Peptides (v.49, #C)

Apelin and copeptin: Two opposite biomarkers associated with kidney function decline and cyst growth in autosomal dominant polycystic kidney disease by Antonio Lacquaniti; Valeria Chirico; Rosaria Lupica; Antoine Buemi; Saverio Loddo; Chiara Caccamo; Paola Salis; Tullio Bertani; Michele Buemi (1-8).
Vasopressin (AVP) plays a detrimental role in autosomal dominant polycystic kidney disease (ADPKD). Copeptin represents a measurable substitute for circulating AVP whereas apelin counteracts AVP signaling. The aim of this study was to investigate the predictive value of apelin and copeptin for the progression of ADPKD disease. 52 ADPKD patients were enrolled and followed until the end of the observation period or the primary study endpoint was reached, defined by the combined outcome of decrease of glomerular filtration rate associated with a total renal volume increase. Receiver operating characteristics (ROC) analysis was employed for identifying the progression of renal disease and Kaplan–Meier curves assessed the renal survival. Adjusted risk estimates for progression endpoint and incident renal replacement therapy (RRT) were calculated using Cox proportional hazard regression analysis. ADPKD patients were characterized by lower apelin levels and higher copeptin levels when compared with healthy subjects. These biomarkers were strictly correlated with osmolality and markers of renal function. At ROC analysis, apelin and copeptin showed a very good diagnostic profile in identifying ADPKD progression. After the follow up of 24 months, 33 patients reached the endpoint. Cox proportional hazard regression analysis showed that apelin predicted renal disease progression and incident RRT independently of other potential confounders. Apelin is associated with kidney function decline in ADPKD, suggesting that it may be a new marker to predict kidney outcome.
Keywords: Apelin; Copeptin; Vasopressin; Autosomal dominant polycystic kidney disease; Total kidney volume; Renal cyst growth;

New satiety hormone nesfatin-1 protects gastric mucosa against stress-induced injury: Mechanistic roles of prostaglandins, nitric oxide, sensory nerves and vanilloid receptors by Alexandra Szlachcic; Zbigniew Sliwowski; Gracjana Krzysiek-Maczka; Jolanta Majka; Marcin Surmiak; Robert Pajdo; Danuta Drozdowicz; Stanislaw J. Konturek; Tomasz Brzozowski (9-20).
Nesfatin-1 belongs to a family of anorexigenic peptides, which are responsible for satiety and are identified in the neurons and endocrine cells within the gut. These peptides have been implicated in the control of food intake; however, very little is known concerning its contribution to gastric secretion and gastric mucosal integrity. In this study the effects of nesfatin-1 on gastric secretion and gastric lesions induced in rats by 3.5 h of water immersion and restraint stress (WRS) were determined. Exogenous nesfatin-1 (5–40 μg/kg i.p.) significantly decreased gastric acid secretion and attenuated gastric lesions induced by WRS, and this was accompanied by a significant rise in plasma NUCB2/nefatin-1 levels, the gastric mucosal blood flow (GBF), luminal NO concentration, generation of PGE2 in the gastric mucosa, an overexpression of mRNA for NUBC2 and cNOS, as well as a suppression of iNOS and proinflammatory cytokine IL-1β and TNF-α mRNAs. Nesfatin-1-induced protection was attenuated by suppression of COX-1 and COX-2 activity, the inhibition of NOS with L-NNA, the deactivation of afferent nerves with neurotoxic doses of capsaicin, and the pretreatment with capsazepine to inhibit vanilloid VR1 receptors. This study shows for the first time that nesfatin-1 exerts a potent protective action in the stomach of rats exposed to WRS and these effects depend upon decrease in gastric secretion, hyperemia mediated by COX-PG and NOS-NO systems, the activation of vagal and sensory nerves and vanilloid receptors.
Keywords: Nesfatin-1; Gastroprotection; Stress; Gastric blood flow; Prostaglandins; Nitric oxide; Sensory nerves; Calcitonin gene-related peptide; Vagal nerves;

