Rapid Separation Technique Research Articles

Decreased cisplatin uptake by resistant L1210 leukemia cells

Cisplatin resistance remains poorly understood compared to other forms of anti-neoplastic drug resistance. In this report radiolabeled cisplatin and rapid separation techniques were used to compare drug uptake by L1210 leukemia cells that are sensitive (K25) or resistant (ZCR9) to cisplatin. Uptake of cisplatin by both cell lines was linear without saturation kinetics up to 100 μM. The resistant ZCR9 cells had 36–60% reduced drug uptake as compared to its sensitive parent line, K25. In contrast, there was no difference in the rate of efflux. We conclude that a decreased rate of uptake is one possible mechanism of cellular cisplatin resistance.

Isotope effect in retention value between55Cr and51Cr in recoll chemistry of Cr/acac/3

Recoiling behaviour of55Cr produced by54Cr/n, γ/55Cr reaction was studied by choosing solid Cr/acac/3 or its benzene solution as target material and by using rapid separation technique. The approximate retention value /“R-value”/ of55Cr, defined as the percentage of the activity retained in the benzene phase which was not extracted with an oxalic acid solution, was found to be higher than that of55Cr for practically all experimental runs tested /15 runs/. The ratio (55Cr/51Cr) was 1.115±0.027.

A two-site immunoradiometric assay with magnetizable particles: human liver ferritin as a model.

Using human liver ferritin as a model, we have developed a two-site immunoradiometric assay. Sample (or standard) is incubated with excess rabbit antiserum and 125I-labeled specific sheep antibodies directed against different antigenic determinants until equilibrium is reached. Then excess sheep anti-rabbit immunoglobulin G serum, covalently linked to magnetizable particles, is added. The radioactivity bound to these particles is directly proportional to the amount of ferritin present. The assay is reproducible and precise throughout the clinically important range for ferritin in serum. Late addition of second antibodies coupled to magnetizable particles is a novel, simple, rapid, and universally-applicable separation technique for two-site immunoradiometric assays. By this approach, the first immunological reaction can occur in liquid phase, which shortens the reaction time, maximizes sensitivity, and avoids the need for continuous mixing, as compared with methods in which one of the first antibodies is immobilized on a solid phase.

A Quarter Century of Thin‐Layer Chromatography—An Interim Report

AbstractThin‐layer chromatography (TLC) has been in general use since 1958. The prerequisites were the use of very small particle size material in the layers and the demonstration that not only lipophilic mixtures but also mixtures of cardiac glycosides, alkaloids, sugars, and amino acids could be separated quickly and simply on silica gel layers. The impressive separations obtained using the method later encouraged attempts to use adsorbants having such, narrow‐range small particles in column chromatography. In order to achieve sufficient flow rates the use of high pressures was necessary. Thus, high pressure or high performance liquid chromatography (HPLC) was born, whose godfather, as far as instrumental development is concerned, was gas chromatography (GC). Today, chromatography is classified according to the nature of the transport phase: liquid, gas, or “electro” chromatography. Sample preparation often requires more time than the actual separation. rapid thermal separation and application techniques (TAS, TFG) have brought advantages to TLC. As before, the art of chromatography consists in choosing the appropriate mobile phase, stationary phase, and separation technique. Gradient TLC offers new possibilities; for example, gradient layers make it possible to obtain selective information on the individual components of a mixture and also allow significant focussing effects to be achieved. Sequential and centrifugal TLC have paved the way for further advances. The questions arise whether the standard conditions, formulated 25 years ago, are still applicable and how the variations which have been introduced should be judged. Group‐ and substance specific methods of detection are discussed in addition to universal methods. Quantitation is critically discussed and it is concluded that HPLC has the better chance in the furture. Combination and coupling techniques ease the positive identification of individual substances. Finally, it is discussed why TLC is the simplest, most versatile, most cost effective, and most widely used chromatographic method and reasons are proposed as to why it will remain so for the foreseeable future.

Paradoxical response of malignant melanoma to methotrexate in vivo and in vitro.

