Peptides (v.26, #2)

RNAIII-inhibiting peptide improves efficacy of clinically used antibiotics in a murine model of staphylococcal sepsis by Andrea Giacometti; Oscar Cirioni; Roberto Ghiselli; Giorgio Dell’Acqua; Fiorenza Orlando; Giuseppina D’Amato; Federico Mocchegiani; Carmela Silvestri; Maria Simona Del Prete; Marco Rocchi; Naomi Balaban; Vittorio Saba; Giorgio Scalise (169-175).
RNAIII-inhibiting peptide (RIP, YSPWTNF-NH2) is a quorum-sensing peptide inhibitor that prevents Staphylococcus aureus toxin production and biofilm formation. A mouse sepsis model was used to test the efficacy of RIP alone or in combination with conventional antibiotics in suppressing S. aureus-induced sepsis. Mice were injected intravenously with 3.0 × 106  CFU of S. aureus ATCC 25923 or with 3.0 × 106  CFU of S. aureus strain Smith diffuse. All animals were randomized to receive intravenously isotonic sodium chloride solution as a control, or 20 mg/kg RIP alone or combined with 20 mg/kg cefazolin, 10 mg/kg imipenem, or 10 mg/kg vancomycin immediately or 6 h after bacterial challenge. Main outcome measures were bacteremia and lethality. All compounds reduced lethality when compared to controls. Although, in general combined-treated groups had significant lower bacterial counts when associated to singly-treated groups only the combination between RIP and vancomycin with respect to cefazolin gave a statistically significant decrease in the lethality rate. Lowest lethality rates (10%) and bacteremia (<102  CFU/ml) were obtained when RIP was administered in combination with vancomycin. Because RIP can be synergistic with current antibiotic therapies and help to reduce S. aureus exotoxins production, it can be considered a promising agent to associate with antibiotics for further clinical research into treatment of sepsis.
Keywords: Vancomycin; Staphylococcus aureus; RNAIII-inhibiting peptide; Bacteremia; Exotoxins;

A protein designated alliumin, with a molecular mass of 13 kDa and an N-terminal sequence similar to a partial sequence of glucanase, and demonstrating antifungal activity against Mycosphaerella arachidicola, but not against Fusarium oxysporum, was isolated from multiple-cloved garlic (Allium sativum) bulbs. The protein, designated as alliumin, was purified using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Mono S, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75. Alliumin was unadsorbed on DEAE-cellulose, but was adsorbed on Affi-gel blue gel, CM-cellulose and Mono S. Its antifungal activity was retained after boiling for 1 h and also after treatment with trypsin or chymotrypsin (1:1, w/w) for 30 min at room temperature. Alliumin was inhibitory to the bacterium Pseudomonas fluorescens and exerted antiproliferative activity toward leukemia L1210 cells. However, it was devoid of ribonuclease activity, protease activity, mitogenic activity toward mouse splenocytes, and antiproliferative activity toward hepatoma Hep G2 cells.
Keywords: Garlic bulbs; Antifungal protein; Alliumin;

A family of acyclic brevinin-1 peptides from the skin of the Ryukyu brown frog Rana okinavana by J. Michael Conlon; Agnes Sonnevend; Thierry Jouenne; Laurent Coquet; David Cosquer; Hubert Vaudry; Shawichi Iwamuro (185-190).
The 24 amino-acid residue antimicrobial peptide, brevinin-1 is synthesized in the skins of a wide range of species of Eurasian and North American frogs belonging to the genus Rana. All previously characterized brevinin-1 peptides contain the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule. Four structurally related peptides were isolated from an extract of the skin of the Ryukyu brown frog Rana okinavana. The amino acid sequences of the peptides [Phe-(Xaa)4-Ile-(Xaa)2-Leu-Ala-Lys-Gly-Leu-Pro-Ser-Leu-Ile-Xaa-Leu-Xaa-Lys-Lys·NH2] identified them as members of the brevinin-1 family that lacked the COOH-terminal cyclic domain but contained a C-terminally α-amidated residue. It is suggested, as one possibility, that the Cys18 in the brevinin-1 consensus sequence has been deleted and the Cys24 residue has mutated to a glycine that acts as substrate for peptidyl-glycine α-amidating monooxygenase. The peptides potently inhibited the growth of Escherichia coli and Staphylococcus aureus confirming that a cyclic domain is not necessary for antimicrobial activity. A fifth peptide (SFLNFFKGAA10KNLLAAGLDK20LKCKISGTQC30), that also displayed broad-spectrum antimicrobial activity, was isolated from the skin extract and showed structural similarity with members of the ranatuerin-2 family previously isolated from the skin of North American ranid frogs.
Keywords: Brevinin-1; Ranatuerin-2; Frog skin; Antimicrobial;

