Peptides (v.28, #11)
Editorial Board (CO2).
Characterization of the N-terminal segment used by the barley yellow dwarf virus movement protein to promote interaction with the nuclear membrane of host plant cells by Sarah Rachel Dennison; Frederick Harris; Klaus Brandenburg; David Andrew Phoenix (2091-2097).
The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic α-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be α-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5–1.0 cm−1) and induced surface pressure changes of 2 mN m−1 in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic α-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.
Keywords: Nuclear envelope; Movement protein; N-terminal amphiphilic helix; Barley yellow dwarf virus;
Purification and identification of adipogenesis inhibitory peptide from black soybean protein hydrolysate by Hyun Jeong Kim; In Young Bae; Chang-Won Ahn; Suyong Lee; Hyeon Gyu Lee (2098-2103).
Adipogenesis inhibitory peptide was isolated and identified from black soybean (Rhynchosia volubilis Lour.) hydrolysate. An adipogenesis inhibitor was purified using consecutive methods including: ultrafiltration (MWCO; 3 and 10 kDa), gel filtration chromatography (Superdex Peptide 10/300 GL column), and reverse-phase high-performance liquid chromatography (μBondapak™ C18 column). Also, the adipogenesis inhibition effect of the purified peptide was measured by observation of droplet of 3T3-L1 adipocyte by Oil Red O staining in the highest active fraction in each step. The peptide was shown to inhibit the differentiation of the 3T3-L1 pre-adipocyte, which was confirmed by morphological study. The adipogenesis inhibitory peptide was purified 71.43-fold from black soybean hydrolysate throughout a five-step purification procedure. The adipogenesis inhibitor was identified to be a tripeptide, Ile-Gln-Asn, having an IC50 value of 0.014 mg protein/ml. Furthermore, the synthetic tripeptide (Ile-Gln-Asn) exhibited the similar adipogenesis effects to the purified peptide. Thus, these results showed the potential anti-obesity effect of the purified peptide through control of adiposity.
Keywords: Black soybean peptide; Adipogenesis inhibitor; 3T3-L1 cell;
High-mass-resolution direct-tissue MALDI-FTMS reveals broad conservation of three neuropeptides (APSGFLGMRamide, GYRKPPFNGSIFamide and pQDLDHVFLRFamide) across members of seven decapod crustaean infraorders by Elizabeth A. Stemmler; Christopher R. Cashman; Daniel I. Messinger; Noah P. Gardner; Patsy S. Dickinson; Andrew E. Christie (2104-2115).
Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) has become an important method for identifying peptides in neural tissues. The ultra-high-mass resolution and mass accuracy of MALDI-FTMS, in combination with in-cell accumulation techniques, can be used to advantage for the analysis of complex mixtures of peptides directly from tissue fragments or extracts. Given the diversity within the decapods, as well as the large number of extant species readily available for analysis, this group of animals represents an optimal model in which to examine phylogenetic conservation and evolution of neuropeptides and neuropeptide families. Surprisingly, no large comparative studies have previously been undertaken. Here, we have initiated such an investigation, which encompasses 32 species spanning seven decapod infraorders. Two peptides, APSGFLGMRamide and pQDLDHVFLRFamide, were detected in all species. A third peptide, GYRKPPFNGSIFamide, was detected in all species except members of the Astacidean genus Homarus, where a Val1 variant was present. Our finding that these peptides are ubiquitously (or nearly ubiquitously) conserved in decapod neural tissues not only suggests important conserved functions for them, but also provides an intrinsic calibrant set for future MALDI-FTMS assessments of other peptides in this crustacean order.
Keywords: Cancer borealis tachykinin-related peptide I (CabTRP I); Crustacean myosuppressin (Crust-MS); Glycine1-SIFamide (Gly1-SIFamide); Valine1-SIFamide (Val1-SIFamide); Orcomyotropin (OMT); Commissural ganglion (CoG); Neurotransmitter; Neuromodulator; Neurohormone; Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS); Sustained off-resonance irradiation collision-induced dissociation (SORI-CID); Neuropeptidomics;
Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning by Can Peng; Xuechen Wu; Yuhong Han; Duoduo Yuan; Chengwu Chi; Chunguang Wang (2116-2124).
