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At least 865 records · Page 48Linked to original sources

PubMed1988

Mechanisms of pathologic calcification.

Pathologic calcification usually is initiated by the biologic membranes of mitochondria or matrix vesicles. Mitochondria frequently initiate intracellular calcification. Matrix vesicles, derived from the outer membrane of cells by budding or cell disruption, initiate extracellular calcification in calcific tendonitis, apatite-deposition osteoarthritis, atherosclerosis, cardiac valvular calcification, tympanosclerosis, and other calcific diseases. Matrix vesicles and mitochondria usually initiate calcification through the interaction of phosphatase enzymes with calcium-binding phospholipids, both of which are membrane-bound. Hydroxyapatite (HA) crystals are formed first within the protective microenvironment of the membrane-enclosed microspace. Once formed and exposed to the extracellular fluid, HA crystals can serve as nuclei or templates, thus supporting progressive, autocatalytic mineral crystal proliferation.

Arthritis↗

PubMed1991

Altered ultrastructural morphology of self-aggregated low density lipoproteins: coalescence of lipid domains forming droplets and vesicles.

Lipid droplets and vesicles can presumably be formed directly from lipoproteins in the extracellular space in atherosclerosis, but an in vitro demonstration of the phenomenon in the absence of cellular pathways has been lacking. Low density lipoproteins (LDL) are known to undergo self-aggregation after brief vortexing in vitro. In the present study, LDL aggregates were examined by electron microscopy, using new mordant techniques for lipid visualization, and by chemical analysis. Aggregation of LDL by vortexing is regularly accompanied by the formation of comparatively large lipid droplets (up to 600 nm diameter) and vesicles. Aggregates containing droplets and vesicles were formed after as little as 5 sec of vortexing, and LDL protein and cholesteryl ester were almost completely (95%) incorporated into aggregates after 4 min vortexing. Substantial fractions of phospholipid and unesterified cholesterol from the original LDL remained in solution even after 4 min vortexing, forming large multilamellar vesicles that did not adhere to the aggregated material. Spontaneous aggregates retrieved from LDL solutions after prolonged storage were also examined by electron microscopy, revealing similar lipid droplets and vesicles. The ultrastructural appearance of LDL aggregated in vitro is remarkably similar to the appearance of extracellular lipid deposits in atherosclerosis, lending credence to the hypothesis of direct extracellular formation of these deposits from lipoproteins.

Arteriosclerosis↗

PubMed2002

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles.

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.

Adrenal Medulla↗

PubMed1977

Ultrastructural tracer studies on the permeability of the Malpighian tubule of the pill millipede, Glomeris marginate (Villers).

The electron-dense tracers ferritin, and iron-dextran, and the protein horseradish peroxidase, have been used to investigate the ultrastructural basis of permeability in the upper and lower segments of the Malpighian tubules of Glomeris marginata. All these materials were able to cross the basal lamina and enter the tubule lumen of the upper segment, and it was established that horseradish peroxidase was able to enter the channels which interrupt the apical junctions. In the upper segment, ferritin, iron-dextran, and horseradish peroxidase are taken up and accumulated within intracellular vesicles. In the lower segment ferritin and iron-dextran enter the cells but become generally distributed over the cytoplasm, as well as entering membrane-bounded vacoules. The behaviour of horseradish peroxidase could not be assessed by owing to the presence of endogenous peroxidase activity in the cells. After fixation by direct application of glutaraldehyde to the undissected tubules, the extracellular spaces contained large numbers of membrane-bounded vesicles. The significance of these observations is discussed in relation to the physiological activities of the tubules.

Animals↗

PubMed2002

Topology of the anion exchange protein AE1: the controversial sidedness of lysine 743.

