Exogenous Peptide Research Articles

Increases in transmembrane glycoprotein NMB (GPNMB), phospho‐ERK1/2, and matrix metallopeptidase (MMP)‐9 follow decline in arylsulfatase B in cystic fibrosis

In previous reports, decline in the enzyme arylsulfatase B (ARSB; N‐acetylgalactosamine‐ 4‐sulfatase) was identified in cystic fibrosis (CF) cells in culture and in circulating leukocytes from CF patients. Recently, the transmembrane glycoprotein NMB (GPNMB) was shown to be increased in a CF gene array and in Arylsulfatase B (ARSB; N‐acetylgalactosamine‐4‐sulfatase)‐null mice. These findings are consistent with previous reports that ARSB is reduced in cystic fibrosis (CF). A mechanism by which decline in ARSB, and the resultant increase in chondroitin 4‐sulfate, increased GPNMB expression has been reported. Subsequently, experiments were designed to assess the potential role of GPNMB in CF and the mechanisms by which GPNMB might contribute to CF pathophysiology. In serum and circulating leukocytes from CF and control patients, GPNMB levels were determined by ELISA. GPNMB was also measured in cells and spent media of CFTR‐corrected, CFTR‐uncorrected, and normal human bronchial epithelial cells (BEC) following ARSB siRNA or control siRNA. The GPNMB which co‐immunoprecipitated with β‐1 integrin was determined by ELISA. Matrix metalloproteinase (MMP)‐9 was measured, and the correlations between GPNMB and MMP‐9 in serum and spent media were determined. Phospho‐ERK1/2 in the circulating leukocytes and in the cultured cells was measured. The impact of excess exogenous RGD peptide and GPNMB siRNA on phospho‐ERK1/2 content was determined, and the impact of the RGD peptide, GPNMB siRNA, and ERK phosphorylation inhibitor on MMP‐9 mRNA expression was determined. GPNMB was markedly increased in cells and serum of CF patients compared to controls (p<0.0001, unpaired t‐test, two‐tailed). In uncorrected CF cells and in BEC following ARSB silencing, the GPNMB was significantly increased (p<0.0001; p<0.001). Silencing GPNMB or treatment with excess RGD peptide significantly inhibited the ARSB‐silencing induced increase in phospho‐ERK1/2 in the BEC. Silencing GPNMB, treatment with excess RGD peptide, or treatment with ERK phosphorylation inhibitor blocked the ARSB silencing‐induced increases in MMP‐9 expression in the normal BEC. Findings suggest that decline in ARSB activity caused by decline in CFTR function leads to increased GPNMB, phospho‐ERK1/2, and MMP‐9 expression, which may contribute to disruption of cell signaling and to organ dysfunction in CF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dynamic rearrangement of the intrinsic ligand regulates KCNH potassium channels

KCNH voltage-gated potassium channels (EAG, ERG, and ELK) play significant roles in neuronal and cardiac excitability. They contain cyclic nucleotide-binding homology domains (CNBHDs) but are not directly regulated by cyclic nucleotides. Instead, the CNBHD ligand-binding cavity is occupied by an intrinsic ligand, which resides at the intersubunit interface between the N-terminal eag domain and the C-terminal CNBHD. We show that, in Danio rerio ELK channels, this intrinsic ligand is critical for voltage-dependent potentiation (VDP), a process in which channel opening is stabilized by prior depolarization. We demonstrate that an exogenous peptide corresponding to the intrinsic ligand can bind to and regulate zebrafish ELK channels. This exogenous intrinsic ligand inhibits the channels before VDP and potentiates the channels after VDP. Furthermore, using transition metal ion fluorescence resonance energy transfer and a fluorescent noncanonical amino acid L-Anap, we show that there is a rearrangement of the intrinsic ligand relative to the CNBHD during VDP. We propose that the intrinsic ligand switches from antagonist to agonist as a result of a rearrangement of the eag domain-CNBHD interaction during VDP.

Correction: Metabolic changes and inflammation in cultured astrocytes from the 5xFAD mouse model of Alzheimer's disease: Alleviation by pantethine.

[This corrects the article DOI: 10.1371/journal.pone.0175369.].

