Peptides (v.25, #12)

BisEDT and RIP act in synergy to prevent graft infections by resistant staphylococci by Philip Domenico; Ellen Gurzenda; Andrea Giacometti; Oscar Cirioni; Roberto Ghiselli; Fiorenza Orlando; Moshe Korem; Vittorio Saba; Giorgio Scalise; Naomi Balaban (2047-2053).
Staphylococci are a major cause of infections associated with indwelling medical devices. Biofilm formation on these devices adds to the antibiotic resistance seen among clinical isolates. RNAIII-inhibiting peptide (RIP) is a heptapeptide that inhibits staphylococcal pathogenesis, including biofilm formation, by obstructing quorum sensing mechanisms. Bismuth ethanedithiol (BisEDT) also prevents biofilm formation at subinhibitory concentrations. RIP and BisEDT were combined to prevent infections in a rat graft model, using antibiotic sensitive and resistant strains of Staphylococcus aureus and Staphylococcus epidermidis. BisEDT, RIP, or rifampin, or their combinations reduced the graft associated bacterial load over seven days. BisEDT–RIP was the best combination, reducing bacterial load to undetectable levels. BisEDT–RIP may prove useful for coating medical devices to prevent staphylococcal infections.
Keywords: Bismuth thiols; Staphylococci; Antimicrobial peptides; RNAIII inhibiting peptide; Biofilms;

Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates by Monica Benincasa; Marco Scocchi; Elena Podda; Barbara Skerlavaj; Lucilla Dolzani; Renato Gennaro (2055-2061).
Ten peptides from 13 to 35 residues in length and covering the whole sequence of the Pro-rich peptide Bac7 were synthesized to identify the domain responsible for its antimicrobial activity. At least 16 residues of the highly cationic N-terminal sequence were required to maintain the activity against Gram-negative bacteria. The fragments Bac7(1–35) and, to a lesser extent, Bac7(1–16) proved active against a panel of antibiotic-resistant clinical isolates of Gram-negative bacteria, with the notable exception of Burkholderia cepacia. In addition, when tested against fungi, the longer fragment was also active against collection strains and clinical isolates of Cryptococcus neoformans, but not towards clinical isolates of Candida albicans.
Keywords: Pro-rich peptide; Bactenecin 7; Cathelicidin peptide; Synthetic analog; Antimicrobial activity; Clinical isolate; Multidrug-resistance;

An antifungal peptide, designated coccinin, with a molecular mass of 7 kDa and an N-terminal sequence resembling those of defensins, was purified from the seeds of large scarlet runner beans (Phaseolus coccineus cv. ‘Major’). The peptide isolated was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. The peptide excerted antifungal activity on a number of fungal species including Botrytis cinerea, Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola, and Rhizoctonia solani. It also inhibited proliferation in the leukemia cell lines HL60 and L1210, and reduced the activity of HIV-1 reverse transcriptase. However, it did not affect proliferation of mouse splenocytes.
Keywords: Peptides; Beans; Antifungal; Antiproliferative;

Structural and functional characterization of N-terminally blocked peptides isolated from the venom of the social wasp Polybia paulista by Susan Pereira Ribeiro; Maria Anita Mendes; Lucilene Delazari dos Santos; Bibiana Monson de Souza; Maurício Ribeiro Marques; Walter Filgueira de Azevedo; Mario Sergio Palma (2069-2078).
Two novel peptides were isolated from the crude venom of the social wasp Polybia paulista, by using RP-HPLC under a gradient of MeCN from 5 to 60% (v/v) and named Polybine-I and -II. Further purification of these peptides under normal phase chromatography, rendered pure enough preparations to be sequenced by Edman degradation chemistry. However, both peptides did not interact with phenylisothiocyanate reagent, suggesting the existence of a chemically blocked N-terminus. Therefore, the sequences of both peptides were assigned by ESI-MS/MS under CID conditions, as follows: Polybine-I Ac-SADLVKKIWDNPAL-NH2 (Mr 1610 Da) and Polybine-II Ac-SVDMVMKGLKIWPL-NH2 (Mr 1657 Da). During the tandem mass spectrometry experiments, a loss of 43 a.m.u. was observed from the N-terminal residue of each peptide, suggesting the acetylation of the N-terminus. Subsequently, the peptides with and without acetylation were synthesized on solid phase and submitted to functional characterizations; the biological activities investigated were: hemolysis, chemotaxis of polymorphonucleated leukocytes (PMNL), mast cell degranulation and antibiosis. The results revealed that the acetylated peptides exhibited more pronounced chemotaxis of PMNL cells and mast cell degranulation than the respective non-acetylated congeners; no hemolytic and antibiotic activities were observed, irrespective to the blockage or not of the α-amino groups of the N-terminal residues of each peptide. Therefore, the N-terminal acetylation may be related to the increase of the inflammatory activity of both peptides.
Keywords: Polybia paulista; N-terminally blocked peptides; Tandem mass spectrometry; Inflammatory peptides; Wasp venom toxins;

