Organotypic hippocampal slice cultures can be used to study hippocampal biochemistry and physiology over a chronic period on the days to weeks timescale. In order to validate the organotypic hippocampal slice culture for our ongoing studies of synaptic function, we have compared, using Western blotting, the levels of a number of synaptic proteins from in vitro organotypic hippocampal slice cultures with those from in vivo hippocampal slices prepared from age-matched controls. We chose to follow the developmental expression of the neuroplastin (np) family of immunoglobulin related cell adhesion molecules (CAMs), np65, a brain specific isoform highly expressed in hippocampal neurones and np55 a more widely expressed isoform and two synaptic marker proteins, synaptophysin, a pre-synaptic marker and post-synaptic density protein-95, PSD95, a post-synaptic marker. All showed increasing expression over the developmental time period, both in vivo and in vitro. The level of both neuroplastins was also consistent between the in vivo and in vitro preparations, whereas the level of PSD95 was markedly increased in the organotypic hippocampal slice cultures while the level of synaptophysin was slightly decreased. Whilst these findings may indicate some differences in the composition and organisation of synapses, the developmental expression profiles of these synaptic proteins within organotypic hippocampal slice cultures suggests they are a valid model for the study of synapse function and development in vitro.

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.

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.

The distribution of a glycoprotein component of the muscle dystrophin complex, beta-dystroglycan, has been determined in subcellular fractions of adult rat forebrain. The results show that beta-dystroglycan is enriched in several membrane fractions, including synaptic membranes, but in marked contrast to dystrophin is not detectable in the postsynaptic density fraction. The antiserum also recognises a second molecular species of apparent molecular mass of 164 kDa which is highly enriched in the postsynaptic density fraction. Preabsorption of the antiserum with the antigen (a 22-mer peptide corresponding to the C-terminal sequence of rabbit skeletal muscle beta-dystroglycan) abolished reactivity against both beta-dystroglycan and the 164-kDa postsynaptic density-enriched protein, confirming that the two species are immunologically related. Enzymatic removal of N-linked oligosaccharide lowered the apparent molecular mass of beta-dystroglycan by 3 kDa but did not alter the mass of the 164-kDa species.

Conference Article| February 01 1995 The post-synaptic density: putative involvement in synapse stabilization via cadherins and covalent modification by ubiquitination P. W. Beesley; P. W. Beesley ‡ *Department of Biochemistry, Royal Holloway, University of London, Egham, Surrey TW20 0EX, U.K. ‡To whom all correspondence should be addressed. Search for other works by this author on: This Site PubMed Google Scholar R. Mummery; R. Mummery *Department of Biochemistry, Royal Holloway, University of London, Egham, Surrey TW20 0EX, U.K. Search for other works by this author on: This Site PubMed Google Scholar J. Tibaldi; J. Tibaldi *Department of Biochemistry, Royal Holloway, University of London, Egham, Surrey TW20 0EX, U.K. Search for other works by this author on: This Site PubMed Google Scholar A. P. Chapman; A. P. Chapman § *Department of Biochemistry, Royal Holloway, University of London, Egham, Surrey TW20 0EX, U.K. Search for other works by this author on: This Site PubMed Google Scholar S. J. Smith; S. J. Smith †Department of Neurology, SmithKline Beecham, Harlow, Herts CN19 5AW, U.K. Search for other works by this author on: This Site PubMed Google Scholar C. C. Rider C. C. Rider *Department of Biochemistry, Royal Holloway, University of London, Egham, Surrey TW20 0EX, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (1): 59–64. https://doi.org/10.1042/bst0230059 Article history Received: September 12 1994 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation P. W. Beesley, R. Mummery, J. Tibaldi, A. P. Chapman, S. J. Smith, C. C. Rider; The post-synaptic density: putative involvement in synapse stabilization via cadherins and covalent modification by ubiquitination. Biochem Soc Trans 1 February 1995; 23 (1): 59–64. doi: https://doi.org/10.1042/bst0230059 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: con A, concanavalin A, mab, monoclonal antibody, NMDA, N-methyl-D-aspartate, P, postnatal day, PAC 1, post-synaptic density and cytoskeleton-enriched epitope, PSD, post-synaptic density, pgp, post-synaptic density-enriched glycoprotein, SM, synaptic membrane This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

We have previously described a monoclonal antibody, PAC 1, that recognises two postsynaptic density (PSD)-enriched glycoproteins (pgps) of apparent M(r) 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 M(r) 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 M(r) 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.

A bitter tasting cyanoglucoside, sarmentosin, was isolated from the Magpie moth. The high concentration in the imago (650,μg insect−1 suggests a defensive role for this substance. The concentration of carotenoids was also found to be exceptionally high.