Cone snails are a group of ancient marine gastropods with highly sophisticated defense and prey strategies using conotoxins in their venom. Conotoxins are a diverse array of small peptides, mostly with multiple disulfide bridges. Using a 3′ RACE approach, we identified six novel peptides from the venom ducts of a worm-hunting cone snail Conus pulicarius. These peptides are named Pu5.1–Pu5.6 as their primary structures show the typical pattern of T-1 conotoxin family, a large and diverse group of peptides widely distributed in venom ducts of all major feeding types of Conus. Except for the conserved signal peptide sequences in the precursors and unique arrangement of Cys residues (CC-CC) in mature domains, the six novel T-1 conotoxins show remarkable sequence diversity in their pro and mature regions and are, thus, likely to be functionally diversified. Here, we present a simple and fast strategy of gaining novel disulfide-rich conotoxins via molecular cloning and our detailed sequence analysis will pave the way for the future functional characterization of toxin-receptor interaction.
Keywords: Cone snail; Conus pulicarius; T-1 conotoxin; cDNA cloning; Sequence diversity;
Heparin-mimetic sulfated peptides with modulated affinities for heparin-binding peptides and growth factors by Sung Hye Kim; Kristi L. Kiick (2125-2136).
Heterogeneity in the composition and in the polydispersity of heparin has motivated the development of homogeneous heparin mimics, and peptides of appropriate sequence and chemical function have therefore recently emerged as potential replacements for heparin in selected applications. Here, we report the assessment of the binding affinities of multiple sulfated peptides (SPs) for a set of heparin-binding peptides (HBPs) and for vascular endothelial growth factor isoform 165 (VEGF165); these binding partners have application in the selective immobilization of proteins and in hydrogel formation through non-covalent interactions. Sulfated peptides were produced via solid-phase methods, and their affinity for the HBPs and VEGF165 was assessed via affinity liquid chromatography (ALC), surface plasmon resonance (SPR), and in selected cases, isothermal titration calorimetry (ITC). The shortest peptide, SPa, showed the highest affinity binding of HBPs and VEGF165 in both ALC and SPR measurements, with slight exceptions. Of the investigated HBPs, a peptide based on the heparin-binding domain of human platelet factor 4 showed greatest binding affinities toward all of the SPs, consistent with its stronger binding to heparin. The affinity between SPa and PF4ZIP was indicated via SPR (K D = 5.27 μM) and confirmed via ITC (K D = 8.09 μM). The binding by SPa of both VEGF and HBPs suggests its use as a binding partner to multiple species, and the use of these interactions in assembly of materials. Given that the peptide sequences can be varied to control binding affinity and selectivity, opportunities are also suggested for the production of a wider array of matrices with selective binding and release properties useful for biomaterials applications.
Keywords: Sulfated peptide; Heparin mimic; Heparin-binding peptides; Affinity chromatography; Growth factor delivery; Hydrogel;
Interaction of antiflammin-1 with uteroglobin-binding protein induces phosphorylation of ERK1/2 in NIH 3T3 cells by Chen Li; Jianzhong Han; Lian Li; Shaojie Yue; Jinfeng Li; Dandan Feng; Huijun Liu; Dejian Jiang; Xiaoqun Qin; Ziqiang Luo (2137-2145).
Previously, it has been suggested that uteroglobin (UG)-binding protein functions as a putative receptor of UG; however, the specific epitope of UG that interacts with this receptor has not yet been identified. The downstream events of UG-binding protein signaling remain unclear. Here we report that antiflammin-1 (AF-1, a bioactive C-terminal peptide of UG) specifically binds to UG-binding protein and has a cellular signaling consequence. We reduced the level of endogenous UG-binding protein expression in murine fibroblast cell line NIH 3T3 by RNA interference and found that knockdown of UG-binding protein inhibited AF-1-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Meanwhile, the interaction between AF-1 and UG-binding protein was confirmed by flow cytometry-based binding assays and co-localization of AF-1 and enhanced green fluorescent protein (EGFP)-tagged UG-binding protein. The present study provides evidence for the first time for AF-1 binding with UG-binding protein, and preliminarily characterized UG-binding protein as a point downstream of AF-1 in mediating ERK phosphorylation.
