SEARCH · PubMed Health

Results for “Extracellular vesicles”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources

PubMed2005

Developmental appearance of dentin matrix protein 1 during the early dentinogenesis in rat molars as identified by high-resolution immunocytochemistry.

Dentin matrix protein 1 (DMP 1) is an acidic phosphoprotein that has been postulated to play an important role in mineralized tissue formation. We have examined rat molar tooth germs by applying a high-resolution immunocytochemical approach with the purpose to identify the temporal and spatial localization of DMP 1 at the onset of dentinogenesis. Upper molar tooth germs of 2- to 3-day-old Wistar rats were fixed in a cacodylate-buffered 0.1% glutaraldehyde + 4% formaldehyde fixative, left unosmicated and embedded in LR White resin. The sections were incubated with a polyclonal DMP 1 antibody for postembedding colloidal gold immunolabeling and examined in a Jeol 1010 transmission electron microscope. The earliest localization of DMP 1 was in the Golgi region as well as in the nucleus of differentiating odontoblasts. When mineralization spread from matrix vesicles to the surrounding matrix, DMP 1 was extracellularly detected around the mineralizing globules. In the regions of fully mineralized mantle dentin, it was present in the mineralized regions, mainly around the peritubular dentin. The appearance of DMP 1 during early dentinogenesis implies a direct role for this protein in both odontoblast differentiation and matrix mineralization.

Animals↗

PubMed2006

Dinucleoside polyphosphates and their interaction with other nucleotide signaling pathways.

Dinucleoside polyphosphates or Ap(n)A are a family of dinucleotides formed by two adenosines joined by a variable number of phosphates. Ap(4)A, Ap(5)A, and Ap(6)A are stored together with other neurotransmitters into secretory vesicles and are co-released to the extracellular medium upon stimulation. These compounds can interact extracellularly with some ATP receptors, both metabotropic (P2Y) and ionotropic (P2X). However, specific receptors for these substances, other than ATP receptors, have been described in presynaptic terminals form rat midbrain. These specific dinucleotide receptors are of ionotropic nature and their activation induces calcium entry into the terminals and the subsequent neurotransmitter release. Calcium signals that cannot be attributable to the interaction of Ap(n)A with ATP receptors have also been described in cerebellar synaptosomes and granule cell neurons in culture, where Ap(5)A induces CaMKII activation. In addition, cerebellar astrocytes express a specific Ap(5)A receptor coupled to ERK activation. Ap(5)A engaged to MAPK cascade by a mechanism that was insensitive to pertussis toxin and required the involvement of src and ras proteins. Diadenosine polyphosphates, acting on their specific receptors and/or ATP receptors, can also interact with other neurotransmitter systems. This broad range of actions and interactions open a promising perspective for some relevant physiological roles for the dinucleotides. However, the physiological significance of these compounds in the CNS is still to be determined.

Adenosine Triphosphate↗

PubMed1992

Human Chang liver cells show large surface openings and endocytic channels that resemble those in the amoeba.

Cytolysis of host cells by pathogenic Entamoeba histolytica can be blocked by specific lysozyme inhibitions and is recently reported to be enhanced by phosphoinositide (PI) signal transduction activation. However the mechanistic relationship between PI second messenger targets and massive lysosomal secretion needed to achieve rapid host cell lysis is unclear. We have previously shown that intracellular alkalinization associated with activated PI hydrolysis produces a massive endocytosis of huge proportions which would force a corresponding exocytosis for the maintenance of overall cell dimensions. These endosomes are processed by primary lysosomes. Apparently then, the massive exocytosis secretory pathway could provide the means for the ejection of lysozymes over target cells. We show here using human Chang liver cells that intracellular alkalinization produced large surface pittings similar to those seen in pathogenic E. histolytica in a rounded state. The SEM profile is correlated with the TEM profile of large endosomes containing extracellular debris and endosomes associated with primary lysosomal vesicles, which could support the notion that some of the pittings seen in the rounded Chang cells and the pathogenic amoebae are exit portals for endosome-lysosomes.

