Peptides (v.23, #12)

Development of novel antibacterial peptides that kill resistant isolates by Mare Cudic; Barry A. Condie; Daniel J. Weiner; Elena S. Lysenko; Zhi Q. Xiang; O. Insug; Philippe Bulet; Laszlo Otvos (2071-2083).
The rapid emergence of bacterial strains that are resistant to current antibiotics requires the development of novel types of antimicrobial compounds. Proline-rich cationic antibacterial peptides such as pyrrhocoricin kill responsive bacteria by binding to the 70 kDa heat shock protein DnaK and inhibiting protein folding. We designed and synthesized multiply protected dimeric analogs of pyrrhocoricin and optimized the in vitro antibacterial efficacy assays for peptide antibiotics. Pyrrhocoricin and the designed dimers killed β-lactam, tetracycline- or aminoglycoside-resistant strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the submicromolar or low micromolar concentration range. One of the peptides also killed Pseudomonas aeruginosa. The designed dimers showed improved stability in mammalian sera compared to the native analog. In a murine H. influenzae lung infection model, a single dose of a dimeric pyrrhocoricin analog reduced the bacteria in the bronchoalveolar lavage when delivered intranasally. The solid-phase synthesis was optimized for large-scale laboratory preparations.
Keywords: Antimicrobial peptides; Bacteriocidal analogs; In vivo protection; Proline-rich; Serum stability; Solid-phase synthesis;

In this data-based theoretical analysis, we use a random approach to estimate amino acid pairs in human phenylalanine 4-hydroxylase (PAH) protein in order to determine which amino acid pairs are more sensitive to 187 variants in human PAH protein. The rationale of this study is based on our hypothesis and previous findings that the harmful variants are more likely to occur at randomly unpredictable amino acid pairs rather than at randomly predictable pairs. This is reasonable to argue as randomly predictable amino acid pairs are less likely to be deliberately evolved, whereas randomly unpredictable amino acid pairs are probably deliberately evolved in connection with protein function. 94.12% of 187 variants occurred at randomly unpredictable amino acid pairs, which accounted for 71.84% of 451 amino acid pairs in human PAH protein. The chance of a variant occurring is five times higher in randomly unpredictable amino acid pairs than in predictable pairs. Thus, randomly unpredictable amino acid pairs are more sensitive to variance in human PAH protein. The results also suggest that the human PAH protein has a natural tendency towards variants.
Keywords: Phenylalanine hydroxylase; Probability; Randomness; Variants;

Immunoconjugates are widely used for indirect detection of analytes (such as antibodies or antigens) in a variety of immunoassays. However, the availability of functional groups such as primary amines or free sulfhydryls in an immunoglobulin molecule is the limiting factor for optimal conjugation and, therefore, determines the sensitivity of an assay. In the present study, an N-terminal bromoacetylated 20 amino acid peptide containing 20 lysine residues was conjugated to N-succinimidyl-S-acetylthioacetate (SATA)-modified IgG or free sulfhydryl groups on 2-mercaptoethylamine (2-MEA)-reduced IgG molecules via a thioether (SCH2CONH) linkage to introduce multiple reactive primary amines per IgG. These primary amines were then covalently coupled with maleimide-activated horseradish peroxidase (HRP). The poly-HRP–antibody conjugates thus generated demonstrated greater than 15-fold signal amplification upon reaction with orthophenyldiamine substrate. The poly-HRP–antibody conjugates efficiently detected human immunodeficiency virus (HIV)-1 antibodies in plasma specimens with significantly higher sensitivity than conventionally prepared HRP–antibody conjugates in an HIV-1 solid-phase enzyme immunoassay and Western blot analysis. The signal amplification techniques reported here could have the potential for development of highly sensitive immunodiagnostic assay systems.
Keywords: Peptide; Lysine; Signal amplification; Enzyme conjugates;