Keywords: Antiflammin-1; Uteroglobin; Uteroglobin-binding protein; Extracellular signal-regulated kinase;
Fluorescence resonance energy transfer (FRET) peptides and cycloretro-inverso peptides derived from bradykinin as substrates and inhibitors of prolyl oligopeptidase by Silvia S. Gorrão; Jefferson P. Hemerly; Aurelio R. Lima; Robson L. Melo; Zoltán Szeltner; László Polgár; Maria A. Juliano; Luiz Juliano (2146-2154).
Prolyl oligopeptidase (POP, EC 184.108.40.206) is a member of a family of serine peptidases with post-proline cleaving activity towards peptides. It is located in the cytosol in active form but without hydrolytic activity on proteins or peptides higher than 30 amino acids. Its function is not well defined, but it is involved in central nervous system disorders. Here, we studied the substrate specificity of wild type POP (POPwt) and its C255T variant lacking the non-catalytic Cys255 . This residue is located in the seven-bladed β-propeller domain that regulates the activity of POP. Fluorescence resonance energy transfer (FRET) peptides were used with sequences derived from bradykinin-containing region of human kininogen and flanked by Abz (ortho-aminobenzoic acid) and EDDnp [N-ethylenediamine-(2,4-dinitrophenyl)]. The peptide Abz-GFSPFRQ-EDDnp was taken as leader substrate for the synthesis of five series of peptides modified at the P3, P2, P ′ 1 , P ′ 2 and P ′ 3 residues. The optimal amino acids in each position for POPwt resulted in the sequence RRPYIR that is very similar to the C-terminal sequence of neurotensin. The cyclic peptides c(G(n)FSPFR) (n = 1–4) were hydrolyzed by POP; their cycloretro and cycloretro-inverso analogues were inhibitors in the micromolar range. The differences between POPwt and its C255T mutant in the hydrolysis of the series derived from Abz-GFSPFRQ-EDDnp were restricted to the non-prime site of the substrates. The kinetic data of hydrolysis and inhibition by the cyclic peptides are consistent with the structures of POP–substrate/inhibitor complexes and with the substrate specificity data obtained with linear FRET peptides. All together, these results give information about the POP–substrate/inhibitor interactions that further complete knowledge of this important oligopeptidase.
Keywords: Peptidases; Bradykinin; Neurotensin; Prolylpeptidase;
Genomic analyses and cloning of novel chicken natriuretic peptide genes reveal new insights into natriuretic peptide evolution by Sofie Trajanovska; Koji Inoue; Yoshio Takei; John A. Donald (2155-2163).
The natriuretic peptide (NP) family consists of multiple subtypes in teleosts, including atrial, B-type, ventricular, and C-type NPs (ANP, BNP, VNP, CNP-1–4, respectively), but only ANP, BNP, CNP-3, and CNP-4 have been identified in tetrapods. As part of understanding the molecular evolution of NPs in the tetrapod lineage, we identified NP genes in the chicken genome. Previously, only BNP and CNP-3 have been identified in birds, but we characterized two new chicken NP genes by cDNA cloning, synteny and phylogenetic analyses. One gene is an orthologue of CNP-1, which has only ever been reported in teleostei and bichir. The second gene could not be assigned to a particular NP subtype because of high sequence divergence and was named renal NP (RNP) due to its predominant expression in the kidney. CNP-1 mRNA was only detected in brain, while CNP-3 mRNA was expressed in kidney, heart, and brain. In the developing embryo, BNP and RNP transcripts were most abundant 24 h post-fertilization, while CNP mRNA increased in a stage-dependant manner. Synthetic chicken RNP stimulated an increase in cGMP production above basal level in chicken kidney membrane preparations and caused a potent dose-dependant vasodilation of pre-constricted dorsal aortic rings. From conserved chromosomal synteny, we propose that the CNP-4 and ANP genes have been lost in chicken, and that RNP may have evolved from a VNP-like gene. Furthermore, we have demonstrated for the first time that CNP-1 is retained in the tetrapod lineage.
