Peptides (v.29, #1)

Evaluation of the inhibitory effects of human serum components on bactericidal activity of human beta defensin 3 by Giuseppantonio Maisetta; Mariagrazia Di Luca; Semih Esin; Walter Florio; Franca Lisa Brancatisano; Daria Bottai; Mario Campa; Giovanna Batoni (1-6).
Naturally occurring cationic antimicrobial peptides (CAPs) are an essential component of the innate immune system of multicellular organisms. At concentrations generally higher than those found in vivo, most CAPs exhibit strong antibacterial properties in vitro, but their activity may be inhibited by body fluids, a fact that could limit their future use as antimicrobial and/or immunomodulatory agents. In the present study, we evaluated the effects of human serum components on bactericidal activity of the human β-defensin 3 (hBD-3), a CAP considered particularly promising for future therapeutic employment. Human serum diluted to 20% strongly inhibited the bactericidal activity of the peptide against both the Gram-positive species Staphylococcus aureus and the Gram-negative species Acinetobacter baumannii. Such activity was not restored in serum devoid of salts (dialyzed), pre-treated with protease inhibitors, or subjected to both of these treatments. The addition of physiological concentrations of NaCl, CaCl2, and human albumin in the bactericidal assay abolished bactericidal activity of hBD-3 against S. aureus, while it only partially inhibited the activity of the peptide against A. baumannii. Although a proteolytic activity of serum on hBD-3 was demonstrated at the protein level by Western blot, addition of physiological concentrations of trypsin to the bactericidal assay only partially affected the antibacterial properties of the peptide. Altogether, these results demonstrate a major role of mono-divalent cations and serum proteins on inhibition of hBD-3 antibacterial properties and indicate a relative lack in sensitivity of the bactericidal activity of this peptide to trypsin and trypsin-like proteases.
Keywords: Antimicrobial peptides; Defensins; Human beta-defensin 3; Inhibition; Serum;

Synthetic peptides Phd1–3 spanning the cationic carboxy-terminal region of human β-defensins HBD-1–3 have been shown to have antibacterial activity. Gross morphological changes were seen in E. coli cells treated with these peptides. In this paper, we have studied the surface-active properties of peptides Phd1–3 and their interactions with different phospholipids using Langmuir–Blodgett monolayers. Compression isotherms and increase in pressure on insertion of peptides into lipid monolayers at different initial pressures indicate the affinity of these peptides for negatively charged lipids. Phd3 inserted less effectively into monolayers as compared to Phd1 and Phd2. The peptides differed in their ability to permeabilize the inner membrane of E. coli, with Phd3 being least effective. It is likely that the peptides kill Gram-negative bacteria by more than one mechanism. When hydrophobicity and net charge favor insertion into lipid membranes, then membrane permeabilization could be the primary event in the killing of bacteria. In cases where membrane insertion does not occur, interaction with phospholipid interface induces highly selective stress that leads to stasis and cell death, as proposed for polymyxin B and bactenecin.
Keywords: Human β-defensins; Single disulfide analogs; Antibacterial activity; Lipid monolayers; Inner-membrane permeabilization;

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes by Rodrigo M. Verly; Magali A. Rodrigues; Katia Regina P. Daghastanli; Angelo Márcio L. Denadai; Iolanda M. Cuccovia; Carlos Bloch; Frédéric Frézard; Marcelo M. Santoro; Dorila Piló-Veloso; Marcelo P. Bemquerer (15-24).
DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L−1) than in its absence (93 μmol L−1). Apparent binding constant values were 2160 and 10,000 L mol−1, respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.
Keywords: Antimicrobial peptide; Cholesterol; Dermaseptin; Isothermal titration calorimetry; Liposomes;