Animals↗

PubMed1988

Culture of proliferating and differentiating fat-storing cells in 3T3-conditioned medium.

There is growing evidence suggesting that hepatic fat-storing cells (FSC) or Ito cells have an important function in vitamin A storage and metabolism and in the synthesis of connective tissue components in normal liver and during fibrogenesis. The purified FSC acquire a fibroblastic morphology and their vitamin A content decreases in culture. We cultivated cells under in vitro conditions that allowed the expression of FSC morphological and functional characteristics for 3-4 weeks of primary culture. Cells were isolated from rat liver by the collagenase-perfusion method without further purification and cultured with 3T3-conditioned medium, which seemed to stimulate the selective proliferation of the FSC. After 8-10 days, round and stellate cells grew actively from a few precursor cells in the primary culture and were not subcultivated; the stellate cells had the ability to become round and vice versa and were highly motile. The cells had intracytoplasmic lipid droplets, a well developed rough endoplasmic reticulum, Golgi complex, numerous vesicles filled with electron-dense material, and extracellular matrix (ECM) components on their surface. Both stellate and round cells showed the presence of desmin by immunofluorescence and vitamin A autofluorescence, but lacked peroxidase activity. The culture conditions we describe allowed the selective proliferation of cells with morphological and functional characteristics of the FSC in the normal liver, raising the possibility of studying FSC proliferation and differentiation.

Animals↗

PubMed1994

Avipox virus-vectored Japanese encephalitis virus vaccines: use as vaccine candidates in combination with purified subunit immunogens.

An avipox virus, canarypox (ALVAC), which is naturally host-range restricted, was used to construct recombinants encoding the Japanese encephalitis virus (JEV) prM, E and NS1 genes (vCP107) and prM and E genes (vCP140). Mice immunized with these recombinant viruses produced JEV neutralizing antibodies and were protected from lethal JEV challenge. Protection was also observed in mice immunized with a subunit vaccine candidate, consisting of extracellular particles (EPs; RNA-free subviral membrane vesicles containing prM/M and E proteins) derived from HeLa cell cultures infected with a JEV-vaccinia recombinant. Mice primed with vCP107 and boosted with EPs had higher antibody levels than mice immunized twice with EPs alone, although the levels were comparable to that obtained in mice immunized twice with the recombinant virus. Mice immunized with a mixture of recombinant virus (vCP107) plus EPs had neutralizing antibody titres higher than mice immunized with the recombinant virus or EPs alone.

Animals↗

PubMed2001

Metalloproteinase activity in growth plate chondrocyte cultures is regulated by 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) and mediated through protein kinase C.

