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PubMed1982

Ultrastructural changes in the neurohypophysis of the aged male rat.

The neurohypophyses (neural lobes of the pituitary) of young adult (3-6 months) and aged (12-30 months) male rats were studied by means of electron microscopy. Stereomorphometric analyses were performed to determine the size, number and relative volume of the hormone-containing vesicles. The principal observations included: 1) the conspicuous depletion in aged rats of the granular hormone-containing vesicles from the axon terminals and the Herring bodies, with a decrease in the relative volume fraction of the vesicles from 4.8 Vv% in the control animals to 1.1 Vv% in the aged rats; 2) a change in the size-distribution of the hormone vesicles; 3) an increase in the extracellular space around the nerve terminals, axons and capillaries; and 4) lipid accumulations and signs of activation in the pituicytes. The possible physiological significance of the findings is discussed in the light of several regulatory functions known to be altered during the process of ageing.

Aging↗

PubMed1991

Parameters of growth in primary cultures and cell lines established from Drosophila imaginal discs.

We have further characterised our tissue culture system for the growth in vitro of Drosophila imaginal disc cells, including the culture medium requirements for optimum growth and we have adjusted the protocol recommended for the initiation of cultures. Many imaginal disc fragments become organised into vesicles, and some of these secrete extracellular material into the lumen. Sensory axons differentiate in primary disc cultures, in the absence of bristle formation. The early stages of cell division to form a cell line are recorded.

Animals↗

PubMed1997

On the release of glutamate and aspartate in the basal ganglia of the rat: interactions with monoamines and neuropeptides.

Using highly sensitive analytical procedures, glutamate (Glu), aspartate (Asp) and several putative neurotransmitters and metabolites can be monitored simultaneously in the extracellular space of neostriatum, substantia nigra and cerebral cortex of the rat by in vivo microdialysis. Glu and Asp are found at sub-micromolar concentrations in all investigated brain regions. In order to ascertain their neuronal origin, we have extensively studied the sensitivity of extracellular Glu and Asp levels to: (i) K(+)-depolarization, (ii) Na(+)-channel blockade, (iii) removal of extracellular Ca2+, (iv) depletion of presynaptic vesicles, and (v) integrity of neuronal pathways. The relevance of these criteria for several neurotransmitters monitored simultaneously or in parallel experiments has also been examined. The functional interactions among different neuronal pathways in the basal ganglia are studied by using selective pharmacological treatments, administered systemically, or locally via intracerebral injections or the microdialysis perfusion medium. Immunohistochemical evidence for the existence of Glu and/or Asp neuronal pathways in the basal ganglia of the rat is presented, discussing especially new findings indicating the existence of a Glu-independent Asp system, intrinsic to the neostriatum of the rat. The clinical relevance of these interactions is discussed, focusing on the implications for the treatment of neurodegenerative disorders affecting the basal ganglia.

Animals↗

PubMed2003

Structural insights into the SNARE mechanism.

Eukaryotic cells distribute materials among intracellular organelles and secrete into the extracellular space through cargo-loaded vesicles. A concluding step during vesicular transport is the fusion of a transport vesicle with a target membrane. SNARE proteins are essential for all vesicular fusion steps, thus they possibly comprise a conserved membrane fusion machinery. According to the "zipper" model, they assemble into stable membrane-bridging complexes that gradually bring membranes in juxtaposition. Hence, complex formation may provide the necessary energy for overcoming the repulsive forces between two membranes. During the last years, detailed structural and functional studies have extended the evidence that SNAREs are mostly in accord with the zipper model. Nevertheless, it remains unclear whether SNARE assembly between membranes directly leads to the merger of lipid bilayers.

Animals↗

PubMed1997

Cellular retention of liposome-delivered anionic compounds modulated by a probenecid-sensitive anion transporter.

