Peptides (v.31, #11)

Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli by B. Bommarius; H. Jenssen; M. Elliott; J. Kindrachuk; Mukesh Pasupuleti; H. Gieren; K.-E. Jaeger; R.E.W. Hancock; D. Kalman (1957-1965).
▶ Novel bacterial expression system for host defense peptides. ▶ Scalable, simplified 2-step purification. ▶ Large-scale expression of peptides as small as 9 amino acids in length. ▶ Successful application of this method to peptides already in clinical trials. ▶ Suitable for large-scale production of antimicrobial peptides and host defense peptides under GMP for therapeutic use.Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for large-scale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.
Keywords: Antimicrobial peptide; Host defense peptide; Bacterial expression system;

A carpet-based mechanism for direct antimicrobial peptide activity against vaccinia virus membranes by R.E. Dean; L.M. O’Brien; J.E. Thwaite; M.A. Fox; H. Atkins; D.O. Ulaeto (1966-1972).
▶ Peptides attack double-membraned extracellular virions of vaccinia virus. ▶ Peptides remove the outer membrane entirely in a single event. ▶ Peptides expose antigens sequestered beneath the outer membrane. ▶ Direct killing is consistent with a carpet model.Antimicrobial peptides have activity against a wide variety of biological membranes and are an important component of innate immunity in vertebrate as well as invertebrate systems. The mechanisms of action of these peptides are incompletely understood and a number of competing but not necessarily mutually exclusive models exist. In this study we examined the virucidal activity of four peptides, the human cathelicidin derived LL37, Xenopus alanine-substituted Magainin-2 amide, uperin-3.1, and a cecropin-LL37 hybrid against vaccinia virus. The peptides were shown to be differentially virucidal but all were shown to attack the viral envelope, with LL37 being the most effective and uperin-3.1 the least. Density gradient analysis of the treated virions indicated the virus outer membrane was efficiently removed by peptide action and suggests a mechanism of direct virus inactivation that is consistent with the carpet model for peptide-mediated membrane disruption. Interestingly, the least effective peptide uperin-3.1 was equally effective as the others at inducing susceptibility to neutralizing antibody. This suggests that in addition to direct killing by a carpet-based mechanism, the peptides may simultaneously operate a different mechanism that exposes sequestered antigen without membrane removal.
Keywords: Antimicrobial peptide; Vaccinia virus; Carpet; Membrane;

Structure–activity studies of RALF, Rapid Alkalinization Factor, reveal an essential – YISY – motif by Gregory Pearce; Yube Yamaguchi; Gerhard Munske; Clarence A. Ryan (1973-1977).
▶ RALF contains a – YISY– motif that is essential for alkalinization and signal perception. ▶ Substitution of alanine for the isoleucine in the – YISY – motif abolishes activity. ▶ The – YISY – motif is highly conserved among RALFs from a wide range of plant species.Rapid Alkalinization Factor (RALF) is a 49-amino acid peptide initially isolated from tobacco leaves that is capable of arresting both root and pollen tube growth. With suspension cells, addition of RALF causes an elevation of the pH of the extracellular media, caused by the blockage of a proton pump. RALF associates with a putative receptor(s) on the surface of the plant cell, initiating a signal transduction pathway. Although the exact function(s) of RALFs are unknown, its presence throughout the plant kingdom attests to its importance in some type of basic regulatory role. In the present study, deletion and substitution analyses of RALF reveal a specific – YISY – motif located at positions 5 through 8 from the N-terminus, highly conserved within the plant kingdom, which is a requirement for productive binding of RALF to its putative receptor. Replacement of isoleucine with alanine in the – YISY – motif caused a severe reduction in alkalinization of suspension cell media and a loss of root growth inhibition with tomato seedlings.
Keywords: RALF; Plant peptide signals; Alkalinization assay;

Lipopeptide induces apoptosis in fungal cells by a mitochondria-dependent pathway by Gaofu Qi; Fayin Zhu; Peng Du; Xiufen Yang; Dewen Qiu; Ziniu Yu; Jingyuan Chen; Xiuyun Zhao (1978-1986).
▶ Lipopeptide played the anti-fungal role by two models: high concentration to elicit pores on cell membrane, and low concentration to induce apoptosis. ▶ Lipopeptide-induced apoptosis could be enhanced by Oligomycin. ▶ Lipopeptide could bind with ATPase on the mitochondrial membrane and result in a decreased ATPase activity in fungal cells. ▶ In lipopeptide-induced apoptosis, Cytochrome C was released from the mitochondria, and then acted with caspase 9 to induce apoptosis by an intracellular pathway. ▶ High caspase 8 activity was also detectable in apoptotic fungal cells, indicated that an extracellular pathway might also be responsible for this lipopeptide-induced apoptosis. Bacillus amyloliquefaciens WH1 inhibit the growth of fungi by producing a new surfactin called as WH1fungin. WH1fungin plays an anti-fungal role by two models: high concentration to elicit pores on cell membrane and low concentration to induce apoptosis. WH1fungin can also inhibits the glucan synthase resulting in a decreased synthesis of callose on fungal cell wall. Further detection revealed that classical apoptotic markers such as reactive oxygen species (ROS) accumulation, phosphatidylserine (PS) externalization, DNA strand breaks and caspase-like activities could be found in fungal cells after treated by WH1fungin. Oligomycin was used as an inhibitor to block the mitochondria-dependent apoptosis in fungal cells, and results showed it could not inhibit but enhance the apoptosis induced by WH1fungin. After isolation by affinity chromatography, WH1fungin was found to bind with ATPase on the mitochondrial membrane and result in a decreased ATPase activity in fungal cells. This was further verified by treating fungal cells with FITC-labeled WH1fungin, which could bind to the mitochondrial membrane showing green fluorescence in fungal cells. After that, cytochrome C was released from the mitochondria, which then acted with caspase 9 to induce apoptosis by an intracellular pathway. High caspase 8 activity was also detectable in apoptotic fungal cells, indicating that an extracellular pathway might also be responsible for apoptosis induced by WH1fungin. Taken together, we report that lipopeptide can induce apoptosis in fungal cells, and induction of apoptosis by lipopeptide might be a common anti-fungal mechanism of Bacillus in the natural habitat.
Keywords: Lipopeptide; Fungi; Apoptosis; ATPase; Cytochrome C;

