Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Mechanisms of cellular detoxication, nuclear aluminum concentration and hepatocyte protection after experimental overload in rats]

A single injection of a massive dose of Al-gluconate (4 mg.kg-1) into the saphenous vein of the rat did not provoke any ultrastructural damage in the liver cells, from 2 min. to 8 days after the injection. Al hepatocytes overload appeared only in nuclei and not in nuclei and not in lysosomes, contrarily to chronic intoxications. Nuclear Al concentration concerned only a small fraction of the quantity injected; another part was sequestered by the macrophage system after precipitation in the blood as phosphates chemically transformed during their incorporation in lysosomes. This effective detoxication mechanism which functioned probably after a first absorption by the hepatocyte, was likely to depend upon the form of gluconate and would explain the resistance of liver cells.     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and identification of β 1 → 2-d-glucans in extracellular polysaccharides of Rhizobium meliloti strain L5-30

Abstract Exopolysaccharides have been found as bound to R. meliloti cells (strain L5-30) or free in the culture medium; they consist of acidic and neutral polysaccharides. Bound and soluble neutral polysaccharides were identified as β 1 → 2 linked glucans with an average M _r value of 4000 to 6000.     

Characterization of the anomalous infection and nodulation of subterranean clover roots by Rhizobium leguminosarum 1020

Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis. Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain. R. leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A. This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs. However, R. leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs. The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence. The anomalous nodulation was inhibited by nitrate. Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis. The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones. Infection threads were readily found in the infection zone of the nodule. However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R. trifolii 0403. This anomalous nodulation of subterranean clover by R. leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.     

Characterization of the antiproliferative activity of Xylopia aethiopica

Background

Xylopia aethiopica, a plant found throughout West Africa, has both nutritional and medicinal uses. The present study aims to characterize the effects of extracts of this plant on cancer cells.

Results

We report that X. aethiopica extract prepared with 70% ethanol has antiproliferative activity against a panel of cancer cell lines. The IC50 was estimated at 12 ??g/ml against HCT116 colon cancer cells, 7.5 ??g/ml and > 25 ??g/ml against U937 and KG1a leukemia cells, respectively. Upon fractionation of the extract by HPLC, the active fraction induced DNA damage, cell cycle arrest in G1 phase and apoptotic cell death. By using NMR and mass spectrometry, we determined the structure of the active natural product in the HPLC fraction as ent-15-oxokaur-16-en-19-oic acid.

Conclusion

The main cytotoxic and DNA-damaging compound in ethanolic extracts of Xylopia aethiopica is ent-15-oxokaur-16-en-19-oic acid.     

Localisation of caveolin in mammary tissue depends on cell type

Caveolins, components of caveolae, are expressed in mammary tissue. In order to determine whether caveolins are present in different mammary cell types and whether their localisation depends on the physiological stage or species, cav-1 and cav-2 were characterised by immunoblotting in mammary tissues from the mouse, ewe and rabbit and localised, by immunofluorescence and electron microscopy, in mammary tissues from the mouse and ewe. At all the physiological stages studied, cav-1 and cav-2 were present in endothelial and myoepithelial cells in which flask-shaped caveolae were abundant. However, labelling of cav-1 and cav-2 associated with small vesiculo-tubular structures (including those close to lipid droplets) was low in epithelial cells. To study the possible association of cav-1 with lipid droplets, lactating ewe mammary fragments were treated in vitro with brefeldin A. This treatment did not modify the association of cav-1-labelled structures with lipid droplets. Finally, HC11 and MCF-10A mammary cell lines were treated with oleic acid. The total quantity of cav-1 was little affected by the treatment, although the lipid droplet labelling of cav-1 was amplified in MCF-10A cells. Thus, the synthesis and localisation of caveolins are mostly dependent upon the cell types of mammary tissue and upon their state of differentiation.     

Lactoferrin at basal side of mouse mammary epithelium derives in part from stroma cells

Lactoferrin is synthesized by glandular epithelial cells and neutrophils and is also present on both sides of the mammary epithelium. We have studied the origin of lactoferrin detected in the various compartments of mouse mammary tissue. As revealed by immunogold electron microscopy, lactoferrin is present in mammary epithelial cells and in the basal region of the epithelium, associated with connective tissue and stroma cells at all physiological stages studied. A perturbation of protein synthesis or transport after in vitro treatment with cycloheximide or brefeldin A does not abrogate lactoferrin labelling in the basal region of the epithelium. The expression of lactoferrin has also been observed in the fat pads of mammary glands from mice surgically depleted of epithelial cells. The sealing of one teat for 24 h is accompanied by an increase in both the number of stroma cells and the labelling of myoepithelial cells. Thus, the lactoferrin present in the interstitial space of the mouse mammary epithelium originates in part from stroma cells. Possible roles of lactoferrin at the basal side of the mammary epithelium are discussed.