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At least 649 records · Page 36Linked to original sources

PubMed2005

Mutation analysis of the flp operon in Actinobacillus actinomycetemcomitans.

Fresh clinical isolates of the periodontal pathogen Actinobacillus actinomycetemcomitans live as autoaggregates, in which cells are densely packed and embedded in an extracellular matrix composed of bundled fimbriae, exopolymers, and vesicles. The expression of fimbriae is known to be determined by the flp operon of 14 genes, flp-1-flp-2-tadV-rcpCAB-tadZABCDEFG. We generated mutations of each gene of this operon in A. actinomycetemcomitans strain D7S. All mutants expressed some changes in the production of extracellular matrix materials that include vesicles, exopolymers, and fimbriae. The expression of fimbriae required the function of flp-1, rcpA, rcpB, tadB, tadD, tadE, and tadF. Mutants of flp-2, tadZ, tadA, tadC, and tadG expressed reduced levels of fimbriae, or fimbriae that had different gross appearance. Importantly, the expression of the non-fimbrial matrix materials was affected by all mutations, suggesting that the flp operon was involved in production of these materials. The flp locus apparently plays a central role in autoaggregation of A. actinomycetemcomitans, which may be the primary survival strategy of this bacterium in vivo.

Aggregatibacter actinomycetemcomitans↗

PubMed1987

Extracellular protein phosphorylation systems in the regulation of cellular responsiveness.

In cells which store ATP within secretory vesicles and release it by exocytosis upon cell stimulation, extracellular protein phosphorylation systems may play an important role in cellular activation and intercellular communication. Secretion of ATP has been well documented in three cell types: neurons, chromaffin cells of the adrenal medulla and platelets. In each of these cell types we have found extracellular protein phosphorylation systems and surface proteins phosphorylated by extracellular ATP. Two types of extracellular protein kinases have been detected: a membrane bound ecto-protein kinase and a soluble, secreted exo-protein kinase. The ecto-protein kinase has properties consistent with involvement in the feedback regulation of cellular activation. The secreted protein kinase can phosphorylate proteins of the extracellular matrix and surface proteins of target cells, and thus may serve as an enzyme with the role of first messenger in cell-cell communication.

Adenosine Triphosphate↗

PubMed2001

Direct observation of membrane retrieval in chromaffin cells by capacitance measurements.

This study was focussed on the identification of the endocytic organelles in chromaffin cells which retrieve large, dense core vesicle (LDCV)-membrane components from the plasma membrane. For this purpose, 'on-cell' capacitance measurements and electron microscopy were employed. We found capacitance steps and capacitance flickers, corresponding to single exo- and endocytic events. The analysis revealed that the total membrane surface of completely fused LDCVs is recycled by large endocytic vesicles and smaller, most likely clathrin-coated vesicles, at approximately the same ratio. These results were confirmed by rapid-freeze immuno-electron microscopy, where an extracellular marker was rapidly internalized into endocytic vesicles that morphologically resembled LDCVs.

Animals↗

PubMed1987

Maturation of myocardial capillaries in the fetal and neonatal rat: an ultrastructural study with a morphometric analysis of the vesicle populations.

