Peptides (v.30, #5)

Protective effects of a cysteine proteinase propeptide expressed in transgenic soybean roots by Brener M. Marra; Djair S.L. Souza; João N. Aguiar; Alexandre A.P. Firmino; Rafael P.D. Sarto; Francine B. Silva; Charles D.S. Almeida; Juvenil E. Cares; Marise V. Continho; Cezar Martins-de-Sa; Octavio L. Franco; Maria F. Grossi-de-Sa (825-831).
Sedentary endoparasitic nematodes cause extensive damage to a large number of ornamental plants and food crops, with estimated economical losses over 100 billion US$ worldwide. Various efforts have put forth in order to minimize nematode damage, which typically involve the use of nematicides that have high cost and enhanced toxicity to humans and the environment. Additionally, different strategies have been applied in order to develop genetically modified plants with improved nematode resistance. Among the strategies are anti-invasion and migration, feeding-cell attenuation, and anti-nematode feeding. In the present study, we focus on anti-nematode feeding, which involves the evaluation and potential use of the cysteine proteinase (CPs) propeptide as a control alternative. The cysteine proteinase prodomain, isolated from Heterodera glycines (HGCP prodomain), is a natural inhibitory peptide used to transform soybean cotyledons using Agrobacterium rhizogenes. Genetically modified soybean roots expressing the propeptide were detected by Western blot and expression levels were measured by ELISA (around 0.3%). The transgenic roots expressing the propeptide were inoculated with a thousand H. glycines at the second juvenile stage, and a remarkable reduction in the number of females and eggs was observed. A reduction of female length and diameter was also observed after 35 days post-inoculation. Furthermore, the H. glycines mature protein was detected in females fed on soybean transformed root expressing or not expressing the propeptide. The data presented here indicate that the HGCP propeptide can reduce soybean cyst nematode infection and this strategy could be applied in the near future to generate resistant crop cultivars.
Keywords: Nematode; Cysteine proteinase; Propeptide; Transgenic plants; Heterodera glycines;

Indolicidin (IN) is a 13-residue Trp-rich antimicrobial peptide isolated from bovine neutrophils. To develop novel IN-derived antimicrobial peptides with enhanced cell specificity (therapeutic index) and potent anti-inflammatory activity, several IN analogs were synthesized by Pro → Lys substitution. All IN analogs displayed an increase in therapeutic index by 3- to 15-fold relative to parental IN. IN and its analogs induced a significant membrane depolarization against intact Staphylococcus aureus in a dose-dependent manner and depolarized membrane potential at 5 μg/ml (MIC for S. aureus) almost completely. However, these peptides caused less than 40% calcein leakage from negatively charged EYPG/EYPE liposomes mimicking bacterial membranes at 10 μg/ml. Based on these results, we hypothesize that IN and its analogs kill microorganisms via the formation of small ion channels that permit transit of ions or protons, but not molecules as large as calcein. Furthermore, IN and its analogs induced a remarkable suppression in nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression in LPS-stimulated mouse macrophage RAW264.7 cells. All IN analogs showed LPS-binding activity comparable to that of IN. Taken together, their potent antimicrobial, anti-inflammatory and LPS-neutralizing activities similar to those of IN, coupled with their no cytotoxicity, our designed IN analogs make excellent candidates for novel antimicrobial and anti-sepsis agents.
Keywords: Antimicrobial peptide; Indolicidin analogs; Cell specificity; Anti-inflammatory activity; LPS-binding activity; DNA binding activity;

Structure and function of a custom anticancer peptide, CB1a by Jiun-Ming Wu; Pey-Shynan Jan; Hui-Chen Yu; Hsu-Yuang Haung; Huey-Jen Fang; Yuan-I Chang; Jya-Wei Cheng; Hueih Min Chen (839-848).
Several natural antimicrobial peptides including cecropins, magainins and melittins have been found to kill cancer cells. However, their efficacy may not be adequate for their development as anticancer agents. In this study, we used a natural antimicrobial peptide, cecropin B (CB), as a template to generate a novel anticancer peptide. Cecropin B is an amphipathic and polycationic peptide derived from the hemolymph of Hyalophora cecropia with well-known antimicrobial and cytolytic properties. The signature pattern of cecropins is W-x-(0,2)-[KDN]-x-{L}-K-[KRE]-[LI]-E-[RKN] (PROSITE: PS00268), and this signature sequence is located at N-terminus of CB. CB1a was constructed by repeating the N-terminal ten amino acids of CB three times and including a hinge near C-terminus. The circular dichroism spectra showed that CB1a is unstructured in aqueous solution, but adopt a helical conformation in membrane-like environment. The solution structure of CB1a in a polar solvent was also studied by NMR. CB1a formed a helix–hinge–helix in 20% HFIP solution, and it was found the bent angle between two helical segments was induced ranging from 60° to 110°. A heparin-binding motif is located in the central part of helix 1. Isothermal titration calorimetry reveals the association constant of CB1a bound to low molecular weight heparin is 1.66 × 105  M−1 at physiological ionic strength at 25 °C. Binding of CB1a to heparin produces a large conformational change toward a more structural state. CB1a demonstrated promising activity against several cancer cells but low toxicity against non-cancer cells. The IC50 of CB1a on leukemia and stomach carcinoma cells were in the range of 2–8-fold lower than those of CB. Besides, CB1a exhibited low hemolytic activity against human red blood cells. Due to these properties, CB1a has the potential to become a promising anticancer agent.
Keywords: Cecropin B; Antimicrobial peptide; Anticancer activity; Heparin-binding motif; CD; NMR solution structure; ITC;