Keywords: Chicken; C-type natriuretic peptide; Comparative genomics; Molecular evolution; Synteny; Renal natriuretic peptide;
Mice heterozygous for adrenomedullin exhibit a more extreme inflammatory response to endotoxin-induced septic shock by Ryan Dackor; Kathleen Caron (2164-2170).
Adrenomedullin (AM) is a highly conserved peptide that can act as a potent vasodilator, anti-microbial factor and anti-inflammatory factor. Several studies have implicated diverse roles for AM in regulating the inflammatory and hemodynamic responses to septic shock. Moreover, during sepsis the receptors that mediate AM signaling [calcitonin receptor-like receptor (calcrl) and receptor activity modifying proteins (RAMP) 2 and 3] undergo dynamic and robust changes in their expression. Although numerous studies have used animal models to study the role of administered or increased AM in septic animals, genetic studies to determine the consequences of reduced AM during septic shock have not yet been performed. Here, we used a murine model of lipopolysaccharide (LPS)-induced septic shock to assess the inflammatory response in mice heterozygous for the AM gene. Following LPS challenge, AM +/− mice had higher expression of TNF-α and IL-1β than LPS-treated wild-type (WT) controls. Consequently, serum TNF-α was also significantly elevated in LPS-treated AM +/− mice compared to WT LPS-treated mice. We also observed higher serum levels of liver enzymes, suggesting more advanced end-organ damage in mice with genetically reduced AM. Finally, we found that RAMP2 and calcrl expression levels were markedly reduced in LPS-treated mice, whereas RAMP3 expression was significantly elevated. Importantly, these changes in receptor gene expression were conserved in AM +/− mice, demonstrating that AM peptide itself does not impact directly on the expression of the genes encoding its receptors. We, therefore, conclude that during septic shock the dynamic modulation of AM and its receptors primarily functions to dampen the inflammatory response.
Keywords: Adrenomedullin; Septic shock; Inflammation; Genetic model; Mice;
Intermedin 17–47 does not function as a full intermedin antagonist within the central nervous system or pituitary by Meghan M. White; Willis K. Samson (2171-2178).
A fragment of intermedin (IMD), IMD17–47, has been shown to antagonize the hypotensive effects of intravenous IMD administration; however, the effects of IMD17–47 have not been studied in other systems such as brain and pituitary gland. IMD17–47 was administered intracerebroventricularly (i.c.v.) into male rats alone or prior to administration of IMD; and blood pressure and food and water intakes measured. Multiple doses of IMD17–47 failed to alter basal blood pressure and heart rate, but did partially reverse the stimulatory effects of IMD given i.c.v. on blood pressure and heart rate. A low dose of IMD17–47 by itself significantly increased basal food and water intake. However, a higher dose of the antagonist did not alter food or water intake compared to control treated rats. No dose of IMD17–47 was able to reverse the inhibitory effects of IMD administered i.c.v. on food and water intake. Furthermore, IMD17–47 failed to significantly alter the inhibitory effects of IMD on growth hormone releasing hormone-stimulated growth hormone release from dispersed anterior pituitary cells in culture. A siRNA molecule designed to compromise IMD production was able to reduce brain IMD levels and did, upon i.c.v. administration, cause increased water drinking in male rats. This tool may provide a better method than the use of the IMD17–47 compound to study the role of endogenous IMD within the CNS and pituitary.
Keywords: Intermedin; Adrenomedullin; Calcitonin gene-related peptide; Blood pressure; Food intake;
Adrenomedullin reduces the severity of cerulein-induced acute pancreatitis by Ozge Ecmel Onur; Ozlem Guneysel; Haldun Akoglu; Arzu Denizbasi; Ender Onur (2179-2183).
We investigated the effect of Adrenomedullin (AM) on cerulein-induced acute pancreatitis in rats. AM treatment (100 ng/kg per rat, subcutaneous) after one hour of cerulein injection reduced the plasma amylase levels, pancreatic weight, pancreatic malondialdehyde (MDA) levels, and the severity of the lesions microscopically. These data suggest that AM has a protective effect on cerulein-induced acute pancreatitis. These could be due to anti-inflammatory properties of AM, inhibition of proinflammatory cytokine secretion, reducing the endothelial permeability increased by reactive oxygen species, endotoxins or cytokines.
