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Yolk transport in the ovarian follicle of the hen (Gallus domesticus): lipoprotein-like particles at the periphery of the oocyte in the rapid growth phase.

Thin sections of the oocyte periphery and surrounding granulosa layer from 1--5 day preovulatory follicles were examined by transmission electron microscopy. With the use of certain procedures in tissue preparation, notably the tannic acid method, numerous particles in the range of 15--40 nm with a mean diameter of 27 nm were observed in both extra- and intracellularly. The particles were abundant in the granulosa basal lamina, in the spaces between the granulosa cells and in the perivitelline space. They appeared to adhere to the oolemma as a continuous double layer which was also observed to line the coated vesicles, 200--350 nm in diameter, invaginating from the oolemma. The layer of particles was not found on the plasma membranes of the granulosa cells, nor were particles present within the cells. In the peripheral cytoplasm of the oocyte the yolk spheres, ranging upwards from 250 nm diameter, were membrane-bound and contained tightly packed particles similar to those on the oolemma. Bodies displaying features intermediate between coated vesicles and yolk spheres suggested that, on entry into the cell, loss of the cytoplasmic coat and obliteration of the vesicular lumen gave rise to nascent yolk spheres which then fused together to form the larger spheres. The extracellular layer, coated vesicles and smaller yolk spheres were absent in oocytes fixed after a 10-min delay. The evidence indicated that 27-nm particles were transferred from the basal lamina to the oocyte surface via the intergranulosa cell channels, incorporated into the cell by adsorptive endocytosis and then transferred to the yolk spheres with little morphological alteration. The identity of the particles with very low density lipoproteins, the major components of the yolk solids, was discussed.

Animals↗

Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles.

In the exocytosis of neurotransmitter, fusion pore opening represents the first instant of fluid contact between the vesicle lumen and extracellular space. The existence of the fusion pore has been established by electrical measurements, but its molecular composition is unknown. The possibility that synaptotagmin regulates fusion pores was investigated with amperometry to monitor exocytosis of single dense-core vesicles. Overexpression of synaptotagmin I prolonged the time from fusion pore opening to dilation, whereas synaptotagmin IV shortened this time. Both synaptotagmin isoforms reduced norepinephrine flux through open fusion pores. Thus, synaptotagmin interacts with fusion pores, possibly by associating with a core complex of membrane proteins and/or lipid.

Animals↗

The glutamate transporter GLAST-1 (EAAT-1) is expressed in the plasma membrane of osteocytes and is responsive to extracellular glutamate concentration.

The glutamate/aspartate transporter GLAST-1 is expressed in bone in vivo and also exists as a splice variant (GLAST-1a) in which exon 3 is excluded. Since GLAST-1 expression is regulated in bone in response to osteogenic mechanical stimuli in vivo and binding of glutamate to receptors on osteoblasts increases osteoblast number and activity in vitro, control of extracellular glutamate concentrations may be critical for balanced bone remodelling. To determine whether GLAST isoforms may act to regulate extracellular glutamate concentration in bone we investigated whether their pattern or level of expression is responsive to glutamate concentration in bone cells. GLAST-1a mRNA is expressed at lower levels than GLAST-1 mRNA in all cells examined. The GLAST-1a/GLAST-1 mRNA ratio is greater in MLO-Y4 osteocytes than in SaOS-2 osteoblast-like cells, although this does vary in SaOS-2 cells in response to extracellular glutamate concentration. Transfection of MLO-Y4 cells with green fluorescent protein (GFP)-tagged GLAST isoforms revealed a plasma membrane localization of GLAST-1, consistent with its transporter function, whereas GLAST-1a appeared to be expressed within internal vesicles. Interestingly, low extracellular glutamate concentrations redistributed GLAST-1-GFP into a similar internal expression pattern. Regulation of the expression and distribution of GLAST-1 by extracellular glutamate in bone cells indicates that it may regulate glutamate signalling in bone, consistent with its operation in the central nervous system.