Amyloid-beta peptide Aβp3-42 affects early aggregation of full-length Aβ1-42 by Hiromi M. Sanders; Robert Lust; Jan K. Teller (849-854).
The major amyloid-beta (Aβ) peptides found in the brain of familial and late onset Alzheimer's disease include the full-length Aβ1-42 and N-terminally truncated, pyroglutamylated peptides Aβp3-42 and Aβp11-42. The biophysical properties of Aβ1-42 have been extensively studied, yet little is known about the other modified peptides. We investigated the aggregation kinetics of brain-specific Aβ peptides to better understand their potential roles in plaque formation. Synthetic peptides were analyzed individually and in mixtures representing various ratios found in the brain. Spectrofluorometric analyses using Thioflavin-T showed that the aggregation of Aβ1-42 was faster compared to Aβp3-42; however, Aβp11-42 displayed similar kinetics. Surprisingly, mixtures of full-length Aβ1-42 and Aβp3-42 showed an initial delay in β-sheet formation from both equimolar and non-equimolar samples. Electron microscopy of peptides individually and in mixtures further supported fluorescence data. These results indicate that Aβ–Aβ peptide interactions involving different forms may play a critical role in senile plaque formation and maintenance of the soluble Aβ pool in the brain.
Keywords: Alzheimer's disease; Amyloid-beta peptides; Aggregation; Pyroglutamate;

Allatostatins (ASTs), with a C-terminal sequence Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-amide, are multifunctional neuropeptides that were first discovered by their ability to inhibit juvenile hormone (JH) synthesis by the corpora allata (CA) in cockroaches. These A-type ASTs have since been demonstrated to inhibit JH synthesis in crickets, termites and more recently locusts. The gene for the precursor of A-type ASTs has been identified in several species of cockroaches, in crickets and in locusts, but not yet in termites, although 5 AST peptides were isolated from the lower termite Reticulitermes flavipes that are identical to known cockroach ASTs. In this study, primers designed from AST amino acid sequences of cockroaches are used to identify the gene for the preproAST peptides in R. flavipes. In addition, the expression of the gene in brain tissues is demonstrated for egg-laying and non-egg-laying neotenic reproductives. The gene codes for 14 individual peptides and its sequence is closer to that of cockroaches and the cricket than to that of other insect orders in which these peptides do not act as allatostatins. Among the known cockroach AST genes, the termite AST gene is most similar to that of Periplaneta americana, a species belonging to the primitive family Blattidae.
Keywords: Reticulitermes flavipes; Allatostatin; Termite; Cockroach;

New conotoxins define the novel I3-superfamily by Duo-Duo Yuan; Li Liu; Xiao-Xia Shao; Can Peng; Cheng-Wu Chi; Zhan-Yun Guo (861-865).
We purified two novel conotoxins, designated as ca11a and ca11b, from the venom of Conus caracteristicus. Based on the amino acid sequence of mature ca11a, we cloned its full-length cDNA. Based on the signal peptide of ca11a, several ca11a-like conotoxins were cloned from C. caracteristicus and C. pulicarius. These novel conotoxins have an I-superfamily cysteine pattern but with a novel signal peptide sequence, suggesting they belong to a new branch of I-superfamily, designated as I3-superfamily. Additionally, two O-superfamily conotoxins were also cloned based on the signal peptide of ca11a, suggesting a possible evolutionary relationship between O- and I-superfamilies.
Keywords: Conotoxin; I-superfamily; cDNA cloning;

Non-strict strand orientation of the Ca2+-induced dimerization of a conantokin peptide variant with sequence-shifted γ-carboxyglutamate residues by Qiuyun Dai; Cai Xiao; Mingxin Dong; Zhuguo Liu; Zhenyu Sheng; Francis J. Castellino; Mary Prorok (866-872).
We have previously found a new mode of metal ion-induced helix–helix assembly for the γ-carboxyglutamate (Gla)-containing, neuroactive conantokin (con) peptides that is independent of the hydrophobic effect. In these unique “metallo-zipper” assemblies of con-G and con-T[K7γ], interhelical Ca2+ coordination induces dimer formation with strictly antiparallel chain orientation in conantokin peptides in which Gla residues are positioned at “i, i + 4, i + 7, i + 11” intervals. In order to probe the property of self-assembly in conantokin peptides with an extended Gla network, a con-T variant (con-T-tri) was synthesized that contains five Gla residues spaced at “i, i + 4, i + 7, i + 11, i + 14” intervals. Sedimentation equilibrium analyses showed that Ca2+, but not Mg2+, was capable of promoting con-T-tri self-assembly. Oxidation and rearrangement assays with Cys-containing con-T-tri variants revealed that the peptide strands in the complex can orient in both parallel and antiparallel forms. Stable parallel and antiparallel dimeric forms of con-T-tri were modeled using disulfide-linked peptides and the biological viability of these species was confirmed by electrophysiology. These findings suggest that small changes within the helix–helix interface of the conantokins can be exploited to achieve desired modes of strand alignment.
Keywords: Conantokin peptides; Self-association; γ-Carboxyglutamic acid; Dimerization; Strand alignment;