The satiety signal oleoylethanolamide stimulates oxytocin neurosecretion from rat hypothalamic neurons by Adele Romano; Tommaso Cassano; Bianca Tempesta; Silvia Cianci; Pasqua Dipasquale; Roberto Coccurello; Vincenzo Cuomo; Silvana Gaetani (21-26).
The anandamide monounsaturated analogue oleoylethanolamide (OEA) acts as satiety signal released from enterocytes upon the ingestion of dietary fats to prolong the interval to the next meal. This effect, which requires intact vagal fibers and intestinal PPAR-alpha receptors, is coupled to the increase of c-fos and oxytocin mRNA expression in neurons of the paraventricular nucleus (PVN) and is prevented by the intracerebroventricular administration of a selective oxytocin antagonist, thus suggesting a necessary role of oxytocinergic neurotransmission in the pro-satiety effect of OEA. By brain microdialysis and immunohistochemistry, in this study we demonstrate that OEA treatment can stimulate oxytocin neurosecretion from the PVN and enhance oxytocin expression at both axonal and somatodendritic levels of hypothalamic neurons. Such effects, which are maximum 2 h after OEA administration, support the hypothesis that the satiety-inducing action of OEA is mediated by the activation of oxytocin hypothalamic neurons.

Salivary VIP concentrations are elevated in humans after acute stress by Giovanni Ventre; Caitlin Colonna; Jennifer Smith; Denise Alfano; Roberta Moldow (27-31).
Salivary (s)-cortisol, s-amylase, s-DHEA are used extensively in stress research. Vasoactive Intestinal Peptide (VIP) is also detectable in saliva using a standard RIA kit. VIP is a 28 amino acid neuropeptide that belongs to the secretin/glucagon family of peptides and acts as a neurotransmitter/neuromodulator. VIP has also been detected in the parasympathetic nerves enervating the salivary glands. Here we measured the level of s-cortisol, s-DHEA, s-amylase and s-VIP in three different stress exercises of different duration and intensity. The results indicate that a brief intense exercise lasting minutes elicited a stress response with significant increases in s-cortisol, s-DHEA, s-amylase and s-VIP. A less rigorous exercise did not elicit a stress response with no significant increases in s-cortisol, s-DHEA, s-amylase and s-VIP. A longer intense exercise lasting hours elicited a stress response with significant increases only in s-cortisol.
Keywords: Salivary VIP; Cortisol; Amylase; DHEA; Stress; Saliva;

NMR solution structure and SRP54M predicted interaction of the N-terminal sequence (1-30) of the ovine Doppel protein by Jorge Pimenta; Aldino Viegas; João Sardinha; Ivo C. Martins; Eurico J. Cabrita; Carlos M.G.A. Fontes; José A. Prates; Rosa M.L.N. Pereira (32-40).
Prion protein (PrPC) biosynthesis involves a multi-step process that includes translation and post-translational modifications. While PrP has been widely investigated, for the homolog Doppel (Dpl), limited knowledge is available. In this study, we focused on a vital step of eukaryotic protein biosynthesis: targeting by the signal recognition particle (SRP). Taking the ovine Dpl (OvDpl(1-30)) peptide as a template, we studied its behavior in two different hydrophobic environments using CD and NMR spectroscopy. In both trifluoroethanol (TFE) and dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC), the OvDpl(1-30) peptide revealed to fold in an alpha-helical conformation with a well-defined central region extending from residue Cys8 until Ser22. The NMR structure was subsequently included in a computational docking complex with the conserved M-domain of SRP54 protein (SRP54M), and further compared with the N-terminal structures of mouse Dpl and bovine PrPC proteins. This allowed the determination of (i) common predicted N-terminal/SRP54M polar contacts (Asp331, Gln335, Glu365 and Lys432) and (ii) different NC orientations between prion and Dpl peptides at the SRP54M hydrophobic groove, that are in agreement with each peptide electrostatic potential. Together, these findings provide new insights into the biosynthesis of prion-like proteins. Besides they also show the role of protein conformational switches in signalization toward the endoplasmic membrane, a key event of major significance in the cell cycle. They are thus of general applicability to the study of the biological function of prion-like as well as other proteins.
Keywords: CD; NMR; Prion-like; Signal peptide; SRP54M;

