Peptides (v.38, #2)
Gayle Richard Olson prize pages (III-IV).
Editorial Board (CO2).
The antimicrobial peptide, shrimp anti-lipopolysaccharide factor (SALF), inhibits proinflammatory cytokine expressions through the MAPK and NF-κB pathways in Trichomonas vaginalis adherent to HeLa cells by Ming-Ching Lin; Cho-Fat Hui; Jyh-Yih Chen; Jen-Leih Wu (197-207).
► Shrimp anti-lipopolysaccharide factor (SALF) inhibits proinflammatory cytokine expressions. ► SALF inhibits through the MAPK and NF-κB pathways to inhibit gene expression. ► SALF downregulated the release of proinflammatory cytokines secreted by T. vaginalis-infected HeLa cells. Trichomonas vaginalis is a parasitic protozoan that causes sexually transmitted infections (STIs) worldwide. The infection is dangerous and easily spreads within a community. Also, some cases of drug resistance were reported. Previously, we reported that the shrimp anti-lipopolysaccharide factor (SALF), an antimicrobial peptide of 24 amino acids, modulates inflammatory responses and inhibits T. vaginalis growth. To date, there is no report on the mechanism of SALF's actions in T. vaginalis’ adherence to HeLa cells. In this research using an ELISA, we found that the SALF downregulated the release of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1) secreted by T. vaginalis which was adhering to HeLa cells. We also performed real-time PCR experiments to determine the roles of the SALF in the expressions of several proinflammatory genes. Through a Western blot analysis, we determined that SALF treatment inhibited T. vaginalis-treated HeLa cells through the p38 and NF-κB pathways. Furthermore, we used different inhibitors to confirm the pathway by ELISA and Western blotting. Taken together, it is apparent that the SALF suppresses T. vaginalis-induced secretion of proinflammatory cytokines by HeLa cells by acting through the p38 and NF-κB pathways.
Keywords: Antimicrobial peptide; SALF; T. vaginalis; Inflammatory response;
Mycobacterium tuberculosis surface protein Rv0227c contains high activity binding peptides which inhibit cell invasion by Diana Marcela Rodríguez; Marisol Ocampo; Hernando Curtidor; Magnolia Vanegas; Manuel Elkin Patarroyo; Manuel Alfonso Patarroyo (208-216).
► This study has revealed high activity binding peptides, from Rv0227c, for U937 and A549 cells. ► It was found HABPs which were able to inhibit mycobacterial entry to target cells. ► The results have shown Rv0227c localization on mycobacterial surface. ► Rv0227c HABPs are peptide candidates for inclusion in an anti-tuberculosis vaccine. Mycobacterium tuberculosis surface proteins involved in target cell invasion may be identified as a strategy for developing subunit-based, chemically-synthesized vaccines. The Rv0227c protein was thus selected to assess its role in the invasion and infection of Mycobacterium tuberculosis target cells. Results revealed Rv0227c localization on mycobacterial surface by immunoelectron microscopy and Western blot. Receptor–ligand assays using 20-mer, non-overlapping peptides covering the complete Rv0227c protein sequence revealed three high activity binding peptides for U937 phagocytic cells and seven for A549 cells. Peptide 16944 significantly inhibited mycobacterial entry to both cell lines while 16943 and 16949 only managed to inhibit entrance to U937 cells and 16951 to A549 cells. The Jnet bioinformatics tool predicted secondary structure elements for the complete protein, agreeing with elements determined for such chemically-synthesized peptides. It was thus concluded that high activity binding peptides which were able to inhibit mycobacterial entry to target cells are of great importance when selecting peptide candidates for inclusion in an anti-tuberculosis vaccine.
Keywords: Mycobacterium tuberculosis; Rv0227c protein; Synthetic peptide; Invasion; Vaccine; Receptor–ligand interaction;
Ghrelin protects against cobalt chloride-induced hypoxic injury in cardiac H9c2 cells by inhibiting oxidative stress and inducing autophagy by Xin-Xin Tong; Dan Wu; Xue Wang; Hua-Li Chen; Jia-Xiang Chen; Xiao-Xiao Wang; Xu-Lei Wang; Lu Gan; Zhi-Yun Guo; Gui-Xiu Shi; Yi-Zheng Zhang; Wei Jiang (217-227).
► It has been observed that the protection of ghrelin in H9c2 cells against hypoxic injury induced by CoCl2 insult. ► It has been observed that CoCl2 insult significantly increased autophagy in H9c2 cells. ► It has been revealed that ghrelin decreased intracellular reactive oxygen species content by inhibiting NADPH oxidases 1 and increasing the mRNA expression and activities of catalase and Mn-SOD. ► It has been proved that ghrelin induced protective autophagy in an AMPK-dependent manner.Ghrelin is a multifunctional peptide that actively protects against cardiovascular ischemic diseases, but the underlying mechanisms are unclear. We used CoCl2 to mimic hypoxic conditions in cardiac H9c2 cells in order to study the mechanism by which ghrelin protects cardiac myocytes against hypoxic injury by regulating the content of intracellular ROS and autophagy levels. Cell apoptosis and necrosis were evaluated by the flow cytometry assay, Hoechst staining, and LDH activity. Cell viability was detected by the WST-1 assay; ROS levels were assessed using DCFH2-DA; and Nox1, catalase and Mn-SOD were assayed by real-time PCR and activity assays. LC3II was measured by Western blot analysis. We observed that CoCl2 induced apoptosis and death of H9c2 cells in a dose- and time-dependent manner. This was characterized by an increase in cell apoptosis, LDH activity, ROS content, Nox1 expression, and autophagy levels and a decrease in cell viability, catalase, and Mn-SOD activities. Ghrelin treatment significantly attenuated CoCl2-induced hypoxic injury by decreasing cell apoptosis, LDH activity, ROS content, and Nox1 expression and increasing cell viability, autophagy levels, catalase, and Mn-SOD mRNA levels and activities. Further experiments revealed that inhibiting autophagy using 3-MA or AMPK pathway with compound C almost abrogated the induction of ghrelin in autophagy. This was associated with a decrease in cell viability and an increase in LDH activity. Our results indicate that ghrelin protected cardiac myocytes against CoCl2-induced hypoxic injury by decreasing Nox1 expression, increasing the expression and activity of endogenous antioxidant enzymes, and inducing protective autophagy in an AMPK-dependent manner.
Keywords: Ghrelin; Hypoxia; Autophagy; Oxidative stress;
Fruit fly behavior in response to chemosensory signals by Pilar Herrero (228-237).
► Taste perception is mediated by 68 types of gustatory receptors in Drosophila. ► Neuropeptides and metabolic hormones are involved in regulation of food intake and meal termination. ► Odorants detection is mediated by 62 types of olfactory receptors in Drosophila. ► Neuropeptides of brain modulate odor search behavior. ► Pheromone perception in Drosophila through gustatory and olfactory receptors trigger aggregation and mating behavior.An important question in contemporary sensory neuroscience is how animals perceive their environment and make appropriate behavioral choices based on chemical perceptions. The fruit fly Drosophila melanogaster exhibits robust tastant and odor-evoked behaviors. Understanding how the gustatory and olfactory systems support the perception of these contact and volatile chemicals and translate them into appropriate attraction or avoidance behaviors has made an unprecedented contribution to our knowledge of the organization of chemosensory systems. In this review, I begin by describing the receptors and signaling mechanisms of the Drosophila gustatory and olfactory systems and then highlight their involvement in the control of simple and complex behaviors. The topics addressed include feeding behavior, learning and memory, navigation behavior, neuropeptide modulation of chemosensory behavior, and I conclude with a discussion of recent work that provides insight into pheromone signaling pathways.