The topology of the band 3 (AE1) polypeptide of the erythrocyte membrane is not fully established despite extensive study. Residues near lysine 743 (K743) have been reported to be extracellular in some studies and cytoplasmic in others. In the work presented here, we have attempted to establish the sidedness of K743 using in situ proteolysis. Trypsin, papain, and proteinase K do not cleave band 3 at or near K743 in intact red cells, even under conditions that cause cleavage on the C-terminal side of the glycosylation site (N642) in extracellular loop 4. In contrast, trypsin sealed inside red cell ghosts cleaves at K743, as does trypsin treatment of inside-out vesicles (IOVs). The transport inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H(2)DIDS), acting from the extracellular side, blocks trypsin cleavage at K743 in unsealed membranes by inducing a protease-resistant conformation. H(2)DIDS added to IOVs does not prevent cleavage at K743; therefore, trypsin cleavage at K743 in IOVs is not a consequence of cleavage of right-side-out or leaky vesicles. Finally, microsomes were prepared from HEK293 cells expressing the membrane domain of AE1 lacking the normal glycosylation site. This polypeptide does not traffic to the surface membrane; trypsin treatment of microsomes containing this polypeptide produces the 20 kDa fragment, providing further evidence that K743 is exposed at the cytoplasmic surface. Therefore, the actions of trypsin on intact cells, resealed ghosts, unsealed ghosts, inside-out vesicles, and microsomes from HEK293 cells all indicate that K743 is cytoplasmic and not extracellular.

Adult↗

PubMed1995

Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive channels.

Hippocampal neurons maintained in primary culture recycle synaptic vesicles and express functional glutamate receptors since early stages of neuronal development. By analyzing glutamate-induced cytosolic calcium changes to sense presynaptically released neurotransmitter, we demonstrate that the ability of neurons to release glutamate in the extracellular space is temporally coincident with the property of synaptic vesicles to undergo exocytotic-endocytotic recycling. Neuronal differentiation and maturation of synaptic contacts coincide with a change in the subtype of calcium channels primarily involved in controlling neurosecretion. Whereas omega-agatoxin IVA-sensitive channels play a role in controlling neurotransmitter secretion at all stages of neuronal differentiation, omega-conotoxin GVIA-sensitive channels are primarily involved in mediating glutamate release at early developmental stages only.

2-Amino-5-phosphonovalerate↗

PubMed1990

Dermatitis herpetiformis and gluten sensitive enteropathy (including celiac disease)--increased subepithelial extracellular matrix viscosity due to gliadin.

It is now firmly established that dermatitis herpetiformis (DH) is associated with gluten sensitive enteropathy (GSE), although the GSE of DH is generally milder than that form which occurs in celiac disease. The toxic fraction of gluten is in the gliadin, a protein fraction. Gliadin is absorbed in GSE and antigliadin antibodies are present in both DH and celiac disease. There is a question of a cross reactivity between reticulin and gliadin. Gliadin is reported to bind to reticulin. Reticulin has a glycosaminoglycan component. Fibronectin, another component of ground substance, also binds to reticulin. Reticulin and fibronectin are important in basement membrane areas and play a role in basement membrane attachment. Gluten may also exert a lectin effect on gastrointestinal mucosa which contributes to the underlying extracellular matrix. DH and GSE have different pathologies because of their different anatomical sites. The pathomechanisms of both diseases can be explained by one mechanism. Gliadin, or a peptide fraction from it, enters or combines with the extracellular matrix and increases tissue viscosity. The protein fraction of glycosaminoglycans in the extracellular matrix is known to control viscosity. In DH the increased extracellular matrix viscosity would interfere with the diffusion of tissue fluid in the dermal papillae and leads to vesicle formation. The intestinal villi serve a very different function. In GSE the increased extracellular matrix viscosity would decrease adsorption from the intestinal tract producing villi with less volume (shortened or atrophic). The shortened villi decrease the production of digestive enzymes and the absorptive surface. The decreased movement of nutrient tissue fluid supplied to the intestinal epithelial cells also eliminates the microvilli.(ABSTRACT TRUNCATED AT 250 WORDS)

Celiac Disease↗

PubMed2004

Screening populations of individual cells for secretory heterogeneity.

