Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins

The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8–12, while the bulk of the plasma proteins was present in fractions 11–28. Vesicle markers peaked in fractions 7–11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.     

Accumulation of 2-micron latex particles in mouse Peyer's patches during chronic latex feeding

2-micron latex particles accumulated in macrophages in intestinal Peyer's patches of mice given latex suspensions as drinking fluid for 2 months. The number of particles accumulating was a direct (but nonlinear) function of the number ingested. Some of the latex particles were still present in Peyer's patches 6 weeks after the cessation of latex feeding.     

Cytomegalovirus-like particles in the red-backed vole submandibular gland

Summary Cytomegalovirus-like particles were observed in the acinarcells and in the intralobular ducts, of the submandibular gland of the red-backed vole. The particles were target-shaped, had a glycoprotein-positive, electron-dense central core, and the core was surrounded by a protein-rich layer and glycoprotein-positive outer layer.     

Evidence for the presence of virus in clonal insulin-producing RINm5F cells

The ultrastructural morphology of the clonal insulin-producing cell line, RINm5F, was investigated. Virus-like particles, probably C type viruses, were identified both intra- and extracellularly. Because these particles could not be found in the original transplantable tumor, it is probable that viruses were induced at some later stage in the development of the RINm5F cell line. All investigators using the RINm5F cells should be aware of the fact that these cells may contain one or several types of viruses, and of the possibility that these particles may interfere with a variety of cellular functions.     

Virus-like particles in the opossum submandibular gland

Summary Numerous inclusion bodies (virus-like particles) were observed in the lumina of the intercellular canaliculi, mucous tubules and intralobular ducts of the opossum submandibular gland. The particles are spherical in outline, show an electron dense core, and are surrounded by a peripheral membrane.     

Binding of ferritin-lectin conjugates to C-type virus in intact cells.

Ferritin-Ricin II and Ferritin-Concanavalin A bound to budding as well as mature C-type viral particles. No differencies in binding between the viral coat and adjacent plasma membrane were detected with either lectin conjugate. Aggregation of viral particles by lectin conjugates was observed, and linking of virus to the plasma membrane resulted in phagocytosis of viral particles.     

Application of immunoelectron microscopy to the detection of viruses in a water medium]

Adenoviruses were immersed in demineralyzed and deionized water for 5 days. The water was subsequently analyzed by Immuno-Electron-Microscopy for detection of viral particles. An attempt of quantation was also made on control, untreated particles. Viruses dispersed in water can be detected by the technique employed but quantitation is limited by the heterogeneous dispersion of the particles on the grids.     

Presence of oncornavirus-like particles in the P388 murine leukemic cell line

A culture of P388 murine lymphoblastoid cells has been shown to contain type C oncornavirus-like particles budding at the plasma membrane. Occasionally intracytoplasmic type A and immature type B particles were also observed by electron microscope techniques. The discovery of oncornavirus-like particles in the P388 cell line increases the utility of this neoplastic system for detecting potential antineoplastic agents.     

Vasoactive intestinal peptide (VIP): specific receptors and adenylate cyclase activation in a human prolactin-secreting pituitary tumor]

Receptors for the Vasoactive Intestinal Peptide (VIP) were characterized in particles enriched in plasma membranes obtained from a human prolactin-secreting pituiatry tumor. Native VIP inhibited competitively the binding of 125I-VIP to the particles and stimulated cyclic AMP production; both these effects were observed at concentrations of VIP as low as 10(-11)-10(-10) M, which are compatible with VIP concentrations in the hypothalamopituitary portal blood.     

