Evidence for two waves of induction of DNA enzymes in stimulated human lymphocytes

The stimulation of human lymphocytes with phytohaemoagglutinin induces the appearance or increase of several enzymes of DNA metabolism [Pedrini etal., Biochem. Biophys. Res. Comm., 47:1221(1972)]. With long times of stimulation, two phenomena are observed; an increase in the levels of DNA polymerase, of a DNase acting on single-stranded DNA, and of an endonuclease, occurring between the third and fourth day, in parallel with a wave of DNA synthesis;a second wave of increase of the same enzymes and of DNA ligase,occurring between the fifth and eight day when the DNA replication rate, as measured by thymidine-pulses, has decreased to values close to the background.     

Mechanism for binding of fatty acids to hepatocyte plasma membranes

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.     

The interactions of bilirubin with model and biological membranes

The partitioning of bilirubin between albumin and model and biological membranes and the differential partitioning of bilirubin between membranes with different lipid and protein compositions were measured. Partition coefficients were independent of the concentration of bilirubin in membranes up to at least 7 mol of bilirubin/mol of phospholipid. The avidity of albumin for bilirubin was greater than that of membranes, but the avidity of the latter for bilirubin depended on the composition of the membrane. Bilirubin partitioned preferentially into model membranes comprised of microsomal lipids greater than dioleoylphosphatidylcholine = plasma membrane lipids much greater than egg phosphatidylcholine = dimyristoylphosphatidylcholine. Partitioning into membranes was increased if these contained proteins, but the effect of proteins could not be attributed to specific binding to sites on proteins, as reflected by the temperature independence of partition coefficients. Differential partitioning of bilirubin into different membranes of pure lipids also was independent of temperature. Differences in the bulk phase fluidity of membranes does not appear to account for the preferential partitioning of bilirubin into some membranes. It appears that bilirubin partitions into elements of free volume of differing sizes in membranes with variable lipid compositions and that the size of these elements can be increased by adding proteins to membranes.     

Interactions of retinol with binding proteins: studies with retinol-binding protein and with transthyretin.

The interactions within the molecular complex in which retinol circulates in blood were studied. To monitor binding between retinol-binding protein (RBP) and transthyretin (TTR), TTR was labeled with a long-lived fluorescence probe (pyrene). Changes in the rotational volume of TTR following its association with RBP were monitored by fluorescence anisotropy of the probe. Titration of TTR with holo-RBP revealed the presence of 1.5 binding sites characterized by a dissociation constant Kd = 0.07 microM. At 0.15 M NaCl, binding of RBP to TTR showed an absolute requirement for the native ligand, retinol. At higher ionic strength (0.5 M NaCl), RBP complexed with retinal also bound to TTR with high affinity (Kd = 0.134 microM). RBP containing retinoic acid did not bind to TTR even at the high salt concentration. The data suggest that the TTR binding site on RBP is in close proximity to the retinoid binding site and that the head group of retinoic acid, when bound to RBP, presents steric hindrance for the interactions with TTR. The implications of the data for selectivity in retinoid transport in the circulation are discussed. The kinetics of the steps leading to complete dissociation of the retinol-RBP-TTR complex was also studied. The first step of this process was dissociation of retinol, which had a rate constant of 0.06/min. Following loss of retinol, the two proteins dissociate. The rate of dissociation is slow (k = 0.055/h), however, indicating that the complex apo-RBP-TTR will be an important factor in regulating serum levels of retinol.     

Repression of ant synthesis early in the lytic cycle of phage P22

Summary Using SDS-polyacrylamide gel electrophoresis to study the early expression of P22 genes we show that early expression of the ant-gene (imm I region) is turned off after 6–8 min, independent of the late acting mnt-repressor. A semi-clear mutant called cir5 is defective for this early ant turn-off. The mutation cir5 maps in the imm I region of P22 between genes mnt and ant. P22 cir5 mutants are defective for a repressor which acts in trans to regulate early ant synthesis. There appears to be no absolute requirement of the cir5 allele for the establishment of lysogeny. The overproduction of ant in the P22 cir5 mutant leads to a marked increase in abortive infections, killing the infected cells. The cir5-phenotype can be suppressed by an ant ^- mutation.     

A microscopically motivated model for the remodeling of cardiomyocytes

Biomechanics and Modeling in Mechanobiology - We present a thermodynamically based model that captures the remodeling effects in cardiac muscle cells. This work begins with the formulation of the...