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PubMed1982

[Lipid concentrations in liver cell fractions in the rat in diet induced obesity].

The changes of lipid parameters induced in male Wistar rats by feeding a high-fat diet for several weeks were studied in cell fractions of the liver. For this purpose subcellular fractionations of liver tissue from 12-week-old animals receiving food containing, respectively, 3% fat (controls) and 50% fat, were performed by means of differential centrifugation, and the lipids in the cell fractions were determined quantitatively. The levels of triglycerides and cholesteryl esters both in total liver and in the cell fractions had risen several fold against the controls. Most of the accumulated lipids were retrieved in the cytosolic supernatant. In contrast, for the phospholipids and the free cholesterol only a slight increase in free cholesterol was observed in total liver, and increases of both lipids had occurred in the cytosolic supernatant, while the particulate fractions showed no alterations. It is pointed out in the discussion that the accumulation of triglycerides and cholesteryl esters in the liver largely concerns the particulate fractions too (observed so far in the literature only for single particulate fractions), which might be of interest for the function of the cell nucleus, the mitochondria and the endoplasmatic reticulum. The increase of phospholipids and free cholesterol in the supernatant has to be considered not only as a passive process of deposition, but could represent necessary contributions to the building up of lipid droplets in the cytosol during deposition of triglycerides and cholesterol in this region of the cell. The studies confirm that the fatty degeneration of the liver, true, is always the result of triglyceride accumulation but that, under certain conditions, cholesterol is also enriched in the form of its esters.

Animals↗
PubMed1979

Tissue-culture cell fractionation. Fractionation of membranes from tissue-culture cells homogenized by glycerol-induced lysis.

1. The disruption of various types of tissue-culture cells by (a) incubation in solutions of 1.2 M-glycerol and (b) transfer of the glycerol-loaded cells to relatively hypo-osmotic solutions of 0.25 M-sucrose was studied. 2. Bivalent cations (2mM-Mg2+) were generally included to preserve the nuclei, but some cells (polyoma-virus-transformed baby-hamster kidney cells) failed to be disrupted adequately under these conditions. 3. Other cells (mouse-embryo fibroblasts) required additional gentle Dounce homogenization to effect complete cell breakage. 4. Purification of the whole homogenate was carried out by a combination of differential centrifugation and sedimentation or flotation through sucrose gradients. 5. Enzyme analysis showed that plasma-membrane, endoplasmic-reticulum and mitochondrial fractions were obtained in good yield and purity.

Animals↗
PubMed1979

Tissue-culture cell fractionation. Fractionation of cellular membranes from 125I/lactoperoxidase-labelled Lettrée cells homogenized by bicarbonate-induced lysis: resolution of membranes by zonal centrifugation and in sucrose and metrizamide gradients.

1. Lettrée cells were grown intraperitoneally in MF-1 mice and labelled extrinsically by the 125I/lactoperoxidase technique. 2. The cells were swollen in 1 mM-NaHCO3 and disrupted in a Dounce homogenizer. 3. Crude fractions of endoplasmic reticulum, plasma membrane and mitochondria were separated from a post-nuclear supernatant by sedimentation-rate gradient centrifugation in a BXIV zonal rotor. 4. Further resolution of these membranes was carried out in isopycnic sucrose gradients. 5. Bands of material from the latter were subfractionated in gradients of metrizamide. Some very pure subfractions of plasma membrane and endoplasmic reticulum were obtained. In addition, one subfraction containing 125I and NADPH-cytochrome c reductase but no Na++K+-stimulated adenosine triphosphatase and another containing these two enzymes but no 125I were resolved.

Animals↗
PubMed1989

Relationship between energy status, hypoxic cell fraction, and hyperthermic sensitivity in a murine fibrosarcoma.

The energy status, radiobiological hypoxic cell fraction, and hyperthermic sensitivity of a spontaneous murine fibrosarcoma, FSa-II, have been evaluated as a function of tumor size. Tumors were evaluated over the size range of 70 to 800 mm3. The concentration of the high-energy phosphate reservoir creatine phosphate progressively decreased by a factor of 5 with increasing tumor volume, and was matched by an increase in creatine. The concentration of ATP also decreased with increasing tumor size, although this decrease was substantially less pronounced. The sum of ATP, ADP, and AMP did not vary with tumor size, suggesting that the necrotic fraction remained constant. The decrease in energy status occurred in parallel with an increase in the size of the hypoxic cell fraction and with increasing thermal sensitivity. The results suggest that energy status may be an important modifier of hyperthermic sensitivity in vivo and reflect tissue oxygen concentration.

Adaptation, Physiological↗
PubMed1989

Automated quantitation of cell density distribution and hyperdense cell fraction in RBC disorders.

