Inactive Control Peptide Research Articles

A systematic investigation of the cardiac intercalated disk in health and disease.

During each heartbeat, action potential (AP) propagation at intercalated discs (ID), specialized structures that electrically (gap junctions [GJ]) and mechanically (adherens junctions [AJ], desmosomes [Des]) couple cardiomyocytes, coordinates the contraction of billions of cardiomyocytes. Electrogenic proteins underlying excitability (cardiac sodium channels [NaV1.5], inward-rectifier potassium channels [Kir2.1] and sodium-potassium ATPase [NKA]) enriched within ID nanodomains are emerging as vital machinery for AP propagation. ID disruption is a hallmark of arrhythmic disease, yet, IDs are omitted from physiological models due to lack of structural data. We used indirect correlative light and electron microscopy [iCLEM] to quantify ID ultrastructure and molecular organization in health and disease and link them with functional properties (conduction velocity, arrhythmia vulnerability). Additional studies in hearts treated with adhesion inhibitor peptides targeting different ID nanodomains helped bridge health with complex disease. In healthy mouse hearts, transmission electron microscopy uncovered smaller, more frequent junctions and closer intermembrane distance at peri-junctional nanodomains in atria vs. ventricles. Confocal microscopy revealed greater NaV1.5, Kir2.1, NKA expression with more preferential ID enrichment in atrial myocytes than ventricular. STORM single molecule localization microscopy, a super-resolution technique, identified chamber-specific differences in NaV1.5, Kir2.1 and NKA clustering and distribution relative to AJ, Des, GJ as well as each other. Adhesion inhibitor peptides Nadp1 (target: AJ), dadp1 (target: Des) and βadp1 (target: GJ-adjacent perinexi), but not corresponding inactive control peptides, acutely (60 minutes) disrupted their respective target nanodomains and dispersed NaV1.5 clusters from those sites. These structural impacts led to conduction slowing and reentrant arrhythmias with severity proportional to the amount of NaV1.5 present at the target site. These data parallel our previous observations in atrial fibrillation (mouse models and human patients) and highlight NaV1.5-rich ID nanodomains as potential targets for antiarrhythmic therapy.

Corneal Application of R9-SOCS1-KIR Peptide Alleviates Endotoxin-Induced Uveitis.

PurposeUveitis is an ocular inflammation that can affect individuals of all ages and is a major cause of blindness. We have tested the therapeutic efficacy of a cell penetrating peptide from the kinase inhibitory region of suppressor of cytokine signaling 1, denoted as R9-SOCS1-KIR.MethodsWe stimulated J774A.1 cells with lipopolysaccharide (LPS) in the presence of R9-SOCS1-KIR or its inactive control peptide. Effect on inflammatory pathways was followed by the nuclear translocation of nuclear factor κB p65 subunit and phosphorylated-p38. Synthesis of inflammatory markers induced by LPS was tested by reverse transcriptase polymerase chain reaction, Western blot analysis, and ELISA of cell supernatants. We monitored effects on the barrier properties of a differentiated ARPE-19 monolayer treated with LPS. We treated C57BL/6 mice topically with either R9-SOCS1-KIR or vehicle and injected their eyes intravitreally with LPS. Eyes were analyzed by fundoscopy, fluorescein angiography, optical coherence tomography, histology, Western blotting, multiplex enzyme-linked immunosorbent assay, and flow cytometry.ResultsTreatment with R9-SOCS1-KIR resulted in suppression of signaling through nuclear factor κB and p-p38 pathways. R9-SOCS1-KIR suppressed the expression of inflammatory genes, the secretion of inflammatory makers such as nitric oxide, and IL-1β induced by LPS. Increased permeability of retinal pigment epithelial cell monolayers was prevented. Corneal administration of R9-SOCS1-KIR blocked the acute inflammation observed in LPS-injected mouse eyes.ConclusionsTreatment with R9-SOCS1-KIR alleviated the inflammatory responses in cell culture. Topical delivery of this peptide on mouse eyes protected against LPS-induced damage.Translational RelevanceTopical delivery of R9-SOCS1-KIR peptide allows the patient to self-administer the drug, while preventing any systemic effects on unrelated organs.

The Nanoscale Basis of Atrial Fibrillation: Functional Impact of Disrupting NaV1.5-rich Intercalated Disk Nanodomains.