Comparative immunohistochemical analysis of the distribution of orexins (hypocretins) in the brain of amphibians by Jesús M. López; Laura Domínguez; Nerea Moreno; Agustín González (873-887).
The orexins (hypocretins) are peptides found primarily in neurons of the hypothalamus of all vertebrates. Many differences were reported about the precise location of orexin containing cells and their projections throughout the brain in different species. However, there are few direct cross-species comparisons. Previous studies in anuran amphibians have also reported notable species differences. We examined and directly compared the distribution of orexinergic neurons and fibers within the brains of representatives of the three amphibian orders, anurans, urodeles and gymnophionans. Simultaneous detection of orexins and tyrosine hydroxylase was used to assess the precise location of the orexins in the brain and to evaluate the possible influence of the orexin system on the catecholaminergic cell groups. Although some differences were noted, a common pattern for the distribution of orexins in amphibians was observed. In all species, most immunoreactive neurons were observed in the suprachiasmatic nucleus, whereas the cells in the preoptic area and the tuberal region were more variable. Orexin immunoreactive fibers in the brain of all species included abundant fibers throughout the preoptic area and hypothalamus, whereas moderate amounts of fibers were present in the pallium, striatum, septum, thalamus, optic tectum, torus semicircularis, rhombencephalon and spinal cord. The use of double immunohistochemistry in amphibians revealed orexinergic innervation in dopaminergic and noradrenergic cell groups, such as the midbrain tegmentum, locus coeruleus and nucleus of the solitary tract, as was previously reported in mammals.
Keywords: Orexin; TH; Immunohistochemistry; Suprachiasmatic nucleus; Hypothalamus; Evolution;

A glycine-leucine-rich peptide structurally related to the plasticins from skin secretions of the frog Leptodactylus laticeps (Leptodactylidae) by J. Michael Conlon; Yasser H.A. Abdel-Wahab; Peter R. Flatt; Jérôme Leprince; Hubert Vaudry; Thierry Jouenne; Eric Condamine (888-892).
A glycine-leucine-rich peptide was isolated from norepinephrine-stimulated skin secretions of the Sante Fe frog Leptodactylus laticeps (Leptodactylidae) whose primary structure (Gly-Leu-Val-Asn-Gly-Leu-Leu-Ser-Ser-Val-Leu-Gly-Gly-Gly-Gln-Gly-Gly-Gly-Gly-Leu-Leu-Gly-Gly-Ile-Leu) contains the (GXXXG)3 motif found in the plasticins, previously identified only in phyllomedusid frogs (Hylidae). Circular dichroism studies showed that the secondary structure of the peptide, termed plasticin-L1, was markedly solvent-dependent displaying a random coil conformation in water, a β-sheet structure in methanol, and an α-helical conformation in 50% trifluoroethanol–water. A synthetic replicate of the peptide did not inhibit the growth of Escherichia coli or Staphylococcus aureus or lyse human erythrocytes at concentrations up to 500 μM. At relatively high concentrations (≥1 μM), the peptide produced a significant (P  < 0.05), although modest (139% of basal rate at 3 μM), increase in the rate of glucose-induced release of insulin from rat clonal BRIN-BD11 β cells without increasing the rate of release of lactate dehydrogenase. A peptide, termed ocellatin-L2 was also identified in the skin secretion that was identical to the previously described ocellatin-L1 except for the substitution Asn23  → Asp. Ocellatin-L2 was devoid of antimicrobial and hemolytic activity but also showed significant activity in stimulating insulin release from BRIN-BD11 cells (181% of basal rate at 3 μM).
Keywords: Frog skin; Leptodactylidae; Phyllomedusinae; Plasticin; Ocellatin;

Bradykinin-related peptides (BRPs) represent one of the most widespread and closely studied families of amphibian defensive skin secretion peptides. Apart from canonical bradykinin (RPPGFSPFR) that was first reported in skin extracts of the European brown frog, Rana temporaria, many additional site-substituted, N- and/or C-terminally extended peptides have been isolated from skin extracts and secretions from representative species of the families Ranidae, Hylidae, Bombinatoridae and Leiopelmatidae. The most diverse range of BRPs has been found in ranid frog skin secretions and this probably reflects the diversity and number of species studied and their associated life histories within this taxon. Amolops (torrent or cascade frogs) is a genus within the Ranidae that has been poorly studied. Here we report the presence of two novel BRPs in the skin secretions of the Chinese Wuyi Mountain torrent frog (Amolops wuyiensis). Amolopkinins W1 and W2 are dodecapeptides differing in only one amino acid residue at position 2 (Val/Ala) that are essentially (Leu1, Thr6)-bradykinins extended at the N-terminus by either RVAL (W1) or RAAL (W2). Amolopkinins W1 and W2 are structurally similar to amolopkinin L1 from Amolops loloensis and the major BRP (Leu1, Thr6, Trp8)-bradykinin from the skin of the Japanese frog, Rana sakuraii. A. wuyiensis amolopkinins were separately encoded as single copies within discrete precursors of 61 amino acid residues as deduced from cloned skin cDNA. Synthetic replicates of both peptides were found to potently antagonize the contractile effects of canonical bradykinin on isolated rat ileum smooth muscle preparations. Amolopkinins thus appear to represent a novel sub-family of ranid frog skin secretion BRPs.
Keywords: Bradykinin; Cloning; Peptide synthesis; Smooth muscle; Antagonist;