PURPOSE: Drug carriers such as liposomes may enhance the intracellular delivery of therapeutic agents for infectious or neoplastic diseases. However, the mechanisms affecting cellular retention of liposome contents are understood poorly. We tested the hypothesis that retention of anionic compounds may be modulated by a nonspecific probenecid-sensitive anion transport mechanism, and that liposome composition may determine the impact of such transporters on drug retention by cells. METHODS: The fluorescent anionic dye hydroxy-pyrene-[1,3,6]-trisulfonate (HPTS) was transferred to the cytoplasm of cultured CV-1 or J774 cells by direct needle-microinjection or by ATP-induced permeabilization of the-plasma membrane, respectively, to investigate whether the cells have anion transport mechanisms capable of extruding HPTS from the cytoplasm. Cellular retention of dye was monitored in the presence and absence of the anion transport inhibitors probenecid or sulfinpyrazone. Liposomes containing HPTS were co-labeled with tetramethylrhodamine-labeled phosphatidylethanolamine (Rho-PE) as a marker of liposome membrane fate, and uptake was investigated using J774 cells. RESULTS: Needle-injected HPTS underwent both sequestration in early endocytic vesicles and rapid extrusion into the extracellular medium. Probenecid or sulfinpyrazone reduced the extrusion of HPTS. Thus HPTS is a substrate for a probenecid-sensitive anion transporter in J774 and CV1 cells. After delivery via fluid liposomes composed of phosphatidylglycerol:phosphatidylcholine:cholesterol (3:7:5 mole ratio) and co-labeled with Rho-PE, cell-associated HPTS declined more rapidly than did Rho-PE. Exposure of cells to 5 mM probenecid doubled the quantity of HPTS retained by cells, without changing the retention of the Rho-PE membrane marker. In contrast, the effect of probenecid was negligible when gel-phase liposomes of distearoylphosphatidylglycerol:cholesterol (10:5 mole ratio) were used. CONCLUSIONS: Probenecid-sensitive nonspecific anion transporters can mediate the extrusion of model anions delivered via liposomes. However, liposome composition modulates the amount of material subject to extrusion from cells, possibly by altering the endocytic compartment in which liposomes release their contents.

Animals↗

PubMed2000

Signal transduction pathways triggered by fibroblast growth factor receptor 1 expressed in Xenopus laevis oocytes after fibroblast growth factor 1 addition. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cgamma.

Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) were used as a biological model system to analyse the signal transduction pathways that are triggered by fibroblast growth factor 1 (FGF1). Germinal vesicle breakdown (GVBD) and phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) occured 15 h after FGF1 addition. These events were Ras-dependent as they were blocked by a Ras dominant negative form. The Ras activity was promoted by three upstream effectors, growth factor-bound protein 2 (Grb2), phosphatidylinositol 3-kinase (PI3K) and Src cytoplasmic kinase. Ras activation was inhibited by a Grb2 dominant negative form (P49L), by PI3K inhibitors, including wortmannin, LY294002, the N-SH2 domain of p85alpha PI3K and by the SH2 domain of Src. Src activation induced by FGF1 was blocked by the SH2 domain of Src and PP2, a specific inhibitor of Src. The Grb2 adaptor was recruited by the upstream Src homology 2/alpha-collagen-related (Shc) effector, as the SH2-Shc domain prevented the GVBD and the ERK2 phosphorylation induced by FGF1. The importance of another signalling pathway involving phospholipase Cgamma (PLCgamma) was also investigated. The use of the PLCgamma inhibitory peptide, neomycin and the calcium chelator BAPTA-AM on oocytes expressing FGFR1 or the stimulation by PDGF-BB of oocytes expressing PDGFR-FGFR1 mutated on the PLCgamma binding site, prevented GVBD and ERK2 phosphorylation. This study shows that the transduction cascade induced by the FGFR1-FGF1 interaction in Xenopus oocytes represents the sum of Ras-dependent and PLCgamma-dependent pathways. It emphasizes the role played by PI3K and Src and their connections with the Ras cascade in the FGFR1 signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed1993

Reactive oxygen species and neutrophil respiratory burst cytochrome b558 are produced by kidney glomerular cells in passive Heymann nephritis.

Reactive oxygen species (ROS) have been implicated in the production of glomerular damage in passive Heymann nephritis (PHN), an experimental form of membranous nephropathy with neutrophil-independent proteinuria. Immunohistochemistry with monoclonal antibodies specific for cytochrome b558 (a major component of the oxidoreductase complex of the respiratory burst in stimulated neutrophilic granulocytes) showed that this enzyme is localized within visceral glomerular epithelial cells (GECs) in a dense, granular pattern in rats with PHN and proteinuria. By immunoelectron-microscopy, the cytochrome was found in membrane vesicles within the GEC and also extracellularly on the GEC membranes facing the glomerular basement membrane (GBM). By immunoblotting, cytochrome b558 was detected in highest concentration in lysates of isolated glomeruli from proteinuric rats. By contrast, only traces were found in normal glomeruli by immunohistochemistry. Depletion of complement abolished the expression of the cytochrome. Using an ultrastructural cerium-H2O2 histochemistry technique, the functional activity of the glomerular ROS-generating system was demonstrated exclusively in proteinuric PHN, where H2O2 was found in highest concentration within the GBM. These results provide evidence that in rats with PHN and proteinuria, the GECs express and externalize respiratory-burst enzymes that generate ROS in a manner similar to neutrophilic granulocytes, which could then lead to glomerular damage.