Background:Atrial fibrillation (AF), which is characterized by chaotic patterns of electrical activation of the atria, affects over 4 million people in the US alone. We previously identified nanoscale structural abnormalities in the hearts of AF patients. Specifically, they displayed swelling of gap junction (GJ) –adjacent perinexi, specialized nanodomains rich in cardiac sodium channels (NaV1.5) and located within intercalated disks (IDs; sites of electromechanical contact between adjacent cells). However, the functional consequences of these nanoscale structural changes remain unclear.Objective:We assessed the structural and functional impacts of selectively disrupting different NaV1.5-rich ID nanodomains.Methods and Results:We utilized peptide mimetics of adhesion domains to selectively inhibit adhesion within different ID nanodomains: 1) Nadp1 (target: N-cadherin), 2) dadp1 (target: Desmoglein-2), and 3) βadp1 (target: sodium channel β1 subunit [SCN1b]). Each active peptide was compared against a corresponding inactive control peptide (Nadp1-c, dadp1-c, βadp1-scr). Sub-diffraction confocal imaging revealed ID enrichment of active peptides, but not inactive controls. Furthermore, each active peptide was preferentially localized in ID regions rich in its corresponding protein target. Peptide treatment (100 μM; 60 minutes) of ex vivo mouse hearts revealed profound widening of perinexi by βadp1 and of mechanical junctions by Nadp1. Dadp1 also induced widening of mechanical junctions albeit to a lower degree. STORM single molecule localization microscopy identified about 50&per; of ID-localized NaV1.5 within GJ-adjacent perinexi, while an additional ∼35&per; was located within N-cad-rich ID sites. Nadp1 and βadp1 induced redistribution of ID localized NaV1.5 away from perinexi and mechanical junctions respectively. Dadp1, again, had similar but milder effects compared to Nadp1. Western blot revealed the expression levels of NaV1.5, connexin 43 (Cx43), connexin 40 (Cx40), β1 in peptide treated hearts to be within 10&per; of levels in untreated controls. Optical mapping revealed atrial conduction slowing in hearts treated with Nadp1 (17cm/s, 70.83&per; of control) and βadp1 (13 cm/s, 54.17&per; of control), but not inactive control peptides (24 cm/s). Volume-conducted electrocardiograms (ECG) revealed P wave prolongation in active peptide treated hearts (Nadp1: 26.5ms, βadp1: 31ms), consistent with conduction slowing compared to the inactive control peptides (16ms). Importantly, burst pacing elicited atrial arrhythmias in all hearts treated with Nadp1 and βadp1. Arrhythmia burden (duration, number of arrhythmias) was highest with βadp1.Conclusions:These results suggest that disruption of NaV1.5-rich ID nanodomains impairs electrical impulse propagation and promotes arrhythmias in the atria. Furthermore, the magnitude of functional impacts are likely determined by the amount of sodium channels contained within the nanodomains disrupted.

α5β1 integrins in hepatocytes act as receptors for bile acids with a (nor)ursodeoxycholane scaffold

Ursodeoxycholic acid (UDCA) is a standard treatment in several cholestatic liver diseases (Figure 1A) [1]. In vivo, conjugation with taurine occurs rapidly and yields tauroursodeoxycholic acid (TUDC), which has been shown to promote choleresis by triggering the insertion of ATP-dependent transport proteins (e.g., the bile salt export pump (Bsep) and the multidrug resistance protein-2 (Mrp2)) into the canalicular membrane [2]. TUDC-induced recruitment of Bsep results from activation of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3 kinase), and c-Src, which leads to downstream activation of extracellular signal-regulated kinases (Erks) and p38 mitogen-activated protein kinase (p38). Upon hepatocyte swelling, either induced by exposure to a hypoosmotic environment or insulin, a5b1 integrins become activated and trigger similar signaling events towards choleresis. a5b1 Integrins may also become activated by a swelling-independent way as previously shown by exposing hepatocytes to pathophysiological concentrations of urea [3]. Both TUDC-induced and swelling-dependent signaling were abolished in the presence of an antagonistic, RGD-motif containing hexapeptide (GRGDSP). These findings led us to hypothesize that a5b1 integrin will act as a receptor for TUDC in hepatocytes. We tested this hypothesis in a combined experimental and computational study [4]. Immunofluorescence staining on cryosections of isolated perfused rat liver (IPRL) revealed the active conformation of b1 integrin within 1 min after addition of TUDC at a concentration of 20 μM. Furthermore, phosphorylation of Erk-1 and -2 as well as activation of the epidermal growth factor receptor were induced by TUDC within the same time span. These effects were sensitive to inhibition by GRGDSP but insensitive to the presence of an inactive control peptide (GRADSP). As TUDC does not affect hepatocyte volume, which excludes that TUDC triggers integrin activation osmotically, these findings demonstrated that TUDC directly activates a5b1 integrins and triggers signaling events towards choleresis. While swelling-induced b1 integrin activation occurs primarily in the plasma membrane, TUDC-induced b1 integrin activation occurs primarily in the cytosol of hepatocytes. We demonstrated that the presence of the Na/taurocholate cotransporting polypeptide (Ntcp) is required for the latter. The need to uptake and/or concentrate TUDC inside the hepatocyte for b1 integrin activation to occur may explain why TUDC primarily acts in the liver. In order to provide insights at a molecular level as to how TUDC activates a5b1-integrin, a complex structure of a homology model of the ectodomain of a5b1-integrin and TUDC was generated by molecular docking and subsequently subjected to molecular dynamics (MD) simulations of 200 ns length [4]. These simulations revealed pronounced conformational changes in three regions of the bA domain of the integrin (Figure 1B): I) Helix a1 straightens and becomes continuous; II) this leads to a tighter packing between the top of helix a7 and the center of a1, which has been characterized as “T-junction formation” in an X-ray structure of integrin aIIbb3 bound to a ligand as well as in computational studies of agonistbound integrins; III) as a result, helix a7 moves downwards and outwards, which imposes a torque on the hybrid domain. The induced rotational motion of the hybrid domain is a prerequisite for the unbending of the integrin ectodomain, which, in turn, is required for integrin activation according to current models. Neither did MD simulations of the ectodomain of a5b1 integrin bound to GRGDSP nor to taurocholic acid (TC) (Figure 1A) reveal such conformational changes, in line with results * Correspondence: gohlke@uni-duesseldorf.de Department of Mathematics and Natural Sciences, Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, 40225 Dusseldorf, Germany Full list of author information is available at the end of the article Bonus et al. European Journal of Medical Research 2014, 19(Suppl 1):S13 http://www.eurjmedres.com/content/19/S1/S13 EUROPEAN JOURNAL OF MEDICAL RESEARCH

