Similar Intracellular Peptide Profile of TAP1/beta 2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

Cells produce and use peptides in distinctive ways. In the present report, using isotope labeling plus semi-quantitative mass spectrometry, we evaluated the intracellular peptide profile of TAP1/beta 2m(-/-) (transporter associated with antigen-processing 1/beta 2 microglobulin) double-knockout mice and compared it with that of C57BL/6 wild-type animals. Overall, 92 distinctive peptides were identified, and most were shown to have a similar concentration in both mouse strains. However, some peptides showed a modest increase or decrease (similar to 2-fold), whereas a glycine-rich peptide derived from the C-terminal of neurogranin (KGPGPGGPGGAGGARGGAGGGPSGD) showed a substantial increase (6-fold) in TAP1/beta 2m(-/-) mice. Thus, TAP1 and beta 2microglobulin have a small influence on the peptide profile of neuronal tissue, suggesting that the presence of peptides derived from intracellular proteins in neuronal tissue is not associated with antigens of the class I major histocompatibility complex. Therefore, it is possible that these intracellular peptides play a physiological role.

Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

Peptides constitute the largest group of Hymenoptera venom toxins; some of them interact with GPCR, being involved with the activation of different types of leukocytes, smooth muscle contraction and neurotoxicity. Most of these toxins vary from dodecapeptides to tetradecapeptides, amidated at their C-teminal amino acid residue. The venoms of social wasps can also contains some tetra-, penta-, hexa- and hepta-peptides, but just a few of them have been structurally and functionally characterized up to now. Protonectin (ILG-TILGLLKGL-NH(2)) is a polyfunctional peptide, presenting mast cell degranulation, release of lactate dehydrogenase (LDH) from mast cells, antibiosis against Gram-positive and Gram-negative bacteria and chemotaxis for polymorphonucleated leukocytes (PMNL), while Protonectin (1-6) (ILGTIL-NH(2)) only presents chemotaxis for PMNL However, the mixture of Protonectin (1-6) with Protonectin in the molar ratio of 1:1 seems to potentiate the biological activities dependent of the membrane perturbation caused by Protonectin, as observed in the increasing of the activities of mast cell degranulation, LDH releasing from mast cells, and antibiosis. Despite both peptides are able to induce PMNL chemotaxis, the mixture of them presents a reduced activity in comparison to the individual peptides. Apparently, when mixed both peptides seems to form a supra-molecular structure, which interact with the receptors responsible for PMNL chemotaxis, disturbing their individual docking with these receptors. In addition to this, a comparison of the sequences of both peptides suggests that the sequence ILGTIL is conserved, suggesting that it must constitute a linear motif for the structural recognition by the specific receptor which induces leukocytes migration. (C) 2010 Elsevier Ltd. All rights reserved.

Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1

Crajoinas RO, Oricchio FT, Pessoa TD, Pacheco BP, Lessa LM, Malnic G, Girardi AC. Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1. Am J Physiol Renal Physiol 301: F355-F363, 2011. First published May 18, 2011; doi: 10.1152/ajprenal.00729.2010.-Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone considered a promising therapeutic agent for type 2 diabetes because it stimulates beta cell proliferation and insulin secretion in a glucose-dependent manner. Cumulative evidence supports a role for GLP-1 in modulating renal function; however, the mechanisms by which GLP-1 induces diuresis and natriuresis have not been completely established. This study aimed to define the cellular and molecular mechanisms mediating the renal effects of GLP-1. GLP-1 (1 mu g.kg(-1).min(-1)) was intravenously administered in rats for the period of 60 min. GLP-1-infused rats displayed increased urine flow, fractional excretion of sodium, potassium, and bicarbonate compared with those rats that received vehicle (1% BSA/saline). GLP-1-induced diuresis and natriuresis were also accompanied by increases in renal plasma flow and glomerular filtration rate. Real-time RT-PCR in microdissected rat nephron segments revealed that GLP-1 receptor-mRNA expression was restricted to glomerulus and proximal convoluted tubule. In rat renal proximal tubule, GLP-1 significantly reduced Na(+)/H(+) exchanger isoform 3 (NHE3)-mediated bicarbonate reabsorption via a protein kinase A (PKA)-dependent mechanism. Reduced proximal tubular bicarbonate flux rate was associated with a significant increase of NHE3 phosphorylation at the PKA consensus sites in microvillus membrane vesicles. Taken together, these data suggest that GLP-1 has diuretic and natriuretic effects that are mediated by changes in renal hemodynamics and by downregulation of NHE3 activity in the renal proximal tubule. Moreover, our findings support the view that GLP-1-based agents may have a potential therapeutic use not only as antidiabetic drugs but also in hypertension and other disorders of sodium retention.