Comparative efficacy of VIP and analogs on activation and internalization of the recombinant VPAC2 receptor expressed in CHO cells by Christelle Langlet; Nathalie Gaspard; Ingrid Nachtergael; Patrick Robberecht; Ingrid Langer (2079-2086).
Using a monoclonal antibody interacting with the extracellular amino-terminus of the human VPAC2 receptor but that did not interfere with ligand binding, we measured by flow cytometry receptor internalization and trafficking induced by full agonists, partial agonists and an antagonist in Chinese hamster ovary cells expressing the recombinant receptor. The agonists, but not the antagonist, induced a rapid, dose-dependent receptor internalization blocked by hypertonic sucrose that was more pronounced for the VIP analog N-hexanoyl-VIP (80%) than for VIP and Ro 25-1553 (50%) and the [A11]-VIP (20%). Re-expression of the receptors at the membrane was achieved within two hours after exposure to VIP and Ro 25-1553 was blocked by 25 μM monensin but not by 10 μg/ml cycloheximide. Re-expression was much slower after exposure to the acylated peptide and was blocked by preincubation with 25 μM monensin and 10 μg/ml cycloheximide.
Keywords: VPAC2 receptor; Receptor internalization; Receptor trafficking; VIP analogs;

Derivatives of the somatostatin analogues octreotide and octreotate labeled with radioiosotopes are used in the diagnosis and therapy of somatostatin receptor (SSTR)-positive tumors. A method has been devised to synthesize N-(4-guanidinomethyl-3-iodobenzoyl)-Phe1-octreotate (GMIBO). Receptor binding assay and scatchard analysis yielded a K d of 4.83 ± 0.19 nM for this peptide. Derivatives of this peptide labeled with radioiodine ([*I]GMIBO) and the α-particle-emitting radiohalogen 211At {N-(3-[211At]astato-4-guanidinomethylbenzoyl)-Phe1-octreotate; [211At]AGMBO} were prepared in a single step from a tin precursor in radiochemical yields of 30–35% and 15–20%, respectively. Paired-label internalization assays performed with the SSTR-positive D341 Med human medulloblastoma cell line demonstrated that [125I]GMIBO and [211At]AGMBO were specifically internalized 20–40% more than N α-(1-deoxy-d-fructosyl)-[131I]I-Tyr3-octreotate ([131I]I-Glu-TOCA), the radioiodinated octreotide derivative previously shown to exhibit maximum internalization in this cell line. Uptake of [131I]GMIBO in D341 Med subcutaneous xenografts in a murine model (8.34 ± 1.82 versus 8.10 ± 2.23% ID/g at 1 h) and SSTR-expressing normal tissues was comparable to that of [125I]I-Glu-TOCA and was shown to be specific. However, the uptake of [131I]GMIBO also was substantially higher in liver (16.9 ± 3.15 versus 1.39 ± 0.45% ID/g at 1 h) and in kidneys (44.33 ± 6.47 versus 3.44 ± 0.68% ID/g at 1 h) compared to that of [125I]I-Glu-TOCA. These data suggest that these novel peptide conjugates retain their specificity for SSTR both in vitro and in vivo; however, because of their higher accumulation in normal tissues they would be best applied in settings amenable to loco-regional administration such as medulloblastoma neoplastic meningitis.
Keywords: SSTR (somatostatin receptor); Octreotide; Octreotate; Glu-TOCA (N α-(1-deoxy-d-fructosyl)-Tyr3-octreotate); Medulloblastoma; Internalization; Externalization; Radiotherapy; Astatine-211;

