS was induced by sucrose ingestion during 6, 12 and 18 months. During aging, an increase in body weight, abdominal fat and dyslipidemia were found but increases in aging MS rats were higher. Control and MS concentrations of serum Ang II from 6-month old rats were similar. Aging did not modify Ang II seric concentration in control rats but decreased it in MS rats. Ang II levels increased in WAT from both groups of rats. Serum and adipose tissue Ang 1-7 increased during aging in MS rats. Western blot analysis revealed that AT1 expression increased in the control group during aging while AT2 and Mas remained unchanged. In MS rats, AT1 and AT2 expression decreased significantly in aged rats. The high concentration of Ang 1-7 and adiponectin in old MS rats might be associated to an increased expression of PPAR-γ. PPAR-γ was increased in adipose tissue from MS rats. It decreased with aging in control rats and showed no changes during aging in MS rats. Ang 1-7/Mas axis was the predominant pathway in WAT from old MS animals and could represent a potential target for therapeutical strategies in the treatment of MS during aging.
Keywords: Renin-Angiotensin System; Age; White adipose tissue; Metabolic Syndrome;

Soluble DPP4 originates in part from bone marrow cells and not from the kidney by Zhendi Wang; Christina Grigo; Julia Steinbeck; Stephan von Hörsten; Kerstin Amann; Christoph Daniel (109-117).
Dipeptidyl peptidase 4 (DPP4) is known to inactivate incretins as well as important chemokines and neuropeptides. DPP4 is expressed as a transmembrane protein but also occurs as a soluble enzyme circulating in the blood. However, the origin of the soluble DPP4 (sDPP4) is still unknown. In this study, DPP4 activity was quantified in plasma and extracted from different rat organs. Then, in order to see if the kidney or the bone marrow was the source of sDPP4, kidney or bone marrow transplantation was performed between wildtype (wt) Dark Agouti (DA) and DPP4 deficient congenic rats (n  = 6–9). Kidney was verified to have the highest DPP4 activity, followed by spleen and lung. In the following three weeks after successful kidney transplantation only transient trace plasma DPP4 activity was detected in DPP4 deficient rats receiving wt kidneys. In addition, DPP4 activity was not diminished in DA wt rats receiving DPP4 deficient kidneys. Both findings indicated that sDPP4 did not originate from the kidney. In contrast, 43 ± 14% (compared to wt) sDPP4 activity was detected in the plasma of DPP4 deficient DA rats that were reconstituted with wt bone marrow cells. Not only leukocyte but also macrophage subpopulations express DPP4 in bone marrow as well as in blood as assessed by flow cytometry. Thus, bone marrow derived cells but not the kidney represent at least one source of sDPP4. And leukocyte or macrophage subpopulations could be potential candidates.
Keywords: Soluble DPP4; CD26; Kidney transplantation; Bone marrow; DPP4 deficient rat;

Biological properties of adrenomedullin conjugated with polyethylene glycol by Keishi Kubo; Mariko Tokashiki; Kenji Kuwasako; Masaji Tamura; Shugo Tsuda; Shigeru Kubo; Kumiko Yoshizawa-Kumagaye; Johji Kato; Kazuo Kitamura (118-121).
Adrenomedullin (AM) is a vasodilator peptide with pleiotropic effects, including cardiovascular protection and anti-inflammation. Because of these beneficial effects, AM appears to be a promising therapeutic tool for human diseases, while intravenous injection of AM stimulates sympathetic nerve activity due to short-acting potent vasodilation, resulting in increased heart rate and renin secretion. To lessen these acute reactions, we conjugated the N-terminal of human AM peptide with polyethylene glycol (PEG), and examined the biological properties of PEGylated AM in the present study. PEGylated AM stimulated cAMP production, an intracellular second messenger of AM, in cultured human embryonic kidney cells expressing a specific AM receptor in a dose-dependent manner, as did native human AM. The pEC50 value of PEGylated AM was lower than human AM, but no difference was noted in maximum response (Emax) between the PEGylated and native peptides. Intravenous bolus injection of 10 nmol/kg PEGylated AM lowered blood pressure in anesthetized rats, but the acute reduction became significantly smaller by PEGylation as compared with native AM. Plasma half-life of PEGylated AM was significantly longer than native AM both in the first and second phases in rats. In summary, N-terminal PEGylated AM stimulated cAMP production in vitro, showing lessened acute hypotensive action and a prolonged plasma half-life in comparison with native AM peptide in vivo.
Keywords: Adrenomedullin; Polyethylene glycol; Molecular modification;

