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PubMed1978

[Possibility of using enriched glial and neuronal cell fractions to study transport of neurotransmitter amino acids in old age].

Calculated on a protein basis the yield of neuronal and glial cell-enriched fractions obtained from the brain of aged and adult rats was found to be equal. In aged animals the high affinity uptake of 14C-GABA by neuronal fractions is reduced by 27.34% while that of glial fractions by 11.87%. At 0 degrees both age groups studied the uptake of 14C-GABA is greatly inhibited--by 66% in neurones and 92% in glia. The conclusion is drawn that glial cells are changed much less with age than neurones and thus their role as modulators of neuronal function increases with age.

Aging↗
PubMed1974

Changes in free and membrane-bound ribosomes during the development of chick liver. A new cell-fractionation approach.

A major difficulty in studying quantitative changes in free and membrane-bound ribosomes in a tissue under different physiological conditions is that membrane-bound ribosomes are not usually recovered quantitatively in a conventional microsomal fraction. This problem was resolved for developing chick liver by determining the conditions for the isolation of a microsomal fraction containing the highest practicable yield of rough vesicles, and then separating it into free-ribosome- and rough-vesicle-containing fractions. With the aid of a marker enzyme for the microsomal membranes and the RNA content of the recovered membrane-bound ribosomes, it was possible to correct for the recovery of rough vesicles and hence to determine the concentration of membrane-bound ribosomes in the homogenate. Despite the fact that morphological studies have suggested that most of the cellular ribosomes are not bound to membrane in chick liver cells at the earliest developmental age studied (6 days of egg incubation), 49% of the total ribosomes were found to be membrane-bound by using the new fractionation technique. This fraction increased (to 66%) during development. The discrepancy between the cell-fractionation and morphological approaches could not be attributed to artifacts of the separation method but rather to difficulties inherent in the morphological approach.

Age Factors↗
PubMed1984

Responsiveness of human T-lymphocyte subpopulations in autologous mixed-lymphocyte reaction using xenoprotein-free separated cells: autologous reactivity lies chiefly in a low density T-cell fraction.

The responsiveness in the autologous mixed-lymphocyte reaction (AMLR) by human T cells separated using two different methods not involving xenoantigen contract was examined. Although T cells from nylon-wool columns were active in AMLR, T cells separated by a Percoll gradient method responded poorly. Further separation of T cells from nylon-wool columns into low density (TL) and high density (TH) fractions by Percoll revealed that TL was enriched, while TH was depleted, in AMLR responsiveness. This difference could not be accounted for by differences in the helper or suppressor cells in the fractions. Moreover, TH responded well in secondary AMLR. Therefore the T cells reactive in AMLR reside chiefly, although not exclusively, in the low density fraction.

Cell Separation↗
PubMed1985

Deficient natural killer (NK) cells in paroxysmal nocturnal haemoglobinuria (PNH): studies of lymphoid cells fractionated by discontinuous density gradient centrifugation.

Non-adherent, non-B lymphoid cells from six patients with PNH and six healthy subjects were fractionated by Percoll discontinuous density gradient centrifugation (DDGC). The cell distribution pattern, NK cell activity (NKA), large granular lymphocytes (LGL) count and surface marker phenotypes were studied. The distribution patterns of patients' cells did not significantly differ from the controls. The peak of the NKA was found in low density fractions where the maximum counts of LGL, Leu-7+2- cells and Leu-11+ cells were present. The NKA and the proportion of Leu-7+2- cells and Leu-11+ cells were significantly lower in patients with PNH (P less than 0.001 for NKA and surface phenotypes; P less than 0.02 for LGL counts). NKA in the Percoll fractions was correlated with the counts of LGL (r=0.69, P less than 0.001), Leu-7+2- cells (r = 0.75, P less than 0.001) and Leu-11+ cells (r = 0.89, P less than 0.001). Therefore, we concluded that NKA is deficient in PNH because of decreased NK cell counts.

Adult↗
PubMed1988

Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor.

Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.

Adenovirus Early Proteins↗
PubMed2005

Isolation and characterization of a putative intestinal stem cell fraction from mouse jejunum.

BACKGROUND & AIMS: Although there have been many recent advances regarding the biology of intestinal stem cells, the field has been hampered significantly by the lack of a method to isolate these cells. Therefore, the aim of this study was to explore the hypothesis that viable intestinal stem cells can be isolated as a side population (SP) by fluorescence-activated cell sorting after staining with the DNA-binding dye Hoechst 33342. METHODS: Preparations of individual cells from either whole mucosa or epithelium of mouse jejunum were stained with Hoechst 33342 and propidium iodide and then sorted using fluorescence-activated cell sorting. Cells were characterized using fluorochrome-labeled antibodies to surface markers, intracellular markers, and annexin V to detect early apoptosis. Total RNA was isolated from sorted fractions and used for quantitative real-time reverse-transcription polymerase chain reaction to evaluate the expression of cell lineage markers and the intestinal stem-cell marker, Musashi-1. RESULTS: Adult and neonatal jejunum contain a viable population of cells that shows the SP phenotype and is sensitive to verapamil. This population of cells (from both mucosal and epithelial preparations) includes a CD45-negative fraction corresponding to nonhematopoietic cells, which shows minimal expression of surface markers typically found on stem cells from other tissues and of intracellular markers found in mesenchymal cells. Additionally, these cells were enriched for Musashi-1 and beta1-integrin, were cytokeratin positive, and survived in culture for up to 14 days. CONCLUSIONS: The CD45-negative SP fraction, although not pure, represents the successful isolation of a viable population significantly enriched in small intestinal epithelial stem cells.