Altered reactivity of gastric fundus smooth muscle in the mouse with targeted disruption of the kinin B1 receptor gene by Ana M.R.B. Barbosa; Sandra A. Felipe; Ronaldo C. Araujo; Elisa M. Kawamoto; Maria H.C. Carvalho; Cristoforo Scavone; João B. Pesquero; Suma I. Shimuta (901-905).
Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B1 receptor (B1 −/−). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B1 −/− when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ (10 μM). It was also found that Ca2+-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca2+-independent inducible NOS (iNOS) activity was greater in B1 −/− mice than in WT animals. Zaprinast (100 μM), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B1 −/− stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B1 −/− mice. In addition, it can be suggested that functional B2 receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B1 receptor in the gastric tissue of the transgenic mice.
Keywords: Kinin B1 receptor knockout mouse; Relaxant responses; Mouse gastric fundus; cGMP; Phosphodiesterase;

Lipopolysaccharides stimulate adrenomedullin synthesis in intestinal epithelial cells: Release kinetics and secretion polarity by Hiroshi Kishikawa; Jiro Nishida; Hitoshi Ichikawa; Shogo Kaida; Tetsuo Morishita; Soichiro Miura; Toshifumi Hibi (906-912).
Adrenomedullin (AM), a potent vasodilator peptide initially isolated from a human pheochromocytoma, functions as an antimicrobial peptide in host defense. In this study, we investigated changes in AM levels in intestinal epithelial cells and the mechanism of its secretion and cellular polarity after exposure to lipopolysaccharides (LPS). When a rat small intestinal cell line (IEC-18 cells) was exposed to LPS, enzyme-linked immunosorbent assay revealed a dose-dependent increase in AM together with an increase in AM mRNA expression, as determined by real-time polymerase chain reaction. Up-regulation of AM by LPS was dose-dependently inhibited by LY294002, PD98059, SP600125 and calphostin-C, suggesting the involvement of the phosphatidylinositol 3 kinase, extracellular signal-regulated kinase, c-Jun NH2-terminal kinase and protein kinase C pathways, respectively, in this process. When polarized IEC-18 cells in a Transwell chamber were stimulated with LPS, AM secretion was directed primarily toward the side of LPS administration (either the apical or basolateral side). In situ hybridization revealed that AM mRNA was expressed in epithelial cells and in the connective tissue in the lamina propria of the jejunum after intraperitoneal or oral administration of LPS. Higher levels of AM mRNA expression were observed in rats treated with LPS via the intraperitoneal route, compared with those treated via the oral route. These findings suggest that intestinal AM plays an important role in mucosal defense in the case of intestinal luminal infection, as well as in the modulation of hemodynamics in endotoxemia.
Keywords: Adrenomedullin; Small intestine; Antimicrobial peptides; Polarity; In situ hybridization;

Nitric oxide mediates feedback inhibition in angiotensin II-induced upregulation of vasopressin mRNA by Luqing Zhang; Ming Tong; Ming Xiao; Lei Li; Jiong Ding (913-917).
Angiotensin II (Ang II) stimulates hypothalamic magnocellular neurons to release arginine vasopressin (AVP) via Ang II type 1 (AT1) receptors during chronic hyperosmotic condition. On the other hand, endogenous nitric oxide (NO) tonically inhibits the activity of AVP producing neurons; and system infusion of Ang II elicits the activity of NO producing neurons in the hypothalamus. These studies suggest that NO may mediate feedback inhibition in Ang II modulation of AVP neuronal excitability. To confirm this hypothesis, we first investigated colocalization of neuronal NO synthase (nNOS) and AT1 receptors in the hypothalamic magnocellular nuclei of adult male rats by using double immunofluorescence. We found that 60% and 65% of AT1 receptors immunoreactive neurons coexpressed nNOS in the hypothalamic paraventricular nucleus and supraoptic nucleus, respectively. We then demonstrated that intracerebroventricular administration of nNOS inhibitor N-omega-nitro-l-arginine methyl ester further enhanced upregulation of AVP mRNA level but totally abolished upregulation of nNOS mRNA level in the paraventricular and supraoptic nuclei of anesthetized rats induced by a prior administration of Ang II. Theses morphological and pharmacological data demonstrate that NO mediates negative feedback regulation of Ang II-induced upregulation of AVP mRNA.
Keywords: Angiotensin II; Angiotensin II type 1 receptors; Arginine vasopressin; mRNA; Neuronal nitric oxide synthase; Nitric oxide; Hypothalamus;

