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PubMed1977

[Extracellular material of some representatives of the genus Corynebacterium (the electron microscopic aspect)].

At the active developmental phases (up to 2 days) cells of toxigenic and nontoxigenic corynebacteria form extracellular vesicle-like material of two types which can be revealed both on whole cells set off by metal and stained negatively, and in ultrathin sections. Extracellular material of the first type is a derivative of an extensive membranous coat of corynebacteria and is formed as a result of its fragmentation. Vesicles of this type are devoid of electron microscope-dense content, have no tendency to coalescence, and fail to promote cell agglutination. Extracellular material of the second type is primarily formed by local thickening of the surface wall layer limited by its external dense layer and the main massif. These thickenings are filled with a microgranular substance of medium electron optic density; after accumulation this substance is released into the external environment. Vesicles of this type promote cell agglutination. Undoubtedly, extracellular material of the second type has a direct relation to the cell metabolic processes. Extracellular material of both types is encountered in all the cultures grown both on hard and in fluid nutrient media. However, in the latter case the process is apparently much more intensive. There is strict correlation between the morphology of the extracellular material and strain signs of the culture, although material of the second type is found to prevail in the cells from cultures possessing toxigenic activity.

Agglutination↗
PubMed1987

The Effect of Extracellular Components from Colletotrichum lindemuthianum on Membrane Transport in Vesicles Isolated from Bean Hypocotyl.

Extracellular components released from mycelia of the alpha and beta races of the bean pathogen, Colletotrichum lindemuthianum, inhibited proton uptake in sealed vesicles prepared from bean hypocotyls. Differential sensitivity of ATP-driven proton transport to nitrate, vanadate, N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, and oligomycin suggested the vesicles were enriched for tonoplast. Anion stimulation of proton transport, by enhancement of ATPase activity and dissipation of the membrane potential, was consistent with this conclusion. Although fungal components inhibited the formation of a pH gradient, the membrane potential was unaffected and the ATPase activity slightly stimulated. These data suggest that the fungal components produce an electroneutral proton exchange. Proton transport in Dark Red Kidney bean tonoplast vesicles was inhibited by mycelial preparations from the incompatible alpha race and compatible beta race. Elicitor activity, however, was greater in the alpha race fractions. Elicitor purified from alpha race culture filtrate did not inhibit proton transport in vesicles isolated from Dark Red Kidney bean. Consequently, elicitor activity need not be associated with an ability to impair tonoplast function.

Journal Article↗
PubMed1996

Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C.

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)2D3 and 24,25-(OH)2D3. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1 alpha, 25-(OH)2D3 (1,25) and 24R,25-(OH)2D3 (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1, 25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKC alpha and zeta as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [gamma 32 P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A1 (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25 but not 1, 25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase.

24,25-Dihydroxyvitamin D 3↗
PubMed1991

Presence of vesicles containing lactate dehydrogenase in the dentin of bovine tooth germs.

The dentin was removed from bovine tooth germs, followed by the separation of the extracellular matrix vesicle fraction after collagenase treatment. Lactate dehydrogenase (LDH)-containing vesicles with a density different from that of matrix vesicles were detected in the matrix vesicle fraction. LDH in these vesicles did not result from cell lysis and vesicle capture during the preparation of the matrix vesicle fraction. The isoenzyme pattern of LDH in LDH-containing vesicles was similar to that of cytosolic LDH of odontoblasts. Other cytosolic enzymes were not detected in LDH-containing vesicles, suggesting the presence of a mechanism for specific uptake of cytosolic LDH during the in vivo formation of the vesicles.

Alkaline Phosphatase↗
PubMed1980

The structure of newly synthesized intracellular thyroglobulin molecules.

Thyroglobulin molecules from normal rats and rats treated with propylthiouracil (PTU) were studied in the electron microscope by the negative staining technique. Intracellular molecules were prepared from exocytotic vesicles, and extracellular molecules from the high-speed centrifugation supernatant. In accord with our previous findings, extracellular thyroglobulin molecules from normal glands had an ovoid conformation, whereas extracellular molecules from PTU-treated glands had a different, cylindrical shape. Thyroglobulin molecules from exocytotic vesicles were of the ovoid variety in normal as well as in PTU-exposed thyroids. Two interpretations are discussed. One would be that PTU exerts a non-physiological effect on ovoid molecules in connection with exocytosis of thyroglobulin. The other interpretation is that thyroglobulin molecules normally undergo 2 transformations (ovoid-cylinder-ovoid) during the process of exocytosis; PTU would inhibit the second transformation and cause accumulation of cylinders in the colloid.

Animals↗
PubMed1982

Receptor-mediated endocytosis and the cellular uptake of low density lipoprotein.

