Structure-activity relationships of alpha-conotoxins targeting neuronal nicotinic acetylcholine receptors

alpha-Conotoxins that target the neuronal nicotinic acetylcholine receptor have a range of potential therapeutic applications and are valuable probes for examining receptor subtype selectivity. The three-dimensional structures of about half of the known neuronal specific alpha-conotoxins have now been determined and have a consensus fold containing a helical region braced by two conserved disulfide bonds. These disulfide bonds define the two-loop framework characteristic for alpha-conotoxins, CCXmCXnC, where loop 1 comprises four residues (m = 4) and loop 2 between three and seven residues (n = 3, 6 or 7). Structural studies, particularly using NMR spectroscopy have provided an insight into the role and spatial location of residues implicated in receptor binding and biological activity.

Subtype-selective noncompetitive or competitive inhibition of human alpha(1)-adrenergic receptors by rho-TIA

The 19-amino acid conopeptide (rho-TIA) was shown previously to antagonize noncompetitively alpha(1B)-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human alpha(1)-AR subtypes (alpha(1A), alpha(1B), and alpha(1D)). Radioligand binding assays showed that rho-TIA was 10-fold selective for human alpha(1B)- over alpha(1A)- and alpha(1D)-ARs. As observed with hamster alpha(1B)-ARs, rho-TIA decreased the number of binding sites (B-max) for human alpha(1B)-ARs without changing affinity (K-D), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, rho-TIA had opposite effects at human alpha(1A)-ARs and alpha(1D)-ARs, decreasing KD without changing Bmax, suggesting it acts competitively at these subtypes. rho-TIA reduced maximal NE-stimulated [H-3] inositol phosphate formation in HEK293 cells expressing human alpha(1B)-ARs but competitively inhibited responses in cells expressing alpha(1A)- or alpha(1D)-ARs. Truncation mutants showed that the amino-terminal domains of alpha(1B)- or alpha(1D)-ARs are not involved in interaction with rho-TIA. Alanine-scanning mutagenesis of rho-TIA showed F18A had an increased selectivity for alpha(1B)-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at alpha(1B)-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus rho-TIA noncompetitively inhibits alpha(1B)-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at alpha(1B)-ARs.

Association of tumor necrosis factor-alpha polymorphisms and ozone-induced change in lung function

Ozone is a major air pollutant with adverse health effects which exhibit marked inter-individual variability. In mice, regions of genetic linkage with ozone-induced lung injury include the tumor necrosis factor-alpha (TNF), lymphotoxin-alpha (LTA), Toll-like receptor 4 (TLR4), superoxide dismutase (SOD2), and glutathione peroxidase (GPX1) genes. We genotyped polymorphisms in these genes in 51 individuals who had undergone ozone challenge. Mean change in FEV1 with ozone challenge, as a percentage of baseline, was -3% in TNF -308G/A or A/A individuals, compared with -9% in G/G individuals (p = 0.024). When considering TNF haplotypes, the smallest change in FEV1 with ozone exposure was associated with the TNF haplotype comprising LTA +252G/TNF -1031T/TNF -308A/TNF -238G. This association remained statistically significant after correction for age, sex, disease, and ozone concentration (p = 0.047). SOD2 or GPX1 genotypes were not associated with lung function, and the TLR4 polymorphism was too infrequent to analyze. The results of this study support TNF as a genetic factor for susceptibility to ozone-induced changes in lung function in humans, and has potential implications for stratifying health risks of air pollution.

Interferon-gamma is required for killing keratinocytes in vitro and in vivo

Epithelial malignancies are common in immunosuppressed individuals and the general population. However the mechanisms by which the adaptive immune system can eliminate immunogenic epithelial cells remain undefined. The aim of this project was to determine the effector molecules required for induction of apoptosis in murine epidermal keratinocytes (MEKs) in vitro and in vivo. HPV16E7-specific CTL lines and T cell receptor transgenic (E7TCRtg) effector cells were obtained from wild type (wt)-C57 and syngeneic mice rendered functionally inactive for perforin (Pfp), interferon-g (IFN-g) or FasL. CTLs or E7TCRtg spleen cells were co-cultured with primary MEKs in vitro or transferred into skin graft recipients. Inhibition of colony formation and skin graft rejection were used as indicators of T cell:KC interaction. Wt E7-specific CTLs and CTLs deficient in perforin, FasL or IFN-g produced mean reductions in colony formation of 67% (62.4–71.3%), 72% (71.1–72%), 76% (73–78%) and 21.5% (14– 34%) respectively. Wt, perforin deficient or FasL deficient CTLs all induced rejection of skin grafts (wt: 6/12; Pfp: 9/15; FasL: 3/13 survival). Transfer and immunisation of wt E7TCRtg spleen cells induces rejection of 50% of grafts (4/8 survival). In contrast, perforin or IFN-g deficient E7TCRtg failed to induce graft rejection (5/6; 4/4 survival). FasL deficient E7TCRtg induced nonspecific rejection of grafts (E7- 2/6 survival; C57- 4/7 survival). Therefore IFN-g production by CTL is necessary and sufficient in vitro and in vivo to kill epithelial cells which express a nonself antigen. Assessment of immunotherapies directed against epithelial tissues may be more effectively achieved by assaying the amount of IFN-g production by CD8 T cells, and the number and affinity of those cells, in conjunction with quantitation of perforin mediated effects in short term assays.