During endochondral development, growth plate chondrocytes must remodel their matrix in a number of ways as they differentiate and mature. In previous studies, we have shown that matrix metalloproteinases (MMPs) extracted from matrix vesicles can extensively degrade aggrecan and that this is modulated by vitamin D metabolites in a manner involving protein kinase C (PKC). Matrix vesicles represent only a small component of the extracellular matrix, however, and it is unknown if the total metalloproteinase complement, including the MMPs and aggrecanases in the culture, is also regulated in a similar way. This study tested the hypothesis that vitamin D metabolites regulate the level of metalloproteinase activity in growth plate chondrocytes via a PKC-dependent mechanism and play a role in partitioning this proteinase activity between the media and cell layer (cells+matrix) in these cultures. To do this, resting zone cells (RC) were treated with 10(-9)-10(-7) M 24R,25-(OH)(2)D(3), while growth zone cells (GC) were treated with 10(-10)-10(-8) M 1alpha,25-(OH)(2)D(3). Cultures of both cell types were also treated with the PKC inhibitor chelerythrine in the presence and absence of vitamin D metabolites. At harvest, the media were either left untreated or treated to destroy metalloproteinase inhibitors, while enzyme activity in the cell layers was extracted with buffered guanidine and then treated like the media to destroy metalloproteinase inhibitors. Neutral metalloproteinase (aggrecan-degrading activity) activity was assayed on aggrecan-containing polyacrylamide gel beads and collagenase activity was measured on telopeptide-free type I collagen. Neutral metalloproteinase activity was found primarily in the cell layer of both cell types; however, activity was greater in extracts of GC cell layers. No collagenase activity could be detected in RC extracts until the metalloproteinase inhibitors were destroyed. In contrast, extracts of GC cell layers contained measurable activity without removing the inhibitors, and destroying the inhibitors resulted in a greater than two-fold increase in activity. No collagenase activity was found in the media of either cell type. 24,25-(OH)(2)D(3) caused a dose-dependent increase in neutral metalloproteinase activity in extracts of RC cells, but had no effect on collagenase activity. In contrast, 1,25-(OH)(2)D(3) caused a dose-dependent decrease in collagenase activity in extracts of GC cells, but had no effect on neutral metalloproteinase activity. In both cases, the effect of the vitamin D metabolite was mediated through the activation of PKC. These results support the hypothesis that metalloproteinases are involved in regulating the bulk turnover of collagen and aggrecan in growth plate chondrocytes and that the amount of metalloproteinase activity found is a function of the cell maturation state. Furthermore, 83-93% of neutral metalloproteinase activity and 100% of collagenase activity is localized to the cell layer. Moreover, the regulation of metalloproteinase activity by 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) involves a PKC-dependent pathway that is controlled by the target cell-specific vitamin D metabolite.

24,25-Dihydroxyvitamin D 3↗

PubMed2005

Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic.

The anterior/posterior (A/P) and dorsal/ventral (D/V) compartment borders that subdivide the wing imaginal discs of Drosophila third instar larvae are each associated with a developmental organizer. Decapentaplegic (Dpp), a member of the transforming growth factor-beta (TGF-beta) superfamily, embodies the activity of the A/P organizer. It is produced at the A/P organizer and distributes in a gradient of decreasing concentration to regulate target genes, functioning non-autonomously to regulate growth and patterning of both the anterior and posterior compartments. Wingless (Wg) is produced at the D/V organizer and embodies its activity. The mechanisms that distribute Dpp and Wg are not known, but proposed mechanisms include extracellular diffusion, successive transfers between neighbouring cells, vesicle-mediated movement, and direct transfer via cytonemes. Cytonemes are actin-based filopodial extensions that have been found to orient towards the A/P organizer from outlying cells. Here we show that in the wing disc, cytonemes orient towards both the A/P and D/V organizers, and that their presence and orientation correlates with Dpp signalling. We also show that the Dpp receptor, Thickveins (Tkv), is present in punctae that move along cytonemes. These observations are consistent with a role for cytonemes in signal transduction.

Actins↗

PubMed1989

Human endomembrane H+ pump strongly resembles the ATP-synthetase of Archaebacteria.

Preparations of mammalian H+ pumps that acidify intracellular vesicles contain eight or nine polypeptides, ranging in size from 116 to 17 kDa. Biochemical analysis indicates that the 70- and 58-kDa polypeptides are subunits critical for ATP hydrolysis. The amino acid sequences of the major catalytic subunits (58 and 70 kDa) of the endomembrane H+ pump are unknown from animal cells. We report here the complete sequence of the 58-kDa subunit derived from a human kidney cDNA clone and partial sequences of the 70- and 58-kDa subunits purified from clathrin-coated vesicles of bovine brain. The amino acid sequences of both proteins strongly resemble the sequences of the corresponding subunits of the vacuolar H+ pumps of Archaebacteria, plants, and fungi. The archaebacterial enzyme is believed to use a H+ gradient to synthesize ATP. Thus, a common ancestral protein has given rise to a H+ pump that synthesizes ATP in one organism and hydrolyzes it in another and is highly conserved from prokaryotes to humans. The same pump appears to mediate the acidification of intracellular organelles, including coated vesicles, lysosomes, and secretory granules, as well as extracellular fluids such as urine.