Many common metabolic and neurological disorders are related to defective regulation of exocytosis at the level of single cells. In exocytosis, vesicles containing the secretory product of a given cell type fuse with the plasma membrane allowing release of the vesicular contents into the extracellular environment where the physiological action can be exerted. The typical secretory vesicle contains between 0.15 and 10 attomoles of material that is released on a millisecond timescale. Hence, detection of this process presents several chemical and analytical challenges. In this work, we utilize the native ATP, stored at high concentrations within the secretory vesicles of most neuroendocrine cells and co-released during exocytosis and during cell lysis, as a universal tracer of cellular secretion events. Organisms studied include pancreatic islets, mast cells, and Escherischia coli. Cellular processes investigated include exocytotic release, stimulated cell lysis, and programmed cell lysis.

Adenosine Triphosphate↗

PubMed1985

Differentiation of extracellular matrix in the cellular cartilage ("Zellknorpel") of the mouse pinna.

Differentiation of cellular cartilage was studied in the mouse pinna with particular reference to matrix material. Fixation of glycosaminoglycans was performed by the use of acridine orange and elastin was identified by staining thin sections with tannic acid and uranyl acetate. Condensation of mesenchymal cells ("prechondroblasts") initiates the formation of a blastema of cartilaginous tissue at postnatal day 4. The synthesis of acidic glycosaminoglycans begins at postnatal day 8 when prechondroblasts transform to chondroblasts. Glycosaminoglycans can be detected within secretory vesicles of chondroblasts at postnatal day 8, in the extracellular space at postnatal day 13. Delicate collagen fibrils and elastic fiber microfibrils are seen between prechondroblasts and chondroblasts. Deposition of elastin begins at postnatal day 11. A network of elastic fibers and lamellae is formed, which replaces both collagen fibrils and elastic fiber microfibrils. In the interstice of mature cellular cartilage only elastin and proteoglycans are present (postnatal day 21). These findings indicate that cellular cartilage represents an independent kind of supporting tissue, which may serve as a progenitor of hyaline or elastic cartilage ("transitional cellular cartilage") but does not differentiate from hyalin cartilage.

Animals↗

PubMed1989

Vesicle- and collagen-mediated calcification in the turkey leg tendon.

The distribution, organization, and orientation of apatitic mineral associated with vesicles and collagen in normal calcifying turkey leg tendon have been examined to determine inorganic-organic interactions in this vertebrate tissue. The study utilized selected area electron diffraction and bright or dark field transmission electron microscopy of thin sections (approximately 70 nm) treated by anhydrous means. Bright field microscopy revealed crystals randomly disposed in radial clusters related to vesicles or highly oriented related to collagen. Dark field images of the crystal c-axis were localized within some hole zone regions of collagen but were generally sparse in vesicles. Images of mineral a,b-axes were associated with vesicles and also appeared in collagen hole zones complementary to those containing c-axis images. Where mineralization of vesicles occurred adjacent to that of collagen, bright and dark field images provided suggestive evidence that the crystals could be contiguous at some sites, but the bulk of extracellular mineralization was spatially discontinuous. The varying disposition of mineral in vesicles and collagen is likely indicative of distinct mechanisms of nucleation in these components, the complementary orientation of mineral c- and a,b-axes in collagen may provide insight into the structure and organization of the protein, and the distribution of vesicle-mediated mineralization appears to contribute to apatite accumulation between collagen fibrils.

Animals↗

PubMed1986

A nerve terminal anchorage protein from electric organ.