Spherical polystyrene microparticles expressing a large number of highly reactive functional groups were chemically engineered to generate antibody–enzyme conjugates as novel signal amplification systems. Chemically modified goat anti-human IgG and horseradish peroxidase (HRP) were combined in a 1:5 ratio and attached to 0.44 μm streptavidin microparticles or N-succinimidyl-S-acetylthioacetate (SATA)-activated 0.29 μm amino microparticles with highly reactive free sulfhydryl groups on their surface. The numbers of HRP molecules/microparticle were further increased by coupling HRP to primary amines on N-terminal biotinylated or bromoacetylated polypeptides containing 20 lysine residues prior to conjugation with streptavidin or sulfhydryl groups-containing microparticles. The antibody–poly-HRP immunoconjugates contained an estimated number of 105  HRP/streptavidin microparticle and 106  HRP/amino microparticle, respectively. These microparticle immunoconjugates efficiently bound to plasma anti-HIV-1 antibodies that had been captured by HIV antigens on 5 μm carboxyl magnetic microparticles and, upon reaction with orthophenyldiamine substrate, produced a detection signal with 5–8 times more sensitivity as compared to conventional HRP-conjugated goat anti-human IgG. The signal amplification technique by microparticle immunoconjugates may provide potentially novel tools for the development of highly sensitive diagnostic systems.
Keywords: Peptide; Microparticle; Signal amplification; Enzyme conjugates; Diagnostic assays;

Solution structure of paralytic peptide of silkworm, Bombyx mori by Kazunori Miura; Manabu Kamimura; Tomoyasu Aizawa; Makoto Kiuchi; Yoichi Hayakawa; Mineyuki Mizuguchi; Keiichi Kawano (2111-2116).
Paralytic peptide of Bombyx mori (BmPP) is one of the multifunctional ENF-peptides; the name of “ENF” is the consensus N-terminal amino acid sequence of the family peptides. We revealed that BmPP significantly possesses growth-blocking activity and plasmatocyte-spreading activity and that its activity profiles are different from those of another ENF-family peptide, namely, the growth-blocking peptide of Pseudaletia separata (PsGBP). We also determined the NMR structures of BmPP and PsGBP under the same conditions, which revealed the structural differences of the first and second β-turn regions between the two peptides. On the basis of our results, it can be considered that the tertiary structural difference in these peptides may cause their different profiles of growth-blocking activity.
Keywords: Solution structure; Nuclear magnetic resonance; Paralytic peptide; Bombyx mori; BmPP; Growth-blocking activity; Plasmatocyte-spreading activity;

A myomodulin peptide has been suggested to mediate the response of the giant glial cells to stimulation of the Leydig interneuron in the central nervous system of the leech Hirudo medicinalis [Eur. J. Neurosci. 11 (1999) 3125]. We have now studied the glial response to the endogenous leech MM peptide (GMGA LRL-NH2 , MMHir). The peptide evokes a membrane outward current (EC50 ∼2 μM), which neither desensitizes nor shows any sign of run-down, and elicits a K+ conductance increase of the glial cell membrane. The peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF) enhances the glial current response, suggesting the presence of endogenous extracellular peptidases.
Keywords: Neuron–glia communication; Leech giant glial cells; Leydig neuron; Central nervous system; Hirudo medicinalis;

Activity and NMR structure of synthetic peptides of the bovine ATPase inhibitor protein, IF1 by Cesira de Chiara; Giuseppe Nicastro; Alberto Spisni; Franco Zanotti; Tiziana Cocco; Sergio Papa (2127-2141).
The protein IF1 is a natural inhibitor of the mitochondrial FoF1-ATPase. Many investigators have been prompted to identify the shortest segment of IF1, retaining its native activity, for use in biomedical applications. Here, the activity of the synthetic peptides IF1-(42–58) and IF1-(22–46) is correlated to their structure and conformational plasticity determined by CD and [ 1 H ]-NMR spectroscopy. Among all the IF1 segments tested, IF1-(42–58) exerts the most potent, pH and temperature dependent activity on the FoF1 complex. The results suggest that, due to its flexible structure, it can fold in helical and/or β-spiral arrangements that favor the binding to the FoF1 complex, where the native IF1 binds. IF1-(22–46), instead, as it adopts a rigid α-helical conformation, it inhibits ATP hydrolysis only in the soluble F1 moiety.
Keywords: NMR; Mitochondrial FoF1-ATPase; ATPase inhibitor; IF1; Peptide; Circular dichroism; Sodium dodecyl sulfate;