INFLORESCENCE DEFICIENT IN ABSCISSION-like is an abscission-associated and phytohormone-regulated gene in flower separation of Lupinus luteus

Abscission is a natural process that occurs to facilitate shedding of no longer needed organs, but on the other hand, can be triggered by certain environmental conditions, e.g. biotic or abiotic stresses. Regardless of the stimuli, organ shedding takes place specifically at the abscission zone (AZ). A signaling pathway that controls this process in Arabidopsis thaliana from ligand to receptors has been proposed. However, knowledge concerning the influence of plant hormones on these molecular elements still remains enigmatic. Excessive and premature flower abscission in the crop species Lupinus luteus L. is a process of substantial interest to the agricultural industry, as it can affect yield. Our strategy combined molecular studies, comprehensive ultrastructural and histological analysis, as well as exogenous hormone treatment to describe the contribution of the Lupinus IDA-like gene in flower abscission. In the AZ of the naturally abscised flowers, the differentiation of morphologically distinct cells characterized by progressive degradation processes was accompanied by LlIDL mRNA accumulation. A similar effect was observed following early steps of AZ activation and after abscisic acid or ethylene treatments. These phytohormones, previously pointed out as key stimulators of flower separation, altered the temporal expression pattern of LlIDL. Exogenous EPIP peptide synthesized on the basis of LlIDL sequence, significantly increased flower abortion rate, which indicates that this motif governs protein activity. In conclusion, our data provide new evidence for LlIDA involvement in both the early and late events of flower abscission supported by detailed spatiotemporal characterization of AZ cell structure and ultrastructure.

Neuroglobin boosts axon regeneration during ischemic reperfusion via p38 binding and activation depending on oxygen signal

Cerebral ischemia causes severe cell death or injury including axon breakdown or retraction in the brain. Axon regeneration is crucial for the functional recovery of injured neurons or brains after ischemia/reperfusion (I/R); however, this process has been proved extremely difficult in adult brains and there is still no effective therapy for it. Here we reported that neuroglobin (Ngb), a novel oxygen-binding or sensor protein existing predominantly in neurons or brains, functions as a driving factor for axon regeneration during I/R. Ngb was upregulated and accumulated in growth cones of ischemic neurons in primary cultures, rat, and human brains, correlating positively to the elevation of axon-regeneration markers GAP43, neurofilament-200, and Tau-1. Ngb overexpression promoted while Ngb knockdown suppressed axon regeneration as well as GAP43 expression in neurons during oxygen-glucose deprivation/reoxygenation (OGD/Re). By using specific pharmacological inhibitors, we identified p38 MAPK as the major downstream player of Ngb-induced axon regeneration during OGD/Re. Mechanistically, Ngb directly bound to and activated p38 in neurons upon OGD/Re. Serial truncation and point mutation of Ngb revealed that the 7–105 aa fragment of Ngb was required and the oxygen-binding site (His64) of Ngb was the major regulatory site for its p38 interaction/activation. Finally, administration of exogenous TAT-Ngb peptides significantly enhanced axon regeneration in cultured neurons upon OGD/Re. Taken together, Ngb promotes axon regeneration via O2-Ngb-p38-GAP43 signaling during I/R. This novel mechanism suggests potential therapeutic applications of Ngb for ischemic stroke and other related axonopathy.

A Common Pathway of Root Growth Control and Response to CLE Peptides Through Two Receptor Kinases in Arabidopsis.

Cell-cell communication is essential for plants to integrate developmental programs with external cues that affect their growth. Recent advances in plant signaling have uncovered similar molecular mechanisms in shoot, root, and vascular meristem signaling that involve receptor-like kinases and small, secreted peptides. Here, we report that the receptor-like kinases TOAD2/RPK2 and RPK1 regulate root growth by controlling cell proliferation and affecting meristem size. Two types of developmental alterations were observed upon exogenous CLE peptide application. The first type was detected in all plants treated, and comprise increased proliferative activity of cells in the stem cell niche and a delay of progression in differentiation of daughter cells. The second type was changes specific to the genotypes that are sensitive to CLE-driven root meristem inhibition and include a large decrease in the occurrence of cell divisions in longitudinal files, correlating with shorter meristems and cessation of root growth. The root meristems of toad2/rpk2 mutant plants are insensitive to the inhibitory effect of CLE17 peptide treatment, consistent with TOAD2/RPK2 function as a receptor for CLE peptides. In addition, a strong reduction in the expression of RPK1 protein upon CLE treatment, dependent on TOAD2/RPK2, suggests that these two RLKs mediate CLE signaling in a common pathway to control root growth.