An altered state of cell hydration is a hallmark of a number of RBC disorders. The two parameters most often used to characterize the state of cell hydration are (a) the complete cell density distribution profile, and (b) the fraction of cells with densities greater than a defined value. Buoyant density cell fractionation with a variety of polymers has long been the preferred method for obtaining data on cell density distribution. Although this method provides accurate and quantitative information on the state of RBC hydration, its applicability has been limited due to the time-consuming experimental procedure involved in generating the data. A recently developed light-scattering method has been used in the present study to quantitate RBC density distribution. This new method accurately quantitates both the cell density distribution profile and the fraction of dense, dehydrated cells in various RBC disorders. The ability of the automated method to generate this information rapidly makes possible objective testing of different hypotheses concerning the contributions of cell hydration and dehydration to the pathophysiology of various RBC disorders.

Anemia, Sickle Cell↗
PubMed1991

Cell fractionation and electron microscope studies of kidney folate-binding protein.

The subcellular distribution of folate-binding protein (FBP) and [3H]folate in the proximal tubule was examined using cell fractionation and different electron microscope (EM) techniques. Cell fractionation of rabbit proximal tubules revealed that FBP distributed into two modes: 50% of FBP distributed with alanylaminopeptidase activity (brush border), and the remaining FBP distributed with organelles of lower density that did not show a large digitonin-induced shift to greater density. Infusion of [3H]folate into the kidney followed by isolation and fractionation of the proximal tubules revealed a time-dependent shift of [3H]folate from the heavy (brush border) mode to the lighter organelle mode. By EM immunocytochemistry, rat kidney FBP locates in the brush border, endocytic invaginations, endocytic vacuoles, and dense apical tubules of proximal tubule cells. EM autoradiography of rat kidney 10 min after intravenous infusion of [3H]folate revealed that the label was significantly concentrated only in the brush border, endocytic vesicles, and lysosomes. These data support a mechanism of receptor-mediated endocytosis for the process of FBP-mediated folate transport in the kidney.

Animals↗
PubMed2006

Lack of correlation of hypoxic cell fraction and angiogenesis with glucose metabolic rate in non-small cell lung cancer assessed by 18F-Fluoromisonidazole and 18F-FDG PET.

UNLABELLED: PET offers a noninvasive means to assess neoplasms, in view of its sensitivity and accuracy in staging tumors and potentially in monitoring treatment response. The aim of this study was to evaluate newly diagnosed non-small cell lung cancer (NSCLC) for the presence of hypoxia, as indicated by the uptake of (18)F-Fluoromisonidazole ((18)F-FMISO), and to examine the relationship of hypoxia to the uptake of (18)F-FDG, microvessel density, and other molecular markers of hypoxia. METHODS: Twenty-one patients with suspected or biopsy-proven NSCLC were enrolled prospectively in this study. All patients had PET studies with (18)F-FMISO and (18)F-FDG. Seventeen patients subsequently underwent surgery, with analysis performed for tumor markers of angiogenesis and hypoxia. RESULTS: In the 17 patients with resectable NSCLC (13 men, 4 women; age range, 51-77 y), the mean (18)F-FMISO uptake in tumor was significantly lower than that of (18)F-FDG uptake (P < 0.0001) and showed no correlation with (18)F-FDG uptake (r = 0.26). The mean (95% confidence interval [CI]) (18)F-FMISO SUV(max) (maximum standardized uptake value) was 1.20 [0.95-1.45] compared with the mean [95% CI] (18)F-FDG SUV(max) of 5.99 [4.62-7.35]. The correlation between (18)F-FMISO uptake, (18)F-FDG uptake, and tumor markers of hypoxia and angiogenesis was poor. A weakly positive correlation between (18)F-FMISO and (18)F-FDG uptake and Ki67 was found. CONCLUSION: The hypoxic cell fraction of primary NSCLC is consistently low, and there is no significant correlation in NSCLC between hypoxia and glucose metabolism in NSCLC assessed by (18)F-FDG. These findings have direct implications in understanding the role of angiogenesis and hypoxia in NSCLC biology.

Aged↗
PubMed2000

Detection of bovine herpesvirus 4 (BoHV-4) DNA in the cell fraction of milk of dairy cattle with history of BoHV-4 infection.

We have demonstrated, by PCR and restriction enzyme analysis of the PCR product, the presence of bovine herpesvirus 4 (BoHV-4) DNA in the cell fraction of milk from dairy cattle with a history of BoHV-4 infection. We next evaluated the infectious nature of BoHV-4 DNA in those cells. Cocultivation of a BoHV-4-sensitive cell line with BoHV-4 DNA-positive milk cell samples produced cytopathic effects. The same result was obtained from frozen and thawed milk cell fraction coming from the cell milk fraction PCR-positive cows, ensuring that cells were killed and only infectious virus could be recovered after cocultivation with sensitive cells. This report shows that infectious BoHV-4 can be present in milk cells and that therefore nursing may be one of the transmission routes of BoHV-4.