Proteinase-activated receptor-1 (PAR1) and PAR2 mediate relaxation of guinea pig internal anal sphincter

Activation of proteinase-activated receptor-1 (PAR1) and PAR2 stimulates contraction of the rat but relaxation of the guinea pig colon. The aim of the present study was to investigate PAR effects on internal anal sphincter (IAS) motility. We measured relaxation of isolated muscle strips from the guinea pig IAS caused by PAR agonists using isometric transducers. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the existence of PAR. In the IAS, thrombin and PAR1 peptide agonists TFLLR-NH2 and SFLLRN-NH2 evoked moderate to marked relaxation in a concentration-dependent manner. In addition, trypsin and PAR2 peptide agonists 2-furoyl-LIGRLO-NH2, SLIGRL-NH2 and SLIGKV-NH2 produced relaxation. In contrast, both PAR1 and PAR2 inactive control peptides did not elicit relaxation. Furthermore, the selective PAR1 antagonist vorapaxar and PAR2 antagonist GB 83 specifically inhibited thrombin and trypsin-induced relaxations, respectively. RT-PCR revealed the presence of PAR1 and PAR2 in the IAS. This indicates that PAR1 and PAR2 mediate the IAS relaxation. The relaxant responses of TFLLR-NH2 and trypsin were attenuated by N(omega)-Nitro-L-arginine (L-NNA), indicating involvement of NO. These responses were not affected by tetrodotoxin, implying that the PAR effects are not neurally mediated. On the other hand, PAR4 agonists GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2 did not cause relaxation or contraction, suggesting that PAR4 is not involved in the sphincter motility. Taken together, these results demonstrate that both PAR1 and PAR2 mediate relaxation of the guinea pig IAS through the NO pathway. PAR1 and PAR2 may regulate IAS tone and might be potential therapeutic targets for anal motility disorders.

Link Protein N-terminal Peptide Binds to Bone Morphogenetic Protein (BMP) Type II Receptor and Drives Matrix Protein Expression in Rabbit Intervertebral Disc Cells

Intervertebral disc (IVD) degeneration and associated spinal disorders are leading sources of morbidity, and they can be responsible for chronic low back pain. Treatments for degenerative disc diseases continue to be a challenge. Intensive research is now focusing on promoting regeneration of degenerated discs by stimulating production of the disc matrix. Link protein N-terminal peptide (LPP) is a proteolytic fragment of link protein, an important cross-linker and stabilizer of the major structural components of cartilage, aggrecan and hyaluronan. In this study we investigated LPP action in rabbit primary intervertebral disc cells cultured ex vivo in a three-dimensional alginate matrix. Our data reveal that LPP promotes disc matrix production, which was evidenced by increased expression of the chondrocyte-specific transcription factor SOX9 and the extracellular matrix macromolecules aggrecan and collagen II. Using colocalization and pulldown studies we further document a noggin-insensitive direct peptide-protein association between LPP and BMP-RII. This association mediated Smad signaling that converges on BMP genes leading to expression of BMP-4 and BMP-7. Furthermore, through a cell-autonomous loop BMP-4 and BMP-7 intensified Smad1/5 signaling though a feedforward circuit involving BMP-RI, ultimately promoting expression of SOX9 and downstream aggrecan and collagen II genes. Our data define a complex regulatory signaling cascade initiated by LPP and suggest that LPP may be a useful therapeutic substitute for direct BMP administration to treat IVD degeneration and to ameliorate IVD-associated chronic low back pain.