Amino Acid Sequence↗

PubMed2002

Function of Lactococcus lactis nisin immunity genes nisI and nisFEG after coordinated expression in the surrogate host Bacillus subtilis.

Nisin-producing Lactococcus lactis strains show a high degree of resistance to the action of nisin, which is based upon expression of the self-protection (immunity) genes nisI, nisF, nisE, and nisG. Different combinations of nisin immunity genes were integrated into the chromosome of a nisin-sensitive Bacillus subtilis host strain under the control of an inducible promoter. For the recipient strain, the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes. But either the lipoprotein NisI or the ABC transporter-homologous system NisFEG, respectively, were also able to protect the Bacillus host cells. The acquired immunity was specific to nisin and provided no tolerance to subtilin, a closely related lantibiotic. Quantitative in vivo peptide release assays demonstrated that NisFEG diminished the quantity of cell-associated nisin, providing evidence that one role of NisFEG is to transport nisin from the membrane into the extracellular space. NisI solubilized from B. subtilis membrane vesicles and recombinant hexahistidine-tagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.

ATP-Binding Cassette Transporters↗

PubMed1989

Dynamic cytoskeleton-integrin associations induced by cell binding to immobilized fibronectin.

We have examined the early events of cellular attachment and spreading (10-30 min) by allowing chick embryonic fibroblasts transformed by Rous sarcoma virus to interact with fibronectin immobilized on matrix beads. The binding activity of cells to fibronectin beads was sensitive to both the mAb JG22E and the GRGDS peptide, which inhibit the interaction between integrin and fibronectin. The precise distribution of cytoskeleton components and integrin was determined by immunocytochemistry of frozen thin sections. In suspended cells, the distribution of talin was diffuse in the cytoplasm and integrin was localized at the cell surface. Within 10 min after binding of cells and fibronectin beads at 22 degrees C or 37 degrees C, integrin and talin aggregated at the membrane adjacent to the site of bead attachment. In addition, an internal pool of integrin-positive vesicles accumulated. The mAb ES238 directed against the extracellular domain of the avian beta 1 integrin subunit, when coupled to beads, also induced the aggregation of talin at the membrane, whereas ES186 directed against the intracellular domain of the beta 1 integrin subunit did not. Cells attached and spread on Con A beads, but neither integrin nor talin aggregated at the membrane. After 30 min, when many of the cells were at a more advanced stage of spreading around beads or phagocytosing beads, alpha-actinin and actin, but not vinculin, form distinctive aggregates at sites along membranes associated with either fibronectin or Con A beads. Normal cells also rapidly formed aggregates of integrin and talin after binding to immobilized fibronectin in a manner that was similar to the transformed cells, suggesting that the aggregation process is not dependent upon activity of the pp60v-src tyrosine kinase. Thus, the binding of cells to immobilized fibronectin caused integrin-talin coaggregation at the sites of membrane-ECM contact, which can initiate the cytoskeletal events necessary for cell adhesion and spreading.

Animals↗

PubMed2001

The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter.

Legionella pneumophila requires the dot/icm genes to create an organelle inside eukaryotic host cells that will support bacterial replication. The dot/icm genes are predicted to encode a type IV-related secretion apparatus. However, no proteins have been identified that require the dot/icm genes for secretion. In this study we show that the DotA protein, which was previously found to be a polytopic membrane protein, is secreted by the Dot/Icm transporter into culture supernatants. Secreted DotA protein was purified and N-terminal sequencing of the purified protein revealed that a 19 amino acid leader peptide is removed from DotA prior to secretion. Extracellular DotA protein did not fractionate in membrane vesicles. Structures containing secreted DotA protein were visualized by electron microscopy and were shaped like hollow rings. These data indicate that the large poly topic membrane protein DotA is secreted from L.pneumophila by a unique process. This represents the first target secreted by the dot/icm-encoded apparatus and demonstrates that this transporter is competent for protein secretion.

Amino Acid Sequence↗

PubMed2005

Internalized GABA-receptor subunits are transferred to an intracellular pool associated with the postsynaptic density.