Keywords: Drosophila melanogaster; Tastant-evoked behavior; Odor-evoked behavior; Feeding regulation; Neuromodulation; Pheromone-driven behavior;
Teduglutide, a glucagon-like peptide 2 analogue: A novel protective agent with anti-apoptotic and anti-oxidant properties in mice with lung injury by Pelin Arda-Pirincci; Fusun Oztay; Bertan Boran Bayrak; Refiye Yanardag; Sehnaz Bolkent (238-247).
► TNF-α treatment combined with Act D makes the lung a vulnerable sensitive organ to damage. ► We demonstrated here for the first time the localization of GLP-2 receptors in the lung. ► Teduglutide is as a novel protective agent with anti-apoptotic and anti-oxidant properties against acute lung injury.Teduglutide is a long-acting synthetic analogue of human glucagon-like peptide-2 (GLP-2). GLP-2 regulates cell proliferation and apoptosis as well as normal physiology in the gastrointestinal tract. In the present study, possible cytoprotective and reparative effects of teduglutide were analyzed on a mouse model with lung injury induced by tumor necrosis factor-alpha (TNF-α) and actinomycin D (Act D). BALB/c mice were divided into six groups: control mice (I), mice injected intraperitoneally with 15 μg/kg TNF-α (II), 800 μg/kg Act D (III), Act D 2 min prior to TNF-α administration with the same doses (IV), mice injected subcutaneously with 200 μg/kg teduglutide every 12 h for 10 consecutive days (V), and mice given Act D 2 min prior to TNF-α administration on day 11 after receiving teduglutide for 10 days (VI). The TNF-α/Act D administration made the lung a sensitive organ to damage. Mice lung subjected to TNF-α/Act D were characterized by the disruption of alveolar wall, induced pulmonary endothelial/epithelial cell apoptosis and expression of active caspase-3. These mice exhibited an increase in lipid peroxidation, glutathione levels, and activities of myeloperoxidase, superoxide dismutase, catalase, glutathione peroxidase and xanthine oxidase, as well as reduced tissue factor and sodium–potassium/ATPase activities. Teduglutide pretreatment regressed the structural damage, cell apoptosis and oxidative stress by reducing lipid peroxidation in mice received TNF-α/Act D. GLP-2 receptors were present on the cell membrane of type II pneumocytes and interstitial cells. Thus, teduglutide can be suggested as a novel protective agent, which possesses anti-apoptotic and anti-oxidant properties, against lung injury.
Keywords: Teduglutide; TNF-α/actinomycin D-induced injury; Apoptosis; Oxidant–antioxidant system; Mice lung;
D-Lys3-GHRP-6 antagonizes the effect of unacylated but not of acylated ghrelin on the growth of HECa10 murine endothelial cells by Joanna Polowinczak-Przybylek; Agnieszka Siejka; Gabriela Melen-Mucha (248-254).
► Both acylated (AG) and unacylated (UAG) ghrelin inhibit proliferation of HECa10 murine endothelial cells in vitro. ► D-Lys3-GHRP-6 (antagonist of GHS-R1a) also inhibits HECa10 cells’ growth when applied alone. ► D-Lys3-GHRP-6 counteracts the effects of UAG but not of AG on HECa10 cells. ► Similar effects of the substances on HECa10 cells suggest that they are not mediated by GHS-R1a. ► D-Lys3-GHRP-6 seems not to be an appropriate antagonist in this experimental condition.Recent studies demonstrate that ghrelin can be an endogenous regulator of angiogenesis. We studied direct effects of human acylated (hAG) and unacylated (hUAG) ghrelin, as well as of rat acylated ghrelin (rAG) on the growth of HECa10 murine endothelial cells. Ghrelin was applied separately or together with D-Lys3-GHRP-6, which is commonly used as an antagonist of ghrelin receptor type 1a – GHS-R1a. The growth of HECa10 cells was assessed with Mosmann and in selected study conditions also with BrdU and TUNEL methods. Both hAG and hUAG (10−5 M to 10−12 M) inhibited the growth of HECa10 cells in 24 h and 72 h cultures. Similarly, rAG decreased the growth of the cells after 24 h (10−7 M and 10−11 M), and after 72 h (10−7 M, 10−8 M and 10−11 M). Unexpectedly, D-Lys3-GHRP-6 itself also inhibited the growth of these cells at 10−4 to 10−6 M in 24 h, 48 h (dose–response effect) and 72 h cultures. D-Lys3-GHRP-6 did not modify the inhibitory effect of rAG. However, D-Lys3-GHRP-6 at the concentration of 10−4 M diminished, abolished or even reversed the inhibitory effect of hUAG in 72 h culture and this was dependent on ghrelin concentrations. These data indicate that both AG and UAG have antiangiogenic properties at least at the level of endothelial growth, through decreased metabolic activity of the cells or stimulation of apoptosis. D-Lys3-GHRP-6 (inhibitor of GHS-R1a) seems not to be an appropriate antagonist in this experimental condition. Similar effects of these substances on HECa10 cells suggest that they are not mediated by GHS-R1a.
Keywords: Ghrelin; Ghrelin receptor antagonist; HECa10 endothelial cells;
Antimicrobial activity of human β-defensin 4 analogs: Insights into the role of disulfide linkages in modulating activity by Himanshu Sharma; Ramakrishnan Nagaraj (255-265).
► Antimicrobial activity of human β-defensin 4 analogs was investigated. ► Activity depends on the position and number of disulfide bonds. ► Analogs do not permeabilize microbial membranes. ► Analogs rapidly accumulate inside microbial cells.Human β-defensins (HBDs) are cationic antimicrobial peptides that are components of the innate immune system. They are characterized by three disulfide bridges. However, the number of cationic residues as well as the presence of lysine and arginine residues vary. In HBD4, the cationic residues occur predominantly in the N-terminal segment, unlike in HBD1–3. We have examined the antimicrobial activity of peptides spanning the N- and C-terminal segments of HBD4. We have introduced one, two and three disulfide bridges in the peptides corresponding to the N-terminal segments. Peptides corresponding to the N-terminal segment had identical sequences and variation was only in the number and spacing of cysteines and disulfide bridges. Antimicrobial activity to varying extents was observed for all the peptides. When two disulfide bridges were present, decrease in antimicrobial potency as well as sensitivity of activity to salt was observed. Enhanced antimicrobial activity was observed when three disulfide bridges were present. The antimicrobial potency was similar to HBD4 except against Escherichia coli and was attenuated in the presence of salt. While the presence of three disulfide bridges did not constrain the peptide to a rigid β-sheet, the activity was considerably more as compared to the peptides with one or two disulfide bridges. The peptides enter bacterial and fungal cells rapidly without membrane permeabilization and appear to exert their activity inside the cells rather than at the membrane.
Keywords: Antimicrobial activity; Disulfide bonds; Human β-defensins; Membrane permeabilization; Salt sensitivity;
PEGylation of a proprotein convertase peptide inhibitor for vaginal route of drug delivery: In vitro bioactivity, stability and in vivo pharmacokinetics by Huiting Ho; Tracy L. Nero; Harmeet Singh; Michael W. Parker; Guiying Nie (266-274).