Animals↗

Ionic channels in synaptic vesicles: are they involved in transmitter release?

Synaptic vesicles were isolated from the nerve terminals of Torpedo electric organ. After fusion, 'giant' vesicles were formed which could be examined by the patch clamp technique. One of the cationic channels, the P channel, shows a small preference for K+ compared to Na+ and has multiple conductance levels. Its rate of opening is voltage and calcium dependent. Fractal analysis of the P channels reveals that its behaviour does not seem to be fractal in nature. At voltages where only one conductance level is observed, fractal analysis shows at least one discrete open state and at least two discrete closed states. There are considerable similarities between the P channel and channels found in granules from the hypophysis. These channels resemble, in turn, the channels found in gap junctions. Therefore, it is not unwarranted to speculate that a gap-junction-like communication between the secretory vesicle and the extracellular space may occur during exocytosis.

Animals↗

Cortisone induced alterations of costal cartilage in single and in parabiosed rats.

Cortisone-treated Buffalo rats have been parabiosed with untreated controls of the same age. The optical and electron microscopy, including histochemistry, of costal cartilage of these rats has been compared with that in single cortisone treated rats, single controls, and control parabiosed with control rats, at 14 and 28 days after parabiosis. Single cortisone-treated rats, in comparison to controls, have shown the greatest alteration in cellular morphology and in the extracellular matrix both at 14 and at 28 days. Cortisone-treated parabiosed rats demonstrate a gradation of these alterations. Cellular alterations include enhancement of lipid and glycogen deposition concrurently with the presence of numerous large cytoplasmic vacuoles containing beaded irregularly-shaped filaments, banded or unbanded collagen-like fibrils, and/or electron dense lamellar bodies. In the extracellular matrix, matrix vesicles, amianthoid fibers, randomly oriented unbanded fibrillar materials, and filament-like materials are most prominent in the single cortisone-treated rats and they are progressively less prominent in the cortisone-treated parabiosed rats, and in the parabiosed and single controls. Calcification of the extracellular matrix follows a similar pattern and is observed initially in pericellular halos of the single cortisone and in cortisone-treated rats parabiosed with controls. Histochemical techniques have shown that chondroitin sulfate is less demonstrable in the single cortisone and in the cortisone-treated parabiosed rats than it is in the single or parabiosed controls at 14 days but, at 28 days, all untreated or treated rats, single or parabiosed are basically comparable. Glycoproteins are prominent in the single cortisone-treated rats both at 14 and at 28 days and, at these same times, they are progressively less prominent in the cortisone-treated parabiosed rats and in the single or parabiosed controls. Many of the cortisone induced alterations in costal cartilage are suggestive of enhancement of the aging process.

Animals↗

Synaptic vesicles in electromotoneurones. II. Heterogeneity of populations is expressed in uptake properties; exocytosis and insertion of a core proteoglycan into the extracellular matrix.

The three populations of synaptic vesicles in electromotor nerve terminals were analysed quantitatively. Empty vesicles (VP0), fully charged vesicles (VP1) and charged but smaller VP2-type vesicles are present in approximately equal amounts in the nerve terminal. The populations show differences in the kinetics of in vitro uptake of acetylcholine, ATP and Ca2+. VP0 and VP2 accumulate acetylcholine and ATP but no Ca2+, whereas VP1 shows negligible acetylcholine and ATP but high Ca2+ uptake. Thus the expression of uptake properties of this secretory organelle depend on the stage it has reached in its life cycle and might constitute a signal for processing. VP2 was found to contain much less core proteoglycan than VP0 and VP1 indicating that part of it has been lost by exocytosis. In synaptic extracellular matrix containing fractions an antigen is detectable that cross-reacts with an antiserum against the vesicle proteoglycan. This material elutes upon gel filtration in a position similar to a smaller form of proteoglycan found in vesicles. We conclude that the electromotor nerve terminal releases a proteoglycan by the regulated secretory pathway that is deposited in the extracellular matrix. It might have a function in keeping pre- and postsynaptic structures in alignment constituting a transsynaptic signal. Based on the findings described, a model of the vesicles' life-cycle is discussed, whereby the VP2 population is the major source of quantal release of acetylcholine.