Conserved high activity binding peptides from the Plasmodium falciparum Pf34 rhoptry protein inhibit merozoites in vitro invasion of red blood cells by Gabriela Arévalo-Pinzón; Hernando Curtidor; Magnolia Vanegas; Carolina Vizcaíno; Manuel A. Patarroyo; Manuel E. Patarroyo (1987-1994).
* Conserved Pf34 high activity binding peptides (HABPs) interact with receptors on the RBC. * Specific binding of Pf34 HABPs is affected by enzymatic treatment of RBCs. * HABPs and mixtures of them inhibit merozoite invasion to RBCs in vitro.Rhoptries are specialized secretory organelles found in all members of the genus Plasmodium whose proteins have been considered as promising vaccine candidates due to their involvement in cell invasion and the formation of the parasitophorous vacuole (PV). The Plasmodium falciparum Pf34 protein was recently identified as a rhoptry-neck protein located in detergent-resistant microdomains (DRMs) that is expressed in mature intraerythrocytic parasite stages, but its biological function is still unknown. Receptor–ligand assays carried out in this study found that peptides 36,051 (101DKKFSESLKAHMDHLKILNN120Y), 36,053 (141KKYIIKEIQNNKYLNKEKKS160), 36,055 (181WLESVNNIEEKSNILKNIKS200Y) and 36,056 (201QLLNNIASLNHTLSEEIKNI220Y), located in the central portion of Pf34, were found to establish protease-sensitive interactions of high affinity and specificity with receptors on the surface of red blood cell (RBCs). In vitro assays showed that Pf34 high activity binding peptides (HABPs) inhibit invasion of RBCs by P. falciparum merozoites, therefore suggesting that Pf34 could act as an adhesin during invasion and supporting the inclusion of Pf34 HABPs in further studies to develop antimalarial control methods.
Keywords: Detergent-resistant microdomains; GPI-anchored; Peptides; Receptors; Vaccine;

Antimicrobial activity of human hepcidin 20 and 25 against clinically relevant bacterial strains: Effect of copper and acidic pH by Giuseppantonio Maisetta; Raffaele Petruzzelli; Franca Lisa Brancatisano; Semih Esin; Alberto Vitali; Mario Campa; Giovanna Batoni (1995-2002).
▶ Hepcidin 20 and hepcidin 25 are bactericidal against a variety of clinical isolates. ▶ Bactericidal activity of hepcidin 25 is enhanced by copper. ▶ Bactericidal activity of hepcidin 20 and hepcidin 25 is highly enhanced at acidic pH. ▶ At pH 5.0 the killing time of hepcidin 20 and hepcidin 25 is shorter than at pH 7.4. ▶ Combinations of low concentrations of hepcidin 20 and 25 are bactericidal at pH 5.0.Hepcidin 25 (hep-25) is a peptide primarily produced by human liver with a central role in iron homeostasis. Its isoform, hepcidin 20 (hep-20), has an unknown function and lacks the first five aminoacids of the amino-terminal portion. This sequence is crucial for iron regulation by hep-25 and contains a molecular motif able to bind metals. Aim of this study, was to evaluate the antibacterial properties of both peptides in vitro, against a wide range of bacterial clinical isolates and in different experimental conditions. Although both peptides were found to be bactericidal against a variety of clinical isolates with different antibiotic resistance profiles, hep-20 was active at lower concentrations than hep-25, in most of the cases. Killing kinetics, carried on in sodium-phosphate buffer at pH 7.4, demonstrated that bactericidal activity occurred not earlier than 30–90 min of incubation. Bactericidal activity of hep-25 was slightly enhanced in the presence of copper, while the same metal did not affect the activity of hep-20. Interestingly, bactericidal activity of both hepcidins was highly enhanced at acidic pH. Acidic pH (pH 5.0 and 6.6) not only reduced the microbicidal concentrations of hepcidins, but also shortened the killing times of both peptides, as compared to pH 7.4. Combining hep-20 and hep-25 at pH 5.0 a bactericidal effect could be obtained at very low concentrations of both peptides. These results render hepcidins interesting for the design of new drugs for the treatment of infections occurring in body districts with physiologic acidic pH.
Keywords: Antimicrobial peptides; Hepcidin 20; Hepcidin 25; Copper; pH; Multi-drug-resistance;

Nisin inhibits dental caries-associated microorganism in vitro by Zhongchun Tong; Liping Dong; Lin Zhou; Rui Tao; Longxing Ni (2003-2008).
Some important results were found about nisin in the present study. ▶ Nisin played a certain role in resistance against the nine common cariogenic microorganisms. ▶ Nisin antibacterial activity differed among cariogenic microorganisms. ▶ The ingredients in saliva did not affect antimicrobial activity of nisin. ▶ Morphology of S. sanguinis, S. mutans, L. fermenti and L. acidophilus with nisin treatment showed different degrees of variation. Therefore, our findings suggested that nisin has considerable potential for prevention of dental caries.Nisin, produced by Lactococcus lactis, is an antibiotic peptide to effectively antagonize a broad spectrum of Gram-positive bacteria, and is widely used as a safe food antimicrobial agent. In the present study, we investigated whether nisin could be used as an effective antibiotic peptide against the nine common cariogenic microorganisms, and its antimicrobial activity could be affected by the ingredients of saliva in oral cavity. In the minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) and spot-on-lawn assay, nisin displayed different MIC, MBC and antimicrobial activity against the nine tested strains. There was statistical difference between the inhibitory zone diameters of nisin against the different tested bacteria (p  < 0.05), but no statistical difference between the inhibitory zone diameters of nisin dissolved in PBS and saliva (p  > 0.05). Furthermore, morphology and membranes of Streptococcus sanguinis, Streptococcus mutans, Lactobacillus fermenti and Lactobacillus acidophilus with nisin treatment were observed and showed different degrees of variation by a Field Emission Scanning Electron Microscope (FE-SEM). Our findings suggested that nisin has considerable potential for prevention and treatment of dental caries.
Keywords: Nisin; MIC; MBC; Spot-on-lawn; Inhibitory zone;

A new subfamily of conotoxins belonging to the A-superfamily by Can Peng; Mingyu Ye; Yanfang Wang; Xiaoxia Shao; Duoduo Yuan; Jing Liu; Edward Hawrot; Chunguang Wang; Chengwu Chi (2009-2016).
▶ Pu14a and ts14a represent a novel α1-family of A-superfamily conotoxin. ▶ Pu14a and ts14a share homologous sequence with a novel cysteine pattern. ▶ α1-conotoxins may also target nAChR.Two novel conotoxins from vermivorous cone snails Conus pulicarius and Conus tessulatus, designated as Pu14.1 and ts14a, were identified by cDNA cloning and peptide purification, respectively. The signal sequence of Pu14.1 is identical to that of α-conotoxins, while its predicted mature peptide, pu14a, shares high sequence similarity with ts14a, with only one residue different in their first intercysteine loop, which contains 10 residues and is rich in proline. Both pu14a and ts14a contain four separate cysteines in framework 14 (C-C-C-C). Peptide pu14a was chemically synthesized, air oxidized, and the connectivity of its two disulfide bonds was determined to be C1-C3, C2-C4, which is the same as found in α-conotoxins. The synthetic pu14a induced a sleeping symptom in mice and was toxic to freshwater goldfish upon intramuscular injection. Using the Xenopus oocyte heterologous expression system, 1 μM of pu14a demonstrated to inhibit the rat neuronal α3β2-containing as well as the mouse neuromuscular α1β1γδ subtypes of nicotinic acetylcholine receptors, and then rapidly dissociated from the receptors. However, this toxin had no inhibitory effect on potassium channels in mouse superior cervical ganglion neurons. According to the identical signal sequence to α-conotoxins, the unique cysteine framework and molecular target of pu14a, we propose that pu14a and ts14a may represent a novel subfamily in the A-superfamily, designated as α1-conotoxins.
Keywords: Conotoxins; A-superfamily; Cysteine framework 14; Nicotinic acetylcholine receptors;