► We PEGylated a peptide inhibitor of proprotein convertase, nona-d-arginine (Poly R). ► Poly R tolerates PEGylation at C-terminus regardless PEG size 1239 Da or 30 kDa. ► C-PEGylated Poly Rs showed enhanced vaginal absorption and mucosal penetration. ► First report a peptide with improved properties for vaginal delivery after PEGylation. ► Findings provide important insights into future design of Poly R derivatives.Uterine proprotein convertase (PC) 6 is critical for embryo implantation in mice and women. It is also one of the PC family members that play a vital role in HIV infectivity. We hypothesized that inhibiting PC6 in the female reproductive tract (vagina, cervix and uterus), may protect women from both pregnancy and HIV infection. One key requirement to prove this concept in an animal model is a vaginally deliverable PC6 inhibitor. Nona-d-arginine (Poly R) is a potent peptide PC inhibitor and is able to inhibit HIV in cell culture. We modified Poly R by PEGylation with different strategies and determined their biochemical properties in vitro and in vivo. PEGylation at the C-terminus, regardless of the PEG size (30 kDa or 1239 Da) did not compromise the inhibitory potency of Poly R. In contrast, PEGylation at both termini (1239 Da) dramatically reduced its inhibitory activity. Poly R and C-PEGylated Poly Rs also showed equal potency in inhibiting a PC6-dependent cellular process critical for embryo implantation. Poly R and the equipotent C-PEGylated Poly Rs were further tested for their serum stability in vitro and pharmacokinetics in vivo following vaginal administration in mice. All Poly Rs were equally stable in mouse serum in vitro for 24 h; C-PEGylated Poly Rs showed enhanced vaginal absorption and penetration across the vaginal mucosa/epithelium. This is the first report that C-terminal PEGylation significantly enhances the therapeutic properties of Poly R for vaginal drug delivery. Our findings also provide important insights into future design of Poly R derivatives.
Keywords: Proprotein convertase; PEGylation; Decidualization; Pharmokinetic;
Antioxidant activity of vasoactive intestinal peptide in HK2 human renal cells by Eva Vacas; Ana M. Bajo; Andrew V. Schally; Manuel Sánchez-Chapado; Juan C. Prieto; María J. Carmena (275-281).
► VIP decreases ROS levels reached by H2O2-induced oxidative stress. ► VIP increases Bcl-2 and decreases Bax in H2O2-injured HK2 human renal cells. ► The antiapoptotic effect of VIP is initiated through FPLR-1. ► VIP might exert a renoprotective effect by suppressing oxidative stress.Oxidative stress is a major mediator of tissue and cell injuries. The injury in chronic nephrotic syndrome, acute renal failure, myeloma kidney injury and other kidney diseases is initiated by oxidative stress. We have previously demonstrated that vasoactive intestinal peptide (VIP) acts as an antiproliferative agent in renal cancer cells. This study was designed to evaluate the renoprotective activity of VIP against H2O2-induced oxidative damage in a proximal tubule kidney cell line (human, non-tumor, HK2 cells) in order to investigate the potential usefulness of this peptide in the treatment of oxidative-stress related kidney diseases. HK2 cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Propidium iodide was used to identify cells undergoing apoptosis. Western blotting was performed with anti-Bcl-2, anti-Bax and anti-formyl peptide receptor (low-affinity variant FPRL-1) monoclonal antibodies whereas 2,7-dichlorofluorescein diacetate was used for measurement of levels of intracellular reactive oxygen species (ROS). HK2 cells were injured with H2O2 in order to induce apoptosis: the effect was time- and dose-dependent. VIP increased the levels of the antiapoptotic protein Bcl-2 and decreased those of the proapoptotic protein Bax. VIP decreased the intracellular ROS levels reached by H2O2-induced oxidative stress. VIP effect on ROS levels involved FPLR-1 but not VPAC1,2 receptors as evidenced by the use of the respective antagonists WRW4 and JV-1-53. Thus, VIP protects HK2 cells from apoptosis by increasing Bcl-2 levels and this effect is initiated through FPLR1 receptor. In conclusion, VIP might exert a renoprotective effect by the suppression of oxidative stress.
Keywords: VIP; FPRL-1; H2O2; Oxidative stress; Renal cells;
Activation of tachykinin, neurokinin 3 receptors affects chromatin structure and gene expression by means of histone acetylation by Amit Thakar; Elise Sylar; Francis W. Flynn (282-290).
► Application of senktide, a NK3R agonist, modified chromatin and gene expression. ► Senktide (1 or 10 nM) increased histone acetylation and gene expression. ► Increased gene expression was largely prevented by pretreatment with HAT inhibitors. ► Senktide (100 nM) decreased acetylation and gene expression, and compacted chromatin.The tachykinin, neurokinin 3 receptor (NK3R) is a g-protein coupled receptor that is broadly distributed in the nervous system and exerts its diverse physiological actions through multiple signaling pathways. Despite the role of the receptor system in a range of biological functions, the effects of NK3R activation on chromatin dynamics and gene expression have received limited attention. The present work determined the effects of senktide, a selective NK3R agonist, on chromatin organization, acetylation, and gene expression, using qRT-PCR, in a hypothalamic cell line (CLU 209) that expresses the NK3R. Senktide (1 nM, 10 nM) caused a relaxation of chromatin, an increase in global acetylation of histone H3 and H4, and an increase in the expression of a common set of genes involved in cell signaling, cell growth, and synaptic plasticity. Pretreatment with histone acetyltransferase (HAT) inhibitor (garcinol and 2-methylene y-butylactone), that inhibits p300, p300/CREB binding protein (CBP) associated factor (PCAF), and GCN 5, prevented the senktide-induced increase in expression of most, but not all, of the genes upregulated in response to 1 nM and 10 nM senktide. Treatment with 100 nM had the opposite effect: a reduction in chromatin relaxation and decreased acetylation. The expression of four genes was significantly decreased and the HAT inhibitor had a limited effect in blocking the upregulation of genes in response to 100 nM senktide. Activation of the NK3R appears to recruit multiple pathways, including acetylation, and possibly histone deactylases, histone methylases, or DNA methylases to affect chromatin structure and gene expression.
Keywords: Hypothalamus; Neurokinin 3 receptor; Receptor signaling; Chromatin; Acetylation;
Molecular cloning of two distinct precursor genes of NdWFamide, a d-tryptophan-containing neuropeptide of the sea hare, Aplysia kurodai by Fumihiro Morishita; Yasuo Furukawa; Osamu Matsushima (291-301).
Expression of NWF90 (left) and NWF87 (right) genes on the ventral surface of the abdominal ganglion of Aplysia.Display Omitted► NdWFamide is a d-Trp-containing cardioexcitatory tripeptide of Aplysia. ► We have cloned two distinct precursor genes of the peptide. ► Both of the predicted precursor (NWF90/NWF87) encoded a single copy of NdWFamide. ► Expression of NWF90 gene is dominant over NWF87 gene in the abdominal ganglia.NdWFamide (NdWFa) is a d-tryptophan-containing cardioexcitatory neuropeptide in gastropod mollusks, such as Aplysia kurodai and Lymanea stagnalis. In this study, we have cloned two cDNA encoding distinct precursors for NdWFa from the abdominal ganglion of A. kurodai. One of the predicted precursor proteins consisted of 90 amino acids (NWF90), and the other consisted of 87 amino acids (NWF87). Both of the predicted precursor proteins have one NWFGKR sequence preceded by the N-terminal signal peptide. Sequential double staining by in situ hybridization (ISH) and immunostaining with anti-NdWFa antibody suggested that NdWFa-precursor and NdWFa peptide co-exist in neurons located in the right-upper quadrant region of the abdominal ganglion. In ISH, NWF90-specific signal and NWF87-specific one were found in different subsets of neurons in the abdominal ganglia of Aplysia. The expression level of NWF90 gene estimated by RT-PCR is much higher than that of NWF87 gene. These results suggest that NWF90 precursor is the major source of NdWFa in Aplysia ganglia.
Keywords: RT-PCR; In situ hybridization; Nervous tissue; d-Amino acid;
Nullomer derived anticancer peptides (NulloPs): Differential lethal effects on normal and cancer cells in vitro by Abdelkrim Alileche; Jayita Goswami; William Bourland; Michael Davis; Greg Hampikian (302-311).