Human apolipoprotein A-I binds amyloid-beta and prevents A beta-induced neurotoxicity

Aggregates of the amyloid-P peptide (A beta) play a central role in the pathogenesis of Alzheimer`s disease (AD). Identification of proteins that physiologically bind A beta and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated A beta(1-42), We isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A-I (apoA-I). We show that purified human apoA-I and A beta form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by A beta. Significantly, A beta/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from A beta-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates A beta aggregation and A beta-induced neuronal damage and that the A beta-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of A beta toxicity. (C) 2009 Published by Elsevier Ltd.

Expression profiles of the glucose-dependent insulinotropic peptide receptor and LHCGR in sporadic adrenocortical tumors

Glucose-dependent insulinotropic peptide receptor (GIPR) and LHCGR are G-protein-coupled receptors with a wide tissue expression pattern. Aberrant expression of these receptors has rarely been demonstrated in adult sporadic adrenocortical tumors with a lack of data on pediatric tumors. We quantified the GIPR and LHCGR expression in a large cohort of 55 patients (25 children and 30 adults) with functioning and non-functioning sporadic adrenocortical tumors. Thirty-eight tumors were classified as adenomas whereas 17 were carcinomas. GIPR, and LHCGR expression were analyzed by real-time PCR and normal human pancreatic and testicular tissue samples were used as positive controls. Mean expression values were determined by fold increase in comparison with a normal adrenal pool. GIPR mRNA levels were significantly higher in adrenocortical carcinomas than in adenomas from both pediatric and adult groups. LHCGR expression was similar in both carcinomas and adenomas from the pediatric group but significantly lower in carcinomas than in adenomas from the adult group (median 0.06 and 2.3 respectively, P<0.001). GIPR was detected by immunohistochemistry in both pediatric and adult tumors. Staining and real-time PCR results correlated positively only when GIPR in RN A levels were increased at least two-fold in comparison with normal adrenal expression levels. In Conclusion, GIPR overexpression was observed in pediatric and adult adrenocortical tumors and very low levels of LHCGR expression were found in all adult adrenocortical carcinomas.

Enterohormonal changes after digestive adaptation: Five-year results of a surgical proposal to treat obesity and associated diseases

Background Recent physiological knowledge allows the design of bariatric procedures that aim at neuroendocrine changes instead of at restriction and malabsorption. Digestive adaptation is a surgical technique for obesity based in this rationale. Methods The technique includes a sleeve gastrectomy, an omentectomy and a jejunectomy that leaves initial jejunum and small bowel totaling at least 3 m (still within normal variation of adult human bowel length). Fasting ghrelin and resistin and fasting and postprandial GLP-1 and PYY were measured pre- and postoperatively. Results Patients: 228 patients with initial body mass index (BMI) varying from 35 to 51 kg/m(2); follow-up: I to 5 years; average EBMIL% was 79.7% in the first year; 77.7% in the second year; 71.6% in the third year; 68.9% in the fourth year. Patients present early satiety and major improvement in presurgical comorbidities, especially diabetes. Fasting ghrelin and resistin were significantly reduced (P<0.05); GLP-1 and PYY response to food ingestion was enhanced (P<0.05). Surgical complications (4.4%) were resolved without sequela and without mortality. There was neither diarrhea nor detected malabsorption. Conclusions Based on physiological and supported by evolutionary data, this procedure creates a proportionally reduced gastrointestinal (GI) tract that amplifies postprandial neuroendocrine responses. It leaves basic GI functions unharmed. It reduces production of ghrelin and resistin and takes more nutrients to be absorbed distally enhancing GLP-1 and PYY secretion. Diabetes was improved significantly without duodenal exclusion. The patients do not present symptoms nor need nutritional support or drug medication because of the procedure, which is safe to perform.