The ultrastructural morphology of the cellular and extracellular components of the developing myocardial capillary wall--from the 16-day-gestation fetus of the rat to the 21-day neonate--was examined. A morphometric analysis of plasmalemmal vesicles and of coated vesicles and pits of capillary endothelial cells was performed during the same developmental period. As the lateral extensions of the capillary endothelial cells change from irregular to regular in their thickness during development, there is an increase in the number of plasmalemmal vesicles and a progression from clusters of plasmalemmal vesicles to a uniform distribution in the endothelial cell. The ratio of vesicles which are open to the luminal front, which are "free" in the cytoplasm, or which are open to the abluminal front of the endothelial cell was consistent throughout development. The numerical density of plasmalemmal vesicles demonstrates a gradual and significant increase. In contrast, the numbers of coated vesicles and pits are variable within a very narrow range, and no pattern of increase or decrease is discernible during development. Similarly, there is no change in interendothelial cell junctions, which consist of occluding and primitive adhesive junctional types, during development. The lamina densa of the basal lamina gradually develops from discontinuous, patchy densities along the abluminal surface of the endothelial cells to a continuous and distinct layer by 21 days gestation. The presence of the proteoglycan species in the developing basal lamina was assessed with the cationic dye ruthenium red (RR), and the appearance of RR-marked proteoglycans was found to parallel the appearance of lamina densa material. found to parallel the appearance of lamina densa material. The RR sites appear discontinuously in patches; and later, the RR sites appear in a continuous and regular planar lattice in the lamina rara interna and externa at 21 days gestation. A complete array of RR-stainable anionic sites outside a continuous lamina densa near birth indicates that the basal laminae of developing capillaries in the heart are morphologically, and in part biochemically, mature by the end of the first neonatal week. Our results show that the endothelial cells and the subtending basal lamina of myocardial capillaries gradually mature morphologically during the final days of gestation and the first neonatal week.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMedDate not supplied

Extracellular acidification alters lysosomal trafficking in human breast cancer cells.

Cancer cells invade by secreting degradative enzymes, which are sequestered in lysosomal vesicles. In this study, the impact of an acidic extracellular environment on lysosome size, number, and distance from the nucleus in human mammary epithelial cells (HMECs) and breast cancer cells of different degrees of malignancy was characterized because the physiological microenvironment of tumors is frequently characterized by extracellular acidity. An acidic extracellular pH (pH(e)) resulted in a distinct shift of lysosomes from the perinuclear region to the cell periphery irrespective of the HMECs' degree of malignancy. With decreasing pH, larger lysosomal vesicles were observed more frequently in highly metastatic breast cancer cells, whereas smaller lysosomes were observed in poorly metastatic breast cancer cells and HMECs. The number of lysosomes decreased with acidic pH values. The displacement of lysosomes to the cell periphery driven by extracellular acidosis may facilitate exocytosis of these lysosomes and increase secretion of degradative enzymes. Filopodia formations, which were observed more frequently in highly metastatic breast cancer cells maintained at acidic pH(e), may also contribute to invasion.

Antigens, CD↗

PubMed2007

Lipid raft-dependent endocytosis of metallothionein in HepG2 cells.

Human hepatocellular carcinoma (HepG2) cells take up metallothionein (MT) by endocytosis. MT co-localizes with albumin but not with transferrin, indicating uptake via a non-classical pathway rather than via clathrin-mediated endocytosis. A lipid raft-dependent uptake is indicated by pravastatin inhibition of cholesterol synthesis and methyl-beta-cyclodextrin inhibition of cholesterol translocation to the plasma membrane, reducing MT uptake by 29% and 69%, respectively. Subcellular fractionation after MT uptake reveals significant amounts of MT in vesicular fractions including lysosomes but virtually no MT in the cytosol. Metals bound to MT are released into the cytosol, however. The findings define a pathway for cellular metal acquisition. Together with results from other studies demonstrating secretion of MT from different cells and the presence of MT in extracellular fluids, the results suggest a function of MT in intercellular communication.

Cell Communication↗

PubMed2005

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forster resonance energy transfer suggest weak interactions between fibroblast growth factor receptor 3 (FGFR3) transmembrane domains in the absence of extracellular domains and ligands.

Lateral dimerization of membrane proteins has evolved as a means of signal transduction across the plasma membrane for all receptor tyrosine kinases (RTKs). The transmembrane (TM) domains of RTKs are proposed to play an important role in the dimerization process. We have investigated whether the TM domains of one RTK, fibroblast growth factor receptor 3 (FGFR3), dimerize in lipid vesicles in the absence of the extracellular domains and ligands. We have performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with peptides produced via solid-phase peptide synthesis that correspond to the TM domain of FGFR3. We have carried out Forster resonance energy transfer (FRET) measurements using two donor-acceptor pairs, fluorescein/rhodamine and Cy3/Cy5, as a function of peptide concentration and donor-to-acceptor mole ratios. Our results suggest that FGFR3 TM domains form sequence-specific dimers in lipid bilayers. However, the dimerization propensity of FGFR3 TM domain is much weaker than the dimerization propensity of glycophorin A (GpA), the well-characterized "membrane dimer standard". We discuss our findings in the context of cell signaling across the plasma membrane and diseases or disorders that occur due to single amino acid mutations in the TM domain of FGFR3.