Agrocybin, an antifungal peptide from the edible mushroom Agrocybe cylindracea by Patrick H.K. Ngai; Zheng Zhao; T.B. Ng (191-196).
An antifungal peptide with a molecular mass of 9 kDa was isolated from fresh fruiting bodies of the mushroom Agrocybe cylindracea. The isolation procedure comprised ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and FPLC-gel filtration on a Superdex 75 column. The antifungal peptide, designated as agrocybin, was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. Agrocybin exerted antifungal activity against several fungal species but lacked inhibitory activity against bacteria when tested up to 300 μM. The activity of HIV-1 reverse transcriptase was attenuated in the presence of agrocybin. It exhibited weaker mitogenic activity than Con A on isolated murine splenocytes, but was devoid of antiproliferative activity on Hep G2 (hepatoma) cells when tested at 110 μM.
Keywords: Isolation; Antifungal; Peptide; Mushroom;

The protegrin family of antimicrobial peptides is among the shortest in sequence length while remaining very active against a variety of microorganisms. The major goal of this study is to characterize easily calculated molecular properties, which quantitatively show high correlation with antibacterial activity. The peptides studied have high sequence similarity but vary in activity over more than an order of magnitude. Hence, sequence analysis alone cannot be used to predict activity for these peptides. We calculate structural properties of 62 protegrin and protegrin-analogue peptides and correlate them to experimental activities against six microbe species, as well as hemolytic and cytotoxic activities. Natural protegrins structures were compared with synthetic derivatives using homology modeling, and property descriptors were calculated to determine the characteristics that confer their antimicrobial activity. A structure–activity relationship study of all these peptides provides information about the structural properties that affect activity against different microbial species.
Keywords: Antimicrobial peptide; Homology modeling; Structure–activity relationships;

Effects of pexiganan alone and combined with betalactams in experimental endotoxic shock by Andrea Giacometti; Oscar Cirioni; Roberto Ghiselli; Fiorenza Orlando; Wojciech Kamysz; Marco Rocchi; Giuseppina D’Amato; Federico Mocchegiani; Carmela Silvestri; Jerzy Łukasiak; Vittorio Saba; Giorgio Scalise (207-216).
To investigate the efficacy of pexiganan, a 22-residue magainin analog, alone and combined with betalactmas antibiotics in three experimental rat models of Gram-negative septic shock. Adult male Wistar rats were given (i) an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS; (ii) 2 × 1010  CFU of E. coli ATCC 25922; and (iii) intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intraperitoneally isotonic sodium chloride solution, 1 mg/kg pexiganan, 1 mg/kg polymyxin B, 20 mg/kg imipenem, 60 mg/kg piperacillin alone and combined with 1 mg/kg pexiganan. Each group included 15 animals. Lethality, bacterial growth in blood or intra-abdominal fluid, endotoxin and TNF-α concentrations in plasma. All compounds reduced the lethality when compared to controls. Piperacillin and imipenem significantly reduced the lethality and the number of E. coli in abdominal fluid compared with saline treatment. Pexiganan showed a slightly lower antimicrobial activity than betalactams even though it achieved a substantial higher decrease in endotoxin and TNF-α plasma concentrations than imipenem and piperacillin. No statistically significant differences were noted for antimicrobial and antiendotoxin activities between pexiganan and polymyxin B. Combination between pexiganan and betalactams showed to be the most effective treatment in reducing all variables measured. The use of a novel antimicrobial compound able to bind to LPS associated to potent antibiotics such as betalactams may become an important future consideration for sepsis treatment.
Keywords: Pexiganan; Antimicrobial peptides; Endotoxin; Lipopolysaccharide; Sepsis;