Animals↗

1alpha,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases.

Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1alpha,25(OH)(2)D(3) and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1alpha,25(OH)(2)D(3) regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1alpha,25(OH)(2)D(3)-binding protein ERp60, phospholipase A(2) (PLA(2)), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1alpha,25(OH)(2)D(3) (10(-8)M), which binds ERp60, activating PLA(2), and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-beta1 stored in the ECM as large latent TGF-beta1 complexes, consisting of latent TGF-beta1 binding protein, latency associated peptide, and latent TGF-beta1. Others have shown that MMP-2 specifically activates TGF-beta2. TGF-beta1 regulates 1alpha,25(OH)(2)D(3)-production, providing a mechanism for local control of growth factor activation. 1alpha,25(OH)(2)D(3) activates PKCalpha in the PM via ERp60-signaling through PLA(2), lysophospholipid production, and PLCbeta. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCzeta. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1alpha,25(OH)(2)D(3), PKCzeta activity is decreased and PKCalpha is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.

Animals↗

Placental calcification: ultrastructural and X-ray microanalytic studies.

Calcification is common in human placentas and is widely recognized as a normal part of maturation and aging of this organ. Eleven human placentas were studied by light and electron microscopy to elucidate the mechanism of placental calcification. Earliest mineral deposits were seen along the trophoblastic basement membrane of the chorionic villi undergoing fibrinoid degeneration. Transmission electron microscopic examination revealed crystalline deposits within small membrane-bound vesicles; the latter appear to be derived from degenerating cells and were particularly numerous within the basement membrane. X-ray microanalysis of these deposits revealed calcium and phosphorus peaks and the pattern of calcium hydroxyapatite was noted by electron diffraction. This pattern of calcification, i.e., precipitation of calcium hydroxyapatite in association with extracellular membrane bound vesicles, is similar to that seen in physiologic and pathologic calcifications of other tissues.

Calcification, Physiologic↗

The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia.

Giardia is an intestinal parasite that belongs to the earliest diverging branch of the eukaryotic lineage of descent. Giardia undergoes adaptation for survival outside the host's intestine by differentiating into infective cysts. Encystation involves the synthesis and transport of cyst wall constituents to the plasma membrane for release and extracellular organization. Nevertheless, little is known about the molecular events related to cyst wall biogenesis in Giardia. Among the components of the cyst wall there are two proteins that we have previously identified and characterized: CWP1 (26 kDa) and CWP2 (39 kDa). Expression of these proteins is coordinately induced, and both concentrated within encystation-specific secretory vesicles before their extracellular polymerization. Although highly similar to each other at the amino terminus, CWP2 includes a COOH-terminal 121-amino acid extension. Here, we show that this extension, rich in basic residues, is cleaved from CWP2 before cyst wall formation by an intracellular cysteine proteinase activity, which is induced during encystation like CWPs. Specific inhibitors prevent release of cyst wall materials, abolishing cyst wall formation. We also report the purification, cloning, and characterization of the encystation-specific cysteine proteinase responsible for the proteolytic processing of CWP2, which is homologue to lysosomal cathepsin C. Encystation-specific cysteine proteinase ESCP possesses unique characteristics compared with cathepsins from higher eukaryotes, such as a transmembrane domain and a short cytoplasmic tail. These features make this enzyme the most divergent cathepsin C identified to date and provide new insights regarding cyst wall formation in Giardia.

Amino Acid Sequence↗

Pathways of transcytosis in the fenestrated endothelium of pancreatic capillaries.