► First use of the nullomer (absent sequences) approach to drug development. ► NulloP 9S1R causes mitochondrial impairment, and ultimately cell death. ► NulloPs effects increase on cancer cells over time, and decrease on normal cells.We demonstrate the first use of the nullomer (absent sequences) approach to drug discovery and development. Nullomers are the shortest absent sequences determined in a species, or group of species. By identifying the shortest absent peptide sequences from the NCBI databases, we screened several potential anti-cancer peptides. In order to improve cell penetration and solubility we added short poly arginine tails (5Rs), and initially solubilized the peptides in 1 M trehalose. The results for one of the absent sequences 9R (RRRRRNWMWC), and its scrambled version 9S1R (RRRRRWCMNW) are reported here. We refer to these peptides derived from nullomers as PolyArgNulloPs. A control PolyArgNulloP, 124R (RRRRRWFMHW), was also included. The lethal effects of 9R and 9S1R are mediated by mitochondrial impairment as demonstrated by increased ROS production, ATP depletion, cell growth inhibition, and ultimately cell death. These effects increase over time for cancer cells with a concomitant drop in IC-50 for breast and prostate cancer cells. This is in sharp contrast to the effects in normal cells, which show a decreased sensitivity to the NulloPs over time.
Keywords: Absent sequences; Nullomers; NulloPs; PolyArgNulloPs; Pentapeptide; Cancer; LnCap; MDA-MB-231; Prostate cancer; Breast cancer; Trehalose; Solubility; Polyargenine; Cell penetrating peptide (CPP); 9R; 9S1R; 124R;
Amelioration of rotenone-induced dopaminergic cell death in the striatum by oxytocin treatment by Oytun Erbaş; Fatih Oltulu; Dilek Taşkiran (312-317).
► The protective effects of oxytocin were investigated in a Parkinson's disease model. ► The behavioral, histological and immunohistochemical parameters were evaluated. ► Oxytocin lessened cell loss in dopaminergic neurons against rotenone toxicity. ► The beneficial effects of oxytocin were achieved via inhibition of apoptotic pathways.Oxytocin (OT) is essentially associated with uterine contraction during parturition and milk ejection reflex. Although several studies implicate the role of OT in anti-inflammatory, anti-oxidative and anti-apoptotic pathways, there is a lack of data with regard to the protective effects of oxytocin in neurodegenerative models such as Parkinson's disease (PD). The present study was undertaken to investigate the neuroprotective effects of oxytocin (OT) on rotenone-induced PD in rats. Twenty adult Sprague-Dawley rats were injected with rotenone (3 μg/μl in DMSO) or vehicle (1 μl DMSO) into the left substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) under stereotaxic surgery, and PD model was assessed by rotational test ten days after drug infusion. The valid PD rats were randomly divided into two groups; Group 1 (n = 7) and Group 2 (n = 7) were administered saline (1 ml/kg/day, i.p.) and oxytocin (160 μg/kg/day, i.p.) through 20 days, respectively. The effects of OT treatment were evaluated by behavioral, histological and immunohistochemical parameters. Apomorphine-induced stereotypic rotations in PD rats were significantly inhibited by OT treatment (p < 0.05). In addition, immunohistochemical studies clearly demonstrated the suppression of Bax, caspase-3, caspase-8 and elevation of Bcl-2 and tyrosine hydroxylase immunoexpression in OT-treated rats compared to saline group. Our findings suggest that oxytocin may have cytoprotective and restorative effects on dopaminergic neurons against rotenone-induced injury. The underlying mechanism may be associated with the inhibition of apoptotic pathways.
Keywords: Parkinson's disease; Rotenone; Oxytocin; Neuroprotection; Animal models;
The substance P/neurokinin-1 receptor system in lung cancer: Focus on the antitumor action of neurokinin-1 receptor antagonists by Miguel Muñoz; Ana González-Ortega; Marisa Rosso; María José Robles-Frias; Andres Carranza; Manuel Vicente Salinas-Martín; Rafael Coveñas (318-325).
► SP/NK-1R antagonists respectively induces/inhibit proliferation in lung cancer cells. ► NK-1 receptor antagonists induce apoptosis in human lung cancer cells. ► Human lung cancer cells overexpress the tachykinin 1 gene. ► The NK-1 receptor is involved in the viability of human lung cancer cells. ► Substance P and NK-1 receptors are expressed in human lung cancer samples.The last decades have seen no significant progress in extending the survival of lung cancer patients and there is an urgent need to improve current therapies. The substance P (SP)/neurokinin-1 receptor (NK-1R) system plays an important role in the development of cancer: SP and NK-1R antagonists respectively induce cell proliferation and inhibition in human cancer cell lines. No study of the involvement of this system in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells has been carried out in depth. Here, we demonstrate the involvement of the SP/NK-1R system in human H-69 (SCLC) and COR-L23 (NSCLC) cell lines: (1) they express isoforms of the NK-1R and mRNA for the NK-1R; (2) they overexpress the tachykinin 1 gene; (3) the NK-1R is involved in their viability; (4) SP induces their proliferation; (5) NK-1R antagonists (Aprepitant (Emend), L-733,060, L-732,138) inhibit the growth of both cell lines in a concentration-dependent manner; (6) the specific antitumor action of these antagonists against such cells occurs through the NK-1R; and (7) lung cancer cell death is due to apoptosis. We also demonstrate the presence of NK-1Rs and SP in all the human SCLC and NSCLC samples studied. Our findings indicate that the NK-1R may be a promising new target in the treatment of lung cancer and that NK-1R antagonists could be new candidate antitumor drugs in the treatment of SCLC and NSCLC.
Keywords: Apoptosis; Aprepitant; L-732,138; L-733,060; Non-small cell lung cancer; Small cell lung cancer;
Leptin and leptin receptor: Analysis of a structure to function relationship in interaction and evolution from humans to fish by J.W. Prokop; R.J. Duff; H.C. Ball; D.L. Copeland; R.L. Londraville (326-336).
Informative docking of Lep and LepR based on sequence alignments and functional data. Amino acids in red are known natural variants associated with disease phenotype, green are post translational modifications, magenta are known mutations that lead to altered function, cyan are amino acids highly conserved in multiple species, and yellow are amino acids with functional groups conserved.Display Omitted► Interaction between leptin and the leptin receptor is conserved in land vertebrates. ► Avian leptin sequences appear artifactual as evolution rates of Lep do not match LepR. ► Fish contain multiple amino acid variations in the interaction of Lep to LepR. ► Predicted binding for Lep are stronger for A vs. B proteins in zebrafish.Leptin is a circulating protein which regulates dietary intake through binding the leptin receptor. Numerous labs have used known structures and mutagenesis to study this binding process in common animal models (human, mouse and rat). Understanding this binding process in other vertebrate species will allow for a better understanding of leptin and leptin receptor function. The binding site between leptin and leptin receptor is highly conserved in mammals as confirmed through sequence alignments mapped onto structures of both leptin and leptin receptor. More variation in this interaction is found in lizard and frog sequences. Using our models, we show that the avian leptin sequences have far less variation in the binding site than does the leptin receptor. This analysis further suggests that avian leptins are artifactual. In fish, gene duplication events have led to the expression of multiple leptin proteins. These multiple leptin proteins have variation in the regions interacting with leptin receptor. In zebrafish and the Japanese rice fish, we propose that leptin A has a higher binding energy than does B. Differing binding energies are evidence of either divergent functions, different binding confirmations, or other protein partners of leptin B.
Keywords: Leptin; Leptin receptor; Protein interaction; Bioinformatics;
The expression of PAC1 increases in the degenerative thymus and low dose PACAP protects female mice from cyclophosphamide induced thymus atrophy by Huahua Zhang; Rongjie Yu; Xiaofei Liu; Xiaoling Guo; Zhixing Zeng (337-343).