P38MAP kinase, but not phosphoinositol-3 kinase, signal downstream of glutamine-mediated fibronectin-integrin signaling after intestinal injury

BackgroundGlutamine appears to mediate protection against gut injury via multiple pathways. These include fibronectin-integrin, PI3-K/MAPK pathways, and activation of heat shock protein (HSP) response. We hypothesize there may be a relationship between these pathways mediating glutamine’s protection in intestinal epithelial-6 cells after heat stress. We assessed whether p38MAPK and PI3-K/Akt signaling are involved in glutamine’s cytoprotective mechanism and the role they play in glutamine-mediated protection in conjunction with fibronectin-integrin osmosignaling after hyperthermia.MethodsIntestinal epithelial cells were treated for 15 min with glutamine, with/without the fibronectin-integrin interaction inhibitor GRGDSP, inactive control peptide GRGESP, p38MAPK inhibitor SB203580, or PI3-K/Akt inhibitor LY294002 under basal (37°C) and stressed (43°C or 44°C) conditions. Cell survival was measured via MTS assay 24 h post-heat stress (44°C × 50 min). Total p38MAPK, [T(P)180/Y(P)182]p38MAPK, total Akt, [S(P)473]Akt, HSP70, FN, and caspase-3 levels were determined via Western blot after non-lethal HS (43°C × 50 min). Additionally, HSP70 levels were assessed via Western blot and ELISA.ResultsWe were able to show that GRGDSP and LY294002 attenuated glutamine’s protective effect. However, SB203580 increased cell survival after heat stress. LY294002 attenuated glutamine-mediated increases in fibronectin and in HSP70 expression after hyperthermia. GRGDSP increased glutamine-mediated attenuations in p38MAPK phosphorylation, but had no effect on glutamine-mediated augmentations in Akt phosphorylation.ConclusionsThese data suggest that glutamine is protective after heat stress by activating PI3-K/Akt signaling preventing fibronectin-integrin expression and increasing HSP70 expression. Furthermore, dephosphorylation of p38MAPK after heat stress plays an important role in glutamine-mediated cellular protection. However, p38MAPK, but not PI3-K/Akt, signals downstream of glutamine-mediated fibronectin-integrin signaling after hyperthermia.

Effects of trypsin, thrombin and proteinase-activated receptors on guinea pig common bile duct motility

Trypsin and thrombin activate proteinase-activated receptors (PARs), which modulate gastrointestinal motility. The common bile duct is exposed to many proteinases that can activate PARs, especially during infection and stone obstruction. We investigated PAR effects on common bile duct motility in vitro. Contraction and relaxation of isolated guinea pig common bile duct strips caused by PAR1, PAR2 and PAR4 agonists were measured using isometric transducers. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the expression of PAR1 and PAR2. Thrombin and two PAR1 peptide agonists, TFLLR-NH2 and SFLLRN-NH2, evoked moderate relaxation of the carbachol-contracted common bile duct in a concentration-dependent manner. Trypsin and three PAR2 peptide agonists, 2-furoyl-LIGRLO-NH2, SLIGKV-NH2 and SLIGRL-NH2, generated moderate to marked relaxation as well. The existence of PAR1 and PAR2 mRNA in the common bile duct was identified by RT-PCR. Moreover, two PAR4-selective agonists, AYPGKF-NH2 and GYPGQV-NH2, produced relaxation of the common bile duct. In contrast, all PAR1, PAR2 and PAR4 inactive control peptides did not elicit relaxation. This indicates that PAR1, PAR2 and PAR4 mediate common bile duct relaxation. The thrombin, TFLLR-NH2, trypsin, and AYPGKF-NH2-induced responses were not affected by tetrodotoxin, implying that the PAR effects are not neurally mediated. Our findings provide the first evidence that PAR1 and PAR2 mediate whereas agonists of PAR4 elicit relaxation of the guinea pig common bile duct. Trypsin and thrombin relax the common bile duct. PARs may play an important role in the control of common bile duct motility.