BhSGAMP-1, a Gene That Encodes an Antimicrobial Peptide, Is Developmentally Regulated by the Direct Action of 20-OH Ecdysone in the Salivary Gland of Bradysia hygida (Diptera, Sciaridae)

Recently we have shown that BhSGAMP-1 is a developmentally regulated reiterated gene that encodes an antimicrobial peptide (AMP) and is expressed exclusively in the salivary glands, at the end of the larval stage. We show, for the first time, that a gene for an AMP is directly activated by 20-OH ecdysone. This control probably involves the participation of short-lived repressor(s). We also found that the promoter of BhSGAMP-1 is not equipped with elements that respond to infection, provoked by the injection of microorganisms, in the salivary glands or in the fat body. We produced polyclonal antibodies against the synthetic peptide and found that the BhSGAMP-1 peptide is secreted in the saliva. The BhSGAMP-1 gene was also activated during the third larval molt. These facts confirm our hypothesis that this preventive system of defense was selected to produce an environment free of harmful microorganisms in the insect`s immediate vicinity, during molts. genesis 47:847-857, 2009. (C) 2009 Wiley-Liss, Inc.

Insulin-like peptide genes in honey bee fat body respond differently to manipulation of social behavioral physiology

Nutrient sensitive insulin-like peptides (ILPs) have profound effects on invertebrate metabolism, nutrient storage, fertility and aging. Many insects transcribe ILPs in specialized neurosecretory cells at changing levels correlated with life history. However, the major site of insect metabolism and nutrient storage is not the brain, but rather the fat body, where functions of ILP expression are rarely studied and poorly understood. Fat body is analogous to mammalian liver and adipose tissue, with nutrient stores that often correlate with behavior. We used the honey bee (Apis mellifera), an insect with complex behavior, to test whether ILP genes in fat body respond to experimentally induced changes of behavioral physiology. Honey bee fat body influences endocrine state and behavior by secreting the yolk protein precursor vitellogenin (Vg), which suppresses lipophilic juvenile hormone and social foraging behavior. In a two-factorial experiment, we used RNA interference (RNAi)-mediated vg gene knockdown and amino acid nutrient enrichment of hemolymph (blood) to perturb this regulatory module. We document factor-specific changes in fat body ilp1 and ilp2 mRNA, the bee`s ILP-encoding genes, and confirm that our protocol affects social behavior. We show that ilp1 and ilp2 are regulated independently and differently and diverge in their specific expression-localization between fat body oenocyte and trophocyte cells. Insect ilp functions may be better understood by broadening research to account for expression in fat body and not only brain.

Gastrointestinal transit, appetite, and energy balance in gastrectomized patients

