Morphometric study of the aortic body types I cell

Summary The subclavian glomus (aortic body) of New Zealand white rabbits was examined ultrastructurally using stereological morphometric analysis. The Type I cells of the glomus possess numerous electron-opaque vesicles which occupy approximately 12% of the cytoplasmic volume of the cells. The amine-containing vesicles comprise a heterogeneous population of vesicles with a mean caliper diameter of 113.5 nm. Differences in vesicle diameters may indicate the storage of different biogenic amines, different secretion or maturation states between glomera and/or additional physiological functions for the glomera.Supported by NIH Grant 5-TO1-GM793 and The University of Texas.     

The acetylcholinesterase reaction and catecholamine fluorescence in the glomus cells of rat carotid body

Summary The presence of both acetylcholinesterase reaction and glyoxylic acid-induced fluorescence of catecholamines in the same glomus cells of rat carotid body was demonstrated using combined histochemical methods. A suggestion is made that the glomus cells have both excitatory and inhibitory effects on the chemosensory nerve via acetylcholine and catecholamines, respectively.Acknowledgments. The present study was supported by grants from the Emil Aaltonen Foundation and the Finnish Cancer Foundation to Timo Waris.     

Trans-Golgi network sorting

The trans-Golgi network (TGN) is a major secretory pathway sorting station that directs newly synthesized proteins to different subcellular destinations. The TGN also receives extracellular materials and recycled molecules from endocytic compartments. In this review, we summarize recent progress on understanding TGN structure and the dynamics of trafficking to and from this compartment. Protein sorting into different transport vesicles requires specific interactions between sorting motifs on the cargo molecules and vesicle coat components that recognize these motifs. Current understanding of the various targeting signals and vesicle coat components that are involved in TGN sorting are discussed, as well as the molecules that participate in retrieval to this compartment in both yeast and mammalian cells. Besides proteins, lipids and lipid-modifying enzymes also participate actively in the formation of secretory vesicles. The possible mechanisms of action of these lipid hydrolases and lipid kinases are discussed. Finally, we summarize the fundamentally different apical and basolateral cell surface delivery mechanisms and the current facts and hypotheses on protein sorting from the TGN into the regulated secretory pathway in neuroendocrine cells. Received 2 November 2000; received after revision 19 February 2001; accepted 19 February 2001     

Multiple roles for the actin cytoskeleton during regulated exocytosis

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e., secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane, and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules.     

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342

Dictyostelium discoideum cells are highly resis tant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size >21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism. Received 14 November 1997; received after revision 16 March 1998; accepted 16 March 1998     

Kinetic analysis and simulation of glucose transport in plasma membrane vesicles of glucose-repressed and derepressed Saccharomyces cerevisiae cells

In this study experimental data on the kinetic parameters investigated by other authors 1-5, 11 together with own data on plasma membrane vesicles, have been subjected to a computer simulation based on the equations describing facilitated diffusion. The simulation led to an ideal fit describing the above data. From this it can be concluded that glucose is transported by facilitated diffusion, and not by active transport as was postulated by Van Steveninck 14,15. The simulation method also demonstrates that the fast sampling technique used by these authors 1-5, 11 underestimated the fluxes. Thus, the parameters given do not contribute to the understand of glucose transport under different metabolic conditions. The K value of plasma membrane vesicles prepared from glucose-repressed cells is around 7 mM. Derepression, particularly by galactose, causes a highly significant increase in affinity as shown by a decrease in the K value to 2 mM. The highest affinity was measured in a triple kinaseless mutant grown on glycerol with a K value of 1 mM. It seems, therefore, that the kinetic parameters derived from initial uptake rates of glucose in intact cells 1-5, 11 using single flux analysis, such as Eadie-Hofstee- or Lineweaver-Burk-plots, are in error.     

Cytomorphologische Voraussetzung des Vesikulardrüsenepithels für die Wirkung von Testosteron

Summary Studies were carried out on the influence of testosterone on the growth of seminal vesicles in relation to age. Moreover, the postnatal development of the secretory cells in seminal vesicles was investigated by electron microscopy, and the functional morphology of the epithelial cells at the moment of first effect of testosterone was discussed.     

Non-synaptic chemical neurotransmission.

Images with apparently gemmulofugal polarity in the EPL of the olfactory bulb are the result of sectioning, along misleading planes, gemmulopetal synapses containing postsynaptic vesicles. Unless one accepts a bidirectional conduction for chemical synapses, the internal granule cells lack actual gemmulofugal synapses and the neurotransmitter contained on their vesicles must act at non-synaptic membranes.     

