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Effect of hemin on the physiology and virulence of Bacteroides gingivalis W50.

Bacteroides gingivalis W50 was grown in a chemostat under steady-state conditions at pH 7.5 +/- 0.2 and a constant growth rate of 6.9 h for periods of up to 6 weeks (146 bacterial generations) in a complex medium. Hemin was capable of limiting the growth of cells up to a concentration of approximately 0.5 micrograms/ml since higher concentrations of hemin did not increase cell yields; cells grew in the absence of exogenously added vitamin K1. Only a limited number of amino acids was metabolized during growth, but because none of these was totally depleted, the limiting nutrient under hemin excess conditions was probably a peptide. A range of fermentation products was produced under all conditions of growth; higher concentrations of cytotoxic metabolites such as propionate and butyrate were formed under hemin excess conditions, although more ammonia was released under hemin limitation. When viewed by electron microscopy, cells grown under hemin limitation appeared to be either coccobacillary or short rods and possessed few fimbriae per cell, but large numbers of extracellular vesicles could be seen both surrounding the cell surface and free in the environment. In contrast, cells grown under hemin excess conditions were more commonly coccus shaped and were more heavily fimbriated but had fewer extracellular vesicles. Marked differences were found in the susceptibility of mice to infection with cells grown under different concentrations of hemin. Cells transferred to media without any added hemin were avirulent, whereas those grown under conditions of hemin limitation (0.33 and 0.40 micrograms/ml) produced a 20 and 50% mortality in mice, respectively. In contrast cells grown under hemin excess always caused 100% mortality in mice, although this virulence was dose dependent. When virulent, the bacteria caused an extensive, spreading infection with necrosis of the skin and subcutaneous tissues. Collagen disintegration was seen histologically, implying a role for collagenase production in the pathogenicity of these bacteria.

Animals↗

Early plasma membrane events occurring in ultraviolet-B-induced apoptosis.

Whereas nonsolar ultraviolet C radiation primarily affects nuclei (i.e., where it is absorbed by nucleic acids) of eukaryotic cells, ultraviolet radiation of long (320-380 nm) wavelengths (ultraviolet A) and intermediate (290-320 nm) wavelengths (ultraviolet B) primarily affects lipid membranes. We have previously demonstrated that ultraviolet B irradiation alters the surface architecture of human B cells and impairs expression of an erythroid growth factor on their surface and on extracellular vesicles. Here, we examined the effects of ultraviolet B irradiation on the capacity of Chinese hamster ovary cells to undergo the process of exfoliation, and on the capacity of Chinese hamster ovary cells transfected with flt3/flk2 cDNA to express the cytokine flt3/flk2. Our results indicate that the rate of release of shed vesicles from untransfected Chinese hamster ovary cells is decreased after one to two h, at a time when there is electron microscopic evidence for retention of vesicles at the cell surface. These changes at the cell surface precede all other apparent morphological changes (including DNA condensation in the nucleus, swelling of the mitochondria and appearance of apoptotic bodies). Furthermore, plasma membranes and shed extracellular vesicles from ultraviolet B irradiated Chinese hamster ovary cells that have been transfected with flt3/flk2 cDNA fail to express the protein.

Animals↗

Ultrastructural characterization of extracellular matrix vesicles in the mineralizing fronts of apical cementum in cats.

Fine structure and the organic-inorganic relationship in mineralized fronts of cementum was studied with post-embedding demineralization and staining method. Typical matrix vesicles were observed between apical cementum and cementoblasts. Some vesicles were enclosed by a unit membrane; others contained needle-like crystals. Cementum crystals were found on unstained sections as filament- and needle-like structures which disappeared completely on EDTA treatment; similarly-shaped structures re-appeared following uranyl and lead staining. These findings suggest that matrix vesicles might act as initiators of additional cementogenesis and that crystal ghosts seen in cementum consist of organic materials.

Animals↗

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342.

Dictyostelium discoideum cells are highly resistant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size > 21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism.

Animals↗

Possible role of extracellular matrix vesicles in initial calcification of healing rachitic cartilage.

