Peptides (v.24, #2)

IgE binding synthetic peptides of Alt a 1, a major allergen of Alternaria alternata by Viswanath P. Kurup; Hari M. Vijay; Veena Kumar; Laura Castillo; Nancy Elms (179-185).
Alternaria alternata protein, Alt a 1 is a major allergen associated with allergy in atopic patients. Although the molecule binds strongly to IgE antibody from patients, the epitopes involved have not been identified or defined. In the present study, we synthesized overlapping peptides spanning the whole sequence and evaluated their IgE binding with sera from patients with Alternaria-induced allergy. The results identified four IgE binding linear regions. Two of these regions K41-P50 and Y54-K63 showed consistent reactivity with all four patients studied. The specific epitopes involved in the immune response may be of value in the immunodiagnosis and probably also in specific immunotherapy.
Keywords: Alternaria allergen; B-cell epitope; IgE binding; Asthma; Synthetic peptides; ELISA;

Expression, purification and functional characterization of a recombinant scorpion venom peptide BmTXKβ by Zhijian Cao; Fan Xiao; Fang Peng; Dahe Jiang; Xin Mao; Hui Liu; Wenxin Li; Dan Hu; Teng Wang (187-192).
BmTXKβ, a scorpion toxin isolated from the Chinese scorpion Buthus martensii Karsch (BmK), was expressed as a GST fusion protein in BL21 (DE3) strain. The recombinant GST-BmTXKβ protein was purified by affinity chromatography. When treated with enterokinase, the GST-BmTXKβ fusion protein released an approximate 6.5 kDa protein which was the expected size for correctly processed. About 2 mg purified recombinant BmTXKβ protein (rBmTXKβ) was produced from 1 l bacterial culture, using this expression and purification system. The function of rBmTXKβ was studied on the rabbit atrial myocyte by whole-cell patch clamp technique. The results showed that rBmTXKβ inhibited the transient outward current (I to) of rabbit atrial myocyte with recovery after washout and the inhibition was concentration-dependent. The rBmTXKβ prolonged the action potential duration of rabbit atrial myocyte in a concentration-dependent manner, whereas it did not affect the action potential amplitude.
Keywords: Expression; BmTXKβ; Whole-cell patch clamp; Atrial myocyte;

Role of MHC II affinity and molecular mimicry in defining anti-HER-2/neu MAb-3 linear peptide epitope by Alberta Lucchese; Stefan Stevanovic; Animesh A Sinha; Abraham Mittelman; Darja Kanduc (193-197).
With the aid of computational biology, we have studied the possibility of predicting the peptides able to evoke humoral immune response by using as experimental model the human HER-2/neu breast cancer-associated antigen. We already demonstrated that HER-2/neu peptides, that are the target of humoral human and mouse immune responses, correspond to those sequences having a low degree of sequence similarity to host’s proteome. Here we report that the linear peptide determinant of the anti-HER-2/neu MAb-3 is characterized by a low degree of sequence similarity to mouse proteome in combination with high binding potential to specific MHC II molecule.
Keywords: HER-2/neu; Epitope prediction; MHC binding potential; Molecular mimicry; Computational biology;

A novel 28-amino acid peptide, termed bombinakinin-GAP, was purified and characterized from skin secretions of the toad Bombina maxima. Its primary structure was established as DMYEIKQYKTAHGRPPICAPGEQCPIWV-NH2, in which two cysteines form a disulfide bond. A FASTA search of SWISS-PROT databank detected a 32% sequence identity between the sequences of the peptide and a segment of rat cocaine- and amphetamine-regulated transcript (CART). Intracerebroventricular (i.c.v.) administration of the peptide induced a significant decrease in food intake in rats, suggesting that it played a role in the control of feeding by brain. Analysis of its cDNA structure revealed that this peptide is coexpressed with bombinakinin M, a bradykinin-related peptide from the same toad. Bombinakinin-GAP appears to be the first example of a novel class of bioactive peptides from amphibian skin, which may be implicated in feeding behavior.
Keywords: Amphibian; Bombina maxima; Peptide; Food intake inhibition; Bradykinin; Bombinakinin-GAP;