Extended-spectrum β-lactamase (ESBL)-producing Gram-negative bacteria are becoming increasingly prevalent and their antibiotic resistance necessitates novel therapeutic intervention. Ascaphin-8 is a cationic α-helical peptide that shows broad-spectrum antibacterial activity but is also toxic to human erythrocytes (LC50  = 55 μM). This study assesses the activity of ascaphin-8, and a series of l-lysine-substituted analogs, against a range of clinical isolates of ESBL-producing bacteria. All ESBL-producing Escherichia coli (MIC = 1.5–6 μM) and Klebsiella pneumoniae (MIC = 12.5–25 μM) strains tested were susceptible to ascaphin-8, as well as a group of miscellaneous ESBL-producing strains (Citrobacter, Salmonella, Serratia, Shigella spp.) (MIC ≤ 25 μM). The Lys4- and Lys8-substituted analogs were generally the most potent against bacteria but showed the highest hemolytic activity. However, the Lys10, Lys14, and Lys18 analogs also displayed potent antibacterial activity while showing very low hemolytic activity (LC50  > 500 μM). An unexpected finding was the susceptibility of ESBL-producing Proteus mirabilis strains to ascaphin-8 (MIC = 12.5–25 μM) and its Lys4-substituted analog (MIC = 6 μM), although non-ESBL isolates of this organism were resistant to these peptides (MIC > 100 μM).
Keywords: Antibacterial peptide; Ascaphin-8; Extended-spectrum β-lactamases; Hemolysis;

Tachyplesin III and granulocyte-colony stimulating factor enhance the efficacy of tazobactam/piperacillin in a neutropenic mouse model of polymicrobial peritonitis by Oscar Cirioni; Roberto Ghiselli; Wojciech Kamysz; Fiorenza Orlando; Carmela Silvestri; Federico Mocchegiani; Fabio Di Matteo; Elzbieta Kamysz; Alessandra Riva; Marco Rocchi; Vittorio Saba; Giorgio Scalise; Andrea Giacometti (31-38).
We investigated the efficacy of tazobactam/piperacillin (TZP), tachyplesin III and granulocyte-colony stimulating factor (G-CSF) in an experimental murine neutropenic intraabdominal infection. BALB/c male mice were rendered neutropenic by intraperitoneal administration of cyclophosphamide on days −4 and −2 pre-infection. Septic shock was induced by cecal ligation and puncture. Animals received intravenously isotonic sodium chloride solution (control group C1), 1 mg/kg of tachyplesin III, 120 mg/kg of TZP, 0.1 mg/kg of G-CSF, tachyplesin III plus TZP, G-CSF plus TZP and finally tachyplesin III plus G-CSF plus TZP, respectively. Lethality, bacterial growth in blood, peritoneum, spleen, liver, and mesenteric lymph nodes, endotoxin, IL-6 and TNF-alpha concentrations in plasma were evaluated. All compounds reduced the lethality when compared to controls. Endotoxin and cytokine plasma levels were significantly higher in TZP-treated animals compared to tachyplesin III-treated animals. Finally, all drug combinations showed to be the most effective treatment in reducing all variables measured. Interestingly, the strongest results concerning the bacterial growth inhibition, lethality and endotoxemia were obtained when the three compounds were contemporaneously administered. The presence of their positive interaction makes tachyplesin III and G-CSF potentially valuable as an adjuvant for antimicrobial chemotherapy of sepsis.
Keywords: Antibiotics; Granulocyte-colony stimulating factor; Tachyplesin III; Antimicrobial peptides; Sepsis; Endotoxin; TNF-alpha;

Comparison of the effects of serpin A1, a recombinant serpin A1-IGF chimera and serpin A1 C-terminal peptide on wound healing by Luis Fernando Congote; Nyssa Temmel; Gulzhakhan Sadvakassova; Monica C. Dobocan (39-46).
Serpin A1 (α1-antitrypsin, α1-proteinase inhibitor), a potent neutrophil elastase inhibitor, has therapeutic potential as a wound-healing agent. We compared the in vitro wound-healing action of serpin A1-IGF, a recombinant fusion protein of serpin A1(M351E-M358L) and insulin-like growth factor I with that observed in the presence of natural serpin A1 or A1-C26, the synthetic C-terminal 26 residue peptide of serpin A1, previously shown to have mitogenic and antiviral activities. All agents reduced wound sizes in monolayers of the kidney epithelial cell line LLC-PK1 and in primary cultures of human skin fibroblasts. Wound reduction in primary human keratinocytes was only observed with the serpin A1-IGF chimera. None of the factors stimulated cell proliferation using a colorimetric assay, with the exception of the serpin A1-IGF chimera, which caused a significant increase of cell proliferation and thymidine incorporation in human skin fibroblasts. However, wound healing by the A1-IGF chimera was reduced in keratinocytes in the presence of mitomycin C, suggesting a role of cell proliferation in wound reduction. The hydrophobic A1-C26 peptide significantly increased the production of collagen I in skin fibroblasts, an appealing asset for skin care applications.
Keywords: Antitrypsin; IGFs; Skin fibroblasts; Fusion proteins; Serpins;