Fluid (20-30 nl) was aspirated by a modified renal micropuncture technique from vitamin D-, phosphate-deficient rats. The fluid revealed a mineral forming agent which could be sedimented at 140,000 X g for 8 hours, was resistant to acid demineralization, but was destroyed by heating, freezing and thawing as well as sonication, and blocked by phospholipase A at 10-5 M but not at 10-7 M. Electron microscopic study of the fluid sediment revealed images consistent with matrix vesicles. These data are consonant with the view that matrix vesicles, their remnants, or closely associated structures comprised the mineral forming agent.

Animals↗

Monoclonal antibodies to lipopolysaccharide of four oral bacteria associated with periodontal disease.

Periodontal disease is a common inflammatory disease which erodes the supporting structures of the teeth, and is initiated by a subgingival infection with selected Gram-negative bacteria. Monoclonal antibodies (mAb) to lipopolysaccharide (LPS) of four periodontal pathogens, A. actinomycetemcomitans, P. intermedia, F. nucleatum and P. gingivalis were examined for specificity and their ability to bind these pathogens in a particle concentration fluorescence immunoassay (PCFIA). The mAb selected were specific for their homologous bacteria and when tested against a large battery of other bacteria, including 16 genera and 46 species, were found not to cross-react with heterologous species. When each of the mAb was challenged with 40 or more homologous freshly isolated bacteria, more than 90% were positive. Non-cellular antigens in the form of soluble LPS and extracellular vesicles were examined for their ability to bind to assay components and alter the apparent results of the assay. LPS was found to have potential as an interfering agent if bound to assay components prior to sample treatment, but this non-specific binding was significantly reduced when a surfactant was added to the buffers. Extracellular vesicles had no significant effect on the estimation of P. gingivalis by the assay.

Aggregatibacter actinomycetemcomitans↗

Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells.

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.

Animals↗

The effect of bone injury on extracellular matrix vesicle proliferation and mineral formation.

Removal of tibial bone marrow in rats is followed by primary bone formation, resorption and marrow restitution. The first week of healing is characterized by partially calcified trabeculae. After 2 weeks, a higher degree of calcification and partial resorption are observed. The third week is characterized by massive resorption of the trabeculae, which are replaced in the fourth week by new bone marrow tissue. This model was used to study primary calcification. Transmission electron micrographs of the young bone revealed osteoblasts, matrix vesicles and calcified fronts. The different vesicular types were defined as 'empty', 'amorphous', 'crystal', and 'rupture'. The vesicles were studied on days 3, 6, 8, 12, 14, 18, 21, 23 and 28 after injury. The mean diameters of most vesicles ranged between 100.3 and 121.9 nm, and their mean distance from the calcified front was less than 976.6 nm. Vesicular density, calculated as number per 10 m2, increased on the eighth day and decreased from the fourteenth day onwards. Highest diameter values were recorded on the sixth day, and decreased onward. Vesicular distance from the calcified front decreased continuously. Distribution of vesicle number, diameter, and distance in each class showed that numbers of empty and amorphous vesicles decreased and of crystal and rupture increased throughout the experiment. Distances from the calcified front and vesicular diameters varied as follows: 'rupture', 'crystal', amorphous', and 'empty', the 'rupture' type being the closest to the front and of the largest diameter. The results confirm the hypothesis that the cell is responsible for the secretion of electron lucent vesicles that accumulate Ca and Pi to form amorphous calcium phosphate complexes that convert to hydroxyapatite. Crystal growth is followed by membrane rupture.

Animals↗

Antibodies to 100- and 60-kDa surface proteins inhibit substratum attachment and differentiation of rodent skeletal myoblasts.