Assessing activation of the human neuropeptide FF2 receptor with a non-radioactive GTP binding assay by Mia Engström; Ale Närvänen; Juha-Matti Savola; Siegfried Wurster (2099-2104).
We have evaluated a novel, time-resolved fluorometric GTP binding assay for its suitability for functional screening of neuropeptide FF (NPFF) receptor ligands. Our results suggest that this assay, which relies on the use of a europium-labeled GTP analogue, Eu-GTP, provides a powerful alternative to the [35S]guanosine-5′-O-(3-thio)triphosphate binding assay for assessing the functional properties of NPFF analogs. Further, we demonstrate that the tetrapeptide PMRF-NH2 exhibited high agonist potency at the NPFF2 receptor, and that the efficacies of this peptide and another shortened NPFF analog were greater than that of NPFF.
Keywords: Neuropeptide FF; Eu-GTP binding assay; [35S]GTPγS binding assay; NPFF2 receptor; Neuropeptides; Structure–activity relationship;

Peptidergic and non-peptidergic innervation and vasomotor responses of human lenticulostriate and posterior cerebral arteries by Inger Jansen-Olesen; Sergio Gulbenkian; Ulla Engel; Manuel Cunha e Sá; Lars Edvinsson (2105-2114).
The aim of the present study was to compare in man the innervation pattern and the functional responses to neuronal messengers in medium sized lenticulostriate and branches of the posterior cerebral arteries (PCA). The majority of the nerve fibers found were sympathetic and displayed specific immunoreactivity for tyrosine hydroxylase (TH) and neuropeptide Y (NPY). Only few nerve fibers displayed vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity. In both arteries, the contractions induced by noradrenaline (NA), NPY and 5-hydroxytryptamine (5-HT) and the relaxant responses induced by acetylcholine (ACh), VIP and pituitary adenylate cyclase activating peptide-27 (PACAP) as well as CGRP and SP were compared in vitro. In conclusion, there was no major difference in innervation pattern or vasomotor sensitivity (pEC50 and pIC50 values) between the two vessels. However, the general pattern indicates stronger vasomotor responses (E max and I max) in the PCA branches as compared to the lenticulostriate arteries which may lend support for the clinical observation of a difference in stroke expression between the two vascular areas.
Keywords: Cerebrovascular; Parasympathetic; Sympathetic; Sensory; PACAP; CGRP; VIP; NPY; SP;

Secretoneurin in the human aqueous humor and the absence of an effect of frequently used eye drops on the levels by Katrin Stemberger; Julia Pallhuber; Alfred Doblinger; Josef Troger; Rudolf Kirchmair; Martina Kralinger; Reiner Fischer-Colbrie; Gerhard Kieselbach (2115-2118).
Secretoneurin (SN) was detected in the aqueous humor (AH) of patients treated topically with tobramycine eye drops alone or tobramycine and cyclosporine A, tobramycine and diclofenac or tobramycine and rimexolone. The levels of the peptide were found to be higher in the uninflamed human than in the rabbit aqueous humor which may be the result of species differences and/or age-related circumstances. Furthermore, they are approximately one hundred times higher than those of classical neuropeptides indicating release from nerve fibers and/or secretion from non-pigmented ciliary epithelium cells. Despite a slight tendency by rimexolone to decrease the levels, there was no significant effect seen for either of the drops. It must be considered that aminoglycosides are known to have toxic side effects and that they can influence the levels of SN which may be not diminished by low topical doses of corticosteroids or nonsteroidal antiinflammatory drugs. The high levels of the peptide are of relevance and may indicate a significant role of secretoneurin in the anterior segment of the eye. This should encourage performing functional studies.