Impaired effect of relaxin on vasoconstrictor reactivity in spontaneous hypertensive rats by Joris van Drongelen; Arianne van Koppen; Jeanne Pertijs; Jonathan H. Gooi; Fred C.G.J. Sweep; Fred K. Lotgering; Marc E.A. Spaanderman; Paul Smits (41-48).
Relaxin is thought to be involved in vasodilation to pregnancy by increasing endothelium-dependent vasodilation and compliance, and decreasing myogenic reactivity. Primary (essential) hypertension predisposes to circulatory maladaptation and subsequent gestational hypertensive disease. This study aimed to determine that vascular responses to chronic exposure to relaxin are impaired in young female rats with primary hypertension. In 10–12 weeks old Wistar-Hannover rats (WHR) and spontaneous hypertensive rats (SHR), we determined vascular responses in isolated kidney and mesenteric arteries after 5-days of chronic exposure to relaxin (4 μg/h) or placebo. SHR show decreased sensitivity to phenylephrine (by 67%, p  < 0.01) and renal perfusion flow (RPFF, by 19%, p  < 0.01), but no changes in flow-mediated vasodilation, myogenic reactivity or vascular compliance. In WHR, relaxin stimulated flow-mediated vasodilation (2.67 fold, from 48 ± 9 to 18 ± 4 μl/min, p  = 0.001), inhibited myogenic reactivity (from −1 ± 2 to 7 ± 3 μm/10 mm Hg, p  = 0.01), and decreased sensitivity to phenylephrine (28%, from 1.39 ± 0.08 to 1.78 ± 0.10 μM, p  < 0.01), but left compliance and RPFF unchanged. NO-blockade by l-NAME diminished most relaxin-mediated responses. In SHR, the vasodilator effects of relaxin were blunted for myogenic reactivity and sensitivity to phenylephrine, with similar effects on flow-mediated vasodilation, compliance, RPFF and equal Rxfp1 (relaxin family peptide receptor) gene expression, as compared to WHR. Primary hypertension blunts both the relaxin-induced inhibition of myogenic reactivity and α-adrenergic vasoconstrictor response, independent from Rxfp1 gene expression, while the relaxin-dependent enhanced flow-mediated vasodilation remains intact. This implies selective resistance to relaxin in young subjects suffering from primary hypertension.
Keywords: Relaxin; Wistar-Hannover rats; Spontaneous hypertensive rats; Mesenteric arteries; Isolated perfused rat kidney; Flow-mediated vasodilation; Myogenic reactivity; Phenylephrine;

Resistin levels are related to fat mass, but not to body mass index in children by Lorena Ortega; Pía Riestra; Pilar Navarro; Teresa Gavela-Pérez; Leandro Soriano-Guillén; Carmen Garcés (49-52).
The relationship of resistin levels with obesity remains unclear. The aim of this study was to determine resistin levels in prepubertal children and adolescents and evaluate their association with anthropometric parameters and body composition. The study population included 420 randomly selected 6–8-year-old children and 712 children aged 12–16 years. Anthropometric data were measured and body mass index (BMI) and waist-to-hip and waist-to-height ratios were calculated. Body composition was assessed using an impedance body composition analyzer. Serum resistin levels were determined using a multiplexed bead immunoassay. Resistin levels were not significantly different between sexes. No significant differences in serum resistin concentrations were found between obese, overweight, and normal weight children at any age, and no significant correlations were observed between resistin concentrations and weight or BMI. However, resistin levels showed a significant positive correlation with fat mass in 12–16-year-old children, particularly in girls. In addition to describing serum resistin levels in prepubertal children and adolescents, our study suggests that resistin is related to body fat rather than to BMI in adolescents.
Keywords: Body mass index; Children; Fat mass; Resistin levels;

Antibiotic therapy for methicillin-resistant Staphylococcus aureus (MRSA) infections is becoming more difficult in hospitals and communities because of strong biofilm-forming properties and multidrug resistance. Biofilm-associated MRSA is not affected by therapeutically achievable concentrations of antibiotics. Therefore, we investigated the in vitro pharmacokinetic activities of antimicrobial cationic peptides (AMPs; indolicidin, cecropin [1–7]-melittin A [2–9] amide [CAMA], and nisin), either alone or in combination with antibiotics (daptomycin, linezolid, teicoplanin, ciprofloxacin, and azithromycin), against standard and 2 clinically obtained MRSA biofilms. The minimum inhibitory concentrations (MIC) and minimum biofilm-eradication concentrations (MBEC) were determined by microbroth dilution technique. The time-kill curve (TKC) method was used to determine the bactericidal activities of the AMPs alone and in combination with the antibiotics against standard and clinically obtained MRSA biofilms. The MIC values of the AMPs and antibiotics ranged between 2 to 16 and 0.25 to 512 mg/L, and their MBEC values were 640 and 512 to 5120 mg/L, respectively. The TKC studies demonstrated that synergistic interactions occurred most frequently when using nisin + daptomycin/ciprofloxacin, indolicidin + teicoplanin, and CAMA + ciprofloxacin combinations. No antagonism was observed with any combination. AMPs appear to be good candidates for the treatment of MRSA biofilms, as they act as both enhancers of anti-biofilm activities and help to prevent or delay the emergence of resistance when used either alone or in combination with antibiotics.
Keywords: MRSA; Biofilm; Time kill curves; Antimicrobial cationic peptides; Antibiotic;