Conformation and activity of δ-lysin and its analogs by Vishnu M Dhople; Ramakrishnan Nagaraj (217-225).
δ-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, δ-lysin does not exhibit antibacterial activity. We have synthesized δ-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7–26 and 5–26 of δ-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike δ-lysin, did not self-associate to adopt α-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on δ-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.
Keywords: Alpha helix; Antibacterial activity; Hemolytic activity; Peptide conformation; Toxin; Synthetic peptides;

Solution structure of a peptide derived from the oncogenic protein β-Catenin in its phosphorylated and nonphosphorylated states by Simon Megy; Gildas Bertho; Josyane Gharbi-Benarous; Françoise Baleux; Richard Benarous; Jean-Pierre Girault (227-241).
β-Catenin plays an essential role in the Wingless/Wnt signaling cascade. Phosphorylation of β-Catenin in its N-terminal region by the kinase GSK-3β is required for the interaction with the SCF-β-TrCP protein complex that targets β-Catenin for proteasome degradation. In the present work, we used two peptides of 32 amino acids referred to β-Cat17–48 and P-β-Cat17–48 for the phosphorylated peptide at the two sites Ser33 and Ser37. Circular dichroism and NMR techniques were used to assess the influence of the phosphorylation. The spectra of the peptides at pH 7.2 were completely assigned. Analysis of the medium-range NOE connectivities indicated that β-Cat17–48 seems to be only poorly folded. These data are in agreement with the result of structure calculations. P-β-Cat17–48 possesses two helical segments around the DpSGXXpS motif, which forms a large bent with the phosphate groups pointing out of the structure. On the contrary, β-Cat17–48 shows less well-defined secondary structures and appears as a more flexible peptide, but adopts in the motif DSGXXS a more compact conformation than P-β-Cat17–48. Differences in this molecular region suggest that conformational changes of phosphorylated β-Catenin play an important role for the interaction with the SCF-β-TrCP protein complex.
Keywords: β-Catenin; β-TrCP; Molecular modeling; NMR spectroscopy; Phosphorylated peptide structure; Wingless/Wnt signals;

Heterologous expression, characterization and structural studies of a hydrophobic peptide from the HIV-1 p24 protein by Priscila V. Castilho; Patricia T. Campana; Assuero F. Garcia; Leila M. Beltramini; Ana Paula U. Araújo (243-249).
Proteins from the inner core of HIV-1, such as the capsid protein (p24), are involved in crucial processes during the virus life cycle. The p24 protein plays an active structural role in the Gag protein and in its mature form. This work describes the production of a peptide derived from the p24 C-terminal, TLRAEQASQEVKNWMTETLLVQNA, using recombinant technology. This region (p24-3) is involved in interfaces during the p24 dimerization, which occurs during capsid assembly. The p24-3 sequence was obtained by a synthetic gene strategy and inserted into the pET 32a expression vector to produce soluble fusion protein in Escherichia coli BL21(DE3). This strategy leads to an incorporation of three amino acid residues (AMA) in the N-terminal of the native sequence to form the recombinant p24-3 (rp24-3). The rp24-3 was purified by reverse phase chromatography to homogeneity, as inferred by mass spectrometry and protein sequence analysis. Structural studies using circular dichroism and steady-state fluorescence showed that the rp24-3 is structured by helical and beta elements. As a function of its hydrophobic character it can self-associate forming oligomers. We present in this paper the first development of a suitable expression system for rp24-3, which provides high amounts of the peptide. This strategy will allow the development of new antiviral (HIV) agents.
Keywords: Recombinant peptide; p24 HIV-1; Synthetic gene; Circular dichroism; Fluorescence spectroscopy;