The volcanoes of Auvergne

It is with good reason that the name Rutherford is closely linked with the early history of the alpha particle. He discovered them, determined their nature, and from 1909 used them to probe the structure of the atom. From 1898 to 1902 Rutherford construed alpha radiation as a type of non-particulate Röntgen radiation. On his theory of the locomotion of radioactive particles Rutherford proposed that alpha radiation consisted of negatively charged particles. During 1902 he confirmed the particulate nature of alpha radiation but discovered that these alpha particles were positively charged. Although Rutherford suspected from 1903 that these alpha particles were related somehow with helium, the proof required six long years of investigation. By mid-1908 it seemed certain that the alpha particle possessed two units of the elementary charge. Since the e/m ratio had already been determined for alpha particles, this evidence enhanced the suspected connection with helium. However, this gain and loss of charge was still construed as an ionization effect. Since as late as 1908 gaseous ionization was assumed to involve the gain or loss of a single unit of charge, Rutherford's alleged case of doubly ionized alpha particles was presumably an exception. Yet helium was known to be an inert gas and thus hardly a likely candidate for such exceptional ionization behaviour. To establish the connection, therefore, Rutherford resorted to a spectroscopic test. He collected spent alpha particles shot into a thin glass tube and gradually observed the spectrum of helium. Rutherford had thus been correct in his assumption, but a proper explanation was possible only after the confirmation of the nuclear structure of the atom.     

Capping of concanavalin A- or ricin-binding sites does not influence phagocytosis in polymorphonuclear leukocytes

Summary Rabbit polymorphonuclear leukocytes (PMNs) were capped with territin-conjugated concanavalin A or ricin, and then allowed to phagocytose yeast cells. Phagocytic activity and lectin distribution were determined by ultrastructural morphometry. Capped PMNs were found to phagocytose as efficiently as control PMNs, and always to ingest the particles with a lectin-free portion of their plasma membrane. This clearly indicates that concanavalin A- and ricin-binding sites of the PMN membrane are not involved in the recognition and phagocytosis of yeast particles.     

Intracisternal A type particles of the extraocular muscle of hereditary muscular dystrophy mouse.

Intracisternal A type virus (IA) particles were observed in the extraocular muscle fiber of hereditary muscular dystroph mouse. The particles appeared approximately 65-75 mmu in diameter, with electron lucent cores.     

Relationship between A-type and C-type particles in Ehrlich ascites tumor cells

Summary Intracisternal and intracytoplasmic A-type particles were discovered in Ehrlich ascites tumor cells. In addition, mature and immature C-type particles were also seen in the intercellular space. It is believed that A particles may represent a precursor or a formative stage of the C particles.Acknowledgment. The author is grateful to Professor F. P. Lisowski for his advice and helpful criticisms and to Mr Y. S. Tong for his expert technical assistance. This project was supported by a grant from the Hong Kong University Research Grants Committee.     

Binding of ferritin-lectin conjugates to C-type virus in intact cells

Summary Ferritin-Ricin II and Ferritin-Concanavalin A bound to budding as well as mature C-type viral particles. No differencies in binding between the viral coat and adjacent plasma membrane were detected with either lectin conjugate. Aggregation of viral particles by lectin conjugates was observed, and linking of virus to the plasma membrane resulted in phagocytosis of viral particles.Acknowledgment. The authors thank Phyllis Johnson for her excellent technical assistance. This work was supported by NIH grant CA 15044-03 and NS 06208. LM is the recipient of an NINDS Research Career Development Award 1 KO4-NS 00101.     

Improvement of warm formability of Al-Mg sheet alloys containing coarse second-phase particles

Several alloying elements involving Zr,Cu,Zn and Sc were added to Al-Mg sheet alloys in order to obtain an excellent combination of high strength and good high-temperature formability.Microstructural examination showed that coarse intermetallic particles were formed in the microstructure and their amounts changed with variations of the alloying elements. During warm rolling of thermomechanical treatments prior to warm deformation,the coarse particles initiated cracks,decreasing the warm formability.For healing the crack damage and further improving the warm formability,a process of hot isothermal press was developed and optimized to the sheet alloys.With this process,the biaxial stretch formability at 350°C was improved by 22%for an aluminum alloy containing a large amount of coarse particles.     