A rabbit polyclonal antiserum was raised against membrane vesicles shed from the surface of fusing L6 rat myoblasts. In immunoblots the antiserum recognized fibronectin, a protein of approximately 100,000 Da (100-kDa), and a protein of approximately 60,000 Da (60 kDa). If added prior to cellular alignment, immunoglobulins from this serum inhibited fusion of both rat (L6) and mouse (C2) myoblasts in a dose-dependent fashion. To determine which component of this serum was responsible for fusion inhibition, antibodies against fibronectin, the 100- and 60-kDa proteins were microaffinity purified and tested, individually, for their effects on myoblast fusion. Antibodies against fibronectin had no effect on fusion. Antibodies against the 100-kDa protein released most cells from the substratum. Antibodies against the 60-kDa protein completely inhibited fusion. Fusion inhibition was accompanied by a corresponding inhibition of expression of two differentiation markers, creatine phosphokinase and the acetylcholine receptor. The 60-kDa protein was found, by immunoblot analysis, in smooth muscle-like cells (BC3H1 cells) and in variant L6 cells that do not differentiate and do not fuse. However, in the differentiation incompetent cells, the 60-kDa antigen appeared to be present in reduced amount. Indirect immunofluorescence of unpermeabilized L6 cells revealed alterations in the distribution of all three antigens during development. Fibronectin first appeared in long fibrillar arrays above the surface of cells that were beginning to align and fuse; fibronectin was not present on myotubes. The 100-kDa protein was seen initially in prominent fibrillar projections at the tips of prefusion myoblasts. During fusion the antigen was observed at sites of cell-cell contact and on extracellular vesicles. The 100-kDa protein appeared to be less abundant on myotubes. The 60-kDa protein first appeared in regions of cell-cell contact on cells that were beginning to align and fuse. As. fusion progressed, the 60-kDa protein was also found in extracellular vesicles. The 60-kDa protein was not observed on myotubes. As a result of this study we have identified two previously undescribed cell surface proteins involved in rodent skeletal myogenesis. The first is an approximately 100-kDa protein involved in early interactions of skeletal myoblasts with their substratum. The second is an approximately 60-kDa protein involved in myoblast differentiation. Both proteins are shed from the myoblast surface during myotube formation.

Animals↗

Modulation of vesicle shedding in 8701 BC human breast carcinoma cells.

Vesicles, shed in the extracellular medium by several kinds of normal and tumoral cells, are known to play important roles in cell-cell and cell-matrix interactions and to participate in mechanisms by which tumoral cells acquire metastatic capability and evade immune surveillance. Regulation of the shedding phenomenon and molecular mechanisms involved in extracellular vesicle production are not known and are the subject of this investigation. Fetal calf serum stimulated shedding short after its addition and its stimulatory effect was dose dependent. This effect was reduced after gelatin-Sepharose adsorption indicating a possible involvement of gelatinases on its stimulatory effect. This conclusion was confirmed by the inhibitory effect of bathophenanthroline. Shedding of membrane vesicles decreased after treatment with all trans retinoic acid, a molecule known for its capability to induce cell differentiation. Brefeldin A, an inhibitor of intracellular vesicle movements, and methylamine, an inhibitor of exocytosis, did not abolish shedding. Quercetin, an inhibitor of phosphatidyl inositol 4 kinase and 1,4 phosphatidyl inositol 5 kinase, and 8-Cl-cAMP, a site selective cAMP analogous which induces growth inhibition and differentiation, significantly decreased the amount of shed vesicles.

8-Bromo Cyclic Adenosine Monophosphate↗

Extracellular matrix vesicles in rat bone after parathyroidectomy.

The enzymatic activity of bone matrix vesicles from parathyroidectomized rats was determined and compared to the activity of vesicles from sham operated and normal animals. The vesicles were isolated from the alveolar bone by collagenase digestion and differential centrifugation and further purified on a discontinuous sucrose density gradient. The amount of extractable protein and the activity of alkaline phosphatase, acid phosphatase, and ATPase in the vesicle fractions thus obtained did not differ significantly from the values characteristic of preparations from control rats. It may therefore be suggested that parathyroid hormone depletion and the associated hypocalcemia have no significant effect on the occurrence and phosphatase activity of bone matrix vesicles.

Acid Phosphatase↗

A comparative study on the occurrence and activity of extracellular matrix vesicles in young and adult rat maxillary bone.

The occurrence of matrix vesicles in the maxillary bone of normal young and adult rats was demonstrated by both ultrastructural and enzymatic studies. Transmission electron microscopy revealed that the vesicles are invested in trilaminar membranes. Occasionally, crystals were found both within the vesicles and in the surrounding matrix. Separated fractions of vesicles, membranes, and cells were studied biochemically. The amounts of vesicular protein and enzymatic activity per gram bone in the adult rats were significantly lower than in the young. This coincides with the higher number of vesicles observed in the TEM specimens of young rats when compared to that in the old ones. The specific activities of all enzymes examined in the three bone fractions obtained from both young and adult rats were similar. This indicates that no significant difference exists in the enzymatic characteristics of matrix vesicles from the maxillary bone of young and adult rats.

Acid Phosphatase↗