Binding and internalization of the melanocyte stimulating hormone receptor ligand [Nle(4), D-Phe(7)]alpha-MSH in B16 melanoma cells

The current study aims to ascertain the fate of the melanocyte stimulating hormone (MSH) receptor and its ligand [Nle(4), D-Phe(7)]alpha-MsH (NDP-MSH) following binding to murine B16 melanoma cells. Cells were incubated with [I-125]-NDP-MSH for up to 180 min and binding, internalization and degradation determined. Intracellular trafficking of the radiolabel was assessed !using Percoll density gradient centrifugation of homogenized cells. Receptor down-regulation and receptor mRNA levels were also measured over 96 hr after exposure to 1 mu M ligand. NDP-MSH accumulation increased with time in a temperature-dependent manner and was inhibited by excess peptide. The ligand was rapidly internalized and translocated to the lysosomal compartment where it was degraded. Internalization was accompanied by a loss or down-regulation of cell surface receptors, suggesting internalization of the NDP-MSH-receptor complex. No recycling of the receptors between the plasma membrane and intracellular compartments could be detected in this cell-hue. Approximately 15% of the surface receptors were resistant to down-regulation, possibly indicating receptor heterogeneity. Down-regulation persisted ibr up to 96 hr and was accompanied by a decrease in MSH receptor mRNA levels 48 hr after treatment. However, before this time, transcript levels were the same in treated and control cells. In contrast to what was seen with NDP-MSH, cell surface receptors removed with trypsin wc:re rapidly replaced. These results show that NDP-MSH not only induced MSH receptor :internalization but also inhibited receptor turnover, resulting in a prolonged down-regulation. It is concluded that, in B16 cells, the MSH receptor undergoes ligand-dependent internalization, resulting in a prolonged down-regulation. Copyright (C) 1996 Elsevier Science Ltd

Identification of the alpha(6) integrin as a candidate receptor for papillomaviruses

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction bf human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [S-35]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha(6) beta(4) integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha(6) or beta(4) integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha(6) integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta(4) integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha(6) beta(4) integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha(6), integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha(6) integrin subunit and that integrin complexes containing alpha(6) integrin complexed with either beta(1) or beta(4) integrins may act as a receptor for PV binding and entry into epithelial cells.

Synthesis of alpha-aspartyl-containing cyclic peptides

alpha-Aspartyl-containing cyclic pentapeptides were synthesised in high yields using a strategy that maintained fluorenylmethyl protection on the aspartic acid side chain during chain assembly, resin cleavage and cyclisation of the linear precursors. Tetra-n-butylammonium fluoride treatment of the fluorenylmethyl-protected cyclic peptides catalysed imide formation, whereas piperidine-induced deprotection resulted in good yields of the target cyclic peptides.

Increased tissue resistance in the nude mouse against Candida albicans without altering strain-dependent differences in susceptibility

Strain differences in tissue responses to infection with Candida albicans were examined in nude mice having susceptible (CBA/CaH) and resistant (BALB/c) parentage. Homozygous (nu/nu) mice of both strains were more resistant to systemic infection with C. albicans than heterozygous (nu/+) littermates as indicated by a reduction in both the severity of tissue damage and colony counts in the brain and kidney. However, the tissue lesions in nu/nu CBA/CaH mice were markedly more severe than those in nu/nu mice with the BALB/c background. This pattern was reflected in the greater fungal burden in the CBA/CaH strain. Analysis of cDNA from infected tissues using a competitive polymerase chain reaction excluded interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), and interleukin 6 (IL-6) as mediators of the enhanced resistance of the nude mice. The results confirm that the different patterns of lesion severity in BALB/c and CBA/CaH mice do not involve T lymphocyte-mediated pathology, and are consistent with the hypothesis that strain-dependent tissue damage is not dependent on the effector function of macrophages or their precursors.

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.

In vivo effects of chicken interferon-gamma during infection with Eimeria

Newly hatched chickens are highly susceptible to infection by opportunistic pathogens during the first 1 or 2 weeks of life, The use of cytokines as therapeutic agents has been studied in animal models as well as in immunosuppressed patients, This approach has become more feasible in livestock animals, in particular poultry, with the recent cloning of cytokine genes and the development of new technologies, such as live delivery vectors, We have recently cloned the gene for chicken interferon-gamma (Ch-IFN-gamma), Poly-HIS-tagged recombinant Ch-IFN-gamma was expressed in Escherichia coil, was purified by Ni chromatography, and was found to be stable at 4 degrees C and an ambient temperature for at least several months and Several weeks, respectively, Ch-IFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis, induced nitrite secretion from chicken macrophages in vitro, and enhanced MHC class II expression on macrophages, Administration of recombinant Ch-IFN-gamma to chickens resulted in enhanced weight gain over a 12-day period, Furthermore, the therapeutic potential of Ch-IFN-gamma was assessed using a coccidial challenge model, Birds were treated with Ch-IFN-gamma or a diluent control and then infected with Eimeria acervulina. Infected birds treated with Ch-IFN-gamma showed improved weight gain relative to noninfected birds, The ability of Ch-IFN-gamma to enhance weight gain in the face of coccidial infection makes it an excellent candidate as a therapeutic agent.