Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.

High-pressure freezing and freeze-substitution were used to study Golgi ultrastructure and its brefeldin A-induced transformations in HepG2 human hepatoma cells. Cryoimmobilization arrested subcellular dynamics within milliseconds, thus considerably improving the temporal resolution in monitoring the very early effects of high brefeldin concentrations at the ultrastructural level (i.e., 20 microg/ml brefeldin applied for 35 s to 8 min). Moreover, this approach ruled out possible cumulative and/or synergistic effects of the drug and fixatives. Several findings differed from studies based on chemical fixation. In particular, Golgi breakdown did not proceed gradually but occurred in distinct steps. We found a conspicuous lag between the absence of nonclathrin coats on Golgi membranes after 30 s of brefeldin treatment and the disassembly of the stacks, which did not start until after 90 to 120 s. At this time, domains at the trans and cis faces separated from the stacks, starting tubulation and fragmentation. After 3-5 min the Golgi apparatus was completely replaced by loose meshworks of straight tubules of different sizes and staining properties; also frequent were bent tubules and vesicles forming glomerule-like structures. After 8 min all kinds of Golgi-derived structures had aggregated within huge clusters. The morphologically highly distinct structures found after brefeldin treatment could in part be correlated with particular Golgi domains in the control cells.

An almost intact, naturally mummified late neolithic human was discovered in 1991 at a glacial field in the Ötztal Alps. Not only the clothing and equipment of the ‘Tyrolean Ice Man’, but also the frozen mummy itself, appeared in good overall condition [[1]Seidler H Bernhard W Teschler-Nicola M Platzer W zur Nedden D Henn R et al.Some anthropological aspects of the prehistoric Tyrolean Ice Man.Science. 1992; 258 (93030753): 455-457Crossref PubMed Scopus (71) Google Scholar]. At the subcellular and molecular level, however, considerable decay was observed ([[2]Handt O Richards M Trommsdorff M Kilger C Simanainen J Georgiev O et al.Molecular genetic analyses of the Tyrolean Ice Man.Science. 1994; 264 (94269642): 1775-1778Crossref PubMed Scopus (176) Google Scholar]; we examined more than 50 tissue samples from all major organ systems, except for the heart and the urogenital system, data not shown). Here, we describe the one extraordinarily well preserved subcellular constituent in the Ice Man: the myelin sheaths. After millenia they still display outstanding structural and molecular integrity, a feature never before demonstrated from any ancient remains [3Doran GH Dickel DN Ballinger Jr, WE Agee OF Laipis PJ Hauswirth WW Anatomical, cellular and molecular analysis of 8,000-yr-old human brain tissue from the Windover archeological site.Nature. 1986; 323: 803-806Crossref PubMed Scopus (105) Google Scholar, 4Gerszten PC Martínez AJ The neuropathology of South American mummies.Neurosurgery. 1995; 36 (95319612): 756-761Crossref PubMed Scopus (29) Google Scholar]. Our approach required only extremely small tissue pieces from this unique specimen. Autopsy samples of 1 mm3[[5]Gunkel AR Freysinger W Thumfart WF Truppe MJ Gaber O Künzel KH et al.Otorhinolaryngologic computer-assisted biopsies of the Iceman.Arch Otolaryngol Head Neck Surg. 1997; 123 (97230798): 253-256Crossref PubMed Scopus (8) Google Scholar] were subjected to freeze-substitution [[6]Steinbrecht RA Müller M Freeze-substitution and freeze-drying.in: Steinbrecht RA Zierold K Cryotechniques in Biological Electron Microscopy. Springer Berlin Heidelberg, 1987: 149-172Crossref Google Scholar], followed by post-embedding immuno-gold and lectin-gold labelling [7Hess MW Mittermann I Luschnig C Valenta R Immunocytochemical localisation of actin and profilin in the generative cell of angiosperm pollen: TEM studies on high-pressure frozen and freeze-substituted Ledebouria socialis Roth (Hyacinthaceae).Histochem Cell Biol. 1995; 104 (96256062): 443-451Crossref PubMed Scopus (17) Google Scholar, 8Debbage PL A systematic histochemical investigation in mammals of the dense glycocalyx glycosylations common to all cells bordering the interstitial fluid compartment of the brain.Acta Histochemica. 1996; 98: 9-28Crossref PubMed Scopus (11) Google Scholar]. The femoral nerve and sympathetic trunk locally exhibited ultrastructural patterns strongly resembling myelin sheaths (see Figure 1): major dense lines alternate with intraperiodal lines, their periodicity being similar to present-day samples [[9]Malhotra SK Van Harreveld A Dorsal roots of the rabbit investigated by freeze-substitution.Anat Rec. 1965; 152 (66076481): 283-292Crossref PubMed Scopus (16) Google Scholar]. (Note that the Ice Man's cerebral cortex also showed recognisable myelin remains, but their chemical preservation was inferior to that of the femoral nerve; data not shown.) The remarkable resistance of myelin to freezing and thawing is already known from experimental studies in which only minor alterations of myelin periodicity were observed [[10]Fernéndez-Morén H Finean JB Electron microscope and low-angle x-ray diffraction studies of the nerve myelin sheath.J Biophys Biochem Cytol. 