Comparative distribution of urocortin- and CRF-like immunoreactivities in the nervous system of the earthworm Lumbricus terrestris by Andrea Lubics; Dóra Reglődi; Márta Szelier; István Lengvári; Tamás Kozicz (205-213).
Corticotropin-releasing factor (CRF) and urocortin (Ucn) are both members of the CRF neuropeptide family. The distribution of Ucn- and CRF-like immunoreactive (ir) structures in the central nervous system of several vertebrate species has been studied, but little is known about that in non-vertebrates. We used a highly specific polyclonal antibody against rat Ucn and CRF to determine and compare the distribution of Ucn- and CRF-like immunoreactivity in the earthworm nervous system. Several Ucn- and CRF-like ir perikarya were described in the cerebral ganglion, subesophageal and ventral cord ganglia. The majority of Ucn-like ir cells were found in the ventral ganglia, whereas CRF-like ir cells were most abundant in the cerebral ganglion. Scattered Ucn- and CRF-like ir varicose fiber terminals were seen in all areas of the earthworm central nervous system. Ucn-like ir cell bodies and fiber terminals were also demonstrated in the pharyngeal wall. No co-localization of Ucn- and CRF-like ir nervous structures were observed. This study provided morphological evidence that Ucn- and CRF-like neurosecretory products exist in the earthworm central nervous system. Furthermore, both the distribution and morphology of Ucn- and CRF-like ir structures were distinct, therefore, it can be hypothesized that these neuropeptides exert different neurendocrine functions in the earthworm nervous system.
Keywords: Urocortin; CRF; Annelid; Earthworm; Nervous system; Immunohistochemistry;

Tissue distribution of pneumadin immunoreactivity in the rat by Jerzy Kosowicz; Bogdan Miskowiak; Aneta Konwerska; Cinzia Tortorella; Gastone G. Nussdorfer; Ludwik K. Malendowicz (215-220).
Pneumadin (PNM) is a decapeptide, originally isolated from mammalian lungs, which exerts a potent stimulating effect on arginine-vasopressin (AVP) release, thereby evoking an antidiuretic effect. We have established a specific radioimmunoassay (RIA) method for rat PNM determination, the sensitivity of which is sufficient for measuring tissue content of the peptide. Moreover, raised antibodies have been used for the immunocytochemical detection of PNM in several rat organs. As expected, high concentrations of PNM were detected by RIA in newborn and adult rat lungs and immunocytochemistry (ICC) localized PNM immunoreactivity (IR) in the bronchial and bronchiolar epithelium. Very high concentrations of PNM were measured by RIA in the prostate, and ICC showed that PNM-IR is contained in the epithelial cells. RIA and ICC demonstrated the presence of low amounts of PNM in the thymus. The highest content of radioimmunoassayable PNM was found in the kidneys and intestinal tract, but dilution test suggested the presence of some interfering substances in these tissues. Accordingly, ICC-detectable PNM-IR was absent in the kidneys and present only in the duodenal criptae and Brunner’s glands of the intestinal tract. RIA did not measure sizeable PNM concentrations in the thyroid gland, but ICC showed PNM-IR in C-cells. RIA and ICC did not detected PNM in testes, seminal vesicles, ovaries, uterus, pancreas, liver, spleen, adrenal glands, and heart. Taken together, our findings suggest that PNM, in addition to its role as hypothalamo–pituitary AVP secretagogue, may be involved in the autocrine–paracrine functional regulation of other peripheral organs, like lungs and prostate and perhaps duodenum, thymus and thyroid gland.
Keywords: Duodenum; Thymus; Thyroid gland;

Pineal peptide with an inhibiting effect on growth of HeLa S3 tumor cells by Petrelli Cristina; Lupidi Giulio (221-225).
The aim of the present study was to assess whether a peptide fraction isolated from calf pineal glands has an effect on proliferation and morphology of HeLa S3 tumor cells. Under the experimental conditions adopted, the results showed that the peptide has a marked inhibitory effect on proliferation of HeLa S3 cells and that permeabilization with calcium phosphate of the plasmatic membrane increases this effect. Moreover, the pineal peptide affects the cytoskeletal morphology of HeLa cells by modifying the distribution of actin. The peptide is probably internalized by the cells and irreversibly modifies the cytoskeletal morphology with consequent inhibition of cellular proliferation.
Keywords: Permeabilization; Pineal peptide; HeLa cells;