Short term effects of liraglutide versus vildagliptin on insulin secretion and sensitivity in type 2 diabetes (Liravis study)

Introduction : incretinomimetics are new type 2 diabetes mellitus drugs that have the ability to increase glucose-induced insulin production. This drug class has two subclasses: Exogenous Glucagon-Like Peptide analogs (GLP1a) such as Liraglutide, and the inhibitors of Dipeptidyl peptidase IV (DPP4i) that prolong the half-life of endogenous GLP1 such as vildagliptin.We aimed to evaluate the short term metabolic effects of a GLP-1a, liraglutide versus a DPP-4i, vildagliptin on type 2 diabetes patients. Methods : we conducted a randomized controlled single blind clinical trial on 14 uncontrolled type 2 diabetes patients (HbA1c ≥ 53mmol/mol). Participants were randomly allocated in 2 groups. Anthropometric measurements, hyperinsulinemic-euglycemic clamp at 80mU/m 2 /min and a mixed meal tolerance test (to assess insulin sensitivity/secretion) were carried out before and after the different interventions. In group 1, 0.6mg/day of liraglutide was administered subcutaneously and increased to 1.2mg the 2nd week whereas, 100mg/day of oral vildagliptin was administered in group 2 for 2 weeks. Results : after the intervention insulin sensitivity remained unchanged: liraglutide (6.6 [4.2-7.9] to 6.9 [4.3-10.8] mg/kg/min; p=0.61) and vildagliptin (7.1 [5.3- 9.0] to 6.5 [5.6-9.4] mg/kg/min; p = 0.86). LDL Cholesterol levels decreased significantly in group 1(0.85 [0.51-1.02] to 0.54 [0.50-0.73] g/L, p=0.04) but not in group 2 (p= 0.23). Concerning insulin secretion, the area under the C-peptide curve varied from (5.5[1.0-10.9] to 14.9 [10.8-17.2] nmol/L/120min, p=0.09) in group 1 and from 1.1 [0.5-14.1] to 13.0 [9.6-16.9] nmol/L/120min, p=0.17) in group 2, showing a non-significant trend towards improvement. A variation in body weight was remarked amongst the groups, being, - 0.6kg and +1.1kg in group 1 and 2 respectively. Conclusion : there are no significant differences on insulin sensitivity and insulin secretion after two weeks of treatment using liraglutide and vildagliptin, in type 2 diabetes mellitus patients.

Major Histocompatibility Complex Dmα/β Genes and Their Expressional Diversity in Healthy and Diseased Water Buffalo Bubalus bubalis

Background/Aims: The major histocompatibility complex (MHC) categorized into three (I, II and III) classes elicits the immunogenic response by presenting exogenous peptides to T cells. The MHC-II DM is composed of DMα and DMβ, two polypeptide chains, both are encoded by separate MHC genes involved in antigen processing and presentation. Despite the acknowledged role of MHC complex in humans, the literature is silent on the organization and expression of these genes in water buffalo Bubalus bubalis, an agriculturally important animal species. Methods: We deduced the full-length mRNA sequences of DMα and DMβ genes, localized them onto the chromosome 2, assessed their copy number per haploid genome and studied tissue and disease specific expression. Results: The Real Time PCR showed higher expression of both the genes and their seven interacting partners in spleen, gonads and spermatozoa. Significantly, upregulation of DMα and DMβ genes and their interacting partners were detected in diseased group of buffaloes as compared to that in healthy ones. Conclusion: The upregulation of Bubalus bubalis (BuLA)-DMα and DMβ genes and their interacting partners reflect their role in regulating immune responses towards the amelioration of diseases. Work on this line would enhance our understanding on the overall roles of MHC locus, allowing development of possible therapeutic treatment strategies.

