Cell fractionation studies

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... 

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... 

Cell fractionation studies of five strains of cyanobacteria indicate that MAAs are located primarily (>90%) within the cytoplasm and not the cell sheaths, walls, or membranes.132 Extracellular placement of MAAs does occur in some cyanobacterial species that posses cellular sheath layers.134135 Extracellular MAAs are covalently bonded to oligosaccharide molecules embedded in the cyanobacterial sheath matrix and provide substantial protection to prevent photobleaching of chlorophyll within the cell. Intracellular or extracellular distributions of MAAs in eukaryotic cells have not been investigated. Based on the high MAA concentrations of Phaeocystis antarctica colonies, it has been suggested that MAAs are associated with the extracellular mucopolysaccharide matrix of the colony.125 This may be a more common phenomenon than currently recognized, and future research efforts will be necessary to further document extracellular occurrence of MAAs in cyanobacteria and algae. 

Trichomonad flagellates seemed to be a good first choice. The human pathogen Trichomonas vaginalis and the cattle pathogen Tritrichomonas foetus were much-studied species available in bacterium-free cultures and were thus amenable to biochemical and cell fractionation studies, approaches extensively practiced by our group. The available physiological data showed that the respiration of these species was not of mitochondrial type, because it could not be inhibited with cyanide and other mitochondrial inhibitors. Furthermore no cytochromes were detected in these trichomonads (Ryley 1955). 

It is well known that occupied VDR is predominantly in the nucleus, but how the VDR localizes to the nucleus has long been a controversial issue and is still poorly understood. Cell fractionation studies show the VDR to be exclusively nuclear [228]. The presence of VDR in both nuclear and cytosolic fractions, however, has been reported with the use of physiological ionic strength buffers or by gentle fractionation methods [261, 262]. The work of Barsony et al. [263] has used immunocytochemical detection to study the intracellular compartmentalization of VDR. They report the transcellular movement of VDR from the cytoplasm to the nucleus in microwave-fixed cells. In the presence of l,25-(OH)2D3, VDR becomes organized on cytoplasmic fibres that resemble or are microtubules [264] and are oriented in... 

Several studies have shown that PLDl and yeast PLD (Spol4) are phosphorylated under basal conditions. The phosphorylation involves Ser and Thr residues and results in relocalization of the enzymes and the appearance of slower migrating enzyme species on SDS polyacrylamide-gel electrophoresis [18, 24, 43, 44] (Fig. 4.3). The phosphorylation of PLDl occurs predominantly in the N-terminal half of the enzyme and appears to have little effect on the intrinsic catalytic activity of the enzyme [18]. However, cell fractionation studies have shown that the phosphorylated form of overexpressed PLDl is not present in the cytosol, in contrast to the non-phosphorylated form, indicating that phosphorylation increases membrane association of the enzyme [18]. Treatment of cells with phorbol ester or overexpression of PKCa results in further phosphorylation of PLDl, but this is associated with a loss of activity [45]. Basal Ser/Thr phosphorylation of PLD2 is low compared with that of PLDl. However, addition of phorbol ester or expression of PKCa greatly increases this. 

Within mammalian cells, HMG-CoA reductase activity is associated almost exclusively with the microsomal membrane fraction [114]. Cell fractionation studies [114,115] and the pronounced proliferation of the smooth endoplasmic reticulum in a cell line which overproduces HMG-CoA reductase 500-fold [116] indicate that the majority of the enzyme resides in the smooth endoplasmic reticulum. An exception is the adrenal gland, in which a major fraction is associated with mitochondria [117,118]. 

Cell fractionation studies in bovine carotid artery first demonstrated that phorbol esters, histamine, angiotensin, and endothelin, but not KCl, induced a translocation of PKC from the cytosol to the membrane fraction (Haller et al., 1990), although the identity of the membranes involved was not determined (Table I). Work with the non-isoform-specific fluorescent PKC probe Bodipy phorbol (Khalil and Morgan,... 

Fig. 14. Localization of PhoE-LacZ hybrid proteins in Escherichia coli by means of cell fractionation studies (A) showing clearly the presence of the hybrid protein in the total cellular proteins of induced cells (lane e), the cell envelopes of induced cells (lane f) and the Triton X-100 insoluble protein fraction of the cell envelopes (lane h) and with the immunogold technique on ultrathin cryosections an accumulation of hybrid protein in the cytoplasm is clearly shown (B). Bar = 0.2 jam. From [165],...

Cell fractionation studies of homogenates of the gastric mucosa have demonstrated that the intrinsic factor is found both in mitochondria and the supernatant fluid. The factor remains difficult to assay a variety of methods have been used, but none is entirely satisfactory. These methods include (1) administration of intrinsic factor preparation to gastrectomized animals (rats or hogs) (2) measurement of vitamin 6 2 binding capacity of the preparation (3) measurement of vitamin Bi2 uptake in tissue and (4) evaluation of the inhibition of cobamide coenzyme activity in the glutamate isomerase reaction. 

Isoforms similar to those reported in the leaves of angiosperms have been reported in unicellular (Florencio and Vega, 1983 Sumar et ai, 1984 Ahmad and Hellebust, 1987) and multicellular algae (Casselton et ai, 1986) and in fern fronds (Stewart et ai, 1986). Cell fractionation studies have shown GS activity to be in the chloroplasts and cytosol of Chlamydomonas reinhardtii (Fischer and Klein, 1988), suggesting that the algal isoforms separated by ion-exchange chromatography are indeed the chloroplastic and cytosolic forms of GS. 

Electron microscopy of soybean nodules (Newcomb and Tandon, 1981) revealed that uninfected cells undergo a pronounced differentiation not seen in infected cells. This differentiation includes enlargement of the microbodies and proliferation of smooth endoplasmic reticulum (Fig. 8A and C). Within uninfected cells, peroxisomes are closely associated with the intercellular free spaces (Fig. 8C). This may be important in facilitating the uptake of O2 required for uricase activity. In the light of the identification of peroxisomes (a type of microbody) as the site of uricase and the endoplasmic reticulum as the probable site of allantoinase, Newcomb and Tandon (1981) proposed that uninfected cells played a key role in the final steps of ureide biogenesis. Immunogold labeling and cell fractionation studies have substantiated this proposal. 

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