A method based upon a combination of fast high-performance liquid chromatography (HPLC) and electrospray ionization (ESI)–mass spectrometry (MS) is developed for the analysis of bioactive peptides in bovine adrenal medulla. The fast HPLC uses a short column (33  mm×4.6  mm ) packed with nonporous silica-based C-18 stationary phase. Prior to HPLC separation, the medulla was homogenized and the peptide-rich fraction was isolated from it by solid-phase extraction. In-source collision-induced dissociation and tandem MS were used to obtain the sequence of the suspected peptides. Several peptides, including Met–Enk, Leu–Enk, Leu–Enk–Lys, bovine adrenal medullary (BAM)-12 (Met–Enk–RRVGRPE), Leu–Enk–Arg, and YGGT, were unambiguously identified. The first four peptides are the products of proenkephalin A precursor protein and Leu–Enk–Arg belongs to the dynorphin family and is derived from proenkephalin B (prodynorphin) precursor. The database search revealed that YGGT is a part of the sequence of five different precursor proteins.
Keywords: Bioactive peptides; Enkephalins; Bovine adrenal medulla; Fast HPLC; LC/ESI–MS; Tandem MS;

N-CAM expression and localization in PC12 cells modulated by extracellular peptides by Maria A Mariggiò; Simone Guarnieri; Stefania Mariggiò; Caterina Morabito; Gian Luigi Gianfranceschi; Giorgio Fanò (2151-2161).
The neural cell adhesion molecules (N-CAMs) play an important role in mediating cell–cell interactions in the nervous system. Different isoforms of these membrane proteins are involved in the formation of the neuronal network and in the dynamic phases of neuronal plasticity.We studied the early stages of the pseudo neuronal differentiation of PC12 cells induced by a class of small acidic peptides capable of modulating gene expression in these cells.The data presented here indicate that peptides with specific sequences induce an increase in N-CAM mRNA expression and protein translocation to the plasma membrane to a comparable degree as NGF.
Keywords: Acidic small peptides; Neuronal differentiation; N-CAM; PC12; NGF;

Adrenomedullin (AM) is a 52 amino acid peptide that is synthesized in a variety of tissues, including the vessels and bones. This study showed that normal human osteoblast (NHOst) secreted immunoreactive AM and that AM stimulated intracellular cAMP production in these cells. An anti-AM monoclonal antibody, which inhibited endogenous AM, caused the number of NHOst to decrease. The effect of a low concentration AM was inhibited by addition of a cAMP-dependent protein kinase A inhibitor (H89). These data suggest that AM is an autocrine or paracrine regulator that promotes the proliferation of NHOst via the cAMP pathway.
Keywords: Adrenomedullin; Osteoblast; cAMP; Proliferation;

Paraventricular noradrenergic systems participate in angiotensin II-induced drinking by Akihiko Ushigome; Junichi Tanaka; Katsuhide Kariya; Masahiko Nomura (2169-2175).
The present study was designed to examine the role of noradrenergic systems in the hypothalamic paraventricular nucleus (PVN) in the drinking response induced by microinjection of angiotensin II (ANG II) into the subfornical organ (SFO) in the awake rat. Intracerebral microdialysis techniques were utilized to quantify the extracellular concentration of noradrenaline (NA) in the region of the PVN. Injections of ANG II (10−6  M, 0.2 μl) into the SFO significantly increased NA release in the PVN area. The increase in the NA concentration caused by the ANG II injection was significantly attenuated by water ingestion. In urethane-anesthetized rats, injections of ANG II into the SFO elicited an elevation in mean arterial pressure (MAP). On the other hand, intravenous injections of the α-agonist metaraminol (5 μg) slightly decreased the release of NA in the PVN area that accompanied an elevation in MAP. These results show that the noradrenergic system in the PVN area may be involved in the dipsogenic response induced by ANG II acting at the SFO.
Keywords: Drinking; Subfornical organ; Paraventricular nucleus; Angiotensin II; Noradrenaline; Blood pressure; Rat;