1957; 3: 725-748Crossref PubMed Scopus (72) Google Scholar]. One plausible interpretation relates to the specific chemical composition and biophysical properties of myelin. Low water content, with high proportions of lipids, together with the highly ordered configuration of the multi-lamellar myelin membranes may have limited ice nucleation. Note, however, that repeated freezing and thawing of the Ice Man during the preceding 5000 years (including the recovery, subsequent storage of the corpse at −6°C and sampling) also caused local structural breakdown and rearrangement of myelin components. This is indicated by nerve fibre remnants displaying disordered ultrastructure. Interestingly, structural breakdown was not necessarily associated with severe chemical alterations: adipocere — an indicator for certain post-mortal transformations [[11]Mayer BX Reiter C Bereuter TL Investigation of the triacylglycerol composition of iceman's mummified tissue by high-temperature gas chromatography.J Chromatogr B Biomed Sci Appl. 1997; 692: 1-6Crossref PubMed Scopus (23) Google Scholar] — is almost absent in the Ice Man's peripheral nerves, in contrast to its predominance in his other tissues (data not shown) and in other glacier corpses; moreover, structurally disordered myelin could be stained specifically. We revealed antigenic reactivity and lectin-binding capacity of major myelin constituents in the Ice Man's femoral nerve (see Figure 1). Moderate, though distinct, immunolabelling of both intact and disordered myelin was consistently obtained with antibodies recognising three groups of myelin constituents: galactocerebroside, sulfatide and other, structurally related, glycolipids (26% of total myelin lipid, detected with anti-GalC [12Ranscht B Clapshaw PA Price J Noble M Seifert W Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside.Proc Natl Acad Sci USA. 1982; 79 (82222209): 2709-2713Crossref PubMed Scopus (729) Google Scholar, 13Bansal R Warrington AE Gard AL Ranscht B Pfeiffer SE Multiple and novel specificities of monoclonal antibodies O1, O4, and R-mAb used in the analysis of oligodendrocyte development.J Neurosci Res. 1989; 24 (90096198): 548-557Crossref PubMed Scopus (386) Google Scholar]); P0-glycoprotein (50% of peripheral myelin protein [[14]Archelos JJ Roggenbuck K Schneider-Schaulies J Linington C Toyka KV Hartung HP Production and characterization of monoclonal antibodies to extracellular domain of P0.J Neurosci Res. 1993; 35 (93287152): 46-53Crossref PubMed Scopus (98) Google Scholar], antibody courtesy of H. Lassmann, Vienna); and myelin basic protein [[15]Sires LR Hruby S Alvord Jr, EC Hellström I Hellstöm KE Kies MW et al.Species restrictions of a monoclonal antibody reacting with residues 130 to 137 in encephalitogenic myelin basic protein.Science. 1981; 214 (82017194): 87-89Crossref PubMed Scopus (47) Google Scholar]. Reaction specificity was assessed by observing low background labelling on collagen fibrils and cells of the perineurium and endoneurium serving as internal negative controls, and by omitting primary antibodies or replacing them with irrelevant antibodies. Myelin oligosaccharides bound the lectin RCA120, confirming the presence of terminal galactosides as found, for example, on galactocerebroside and on the glycan chains of glycoproteins. Positive, though weaker, labelling with other lectins (WGA, HPA, and UEA-I) strongly suggested the presence of terminal and subterminal galactosides, fucosides and the polylactosamines forming intermediate links in the glycan structures. Furthermore, Con A, a sensitive indicator for disruption of these structures, did not react with myelin. Together, as far as antigenic and lectin-binding sites of major myelin constituents are concerned, the amino-acid and sugar composition, and even the spatial arrangement of these components, appear remarkably well preserved. Thus, the molecular architecture of myelin in the peripheral nervous system of the Tyrolean Ice Man can be considered essentially undamaged; furthermore, we depicted structures probably representing intact fragments of myelin sheaths, features never before reported from ancient remains. We are indebted to W. Platzer, O. Gaber and K.H. Künzel (Innsbruck) for tissue sampling, B. Afzelius and B. Fundin (Stockholm), H. Lassmann and R. Valenta (Vienna), G. Rutter (Hamburg), M. Pavelka, W. Pfaller, W. Schmidt and G. Sperk (Innsbruck) for helpful discussions, and C. Huckhagel, K. Gutleben and R. Haring for technical assistance. MW Hess, K Pfaller, PL Debbage and G Klima, Institute of Histology and Embryology, University of Innsbruck, Müllerstrasse 59, A-6010 Innsbruck, Austria. E-mail address for MW Hess (corresponding author): [email protected] E Kirschning and H Hohenberg, Heinrich-Pette-Institute for Experimental Virology and Immunology at the University of Hamburg, Martinistrasse 52, D-20252 Hamburg, Germany.

