Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity

A conformationally biased decapeptide agonist of human C5a (C5a(65-74)Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a(12-20)-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.

The glycine receptor

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. The GlyR comprises a pentameric complex that forms a chloride-selective transmembrane channel, which is predominantly expressed in the spinal cord and brain stem. We review the pharmacological and physiological properties of the GlyR and relate this information to more recent insights that have been obtained through the cloning and recombinant expression of the GlyR subunits. We also discuss insights into our understanding of GlyR structure and function that have been obtained by the genetic characterisation of various heritable disorders of glycinergic neurotransmission. (C) 1997 Elsevier Science Inc.

Presence of novel N-CAM glycoforms in the rat olfactory system

The functional activity of the neural cell adhesion molecule N-CAM can be modulated by posttranslational modifications such as glycosylation. For instance, the long polysialic acid side chains of N-CAM alter the adhesion properties of the protein backbone. In the present study, we identified two novel carbohydrates present on N-CAM, NOC-3 and NOC-4. Both carbohydrates were detected on N-CAM glycoforms expressed by subpopulations of primary sensory olfactory neurons in the rat olfactory system. Based on the expression of NOC-3 and NOC-4 and the olfactory marker protein (OMP), four independent subpopulations of primary sensory olfactory neurons were characterized. These neurons expressed: both NOC-3 and NOC-4 but not OMP; both NOC-4 and OMP but not NOC-3; NOC-3, NOC-4, and OMP together; and OMP alone. The NOC-3- and NOC-4-expressing neurons were widely dispersed in the olfactory neuroepithelium lining the nasal cavity. The axons of NOC-4 expressing neurons innervated all glomeruli in the olfactory bulb, whereas the NOC-3 expressing axons terminated in a discrete subset of glomeruli scattered throughout the whole olfactory bulb. We propose that both NOC-3 and NOC-4 are part of a chemical code of olfactory neurons which is used in establishing the topography of connections between the olfactory neuroepithelium and the olfactory bulb. (C) 1997 John Wiley & Sons, Inc.

Concentration dependence of sodium permeation and sodium ion interactions in the cyclic AMP-gated channels of mammalian olfactory receptor neurons

The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 mu M) of adenosine 3', 5'-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mM. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to O mV. When the external NaCl concentration was maintained at 150 mM and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na+ equilibrium potential indicating that P-Cl/P-Na approximate to 0. However, at low external NaCl concentrations (less than or equal to 100 mM) there was some significant chloride permeability. Our results further indicated that Na+ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K(m)s in the range of 100-150 mM and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels.

Infrared seeded parametric four-wave mixing for sensitive detection of molecules

We have developed a sensitive resonant four-wave mixing technique based on two-photon parametric four-wave mixing with the addition of a phase matched ''seeder'' field. Generation of the seeder field via the same four-wave mixing process in a high pressure cell enables automatic phase matching to be achieved in a low pressure sample cell. This arrangement facilitates sensitive detection of complex molecular spectra by simply tuning the pump laser. We demonstrate the technique with the detection of nitric oxide down to concentrations more than 4 orders of magnitude below the capability of parametric four-wave mixing alone, with an estimated detection threshold of 10(12) molecules/cm(3).

Potential GABAB receptor antagonists .9. The synthesis of 3-amino-3-(4-chlorophenyl)propanoic acid, 2-amino-2-(4-chlorophenyl)ethylphosphonic acid and 2-amino-2-(4-chlorophenyl)ethanesulfonic acid

This paper describes the synthesis of 3-amino-3-(4-chlorophenyl)propanoic acid and the corresponding phosphonic and sulfonic acids, lower homologues of baclofen, phaclofen and saclofen respectively. The chlorinated acids were all weak specific antagonists of GABA at the GABAB receptor, with the sulfonic acid (pA(2) 4.0) being stronger than the phosphonic acid (pA(2) 3.8) and carboxylic acid (pA(2) 3.5).

