Measurement variability in DNA flow cytometry of replicate samples

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.     

Induction of reversible changes in cell-surface glycoconjugates and lung colonization potential by 13-cis retinoic acid

Murine squamous carcinoma cells (KLN205) grown in a medium supplemented with the retinoid, 13-cis retinoic acid (RA), had dose-dependent, selective increases in the expression of certain lectin receptors, which correlated with a dramatic decrease in the ability to form pulmonary colonies (P ?.0003) (Couch MJ, Pauli BU, Weinstein RS, Coon JS: JNCI, 78:971 ?977, 1987). These findings suggest a possible relationship between the RA-induced glycoconjugate alterations and the decreased experimental metastatic behavior. We further define the mechanism of RA's action. The finding that RA treatment (5 × 10^?6 M, 5 × 10^?7 M) did not perturb the cell cycle of KLN205 cells provides further proof that the decreased metastatic behavior is not attributable to any inhibition in the rate of growth or to alterations in the cell cycle. Furthermore, since stable subpopulations with variable lectin binding could not be detected, the mechanism of RA's action does not appear to be due to selection of variant tumor-cell subpopulations. Finally, in a scries of experiments designed to determine the reversibility of the RA treatment, the RA-induced decrease in metastatic behavior reverted back to a more metastatic state in the same time frame (3 days) as the reversion of the RA-induced changes in cell-surface glycoconjugate expression. This reversion provides further evidence for a close relationship between the RA-induced modulation of tumor cell-surface glycoconjugate expression and the decreased metastatic behavior; it suggests that transient, reversible modulation of the tumor cell surface may play a role in determining metastatic behavior.     

Further characterization of the magnesium chelatase in isolated developing cucumber chloroplasts : substrate specificity, regulation, intactness, and ATP requirements

Mg-chelatase catalyzes the first step unique to the chlorophyll branch of tetrapyrrole biosynthesis, namely the insertion of Mg into protoporphyrin IX (Proto). Mg-chelatase was assayed in intact chloroplasts from semi-green cucumber (Cucumis sativus, cv Sumter) cotyledons. In the presence of Proto and MgATP, enzyme activity was linear for 50 minutes. Plastid intactness was directly related to (and necessary for) Mg-chelatase activity. Uncouplers and ionophores did not inhibit Mg-Chelatase in the presence of ATP. The nonhydrolyzable ATP analogs, β,γ-methylene ATP and adenylylimidodiphosphate, could not sustain Mg-chelatase activity alone and were inhibitory in the presence of ATP (I₅₀ 10 and 3 millimolar, respectively). Mg-chelatase was also inhibited by N-ethylmaleimide (I₅₀, 50 micromolar) and the metal ion chelators 2,2′-dipyridyl and 1, 10 phenanthroline (but not to the same degree by their nonchelating analogs). In addition to Proto, the following porphyrins acted as Mg-chelatase substrates, giving comparable specific activities: deuteroporphyrin, mesoporphyrin, 2-ethyl, 4-vinyl Proto and 2-vinyl, 4-ethyl Proto. Mg-chelatase activity and freely exchangeable heme levels increased steadily with greening, reaching a maximum and leveling off after 15 hours in the light. Exogenous protochlorophyllide, chlorophyllide, heme, and Mg-Proto had no measurable effect on Mg-chelatase activity. The potent ferrochelatase inhibitors, N-methylmesoporphyrin and N-methylprotoporphyrin, inhibited Mg-chelatase at micromolar concentrations.     

Band 3 protein of the red cell membrane of the llama: crosslinking and cleavage of the cytoplasmic domain

Comparative studies were done on the cytoplasmic domain of the band 3 protein in the red cell membranes of the the human and the llama. Two approaches were used: crosslinking with o-phenanthroline/CuSO4, and cleavage with 2-nitro-5-thiocyanobenzoate. o-Phenanthroline/CuSO4 crosslinks the band 3 polypeptide chains in the human; in contrast band 3 in the llama is minimally crosslinked by this agent. 2-Nitro-5-thiocyanobenzoate cleaves band 3 in the human into a 23,000-dalton fragment; a similar fragment is not generated from the llama band 3. These studies show that the cysteine residue located 23,000 daltons from the N-terminus of band 3 in the human involved in these reactions is unavailable for crosslinking and cleavage in the llama. Species differences in the cytoplasmic domain of band 3 may contribute to the unusual resistance of llama red cells to osmotic, chemical and physically-induced deformation.     