Methotrexate (MTX) shows consistent cytotoxicity for melanoma cells in vitro but it is ineffective in clinical use at equivalent concentrations in vivo. This apparent paradox has been investigated by cell culture techniques and results quantified by cell number. In an in vitro model of high dose MTX therapy followed by leucovorin rescue (HD-MTX-LCR) there was survival of both melanoma and choriocarcinoma cell lines but not of an acute lymphocytic leukaemia cell line. The 70H metabolite of MTX was identified by HPLC in plasma samples of melanoma patients treated by HD-MTX-LCR, in which MTX concentrations approximately 10(-5) M were maintained for 24 h. However, metabolism per se is unlikely to account for the lack of response to MTX clinically. In vitro 70H MTX (10(-7) - 10(-6) M) was two orders of magnitude less cytotoxic for melanoma than MTX (10(-9) - 10(-8) M). The cellular accumulation of [3H]-MTX, using a rapid gradient centrifuge technique for separation of melanoma cells from medium, was reduced in the presence of 70H-MTX. The results suggest that reduced cellular uptake of MTX combined with biochemical rescue of tumour cells may partially explain the paradoxical lack of clinical response of melanoma to the drug.

Single-step, rapid separation of acidic and basic isoenzymes from commercial horseradish peroxidase

A simple and rapid technique for simultaneous separation of the acidic and basic isoenzymes of horseradish peroxidase is described. Upon application of a potential gradient on the electrolyte system composed of Pharmalyte (pH 2.5-5.0) histidine (pI 7.64), and Ampholyte (pH 9-11), the acidic and basic isoenzymes with pI 4.0-8.4 of horseradish peroxidase accumulated at the two interphases generated by this arrangement, with a recovery of 75 +/- 5%. Some advantages, such as rapidity, simplicity, low cost, good yields, and others, of this system over existing ones are outlined.

A rapid rosette technique for quantitation and separation of mononuclear cell subsets using monoclonal antibodies

We present a rapid and simple method for simultaneous quantitation and separation of mononuclear cell (MNC) subsets. When lymphoid cells are sensitized with monoclonal antibodies of the OK and Leu series, they rapidly form rosettes with ox erythrocytes (ORBC) coated with affinity-purified rabbit IgG against mouse IgG. Rosette-forming cells (RFC) may then be counted and separated from non-rosetting MNC by Isopaque-Ficoll gradient centrifugation. The yield and viability are close to 100% after ORBC lysis. Adherent cells do not interfere. Isolated T8 + and Leu3a + cells were further tested: the purity was 97–99%, and the cells were functionally intact with respect to their modulating activity on the generation of immunoglobulin-secreting cells by MNC after stimulation with pokeweed mitogen.

Rapid chromatofocusing of proteins

Chromatofocusing, a chromatographic technique whereby proteins are selectively eluted from an ion-exchange support according to their p I values, has been adapted to high-performance liquid chromatography. It was found that chromatofocusing can detect heterogeneity in protein preparations not demonstrated by reverse-phase or size exclusion chromatography and that the resolution of chromatofocusing is comparable to ion-exchange chromatography. Although chromatofocusing may not resolve proteins as well as isoelectric focusing, it has advantages over this technique in both speed and capacity. The usefulness of chromatofocusing as an additional technique in the analysis and preparation of proteins is discussed. The rapid separation technique described here is able to resolve protein mixtures in the chromatofocusing mode in approximately 30 min.

Rapid radioimmunoassay for serum myoglobin.

A rapid radioimmunoassay for serum myoglobin concentration has been developed, capitalizing on a technique for rapid separation of antibody-bound antigen from free antigen using Sepharose-bound sheep anti-rabbit gamma-globulin. Total time required for the assay is 2.75 h, making it suitable for clinical approaches employing myoglobin for rapid identification of patients in the active phase of myocardial injury.

The on-line mass separator OSIRIS and the study of short-lived fission products

A review of the OSIRIS isotope-separator-on-line facility is given describing, in particular, the integrated target-ion source system, the rapid chemical separation techniques, and the study of the delay between the production and the measurement of the nuclides. The experimental programme for OSIRIS is also briefly sketched.

A rapid assay technique for RNA ribose methylases

A rapid technique for quantitative separation of ribose-methylated nucleosides from base-methylated and non-methylated nucleosides by chromatography on DEAE-cellulose paper in the presence of borate is described. The method has been used as an assay for tRNA ribose methylases from yeast, using under methylated Escherichia coli tRNA as substrate. The main product formed with a partly purified yeast enzyme was characterized as 2'-O-methylcytidine.

Studies on vitamin B 12 binders of human serum. (I) Isolation and characterization.

SummaryA DEAE-cellulose column chromatographic technique for rapid separation of TC II, TC III and TC I from 0.5 ml of normal human serum is described. The molecular weights of TC II, TC III and TC I, as determined by gel-filtration on Sephadex G-200 columns were found to be 40,000, 100,000 and 120,000 respectively. On electrophoresis, TC II moves as a β-globulin, TC I between α1 and α2-globulin, and TC III between β and α2-globulin.

