Peptides (v.30, #10)

Antimicrobial peptides from the skin secretions of the New World frogs Lithobates capito and Lithobates warszewitschii (Ranidae) by J. Michael Conlon; Mohammed A. Meetani; Laurent Coquet; Thierry Jouenne; Jérôme Leprince; Hubert Vaudry; Jolanta Kolodziejek; Norbert Nowotny; Jay. D. King (1775-1781).
Taxonomic revisions within the anuran family Ranidae have established the genus Lithobates that currently comprises 49 species of frogs from the New World. Peptidomic analysis, using reversed-phase HPLC with on-line detection by electrospray mass spectrometry, has led to the identification of multiple antimicrobial peptides in norepinephrine-stimulated skin secretions of the North American frog Lithobates capito and the Central American frog Lithobates warszewitschii. Structural characterization of the peptides demonstrated that the L. capito secretions contained brevinin-1 (1), esculentin-1 (1), esculentin-2 (1), ranatuerin-2 (3), and temporin (2) peptides. L. warszewitschii secretions contained brevinin-1 (1), esculentin-2 (1), ranatuerin-2 (2), and temporin (1) peptides. Values in parentheses indicate number of peptides in each family. Temporin-CPa from L. capito, with the atypical structure IPPFIKKVLTTVF·NH2, also showed atypical growth-inhibitory activity having greater potency against Escherichia coli (MIC = 25 μM) and Candida albicans (MIC = 25 μM) than against Staphylococcus aureus (MIC = 50 μM). Phylogenetic analysis based upon the amino acid sequences of 37 ranatuerin-2 peptides from 17 species belonging to the genus Lithobates provides support for currently accepted taxonomic relationships. L. capito is sister-group to Lithobates sevosus in a clade that also contains Lithobates areolatus, and Lithobates palustris. L. warszewitschii is most closely related to the Central American species Lithobates tarahumarae and Lithobates vaillanti.
Keywords: Frog skin; Antimicrobial peptide; Ranatuerin-2; Temporin; Lithobates;

Identification of novel I-superfamily conopeptides from several clades of Conus species found in the South China Sea by Zhuguo Liu; Ning Xu; Jie Hu; Chongjia Zhao; Zheng Yu; Qiuyun Dai (1782-1787).
The I-superfamily of Conus peptides represents a new class of peptides with four disulfide bridges (-C-C-CC-CC-C-C-) that falls into three (I1, I2 and I3) categories according to the different signal peptide sequences. The I-superfamily has received increasing attention because it targets K+ ion channels, a function that is relatively rare in conotoxins. Herein we report 11 novel I-superfamily conotoxins from the venom ducts of five Cone snails (Conus eburneus, Conus imperialis, Conus vitulinus, Conus emaciatus and Conus litteratus) native to the South China Sea using a primer designed according to the N-terminus of the signal sequence of I2-superfamily conotoxins. The alignment of sequences revealed that signal regions exhibited moderate conservation with the exception of Eb11.3 from C. eburneus with homologies of 21.1%, 38.5% and 30.0% to the signal peptides of I1, I2 and I3 superfamily conotoxins, respectively. The mature peptides ranged from almost identical to highly divergent between species. Analyses of the evolutionary trees of these peptides with those of reported I-superfamily conotoxins showed that nine of them fall in I2 superfamily clades, but two of them were neither I1- and I2- nor I3-superfamily clades. Notably, some peptides exhibited significantly different amino acid residues in the intercysteine loops compared with group A, B and C of I-superfamily conopeptides, suggesting that they may have different bioactivities and functions.
Keywords: I-superfamily conopeptides; Clone; Diversity; South China Sea;

Defensins are essentially ancient natural antibiotics with potent activity extending from lower organisms to humans. They can inhibit the growth or virulence of micro-organisms directly or indirectly enhance the host's immune system. The successful prediction of defensin peptides will provide very useful information and insights for the basic research of defensins. In this study, by selecting the N-peptide composition of reduced amino acid alphabet (RAAA) obtained from structural alphabet named Protein Blocks as the feature parameters, the increment of diversity (ID) is firstly developed to predict defensins family and subfamily. The jackknife test based on 2-peptide composition of reduced amino acid alphabet (RAAA) with 13 reduced amino acids shows that the overall accuracy of prediction are 91.36% for defensin family, and 94.21% for defensin subfamily. The results indicate that ID_RAAA is a simple and efficient prediction method for defensin peptides.
Keywords: Defensins family and subfamily; Increment of diversity; Reduced amino acid alphabet; N-Peptide composition; Prediction performance;

In vitro activity of Tachyplesin III alone and in combination with terbinafine against clinical isolates of dermatophytes by O. Simonetti; G. Ganzetti; D. Arzeni; A. Campanati; B. Marconi; C. Silvestri; O. Cirioni; E. Gabrielli; I. Lenci; W. Kamysz; E. Kamysz; A. Giacometti; G. Scalise; F. Barchiesi; A. Offidani (1794-1797).
Aim of our study was to investigate the in vitro effects of Tachyplesin III (TP), a potent disulfide-linked peptide, in dermatophytes infections, with respect to or in combination with terbinafine (TERB), against 20 clinical isolates of dermatophytes belonging to four species. A broth microdilution method following the CLSI recommendations (M38-A) was used for testing drugs alone and in combination. TERB MICs were significantly lower than those observed for TP (p  < 0.001). Testing for antifungal agents in combination was performed for TERB with TP for all the 20 isolates. TERB activity in combination with TP showed indifferent activity for 14 of the 20 isolates (70%); synergic activity for 6 of the 20 isolates (30%); no antagonistic activity was observed. Further experiments were conducted with Microsporum canis 133, Trichophyton rubrum 62 and Trichophyton mentagrophytes 91 for fungal biomass. TP and TERB did not show a significant growth reduction compared to the control against T. mentagrophytes and T. rubrum. A significant difference of growth reduction both for TP and TERB compared to controls was observed for M. canis (p  < 0.01). In conclusion our study demonstrated that Tachyplesin III has potential activity against dermatophytes. In addition, we observed that the in vitro activity of Tachyplesin III can be enhanced upon combination with terbinafine. Synergy could permit lower doses of the individual antifungal agents to be used more effectively and/or safely.
Keywords: Tachyplesin III; Terbinafine; Dermatophytes; Fungal biomass; Sinergy testing;