Synthesis and evaluation of eight-membered cyclic pseudo-dipeptides by Andrew D. Abell; Karina M. Brown; James M. Coxon; Matthew A. Jones; Sigeru Miyamoto; Axel T. Neffe; Janna M. Nikkel; Blair G. Stuart (251-258).
In the course of the development of calpain inhibitors, we report the synthesis of eight-membered cyclic pseudo dipeptides closely related to the known inhibitor SJA6017. The ring closure was effected by metathesis of the diallyl-substituted dipeptides 6 and 7. The formation of the dipeptides under kinetic control leads to the preferential formation of the unlike diastereomer 7 over the like diastereomer 6. The relative configuration of the diastereomers was determined by NMR and modeling studies of the related cyclic compounds 8 and 9 and their derivatives. The compounds proved not to inhibit calpain.
Keywords: Peptidomimetics; Molecular modeling; Metathesis; Cataract; Calpain;

Cloning and characterization of a molt-inhibiting hormone-like peptide from the prawn Marsupenaeus japonicus by Tsuyoshi Ohira; Hidekazu Katayama; Satoshi Tominaga; Tetsu Takasuka; Teruaki Nakatsuji; Haruyuki Sonobe; Katsumi Aida; Hiromichi Nagasawa (259-268).
Recently, it was demonstrated by PCR amplification that an additional molt-inhibiting hormone (MIH)-like peptide was present in the kuruma prawn Marsupenaeus japonicus. In this study, a cDNA encoding this peptide designated Pej-MIH-B was cloned. The Pej-MIH-B gene was expressed strongly in the nerve cord, and weakly in the eyestalk. It was possible to isolate Pej-MIH-B from the sinus glands in the eyestalks. The recombinant Pej-MIH-B expressed in Escherichia coli showed low molt-inhibiting activity, but did not exhibit hyperglycemic activity. These results suggest that Pej-MIH-B does not function as MIH or CHH intrinsically, but may have some unknown functions.
Keywords: Crustacea; Crustacean hyperglycemic hormone family; Kuruma prawn; Marsupenaeus japonicus; Molt; Molt-inhibiting hormone;

The Drosophila FMRFamide-related peptide, DPKQDFMRFamide modulates synaptic transmission at the larval neuromuscular junction. The amplitude of excitatory junctional potentials (EJPs) produced by the selective stimulation of motor neuron MN6/7-Ib increases following application of 1 μM DPKQDFMRFamide. EJPs elicited by stimulating motor neuron MNSNb/d-Is, however, exhibit no significant increase with the same concentration of neuropeptide. The mechanisms underlying the modulatory effects of DPKQDFMRFamide were examined using a combination of pharmacological and genetic methods. Three independent lines of evidence implicate CaMKII as an essential effector protein or part of the signal transduction pathway. The effect of the neuropeptide is suppressed by 1 μM KN-93 (CaMKII inhibitor) and by heat-shock induced expression of a CaMKII inhibitor. A heterozygous CaM kinase mutant responds poorly to the peptide.
Keywords: FMRFamide; Drosophila; Neuromuscular junction; CaMKII; DPKQDFMRFamide;

Peptidergic neuromodulation of the lumbar locomotor network in the neonatal rat spinal cord by Grégory Barrière; Sandrine Bertrand; Jean-René Cazalets (277-286).
It is now well established that a dynamic balance of neurotransmitters and neuromodulators finely influence the output of neuronal networks and subsequent behaviors. In the present study, to further understand the modulatory processes that control locomotor behavior, we investigated the action of 11 neuropeptides, chosen among the various peptide subfamilies, on the lumbar neuronal network in the in vitro neonatal rat spinal cord preparation. Peptides were bath-applied alone, in combination with N-methyl-d,l-aspartate (NMA) or with the classical ‘locomotor cocktail’ of NMA and serotonin. Using these different experimental paradigms, we show that each peptide can neuromodulate the lumbar locomotor network and that peptides exhibit different neuromodulatory profiles and potencies even within the same family. Only vasopressin, oxytocin, bombesin and thyrotropin releasing hormone triggered tonic or non-organized rhythmic activities when bath-applied alone. All the neuropeptides modulated NMA induced activity and/ or ongoing sequences of fictive locomotion to varying degrees. These results suggest that neuropeptides play an important role in the control of the neural network for locomotion in the neonatal rat. Their various profiles of action may account in part for the great flexibility of motor behaviors.
Keywords: Peptides; Fictive locomotion; Newborn rat; Neuromodulation; Spinal cord;