Endogenous corticotropin releasing factor regulates adrenergic and opioid receptors by Richard B. Rothman; Nga Vu; Heng Xu; Michael H. Baumann; Yi-Feng Lu (2177-2180).
Previous work from this laboratory demonstrated that intracerebroventricular (i.c.v.) administration of IgG antibodies directed against selected neuropeptides changed the density of opioid receptors, suggesting that neuropeptides in the CNS can perform a regulatory role. To further test this hypothesis, we administered anticorticotropin (CRF) IgG to rats via the i.c.v. route and measured the density of opioid mu and delta receptors and also beta- and alpha2-adrenergic receptors. The results demonstrated that anti-CRF IgG upregulates mu and beta-adrenergic receptors. We conclude that CRF in the cerebrospinal fluid may exert regulatory effects throughout the brain.
Keywords: Corticotropin releasing factor (CRF); Adrenergic receptors; Opioid receptors;

Intraventricular insulin decreases kappa opioid-mediated sucrose intake in rats by A.J. Sipols; J. Bayer; R. Bennett; D.P. Figlewicz (2181-2187).
The hormone insulin acts in the central nervous system (CNS) as a regulator of body adiposity and food intake. Recent work from our laboratory has provided evidence that one way by which insulin may decrease food intake is by decreasing the rewarding properties of food. Evidence from others suggests that endogenous opioids may mediate the palatable properties of foods, and insulin may decrease nonfood-related reward via interaction with some CNS kappa opioid systems. In the present study we examined the ability of insulin to interact with exogenous or endogenous kappa opioids to modulate feeding of palatable sucrose pellets by nondeprived rats. Insulin (5 mU intracerebroventricular (i.c.v.), t=−3 h) completely reversed the ability of the exogenous kappa agonist U50,488 (26 μg, i.c.v., t=−15 min) to stimulate 90-min sucrose feeding (211±32% reduced to 125±23% of 90-min baseline intake). Further, i.c.v. insulin (5 mU, t=−3 h) interacted with a subthreshold dose of the kappa receptor antagonist norbinaltorphimine (5 μg, i.c.v., t=−15 min) to decrease the 90-min sucrose intake baseline (77±11% versus 109±10% of 90 min baseline intake, insulin/norbinaltorphimine versus norbinaltorphimine). Together these studies provide new evidence that insulin in the CNS may decrease the action of CNS kappa opioid system(s) that mediate palatable feeding.
Keywords: Insulin; Kappa opioid; Palatability; Food intake;

Novel peptide–peptide cooperation may transform feeding behavior by Abba J. Kastin; Weihong Pan; Victoria Akerstrom; Laszlo Hackler; Chuanfeng Wang; Catherine M. Kotz (2189-2196).
There is need for a new approach to the suppression of feeding. Here, we show that two of the most potent endogenous satiety peptides interact in a novel way to cross the blood–brain barrier (BBB) and to suppress food intake. Combined peripheral administration of leptin and urocortin (UCN) significantly decreased food intake, whereas neither one showed an effect when given alone in the same doses. We further provide a mechanism whereby this novel cooperativity can occur by demonstrating that UCN, which by itself does not cross the BBB, can readily enter the brain by associating with leptin. Such a novel interaction between two peptides at the BBB opens new approaches for general study of the dynamic regulatory role of the BBB in brain–body communication as well as the specific study of obesity.
Keywords: Peptide–peptide cooperation; Feeding behavior; Leptin; Urocortin; Blood–brain barrier; Transport;

