Rosemary S. Mummery

Characterization of the Heparin-Binding Properties of IL-6

We establish, using an ELISA approach, that recombinant human and murine IL-6 bind to an immobilized heparin-BSA complex. In the case of human IL-6, this binding is displaceable by soluble heparin, IC50 ∼2 μg/ml, corresponding to ∼200 nM. This binding is specific because chondroitin sulfates B and C fail to compete, whereas chondroitin sulfate A and several heparan sulfates are weak inhibitors. Of a range of chemically modified heparins examined, the strongest competitor was the 2-O-desulfated product, but even this showed a considerably reduced IC50 (∼30 μg/ml). The epitopes of five IL-6-specific mAbs were still accessible in heparin-bound IL-6, and the dimer formed from the association of rIL-6 with its truncated soluble receptor polypeptide, srIL-6α, still bound to heparin. Further analysis showed that heparin competed partially and weakly with the binding of srIL-6 to IL-6; however, it competed strongly for the binding of the rIL-6/srIL-6Rα dimer, to soluble glycoprotein 130. In studies of the proliferation of IL-6-sensitive Ba/F3 cells expressing glycoprotein 130, we were unable to detect any effect of either the removal of cell surface heparan sulfate, or addition of soluble heparin. By contrast, heparin was able to protect IL-6 from digestion by the bacterial endoproteinase Lys-C. Overall, our findings show that IL-6 is a heparin-binding cytokine. This interaction will tend to retain IL-6 close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine mode of activity. Moreover, this binding may serve to protect the IL-6 from proteolytic degradation.

N-Cadherin Is a Major Glycoprotein Component of Isolated Rat Forebrain Postsynaptic Densities

Abstract: We have previously described a monoclonal antibody, PAC 1, that recognises two postsynaptic density (PSD)‐enriched glycoproteins (pgps) of apparent Mr 130,000 (pgp130) and 117,000 (pgp117). Immunodevelopment of western blots of rat forebrain homogenate, synaptic membrane (SM), and PSD samples with PAC 1 and an N‐cadherin antiserum shows that pgp130 and N‐cadherin are of identical apparent Mr and show identical patterns of enrichment in these fractions. The apparent molecular masses of pgp130 and N‐cadherin are both lowered by 11 kDa following removal of N‐linked carbohydrate with endoglycosidase‐F containing N‐glycopeptidase. The two molecules show an identical pattern of migration when separated by two‐dimensional electrophoresis. A single 130‐kDa band immunoprecipitated from solubilised PSD preparations by the N‐cadherin antiserum is recognised by PAC 1 on western blots. We conclude that pgp130 is N‐cadherin. Development of western blots of two‐dimensional gel separations of SM and PSD glycoproteins shows that N‐cadherin is a major glycoprotein component of PSDs. The immunoprecipitation experiments show that the Mr of N‐cadherin is greater than that of the major pgp, PSD gp116. The PAC 1 antibody recognises two concanavalin A‐binding glycoproteins with apparent molecular masses of 136 and 127 kDa in liver samples. The 136‐kDa band is also recognised by the N‐cadherin antiserum. These observations, together with data showing that the PAC 1 epitope is intracellular, suggest that PAC 1 is a pan‐cadherin antibody and recognises an epitope on the conserved cadherin intracellular carboxyl‐terminal domain.

The major determinant of the heparin binding of glial cell-line-derived neurotrophic factor is near the N-terminus and is dispensable for receptor binding

GDNF (glial cell-line-derived neurotrophic factor), and the closely related cytokines artemin and neurturin, bind strongly to heparin. Deletion of a basic amino-acid-rich sequence of 16 residues N-terminal to the first cysteine of the transforming growth factor beta domain of GDNF results in a marked reduction in heparin binding, whereas removal of a neighbouring sequence, and replacement of pairs of other basic residues with alanine had no effect. The heparin-binding sequence is quite distinct from the binding site for the high affinity GDNF polypeptide receptor, GFRalpha1 (GDNF family receptor alpha1), and heparin-bound GDNF is able to bind GFRalpha1 simultaneously. The heparin-binding sequence of GDNF is dispensable both for GFRalpha1 binding, and for activity for in vitro neurite outgrowth assay. Surprisingly, the observed inhibition of GDNF bioactivity with the wild-type protein in this assay was still found with the deletion mutant lacking the heparin-binding sequence. Heparin neither inhibits nor potentiates GDNF-GFRalpha1 interaction, and the extracellular domain of GFRalpha1 does not bind to heparin itself, precluding heparin cross-bridging of cytokine and receptor polypeptides. The role of heparin and heparan sulfate in GDNF signalling remains unclear, but the present study indicates that it does not occur in the first step of the pathway, namely GDNF-GFRalpha1 engagement.

Carotenoids of certain compositae flowers

Abstract Eleven species and varieties of flowers of Compositae have been investigated for their carotenoid contents. Epoxy-carotenes and xanthophylls were found in fairly large amounts and were the main pigments in some cases. Carotenoid compositions are reported for the first time for flowers of Gerbera jamesonii, Hypochoeris radicata and Senecio scandens. Yellow flowers tended to have more xanthophylls while orange ones had a large amount of one carotene, except in Tagetes erecta where the only difference between the yellow and orange seemed to be in the total carotenoids. Lutein, universally present in leaves, was not always found in the flowers studied, while β-carotene was always found in fairly small quantities; flavoxanthin and chrysanthemaxanthin were usually present in fairly large amounts.