Glucose and calcium ions may modulate the efficiency of bovine β-casomorphin-7 permeability through a monolayer of Caco-2 cells by Beata Jarmołowska; Małgorzata Teodorowicz; Ewa Fiedorowicz; Edyta Sienkiewicz-Szłapka; Michał Matysiewicz; Elżbieta Kostyra (59-67).
Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including β-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the β-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca2+ concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using μ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.
Keywords: β-Casomorphin-7; Caco-2 cells monolayer; Food allergy; Infant formula; Permeability coefficient;

Increased expression of (pro)renin receptor in aldosterone-producing adenomas by Hajime Yamamoto; Kiriko Kaneko; Koji Ohba; Ryo Morimoto; Takuo Hirose; Fumitoshi Satoh; Kazuhito Totsune; Kazuhiro Takahashi (68-73).
(Pro)renin receptor ((P)RR) is a specific receptor for renin and prorenin. The aim of the present study is to clarify expression and possible pathophysiological roles of (P)RR in aldosterone-producing adenomas (APAs) and other adrenal tumors. Expression of (P)RR was studied by immunocytochemistry, western blot analysis and real-time RT-PCR in adrenal tumor tissues obtained at surgery. Immunocytochemistry showed that (P)RR was expressed in normal adrenal glands and tumor tissues of adrenocortical tumors including APAs. In the normal adrenal glands, positive (P)RR immunostaining was observed in both adrenal cortex and medulla, with higher (P)RR immunostaining observed in zona glomerulosa and zona reticularis. Positive (P)RR immunostaining was also observed in the adrenocortical tumors, with elevated (P)RR immunostaining found in APAs, particularly in compact cells. By contrast, no apparent (P)RR immunostaining was observed in pheochromocytomas. Western blot analysis showed a band of (P)RR protein in normal adrenal glands and adrenocortical tumors at the position of 35 kDa. The relative expression levels of (P)RR protein were higher in tumor tissues of APAs than in attached non-neoplastic adrenal tissues of APAs. Real-time RT-PCR showed that expression levels of (P)RR mRNA were significantly increased in tumor tissues of APAs compared with other adrenal tumor tissues and attached non-neoplastic adrenal tissues of APAs. The present study has shown for the first time that expression of (P)RR is elevated in tumor tissues of APAs, raising the possibility that (P)RR may play pathophysiological roles in APAs, such as aldosterone secretion and cell proliferation.
Keywords: (Pro)renin receptor; Prorenin; Adrenal; Aldosterone;

Effects of thyroid status on NEI concentration in specific brain areas related to reproduction during the estrous cycle by Carolina Ayala; Gisela Erika Pennacchio; Marta Soaje; Norma Beatriz Carreño; Jakson Cioni Bittencourt; Graciela Alma Jahn; María Ester Celis; Susana Ruth Valdez (74-80).
We previously showed that short-term hypo- and hyperthyroidism induce changes in neuropeptide glutamic-acid-isoleucine-amide (NEI) concentrations in discrete brain areas in male rats. To investigate the possible effects of hypo- and hyperthyroidism on NEI concentrations mainly in hypothalamic areas related to reproduction and behavior, female rats were sacrificed at different days of the estrous cycle. Circulating luteinizing hormone (LH), estradiol and progesterone concentrations were measured in control, hypothyroid (hypoT, treated with PTU during 7–9 days) and hyperthyroid (hyperT, l-T4 during 4–7 days) animals. Both treatments blunted the LH surge. Hypo- and hyperthyroidism increased estradiol concentrations during proestrus afternoon (P-PM), although hypoT rats showed lower values compared to control during proestrus morning (P-AM). Progesterone levels were higher in all groups at P-PM and in the hyperT during diestrus morning (D2). NEI concentrations were lower in hypoT rats during the estrous cycle except in estrus (E) in the peduncular part of the lateral hypothalamus (PLH). They were also reduced by both treatments in the perifornical part of the lateral hypothalamus (PeFLH) during P-PM. Hypothyroidism led to higher NEI concentrations during P-PM in the organum vasculosum of the lamina terminalis and anteroventral periventricular nucleus (OVLT + AVPV). The present results indicate that NEI concentration is regulated in a complex manner by hypo- and hyperthyroidism in the different areas studied, suggesting a correlation between NEI values and the variations of gonadal steroid levels during estrous cycle. These changes could be, in part, responsible for the alterations observed in the hypothalamic–pituitary–gonadal axis in these pathologies.
Keywords: Neuropeptide glutamic-acid-isoleucine-amide (NEI); Hypothalamus; Hypothyroidism; Hyperthyroidism; Estrous cycle; Reproduction;