Microanatomical evidence is presented that the intercellular fluid (ICF) compartment of the central nervous tissue is lined entirely and exclusively by heavily glycosylated cells, with glycoconjugates exposed primarily at the apical cell surface, fronting the CSF or blood. On both common ependymal cells and on those specialised to form the choroid plexus epithelium, oligosaccharides coat the cilia and microvilli at the apical surface, and also the smoother lateral and basal cell surfaces. In the ependyma, folded and wrinkled structures seem especially associated with freely exposed carbohydrates. On cerebral endothelial cells, oligosaccharides coat the luminal surface densely and the basal surface lightly. The patterns of carbohydrate distribution thus vary from one cell type to another, but the different cell types all bear essentially the same set of oligosaccharides, variations being due largely to degree of terminal sialylation. Furthermore, the same set of oligosaccharides borders the brain in a broad spectrum of mammals, including pouched and placental mammals. In both epithelia and endothelia, the lectin binding sites visualised in fixed and embedded preparations were shown to be exposed likewise at the cell surfaces in unfixed tissues and so able to bind molecules present in the fluid (CSF or blood) bathing the cells in vivo. This phylogenetically ancient enclosure of the ICF compartment in a "ring of sugars" is suggested to relate to regulation of the central neuronal microenvironment.

Proteins antigenically cross-reactive with lectins were sought in the placenta by immunohistochemistry using polyclonal antibodies raised in rabbit against four well-known lectins: Concanavalin A, Wheat germ agglutinin, Ulex europaeus agglutinin, and Phaseolus vulgaris leukoagglutinin (PHA-L), as well as one antibody raised in goat against PHA-L. Even at high dilutions of the primary antibody, strong staining was obtained after short incubations, in patterns generally resembling those obtained for placental lectins by other means, such as those based on binding capacity for glycosylated probes. One of the immunohistochemical patterns distinguishes with great clarity between the trophoblast cell layers, thus relating to developmental and functional parameters; another localises PHA-L-immunoreactivity to the syncytiotrophoblast. These results underline the validity of the immunohistochemical screening as an approach in its own right. Both positive and negative controls were applied to the immunohistochemical methodology. These controls showed that the staining patterns obtained relate to the specificities of the primary antibodies employed; i.e. to lectins. The PHA-Llike cross-reactivity was analysed immunochemically. In electrophoretically separated and Western-blotted placental extracts there were found anti-PHA-L-binding fractions of apparent molecular weights 30 kDa, 58 kDa and 67 kDa. Control studies of the PHA-L antigen showed anti-PHA-L-binding fractions of approximate molecular weights 32 kDa and 60 kDa. The 30 kDa fraction from placenta and the 32 kDa fraction from PHA-L antigen bound lactosylated BSA but not fucosylated BSA. Taken together, the immunohistochemical and biochemical data reveal the presence in the placenta of lectins, one of which resembles PHA-L not only antigenically but also in molecular weight and in sugar-binding specificity.