Antimicrobial cyclic decapeptides with anticancer activity by Lidia Feliu; Glòria Oliveras; Anna D. Cirac; Emili Besalú; Cristina Rosés; Ramon Colomer; Eduard Bardají; Marta Planas; Teresa Puig (2017-2026).
▶ Anticancer activity of a library of cyclic antimicrobial decapeptides. ▶ Induction of apoptosis by cyclic decapeptides. ▶ Synergism with cisplatin.Antimicrobial peptides have been considered as potential candidates for cancer therapy. We report here the cytotoxicity of a library of 66 antibacterial cyclodecapeptides on human carcinoma cell lines, and their effects on apoptosis [as assessed by cleavage of poly(ADP-ribose) polymerase (PARP)] and cell signaling proteins (p53 and ERK1/2) in cultured human cervical carcinoma cells. A design of experiments approach permitted to analyze the results of a subset of 16 peptides and define rules for high anticancer activity against MDA-MB-231 breast carcinoma cells. Eight peptides were identified with IC50 values ranging from 18.5 to 57.5 μM against the five cell lines tested, being HeLa cells the most sensitive. Among these sequences, BPC88, BPC96, BPC98, and BPC194 displayed specificity and high cytotoxicity against HeLa cells (IC50 of 22.5–38.5 μM), showed low hemolytic activity and low cytotoxicity to non-malignant fibroblasts, and were stable to proteases in human serum. Induction of apoptosis by these peptides was observed and the apoptotic effect of BPC88 and BPC96 caused a marked decrease on the activated form of ERK1/2 kinase and an induction of p53. We further showed that BPC96 at low doses synergized the cytotoxic effect of cisplatin. These findings suggest that cyclic decapeptides may represent novel anticancer agents providing a new strategy in cancer therapy.
Keywords: Apoptosis; Cervical carcinoma; Cyclopeptides; Pharmacological synergism; Design of experiments;

Novel peptide ligands that bind specifically to mouse embryonic stem cells by Saijuan Zhao; Wenxiu Zhao; Lan Ma (2027-2034).
Display Omitted▶ The search for new embryonic stem (ES) cell markers is critical for ES cells researches. ▶ Phage display is a novel technology used to find the new proteins in cells. ▶ Peptides obtained from phage display panning could bind mouse ES cells with specificity. ▶ Western blot analysis reveals unique receptors on the mouse ES cell membrane. ▶ The selected peptides may provide a way to label, identify, and characterize ES cells.The search for new ES cell markers is critical not only for identification, isolation and visualization of embryonic stem (ES) cells, but also for potential clinical treatment as a targeting agent. Here, by using phage display technology, 12-mer peptide ligands that bind specifically to mouse ES cells were isolated. After four rounds of negative-positive selection, nine sequences in 20 random samples from the chosen clones were selected. Enzyme-linked immunosorbent assay results suggested the Seq2 peptide (KHMHWHPPALNT) had high affinity and specificity to the mouse ES cells. The binding capability of the Seq2 phage could be matched with that of a chemically synthesized peptide with a sequence identical to that displayed by the phage, indicating that this ability was due to the peptide sequence itself. Immunofluorescence analysis confirmed that Seq2 phage selectively bound to the mouse ES cells but not to the differentiated mouse ES cells. Western blot analysis proved the Seq2 phage was bound to two mouse ES membrane proteins which were about 18/20KD, suggesting that the selected peptide targeted to a unique receptor expressed on the mouse ES cells with specificity. Peptides obtained from the study may provide a way to label, identify, and characterize ES cells.
Keywords: Mouse embryonic stem cells; Differentiated cells; Phage-displayed peptide library screening; Cell-specific peptides;

Nuclear localization of vasoactive intestinal peptide (VIP) receptors in human breast cancer by Ana Valdehita; Ana M. Bajo; Ana B. Fernández-Martínez; M. Isabel Arenas; Eva Vacas; Pedro Valenzuela; Antonio Ruíz-Villaespesa; Juan C. Prieto; María J. Carmena (2035-2045).
▶ Endocytosis and nuclear translocation of peripheral GPCRs. ▶ VPAC1 receptor is localized in cell nuclear fraction in human breast. ▶ VPAC1 receptor is functional in plasma membrane and nucleus from breast cancer cells.Vasoactive intestinal peptide (VIP) and its receptors (VPACs) are involved in proliferation, survival, and differentiation in human breast cancer cells. Its mechanism of action is traditionally thought to be through specific plasma membrane receptors. There is compelling evidence for a novel intracrine mode of genomic regulation by G-protein-coupled receptors (GPCRs) that implies both endocytosis and nuclear translocation of peripheral GPCR and/or the activation of nuclear-located GPCRs by endogenously-produced, non-secreted ligands. Regarding to VPAC receptors, which are GPCRs, there is only a report suggesting them as a dynamic system for signaling from plasma membrane and nuclear membrane complex. In this study, we show that VPAC1 receptor is localized in cell nuclear fraction whereas VPAC2 receptor presents an extranuclear localization and its protein expression is lower than that of VPAC1 receptor in human breast tissue samples. Both receptors as well as VIP are overexpressed in breast cancer as compared to non-tumor tissue. Moreover, we report the markedly nuclear localization of VPAC1 receptors in estrogen-dependent (T47D) and independent (MDA-MB-468) human breast cancer cell lines. VPAC1 receptors are functional in plasma membrane and nucleus as shown by VIP stimulation of cAMP production in both cell lines. In addition, VIP increases its own intracellular and extracellular levels, and could be involved in the regulation of VPAC1-receptor traffic from the plasma membrane to the nucleus. These results support new concepts on function and regulation of nuclear GPCRs which could have an impact on development of new therapeutic drugs.
Keywords: VIP receptors; Nuclear VPAC receptors; Human breast cancer; cAMP;