Exogenous murine antimicrobial peptide CRAMP significantly exacerbates Ovalbumin-induced airway inflammation but ameliorates oxazolone-induced intestinal colitis in BALB/c mice

ABSTRACTCathelicidin has been reported to be multifunctional. The current study aimed to investigate the influences of exogenous cathelicidin-related antimicrobial peptide (CRAMP) on inflammatory responses in different disease models. In OVA-induced allergic airway inflammation, CRAMP significantly enhanced the infiltration of inflammatory cells and accumulation of proinflammatory Th2 cytokine IL-13 and IL-33 in bronchial alveolar lavage fluid (BALF), exacerbated lung tissue inflammation and airway goblet cell hyperplasia, and elevated OVA-specific IgE level in serum. In oxazolone-induced intestinal colitis, the expression levels of CRAMP and its receptor FPR2 significantly increased in comparison with those of TNBS-induced mice, vesicle and normal controls. Exogenous CRAMP significantly prevented the development of ulcerative colitis, evidenced by improved body weight regain, decreased colons weight/length ratio, elevated epithelial integrity, and ameliorated colon tissue inflammation. In addition, pro-inflammatory cytokines TNF-α, IL-1β, IL-4 and IL-13, as well as chemokines CXCL2 and CXCL5 for neutrophils recruitment were significantly decreased in CRAMP-treated mice, and epithelial repair-related factors MUC2 and Claudin1 were increased, determined by real time-PCR and ELISAs. The results indicated that although CRAMP has pro-inflammatory effects in airway, local application of exogenous CRAMP might be a potential approach for the treatment of ulcerative colitis.

Aging-Induced Modulation of Pituitary Adenylate Cyclase-Activating Peptide- and Vasoactive Intestinal Peptide-Induced Vasomotor Responses in the Arteries of Mice

Pituitary adenylate cyclase-activating peptide (PACAP; 1-38 and 1-27) and vasoactive intestinal peptide (VIP) are related neuropeptides of the secretin/glucagon family. Overlapping signaling through G-protein-coupled receptors mediates their vasomotor activity. We previously showed that PACAP deficiency (PACAP-KO) shifts the mechanisms of vascular response and maintains arterial relaxation through the VIP backup mechanism and (mainly) its VPAC1R, but their age-dependent modulation is still unknown. We hypothesized that backup mechanisms exist, which maintain the vasomotor activity of these peptides also in older age. Thus, we investigated the effects of exogenous VIP and PACAP peptides in isolated carotid arteries of 2- and 15-month-old wild-type (WT) and PACAP-KO mice. All peptides induced relaxation in the arteries of young WT mice, whereas in young PACAP-KO mice PACAP1-27 and VIP, but not PACAP1-38, induced relaxation. Unlike VIP, PACAP-induced vasomotor responses were reduced in aging WT mice. However, in the arteries of aging PACAP-KO mice, PACAP1-27- and VIP-induced responses were reduced, but PACAP1-38 showed a greater vasomotor response compared to that of young PACAP-KO animals. There were no significant differences between the vasomotor responses of aging WT and PACAP-KO mice. Our data suggest that, in the absence of PACAP both in young and old ages, the vascular response is mediated through backup mechanisms, most likely VIP, maintaining proper vascular relaxation in aging-induced PACAP insufficiency.

Apolipoprotein E as a novel therapeutic neuroprotection target after traumatic spinal cord injury

Apolipoprotein E (apoE), a plasma lipoprotein well known for its important role in lipid and cholesterol metabolism, has also been implicated in many neurological diseases. In this study, we examined the effect of apoE on the pathophysiology of traumatic spinal cord injury (SCI). ApoE-deficient mutant (apoE−/−) and wild-type mice received a T9 moderate contusion SCI and were evaluated using histological and behavioral analyses after injury. At 3days after injury, the permeability of spinal cord-blood-barrier, measured by extravasation of Evans blue dye, was significantly increased in apoE−/− mice compared to wild type. The inflammation and spared white matter was also significantly increased and decreased, respectively, in apoE−/− mice compared to the wild type ones. The apoptosis of both neurons and oligodendrocytes was also significantly increased in apoE−/− mice. At 42days after injury, the inflammation was still robust in the injured spinal cord in apoE−/− but not wild type mice. CD45+ leukocytes from peripheral blood persisted in the injured spinal cord of apoE−/− mice. The spared white matter was significantly decreased in apoE−/− mice compared to wild type ones. Locomotor function was significantly decreased in apoE−/− mice compared to wild type ones from week 1 to week 8 after contusion. Treatment of exogenous apoE mimetic peptides partially restored the permeability of spinal cord-blood-barrier in apoE−/− mice after SCI. Importantly, the exogenous apoE peptides decreased inflammation, increased spared white matter and promoted locomotor recovery in apoE−/− mice after SCI. Our results indicate that endogenous apoE plays important roles in maintaining the spinal cord-blood-barrier and decreasing inflammation and spinal cord tissue loss after SCI, suggesting its important neuroprotective function after SCI. Our results further suggest that exogenous apoE mimetic peptides could be a novel and promising neuroprotective reagent for SCI.