Trophoblastic cells with their strictly controlled propensity for invasive growth hereby bear limited resemblance to malignant cells. Therefore, detailed description of molecular characteristics of this cell type in comparison to histologically related tumor cells may aid in defining physiologically relevant differences. In view of the potential importance of selective protein-carbohydrate interactions in recognitive processes, labelled neoglycoproteins were employed to evaluate systematically the presence and distribution of sugar receptors in tumor cells of chorionepithelioma and in trophoblastic cells of an early stage of gestation. Control reactions in glycohistochemistry, involving prolonged incubation, pH variation and use of nucleotides excluded the possiblity that glycosidases or glycosyltransferases grovern the sugar-specific binding to the tissue sections. Pronounced differences were detected in the expression of receptors (lectins) for α- and β-glucosides as well as for N-acetylgalactosamine and N-acetylglucosamine. Further differences were revealed by probing both types of tissue with lactosylated, fucosylated, xylosylated and sialylated carrier protein. Upon developmental maturation of the normal cells in placenta the extent of binding of the neoglyco-proteins decreased. The glycohistochemical differences between malignant and normally developing trophoblastic cells were found to be reflected in the alteration of expression of certain endogenous lectins, as ascertained by affinity chromatography and gel electrophoretic analysis. Consequently, we assume that the transient presence of certain endogenous lectins during early stages of gestation and their differential expression in relation to tumor cells of chorionepithelioma may be relevant for the special invasive and adhesive behavior of human trophoblastic cells.

The presence and location of specific endogenous lectins in the anterior epithelia of the porcine eye were determined by histochemical staining using biotinylated glycosylated carrier molecules. Endogenous lectins with specificities for galactosides, alpha-mannosides and beta-xylosides were present in the conjunctival epithelium, but the corneal anterior epithelium appeared essentially free of histochemically demonstrable endogenous lectins. The corneal posterior epithelium contained a rich and complex set of endogenous lectins. The transitional area between conjunctival and corneal anterior epithelia at the limbus cornea contained, in striking contrast to either of the epithelia bordering it, cells expressing endogenous lectins in considerable wealth and variety.

Protein-carbohydrate interactions are supposed to play a pivotal role in mediation of recognitive interactions, relevant to cellular interactions and transport. The brain microvasculature is the site of numerous cell-cell and cell-matrix recognitive interactions. Assuming carbohydrate-protein interactions play important physiological roles here, then specific carbohydrate-binding proteins should be prominent components of this microvasculature, in addition to the wealth of endogenous glycoconjugates reported by other authors. Human brain and brain tumor microvessels were analyzed histochemically for expression of endogenous sugar-binding proteins using a panel of biotin-conjugated, chemically glycosylated probes with specificities for alpha-/beta-D-galactosides and -D-glucosides, alpha-L-fucosides, alpha-D-mannosides, and beta-D-xylosides, and for expression of endogenous glycoconjugates using a panel of biotin-conjugated plant lectins with specificities for fucoside, galactoside, mannoside and glucoside moieties. Wax-embedded aldehyde- or Bouin-fixed tissues or acetone-fixed frozen sections were examined. Blood-brain barrier function was checked by ascertaining immunohistochemically the extravasation of serum albumin in these tissues. Microvessels in normal human brain tissues (with intact blood-brain barrier) contained abundant endogenous sugar-binding proteins with specificities for beta-galactosides, alpha-mannosides and beta-xylosides, and lesser amounts of proteins binding alpha-galactosides, alpha-fucosides and glucosides. Endogenous glycoconjugates bearing beta-galactoside, alpha-D-mannoside/alpha-D-glucoside or alpha-L-fucoside moieties were also abundant. In brain tumors (with defective blood brain barrier) the microvessels contained altered patterns of endogenous sugar-binding proteins, particularly noticeable in glioblastomas, in which there were notable alterations in galactoside-binding proteins in the microvessels.

Histochemical detection of binding sites for sulfated polysaccharides believed to be important mediators within recognitive interactions was carried out by application of biotinylated probes such as heparin, native and desulfated fucoidan, dermatan sulfate, and two types of carrageenans. The probes were derivatized by mild cyanogen bromide activation and subsequent aminoalkylation to allow incorporation of biotin, inserted with an epsilon-aminocaproic acid spacer to reduce charge-related and steric impediments. Specific labeling could be detected in different cell types of human placenta, dependent on the developmental stage. Sulfated polysaccharides bound predominantly to leucocytes in full-term placenta, whereas demonstration of specific binding sites in decidua, syncytiotrophoblasts, and cytotrophoblasts was restricted primarily to heparin and, less intensely, fucoidan, although not desulfated fucoidan. Heparin binding in the placenta after 8 weeks of gestation was reduced for epithelia that, at this stage of development, revealed carrageenan binding sites. Fucoidan binding was at this developmental stage measurable only for leucocytes. These results provide definite histochemical evidence for the presence and developmental regulation of expression of receptors for sulfated polysaccharides in different cell types of human placenta.