Regional differences in PACAP transport across the blood–brain barrier in mice: a possible influence of strain, amyloid β protein, and age by Naoko Nonaka; William A. Banks; Hidekatsu Mizushima; Seiji Shioda; John E. Morley (2197-2202).
The blood–brain barrier (BBB) controls the exchange of peptides and regulatory proteins between the central nervous system (CNS) and the blood. Transport across the BBB of such regulatory substances is altered in animal models of Alzheimer’s disease. These alterations could lead to cognitive impairments or diminish their therapeutic potential. Here, we measured the transport rate of radioactively labeled pituitary adenylate cyclase-activating polypeptide (PACAP) from blood into whole brain and into 11 brain regions in three groups of mice: young (2 months old) ICR, young (2 months old) SAMP8, and aged (12 months old) SAMP8 mice. The SAMP8 is a strain which develops impaired learning and memory with aging that correlates with an age-related increase in brain levels of amyloid β protein (AβP). PACAP is a powerful neurotrophin that may have a therapeutic role in neurodegenerative diseases. We found that I-PACAP crossed the BBB fastest at the hypothalamus and the hippocampus in all three groups. Slower transport rates into the whole brain, the olfactory bulb, the hypothalamus, and the hippocampus for aged SAMP8 mice was likely related to differences both from strain and expression of AβP with aging.
Keywords: Blood–brain barrier; PACAP; SAMP8; SAM; Aging; Amyloid β protein; Hypothalamus; Hippocampus; Olfactory bulb; Transport;

Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time, age and genetic background by L. Lin; J. Wisor; T. Shiba; S. Taheri; K. Yanai; S. Wurts; X. Lin; M. Vitaterna; J. Takahashi; T.W. Lovenberg; M. Koehl; G. Uhl; S. Nishino; E. Mignot (2203-2211).
The hypocretins (1 and 2) have emerged as key regulators of sleep and wakefulness. We developed a high-throughput enzyme immunoassay (EIA) to measure total brain hypocretin levels from large numbers of mice. Hypocretin levels were not altered by circadian time or age. However, significant differences in one or both hypocretin peptides were observed between different mouse strains. We studied hypocretin levels in knockout and transgenic mouse models with obesity, circadian gene mutations or monoaminergic defects. Compared to controls, only histamine receptor knockouts had lower hypocretin levels. This was most pronounced in H1 receptor knockouts suggesting the existence of a positive feedback loop between hypocretin and histaminergic neurons.
Keywords: Hypocretin; Orexin; Narcolepsy; Enzyme immunoassay; Histamine receptor; Knockout;

Melanin-concentrating hormone, hippocampal nitric oxide levels and memory retention by Mariana Varas; Mariela Pérez; Marı́a Elena Monzón; Susana Rubiales de Barioglio (2213-2221).
The present study attempts to determine, if the effect of melanin-concentrating hormone (MCH) upon memory retention is correlated with changes in nitric oxide synthase (NOS) activity and tissue levels of nitric oxide (NO) and cGMP. We used a behavioral experiment using a step-down inhibitory avoidance test, the biochemical determinations of NO and cGMP, and electrophysiological model. Results of behavioral studies (step-down test) showed that MCH administration reverts the amnesic effects induced by N G-nitro-l-arginine (l-NOArg). Moreover, electrophysiological studies demonstrated that l-NOArg did not block the potentiation induced by the peptide. Hippocampal NO and cGMP levels increased after MCH injection.
Keywords: MCH; Hippocampus; Nitric oxide; cGMP; Synaptic transmission; Behavior;

Passage of amyloid β protein antibody across the blood–brain barrier in a mouse model of Alzheimer’s disease by William A. Banks; Brie Terrell; Susan A. Farr; Sandra M. Robinson; Naoko Nonaka; John E. Morley (2223-2226).
Vaccinations against amyloid β protein (AβP) reduce amyloid deposition and reverse learning and memory deficits in mouse models of Alzheimer’s disease. This has raised the question of whether circulating antibodies, normally restricted by the blood–brain barrier (BBB), can enter the brain [Nat. Med. 7 (2001) 369–372]. Here, we show that antibody directed against AβP does cross the BBB at a very low rate. Entry is by way of the extracellular pathways with about 0.11% of an intravenous (i.v.) dose entering the brain by 1 h. Clearance of antibody from brain increasingly dominates over time, but antibody is still detectable in brain 72 h after i.v. injection. Uptake and clearance is not altered in mice overexpressing AβP. This ability to enter and exit the brain even in the presence of increased brain ligand supports the use of antibody in the treatment of Alzheimer’s and other diseases of the brain.
Keywords: Alzheimer’s disease; Amyloid β protein; Amyloid precursor protein; Antibody; Blood–brain barrier; Mice;

Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia by D. Reglodi; A. Tamás; A. Somogyvári-Vigh; Z. Szántó; E. Kertes; L. Lénárd; A. Arimura; I. Lengvári (2227-2234).
PACAP exerts neuroprotective effects under various neurotoxic conditions in vitro. In vivo, it reduces brain damage after global and transient focal ischemia. The present study investigated whether PACAP has neuroprotective effects when applied before the onset of permanent ischemia. Rats were given bolus injections of PACAP38 intracerebroventricularly, and then underwent permanent middle cerebral artery occlusion. The results show that 2 μg of PACAP significantly reduced the infarct size measured 12 and 24 h after the onset of ischemia. No further reduction was obtained by a 7-day pretreatment. PACAP also ameliorated certain sensorimotor deficits. Our present study provides further evidence for the neuroprotective effects of PACAP, and implies that it might be a promising preventive therapeutic agent in ameliorating ischemic brain damage.
Keywords: Permanent focal cerebral ischemia; Neuroprotection; Rat; Neuropeptides; PACAP;

Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure by Chris J. Pemberton; Tim G. Yandle; Eric A. Espiner (2235-2244).
In order to elucidate how brain natriuretic peptides (NPs) are affected by experimentally induced heart failure, we have measured the immunoreactive (IR) levels of the NP in extracts from 10 regions of ovine brain, including pituitary, and clarified their molecular forms using high performance liquid chromatography (HPLC). Using species-specific radioimmunoassay (RIA), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were all detected in extracts taken from control animals and sheep that had undergone rapid ventricular pacing for 7 days to induce heart failure. CNP was the most abundant NP as assessed by specific RIA, and the pituitary contained the highest IR levels for all three NP. Compared with control animals, the pituitary content of BNP in animals with heart failure was reduced by 40% (control, 0.26±0.02 pmol/g wet weight versus heart failure 0.16±0.01; P<0.01, n=7). No other significant changes were observed. The molecular forms of ANP and CNP in whole brain extracts as assessed by HPLC were proANP and CNP22, CNP53 and proCNP, respectively. BNP in pituitary extracts was assessed to be primarily proBNP with a minor component of mature BNP26.
Keywords: Natriuretic peptide; Heart failure; Brain concentrations; Molecular form; HPLC;

Colocalization of NO and VIP in neurons of the submucous plexus in the rat intestine by Yoshihide Chino; Masaki Fujimura; Kunio Kitahama; Mineko Fujimiya (2245-2250).
Since very few previous studies have carried out the quantitative analysis for the colocalization of nitric oxide (NO) and vasoactive intestinal peptide (VIP) in the submucous neurons in the rat digestive tract, we applied in vivo treatment of colchicine to enhance the immunoreactivity and examined the colocalization of NO synthase (nNOS) and VIP in neurons of the submucous plexus throughout the rat digestive tract. The density of nNOS-containing neurons in the submucous plexus in the stomach corpus (103±25 cells/cm2, n=3) and that in the antrum (157±9 cells/cm2, n=3) were significantly lower than those in small and large intestine. However no difference was detected in the cell density among duodenum (1967±188 cells/cm2, n=3), jejunum (2640±140 cells/cm2, n=3), ileum (2070±42 cells/cm2, n=3), proximal colon (2243±138 cells/cm2, n=3) and distal colon (2633±376 cells/cm2, n=3). The proportion of nNOS-immunoreactive (IR), nNOS/VIP-IR and VIP-IR neurons to the total number of submucous neurons was examined. nNOS/VIP-IR neurons comprised 45–55% of total number of submucous neurons from the duodenum to the proximal colon, however those comprised 66.4±5.1% in the distal colon. The results showed that the dense distribution of nNOS-containing neurons was found in the submucous plexus throughout the small and large intestine, and large population of submucous neurons co-stored nNOS and VIP.
Keywords: Niric oxide; Nitric oxide synthase; Immunohistochemistry; Localization; VIP; Submucous plexus; Rat;

