Cleavage of dT8 and dT8 phosphorothioyl analogues by Escherichia coli DNA topoisomerase I: product and rate analysis

Escherichia coli DNA topoisomerase I catalyzes the cleavage of short, single-stranded oligodeoxynucleotides with dT8 as the shortest cleavable oligo(thymidylic acid). The 5'-32P-labeled products formed from the cleavage of [5'-32P]dT8 are dT5, dT4, and dT3 with over 70% of the substrate cleaved to dT4. Mg(II) ions affect this product distribution by increasing the percentage of dT4 formed. The substitution of a sulfur atom for a nonbridging oxygen atom in a phosphodiester linkage yields oligodeoxynucleotide phosphorothioyl (PS) analogues. The epimers of the analogues were separated, and the position and stereochemistry of the phosphorothiodiester bond were determined. Topoisomerase I is stereospecific in its reactivity toward these analogues. With the oligodeoxynucleotide PS analogue substrates, the rate of cleavage, the stereospecificity, and the product distribution depend upon the position and the stereochemistry of the phosphorothiodiester linkage.     

Coordinate regulation of ribosomal protein mRNA level in regenerating rat liver. Study with the corresponding mouse cloned cDNAs.

Cloned mouse ribosomal protein (rp) cDNAs exhibit extensive homology with the corresponding rat sequences. The size of the rp-mRNAs and complexity of the rp-genes are very similar in the two species. Using the mouse rp-recombinant DNAs we find that the relative abundance of rat L7, L13, L18, L30, L32/33 and S16 mRNAs increases after partial hepatectomy. Their maximal level is about twice that of normal rat liver, and is achieved 12-18 h after the operation, while the relative abundance of albumin mRNA decreases to half the normal values 12 h after partial hepatectomy. This concomitant increase in the relative content of these rp-mRNAs indicates coordinate regulation of their level in the rat. The dissimilar behavior of L10 and L19 rp-mRNA suggests additional control mechanisms of rp-mRNA levels in the regenerating rat liver.     

Experimental investigations in prolonged myocardial ischaemia. Note I. Biology of the myocardial fiber after transient myocardial fiber after transient myocardial ischaemia

The first ten days' evolution of post-ischaemic lesions of the premonitory or angina pectoris syndrome type was experimentally studied by the challenge of a short-term (10 and 15 min) ischaemia, of an adaptation to ischaemia and an adaptation followed by prolonged ischaemia (20 and 35 min). Worthy of note was the persistence of reversible lesions after short-term ischaemia and adaptation, and the progressive evolution towards cytolysis and cicatrization of some pancicellular foci after adaptation followed by prolonged ischaemia. The role of mitochondrial lesions, of lysosomal hydrolases, the inefficiency of renewed circulation, as well as problems of diagnosis are discussed.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

The effect of sulfated polysaccharides on the free intracellular calcium ion concentration of lymphocytes

Recent studies have demonstrated that murine lymphocytes express specific cell-surface receptors for a range of sulfated polysaccharides. In order to determine whether polysaccharide binding induces transmembrane signaling, the effects of sulfated polysaccharides on the free intracellular calcium ion concentration [( Ca2+]i) of mouse thymocytes and spleen cells were determined. Cells were loaded with Indo-I, a fluorescent indicator of calcium ion concentration. The validity and limitations in the use of this indicator in the determination of [Ca2+]i are documented. Dextran sulfate (Mn = 500,000), iota-carrageenan, lambda-carrageenan and kappa-carrageenan all cause relatively large changes in the [Ca2+]i of thymocytes (change in [Ca2+]i greater than 50 nM). Of these, dextran sulfate (Mn = 500,000) always had the greatest effect on [Ca2+]i. Smaller responses were obtained with heparin and dextran sulfate (Mn = 5000), while no response was obtained with chondroitin 4-sulfate, chondroitin 6-sulfate, pentosan sulfate or fucoidin. This response pattern (with the exception of fucoidin and pentosan sulfate) corresponds with the expression of thymocyte receptors for these polysaccharides. The increase in [Ca2+]i caused by the sulfated polysaccharides requires extracellular Ca2+ ions however, it is unlikely that voltage-dependent ion channels are involved in these responses. In contrast to thymocytes, although spleen cells express receptors for sulfated polysaccharides, they were unresponsive to all of the sulfated polysaccharides tested, suggesting a basic difference between thymocytes and peripheral T and B lymphocytes in their response to the binding of sulfated polysaccharides.     

Developmental Patterns of Catechol-O-Methyltransferase in Genetically Different Rat Strains: Enzymatic and Immunochemical Studies

Abstract: Catechol- O -methyltransferase (COMT) activity in the liver and kidneys of adult Fischer-344 (F-344) rats is only half of that in the same organs of Wistar-Furth (W-F) rats. The trait of low COMT activity in these animals is inherited in an autosomal recessive fashion. A comprehensive study of patterns of change in COMT activity during growth and development was performed to determine whether "temporal gene" effects might play a role in the inherited differences in enzyme activity present in adult animals. The COMT activity expressed per mg protein in liver and kidneys of newborn F-344 rats is only 50–60% of that in the same organs of W-F animals. The liver and the kidneys of newborn rats of both strains have COMT activity an order of magnitude higher than those in brain, heart, or blood. In addition, in both strains there are much larger increases in liver and kidney COMT activities during growth and development (5–10 fold) than in blood, brain, or heart (one- to twofold). Immunotitration with antibodies against rat COMT demonstrates that differences in immunoreactive COMT parallel differences in COMT activity, both between strains and within strains during growth and development. However, when the temporal patterns of change in enzyme activities in the liver and the kidneys of the two strains of rat are compared at multiple times during growth and development, no differences in the patterns are present. These results make it unlikely that temporal gene effects can explain the inherited differences in COMT activity in liver and kidneys of F-344 and W-F rats.