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T1 - Extracellular Vesicles and Their Role in Urologic Malignancies

We next investigated which metabolites were most concentrated in the EVs. In order to get an approximation of the intra-EV concentration range, we calculated the concentrations of the metabolites in the EVs by dividing the mole amount of metabolites with the total EV volumes in the samples (Table ). The total volume of EVs ranged between 60-830 nl within the uEV preparations and was 372 nl in the pEVs. These calculations indicated that the intra-EV concentrations of the metabolites varied from sub micromolar up to > 10 mM in both uEVs and pEVs (Table ). EVs from both sources were rich in D-Ribose 5-phosphate, the most abundant metabolite in pEVs and 3rd most abundant in uEVs, and other metabolites involved in nucleotide metabolism in mostly > 10 µM to mM range. Amino acid ornithine, with the highest concentration of metabolites in the uEVs and 15th highest in pEVs, and several other members of the urea cycle were present in > 50 µM to mM concentrations in the EVs. Ornithine serves also as a precursor for the biosynthesis of spermidine, a multifunctional polyamine that stabilizes nucleic acids and membranes, present in > 10 µM quantities in both EV types. In conclusion, our data indicated that the metabolite profiles of EVs from different sources contained similarities, but also distinct differences. The EVs carried a subset of metabolites from several pathways, with high intra-EV concentrations of some specific metabolites.

The nucleolus and ribosomes form part of the proteinsynthesizing machinery of the cell

AB - Context: Research has increased significantly on small vesicles secreted by healthy and diseased cells. Recent discoveries have revealed their functional and biomarker roles in urologic diseases. Whether and how this knowledge of extracellular vesicles (EVs) affects translational research and clinical practices have become pertinent questions. Objective: To provide an overview of the currently available literature on the rising field of EVs, focusing on function and pathogenesis in urologic cancers and the usefulness of EVs as biomarkers. Evidence acquisition: A systematic literature search was conducted using PubMed to identify original articles, review articles, and editorials regarding EVs in different types of urologic tumor diseases. Articles published between 2005 and 2015 were reviewed and selected with the consensus of all authors. Evidence synthesis: Besides soluble factors, different types of EVs are involved in the complex cross talk between different cell types. EVs regulate normal physiologic processes like spermatogenesis and renal function, as well as disease-specific processes including bladder, kidney, and prostate cancer. The content of EVs is derived from the cytoplasm of the donor cell. The proteins and RNAs within these EVs can be isolated from body fluids (eg, urine and blood) and represent potential diagnostic and prognostic biomarkers. EVs are also candidate therapeutic targets and potentially useful as therapeutic vehicles. Conclusions: The current data suggest that EVs are important regulators of cell-cell communication. The growing knowledge about their roles in urologic malignancies provides the basis for novel therapeutic strategies. In addition, nucleic acid and the protein content of EVs holds promise for the discovery of urine- or serum-based biomarkers for kidney, bladder, and prostate cancer. Patient summary: Normal and cancer cells secrete small vesicles that contain proteins and RNAs from the cell of origin. Changes in the diseased cells can be detected by examining the altered content of these vesicles when secreted in body fluids, for example, blood and urine. The recently discovered roles of extracellular vesicles (EVs) provide new options to detect malignancy in the urine and blood. The uptake of EVs may be blocked therapeutically and thereby potentially impede cancer progression. Extracellular vesicles regulate many cellular processes by exchange of nucleic acids and proteins. Their cell-specific content provides the basis for a new class of biomarkers in urologic malignancies.

A key role for vesicles in fungal secondary metabolism

This review attempts to demonstrate the role of seminal vesicles on male fertility

It took a long time to realize that glutamate is a neurotransmitter
It may sound astonishing, but it took the scientific community a long time to realize that glutamate is a neurotransmitter although it was noted already 70 years ago that glutamate is abundant in the brain and that it plays a central role in brain metabolism. Ironically, the reason for the delay seems to have been its overwhelming importance. Brain tissue contains as much as 5 - 15 mmol glutamate pr kg, depending on the region, more than of any other amino acid. Glutamate is one of the ordinary 20 amino acids which are used to make proteins and takes parts in typical metabolic functions like energy production and ammonia detoxification in addition to protein synthesis. It was hard to believe that a compound with so many functions and which is present virtually everywhere in high concentrations could play an additional role as transmitter.