Identification of gastrin and multiple cholecystokinin genes in teleost by Tadahide Kurokawa; Tohru Suzuki; Hisashi Hashimoto (227-235).
To identify the teleost gastrin, CCK/gastrin family genes were isolated from puffer and flounder. The cDNA of puffer gastrin, CCK1 and CCK2 were 678, 752 and 533 bp, respectively. Puffer gastrin gene consists of three exons, in contrast, CCKs consist of four exons. Flounder gastrin mRNA (526 bp) was expressed in the intestine but not in the brain. It was developed synchronously with the stomach differentiation in the larval stage. The phylogenetic analysis shows that puffer and flounder gastrin classified into the vertebrate gastrin cluster and two types of CCK were probably produced by the genome duplication occurred in teleost phylogeny.
Keywords: Gastrin; CCK; cDNA cloning; Tissue expression; Larvae; Flounder; Puffer; Teleost;

Cholecystokinin (CCK) is a peptide hormone released from the I-cells of the upper small intestine. CCK evokes a variety of physiological responses, such as stimulation of pancreatic secretion, reduction of food intake and inhibition of gastric emptying. Previously, we reported that CCK activates enteric neurons in the rat. However the specific subpopulations of enteric neurons activated by CCK have not been identified. In the work reported here, we utilized immunohistochemical detection of nuclear Fos, a marker for neuronal activation, and selected phenotypic markers to identify some of the neuronal subpopulations activated by CCK. The phenotypic markers that we examined were: nitric oxide synthase (NOS), neurokinin-1 receptor (NK-1R), calbindin (Cal), Calretinin (Calr), and neurofilament-M (NF-M). We found that in the myenteric plexus of the rat duodenum and jejunum, CCK activated NOS immunoreactive neurons. In the submucosal plexus of duodenum and jejunum, CCK activated Cal, Calr and NF-M immunoreactive neurons. CCK failed to activate NK-1R immunoreactive neurons in either plexus. Our results indicate that CCK activates distinct enteric neurons in the rat upper small intestine. Furthermore the fact that NOS immunoreactive neurons were activated suggests that CCK modulates the activity of inhibitory motor neurons in the myenteric plexus. Expression of Fos immunoreactivity in Calr and Cal immunoreactive neurons is consistent with a role for CCK in modulation of intrinsic sensory and/or secretomotor neuronal activity in the submucosal plexus.
Keywords: Myenteric plexus; Submucosal plexus; Enteric nervous system; Neurokinin-1 receptor; Neurofilament-M; Calbindin; Calretinin; Nitric oxide synthase; c-fos;

Hypothalamic administration of cAMP agonist/PKA activator inhibits both schedule feeding and NPY-induced feeding in rats by Sulaiman Sheriff; William T. Chance; Sabahat Iqbal; Tilat A. Rizvi; Chun Xiao; John W. Kasckow; A. Balasubramaniam (245-254).
Following central administration, neuropeptides that decrease the level of cAMP induce feeding. Conversely, cAMP activating neuropeptides tend to elicit satiety. When the inhibitory effect of neuropeptide Y (NPY) on the hypothalamic cAMP production was blocked by pertussis toxin, the potent orexigenic effect of NPY was lost. These findings suggest that there may be a link between hypothalamic cAMP and the central regulation of food intake. In this report, we show that the injection of the membrane-permeable cAMP agonist, adenosine-3′,5′-cyclic monophosphorothioate Sp-isomer (Sp-cAMP), into perifornical hypothalamus (PFH) significantly inhibited schedule-induced and NPY-induced food intake for up to 4 h. This inhibitory effect was normalized within 24 h. A taste aversion could not be conditioned to Sp-cAMP treatment, suggesting that the anorectic response was not due to malaise. Sp-cAMP administration significantly increased the active protein kinase A (PKA) activity in dorsomedial (DMH) and ventromedial (VMH), but not in lateral (LH) hypothalamus. Consistently, food deprivation lowered, while refeeding normalized endogenous cAMP content in DMH and VMH, but not in LH areas. No significant effect of adenosine-3′,5′-cyclic monophosphorothioate Rp-isomer (Rp-cAMP, cAMP antagonist) was observed on hypothalamic PKA activity, schedule-induced, or NPY-induced food intake. These findings suggest that the increase in cAMP level and PKA activity in DMH and VMH areas may trigger a satiety signal.
Keywords: Satiety; cAMP; PKA; Hunger; Hypothalamus;