Age Factors↗
PubMedDate not supplied

Rearranging antigen-receptor genes in enriched Reed-Sternberg cell fractions of Hodgkin's disease.

Molecular genetic analysis of rearranging antigen-receptor genes in non-Hodgkin's lymphomas has revealed the clonality and lineage in the majority of cases. In an analogous approach, we sought to apply gene rearrangement analysis to Hodgkin's disease to understand better the clonality and origin of this disorder. However, the putative neoplastic cell of Hodgkin's disease, the Reed-Sternberg cell and its variants, is extraordinarily rare in most cases of Hodgkin's disease. On the average, Reed-Sternberg cells and variants represent 0.1 per cent of total cell suspensions of nodular sclerosing Hodgkin's disease. As this frequency is below the minimum threshold of sensitivity of the Southern blot assay, we attempted to enrich for Reed-Sternberg cells before DNA extraction and analysis. Using either elutrition or Percoll density gradient centrifugation, we were able to enrich the percentage of Reed-Sternberg cells and variants to above 1 per cent in five cases of nodular sclerosing Hodgkin's disease. In three of these cases, immunoglobulin gene rearrangements were identified, but no T cell receptor gene rearrangements were seen. No rearrangements were detected in unseparated cells or in the Reed-Sternberg cell-depleted fractions. In addition, the L428 Hodgkin's disease cell line was found to have one rearranged and one deleted heavy-chain gene, a rearranged kappa gene, a rearranged lambda gene, and a single rearranged beta allele. No rearrangements of the T gamma gene were found in L428. Taken together, these findings indicate that clonal cell populations are present in Hodgkin's disease and suggest the possibility of a clonally expanded lymphoid cell in this disorder.

Genes, Immunoglobulin↗
PubMed2004

Development of a rare cell fractionation device: application for cancer detection.

Isolating rare cells from biological fluids including whole blood or bone marrow is an interesting biological problem. Characterization of a few metastatic cells from cancer patients for further study is desirable for prognosis/diagnosis. Traditional methods have not proven adequate, due to the compositional complexity of blood, with its large numbers of cell types. To separate individual cells based on their mechanical characteristics, we have developed a series of massively parallel microfabricated sieving device. These devices were constructed with four successively narrower regions of channels numbering approximately 1800 per region. As cells traversed the device, they encountered each region and stopped at a gap width that prohibited passage due to their size. Cultured neuroblastoma cells, when mixed with whole blood and applied to the device, were retained in the 10-microm-wide by 20-microm-deep channels. All other cells migrated to the output. A derivative of the same device was utilized to characterize migration of whole blood. Adult white blood cells were retained at the 2.5-microm-wide by 5-microm-deep channels, while red blood cells passed through these channels. Devices designed to capture rare cells in peripheral circulation for downstream analysis will provide an important tool for diagnosis and treatment.

Cell Separation↗
PubMed1997

Detection of malignant cells of epithelial origin in mononuclear cell fractions.

Qualitative and quantitative detection of contaminating malignant cells in stem cell apheresis products intended for supportive therapy after high-dose chemotherapy of sensitive solid tumors is a demanding task for the laboratory. Human breast cancer cells in differing concentrations among mononuclear cells were quantitated by multicolor flow cytometry and immunocytochemistry. Comparing specificity, sensitivity, and laboratory processing time, the immunocytochemical detection of the above-mentioned malignant cells of epithelial origin was demonstrated to be the superior method.

Breast Neoplasms↗
PubMed2005

Monitoring dioxins in food and feedstuffs using accelerated solvent extraction with a novel integrated carbon fractionation cell in combination with a CAFLUX bioassay.

The concentrations of dioxins in fish oil and fish meal were determined with accelerated solvent extraction, using a novel integrated carbon fractionation extraction cell followed by a miniturized multilayer silica column and bioanalysis on a recently-developed chemically-activated fluorescent gene expression cell bioassay. The developed method allows for simultaneous gravimetric lipid weight determination, which was shown for both matrices under study (about 100% lipid recovery of each sample). Initial results practically meet the quality criteria on screening methods for control of dioxins in food and feedstuffs laid down in the EU Commission Directives 2002/69/EC (food) and 2002/70/EC (feed). This demonstrates that the developed method can be used as a screening tool for monitoring dioxins in food and feed after some additional improvements and testing on a greater number of matrices.

Animals↗