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| 24. |
- Ronquist, Göran, et al.
(författare)
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Human Prostasomes Contain Chromosomal DNA
- 2009
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Ingår i: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 69:7, s. 737-743
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND. The aim of this study was to perform a comprehensive evaluation of the occurrence of DNA in human prostasomes. METHODS. Prostasomes were purified from seminal fluid (seminal prostasomes) and from PC-3-cells (PC-3 cell prostasomes). DNA extracted from both sources of prostasomes was visualized on agarose gels. Further, theDNAwas cloned and sequenced (13 clones from seminal prostasomal DNAand 16 clones from PC-3 cell prostasomal DNA) and identified by alignment in the BLAST-nucleotide search database. In order to decide if the DNA was internally or externally located in/on prostasomes, prostasomes were treated with nuclease (DNase) and A260 was measured before and after treatment. Additionally, flow cytometric studies were performed with membrane permeable and membrane impermeable DNA stains. RESULTS. We identified human chromosomal DNA in purified prostasomes from both sources and treatment with DNase demonstrated that the prostasome-shielded DNA was protected from enzyme attack. Membrane-permeable DNA stain raised the fluorescence contrary to membrane-impermeable stain. Clearly discernible nucleic acid of prostasomes was separated on 1% agarose gel yieldingDNAfragments of about 13 kbp and below with a marked band at about 1 kbp. Cloning and sequencing of 13 fragments from seminal prostasomes and 16 from PC-3 cell prostasomes revealed a chromosomal origin of the DNA. In purified seminal prostasomes, 4 out of 13 DNA clones featured gene sequences (31%). The corresponding figure for PC3-derived prostasomes was 4 out of 16 clones featuring gene sequences (25%). CONCLUSION. Human prostasomes contain chromosomal DNA. Both nuclease treatment and differential DNA stainings indicated an inside location of the prostasomal DNA. Our findings suggest a DNA-delivery function of prostasomes to sperm cells.
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| 26. |
- Ronquist, Göran K., et al.
(författare)
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Prostasomal DNA Characterization and Transfer Into Human Sperm
- 2011
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Ingår i: Molecular Reproduction and Development. - : Wiley. - 1040-452X .- 1098-2795. ; 78:7, s. 467-476
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Tidskriftsartikel (refereegranskat)abstract
- Human prostasomes, exosome-like microvesicles secreted by acinar cells of the prostate gland, contain chromosomal DNA. Agarose gel electrophoresis of DNA from seminal prostasomes displayed fragments of over 12 kb and smaller, with a distinct band around 1 kb that was excised, cloned, and sequenced. The sequences showed 8 out of 25 clones (32%) originating from genes. We elaborated the concept further by carrying out a genome-wide DNA copy number analysis of prostasomal DNA, hypothesizing that human prostasomes contain fragments of DNA randomly selected from the entire genome. Acridine orange-stained prostasomes were incubated with freshly prepared sperm for different times, and a transfer of acridine orange-stained prostasomal DNA to sperm (preferentially the head region) was observed. Fluorescence microscopy of slices in the center of 14 optical slides of the sperm head displayed an even fluorescence rather than a halo-like one, indicating DNA-uptake rather than just binding along the sperm head membrane.
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| 27. |
- Ronquist, Göran K, et al.
(författare)
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Prostasomes are heterogeneous regarding size and appearance but affiliated to one DNA-containing exosome family
- 2012
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Ingår i: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 72:16, s. 1736-1745
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:Prostate acinar epithelial cells release microvesicles (prostasomes) that possess pleiotropic biological effects relevant for successful fertilization. Prostasomes are formed in a similar way as exosomes but are heterogeneous as regards size and appearance. Like exosomes they are thought to be mediators of intercellular communication.METHODS:We prepared seminal prostasomes in accordance with the prevailing protocol for exosome preparation including passage through a 0.2 µm filter and centrifugation in a sucrose gradient.RESULTS:We compared the "filterable prostasomes" with those trapped on the filter ("nonfilterable prostasomes") and, qualitatively, no conspicuous differences were apparent regarding ultrastructure and SDS-PAGE banding pattern. Moreover, both types of prostasomes contained DNA fragments and Western blot revealed presence of prostate specific membrane antigen (PSMA), CD38, and annexin A1.CONCLUSIONS: Reasonably, prostasomes could be included in the exosome family and be regarded as one entity containing chromosomal DNA.
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| 28. |
- Ronquist, Göran, et al.