Distribution of neuropeptide Y Y1 and Y2 receptors in the postmortem human heart by Ann-Cathrine Jönsson-Rylander; Margareta Nordlander; Aud Svindland; Arnfinn Ilebekk (255-262).
In the present study, we present for the first time the presence and distribution of neuropeptide Y (NPY) receptors Y1 and Y2 in the human postmortem heart using specific antibodies raised against extracellular parts of the receptors. A more intensive staining against the Y2 than against the Y1 receptors was detected on both atrial and ventricular cardiomyocytes. Immunoreactivity against both receptors was identified on both conducting fibers and cardiac nerves. More vessels stained positively for the Y2 than for the Y1 receptor, but the Y1 receptors were more abundant in subendocardial than subepicardial vessels of the left ventricular wall.
Keywords: Cardiomyocytes; Conducting fibers; Human myocardium; Nerve fibers; Neuropeptide Y Y1 and Y2 receptors; Smooth muscle cells; Subendo- and subepicardial receptor distribution;

Elongation studies of the human agouti-related protein (AGRP) core decapeptide (Yc[CRFFNAFC]Y) results in antagonism at the mouse melanocortin-3 receptor by Christine G Joseph; Rayna M Bauzo; Zhimin Xiang; Amanda M Shaw; William J Millard; Carrie Haskell-Luevano (263-270).
The agouti-related protein (AGRP) is an endogenous antagonist of the brain melanocortin receptors (MC3R and MC4R) and is believed to be involved in feeding behavior and energy homeostasis. Previous results identified that the human AGRP decapeptide Yc[CRFFNAFC]Y binds to the MC3R and MC4R and acts as an antagonist at the MC4R but not at the MC3R. We have synthesized the amidated version of this decapeptide as well as performed elongation studies at both the N-and C-terminus of the monocyclic hAGRP(109–118) peptide. This study was designed to identify monocyclic peptide fragments of the hAGRP(86–132) to determine the minimal active monocyclic sequence necessary for antagonism at the MC3R. For binding studies, radiolabeled 125 I -NDP-MSH versus 125 I -hAGRP(86–132) were utilized to determine if there were differences in the ability of the AGRP fragments prepared herein to competitively displace the 125 I -NDP-MSH versus AGRP(86–132) radiolabel. The binding IC50 values of radiolabeled hAGRP(86–132) versus NDP-MSH were identical within experimental error, supporting the hypothesis that AGRP and NDP-MSH interact with overlapping binding epitopes at the MC3R and MC4R. The most notable results include identification of the TAYc[CRFFNAFC]YAR-NH2 (pA 2=6.1, K i=790 nM, mMC3R) and the Yc[CRFFNAFC]YARKL-NH2 (pA 2=6.2, K i=630 nM, mMC3R) peptides as the minimal monocyclic AGRP-based fragments possessing antagonist pharmacology at the MC3R. Interestingly, extension of the AGRP(109–118) decapeptide at both the N- and C-terminus by two amino acids conferred detectable mMC3R antagonism, while retaining high nanomolar MC4R antagonist and micromolar MC1R agonist pharmacological properties. These data support the hypothesis that elongation of the hAGRP(109–118) decapeptide results in antagonism at the MC3R while retaining MC1R agonist activity and MC4R antagonist activity.
Keywords: Melanocortin; Melanotropin; Agouti; Agouti-related protein; α-Melanocyte stimulating hormone; Obesity; MC3R; MC4R;