Новий погляд на ендометріоз: роль відновлення повноцінного імунного нагляду над ектопічними клітинами ендометрія

The article deals with the role of immunological disorders in the occurrence of genital endometriosis recurrences, as well as the results of treatment of this disease. The objective: the study of the effect of restoring immune homeostasis with the help of exogenous peptides on the effectiveness of the treatment of endometriosis. Patients and methods. Endometriosis was treated in 68 women of childbearing age. In 48 patients, uterine endometriosis (adenomyosis) was diagnosed, and 20 cases were endometriosis of the vaginal part of the cervix. In order to study the effectiveness of Camelyn-Bio in the treatment of endometriosis, patients were divided into two groups. Results. In the group of patients who were prescribed hormonal monotherapy, 3 months after the end of the course of hormonal treatment, recurrence of endometriosis was observed. They arose due to systemic and local immunological disorders, which probably led to ectopic endometrial cells from immune surveillance. Due to the properties of the exogenous peptides of the Camelyn-Bio preparation, reacting with the regulatory peptides of the ectopic cells of the endometrium, there are conditions for a complete immunological surveillance of the endometrium foci. In the group of patients treated with Camelyn-Bio, the incidence of relapse was 4 times lower than that of the control group. Conclusion. Treatment of endometriosis with the combined progestogen and estragon drug is effective and contributes to a sustained regression of the clinical symptoms of the disease. A break in the use of basic hormonal therapy leads to relapses of endometriosis, which is confirmed by the instrumental methods of research. Clinical experience confirms that the restoration of the immune homeostasis of patients with the Camelyn-Bio preparation is an important factor in increasing the effectiveness of endometriosis treatment, leads to clinical remission and improves the quality of life. Key words: endometriosis, ectopic cells of the endometrium, apoptosis, Camelyn-Bio, immune homeostasis, relapses, treatment.

Chlamydia trachomatis Prevents Apoptosis Via Activation of PDPK1-MYC and Enhanced Mitochondrial Binding of Hexokinase II

The intracellular human bacterial pathogen Chlamydia trachomatis pursues effective strategies to protect infected cells against death-inducing stimuli. Here, we show that Chlamydia trachomatis infection evokes 3-phosphoinositide-dependent protein kinase-1 (PDPK1) signaling to ensure the completion of its developmental cycle, further leading to the phosphorylation and stabilization of MYC. Using biochemical approaches and imaging we demonstrate that Chlamydia-induced PDPK1-MYC signaling induces host hexokinase II (HKII), which becomes enriched and translocated to the mitochondria. Strikingly, preventing the HKII interaction with mitochondria using exogenous peptides triggers apoptosis of infected cells as does inhibiting either PDPK1 or MYC, which also disrupts intracellular development of Chlamydia trachomatis. These findings identify a previously unknown pathway activated by Chlamydia infection, which exhibits pro-carcinogenic features. Targeting the PDPK1-MYC-HKII-axis may provide a strategy to overcome therapeutic resistance of infection.

Intercellular Genetic Interaction Between Irf6 and Twist1 during Craniofacial Development