Effects of a behaviorally active antibody on the brain uptake and clearance of amyloid beta proteins by William A. Banks; Patrizia Pagliari; Ryota Nakaoke; John E. Morley (287-294).
Antibodies directed against amyloid beta protein (AßP) have been suggested to be effective in the treatment of Alzheimer's disease (AD). Here, we used in vivo and in vitro models to test some of the mechanisms by which antibodies may produce their effects. We found that the blood-to-brain uptake of murine AßP1–42 was significantly reduced when co-injected peripherally with an antibody known to reverse cognitive defects in the SAMP8, an mouse model of AD. This antibody was not effective when tested against the more slowly transported human AßP1–42. Antibody given by intracerebroventricular (icv) injection did not improve the clearance of murine AßP1–42 from the brains of young healthy mice, which already rapidly clear AßP by saturable and non-saturable mechanisms. Antibody given icv also did not improve the clearance of human AßP1–42 from the brains of aged SAMP8 mice, a combination in which the AßP is only poorly cleared from brain. IV antibody also did not affect retention of murine AßP in young mice. In vitro transwell studies with monolayers of mouse brain endothelial cells (MBEC) found no evidence that antibody in the vascular chamber would retard the reuptake of AßP which had been effluxed from the brain-side chamber. A statistical trend suggested that antibody might decrease the association of AßP with brain vasculature. In conclusion, we found that icv administration of antibody was not effective in aiding clearance of AßP already in brain, but that blood-borne antibody can inhibit the entry of AßP into brain and might prevent AßP from associating with the brain vasculature.
Keywords: Amyloid beta proteins; Antibody; Brain; Blood–brain barrier; Cerebrospinal fluid; Peptide;

Characterization of the [125I]endomorphin-2 binding sites in the MCF7 breast cancer cell line by Jakub Fichna; Urszula Krajewska; Marek Rozalski; Marek Mirowski; Anna Janecka (295-299).
In the present study, the expression of the μ-opioid receptor on protein level has been demonstrated in MCF7 breast cancer cells. Binding of the [125I]-labeled μ-opioid receptor selective ligand endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) was examined in vitro using a cross-linking assay followed by a Western blot technique. The radioactive complex had a molecular weight of about 65 kDa and was detectable by anti-μ-opioid receptor antibody, indicating the presence of μ-opioid receptors in MCF7 cell membranes. Characterization of endomorphin-2 binding to the membranes obtained from MCF7 cells was performed. Cold saturation experiments with [125I]endomorphin-2 showed biphasic binding curves in Scatchard coordinates. One component represents a high affinity and low capacity, and the other low affinity and higher capacity binding sites. The obtained B max values for [125I]endomorphin-2 binding to MCF7 membranes were much higher than those obtained for mouse brain. Pharmacological characterization of the [125I]endomorphin-2 binding sites was made using endomorphin-2 and two other μ selective ligands, morphiceptin, and [d-1-Nal3]morphiceptin on MCF7 cell membrane preparations and whole MCF7 cells. In both cases, the rank order of potency was [d-1-Nal3]morphiceptin > endomorphin-2 > morphiceptin, but in case of whole MCF7 cells the IC50 values were about 40 times higher.
Keywords: Opioid analog; Binding studies; μ-Opioid receptor;