Fundamental cellular processes such as proliferation and differentiation and cell-matrix interactions are considered to be influenced by recognition mechanisms often involving specific binding of proteins to carbohydrate-bearing proteins or lipids (Gabius, 1987). The cell-cell and cell-matrix interactions at the endothelial surfaces of the brain vasculature are of interest because in normal brain these cells not only maintain the “blood-brain barrier” but also form the interface between the blood and the immunologically-privileged brain tissues; the regulatory mechanisms that operate here are profoundly distrubed in and near brain tumours. We have previously screened the human cerebral microvasculature by glycohistochemical methods, finding no systematic differences in lectin binding or (neo) glycoprotein binding sites between tumour-free brain tissues and tumour tissues (Debbage et al., 1987a, b, c).

Human placentas of different stages of development were histochemically analyzed for expression of endogenous sugar-binding proteins using a panel of biotin-conjugated, chemically glycosylated probes with specificity for beta-galactosides, alpha-galactosides, alpha-mannosides, alpha-fucosides and alpha-glucosides. Temporal differences in the expression of sugar-binding proteins and different patterns of staining of the component cell types of human placenta were discerned, especially pronounced for alpha-fucoside-specific binding in the trophoblast and alpha-glucoside-specific binding in fetal and maternal macrophages. Fractionation of salt and detergent extracts from human placentas by affinity chromatography on columns with immobilized carbohydrates or glycoproteins substantiated the histochemically detectable temporal changes on the basis of alterations in the pattern of individual sugar-binding proteins, as determined by gel electrophoresis under denaturing conditions. Analysis of the trophoblastic layer primarily disclosed the presence of several additional sugar-binding proteins (lectins) in comparison to full-term placenta. The presence and developmental changes of such endogenous sugar receptors may lead to specific carbohydrate-protein interactions of physiological significance with similarly developmentally regulated carbohydrated portions of glyco-conjugates, already detected in human placenta by plant lectins.

Conference Article| February 01 1982 Binding of concanavalin A and ricin 120 to parenchymal and non-parenchymal cells of rat liver L. G. WILLIAMS; L. G. WILLIAMS *Department of Biochemistry, University College, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar D. J. WILLIAMS; D. J. WILLIAMS *Department of Biochemistry, University College, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar D. S. O'DELL; D. S. O'DELL †Department of Zoology, University College, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar P. L. DEBBAGE P. L. DEBBAGE ‡Department of Anatomy, University College, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1982) 10 (1): 38. https://doi.org/10.1042/bst0100038 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation L. G. WILLIAMS, D. J. WILLIAMS, D. S. O'DELL, P. L. DEBBAGE; Binding of concanavalin A and ricin 120 to parenchymal and non-parenchymal cells of rat liver. Biochem Soc Trans 1 February 1982; 10 (1): 38. doi: https://doi.org/10.1042/bst0100038 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 This content is only available as a PDF. © 1982 Biochemical Society1982 Article PDF first page preview Close Modal You do not currently have access to this content.

The distribution of binding sites for ricin 120 in cell cultures of spinal cord from the chick embryo was examined. A characteristic pattern was observed, which remained similar in cultures fixed by a variety of methods. Light microscopy demonstrated that the most prominent staining was of small rounded or amoeboid cells. Electron microscopy showed that ricin was bound over their entire surfaces. The ultrastructural characteristics of these cells suggest their identification as microglia.

We report here the results observed when tubulin fluorescence immunohistochemistry is performed upon dissociated cultures of nervous tissue, principally of chick embryo spinal cord. When fixation includes nonpolar solvents or detergents, a uniform fluorescence is seen in neuron perikarya (with the exception of their nuclei), and the processes to which they give rise. Fixation with formaldehyde or glutaraldehyde alone, however, results in a discontinuous staining of neurites, and a less regular staining of their perikarya. The former pattern of response can be elicited if aldehyde fixation is followed by exposure to non-polar solvents. Such results are obtained both in thinly spread regions of the cultures, where neurons and their processes can easily be seen, and in the cell aggregates that also characterise them. Possible interpretations of these results are discussed.