Pulmonary peptidergic innervation remodeling and development of airway hyperresponsiveness induced by RSV persistent infection by Yu-Rong Tan; Tao Yang; Shui-Ping Liu; Yang Xiang; Fei Qu; Hui-Jun Liu; Xiao-Qun Qin (47-56).
Respiratory syncytial virus (RSV) infection causes bronchiolitis in infants and children, which is an important risk factor for the development of chronic asthma. To probe the underlying mechanisms that RSV infection increases the susceptibility of asthma, this present study was designed to establish a RSV persistent infection animal model by cyclophosphamide (CYP) pretreatment that more closely mimic human RSV infection. CYP is an immunosuppressant, which induced deficiency in cellular and humoral immunity. Pulmonary RSV titers, airway function and peptidergic innervation were measured on 7 d, 28 d, 42 d and 60 d postinfection. The results showed that during RSV persistent infection, the lungs of RSV-inoculated animals pretreated with CYP showed higher RSV titers and exhibited obvious chronic inflammation. The results also showed that protein gene product 9.5 (PGP9.5), substance P (SP) and calcitonin gene-related peptide (CGRP)-immunoreactive fibers increased and vasoactive intestinal peptide (VIP)-immunoreactive fibers decreased during RSV persistent infection. These results demonstrate that RSV persistent infection induces significant alterations in the peptidergic innervation in the airways, which may be associated with the development of altered airway function.
Keywords: Respiratory syncytial virus; Airway hyperresponsiveness; Peptidergic innervations;

Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish by R. van Aerle; P. Kille; A. Lange; C.R. Tyler (57-64).
In mammals, the Kiss1 receptor (Kiss1r) and its kisspeptin ligands are key factors regulating the onset of puberty. In fish, however, the mechanisms underlying the initiation of puberty are poorly understood and the role of the Kiss1r/kisspeptin pathway in this process has not been established. In this study, a bioinformatics approach was used to identify the genes for Kiss1 and Kiss1r in five teleost genomes and the information used to clone the corresponding transcripts from zebrafish. Zebrafish kiss1r was expressed predominantly in the brain, with a minor level of expression in the eye, and zebrafish kiss1 was expressed in brain, intestine, adipose tissue and testis. Analysis of the chromosome region containing the kiss1 locus showed high synteny across vertebrate genomes. In contrast to their mammalian homologues, teleost Kiss1 protein sequences were poorly conserved with the exception of the region representing kisspeptin-10. Signal peptide sequences and likely cleavage and amidation sites in the teleost Kiss1 sequences were determined and found to be similar to those in mammalian Kiss1. This is the first report of the existence and characterization of the Kiss1 gene outside the mammalian taxa, suggesting that a functional Kiss1/Kiss1 receptor pathway is conserved across vertebrate species.
Keywords: Kiss1; Kisspeptins; Kiss1 receptor; GPR54; Puberty; Brain–pituitary–gonad axis; Danio rerio;

Helokinestatin: A new bradykinin B2 receptor antagonist decapeptide from lizard venom by Hang Fai Kwok; Tianbao Chen; Martin O’Rourke; Craig Ivanyi; David Hirst; Chris Shaw (65-72).
Synthetic bradykinin antagonist peptides/peptoids have been powerful tools for delineating the roles of kinins in both normal physiology and in pathological states. Here, we report the identification of a novel, naturally occurring bradykinin B2 receptor antagonist peptide, helokinestatin, isolated and structurally characterized from the venoms of helodermatid lizards—the Gila monster (Heloderma suspectum) and the Mexican beaded lizard (Heloderma horridum). The primary structure of the peptide was established by a combination of microsequencing and mass spectroscopy as Gly-Pro-Pro-Tyr-Gln-Pro-Leu-Val-Pro-Arg (M r 1122.62). A synthetic replicate of helokinestatin was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle, mediated by the B2 receptor sub-type, in a dose-dependent manner. Natural selection, that generates functional optimization of predatory reptile venom peptides, can potentially provide new insights for drug lead design or for normal physiological or pathophysiological processes.
Keywords: Bradykinin; Lizard; Helokinestatin; Venom peptides; Mass spectroscopy;