During receptor-mediated endocytosis various extracellular nutritional and regulatory molecules bind to plasma membrane receptors and rapidly enter target cells. In many systems (including those for certain plasma transport proteins, protein hormones, glycoproteins, toxins and viruses, and other plasma proteins) the receptors cluster in discrete regions of the surface membrane called coated pits, which invaginate into the cell to form endocytic vesicles. The extracellular ligand enclosed in the endocytic vesicle is delivered to intracellular sites, frequently to lysosomes, where it is degraded. In one system of receptor-mediated endocytosis, namely the one for plasma low density lipoprotein (LDL), the receptor functions to internalize LDL. The LDL is delivered to lysosomes where it is degraded and its cholesterol is released for use in the synthesis of membranes, steroid hormones and bile acids. Three recent advances in the LDL receptor system are reviewed: (1) the development of a method for purifying the receptor to apparent homogeneity and the demonstration that the LDL-binding site is contained within a glycoprotein of relative molecular mass 164000 and an acidic isoelectric point of 4.6; (2) the production of monoclonal antibodies directed against the receptor and the use of these antibodies as probes for receptor-mediated endocytosis; and (3) the use of monovalent carboxylic ionophores (such as monensin) to demonstrate by immunofluorescence that the LDL receptor enters the cell together with LDL, after which it recycles to the surface.

Animals↗
PubMed1992

Studies of matrix vesicle-induced mineralization in a gelatin gel.

Matrix vesicles isolated from fourth-passage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and beta-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicles is to transport enzymes for matrix modification.

Adenosine Triphosphate↗
PubMed1994

Localization of cathepsins B, D, and L in the rat osteoclast by immuno-light and -electron microscopy.

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and -electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabeled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Nongenomic regulation of extracellular matrix events by vitamin D metabolites.

Vitamin D metabolites appear to regulate chondrocytes and osteoblasts via a combination of genomic and nongenomic mechanisms. Specificity of the nongenomic response to either 1,25-(OH)2D3 or 24,25-(OH)2D3 may be conferred by the chemical composition of the target membrane and its fluid mosaic structure, by the presence of specific membrane receptors, or by the interaction with classic vitamin D receptors. Nongenomic effects have been shown to include changes in membrane fluidity, fatty acid acylation and reacylation, arachidonic acid metabolism and prostaglandin production, calcium ion flux, and protein kinase C activity. Chondrocytes metabolize 25-(OH)D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3; production of these metabolites is regulated by both growth factors and hormones and is dependent on the state of cell maturation. 1,25-(OH)2D3 and 24,25-(OH)2D3 may interact directly with extracellular matrix vesicles to regulate their function in the matrix, including protease activity, resulting in matrix modification and calcification. Isolated matrix vesicles, produced by growth zone chondrocytes, can activate latent transforming growth factor-beta when incubated with exogenous 1,25-(OH)2D3. These observations suggest that nongenomic regulation of matrix vesicle structure and function may be a mechanism by which mesenchymal cells, like osteoblasts and chondrocytes, may modulate events in the extracellular matrix at sites distant from the cell surface.

Animals↗
PubMed1996

Vitamin D metabolites regulate matrix vesicle metalloproteinase content in a cell maturation-dependent manner.

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcification in vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-beta. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1, 25(OH)2D3 and 24,25(OH)2D3. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10(-10)-10(-7) M24, 25(OH)2D3 or growth zone chondrocytes incubated with 10(-11)-10(-8) M 1,25(OH)2D3, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)2D3 increased alkaline phosphatase by 35-60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)2D3 increased alkaline phosphatase by 35-60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)2D3 regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A2 specific activities as well as metalloproteinases which degrade proteoglycans.

24,25-Dihydroxyvitamin D 3↗
PubMed1982

Transplantation of free perichondrial grafts into rabbit articular cartilage is associated with matrix vesicle calcification.

Autogenous perichondrial grafts from rabbit ear were transplanted into articular cartilage defects in the knee joint. Electron micrographs performed after 2 months revealed calcifications associated with cartilaginous-forming cells and extracellular matrix vesicles. Many of these vesicles contained apatite crystals, particularly in areas adjacent to the calcifying fronts. In these areas the vesicular membrane was occasionally ruptured by the crystals. Many crystals in the calcifying front proper were organized alongside collagen fibers. Examination of sham-operated and normal controls revealed the occurrence of non-calcifying matrix vesicles. The results of the present and previous studies suggest that matrix vesicles occur in normal articular cartilage in a latent form, their calcification being associated with changes of the metabolic state of the tissue.

Animals↗
PubMed2006

Extracellular chelation of zinc does not affect hippocampal excitability and seizure-induced cell death in rats.

In the nervous system, zinc can influence synaptic responses and at extreme concentrations contributes to epileptic and ischaemic neuronal injury. Zinc can originate from synaptic vesicles, the extracellular space and from intracellular stores. In this study, we aimed to determine which of these zinc pools is responsible for the increased hippocampal excitability observed in zinc-depleted animals or following zinc chelation. Also, we investigated the source of intracellularly accumulating zinc in vulnerable neurons. Our data show that membrane-permeable and membrane-impermeable zinc chelators had little or no effect on seizure activity in the CA3 region. Furthermore, extracellular zinc chelation could not prevent the accumulation of lethal concentrations of zinc in dying neurons following epileptic seizures. At the electron microscopic level, zinc staining significantly increased at the presynaptic membrane of mossy fibre terminals in kainic acid-treated animals. These data indicate that intracellular but not extracellular zinc chelators could influence neuronal excitability and seizure-induced zinc accumulation observed in the cytosol of vulnerable neurons.