GSK3 involvement in amylin signaling in isolated rat soleus muscle by Tatjana Abaffy; Garth J.S. Cooper (2119-2125).
Amylin can evoke insulin resistance by antagonizing insulin in a non-competitive manner. Here, we investigated the glycogenolytic effect of amylin in isolated skeletal muscle and compared it to the effects of a calcitonin gene-related peptide (CGRP). Amylin alone had no statistically significant effect on glucose transport. However, amylin decreased insulin-stimulated glucose transport by about 30%. The involvement of cAMP could not be detected at the concentrations shown to promote glycogenolysis. Previously, it has been shown that increased glycogen synthase kinase 3 (GSK3) activity plays a role in insulin resistance. Here, the ratio of GSK3 α:β isoforms in rat soleus was found to be 1.2:1. We found that amylin increased GSK3α activity, which in turn led to increased phosphorylation of glycogen synthase and decreased glycogen synthesis de novo.
Keywords: Glycogen; cAMP; 2-deoxy-glucose; CGRP;

The F1-ATPase β-subunit is the putative enterostatin receptor by MieJung Park; Ling Lin; Sonyja Thomas; Hugh D. Braymer; Pamela M. Smith; David H.T. Harrison; David A. York (2127-2133).
It has been suggested that the F1-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin1–7, in the absence and presence of cold enterostatin. 125I-β-casomorphin1–7 weakly binds to the rat F1-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin1–7 binding to the F1-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F1-ATPase complex. Western blot analysis showed the F1-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F1-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin1–7 bind to distinct sites on the protein.
Keywords: Enterostatin; β-Casomorphin; F1-ATPase β-subunit; Food intake; Enterostatin receptor;

The anorectic effect of neurotensin is mediated via a histamine H1 receptor in mice by Kousaku Ohinata; Tomoko Shimano; Rena Yamauchi; Shinobu Sakurada; Kazuhiko Yanai; Masaaki Yoshikawa (2135-2138).
Neurotensin (NT), a tridecapeptide found in the mammalian brain and peripheral tissues, induces a decrease in food intake after central administration. In this investigation, we examine whether the histaminergic system is involved in NT-induced suppression of feeding. Intracerebroventricular injection of NT (0.1–1 nmol/mouse) led to dose-dependent inhibition of food intake in fasted ddY mice. The anorectic effect induced by NT (0.1 nmol/mouse) was ameliorated upon co-administration of pyrilamine (3 nmol/mouse), an antagonist for histomine H1 receptor. The NT-induced anorectic effect was partially ameliorated in H1 knockout mice. The findings suggest that the H1 receptor in part mediates the NT-induced suppression of food intake.
Keywords: Neurotensin; Histamine; H1 receptor; Food intake; Leptin;

Guide cannula were implanted in rats aimed at the paraventricular nucleus (PVN) of the hypothalamus for microinjection of neuropeptide Y (NPY), D-NPY27–36, or vehicle. In the Wistar rat, there was no significant effect on the consumption of ethanol. In Myers’ high ethanol preferring (mHEP) rats, D-NPY27–36 caused a significant 54% decrease in ethanol consumption from baseline, but the response was not different from vehicle. NPY-induced feeding in satiated Wistar rats, was blocked by a Y1 receptor antagonist, D-NPY27–36. D-NPY27–36 decreased 78% feeding in food-deprived rats. Thus, neither the Wistar nor the mHEP rat perceives ethanol as a source of calories comparable to food.
Keywords: NPY or neuropeptide Y; Caloric intake; Drinking behavior; PVN or paraventricular nucleus;