Crystal structure at 1.1 angstrom resolution of alpha-conotoxin PnIB: Comparison with alpha-conotoxins PnIA and GI

Conotoxins are small, cysteine-rich peptides isolated from the venom of Conus spp. of predatory marine snails, which selectively target specific receptors and ion channels critical to the functioning of the neuromuscular system. alpha-Conotoxins PnIA and PnIB are both 16-residue peptides (differing in sequence at only two positions) isolated from the molluscivorous snail Conus pennaceus. In contrast to the muscle-selective alpha-conotoxin GI from Conus geographus, PnIA and PnIB block the neuronal nicotinic acetylcholine receptor (nAChR). Here, we describe the crystal structure of PnIB, solved at a resolution of 1.1 Angstrom and phased using the Shake-and-Bake direct methods program. PnIB crystals are orthorhombic and belong to the space group P2(1)2(1)2(1) with the following unit cell dimensions: a = 14.6 Angstrom, b = 26.1 Angstrom, and c = 29.2 Angstrom. The final refined structure of alpha-conotoxin PnIB includes all 16 residues plus 23 solvent molecules and has an overall R-factor of 14.7% (R-free of 15.9%). The crystal structures of the alpha-conotoxins PnIB and PnIA are solved from different crystal forms, with different solvent contents. Comparison of the structures reveals them to be very similar, showing that the unique backbone and disulfide architecture is not strongly influenced by crystal lattice constraints or solvent interactions. This finding supports the notion that this structural scaffold is a rigid support for the presentation of important functional groups. The structures of PnIB and PnIA differ in their shape and surface charge distribution from that of GI.

Generation and phenotypic characterization of new human ovarian cancer cell lines with the identification of antigens potentially recognizable by HLA-restricted cytotoxic T cells

This study describes a simple method for long-term establishment of human ovarian tumor lines and prediction of T-cell epitopes that could be potentially useful in the generation of tumor-specific cytotoxic T lymphocytes (CTLs), Nine ovarian tumor lines (INT.Ov) were generated from solid primary or metastatic tumors as well as from ascitic fluid, Notably all lines expressed HLA class I, intercellular adhesion molecule-1 (ICAM-1), polymorphic epithelial mucin (PEM) and cytokeratin (CK), but not HLA class II, B7.1 (CD80) or BAGE, While of the 9 lines tested 4 (INT.Ov1, 2, 5 and 6) expressed the folate receptor (FR-alpha) and 6 (INT.Ov1, 2, 5, 6, 7 and 9) expressed the epidermal growth factor receptor (EGFR); MAGE-1 and p185(HER-2/neu) were only found in 2 lines (INT.Ov1 and 2) and GAGE-1 expression in 1 line (INT.Ov2). The identification of class I MHC ligands and T-cell epitopes within protein antigens was achieved by applying several theoretical methods including: 1) similarity or homology searches to MHCPEP; 2) BIMAS and 3) artificial neural network-based predictions of proteins MACE, GAGE, EGFR, p185(HER-2/neu) and FR-alpha expressed in INT.Ov lines, Because of the high frequency of expression of some of these proteins in ovarian cancer and the ability to determine HLA binding peptides efficiently, it is expected that after appropriate screening, a large cohort of ovarian cancer patients may become candidates to receive peptide based vaccines. (C) 1997 Wiley-Liss, Inc.

Non-expression of insulin-like growth factor-I receptor is associated with apoptosis: an ultrastructural study on rat ameloblasts

Insulin-like growth factor-I (IGF-I) is a preiotrophic polypeptide which appears to have roles both as a circulating endocrine hormone and as a locally synthesized paracrine or autocrine tissue factor. IGF-I plays a major role in regulating the growth of cells in vivo and in vitro and initiates metabolic and mitogenic processes in a wide variety of cell types by binding to specific type I receptors in the plasma membrane, In this study, we report the distribution of IGF-I receptors in odontogenic cells at the ultrastructural level using the high resolution protein A-gold technique, In the pre-secretory stage, very little gold label was visible over the ameloblasts and odontoblasts, During the secretory stage the label was mostly seen in association with the cell membranes and endoplasmic reticulum of the ameloblasts. Lysosome-like elements in the post-secretory stage were labelled as well as multivesicular dense bodies, Very little labelling was encountered in the ameloblasts in the transitional stage, where apoptotic bodies were clearly visible, The maturation stage also exhibited labelling of the secretory-like granules in the distal surface. The presence of gold particles over the plasma membrane is an indication that IGF-I receptor is a membrane-bound receptor. Furthermore, the intracellular distribution of the label over the endoplasmic reticulum supports the local synthesis of the IGF-I receptor. The absence of labelling over the transitional ameloblasts suggests that the transitional stage may require the non-expression of IGF-I as a prerequiste or even a trigger for apoptosis.