Accumulation of 2-μm latex particles in mouse Peyer's patches during chronic latex feeding

Summary 2-m latex particles accumulated in macrophages in intestinal Peyer's patches of mice given latex suspensions as drinking fluid for 2 months. The number of particles accumulating was a direct (but nonlinear) function of the number ingested. Some of the latex particles were still present in Peyer's patches 6 weeks after the cessation of latex feeding.Acknowledgments. Research supported by the U. S. Energy Research and Development Administration. We thank Mr W. Marin, Division of Photography and Graphic Arts, Brookhaven National Laboratory, for figure 2.The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

The Last Century of Physics

The mesotrons, or mesons, were the first elementary particles observed to be inherently unstable. This essay offers a reconstruction of the stream of researches related to mesotron decay, and examines how these researches shaped some of the basic concepts and practices of the emerging field of particle physics. Mass measurements could not settle the question of whether the mesons were a homogeneous kind of particles or an assortment of particles with different masses. The assumption of a single mass prevailed not on experimental grounds but because the mesons were identified tentatively with the carriers of the nuclear force according to a theory formulated by Hideki Yukawa. The identification gained currency because it entailed the prediction of meson decay, and thereby upheld the promise of a unified explanation of nuclear and cosmic-ray phenomena. In turn, the observation of decay and the measurement of the mean lifetime created the conditions for investigating the nuclear interactions of mesons at rest. Interest in these interactions was heightened, immediately after WWII, by the prospect of building and using accelerators to acquire knowledge about fundamental nuclear processes. Using decay to study nuclear capture, however, led to the realization that there exist not only different kinds of mesons but also two nuclear forces.     

A note on the prehistory of indistinguishable particles

In modern terms, quantum statistics differs from classical statistics for the indistinguishability of its elementary entities. An historical investigation of the emergence of Bose–Einstein statistics, however, shows that quantum statistics was initially interpreted as a statistics of non-independence, for it extended to gas particles the statistical correlation that was a long-recognized characteristic of light quanta. At the same time, the development of a quantum–statistical theory of the ideal gas was riddled with the question of the statistical significance of the exchange symmetry of a system of equal particles. Indistinguishability combines exchange symmetry and statistical correlation, and relates them to the loss of identity of particles in quantum mechanics. It is instructive, however, not to conflate these properties when analysing the historical emergence of quantum statistics. The statistical correlation of light quanta and the exchange symmetry of gas molecules remained two separate problems even though quantum gas theory and Bose–Einstein statistics were born from gas-radiation analogies in statistical theory.     

Evidence for the presence of virus in clonal insulin-producing RINm5F cells

Summary The ultrastructural morphology of the clonal insulin-producing cell line, RINm5F, was investigated. Virus-like particles, probably C type viruses, were identified both intra- and extracellularly. Because these particles could not be found in the original transplantable tumor, it is probable that viruses were induced at some later stage in the development of the RINm5F cell line. All investigators using the RINm5F cells should be aware of the fact that these cells may contain one or several types of viruses, and of the possibility that these particles may interfere with a variety of cellular functions.This work was supported by grants from the Lake County Medical Center Developmental Agency (to VH and PWB) and the American Heart Association, Indiana Affiliate (to PWB,^*USA) and by the Swedish Medical Research Council (12X-562), the Swedish Diabetes Association, the Nordic Insulin Foundation and Åke Wibergs Foundation (^**Sweden). Per-Olof Berggren is a recipient of a postdoctoral fellowship from the Swedish Medical Research Council.     

Intranukleäre virusähnliche Partikeln in einem Mammakarzinom

Summary By electron microscopic examination of a solid, chiefly scirrhous mammary cancer, 3 kinds of unusual nuclear inclusions were found: (a) Electron dense particles of a 54–80 nm diameter, whose outlines appear mainly hexagonal, which points at an icosahedral structure, (b) Clusters of granules with a diameter of 200–300å, respectively 300–400å, at whose circumference the larger particles appear, (c) Bundles of filaments in close association and continuous with the granules. In all 3 nuclear inclusions there are subunits of 40–45å. The comparison between these results and experiments published suggest that these nuclear inclusions are (a) virus particles, (b) virus at an early stage of development and (c) virus protein subunits.