Vasoactive intestinal peptide binding autoantibodies in autoimmune humans and mice by Yogesh Bangale; Dana Cavill; Tom Gordon; Stephanie Planque; Hiroaki Taguchi; Gita Bhatia; Yasuhiro Nishiyama; Frank Arnett; Sudhir Paul (2251-2257).
Autoantibodies capable of binding the immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) were detected in the sera of a mouse strain prone to autoimmune disease due to the lpr mutation (MRL/lpr). The autoantibodies were not present in control wildtype MRL/lpr mice, but they were readily detected in humans without autoimmune disease. The binding was due to low affinity VIP recognition. Increased VIP binding activity was evident in patients with systemic lupus erythematosus but not systemic sclerosis, Sjögren’s syndrome (SS), rheumatoid arthritis or autoimmune thyroiditis. Recombinant VIP binding Fv clones (fragment variable; the variable domains of the light and heavy chains antibody subunits joined with a peptide linker) were isolated from a phage display library prepared from lupus patients. One Fv clone displaying VIP-selective binding and several clones displaying cross-reactivity with unrelated peptides were identified. Replacement mutations in the VIP-selective clone were preferentially localized in the regions known to make contacts with the antigen, i.e. the complementarity determining regions, suggesting that the selective binding activity is due to immunological maturation of the antibodies. Frequent occurrences of autoantibody responses to VIP indicate that immunological tolerance to this neuropeptide can be readily broken. The depletion of VIP by specific antibodies in autoimmune disease may interfere with VIP regulation of T cells and inflammatory cells and result in further amplification of autoreactive immunological responses.
Keywords: Neuroimmune interactions; Lupus; Autoantibodies; Vasoactive intestinal peptide;

Structural requirements at the N-terminus of urotensin II octapeptides by D.H. Coy; W.J. Rossowski; B.L. Cheng; J.E. Taylor (2259-2264).
Urotensin II is the latest of a growing list of peptides exhibiting potent cardiovascular effects. It is an extremely potent vasoconstrictor in primates; its excretion is elevated in hypertensive patients thus suggesting therapeutic potential for urotensin II analogues, particularly receptor antagonists. In the present study, a number of interesting structural features pertaining to the N-terminus of urotensin II have been evaluated for binding to cloned human and rat urotensin II receptors and functional effects on rat upper thoracic aorta smooth muscle preparations. Shortened octapeptides retained full binding affinities and functional activities, did not require a free N-terminal amino group, and could tolerate an amidated C-terminus. The N-terminal Asp residue present in the octapeptides did not require a negatively charged side chain, merely one which contained a hydrogen bond acceptor CO group which could be present at a variety of positions on the side chain. The side chain could be constrained into a trans-olefinic configuration with full retention of potency, but potency was lost in the cis configuration. N-terminal aromatic amino substituted with polar groups such as OH and NO2 also resulted in high affinity analogues. Overall, the correlation between binding affinities for the human and rat receptors was quite good. These findings could be of value in the development of more potent urotensin II receptor antagonists based on the previously identified somatostatin antagonist octapeptides which we have recently found, function as relatively weak urotensin II antagonists.
Keywords: Urotensin II structure–activity relationships; Human and rat urotensin II receptors; Rat aorta;