Gap junctional intercellular communication of cultured rat liver parenchymal cells is stabilized by epithelial cells and their isolated plasma membranes

The gap junctional intercellular communication (GJIC) determined by measuring dye coupling with Lucifer yellow, decreased within 3 d from 66% to 28% in monocultures of rat liver parenchymal cells. Coculturing of the parenchymal cells with a nonparenchymal epithelial cell line from rat liver resulted in increased and stabilized intercellular communication (83% after 3 d). The presence of isolated plasma membrane vesicles of the nonparenchymal epithelial cells also stabilized the intercellular communication between the liver parenchymal cells (70% after 3 d). When liver parenchymal cells were cocultured with a rat liver fibroblast cell line the gap junctional communication between the parenchymal cells was not stabilized (43% after 3 d), and isolated plasma membrane vesicles of the fibroblast were also unable to support the GJIC in parenchymal cells (35% after 3 d). It is concluded that plasma membrane constituents of the nonparenchymal epithelial cells were responsible for the stabilization of the GJIC between parenchymal cells. A heterotypic gap junctional communication between parenchymal and nonparenchymal cells was not observed.     

Accumulation of 3H-dopamine by synaptic vesicles from rat striatum in an impermeant medium

The accumulation of 3H-dopamine by synaptic vesicles from rat striatum was significantly stabilized in a membrane impermeant medium. The characteristics of dopamine accumulation by striatal vesicles were quite similar to those reported for dopamine accumulation by a whole brain vesicle preparation in the same medium, and were significantly different from the characteristics previously reported for vesicular accumulation of norepinephrine.     

Non-synaptic chemical neurotransmission

Summary Images with apparently gemmulofugal polarity in the EPL of the olfactory bulb are the result of sectioning, along misleading planes, gemmulopetal synapses containing postsynaptic vesicles. Unless one accepts a bidirectional conduction for chemical synapses, the internal granule cells lack actual gemmulofugal synapses and the neurotransmitter contained in their vesicles must act at non-synaptic membranes.Supported by grant MA4183 of the Medical Research Council of Canada.     

Serglycin – Structure and biology

Serglycin is a proteoglycan found in hematopoietic cells and endothelial cells. It has important functions related to formation of several types of storage granules. In connective tissue mast cells the covalently attached glycosaminoglycan is heparin, whereas mucosal mast cells and activated macrophages contain oversulfated chondroitin sulfate (type E). In mast cells, serglycin interact with histamine, chymase, tryptase and carboxypeptidase, in neutrophils with elastase, in cytotoxic T cells with granzyme B, in endothelial cells with tissue-type plasminogen activator and in macrophages with tumor necrosis factor-α. Serglycin is important for the retention of key inflammatory mediators inside storage granules and secretory vesicles. Serglycin can further modulate the activities of partner molecules in different ways after secretion from activated immune cells, through protection, transport, activation and interactions with substrates or target cells. Serglycin is a proteoglycan with important roles in inflammatory reactions. Received 2 October 2007; received after revision 7 November 2007; accepted 12 November 2007     

Platelets as crucial partners for tumor metastasis: from mechanistic aspects to pharmacological targeting

Platelets are anucleated cells that circulate in the blood as sentinels of tissue integrity. In fact, they are rich in a plethora of proteins and other factors stored in different granules which they selectively release upon stimulation. Moreover, platelets synthesize a vast number of lipids and release various types of vesicles, including exosomes which are rich in genetic material. Platelets possess a central function to interact with other cell types, including inflammatory cells and cancer cells. Recent findings have enlightened the capacity of platelets to induce changes in the phenotype of cancer cells which acquire invasiveness thus enhancing their metastatic potential. Thus, it has been hypothesized that targeting the platelet may represent a novel strategy to prevent the development and progression of cancer. This is supported by the efficacy of the antiplatelet agent low-dose aspirin. Studies are ongoing to verify whether other antiplatelet agents share the anticancer effectiveness of aspirin.     

Incorporation of phospholipids in Ehrlich ascites tumor cells

Ehrlich ascites tumor cells (EATC) were incubated with unilamellar vesicles (UV) or multilamellar vesicles (MV). UV and MV were incorporated differently into EATC. The increase in 32P-phospholipid in EATC in the presence of UV was 12% in 300 min. Absorption of phospholipid from MV could account for only 3%. About 50% of the UV incorporation of 32P was by endocytosis and/or fusion.     

