Herman Amnéus

Resolution in Affinity Chromatography the Effect of the Heterogeneity of Immobilized Soybean Trypsin Inhibitor on the Separation of Pancreatic Proteases

By affinity chromatography, trypsins and chymotrypsins from mouse pancreas homogenates have been separated using soybean trypsin inhibitor immobilized on Sepharose. The effects of the functional heterogeneity of the adsorbent have been investigated in terms of the resolution obtained. Heterogeneity of the adsorbent have been investigated in terms of the resolution obtained. Heterogeneity has been found to originate from the following sources: heterogeneity of the ligand before immobilization; alteration of the ligand by immobilization; and modification of the ligand after immobilization by molecules to be fractionated. Only when the heterogeneity of the adsorbent was minimized could the resolution of closely related enzyme species be achieved. The elution conditions for different enzymes depended on the amount of enzyme applied, as no complete homogeneity could be obtained. In addition, it was found that the adsorbent was partly degraded by the pancreas extract, reducing its fractionating capacity.

Resistance to 6-thioguanine in spontaneously cycling and in mitogen-stimulated human peripheral lymphocytes

Spontaneously cycling lymphocytes (in cell division in cultures without addition of phytohemagglutinin, PHA) go through the various phases of the first division with the same kinetics as PHA-stimulated cells. In samples from 10 referents, the frequency of spontaneously cycling lymphocytes varied from 8.9 X 10(-5)-9.5 X 10(-3) as indicated with autoradiography on cells in (S + G2) phase determined by flow sorting. In PHA-stimulated samples from the same persons the frequency of 6-thioguanine (TG)-resistant variants was between 4 X 10(-7) and 2.6 X 10(-6), which indicates that most of the spontaneously cycling cells were TG-sensitive. With lymphocytes from one of the referents it was found that: (i) in the presence of 2 X 10(-4) M TG, more than 97% of the spontaneously cycling cells were inhibited before or in early S phase, and (ii) when a flow cytometer was set to sort out TG-resistant cells in late S + G2 phase after 48 h incubation in medium with PHA, the contribution of TG-resistant cells from the spontaneously cycling fraction amounted to less than 2% of the total number of resistant cells.

The frequency of 6-thioguanine-resistant human peripheral blood lymphocytes as determined by flow cytometry and by clonal propagation.

Human peripheral blood lymphocytes were selected for 6-thioguanine resistance in short-term cultures. Resistant cells, defined as cells capable of incorporating tritiated thymidine under the selective conditions, were flow-cytometrically differentiated with respect to their DNA content. This was carried out by sorting at two stages of the cell cycle, before and after mid-S-stage, yielding frequencies of resistant cells in the range of 10(-4) and 10(-5), respectively. Observed frequencies for cells from the whole cell cycle spectrum and for cells cultured according to the long-term protocol, the clonal assay, were in the range of 10(-4) and 10(-5), respectively. Our interpretation of these results is that the over-representation of tritiated thymidine-labelled cells occurring before mid-S-stage after short-term culture reflects less resistant cells or phenocopies which are probably eliminated during long-term culture with the clonal assay, hence leading to a decreased frequency of 6-thioguanine-resistant cells. We conclude, therefore, that short-term culture in combination with flow cytometric sorting after mid-S-stage in the cell cycle can be used as an alternative to the clonal assay for the determination of fully 6-thioguanine-resistant human peripheral lymphocytes.

Attempts to use the HPRT-assay as an automated short-term monitor for an acute exposure to mutagens

Attempts have been made to use the hypoxanthine-guanine-phospho-ribosyl-transferase-assay as a method for automated screening of agent-induced phenotypic variants of human peripheral lymphocytes reflecting 6-thioguanine resistance and assumed to indicate genotoxic action. Different protocols of the hypoxanthine-guanine-phospho-ribosyl-transferase-system were used in this study in order to investigate whether the system can be a candidate for a short-term test for a rapid and reliable identification of biological systems exposed to agents. The current protocols were based on: 1) fluoresceinated monoclonal antibodies against bromodeoxyuridine-DNA for labelling of 6-thioguanine-resistant human lymphocytes and direct flow-cytometric enumeration of bromodeoxyuridine-positive events and: 2) indirect flow-cytometric enrichment of 6-thioguanine-resistant cells labelled with 3H-thymidine followed by autoradiographic enumeration of positive events. Both the direct and the indirect enumeration method yielded similar results down to the range 10−4 with respect to frequency of variants. For the less time-consuming direct enumeration method the resolution was limited due to non-specific binding of the antibody and false positives. It was, nevertheless, sufficient to score variants induced in vitro with the mutagens EMS, MMC and TT in the same range as e.g. that of cancer patients during and after chemotherapy or radiotherapy, or that of psoriasis patients during and after PUVA (8-methoxypsoralen and long range UV light)-therapy. We conclude that the direct enumeration protocol can be used for a rapid screening of so called outliers, but a more sensitive test, such as the more time-consuming enrichment protocol based on autoradiography, must be used in order to score variants in the range 10−5–10−6