▶ VIP elevates SP-A expression in ATII cells. ▶ C-Fos protein is essential for VIP induced SP-A expression in ATII cells. ▶ Activation of c-Fos expression by PKC is required for VIP induced SP-A expression.Vasoactive intestinal peptide (VIP) is a major neurotransmitter in the lungs and regulates many aspects of pulmonary functions. Pulmonary surfactant (PS), a complex mixture of lipids and proteins, produced by the alveolar type II (ATII) cells maintains alveolar integrity and plays important roles in the control of host defense and inflammation in the lungs. Surfactants deficiency or dysfunction is associated with occurrence and development of many pulmonary diseases. We reported previously that VIP enhanced the synthesis of pulmonary surfactants phospholipid in ATII cells. In this study, the effect of VIP on the expression of pulmonary surfactant protein A (SP-A) in lung explants was investigated. Firstly, we found that VIP elevated SP-A expression in ATII cells which was mediated by enhanced sp-a gene transcription. Furthermore, we identified that c-Fos protein was essential for VIP induced SP-A expression in ATII cells. Finally, we provided evidence to show that activation of c-Fos expression by PKC was required for VIP induced SP-A expression. Altogether, our work showed that VIP regulated the function of pulmonary surfactant system in the lungs and further investigation of the underlying mechanism would provide important clues for better therapeutic strategy design for pulmonary disorders caused by surfactant deficiency.
Keywords: Vasoactive intestinal peptide; Pulmonary surfactant; Pulmonary surfactant protein A; c-Fos; PKC;

Substance P (SP) and vasoactive intestinal polypeptide (VIP) in the lower uterine segment in first and repeated cesarean sections by Antonio Malvasi; Andrea Tinelli; Carlo Cavallotti; Stefano Bettocchi; Gian Carlo Di Renzo; Michael Stark (2052-2059).
▶ The SP amount was higher in repeat CS scars, while the VIP amount was lower. ▶ This could be due to the inflammatory activity of SP, so its increase is likely to affect the recovery of the inflamed tissue in CS scars. ▶ The reduction of VIP in the repeat CS scars could be related to its anti-inflammatory activity, so its decrease is likely to affect the recovery of the inflamed tissue in CS scars.The authors studied the presence of substance P (SP) and vasoactive intestinal polypeptide (VIP) and their related fibers in the lower uterine segment (LUS) in 133 women undergoing cesarean sections (CS) during active labor. These were divided into 2 groups: women undergoing repeat or first CSs. Specimens were collected from the LUS and were evaluated by light microscopy and by immunohistochemistry, for the morphometrical quantification of the SP and VIP fibers in the LUS. The SP amount was higher in the post-CS scar, while the VIP amount decreased: nerve fibers contained an SP amount of up to 13 ± 2.6 C.U., while nerve fibers contained a VIP amount of up to 7 ± 1.9 C.U. The SP amount counts 10 ± 1.5% of the total Bodian fibers, and the ratio of the VIP is 10 ± 1.8% of their total amount. In normal conditions only 6.61 C.U. of the Bodian surface is occupied by SP related nerve fibers in contrast to 6.63 C.U. of the total surface by VIP; the amount of SP increased up to 13 ± 2.6 C.U., while it decreased in the LUS previous scars. The SP levels are higher in repeat CS, while the VIP levels are reduced in the LUS. The increase of SP is probably linked to the attempt to achieve cervical ripening in a post-CS LUS, with the possible consequences of dystocia during vaginal birth after CS. Nevertheless, the decrease of VIP probably affects the relaxation of the internal uterine orifice, compromising the LUS formation and cervical ripening.
Keywords: Cesarean section; Substance P (SP); Vasoactive intestinal polypeptide (VIP); Labor; Neurotransmitters; Neuropeptides; Delivery; VBAC; Misgav Ladach cesarean section;

▶ L-type Ca2+ channel agonist stimulates Ca2+ influx into smooth muscle cells. ▶ Trp-His prevents the elevation of intracellular Ca2+ concentration. ▶ Trp-His binds to the extracellular site of L-type Ca2+ channel proteins.Our previous findings regarding the biological activities of small peptides revealed that a di-peptide, Trp-His (WH), could play a role in the prevention of vascular lesions, including cell proliferation and atherosclerosis. Its vasoprotective effects could be associated with suppression of the vasocontraction signaling cascade, but the underlying mechanism(s) remains obscure. In this study, we attempted to elucidate the vasoprotective mechanism of WH, in opposing the proliferation of rat vascular smooth muscle cells (VSMCs). In VSMCs from 8 week-old male Wistar rat thoracic aortae, WH evoked a significant dose-dependent anti-proliferation effect, without cytotoxicity. In mitogen-stimulated cell experiments, 300 μM WH inhibited cytosolic Ca2+ elevation in VSMCs induced by 10 μM angiotensin II (Ang II). Furthermore, WH suppressed extracellular Ca2+ entry into CaCl2-stimulated VSMCs. The biological capacity of WH as an intracellular Ca2+ ([Ca2+]i) suppressor was also proven when 50 μM Bay K8644 was used to enhance Ca2+ entry via a voltage-dependent l-type Ca2+ channel (VDCC) and 300 μM WH elicited a 23% reduction in [Ca2+]i. The absence of a reduction of the [Ca2+]i by the mixture of tryptophan and histidine revealed the importance of the peptide backbone in the [Ca2+]i reduction effect. Furthermore, the WH-induced [Ca2+]i reduction was abolished by verapamil, but not by nifedipine, indicating that WH likely binds to an extracellular site of the VDCC at a site similar to that of the dihydropyridine type-Ca2+ channel blockers.
Keywords: Ca2+ channel blocker; Small peptide; Smooth muscle cell; Anti-proliferation; Anti-atherosclerosis;

Human RFamide-related peptide-1 diminishes cellular and integrated cardiac contractile performance by R. Nichols; L.A. Demers; B.M. Larsen; D. Robinson; K. Converso; M.W. Russell; M.V. Westfall (2067-2074).
▶ Human RFRP-1 peptide (hRFRP-1) dramatically decreases contractility in isolated cardiac myocytes. ▶ The structurally-related peptide 26RFa does not affect contractility in isolated cardiac myocytes. ▶ Pertussis toxin did not affect the influence of hRFRP-1 on cardiac function in isolated cardiac myocytes suggesting the peptide activates PKC to impact contractility. ▶ Human RFRP-1 decreases cardiac function in whole animal as well as in the isolated cardiac myocyte.Peptides influence cardiac dysfunction; however, peptidergic modulation of contractile performance remains relatively uncharacterized. We identified a novel human peptide that modulates mammalian contractile performance. Members of the FMRFamide-related peptide (FaRP) family contain a C-terminal RFamide but structurally variant N-terminal extension. We report human RFamide-related peptide-1 (hRFRP-1) and rat RFRP-1 rapidly and reversibly decreased shortening and relaxation in isolated mammalian cardiac myocytes in a dose dependent manner. The mammalian FaRP, 26RFa, structurally related to RFRP-1 by only an RFamide did not influence myocyte contractile function. The protein kinase C (PKC) inhibitor bisindolylmaleimide-1 blocked hRFRP-1 activity. Pretreatment with pertussis toxin (PTX) did not diminish hRFRP-1 influence on contractile function. In addition, intravenous injection of hRFRP-1 in mice decreased heart rate, stroke volume, ejection fraction, and cardiac output. Collectively these findings are consistent with the conclusion RFRP-1 is an endogenous signaling molecule that activates PKC and acts through a PTX-insensitive pathway to modulate cardiac contractile function. Taken together these negative chronotropic, inotropic, and lusitropic effects of hRFRP-1 are significant; they suggest direct acute cellular and organ-level responses in mammalian heart. This is the first known study to identify a mammalian FaRP with cardio-depressant effects, opening a new area of research on peptidergic modulation of contractile performance. The high degree of RFRP structure conservation from amphibians to mammals, and similarity to invertebrate cardioinhibitory peptides suggests RFRP-1 is involved in important physiological functions. Elucidation of mechanisms involved in hRFRP-1 synthesis, release, and signaling may aid the development of strategies to prevent or attenuate cardiac dysfunction.
Keywords: 26RFa; Brain; Cardiovascular; Drosophila; FMRFamide; Heart; Myosuppressin; Protein kinase C (PKC);