A procedure which covalently attaches an electron dense tracer to cell surface sialoglycoconjugates was used in order to label the luminal surface of capillary endothelium in rat pancreas. Mild oxidation of the vasculature by perfusion with 1 mM sodium periodate, followed by a 30-90 min incubation with ferritin hydrazide (FH) at 37 degrees C, led to a characteristic decoration of the endothelial surface. FH was taken up by the endothelium in coated vesicles and, to a much lesser extent, in plasmalemmal vesicles or vacuoles. Though rarely, FH was found at the abluminal front of the endothelium in coated vesicles opened to the extracellular space either directly or via a smooth-surfaced vesicle, thus suggesting the involvement of coated structures in transendothelial transport (transcytosis). A conjugate of wheat germ agglutinin with ferritin, which binds noncovalently to cell surface glycoconjugates containing N-acetylneuraminyl and N-acetylglucosaminyl residues, was transported transcellularly in monomeric form, mainly through smooth-surfaced (plasmalemmal) vesicles and transendothelial channels. Cationized ferritin (CF), pI approximately 8.4, which binds to cell surface acidic sites via an electrostatic noncovalent interaction, was rapidly internalized by the oxidized endothelium in coated structures and in vacuoles. Big aggregates of CF were transported and delivered to the abluminal front of the endothelium via large vacuoles, vesicles, and through coated structures and multivesicular bodies. These results show that the capillary endothelial cell possesses multiple mechanisms for transporting macromolecules across the cell, involving vacuoles, smooth or coated vesicles, channels, and lysosomal-related compartments. They also indicate that, besides molecular weight and electric charge of macromolecules, other characteristics of the ligands (e.g., type of bond with cell surface components, state of aggregation) are factors influencing the transendothelial transport.

Animals↗

[Secretion of periplasmic PhoA protein during its supersynthesis into the medium using outer membrane vesicles. Features of chemical composition of the vesicles and membranes of Escherichia coli secreting cells].

E. coli K12802 cells transformed by multicopy plasmid with phoA gene acquire the ability to oversynthesize alkaline phosphatase, secrete it into the cultural medium, and accumulate the precursor of this enzyme. The dynamics of enzyme production and secretion as well as cytomorphological changes revealed the existence of a mechanism of selective enzyme secretion into the medium. It is characterized by a decrease of enzyme specific activity in periplasm and its increase in cultural medium, appearance of numerous local zones of adhesion of cytoplasmic and outer membranes, formation of large extracellular outer membrane vesicles containing PhoA protein on the cell poles, and their release into the medium. We isolated the vesicles and found that they contain PhoA (in dominating quantity), several other periplasmic proteins, and matrix proteins of outer membranes. By their phospholipid and protein composition, they correspond to the fraction of outer membranes which have the largest density and sedimentation rate and, apparently, contain no lipoprotein.

Alkaline Phosphatase↗

Neuro-epithelial bodies in the lung of the rat and the mouse.

Histochemical and ultrastructural examinations were performed on neuroepithelial bodies (N. E. B.) in the lung of the mouse and rat. The N. E. B. were identified as specialized groups of pale, columnar cells. They were located throughout the intrapulmonary airway. These cells displayed some special cytochemical properties also seen in the APUD (Amine Precursors Uptake and Decarboxylation) endocrine system, such as cytoplasmic argyrophilia and the capability of selective uptake of amine precursors. Ultrastructrally the N. E. B. were composed of a kind of granulated cell which had concentrations of specific cored vesicles in the basal cytoplasm. It seemed likely that the cells released the contents of the cored vesicles into the extracellular space by exocytosis. These cells made extensive and intimate contact with the mitochondria-rich nerve terminals which occurred mainly at the level of the supranuclear portions of the granulated cells. In this contact area, the membrane of the nerve terminals were apposed to that of the granulated cells with a consistent gap of about 20 nm. Among these membrane appositions, some membrane thickenings were observed in which cored vesicles were closely associated in the granulated cell cytoplasm. From these observations, it was conceived that the lung N. E. B. function both as receptor and endocrine organs and that their specific cored vesicles may have an intimate correlation with these dual functions.