A Two-Step Procedure for Quantitative Isolation of Pure Double Strand DNA from Animal Tissues and Cell Cultures

Abstract A two step procedure for the quantitative isolation of protein- and RNA-free double-strand DNA from animal tissue and cell homogenates is described. In the first step proteins not complexed with DNA are hydrolyzed with an immobilized protease (Proteinase K) that is separated by filtration after the de-proteinization. Then the DNA is adsorbed to hydroxylapatite (HA) and desorbed from the adsorbent by stepwise elution with buffers of increasing ionic strength. The DNA content was determined directly from the absorption at 260 nm. The melting curve of the isolated DNA showed that it was double stranded. The protein content in the DNA was determined from the ratio of the adsorbance at 260 to 230 nm. Non-histone proteins complexed to DNA determined the rate of deproteinization that was found to be tissue specific. These proteins were found to have a larger influence on the ratio A260/A230 than histones, indicating that their absorption (at 230 nm) is markedly perturbed when they are bound to DNA.

Intraarterial and intraportal in vivo catheterization of the regenerating rat liver. Effects upon body and liver weights and DNA synthesis.

A method for concomitant partial hepatectomy and catheterization of the arterial and portal systems of the liver in the rat is described. Catheters were inserted into the gastroduodenal artery and the ileocolic vein. Continuous saline perfusion was performed during 36 hours. In catheterized rats recovery of liver and body weight lagged behind that of non-catheterized rats. The more extensive surgery and the presence of catheters also caused decreased incorporation of 3H-thymidine into liver DNA 24 hours postoperatively. The variation in thymidine incorporation between animals was large. It was shown that by pre-labelling liver DNA with 14C-thymidine the rats can serve as their own controls during acute experiments involving 3H-thymidine, thus reducing the inconsistency of individual variation.

Biospecific sample preparation and its application in isoelectric focusing

Summary Proteases have been isolated and analyzed by biospecific adsorption combined with isoelectric focusing. Soybean trypsin inhibitor immobilized in Sepharose, a biospecific adsorbent for chymotrypsins and trypsins, was found to isolate - and concentrate — these enzymes quantitatively from both dilute solutions and pancreatic tissue. The proteases bound to the adsorbent were then layered on focusing gel plates at a pH value permitting desorption by H+-ions. This sample application procedure permitted simultaneous desorption and focusing of the enzymes. Preparative and analytical isoelectric focusing were then performed in the absence of interfering substances.

Irradiation Effects upon Ischemic Regenerating Rat Liver Cells

Starch particles injected into the arterial and portal systems of the liver of the rat caused a temporary blockage of the liver circulation and consequent hypoxia in the liver cells. In the regenerating liver this resulted in a 30-40% decrease of thymidine incorporation into DNA, when analysed 1.5 hours after injection. Irradiation-induced cell damage, evaluated by thymidine incorporation 1.5 hours after irradiation with a single dose of X-rays, was not ameliorated by the ischemic condition. It is suggested that this depends on an inhibited nucleotide metabolism and DNA synthesis leading to an additive metabolic hypoxic effect of the starch particles on radiation damage. An equal level of thymidine incorporation, however, was found in an ischemic and a non-ischemic group of animals 16 hours after irradiation. In this case the liver cells in the ischemic group had overcome the additional inhibition of DNA synthesis caused by temporary hypoxia.

Physical-Chemical Factors Influencing the Resolution in Affinity Chromatography

When affinity chromatography is used for analytical or preparative purposes, such as for the separation of modified enzyme molecules from native enzymes, a biospecific adsorbent with a high resolution is required. The resolution is determined by the following physical-chemical factors: (a) distribution coefficient in the sorption chromatographic procedure, (b) specificity of the binding between adsorbent and enzyme and its modulation by desorbing ligands, and (c) kinetics of the adsorption and desorption. The influence of (a) and (b) on the resolution has been reported previously (1–3). The kinetics of adsorption and desorption are reported here. The number of adsorbent molecules, determined by stationary methods, is generally higher than the corresponding number determined by dynamic methods (loading a chromatographic column).