Peptides (v.25, #4)


Neuropeptide chronomics in clinically healthy young adults: circaoctohoran and circadian patterns by Alex Löckinger; Dieter Köberle; Paul St König; Alois Saria; Manfred Herold; Germaine Cornélissen; Franz Halberg (533-542).
Endothelin-1 (ET-1) undergoes an about 8-h (circaoctohoran) rather than a circadian variation in clinical health. Herein, 24 h plasma concentrations of vasoactive intestinal peptide (VIP), substance P (SP), neuropeptide Y (NpY), and cortisol used as reference, were obtained from 20 healthy young adults starting at 07:00 or 19:00 h. Like ET-1, SP and NpY undergo a circaoctohoran variation, whereas VIP is circadian rhythmic, peaking during the night, some 8 h prior to the circadian acrophase of cortisol. Maps of circadian and extra-circadian patterns may serve for screening, diagnosis and a better understanding of mechanisms underlying the etiology of various diseases.
Keywords: Circadian; Circaoctohoran; Plasma concentrations; Vasoactive intestinal peptide (VIP); Substance P (SP); Neuropeptide Y (NpY); Endothelin-1; Cortisol;

A potential antitumor peptide therapeutic derived from antineoplastic urinary protein by Kathleen M Hehir; Alexander Baguisi; Sarah E Pennington; Janna M Bates; Paul A DiTullio (543-549).
New therapies in cancer treatment are focusing on multifaceted approaches to starve and kill tumors utilizing both antiangiogenic and chemotherapeutic compounds. Antineoplastic Urinary Protein (ANUP), a 32 kDa protein normally secreted in human urine, has been previously described as a molecule possessing both antiproliferative and antiangiogenic activities. Two synthetic peptides complimentary to the N-terminus of ANUP were designed to test their ability to reproduce these beneficial effects but ultimately to provide a more useful small molecule therapeutic. The results show that the peptides reduced tumor burden by up to 70% in a nude mouse model and demonstrated the ability to inhibit blood vessel formation in a chick chorioallantoic membrane assay (CAM).
Keywords: Angiogenesis; Antineoplastic urinary protein; ANUP; CAM;

Identification and characterization of peptides that bind to cyanovirin-N, a potent human immunodeficiency virus-inactivating protein by Zhaozhong Han; John T Simpson; Matthew J Fivash; Robert Fisher; Toshiyuki Mori (551-561).
Cyanovirin-N (CV-N) exerts a potent human immunodeficiency virus (HIV)-inactivating activity against diverse strains of HIV by binding to the viral surface envelope glycoprotein gp120 and blocking its essential interactions with cellular receptors. Based on previous thermodynamic analyses, it has been speculated that discrete protein–protein interactions might play an important ancillary role in the CV-N/gp120 binding event, in addition to the interactions of CV-N with specific oligosaccharides present on gp120. Here, we report the identification and characterization of CV-N-binding peptides, which were isolated by screening of M13 phage-displayed peptide libraries. After performing three rounds of biopanning of the libraries against biotinylated CV-N, a CV-N-binding motif, X3CX6(W/F)(Y/F)CX2(Y/F), was evident. A vector was designed to express CV-N-binding peptides as a fusion with thioredoxin (Trx) containing a penta-His affinity tag. The CV-N-binding peptides fused with His-tagged Trx inhibited binding of the corresponding peptide-bearing phages to CV-N, confirming that the peptides possessed CV-N-binding activity. Optical biosensor binding studies showed that the one of the CV-N-binding peptide, TN10-1, bound to CV-N with a K D value of 1.9 μM. The results of alanine scanning mutagenesis of the peptide showed that aromatic residues at positions 11, 12, and 16, as well as the conformational structure of the peptide secured by a disulfide bond, were important for the binding interactions. A series of competitive binding assays confirmed that gp120 inhibited CV-N binding of the corresponding peptide-bearing phages, and suggested that TN10-1 peptides were mimicking the protein component of gp120 rather than mimicking specific oligosaccharides present on gp120.
Keywords: Cyanovirin-N; CV-N; Peptide; Phage display; Library; HIV; gp120;