Evolution of the thermotropic properties of large unilamellar vesicles (LUV)

Summary The evolution of the thermotropic properties of large unilamellar vesicles (LUV) made by the reverse-phase evaporation technique has been studied by differential scanning calorimetry (DSC) and by fluorescence polarization of the diphenylhexatriene probe inserted in the lipid phase. Lipid fluidity and transition temperatures of DL-a-dimyristoyl-and DL-a-dipalmitoyl-phosphatidylcholine vesicles were practically not modified at room temperature (19–20°C), even after several days. Because a better knowledge of the physico-chemical properties of LUV seems essential for its use as a model membrane or as a carrier of exogenous material into cells, we compare it with the stability of the widely used multilamellar (MLV) and sonicated unilamellar vesicles (SUV).     

How cancer cells dictate their microenvironment: present roles of extracellular vesicles

Intercellular communication plays an important role in cancer initiation and progression through secretory molecules, including growth factors and cytokines. Recent advances have revealed that small membrane vesicles, termed extracellular vesicles (EVs), served as a regulatory agent in the intercellular communication of cancer. EVs enable the transfer of functional molecules, including proteins, mRNA and microRNAs (miRNAs), into recipient cells. Cancer cells utilize EVs to dictate the unique phenotype of surrounding cells, thereby promoting cancer progression. Against such “education” by cancer cells, non-tumoral cells suppress cancer initiation and progression via EVs. Therefore, researchers consider EVs to be important cues to clarify the molecular mechanisms of cancer biology. Understanding the functions of EVs in cancer progression is an important aspect of cancer biology that has not been previously elucidated. In this review, we summarize experimental data that indicate the pivotal roles of EVs in cancer progression.     

Role of FGFRL1 and other FGF signaling proteins in early kidney development

The mammalian kidney develops from the ureteric bud and the metanephric mesenchyme. In mice, the ureteric bud invades the metanephric mesenchyme at day E10.5 and begins to branch. The tips of the ureteric bud induce the metanephric mesenchyme to condense and form the cap mesenchyme. Some cells of this cap mesenchyme undergo a mesenchymal-to-epithelial transition and differentiate into renal vesicles, which further develop into nephrons. The developing kidney expresses Fibroblast growth factor (Fgf)1, 7, 8, 9, 10, 12 and 20 and Fgf receptors Fgfr1 and Fgfr2. Fgf7 and Fgf10, mainly secreted by the metanephric mesenchyme, bind to Fgfr2b of the ureteric bud and induce branching. Fgfr1 and Fgfr2c are required for formation of the metanephric mesenchyme, however the two receptors can substitute for one another. Fgf8, secreted by renal vesicles, binds to Fgfr1 and supports survival of cells in the nascent nephrons. Fgf9 and Fgf20, expressed in the metanephric mesenchyme, are necessary to maintain survival of progenitor cells in the cortical region of the kidney. FgfrL1 is a novel member of the Fgfr family that lacks the intracellular tyrosine kinase domain. It is expressed in the ureteric bud and all nephrogenic structures. Targeted deletion of FgfrL1 leads to severe kidney dysgenesis due to the lack of renal vesicles. FgfrL1 is known to interact mainly with Fgf8. It is therefore conceivable that FgfrL1 restricts signaling of Fgf8 to the precise location of the nascent nephrons. It might also promote tight adhesion of cells in the condensed metanephric mesenchyme as required for the mesenchymal-to-epithelial transition.     

Post-translational add-ons mark the path in exosomal protein sorting

Extracellular vesicles (EVs) are released by cells to the extracellular environment to mediate inter-cellular communication. Proteins, lipids, nucleic acids and metabolites shuttled in these vesicles modulate specific functions in recipient cells. The enrichment of selected sets of proteins in EVs compared with global cellular levels suggests the existence of specific sorting mechanisms to specify EV loading. Diverse post-translational modifications (PTMs) of proteins participate in the loading of specific elements into EVs. In this review, we offer a perspective on PTMs found in EVs and discuss the specific role of some PTMs, specifically Ubiquitin and Ubiquitin-like modifiers, in exosomal sorting of protein components. The understanding of these mechanisms will provide new strategies for biomedical applications. Examples include the presence of defined PTM marks on EVs as novel biomarkers for the diagnosis and prognosis of certain diseases, or the specific import of immunogenic components into EVs for vaccine generation.     

Calcium transport by red blood cell membranes from young and adult cattle

It is shown using inside-out membrane vesicles that cattle red cells extrude calcium by means of a calmodulin sensitive Ca-pump whose activity is high in calves and extremely low in adult cows. The decline is not due to loss of calmodulin susceptibility nor to a drop in Ca-affinity.