An abundantly secreted thermostable peptide (designed AcAFP) with a molecular mass of 5777 Da was isolated and purified in a previous work from a local strain of A. clavatus (VR1). Based on the N-terminal amino acid (aa) sequence of the AcAFP peptide, an oligonucleotide probe was derived and allowed the amplification of the encoding cDNA by RT-PCR. This cDNA fragment encodes a pre-pro-protein of 94 aa which appears to be processed to a mature product of 51 aa cys-rich protein. The deduced aa sequence of the pre-pro-sequence reveals high similarity with ascomycetes antifungal peptide. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clone revealed the presence of an open reading frame of 282 bp interrupted by two small introns of 89 and 56 bp with conserved splice site. The three-dimensional (3D) structure modeling of AcAFP exhibits a compact structure consisting of five anti-parallel β barrel stabilized by four internal disulfide bridges. The folding pattern revealed also a cationic site and spatially adjacent hydrophobic stretch. The antifungal mechanism was investigated by transmission and confocal microscopy. AcAFP cause cell wall altering in a dose-dependent manner against the phytopathogenic fungus Fusarium oxysporum.
Keywords: Aspergillus clavatus; Antifungal peptide; Gene organization; Cellular morphology; 3D structure;

Clematis montana lectin, a novel mannose-binding lectin from traditional Chinese medicine with antiviral and apoptosis-inducing activities by Hao Peng; Hui Lv; Ying Wang; Yan-hong Liu; Chun-yang Li; Liang Meng; Fang Chen; Jin-ku Bao (1805-1815).
A novel mannose-binding lectin (designated CML) was isolated from Clematis montana Buch.-Ham stem (Ranunculaceae) using ion exchange and gel filtration chromatographies on DEAE-Sepharose and Sephacryl S-100. The purified C. montana lectin was a homodimer of 11,968.9 Da subunits as determined by gel filtration and MS. The hemagglutinating activity of CML was inhibited by branched oligomannosides. The N-terminal 15-amino acid sequence of CML, DNVKYSGQVKNTGSA, has not been reported for other lectins. Also, the peptide mass fingerprinting assay confirmed that there is no match result of similar plant lectins for CML, indicating CML may be a novel plant lectin. CML showed marked antiviral activity against various viruses in cell culture. Subsequently, CML was also found to exhibit remarkable inhibitory effect on L929, HeLa, MCF7 and HepG2 cells. Furthermore, CML specially induced L929 cell apoptosis in dose-dependent manner as evidenced by MTT, fluorescent microscopy, LDH activity-based cytotoxicity assays and DNA ladder. Moreover, due to both caspase inhibitors and Western blot analyses, caspase was also found to play the important role in the potential apoptotic mechanism of CML. When the carbohydrate-binding site was fully inhibited by sugars, cytotoxicity was abruptly decreased and apoptotic phenomenon in L929 cells was not observed, suggesting a significant correlation between mannose-binding-specific activity and the antineoplastic mechanism.
Keywords: Clematis montana lectin; Mannose-binding-specific activity; Antiviral activity; Apoptosis; Caspase;

An adenovirus-delivered peptide aptamer C1-1 targeting the core protein of hepatitis B virus inhibits viral DNA replication and production in vitro and in vivo by Wei Zhang; Wei Ke; Si-Si Wu; Lu Gan; Rui Zhou; Chang-Yan Sun; Qing-Shan Long; Wei Jiang; Hong-Bo Xin (1816-1821).
Peptide aptamers are molecules which can specifically bind to a given target protein and have the potential to selectively block the function of the target protein. It has been reported that a peptide aptamer (C1-1) identified from a randomized expression library specifically bound to the core protein of hepatitis B virus and inhibited viral capsid formation and DNA replication in vitro. Adenoviral systems are popular platforms for reliable gene delivery and high-level transient expression in any mammalian cell type in vitro, and have a natural tropism for the liver after systemic administration. In the present study, we explored the feasibility of gene therapy against HBV infection with adenoviral system, and found that systematic administration of recombinant adenovirus encoding the peptide aptamer (C1-1) significantly inhibited viral capsid formation, HBV DNA replication and virion production in vivo. These results suggest an efficient antiviral treatment against HBV infection by delivery of anti-HBV peptide aptamer with recombinant adenovirus.
Keywords: Peptide aptamer; Hepatitis B virus (HBV); Core protein; Adenovirus;

