Erythrocytes extraction

Proteasome research began in the late 1960s [19] when Harris discovered a barrel-shaped particle in erythrocyte extracts. From then on knowledge about the proteasome and the regulating factors of this protease has been added to step by step. Today it is generally accepted that the ubiquitin-proteasome system is responsible for the turnover of the bulk of intracellular cytosolic and nuclear proteins [20]. It is known that this protease is located in the cytosol, the nucleus, and that it is attached to the ER and other cell membranes. 

Trager, W. (1950). Studies on the extracellular cultivation of an intracellular parasite. I. Development of the organism in erythrocyte extracts and the favoring effect of adenosine triphosphate. J. Exp. Med. 92,349-366. 

Rabbit liver was perfused with phTj-hypoxanthine, as previously described, followed by washout perfusion for ten minutes with an isotonic solution of salts containing glucose, to remove extracellular label. An oxygenated washed human erythrocyte suspension was then perfused through the liver for 1 hr. Erythrocytes were collected and washed in an isotonic solution of salts. Liver and erythrocyte extracts were chromatographed and assayed. Purine bases, prepared by hydrolysis of the nucleotide fractions, separated by chromatography on Dowex 50. 

Figure 4. Neutralization of erythrocyte PP-ribose-P synthetase activity by concentrated serum IgG from rabbits immunized with purified normal PP-ribose-P synthetase. To a constant amount of erythrocyte extract, increasing amounts of antiserum were added and after incubation at 37° and 4° each for 30 minutes, enzyme activity was measured. Enzyme from erythrocytes of... 

Lee, T. J. F, Mcllhany, M. R, Sarwinski, S., 1984 Erythrocyte extracts enhance neurogenic vasoconstriction of dog cerebral arteries in vitro, J. Cereb. Blood Flow Metab. 4, 474-476. 

In vitro cytotoxicity assays using isolated cells have been applied intermittently to cyanobacterial toxicity testing over several years." Cells investigated for suitability in cyanobacterial toxin assays include primary liver cells (hepatocytes) isolated from rodents and fish, established permanent mammalian cell lines, including hepatocytes, fibroblasts and cancerous cells, and erythrocytes. Earlier work suggested that extracts from toxic cyanobacteria disrupted cells of established lines and erythrocytes," but studies with purified microcystins revealed no alterations in structure or ion transport in fibroblasts or erythrocytes,... 

Homogenates of MetruUum senile, possibly the world s most common large sea anemone, yield extracts that are powerfully hemolytic for washed mammalian erythrocytes (22). The active substance, metridiolysin, is a protein of molecular weight approximately 80,000. In contrast to the sphingomyelin-inhibitable toxins, metridiolysin is an acidic protein having a pi of about 5. It is thermolabile and is inactivat by proteolytic enzymes. The optimal pH for hemolysis is between 5 and 6, and at pH 8 the lysin is inactive. It can be dissociated into two subunits of unequal size. Besides being cytolytic in vitro, metridiolysin is lethal when injected intravenously into mice. As shown in Table IV erythrocytes from the horse or dog are about a hundred times as sensitive to lysis as those from the mouse, and erythrocytes from other animals tested are intermediate in sensitivity. 

Suberitine, a small protein from the sponge Suberites domcuncula, has a variety of actions. It is not very toxic but causes hemolysis in human erythrocytes, flaccid paralysis in crabs and depolarization of squid axon and abdominal nerve of crayfish. A variety of extracts from Porifera have been shown to be toxic to fish and generally have cytotoxic and hemolytic actions (62,63). As discussed previously, a variety of sponges exude substances that are toxic to fish. 

Kuriki, K., Tajima, K., and Tokudome, S., Accelerated solvent extraction for quantitative measurement of fatty acids in plasma and erythrocytes. Lipids, 41, 605, 2006. 