The monoclonal antibody PAC 1 (postsynaptic density and cytoskeleton enriched) recognizes an epitope present on two postsynaptic density-enriched glycoproteins of 130,000 (postsynaptic density-enriched glycoprotein 130) and 117,000 mol. wt (postsynaptic density-enriched glycoprotein 117), and a cytoskeleton-enriched polypeptide of 155,000 mol. wt (cp155). The PAC 1 antibody has been used to study the development of the PAC 1 antigens in the developing rat forebrain in vivo and in tissue culture. cp155 is detected by embryonic day 14 and its level continues to rise until the sixth postnatal week. Postsynaptic density-enriched glycoproteins 130 and 117 are also expressed in embryonic brain although the level of postsynaptic density-enriched glycoprotein 130 initially increases more rapidly than that of postsynaptic density-enriched glycoprotein 117. Peak values are observed at postnatal days 4 (postsynaptic density-enriched glycoprotein 117) and 9 (postsynaptic density-enriched glycoprotein 130). The level of post synaptic density-enriched glycoprotein 117 subsequently decreases to some 50% of the peak value by postnatal day 42. Immunocytochemical studies show that PAC 1 immunoreactivity in developing cerebral cortex, detectable by postnatal day 0, is primarily associated with the perikarya and dendrites of pyramidal cells. The immunoreactivity develops as patches of PAC 1-positive neurons, uniform staining of the cortex only being fully established after postnatal day 9. Double-immunofluorescence labelling studies of forebrain cultures prepared from embryonic day 18 animals shows that many, but not all, growth-associated protein 43-positive neurons exhibit PAC 1 immunoreactivity. Some non-neuronal cells also stain with the PAC 1 monoclonal antibody. The growth cones of cultured neurons exhibit PAC 1 immunoreactivity and the PAC 1 antigens are detected on immunodeveloped western blots of isolated growth cones. The PAC 1 epitope is intracellular, but immunoreactivity does not co-localize with F-actin as detected by rhod-amine-phalloidin or with tubulin immunoreactivity. Postsynaptic density-enriched glycoprotein 130 is readily detected on PAC 1 immunodeveloped western blots of forebrain cultures maintained for up to 14 days in vitro. Postsynaptic density-enriched glycoprotein 117 is only poorly expressed by these cultures. The PAC 1 glycoproteins are present in forebrain synaptic membranes and postsynaptic densities at an early stage of development. The synaptic membrane level of postsynaptic density-enriched glycoprotein 130 and postsynaptic density-enriched glycoprotein 117 increases markedly between postnatal days 3 and 8. The level of both glycoproteins detected in postsynaptic densities remain virtually constant from postnatal days 9-90. These results are consistent with functional roles for these molecules in neuronal and synapse development.

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.

Carotenoid concentration in model swallowtail butterflies is exceptionally high; it is suggested that they may exert a photo-protective function in species storing nitrophenanthrenes. Carotenoid concentration in the viceroy is also high. Again, it is suggested that this represents a protective mechanism for Lepidoptera feeding on Salix which contains salicin. Such a mechanism would not be required by the monarch storing cardenolides.

The carotenoids found in adult kite swallowtails (Graphium spp.) and the Danaid glassy tigers (Ideopsis, Tirumala and Parantica), butterflies with exposed blue/green pigmented wing membranes (which contain pterobilins) and a modified scale vestiture, were compared with those of related species of Papilios and Danaids with concealed wing membranes an an entire scale vestiture. It was found that both the kite swallowtails contained only lutein, and even lacked β-carotene, the first known instance among butterflies. This was presumably due to selective storage by the larva since the food-plant was found to be rich in varied carotenoids. A possible link between the high concentration of pterobilins in the wing membrane and the lack of carotenoids is discussed.

Silver replaces the golden areas of danaid pupae if the larvae are reared on a carotenoid‐free diet. These pigments are therefore responsible for the yellow colouring of the “golden glance”. Lutein is the dominant carotenoid present in pupal exuviae. In response to light cues, these pigments can be re‐routed into deeper tissues, thus pupae of Euploea core become silver instead of gold if reared against a dull white background.

The distribution, concentration and excretion of different carotenoids in the tissues of locust hoppers were investigated. Those reared in bright (1300 lx) light stored more and excreted less than specimens reared in dim (450 lx) light: 70% of the former were homo‐chromic and only 2% of the latter matched their background. Unlike the specimens investigated by Goodwin (1952) our insects stored xanthophylls in their body tissues, but lacked astaxanthin. Exuviae contained four carotenoids.

The light cues received by the larvae of Pieris brassicae which determine diapause can also influence the carotenoid distribution (and hence the colour) in the epidermis and cuticle of the diapausing pupae. Irrespective of background or light cues received during the “sensitive period” of the pharate pupa, these diapausing pupae are coloured green. They then contain more than double the concentration of carotenoids in their epidermis than the non‐diapausing pupae. This green colour can be somewhat modified by switching the full grown larvae to long day regimes immediately after feeding ceases.The Large White and Small White butterfly each has a characteristic carotenoid storage pattern, which can be demonstrated by feeding the larvae on portions of the same cabbage leaves. The braconid parasite Apanteles glomeratus mirrors the carotenoids in its host.

ABSTRACTThe carotenoids of a blue mutant and normal Hyalophora cecropia (L.) (both reared on apple leaves) were compared. Pupae derived from blue larvae contained 3·78 μg of carotenoids per individual, compared with 266·0 μg in pupae derived from normal green larvae.

The black mutant larva of Manduca sexta contains approximately the same quantity of carotenoids as the normal green type, but there is possibly a slight qualitative difference between them, the former storing more β-carotene and less violaxanthin.