Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D H-1 NMR data, Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1-15, 8-20, 14-31 and 16-42 (a 1-4/2-6/3-7/5-8 pattern), The structure consists of a small three-stranded, anti-parallel beta-sheet and a series of interlocking gamma-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the omega-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues, Both of these structural features may play a role in its binding to the voltage-gated sodium channel. (C) 1997 Federation of European Biochemical Societies.

Membrane-bounded nucleoids in microbial symbionts of marine sponges

In thin sections of resin-embedded samples of glutaraldehyde- and osmium tetroxide-fixed tissue from five genera of marine sponges, Stromatospongia, Astrosclera, Jaspis, Pseudoceratina and Axinyssa, cells of a bacteria-like symbiont microorganism which exhibit a membrane-bounded nuclear region encompassing the fibrillar nucleoid have been observed within the sponge mesohyl. The nuclear region in these cells is bounded by a single bilayer membrane, so that the cell cytoplasm is divided into two distinct regions. The cell wall consists of subunits analogous to those in walls of some Archaea. Cells of the sponge symbionts observed here are similar to those of the archaeal sponge symbiont Cenarchaeum symbiosum. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Dendritic cells in rheumatoid arthritis - A model autoimmune inflammatory site

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease in which unknown arthrogenic autoantigen is presented to CD4+ T cells. The strong association of the disease with an epitope within the HLA-DR chain shared between various alleles of HLA-DR4 and DR1 emphasizes the importance of antigen presentation. This immune response predominantly occurs in the synovial tissue and fluid of the joints and autoreactive T cells are readily demonstrable in both the synovial compartment and blood. Circulating dendritic cells (DC) are phenotypically and functionally identical with normal peripheral blood (PB) DC. In the synovial tissue, fully differentiated perivascular DC are found in close association with T cells and with B cell follicles, sometimes containing follicular DC. These perivascular DC migrate across the activated endothelium from blood and receive differentiative signals within the joint from monocyte-derived cytokines and CD40-ligand+ T cells. In the SF, DC manifest an intermediate phenotype, similar to that of monocyte-derived DC in vitro. Like a delayed-type hypersensitivity response, the rheumatoid synovium represents an effector site. DC at many effector sites have a characteristic pattern of infiltration and differentiation. It is important to note that the effector response is not self-limiting in RA autoimmune inflammation. In this article, we argue that the presentation of self-antigen by DC and by autoantibody-producing B cells is critical for the perpetuation of the autoimmune response. Permanently arresting this ongoing immune response with either pharmaceutical agents or immunotherapy is a major challenge for immunology.

Proliferation in monocyte-derived dendritic cell cultures is due to cells capable of myeloid differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [H-3]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor-alpha led to maturation of the DC, but did not inhibit proliferation. Ki67(+) cells were observed in cytospins of these DC, and by double staining were CD3(-)CD19(-)CD11c(-)CD40(-) and myeloperoxidase(+), suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34(+)CD33(-)CD14(-) progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes. (C) 1998 by The American Society of Hematology.

Adverse effects of cannabis

Cannabis is the most widely used illicit drug in many developed societies. Its health and psychological effects are not well understood and remain the subject of much debate, with opinions on its risks polarised along the lines of proponents' views on what its legal status should be. An unfortunate consequence of this polarisation of opinion has been the absence of any consensus on what health information the medical profession should give to patients who are users or potential users of cannabis. There is conflicting evidence about many of the effects of cannabis use, so we summarise the evidence on the most probable adverse health and psychological consequences of acute and chronic use. This uncertainty, however, should not prevent medical practitioners from advising patients about the most likely ill-effects of their cannabis use. Here we make some suggestions about the advice doctors can give to patients who use, or are contemplating the use, of this drug.