Urotensin II alters vascular reactivity in animals subjected to volume overload by Gregory S. Harris; Robert M. Lust; Laxmansa C. Katwa; Christopher J. Wingard (2075-2082).
▶ Volume overload alters vascular reactivity and up regulates urotensin II. ▶ Changes in thoracic aorta reactivity were sensitive to Rho-kinase inhibition. ▶ Sensitivity changes were opposite of UTII receptor or Rho-kinase protein expression.Congestive heart failure (CHF) alters vascular reactivity and up regulates in urotensin II (UTII), a potent vasoactive peptide. The aim of this study was to investigate the interaction between CHF and UTII in altering vascular reactivity in a rat model of volume overload heart failure. Animals were divided into 4 groups: control, UTII infused (UTII), volume overload only (VO) or volume overload + UTII (VO + UTII). Volume overload was established by the formation of an aortocaval fistula. Following fistula formation animals were administered UTII at a rate of 300 pmol/kg/h for 4 weeks subcutaneously with mini-osmotic pumps. Thoracic aorta rings, with/without endothelium, were subjected to cumulative dose–responses to phenylephrine, sodium nitroprusside (SNP), acetylcholine (ACH), UTII, and the Rho-kinase inhibitor HA-1077. Aortas from VO animals exhibited increased sensitivity to phenylephrine and UTII with a decreased relaxation response to ACH and HA-1077. Aortas from animals subjected to chronic UTII with volume overload (VO + UTII) retained their sensitivity to phenylephrine and UTII while they improved their relaxation to HA-1077 but not ACH. The constrictive response to UTII was dose-dependent and augmented at concentrations <0.01 μM in VO animals. The changes in vascular reactivity paralleled an elevation of both the UTII and α1A-adrenergic receptor while the Rho and Rho-kinase signalling proteins were diminished. We found that volume overload increased sensitivity to the vasoconstrictor agents that was inversely related to changes in the Rho-kinase expression. The addition of UTII with VO reversed the constrictive vascular response through alterations in the Rho-kinase signalling pathway.
Keywords: Vascular smooth muscle; Endothelial dependent relaxation; Rho-kinase; Thoracic aorta; Rat;

A simple approach for the preparation of mature human relaxin-3 by Xiao Luo; Ya-Li Liu; Sharon Layfield; Xiao-Xia Shao; Ross A.D. Bathgate; John D. Wade; Zhan-Yun Guo (2083-2088).
Display Omitted▶ A single-chain precursor of relaxin-3 with a mini C-peptide and a 6×His tag was designed. ▶ The precursor was expressed well in E. coli cells. ▶ After purification, in vitro refolding, and enzymatic digestion, fully active human relaxin-3 was obtained in high yield and at low cost. ▶ Our present work provides a highly efficient approach for the preparation of relaxin-3 as well as its analogues for functional and structural analyses.Relaxin-3 (also known as INSL7) is the most recently identified member of the insulin-like family. It is predominantly expressed in the nucleus incertus of the brain and involved in the control of stress response, food intake, and reproduction. In the present work, we have established a simple approach for the preparation of the mature human relaxin-3 peptide. We first designed and recombinantly expressed a single-chain relaxin-3 precursor in E. coli cells. After purification by immobilized metal ion affinity chromatography, refolding in vitro through disulfide reshuffling, and digestion by endoproteinase Asp-N, mature human relaxin-3 was obtained in high yield and at low cost. Peptide mapping and circular dichroism spectroscopy studies suggested that the recombinant relaxin-3 adopted an insulin-like fold with the expected disulfide linkages. The recombinant mature relaxin-3 was fully active in both RXFP3 binding and activation assays. The activity of the single-chain precursor was very low, suggesting that a free C-terminus of the B-chain is necessary for receptor-binding and activation of relaxin-3. Our present work provides a highly efficient approach for the preparation of relaxin-3 as well as its analogues for functional and structural analyses.
Keywords: Insulin-like peptide; Recombinant expression; Relaxin-3;

Diurnal changes of arginine vasopressin-enhanced green fluorescent protein fusion transgene expression in the rat suprachiasmatic nucleus by Takashi Maruyama; Toyoaki Ohbuchi; Hiroaki Fujihara; Minori Shibata; Koji Mori; David Murphy; Govindan Dayanithi; Yoichi Ueta (2089-2093).
▶ AVP-eGFP transgenic rats can be used to observe diurnal AVP gene expression. ▶ AVP-eGFP gene expression can be used to observe the response to a light stimulus. ▶ A single AVP-eGFP neuron isolated from the SCN is used for electrophysiology.We have recently developed a new transgenic rat line expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The AVP-eGFP transgene is expressed in the paraventricular (PVN) and supraoptic (SON) nuclei and the suprachiasmatic nucleus (SCN) of the hypothalamus. Transgene expression in the PVN and SON showed an exaggerated response to salt loading and nociceptive stimulation. However, the expression of the AVP-eGFP transgene in the SCN did not change under these stressful conditions. Here, we examined daily profiles of the expression of the AVP-eGFP transgene in the SCN in comparison with the endogenous AVP and Period (Per1 and Per2) genes. While all of these genes elicited diurnal patterns of expression in the SCN, the rate of rhythmic change of transgene expression was significantly greater than that of the endogenous AVP gene. We also examined the effect of a light stimulus on the expression of the AVP-eGFP, AVP, Per1 and Per2 genes in the SCN of transgenic rats. Ninety minutes after a light stimulus, AVP-eGFP mRNA and AVP hnRNA levels in the SCN were significantly decreased, while Per2 mRNA levels were significantly increased. In addition, we observed the eGFP fluorescence in the SCN and recorded the electrophysiological properties of a dissociated SCN eGFP-positive neuron. The AVP-eGFP transgenic rat is a useful animal model to study the diurnal change and dynamics of the AVP system, and enables the facile identification of SCN AVP neurons both in vivo and in vitro.
Keywords: Hypothalamus; GFP; Period gene; Transgenic rats; Suprachiasmatic nucleus; Arginine vasopressin;