N2 - Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B1; these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.

role of endoplasmic reticulum in protein synthesis

Effects of testosterone on messenger ribonucleic acid and protein synthesis in rat seminal vesicle.

We isolated EVs with differential centrifugation from 10-53 ml of urine or 5 ml of platelet concentrate to include the EV populations with both exosomes and larger EVs (Fig. and ). Ultracentrifugation supernatants were carefully removed before and after the washing step to ascertain that the EV pellets would not contain any extra-vesicular urine or platelet concentrate supernatant-derived metabolites.

We previously demonstrated that interfering with the activity of two HOPS (class C) tethering complex proteins encoded by avaA (homologue of yeast ypt7) and vps16 results in accumulation of large numbers of small, membrane bound organelles presumably because they fail to fuse with each other. This increase in organelle number accompanies increases in synthesis, storage, and export of aflatoxin. Because ypt7 in yeast mediates fusion of late endosomes with vacuoles, these data confirmed that V fraction is composed primarily of transport vesicles and endosomes; a small number of vacuoles was also observed in V fraction. Treatments that block endosome fusion with vacuoles increased the number of small sub-cellular compartments (and not larger compartments) as well as the synthesis, storage, and export of aflatoxin which suggests that transport vesicles, endosomes, but not vacuoles play a major role in aflatoxin synthesis. V fraction proteome data support this idea.

Although the iron-chelating protein lactoferrin is secreted by the seminal vesicles, the precise role of lactoferrin in semen is unclear.
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The role of vesicles in aflatoxin biosynthesis and export was ..

We distributed proteins identified in V fraction among 8 groups () based on predicted biological function and a small number were placed in a ninth category when their function was unknown. Specific enzyme markers typical of a fraction containing transport vesicles, enodosomes, and vacuoles were consistently identified in V fraction samples including vpsA (Ras-like GTPase involved in transport vesicle fusion), vacuolar transporters, stomatin, coatamer, clathrin, autophagic serine protease, metallopeptidase, calnexin (an endoplasmic reticulum membrane marker), woronin body major protein, 2 catalases, SNARE domain protein, 3 aminopeptidases, secretory pathway gdp dissociation protein, sorbitol and xylulase reductase, trehalose synthase, and 3 superoxide dismutases, (Mn, Zn, Fe),,. Of particular interest, enzymes involved in secondary metabolism were detected primarily at 36 h in YES (including a group of aflatoxin enzymes discussed below), although progressively smaller numbers of peptides associated with secondary metabolic enzymes could be detected at 24 h in YES and at 24 and 36 h in YEP, respectively.

Outline the role of ribosomes in protein synthesis

One primary function of transport vesicles is to carry protein cargo to and from sub-cellular organelles within the cell, . Consistent with this role, a large number of proteins detected in V fraction were assigned to nuclei, mitochondria, and peroxisomes. We previously demonstrated that intact nuclei and mitochondria are not present in V fraction and key cytoplasmic (lactate dehydrogenase) and mitochondrial (succinate dehydrogenase) marker enzymes are barely detected. These data confirm that many of the membrane bound sub-cellular compartments in V fraction are transport vesicles which transport a complex mixture of enzymes involved in primary and secondary metabolism to their destination within the cell. At present, it is not clear if these enzymes co-exist in the same membrane bound compartment or if separate compartments carry out unique functions.

What Are the Roles of Ribosomes in Protein Synthesis?

A. parasiticus was grown for 24 or 36 h under standard aflatoxin inducing (YES) and non-inducing conditions (YEP),. Aflatoxin synthesis initiates at approximately 30 h in YES medium, reaches maximum rates between 36 and 48 h, and then declines; neither aflatoxin nor aflatoxin enzymes are synthesized at detectable levels at any time in YEP or at 24 h in YES,,,. In contrast, aflatoxin and aflatoxin enzymes are detected at high levels by 36 h in YES,,,. A purified fraction containing transport vesicles, endosomes, and vacuoles which we designated V fraction was prepared from cultures incubated for 24 or 36 h in YEP or YES and high throughput LC/MS/MS analysis was conducted (see Methods). Proteome analysis for 2 biological replicates for YES 24 h, YEP 24 h, YEP 36h samples and 4 biological replicates for YES 36 h sample (including 2 V fraction reband samples) showed similar trends; representative data from one biological replicate are presented to illustrate the analyses.

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