Previous studies performed in this laboratory have demonstrated that the fetal lung contains immunoreactive adrenocorticotropin (irACTH), and that the lung both clears and secretes irACTH under basal and stimulated conditions. Furthermore, we have demonstrated that the irACTH in fetal lung is accounted for by proopiomelanocortin (POMC), and that there is an evidence of post-translational processing that is distinct from the pattern of processing typical of the anterior pituitary. The present study was designed to test the hypothesis that POMC is synthesized in the fetal lung, and that there is decreased synthesis in the late-gestation ovine fetal lung. Lungs were collected from fetal sheep at 80, 100, 120, 130, and 145 days gestation (n  = 4/group; term = 147 days). POMC mRNA was measured using reverse transcription and real-time polymerase chain reaction with probe and primers designed in this laboratory. The greatest abundance of POMC mRNA was in the 80-days fetal sheep, and the relative abundance decreased as a function of fetal gestational age. POMC protein was measured using immunoblot analysis in lungs from 80, 120, and 145-days fetal sheep. The pattern of POMC protein abundance was consistent with that of the mRNA (highest at 80 days, lowest at 145 days). The POMC immunoblot revealed specific staining of a peptide with molecular weight of 27 kDa and another peptide with a molecular weight slightly higher than that of native POMC (32 kDa). For comparison, we measured POMC mRNA in skeletal muscle and small intestine. We found POMC expression in both fetal tissues, but no statistically significant ontogenetic pattern of expression. We conclude that POMC is synthesized in the ovine fetal lung, and that the rate of synthesis decreases as the fetus matures in utero. We speculate that the decreasing abundance of POMC mRNA and protein reflects decreased release of POMC and POMC-related peptides into the fetal bloodstream.
Keywords: Gestation; Parturition; POMC mRNA;

Angiotensin-induced vasopressin release and activation of hypothalamic neuron in pre-term fetuses by Zhice Xu; Fang Hu; Lijun Shi; Wanping Sun; Jiawei Wu; Paul Morrissey; Jiaming Yao (307-314).
Our previous studies have shown that central administration of angiotensin II (ANG II) causes vasopressin release in the near-term fetus in utero as evidence that the hypothalamic-neurohypophysial system has relatively matured before birth. However, it is still unknown whether the vasopressin controlling centers have been functionally developed in younger fetuses. This study determined fetal plasma vasopressin levels and hypothalamic vasopressin neuron activity in the chronically instrumented pre-term ovine fetuses. Introcerebroventricular (i.c.v.) administration of ANG II did not affect fetal plasma osmolality and sodium concentrations. However, fetal plasma vasopressin levels were significantly increased (∼3-fold) in response to central injection of ANG II. Central ANG II also induced vasopressin-neuron activity marked with c-fos expression in the fetal hypothalamus at pre-term. In addition, the fetal organum vasculosum of the lamina terminalis and the subfornical organ were activated. The results suggest that hypothalamic-neurohypophysial system has been relatively intact and functional at 70% gestational age, and that central angiotensin is important in inducing fetal vasopressin release in utero.
Keywords: Fetal vasopressin; Central angiotensin; Hypothalamic nuclei; Pre-term;

Hemodynamic and metabolic effects of angiotensin II on the liver by Écio Alves Nascimento; Luciana Gioli-Pereira; Leda Teixeira Carvalho; Edson Lucas Santos; João Bosco Pesquero; Maria Kouyoumdjian; Durval Rosa Borges (315-322).
To ascertain the mechanism of interaction between angiotensins (AI and AII) and the liver, an angiotensin-converting enzyme inhibitor (captopril) and a receptor antagonist (losartan) were used. Monovascular or bivascular liver perfusion was used to assess both hemodynamic (portal and arterial hypertensive responses) and metabolic (glucose production and oxygen consumption) effects. Microphysiometry was used for isolated liver cell assays to assess AII or losartan membrane receptor-mediated interaction. Captopril abolishes portal hypertensive response (PHR) to AI but not the AII effect. AII infused via the portal pathway promotes calcium-dependent PHR but not a hypertensive response in the arterial pathway (AHR); when infused into the arterial pathway AII promotes calcium-dependent PHR and AHR. Losartan infused into the portal vein abolishes PHR to AII but not the metabolic response; when infused via both pathways it abolishes the hypertensive responses and inhibits the metabolic effects. Isolated liver cells specifically respond to AII. Sinusoidal cells, but not hepatocytes, respond to 10 nM losartan. We conclude that AI has to be converted to AII to produce PHR. Quiescent stellate cells interacts in vitro with AII and losartan. Hemodynamic responses to AII are losartan-dependent but metabolic responses are partially losartan-independent. AII hemodynamic actions are mainly presinusoidal.
Keywords: Angiotensin; Angiotensin-converting enzyme; Bivascular liver perfusion; Portal hypertension; Angiotensin receptor; Losartan;