A mouse model for interstitial cystitis/painful bladder syndrome based on APF inhibition of bladder epithelial repair: a pilot study

BackgroundInterstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic bladder disorder with bladder epithelial thinning or ulceration, pain, urinary frequency and urgency. There is no reliably effective therapy for IC/PBS, and no generally accepted animal model for the disorder in which potential therapies can be tested. Bladder epithelial cells from IC/PBS patients make a small glycopeptide antiproliferative factor or "APF" that inhibits proliferation, decreases tight junction protein expression, increases paracellular permeability, and induces changes in gene expression of bladder epithelial cells in vitro that mimic abnormalities in IC/PBS patient biopsy specimens in vivo. We therefore determined the ability of a synthetic APF derivative to inhibit bladder epithelial repair in mice.MethodsThe bladder epithelium of female CBA/J mice was stripped by transurethral infusion of 3% acetic acid, and mice were subsequently treated daily with one of three intravesical treatments [synthetic as-APF, inactive unglycosylated control peptide, or phosphate buffered saline carrier (PBS)] for 1–21 days. Fixed bladder sections were either stained with haematoxylin and eosin for determination of epithelial area by image analysis, or incubated with anti-uroplakin III (UPIII) or anti-zonula occludens type 1 (ZO-1) antibodies for immunofluorescence microscopy. Epithelial measurement data were analyzed by a two-way analysis of variance (ANOVA); post hoc comparisons of multiple groups were carried out using the Tukey-Kramer method.ResultsBladder epithelial repair was significantly attenuated in as-APF-treated mice as compared to control mice on days 3–21 (p < 0.05); the mean epithelial/total area over all measured days was also significantly lower in as-APF-treated mice vs. mice in either control group by post hoc analysis (p < 0.0001 for both comparisons). UPIII and ZO-1 expression was also decreased in as-APF-treated mice as compared to mice in either control group by day 7 (UPIII) or day 14 (ZO-1).ConclusionsThis model demonstrates in vivo effects of as-APF which abrogates bladder epithelial repair and expression of UPIII and ZO-1 in CBA/J mice following transurethral acetic acid infusion. As bladder epithelial thinning, decreased UPIII expression, and decreased ZO-1 expression are histopathologic features of IC/PBS patient biopsies, this model may be useful for studying the pathophysiology of IC/PBS and the effect of potential therapies.

Neuromedin S Increases L-type Ca2+ Channel Currents Through Giα-protein and Phospholipase C-dependent Novel Protein Kinase C Delta Pathway in Adult Rat Ventricular Myocytes

Neuromedin S (NMS), a peptide structurally related to NMU, has been identified in the mammalian heart tissues. However to date, any role of NMS in cardiomyocytes and the relevant mechanisms still remain unknown. In this study, we identified a novel functional role of NMS in modulating L-type Ca²⁺ channels in adult rat ventricular myocytes, in which NMU type 2 receptors (NMUR2), but not NMUR1, are endogenously expressed. We found that NMS at 0.1 µM reversibly increased I_Baby ∼29.7%. Intracellular infusion of GDP-β-S or a selective antibody raised against the G_i-protein blocked the stimulatory effects of NMS. The classical and novel protein kinase C (nPKC) antagonist calphostin C or chelerythrine chloride, as well as the phospholipase C (PLC) inhibitor U73122, abolished NMS responses, whereas a classical PKC antagonist Gö6976 or a PKA antagonist PKI 5-24 had no such effects. Pretreatment of cells with PKC-δ specific inhibitor rottlerin or intracellular application of a PKC-δ-derived inhibitory peptide, δV1-1, abolished NMS responses, while an inactive control peptide had no effects. In summary, NMS acting through NMUR2 increases I_Bavia a G_iα-protein-dependent PKC-δ pathway in rat ventricular myocytes.

Identification of Platelet Releasate Proteins that Bind to Mastoparan, a Peptide that Inhibits Shear-Induced TGF-β1 Activation,

