Identification and quantitation of ascorbic acid in extracts of human lymphocytes by high-performance liquid chromatography.
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Ascorbic Acid↗
Biomedical subjects
L F Liebes
Publications and source records attributed to L F Liebes.
Electrophoretic mobility distributions distinguish hairy cells from other mononuclear blood cells and provide evidence for the heterogeneity of normal monocytes.
The electrophoretic mobility distributions of hairy cells, normal monocytes. CLL, and normal lymphocytes isolated from blood were determined by electrophoretic light scattering. Values obtained for hairy cells, 1.52 X 10(-4) cm2/V . sec, were indistinguishable from that of normal monocytes. The mobility of CLL lymphocytes was similar to that of normal B cells. After exposure to neuraminidase, hairy cells revealed a homogeneous distribution with a reduced mobility of 0.55 X 10(-4) cm2/V . sec, while normal monocytes showed a heterogeneous distribution of electrophoretic mobilities suggestive of subpopulations. The electrokinetic behavior of hairy cells thus differs from that or normal and CLL lymphocytes before, and from that of monocytes after, treatment with neuraminidase. The hairy cell therefore possesses a distinct pattern of surface charge properties that clearly distinguish it from the circulating B cells, T cells, or monocytes.
B-Lymphocytes↗
Comparison of lipid composition and 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization measurements of hairy cells with monocytes and lymphocytes from normal subjects and patients with chronic lymphocytic leukemia.
In this report, we compare the lipid composition and fluorescence polarization properties of hairy cells with those of monocytes and lymphocytes from normal subjects and of lymphocytes from patients with chronic lymphocytic leukemia. For hairy cells, the cholesterol content was 4.66 +/- 1.49 (S.D.) mumol/10(9) cells, and the cholesterol/phospholipid ratio was 0.60 +/- 0.09. These were significantly higher than the values of normal lymphocytes, (cholesterol content, 2.75 +/- 0.65 mumol; cholesterol/phospholipid ratio, 0.50 +/- 0.07) or of chronic lymphocytic leukemia lymphocytes (cholesterol content, 1.76 +/- 0.43 mumol; cholesterol/phospholipid ratio, 0.44 +/- 0.07). Normal monocyte values (cholesterol content, 5.81 +/- 2.08 mumol; cholesterol/phospholipid ratio, 0.59 +/- 0.06) were similar to those of hairy cells. Using the probe 1,6-diphenyl-1,3,5-hexatriene, the fluorescence polarization value at 25 degrees for hairy cells was 0.302, compared to the value of 0.259 obtained with chronic lymphocytic leukemia lymphocytes. Intermediate values (0.294) were obtained with normal lymphocytes and monocytes. Fluorescence polarization values were higher in hairy cell membranes than in chronic lymphocytic leukemia lymphocyte membranes, indicating a low fluidity in the former cell, compatible with their higher cholesterol content and cholesterol/phospholipid ratio. These studies show that two neoplastic cells, hairy cells and chronic lymphocytic leukemia lymphocytes, differ markedly in membrane fluidity and that a high membrane fluidity does not necessarily occur in neoplasia.
Aged↗
Human lymphocyte tubulin. Purification and characterization in normal and leukemic cells.
Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119000. The protein was shown to consist of two subunits, with molecular weights of 61000 and 58000 comparable to the alpha and beta polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 +/- 0.86 . 10(6) M-1 and a level in normal lymphocytes of 1.21 . 10(2) +/- 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and Ka for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocytes tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity for function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.
B-Lymphocytes↗
Differences in the behavior of the membrane and membrane-associated filamentous structures in normal and chronic lymphocytic leukemia (CLL) lymphocytes.
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Antilymphocyte Serum↗
Fluorescent labeling of DNA in solution with covalently bound acriflavin.
We have prepared a fluorescent derivative of DNA based on the acriflavin-Feulgen histological procedure for staining DNA. Our procedure involved binding acriflavin to DNA in solution by reacting the acriflavin with aldehydes formed on the deoxyribose of DNA by controlled removal of a few percent of the purine bases of the DNA. Partially depurinated DNA was reacted with the acriflavin reagent, and unbound acriflavin was removed by chromatography on Sephadex G-25 eluted with phosphate buffered guanidine -HCl. Such single-stranded depurinated DNA bound 0.36 acriflavin molecules per 100 purine bases per h of depurination. DNA containing one bound acriflavin per 200 bases reassociated at 85% of the value of control DNA. The acriflavin - DNA complex showed new absorption maxima at 466 and 370 nm. The fluorescent product had excitation maxima at 304 and 465 nm and an emission maximum at 502 nm. This labeling procedure should be useful in place of or in addition to radioactive labeling for DNA.
Acridines↗
The solution behavior of the bovine myelin basic protein in the presence of anionic ligands. Binding behavior with the red component of trypan blue and sodium dodecyl sulfate.