Discovery and in vivo evaluation of new melanocortin-4 receptor-selective peptides by Wouter A.J. Nijenhuis; John A.W. Kruijtzer; Nienke Wanders; Dorien H. Vrinten; Keith M. Garner; Wim M.M. Schaaper; Rob H. Meloen; Willem Hendrik Gispen; Rob M. Liskamp; Roger A.H. Adan (271-280).
The melanocortin-4 receptor (MC4R) is involved in several physiological processes, including body weight regulation and grooming behaviour in rats. It has also been suggested that the MC4R mediates the effects of melanocortin ligands on neuropathic pain. Selective compounds are needed to study the exact role of the MC4R in these different processes. We describe here the development and evaluation of new melanocortin compounds that are selective for the MC4R as compared with the other centrally expressed receptors, MC3R and MC5R. First, a library of 18 peptides, in which a melanocortin-based sequence was systematically point-mutated, was screened for binding to and activity on the MC3R, MC4R and MC5R. Compound Ac-Nle-Gly-Lys-d-Phe-Arg-Trp-Gly-NH2 (JK1) appeared to be the most selective MC4R compound, based on affinity. This compound is 90- and 110-fold selective for the MC4R as compared to the MC3R and MC5R, respectively. Subsequent modification of JK1 yielded compound Ac-Nle-Gly-Lys-d-Nal(2)-Arg-Trp-Gly-NH2 (JK7), a selective MC4R antagonist with 34-fold MC4R/MC3R and 109-fold MC4R/MC5R selectivity. The compounds were active in vivo as determined in a grooming assay and a model for neuropathic pain in rats. Intravenous (i.v.) injections suggested that they were able to pass the blood–brain barrier.The compounds identified here will be useful in further research on the physiological roles of the MC4R.
Keywords: Melanocortin receptor; Selective ligand; PEPSCAN; Grooming; Neuropathic pain;

Interactions of VIP with rigid phospholipid bilayers: implications for vasoreactivity by Hayat Önyüksel; Beena Ashok; Sumeet Dagar; Varun Sethi; Israel Rubinstein (281-286).
The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), a pleiotropic amphipathic peptide, interacts with rigid liposomes composed of gel phase phospholipids. We found that incubation of VIP with small unilamellar gel phase liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and egg phosphatidylglycerol (ePG) for 2 h at room temperature had no significant effects on VIP secondary structure. Moreover, suffusion of VIP (0.01, 0.1 and 1.0 nmol) incubated in saline or with DPPC/ePG liposomes (size, 30 and 100 nm) for 2 h at room temperature or 4 °C onto the intact hamster cheek pouch microcirculation elicited a similar concentration-dependent vasodilation except for 0.01 nmol VIP (P<0.05). By contrast, incubation of VIP with gel phase liposomes overnight at 4 °C significantly potentiated vasodilation evoked by all three concentrations of the peptide in comparison to aqueous VIP (P<0.05). VIP-induced vasodilation was liposome size-independent. The ratio of VIP to phospholipids in DPPC/ePG liposomes was concentration-independent. Collectively, these data indicate that short-term interactions of VIP with rigid phospholipid bilayers are limited resulting in only modest effects on VIP vasoreactivity in vivo.
Keywords: Neuropeptide; DPPC; PG; Circular dichroism; Peripheral microcirculation; Vasodilation; Hamster cheek pouch;

Changes in amount of ADM mRNA and RAMP2 mRNA in calcified vascular smooth muscle cells by Yong Fen Qi; Shu Heng Wang; Bao Hong Zhang; Ding Fang Bu; Tang Chao Shu; Jun Bao Du (287-294).
This work was aimed to explore the changes and significance of adrenomedullin (ADM) mRNA and receptor activity modifying protein 2 (RAMP2) mRNA in calcified vascular smooth muscle cells (VSMCs). Calcification of cultured rat VSMCs was produced by incubation with β-glycerophosphate. Content of ADM released by VSMCs was measured by radioimmunoassay (RIA). The amount of ADM mRNA and RAMP2 mRNA was determined by competitive quantitative RT-PCR. The intracellular calcium content, alkaline phosphatases activity and cellular 45Ca2+-uptake were determined. The results showed that the content of calcium, 45Ca2+-uptake and alkaline phosphatases activity in calcified VSMCs were increased by 118%, 174% and seven-fold (all P<0.01), respectively, compared with control VSMCs. Content of ADM in medium was increased by 99% (P<0.01). Furthermore, it was found that the amount of ADM mRNA and RAMP2 mRNA in calcified cells was elevated by 78 and 56% (all P<0.05), respectively, compared with control. The elevated levels of RAMP2 mRNA were in positive correlation with ADM mRNA (r=0.76, P<0.05) in calcified VSMCs. In conclusion, calcified VSMCs generated an increased amount of ADM, and up-regulated gene expressions of ADM and RAMP2.
Keywords: Vascular smooth muscle cells (VSMCs); Adrenomedullin (ADM); Receptor activity modifying protein 2 (RAMP2); mRNA;