Identification and polymorphism of Plasmodium vivax RBP-1 peptides which bind specifically to reticulocytes by Mauricio Urquiza; Manuel A. Patarroyo; Viviana Marı&#x0301;n; Marisol Ocampo; Jorge Suarez; Ramses Lopez; Alvaro Puentes; Hernando Curtidor; Javier Garcı&#x0301;a; Luis E. Rodrı&#x0301;guez; Ricardo Vera; Angela Torres; Marilu Laverde; Ana P. Robles; Manuel E. Patarroyo (2265-2277).
Plasmodium vivax merozoite preferentially invades reticulocytes probably using PvRBP-1 as ligand. One hundred and ninety-five, 15-mer peptides has been synthesised from PvRBP-1 sequence; tested in reticulocyte- or erythrocyte-binding assays. Twenty-five peptides (K d=76–380 nM) specifically defined four reticulocyte-binding regions. It has been reported that a highly conserved Region-I recombinant fragment binds specifically to reticulocytes. HABP-critical residues for reticulocyte-binding were highly conserved in 20 Colombian P. vivax clinical isolates, suggesting an important biological function. There were six overlapping reticulocyte-binding sites for these peptides according to enzyme sensitivity and mutual competition-binding assays; located on 26- and 41-kDa reticulocyte membrane surface proteins.
Keywords: PvRBP-1; Plasmodium vivax; Reticulocyte-binding peptides; P. vivax merozoite invasion;

Peptide T bolus normalizes the growth hormone secretion pattern in two children with AIDS by Charlotte Barbey-Morel; Kevin McDonnell; Candace B. Pert; MerriBeth Adams; Dean Farrand; Michael R. Ruff; Michael D. Lumpkin (2279-2281).
In humans, HIV infection reduces growth hormone (GH) secretion contributing to AIDS wasting. In rats, the HIV envelope protein gp120 alone blocks GH secretion both in vitro and in vivo through GH-releasing hormone receptors. Peptide T, a modified octapeptide derived from gp120, normalizes GH secretion. We now report that an intravenous bolus of peptide T normalizes nocturnal GH secretion in two out of three children with AIDS. These results, coupled with the lack of toxicity of this experimental AIDS therapeutic, justify clinical trials for AIDS wasting and pediatric AIDS. A clinical and basic science update on peptide T appears in Current HIV Research.
Keywords: Peptide T; AIDS wasting; gp120; Growth hormone; Growth hormone releasing hormone receptor; Pediatric AIDS; HIV envelope protein;

Neuropeptides, food intake and body weight regulation: a hypothalamic focus by J.J.G. Hillebrand; D. de Wied; R.A.H. Adan (2283-2306).
Energy homeostasis is controlled by a complex neuroendocrine system consisting of peripheral signals like leptin and central signals, in particular, neuropeptides. Several neuropeptides with anorexigenic (POMC, CART, and CRH) as well as orexigenic (NPY, AgRP, and MCH) actions are involved in this complex (partly redundant) controlling system. Starvation as well as overfeeding lead to changes in expression levels of these neuropeptides, which act downstream of leptin, resulting in a physiological response. In this review the role of several anorexigenic and orexigenic (hypothalamic) neuropeptides on food intake and body weight regulation is summarized.
Keywords: Neuropeptides; Food intake; Body weight; Energy homeostasis; Hypothalamus;

Endogenous opiates and behavior: 2001 by Richard J Bodnar; Maria M Hadjimarkou (2307-2365).
This paper is the twenty-fourth installment of the annual review of research concerning the opiate system. It summarizes papers published during 2001 that studied the behavioral effects of the opiate peptides and antagonists. The particular topics covered this year include the molecular–biochemical effects and neurochemical localization studies of endogenous opioids and their receptors (), and the roles of these opioid peptides and receptors in pain and analgesia (); stress and social status (); tolerance and dependence (); learning and memory (); eating and drinking (); alcohol and drugs of abuse (); sexual activity and hormones, pregnancy, development and endocrinology(); mental illness and mood (); seizures and neurologic disorders (); electrical-related activity and neurophysiology (); general activity and locomotion (); gastrointestinal, renal and hepatic functions (); cardiovascular responses (); respiration and thermoregulation (); and immunological responses ().
Keywords: Endogenous opiates; Opiate system; Behavioral effects;