(författare)
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Proteomic analysis of prostate cancer metastasis : derived prostasomes
- 2010
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Ingår i: Anticancer Research. - 0250-7005 .- 1791-7530. ; 30:2, s. 285-290
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Tidskriftsartikel (refereegranskat)abstract
- The secretory epithelial cells of the prostate gland use sophisticated vehicles in the form of prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. We investigated prostasomes from vertebral metastases of prostate cancer, taken from the operating field at surgery, directly taken care of under protease inhibitory conditions for later 2-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) protein characterization. A total of 104 spots were punched out for identification. Twenty five unique protein spots had a MALDI-TOF above 49 and another 5 proteins were determined by MS/MS. The remaining 74 spots were either identical to already determined proteins or had no reliable score. Annexins A1, A3, and A5 as well as dimethylarginine dimethylaminohydrolase 1 were among the identified proteins. The annexins and dimethylarginine dimethylaminohydrolase 1 found in cancer-derived prostasomes can act, among other things, as angiogenic factors and can increase the vascular development in the neighborhood of the tumor. Cancer-derived prostasomes may play an important role in the interaction between tumor cells and their environment.
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| 29. |
- Ronquist, Göran, et al.
(författare)
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Serum antibodies against prostasomal clusterin in prostate cancer patients
- 2008
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Ingår i: Scandinavian Journal of Clinical and Laboratory Investigation. - : Informa UK Limited. - 0036-5513 .- 1502-7686. ; 68:3, s. 219-227
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Tidskriftsartikel (refereegranskat)abstract
- Objective. Clusterin is a ubiquitous secretory sulphated glycoprotein present in prostasomes. It is an antiapoptotic mediator in prostate cancer and is among the most frequently occurring prostasomal proteins immunogenic in prostate cancer patients. The aim of the present study was to investigate the occurrence of anticlusterin antibodies in the serum of patients with prostate cancer and whether there is a relationship between anticlusterin antibody titres and other clinico-pathological variables. Material and methods. Serum samples were collected from 391 consecutive patients with suspected prostate cancer (150 benign prostate and 241 prostate cancer). The patients’ serum samples were used in an ELISA where microtitre wells were coated with purified clusterin from serum of a healthy volunteer. Flow cytometric studies of clusterin and prostasomes were performed. Results. Flow cytometric analyses revealed the presence of clusterin on the surface of seminal prostasomes. Anti-clusterin ELISA titres in sera of patients did not differ significantly from those of a control group. A significant ‘‘inverse’’ correlation existed between anti-clusterin ELISA titres and lymph node metastases (p50.047), but only 11 out of 161 patients had metastases. These titres correlated significantly with total prostate (p50.021) and transitional zone (p50.015) volumes of the patients. Conclusions. The correlation between serum anti-clusterin antibody titres and other clinico-pathological variables was generally weak in prostate cancer patients, although clusterin has been assigned an important role in tumourigenesis and progression of prostate cancer. However, the anti-clusterin antibody titre appeared to be related to prostate volume, correlating to both transitional zone volume and total volume of the prostate.
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| 30. |
- Ronquist, K Göran, et al.
(författare)
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Human prostasomes express glycolytic enzymes with capacity for ATP production
- 2013
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Ingår i: American Journal of Physiology. Endocrinology and Metabolism. - : American Physiological Society. - 0193-1849 .- 1522-1555. ; 304:6, s. E576-E582
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Tidskriftsartikel (refereegranskat)abstract
- Prostasomes are prostate-derived, exosome-like microvesicles that transmit signaling complexes between the acinar epithelial cells of the prostate and sperm cells. A vast majority of prostasomes has a diameter of 30 - 200 nm and they are generally surrounded by a classical membrane bilayer. Using a selected proteomic approach, it became increasingly clear that prostasomes harbor distinct subsets of proteins that may be linked to adenosine triphosphate (ATP) metabolic turnover that in turn might be of importance in the role of prostasomes as auxiliary instruments in the fertilization process. Among the 21 proteins identified most of the enzymes of anaerobic glycolysis were represented and three of the glycolytic enzymes present are among the ten top proteins found in most exosomes, once again linking prostasomes to the exosome family. Other prostasomal enzymes involved in ATP turnover were adenylate kinase, ATPase, 5'-nucleotidase and hexose transporters. The identified enzymes in their prostasomal context were operational for ATP formation when supplied with substrates. The net ATP production was low due to a high prostasomal ATPase activity that could be partially inhibited by vanadate that was utilized in order to profile the ATP forming ability of prostasomes. Glucose and fructose were equivalent as glycolytic substrates for prostasomal ATP formation and the enzymes involved were apparently surface-located on prostasomes, since an alternative substrate not being membrane-permeable (glyceraldehyde 3-phosphate) was operative, too. There is no clear cut function linked to this subset of prostasomal proteins but some possible roles are discussed.
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