APUD Cells↗

Novel secretory vesicle proteins essential for membrane fusion display extracellular-matrix domains.

Exocytotic mutants can be obtained in Paramecium that affect the organization of the fusion machinery, visible by electron microscopy. The site of action of the genes in the plasma membrane, cytosol or secretory compartment can easily be determined in such mutants. Functional complementation cloning of exocytotic mutants specifically affected in the secretory compartment, nd2-1 and nd169-1, reported here, and the previously studied nd7-1, led to the discovery of a set of novel proteins that display PSI and EGF domains, normally found in extracellular matrix proteins and involved in transmembrane signaling. The structure of one of these proteins, Nd2p, and of the product of a paralog found in the genome Nd22p, corresponds to that of type I membrane receptors, generally involved in protein and vesicle sorting. Our characterization suggests that the proteins we have identified are required to indicate the presence of a mature secretory vesicle to the plasma membrane, to prepare the machinery for fusion. We propose to name this novel subclass of receptors VEMIF, for Vesicular Extracellular-Matrix-like proteins Involved in preparing membrane Fusion.

Amino Acid Sequence↗

A fusion pore phenotype in mast cells of the ruby-eye mouse.

Using patch-clamp capacitance and amperometric techniques, we have identified an exocytotic phenotype that affects the function of the fusion pore, the molecular structure that connects the lumen of a secretory vesicle with the extracellular environment during exocytosis. Direct observation of individual exocytotic events in mast cells from the ruby-eye mouse (ru/ru) showed a 3-fold increase in the fraction and duration of transient fusion events with respect to wild-type mice. The fraction of the total fusion events that were transient increased from 0.22 +/- 0.02 (wild type) to 0.65 +/- 0.02 (ru/ru), and the average duration of these events increased from 418 +/- 32 ms (wild type) to 1207 +/- 89 ms (ru/ru). We also show that this phenotype can reduce and delay an evoked secretory response by causing the fusion of vesicles that have been previously emptied by repeated cycles of transient fusion. The exocytotic phenotype that we describe here may be a cause of diseases like platelet storage pool deficiency and prolonged bleeding times for which the ruby-eye mouse serves as an animal model. Furthermore, the identification of the gene causing the fusion pore phenotype reported here will illuminate the molecular mechanisms regulating exocytotic fusion.

Animals↗

PlcR1 and PlcR2 are putative calcium-binding proteins required for secretion of the hemolytic phospholipase C of Pseudomonas aeruginosa.

The plcHR operon of Pseudomonas aeruginosa includes the structural gene for the hemolytic phospholipase C,plcH (previously known as plcS), and two overlapping, in-phase, genes designated plcR1 and plcR2. Hemolytic and phospholipase C (PLC) activities produced by Escherichia coli and P. aeruginosa T7 expression systems were measured in strains carrying both plcH and plcR genes and in strains carrying each gene separately. When plcH was expressed by itself in the E. coli T7 system, the area of the hemolytic zone on blood agar was less than twice the area of growth. By contrast, when plcR was coexpressed with plcH in this system, the area of the hemolytic zone was approximately 10 times that of the area of the growth on blood agar. Native polyacrylamide gel electrophoretic analyses of PlcH activity expressed in either the E. coli or the P. aeruginosa T7 system carrying plcH alone, or along with the plcR genes, suggest that PlcR either posttranslationally alters the physical or biochemical nature of PlcH or releases PlcH from a complex in the cell so that it can be secreted. The hypothesis that PlcR is involved in the secretion of PlcH is supported by the observation that the ratio of extracellular to cell-associated PlcH activity produced by P. aeruginosa strains containing an in-frame deletion in the chromosomal plcR genes is significantly reduced in comparison with this ratio seen with the wild-type parental strain. This defect in the secretion of PlcH can be complemented by the plcR genes in trans. Additional data suggest that PlcR does not directly affect the secretion of the nonhemolytic phospholipase C (PlcN). PlcR is highly similar to a calcium-binding protein (CAB) from Streptomyces erythraeus. PlcR and CAB contain typical motifs (EF hands) characteristic of eucaryotic calcium-binding proteins, including calmodulin. P. aeruginosa naturally produces membrane vesicles (MVs) containing extracellular proteins including PLC. MVs from the PAO1WT strain contained at least 10-fold more PLC specific activity than those isolated from a strain carrying a deletion of plcR (PAO1 deltaR). Immunogold electron microscopy of PAO1WT and PAO1 deltaR whole cells revealed a distribution of PlcH in these strains consistent with the hypothesis that PlcR is required for the secretion of PlcH.