Endocytosis represents an important mechanism regulating cell-surface expression of neurotransmitter receptors, including GABAA receptors, in neurons. Little is known, however, about trafficking of internalized receptors. Here, we used antibody tagging in living rat hippocampal neurons in culture to monitor GABAA receptor internalization. We show that cell-surface receptors have a homogeneous distribution reflecting their mobility in the membrane. Unexpectedly, internalized GABAA receptors were detected mainly in a subsynaptic pool associated with gephyrin at postsynaptic sites, whereas AMPA-type glutamate receptors were accumulated in the soma. This process was time-dependent and could be prevented by blocking clathrin-coated vesicle endocytosis. In control experiments, the existence of an intracellular pool of GABAA receptors associated with gephyrin was confirmed independently of internalization of surface receptors, and constitutive endocytosis, unrelated to antibody-tagging, could be demonstrated for both AMPA and GABAA receptors using a biotinylation assay. These results suggest that cycling of GABAA receptors between the cell surface and the subsynaptic pool provides a mechanism for the short-term regulation of GABAergic neurotransmission. Furthermore, the close association of gephyrin with internalized GABAA receptors suggests a role in intracellular receptor trafficking.

Animals↗

PubMed1987

ATP release from affinity-purified rat cholinergic nerve terminals.

Cholinergic nerve terminals were affinity purified from rat caudate nucleus. On stimulation with both 22.6 mM KCl and 50 microM veratridine, ATP was released in a Ca2+-dependent manner. The molar ratio of released acetylcholine to ATP (9:1) was closer to that found in isolated cholinergic vesicles (7:1) than whole terminals (3:1). Extracellular [14C]ATP was rapidly metabolized by these terminals to adenosine and inosine via ectonucleotidases. The terminals had a saturable, high-affinity uptake mechanism for adenosine (Km = 16.6 microM). Veratridine stimulation also caused the Ca2+-dependent release of nucleosides in a dipyridamole-sensitive manner. Both theophylline treatment and inhibition of extracellular ATP breakdown resulted in increased ATP and nucleoside release. Extracellular adenosine was shown to inhibit acetylcholine release, probably via the A1 receptor. The role of extracellular purines at the cholinergic nerve terminal is discussed.

1-Methyl-3-isobutylxanthine↗

PubMed1985

Ultracytochemical localization of calcium in the superficial pineal gland of the Mongolian gerbil.

In order to demonstrate fine localization of Ca2+ in the superficial pineal gland of Meriones unguiculatus, the pyroantimonate technique was employed. Control experiments were performed with EGTA and analysis of obtained reaction product using an energy-dispersive x-ray microanalysis. Precipitates of calcium antimonate were formed almost exclusively in swollen clear pinealocytes, in and along their cell membranes, over their nuclei, in mitochondria, the Golgi apparatus, endoplasmic and integrade reticulums, acervuli, in vesicles surrounding synaptic bars, cytoplasmic matrix, and flocculent extracellular material. It has been concluded that the swollen pinealocytes represent a degenerative cell form probably occurring by impairment of plasmalemmal Ca2+-ATPase's ability to eliminate Ca2+ ion from the cell, with consecutive increase of calcium in the cytoplasmic matrix, followed by increase of intramitochondrial Ca2+ concentration, gradual arrest of mitochondrial function, depolymerization of cytoskeletal microtubuli, loss of the cell form, and cell death. Decrease of function of pinealocyte plasmalemma seems to be related to aging.

Animals↗

PubMed1969

Entry and release of poliovirus as observed by electron microscopy of cultured cells.