► The expression of PAC1 in the thymus was higher in female mice than in male mice. ► The expression of PAC1 and PACAP increased in the thymus damaged by CPS. ► Low dose of PACAP displayed significant protective effects against CPS-induced thymus atrophy.Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with cytoprotective ability mediated by its specific receptor PAC1. In this research, firstly the thymus index and the expression of PAC1 in the normal and degenerative thymus with different gender were assayed; secondly PACAP in different dose was used to treat the female mice with cyclophosphamide (CPS) and the changes in thymus index, the expression of PAC1, histopathology, apoptosis, oxidative status and the caspase 3 activity in thymus were determined and compared. It was found that in the mice of age from 1 to 9 weeks in the stage of sex development, the thymus index was significantly higher in female mice than in male mice. And it was found for the first time that the PAC1 expression level in thymus of female mice was significantly higher than that of male mice and the expression of the PAC1 and PACAP increased significantly in the degenerative thymus induced by CPS. After PACAP was co-injected with CPS to the female mice, it was shown that only low dose (1 nmol/kg) of PACAP promoted the thymus index, inhibited the cell apoptosis, ameliorated the oxidative status and decreased the caspase activity significantly, while high dose (10 nmol/kg) of PACAP had no significant protective effects against CPS-induced thymus atrophy. It was concluded that the expression of PAC1 in the thymus changes in reverse ratio with thymus index and in direct ratio with cell apoptosis and only low dose of PACAP had positive effects against the CPS-induced thymus atrophy.
Keywords: Pituitary adenylate cyclase activating polypeptide (PACAP); Cyclophosphamide; Thymus; PAC1;
Purification and identification of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates by Ning Chen; Hongmei Yang; Yi Sun; Jun Niu; Shuying Liu (344-349).
► Antioxidant hydrolysates from walnut proteins were prepared using neutrase, alcalase and pepsin. ► Walnut protein hydrolysates were purified sequentially by ultrafiltration, gel filtration and RP-HPLC. ► The activities of peptide were evaluated by radicals scavenging, reducing power and lipid peroxidation inhibitory activity. ► The structure of purified peptide was determined as Ala-Asp-Ala-Phe.Walnut proteins were hydrolyzed separately using three different proteases to obtain antioxidant peptides. The antioxidant activities of the hydrolysates were measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. Among hydrolysates, pepsin hydrolysate obtained by 3 h exhibited the highest antioxidant activities, which could also quench the hydroxyl radical, chelate ferrous ion, exhibit reducing power and inhibit the lipid peroxidation. Then, 3-h pepsin hydrolysates were purified sequentially by ultrafiltration, gel filtration and RP-HPLC. The sequence of the peptide with the highest antioxidative activity was identified to be Ala-Asp-Ala-Phe (423.23 Da) using RP-HPLC-ESI-MS, which was identified for the first time from walnut protein hydrolysates. Last, the inhibition of the peptide on lipid peroxidation was similar with that of reduced glutathione (GSH). These results indicate that the protein hydrolysates and/or its isolated peptides may be effectively used as food additives.
Keywords: Walnut protein hydrolysates; Free radical scavenging activity; Purification; Antioxidant peptide;
Enhanced expression of human β-defensin 2 in peripheral lungs of patients with chronic obstructive pulmonary disease by Zenglin Liao; Jiajia Dong; Xiaorong Hu; Tao Wang; Chun Wan; Xiao’ou Li; Lin Li; Lingli Guo; Dan Xu; Fuqiang Wen (350-356).
► We measured hBD-2 mRNA and protein levels in peripheral lung tissue of COPD patients. ► Among COPD patients, hBD-2 mRNA levels were higher in current smokers than in non-smokers. ► hBD-2 mRNA levels positively correlated with IL-8 mRNA levels. ► hBD-2 mRNA levels negatively correlated with the FEV1/FVC ratio and predicted FEV1%. ► Smoking may further elevate hBD-2 in peripheral lung tissue of COPD patients.Human β-defensin 2 (hBD-2) has antimicrobial activity and may play a role in airway mucosal defense, but studies have not yet examined its expression in lung tissue of patients with chronic obstructive pulmonary disease (COPD). Here we investigated hBD-2 levels in lung tissues of COPD patients and analyzed their correlations with IL-8, IL-1β, cigarette smoking and lung function in order to see whether the protein may be involved in pathogenesis of the disease. Peripheral lung tissue specimens were obtained from 51 patients who underwent lung resection for peripheral lung cancer: healthy non-smokers (n = 8), healthy current smokers (n = 7), non-smokers with COPD (n = 11), and current smokers with COPD (n = 25). RT-PCR and immunohistochemical staining were used to detect expression levels of hBD-2, IL-8 and IL-1β. Expression of hBD-2 mRNA was significantly higher in COPD patients than in healthy controls, and significantly higher in current smokers than in non-smokers (p < 0.05). Among healthy controls, hBD-2 mRNA levels were similar between current smokers and non-smokers. Immunohistochemistry showed hBD-2 protein to be expressed mainly in epithelia of distal bronchioles and its expression pattern among our patient groups mirrored that of the mRNA. IL-8 mRNA levels were significantly higher in COPD patients than in healthy controls (p < 0.05), while IL-1β mRNA levels did not differ significantly among the groups. Levels of hBD-2 mRNA positively correlated with levels of IL-8 mRNA (r = 0.545, p = 0.002), and negatively correlated with FEV1/FVC ratios and with predicted FEV1% values (r = −0.406, p = 0.011). Our results indicate that hBD-2 expression is elevated in distal airways of COPD patients and that it may be involved in pathogenesis of the disease. Our data implicate cigarette smoking as a factor that may elevate hBD-2 levels in lung tissues of COPD patients.
Keywords: Human β-defensin 2; COPD; Cigarette smoking; IL-8; Lung function;
Insights into buforin II membrane translocation from molecular dynamics simulations by Donald E. Elmore (357-362).
► Performed molecular dynamics simulations of buforin II membrane entry. ► First pore-forming simulations for a non-lytic antimicrobial peptide. ► Showed relationship between membrane entry, helical deformation and lipid interaction. ► Observed toroidal pore formation proposed in previous experimental studies. ► Considered changes to MD simulation conditions necessary to promote membrane entry.Buforin II is a histone-derived antimicrobial peptide that readily translocates across lipid membranes without causing significant membrane permeabilization. Previous studies showed that mutating the sole proline of buforin II dramatically decreases its translocation. As well, researchers have proposed that the peptide crosses membranes in a cooperative manner by forming transient toroidal pores. This paper reports molecular dynamics simulations designed to investigate the structure of buforin II upon membrane entry and evaluate whether the peptide is able to form toroidal pore structures. These simulations showed a relationship between protein–lipid interactions and increased structural deformations of the buforin N-terminal region promoted by proline. Moreover, simulations with multiple peptides show how buforin II can embed deeply into membranes and potentially form toroidal pores. Together, these simulations provide structural insight into the translocation process for buforin II in addition to providing more general insight into the role proline can play in antimicrobial peptides.
Keywords: Antimicrobial peptide; Cell-penetrating peptide; Histone; Membrane translocation; Molecular dynamics;
Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum by Aline Rifflet; Sabine Gavalda; Nathan Téné; Jérôme Orivel; Jérôme Leprince; Laure Guilhaudis; Eric Génin; Angélique Vétillard; Michel Treilhou (363-370).
► First study describing the antibacterial activity of the venom of the Tetramorium bicarinatum ant against different referenced and wild Staphylococcus strains. ► The compound responsible for antibacterial activity is a novel amidated linear cationic peptide. ► This peptide, named Bicarinalin, is composed of 20 amino-acids and her sequence is totally original. ► Bicarinalin is weakly hemolytic, so it could be an interesting alternative drug.A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.
