Beiträge zur Morphologie der Peripheren Nervenfaser

Zusammenfassung Die Ansicht vonTheodor Boveri (1885), wonach sich dieSchwann-sche Scheide an denRanvierschen Schnürringen von der Außenseite des Markes auf dessen Innenseite umschlägt und so das Axolemm bildet, kann an Hand von formalinfixierten und gefärbten Präparaten (Serienlängsschnitten) bestätigt und durch entsprechende Mikroaufnahmen belegt werden.Der Begriff derSchwannschen Zelle (= Neurolemmzelle) ist demnach insofern jetzt weiter zu fassen, als diese einröhrenförmiges Gebilde darstellt, das die Markscheide in sich einschließt.Es wird deshalb erneut vorgeschlagen, dieSchwannsche Scheide fortan als äußeres Neurolemm und das Axolemm als inneres Neurolemm zu bezeichnen und auch die jeweils zugehörigen, bereits an anderer Stelle (R. Sulzmann 1955) beschriebenen Leisten dementsprechend zu benennen.Die Tatsache, daß die Schnürringe von Fasern nahezu gleicher Stärke in ein und demselben Faserbündel stets auf annähernd gleicher Höhe anzutreffen sind, wird lediglich als eineFolge gleicher Wachstumsgeschwindigkeit ohne besondere funktionelle Bedeutung aufgefaßt. Bezüglich der Funktion der Schnürringe wird vermutet, daß sie in erster Linie derErnährung der jeweils angrenzendenSchwannschen Segmente, insbesondere aber dem Stoffaustausch der betreffenden Achsenzylinderabschnitte dienen.Weiterhin wird angenommen, daß sich die Besonderheiten im Bau desRanvierschen Schnürrings, die zweifelsohne von großer praktischer Bedeutung sind, aus derEntwicklungsgeschichte der peripheren markhaltigen Nervenfaser ableiten lassen.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

The determination of the electron density profile of the human erythrocyte ghost membrane by small-angle x-ray diffraction.

Diffraction patterns of stacked hemolyzed erythrocyte ghosts in the wet state were recorded. Three orders of a surprisingly high first-order periodicity of 600 A were detected. The scattering curves were evaluated by the Q-function method, including lattice distortions of the one-dimensional multilamellar system. The resulting electron density profile of the membrane in the wet state is strongly asymmetric. It consists of an asymmetrical bilayer-type part and an excess of positive relative electron density at the inner, cytoplasmic, side of the membrane. The extension of the whole membrane profile in the wet state is 100--120 A. We suggest that the innermost positive density peak mainly represents the loosely bound protein components spectrin and actin, located at the cytoplasmic side of the membrane and sometimes seen as "fuzzy" material on electron micrographs.     

Mechanism of effect of aging on membrane transport in leaf strips of Centranthus ruber: Possible ethylene involvement in cutting shock

Cerulenin has been used to investigate the mechanism by which leaf strips of Centranthus ruber (L.) Lam et D.C. increase their capacity for solute uptake during a period of incubation in CaSO₄ (aging). -Aminoisobutyric acid was used to assess uptake capability. The leaf strips developed their uptake capacity for at least 8–10 h after excision. Cerulenin, if added to the aging medium immediately after cutting or at any time during the aging process, almost completely halted this development, but did not bring about loss of the uptake capacity already achieved. That cerulenin was specifically interfering with fatty-acid biosynthesis was indicated by the fact that it drastically depressed incorporation of labelled acetate into the lipid fraction but did not affect the incorporation of labelled alanine. Cycloheximide strongly inhibited the development of uptake capacity, but sensitivity to actinomycin D was not evident for at least 2 h. The results are consistent with the concept that slicing leads to immediate damage to tissue membranes and that aging is a process of membrane repair, involving renewed synthesis of membrane lipids and membrane proteins.The damage to membranes associated with cutting may involve wound ethylene. This is indicated by the following findings: Treatment of aged leaf strips with Ethrel (2-chloroethylphosphonic acid) resulted in drastic loss of their acquired uptake capacity. The strips recovered from such Ethrel-induced loss of uptake capacity while aging in CaSO₄ as they can after cutting. Cerulenin halted recovery of uptake capacity after ethrel treatment just as it did after cutting. Treatment of leaves before cutting with amino-ethoxyvinylglycine somewhat improved the uptake performance of leaf strips immediately after excision.     