Blood Chemistry Data. All blood specimens were obtained by venipuncture. The erythrocyte protoporphyrin (EP) was measured by the extraction method. Blood lead determinations were done In quadruplicate and are presented as an arithmetic mean of the four replicates. 

Different tissues have different lipid compositions. The most common lipid components of membranes are PC and PE. Lipid extracts from brain and lung are also rich in PS heart tissue is rich in PG, and liver is rich in PI [567]. Human blood cells, as ghost erythrocytes (with cytoplasm contents removed), are often used as membrane models. These have different compositions between the inner and outer leaflets of the bilayer membrane. Phospholipids account for 46% of the outer leaflet membrane constituents, with PC and Sph about equal in amount. The inner leaflet is richer in phospholipids (55%), with the mix 19% PE, 12% PS, 7% PC and 5% Sph [567],... 

Inhibition of ferrochelatase in the heme pathway causes accumulation of protoporphyrin in erythrocytes (CDC 1985). Most protoporphyrin in erythrocytes (about 90%) exists as zinc protoporphyrin (ZnPP). This fraction is preferentially measured by hematofluorometers. Extraction methods measure all the protoporphyrin present, but strip the zinc from the ZnPP during the extraction process. For this reason,... 

Little is known of the chemical nature of the group specific substances proper for they are extremely difficult to isolate. Active alcoholic extracts have been obtained from erythrocytes by various workers11 and in some instances these extracts were shown to contain carbohydrates. 

There is a great deal of interest in the determination of lead, particularly micromethods applicable to the analysis blood lead in children. Consequently, reports continue to appear on the atomic absorption determination of lead in blood and urine. Ninety percent of blood lead is found in the erythrocytes and, therefore, whole blood is analyzed rather than serum or plasma. Berman etal. 134) have described a procedure for determining normal lead levels in which only 250 fd of blood are taken. The blood is deproteinized with 1 ml of 10 % trichloroacetic acid and then the lead is extracted with APDC into 1 ml of MIBK, at pH 3.5. 

Another group of non-histone proteins have been identified as essential components for the formation of the condensed chromosome (Table 1). Topoisomerase II (topo II) localizes in the scaffold/matrix fraction of the interphase nuclear (Berrios et al., 1985) and the mitotic chromosome (Maeshima and Laemmli, 2003) (see section 3.1). Topo II forms a ring-shaped homodimer (Berger et al, 1996 Nettikadan et al, 1998) and catalyzes the decatenation and relaxation of DNA double strand (Wang, 2002). In fission yeast, chromosomes cannot be condensed without functional topo II (Uemura et al, 1987). In addition, in in vitro experiment, mitotic extracts containing topo II induce chromatin condensation in the isolated nuclei from HeLa and chicken erythrocyte cells (Adachi et al., 1991). 

Using optical traps, Cui and Bustamante [76] stretched isolated chicken erythrocyte fibers, and Bennink et al. [77] pulled on fibers directly reconstituted in the flow cell from X-DNA and purified histones with the help of Xenopus extracts (see Fig. 10a for a schematic of the latter experiment). Up to 20 pN, the fibers underwent reversible stretching, but applying stretching forces above 20 pN led to irreversible alterations, interpreted in terms of removal of histone octamers from the fibers with recovery of the mechanical properties of naked DNA. 

Animals, usually rodents, are exposed to the test substance by an appropriate route, usually by gavage or by intraperitoneal injection. Each treated and control group must include at least five animals per sex. Positive controls should produce micronuclei in vivo at exposure levels expected to give a detectable increase over background. No standard treatment schedule (i.e., 1, 2, or more treatments at 24-h intervals) has been recommended. Three dose levels are generally used these should cover a range from the maximum to little or no toxicity. The erythrocytes are sampled from the bone marrow and/or peripheral blood of the animals. If bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained. When peripheral blood is used, the blood is collected at appropriate times after treatment and smear preparations are made and stained. Preparations are analyzed for the presence of micronuclei. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. 

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