Interferon Regulatory Factor 6 (IRF6) and TWIST1 are transcription factors necessary for craniofacial development. Human genetic studies showed that mutations in IRF6 lead to cleft lip and palate and mandibular abnormalities. In the mouse, we found that loss of Irf6 causes craniosynostosis and mandibular hypoplasia. Similarly, mutations in TWIST1 cause craniosynostosis, mandibular hypoplasia and cleft palate. Based on this phenotypic overlap, we asked if Irf6 and Twist1 interact genetically during craniofacial formation. While single heterozygous mice are normal, double heterozygous embryos (Irf6+/−; Twist1+/−) can have severe mandibular hypoplasia that leads to agnathia and cleft palate at birth. Analysis of spatiotemporal expression showed that Irf6 and Twist1 are found in different cell types. Consistent with the intercellular interaction, we found reduced expression of Endothelin1 (EDN1) in mandible and transcription factors that are critical for mandibular patterning including DLX5, DLX6 and HAND2, were also reduced in mesenchymal cells. Treatment of mandibular explants with exogenous EDN1 peptides partially rescued abnormalities in Meckel’s cartilage. In addition, partial rescue was observed when double heterozygous embryos also carried a null allele of p53. Considering that variants in IRF6 and TWIST1 contribute to human craniofacial defects, this gene-gene interaction may have implications on craniofacial disorders.

Erratum to: Exogenous Adipokine Peptide Resistin Protects Against Focal Cerebral Ischemia/Reperfusion Injury in Mice.

Previous studies have demonstrated that plasma resistin levels were increased in patients with acute ischemic stroke. However, the role of resistin after ischemic brain injury is still unclear. In this study, we investigated the protective effects of resistin on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that resistin (i.c.v.) significantly reduced infarct volume and improved neurological deficits after 45 min of ischemia and 24 h of reperfusion. Furthermore, our data demonstrate that intraperitoneal administration of resistin (10 µg/kg body weight) also had protective effects on infarct volume, indicating the crossing of resistin through the impaired BBB after ischemia injury. Resistin treatment reduced cleaved protein level of Poly(ADP-ribose)polymerase-1 (PARP-1), a marker of cellular apoptosis, showing the anti-apoptotic activity of resistin. Resistin increased the level of phosphorylated Akt after ischemic brain injury. The neuroprotective effect of resistin was partially reversed by a PI3K inhibitor wortmannin, demonstrating that the PI3K/Akt signal pathway is involved in the anti-apoptotic mechanisms of resistin. Finally, we found that resistin treatment improved neurological function recovery at 14 days after treatment, including balance ability and muscle strength. Given these findings, resistin may have therapeutic potential for the treatment of stroke.

Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection

The ability to analyze biological samples for multitudinous exogenous peptides with a single analytical method is desired for doping control in horse racing. The key to achieving this goal is the capability of extracting all target peptides from the sample matrix. In the present study, theory of mixed-mode solid-phase extraction (SPE) of peptides from plasma is described, and a generic mixed-mode SPE procedure has been developed for recovering multitudinous exogenous peptides with remarkable sequence diversity, from equine plasma and urine in a single procedure. Both the theory and the developed SPE procedure have led to the development of a novel analytical method for comprehensive detection of multitudinous bioactive peptides in equine plasma and urine using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Thirty nine bioactive peptides were extracted with strong anion-exchange mixed-mode SPE sorbent, separated on a reversed-phase C18 column and detected by HRMS and data-dependent tandem mass spectrometry. The limit of detection (LOD) was 10–50 pg mL−1 in plasma for most of the peptides and 100 pg mL−1 for the remaining. For urine, LOD was 20–400 pg mL−1 for most of the peptides and 1–4 ng mL−1 for the others. In vitro degradation of the peptides in equine plasma and urine was examined at ambient temperature; the peptides except those with a D-amino acid at position 2 were unstable not only in plasma but also in urine. The developed method was successful in analysis of plasma and urine samples from horses administered dermorphin. Additionally, dermorphin metabolites were identified in the absence of reference standards. The developed SPE procedure and LC-HRMS method can theoretically detect virtually all peptides present at a sufficient concentration in a sample. New peptides can be readily included in the method to be detected without method re-development. The developed method also generates such data that can be retrospectively analyzed for peptides unknown at the time of sample analysis. It is the first generic analytical method for comprehensive detection of multitudinous exogenous peptides in biological samples, to the authors' knowledge.