Adrenomedullin is increased by pulsatile shear stress on the vascular endothelium via periodic acceleration (pGz) by Alfredo Martínez; Jaqueline Arias; Jorge A. Bassuk; Heng Wu; Paul Kurlansky; Jose A. Adams (73-78).
Periodic acceleration (pGz) is produced by a platform which moves the supine body repetitively in a headward to footward direction. The imparted motion produces pulsatile shear stress on the vascular endothelium. Pulsatile shear stress on the vascular endothelium has been shown to elicit production of a host of cardioprotective, cytoprotective mediators. The purpose of this study was to ascertain if pGz also enhances production of adrenomedullin (AM) in normal healthy swine. Twelve pigs (weight range 20–30 kg) were anesthetized, intubated and placed on conventional mechanical ventilation. All animals were secured to the motion platform. In one group (pGz) (n  = 7) was activated for 1 h, and monitored for an additional 3 h. A control group (CONT) (n  = 5) served as time control. Arterial blood gases, hemodynamic measurements, and serum for AM, interleukin 4, 6 and thromboxane B2 (TBXB2) were measured at baseline, immediately after pGz, and 3 h after pGz had been discontinued. There was no significant change from baseline value in IL-4, IL-6 or TBXB2. Mean arterial blood pressure decreased in pGz-treated animals from 115 ± 10 at baseline to 90 ± 8 after 60 min of pGz (p  < 0.01). AM levels increase from 776 ± 176 pg/ml baseline to 1160 ± 68 pg/ml immediately after pGz, and remained elevated to 1584 ± 160 pg/ml, 3 h after pGz (p  < 0.01 vs. BL). This is the first report of AM-enhanced production using a non-invasive method of increasing pulsatile shear stress on the vascular endothelium. pGz increases production of AM in normal healthy swine. These changes are independent of IL-4, IL-6 or TBXB2 production.
Keywords: Adrenomedullin; Pulsatile shear stress; Periodic acceleration; Nitric oxide; Prostaglandins; Endothelium;

C-type natriuretic peptide (CNP) significantly increases in chronic heart failure (CHF) patients as a function of clinical severity. Aim of this study was to evaluate in CHF patients the relationship between circulating CNP concentrations and echo-Doppler conventional indices of left ventricular (LV) function as well as less load independent parameters as dP/dt. LV ejection fraction (EF), left ventricular end-diastolic dimension (LVEDD) and LV dP/dt were evaluated together with plasma CNP levels in 38 patients with CHF and in 63 controls. CNP levels resulted significantly higher in CHF patients than in controls (7.19 ± 0.59 pg/ml vs. 2.52 ± 0.12 pg/ml, p  < 0.0001). A significant correlation between dP/dt and CNP levels (r  = −0.61, p  < 0.0001) was observed. A good correlation with EF (r  = −0.55, p  < 0.001) and a less significant relation with LVEDD (r  = 0.316, p  < 0.05) were also reported. When patients were divided according to dP/dt values a very significant difference in CNP levels was observed: Group I (<600, n  = 25) vs. Group II (>600, n  = 13): 8.46 ± 0.69 and 4.75 ± 0.75 pg/ml, respectively, p  < 0.001. This is the first study that reports a correlation between CNP and dP/dt in CHF patients, thus suggesting a possible role on cardiac contractility.
Keywords: Chronic heart failure (CHF); C-type natriuretic peptide (CNP); Radioimmunoassay (RIA); Cardiac echo-Doppler; dP/dt;

Proenkephalin expression and enkephalin release are widely observed in non-neuronal tissues by Gerene M. Denning; Laynez W. Ackermann; Thomas J. Barna; John G. Armstrong; Lynn L. Stoll; Neal L. Weintraub; Eric W. Dickson (83-92).
Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. Studies have shown that enkephalins play an important role in behavior, pain, cardiac function, cellular growth, immunity, and ischemic tolerance. Our global hypothesis is that enkephalins are released from non-neuronal tissues in response to brief ischemia or exercise, and that this release contributes to cardioprotection. To identify tissues that could serve as potential sources of enkephalins, we used real-time PCR, Western blot analysis, ELISA, immunofluorescence microscopy, and ex vivo models of enkephalin release. We found widespread expression of preproenkephalin (pPENK) mRNA and production of the enkephalin precursor protein proenkephalin (PENK) in rat and mouse tissues, as well as in tissues and cells from humans and pigs. Immunofluorescence microscopy with anti-enkephalin antisera demonstrated immunoreactivity in rat tissues, including heart and skeletal muscle myocytes, intestinal and kidney epithelium, and intestinal smooth muscle cells. Finally, isolated tissue studies showed that heart, skeletal muscle, and intestine released enkephalins ex vivo. Together our studies indicate that multiple non-neuronal tissues produce PENK and release enkephalins. These data support the hypothesis that non-neuronal tissues could play a role in both local and systemic enkephalin-mediated effects.
Keywords: Proenkephalin; Enkephalins; Gene expression; Skeletal muscle;