Evidence on the relative roles of endothelin ETA and ETB receptors in mediating the nociceptive and hyperalgesic actions of endothelin-1 is still fragmented and conflicting, due to variations between species and/or models. This study assesses the participation of ETA and ETB receptors on the nociceptive behavior and hyperalgesia to chemical (formalin), mechanical and thermal stimuli evoked by endothelin-1 injected into the rat hind-paw. Intraplantar ( injection of endothelin-1 (1-30 pmol, 50 μl) induced dose-dependent nociceptive behaviors over the first hour. Endothelin-1 (3–30 pmol) also potentiated both phases of nociception induced by a subsequent ipsilateral injection of formalin (0.5%, 50 μl). Endothelin-1, at 10 pmol, increased responses of the first phase (0–10 min) by 97% and of the second phase (15–60 min) by 120%, and similar degrees of potentiation were observed following 30 pmol of the peptide. Endothelin-1 (1–30 pmol) caused slowly developing long-lasting thermal and mechanical hyperalgesia with maximum effects at 10 and 30 pmol, respectively, reaching significance at 2–3 h and remaining elevated for up to at least 8 h after injection. Treatment with the selective ETA and ETB peptidic antagonists BQ-123 and BQ-788 (, both at 10 nmol, 3.5 h after ET-1 injection) or with the non-peptidic antagonists atrasentan and A-192621 systemically (i.v., 10 and 20 mg/kg, respectively) each caused significant reductions in endothelin-1-induced nociception, as well as chemical, thermal and mechanical hyperalgesia. Thus, the nociceptive and hyperalgesic effects induced by endothelin-1 seem to be mediated by both ETA and ETB receptors.
Keywords: Endothelin; Nociception; Hyperalgesia; ETA receptor; ETB receptor;

Nociceptin-induced modulation of human T cell function by Kate H. Easten; Rachel A. Harry; Wendy M. Purcell; Julie D. McLeod (926-934).
There is an accumulating evidence for the immunoregulatory role of the neuropeptide, nociceptin/orphanin FQ (N/OFQ) however its role on T cell function requires elucidation. This study has demonstrated an inhibitory role for N/OFQ on SEB-activated T cell function. N/OFQ decreases T cell proliferation, which is abrogated when the costimulatory receptors CD80 and CD86 are blocked. In addition, evidence suggests that the immunoregulatory cytokines TGF-β, IFN-γ and nitric oxide (NO) are involved in the N/OFQ effect. N/OFQ also, through involvement of IFN and NO, induces the expression of the immunosuppressive modulator indoleamine 2,3-dioxygenase (IDO), suggesting a central role for IDO in the N/OFQ effect on T cell proliferation. The data presented in this report indicate a multi-faceted mechanism of action used by N/OFQ to modulate T cell function.
Keywords: Nociceptin; IDO; IFN-γ; T cells; Neuropeptides; SEB;

Neuromedin-U inhibits unilateral adrenalectomy-induced compensatory adrenal growth in the rat by Ludwik K. Malendowicz; Diego Guidolin; Marcin Trejter; Marcin Rucinski; Andrea Porzionato; Raffaele de Caro; Magdalena Nowak (935-939).
Neuromedin-U (NMU) is a brain–gut peptide, which has been previously found to stimulate hypothalamic–pituitary–adrenal axis in the rat and to control the growth of the rat adrenal cortex. The present study aimed to investigate the possible involvement of NMU in the regulation of unilateral adrenalectomy-induced compensatory adrenal growth, a phenomenon known to be neurally mediated. The right adrenal gland of mature female rats was removed, the contralateral gland was then analyzed at 24 and 72 h following surgery. Groups of rats were given 3 subcutaneous injections (24, 16 and 8 h before decapitation) of NMU8 (1.5 or 3.0 nmol/100 g/per injection). Three hours before sacrifice all rats received an intraperitoneal injection of 0.1 mg/100 g body weight of vincristin. By means of RT-PCR the presence of NMUR1 mRNA was detected in adrenal cortex of both intact and hemiadrenalectomized rats. As expected, unilateral adrenalectomy-induced an increase in adrenal weight, associated with increased plasma ACTH, aldosterone and corticosterone levels. The administration of NMU to hemiadrenalectomized rats did not significantly affect these parameters. NMU administration, however, notably inhibited the unilateral adrenalectomy-induced adrenocortical cell proliferation in both zona glomerulosa and zona fasciculata, as assessed by the metaphase index and the number of parenchymal cell nuclei per unit area of the tissue. When compared to hemiadrenalectomized animals receiving saline, a significant decrease of blood corticosterone levels was observed after 24 h in rats treated with the highest dose of NMU. Since these effects were independent on changes in blood ACTH, they could reflect an interaction of NMU with the neural system innervating the adrenal gland.
Keywords: Neuromedin-U; Neuromedin-U receptor; Compensatory adrenal growth; Unilateral adrenalectomy; Cell proliferation; ACTH; Corticosterone;