Metabolism and absorption enhancement of methionine enkephalin in human nasal epithelium by Remigius U Agu; H Vu Dang; Mark Jorissen; Renaat Kinget; Norbert Verbeke (563-569).
The objective of this study was to investigate absorption enhancing approaches for systemic delivery of methionine enkephalin via the nose. Absorption promotion of methionine enkephalin in the presence of protease inhibitors (bestatin, puromycin) and absorption enhancers (glycocholate, dimethyl-β-cyclodextrin) were investigated in human nasal epithelium. Co-administration of the peptide with protease inhibitors and absorption enhancers resulted in a remarkable increase in Met-Enk permeation (4- to 94-fold). The increase was proportional to transepithelial resistance reduction and permeation of paracellular marker dye. Perturbation of the epithelial tight junctions seen in vitro may not occur in vivo due to mucus protection and mucociliary clearance.
Keywords: Peptides; Methinonine enkephalin; Aminopeptidase; Nasal cell culture; Protease inhibitors; Absorption enhancers; Toxicity;

Opioid peptide response to spinal cord stimulation in chronic critical limb ischemia by Fiorella Fontana; Pasquale Bernardi; Giuseppina Lanfranchi; Santi Spampinato; Rosanna Di Toro; Eleonora Conti; Francesca Bonafè; Sergio Coccheri (571-575).
Twelve patients with chronic critical limb ischemia in whom a spinal cord stimulation (SCS) system had been implanted for at least one year had increased microvascular flow and achieved healing of trophic acral lesions. After switching off the system, the clinical improvement persisted for 10 days and the neurohormonal pattern showed high plasma values of β-endorphin and Met-enkephalin, normal dynorphin B, endothelin-1 and catecholamines, and low nitric oxide. Met-enkephalin levels were further increased (P<0.01) immediately after switching on the electrical stimulation again. The persistence of high plasma opioid levels after switching off the spinal cord stimulation explains the absence of subjective complaints and suggests an involvement of opioids in the regulation and improvement of the microcirculation.
Keywords: Chronic critical limb ischemia; Spinal cord stimulation; β-Endorphin; Met-enkephalin; Dynorphin B; Endothelin-1; Norepinephrine; Epinephrine; Nitric oxide;

Supraspinal anti-allodynic and rewarding effects of endomorphins in rats by Eagle Yi-Kung Huang; Ching-Ming Chen; Pao-Luh Tao (577-583).
Two potent endogenous opioid peptides, endomorphin-1 (EM-1) and -2 (EM-2), which are selective μ-opioid agonists, have been identified from bovine and human brain. These endomorphins were demonstrated to produce a potent anti-allodynic effect at spinal level. In the present study, we further investigated their supraspinal anti-allodynic effects and rewarding effects. In a neuropathic pain model (sciatic nerve crush in rats), EM-1 and -2 (15 μg, i.c.v.) both showed significant suppressive effects in the cold-water allodynia test, but EM-1 showed a longer duration than EM-2. Naltrexone (NTX; 15 μg) and naloxonazine (NLZ; 15 μg) were both able to completely block the anti-allodynic effects of EM-1 and -2. In the tests of conditioned place preference (CPP), only EM-2 at the dose of 30 μg showed significant positive rewarding effect, whereas both endomorphins did not induce any reward at the dose of 15 μg. Due to the low solubility and the undesired effect (barrel rotation of the body trunk), EM-1 was not tested for the dose of 30 μg in the CPP tests. It was also found that acute EM-2 (30 μg) administration increased dopamine turnover in the shell of nucleus accumbens in the microdialysis experiments. From these results, it may suggest that EM-1 and -2 could be better supraspinal anti-allodynic agents compared with the other opioid drugs, although they may also induce rewarding.
Keywords: Endomorphins; Neuropathic pain; Allodynia; Rewarding; Conditioned place preference; Microdialysis; Nucleus accumbens; Dopamine;