Changes of hypothalamic α-MSH and CART peptide expression in diet-induced obese rats by De-Run Tian; Xiao-Dong Li; Yu-Shun Shi; You Wan; Xiao-Min Wang; Jaw-Kang Chang; Jun Yang; Ji-Sheng Han (2147-2153).
Two hypothalamic peptides, cocaine and amphetamine-regulated transcript (CART) and α-melanocyte-stimulating hormone (α-MSH), recognized as anorexigenic neuropeptides to suppress the feeding behavior, were monitored in rats fed with a high-fat (HIF) diet for 14 weeks. While half of the rats developed obesity (diet-induced obese, DIO), some did not (diet resistant, DR). Compared to the DR rats and the control rats (fed with standard chow), DIO rats were accompanied by a markedly higher energy intake and a decrease in the number of neurons carrying α-MSH and CART peptide in the arcuate nucleus of the hypothalamus. Failure of hypothalamic anorexigenic peptides CART and α-MSH to increase their content in response to HIF diet may play a key role for overly high energy consumption, resulting in obesity.
Keywords: Cocaine and amphetamine-regulated transcript peptide; α-Melanocyte-stimulating hormone; Diet-induced obesity; High-fat diet; Hypothalamus;

Phenylpropanolamine (PPA) is an appetite suppressant. The mechanism for the anorectic effect of PPA has been attributed to its action on the site of hypothalamic paraventriculum. Neuropeptide Y (NPY) is an appetite stimulant that is widely distributed in the site of hypothalamus. It is not clear whether hypothalamic NPY is involved in the anorectic action of PPA. This study was aimed to investigate the mechanism underlying the involvement of NPY gene in the anorectic action of PPA. Results revealed that PPA treatment in rats could decrease both NPY content and mRNA level in the hypothalamus. In addition, the expression of NPY immunoreactivity following PPA treatment was decreased in areas of hypothalamic arcuate nucleus, paraventricular nucleus and periventricular area using immunohistochemical staining, suggesting an involvement of NPYergic pathway in the action of PPA anorexia. Our results provided immunohistochemical and genomic evidence to suggest that PPA might reduce feeding by altering NPY gene expression.
Keywords: Anorectic agent; Orexigenic peptide; Food intake; Metabolism; Immunohistochemistry; RT-PCR; Hypothalamus;

Effects of hexarelin against acid-independent and acid-dependent ulcerogens in the rat by V. Sibilia; A. Torsello; F. Pagani; D. Rapetti; N. Lattuada; V. Locatelli; I. Bulgarelli; F. Guidobono; C. Netti (2163-2170).
The effects of intracerebroventricular (icv) or subcutaneous (sc) hexarelin (Hexa) administration, against gastric ulcers induced by ethanol (50%, 1 ml/rat/os) or Indomethacin (20 mg/kg/os) were examined in conscious rats. Hexa at 1 nmol/rat, icv or 10 nmol/kg, sc reduced ethanol-induced ulcers by 47% and 32% respectively. Hexa, but not ghrelin significantly worsened (+40%) Indomethacin-induced ulcers when injected sc. Hexa-gastroprotection against ethanol-induced ulcers was removed by the GHS-R antagonist (d-Lys3)-GRPR-6 and by the inhibitor of NO-synthase (NOS) N ω-nitro-l-arginine methyl ester. Semiquantitative RT-PCR assay of gastric NOS mRNA isoforms revealed that the reduction in iNOS-derived NO and the increase of constitutive-derived NO are relevant for the gastroprotection of Hexa against ethanol-induced gastric damage.
Keywords: Gastric ulcer; Hexarelin; Ghrelin; GHS-R antagonist; Nitric oxide synthase;