Abstract 3271Transforming growth factor β1 (TGF-β1) is a disulfide-bonded, 25 kD homodimeric protein produced by most cell types, including platelets, that functions as a cytokine in many physiologic and pathologic processes. Platelets contain 40–100 times more TGF-β1 than other cells and release it as an inactive large latent complex (LLC) comprised of TGF-β1 non-covalently associated with its latency-associated peptide (LAP), which is, in turn, disulfide-bonded to latent TGF-β binding protein 1 (LTBP-1). Thrombospondin-1 (TSP1), proteases, and reactive oxygen species have all been shown to activate TGF-β1 in vitro and a role for integrins in vivo has been inferred from studies of transgenic mice. Recently, we discovered that shear force can activate latent TGF-β1 released from platelets in vitro and that thiol-disulfide exchange contributes to shear-dependent TGF-β1 activation. A number of thiol isomerase enzymes that can catalyze thiol-disulfide exchange have been identified in platelets, including protein disulfide isomerase (PDI), ERp5, ERp57, ERp72, ERp44, ERp29, and TMX3. As shear-induced activation of TGF-β1 is partially thiol-dependent, we investigated if thiol isomerases can affect this process. Mastoparan is a non-thiol-containing wasp venom peptide known to inhibit the chaperone activity of PDI, ERp5, and perhaps other thiol isomerases. We recently showed that mastoparan, (INLKALAALAKKIL), inhibits stirring-induced TGF-β1 activation by more than 90% (100 μM; n=3, p=0.03), whereas no inhibition was observed with an inactive mastoparan-like control peptide (INLKAKAALAKKLL) at 100 μM (n=3, p=0.66). To identify the proteins that bind to mastoparan, either directly or indirectly, platelet releasates were chromatographed on a mastoparan affinity column prepared from N-hydroxysuccinimide Sepharose. Two control columns were employed: 1. unconjugated Sepharose, and 2. Sepharose conjugated with the mastoparan-like control peptide. Elution of bound proteins was achieved by increasing the NaCl concentration. Proteins identified by mass spectrometry as specifically binding to the mastoparan peptide column included LTBP-1, TGF-β1 precursor, clusterin, coagulation Factor V, multimerin-1, 14-3-3 protein zeta/delta, and α-actinin 4. These results were confirmed by immunoblotting. Furthermore, the thiol isomerases PDI, ERp5, ERp57, and ERp72 were all found to bind specifically to mastoparan as confirmed by immunoblotting. We conclude that mastoparan affinity chromatography identified a number of proteins in platelet releasates that may contribute to shear-induced TGF-β1 activation. Disclosures:Coller:Centocor/Accumetrics/Rockefeller University:.

Glycomimetic Improves Recovery after Femoral Injury in a Non-Human Primate

In adult mammals, restoration of function after peripheral nerve injury is often poor and effective therapies are not available. Previously we have shown in mice that a peptide which functionally mimics the human natural killer cell (HNK)-1 trisaccharide epitope significantly improves the outcome of femoral nerve injury. Here we evaluated the translational potential of this treatment using primates. We applied a linear HNK-1 mimetic or a functionally inactive control peptide in silicone cuffs used to reconstruct the cut femoral nerves of adult cynomolgus monkeys (Macaca fascicularis). Functional recovery was evaluated using video-based gait analysis over a 160-day observation period. The final outcome was further assessed using force measurements, H-reflex recordings, nerve histology, and ELISA to assess immunoreactivity to HNK-1 in the treated monkeys. Gait deficits were significantly reduced in HNK-1 mimetic-treated compared with control peptide-treated animals between 60 and 160 days after injury. Better outcome at 160 days after surgery in treated versus control animals was also confirmed by improved quadriceps muscle force, enhanced H-reflex amplitude, decreased H-reflex latency, and larger diameters of regenerated axons. No adverse reactions to the mimetic, in particular immune responses resulting in antibodies against the HNK-1 mimetic or immune cell infiltration into the damaged nerve, were observed. These results indicate the potential of the HNK-1 mimetic as an efficient, feasible, and safe adjunct treatment for nerve injuries requiring surgical repair in clinical settings.

The mechanism of L‐Glutamine‐mediated protection requires the activation of cell surface receptors

IntroductionL‐Glutamine (GLN), integrin/Src and epidermal growth factor receptor (EGFR) pathway can induce cellular protection.HypothesisGLN activates the O‐glycosylation pathway, HSF‐1 and HSP expression via cell swelling by the activation of integrin/Src and EGFR signaling to prevent apoptosis.MethodsIEC‐6 cells treated with GLN (up to 20 mM), with or without the integrin inhibitor GRGDSP (50 μM), inactive control peptide GRGESP (50 μM), Src‐kinase inhibitor PP‐2 (75 μM), or EGFR inhibitor AG1478 (10 μM). Cell survival via MTS assay 24h post‐HS (44ºCx50min). O‐glycosylated proteins, HSF‐1, HSPs, Bax, cleaved caspase‐3 and PARP levels determined via Western blot, cell area size via fluorescence microscopy after non‐lethal HS (43ºCx45min).ResultsGRGDSP significantly attenuated GLN‐mediated increases in cell area size, HSF‐1 and O‐GlcNAc levels after HS (p&lt; 0.05; n&gt;3). GLN‐mediated increases in HSPs were decreased by GRGDSP and PP2 by 70%–100% after HS (p&lt; 0.05; n=4). GRGDSP, PP2, and AG1478 attenuated GLN's protection by 70%–100% (p&lt; 0.05; n=4). GRGDSP increased GLN‐mediated decreases in cleaved caspase‐3, cleaved PARP and Bax levels after HS, indicating that GLN lost its protection (p&lt;0.05; n=3). GRGESP control peptide had no effect on GLN‐mediated protection pathways.ConclusionIntegrin/Src and EGFR signaling seem to be vital components of GLN's protective mechanism. R01 GM078312

Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells

BackgroundUrinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF) is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4). Because synthetic asialo-APF (as-APF) has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells.MethodsT24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled) double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3β (GSK3β), β-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot.ResultsT24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3β, or β-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3β/β-catenin phosphorylation in response to APF treatment were all specifically abrogated following CKAP4 siRNA knockdown.ConclusionsSynthetic as-APF inhibits cell proliferation in T24 bladder carcinoma cells via the CKAP4 receptor. The mechanism for this inhibition involves regulating phosphorylation of specific cell signaling molecules (Akt, GSK3β, and β-catenin) plus mRNA and protein expression of p53 and MMP2.

Conformational Analysis of a Synthetic Antimicrobial Peptide in Water and Membrane-Mimicking Solvents: A Molecular Dynamics Simulation Study

We have investigated structural and dynamic properties of the synthetic peptide hlF1-11 (GRRRSVQWCA, i.e., the first 11 N-terminal amino acids of the human lactoferrin protein) in water, 250 mM NaCl solution, 50% (V/V) water–trifluoroethanol mixture, and in the membrane mimetic 4:4:1 methanol–chloroform–water mixture. For comparison, we have also performed analogous simulations for the biologically inactive control peptide featuring Ala substitutions in the 2, 3, 6 and 9 positions of the hlF1-11 sequence. Statistical analyses of the trajectories indicate that only in the membrane-mimicking medium hlF1-11 adopts preferentially a conformation suitable to interact effectively with the membrane. In this conformation the peptide cationic region is rather flexible and elongated, while the C-terminal hydrophobic moiety appears as a more rigid hairpin-shaped loop approximately perpendicular to the cationic region. No such conformation is statistically relevant for the control peptide.

Proteinase-Activated Receptor-4 (PAR4) Activation Leads to Sensitization of Rat Joint Primary Afferents Via a Bradykinin B2 Receptor-Dependent Mechanism

The G-protein-linked receptor, proteinase-activated receptor-4 (PAR(4)) is activated by proteinases released into the joint during inflammation. It is unclear whether PAR(4) has a pro- or anti-nociceptive effect and whether it directly affects nerve activity. In this study, we examined the expression of PAR(4) in joints and dorsal root ganglion (DRG) neurons and whether activation of PAR(4) has an effect on nociception in normal rat knee joints. Electrophysiological recordings were made from joint primary afferents in male Wistar rats during both nonnoxious and noxious rotations of the knee. Afferent firing rate was recorded for 15 min post close intra-arterial injection of 10(-9)-10(-5) mol of the PAR(4) activating peptide, AYPGKF-NH(2), or the inactive peptide, YAPGKF-NH(2) (100 mul bolus). Rats were either naive or pretreated with the selective PAR(4) antagonist, pepducin P4pal-10, the transient receptor potential vanilloid-1 (TRPV1) antagonist, SB366791, or the bradykinin B(2) receptor antagonist, HOE140. Immunofluorescence experiments showed extensive PAR(4) expression in the knee joint and in sensory neurons projecting from the joint. AYPGKF-NH(2) significantly increased joint afferent firing during nonnoxious and noxious rotation of the knee. The inactive control peptide, YAPGKF-NH(2) was without effect. Systemic pretreatment with the PAR(4) antagonist, pepducin P4pal-10, inhibited the AYPGKF-NH(2)-induced increase in firing rate. Pretreatment with HOE140, but not SB366791, also blocked this increase in firing rate. These data reveal that in normal rat knee joints, PAR(4) activation increases joint primary afferent activity in response to mechanical stimuli. This PAR(4)-induced sensitization is TRPV1-independent but involves B(2) receptor activation, suggesting a role for kinins in this process.