The bombesin/gastrin releasing peptide receptor antagonist RC-3095 blocks apomorphine but not MK-801-induced stereotypy in mice by Carolina A Meller; João Antônio Pêgas Henriques; Gilberto Schwartsmann; Rafael Roesler (585-588).
Bombesin (BN)-like peptides might be involved in the pathogenesis of neuropsychiatric disorders such as schizophrenia. Stereotyped behaviors induced by the dopamine receptor agonist apomorphine or the N-methyl-D-aspartate glutamate receptor antagonist dizocilpine (MK-801) in rodents have been proposed as animal models of schizophrenic psychosis. In the present study we evaluated the effects of the BN/gastrin-releasing peptide receptor (GRP) antagonist (D-Tpi6, Leu13 psi[CH2NH]-Leu14) bombesin (6–14) (RC-3095) on apomorphine and MK-801-induced stereotyped behavior in mice. An intraperitoneal (i.p.) injection of RC-3095 (1.0, 10.0 or 100.0 mg/kg) blocked apomorphine-induced stereotypy. The inhibitory effect of RC-3095 on apomorhine-induced stereotypy was similar to that induced by haloperidol (0.5 mg/kg). RC-3095 did not affect stereotyped behavior induced by MK-801 (0.5 mg/kg). The results provide the first evidence that BN/GRP receptor antagonism blocks stereotyped behavior induced by a dopamine agonist. Together with previous evidence, the present study indicates that the BN/GRP receptor can be considered a drug target in the investigation of potential new agents for treating neuropsychiatric disorders.
Keywords: RC-3095; Bombesin receptor; Gastrin/releasing peptide; Stereotypy; Antipsychotics; Psychosis; Mice;

Distribution of adrenomedullin-containing perivascular nerves in the rat mesenteric artery by N Hobara; A Nakamura; A Ohtsuka; M Narasaki; K Shibata; Y Gomoita; H Kawasaki (589-599).
Distribution of adrenomedullin (AM)-containing perivascular nerve fibers was studied in rat mesenteric arteries. Many fibers containing AM-like immunoreactivity (LI) were observed in the adventitia. AM-LI fibers were abolished by cold storage denervation or capsaicin but not 6-hydroxydopamine. Double immunostainings showed colocalization of AM-LI with calcitonin gene-related peptide (CGRP)-LI. The dorsal root ganglia had many AM-positive cells and AM mRNA detected by RT–PCR. Electron microscopy study revealed high proportions of immunogold labeling for AM and colocalization of both AM-LI and CGRP-LI in unmyelinated nerve axons. These results suggest that AM-containing perivascular nerves are distributed in the rat mesenteric artery.
Keywords: Adrenomedullin; Perivascular nerve; Rat mesenteric artery; Calcitonin gene-related peptide; Neuropeptide Y; Capsaicin-sensitive nerve;

Adrenomedullin (ADM) is a potent vasodilatory peptide which regulates blood pressure, cell growth and bone formation. Our work was aimed to explore the production of ADM, changes and pathophysiological significance of ADM mRNA and ADM receptor components—calcitonin receptor like receptor (CRLR) and receptor activity modifying proteins (RAMPs) mRNA in calcified myocardium and aorta of rats induced by Vitamin D3 plus nicotine. Contents of ADM in plasma, myocardium and aorta were measured by radioimmunoassay (RIA). The amount of ADM, CRLR and RAMPs mRNA was determined by semi-quantitative RT-PCR. The calcium content and alkaline phosphatase activity in myocardium and aorta of rats were measured. The results showed that the contents of calcium in calcified myocardium and aorta were increased by 3.5- and 6-fold (all P<0.01), respectively, and alkaline phosphatases activity in calcified myocardium and aorta were increased by 66.5 and 82.7% (all P<0.01), respectively, compared with control. Contents of ADM in plasma, myocardium and aorta were increased by 58% (P<0.01), 14.3% (P<0.01) and 27.8% (P<0.05). Furthermore, it was found that the amount of ADM, CRLR and RAMP2 mRNA in calcified myocardium was elevated by 90.6, 157.5 and 119.6% (all P<0.01), RAMP3 mRNA was decreased by 14.1% (P<0.01), respectively, compared with control. The amount of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified aorta was elevated by 37.7% (P<0.01), 41.4% (P<0.01), 60.1% (P<0.05) and 13% (P<0.01), respectively, compared with control. The elevated level of CRLR and RAMP2 mRNA were in positive correlation with that of ADM mRNA (r=0.992 and 0.882, respectively, P<0.01) in calcified myocardium. The elevated level of CRLR and RAMP3 mRNA were also in positive correlation with that of ADM mRNA (r=0.727, P<0.05 and 0.816, P<0.01, respectively) in calcified aorta. These results demonstrated that calcified myocardium and aorta generated an increased amount of ADM, up-regulated gene expressions of ADM, CRLR and RAMP2 mRNA. While the alteration of RAMP3 mRNA in calcified myocardium and aorta was different. These suggested that ADM and its receptor system might involve in the regulation of calcification in heart and aorta.
Keywords: Calcification; Adrenomedullin; Calcitonin receptor like receptor; Receptor activity modifying protein; Heart and vessel;