Keywords: Adrenomedullin; Acute pancreatitis; Cerulein;
Glucagon-like peptide-1 modulates synaptic transmission to identified pancreas-projecting vagal motoneurons by Shuxia Wan; Kirsteen N. Browning; R. Alberto Travagli (2184-2191).
Using a brainstem slice preparation, we aimed to study the pre- and postsynaptic effects of glucagon-like peptide-1 (GLP-1) on synaptic transmission to identified pancreas-projecting vagal motoneurons. Following blockade of GABAergic mediated currents with bicuculline, perfusion with 100 nM GLP-1 increased both amplitude and frequency of excitatory postsynaptic currents (EPSCs) in 21 of 52 neurons. Perfusion with the GLP-1 selective agonist exendin-4 (100 nM), also increased the frequency of spontaneous EPSCs, while pretreatment with the GLP-1 selective antagonist, exendin 9–39, prevented the effects of GLP-1. In the presence of kynurenic acid to block ionotropic glutamatergic currents, perfusion with GLP-1 increased the frequency of inhibitory postsynaptic currents (IPSCs) in 28 of 74 neurons; in 14 of these responsive neurons, GLP-1 also increased IPSC amplitude, indicating actions at both pre- and postsynaptic sites. Perfusion with exendin-4 increased the frequency of spontaneous IPSCs, while pretreatment with exendin 9–39 prevented the effects of GLP-1. These results suggest that GLP-1 modulates both excitatory and inhibitory synaptic inputs to pancreas-projecting vagal motoneurons.
Keywords: Brainstem; Dorsal vagal complex; GLP-1; Pancreas; Electrophysiology;
Comparison of the metabolic effects of GIP receptor antagonism and PYY(3-36) receptor activation in high fat fed mice by N. Irwin; K. Hunter; P.R. Flatt (2192-2198).
Glucose-dependent insulinotropic polypeptide (GIP) and peptide YY (PYY) are secreted from the intestinal K- and L-cells, respectively, following a meal. Both peptides are believed to play a key role in glucose homeostasis and energy expenditure. This study investigated the effects of daily administration of the stable and specific GIP-R antagonist, (Pro3)GIP (25 nmol/kg) and the endogenous truncated form of PYY, PYY(3-36) (50 nmol/kg), in mice fed with a high fat diet. Daily i.p. injection of (Pro3)GIP, PYY(3-36) or combined peptide administration over 24 days significantly (P < 0.05–0.01) decreased body weight compared with saline-treated controls without change in food intake. Plasma glucose levels and glucose tolerance were significantly (P < 0.05) lowered by (Pro3)GIP treatment alone, and in combination with PYY(3-36). These changes were accompanied by a slight improvement of insulin sensitivity in all of the treatment groups. (Pro3)GIP treatment significantly reduced plasma corticosterone (P < 0.05), while combined administration with PYY(3-36) significantly lowered serum glucagon (P < 0.05). No appreciable changes were observed in either circulating or glucose-stimulated insulin secretion in all treatment groups. (Pro3)GIP-treated mice had significantly (P < 0.01) lowered fasting glucose levels and an improved (P < 0.05) glycemic response to feeding. These comparative data indicate that chemical ablation of GIP receptor action using (Pro3)GIP provides an especially effective means of countering obesity and related abnormalities induced by consumption of high fat energy rich diet.
Keywords: Glucose-dependent insulinotropic polypeptide (GIP); Peptide YY (PYY); Glucose homeostasis; High fat feeding; Obesity;
Study of angiotensin-(1–7) vasoactive peptide and its β-cyclodextrin inclusion complexes: Complete sequence-specific NMR assignments and structural studies by Ivana Lula; Ângelo L. Denadai; Jarbas M. Resende; Frederico B. de Sousa; Guilherme F. de Lima; Dorila Pilo-Veloso; Thomas Heine; Hélio A. Duarte; Robson A.S. Santos; Rubén D. Sinisterra (2199-2210).