Social isolation modulates corticotropin-releasing factor type 2 receptor, urocortin 1 and urocortin 2 mRNAs expression in the cardiovascular system of prairie voles by Hossein Pournajafi-Nazarloo; Leila Partoo; Lisa Sanzenbacher; Maryam Esmaeilzadeh; Jamespaul Paredes; Kozo Hashimoto; Fereidoun Azizi; C. Sue Carter (940-946).
The purpose of the present study was to examine the effect of social isolation stress on the expression of messengers ribonucleic acid (mRNAs) for corticotropin-releasing factor receptor type 2 (CRF2 receptor), urocortin 1 (Ucn 1) and urocortin 2 (Ucn 2) in the cardiovascular system of female and male prairie voles (Microtus ochrogaster). Isolation for 1 h (single isolation) or 1 h of isolation every day for 4 weeks (repeated isolation) was followed by a marked increase in plasma corticosterone level. However, continuous isolation for 4 weeks (chronic isolation) did not significantly affect plasma corticosterone level in female or male animals. A single period of isolation did not influence the expression of the CRF2 receptor, however, both repeated and chronic isolation significantly decreased CRF2 receptor mRNA in the ventricle and aorta of both sexes. Neither single nor chronic isolation significantly affected Ucn 1 mRNAs expression; however, repeated isolation increased Ucn 1 mRNA expression in the ventricles of female and male animals. Although, a single isolation produced no effect on cardiac Ucn 2 mRNA expression, both repeated and chronic isolation were followed by increased heart Ucn 2 mRNA expression in both sexes. We speculate that during repeated isolation Ucn 1 along with Ucn 2 are increased, which in turn down-regulates CRF2 receptor mRNA expression, and that Ucn 2 also may be one of factors responsible for the down-regulation of CRF2 receptor mRNA expression in cardiovascular system that is associated with chronic isolation.
Keywords: Cardiovascular system; CRF receptor; Isolation stress; Urocortin;

Corticotropin-releasing factor (CRF) is well known for its role in the hypothalamic-pituitary-adrenocortical (HPA) axis and its involvement in stress and anxiety. CRF acts via two main receptor subtypes, CRF1 and CRF2. Other endogenous CRF-related peptide ligands are the Urocortins 1 and 2 and Stresscopin. While CRF is thought to mediate its anxiogenic-like properties through CRF1, the role of CRF2 and its endogenous ligands Urocortin 2 and Stresscopin are less clear, with a suggested role in mediating the delayed effects of stress. Measurement of local cerebral glucose utilization (LCGU) provides an estimate of neuronal activity, and is of potential use as a translational tool in comparison to FDG PET. We hypothesized that comparison of the patterns of metabolic changes induced by CRF-related peptides could provide further information on their role in the brain. The present studies examined the effects of CRF-related peptides on LCGU, and the role of CRF1 and CRF2 in the CRF-induced LCGU response. CRF induced increases in LCGU in hypothalamic, thalamic, cerebellar and hippocampal regions, and further studies using antagonists or mutant mice lacking a functional CRF1 receptor clearly suggested a role for CRF2 in this effect. Urocortin 1 increased LCGU in a dissected hindbrain region. However, central administration of the CRF2-selective agonists Urocortin 2 and Stresscopin failed to affect LCGU, which may suggest ligand-dependent receptor activation within the CRF system. The present data supports a role for CRF2 in the regulation of neuronal glucose metabolism.
Keywords: Glucose metabolism; Corticotropin-releasing factor; Hypothalamic-pituitary-adrenal axis; 2-Deoxyglucose;

GHTD-amide: A naturally occurring beta cell-derived peptide with hypoglycemic activity by S.G. Paule; B. Nikolovski; R.E. Gray; J.P. Ludeman; A. Freemantle; R.A. Spark; J.B. Kerr; F.M. Ng; P.Z. Zimmet; M.A. Myers (955-961).
In the early 1970s, a peptide fraction with insulin potentiating activity was purified from human urine but the identity and origins of the active constituent remained unknown. Here we identify the active component and characterize its origins. The active peptide was identified as an alpha amidated tetrapeptide with the sequence GHTD-amide. The peptide was synthesized and tested for stimulation of glycogen synthesis and insulin potentiation by insulin tolerance testing in insulin-deficient rats, which confirmed GHTD-amide as the active peptide. Tissue localization using a peptide-specific anti-serum and epifluorescent and confocal microscopy showed decoration of pancreatic islets but not other tissues. Confocal microscopy revealed co-localization with insulin and immunogold and electron microscopy showed localization to dense core secretory granules. Consistent with these observations GHTD-amide was found in media conditioned by MIN6 islet beta cells. Sequence database searching found no annotated protein in the human proteome encoding a potential precursor for GHTD-amide. We conclude that the insulin potentiating activity originally described in human urine is attributable to the tetrapeptide GHTD-amide. GHTD-amide is a novel peptide produced by pancreatic beta cells and no precursor protein is present in the annotated human proteome. Stimulation of glycogen synthesis and co-localization with insulin in beta cells suggest that GHTD-amide may play a role in glucose homeostasis by enhancing insulin action and glucose storage in tissues.
Keywords: Peptide; Alpha amidation; Pancreatic beta cell; Insulin; Islets of Langerhan;

Central leptin gene therapy, a substitute for insulin therapy to ameliorate hyperglycemia and hyperphagia, and promote survival in insulin-deficient diabetic mice by Shinya Kojima; Akihiro Asakawa; Haruka Amitani; Takeo Sakoguchi; Naohiko Ueno; Akio Inui; Satya P. Kalra (962-966).
Long-term benefits of central leptin gene therapy in insulin-deficient diabetes are not known despite its therapeutic effects in obesity animal models such as ob/ob and diet-induced obese mice. Adult male mice were injected intraperitoneally with streptozotocin (STZ, 200 mg/kg) to induce insulitis. A week later, only diabetic STZ-pretreated mice (blood glucose >350 mg/dl) received intracerebroventricularly (icv) an injection of recombinant adeno-associated virus vector (rAAV) encoding either green fluorescent protein (control), or leptin gene (rAAV-lep). Body weight (BW), food intake, blood glucose, insulin and survival rate responses were monitored post-icv injection at regular intervals for 52 weeks. The STZ pre-injected diabetic mice remained hyperphagic, gradually lost BW and died by week 6 after receiving control vector. In marked contrast, injection of rAAV-lep to raise hypothalamic leptin levels, rescued the STZ-pretreated mice from early mortality, gradually curbed hyperphagia to normalize intake by week 20, and maintained BW at significantly lower than the control range. Blood glucose levels in these mice started to recede dramatically by week 2–3 to normalize by week 8, and euglycemia was sustained during the remaining course of the experiment. rAAV-lep injected mice did not exhibit any discernible untoward gross behavioral changes and diabetic complications and showed a partial return of pancreatic β-cell function. These results show for the first time that one time central leptin gene therapy is effective and durable in reinstating euglycemia and energy homeostasis for extended periods in the absence of insulin.
Keywords: Leptin; Gene therapy; Diabetes mellitus; Type 1; Longevity;