Animals↗
PubMed2005

Transdifferentiation of smooth muscle cells into chondrocytes in atherosclerotic arteries in situ: implications for diffuse intimal calcification.

Several hypotheses have been offered to explain the occurrence of arteriosclerotic calcification but the mechanisms involved are still not well understood. Using a combination of electron microscopy and immunohistochemistry, atherosclerotic plaques from human arteries as well as atherosclerotic-like lesions from aortas of apo-E-deficient mice were examined to identify cell type(s) associated with calcification. Electron microscopic analysis showed that, in human atherosclerotic plaques, chondrocyte-like cells were present in areas surrounding the necrotic cores. In these areas, some smooth muscle cells displayed features of their transdifferentiation into chondrocyte-like cells. Immunohistochemical analysis confirmed that smooth muscle cells with a reduced content of alpha-smooth muscle actin expressed Sox-9. Destruction of chondrocytes resulted in the accumulation of numerous membrane-bound vesicles in the extracellular space. Membrane-bound vesicles originating from chondrocytes were found to undergo calcification. Similar processes were found to occur in atherosclerotic-like lesions in apo-E-deficient mice. These observations suggest that transdifferentiation of smooth muscle cells into chondrocytes contributes to atherosclerotic calcification.

Adult↗
PubMed1990

DNA Is Packaged within Membrane-Derived Vesicles of Gram-Negative but Not Gram-Positive Bacteria.

Recently, DNA packaged within nuclease-resistant membrane vesicles of Neisseria gonorrhoeae and Borrelia burgdorferi was described. This study assayed 18 species of gram-negative and gram-positive eubacteria for nuclease-protected DNA associated with extracellular membrane vesicles. Vesicles from only the gram-negative bacteria contained nuclease-protected linear or supercoiled DNAs or both.

Journal Article↗
PubMed1981

Extracellular matrix vessels in human osteogenic neoplasms: an ultrastructural and enzymatic study.

The results of a study of the ultrastructural and enzymatic features of extracellular matrix vesicles in human osteogenic neoplasms are reported. Specimens from three osteosarcomas, a chondrosarcoma, and an osteoblastoma were processed for electron microscopic study and for preparation of vesicular, membrane, and cellular fractions. Electron micrographs of each lesion showed primary mineralization comprised of matrix vesicles and calcifying nodules. There was a distinct pattern of distribution of enzymatic activity among fractions from the osteosarcomas; namely that the highest values for specific activity of alkaline and pyrosphosphatases and adenosine triphosphases (ATPases) in the vesicle fractions and lowest in the cell fractions. This pattern was not consistent in fractions from the other neoplasms. The aforementioned enzymes are considered essential for the onset of mineralization. The data presented establish the role of matrix vesicles in neoplastic calcification and suggest the need for further studies into the diagnostic value of the vesicles.

Acid Phosphatase↗
PubMed1992

Interaction between lymphokine activated killer cells generated in the presence of cisplatin/FK-565 and tumor cells: transmission electron microscopy analysis.

Lymphokine activated killer (LAK) cells generated in the presence of cisplatin/FK-565 along with interleukin-2 (IL-2) showed enhanced binding to tumor cells as compared with LAK cells generated in the presence of IL-2 alone. The LAK cells showed a reorientation of cellular organelles in the presence of tumor cells. LAK-tumor conjugate pairs showed vesicle-like structures in the extracellular pockets. The number of vesicles in the extracellular pockets was found to be increased in the case of LAK cells generated in the presence of cisplatin/FK-565 along with IL-2. The closer interaction between the LAK cell-target cell and the large number of vesicles in the extracellular space of conjugate pairs may explain the increased lysis of target cells by LAK cells generated in the presence of cisplatin/FK-565 along with IL-2 as compared with IL-2 alone.

Antibiotics, Antineoplastic↗
PubMed2002

1alpha,25(OH)2D3 regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis.

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha,25(OH)(2)D(3). Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha,25(OH)(2)D(3) in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha,25(OH)(2)D(3) during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha,25(OH)(2)D(3), PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha,25(OH)(2)D(3) was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1alpha,25(OH)(2)D(3) membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha,25(OH)(2)D(3)-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1alpha,25(OH)(2)D(3) in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G(i)/G(s) had no effect. However, inhibition of G(q) blocked the effect of 1alpha,25(OH)(2)D(3). The rapid effect of 1alpha,25(OH)(2)D(3) also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1alpha,25(OH)(2)D(3) at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.

Animals↗