Background: Alterations in gastrointestinal tract physiology after gastrectomy may affect appetite and energy balance. Objective: The objective of this study was to examine energy balance, appetite, and gastrointestinal transit in subjects with gastrectomy. Design: Seven subjects with total gastrectomy (TG) and 14 subjects with partial gastrectomy (PG), who were free from signs of recurrent disease, and 10 healthy control subjects were studied. Resting energy expenditure (REE) was measured by indirect calorimetry and compared with REE predicted by the Harris-Benedict equation (mREE/pREE%). Gastrointestinal transit was measured by scintigraphy. Habitual food intake was assessed, and appetite was measured during scintigraphy after ingestion of a test meal (361 kcal). Results: Body mass index was not different among the groups. mREE/pREE% was higher in patients with PG (P < 0.01) than in control subjects. The TG group showed higher energy intake (P < 0.05) than the PG group and control subjects. Gastric emptying was faster in the PG group than in control subjects, and gastrointestinal transit was accelerated in both PG and TG groups. An intense, precocious postprandial fullness and a relatively early recovery of hunger and prospective consumption sensations were seen in these patients. Conclusions: Patients with PG or TG have higher than predicted energy expenditure, which in TG seems to be compensated for by increased energy intake. These patients have preserved postprandial appetite responses and precocious postprandial fullness, which seem to be associated with disturbances in gastrointestinal transit of the ingested meal and are likely to be independent of vagal fiber integrity or stomach-released ghrelin. Am J Clin Nutr 2009; 89: 231-9.

Rationale and design of a randomized placebo-controlled trial assessing the effects of etiologic treatment in Chagas` cardiomyopathy: The BENznidazole Evaluation For Interrupting Trypanosomiasis (BENEFIT)

Background Benznidazole is effective for treating acute and chronic (recently acquired) Tryponosoma cruzi infection (Chagas` disease). Recent data indicate that parasite persistence plays a pivotal role in the pathogenesis of chronic Chagas` cardiomyopathy. However, the efficacy of trypanocidal therapy in preventing clinical complications in patients with preexisting cardiac disease is unknown. Study Design BENEFIT is a multicenter, randomized, double-blind, placebo-controlled clinical trial of 3,000 patients with Chagas` cardiomyopathy in Latin America. Patients are randomized to receive benznidazole (5 mg/kg per day) or matched placebo, for 60 days. The primary outcome is the composite of death; resuscitated cardiac arrest; sustained ventricular tachycardia; insertion of pacemaker or cardiac defibrillator; cardiac transplantation; and development of new heart failure, stroke, or systemic or pulmonary thromboembolic events. The average follow-up time will be 5 years, and the trial has a 90% power to detect a 25% relative risk reduction. The BENEFIT program also comprises a substudy evaluating the effects of benznidazole on parasite clearance and an echo substudy exploring the impact of etiologic treatment on left ventricular function. Recruitment started in November 2004, and >1,000 patients have been enrolled in 35 centers from Argentina, Brazil, and Colombia to date. Conclusion This is the largest trial yet conducted in Chagas` disease. BENEFIT will clarify the role of trypanocidal therapy in preventing cardiac disease progression and death.

HMGB1-derived peptide acts as adjuvant inducing immune responses to peptide and protein antigen

There is a need for new adjuvants that will induce immune responses to subunit vaccines. We show that a short peptide, named Hp91, whose sequence corresponds to an area within the endogenous molecule high mobility group box (HMGB1) protein 1 potentiates cellular immune responses to peptide antigen and cellular and humoral immune responses to protein antigen in vivo. Hp91 promoted the in vivo production of the immunomodulatory cytokines, IFN-gamma, TNF-alpha, IL-6, and IL-12 (p70), as well as antigen-specific activation of CD8+ T cells. These results demonstrate the ability of a short immunostimulatory peptide to serve as an adjuvant for subunit vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

Protective Effect of RC-3095, an Antagonist of the Gastrin-Releasing Peptide Receptor, in Experimental Arthritis