Keywords: Tetramorium bicarinatum; Ant venom; Staphylococcus; ESI-MS/MS; Antibacterial peptide; AMP; Bicarinalin;
Molecular cloning of skin peptide precursor-encoding cDNAs from tibial gland secretion of the Giant Monkey Frog, Phyllomedusa bicolor (Hylidae, Anura) by Enrico König; Valerie C. Clark; Chris Shaw; Olaf R.P. Bininda-Emonds (371-376).
► We provide the first targeted analysis of the tibial gland of Phyllomedusa bicolor. ► Our analyses identified two novel peptides for this otherwise well-studied species. ► We hypothesize that defensive compounds might vary between different skin glands. ► We provide more evidence for the sharing of specific AMPs among anuran species.The skins of phyllomedusine frogs have long been considered as being tremendously rich sources of bioactive peptides. Previous studies of both peptides and cloning of their precursor encoding cDNAs have relied upon methanolic skin extracts or the dissected skins of recently deceased specimens and have not considered the different glands in isolation. We therefore focused our attention on the tibial gland of the Giant Monkey Frog, Phyllomedusa bicolor and constructed a cDNA library from the skin secretion that was obtained via mechanical stimulation of this macrogland. Using shotgun cloning, four precursors encoding host-defense peptides were identified: two archetypal dermaseptins, a phyllokinin and a phylloseptin that is new for this species but has been recently described from the Waxy Monkey Leaf Frog, Phyllomedusa sauvagii. Our study is the first to report defensive peptides specifically isolated from anuran tibial glands, confirming the hypothesis that these glands also contribute to chemical defense. Moreover, the discovery of novel compounds for this otherwise very well characterized species suggests that this largely neglected gland might possess a different cocktail of secretions from glands elsewhere in the same animal. We will also discuss some evolutionary implications of our findings with respect to the adaptive plasticity of secretory glands.
Keywords: Phyllokinin; Phylloseptin; Host-defense peptides; Frog skin secretion;
Circulating obestatin is increased in patients with cardiorenal syndrome and positively correlated with vasopressin by Jian-Bo Shi; Zhi-Fu Guo; Xing Zheng; Zhi-Bin Wang; Yuan-Ji Ma (377-380).
► We measured plasma concentration of obestatin and AVP in patients with cardiorenal syndrome (CRS). ► Plasma obestatin and vasopressin were elevated in patients with CRS. ► Plasma concentration of obestatin was positively correlated with AVP. ► Plasma concentration in the overall analysis that included subjects from all disease categories, but not within the CRS group.Obestatin regulates fluid and electrolyte homeostasis mainly by opposing the action of vasopressin (AVP). We measured plasma concentration of obestatin and AVP in patients with cardiorenal syndrome (CRS). Plasma AVP and obestatin concentration were measured in 34 patients with type II CRS. The data were compared to that in 31 patients with chronic kidney disease (CKD), 41 patients with chronic heart failure (CHF) and 30 healthy subjects. Obestatin was significantly higher in the patients with CRS (355.8 ± 85.1 pg/ml) than that in the healthy controls (212.3 ± 37.9 pg/ml, P < 0.01), the patients with CKD (246.7 ± 34.3 pg/ml, P < 0.01) and the patients with CHF (258.4 ± 112.1 pg/ml, P < 0.01). AVP was also significantly higher in the patients with CRS (65.1 ± 36.0 pg/ml) than that in the healthy controls (38.5 ± 20.1 pg/ml, P < 0.01), the patients with CKD (50.4 ± 24.8 pg/ml, P < 0.01) and the patients with CHF (54.6 ± 16.3 pg/ml, P < 0.01). Plasma concentration of obestatin was positively correlated with AVP plasma concentration in the overall analysis that included subjects from all disease categories (r = 0.219, P < 0.05), but not within the CRS group. Plasma obestatin and vasopressin were elevated in patients with CRS. Plasma obestatin concentration seemed to be positively correlated with plasma AVP.
Keywords: Obestatin; Vasopressin; Cardiorenal syndrome; Fluid; Homeostasis;
Mimotope peptides selected from phage display combinatorial library by serum antibodies of pigs experimentally infected with Taenia solium as leads to developing diagnostic antigens for human neurocysticercosis by Karlen Gazarian; Merril Rowlay; Tatiana Gazarian; Jorge Enrique Vazquez Buchelli; Marisela Hernández Gonzáles (381-388).
► Neurocysticercosis diagnosis requires involvement of immunodominant epitopes. ► Phage library screening with sera of infected pigs selects mimics of an immunodominant epitope. ► Net result: antigens for the diagnosis of neurocysticercosis.Neurocysticercosis is caused by penetration of the tapeworm Taenia solium larvae into the central nervous system resulting in a diverse range of neurologic complications including epilepsy in endemic areas that globalization spreads worldwide. Sensitive and specific immunodiagnosis is needed for the early detection and elimination of the parasite, but the lack of standardized, readily obtainable antigens is a challenge. Here, we used the phage display for resolving the problem. The rationale of the strategy rests on the concept that the screening of combinatorial libraries with polyclonal serum to pathogens reveals families of peptides mimicking the pathogen most immunodominant epitopes indispensable for the successful diagnosis. The screening of a 7mer library with serum IgG of four pigs experimentally infected with parasite followed by computer aided segregation of the selected sequences resulted in the discovery of four clusters of homologous sequences of which one presented a family of ten mimotopes selected by three infected pig serum IgGs; the common motif sequence LSPF carried by the family was considered to be the core of an immunodominant epitope of the parasite critical for the binding with the antibody that selected the mimotopes. The immunoassay testing permitted to select a mimotope whose synthetic peptide free of the phage with the amino acid sequence Leu-Ser-Fen-Pro-Ser-Val-Val that distinguished well a panel of 21 cerebrospinal fluids of neurocysticercosis patients from the fluids of individuals with neurological complications of other etiology. This peptide is proposed as a lead for developing a novel molecularly defined diagnostic antigen(s) for the neurocysticercosis.
Keywords: Neurocysticercosis; Taenia solium; Phage display; Epitope; Mimotope; Diagnosis;
A novel class of antimicrobial peptides from the scorpion Heterometrus spinifer by Yao Nie; Xian-Chun Zeng; Ye Yang; Feng Luo; Xuesong Luo; Shifen Wu; Lei Zhang; Jianping Zhou (389-394).
► A novel class of venom peptides (referred to as HsAp, HsAp2, HsAp3 and HsAp4) were identified from Heterometrus spinifer. ► The four peptides are highly basic, cationic and weakly amphipathic. ► HsAp processes antimicrobial activities against both Gram-positive and Gram-negative bacteria, as well as a fungus. ► The genes encoding the four peptides are intronless.The venom peptides from the scorpion Heterometrus spinifer have been poorly characterized so far. Here, we identified a novel class of antimicrobial peptides from the venom gland of H. spinifer, which were referred to as HsAp, HsAp2, HsAp3 and HsAp4, respectively. Each of the four peptides consists of 29 amino acid residues, and is cationic and weakly amphipathic. They display no significant homology to any other known peptides, and thus represent a new family of venom peptides from scorpions. Antimicrobial assay showed that HsAp is able to inhibit the growth of both Gram-negative and Gram-positive bacteria with the MIC values of 11.8–51.2 μM. HsAp is also able to inhibit the growth of the tested fungus. Genomic analysis indicated that the genes of all the four peptides are intronless. Our studies expand the families of antimicrobial peptides from scorpions.