Leptin increases osteoblast-specific osteocalcin release through a hypothalamic relay by Satya P. Kalra; Michael G. Dube; Urszula T. Iwaniec (967-973).
Enhanced long-term expression of leptin by gene therapy selectively in the hypothalamus, without leakage to the systemic circulation, abrogated skeletal abnormalities and reinstated weight and insulin–glucose homeostasis in leptin-deficient ob/ob mice. Whether increases in osteocalcin, a hormone produced by osteoblasts and known to play a role in bone growth and recently in glucose–insulin homeostasis, may link these benefits of central leptin was assessed. The effects of a single intraventricular injection of non-immunogenic, non-pathogenic recombinant adeno-associated virus vector encoding leptin gene (rAAV-lep) or green fluorescent protein gene (rAAV-GFP, control) were studied in three genotypes, wild type (wt), obese diabetic, hyperinsulinemic ob/ob and non-obese, diabetic insulinopenic Akita mice. Selective hypothalamic leptin expression with central rAAV-lep treatment decreased weight, fat mass, food intake, suppressed insulin levels in ob/ob and wt mice, and conferred euglycemia by suppressing blood glucose in all three genotypes. Contemporaneously, rAAV-lep treatment also augmented blood osteocalcin levels. In wt mice, osteocalcin rose by 51% and, whereas, basal osteocalcin levels in ob/ob and Akita mice were significantly lower as compared to those in wt mice (26% and 55%, respectively), gene therapy reinstated levels to the control range in ob/ob mice, and raised 40% above the wt range even in the absence of insulin in Akita mice. These findings demonstrate that the central beneficial effects of leptin on bone growth involve increased hypothalamic relay of signals that augment osteocalcin efflux from osteoblasts into the general circulation, a response that, in turn, may also modulate glucose–insulin and weight homeostasis.
Keywords: Osteocalcin; Osteoblast; Leptin; Hypothalamus; Bone remodeling; Glucose–insulin homeostasis;

Distinct roles of the DRY motif in rat melanin-concentrating hormone receptor 1 in signaling control by Yoshimi Aizaki; Kei Maruyama; Mitsue Nakano-Tetsuka; Yumiko Saito (974-981).
Rhodopsin family (class A) G protein-coupled receptors possess common key residues or motifs that appear to be important for receptor function. To clarify the roles of the highly conserved amino acid triplet Asp3.49–Arg3.50–Tyr3.51 (DRY motif), we examined how single-substitution mutations of the amino acids in the motif influenced specific features of rat melanin-concentrating hormone receptor 1 (MCH1R) activity. Substitution of either Asp1403.49 or Tyr1423.51 to Ala resulted in nonfunctional receptors, despite the retention of apparent potencies for agonist binding. These loss-of-function phenotypes may be caused by the lack of stimulation for GDP-GTP exchange observed in GTPγS-binding assays. On the other hand, substitution of Arg1413.50 to Ala caused a 4-fold reduction in the agonist binding affinity and, concomitantly, a rightward shift of the dose-dependency curve for calcium mobilization and inhibition of cyclic AMP production. Although many experimental studies have suggested that the DRY motif is involved in maintaining the receptor in its ground state, none of the DRY motif substitutions to Ala in MCH1R led to constitutive activation, in terms of the basal signaling level for ERK1/2 activation or GTPγS binding. These data suggest that the major contribution of the DRY motif in MCH1R is to govern receptor conformation and G protein coupling/recognition.
Keywords: G protein-coupled receptor; DRY motif; Melanin-concentrating hormone; Calcium influx; Site-directed mutagenesis; Constitutive activation;