H-1-NMR structural studies of a cystine-linked peptide containing residues 71-93 of transthyretin and effects of a Ser84 substitution implicated in familial amyloidotic polyneuropathy

The Ile-->Ser84 substitution in the thyroid hormone transport protein transthyretin is one of over 50 variations found to be associated with familial amyloid polyneuropathy, a hereditary type of lethal amyloidosis. Using a peptide analogue of the loop containing residue 84 in transthyretin, we have examined the putative local structural effects of this substitution using H-1-NMR spectroscopy. The peptide, containing residues 71-93 of transthyretin with its termini linked via a disulfide bond, was found to possess the same helix-turn motif as in the corresponding region of the crystallographically derived structure of transthyretin in 20% trifluoroethanol (TFE) solution. It therefore, represents a useful model with which to examine the effects of amyloidogenic substitutions. In a peptide analogue containing the Ile84-->Ser substitution it was found that the substitution does not greatly disrupt the overall three-dimensional structure, but leads to minor local differences at the turn in which residue 84 is involved. Coupling constant and NOE measurements indicate that the helix-turn motif is still present, but differences in chemical shifts and amide-exchange rates reflect a small distortion. This is in keeping with observations that several other mutant forms of transthyretin display similar subunit interactions and those that have been structurally analysed possess a near native structure. We propose that the Ser84 mutation induces only subtle perturbations to the transthyretin structure which predisposes the protein to amyloid formation.

Identification of residues involved in binding of IL5 to beta(com) using beta(IL3) and beta(com) chimeras

In mice there are two forms of the beta chain used in the IL3 receptor system, beta(com) and beta(IL3). beta(com) is used by the IL3, ILS and GM-CSF receptors whereas Pns is only used in the IL3 receptor. In this work an assay was developed to identify residues of beta(IL3) that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the beta(IL3) B'-C' loop were replaced with beta(com) sequence a form of beta(IL3) was produced that was able to respond to IL5. This region is also responsible for IL3 binding to beta(IL3) in the absence of alpha chain. It is therefore an important structural motif of beta(com) and beta(IL3) responsible for both ligand interaction and specificity. (C) 1999 Federation of European Biochemical Societies.

Role of chemical cues from cotton in mediating host selection and oviposition behaviour in Helicoverpa armigera (Hubner) (Lepidoptera : Noctuidae)

We investigated the role of chemoreception in the host selection and oviposition behaviour of Helicoverpa armigera in the laboratory using five cotton genotypes and synthetic volatile terpenes. Female moths oviposited on substrates treated with methanol, ethanol, acetone and pentane extracts of leaves, squares and flowers of the cotton genotypes. Phytochemicals soluble in pentane were the most efficient in eliciting oviposition behaviour. In a two-way bioassay, pentane extracts of leaves or squares of a Multiple Host-plant Resistance genotype (MHR11), Deltapine commercial (DP90), and Smith Red Leaf (SRL) received significantly more eggs than solvent-treated controls. Extracts of squares of the native genotype Gossypium nelsonii did not receive more eggs. Females preferred DP90 and MHR11 to SRL and G. nelsonii. Female moths also laid more eggs on pentane extracts of MHR11 flowers than MHR11 leaves from preflowering, early flowering and peak-flowering plants. In a flight chamber, female moths used olfactory cues at short range to mediate oviposition and discrimination between host plants. Egg-laying, mated females were attracted at a distance (1.5 m) to volatile compounds released by whole plants and odours emanating from filter papers treated with synthetic volatile terpenes. Individually, the terpenes did not stimulate any significant oviposition response. However, there was a significant oviposition response to a mixture of equal volumes of the terpenes (trans-beta-caryophyllene, alpha-pinene, beta-pinene, myrcene, beta-bisabolol, and alpha-humulene). Conversely, antennectomised (moths with transected antennae), egg-laying, mated females did not stimulate any significant oviposition response. The significance of these findings in relation to H. armigera hostplant selection are discussed.

Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that Of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity:for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not,necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within I h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

FHA domain boundaries of the Dun1p and Rad53p cell cycle checkpoint kinases

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues), In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks, This indicates that the non-catalytic FI-IA domain plays an important role in the transcriptional function of the Dun1p protein kinase. (C) 2000 Federation of European Biochemical Societies.