Effects of sex and reproductive experience on the number of orexin A-immunoreactive cells in the prairie vole brain by Michael Donlin; Breyanna L. Cavanaugh; Olivia S. Spagnuolo; Lily Yan; Joseph S. Lonstein (122-128).
Large populations of cells synthesizing the neuropeptide orexin (OX) exist in the caudal hypothalamus of all species examined and are implicated in physiological and behavioral processes including arousal, stress, anxiety and depression, reproduction, and goal-directed behaviors. Hypothalamic OX expression is sexually dimorphic in different directions in laboratory rats (F > M) and mice (M > F), suggesting different roles in male and female physiology and behavior that are species-specific. We here examined if the number of hypothalamic cells immunoreactive for orexin A (OXA) differs between male and female prairie voles (Microtus ochrogaster), a socially monogamous species that pairbonds after mating and in which both sexes care for offspring, and if reproductive experience influences their number of OXA-immunoreactive (OXA-ir) cells. It was found that the total number of OXA-ir cells did not differ between the sexes, but females had more OXA-ir cells than males in anterior levels of the caudal hypothalamus, while males had more OXA-ir cells posteriorly. Sexually experienced females sacrificed 12 days after the birth of their first litter, or one day after birth of a second litter, had more OXA-ir cells in anterior levels but not posterior levels of the caudal hypothalamus compared to females housed with a brother (incest avoidance prevents sibling mating). Male prairie voles showed no effect of reproductive experience but showed an unexpected effect of cohabitation duration regardless of mating. The sex difference in the distribution of OXA-ir cells, and their increased number in anterior levels of the caudal hypothalamus of reproductively experienced female prairie voles, may reflect a sex-specific mechanism involved in pairbonding, parenting, or lactation in this species.
Keywords: Hormones; Hypothalamus; Monogamy; Neuropeptides; Reproduction; Reward;

Orexins are hypothalamic neuropeptides, which are involved in several physiological functions of the central nervous system, including anxiety and stress. Several studies provide biochemical and behavioral evidence about the anxiogenic action of orexin A. However, we have little evidence about the underlying neuromodulation. Therefore, the aim of the present study was to investigate the involvement of neurotransmitters in the orexin A-induced anxiety-like behavior in elevated plus maze (EPM) test in mice. Accordingly, mice were pretreated with a non-selective muscarinic cholinergic antagonist, atropine; a γ-aminobutyric acid subunit A (GABA-A) receptor antagonist, bicuculline; a D2, D3, D4 dopamine receptor antagonist, haloperidol; a non-specific nitric oxide synthase (NOS) inhibitor, nitro-l-arginine; a nonselective α-adrenergic receptor antagonist, phenoxybenzamine and a β-adrenergic receptor antagonist, propranolol 30 min prior to the intracerebroventricular administration of orexin A. The EPM test started 30 min after the i.c.v. injection of the neuropeptide. Our results show that orexin A decreases significantly the time spent in the arms (open/open + closed) and this action is reversed by bicuculline, phenoxybenzamine and propranolol, but not by atropine, haloperidol or nitro-l-arginine. Our results provide evidence for the first time that the orexin A-induced anxiety-like behavior is mediated through GABA-A-ergic, α- and β-adrenergic neurotransmissions, whereas muscarinic cholinergic, dopaminergic and nitrergic neurotransmissions may not be implicated.
Keywords: Orexin A; Anxiety; Elevated plus maze; Neurotransmitter; Receptor antagonist;

Corrigendum to “Adrenomedullin and intermedin gene transcription is increased in leukocytes of patients with chronic heart failure at different stages of the disease” [Peptides 55 (2014) 13–16] by Manuela Cabiati; Laura Sabatino; Benedetta Svezia; Raffaele Caruso; Alessandro Verde; Chiara Caselli; Tommaso Prescimone; Daniela Giannessi; Silvia Del Ry (135).

Corrigendum to “A novel single-domain peptide, anti-LPS factor from prawn: Synthesis of peptide, antimicrobial properties and complete molecular characterization” [Peptides 53 (2014) 79–88] by Jesu Arockiaraj; Venkatesh Kumaresan; Prasanth Bhatt; Rajesh Palanisamy; Annie J. Gnanam; Mukesh Pasupuleti; Marimuthu Kasi; Mukesh Kumar Chaurasia (136).