Calcium at the surface of cardiac plasma membrane vesicles: Cation binding,surface charge screening,and Na−Ca exchange

Summary Calcium binding and Na–Ca exchange activity were measured in isolated cardiac plasma membrane vesicles under various ionic conditions. A model was developed to describe the Ca binding characteristics of cardiac sarcolemmal vesicles using the Gouy-Chapman theory of the diffuse double layer with specific cation binding to phospholipid carboxyl and phosphate groups. The surface association constants used for Ca, Na, K and H binding to both of these groups were 7, 0.63, 0.3 and 3800m ^–1, respectively. This model allows the estimation of surface [Ca] under any specific ionic conditions. The effects of the divalent screening cation, dimethonium, on Ca binding and Na–Ca exchange were compared. Dimethonium had no significant effect on Ca binding at high ionic strength (150mm KCl), but strongly depressed Ca binding at low ionic strength. Dimethonium had no significant effect on Na–Ca exchange (Na-inside dependent Ca influx) at either high or low ionic strength. These results suggest that the Ca sites of the Na–Ca exchanger are in a physical environment where they are either not exposed to or not sensitive to surface [Ca].     

A new pollen type,C-banded and fluorochrome counterstained chromosomes,and evolution inGuatteria and related genera (Annonaceae)

Guatteria, Guatteriopsis, Guatteriella andHeteropetalum share the same conspicuous pollen type which is new for theSpermatophyta. It is zonoaperturate with a folded aperture region and an extremely reduced exine. First chromosome counts and karyotype analyses forGuatteriopsis (4 species investigated) andGuatteriella (1 species) are identical with those ofGuatteria (19 species seen): 2n = 28. The genome is characterized by diploidization and partly telocentric chromosomes. Sequentially Giemsa C- and fluorochrome banded chromosomes and interphase nuclei are described. The cuticular folding pattern is distinct forHeteropetalum only. Growth forms and ecology are reported for many species. The evolutionary pattern of theGuatteria group is discussed and compared with other genera and families.     

Three classes of signalling molecules on B-cell membranes

The question of whether surface immunoglobulin and Ia molecules have a signalling function in helper T cell-dependent activation of B cells has been evaluated. Two sources of B cells have been used, one a purified population of hapten-binding B cells, the other a B-cell lymphoma, CH12, with known antigen specificity. Evidence is presented that both immunoglobulin and Ia molecules are receptors actively involved in the initial activation of resting B cells. Nevertheless, the requirements for ligand binding to either receptor can be bypassed under appropriate conditions, and the implications of this result for the function of these molecules is discussed. With respect to B-cell Ia, the authors present data that demonstrate two distinct functions of this molecule, one as a restricting element for T-cell activation, the second as a signalling receptor for B-cell excitation. On the CH12 surface, the I-A molecule fulfills the former function, but T-cell interactions with I-A fail to result in B-cell stimulation, suggesting that B-cell Ia may limit helper T cell-B cell interactions. We suggest that the binding of antigen surface immunoglobulin and binding of helper T-cell receptors to the appropriate Ia molecule(s) results in the activation of genes that encode for a third class of membrane B-cell receptors, those that bind B-cell stimulating factors.     

Cell surface changes in preimplantation mouse embryos during compaction investigated using FITC conjugated lectins after proteolytic enzyme treatment

Summary Fluorescein-conjugated lectins were used to examine the reappearance of glycoproteins on the surface of 8-cell mouse embryos after treatment with proteolytic enzymes. Embryos were decompacted in calcium free medium, treated with various proteases and the process of recompaction monitored. The most effective enzymes in delaying recompaction were subtilopeptidase A and proteinase K at 1 mg/ml; the initiation of recompaction was delayed by about 5 h and 90% recompaction by 14–18 h. Papain and -chymotrypsin were only effective in the absence of calcium. The reappearance of receptors for fluorescein-conjugated Con-A, MPA, RCA-I, FBP, BSL-II and DBA was examined photometrically at 0,8–10 and 17–18 h after proteinase K treatment. There was an increase in binding of MPA, RCA-I, FBP and BSL-II in control embryos during the period of the experiment, between approx. 61 and 80 h post coitum in which embryos passed from the 8-cell stage to the 16–32 cell stage. Con-A binding remained the same and that of DBA decreased. By the time that 50% of enzyme treated embryos had recompacted (8–10 h) binding of Con-A was similar to control embryos. Binding of FBP had almost reached control levels while that of BSL-II, DBA, RCA-I and MPA had reached 60–85% of control levels. When embryos were fully compact (17–18 h) Con-A, FBP and DBA were bound in equal or slightly greater amounts to enzyme treated as to control embryos, and receptors for BSL-II, MPA and RCA-I had recovered almost to control levels. The results clearly show that the recovery of glycoproteins on the surface of 8–16 cell embryos parallels recompaction, providing further evidence for the role of these molecules in compaction.     

Cross-linked collagen surface for cell culture that is stable,uniform, and optically superior to conventional surfaces

Summary A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed. This work was supported by the Leopold Schepp Foundation, the Dysautonomia Foundation, National Institutes of Health Grants NS14768 and NS11237, and Institutional Core Grant HD06276.     