Animals↗
PubMed2006

Continuous flow magnetic cell fractionation based on antigen expression level.

Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry.

Antigens↗
PubMedDate not supplied

[Nucleases of cell fractions of rat brain tissues].

Nucleases are found in different fractions of nerve cells in the rat brain. The nuclease denaturating preferably the one-chain DNA at pH 8.0 is located chiefly in the neuron nuclei. Fractions of cell nuclei containg mainly neuron nuclei or glial nuclei were obtained by the method of ultracentrifugation within the sucrose gradient.

Animals↗
PubMed1982

The pancreatic beta cell fraction in children with errors of amino acid metabolism.

Blocks of pancreas were obtained from the following cases of errors of amino acid metabolism: eight cystinosis, eight tyrosinosis, five phenylketonuria, three hypermethioninemia, two hyperprolinemia and two maple syrup urine disease. Blocks were also obtained from four cases of homocystinuria and 72 control patients of the same age range who had died from a variety of conditions believed not to affect the pancreas. Sections were cut from each pancreatic block and stained with haematoxylin or for insulin and pancreatic polypeptide (PP) by the immunoperoxidase method. Measurements were performed separately in the PP rich and the PP poor regions of all sections. The fractional surface area of section occupied by insulin stained cells (%) and the cellular density (nuclei/100 microgram2) of each specimen were estimated. The beta cell fractional area in the PP poor region of the experimental cases was plotted against the logarithm of gestational age and compared to a reference grid of the 10th, 50th and 90th centile estimates of the control cases (Fig. 1). The distribution of results from the cases of tyrosinosis, phenylketonuria and cystinosis were skewed positively; four of eight tyrosinosis and three of five phenylketonuria cases lying above the 90th centile (P less than 0.001). The beta cell fractional area of the cystinosis cases was also significantly increased (Table 1, P less than 0.05). The results from the cases dying from maple syrup urine disease, hyperprolinemia, hypermethioninemia or homocystinuria were distributed as might be expected to occur by chance.

Amino Acid Metabolism, Inborn Errors↗
PubMed1960

A cytochemical study on the pancreas of the guinea pig. 5. In vivo incorporation of leucine-1-C14 into the chymotrypsinogen of various cell fractions.

CHYMOTRYPSINOGEN SYNTHESIS IN THE EXOCRINE CELL OF THE GUINEA PIG PANCREAS WAS STUDIED UNDER THE FOLLOWING CONDITIONS: Animals fed after a fast of approximately 48 hours received approximately 1 hour after feeding an intravenous injection of DL-leucine-1-C(14). At various time intervals (1 to 45 minutes) after the injection, the glands were removed and fractionated into a series of cell fractions of known cytological significance. Ten to twelve animals were used for each time point. From each cell fraction, the chymotrypsinogen was isolated by acid extraction and purified by (NH(4))(2)SO(4) fractionation, isoelectric precipitation, and chromatography. Because of the minuteness of the quantities involved, chymotrypsinogen amounts were calculated from enzymatic activity figures, and a carrier method was used to precipitate and count the enzyme. The chymotrypsinogen isolated from the attached ribonucleoprotein particles of the microsomal fraction had the highest specific radioactivity at the early time points (1 to 3 minutes). After long intervals (at 15 to 45 minutes), the specific radioactivity of the enzyme increased in the microsomal contents and finally in the zymogen granules. The results are compatible with the view that the chymotrypsinogen is synthesized in or on the attached RNP particles and subsequently transported to other cell compartments.

Animals↗
PubMed1991

HIV-1 gag proteins in virions and in infected cell fractions.

The relation of the initial products of the HIV-1 gag gene to the final products was determined in virus samples and cell fractions of infected H9 and Jurkat-tat cell cultures. The proteins were identified by immunoblotting with pooled sera from AIDS patients or monoclonal antibodies. The proportion in the virions of gag precursor proteins and the products of their proteolytic cleavage varied according to the maturity of the virus particles as determined by electron microscopy. The distribution of viral gag proteins in the cell fractions was determined 2, 4, and 24 h after infection. Treatment of cells with cycloheximide to block de novo protein synthesis did not significantly affect the results. Gag proteins containing the N terminus of the precursor p55 (including p55, the intermediate precursors p41(45) and p39, and mature protein p17) were found in the cell nuclei up to 24 h after infection. The major core protein p24 was located in the cytoplasmic fraction. These data strongly suggest that gag precursors from the p55 N terminus and the matrix protein p17 enter the infected cell separately from the major core protein p24, or become separated from it in the cytoplasm.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Cell fractionation, detergent sensitivity and solubilization of Dictyostelium adenylate cyclase and guanylate cyclase.