Disassociated increases of adrenomedullin in the rat cerebrospinal fluid and plasma after salt loading and systemic administration of lipopolysaccharide by Lei Chen; Seiichi Hashida; Kazuo Kitamura; Tanenao Eto; Kenji Kangawa; Ryota Serino; Bela Kis; Hiroshi Yamashita; Yoichi Ueta (609-614).
To determine the role of adrenomedullin (AM) in the fluid electrolyte homeostasis and endotoxin shock, cerebral spinal fluid (CSF) and plasma were sampled from rats after respective challenges. The AM levels were measured by a highly sensitive immunoassay. The AM levels in the CSF of the rats anesthetized with ether (10.7±0.60 fmol/ml) were significantly higher than those with isoflurane (5.17±0.70 fmol/ml, P<0.01), while the plasma level did not differ significantly. The CSF levels of the rats received 2% saline drinking increased to 3 and 4 folds at day 5 and day 7, respectively, while the plasma levels did not differ from controls at both time points. The AM levels in CSF or plasma increased to 1.5 and 3 folds at 1.5 h after intraperitoneal (i.p.) administration of lipopolysaccharide (LPS, 5 mg/kg), reached 6.5 and 30 folds at 6 h, respectively, while no change was observed in the controls. The present findings suggest that AM in the CSF is regulated independently from that in the plasma, the centrally synthesized AM plays and important role in the regulation of the fluid electrolyte homeostasis. Furthermore, the circulatory AM plays an important role in the endotoxin shock.
Keywords: Adrenomedullin; CSF; Plasma; Salt loading; Endotoxin shock; Stress;

In the central nervous system and the periphery, atrial natriuretic peptide (ANP) and angiotensinII(AngII) play important and opposite roles in regulating blood pressure and fluid electrolyte balance. Their central mechanisms are unclear. In the brain the anteroventral third ventricle region (AV3V) contains the most prominent collection of atriopeptin-like immunoreactive perikarya. Our previous studies show that: (1) AV3V stimulation by glutamate produces a fall in blood pressure; (2) there is an AngII pressor system composed of the lateral hypothalamus/perifornical region (LH/PF), subfornical organ (SFO), nucleus paraventricularis (NPV) and rostral ventrolateral medulla (RVL). The present study was to examine whether ANPergic projections from the AV3V could act on nuclei involved in the above-mentioned AngII pressor system. Here we demonstrate that: (1) Injection of atriopeptinIII into the LH/PF, SFO, NPV, or RVL induces a depressor response; whereas injection of normal saline has no effect. (2) Pre-injection of A 71915 (an atriopeptinIII antagonist) into the LH/PF, SFO, NPV, or RVL reverses the depressor response of the AV3V to glutamate (Glu). The results suggest that excitation of atriopeptinergic neurons in the AV3V by Glu produces an inhibitory effect on each nucleus in the LH/PF-SFO-NPV-RVL AngII pressor system.
Keywords: Anteroventral third ventricle region; Lateral hypothalamus/perifonical region; Subfornical organ; Atrial natriuretic peptide; Blood pressure;

The fruiting body of Tricholoma giganteum has many pharmaceutical uses and has long been utilized as a home remedy in Asia. This study describes the extraction and characterization of the first angiotensin I-converting enzyme (ACE) inhibitory peptide from T. giganteum. The maximum ACE inhibitory activity (IC50: 0.31 mg) was obtained when the fruiting body of T. giganteum was extracted with distilled water at 30 °C for 3 h. After the purification of ACE inhibitory peptides with ultrafiltration, Sephadex G-25 column chromatography, and reverse-phase HPLC, an active fraction with an IC50 of 0.04 mg and a yield of 0.3% was obtained. The ACE inhibitory peptide was a novel tripeptide, showing very low similarity to other ACE inhibitory peptide sequences, and was sequenced as Gly–Glu–Pro. The purified ACE inhibitor from T. giganteum competitively inhibited ACE, and it maintained inhibitory activity even after incubation with proteases. ACE inhibitor from T. giganteum showed a clear antihypertensive effect in spontaneously hypertensive rats (SHR), at a dosage of 1 mg/kg.
Keywords: Tricholoma giganteum; Angiotensin I-converting enzyme inhibitor; Antihypertension;