Cells of differing culture types were inoculated with poliovirus at 37 C, sampled at intervals during the replicative cycle, and examined in thin sections by electron microscopy. The earliest samples, taken at 2 and 5 min postinoculation, showed virus particles adjacent to the exterior of the plasma membrane and others that had apparently penetrated it directly; later samples showed fewer such particles or none. Particles lying in the peripheral cytoplasm frequently appeared swollen and distorted in shape. No sign of virus entry by a pinocytotic process was found at any time. At 3 hr, and subsequently during the replication cycle, particles of progeny virus appeared in the cytoplasm. They were found free in the cytoplasmic matrix, aligned along the elements of filamentous complexes, and enclosed within vesicles. Some of the vesicles were found to be open to the extracellular space, indicating a likely mechanism of virus release.

Amnion↗

PubMed2004

Genome-wide linkage analysis of chromogranin B expression in the CEPH pedigrees: implications for exocytotic sympathochromaffin secretion in humans.

Chromogranin B (CgB), a major member of the chromogranin/secretogranin family of catecholamine storage vesicle secretory proteins, plays both intracellular (vesiculogenic) and extracellular (prohormone) roles in the neuroendocrine system, and its biosynthesis and release are under the control of efferent sympathetic nerve traffic ("stimulus-transcription coupling"). To explore the role of heredity in control of CgB, we conducted a genome-wide linkage analysis of CgB release in 12 extended CEPH (Centre d'Etude du Polymorphisme Humain) pedigrees. Region-specific radioimmunoassays were used to measure five CgB fragments in plasma: CgB1-16, CgB312-331, CgB439-451, CgB568-577, and CgB647-657. Substantial heritability, as measured by h2r, was observed for three of the fragment concentrations, CgB312-331, CgB439-451, and CgB568-577, which yielded h2r estimates ranging from 0.378 (P = 0.002) to 0.910 (P < 0.0000001). Variance-component genome-wide linkage analysis with 654 microsatellite markers at 5 cM spacing identified a major quantitative trait locus for CgB312-331 on chromosome 11q24-q25 with a maximum multipoint LOD score of 5.84. Significant allelic associations between markers in the region and CgB levels were also observed. Although the 2-LOD confidence interval for linkage did not include the CgB locus itself, known trans-activators of the CgB gene promoter, or prohormone cleaving proteases, examination of positional candidate loci within this region yielded novel and plausible physiological candidates for further exploration. Allelic variation in this region may thus influence effects of sympathetic outflow on target organs in humans.

Alleles↗

PubMed1994

Ultrastructural localization of major basic protein in the human eosinophil lineage in vitro.

We examined the ultrastructural localization of (a) a secondary granule matrix protein--eosinophil peroxidase (EPO)--by cytochemistry, (b) a secondary granule core protein (major basic protein, MBP) by immunogold labeling, and (c) a primary granule protein (the Charcot-Leyden crystal protein, CLC protein) by immunogold labeling in eosinophilic myelocytes (EMs) and mature, activated eosinophils that differentiated from umbilical cord blood progenitors cultured in the presence of recombinant human interleukin-5 (rhIL-5). These studies provide the first substructural localization of MBP to condensing cores of immature secondary granules of EMs, as well as identification of unicompartmental, MBP-rich secondary granules that are devoid of matrix compartments and EPO content and are not primary granules by virtue of their lack of CLC protein. These granules occur in quantity in IL-5-activated mature human eosinophils, which have previously been shown to actively transport EPO from the matrix compartments of their secondary granules to the extracellular milieu in smooth membrane-bound cytoplasmic vesicles, a secretory process termed piecemeal degranulation, whereby eosinophils progressively empty cytoplasmic granules of their contents in the absence of classical granule extrusion.

Blood Proteins↗

PubMed1981

Ultrastructural studies on congenital generalized fibromatosis regressed spontaneously.

Cellular components of a subcutaneous nodule from a male infant with congenital generalized fibromatosis consisted of myofibroblasts, fibroblasts, and their various intermediate types. The myofibroblasts were characterized by the presence of bundles of microfilaments and dense bodies similar to those in smooth muscle cells. Membrane-bound vesicles containing dense material were abundant in the extracellular space. They were probably lysosomes released by disintegration of cellular components. A possible role of the myofibroblasts and extracellular lysosomes in the regression of nodular lesions was discussed.

Fibroma↗