Animals↗

PubMed2001

Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

ADP-Ribosylation Factor 1↗

PubMed1993

Involvement of cellular metabolism of calcium and phosphate in calcification of avian growth plate cartilage.

Past work established that matrix vesicles (MV) are primary initiators of extracellular mineral deposition in endochondral calcification. Reviewed here are studies on how direct cellular metabolism of Ca2+ and inorganic phosphate (Pi), and cellular interaction with the matrix, are involved in the formation of calcifiable MV. Presented is a working model of how chondrocytes in growth plate (GP) cartilage are envisioned to induce the formation of calcifiable MV. In part, this model is based on recent laser confocal imaging of living cartilage tissue sections with Indo-I AM, a fluorescent permeant Ca2+ probe. These studies indicate that GP chondrocytes actively acquire Ca2+, concentrate it to the cell periphery and exfoliate it as Ca(2+)-rich MV. Data from direct chemical analysis and 31P-NMR studies on freshly isolated cells show that GP chondrocytes are depleted of ATP and have elevated cytosolic Pi, a condition prerequisite to formation of Ca(2+)-acidic phospholipid (APL)-Pi complex-primed MV. Chondrocyte cell membrane processes from which MV arise have been found to be tightly linked to the cartilage-specific extracellular matrix collagens and proteoglycans. Annexins V and VI, APL-dependent Ca(2+)-binding proteins that form Ca2+ channels in chondrocytes and MV membranes, also bind to the matrix collagens and may serve as mechano-transducers in GP cartilage, gating Ca2+ entrance into the cells and MV. This interaction between the extracellular matrix and chondrocytes appears to facilitate Ca2+ loading of chondrocytes, formation of Ca2+ and Pi-primed MV and rapid induction of mineralization in GP cartilage.

Animals↗

PubMed1987

Ultrastructural study of calcification process in the ligamentum flavum of the cervical spine.

The ultrastructure of the formation of calcified nodules in three cases of symptomatic calcification of the cervical ligamentum flavum were studied. In some areas of the ligament, extracellular plasma membrane-invested matrix vesicles and thick wall-bound matrix giant bodies with or without mineralized deposits were present. These calcified vesicles and bodies were also encountered in the wide mineralized areas among the collagen and elastic fibers in the ligament. The mineralization process of the calcified nodules in the ligamentum flavum implies that matrix vesicles and matrix giant bodies acquire mineralized precipitates; then some gather in clusters. Calcified deposits may spread to collagen and elastic fibers contiguous with the calcified vesicles and bodies, and some eventually coalesce to make a calcified nodule.

Aged↗

PubMed1997

Evidence that agrin directly influences presynaptic differentiation at neuromuscular junctions in vitro.

The synaptic protein agrin is required for aspects of both pre- and postsynaptic differentiation at neuromuscular junctions. Although a direct effect of agrin on postsynaptic differentiation, presumably through the MuSK receptor, is established, it is not clear whether agrin directly affects the presynaptic nerve. To provide evidence on this point, we used anti-agrin IgG to disrupt agrin function in chick ciliary ganglion (CG) neuron/myotube cocultures. In cocultures grown in the presence of 200 microg/ml anti-agrin IgG, clustering of acetylcholine receptors (AChRs), extracellular matrix proteins, and the synaptic vesicle protein synaptotagmin (syt) at nerve-muscle contacts was inhibited. Syt clustering was still inhibited in the presence of 100 microg/ml blocking antibody, while the postsynaptic clustering of AChRs, heparan sulphate proteoglycan, and s-laminin was retained. Additionally, in CG neurons cultured with COS cells expressing agrin A0B0, which lacks the ability to signal postsynaptic differentiation, syt clustering was induced and this clustering was also blocked by anti-agrin IgG. Our results demonstrate that agrin function is acutely required for pre- and postsynaptic differentiation in vitro, and strongly suggest that agrin is directly involved in the induction of presynaptic differentiation.