In Vivo Characterization of High Basal Signaling from the Ghrelin Receptor

The receptor for the orexigenic peptide, ghrelin, is one of the most constitutively active 7TM receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active receptor is associated with change in signaling independent of the endogenous ligand. In the following study, we found that the expression of the ghrelin receptor in the hypothalamus was up-regulated approximately 2-fold in rats both during 48-h fasting and by streptozotocin-induced hyperphagia. In a separate experiment, to probe for the effect of the high basal signaling of the ghrelin receptor in vivo, we used intracerebroventricular administration by osmotic pumps of a peptide [D-Arg(1), D-Phe(5), D-Trp(7,9), Leu(11)]-substance P. This peptide selectively displays inverse agonism at the ghrelin receptor as compared with an inactive control peptide with just a single amino acid substitution. Food intake and body weight were significantly decreased in the group of rats treated with the inverse agonist, as compared with the groups treated with the control peptide or the vehicle. In the hypothalamus, the expression of neuropeptide Y and uncoupling protein 2 was decreased by the inverse agonist. In a hypothalamic cell line that endogenously expresses the ghrelin receptor, we observed high basal activity of the cAMP response element binding protein, an important signaling transduction pathway for appetite regulation. The activation was further increased by ghrelin administration and decreased by administration of the inverse agonist. It is suggested that the high constitutive signaling activity is important for the in vivo function of the ghrelin receptor in the control of food intake and body weight.

PICK1-mediated Glutamate Receptor Subunit 2 (GluR2) Trafficking Contributes to Cell Death in Oxygen/Glucose-deprived Hippocampal Neurons

Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca2+/Zn2+-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.

Triggering of proteinase‐activated receptor 4 leads to joint pain and inflammation in mice

To investigate the role of proteinase-activated receptor 4 (PAR-4) in mediating joint inflammation and pain in mice. Knee joint blood flow, edema, and pain sensitivity (as induced by thermal and mechanical stimuli) were assessed in C57BL/6 mice following intraarticular injection of either the selective PAR-4 agonist AYPGKF-NH(2) or the inactive control peptide YAPGKF-NH(2). The mechanism of action of AYPGKF-NH(2) was examined by pretreatment of each mouse with either the PAR-4 antagonist pepducin P4pal-10 or the bradykinin antagonist HOE 140. Finally, the role of PAR-4 in mediating joint inflammation was tested by pretreating mice with acutely inflamed knees with pepducin P4pal-10. PAR-4 activation caused a long-lasting increase in joint blood flow and edema formation, which was not seen following injection of the control peptide. The PAR-4-activating peptide was also found to be pronociceptive in the joint, where it enhanced sensitivity to a noxious thermal stimulus and caused mechanical allodynia and hyperalgesia. The proinflammatory and pronociceptive effects of AYPGKF-NH(2) could be inhibited by pepducin P4pal-10 and HOE 140. Finally, pepducin P4pal-10 ameliorated the clinical and physiologic signs of acute joint inflammation. This study demonstrates that local activation of PAR-4 leads to proinflammatory changes in the knee joint that are dependent on the kallikrein-kinin system. We also show for the first time that PARs are involved in the modulation of joint pain, with PAR-4 being pronociceptive in this tissue. Thus, blockade of articular PAR-4 may be a useful means of controlling joint inflammation and pain.

The divergent roles of protein kinase C epsilon and delta in simulated ischaemia–reperfusion injury in human myocardium

Experimental studies suggest that cardioprotection can be achieved through either the activation of PKC-ɛ prior to the index ischaemic episode or the inhibition of PKC-δ at the onset of reperfusion. However, whether these PKC isoforms exert such divergent roles in human myocardium, subjected to simulated ischaemia–reperfusion injury, is unclear. Human atrial trabeculae were isolated from right atrial appendages harvested from patients undergoing elective cardiac surgery. These were subjected to 90 min of hypoxia followed by 120 min of reoxygenation, at the end of which the recovery of baseline contractile function was determined. Atrial trabeculae were randomised to receive various treatment protocols comprising a peptide activator of PKC-ɛ, a peptide inhibitor of PKC-δ and their respective inactive control peptides. Administering the PKC-δ peptide inhibitor at reoxygenation improved the recovery of function at all the concentrations tested (39.3 ± 1.4% at 5 nM, 52.4 ± 2.9% at 50 nM and 46.8 ± 2.9% at 500 nM versus the control group, 27.5 ± 1.4%: N ≥ 6/group: P < 0.02). Preconditioning with the PKC-ɛ peptide activator improved the recovery of function (40.0 ± 0.8% at 50 nM and 49.7 ± 3.1% at 500 nM versus the control group 27.5 ± 1.4%: N ≥ 6/group: P < 0.02). This cardioprotective effect was comparable to that achieved by a standard hypoxic preconditioning protocol (52.3 ± 3.2%). Interestingly, administering the PKC-ɛ activator (500 nM) at the onset of reperfusion also improved the recovery of contractile function (40.7 ± 2.1% versus 27.5 ± 1.5%: N ≥ 6/group: P < 0.05). In human myocardium, cardioprotection can be achieved by either inhibiting PKC-δ or activating PKC-ɛ at the onset of reperfusion. In addition, PKC-ɛ activation offers cardioprotection when administered as a preconditioning strategy.