Objective. To evaluate the antiinflammatory effects of RC-3095 in 2 experimental models of arthritis, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA), and to determine the mechanisms of action involved. Methods. RC-3095 was administered daily to mice with CIA and mice with AIA, after induction of disease with methylated bovine serum albumin. Disease incidence and severity were assessed using a clinical index and evaluation of histologic features, respectively. In mice with CIA, gastrin-releasing peptide receptor (GRPR) was detected by immunohistochemical analysis, while in mice with AIA, migration of neutrophils, presence of glycosaminoglycans, and lymphocyte proliferation, determined using the MTT assay, were assessed. Expression of cytokines interleukin-17 (IL-17), IL-1 beta, and tumor necrosis factor alpha (TNF alpha) was evaluated in all mouse knees using enzyme-linked immunosorbent assay. Treg cell production was assessed by flow cytometry in the joints of mice with AIA. Results. In mice with AIA, administration of RC-3095 reduced neutrophil migration, mechanical hypernociception, and proteoglycan loss. These findings were associated with inhibition of the levels of all 3 proinflammatory cytokines, decreased lymphocyte proliferation, and increased Treg cell numbers. In the CIA model, treatment with RC-3095 led to a significant reduction in arthritis clinical scores and the severity of disease determined histologically. Synovial inflammation, synovial hyperplasia, pannus formation, and extensive erosive changes were all dramatically reduced in the arthritic mice treated with RC-3095. Furthermore, arthritic mice treated with RC-3095 showed a significant reduction in the concentrations of IL-17, IL-1 beta, and TNF alpha, and showed a diminished expression of GRPR. Conclusion. These findings suggest that the GRP pathway has a significant role in chronic arthritis, and its inhibition can be explored as a possible therapeutic strategy in rheumatoid arthritis.

Murine and rat cavernosal responses to endothelin-1 and urotensin-II Vasoactive Peptide Symposium

Endothelin-1 (ET-1) and urotensin-II (U-II) are the most potent constrictors of human vessels. Although the cavernosal tissue is highly responsive to ET-1, no information exists on the effects of U-II on cavernosal function. The aim of this study was to characterize ET-1 and U-II responses in corpora cavernosa from rats and mice. Male Wistar rats and C57/BL6 mice were used at 13 weeks. Cumulative concentration-response curves to ET-1, U-II, and IRL-1620, an ET(B) agonist, were performed. ET-1 increased force generation in cavernosal strips from mice and rats, but no response to U-II was observed in the presence or absence of N(omega)-nitro-L-arginine methyl ester (L-NAME), or in strips prestimulated with 20 mM KCI. IRL-1620 did not induce cavernosal contraction even in presence of L-NAME, but induced a cavernosal relaxation that was greater in rats than mice. No relaxation responses to U-II were observed in cavernosal strips precontracted with phenylephrine. mRNA expression of ET-1, ET(A), ET(B), and U-II receptors, but not U-II was observed in cavernosal strips. ET-1, via ET(A) receptors activation, causes contractile responses in cavernosal strips from rats and mice, whereas ET(B) receptor activation produces relaxation. Although the cavernosal tissue expresses U-II receptors, U-II does not induce contractile responses in corpora cavernosa from mice or rats. J Am Soc Hypertens 2008;2(6): 439-447. Published by Elsevier Inc. on behalf of the American Society of Hypertension.

Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta A(12,14)-prostaglandin J(2), reduces neutrophil migration via a nitric oxide pathway

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma), such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ(2)-mediated activation of PPAR-gamma ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ(2) administration decreases leukocyte rolling and adhesion to the inflammated mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS(-/-) mice were not susceptible to 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ(2)-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-I expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ(2), whereas 15d-PGJ(2) inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ(2) suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.

Alterations in cyclic GMP levels in preeclampsia may reflect increased B-type natriuretic peptide levels and not impaired nitric oxide activity

Objectives: We compared nitrite, B-type natriuretic peptide (BNP), and cGMP levels in preeclamptic with those found in healthy pregnant. Methods: We studied 21 healthy pregnant and 27 preeclamptic. Plasma cGMP and BNP levels were determined by ELISA. Nitrite levels were determined by chemiluminescence. Results: Higher cGMP and BNP, and lower nitrite levels were found in preeclamptic versus healthy pregnant Conclusions: Altered cGMP levels reflect increased BNP levels and not impaired nitric oxide activity in preeclampsia. (C) 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.