Thymosin beta 4 mRNA and peptide expression in phagocytic cells of different mouse tissues by Melissa Paulussen; Bart Landuyt; Liliane Schoofs; Walter Luyten; Lut Arckens (1822-1832).
Thymosin beta 4 (Tβ4) is a peptide of 43 amino acids, mainly recognized as a regulator of actin polymerization by sequestering G-actin. Meanwhile, the peptide has been implicated in lymphocyte maturation, carcinogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. The peptide is also involved in lesion-induced neuroplasticity through microglia upregulation and it participates in the growth of neuronal processes. However, its precise cellular localization throughout the entire body of the mouse has not been documented. We therefore initiated a detailed investigation of the tissue distribution and cellular expression of the Tβ4 peptide and its precursor mRNA by immunocytochemistry and in situ hybridization, respectively. In the brain, Tβ4 was clearly present in neurons of the olfactory bulb, neocortex, hippocampus, striatum, amygdala, piriform cortex and cerebellum, and in microglia across the entire brain. We further localized Tβ4 in cells, typically with many processes, inside thymus, spleen, lung, kidney, liver, adrenal gland, stomach and intestine. Remarkably, Tβ4 was thus associated with microglia and macrophages, the differentiated phagocytic cells residing in every tissue. Motility and phagocytosis, two important activities of macrophages, depend on actin, which can explain the presence of Tβ4 in these cells.
Keywords: Thymosin beta 4; Macrophage; Microglia; In situ hybridization; Immunocytochemistry;

A peptidomimetic of NT-3 acts as a TrkC antagonist by Fouad Brahimi; Andrey Malakhov; Hong Boon Lee; Mookda Pattarawarapan; Lubijca Ivanisevic; Kevin Burgess; H. Uri Saragovi (1833-1839).
Neurotrophins are a family of growth factors that regulate the peripheral and central nervous system. We designed and tested a mini-library of small molecules peptidomimetics based on β-turns of the neurotrophin growth factor polypeptides NT-3, which is the natural ligand for TrkC receptors. Biological studies identified a peptidomimetic 2Cl that exhibited selective antagonism of TrkC. 2Cl reduces TrkC activation and signaling promoted by NT-3, and selectively blocks ligand-dependent cell survival. 2Cl also blocks ligand-independent TrkC activation and signals that take place when the receptor is over-expressed. This work adds to our understanding of how the neurotrophins function through Trk receptors, and demonstrates that peptidomimetics can be designed to selectively disturb neurotrophin–receptor interactions, and receptor activation.
Keywords: Neurotrophin; Receptor; Tyrosine kinase; Antagonism;

Peptide array-based analysis of the specific IgE and IgG4 in cow's milk allergens and its use in allergy evaluation by Naoki Matsumoto; Mina Okochi; Miyoko Matsushima; Ryuji Kato; Tomokazu Takase; Yasuko Yoshida; Mitsuo Kawase; Ken-ichi Isobe; Tsutomu Kawabe; Hiroyuki Honda (1840-1847).
Cow's milk (CM) is one of the major causes of food allergies in children. We constructed a peptide array consisting of a linear 16-mer peptide library with an offset of 3-mer, which corresponds to the primary sequences of six major CM allergens. The immune reactivity to cow's milk proteins diminishes with age and clinical tolerance commonly occurs. Although the central role of IgE in allergy is well established, the role of other specific antibody classes in obtaining immunotolerance is not well known. The hypothesis that patients become tolerant when they develop immunological changes particularly with the IgG4 isotype has been proposed. In this study, the binding pattern of the CM protein-specific IgE and IgG4 epitopes was measured using the peptide array with sera of 12 patients with persistent CM allergy (CMA), sera of 5 children who outgrew CMA, and sera of 7 CM-sensitized children without allergy symptoms. In CMA patients the IgG4/IgE fluorescence intensity ratios varied greatly from peptide to peptide, and the scatter plots of IgE versus IgG4 signals using significant IgE-binding peptides showed different distribution patterns. When setting the boundary line based on the IgG4/IgE ratio (IgG4/IgE = 2), patients with persistent CMA and CM-sensitized children can be distinguished by the plot pattern of peptides. Furthermore, the number of peptide plots in these regions was less in children who outgrew CMA. The approach employed in this study will allow for the distinction between CMA and CM-sensitization, and will enable the estimation of CMA outgrow by monitoring the time elapsed data.
Keywords: Peptide array; Piezoelectric ceramic micropump; Cow's milk allergy; IgE-binding epitopes; IgG4/IgE ratio;

Stability to gastrointestinal enzymes and structure–activity relationship of β-casein-peptides with antihypertensive properties by Ana Quirós; María del Mar Contreras; Mercedes Ramos; Lourdes Amigo; Isidra Recio (1848-1853).
Physiological digestion plays a key role in the formation and degradation of angiotensin-converting enzyme (ACE)-inhibitory peptides. In this study, we evaluated the impact of a simulated gastrointestinal digestion on the stability of eight peptides previously identified in fermented milk with antihypertensive activity. Two of these identified peptides with sequences LHLPLP and LVYPFPGPIPNSLPQNIPP, possess ACE-inhibitory activity in vitro and antihypertensive activity in vivo. The results showed that LHLPLP was resistant to digestive enzymes. In contrast, LVYPFPGPIPNSLPQNIPP was totally hydrolyzed and its activity decreased after incubation with pepsin and a pancreatic extract. The peptide LHLPLP was incubated with ACE and was found to be a true inhibitor of the enzyme and to exhibit a competitive inhibitor pattern. A structure–activity relationship study of this peptide was carried out by synthesizing several modified peptides related to the sequence LHLPLP. The substitution of amino acid Leu in the penultimate position by Gly improved the ACE-inhibitory activity twofold and the substitution of Pro at C-terminal position by Arg increased the activity twofold, with an IC50 of LHLPLR as low as 1.8 μM.
Keywords: ACE-inhibitory peptides; Fermented milk; Simulated gastrointestinal digestion; Antihypertensive activity; Competitive inhibitors;