Cell fractionation studies have been performed, in order to obtain insight into the subcellular distribution of Dictyostelium adenylate cyclase and guanylate cyclase and also to provide a starting point for further study and isolation of these enzymes and their regulatory components. Adenylate cyclase and cAMP receptors were found in the same membrane fractions, but were distributed different from the plasma membrane marker alkaline phosphatase. Guanylate cyclase was partially soluble, partially particulate. In isopycnic gradients, particulate guanylate cyclase was present in other fractions than cAMP receptors and adenylate cyclase, but in similar ones to alkaline phosphatase. These observations are consistent with the hypothesis that cell-surface cAMP receptors and adenylate cyclase interact via a membrane-bound G-protein, whereas the receptors activate guanylate cyclase via a cytosolic factor. The adenylate cyclase activity in membranes obtained by sucrose gradient centrifugation was retained in the presence of various detergents, while with the same detergents the activity of particulate guanylate cyclase was lost. This adenylate cyclase was solubilized as assessed by gel filtration and centrifugation experiments, and it behaved heterogeneous in fractionation studies. In gel filtration, the major component eluted at a position corresponding to a Stokes radius of 4-7 nm. A purification of about 70-fold as compared to the cell homogenate was obtained by affinity chromatography of adenylate cyclase on ATP-Sepharose. We conclude that cell fractionation provides useful starting material for isolation and further study of Dictyostelium adenylate cyclase.

Adenylyl Cyclase Inhibitors↗
PubMed1991

Proteolysis of splicing factors during rat and monkey cell fractionation.

We have investigated the ability of various rat and monkey cell lines to yield nuclear extracts that would allow splicing of a model adenovirus pre-mRNA substrate. Extracts from normal FR3T3, rat-1 and CV-1 fibroblasts were unable to assemble splicing complexes and displayed a dramatic reduction in the binding activity of the splicing factor 65 kD U2AF. These results correlated with reduced levels of 65 kD U2AF and the snRNP-associated B protein. When a battery of protease inhibitors was used during cell fractionation, increased levels of 65 kD U2AF and B proteins were detected. Most importantly, U2AF binding and complex formation were dramatically improved in FR3T3, rat-1 and CV-1 extracts. Interestingly, transformation of rat and monkey cells with the SV40 large T antigen yielded extracts active in complex formation. Similar extracts were generated following transformation of rat-1 cells with the Py middle T antigen but not with the v-fos oncogene. Only SV40-transformed FR3T3 extracts displayed splicing activity. Our results indicate that proteolysis is a major obstacle encountered during the preparation of active extracts from normal rat and monkey cells and suggest that cells transformed with T antigens manifest reduced proteolysis during fractionation.

Adenoviridae↗
PubMed1985

Enzymatic activities in cell fractions of mycoplasmalike organisms purified from aster yellows-infected plants.

Mycoplasmalike organisms (MLOs), purified from aster yellows-infected plants were osmotically lysed, and the membranes were separated from the cytoplasmic fraction through differential centrifugation. Electron microscopic examinations of sections of the purified MLOs and the isolated membranes showed pleomorphic bodies and unit membranous empty vesicles, respectively. Cell fractions were tested for NADH oxidase, NADPH oxidase, ATPase, RNase, DNase, and p-nitrophenyl phosphatase activity. NADH oxidase and ATPase were confined to the membrane fraction and NADPH oxidase to the cytoplasmic fraction of the MLOs. para-Nitrophenyl phosphatase, RNase, and DNase activities were detected in both membrane and cytoplasmic fractions, but p-nitrophenyl phosphatase and RNase appeared to be associated with membranes and DNase with the cytoplasmic fraction. Glucose-6-phosphate dehydrogenase was found in the cytoplasmic fraction of the MLO cells. Our findings on the distribution of enzymes in MLO cells and cell fractions are the first basic documentation on nonhelical, nonculturable microbes parasitic to plants.

4-Nitrophenylphosphatase↗
PubMed1979

Uptake of uranium by various cell fractions of Chlorella regularis.

To know what kinds of the cell components of Chlorella regularis are concerned with uranium binding, uptake of uranium by various cell fractions was examined. The uptake value (microgramU/mg starting dry cells) of the hot water-treated cells was almost the same as that of the starting dry Chlorella cells, showing that the cell components extracted with hot water were not so concerned with uranium binding. The cell components extracted with dilute alkali seemed to play an important role in uranium binding, and those extracted with chloroform-methanol seemed to be partly concerned with uranium binding. The cellulose fraction of the cells was scarcely concerned with uranium binding. In the dry cells, 34% of uranium taken up existed in the cell walls. However, in the living cells, 85% existed in the cell walls. The above results showed that the dry or the hot water-treated cells are the most convenient for uranium recovery from the aqueous systems.

Chlorella↗