Delayed-neutron emission from short-lived Br and I isotopes

Abstract Rapid radiochemical separation techniques have been used to investigate the delayed-neutron emission from bromine and iodine isotopes produced by thermal-neutron induced fission of 235 U. The nuclides 0.63 sec 91 Br, 0.26 sec 92 Br and 0.41 sec 141 I have been identified as delayed-neutron precursors. Relative neutron abundances of these and of the known precursors 87–90 Br and 137–140 I have been measured and absolute neutron abundances and neutron emission probabilities have been deduced. The P n values are discussed within a simple semiempirical picture.

A rapid technique for separation of thymocytes from suspensions by centrifugation through silicone oil

A method is described for rapid separation of thymocytes from suspensions by centrifugation of the cells through silicone oil. The usefulness of several radioactive compounds as markers for the trapped extracellular water space and the total water space of the cell pellets was investigated. According to the results obtained with [ 3H]inulin and [ 3H]methoxyinulin, the extracellular water space comprised approximately 12% of the total water space as determined by 3H 2O or C 35S(NH 2) 2. The thymocytes appeared to be undamaged after the separation as judged by their oxygen consumption, sodium content, and dexamethasone binding.

Quantitative Measurement of Ovalbumin Messenger Ribonucleic Acid Activity: LOCALIZATION IN POLYSOMES, INDUCTION BY ESTROGEN, AND EFFECT OF ACTINOMYCIN D

Abstract Procedures are described for the quantitative assay of ovalbumin mRNA in a rabbit reticulocyte lysate protein-synthesizing system. A technique for rapid and efficient separation of labeled immunoprecipitated ovalbumin from nonspecific radioactivity is presented. The capacity of the lysate system for total de novo protein synthesis, determined with the use of aurintricarboxylic acid, was found to be correlated with the capacity for ovalbumin mRNA translation. Hemin, a compound that stimulates hemoglobin synthesis in reticulocyte lysates, also stimulates ovalbumin synthesis. The yield of ovalbumin mRNA activity was used as a criterion in choosing optimal conditions for the isolation of undegraded nucleic acid from oviduct. Phenol extraction of total oviduct homogenate yielded only one-third as much activity as isolation by sodium dodecyl sulfate sucrose gradient. When high concentrations of heparin were used to inhibit ribonuclease, the degradation rate of ovalbumin mRNA activity after homogenization could be slowed enough (half-life = 6.4 hours) to permit nearly quantitative recovery of activity in subcellular fractions. No translation of mRNA fragments could be detected in partially degraded nucleic acid preparations. Seventy-one per cent of the total ovalbumin mRNA activity sedimented with polysomes with only 5% in the 27,000 x g pellet. Administration of estrogen to immature chicks induced ovalbumin mRNA activity, and continued hormone treatment was necessary for the maintenance of activity. In vivo administration of actinomycin D to chicks led to a 65% increase in the rate of ovalbumin synthesis in culture with no change in total or polysomal mRNA levels, providing direct evidence that this drug increases the rate of ovalbumin mRNA translation.

The kinetics of release of 86Rubidium and hemoglobin from erythrocytes damaged by antibody and complement

Sheep erythrocytes carrying 100–200 SAC′ la,4 (rabbit antibody and guinea pig complement) were labelled with 86Rb and treated with varying dilutions of guinea pig C′2 at 30°C. At the peak of SAC't' la, 4,2a generation, C′-EDTA was added and kinetic sampling was begun by means of a simple filtration technique for rapid separation of cells from medium. Release of 86Rb⨥ started after a lag phase of about 0·5–1·0 min, and followed first order kinetics during a prolytic phase lasting 1–15 min, depending on C′2 mulitplicity. Cell lysis then ensued, with first-order release of hemoglobin (Hb). The kinetics of 86Rb⨥ and Hb release form the control populations. A physiological interpreration of the controls is suggested.

Rapid separation technique for mono- and disulfonic acids

Rapid separation technique for mono- and disulfonic acids

Concise Treatment of a Complex Subject

Click to increase image sizeClick to decrease image size Additional informationNotes on contributorsJames L. BrownleeJames L. Brownlee, Jr., has been a member of the faculties of chemistry and nuclear engineering of the University of Illinois since receiving his PhD degree in 1961 in physical analytical chemistry from the University of Michigan. His doctoral work was in the field of activation analysis with emphasis on the use of short-lived isotopes and the development of rapid radiochemical separation techniques. He holds a BSc degree (1953) from the University of California at Los Angeles and a MSc degree (1959) from the University of Michigan.