β-Casomorphins-7 in infants on different type of feeding and different levels of psychomotor development by Natalya V. Kost; Оleg Yu. Sokolov; Оksana B. Kurasova; Alexander D. Dmitriev; Julia N. Tarakanova; Мarina V. Gabaeva; Yuriy A. Zolotarev; Аlexander K. Dadayan; Sergei A. Grachev; Еkaterina V. Korneeva; Inna G. Mikheeva; Аndrey A. Zozulya (1854-1860).
Casomorphins are the most important during the first year of life, when postnatal formation is most active and milk is the main source of both nutritive and biologically active material for infants. This study was conducted on a total of 90 infants, of which 37 were fed with breast milk and 53 were fed with formula containing cow milk. The study has firstly indicated substances with immunoreactivity of human (irHCM) and bovine (irBCM) β-casomorphins-7 in blood plasma of naturally and artificially fed infants, respectively. irHCM and irBCM were detected both in the morning before feeding (basal level), and 3 h after feeding. Elevation of irHCM and irBCM levels after feeding was detected mainly in infants in the first 3 months of life. Chromatographic characterization of the material with irBCM has demonstrated that it has the same molecular mass and polarity as synthetic bovine β-casomorphin-7. The highest basal irHCM was observed in breast-fed infants with normal psychomotor development and muscle tone. In contrast, elevated basal irBCM was found in formula-fed infants showing delay in psychomotor development and heightened muscle tone. Among formula-fed infants with normal development, the rate of this parameter directly correlated to basal irBCM. The data indicate that breast feeding has an advantage over artificial feeding for infants’ development during the first year of life and support the hypothesis for deterioration of bovine casomorphin elimination as a risk factor for delay in psychomotor development and other diseases such as autism.
Keywords: β-Casomorphins-7; Infants; Breast feeding; Formula feeding; Psychomotor development; Muscle tone;

Reproduction and maternal behavior in insulin-regulated aminopeptidase (IRAP) knockout mice by Vi Pham; Peta Burns; Anthony L. Albiston; Holly R. Yeatman; Leelee Ng; Shanti Diwakarla; Siew Yeen Chai (1861-1865).
During human pregnancy, a circulating form of insulin-regulated aminopeptidase (IRAP EC, often termed oxytocinase or placental leucine aminopeptidase (PLAP), is present in plasma. It is proposed that circulating IRAP plays an important role in regulating the circulating levels of oxytocin and/or vasopressin during pregnancy. We assessed the reproductive and maternal profile of global IRAP knock out mice. No differences in the reproductive profile were observed, with normal gestational period, litter size and parturition recorded. However, western blot analysis of pregnant mouse serum, failed to detect IRAP, a result which was confirmed by fluorimetric IRAP enzyme assay. A review of the literature revealed that the presence of IRAP in the maternal circulation during pregnancy has been only reported in humans. Moreover, the sequence, Phe154 Ala155, identified as the cleavage site for the release of soluble IRAP, is restricted to members of the homindae family. Therefore the absence of IRAP from the circulation in mice, and other species during pregnancy, is due to the inability of a secretase to cleave placental IRAP to produce a soluble form of the enzyme. Given the expression of IRAP in areas of the brain associated with oxytocin modulated maternal behavior, we also investigated whether the IRAP global knockout mice had improved maternal responses. Using standard tests to assess maternal behavior, including pup retrieval, feeding and nurturing, no differences between knock out and wild type dams were observed. In conclusion, the physiological significance of circulating IRAP during human pregnancy cannot be addressed by investigations on mice.
Keywords: Oxytocinase; IRAP; Pregnancy; Oxytocin; Vasopressin;

Enterostatin alters protein trafficking to inhibit insulin secretion in Beta-TC6 cells by MieJung Park; Jeffery Farrell; Karalee Lemmon; David A. York (1866-1873).
Enterostatin is a peptide that regulates dietary fat intake in rodents and inhibits insulin secretion from pancreatic beta cells. Microarray studies of the genomic response of both a human hepatoma cell line (HepG2 cells) and a mouse hypothalamic cell line (GT1-7 cells) to enterostatin suggested that it might regulate protein trafficking. Using semi-quantitative real-time PCR and Western blot analysis, we confirmed that enterostatin upregulated Scamp2 and down regulated Dynamin2 in these cell lines. The receptor for enterostatin is the F1-ATPase beta subunit. We transfected HepG2 cells with either a green fluorescent protein (GFP) tagged F1-ATPase beta subunit or a red fluorescent protein (RFP) tagged F1-ATPase alpha subunit to study the effects of enterostatin on translocation of its own receptor protein. Enterostatin induced movement of GFP-beta subunit to the cell periphery area but did not have any effect on the localization of RFP-alpha subunit protein in HepG2. As Scamp2 is involved in glucose uptake in mouse Beta-TC6 insulinoma cells we tested enterostatin's effect in Beta-TC6 cells. Glucose stimulated insulin release was inhibited by enterostatin as reported previously. Using siRNA to Scamp2 did not change glucose stimulated insulin release but siRNA to Dynamin2 and dominant negative Dynamin2 (Dyn K44A) inhibited glucose stimulated insulin release and abolished the response to enterostatin. This suggests enterostatin inhibits glucose stimulated insulin release in pancreatic beta cells through down regulation of Dynamin2. This study also suggests that enterostatin might have a more generalized effect on protein trafficking in various cells.
Keywords: Enterostatin; Dynamin2; Scamp2; Insulin secretion; Protein trafficking;