Amino Acid Sequence↗

PubMed2005

Properties of synaptic vesicle pools in mature central nerve terminals.

Readily releasable and reserve pools of synaptic vesicles play different roles in neurotransmission, and it is important to understand their recycling and interchange in mature central synapses. Using adult rat cerebrocortical synaptosomes, we have shown that 100 mosm hypertonic sucrose caused complete exocytosis of only the readily releasable pool (RRP) of synaptic vesicles containing glutamate or gamma-aminobutyric acid. Repetitive hypertonic stimulations revealed that this pool recycled (and reloaded the neurotransmitter from the cytosol) fully in <30 s and did so independently of the reserve pool. Multiple rounds of exocytosis could occur in the constant absence of extracellular Ca(2+). However, although each vesicle cycle includes a Ca(2+)-independent exocytotic step, some other stage(s) critically require an elevation of cytosolic [Ca(2+)], and this is supplied by intracellular stores. Repetitive recycling also requires energy, but not the activity of phosphatidylinositol 4-kinase, which maintains the normal level of phosphoinositides. By varying the length of hypertonic stimulations, we found that approximately 70% of the RRP vesicles fused completely with the plasmalemma during exocytosis and could then enter silent pools, probably outside active zones. The rest of the RRP vesicles underwent very fast local recycling (possibly by kiss-and-run) and did not leave active zones. Forcing the fully fused RRP vesicles into the silent pool enabled us to measure the transfer of reserve vesicles to the RRP and to show that this process requires intact phosphatidylinositol 4-kinase and actin microfilaments. Our findings also demonstrate that respective vesicle pools have similar characteristics and requirements in excitatory and inhibitory nerve terminals.

1-Phosphatidylinositol 4-Kinase↗

PubMed2003

Phosphoarginine regulation of the squid nerve Na+/Ca2+ exchanger: metabolic pathway and exchanger-ligand interactions different from those seen with ATP.

In squid nerves the Na(+)-Ca(2+) exchanger is up-regulated by ATP and phosphoarginine (PA). ATP regulation involves drastic alterations in the Na(+)(i), H(+)(i) and Ca(2+)(i) interactions with the large intracellular cytoplasmic loop of the exchanger protein. In this work we explored the mechanisms associated with PA regulation in intracellular dialysed squid axons and squid optic nerve membrane vesicles. Dialysed axons were used to measure the four modes of exchange fluxes (Na(+)(o)-Ca(2+)(i) or forward exchange, Ca(2+)(o)-Na(+)(i) or reverse exchange, Ca(2+)(o)-Ca(2+)(i) exchange and Na(+)(o)-Na(+)(i) exchange) under controlled intra- and extracellular conditions. Inside-out membrane vesicles allowed measurement of the Na(+)-gradient-dependent (45)Ca(2+) uptake (forward mode) as influenced by ligands and digestion with chymotrypsin from the intracellular side. The results show that, unlike ATP, PA regulation does not affect the H(+)(i), Na(+)(i) and Ca(2+)(i) interactions with the intracellular 'regulatory' loop, but increases the affinity of the intracellular transport sites, preferentially for Ca(2+)(i) (about 20-fold) over Na(+)(i) (50%); i.e. PA favours the forward mode over the other exchange modes. Intracellular chymotrypsin digestion removed ATP regulation while leaving modulation by PA unmodified. Western blot analysis suggested that chymotrypsin disrupts the large intracellular loop. Together these results indicate that ATP and PA regulations are associated with different structures inside and outside the exchanger protein. Based on these observations we expanded our previous model for metabolic regulation of the Na(+)-Ca(2+) exchanger by adding to the original 'ATP region' a new zone, the 'PA region', related to the intracellular transport sites for Na(+)(i) and Ca(2+)(i). This new model is able to explain most previous and present results.