Keywords: Antimicrobial peptide; Scorpion venom; Heterometrus spinifer; Genomic organization; Weakly amphipathic peptide;
Delayed administration of pituitary adenylate cyclase-activating polypeptide 38 ameliorates renal ischemia/reperfusion injury in mice by modulating Toll-like receptors by Altaf-M. Khan; Min Li; Solange Abdulnour-Nakhoul; Jerome L. Maderdrut; Eric E. Simon; Vecihi Batuman (395-403).
► Bilateral renal ischemia/reperfusion (IR) induced kidney injury in mice. ► IR activated Toll-like receptors (TLRs). ► TLRs induced inflammatory cytokine production. ► Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) modulated TLRs. ► PACAP38 protected kidney even when treatment was started 24 h after IR.We investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) ameliorates kidney injury after ischemia/reperfusion (IR) by modulating Toll-like receptor (TLR)-associated signaling pathways. Male C57BL/6 mice were subjected to bilateral renal ischemia for 45 min. PACAP38, 20 μg in 100 μl of saline, was administered i.p. at 24 and 48 h after IR, and mice were euthanized at 72 h. In IR mice, PACAP38 maintained serum creatinine near control levels (0.81 ± 0.08 vs. 0.69 ± 0.17 mg/dl in controls, p = NS, vs. 1.8 ± 0.03 in saline-treated IR mice, p < 0.01) and significantly reduced the expression of kidney injury biomarkers. PACAP38 significantly reduced the levels of apoptosis and neutrophil infiltration, and protected against tubular damage. With PCR arrays, 59 of 83 TLR-related genes significantly changed their expression after IR. TLR2 increased 162 fold, followed by Fas-associated death domain (37 fold) and TLR6 (24 fold), while ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) decreased 55 fold. PACAP38 given 24 and 48 h after IR injury significantly reversed these changes in 56 genes, including TLR2, TLR3, TLR4, TLR6, and genes in the NF-κB pathways. The alterations in TLR2, TLR3, TLR6, and UBE2V1 were confirmed by RT-PCR. After IR, PACAP38 also suppressed protein levels of TLR-associated cytokines. PACAP38 reversed the changes in IR-activated TLR-associated NF-κB signaling pathways even when treatment was delayed 24 h. Therefore, PACAP38 could be an effective therapeutic for unexpected IR-mediated renal injury. The prominently IR-induced TLR-related genes identified in this study could be novel drug targets.
Keywords: Acute kidney injury; Apoptosis; Cytokines; Gene arrays; Innate immunity; Ischemia/reperfusion; Peptides; Toll-like receptors;
Angiogenesis in the course of enucleation-induced adrenal regeneration—Expression of selected genes and proteins involved in development of capillaries by Marianna Tyczewska; Marcin Rucinski; Marcin Trejter; Agnieszka Ziolkowska; Marta Szyszka; Ludwik K. Malendowicz (404-413).
► It is the first whole transcriptome profiling in enucleation-induced adrenal regeneration. ► In the course of adrenal regeneration about 2000 genes showed more than 2-fold up/down-regulation. ► During the regeneration 62 genes involved in angiogenesis were identified as up/down-regulated.Enucleation-induced rapid proliferation of adrenocortical cells and restoration of adrenals structure requires formation of new blood vessels. The performed studies aimed to select from around 30,000 transcripts, identified by means of Affymetrix® Rat Gene 1.1 ST Array, the genes involved in angiogenesis in the course of enucleation-induced adrenal regeneration and to characterize their expression levels in regenerating gland between days 1 and 15 after surgery. At day 1 of regeneration almost 2000 genes showed more than 2-fold up/down-regulation. At days 1–3 after surgery the highest expression demonstrated genes involved in the development of inflammation and blood clot formation. From around 2000 genes we selected genes involved in angiogenesis. During the regeneration 62 genes involved in angiogenesis were identified as up- or down-regulated. Some data were also validated by QPCR. Levels of Vegfa and Kdr (Vegfr-2) mRNAs were very low at day 1 of regeneration and remained unchanged thereafter. The highest expression of Figf gene was found at day 5 while that of Vwf gene at days 1 and 2 after surgery. Levels of Thy1 mRNA increased notably between days 2 and 5 of the experiment. In comparison to control rats, Mc2r (ACTH receptor) expression was lowered at day 1 of the experiment and remained unchanged thereafter. This suggests that enucleation-induced adrenal neoangiogenesis does not require elevated expression of ACTH receptor. Results of our studies strongly suggest that enucleation-induced adrenal regeneration is an angiogenesis-dependent process. Moreover, immunohistochemistry suggests that regenerating adrenal parenchymal cells release numerous angiogenic factors which paracrinally may regulate formation of new vessels.
Keywords: Adrenal regeneration; Transcriptome analysis; Microarray RNA; Angiogenesis; QPCR; Immunohistochemistry; Rat;
The permeation of dynorphin A 1–6 across the blood brain barrier and its effect on bovine brain microvessel endothelial cell monolayer permeability by Courtney D. Kuhnline Sloan; Kenneth L. Audus; Jane V. Aldrich; Susan M. Lunte (414-417).
► Dynorphin A 1–6 is identified as a metabolite of Dynorphin A 1–17 in the presence of bovine brain microvessel endothelial cells (BBMECs). ► The blood brain barrier (BBB) permeability of the neuropeptide Dyn A 1–6 is investigated using the BBMEC culture model of the BBB. ► The directional and temperature dependent permeation of Dyn A 1–6 is evaluated. ► Dyn A 1–6 pretreatment is found to induce an opening of the BBB, increasing the permeation of fluorescein, a low molecular weight, low permeability control substance.Dynorphin A 1–17 (Dyn A 1–17) is an endogenous neuropeptide known to act at the kappa opioid receptor; it has been implicated in a number of neurological disorders, including neuropathic pain, stress, depression, and Alzheimer's and Parkinson's diseases. The investigation of Dyn A 1–17 metabolism at the blood–brain barrier (BBB) is important since the metabolites exhibit unique biological functions compared to the parent compound. In this work, Dyn A 1–6 is identified as a metabolite of Dyn A 1–17 in the presence of bovine brain microvessel endhothelial cells (BBMECs), using LC–MS/MS. The transport of Dyn A 1–6 at the BBB was examined using this in vitro cell culture model of the BBB. Furthermore, the permeation of the BBB by the low molecular weight permeability marker fluorescein was characterized in the presence and absences of Dyn A 1–6.
The NOP receptor involvement in both withdrawal- and CCk-8-induced contracture responses of guinea pig isolated ileum after acute activation of κ-opioid receptor by Pietro Marini; Luca Romanelli; Daniela Valeri; Maria Grazia Cascio; Paolo Tucci; Pacifico Valeri; Maura Palmery (418-426).
Guinea pig ileum isolated preparation: κ-opioid withdrawal contractures.Display Omitted► In GPI, the NOP receptor system behaves as a potential anti-opioid system.In isolated guinea-pig ileum (GPI), the κ-opioid acute withdrawal response is under the control of several neuronal signaling systems, including the μ-opioid, the A1-adenosine and the CB1 receptors, which are involved in the inhibitory control of the κ-withdrawal response. After κ-opioid system stimulation, indirect activation of μ-opioid, A1-adenosine and CB1 systems is prevented by the peptide cholecystokinin-8 (CCk-8). In the present study, we have investigated whether the NOP system is also involved in the regulation of the acute κ-withdrawal response. Interestingly, we found that in GPI preparation, the NOP system is not indirectly activated by the κ-opioid receptor stimulation, but instead this system is able by itself to directly regulate the acute κ-withdrawal response. Specifically, our results clearly highlight first the existence of an endogenous tone of the NOP system in GPI, and second that it behaves as a functional anti-opioid system. We also found that, the NOP receptor system is involved in the regulation of the CCk-8-induced contracture intensity, only when in the presence of the κ-opioid receptor stimulation. This effect seems to be regulated by an activation threshold mechanism. In conclusion, the NOP system could act as neuromodulatory system, whose action is strictly related to the modulation of both excitatory and inhibitory neurotransmitters released in GPI enteric nervous system.