Agrin↗

PubMed1993

Ultrastructure of wound healing following direct pulp capping with calcium-beta-glycerophosphate (Ca-BGP).

Tissue changes in rat incisors after direct pulp capping with calcium-beta-glycerophosphate (Ca-BGP) have been studied using light and electron microscopy. Immediately after pulp capping, Ca-BGP was converted to hydroxyapatite (HAP) at the cavity floor. At Day 1, the exposure site was covered with Ca-BGP-mediated mineralized tissue. Osteodentine had developed below this mineralized tissue at Day 3. Matrix vesicles (MV) were observed in the extracellular matrix between large cells and osteodentine. At Day 5, tubular dentine was observed below the osteodentine. Three days after the application of Ca(OH)2 in a control experiment, osteodentine had formed below the necrotic zone; however, tubular dentine was not observed at Day 5. These findings suggest that the applied Ca-BGP might be the source of Ca and inorganic phosphate (Pi) through hydrolysis by alkaline phosphatase (ALP), and that Ca-BGP-mediated mineralized tissue induces the early formation of tubular dentine.

Animals↗

PubMed1994

Biological and morphological characteristics of a transplanted cartilaginous tumor derived from a human osteogenic sarcoma.

A cartilaginous tumor derived from a human osteogenic sarcoma of the mandible has been maintained by serial passage to nude mice. Tumor growth was multilobular. Radiopaque spots were seen scattered throughout the tumor at three months after transplantation. Both light and electron microscopic examination at three months revealed that the tumor contained cartilaginous cells at various stages of differentiation. There was metachromasia throughout tumor lobules except in the marginal region. Von Kossa staining was positive in the central region. Ultrastructural study identified four subtypes of chondrocytic cells of a neoplastic nature. In the extracellular matrix around hypertrophic cells, matrix vesicles were observed with mineral deposits. Alkaline phosphatase was found on the plasma membrane and Golgi complexes of hypertrophic cells, and on matrix vesicles. Thus cell lineage and the manner of calcification of the transplanted tumor were similar to those of epiphyseal growth cartilage.

Adult↗

PubMed1986

Ultrastructure of fluoride-induced cysts in the rat molar enamel organ.

Earlier studies have shown that a single high dose of fluoride induces subameloblastic cysts separated by morphologically unaltered ameloblasts in the developing rat molar. In the present investigation, the ultrastructure of both the ameloblasts forming the cystic wall, and the cells within the cystic lumina were studied by means of transmission electron microscopy. It was shown that 24 h after fluoride injection the ameloblasts of the cystic wall showed various degrees of cytoplasmic and nuclear alterations. Some cells displayed signs of necrosis as indicated by condensation of the chromatin. The cytoplasmic changes varied from altered organelle morphology to fragmentation and almost complete shedding of the whole cytoplasm. In the ameloblasts of the cystic wall secretory products accumulated intracellularly, in distended rough endoplasmatic reticulum, in vesicles of the Golgi region and extracellularly between ameloblasts as well as between cells in the stratum intermedium, indicating an altered matrix secretion. Electron lucent material, cell and cell fragments were found in the cystic lumina, the two latter apparently originating from the ameloblastic layer. The degenerative changes seemed to follow the normal pattern of cell degeneration.

Ameloblasts↗

PubMed1972

Nonviral microbodies with viral antigenicity produced in cytomegalovirus-infected cells.

Masses of homogeneous electron-dense material accumulate in the cytoplasmic inclusions of cultured fibroblasts which have been infected with "wild" and "adapted" strains of human cytomegalovirus. The substance appears to be produced by microtubular membranes and the Golgi apparatus; ultrastructural histochemistry suggests that it is not lysosomal in nature nor is it comprised of lipids or polysaccharides. The dense material "buds" into cytoplasmic tubules forming circumscribed bodies having an investing membrane similar to the viral envelope. After transport to the extracellular milieu in cytoplasmic tubules and vesicles, virions and dense bodies can be demonstrated by immune electron microscopy. The homogeneous dense body appears to be a unique product of the cytomegalovirus-infected cell which possesses a limiting membrane having antigenic determinants common with the viral envelope.