Adrenomedullin stimulates proliferation and inhibits apoptosis of immature rat thymocytes cultured in vitro by Anna S. Belloni; Marcin Trejter; Ludwik K. Malendowicz; Gastone G. Nussdorfer (295-300).
Adrenomedullin (AM) is a hypotensive peptide, which derives from the proteolytic cleavage of pro(p)AM, and acts through two subtypes of receptors, named L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR functions as either a calcitonin gene-related peptide (CGRP) receptor or a selective AM receptor depending on which member of a family of receptor-activity-modifying proteins (RAMPs) is expressed: RAMP1 generates CGRP receptors, while RAMP2 and RAMP3 produce AM receptors. Reverse transcription (RT)–polymerase chain reaction (PCR) consistently allowed the detection of pAM and peptidyl-glycine α-amidating monooxygenase (the enzyme converting immature AM to the mature peptide) mRNAs in the thymus cortex of immature (10-day-old) rats. Accordingly, radioimmune assay (RIA) measured low but sizeable AM concentrations in this tissue. RT–PCR also demonstrated the presence of the specific mRNAs of L1-R, CRLR and RAMPs. AM (from 10−9 to 10−7  M) increased proliferation index and lowered apoptotic index of cultured immature rat thymocytes, and the effects were annulled by the AM receptor antagonist AM(22–52). In conclusion, our study demonstrated that (1) immature rat thymus cortex expresses AM and the AM receptors L1-R and CRLR/RAMP; and (2) AM, acting via AM(22–52)-sensitive receptors, exerts a potent growth promoting effect on immature rat thymus, by enhancing proliferation and lowering apoptotic death of thymocytes. Taken together, these findings could suggest that AM may play a role in the development of immunity.
Keywords: Adrenomedullin; Thymocytes; Cell proliferation; Apoptosis; Immature rat;

Expression of urotensin II and its receptor in adrenal tumors and stimulation of proliferation of cultured tumor cells by urotensin II by Kazuhiro Takahashi; Kazuhito Totsune; Osamu Murakami; Zenei Arihara; Takao Noshiro; Yutaka Hayashi; Shigeki Shibahara (301-306).
Urotensin II is a potent vasoactive peptide, which was originally isolated from fish urophysis. We studied expression of urotensin II and its receptor mRNAs in the tumor tissues of adrenocortical tumors, pheochromocytomas and neuroblastomas. Effects of exogenously added urotensin II on cell proliferation were studied in a human adrenocortical carcinoma cell line, SW-13 and a human renal cell carcinoma cell line, VMRC-RCW. The reverse transcriptase polymerase chain reaction (RT-PCR) showed expression of urotensin II and its receptor mRNAs in all the samples examined; seven pheochromocytomas, nine adrenocortical adenomas (four with primary aldosteronism, four with Cushing syndrome and one with non-functioning adenoma), four adrenocortical carcinomas, one ganglioneuroblastoma and five neuroblastomas, as well as four normal portions of adrenal glands (cortex and medulla). Urotensin II-like immunoreactivity was detected in one of eight adrenocortical adenomas, two of four adrenocortical carcinomas, one of six pheochromocytomas, and one of five neuroblastomas by radioimmunoassay, but not in normal portions of adrenal glands (detection limit; 0.2 pmol/g wet weight). Treatment with urotensin II for 24 h significantly increased number of SW-13 cells (at 10−8 and 10−7  mol/l) and VMRC-RCW cells (at 10−8  mol/l). These findings raise the possibility that urotensin II may act as an autocrine/paracrine growth stimulating factor in adrenal tumors.
Keywords: Urotensin II; Adrenal; Tumors; Pheochromocytoma; Neuroblastoma; Adrenocortical; Growth;