Ghrelin is a peptide produced by the stomach and released into the circulation. As a natural ligand of the growth hormone secretagogue (GHS) receptor, it stimulates growth hormone secretion but it also stimulates feeding in humans and rodents. The orexigenic effect of ghrelin has been related to AgRP/NPY and orexin pathways. We proposed that ghrelin might be involved in the susceptibility to diet induced obesity and in the regulation of macronutrient selection. We have investigated these hypotheses in two strains of rat, the Osborne–Mendel (OM) rat that prefers diets high in fat and is sensitive to dietary obesity and the S5B/P1 (S5B) rat that prefers a low fat diet and is resistant to high fat diet induced obesity.OM and S5B rats were adapted to a choice of high fat (HF) and low fat (LF) diet for 2 weeks. GHRP-2, an analogue of ghrelin, was injected intraperitoneally into satiated and 24 h fasted rats at doses of 10, 30 and 90 nmol. Food intake was measured over the next 4 h period. In satiated S5B rats, GHRP-2 stimulated intake of the LF diet in a dose dependent manner but did not affect the intake of the HF diet. In satiated OM rats, 90 nmol of GHRP-2 stimulated HF intake. In contrast, neither fasted OM nor S5B rats increased the intake of either HF or LF diet in response to GHRP-2. Fasting for 18 h induced a large rise in ghrelin mRNA in stomach of OM rats but not in S5B rats. There were no significant differences in plasma total ghrelin. An increase in ghrelin mRNA in stomach immediately before the onset of the dark cycle was observed in OM but not in S5B rats. Active ghrelin level was significantly affected by different feeding conditions in both OM and S5B rats adapted on HF diet with a trend to increase after 48 h of fasting and to decline to basal levels following 10 h of refeeding. These data suggest that ghrelin stimulates the intake of the preferred macronutrient. In addition, a differential regulation of ghrelin gene expression between OM and S5B rats may be important in their differential sensitivity to HF diet-induced obesity.
Keywords: Diet-induced obesity; Ghrelin; Macronutrient selection; Osborne–Mendel rats; S5B/P1 rats;

Ghrelin inhibits FGF-2-mediated angiogenesis in vitro and in vivo by Maria Teresa Conconi; Beatrice Nico; Diego Guidolin; Silvia Baiguera; Raffaella Spinazzi; Piera Rebuffat; Ludwik K. Malendowicz; Angelo Vacca; Gianni Carraro; Pier Paolo Parnigotto; Gastone G. Nussdorfer; Domenico Ribatti (2179-2185).
Recent evidence indicates that ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), is highly expressed in the cardiovascular system, and in this study we addressed the possibility that ghrelin may affect angiogenesis in vitro and in vivo. Reverse transcription-polymerase chain reaction showed that human umbilical vein endothelial cells (HUVECs) express ghrelin and GHS-R mRNAs. Ghrelin inhibited FGF-2-induced proliferation of HUVECs cultured in vitro, the maximal effective concentration being 10−8  M, and this effect was annulled by the GHS-R antagonist d-Lys3-growth hormone releasing peptide-6. FGF-2 stimulated HUVEC cultured on Matrigel to form capillary-like structures, and ghrelin (10−8  M) suppressed this effect. In the chick embryo chorioallantoic membrane in vivo assay, FGF-2 induced a strong angiogenic response, which was counteracted by ghrelin (500 ng). Taken together, these findings suggest that ghrelin acts as an angiostatic molecule and indicate that its activity is comparable to that of a well-known angiostatic agent, i.e., vinblastine. The antiangiogenic activity of ghrelin deserves further investigations, alone or together with other antiangiogenic agents, for the treatment of pathological conditions characterized by enhanced angiogenesis.
Keywords: Angiogenesis; Antiangiogenesis; FGF-2; Ghrelin; Vinblastine;

Leptin fluctuates in intestinal ischemia-reperfusion injury as inflammatory cytokine by Ji Lin; Guang-Tao Yan; Lu-Huan Wang; Xiu-Hua Hao; Kai Zhang; Hui Xue (2187-2193).
As leptin is an active mediator mainly secreted by adipose tissue and is closely related with energy metabolism, we evaluate both the changes of leptin levels in serum and adipose tissue with a concise radioimmunoassay and the changes of leptin mRNA expression in adipose tissue with RT-PCR, during the severe metabolic impediment in rat intestinal ischemia-reperfusion (I/R) injury. Results show that not only leptin levels in serum and adipose tissue but also its mRNA expression in adipose tissue undergo a fluctuation according to different injury times. Therefore, we conclude that leptin has a time-dependent response to acute inflammatory stimuli and acts as an anti-inflammatory cytokine.
Keywords: Leptin; Ischemia-reperfusion, intestinal; Inflammation, acute; Adipose; Cytokine; Radioimmunoassay;