A novel DPP-IV-resistant analog of glucagon-like peptide-1 (GLP-1): KGLP-1 alone or in combination with long-acting PLGA microspheres by Zhihui Gao; Yu Tang; Jiaqi Chen; Ru Bai; Qi Zhang; Yuanyuan Hou; Yaxin Lu; Gang Bai (1874-1881).
Glucagon-like peptide-1 (GLP-1) is an important hormone peptide secreted from the gastrointestinal tract in response to nutrient ingestion. Its multifaceted actions make GLP-1 attractive as a candidate for the treatment of type 2 diabetes mellitus. However, its main limitation is an extremely short half-life, which is due to rapid inactivation by a ubiquitous enzyme, dipeptidyl peptidase-IV (DPP-IV). Therefore, here we describe the development of a novel GLP-1 analog, designated KGLP-1. Initial in vitro experiments revealed that KGLP-1 bound to and activated GLP-1R with similar efficacy as native GLP-1. Importantly, KGLP-1 showed marked resistance to inactivation by DPP-IV. Further in vivo studies confirmed that KGLP-1 had antihyperglycemic and insulinotropic actions after intraperitoneal injection to KM mice and alloxan-induced diabetic mice. Finally, we prepared KGLP-1-loaded poly (d,l-lactic-co-glycolic acid) microspheres (PLGA MS) using the solid in oil in oil (s/o/o) solvent extraction method, which achieved controlled release and biological efficacy over a period of 10 days after a single subcutaneous injection in alloxan-induced diabetic rats.
Keywords: GLP-1; Type 2 diabetes; DPP-IV-resistant analog; Antihyperglycemic; Insulinotropic; Poly (d,l-lactic-co-glycolic acid) microspheres (PLGA MS);

Cathepsin L plays a major role in cholecystokinin production in mouse brain cortex and in pituitary AtT-20 cells: Protease gene knockout and inhibitor studies by Margery C. Beinfeld; Lydiane Funkelstein; Thierry Foulon; Sandrine Cadel; Kouki Kitagawa; Thomas Toneff; Thomas Reinheckel; Christoph Peters; Vivian Hook (1882-1891).
Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK9/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for the production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells.
Keywords: Cholecystokinin; Protease; Cathepsin L; Prohormone convertase; Brain; Pituitary;

Analysis of the therapeutic functions of novel melanocortin receptor agonists in MC3R- and MC4R-deficient C57BL/6J mice by K. Ganesh Kumar; Gregory M. Sutton; Jesse Z. Dong; Pierre Roubert; Pascale Plas; Heather A. Halem; Michael D. Culler; Hyunwon Yang; Vishwa D. Dixit; Andrew A. Butler (1892-1900).
Melanocortin receptor agonists act in the brain to regulate food intake and body weight and, independently of these actions, affect insulin sensitivity. These experiments investigated the function of novel non-selective melanocortin receptor agonists (BIM-22493, BIM-22511) that cross the blood–brain barrier when administered peripherally. Treatment of diet induced obese C57BL/6J (B6) mice with melanocortin agonists administered peripherally improved obesity, hyperinsulinemia (∼50%) and fatty liver disease. Specificity of function was determined using B6 melanocortin-3 and melanocortin-4 receptor knockout mice (MC3RKO, MC4RKO). Chow fed MC4RKO but not MC3RKO used for these tests exhibited obesity, hyperinsulinemia and severe hepatosteatosis associated with increased expression of insulin-stimulated genes involved in lipogenesis. Reduced food intake associated with acute BIM-22493 treatment, and weight loss associated with 14 days of treatment with BIM-22511, required functional MC4R but not MC3R. However, while 14 days of treatment with BIM-22511 did not affect body weight and even increased cumulative food intake in MC4RKO, a significant reduction (∼50%) in fasting insulin was still observed. Despite lowering insulin, chronic treatment with BIM-22511 did not improve hepatosteatosis in MC4RKO, and did not affect hepatic lipogenic gene expression. Together, these results demonstrate that peripherally administered melanocortin receptor agonists regulate body weight, liver metabolism and glucose homeostasis through independent pathways. MC4R are necessary for melanocortin agonist-induced weight loss and improvements in liver metabolism, but are not required for improvements in hyperinsulinemia. Agonists with activity at MC4R improve glucose homeostasis at least partially by causing weight loss, however other melanocortin receptors may have potential for treating aberrations in glucose homeostasis associated with obesity.
Keywords: Obesity; Diabetes; Insulin; Melanocortins; Proopiomelanocortin; Melanocyte-stimulating hormones;