Neuroprotective effects of stanniocalcin 2 following kainic acid-induced hippocampal degeneration in ICR mice by Jong-Seon Byun; Jae-Won Lee; Su Young Kim; Kwang Jun Kwon; Jong-Hee Sohn; Kyunyoung Lee; Dahlkyun Oh; Sung-Soo Kim; Wanjoo Chun; Hee Jae Lee (2094-2099).
▶ STC2 attenuates KA-induced neuronal death. ▶ STC2 attenuates KA-induced microglial activation. ▶ STC2 attenuates KA-induced HO-1 expression in the glial cells. ▶ STC2 inhibits expression levels of NO, TNF-α, and IL-1β in LPS-stimulated BV2 cells.Stanniocalcin 2 (STC2), the paralog of STC1, has been shown to act as a novel target of the mammalian unfolded protein response. We investigated the potential neuroprotective actions of STC2 against kainic acid toxicity in the hippocampus of ICR mice. STC2-treated mice experienced less neuronal cell loss in the CA3 area of the hippocampus. Also, microglial activation and heme oxygenase 1 expression were attenuated in the hippocampus of STC2-treated mice. To confirm whether STC2 regulates microglial activation directly, nitric oxide levels were measured in BV2 cells cultured with and without 10 nM STC2. STC2 decreased the level of nitric oxide induced by lipopolysaccharide (LPS) treatment significantly. Also, STC2 pretreatment significantly decreased TNF-α and IL-1β expression induced by LPS treatment. These observations demonstrated that STC2 exerts neuroprotective actions against excitotoxic insults through the inhibition of microglial activation.
Keywords: Stanniocalcin 2; Kainic acid; Microglial activation; BV2 cells;

Molecular dynamics simulations of Aβ fibril interactions with β-sheet breaker peptides by Neil J. Bruce; Deliang Chen; Shubhra G. Dastidar; Gabriel E. Marks; Catherine H. Schein; Richard A. Bryce (2100-2108).
▶ LPFFD is correctly predicted via computation to bind more tightly to Aβ fibril than LHFFD. ▶ Pro2 in LPFFD restricts its shape to fit Aβ fibril topology. ▶ Asp5 in LPFFD forms a key interaction with Aβ fibril Lys16 side chain.Accumulation and aggregation of the 42-residue amyloid-β (Aβ) protein fragment, which originates from the cleavage of amyloid precursor protein by β and γ secretase, correlates with the pathology of Alzheimer's disease (AD). Possible therapies for AD include peptides based on the Aβ sequence, and recently identified small molecular weight compounds designed to mimic these, that interfere with the aggregation of Aβ and prevent its toxic effects on neuronal cells in culture. Here, we use molecular dynamics simulations to compare the mode of interaction of an active (LPFFD) and inactive (LHFFD) β-sheet breaker peptide with an Aβ fibril structure from solid-state NMR studies. We found that LHFFD had a weaker interaction with the fibril than the active peptide, LPFFD, from geometric and energetic considerations, as estimated by the MM/PBSA approach. Cluster analysis and computational alanine scanning identified important ligand–fibril contacts, including a possible difference in the effect of histidine on ligand–fibril π-stacking interactions, and the role of the proline residue in establishing contacts that compete with those essential for maintenance of the inter-monomer β-sheet structure of the fibril. Our results show that molecular dynamics simulations can be a useful way to classify the stability of docking sites. These mechanistic insights into the ability of LPFFD to reverse aggregation of toxic Aβ will guide the redesign of lead compounds, and aid in developing realistic therapies for AD and other diseases of protein aggregation.
Keywords: Alzheimer's disease; Amyloid-β; Molecular dynamics simulation; Replica exchange molecular dynamics; β-Blocker peptides; Aggregation inhibitors;

Ghrelin and obestatin promote the allergic action in rat peritoneal mast cells as basic secretagogues by Tatsuya Hirayama; Tsutomu Kawabe; Miyoko Matsushima; Yuko Nishimura; Yuko Kobe; Yui Ota; Kenji Baba; Kenzo Takagi (2109-2113).
▶ Ghrelin and obestatin induce histamine release from mast cells as basic secretagogues. ▶ Ghrelin- and obestatin-induced histamine release is mediated via Gαi. ▶ Ghrelin stimulates mast cells independent of GHSR.Ghrelin is an endogenous ligand of the type 1a growth hormone secretagogue receptor (GHSR1a) that regulates energy balance. Ghrelin and obestatin, derived from the post-translational processing of preproghrelin, are involved in a diverse range of biological activities, yet their effect on the immune system is not fully understood. In the present study, we investigated the roles of ghrelin and obestatin on mast cell degranulation and found that both ghrelin and obestatin induce the release of histamine from rat peritoneal mast cells. This induced histamine release was inhibited by the pertussis toxin, an inhibitor of Gαi protein, and extracellular Ca2+. Rat peritoneal mast cells and rat basophilic leukemia (RBL-2H3) cells did not express the ghrelin receptor GHSR1a, suggesting that histamine release induced by ghrelin occurs via a receptor-independent mechanism. We report here that ghrelin and obestatin, belonging to the family of basic secretagogues, stimulate mast cells independent of a receptor, and this may play a crucial role at the site of allergy or inflammation.
Keywords: Ghrelin; Obestatin; Mast cells; Basic secretagogues; Histamine;

Protective effect of ghrelin on acetaminophen-induced liver injury in rat by Masoumeh Golestan Jahromi; Fatemeh Nabavizadeh; Jalal Vahedian; Hossein Nahrevanian; Ahmad-Reza Dehpour; Ali Zare-Mehrjardi (2114-2117).
▶ Acetaminophen activated the cascade of pro-inflammatory cytokine in the liver. ▶ Liver necrosis and liver failure occurs. ▶ Ghrelin prevention of production of cytokine. ▶ Ghrelin inhibits inflammatory response and cell death in the liver.Ghrelin is a peptide that has protective effects on many tissues of the body. It has anti-inflammatory and anti-oxidant effects. Acetaminophen, a commonly used analgesic-antipyretic drug, has hepatotoxic side effects. The aim of this study was to evaluate the protective role of ghrelin in liver toxicity due to acetaminophen overdose. Thirty male rats were used in this study and divided into five groups. They were control, propylene-glycol (as a solvent of acetaminophen), acetaminophen, acetaminophen and NAC, acetaminophen and ghrelin groups. Tumor necrosis factor alpha (TNF-α), and hepatic enzymes, AST (aspartate aminotransferase) and ALT (alanine aminotransferase), were assessed and histologic study of liver were performed as indicators of liver damage following acetaminophen toxicity. Results showed that Ghrelin decreased ALT and AST to the normal level, and also reduced TNF-α. Although NAC (the standard antidote of acetaminophen toxicity) also reduced ALT, AST and TNF-α levels, our results show that ghrelin is more potent than NAC in protecting the liver from acetaminophen-induced liver injury.
Keywords: Ghrelin; Acetaminophen toxicity; Liver injury; Tumor necrosis factor alpha (TNF-α);

