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Virtosomes

Explore the presence and role of virtosomes as possible intracellular messengers involved in inter-cellular signaling. Learn about the structure, composition, and function of virtosomes in various tissues, including their potential applications in fetal disorder diagnosis, tumor markers, and treatment monitoring. Discover the origins of circulating DNA and the implications of virtosomes in cell communication and differentiation. Investigate the differences induced by virtosomes in stimulated and non-stimulated lymphocytes. Gain insights into DNA duplication and changes during differentiation in rat hepatocytes.

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Virtosomes

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  1. Virtosomes Possible intracellular messenger Mariapia Viola Fondazione “Enrico Puccinelli” ONLUS Research Center

  2. Presence in various tissue Virtosomes have been demonstrated in human and other mammalian, avian amphibian and tumour cells They can be isolated from body fluids. They may be involved in inter-cellular signalling

  3. Circulating DNA in body fluids First demonstration in blood in 1948 Many researchers have confirmed these results They are being exploited for: • Fetal disorder diagnosis • Markers for different tumours • Markers for pathological disorders • Monitoring of treatment

  4. Possible origin of circulating DNA Nucleated erythrocytes White blood cell breakdown Breakdown of bacteria and viruses Leucocytes surface DNA Cell and tissue necrosis Cell apoptosis Cellular release of exosomes Cellular release of transposons and retro-transposons Parasite nucleic acids Cellular release of virtosomes

  5. Virtosome The virtosomes were separated: • from the cytoplasm using very mild homogenization; • from the medium in which the cells are placed for at least 4 hrs. The virtosomes were isolated from other components by ultra- centrifugation.

  6. Virtosome structure They are a cytosolic complex formed from newly synthesized: • DNA • RNA • Proteins • Phospolipids

  7. Composition of virtosomes Cytosolic Released Protein: 41.01% 37.91% DNA: 3.45% 4.65% RNA: 35.09% 53.41% PLs: 19.90% 3.94%

  8. Cytosolic v Released Virtosomal Phospholipids Cytosolic Released Mean values % • PS 12.50 14.78 • PI 19.23 16.25 • SM 19.23 27.57 • PC 22.58 20.69 • PE 22.50 20.69

  9. Sphingomyelin of cytosolic and released virtosomes • P-Ls are associated with chromatin structure • SM forms a large percentage • SM’s role in DNA transcription is documented • SM is also linked to an RNase resistant RNA • SM may aid the transfer of RNA to the cytoplasm

  10. Virtosomal Function Appears to act as intercellular messengers. Virtosomes from non-dividing, non-stimulated lymphocytes block cell division in dividing stimulated lymphocytes, and vice versa. We will use non-stimulated and stimulated lymphocytes in this study in order to compare the virtosomes from dividing and non-dividing cells as well as the differences induced on stimulation.

  11. Effect of virtosomes derived from PHA stimulated cells sperim control

  12. Effect of virtosomes from non stimulated cells on PHA Lymphocytes sperim control

  13. Lymphocytes not Stimulated a b d e f c

  14. StimulatedLymphocytes 2 1

  15. Two-dimensional gel electrophoresis (2D-electrophoresis) RESULTS Stimulated lymphocytes, spot 1: NFRKB_HUMAN Nuclear factor related to kappa-B-binding protein Non-stimulated lymphocytes, spot f: RAI1_HUMAN Retinoic acid-induced protein

  16. Gel Mono 50 ug /lane NotStimulated Stimulated 5NS 5S 1NS 2S 3+4S 3+4NS

  17. Specific protein in stimulated lymphocytes The most interesting are: • ZZNF 160 zub-nc finger protein 160 regulating transcription with affinity to the DNA and Zn present in another finger protein, • PNPH purine nucleoside phosphorylase with a central role in purine metabolism

  18. DNA duplication This mobile DNA is most likely synthesized during G1/G0 phases. It is synthesized in non dividing cells. It may be related to differentiation. It is synthesized possibly in relation to cell function

  19. DNA changes during differentiation Rat Hepatocytes : amount of DNA/nucleus • Foetal life 4.7x10-12 • Neonatal period 7.7x10-12

  20. Non premitotic DNA synthesis The synthesis of DNA happens during the first day of life and is accompanied by loss and new synthesis in the second day. In this period the mitotic activity is very low (0.4%) in comparison to labelled cells (20%). The synthesis of specific enzymes, like thyrosin transaminase, start from the second day of life

  21. The virtosomes may explain the hepatocytes expression? In the second day of rat life the hepatocytes are able to express new specific proteins. In which way they comunicate without division? The loss of DNA of the second day may be due to the release of virtosomes which favour the differentiation of the other cells

  22. Contacts: Fondazione ONLUS "Enrico Puccinelli" Presidente/President Prof. Mariapia Viola Sede legale/Legal adress Piazza Università, 1 Perugia 06123 ITALY Sede operativa/ headquarters Via Pietro Cestellini, 3 Ponte San Giovanni  06135 Perugia ITALY Tel. e Fax: 0039 075 572 21 94 Website:    www.fondazionepuccinelli.org/ E-mail:      presidente@fondazionepuccinelli.org Skype:       Mariapia Viola For your 5 x 1000 CF: 94043090540

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