Electrophysiological effects of ghrelin on laterodorsal tegmental neurons in rats: An in vitro study by Shinobu Takano; Juhyon Kim; Yuki Ikari; Masaki Ogaya; Kazuki Nakajima; Yutaka Oomura; Matthew J. Wayner; Kazuo Sasaki (1901-1908).
Ghrelin, a gut and brain peptide, is a potent stimulant for growth hormone (GH) secretion and feeding. Recent studies further show a critical role of ghrelin in the regulation of sleep–wakefulness. Laterodorsal tegmental nucleus (LDT), that regulates waking and rapid eye movement (REM) sleep, expresses GH secretagogue receptors (GHS-Rs). Thus, the present study was carried out to examine electrophysiological effects of ghrelin on LDT neurons using rat brainstem slices, and to determine the ionic mechanism involved. Whole cell recording revealed that ghrelin depolarizes LDT neurons dose-dependently in normal artificial cerebrospinal fluid (ACSF). The depolarization persisted in tetrodotoxin-containing ACSF (TTX ACSF), and is partially blocked by the application of [d-Lys3]-GHRP-6, a selective antagonist for GHS-Rs. Membrane resistance during the ghrelin-induced depolarization increased by about 18% than that before the depolarization. In addition, the ghrelin-induced depolarization was drastically reduced in high-K+ TTX ACSF with a K+ concentration of 13.25 mM. Reversal potentials obtained from IV curves before and during the depolarization were about −83 mV, close to the equilibrium potential of the K+ channel. Most of the LDT neurons recorded were characterized by an A-current or both the A-current and a low threshold Ca2+ spike, and they were predominantly cholinergic. These results indicate that ghrelin depolarizes LDT neurons postsynaptically and dose-dependently via GHS-Rs, and that the ionic mechanisms underlying the ghrelin-induced depolarization include a decrease of K+ conductance. The results also suggest that LDT neurons are implicated in the cellular processes through which ghrelin participates in the regulation of sleep–wakefulness.
Keywords: Ghrelin; Laterodorsal tegmental nucleus; Cholinergic neurons; Sleep; Wakefulness; K+ conductance; [d-Lys3]-GHRP-6;

Ghrelin receptor agonist, GHRP-2, attenuates burn injury-induced MuRF-1 and MAFbx expression and muscle proteolysis in rats by Sulaiman Sheriff; Rashika Joshi; Lou Ann Friend; J. Howard James; Ambikaipakan Balasubramaniam (1909-1913).
Thermal injury results in hypermetabolism, loss of body weight, and skeletal muscle wasting in mice and rats. Our earlier studies have demonstrated that ghrelin injection stimulates food intake and growth hormone release and inhibits skeletal muscle proteolysis in rats with thermal injury. We sought to develop a lower molecular weight, stable and longer acting peptide to combat the catabolic responses caused by thermal injury. Towards this goal, we examined the role of the hexapeptide mimetic of ghrelin, growth hormone-releasing peptide-2 (GHRP-2), on expression of E3 ubiquitin ligases and breakdown of muscle protein in rats with thermal injury. Slow in vivo release of GHRP-2 through minipump for 24 h attenuated the thermal injury-induced increase in mRNA expression of IL-6 and of the E3 ubiquitin ligases, MuRF-1 and MAFbx, in rat skeletal muscle. Furthermore, burn-induced increases in total and myofibrillar protein breakdown from rat EDL muscle were attenuated by GHRP-2. These findings suggest that catabolic responses resulting from thermal injury can be attenuated by GHRP-2.
Keywords: Burn injury; E3 ubiquitin ligases; IL-6; Skeletal muscle; Real-time PCR; Protein breakdown;

Lithium attenuates behavioral and biochemical effects of neuropeptide S in mice by A.A. Castro; T.S. Casagrande; M. Moretti; L. Constantino; F. Petronilho; G.C.B. Guerra; G. Calo’; R. Guerrini; F. Dal-Pizzol; J. Quevedo; E.C. Gavioli (1914-1920).
Neuropeptide S (NPS) and its receptor NPSR comprise a recently deorphaned G-protein-coupled receptor system. There is a body of evidence suggesting the involvement of NPS in wakefulness, anxiety, locomotor activity and oxidative stress damage. Considering that mood stabilizers block the stimulatory effect of psychostimulants in rodents, the present study aimed to investigate the effects of the pretreatment with lithium and valproate on the hyperlocomotion evoked by NPS. Another relevant action induced by lithium and valproate is the neuroprotection against oxidative stress. Thus, aiming to get further information about the mechanisms of action of NPS, herein we evaluated the effects of NPS, lithium and valproate, and the combination of them on oxidative stress damage. Behavioral studies revealed that the pretreatment with lithium (100 mg/kg, i.p.) and valproate (200 mg/kg, i.p.) prevented hyperlocomotion evoked by NPS 0.1 nmol. Importantly, the dose of valproate used in this study reduced mouse locomotion, although it did not reach the statistical significance. Biochemical analyses showed that lithium attenuated thiobarbituric reactive species (TBARS) formation in the striatum, cerebellum and hippocampus. NPS per se reduced TBARS levels only in the hippocampus. Valproate did not significantly affect TBARS levels in the brain. However, the combination of mood stabilizers and NPS blocked, instead of potentiate, the neuroprotective effects of each one. No relevant alterations were observed in carbonylated proteins after all treatments. Altogether, the present findings suggested that mainly the mood stabilizer lithium evoked antagonistic effects on the mediation of hyperlocomotion and protection against lipid peroxidation induced by NPS.
Keywords: Neuropeptide S; Locomotor activity; Oxidative stress; Lipid peroxidation; Protein carbonyls; Lithium; Valproate; Mood stabilizers;