Adenosine Triphosphate↗

PubMed1996

Molecular machinery mediating vesicle budding, docking and fusion.

A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs (SNAP receptors) on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.

Capsid↗

PubMed1996

Molecular machinery mediating vesicle budding, docking and fusion.

A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.

Animals↗

PubMed2005

Extracellular matrix degrading enzymes at the prostasome surface.

Prostasomes, prostatic secretory vesicles found in human ejaculates, were analyzed to verify the existence at their surfaces of enzymes involved in the degradation of the extracellular matrix. Findings were compared with those of prostasomes isolated from two human adenocarcinoma cell lines that reflect clinical features and molecular pathways of androgen-insensitive and hormone-responsive prostate cancer. Our aim was to determine whether neoplastic transformation is accompanied by changes of glycosidase and protease activities. Our results show that decreases of dipeptidyl peptidase IV and increases of urokinase plasminogen activator and cathepsin B are consistent with the clinical features of the cell lines, whereas increases of glycosidase activities seem to be of scarce biological significance.

Cathepsin B↗

PubMed1975

[The development of intramyocardial haemorrhages or fluidaccumulation in the extracellular space].

Isolated rat hearts according to Langendorff and rabbit hearts after orthostatic collapse were studied under the light and electron microscope. The light-micrographs were also quantitatively analysed. Changes in the vessels are noticeable, especially those in the sinusoid's. Vessel ruptures occur, also isolated endothelial ruptures with intact basement membranes and perfusion fluid enters the extracellular space. Numerous 0.06-01 mu large vesicles appear in the cytoplasma of the endothelial cells. A large number of vesiculation processes can be demonstrated along the cell membrane. The extracellular space is strongly-dilated. Changes in the colloid osmotic pressure and a rise in perfusion pressure have no clearly demonstrable influence on the extent of extravasation under the experimental conditions. The mechanism possible inducing such vessel changes is discussed.

Animals↗

PubMed1981

Immuno electron microscopic studies on cells synthesizing elastin.

Embryonic chick and hamster aortas were examined in the electron microscope using ferritin-conjugated, elastin-specific antibodies after etching of the plastic, thin sections with a dilute solution of benzene, methanol and ethanol. Specific staining of intracellular vesicles was observed in both smooth muscle and endothelial cells in addition to extracellular elastin fibers. In the chick cells, these ferritin-stained vesicles had a lipid-laden appearance. In both species the vesicles appeared to fuse with the plasma membrane and discharge their contents into the extracellular space suggesting elastin is secreted via vesicular structures.

Animals↗

PubMed1987

Coated vesicles and pits during enhanced quantal release of acetylcholine at the neuromuscular junction.

Frog neuromuscular junctions were stimulated by different methods to secrete quanta of ACh, and the attendant changes in the ultrastructure of the nerve terminal were assessed by morphometric analysis of electron micrographs. Secretion was stimulated by electrical stimulation at 2 Hz or by application of the secretagogues, lanthanum, ouabain or black widow spider venom, either in the presence or in the absence of extracellular Ca2+. The numbers of synaptic vesicles, coated vesicles and coated pits, and the length of axolemma and area of axoplasm were measured on the micrographs. There was a significant increase (about threefold) in the total number of coated structures (vesicles plus pits) per micron2 of axoplasm, but the fractional increase in the number of coated pits exceeded the fractional increase in the number of coated vesicles. These increases were positively correlated with the increase in the length of axolemma per unit area and negatively correlated with the changes in concentration of synaptic vesicles, suggesting that they were due to the increases in the surface area of the terminal that accompany a loss of vesicles. However, the increase in the concentration of coated structures was not related to the number of quanta secreted or to the estimated number of vesicles recycled. The lack of correspondence between the fractional increases in the coated pits and coated vesicles and the poor correlation between the numbers of these structures and the overall parameters of the secretory process suggest that, in contrast to the situation in other secretory systems, coated pits and coated vesicles may not play a crucial role in maintaining the functional population of synaptic vesicles at rapidly secreting neuromuscular junctions.