Peptide YY (PYY) and ghrelin exhibit a reciprocal association and antagonistic physiological effects in the peripheral circulation. Research has yet to clarify the effect of weight loss on the 24 h profile of PYY or its association to 24 h ghrelin. We sought to determine if diet- and exercise-induced weight loss affects the 24 h profile of PYY and its association with 24 h ghrelin in normal weight, premenopausal women. Participants (n  = 13) were assessed at baseline (BL) and after a 3-month diet and exercise intervention (post). Blood samples obtained q10 min for 24 h were assayed for total PYY and total ghrelin q60 min from 0800 to 1000 h and 2000 to 0800 h and q20 min from 1000 to 2000 h. The ghrelin/PYY ratio was used as an index of hormonal exposure. Statistical analyses included paired t-tests and linear mixed effects modeling. Body weight (−1.85 ± 0.67 kg; p  = 0.02), and body fat (−2.53 ± 0.83%; p  = 0.01) decreased from BL to post. Ghrelin AUC (5252 ± 2177 pg/ml/24 h; p  = 0.03), 24 h mean (216 ± 90 pg/ml; p  = 0.03) and peak (300 ± 134 pg/ml; p  = 0.047) increased from BL to post. No change occurred in PYY AUC (88.2 ± 163.7 pg/ml; p  = 0.60), 24 h mean (4.8 ± 6.9 pg/ml; p  = 0.50) or peak (3.6 ± 6.4 pg/ml; p  = 0.58). The 24 h association between PYY and ghrelin at baseline (p  = 0.04) was weakened at post (p  = 0.14); however, the ghrelin/PYY lunch ratio increased (p  = 0.01) indicating the potential for ghrelin predominance over PYY in the circulation. PYY and ghrelin are reciprocally associated during a period of weight stability, but not following weight loss. An “uncoupling” may have occurred, particularly at lunch, due to factors that modulate ghrelin in response to weight loss.
Keywords: Peptide YY; Ghrelin; Weight loss; Premenopausal women;

The DPP-4 inhibitor MK0626 and exercise protect islet function in early pre-diabetic kkay mice by Yupeng Li; Jing Xiao; Hui Tian; Yu Pei; Yanhui Lu; Xiaofei Han; Yu Liu; Wenwen Zhong; Banruo Sun; Fusheng Fang; Hua Shu (91-99).
Dipeptidyl peptidase-4 (DPP-4) inhibitor and exercise have proven to be effective treatments for diabetes. However, the effects of these interventions in compensatory hyperinsulinemia prediabetic period are unknown. The purpose of this study was to determine if these interventions have protective effects on β-cell function and preventive effects on the onset of diabetes in prediabetic kkay mice. After 2 weeks of high-fat diet feeding, we treated 7-week-old mice with a normal diet, high-fat diet, exercise training, or the DPP-4 inhibitor for 8 weeks. C57BL/6J mice served as a normal control. Kkay mice without intervention developed diabetes at week 15, but no diabetic mice were observed in the DPP-4I or exercise groups as well as the normal control group. The DPP-4I and exercise groups showed improved body weight, blood glucose level, glucose tolerance, insulin sensitivity, islet area, and islet morphology. In addition, the proportion of Ki67-positive β-cells in the treatment groups was obviously higher than that in the untreated groups. MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homolog A) expression in the treated groups increased markedly. However PDX-1 (pancreatic and duodenal homeobox-1) expression did not differ significantly among the groups. The results show that exercise and DPP-4I treatment conducted during the hyperinsulinemic prediabetic stage contribute to the maintenance of β-cell function and morphology, enhance β-cell proliferation, extend the compensatory insulin hypersecretion period, and delay disease onset. The expression of PDX-1 was not altered significantly during the early stages of diabetes. However, the reduced expression of the insulin transcription factor MafA may play an important role in the development of prediabetes.
Keywords: DPP-4 inhibitor; Exercise; Pre-diabetes; Kkay mice; MafA; PDX-1; Proliferation;

Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor by Christina Rye Underwood; Lotte Bjerre Knudsen; Patrick W. Garibay; Günther H. Peters; Steffen Reedtz-Runge (100-108).
The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C-terminally truncated GLP-1R. Single cysteines were initially substituted with alanine, and functionally redundant cysteines were subsequently changed simultaneously. Our results indicate that Cys174, Cys226, Cys296 and Cys403 are important for the GLP-1-mediated response, whereas Cys236, Cys329, Cys341, Cys347, Cys438, Cys458 and Cys462 are not. Extensive deletions were made in the C-terminal tail of GLP-1R in order to determine the limit for truncation. As for other family B GPCRs, we observed a direct correlation between the length of the C-terminal tail and specific binding of 125I-GLP-1, indicating that the membrane proximal part of the C-terminal is involved in receptor expression at the cell surface. The results show that seven cysteines and more than half of the C-terminal tail can be removed from GLP-1R without compromising GLP-1 binding or function.
Keywords: G-protein coupled receptor; Glucagon-like peptide-1; Agonist; Cysteine; Mutagenesis;

Interactive effect of galanin-like peptide (GALP) and spontaneous exercise on energy metabolism by Kazuo Ito; Haruaki Kageyama; Satoshi Hirako; Lihua Wang; Fumiko Takenoya; Tetsuro Ogawa; Seiji Shioda (109-116).
Galanin-like peptide (GALP) is a neuropeptide involved in energy metabolism. The interactive effect of GALP and exercise on energy metabolism has not been investigated. The aim of this study was to determine if energy metabolism in spontaneously exercising mice could be promoted by intracerebroventricular (ICV) GALP administration. Changes in respiratory exchange ratio in response to GALP ICV administration indicated that lipids were primarily consumed followed by a continuous consumption of glucose throughout the dark period in non-exercising mice. In mice permitted to spontaneously exercise on a running-wheel, GALP ICV administration increased the consumed oxygen volume and heat production level from 5 to 11 h after administration. These effects occurred independently from the total running distance. The interaction between GALP ICV administration and spontaneous exercise decreased body weight within 24 h (F (1,16)  = 5.772, p  < 0.05), with no significant interaction observed regarding food and water intake or total distance. Energy metabolism-related enzymes were assessed in liver and skeletal muscle samples, with a significant interaction on mRNA expression between GALP ICV administration and spontaneous exercise observed in phosphoenolpyruvate carboxykinase (F (1,16)  = 18.602, p  < 0.001) that regulates gluconeogenesis and glucose transporter-4 (F (1,16)  = 21.092, p  < 0.001). GALP significantly decreased the mRNA expression of sterol regulatory element-binding protein-1c (p  < 0.05) that regulates fatty acid synthesis regardless of spontaneous exercise with no changes to acetyl-CoA carboxylase a and fatty acid synthetase. These results indicate the GALP ICV administration can further promote energy metabolism when administered to spontaneously exercising mice.
Keywords: RER; Metabolism-related enzyme; PEPCK; GLUT-4; SREBP-1c;