The nerve terminal and the postsynaptic receptor-containing membranes of the electric organ are both linked to the basal lamina that runs between them. We have identified an extracellular matrix protein whose physical properties suggest it anchors the nerve terminal to the basal lamina. The protein was identified because it shares an epitope with a proteoglycan component of electric organ synaptic vesicles. It too behaves like a proteoglycan. It is solubilized with difficulty from extracellular matrix fractions, elutes from DEAE Sephacel at pH 4.9 only at high ionic strength, and binds to a laminin affinity column from which it can be eluted with heparin. Under denaturing conditions it sediments rapidly and has a large excluded volume although it can be included in Sephacryl S-1000 columns. This large, highly charged extracellular matrix molecule can be readily reconstituted into liposomes consistent with the presence of a hydrophobic tail. By immunoelectron microscopy the antigen is found both in synaptic vesicles and on the plasma membrane of the nerve terminal. Since this is the first protein described that links the nerve terminal membrane to the extracellular matrix, we propose calling it terminal anchorage protein one (TAP-1).

Animals↗

PubMed2005

Membrane vesicles traffic signals and facilitate group activities in a prokaryote.

Many bacteria use extracellular signals to communicate and coordinate social activities, a process referred to as quorum sensing. Many quorum signals have significant hydrophobic character, and how these signals are trafficked between bacteria within a population is not understood. Here we show that the opportunistic human pathogen Pseudomonas aeruginosa packages the signalling molecule 2-heptyl-3-hydroxy-4-quinolone (pseudomonas quinolone signal; PQS) into membrane vesicles that serve to traffic this molecule within a population. Removal of these vesicles from the bacterial population halts cell-cell communication and inhibits PQS-controlled group behaviour. We also show that PQS actively mediates its own packaging and the packaging of other antimicrobial quinolines produced by P. aeruginosa into vesicles. These findings illustrate that a prokaryote possesses a signal trafficking system with features common to those used by higher organisms and outlines a novel mechanism for delivery of a signal critical for coordinating group behaviour in P. aeruginosa.

Biological Transport↗

PubMed1995

Markers of primary mineralization are correlated with bone-bonding ability of titanium or stainless steel in vivo.

Critical events in the adaptation of osseous tissues to implant materials involve initial calcification of the newly synthesized bone. Previous studies indicated that bone-bonding but not nonbonding glass ceramics increase the matrix vesicle number, thereby compensating for delayed maturation of the extracellular organelles. The present study assessed whether this was also true for metal implants commonly used in orthopaedics and oral medicine. Bone-bonding titanium (Ti) or nonbonding stainless steel (SS) implants were placed in the right tibias of Sabra rats following ablation of the marrow. At 3, 6, 14, and 21 days postinjury, newly formed endosteal bone in the treated and contralateral limbs was removed and matrix vesicle-enriched membranes isolated. Alkaline phosphatase and phospholipase A2 specific activities and phosphatidylserine (PS) content were determined and compared with those of a nonsurgical control group. Results show that matrix vesicle alkaline phosphatase and phospholipase A2 activity and PS content was increased in the Ti-implanted limbs at 6 (peak), 14, and 21 days, although at levels less than observed in normal healing. Alkaline phosphatase activity remained elevated throughout the healing period. In contrast, these parameters were markedly inhibited in the SS-implanted limbs with respect to Ti or to normal healing. Both implants altered the systemic response associated with marrow ablation, but in an implant-specific manner. The results support the hypothesis that cells adjacent to bone-bonding materials can compensate for negative effects on primary mineralization during osteogenesis, whereas cells adjacent to nonbonding materials either do not compensate or are further depressed. The data support the use of the rat marrow ablation model as a tool for rapid, initial assessment of biomaterials in bone.

Alkaline Phosphatase↗

PubMed1992

Ultrastructural evidence for neurogenically mediated changes in blood vessels of the rat dura mater and tongue following antidromic trigeminal stimulation.