Angiotensin-(1-7) antagonist, A-779, microinjection into the caudal ventrolateral medulla of renovascular hypertensive rats restores baroreflex bradycardia by Luiza Michelle Cangussu; Uberdan Guilherme Mendes de Castro; Raquel do Pilar Machado; Marcelo Eustáquio Silva; Patrícia Maria Ferreira; Robson Augusto Souza dos Santos; Maria José Campagnole-Santos; Andréia Carvalho Alzamora (1921-1927).
In the present study we evaluated the effect of caudal ventrolateral medulla (CVLM) microinjection of the main angiotensin (Ang) peptides, Ang II and Ang-(1-7), and their selective antagonists on baseline arterial pressure (AP) and on baroreceptor-mediated bradycardia in renovascular hypertensive rats (2K1C). Microinjection of Ang II and Ang-(1-7) into the CVLM of 2K1C rats produced similar decrease in AP as observed in Sham rats. In both Sham and 2K1C, the hypotensive effect of Ang II and Ang-(1-7) at the CVLM was blocked, for up to 30 min, by previous CVLM microinjection of the Ang II AT1 receptor antagonist, Losartan, and Ang-(1-7) Mas antagonist, A-779, respectively. As expected, the baroreflex bradycardia was lower in 2K1C in comparison to Sham rats. CVLM microinjection of A-779 improved the sensitivity of baroreflex bradycardia in 2K1C hypertensive rats. In contrast, Losartan had no effect on the baroreflex bradycardia in either 2K1C or Sham rats. These results suggest that Ang-(1-7) at the CVLM may contribute to the low sensitivity of the baroreflex control of heart rate in renovascular hypertensive rats.
Keywords: Caudal ventrolateral medulla; Baroreflex control of heart rate; Angiotensin II; Angiotensin-(1-7); Renovascular hypertension-Goldblatt-2K1C; A-779;

Bolus intravenous injection of obestatin does not change blood pressure level of spontaneously hypertensive rat by Zhao-Feng Li; Shu-Wei Song; Yong-Wen Qin; Jian-Liang Zhang; Xian-Xian Zhao; Bi-Li Zhang; An-Jing Ren; Zhi-Fu Guo; Xing Zheng (1928-1930).
Ghrelin, an endogenous ligand for the GH secretagogue receptor, has been shown to decrease arterial pressure. Obestatin, a sibling of ghrelin derived from preproghrelin, opposes several physiological actions of ghrelin. The aim of this study was to determine the effects of bolus intravenous injection of obestatin on blood pressure in spontaneously hypertensive rats. Three different dosages of obestatin (10, 50, and 100 μg/kg) and one dosage of ghrelin (10 μg/kg) were applied. The mean arterial pressure and heart period were continuously recorded for 30 min after injection of drugs. Baroreflex sensitivity was also investigated. In this study, we first demonstrated that intravenous injection of obestatin showed no significant effects on mean blood pressure (10 μg/kg: 113.8 ± 2.0 mmHg vs. 114.4 ± 1.6 mmHg; 50 μg/kg: 110 ± 2.4 mmHg vs. 109 ± 3.2 mmHg; 100 μg/kg: 115.9 ± 1.5 mmHg vs. 115.8 ± 2.4 mmHg; all P  > 0.05), heart period (10 μg/kg: 184.7 ± 3.9 ms vs. 185.5 ± 4.1 ms; 50 μg/kg: 185.9 ± 4.1 ms vs. 193.4 ± 4.5 ms; 100 μg/kg: 137.7 ± 4.5 ms vs. 143.9 ± 5.6 ms; all P  > 0.05), or baroreflex sensitivity (10 μg/kg: 0.414 ± 0.03 ms/mmHg vs. 0.442 ± 0.02 ms/mmHg; 50 μg/kg: 0.453 ± 0.04 ms/mmHg vs. 0.439 ± 0.01 ms/mmHg; 100 μg/kg: 0.398 ± 0.02 ms/mmHg vs. 0.401 ± 0.01 ms/mmHg; all P  > 0.05), however, intravenous injection of ghrelin could decrease mean arterial pressure (115.9 ± 1.5 mmHg vs. 108.6 ± 3.6 mmHg, P  < 0.01) and increase heart period (132.4 ± 2.8 ms vs. 152.6 ± 7.4 ms, P  < 0.05), but did not change baroreflex sensitivity (0.36 ± 0.009 ms/mmHg, P  > 0.05) in spontaneously hypertensive rats.

Central angiotensin AT1 receptors are involved in metabolic adjustments in response to graded exercise in rats by Laura H.R. Leite; Ana Cristina R. Lacerda; Cláudio H. Balthazar; Umeko Marubayashi; Cândido C. Coimbra (1931-1935).
To investigate the influence of central angiotensin AT1-receptors blockade on metabolic adjustments during graded exercise, Losartan (Los) was intracerebroventricularly injected in rats before running until fatigue. Oxygen consumption (VO2) was measured (n  = 6) and blood samples collected (n  = 7) to determine variations of glucose, lactate and free fatty acids (FFA). Los-rats exhibited a hyperglycemic response, already observed at 20% of maximal work, followed by a higher lactate levels and FFA mobilization from adipose tissue. Despite the reduced total time to fatigue and the higher VO2 associated with reduced mechanical efficiency, exercise led to the attainment of similar levels of effort in both groups. In summary, central AT1-receptor blockade during graded exercise induces hyperglycemia and higher FFA mobilization from adipose tissue at low exercise intensities in rats running at the same absolute exercise intensity. These data suggest that the central angiotensinergic system is involved in metabolic adjustments during exercise since central blockade of AT1-receptors shifts energy balance during graded exercise, similarly to situations of higher and premature sympathetic activation.