We report the complete sequence-specific hydrogen NMR assignments of vasoactive peptide angiotensin-(1–7) (Ang-(1–7)). Assignments of the majority of the resonances were accomplished by COSY, TOCSY, and ROESY peak coordinates at 400 MHz and 600 MHz. Long-side-chain amino acid spin system identification was facilitated by long-range coherence transfer experiments (TOCSY). Problems with overlapped resonance signals were solved by analysis of heteronuclear 2D experiments (HSQC and HMBC). Nuclear Overhauser effects (NOE) results were used to probe peptide conformation. We show that the inclusion of the angiotensin-(1–7) tyrosine residue is favored in inclusion complexes with β-cyclodextrin. QM/MM simulations at the DFTB/UFF level confirm the experimental NMR findings and provide detailed structural information on these compounds in aqueous solution.
Keywords: Angiotensin-(1–7); NMR studies and ROESY; Cyclodextrins; Inclusion compounds; QM/MM; DFTB and UFF simulation;
Further evidence for a C-terminal structural motif in CCK2 receptor active peptide hormones by Shane R. Stone; Craig Giragossian; Dale F. Mierke; Graham E. Jackson (2211-2222).
A comparison of the conformational characteristics of the related hormones [Nle15] gastrin-17 and [Tyr9-SO3] cholecystokinin-15, in membrane-mimetic solutions of dodecylphosphocholine micelles and water, was undertaken using NMR spectroscopy to investigate the possibility of a structural motif responsible for the two hormones common ability to stimulate the CCK2 receptor. Distance geometry calculations and NOE-restrained molecular dynamics simulations in biphasic solvent boxes of decane and water pointed to the two peptides adopting near identical helical C-terminal configurations, which extended one residue further than their shared pentapeptide sequence of Gly-Trp-Met-Asp-Phe-NH2. The C-terminal conformation of [Nle15] gastrin-17 contained a short α-helix spanning the Ala11-Trp14 sequence and an inverse γ-turn centered on Nle15 while that of [Tyr9-SO3] cholecystokinin-15 contained a short 310 helix spanning its Met10 to Met13 sequence and an inverse γ-turn centered on Asp14. Significantly, both the C-terminal helices were found to terminate in type I β-turns spanning the homologous Gly-Trp-Met-Asp sequences. This finding supports the hypothesis that this structural motif is a necessary condition for CCK2 receptor activation given that both gastrin and cholecystokinin have been established to follow a membrane-associated pathway to receptor recognition and activation. Comparison of the conformations for the non-homologous C-terminal tyrosyl residues of [Nle15] gastrin-17 and [Tyr9-SO3] cholecystokinin-15 found that they lie on opposite faces of the conserved C-terminal helices. The positioning of this tyrosyl residue is known to be essential for CCK1 activity and non-essential for CCK2 activity, pointing to it as a possible differentiator in CCK1/CCK2 receptor selection. The different tyrosyl orientations were retained in molecular models for the [Nle15] gastrin-17/CCK2 receptor and [Tyr9-SO3] cholecystokinin-15/CCK1 receptor complexes, highlighting the role of this residue as a likely CCK1/CCK2 receptor differentiator.
Keywords: Gastrin; Cholecystokinin; CCK2; Structural motif; Type I β-turn; NMR; Membrane-mimetic media; Micelles; Dodecylphosphocholine;
Nesfatin-1 crosses the blood–brain barrier without saturation by Weihong Pan; Hung Hsuchou; Abba J. Kastin (2223-2228).
Nesfatin-1 is an 82 amino acid peptide that suppresses food intake after intracerebroventricular injection. Nesfatin-1 and its precursor NUCB2 were identified by subtraction cloning in cell lines of both neuronal and adipocytic origin. This provides a strong basis for studies to determine how peripherally derived nesfatin-1 permeates the blood–brain barrier (BBB) to participate in its CNS actions and whether pharmacological delivery by the peripheral route is feasible. In this study, nesfatin-1 remained stable in blood at least 20 min after intravenous injection and permeated the BBB by a non-saturable mechanism. The influx rate of nesfatin-1 after intravenous delivery was 0.27 ± 0.11 μl/g-min, and 0.3% of nesfatin-1 reached brain parenchyma 10 min after injection. The lack of saturation of influx was shown by use of excess unlabeled nesfatin-1 in multiple-time regression analysis, capillary depletion, and in situ brain perfusion. After intracerebroventricular injection, nesfatin-1 had a half-time disappearance of 23.8 min, which was not significantly different from that of albumin. This indicates that nesfatin-1 exited the brain by bulk absorption of cerebrospinal fluid without a specific efflux transport system. We conclude that the permeation of nesfatin-1 is a non-saturable process in either the blood-to-brain or brain-to-blood direction. Thus, the limited penetration under physiological conditions does not limit the pharmacological delivery of this satiety peptide as a potential therapeutic agent.