Central orexin-A increases gastric motility in rats by Mehmet Bülbül; Reji Babygirija; Kirk Ludwig; Toku Takahashi (2118-2122).
▶ Orexin-A modulates postprandial gastric contractions in non-stressed conditions. ▶ Endogenous central orexin-A mediates augmented gastric motility induced by acute restraint stress. ▶ Central endogenous orexin-A mediates predominantly stress-induced augmented motility via central orexin receptor type-1 (OX1R).Orexin receptor type-1 (OX1R) is expressed in the dorsal motor nucleus of vagi (DMV). Although orexin-A (OXA) plays an important role in mediating stress responses, it remains unclear how central OXA regulates gastric dysmotility induced by stress. Acute restraint stress (ARS) delays solid gastric emptying via the central corticotropin releasing factor (CRF) and peripheral autonomic neural pathways. We have previously shown that ARS impairs postprandial antro-pyloric coordination and delays solid gastric emptying in rats. We also showed that postprandial gastric contractions were augmented in response to ARS in rats. However, the mechanism of augmented postprandial gastric contractions induced by ARS remains unclear. We tested the hypothesis that augmented gastric motility induced by ARS is mediated via the central OX1R. We also assessed the role of endogenous OXA in the mediation of gastric motility under non-stressed conditions in conscious rats. A strain gauge transducer was implanted on the antrum to record postprandial gastric motility. To investigate whether endogenous OXA is involved in ARS-induced augmented gastric motility, selective OX1R antagonist, SB-334867 (16 μg), was administered intracerebroventricularly (icv). Icv-injection of SB-334867 abolished the augmented gastric contractions induced by ARS. Spontaneous postprandial gastric motility was enhanced by icv-injection of OXA (10 μg), while it was attenuated by icv-injection of SB-3334867. It is suggested that central OXA mediates augmented gastric motility induced by ARS in rats. Central OXA also modulates postprandial gastric contractions in non-stressed conditions.
Keywords: Hypocretin; Restraint stress; Dorsal motor nucleus of vagi (DMV); Corticotropin releasing factor (CRF); Gastric emptying; Antro-pyloric coordination;

Post-natal stress-induced endocrine and metabolic alterations in mice at adulthood involve different pro-opiomelanocortin-derived peptides by Stefano Loizzo; Gabriele Campana; Stefano Vella; Andrea Fortuna; Gabriella Galietta; Irene Guarino; Loredana Costa; Anna Capasso; Paolo Renzi; Giovanni V. Frajese; Flavia Franconi; Alberto Loizzo; Santi Spampinato (2123-2129).
▶ Post-natal complex stress induce type-2 diabetes signs in adult male mice. ▶ Some signs depend on the endogenous opioid system upset and are prevented by naloxone. ▶ Other diabetes signs depend on HPA hormones upset. ▶ All diabetes signs are prevented by antisense oligonucleotide versus pro-opiomelanocortin. ▶ Different diabetes signs in our model are modulated through different pathogenetic mechanisms.In previous investigations we added a physical stress (mild pain) to the “classical” post-natal psychological stress in male mice, and we found that this combination produced a series of dysmetabolic signs very similar to mild human type-2 diabetes. Here, for the first time we demonstrate that within this diabetes model at least two groups of signs depend on the unbalance of two different endogenous systems. Newborn male mice were daily exposed to stressful procedures for 21 days (brief mother separation plus sham injection). Other groups underwent the same procedure, and also received naloxone (Na) to block μ–δ endogenous receptors, or a phosphorothioate antisense oligonucleotide (AS) directed against pro-opiomelanocortin (POMC)-mRNA [to block adrenocorticotropin (ACTH)- and POMC-derived opioid peptides]. Adult mice which received only post-natal stress increased body weight (+7.5%), abdominal overweight (+74%), fasting glycemia (+43%), plasma corticosterone (+110%), plasma (+169%) and pituitary (+153%) ACTH levels. Conversely, hypothalamic ACTH and corticotropin-releasing hormone (CRH) were reduced (−70% and −75%, respectively). Neonatal AS administration reverted all parameters to control values. Neonatal naloxone had little or no influence on glucose, corticosterone, ACTH, CRH levels, whereas it prevented body overweight and abdominal overweight. We conclude that, within this type-2 diabetes model in male mice at least two endocrino-neurohumoral systems are damaged, one concerning the opioid system, and the other concerning HPA hormones. The use of the two drugs was of primary importance to demonstrate this statement, and to demonstrate that these two groups of signs could be defined as “separate entities” following our complex post-natal stress model.
Keywords: Psychological stress; Pain stress; Pro-opiomelanocortin; Hypothalamus–pituitary–adrenal (HPA) hormones; Endogenous opioid system; Antisense oligonucleotide (AS);

▶ IP injection of CCK-8s induces anorexigenic action in goldfish. ▶ CCK-8s-induced anorexigenic action is blocked by treatments with capsaicin or the CRH receptor antagonist. ▶ POMC mRNA level in the diencephalon is increased after treatment with CCK-8s. ▶ Anorexigenic action of CCK-8s is mediated by the vagal afferent and subsequently through the melanocortin- and CRH-signaling pathways.We have been extensively investigating the mechanisms by which neuropeptides regulate feeding behavior by using a goldfish (Carassius auratus) model. In this species, the anorexigenic action of melanocortin peptide is centrally mediated via the corticotropin-releasing hormone (CRH)/CRH receptor neuronal system, whereas sulfated cholecystokinin octapeptide (CCK-8s) is involved in the appetite regulation as a peripheral anorexigenic factor. The aim of the present study was to identify the mechanism of the anorexigenic effect of peripherally injected CCK-8s, which has not yet been identified in goldfish. Co-administration of capsaicin, a neurotoxin that destroys primary sensory afferents, at 100 nmol/g BW, blocked the anorexigenic action of intraperitoneally injected CCK-8s (100 pmol/g BW), whereas the anorexigenic action of intracerebroventricularly injected CCK-8s (5 pmol/g BW) was not blocked by co-administration of capsaicin. Pre-treatment with a specific CRH receptor antagonist, α-helical CRH(9–41), attenuated the anorexigenic action of CCK-8s. The expression level of CRH mRNA in the diencephalic tissue of the CCK-8s-injected group was not changed, but the level of proopiomelanocortin mRNA was significantly increased at 1 h after treatment. Therefore, we have identified for the first time that the reduction of appetite induced by peripherally injected CCK-8s in goldfish appears to be mediated by the vagal afferent and subsequently through the melanocortin- and corticotropin-releasing hormone-signaling pathways.
Keywords: Feeding behavior; Sulfated cholecystokinin octapeptide; Vagal afferent; Corticotropin-releasing hormone; Proopiomelanocortin; Anorexigenic action; Goldfish;