Identification of ghrelin in the house musk shrew (Suncus murinus): cDNA cloning, peptide purification and tissue distribution by Yuko Ishida; Satoshi Sakahara; Chihiro Tsutsui; Hiroyuki Kaiya; Ichiro Sakata; Sen-ichi Oda; Takafumi Sakai (982-990).
Ghrelin is the endogenous ligand for the growth hormone (GH) secretagogue receptor, and the sequence of ghrelin has been determined in many species from fish to mammals. In the present study, to reveal the production of ghrelin in the house musk shrew (Suncus murinus, order: Insectivora, suncus is used as a laboratory name), we determined the cDNA sequence and structure of suncus ghrelin and also demonstrated the ghrelin-producing cells in the gastrointestinal tract. Results of cDNA cloning and mass spectrometry analysis revealed that suncus ghrelin is composed of 18 or 26 amino acid residues and that the 3rd Ser was acylated mainly by n-octanoic acid. The 10 amino acids of the N-terminal region of suncus mature ghrelin were consistent with those of other mammals. Quantitative RT-PCR revealed that suncus ghrelin mRNA is highly expressed in the gastric corpus and pyloric antrum, and low expression levels were found in various tissues, including the intestinal tract. Ghrelin cells were found only in the corpus and antrum by immunohistochemistry and in situ hybridization, and most of the ghrelin cells were closed-type cells with relatively rich cytoplasm and scattered in the glandular body and base of the gastric mucosa. The density of ghrelin cells in the corpus was significantly greater than that in the antrum. The results of this study together with our recent results regarding motilin production in the suncus indicate that the suncus will be a useful model animal for study of physiological function of the motilin/ghrelin family.
Keywords: Ghrelin; Suncus murinus; cDNA cloning; Gene expression; IHC; ISH;

Is desacyl ghrelin a modulator of food intake? by Tobias Inhoff; Bertram Wiedenmann; Burghard F. Klapp; Hubert Mönnikes; Peter Kobelt (991-994).
Desacyl ghrelin is produced in the gastric mucosa and plasma by deacylation of ghrelin. It occurs in considerably larger amounts than ghrelin in various regions in the organisms of rats and mice. It exerts biological activities in vitro as different as stimulating adipogenesis or inhibiting glucose output in hepatocytes. In fasted rats, desacyl ghrelin levels decreased under catabolic metabolic conditions and in mice, high desacyl ghrelin concentrations went along with decreased food intake. These observations suggest an influence of the peptide on food intake and energy homeostasis. Behavioral studies led to controversial results, but several suggest an anorexigenic effect. Studies on desacyl ghrelin-induced modulation of food intake indicate the involvement of central nervous pathways, since it is said to cross the blood–brain barrier and to induce increased neuronal activity hypothalamic nuclei. It is likely to be involved in the regulation of the synthesis of anorexigenic hypothalamic mediators. Quite possibly, there might be means of interaction between desacyl ghrelin and its supposable precursor ghrelin.
Keywords: Desacyl ghrelin; Food intake; Rats; Mice;

A new anorexigenic protein, nesfatin-1 by Hiroyuki Shimizu; Aya Ohsaki; Sinsuke Oh-I; Shuichi Okada; Masatomo Mori (995-998).
An anorexigenic peptide, nesfatin-1 was found in rat hypothalamus, and its expression in the paraventricular nucleus of the hypothalamus was reduced by starvation. Intracerebroventricular administration dose-dependently inhibited food intake for 6 h in male Wistar and leptin resistant, Zucker fatty rats. There may be a crosstalk between nesfatin-1 pathway and melanocortin pathway in the brain. Nesfatin-1 neurons co-express with oxytocin, vasopressin and melanin concentrating hormone in the hypothalamus. Intraperitoneal administration of nesfatin-1 and its mid-segment dose-dependently inhibited food intake for 3 h. Mid-segment of nesfatin-1 decreased food intake under leptin-resistant animal models of obesity. Intraperitoneal administration of the mid-segment of nesfatin-1 increased proopiomelanocortin and cocain- and amphetamine-related peptide mRNA expression in the nucleus of the solitary tract, but not in arcuate nucleus of the hypothalamus. In this review, we summarized recent progress in the research about the possible mechanism of nesfatin-1-induced anorexia.
Keywords: Nesfatin-1; Leptin; Melanocortin; Food intake; Body weight;

Mycosis, caused by both filamentous fungi and pathogenic yeasts is a major concern nowadays especially in the immunocompromised patient population. The emergence of pathogenic fungi resistant to current therapies in the last few decades has intensified the search for new antifungals like cationic peptides, which are the key components of innate defense mechanism. The review provides an inventory of different peptides from a diverse array of organisms from bacteria to mammals with proven antifungal activity, their therapeutic options and also about those which are in various stages of preclinical development. Literature, on the total and semi-synthetic variants of the parent peptides that exhibit an improved antifungal activity is also reviewed.
Keywords: Antifungal peptides; Innate immunity; Candida albicans; Aspergillus niger; Defensin; Echinocandin;

Plant defensins—Prospects for the biological functions and biotechnological properties by André de Oliveira Carvalho; Valdirene Moreira Gomes (1007-1020).
Plant defensins are a prominent family of cationic peptides in the plant kingdom. They are structurally and functionally related to defensins that have been previously characterized in mammals and insects. They present molecular masses between 5 and 7 kDa and possess a pattern of eight conserved Cys residues. The three-dimensional structure of plant defensins is small and globular. It has three anti-parallel β-sheets and one α-helix that is stabilized by a structural motif composed of disulfide bridges. This motif is found in other peptides with biological activity and is called the Cys stabilized αβ motif (CSαβ). Based on the growing knowledge on defensin structure, gene expression and regulation, and also their in vitro biological activity, it has become clear that plant defensins are complex and sophisticated peptides whose function extends beyond their role in defense of plants against microbial infection. This review discusses recent data and will present comprehensive information regarding the study of defensins.
Keywords: α-Amylase; Antimicrobial peptide; Antifungal; Ion channel blockers; Protease inhibitors; Structure; Zinc tolerance;