Keywords: Nesfatin-1; Blood–brain barrier; Permeability; Autoradiography;
Copper (II) modulates in vitro aggregation of a tau peptide by Lian-Xiu Zhou; Jin-Tang Du; Zhi-Yang Zeng; Wei-Hui Wu; Yu-Fen Zhao; Kenji Kanazawa; Yasuko Ishizuka; Tadashi Nemoto; Hiroshi Nakanishi; Yan-Mei Li (2229-2234).
Copper (II) has been implicated in the pathology of Alzheimer's disease (AD) for the impaired homeostatic mechanism found in the brains of AD patients. Here we studied the binding properties of Cu(II) with the first microtubule-binding repeat, encompassing residues 256–273 of the human tau441 sequence. Additionally, the effect of Cu(II) on the assembly of this repeat was also investigated. Our results indicate that Cu(II) can bind to this repeat with His268 involved and has an inhibiting effect on the in vitro aggregation of this repeat. This work provides new insight into the role of Cu(II) in Alzheimer's disease.
Keywords: Tau protein; Alzheimer's disease; Copper; Aggregation;
Neuropeptide FF (NPFF) reduces the expression of morphine- but not of ethanol-induced conditioned place preference in rats by Jolanta Kotlinska; Agnieszka Pachuta; Tomasz Dylag; Jerzy Silberring (2235-2242).
Neuropeptide FF (NPFF) has been described as an anti-opioid peptide. It plays a role in opioid antinociception, dependence and tolerance. Previous study has indicated that 1DMe ([D-Tyr1, (NMe)Phe3]NPFF), a stable analog of NPFF, inhibits acquisition of the rewarding effect of morphine but not of ethanol in mice. The rewarding effects of these drugs were measured in the unbiased paradigm of conditioned place preference (CPP). The present study examines the influence of NPFF on the expression of morphine- and ethanol-induced CPP in the biased procedure in rats. Our experiments showed that NPFF, given intracerebroventricularly (i.c.v.) at the doses of 5, 10 and 20 nmol, inhibited the expression of morphine-induced CPP. NPFF gave itself, neither induced place preference nor aversion, although a tendency to aversive effect was seen at the highest dose of 20 nmol. NPFF did not indicate fear behavior in the elevated plus maze test, and did not disturb locomotor activity of rats. However, NPFF was unable to inhibit the expression of ethanol-induced CPP. Probably this effect is due to the fact that ethanol reward is a more complex process and apart from the role of opioids, there are other neurotransmitters also involved in this mechanism. These results suggest that NPFF is involved in the expression of morphine reward. Moreover, our study supports an anti-opioid character of this peptide.
Keywords: NPFF; Morphine; Ethanol; Conditioned place preference; Fear behavior; Locomotor activity;
Regulation of nociceptin/orphaninFQ secretion by immune cells and functional modulation of interleukin-2 by Thomas R. Miller; Allison J. Fulford (2243-2252).
Previous research has demonstrated that numerous populations of immune cell, including lymphocytes, synthesize nociceptin (N/OFQ) precursor mRNA although little is known regarding the immunological role of N/OFQ. In the present study we have demonstrated significant effects of mitogens, pro-inflammatory cytokines, cyclic AMP analogues, glucocorticoids and CRF on N/OFQ secretion by rat splenocytes in vitro. N/OFQ (10−14 to 10−10 M) was also shown to inhibit proliferation of Con A-activated splenocytes and production of IL-2 in vitro. In summary we have shown how a variety of stimuli relevant to inflammation can regulate endogenous N/OFQ secretion by splenocytes in vitro. We also suggest that N/OFQ may promote anti-inflammatory actions via suppression of IL-2 in vivo.
Keywords: Nociceptin; Mitogens; IL-2; Inflammation; Glucocorticoids; Splenocytes;