Immunogenic mimics of Brucella lipopolysaccharide epitopes by Concetta Beninati; Manuela Garibaldi; Carla Lo Passo; Giuseppe Mancuso; Salvatore Papasergi; Gabriella Garufi; Ida Pernice; Giuseppe Teti; Franco Felici (1936-1939).
Brucella melitensis and Brucella abortus are responsible for brucellosis in bovine and ovine species and for Malta fever in humans. The lipopolysaccharide (LPS) of Brucella is an important virulence factor and can elicit protective antibodies. Because of their potential importance in vaccine design and in serological diagnosis, we developed peptides mimicking the antigenic properties of distinctive antigenic determinants of Brucella LPS. These peptides were selected from several phage display random peptide libraries for their ability to bind monoclonal antibodies directed against the A- or C-type epitopes of Brucella LPS. Plasmids encoding for two of the isolated peptides induced, after DNA immunization, LPS-specific antibody responses. Although these responses were only moderate in extent, these data further suggest the feasibility of using peptide mimics of carbohydrate epitopes as immunogens, a property which may be useful in the design of novel anti-Brucella vaccines.
Keywords: Mimotopes; Phage display; Brucella; Monoclonal antibodies;

Codon number shapes peptide redundancy in the universal proteome composition by Anthony Kusalik; Brett Trost; Mik Bickis; Candida Fasano; Giovanni Capone; Darja Kanduc (1940-1944).
The proteomes catalogued in the UniRef100 database were collected into a single proteome set and examined for actual versus theoretical pentapeptide occurrences. We found a highly diversified degree of pentapeptide redundancy. Numerically, 953 pentamers are expressed only once in the protein world, whereas 103 pentamers occur more than 50,000 times. Moreover, it seems that 417 potentially possible pentapeptides are not present in the protein world. On the whole, tracing the redundancy profile of the protein world as a function of pentapeptide occurrences reveals a quasi-Gaussian curve, with tails representing scarcely and repeatedly occurring 5-mers. Analysis of physico-chemical-biological parameters shows that codon number is the main factor influencing and favoring specific pentapeptide frequencies in the universal proteome composition. That is, when compared to the set of never-expressed 5-mers, the pentapeptides frequently represented in the universal proteome are endowed with a higher number of multi-codonic amino acids. In contrast, the bulkiness degree and the hydrophobicity level play a smaller role. Unexpectedly, the heat of formation of pentapeptide appears to have the least influence.

Seven decades of angiotensin (1939–2009) by Ranko Skrbic; Rajko Igic (1945-1950).
Two research groups in both North and South America independently discovered that renin released a novel vasopressor agent. The Argentine group named it hypertensin, and called its plasma protein substrate hypertensinogen. The group from the United States named it angiotonin. In 1958, Braun Menendez and Irvine Page suggested that the peptide should be named angiotensin. The combined name eventually became commonly used to avoid linguistic confusion. Research scientists and physicians today acknowledge that studies of the renin–angiotensin system (RAS) have greatly improved our understanding of several diseases. Certainly, medical practice profited significantly from the synthesis and application of numerous pharmacological agents that antagonize either the biosynthesis or pharmacological responses of endogenously generated angiotensin II. Ultimately, discovery of the renin–angiotensin system led to many studies that resulted in therapies for vascular disease. This article briefly reviews research related to the discovery of angiotensin and indicates the importance of additional studies related to the RAS.
Keywords: Renin–angiotensin system; Angiotensin-converting enzyme; Chymase; Angiotensinogen–angiotensin cascade;

Ghrelin and its therapeutic potential for cachectic patients by Jun-ichi Ashitani; Nobuhiro Matsumoto; Masamitsu Nakazato (1951-1956).
The discovery of ghrelin has resulted in the development of approaches to appetite, enabling a better understanding of the mechanisms regulating appetite through molecular analyses. Ghrelin is a 28-amino acid peptide that was isolated from the stomach only a decade ago, and has recently been investigated as a potential therapeutic endogenous agent. This peptide increases appetite, adjusts energy balance, suppresses inflammation, and enhances the release of growth hormone from the pituitary gland. Although many bioactive substances such as peptide YY, leptin, adiponectin and obestatin are involved in appetite control, ghrelin is the only known peptide to signal starvation information from a peripheral organ to the central nervous system, contributing to an increase in appetite. Clinical trials have revealed the effectiveness of ghrelin in increasing lean body mass and activity in cachectic patients. As shown in clinical research on humans and basic research using animal models, cachexia often occurs in response to excess release of proinflammatory cytokines and induces further appetite loss, which aggravates the physiological status of underlying diseases. Ghrelin functions as a protector against the vicious cycle of the cachectic paradigm through orexigenic, anabolic and anti-inflammatory effects, so administration of ghrelin may be able to improve quality of life in cachectic patients. We show here a significant role of ghrelin in the pathophysiology of cachectic diseases and the possibility of clinical applications.
Keywords: Ghrelin; Clinical trial; Cachexia;

Although its role in energy homeostasis is firmly established, the evidence accumulated over a decade linking the adipocyte leptin–hypothalamus axis in the pathogenesis of diabetes mellitus has received little attention in the contemporary thinking. In this context various lines of evidence are collated here to show that (1) under the direction of leptin two independent relays emanating from the hypothalamus restrain insulin secretion from the pancreas and mobilize peripheral organs – liver, skeletal muscle and brown adipose tissue – to upregulate glucose disposal, and (2), leptin insufficiency in the hypothalamus produced by either leptinopenia or restriction of leptin transport across the blood brain barrier due to hyperleptinemia of obesity and aging, initiate antecedent pathophysiological sequalae of diabetes type 1 and 2. Further, we document here the efficacy of leptin replenishment in vivo, especially by supplying it to the hypothalamus with the aid of gene therapy, in preventing the antecedent pathophysiological sequalae – hyperinsulinemia, insulin resistance and hyperglycemia – in various animal models and clinical paradigms of diabetes type 1 and 2 with or without attendant obesity. Overall, the new insights on the long-lasting antidiabetic potential of two independent hypothalamic relays engendered by central leptin gene therapy and the preclinical safety indicators in rodents warrant further validation in subhuman primates and humans.
Keywords: Leptin gene therapy; Hypothalamus; Diabetes mellitus type 1 and 2;