The incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

The effect of a treatment with 5-fluoro-2'-deoxyuridine (FdUrd) in combination with 2'-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10(-9)-5 X 10(-7) M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70-80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10(-4) M. However, at the highest FdUrd dose tested (10(-7) M), 10(-4) M dUrd could not reverse the FdUrd effect in long-term experiments (20 h, about one cell cycle interval), as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with [6-3H]dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label (at least 3% of the total radioactivity incorporated into DNA) in the form of [6-3H]dUMP. Thus, we conclude that, under our experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2'-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. As far as the formation of SCEs is concerned, analysis of second division chromosomes showed that 2'-deoxyuridine monophosphate (dUMP) residues present in newly-synthesized DNA strands are probably not relevant to SCE formation. However, by analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, we have scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.     

Insulin and IGF receptors are developmentally regulated in the chick embryo eye lens

We have previously reported that insulin-like growth factor (IGF) receptors appear to predominate over insulin receptors in early stages of embryogenesis in the chick (days 2-3 whole embryo membranes). Overall, [125I]IGF I and II binding to specific receptors was maximal when the rate of brain growth is highest. In the present study we used the embryonic chick lens, a well-defined tissue composed of a single type of cell, to analyse whether changes of insulin and IGF I binding are correlated with changes in growth rate and differentiation state of the cells. We show that both insulin receptors and IGF receptors are present in the lens epithelial cells, and that each type is distinctly regulated throughout development. While there is a direct correlation between IGF-binding capability and growth rate of the cells, there is less relation to differentiation status and embryo age. Insulin receptors, by contrast, appear to be mostly related to the differentiated state of cells, decreasing sharply in fibers, irrespective of their developmental age.     

Purification and characterization of glutathione transferase of human thyroid

An anionic (pI 4.6) isoenzyme of glutathione transferase was purified to homogeneity from human thyroid by affinity chromatography followed by isoelectric focusing. The content of enzyme was calculated to constitute about 0.2% of soluble proteins. The enzyme is formed by two identical subunits of 23,000 daltons approximately. The thyroid transferase did not catalyze the reduction of peroxides. Physical, catalytic and immunological analyses demonstrated extensive similarities between the thyroid transferase and the transferase from placenta, erythrocytes and breast. On the other hand, the thyroid transferase appears catalytically different from transferase 7-7, even if both cross-react with the antibodies raised against human placenta transferase.     

DNA-protein flow cytometric analysis in non-Hodgkin lymphoma

A flow cytometric study of DNA and protein contents was performed on cell suspensions obtained from 73 adult patients with non-Hodgkin lymphoma. Bivariate analysis identified a second subpopulation, not revealed by DNA determination, in 25% of the tumors. Protein heterogeneity was more frequently observed in diffuse than in nodular histology according the Rappaport classification and in high-grade than in low-grade malignancy tumors by the Kiel classification and the Working Formulation, but it was not related to ploidy or cell proliferative rate. The presence of an additional subpopulation, detected by protein analysis, defined as monoclonal by DNA analysis, could adversely affect clinical outcome in terms of response to treatment and overall survival.     

Implications of disaggregation procedures on biological representation of human solid tumours

Abstract. This study was designed to define some biological aspects of cell suspensions, obtained by mechanical or enzymatic disaggregations, and to verify whether single cell suspensions are representative of original solid tumours. The study was performed on a series of 25 human solid tumours including breast carcinoma, ovarian carcinoma and malignant melanoma. A higher cell viability and a loss of aneuploid subpopulations, or a lower fraction of aneuploid cells, were observed in enzymatically-released samples than in samples obtained by the mechanical procedure. Moreover, the proliferative activity, which was generally similar for the cell suspensions obtained by the two disaggregation procedures, was always markedly lower in the cell suspensions than in solid samples from the same tumour. In conclusion, the results from this study indicate that many changes, such as selective release of cell populations from the tumour matrix, damage and destruction of aneuploid and proliferating cells can be induced to various extents by different disaggregation procedures.     

The gene for trypsin inhibitor CMe is regulated in trans by the lys 3a locus in the endosperm of barley (Hordeum vulgare L.)

Summary A cDNA encoding trypsin inhibitor CMe from barley endosperm has been cloned and characterized. The longest open reading frame of the cloned cDNA codes for a typical signal peptide of 24 residues followed by a sequence which is identical to the known amino acid sequence of the inhibitor, except for an Ile/Leu substitution at position 59. Southern blot analysis of wheat-barley addition lines has shown that chromosome 3H of barley carries the gene for CMe. This protein is present at less than 2%–3% of the wild-type amount in the mature endosperm of the mutant Risø 1508 with respect to Bomi barley, from which it has been derived, and the corresponding steady state levels of the CMe mRNA are about I%. One or two copies of the CMe gene (synonym Itc1) per haploid genome have been estimated both in the wild type and in the mutant, and DNA restriction patterns are identical in both stocks, so neither a change in copy number nor a major rearrangement of the structural gene account for the markedly decreased expression. The mutation at the lys 3a locus in Risø 1508 has been previously mapped in chromosome 7 (synonym 5H). A single dose of the wild-type allele at this locus (Lys 3a) restores the expression of gene CMe (allele CMe-1) in chromosome 3H to normal levels.     

Influence of charged groups on the conformational stability of succinoglycan in dilute aqueous solution

Viscosity, optical activity, and differential scanning calorimetry data clearly point out that partial and/or total removal of charged substituent groups, i.e. succinate and pyruvyl residues, from succinoglycan lead to water soluble derivatives exhibiting a higher stability order → disorder conformational changes with respect to the native polysaccharide. The new succinoglycan derivatives also exhibit very little, if any, hysteresis upon ‘renaturation’ (cooling) as opposed to the case of the parent polymer. The absence of ionized groups is thus beneficial, thermodynamically and kinetically, to the attainment in dilute aqueous solution of an ordered conformation by the uncharged succinoglycan backbone, as allowed by the regular enchainment of its constituent sugar residues.     

Urinary elastase 1 in chronic pancreatic disease

Serum and urine elastase 1, its renal output and clearance and urinary gamma-glutamyltransferase and ribonuclease excretions were measured in 16 patients with pancreatic cancer, 23 with chronic pancreatitis and in 22 healthy controls in order to evaluate elastase 1 plasma-urine transfer in chronic pancreatic disease and to investigate any factors that might influence the clearance of this enzyme. In an additional group of 17 patients with different pancreatic diseases the serum molecular size distribution of elastase 1 after chromatography was ascertained. An increased urinary elastase 1 output was found in 4/16 patients with pancreatic cancer and in 6/23 with chronic pancreatitis. No correlation was found between circulating elastase 1 and its urinary output; a negative correlation was detected between the serum levels of this enzyme and its clearance. The excretion of ribonuclease and gamma-glutamyltransferase was correlated with elastase 1 output and clearance. While the majority of elastase 1 in serum was accounted for by high molecular forms, probably the expression of complexes with serum inhibitors, free circulating enzyme was present in all patients with high serum elastase 1. Our findings suggest that elastase 1 urinary excretion increases in some patients with chronic pancreatic disease regardless of the neoplastic or inflammatory nature of the illness. Although the availability of different amounts of ultrafiltrable enzyme may play a role in influencing elastase 1 plasma-urine transfer, renal tubular damage appears to be the most important factor influencing the increase in the urinary output of elastase 1.