RNA is required for enzymatic conversion of glutamate to delta-aminolevulinate by extracts of Chlorella vulgaris

Formation of delta-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase). Cell extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components. RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor. The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated. After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C. vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography. Baker's yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E. coli tRNAglu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs with glutamate.     

Interleukins 2 and 3 regulate the in vitro proliferation of two distinguishable populations of 20-alpha-hydroxysteroid dehydrogenase-positive cells

The ability of interleukin 2 (IL 2), interleukin 3 (IL 3), and granulocyte/macrophage colony-stimulating factor (GM-CSF) to induce the proliferation of cells from thymus, spleen, or bone marrow was examined and compared with their ability to induce expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH). In the thymus, the peanut agglutinin agglutinated cells (PNA+) lacked 20 alpha SDH and showed no detectable response to IL 2, IL 3, or GM-CSF in either proliferation or induction of 20 alpha SDH. In contrast, the PNA nonagglutinated (PNA-) subpopulation expressed 20 alpha SDH and proliferated in response to Con A and/or IL 2. The responding cells that could be expanded in vitro with IL 2 expressed high levels of 20 alpha SDH. Neither IL 3 nor GM-CSF in the presence or absence of Con A had a demonstrable effect on the PNA- population. In cultures of bone marrow cells, both IL 3 and GM-CSF induced proliferation, whereas IL 2 had no effect on proliferation in the presence or absence of Con A. Thy-1-depleted bone marrow cells, expanded in tissue culture with IL3, contained cells that co-expressed Thy-1 and 20 alpha SDH. In contrast, cells proliferating in vitro to GM-CSF did not expressed Thy-1 or 20 alpha SDH. In cultures of normal splenic lymphocytes, two populations of cells capable of expressing 20 alpha SDH were detected. One population could be expanded in vitro with IL 2 and Con A, whereas the second was responsive to IL 3. In spleens from athymic mice, only the latter cells were detected. These results demonstrate that IL 3 and IL 2 responsiveness distinguishes two populations of 20 alpha SDH cells. The relevance of these observations to the possible relationship of IL 3 and IL 2 in T cell differentiation is discussed.     

Ultrastructural analysis of pathologic lesions in sterol-deficient Nippostrongylus brasiliensis larvae

A variety of cellular lesions were manifested by the free-living larval stages of Nippostrongylus brasiliensis cultured axenically in medium lacking cholesterol. Pathologic changes developed rapidly and were most apparent in intestinal cells which displayed generalized degradation of membranous organelles. Mitochondria, endoplasmic reticulum, and Golgi complexes became disassociated and vacuolated. Autophagosomes appeared within intestinal cells and contained a wide variety of cellular components. By the 5th day gross vacuolization and degeneration of intestinal cells occurred and the hypodermis and lateral cords displayed lysed cytoplasmic regions. The latter structures are concerned with synthesis of cuticle and their degeneration correlates with the suppression of molting and the abnormal molts that occurred.     

The decolorization of the polymeric dye Poly-Blue (polyvinalamine sulfonate-anthroquinone) by lignin degrading fungi

Summary In this work we have investigated the decolorization of the polymeric dye Poly-B411 by several fungi. Only fungi with known lignin degrading ability were able to decolorize the dye. Pleurotus ostreatus sp. florida decolorized the dye both in solid and liquid media. Decolorizing ability developed in the absence of the dye but only when the fungus had been previously cultivated on lignin containing substrates.The work was supported by a grant from the Charles Wolfson Trust