Anxiolytic- and antidepressant-like activities of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta opioid receptor agonist by Raffaella Vergura; Gianfranco Balboni; Barbara Spagnolo; Elaine Gavioli; David G. Lambert; John McDonald; Claudio Trapella; Lawrence H. Lazarus; Domenico Regoli; Remo Guerrini; Severo Salvadori; Girolamo Caló (93-103).
Knockout and pharmacological studies have shown that delta opioid peptide (DOP) receptor signalling regulates emotional responses. In the present study, the in vitro and in vivo pharmacological profile of the DOP ligand, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512) was investigated. In receptor binding experiments performed on membranes of CHO cells expressing the human recombinant opioid receptors, UFP-512 displayed very high affinity (pK i 10.20) and selectivity (>150-fold) for DOP sites. In functional studies ([35S]GTPγS binding in CHOhDOP membranes and electrically stimulated mouse vas deferens) UFP-512 behaved as a DOP selective full agonist showing potency values more than 100-fold higher than DPDPE. In vivo, in the mouse forced swimming test, UFP-512 reduced immobility time both after intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration. Similar effects were recorded in rats. Moreover, UFP-512 evoked anxiolytic-like effects in the mouse elevated plus maze and light–dark aversion assays. All these in vivo actions of UFP-512 were fully prevented by the selective DOP antagonist naltrindole (3 mg/kg, s.c.). In conclusion, the present findings demonstrate that UFP-512 behaves as a highly potent and selective agonist at DOP receptors and corroborate the proposal that the selective activation of DOP receptors elicits robust anxiolytic- and antidepressant-like effects in rodents.
Keywords: UFP-512; DOP receptor; Receptor binding; Bioassay; Forced swimming test; Light–dark aversion; Elevated plus-maze test;

A derivative of the melanocortin receptor antagonist SHU9119 (PG932) increases food intake when administered peripherally by Gregory M. Sutton; M. Josephine Babin; Xuyuan Gu; Victor J. Hruby; Andrew A. Butler (104-111).
Melanocortin receptors are considered promising candidates for the treatment of behavioral and metabolic disorders ranging from obesity to anorexia and cachexia. These experiments examined the response of mice to peripheral injections of two compounds. PG932 is a derivative of SHU9119 which is non-selective antagonist of melanocortin-3 and melanocortin-4 receptors (Mc3r and Mc4r). PG946 is a derivative of a hybrid of α- and β-MSH, and is a moderately selective Mc3r antagonist. SHU9119 increases food intake when administered intracerebroventricularly but is without effect when injected into the periphery. In contrast, PG932 was found to be highly effective at stimulating food intake when administered peripherally by intraperitoneal injection. The orexigenic effect of PG932 required functional Mc4r, suggesting that inhibition of this receptor is involved in the stimulation of food intake. PG946 did not significantly affect on feeding behavior. PG932 is thus a useful new compound for studies examining the regulation of appetite and energy balance, and may also prove useful for the treatment of cachectic conditions.
Keywords: Melanocortin receptor; Antagonist; Anorexia; Appetite; Endotoxin; Proopiomelanocortin;

