Cells fractionation

Blood can be collected ia the form of whole blood donations. In the United States, one unit, ie, 450 mL, of blood is collected from a healthy volunteer blood donor who is allowed to donate blood once every 10 weeks. A unit of blood is typically separated iato a red cell fraction, ie, red cell concentrate a platelet fraction, ie, random donor platelets (RDP) and plasma. 

Filtering cells and cell fractions from fluid media. These particles, after concentration by filtration, may be examined through subsequent quantitative or qualitative analysis. The filtration techniques also have applications in fields related to immunology and implantation of tissues as well as in cytological evaluation of cerebrospinal, fluid. 

Typical approaches for measuring diffusivities in immobilised cell systems include bead methods, diffusion chambers and holographic laser interferometry. These methods can be applied to various support materials, but they are time consuming, making it onerous to measure effective dififusivity (Deff) over a wide range of cell fractions. Owing to the mathematical models involved, the deconvolution of diffusivities can be very sensitive to errors in concentration measurements. There are mathematical correlations developed to predict DeS as... 

Organophosphates, such as methyl parathion, are known to inhibit cholinesterase activity. A method has been developed to measure the extent of this inhibition and relate it to organophosphate exposure (EPA 1980d Nabb and Whitfield 1967). In this EPA-recommended method, blood is separated into plasma and red blood cell fractions. The fractions are treated with saline solution, brought to pH 8 with sodium hydroxide, and dosed with acetylcholine perchlorate. The ensuing acetic acid releasing enzyme reaction... 

PelZ is a hydrophilic protein of 420 amino acids with a short hydrophobic sequence at its N-terminal end which has Ae characteristics of the signal sequences of exported proteins. The signal peptide may be 24 amino acids long, which would corroborate wiA the usual length encountered in prokaryotes. The molecular cloning of the pelZ gene in an expression vector pT7-6 allowed for the specific 35S-cysteine-methionine raAo-labelling of PelZ in E. coli K38. We could detect, in crude extracts, the presence of a precursor and a mature form of PelZ. After cell fractionation, Ae mature form of PelZ could be localized in Ae periplasm of E. coli. So PelZ appears to be a protein exported by Ae Sec-dependent system of translocation. 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Recently, a lectin made from purified elderberry (Sambucus nigra) has been used in the fractionation of normal bone marrow cells. Fractionation minimizes the effects of radiation on normal tissue. This work has been carried out by Dr. Madeleine Mumcuoglu, who patented the elderberry flu formulas, as well as doctors Daphna Manor and Shimon Slavin. There is hope that elderberry contains properties that may have potential in improving the success of bone marrow transplants. 

Mullen et al. (1989) reported that Bacillus cereus, B. subtilis, E. coli and P. aeruginosa were able to sorb an average of 89% of the total Ag+ and 12-27% of the total Cd2+, Cu2+ and La3+ from a ImM solution. Using polyacrylamide-entrapped cells of Brevibacterium sp strain PBZ, Simine et al. (1998) measured a sorption capacity of 40 mg g-1 and 13 mg g-1 dry biomass for Pb and Cd, respectively. Hall et al. (2001) isolated two bacterial strains of P. syringae that were tolerant to 1000 mg L-1 Cu. Similarly, Amoroso et al. (2001) were able to obtain Streptomyces spp. strains R22 and R25 with a high tolerance to Cr from sediments of the Sail River, Argentina. The cells of R22 and R25 could accumulate 10.0 and 5.6 mg Cr g-1 dry weight, respectively, from a concentration of 50 mg Cr mL 1. Cell fractionation studies with strain R22 showed that most of the chromium... 

Cell fractionation procedures were fundamental to the biochemical identification of steroid and thyroid hormone receptors in brain as well as in other tissues. Isolation of highly purified cell nuclei from small amounts of tissue from discrete brain regions generally is accomplished with the aid of a nonionic detergent, such as Triton X-100 [7],... 

Understanding of the intracellular localization of steroid receptors has gone through a number of phases, beginning with the view that receptors translocated from cytoplasm to nucleus in the presence of hormone. Indeed, with the exception of thyroid hormone receptors, which are exclusively nuclear in location, cell fractionation studies have revealed that in the absence of hormone, steroid receptors are extracted in the soluble or cytosolic fraction. However, when steroid is present in the cell, many occupied receptors are retained by purified cell nuclei. Histological procedures, such as immunocytochemistry, have confirmed the largely nuclear localization of occupied receptors, but... 