A conformation-constrained peptide library based on insect defensin A by An Zhao; Yanning Xue; Jie Zhang; Bo Gao; Jiannan Feng; Canquan Mao; Li Zheng; Nongle Liu; Fang Wang; Huixin Wang (629-635).
Here, we reported a conformation-constrained peptide library, that was constructed based on the scaffold of a 29 amino acids peptide derived from insect defensin A. The peptide scaffold was designed utilizing the InsightII molecular modeling software and then displayed on M13 filamentous bacteriophage by fusion with coat protein III. The library was constructed by randomization of seven positions located within the two loops of the peptide scaffold generating approximately 8.3×108 transformants. Sequences from 14 randomly selected phage clones indicated that the distribution of nucleotides and amino acids paralleled with the expected frequency. Screening against the target proteins: tumor necrosis factor α, TNF receptor 1, TNF receptor 2 and monoclonal antibody against BMP-2 showed significant enrichment in all cases. The results presented here show that the reconstructed insect defensin A domain will be a promising non-antibody protein scaffold for the presentation of a phage-displayed constrained peptide library.
Keywords: Conformation-constrained peptide library; Insect defensin A; Screening;

Kbot1, a three disulfide bridges toxin from Buthus occitanus tunetanus venom highly active on both SK and Kv channels by Basma Mahjoubi-Boubaker; Marcel Crest; Rym Ben Khalifa; Mohamed El Ayeb; Riadh Kharrat (637-645).
On attempts to identify toxins showing original profile of activity among K+ channels, we purified Kbot1, a scorpion toxin that blocks Kv1 and SK potassium channels. With 28 amino-acid residues, Kbot1 is the shortest toxin sequenced in Buthus occitanus scorpion. It is linked by three disulfide bridges and its primary structure is 93% identical to that of BmP02 isolated from the venom of the Chinese scorpion Buthus martensi Karsch [Eur. J. Biochem. 245 (1996) 457]. Kbot1 exhibited a low neurotoxicity in mice after intracerebroventricular injection (LD50≃0.8 μg per mouse). It competes with iodinated apamin for its rat brain synaptosomal membrane-binding site (IC50 of 20 nM). Despite 30% sequence identity between Kbot1 and ChTX, competitive experiments on the [ 125 I ] charybdotoxin, show that Kbot1 inhibits its binding to its rat brain synaptosomes with IC50 of 10 nM. This result was supported by electrophysiological experiments on cloned voltage-dependent K+ channels from rat brain, expressed in Xenopus oocytes. Kbot1 blocks Kv1.1, Kv1.2 and Kv1.3 currents with IC50 of 145, 2.5 and 15 nM, respectively. Based on these data, Kbot1 may be considered as the first member of subfamily 9 of scorpion toxins [Trends Pharmacol. Sci. 20 (1999) 444], highly active on both Kv and SK channels.
Keywords: Scorpion venom; Short scorpion toxins; Potassium channel; Voltage-dependent potassium channel; Calcium-activated potassium channel; Xenopus oocytes;

Valproate modulates TRH receptor, TRH and TRH-like peptide levels in rat brain by A.Eugene Pekary; Albert Sattin; James L. Meyerhoff; Mark Chilingar (647-658).
We have tested our hypothesis that alterations in the levels of TRH receptors, and the synthesis and release of tripeptide TRH, and other neurotropic TRH-like peptides mediate some of the mood stabilizing effects of valproate (Valp). We have directly compared the effect of 1 week of feeding two major mood stabilizers, Valp and lithium chloride (LiCl) on TRH binding in limbic and extra-limbic regions of male WKY rats. Valp increased TRH receptor levels in nucleus accumbens and frontal cortex. Li increased TRH receptor binding in amygdala, posterior cortex and cerebellum. The acute, chronic and withdrawal effects of Valp on brain levels of TRH (pGlu-His-Pro-NH2, His-TRH) and five other TRH-like peptides, Glu-TRH, Val-TRH, Tyr-TRH, Leu-TRH and Phe-TRH were measured by combined HPLC and RIA. Acute treatment increased TRH and TRH-like peptide levels within most brain regions, most strikingly in pyriform cortex. The fold increases (in parentheses) were: Val-TRH (58), Phe-TRH (54), Tyr-TRH (25), TRH (9), Glu-TRH (4) and Leu-TRH (3). We conclude that the mood stabilizing effects of Valp may be due, at least in part, to its ability to alter TRH and TRH-like peptide, and TRH receptor levels in the limbic system and other brain regions implicated in mood regulation and behavior.
Keywords: Valproate; TRH-like peptides; TRH receptors; Depression; Cortex; Limbic system;