Energization of the sugar transport mechanism in the plasmalemma of isolated mesophyll protoplasts

The mechanism of 3-O-methyl-d-glucose transport through the plasmalemma has been investigated in protoplasts isolated from the mesophyll of Pisum sativum L. var. Dan.Analysis of the fluxes after 50 minutes of uptake showed that the gradual decrease in slope of the net uptake curve with time was not due to any decline in uptake capacity; it represented the approach to flux equilibrium of a small compartment of the protoplast, probably the cytoplasm.The energy of activation for initial flux into this compartment was 20 kilocalories per mole between 17 and 27 C. Very high discrimination was shown with regard to sugar isomers. Light strongly promoted flux (by a factor of 2.5 in the case of methyl glucose). Initial flux showed sharply contrasting inhibitor sensitivity in the light and the dark. Light uptake was sensitive to the proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP), but stable for at least the first 10 minutes to the ATPase inhibitors quercetin, rutin, and diethylstilbestrol, as well as to arsenate. Dark uptake, on the other hand, was stable to CCCP but was immediately depressed by quercetin, rutin, diethylstilbestrol, and arsenate.Protoplasts which received a light pretreatment before incubation in the dark took up methyl glucose at the accelerated light rate for the first 7 minutes. Moreover, the light pretreatment sensitized subsequent initial dark uptake to CCCP, and conferred on it the stability to ATPase inhibitors and arsenate characteristic of light uptake. After about 7 minutes the characteristic inhibitor responses of dark uptake were resumed.It is proposed that more than one mode of energy-coupling for sugar transport may operate in these protoplasts.     

Hysteresis in the responses of membrane potential, membrane resistance, and growth rate to cyclic temperature change

Measurements of electrical potential, membrane resistance, and elongation rate have been carried out on the developing pollen tube of Oenothera drummondii.     

N-glycosylation is required for human CD2 immunoadhesion functions.

The T-lymphocyte glycoprotein receptor, CD2, mediates cell-cell adhesion by binding to the surface molecule CD58 (LFA-3) on many cell types including antigen presenting cells. Two domains comprise the CD2 extracellular segment, with all adhesion functions localized to the amino-terminal domain that contains a single N-glycosylation site at Asn65. We have defined an important role for the N-linked glycans attached to Asn65 of this domain in mediating CD2-CD58 interactions and also characterize its N-glycotype structure. Analysis of deglycosylated soluble recombinant CD2 as well as a mutant transmembrane CD2 molecule containing a single Asn65-Gln65 substitution demonstrates that neither deglycosylated CD2 nor the mutant CD2 transmembrane receptor binds CD58 or monoclonal antibodies directed at native CD2 adhesion domain epitopes. Electrospray ionization-mass spectrometry demonstrates that high mannose oligosaccharides ((Man)nGlcNAc2, n = 5-9) are the only N-glycotypes occupying Asn65 when soluble CD2 is expressed in Chinese hamster ovary cells. Based on a model of human CD2 secondary structure, we propose that N-glycosylation is required for stabilizing domain 1 in the human receptor. Thus, N-glycosylation is essential for human CD2 adhesion functions.     

Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. Novel fragments produced by trifluoroacetic acid.

Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.     

Characterization of glycosphingolipids by supercritical fluid chromatography-mass spectrometry

Gangliosides have been characterized by supercritical fluid chromatography-chemical ionization mass spectrometry (SFC-CIMS) as permethyl and pertrimethylsilyl derivatives, using carbon dioxide as the SFC mobile phase and CI reagent gas. Ganglioside classes and ceramide heterogeneity within each class are well resolved by SFC. Direct SFC-interfacing allows the analytical manipulations of single-ion monitoring, total-ion plots, background subtraction, library searches, and spectral reconstruction algorithms. Addition of ammonia to the CI ion chamber (NH3 as a CI reagent gas) yields abundant molecular-weight-related ions, (MH)+ and (MNH4)+ from analyte derivatives. Substitution of methanol for ammonia yields considerable parent-ion fragmentation, providing structural information on carbohydrate sequence, fatty acid, and sphingoid components. Under these latter conditions a unique alpha-cleavage fragment is observed which differentiates fatty acid from sphingosine heterogeneity. For ganglioside samples, the carboxyl group of neuraminyl residue(s) have been esterified with pentafluorobenzyl bromide and the products analyzed by negative ion chemical ionization MS. This modification improves chemical selectivity and greatly enhances detecting sensitivity. These "soft" ionization conditions provide abundant molecular-weight-related anions for collision-induced dissociation and subpicogram detection.