Delta sleep inducing peptide (DSIP): effect on respiration activity in rat brain mitochondria and stress protective potency under experimental hypoxia by Elena M. Khvatova; Victor N. Samartzev; Pavel P. Zagoskin; Igor A. Prudchenko; Inessa I. Mikhaleva (307-311).
Neuromodulatory delta sleep inducing peptide (DSIP) seems to be implicated in the attenuation of stress-induced pathological metabolic disturbances in various animal species and human beings. Mitochondria, as cell organelles, are considered especially sensitive to stress conditions. In this work, the influence of DSIP and Deltaran®—a recently developed product based upon DSIP—on processes of oxidative phosphorylation and ATP production in rat brain mitochondria and rat brain homogenates was studied. A polarographic measurement of oxygen consumption was applied to evaluate the impact of DSIP on maximal rates of mitochondrial respiration and coupling of respiration to ATP production. We provide evidence that DSIP affected the efficiency of oxidative phosphorylation on isolated rat brain mitochondria. This peptide significantly increased the rate of phosphorylated respiration V3, while the rate of uncoupled respiration VDNP remaining unchanged. It enhanced the respiratory control ratio RCR and the rate of ADP phosphorylation. DSIP and Deltaran exhibited the same action in rat brain homogenates. We also examined the influence of DSIP under hypoxia when mitochondrial respiratory activity is altered. In rats subjected to hypoxia, we detected a significant stress-mediated reduction of V3 and ADP/t values. Pretreatment of rats with DSIP at the dose of 120 μg/kg (i.p.) prior to their subjection to hypoxia completely inhibited hypoxia-induced reduction of mitochondrial respiratory activity. The revealed capacity of DSIP to enhance the efficiency of oxidative phosphorylation found in vitro experiments could contribute to understanding pronounced stress protective and antioxidant action of this peptide in vivo.
Keywords: DSIP; Deltaran; Rat brain mitochondria; Respiratory chain; Oxidative phosphorylation;

Pro-Leu-glycinamide and its peptidomimetic, PAOPA, attenuate haloperidol induced vacuous chewing movements in rat: A model of human tardive dyskinesia by S Sharma; P Paladino; J Gabriele; H Saeedi; P Henry; M Chang; R.K Mishra; R.L Johnson (313-319).
In the present experimental paradigm, we examine the effect of l-prolyl-l-leucyl-glycinamide (PLG) co-administration with haloperidol on vacuous chewing movements (VCM) in rats—a model of tardive dyskinesia (TD) in humans. We examined the dose dependent induction of VCM through both injected and orally administered PLG (MIF-1). Our results show significant levels of VCM attenuation (P<0.05) in rats treated with 10 mg/kg of PLG. Doses of 1 and 100 mg/kg were ineffective. Reductions were present in both orally treated and injected rats. We also examined the therapeutic effect of a peptidomimetic of PLG—PAOPA. PAOPA was able to produce similar behavioral effects to PLG at a dose, which was 100-fold lower than the effective dose of PLG. These results suggest that PLG may play a role in D2 receptor expression and function, as well as providing a therapy for neuroleptic induced TD.
Keywords: Tardive dyskinesia; PLG; PAOPA; (MIF-1); Haloperidol; VCM;

This paper discusses the effects of gliadorphin-7 (GD-7) infusion in rats and contrasts them with those of β-casomorphin-7 (βC-7). Both induce FLI in a dose related fashion. Very strong expression in both geniculate nuclei (GN) and the alveus hippocampus follow GD-7 400 μg and βC-7 30 μg/kg BW. GD-7 affects only these three regions while βC-7affects 45. FLI is prevented by Naloxone 2 mg/kg BW in all regions except the GN where it is diminished 60%. βC-7 causes bizarre behavior beginning 60 s after infusion is started. GD-7 causes no behavioral change. These findings suggest GD-7 gains access to brain cells by diffusion through circumventricular organs while βC-7 passes the BBB by carrier facilitation.
Keywords: Gliadorphin; β-Casomorphin (βC-7); Fos-like immunoreactivity; Geniculate nuclei; Naloxone;

Effect of rubiscolin, a δ opioid peptide derived from Rubisco, on memory consolidation by Shuzhang Yang; Yukio Kawamura; Masaaki Yoshikawa (325-328).
Rubiscolin-6 (YPLDLF) is a δ selective opioid peptide isolated from the enzymatic digests of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from spinach leaves. In a step-through type passive avoidance test in ddY mice, rubiscolin-6 enhanced memory consolidation at doses of 3 nmol/mouse after intracerebroventricular administration, and at 100 mg/kg after oral administration. These doses are smaller than the optimal doses for an analgesic effect. The memory enhancing effect of rubiscolin-6 was blocked by pretreatment with the δ antagonist naltrindole, suggesting the involvement of the δ opioid receptor.
Keywords: Rubiscolin; Rubisco; δ opioid peptide; Memory consolidation; Passive avoidance test;