Vasonatrin peptide, a novel protector of dopaminergic neurons against the injuries induced by n-methyl-4-phenylpyridiniums by Hai-Kang Zhao; Bao-Ying Chen; Rui Chang; Jian-Bang Wang; Feng Ni; Lei Yang; Xi-Chao Dong; Shu-Hu Sun; Ge Zhao; Wei Fang; Quan-Rui Ma; Xue-Lian Wang; Jun Yu (117-122).
Vasonatrin peptide (VNP), a novel manmade natriuretic peptide, is known as a cardiovascular active substance. However, its neuroeffects are largely unknown. Here, cultured dopaminergic neurons from ventral mesencephalon of mouse were exposed to N-methyl-4-phenylpyridinium (MPP+), and the effects of VNP on the neurotoxicity of MPP+ were investigated. As a result, MPP+ caused injuries in the dopaminergic neurons. VNP significantly reduced the cytotoxicity of MPP+ by increasing axon number and length of dopaminergic neurons, and by enhancing the cell viability. Also, the MPP+-induced depolymerization of β-Tubulin III was attenuated by the treatment of VNP. In addition, VNP significantly increased the intracellular levels of cGMP. These effects of VNP were mimicked by 8-br-cGMP (a cell-permeable analog of cGMP), whereas inhibited by HS-142-1 (the antagonist of the particulate guanylyl cyclase-coupled natriuretic peptide receptors), or KT-5823 (a cGMP-dependent protein kinase inhibitor). Taken together, VNP attenuates the neurotoxicity of MPP+ via guanylyl cyclase-coupled NPR/cGMP/PKG pathway, indicating that VNP might be a new effective reagent in the treatment of neuron degeneration of dopaminergic neurons in Parkinson's disease (PD).
Keywords: Natriuretic peptides; Dopaminergic neurons; N-methyl-4-phenylpyridinium; Parkinson's disease;

Concentrations of preptin, salusins and hepcidins in plasma and milk of lactating women with or without gestational diabetes mellitus by Suleyman Aydin; Onder Celik; Bilgin Gurates; İbrahim Sahin; Mustafa Ulas; Musa Yilmaz; Mehmet Kalayci; Tuncay Kuloglu; Zekiye Catak; Aziz Aksoy; İbrahim Hanefi Ozercan; Selahattin Kumru (123-130).
This study was undertaken to ascertain whether human milk contains preptin, salusin-alpha (salusin-α) and -beta (salusin-β) and pro-hepcidin and hepcidin-25, and whether there are relationships between plasma and milk preptin, salusin-α and -β and pro-hepcidin and hepcidin-25 concentrations in lactating mothers with and without gestational diabetes mellitus (GDM). Blood was obtained from non-lactating women (n  = 12), non-diabetic lactating women (n  = 12), and GDM lactating women (n  = 12). Colostrum, transitional milk, and mature milk samples were collected just before suckling from healthy and GDM lactating women. Peptides concentrations were determined by ELISA and EIA. Mammary gland tissues were screened immunohistochemically for these peptides. Women with GDM had significantly higher plasma and colostum preptin concentrations than healthy lactating women during the colostral and transitional milk period. Salusin-alpha and -beta levels in milk and plasma were lower in women with GDM. Salusin-α and -β were significantly lower in both plasma and colostrums of GDM than of healthy lactating women. Women with GDM had significantly higher colostum prohepcidin and hepcidin-25 concentrations than healthy lactating women during the colostral period. Plasma prohepcidin was also higher in women with GDM than in healthy lactating women during the colostral period, but plasma prohepcidin and hepcidin-25 levels decreased during mature milk period. Transitional milk pro-hepcidin and hepcidin-25 levels in women with GDM were higher than in healthy lactating women. All these results revealed that the mammary gland produces those peptides, which were present in milk at levels correlating with plasma concentrations.
Keywords: Preptin; Salusin; Hepcidin; Gestational diabetes mellitus;

Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. Biofilm formation by A. baumannii is an additional factor in its ability to resist antimicrobial therapy. The antibacterial and antibiofilm activities of the human antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 16 to 32 μg/mL. The MIC of KS-30 fragment varied from 8.0 to16 μg/mL and the KR-20 fragment MIC ranged from 16 to 64 μg/mL. LL-37 and KS-30 fragment exhibited 100% bactericidal activity against five A. baumannii strains, including four MDR clinical isolates, within 30 min at concentrations of 0.25–1 μg/mL. By 0.5 h, the fragments KR-20 and KR-12 eliminated all tested strains at 8 and 64 μg/mL respectively. LL-37 and its fragments displayed anti-adherence activities between 32-128 μg/mL. A minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-37 inhibited and dispersed A. baumannii biofilms at 32 μg/mL respectively. Truncated fragments of LL-37 inhibited biofilms at concentrations of 64–128 μg/mL. KS-30, the truncated variant of LL-37, effectively dispersed biofilms at 64 μg/mL. At 24 h, no detectable toxicity was observed at the efficacious doses when cytotoxicity assays were performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 and KR-20 adds significance to their efficacy. These peptides can be potential therapeutics against MDR A. baumannii infections.
Keywords: Cathelicidin; Biofilm; Acinetobacter baumannii; Anti-infective;