Hypoxia—Ischemia Induces a Rapid Elevation of Ubiquitin Conjugate Levels and Ubiquitin Immunoreactivity in the Immature Rat Brain

Postnatal rats at 7 and 21 days of age were subjected to unilateral hypoxia—ischemia (H/I) by right carotid artery ligation followed by 1.5 to 2 hours of hypoxia (8% oxygen). Brains were frozen at specific intervals of recovery from 0 to 24 hours. Western blots of samples of right and left forebrain were immunodeveloped with a monoclonal antibody specific for ubiquitin, RHUb 1. An elevation of ubiquitin conjugate levels in the right compared with the left forebrain of 7-day-old animals was detectable immediately following H/I and increased by close to 60% of control level within 1 hour of recovery. The conjugate immunoreactivity remained at this level for 6 hours but had declined to control levels by 24 hours of recovery. No such increase was observed in response to hypoxia alone. Similar changes were observed in samples from the 21-day-old rat brain. However, the elevation of ubiquitin conjugate levels was of slower onset and persisted longer than observed for the 7-day-old animals. Immunocytochemical studies of brain fixed by immersion in formaldehyde/acetone/methanol showed that ubiquitin-like immunoreactivity was increased in the right, but not left, cerebral cortex and hippocampus of animals subjected to H/I. The data suggest that elevated ubiquitination may represent a neuroprotective response to H/I.

IMMUNOGLOBULIN SUPERFAMILY MEMBERS GP65 AND GP55 : TISSUE DISTRIBUTION OF GLYCOFORMS

Gp65 and gp55 are immunoglobulin superfamily members produced by alternative splicing of the same gene transcript, and originally identified as components of synaptic membranes. A monoclonal antibody specific for gp65 and gp55 has been used to detect immunoreactive species in a wide range of tissues. All immunoreactive species bind to concanavalin A and deglycosylation studies show that in all tissues tested other than brain the immunoreactive species are derived from gp55. HEK cells transfected with gp65 or gp55 express different glycoforms from brain showing that the pattern of glycosylation of these molecules is dependent upon the cell type in which they are expressed.

Carotenoids of Lilies and of Red Pepper: Biogenesis of Capsanthin and Capsorubin

Summary Carotenoids of petals of the following lilies: Lilium leichtinii var. Maximowiczii, L. Davidii var. Willmottiae and of their hybrid L. Maxwill were investigated as they all contained capsanthin and capsorubin. The hybrid possessed not only the parent carotenoids but also β -carotene monoepoxide, cryptocapsin and lutein not found in the parents. Carotenoids not present in parents but in hybrids have already been observed in citrus hybrids. Petals of L. amabile , a redder flower, had very much the same carotenoids as the others studied. Capsanthin and capsorubin were also found together when Capsicum annuum var. grossum (pepper) fruits matured but not at the green photosynthetic stage. Our results both with lilies and pepper fruits correlated well with the biosynthetic pathway proposed for capsanthin and capsorubin formation. No new carotenoids were observed in other floral parts of lilies investigated. However, antheraxanthin which had been suggested as a carotenoid specifically associated with reproduction in higher plants was found as the main carotenoid of the anthers investigated.

Molecular Characterisation and Structural Relationship of the Synapse‐Enriched Glycoproteins gp65 and gp55

Abstract: gp65 and gp55 are glycoprotein components of CNS synapses that are recognised by a single monoclonal antibody, SMgp65. This antibody has now been used to investigate the molecular properties of these two glycoproteins and the structural relationship between them. Both gp65 and gp55 occur in most brain regions as doublets of apparent molecular masses of 63 and 67 kDa, and 52 and 57 kDa, respectively. Striatal samples, however, are enriched in a novel gp65 iso‐form of 69 kDa. Removal of oligosaccharide residues from gp65 and gp55 with trifluoromethanesulphonic acid shows that gp65 and gp55 are composed of single polypeptide chains of 40 and 28 kDa, respectively. Removal of sialic acid residues with neuraminidase lowers the apparent molecular mass of both glycoproteins by 5–6 kDa. Triton X‐114 phase partitioning and alkaline extraction of synaptic membranes indicate that both gp65 and gp55 are integral membrane glycoproteins. Treatment of synaptic membranes with phosphatidylinositol‐specific phospholipase C does not solubilise either glycoprotein. One‐dimensional peptide and epitope maps obtained by digestion of gp65 and gp55 with endoproteinase lys C or subtilisin are consistent with a close structural relationship between the two molecules. Tryptic digestion of samples enriched in gp65 and/or gp55 results in the formation of a novel immunoreactive 53‐kDa species that is resistant to further trypsin degradation except in the presence of 0.1% (wt/vol) sodium dodecyl sulphate. Trypsin treatment of cultures of forebrain neurones in situ lowers the apparent molecular mass of gp65 to 53 kDa. These results confirm the structural similarity ofgp6 5 andgp5 5 and suggest that the major difference between the two glycoproteins is likely to be a single 12‐kDa cell surface‐located polypeptide fragment.

Chromatography of carotenoids using papers filled with silica gel and with alumina

Abstract the R f values of twenty-two carotenoids separated on Whatman paper Chromedia AH 81 and SG81 are given. The R f values are readily reproducible and separation of a large number of carotenoids can easily be carried out in about half-an-hour.

PHYTOCHROME MEDIATED CAROTENOID SYNTHESIS IN THE FUNGUS VERTICILLIUM AGARZCZNUM

Abstract— Ten minutes of red irradiation (R) increased carotenogenesis in Verticillium agoricinum and this effect was reversed by 10min of far‐red (FR) irradiation indicating that phytochrome is involved. A far‐red minus red difference spectrum of a crude extract shows a peak at 670 nm and a dip at 750 nm wavelength, values slightly larger than higher plant phytochrome. indicating the presence of phytochrome.