Involvement of cyclooxygenase-dependent pathway in contraction of isolated ileum by urotensin II by Syunji Horie; Yuumi Tsurumaki; Akiyoshi Someya; Tetsuya Hirabayashi; Takeshi Saito; Yasunobu Okuma; Yasuyuki Nomura; Toshihiko Murayama (323-329).
We previously reported that urotensin II induced biphasic (brief- and long-lasting) contractions and the brief contraction was mediated by acetylcholine release from ganglionic cholinergic neurons in a segment of guinea-pig ileum. In the present work, we studied the mechanism contributing to long-lasting contractions induced by urotensin II. Treatment with 0.1 μM tetrodotoxin, 300 nM ω-conotoxin GVIA (an inhibitor of N-type Ca2+ channels) and 10 μM indomethacin (an inhibitor of cyclooxygenases) markedly inhibited 100 nM urotensin II-induced long-lasting contractions. The addition of 1 μM prostaglandin F (PGF) caused a limited brief contraction following long-lasting contraction, while 1 μM PGE2 induced marked biphasic contractions. Treatment with neurotoxins inhibited the long-lasting contractions induced by PGF and PGE2 without changing the PGE2-induced brief contractions. Treatment with 1 μM atropine markedly inhibited the urotensin II- and PGF-induced long-lasting contractions, but was less effective on the PGE2 responses. Treatment with a phospholipase A2 inhibitor decreased the urotensin II-induced contractions. These findings suggest that urotensin II induces, at least partially, long-lasting contractions via PG-sensitive cholinergic neurons and muscarinic acetylcholine receptors in the ileum.
Keywords: Urotensin II; Prostanoids; Cyclooxygenase; Cholinergic neurons; Guinea-pig ileum;

The purpose of this study was to localize sites of calcitonin gene-related peptide binding in neonatal, freshly isolated and dedifferentiated adult cardiac myocytes in order to help us elucidate the mechanisms of action of this neuropeptides. Previous work has shown that treatment with calcitonin gene-related peptide results in dramatic changes in calcium transients, so we carried out multi-channel acquisitions of fluorescently labeled images to reveal where calcitonin gene-related protein and the l-type calcium channel were localized. Calcitonin gene-related protein was sparse and randomly distributed in rod-like adult cardiomyocytes, found in abundance in areas of the cell where striations were apparent and not where adhesion proteins predominated in dedifferentiating adult myocytes, and in a large perinuclear concentration, with some spreading into the cytoplasm in neonatal cells. Subsequent modeling demonstrated that calcitonin gene-related peptide and the l-type calcium channel protein were closely associated in each of the three myocyte types, suggesting that while the peptide has dramatic and different effects on intracellular calcium levels of the various cardiomyocytes, the action is probably via diverse mechanisms as a result of effects on different channels or pump proteins due to alterations in intracellular calcium concentrations.
Keywords: Calcium transients; Fluorescence imaging; Heart failure; Neuropeptides; Calcitonin gene-related peptide;

The intracellular signal transduction pathway by which the yeast Saccharomyces cerevisiae responds to the presence of peptide mating pheromone in its surroundings is one of the best understood signaling pathways in eukaryotes, yet continues to generate new surprises and insights. In this review, we take a brief walk down the pathway, focusing on how the signal is transmitted from the cell-surface receptor-coupled G protein, via a MAP kinase cascade, to the nucleus.
Keywords: Yeast mating pheromone; Mitogen-activated protein kinase; Signal transduction; Saccharomyces cerevisiae;