Acetylcholine↗

PubMed2001

Ultrastructure of Mikrocytos mackini, the cause of Denman Island disease in oysters Crassostrea spp. and Ostrea spp. in British Columbia, Canada.

An ultrastructural study was carried out on Mikrocytos mackini, the cause of Denman Island disease in Pacific oysters Crassostrea gigas in western Canada. Three forms were identified, quiescent cells (QC), vesicular cells (VC) and endosomal cells (EC). QC occurred in the vesicular connective tissue (VCT), haemocytes (hyalinocytes), adductor and heart myocytes, and extracellularly. They had a central round to ovoid nucleus, < 7 cisternae of inactive nuclear membrane-bound Golgi, few vesicles and lysosome-like bodies. VC were rarely extracellular and usually occurred in adductor and heart myocytes, in close association with host cell mitochondria. The contents of the host cell mitochondria appeared to pass through a tubular extension into the cytoplasm of the parasite. Cytoplasmic vesicles resembled the tubular structure in appearance and size. EC occurred in the VCT, in haemocytes and extracellularly. They had a dilated nuclear membrane, sometimes containing a looped membranous structure that appeared to derive from the nucleus, and pass into the cytoplasm. A well-developed anastomosing endoplasmic reticulum connected the nuclear and plasma membranes, and endosomes were present in the cytoplasm. QC and EC cells were frequently observed tightly against, or between, the nuclear membranes of the host cell. Few organelles occurred in all forms of M. mackini, especially QC. The lack of organelles found in most eukaryotic cells, including mitochondria or their equivalents, may be due to obligate parasitism and the utilization of host cell organelles reducing the need for parasite organelles. Alternatively, perhaps M. mackini is a primitive eukaryote. Although phylogenetic affinities could not be determined, it is not a haplosporidian. A developmental cycle is proposed from these findings.

Animals↗

PubMed2005

The potential of exosomes in immunotherapy.

In 1996, B cell-derived exosome immunogenicity was first reported. In 1998, dendritic cell-derived exosomes were also shown to have immunogenic properties in mice, leading to tumour eradication. These observations showed for the first time that exosomes were bioactive vesicles with immuno-regulatory roles and potent antitumour effects. Exosomes are secreted vesicles formed in late endocytic compartments (multivesicular bodies). Vesicles exocytosed from multivesicular bodies into the extracellular medium are referred to as 'exosomes' and should not be confused with the more recently described 'ribonuclease complex' also named exosome. Exosomes are vesicles of 50-100 nm in diameter, formed by inward budding of the endosomal membrane in a process that sequesters particular proteins and lipids. This review will describe the biogenesis, the protein and lipid compositions, and the biological functions of exosomes; this review will also present their immunostimulatory functions in mice and humans, and will discuss their possible use as cancer vaccines.

Animals↗

PubMed2004

'Nectosome': a novel cytoplasmic vesicle containing nectin in the egg of the sea urchin, Temnopleurus hardwickii.

Localization of an extracellular matrix protein, Th-nectin, in the eggs and embryos of the sea urchin Temnopleurus hardwickii was examined by both immunofluorescence and immunoelectron microscopy. The protein is associated with a tubular structure packaged in rod-shaped vesicles that were designated as 'nectosomes'. In unfertilized eggs, nectosomes are distributed uniformly throughout the cytoplasm, but after fertilization, they gradually translocate to the cortical zone where they are arranged perpendicular to the plasma membrane. The migration of the nectosomes was strongly inhibited by cytochalasin B, which suggested that microfilaments play an important role in this process. Immunocytochemical and immunoblotting analyses both ascertained that nectin is secreted into the hyaline layer. Some nectosomes remain in the apical cytoplasm of dermal cells until the gastrula stage. Ultrastructural examination revealed that the accumulation of nectosomes in the oocyte cytoplasm begins quite early in oogenesis, concomitant with the accumulation of cortical vesicles.

Animals↗