The interaction of the azo dye (2,3'-dimethyldiphenyl-7-azo-8-amino-1-napthol 3,6-disulfonic acid (TBR) and sodium dodecyl sulfate with the bovine myelin basic protein has been studied using absorbance, circular dichroism and 220 MHz PMR spectroscopy. Additional analyses of the binding reaction were carried out using light scattering, ultracentrifugal and electrophoretic techniques. A procedure for preparing pure TBR was developed. A modified structure for this synthesized TBR has been suggested. The mechanism of TBR binding to the myelin basic protein was found to be metachromatic. In addition, the interaction of TBR with the basic protein which gives rise to aggregation of the dye bound species was found to be analogous to the model proposed by Schwarz, G. and Seelig-Löffler, A. ((1975) Biochim. Biophys. Acta 379, 125-138) to explain the binding of acridine orange with poly (alpha-L-glutamic acid). PMR spectral analyses suggested that arginine residues provide the majority of primary sites of attachment on the basic protein for TBR. The effect of sodium dodecyl sulfate binding with the bovine myelin basic protein was found to induce a minimal change in the conformation of the protein. The induction of only about 20% alpha helial structure could be demonstrated and the binding was reversed by raising the solution temperature to 73 degrees C. The difference in the observed behavior of basic protein arising from TBR binding as opposed to the binding of sodium dodecyl sulfate is viewed as resulting from two different binding mechanisms. The binding behavior of TBR is primarily a consequence of charge-charge interaction while the binding effects of sodium dodecyl sulfate are a consequence of hydrophobic interaction. The sodium dodecyl sulfate binding acts as a shield which limits charge-charge interaction in the basic protein molecule thus preventing aggregate formation while TBR imposes no such restraints.
Animals↗
Automatic gas tank switching system for CO2 incubators.
The use and construction of an automatic gas tank switching system are described. This device monitors the gas pressure in a CO2 incubator gas system and automatically switches to a reserve tank when the main supply tank is depleted. The unit contains an alarm system that signals either loss of power or gas pressure in the supply system.
Carbon Dioxide↗
Reverse transcriptase activity per virion for avian myeloblastosis virus and Rauscher murine leukemia virus.
We have measured reverse transcriptase enzyme activity per virus particle for samples of avian myeloblastosis virus (BAI strain) and murine leukemia virus (RAUSCHER) USing the synthetic template poly(rC)-oligo(dG). Absolute virus concentrations were determined directly by laser beat frequency spectroscopy. Enzyme activity per virion was determined from the slope of the activity plotted as a function of virus concentration. With this reverse transcriptase assay, the minimum activity (expressed as picomoles of dGTP incorporated/virion per hour) is estimated at (28.1 +/- 4.2) X 10(-7) for avian myeloblastosis virus and (1.1 +/- 0.2) X 10(-7) for murine leukemia virus. The sensitivity of this assay, which is determined by the level of incorporated radioactivity measurable above background, is 2.5 X 10(-4) virions for avian myeloblastosis virus (with dGTP specific activity of 8.9 Ci/mmol) and 88 X 10(-4) virions for murine leukemia virus (with dGTP specific activity of 6.52 CI/mmol). These results show that although reverse transcriptase assays can obviously be used to measure relative virus concentrations of equally purified samples of the same virus, they can be very misleading when used to compare the concentrations of different virus species.
Avian Leukosis Virus↗
Solution behavior, circular dichroism and 22 HMz PMR studies of the bovine myelin basic protein.
Bovine myelin basic protein has been investigated with regard to its solution behavior, circular dichroism and 220 MHz PMR spectral properties. At pH 4.8 gamma/2=0.1 acetate buffer, light scattering yielded a Mr of 17 700 and a virial coefficient of 1.0-10(-4) mol-ml/g2. Above pH 7.0 the protein was found to aggregate to higher mol. wt species. Sedimentation experiments at pH 4.8 yielded s degrees 20,w of 1.27 S at gamma/2=0.1 and 1.46 S at gamma/2=0.35. The diffusion coefficient determined from ultracentrifugal experiments was 7.25-10(-7) cm2/s at gamma/2=0.1 and 0.35. The value of f/f0 from diffusion at pH 4.8 and gamma/2=0.35 was 1.64, corresponding to an axial ratio of 11 to 1. The radius of gyration was calculated as 4.28 nm and the root mean square end to end distance was 10.5 nm. At pH 9.0, gamma/2=0.1, s degrees 20,w was 1.71 S and D degrees 20,w was estimated at 7.4-10(-7) cm2/s. The behavior at pH 9.0 reverted to the behavior at pH 4.8 when the pH was readjusted. The E1%/1cm=5.64 at 276.4 nm and 225 at 196 nm. Titration of the protein with trifluoroethanol elicited three distinct regions of conformation stability having increasing helical content as the mol fraction of trifluoroethanol increased. The results of the present study have permitted some comparison of analogous properties and conformational behavior with the basic membrane protein cytochrome c.
Animals↗
Quantitation of RNA tumor viruses by spectroscopy of density gradient gels.
We have developed a system for virus particle quantitation based on the measurement of the optical absorbance of stained viruses which first have been banded at their buoyant density in an equilibrum 24 to 53% (wt/wt) sucrose density gradient, then fixed in position in the gradient by photopolymerizing an acrylamide-riboflavin mixture in the sucrose, and finally stained and destained. Using plasma from mice infected with leukemia virus (Rauscher) or chickens infected with avian myeloblastosis virus (BAI strain) or suitable controls, we have shown that this technique specifically detects RNA tumor viruses. By using virus stock solutions for which the absolute concentrations were determined by laser beat frequency spectroscopy, we have calibrated the absorbance of the viral bands in terms of virus particle concentration. Using 0.8-ml gradients gels (4 by 45 mm) we can detect as low as 2 x 10(7) viral particles with Coomassie blue staining and 6 x 10(6) viral particles with a more sensitive staining procedure using amido black.
Animals↗