A NOVEL PEPTIDE PIVOTAL IN ALZHEIMER’S DISEASE: TIME-DEPENDENT TROPHIC-TOXIC AXIS IN PRIMARY RAT CORTICAL NEURONAL CULTURES

Acetylcholinesterase (AChE) is present in all neurons susceptible to degeneration in Alzheimer's disease regardless of their respective neurotransmitter. A peptide (T14) has been identified, cleaved from the C-terminus of AChE, which has been shown to be bioactive in both cell culture systems as well as ex vivo preparations. This peptide is toxic to PC12 cells and has a trophic-toxic axis in organotypic cultures. In the light of these findings, it has been suggested that Alzheimer's disease is the result of aberrant, or toxic, signalling by this peptide. Here we investigate the time and dose dependent effects of the peptide in rat neuronal cultures. Primary cortical neurons from 2 day old rats were grown in culture for 14 days. Increasing doses of exogenous AChE peptide applied for specified periods of time, ranging from 24 hours to 288 hours, to the neuronal cultures. The Cell Counting Kit-8 was used to quantify the number of viable cells following the treatment period. The AChE peptide dose response displayed a complex pattern of trophic-toxic effects. The effects of the AChE peptide are both dose and time dependent, suggesting a trophic-toxic pattern as reported previously in other preparations.

Antigenicity of phage clones and their synthetic peptides for the serodiagnosis of canine and human visceral leishmaniasis

In the Americas, Brazil is responsible by 90% of the cases registered of visceral leishmaniasis (VL), and Leishmania infantum is the most common parasite species responsible by disease in Brazilian dogs and humans. A precise diagnosis may allow to a faster and more effective treatment against the disease, which increases the possibility of cure, as well as to induce less toxic effects, due to a lower time exposition for the chemotherapeutics. In a previous study, two L. infantum mimotopes, B10 and C01 clones, were recognized by antibodies in VL dogs sera by a phage display technology, and were well-successfully evaluated as vaccine candidates against visceral and tegumentary leishmaniasis. In the present work, the diagnostic efficacy of these clones, as well as of their exogenous peptides (B10: LSFPFPG and C01: FTSFSPY), was evaluated to diagnose canine and human VL. ELISA assays were performed with the four antigens, and results showed that both clones, as well as their synthetic peptides; showed high sensitivity and specificity values to identify VL samples, presenting an excellent performance to serologically diagnose VL-developing humans and dogs. On the other hand, a wild-type phage, a random non-specific clone and a L. infantum antigenic preparation were used as controls, and showed worst sensitivity and specificity results. In conclusion, besides their biological action as vaccine, B10 and C01 phages and their synthetic peptides could be considered as new markers for the serodiagnosis of canine and human VL.

P1085Regulation of hERG cardiac potassium channels by exogenous peptides

P1085Regulation of hERG cardiac potassium channels by exogenous peptides

Feasibility, safety, and tolerance of subcutaneous synthetic canine B-type natriuretic peptide (syncBNP) in healthy dogs and dogs with stage B1 mitral valve disease

IntroductionAn important aspect of heart failure is the progressive ineffectiveness of the salutary natriuretic peptide system and its secondary messenger, 3′,5′-cyclic guanosine monophosphate (cGMP). In humans with acute heart failure, administration of exogenous natriuretic peptide is associated with improvement in clinical signs and reduction of cardiac filling pressures. This study aimed to determine the feasibility, tolerance, and safety of subcutaneous (SC) synthetic canine B-type natriuretic peptide (syncBNP) administration in dogs. AnimalsSix privately owned dogs. Materials and methodsDogs were enrolled in a modified 3 + 3 phase I trial. Three dogs initially received doses of 2.5 and 5 μg/kg SC syncBNP followed by an additional three dogs dosed at 5 and 10 μg/kg. Hemodynamic monitoring was performed for 120 min after each injection. Blood and urine samples were collected at 45 and 120 min after injection of 5 μg/kg. Major adverse clinical events that would potentially halt testing were pre-defined. ResultsFour healthy dogs and two dogs with stage B1 mitral valve disease were recruited. Synthetic canine B-type natriuretic peptide was well tolerated at all doses. Synthetic canine B-type natriuretic peptide at 5 μg/kg significantly increased median plasma cGMP (baseline cGMP, 131.5 pmol/mL [range, 91.9–183.6 pmol/mL]; 45 min, 153.6 pmol/mL [140.3–214.3 pmol/mL]; 120 min, 192.7 pmol/mL [139.1–240.1 pmol/mL]; p=0.041). Discussion and conclusionsWe report for the first time administration of syncBNP in privately owned dogs. Administration of SC syncBNP was feasible, well tolerated, safe, and increased plasma cGMP concentration. Further studies using exogenous syncBNP for treatment of heart disease are warranted.