Behavioral and neuroendocrine effects of the selective CRF2 receptor agonists urocortin II and urocortin III by Mary Ann Pelleymounter; Margaret Joppa; Nick Ling; Alan C Foster (659-666).
We compared the in vivo efficacy of two selective CRF2 agonists, mouse urocortin II (mUcn II) and human urocortin III (hUcn III), using food intake, anxious behavior, or ACTH release in CD-1 or Balb/c mice as indices of biological stress responses. All three peptides produced anorexia (Minimal Effective Dose (M.E.D.) for CRF and mUcn II=0.03 nmol; M.E.D. for hUcn III=0.3 nmol). Only mUcn II and CRF appeared to increase anxious behaviors in the elevated plus maze test (M.E.D.=0.3 and 0.01 nmol, respectively). CRF increased the release of plasma ACTH (M.E.D. of 0.3 nmol), while mUcn II and hUcn III had no effect on ACTH release. These data suggest that the CRF2 receptor subtype plays a primary role in the activation of behavioral, but not neuroendocrine, stress responses.
Keywords: Corticotropin releasing factor (CRF); Mouse urocortin II (mUcn II); Human urocortin III (hUcn III); Elevated plus maze (EPM); Food Intake; ACTH; Stress; Anxiety;

Increased expression of melanin concentrating hormone (MCH), an orexigenic neuropeptide produced by neurons in the lateral hypothalamic area (LHA), is implicated in the effect of energy restriction to increase food intake. Since melanocortins inhibit Mch gene expression, this effect of energy restriction to increase Mch signaling may involve reduced hypothalamic melanocortin signaling. Consistent with this hypothesis, we detected increased hypothalamic Mch mRNA levels in agouti (A y ) mice (by 102%; P<0.05), a model of genetic obesity resulting from impaired melanocortin signaling, compared to wild-type controls. If reduced melanocortin signaling mediates the effect of energy restriction, hypothalamic Mch gene expression in A y mice should not be increased further by energy restriction, since melanocortin signaling is impaired in these animals regardless of nutritional state. We therefore investigated the effects of energy restriction on hypothalamic Mch gene expression in both A y mice and in wild-type mice with diet-induced obesity (DIO). Responses in these mice were compared to those induced by administration of 17β-estradiol (E2) at a dose previously shown to reduce food intake and Mch expression in rats. In both A y and DIO mice, energy restriction increased hypothalamic Mch mRNA levels (P<0.05 for each) via a mechanism that was fully blocked by E2. However, E2 did not lower levels of Mch mRNA below basal values in A y mice, whereas it did so in DIO mice. Thus, the effect of energy restriction to increase hypothalamic Mch gene expression involves an E2-sensitive mechanism that is not altered by impaired melanocortin signaling. By comparison, impaired melanocortin signaling increases hypothalamic Mch gene expression via a mechanism that is insensitive to E2. These findings suggest that while both energy restriction and reduced melanocortin signaling stimulate hypothalamic Mch gene expression, they do so via distinct mechanisms.
Keywords: Melanin-concentrating hormone; Agouti; Melanocortins; Estrogen; Hypothalamus;