To determine if insulin-like growth factor (IGF)-1 and -2, FSH, or leptin alter IGF-binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and (or) theca cells, granulosa and theca cells were collected from bovine ovarian follicles, plated for 48 h in 10% FCS and then treated for 24 h in serum-free medium containing various hormone treatments arranged in three different experiments. Amounts of IGFBP-2, -3, -4, and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. Neither 100 ng/ml of IGF-1 nor IGF-2 had an effect (P > 0.10) on IGFBP-2, -3, -4, or -5 mRNA levels in small-follicle (1–5 mm; Experiment 1) granulosa cells. In large-follicle (>7.9 mm; Experiment 2) granulosa cells, 100 ng/ml of IGF-1 increased (P < 0.05) IGFBP-2 mRNA levels above controls and 3 ng/ml of IGF-1; 100 ng/ml of IGF-1 also decreased (P < 0.10) IGFBP-5 mRNA levels compared to 3 ng/ml of IGF-1 or FSH or 100 ng/ml leptin, while 100 ng/ml of IGF-2 had no effect (P > 0.10) on IGFBP-2, -3, -4, and -5 mRNA levels (Experiment 2). At the doses tested, leptin and FSH had no effect (P > 0.10) on IGFBP-2, -3, -4, and -5 mRNA levels in large-follicle granulosa cells. In theca cells, IGF-2 decreased (P < 0.05) IGFBP-2 mRNA levels, but had no effect on IGFBP-3 or -4 mRNA expression (Exp. 3); IGF-1 did not affect (P > 0.10) thecal IGFBP-2, -3 or -4 mRNA levels. In contrast, IGF-1 but not IGF-2 increased (P < 0.01) thecal IGFBP-5 mRNA levels. Ligand blotting revealed that both IGF-1 and -2 increased IGFBP-2 and -5 (protein) and had no effect on IGFBP-3 (protein), whereas IGF-1 (but not IGF-2) increased IGFBP-4 (protein), suggesting IGFBP-2, -4, and -5 are post-transcriptionally regulated. These results suggest that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by IGF-1 and -2, therefore discretely modulating the amount of bio-available IGFs to these cells depending upon the specific hormonal milieu.
Keywords: Insulin-like growth factor; Granulosa cell; Theca cell; Ovary; Follicle;

Endogenous opiates and behavior: 2003 by Richard J. Bodnar; Gad E. Klein (2205-2256).
This paper is the 26th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over a quarter-century of research. It summarizes papers published during 2003 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular–biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17).
Keywords: Endogenous opiates; Opioid receptors; Opioid agonists;

Effects of peptides, with emphasis on feeding, pain, and behavior by Yongmei Yu; Ali Jawa; Weihong Pan; Abba J. Kastin (2257-2289).
Novel effects of naturally occurring peptides are continuing to be discovered, and their mechanisms of actions as well as interactions with other substances, organs, and systems have been elucidated. Synthetic analogs may have actions similar or antagonistic to the endogenous peptides, and both the native peptides and analogs have potential as drugs or drug targets. The journal Peptides publishes many leading articles on the structure–activity relationship of peptides as well as outstanding reviews on some families of peptides. Complementary to the reviews, here we extract information from the original papers published during the past five years in Peptides (1999–2003) to summarize the effects of different classes of peptides, their modulation by other chemicals and various pathophysiological states, and the mechanisms by which the effects are exerted. Special attention is given to peptides related to feeding, pain, and other behaviors. By presenting in condensed form the effects of peptides which are essential for systems biology, we hope that this summary of existing knowledge will encourage additional novel research to be presented in Peptides.
Keywords: Peptides; Adrenomedullin; Amylin; Angiotensin; Bombesin; Bradykinin; Cholecystokinin; Opioid peptides; Ingestive peptides; Neurotrophic peptides; Feeding; Pain; Memory; Addiction; Locomotor activity; Electrolyte balance; CNS; Blood–brain barrier;