Cytochemical and scanning electron-microscopic analysis of apoptotic cells and their phagocytosis in mucoid epithelium of the mouse stomach

Summary The formation of apoptotic cells and their phagocytosis by viable neighbouring cells in the gastric epithelium of 2-to 6-day-old mice was analysed. In order to observe the topographic relationship between apoptotic and normal epithelial cells using scanning electron microscope, the critical-point dried tissues was cracked before coating with gold. Cytochemical methods for the identification of surface carbohydrates and different tracers for apical and lateral cell membranes were applied for the analysis using the transmission electron microscope. Apoptotic cells were found on apical and lateral surfaces; this indicates the presence of tight connections with viable cells at some points. Ruthenium red strongly stained all accessible surfaces of normal cells and of apoptotic bodies. The quantity of neutral mucosubstances, as revealed by staining with tannic acid-uranyl acetate, seemed to decrease in the glycocalyx of apoptotic cells. The scanning and transmission electronmicroscopic results suggest that the phagocytotic vacuoles arise at the lateral side of the cells. The phagocytotic activity is not dependent upon a definite differentiation step of the mucoid cell.     

Structural and chemical characterization of S-layers of selected strains ofBacillus stearothermophilus andDesulfotomaculum nigrificans

The structures, amino acid- and neutral sugar compositions of the crystalline surface layers (S-layers) of four selected strains each ofBacillus stearothermophilus andDesulfotomaculum nigrificans were compared. Among the four strains of each species a remarkable diversity in the molecular weights of the S-layer subunits and in the geometry and constants of the S-layer lattices was apparent. The crystalline arrays included hexagonal (p6), square (p4) and oblique (p2) lattices. In vitro self-assembly of isolated S-layer subunits (or S-layer fragments) led to the formation of flat sheets or open-ended cylindrical assembly products. The amino acid composition of the S-layers exhibited great similarities and was predominantly acidic. With the exception of the S-layers of two strains ofB. stearothermophilus (where only traces of neutral sugars could be detected), all other S-layer proteins seemed to be glycosylated. Among these strains significant differences in the amount and composition of the glycan portions were found. Based on this diversity interesting questions may be asked about the biological significance of the carbohydrate units of glycoproteins in prokaryotic organisms.     

Expression of epidermal growth factor receptor and associated glycoprotein on cultured human brain tumor cells

The expression of epidermal growth factor (EGF-R) in normal glial and glioma cells grown in culture was examined by using several independent assays. Immunoprecipitation with the monoclonal antibody R1 of extracts from metabolically labeled glial and glioma cells revealed a protein of Mr approximately 170,000, with a migration in sodium dodecyl sulfate-polyacrylamide gels identical to the EGR-R of A431 epidermal carcinoma cells. Furthermore, in the majority of glioma extracts, a protein of Mr approximately 190,000 was specifically immunoprecipitated by this antibody. Similar results were obtained by immunoblotting with a second antibody directed against a synthetic peptide in the sequence of the v-erb-B oncogene. In cell lines expressing both proteins, each was specifically phosphorylated on tyrosine in immune complex kinase assays. The majority of glioma cells bound between 40,000 to 80,000 125I-labeled epidermal growth factor molecules per cell. These results suggest that the expression of EGF-R is common in cultured human glioma cells. In addition, a structurally related protein, is expressed in some of these cells.     

Leishmania mexicana amazonensis: Surface charge of amastigote and promastigote forms

The surface charge of Leishmania mexicana amazonensis was evaluated by means of the binding of colloidal iron hydroxyde particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, visualizated by electron microscopy and by direct measurements of the electrophoretic mobility of cells suspended in solutions of different pH. The following forms of the parasite were analysed: amastigotes (surrounded or not by the membrane of the endocytic vacuole, isolated from lesions), transitional forms, and infective (5 passages) and noninfective (176 passages) promastigotes. The results obtained indicate that the surface of L. m. amazonensis contains both negatively and positively charged dissociating groups and that changes occur in the surface charge during amastigote-promastigote transformation. Treatment of the parasite with neuraminidase significantly reduced the electrophoretic mobility of the cells. Neuraminidase-treated cells recovered their normal electrophoretic mobility when incubated for 8 hr in fresh culture medium by a process that is inhibited by puromycin.     

Cell Surface Sialoglycoproteins of Cultured Rat Cerebellar Interneurons

Abstract: The sialoglycoproteins of cultures of relatively pure rat cerebellar interneurons were labelled by NaIO₄ oxidation/NaB ³H₄ reduction. The labelled molecules were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate followed by fluorography. Faint labelling could be detected in three components if cells were labelled without any oxidation. In young cultures, oxidation by galactose oxidase alone failed to reveal any additional bands. After oxidation by NaIO₄ or galactose oxidase in the presence of neuraminidase, many more components were labelled. After NaIO₄ oxidation, about 80% of the cell-associated radioactivity could be removed by treating the cells with neuraminidase, which left the cells more than 95% viable. The majority of the bands seen after neuraminidase treatment were substantially reduced when compared with untreated controls, supporting a surface localisation of these molecules. Reproducible developmental changes were seen in the profiles of bands labelled by NaIO₄/NaB ³H₄ in time course studies of cultures up to 8 days in vitro . Some bands became more prominent, and others disappeared. The gel profiles of the neuron cultures were quite distinct from those of cerebellar astrocyte cultures, which contain all the cell types likely to be contaminants of the neuron cultures.