The intrinsic antinociceptive effects of oxycodone appear to be kappa-opioid receptor mediated

Our previous studies in the Sprague-Dawley rat showed that the intrinsic antinociceptive effects of oxycodone are naloxone reversible in a manner analogous to morphine but that in contrast to morphine, oxycodone's antinociceptive effects have a rapid onset of maximum effect (approximate to 5-7 min compared to 30-45 min for morphine), comprise one antinociceptive phase (compared to two phases) and are of relatively short duration (approximate to 90 min compared to approximate to 180 min). In the present study, administration of a range of selective opioid receptor antagonists has shown that the intrinsic antinociceptive effects of oxycodone (171 nmol) are not attenuated by i.c.v. administration of (i) naloxonazine, a mu(1)-selective opioid receptor antagonist, or (ii) naltrindole, a delta-selective opioid receptor antagonist, in doses that completely attenuated the intrinsic antinociceptive effects of equipotent doses of the respective mu- and delta-opioid agonists, morphine and enkephalin-[D-Pen(2,5)] (DPDPE). Although beta-funaltrexamine (beta-FNA) attenuated the antinociceptive effects of oxycodone (171 nmol i.c.v.), it also attenuated the antinociceptive effects of morphine and bremazocine (kappa-opioid agonist) indicative of non-selective antagonism. Importantly, the antinociceptive effects of oxycodone (171 nmol i.c.v.) were markedly attenuated by the prior i.c.v. administration of the selective kappa-opioid receptor antagonist, norbinaltorphimine (nor-BNI), in a dose (0.3 nmol) that did not attenuate the antinociceptive effects of an equipotent dose of i.c.v. morphine (78 nmol). Taken together, these data strongly suggest that the intrinsic antinociceptive effects of oxycodone are mediated by K-opioid receptors, in contrast to morphine which interacts primarily with mu-opioid receptors. (C) 1997 International Association for the Study of Pain. Published by Elsevier Science B.V.

Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB), The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology, The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis, In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

Costimulatory molecules in human periodontal disease tissue

An immunoperoxidase technique was used to examine CD28, CD152, CD80 and CD86 positive cells in gingival biopsies from 21 healthy/gingivitis and 26 periodontitis subjects. The samples were placed into 3 groups (small, intermediate, large) according to the size of the infiltrate. The percent CD28+ T cells in the connective tissue infiltrates was highly variable with no differences between the healthy/gingivitis and periodontitis groups. While there was an increase in positive cells in intermediate infiltrates from both healthy/gingivitis (28.5%) and periodontitis (21.4%) patients compared with small infiltrates (8.6% and 11.8%, respectively), this was not significant, although the percent CD28+ T cells did increase significantly in tissues with increased proportions of B cells relative to T cells (p=0.047). A mean of less than 5% infiltrating T cells were CD152+ which was significantly lower than the mean percent CD28+ T cells in intermediate healthy/gingivitis lesions (p=0.021). The mean percent CD80+ and CD86+ B cells and macrophages was 1–7% and 8–16%, respectively, the difference being significant in intermediate healthy/gingivitis tissues (p=0.012). Analysis of these cells in relation to increasing numbers of B cells in proportion to T cells and also to macrophages, suggested that CD80 was expressed predominantly by macrophages while CD86 was expressed by both macrophages and B cells. Few endothelial cells expressed CD80 or CD86. Keratinocytes displayed cytoplasmic staining of CD80 rather than CD86 although the numbers of positive specimens in the healthy/gingivitis and periodontitis groups reduced with increasing inflammation. In conclusion, percentages of CD28, CD152, CD80 and CD86 did not reflect differences in clinical status. However, the percent CD28+ T cells increased with increasing size of infiltrate and with increasing proportions of B cells suggesting increased T/B cell interactions with increasing inflammation. The percent CD152+ cells remained low indicating that CD152 may not be involved in negative regulation of T cells in periodontal disease. CD80 and CD86 have been reported to promote Th1 and Th2 responses, respectively, and the higher percent CD86+ cells suggests a predominance of Th2 responses in both healthy/gingivitis and periodontitis tissues. Nevertheless, other factors including cytokines themselves and chemokines which modulate T cell cytokine profiles must be monitored to determine the nature of Th1/Th2 responses in periodontal disease.