Peptide YY (3–36) [PYY(3–36)] inhibits feeding in rodents, nonhuman primates and humans, yet the neural circuits underlying this action remain to be determined. Here we assessed whether PYY(3–36) inhibits feeding by activating neurons in forebrain and hindbrain sites containing Y2 receptors and linked to control of food intake, or in hindbrain sites immediately downstream of vagal afferent neurons. Rats received an anorexigenic dose of PYY(3–36), and the number of neurons expressing Fos, an indicator of neuronal activation, was determined in anterior hypothalamus (AH), arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), lateral hypothalamus (LH), ventromedial hypothalamus (VMH), central nucleus of the amygdala (CeA), area postrema (AP), and caudal medial nucleus tractus solitarius (cmNTS), commissural NTS (cNTS), and gelatinosus NTS (gNTS). Expression of tyrosine hydroxylase (TH), an indicator of catecholamine synthesis, was also measured in the cmNTS. PYY(3–36) increased Fos in ARC, cmNTS, gNTS and AP. Approximately 10% of Fos(+) neurons in the cmNTS were TH(+). These results suggest that PYY(3–36) inhibits feeding through direct activation of ARC neurons, and direct and/or indirect activation via vagal afferent nerves of cmNTS, gNTS and AP, including some catecholaminergic neurons in the cmNTS.
Keywords: PYY(3–36); Satiety; Forebrain; Hindbrain; Fos; Tyrosine hydroxylase;

Olanzapine blocks the sympathetic and hyperthermic reactions due to cerebral injection of orexin A by M. Monda; An. Viggiano; Al. Viggiano; R. Mondola; E. Viggiano; G. Messina; D. Tafuri; V. De Luca (120-126).
Since experiments regarding a possible relation between olanzapine and orexin A has been scarcely reported in international literature, this experiment tested the effect of olanzapine on the sympathetic and thermogenic effects induced by orexin A. The firing rates of the sympathetic nerves to interscapular brown adipose tissue (IBAT), along with IBAT, colonic temperatures and heart rate were monitored in urethane-anesthetized male Sprague–Dawley rats before an injection of orexin A (1.5 nmol) into the lateral cerebral ventricle and over a period of 150 min after the injection. The same variables were monitored in rats with an intraperitoneal administration of olanzapine (10 mg/kg bw), injected 30 min before the orexin administration. The results show that orexin A increases the sympathetic firing rate, IBAT, colonic temperatures and heart rate. This increase is blocked by the injection of olanzapine. These findings indicate that olanzapine affects the complex reactions related to activation of orexinergic system.
Keywords: Body temperature; Orexin A; Olanzapine; Rat; Sympathetic activity;

This review critically reappraises recent scientific evidence concerning central leptin insufficiency versus leptin resistance formulations to explain metabolic and neural disorders resulting from subnormal or defective leptin signaling in various sites in the brain. Research at various fronts to unravel the complexities of the neurobiology of leptin is surveyed to provide a comprehensive account of the neural and metabolic effects of environmentally imposed fluctuations in leptin availability at brain sites and the outcome of newer technology to restore leptin signaling in a site-specific manner. The cumulative new knowledge favors a unified central leptin insufficiency syndrome over the, in vogue, central resistance hypothesis to explain the global adverse impact of deficient leptin signaling in the brain. Furthermore, the leptin insufficiency syndrome delineates a novel role of leptin in the hypothalamus in restraining rhythmic pancreatic insulin secretion while concomitantly enhancing glucose metabolism and non-shivering thermogenic energy expenditure, sequelae that would otherwise promote fat accrual to store excess energy resulting from consumption of energy-enriched diets. A concerted effort should now focus on development of newer technologies for delivery of leptin or leptin mimetics to specifically target neural pathways for remediation of diverse ailments encompassing the central leptin insufficiency syndrome.
Keywords: Leptin insufficiency; Brain; Obesity; Metabolic syndrome; Cognition; Alzheimer's disease;

Behavioral controls of food intake by Stephen C. Benoit; Andrea L. Tracy (139-147).
Recent conceptualizations of food intake have divided ingestive behavior into multiple distinct phases. Here, we present a temporally and operationally defined classification of ingestive behaviors. Importantly, various physiological signals including hypothalamic peptides are thought to impact these distinct behavioral phases of ingestion differently. In this review, we summarize a number of behavioral assays designed to delineate the effects of hormone and peptide signals that influence food intake on these ingestive mechanisms. Finally, we discuss two issues that we have encountered in our laboratory which may obstruct the interpretation of results from these types of studies. First, the influence of previous experience with foods used in these behavioral tests and second, the importance of the nutrient composition of the selected test foods. The important conclusion discussed here is that the behavioral analysis of ingestion is accompanied by theoretical constructs and artificial divisions of biological realities and the appreciation of this fact can only increase the opportunities of contemporary behavioral scientists to make significant and novel observations of ingestive behaviors.
Keywords: Learning; Ingestive behavior; Hypothalamus; NPY; Melanocortins;