Acid Phosphatase↗

PubMed1993

Viral liposomes released from insect cells infected with recombinant baculovirus expressing the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) has been found to promote assembly and budding of virions as well as down-regulating of VSV transcription. Large quantities of M protein can be produced in insect cells infected with recombinant baculovirus expressing the VSV M gene under control of the polyhedrin promoter. Analysis by pulse-chase experiments and density gradient centrifugation revealed that the [35S]methionine-labeled M protein synthesized in insect cells is released into the extracellular medium in association with lipid vesicles (liposomes). Electron microscopy and immunogold labeling showed that M protein expressed in insect cells induced the formation on plasma membrane of vesicles containing M protein, which are released from the cell surface in the form of liposomes. The baculovirus vector itself or recombinants expressing VSV glycoprotein (G) or nucleocapsid (N) protein did not produce the formation of vesicles in infected cells. The baculovirus-expressed M protein retains biological activity as demonstrated by its capacity to inhibit transcription when reconstituted with VSV nucleocapsids in vitro. These data suggest that M protein has the capacity to associate with the plasma membrane of infected cells and, in so doing, causes evagination of the membrane to form a vesicle which is released from the cell. This observation leads to the postulate, which requires further proof, that the VSV M protein can induce the formation and budding of liposomes from the cell membrane surface.

Animals↗

PubMed2003

Herpes simplex virus glycoprotein K, but not its syncytial allele, inhibits cell-cell fusion mediated by the four fusogenic glycoproteins, gD, gB, gH, and gL.

A Myc epitope was inserted at residue 283 of herpes simplex virus type 1 (HSV-1) glycoprotein K (gK), a position previously shown not to interfere with gK activity. The Myc-tagged gK localized predominantly to the endoplasmic reticulum, both in uninfected and in HSV-infected cells. gK, coexpressed with the four HSV fusogenic glycoproteins, gD, gB, gH, and gL, inhibited cell-cell fusion. The effect was partially dose dependent and was observed both in baby hamster kidney (BHK) and in Vero cells, indicating that the antifusion activity of gK may be cell line independent. The antifusion activity of gK did not require viral proteins other than the four fusogenic glycoproteins. A syncytial (syn) allele of gK (syn-gK) carrying the A40V substitution present in HSV-1(MP) did not block fusion to the extent seen with the wild-type (wt) gK, indicating that the syn mutation ablated, at least in part, the antifusogenic activity of wt gK. We conclude that gK is part of the mechanism whereby HSV negatively regulates its own fusion activity. Its effect accounts for the notion that cells infected with wt HSV do not fuse with adjacent, uninfected cells into multinucleated giant cells or syncytia. gK may also function to preclude fusion between virion envelope and the virion-encasing vesicles during virus transport to the extracellular compartment, thus preventing nucleocapsid de-envelopment in the cytoplasm.

Alleles↗

PubMed1993

Fluid-phase endocytosis by in situ cardiac myocytes of rat atria.

Fluid-phase endocytosis (FPE) associated with recycling of fused plasmalemma-secretory granules or membranes and/or membrane receptors by in situ cardiac myocytes was studied at 37 degrees C in vitro noncontracting adult rat atrial preparations. Measurements included 1) the volume (VS*) of the compartment consisting of presumptive endocytotic vesicles and the endosomes or lysosomes transiently in continuity with them (S*), which internalizes [14C]-sucrose but is inaccessible to simultaneously measured [methoxy-3H]inulin, 2) the kinetics of [14C]sucrose efflux from S*, and 3) morphometry to quantify interstitial space and non-heart muscle cells. Vs* (0.39 +/- 0.04 ml/g dry atrium for unstretched atria at 37 degrees C) was 1) variable over a 3.7-fold range under various experimental conditions, 2) significantly increased by neomycin or by lowering the temperature to 18 degrees C, and 3) significantly decreased by alpha 1-adrenergic stimulation. Analysis of sucrose efflux kinetics confirmed the presence of an intramyocytic sucrose-containing compartment. A smaller inulin-inaccessible sucrose space (S*) was also present in right ventricle. Thus, during FPE, vesicles and endosomes initially containing high (extracellular) Ca2+ and Cl- concentrations continually enter, circulate within, and undergo exocytosis from myocardial cells.

Animals↗