The intracellular distribution of steroid hormone receptors has long been the object of controversy. The first theoretical formulation on the intracellular location of the ERs was elaborated by Jensen in 1968 and is known as the two-step theory. Its execution was based entirely on biochemical observations obtained by means of tritium-marked estradiol. The ERs, in cells not exposed to hormones, are found abundantly in the soluble cell fraction, or cytosol (Fig. 1.1). Treatment with hormones confines the receptors to the particulated or nuclear fraction and causes their disappearance from the cytosol. The two-step theory established that the receptor is found in the cytoplasm naturally and upon the arrival of a hormone it is transformed into a complex hormone-receptor (first step) capable of translocating itself to the nucleus and of modifying gene expression (second step). 

Simultaneously, Gorski s group (Welshons et al. 1984), utilizing a type of cell fractionation that permits separating the cytoplasm from the nucleus, was also able to detect the presence of nuclear ER, even if the cell had not been exposed to hormones. These findings led to a different theoretical formulation, according to which the native receptors would be found in the cell nucleus, to which the hormone would accede directly. 

There are also numerous enzymes anchored in membranes of the microsomal cell fraction that participate in the metabolism of steroid hormones. Thus, those of the p450 family, which carry out molecular oxidation, or the sulfatases and sulfotransferases, more or less specific to several hormones (Pasqualini et al. 1995). The affinity of steroid hormones for proteins of the membrane (Kd between 10 and 100 nM) is frequently greater than that which some of these enzymes present for their substrates (Luzardo et al. 2000). Therefore, it is unlikely that a part of the proteins of the membrane that bind steroids is in reality enzymes metabolizing these hormones. 

Miyazato, A., et al., "Identification of Myelodysplastic Syndrome-Specific Genes by DNA Microarray Analysis with Purified Hematopoietic Stem Cell Fraction," Blood, 98, 422-427 (2001). 

Cells other than T lymphocytes also appear to be involved in tolerance induction. Depletion of macrophages inhibited tolerance induction and transfer studies with non-T cell fractions from tolerant animals was shown to confer tolerance to naive animals [29] Thus, tolerance induction by low doses of D-penicillamine appears to have a complex mechanism that includes various T cell subsets as well as non-T cells, that may be antigen presenting cells. 

Bowles DJ, Quail PH, Moire DJ, Hartnann GC. Use of markers in plant cell fractionation, in Plant Organelles (Reid E, ed.), Ellis Horwood, Chichester, UK, 1979, pp. 207-227. 

Quail PH. Plant cell fractionation. Ann Rev Plant Physiol 1979 30 425-484. 

It is often necessary to assess the efficiency of cell fractionation procedures. Electron microscopy of the prepared fractions is very informative but gives no quantitative indication of the purity of the fraction. It is often easier to measure the relative concentrations of marker enzymes in each fraction (Table 8.9). 

Some of the results obtained by differential centrifugation showed enzyme distribution between different cell fractions which were difficult to interpret. Enzymes like carbamoyl phosphate synthase or isocitrate dehydrogenase were found both in mitochondria and in the soluble fraction of the cell. This led to detailed kinetic studies with purified enzymes which indicated there might be populations of enzymes with slightly different properties (isozymes) catalyzing similar reactions in different compartments or in different cell types. Variations in kinetic behavior appeared to tailor the enzyme appropriately to the particular compartment or cell where the reaction took place. 

The heyday of differential centrifugation and work on isolated cell fractions was short. In the 1940s and 1950s biochemists attracted much critical comment because of their use of disorganized homogenates and then of separated cell fractions. Many metabolic pathways were, however, successfully analyzed with little reference to their normal setting within the cell. 

In our previous studies on the sulfate conjugation of phenols by fish livers, all the liver slices of the test fish and shellfish exhibited sulfate conjugation activities with phenol(14), and among various liver cell fractions separated by ultracentrifugation, only the soluble fraction displayed the sulfate conjugation activity for phenol and various phenolic compounds(15). 

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

During cell fractionation, it is very important to analyze the purity of the fractions obtained. Whether or not the intended organelle is present in a particular fraction, and whether or not the fraction contains other components, can be determined by analyzing characteristic marker molecules. These are molecules that occur exclusively or predominantly in one type of organelle. For example, the activity of organelle-specific enzymes (marker enzymes) is often assessed. The distribution of marker enzymes in the cell reflects the compartmentation of the processes they catalyze. These reactions are discussed in greater detail here under the specific organelles. 

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