The interaction of an antimicrobial decapeptide with phospholipid vesicles by Myeong-Jun Choi; Sun Hee Kang; Seunghee Kim; Jin-Soo Chang; Sung Soo Kim; Hyeongjin Cho; Keun-Hyeung Lee (675-683).
Previously, by using combinatorial peptide libraries, we have identified activity-optimized decapeptide (KSL, KKVVFKVKFK-NH2), which exhibited a broad spectrum of the activity against bacteria and fungi without hemolytic activity. In order to examine lipid requirements and to understand the mode of KSL action, we investigated interactions of the peptide with vesicles consisting of various lipid compositions. KSL increased the permeability of negatively charged but not zwitterionic phospholipid membranes, and the leakage was independent on the size of encapsulated molecules (calcein, 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS)/N,N′-p-xylene bis(pyridinium) bromide (DPX), and fluorescein isothiocyanate (FITC)–dextran with different molecular weight), indicating that the peptide did not form pores or channels in this leakage process. KSL ability to permeabilize vesicles with negatively charged surface was dramatically reduced upon the addition of zwitterionic phospholipid rather than cholesterol, which revealed that the surface charge of lipid membranes played a major role in the activity and selectivity of KSL. Moreover, KSL diastereomer did not increase the permeability of negatively charged vesicles, indicating that the secondary structure of KSL was also required for membrane perturbation activity. Interestingly, KSL had an ability to cause aggregation and subsequent fusion of the acidic vesicles, which seemed to be related to the biological action. Structural studies performed by circular dichroism (CD) spectroscopy indicated that in the presence of acidic vesicles, the β sheet structure of KSL must be required for the ability to (1) induce a leakage of dye from the acidic vesicles (2) to fuse the acidic vesicles.
Keywords: Antibacterial peptide; Net charge; Permeability; Lipid membranes; Fusion; Vesicles;

A ribonuclease that is co-specific for poly C and poly U has been isolated from the fruiting bodies of the black oyster mushroom. The enzyme possesses a molecular mass of 14 kDa, and is unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-cellulose. A pH of 7 is required for the enzyme to exhibit maximal activity. The activity of the enzyme does not vary appreciably over the temperature range 30–60 °C, but drops when the temperature is reduced to 20 °C or raised to and above 70 °C. The ribonuclease does not exert any inhibitory activity toward HIV-1 reverse transcriptase.

A polypeptide, with a molecular mass of 16.5 kDa as determined by gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, has been isolated from the mushroom Polyporus adusta. The polypeptide, designated as adustin, inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 0.34 μM. It was isolated using a protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. Adustin was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and CM-Sepharose.
Keywords: Mushroom; Translation-inactivating; Protein;

An antifungal polypeptide, with a molecular mass of 28 kDa as judged by gel filtration and appearing as a single band with a molecular mass of 14 kDa in sodium dodecyl suflate-polyacrylamide gel electrophoresis, was isolated from fresh fruiting bodies of the mushroom Polyporus alveolaris. The antifungal polypeptide, designated as alveolarin, demonstrated an inhibitory action on mycelial growth in Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola. Alveolarin was isolated with a procedure that entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75 by fast protein liquid chromatography.
Keywords: Alveolarin; Antifungal polypeptide; Polyporus alveolaris;

This invited review, based on the receipt of the Third Gayle A. Olson and Richard D. Olson Prize for the publication of the outstanding behavioral article published in the journal Peptides in 2002, examines the 30-year historical perspective of the role of the endogenous opioid system in feeding behavior. The review focuses on the advances that this field has made over the past 30 years as a result of the timely discoveries that were made concerning this important neuropeptide system, and how these discoveries were quickly applied to the analysis of feeding behavior and attendant homeostatic processes. The discoveries of the opioid receptors and opioid peptides, and the establishment of their relevance to feeding behavior were pivotal in studies performed in the 1970s. The 1980s were characterized by the establishment of opioid receptor subtype agonists and antagonists and their relevance to the modulation of feeding behavior as well as by the use of general opioid antagonists in demonstrating the wide array of ingestive situations and paradigms involving the endogenous opioid system. The more recent work from the 1990s to the present, utilizes the advantages created by the cloning of the opioid receptor genes, the development of knockout and knockdown techniques, the systematic utilization of a systems neuroscience approach, and establishment of the reciprocity of how manipulations of opioid peptides and receptors affect feeding behavior with how feeding states affect levels of opioid peptides and receptors. The role of G-protein effector systems in opioid-mediated feeding responses, which was the subject of the prize-winning article, is then reviewed.
Keywords: Opioid agonists; G-proteins; Antisense probes; Opioid receptors and genes; Opioid antagonists; Food deprivation; Glucoprivation; Palatability; Taste hedonics; Macronutrient selection; Obesity;