Ghrelin and peptide YY increase with weight loss during a 12-month intervention to reduce dietary energy density in obese women by Brenna R. Hill; Barbara J. Rolls; Liane S. Roe; Mary Jane De Souza; Nancy I. Williams (138-144).
Reducing dietary energy density (ED) promotes weight loss; however, underlying mechanisms are not well understood. The purpose of this study was to determine if low-ED diets facilitate weight loss through actions on ghrelin and peptide YY (PYY), independent of influences of psychosocial measures. Seventy-one obese women (BMI 30–40 kg/m2) ages 22–60 years received counseling to reduce ED. Fasting blood samples were analyzed for total ghrelin and total PYY by radioimmunoassay at months 0, 3, 6, and 12. Restraint, disinhibition, and hunger were assessed by the Eating Inventory. Body weight (−7.8 ± 0.5 kg), BMI (−2.9 ± 0.2 kg/m2), body fat (−3.0 ± 0.3%), and ED (−0.47 ± 0.05 kcal/g or −1.97 ± 0.21 kJ/g) decreased from months 0 to 6 (p  < 0.05) after which no change occurred from months 6 to 12. Ghrelin increased in a curvilinear fashion (month 0: 973 ± 39, month 3: 1024 ± 37, month 6: 1109 ± 44, and month 12: 1063 ± 45 pg/ml, p  < 0.001) and PYY increased linearly (month 0: 74.2 ± 3.1, month 3: 76.4 ± 3.2, month 6: 77.2 ± 3.0, month 12: 82.8 ± 3.2 pg/ml, p  < 0.001). ED, body weight, and hunger predicted ghrelin, with ED being the strongest predictor (ghrelin = 2674.8 + 291.6 × ED − 19.2 × BW − 15 ×  H; p  < 0.05). There was a trend toward a significant association between ED and PYY (PYY = 115.0 − 43.1 × ED; p  = 0.05). Reductions in ED may promote weight loss and weight loss maintenance by opposing increases in ghrelin and promoting increases in PYY.
Keywords: Ghrelin; Peptide YY; Dietary energy density; Weight loss;

A ‘conovenomic’ analysis of the milked venom from the mollusk-hunting cone snail Conus textile—The pharmacological importance of post-translational modifications by Zachary L. Bergeron; Joycelyn B. Chun; Margaret R. Baker; David W. Sandall; Steve Peigneur; Peter Y.C. Yu; Parashar Thapa; Jeffrey W. Milisen; Jan Tytgat; Bruce G. Livett; Jon-Paul Bingham (145-158).
Cone snail venoms provide a largely untapped source of novel peptide drug leads. To enhance the discovery phase, a detailed comparative proteomic analysis was undertaken on milked venom from the mollusk-hunting cone snail, Conus textile, from three different geographic locations (Hawai’i, American Samoa and Australia's Great Barrier Reef). A novel milked venom conopeptide rich in post-translational modifications was discovered, characterized and named α-conotoxin TxIC. We assign this conopeptide to the 4/7 α-conotoxin family based on the peptide's sequence homology and cDNA pre-propeptide alignment. Pharmacologically, α-conotoxin TxIC demonstrates minimal activity on human acetylcholine receptor models (100 μM, <5% inhibition), compared to its high paralytic potency in invertebrates, PD50  = 34.2 nMol kg−1. The non-post-translationally modified form, [Pro]2,8[Glu]16α-conotoxin TxIC, demonstrates differential selectivity for the α3β2 isoform of the nicotinic acetylcholine receptor with maximal inhibition of 96% and an observed IC50 of 5.4 ± 0.5 μM. Interestingly its comparative PD50 (3.6 μMol kg−1) in invertebrates was ∼100 fold more than that of the native peptide. Differentiating α-conotoxin TxIC from other α-conotoxins is the high degree of post-translational modification (44% of residues). This includes the incorporation of γ-carboxyglutamic acid, two moieties of 4-trans hydroxyproline, two disulfide bond linkages, and C-terminal amidation. These findings expand upon the known chemical diversity of α-conotoxins and illustrate a potential driver of toxin phyla-selectivity within Conus.
Keywords: α-Conotoxin; Conopeptides; Conus textile; Mass spectrometry; Milked venom; Radula tooth; Post-translational modifications; nAChR;