Calcium-Binding Proteins↗

A pulse-chase study tracking the conversion of macrophage-endocytosed serum amyloid A into extracellular amyloid.

OBJECTIVE: To determine whether serum amyloid A (SAA) is internalized by and processed in macrophages en route to deposition as extracellular amyloid. METHODS: SAA was tracked in cultures of peritoneal macrophages, using a pulse-chase protocol. Macrophages were pulsed with either fluorescently (with Texas Red) tagged SAA (TxR-SAA) or iodinated SAA ((125)I-SAA). Cells were then rinsed and shifted to chase medium containing unlabeled SAA and amyloid-enhancing factor (AEF) to induce amyloid formation. At selected times, TxR-SAA in living cells was observed by confocal scanning microscopy. (125)I-SAA was visualized and quantified in cell lysates and medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorimaging. The presence of amyloid was confirmed by Congo red staining. RESULTS: Confocal microscopy immediately after the pulse revealed TxR-SAA in endosomal vesicles, with no extracellular or cell surface accumulation. After 24 hours and 72 hours of chase, virtually all TxR-SAA remained intracellular. By 10 days, extracellular fluorescence was very strong, indicating that SAA had moved out of cells. Congo red staining revealed amyloid colocalized with areas of extracellular fluorescence. Experiments using (125)I-SAA showed that while 90-95% of internalized (125)I-SAA was degraded within 24 hours, 5-10% persisted as intact SAA or SAA peptides. Immediately after the pulse, SAA was full-length, but within 24 hours, discrete (125)I-SAA peptides were seen. Each peptide had an intact SAA amino-terminus, as expected for AA protein. Amyloid was detected in cultures as early as 24 hours after initiation of treatment with SAA and AEF and appeared to be intracellular. CONCLUSION: The results of this study provide direct evidence that SAA internalized by and processed in macrophages forms extracellular amyloid. Based on the presence of (125)I-AA protein in macrophage lysates prior to the appearance of extracellular TxR-labeled amyloid, it was concluded that cleavage of SAA to AA occurs intracellularly.

Animals↗

Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis.

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.

Animals↗

A comparative analysis of strategies for isolation of matrix vesicles.

Matrix vesicles (MVs) are extracellular organelles involved in the initial steps of mineralization. MVs are isolated by two methods. The first isolation method of MVs starts with collagenase digestion of osseous tissues, followed by two differential centrifugations. The second isolation method does not use proteases but rather starts with differential centrifugation, followed by a fractionation on a sucrose gradient. The first method results in a homogeneous population of MVs with higher cholesterol/lipid content, alkaline phosphatase activity, and mineral formation rate as compared with MVs isolated by the second method. The second method leads to higher protein diversity as compared with MVs isolated according to the first method. Due to their distinct protein composition, lipid-to-protein and cholesterol-to-phospholipid ratios, and differences in rates of mineral formation, both types of isolated MVs are crucial for proteomic analysis and for understanding the regulation of mineralization process at the molecular level.

Animals↗