We investigated the effects of unilateral electrical trigeminal ganglion stimulation (0.1 or 1.0 mA, 5 Hz, 5 ms, 5 min) on the morphology of blood vessels within the rat dura mater and tongue using light and transmission electron microscopy. Stimulation at both intensities caused changes which were confined to the ipsilateral post-capillary venules except in the tongue where arterioles were affected as well. Changes were more marked after 1.0 mA. Dramatic increases in the numbers of endothelial pinocytotic vesicles were found along the luminal and abluminal surfaces ipsilateral to the stimulation. Tight junctions remained largely intact, except that injected ferritin particles were occasionally trapped inside these junctions. Cytoplasmic microvilli and endothelial blebs were sometimes present as well. Approximately 80% of the examined dural post-capillary venules showed one or more of these endothelial changes. Horseradish peroxidase injected intravenously 5 min prior to stimulation was detected in the extracellular space surrounding dural blood vessels and within pinocytotic vesicles. Ferritin injected similarly, was also localized in post-capillary venule walls, interstitial spaces, intraendothelial vesicles and in vacuoles. Platelet accumulation and aggregation were present in approximately 10% of post-capillary venules in dura and tongue. These changes were associated with mast cell secretion, but neither vascular nor mast cell activation was observed in adult rats in whom C-fibers were destroyed during the neonatal period with capsaicin. The present observations provide morphological evidence which supports findings from previously reported albumin tracer studies suggesting enhanced transport and endothelial activation following electrical stimulation of small caliber afferent fibers.

Animals↗

PubMed1996

Interaction between Na ions and the Cl/HCO3 exchanger in basolateral membranes from rat jejunum.

The jejunal basolateral Cl/HCO3 exchanger is modulated by two Na-dependent regulatory sites located on the inner and outer membrane surfaces. The aim of this work was to focus on the interaction between the anion exchanger and intracellular or extracellular sodium. Uptake studies, performed using basolateral membrane vesicles, provided kinetic parameters as a function of outside or inside Na concentration. The intracellular Na-sensitive modifier site seems to be primarily involved in the modulation of the Cl/HCO3 exchanger.

Animals↗

PubMed1978

Actin--membrane interactions: association of G-actin with the red cell membrane.

Chemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G-actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G-actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside-out vesicles which have been stripped of endogenous actin and spectrin by low-ionic-strength incubation bind little G-actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G-actin can be increased up to 40-fold. Further, this crude spectrin extract can compete for and abolish G-actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G-actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G-actin is added to reconstituted inside-out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G-actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.

Actins↗

PubMed1980

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts.

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollagen type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.

Ascorbic Acid↗

PubMed1989

Role of spermatozeugmata in the spawning ecology of the brooding oyster Ostrea edulis.

Ostrea edulis spawns spermatozeugmata, each composed of radially arrayed sperm cells attached by an extracellular matrix (ECM) to a core of acellular vesicles. The acellular vesicles are formed from excess nuclear and plasma membranes produced during spermatid condensation, and the ECM is topologically restricted to the interstices between acellular vesicles and sperm heads, being absent from the flagellar surface. When released into seawater, spermatozeugmata retain their structural integrity for varying periods (up to 24 hours) and become demersally distributed in still water. The flagella activate, initially beating in a non-synchronized, languid manner; however, both the tempo and amplitude of the flagellar action gradually increase to resemble that of typical "primitive" sperm once the cells are released from the spermatozeugma. This increase in flagellar activity and subsequent gamete release coincide with an erosion of the ECM and suggest that the ECM may modulate sperm motility in addition to adhering the cells to the spermatozeugma. Sperm are capable of fertilizing eggs only after dissociating from the spermatozeugma. The net effect of spermatozeugma formation in Ostrea edulis may be the retention of viable sperm in high concentrations at the benthic/water column interface for prolonged periods after spawning, at least in low current regimes. Unfertilized O. edulis eggs are also concentrated at this location, in the inhalent chambers of female oysters. Spermatozeugmata are entrained by the inhalent current of females and carried into the brood chamber where fertilization occurs. The Ostrea edulis spermatozeugma likely functions as an efficient sperm transfer mechanism if two conditions are met: 1) eggs are retained as broods within benthic females, 2) egg masses are at intermediate distances from spawning males.

Animals↗