The hypocretins are weak agonists at recombinant human orexin-1 and orexin-2 receptors.

The pharmacology of the orexin-like peptides, hypocretin-1 and hypocretin-2, was studied in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX(1)) or orexin-2 (OX(2)) receptors by measuring intracellular calcium ([Ca(2+)](i)) using Fluo-3AM. Orexin-A and orexin-B increased [Ca(2+)](i) in CHO-OX(1) (pEC(50)=7. 99+/-0.05 and 7.00+/-0.10 respectively, n=8) and CHO-OX(2) (pEC(50)=8.30+/-0.05 and 8.21+/-0.07 respectively, n=5). However, hypocretin-1 and hypocretin-2 were markedly less potent, with pEC(50) values of 5.31+/-0.04 and 5.41+/-0.04 respectively in CHO-OX(2) cells (n=5). In CHO-OX(1) cells 10 microM hypocretin-1 only elicited a 37.5+/-3.4% response whilst 10 microM hypocretin-2 elicited a 18.0+/-2.1% response (n=8). Desensitisation of OX(1) or OX(2) with orexin-A (100 nM) abolished the response to orexin-A (10 nM) and the hypocretins (10 microM), but not to UTP (3 microM). In conclusion, the hypocretins are only weak agonists at the orexin receptors.

Nucleotide and amino acid sequences of the metallo-beta-lactamase, ImiS, from Aeromonas veronii bv. sobria.

The Aeromonas veronii bv. sobria metallo-beta-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-beta-lactamases.

Interactions between leptin and hypothalamic neuropeptide Y neurons in the control of food intake and energy homeostasis in the rat.

Leptin acts on the brain to inhibit feeding, increase thermogenesis, and decrease body weight. Neuropeptide Y (NPY)-ergic neurons of the hypothalamic arcuate nucleus (ARC) that project to the paraventricular nuclei (PVN) and dorsomedial nuclei (DMH) are postulated to control energy balance by stimulating feeding and inhibiting thermogenesis, especially under conditions of energy deficit. We investigated whether leptin's short-term effects on energy balance are mediated by inhibition of the NPY neurons. Recombinant murine leptin (11 microg) injected into the lateral ventricle of fasted adult Wistar rats inhibited food intake by 20-25% between 2 and 6 h after administration, compared with saline-treated controls (P < 0.05). Uncoupling protein mRNA levels in brown adipose tissue (BAT) rose by 70% (P < 0.01). Leptin treatment significantly reduced NPY concentrations by 20-50% (P < 0.05) in the ARC, PVN, and DMH and significantly decreased hypothalamic NPY mRNA levels (0.61 +/- 0.02 vs. 0.78 +/- 0.03 arbitrary units; P < 0.01). A second study examined changes in leptin during 5 days' intracerebroventricular NPY administration (10 microg/day), which induced sustained hyperphagia and excessive weight gain. In NPY-treated rats, leptin mRNA levels in epididymal fat were comparable to those in saline-treated controls (0.94 +/- 0.17 vs. 1.0 +/- 0.28 arbitrary units; P > 0.1), but plasma leptin levels were significantly higher (4.88 +/- 0.66 vs. 2.85 +/- 0.20 ng/ml; P < 0.01). Leptin therefore acts centrally to decrease NPY synthesis and NPY levels in the ARC-PVN projection; reduced NPY release in the PVN may mediate leptin's hypophagic and thermogenic actions. Conversely, NPY-induced obesity results in raised circulating leptin concentrations. Leptin and the NPY-ergic ARC-PVN neurons may interact in a homeostatic loop to regulate body fat mass and energy balance.

Conformation of the cytoplasmic domain of phospholamban by NMR and CD.

Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have been used to characterize the conformation of the putative cytoplasmic domain of phospholamban (PLB), an oligomeric membrane-bound protein which regulates the activity of the cardiac sarcoplasmic reticulum Ca(2+)-dependent ATPase. In aqueous solution the 25-residue peptide adopts a number of rapidly interconverting conformers with no secondary structural type obviously predominating. However, in trifluoroethanol (TFE) the conformation, while still highly dynamic, is characterized by a high proportion of helical structures. Evidence for this is provided by alpha CH chemical shifts and low NH chemical shift temperature coefficients, small NH-alpha CH intraresidue scalar coupling constants, a substantial number of distinctive interresidue nuclear Overhauser effects (NOEs) [dNN(i, i + 1), d alpha N(i, i + 3), d alpha beta(i, i + 3) and d alpha N(i, i + 4)] and characteristic CD bands at 190 (positive), 206 (negative) and 222 nm (negative). The helicity is interrupted around Pro-21. The activity of PLB is regulated by phosphorylation at either Ser-16 or Thr-17. CD shows that phosphorylation at Ser-16 by the cAMP-activated protein kinase causes about an 11% decrease in alpha-helical content in TFE.

Electrophoretic techniques for the analysis of synthetic amyloid beta-A4-related peptides.

Two electrophoretic techniques have been developed for the analysis of synthetic beta-A4 related peptides. The first is an acetic acid--urea--polyacrylamide gel electrophoresis denaturing system while the second employs capillary zone electrophoresis at pH 7. Both methods were calibrated to identify the state (monomeric or aggregate) of the longer amyloid fragments beta-A4(1-40) and beta-A4(1-43) during analysis.