Peripheral antinociceptive effects of the cyclic endomorphin-1 analog c[YpwFG] in a mouse visceral pain model by Andrea Bedini; Monica Baiula; Luca Gentilucci; Alessandra Tolomelli; Rossella De Marco; Santi Spampinato (2135-2140).
▶ The endomorphin-1 analog c[YpwFG] binds preferentially to mu2-opioid receptors. ▶ c[YpwFG] is active against visceral pain acting through peripheral opioid receptors. ▶ c[YpwFG] is resistant to peptidases and is a new tool to treat visceral pain.We previously described a novel cyclic endomorphin-1 analog c[Tyr-D-Pro-D-Trp-Phe-Gly] (c[YpwFG]), acting as a mu-opioid receptor (MOR) agonist. This study reports that c[YpwFG] is more lipophilic and resistant to enzymatic hydrolysis than endomorphin-1 and produces preemptive antinociception in a mouse visceral pain model when injected intraperitoneally (i.p.) or subcutaneously (s.c.) before 0.6% acetic acid, employed to evoke abdominal writhing (i.p. ED50  = 1.24 mg/kg; s.c. ED50  = 2.13 mg/kg). This effect is reversed by the selective MOR antagonist β-funaltrexamine and by a high dose of the mu1-opioid receptor-selective antagonist naloxonazine. Conversely, the kappa-opioid receptor antagonist nor-binaltorphimine and the delta-opioid receptor antagonist naltrindole are ineffective. c[YpwFG] produces antinociception when injected i.p. after acetic acid (ED50  = 4.80 mg/kg), and only at a dose of 20 mg/kg did it elicit a moderate antinociceptive response in the mouse, evaluated by the tail flick assay. Administration of a lower dose of c[YpwFG] (10 mg/kg i.p.) apparently produces a considerable part of antinociception on acetic acid-induced writhes through peripheral opioid receptors as this action is fully prevented by i.p. naloxone methiodide, which does not readily cross the blood–brain barrier; whereas this opioid antagonist injected intracerebroventricularly (i.c.v.) is not effective. Antinociception produced by a higher dose of c[YpwFG] (20 mg/kg i.p.) is partially reversed by naloxone methiodide i.c.v. administered. Thus, only at the dose of 20 mg/kg c[YpwFG] can produce antinociception through both peripheral and central opioid receptors. In conclusion, c[YpwFG] displays sufficient metabolic stability to be effective after peripheral administration and demonstrates the therapeutic potential of endomorphin derivatives as novel analgesic agents to control visceral pain.
Keywords: Endomorphin-1; Antinociception; Visceral pain; mu-Opioid receptor; Naloxone methiodide;

α-Melanocyte-stimulating hormone (α-MSH) reverses impairment of memory reconsolidation induced by interleukin-1 beta (IL-1 beta) hippocampal infusions by Ivana Machado; Patricia González; Helgi Birgir Schiöth; Mercedes Lasaga; Teresa Nieves Scimonelli (2141-2144).
▶ IL-1β has a detrimental effect on reconsolidation of contextual memory. ▶ Memory impairment induced by IL-1β is reversed by α-MSH. ▶ α-MSH may exert this protective effect by activating brain MC4-R.Interleukin-1 beta (IL-1β) significantly influences cognitive processes. Treatments which raise the level of IL-1β in the brain impair memory consolidation in contextual fear conditioning. However, the effect of IL-1β on memory reconsolidation has not yet been established. The melanocortin α-melanocyte-stimulating hormone (α-MSH) exerts potent anti-inflammatory actions by antagonizing the effect of proinflammatory cytokines. Five subtypes of melanocortin receptors (MC1R–MC5R) have been identified, of which MC3R and MC4R are predominant in the central nervous system. The present experiments show that the injection of IL-1β (5 ng/0.25 μl) in dorsal hippocampus up to 30 min after re-exposition to the context decreases freezing during the contextual fear test. Impairment of memory reconsolidation was reversed by treatment with α-MSH (0.05 μg/0.25 μl). Administration of the MC4 receptor antagonist HS014 (0.5 μg/0.25 μl) blocked the effect of α-MSH. These results suggest that IL-1β may influence memory reconsolidation and that activation of central MC4R could lead to improve cognitive performance.

Insulin-induced gene: A new regulator in lipid metabolism by Xiao-Ying Dong; Sheng-Qiu Tang (2145-2150).
▶ Insigs play a novel role in cholesterol homeostasis. ▶ Insigs are required for lipogenesis. ▶INSIGs genetic polymorphisms are associated with obesity and hypercholesterolaemia. ▶INSIGs genetic deficiency leads to metabolic diseases.Insulin-induced genes (Insigs) including Insig-1 and Insig-2, are proteins that mediate sterol regulation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). Insigs perform distinct tasks in the regulation of these effectors: they promote the endoplasmic reticulum (ER) retention of SCAP, but ubiquitin-mediated degradation of HMG-CoA reductase. Through these activities, Insig-1 and Insig-2 influence cholesterol metabolism, lipogenesis, and glucose homeostasis in diverse tissues such as adipose tissue and liver. In this article, we focus on the functions, expression and regulation, gene polymorphisms of Insigs, and their deficiency with diseases.
Keywords: Insigs; SCAP; HMG-CoA reductase; Cholesterol metabolism; Lipogenesis; Glucose homeostasis; Disease;

▶ Thymosin alpha 1 (Tα1) has a lot of biological activities. ▶ Tα1 has a broad spectrum of applications in clinic. ▶ Genetic engineering expression of Tα1 has attracted more attention recently. ▶ Tα1 products obtained by genetic engineering may be applied in clinic in the future.Thymosin alpha 1 (Tα1), a 28-amino acid peptide, was first described and characterized from calf thymuses in 1977. This peptide can enhance T-cell, dendritic cell (DC) and antibody responses, modulate cytokines and chemokines production and block steroid-induced apoptosis of thymocytes. Due to its pleiotropic biological activities, Tα1 has gained increasing interest in recent years and has been used for the treatment of various diseases in clinic. Accordingly, there is an increasing need for the production of this peptide. So far, Tα1 used in clinic is synthesized using solid phase peptide synthesis. Here, we summarize the genetic engineering methods to produce Tα1 using prokaryotic or eukaryotic expression systems. The effectiveness of these biological products in increasing the secretion of cytokines and in promoting lymphocyte proliferation were investigated in vitro studies. This opens the possibility for biotechnological production of Tα1 for the research and clinical applications.
Keywords: Thymosin alpha 1; Biological activities; Applications; Genetic engineering production;