Keywords: OFQ/N; UFP-101; NOP receptor; κ-Opioid receptor; CCk-8; GPI; Opioid withdrawal;
Structural and activity changes in three bioactive anuran peptides when Asp is replaced by isoAsp by Antonio N. Calabrese; Katarina Markulic; Ian F. Musgrave; Hui Guo; Lixin Zhang; John H. Bowie (427-436).
► The Asp (and Asn) to isoAsp interconversion is a natural phenomenon. ► In the amphibian peptides studied it affects both their structure and bioactivity. ► The effects are unpredictable, with each system responding differently. ► isoAsp also perturbs protease recognition and consequently proteolysis.The Asp and isoAsp isomers of three bioactive peptides, Crinia angiotensin 11 [APGDRIYHPF(OH)], uperin 1.1 [pEADPNAFYGLM(NH2)] and citropin 1.1 [GLFDVIKKVASVIGGL(NH2)] were tested for changes in (i) susceptibility towards proteolytic cleavage, (ii) activity (smooth muscle activity for Crinia angiotensin 11 and uperin 1.1 isomers, and antimicrobial activity for the two isomers of citropin 1.1), and (iii) 3D structures in water, trifluoroethanol-d3/water (1:1) and DPC micelles as determined by 2D nuclear magnetic resonance spectroscopy. Proteolytic cleavage with trypsin was identical for each pair of Asp/isoAsp isomers. Cleavage with chymotrypsin was the same for the Crinia angiotensin and uperin 1.1 isomeric pairs, but different for the two Asp/isoAsp citropin 1.1 isomers. Chymotrypsin cleaved at Phe3 (adjacent to Asp4) for citropin 1.1, but not at Phe3 (adjacent to isoAsp4) for isoAsp citropin 1.1. The smooth muscle activity of the isoAsp isomer of Crinia angiotensin 11 was less than that of the Asp isomer. The smooth muscle activity of isoAsp3-uperin 1.1 is greater than that of the Asp isomer at low concentration (<10−9 M) but no different from the Asp isomer at concentrations > 10−9 M. Citropin 1.1 is a wide-spectrum antibiotic against Gram positive organisms, while the isoAsp isomer is inactive against the test pathogens Staphylococcus aureus and Bacillus subtilis. The observed changes in activity are accompanied by changes in the 3D structures of isomers as determined by 2D nuclear magnetic resonance spectroscopy.
Keywords: Asp and isoAsp isomers; Crinia angiotensin 11; Uperin 1.1; Citropin 1.1; Proteolytic cleavage; Activity (smooth muscle or antimicrobial); 3D structures by nuclear magnetic resonance;
Subcellular characteristics of functional intracellular renin–angiotensin systems by Peter M. Abadir; Jeremy D. Walston; Robert M. Carey (437-445).
► Compelling evidence now exists for complete, independent, differentially regulated intracellular renin–angiotensin systems in many tissues. ► Angiotensinogen, the enzymes renin and angiotensin converting enzyme and angiotensin peptides can be synthesized and retained intracellularly. ► Angiotensin receptors (AT1 and AT2) are abundant intracellularly mainly at the nuclear and mitochondrial levels. ► Functional intracellular renin–angiotensin system units have been documented both in nuclei and mitochondria.The renin–angiotensin system (RAS) is now regarded as an integral component in not only the development of hypertension, but also in physiologic and pathophysiologic mechanisms in multiple tissues and chronic disease states. While many of the endocrine (circulating), paracrine (cell-to-different cell) and autacrine (cell-to-same cell) effects of the RAS are believed to be mediated through the canonical extracellular RAS, a complete, independent and differentially regulated intracellular RAS (iRAS) has also been proposed. Angiotensinogen, the enzymes renin and angiotensin-converting enzyme (ACE) and the angiotensin peptides can all be synthesized and retained intracellularly. Angiotensin receptors (types I and 2) are also abundant intracellularly mainly at the nuclear and mitochondrial levels. The aim of this review is to focus on the most recent information concerning the subcellular localization, distribution and functions of the iRAS and to discuss the potential consequences of activation of the subcellular RAS on different organ systems.
Keywords: Renin–angiotensin system; Intracellular; Hypertension; Cardiovascular disease; Angiotensin peptides; Angiotensin receptors;
Expression systems for heterologous production of antimicrobial peptides by Nádia Skorupa Parachin; Kelly Cristina Mulder; Antônio Américo Barbosa Viana; Simoni Campos Dias; Octávio Luiz Franco (446-456).
Display Omitted► This review describes the main heterologous systems currently used for AMPs production. ► Strategies for reaching a higher functional peptide production were focused. ► Data revised indicates the challenges of large-scale production of AMPs.Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness.
Keywords: Heterologous expression; Antimicrobial peptide; Escherichia coli; Pichia pastoris;
Advances and prospects of anginex as a promising anti-angiogenesis and anti-tumor agent by Ju Bo Wang; Mao De Wang; En Xiao Li; Dan Feng Dong (457-462).
► Anginex is specific for angiogenically activated ECs and shows no toxicity on normal blood vessels. ► Anginex and compound or recombinant anginex could effectively inhibit formation of new blood vessels and tumor growth. ► Anginex could enhance the anti-tumor effects of chemotherapy and radiotherapy. ► Anginex and compound or recombinant anginex is an effective anti-angiogenic agent and can be used in anti-cancer therapy.Anginex, a novel artificial cytokine-like peptide (βpep-25), is designed by using basic folding principles and incorporating short sequences from the β-sheet domains of anti-angiogenic agents, including platelet factor-4 (PF4), interleukin-8 (IL-8), and bactericidal-permeability increasing protein 1 (BP1). Anginex can specially block the adhesion and migration of the angiogenically activated endothelial cells (ECs), leading to apoptosis and ultimately to the inhibition of angiogenesis and tumor growth. In vitro and in vivo studies have proved its inhibitory effects on the formation of new blood vessels and tumor growth even though the mechanism is not clear. The inhibitory effects of anginex can be enhanced when it is applied in combination with other therapies, such as chemotherapy, radiotherapy and other anti-angiogenic agents. The limitations of anginex, including poor stability, short half life, complicated synthesis and low purity, have been conquered by modifying its structure or designing novel compound anginex and recombinant anginex, which makes possible the clinical application of anginex. Here, we summarize the basic and preclinical trials of anginex and discuss the prospects of anginex in clinical application. We come to the conclusion that anginex and compound or recombinant anginex can be used as effective anti-angiogenic agents.
Keywords: Anginex; Angiogenesis; Tumor growth; β-Sheet; Endothelial cell; βpeptides;
Endogenous opiates and behavior: 2011 by Richard J. Bodnar (463-522).
► This paper is the 34rd annual review of research on the endogenous opioid system. ► It summarizes 2010 behavioral, molecular and pharmacological opioid papers. ► Topics include opioid analgesic, stress, tolerance and dependence effects. ► Topics also include learning, ingestion, alcohol and drugs of abuse. ► Topics also examine sex, mental illness, and neurologic disorders.This paper is the thirty-fourth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2011 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular–biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration (Section 16); and immunological responses (Section 17).
Keywords: Opioid receptors; Enkephalin; Beta-endorphin; Endomorphin; Nociceptin; Dynorphin; Morphine; Naloxone;
Corrigendum to “Adipokinetic hormones (AKHs) of sphingid Lepidoptera, including the identification of a second M. sexta AKH” [Peptides 34 (2012) 44–50] by Robert J. Weaver; Heather G. Marco; Petr Šimek; Neil Audsley; Kevin D. Clark; Gerd Gäde (523).