Acridine-psoralen amines and their interaction with deoxyribonucleic acid

A series of novel compounds in which a 9-acridinyl nucleus is linked to a psoralen nucleus in the 5- or 8-position via polyamines was prepared and examined. Their reversible binding to DNA and their irreversible binding to DNA and DNA cross-linking upon irradiation with UV-A light were examined. It was found that they were all less efficiently photoreactive than 8-methoxypsoralen (8-MOP), both in cross-linking and photobinding to DNA, whereas the ratio between their photobinding and cross-linking was 40-400 times that of 8-MOP. Compounds in which the linker was attached to the 5-position in psoralen showed smaller cross-linking and photobinding efficiencies and larger ratios between photobinding and cross-linking than those of psoralens attached in the 8-position. This strongly indicates that the 9-substituents of the acridines are oriented toward the minor groove. Flow linear dichroism studies showed that the compounds were DNA intercalating with the acridine moiety, whereas the psoralen moiety in no case was clearly intercalating. This conclusion was further supported by viscometry which also strongly indicated monointercalation.     

Kinetics of dissociation of reduced nicotinamide adenine dinucleotide phosphate from its complexes with malic enzyme in relation to substrate inhibition and half-of-the-sites reactivity

Malic enzyme of pigeon liver binds NADPH at four equivalent enzyme sites and binds Mn2+ and malate each at two sets of "tight" and "weak" sites with negative cooperativity [Pry, T. A., & Hsu, R. Y. (1980) Biochemistry 19, 951-962]. Stopped-flow studies on the displacement of NADPH from the malate-enzyme complexes E4-NADPH4, E4-Mn2(2+)-NADPH4, E4-Mn2(2+)-NADPH4-dimalate, and E4-Mn2(2+)-NADPH4-tetramalate by large excess NADP+ or its analogue phosphoadenosine(2')diphospho(5')ribose show that NADPH dissociates from the binary complex rapidly with a first-order rate constant of 427 s-1. Dissociation from the ternary E4-Mn2(2+)-NADPH4 complex containing two tightly bound Mn2+ ions can be described by a single first-order process with a rate constant of 135 s-1, or more satisfactorily by two simultaneous first-order processes attributable to the reactions of Mn2+-deficient (k congruent to 427 s-1) and Mn2+-liganded (k = 96 s-1) subunits. The latter equals twice the maximum steady-state turnover rate of 53.2 + 3.0 s-1 assigned to dissociation of the reduced nucleotide from transient E-Mn2+-NADPH, and this 2:1 ratio strongly supports our proposed "half-of-the-sites" model [Hsu, R. Y., & Pry, T. A. (1980) Biochemistry 19, 962-968]. Dissociation from the E4-Mn2(2+)-NADPH4-dimalate complex (k = 100 s-1) follows only the slower process, suggesting that occupancy of malate at two sites tightens enzyme-bound NADPH on the adjacent sites. Binding of malate at two additional weak sites yields E4-Mn2(2+)-NADPH4-tetramalate and a NADPH dissociation rate constant of 2.69 s-1. The 97% decrease in NADPH dissociation parallels the observed 93% maximal inhibition by malate and is the cause of substrate inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies to neurospora adenylate cyclase

A monoclonal antibody against $\text{Neurospora}$ soluble adenylate cyclase was obtained. The antibody inhibits cyclase activities from several lower eucaryotic organisms but not activities associated to testicular cytosol or turkey erythrocyte membranes.     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Differentiation and vascularization of the metanephric kidney grafted on the chorioallantoic membrane

The origin and development of mouse kidney vasculature were examined in chorioallantoic grafts of early kidney rudiments and of experimentally induced explants of separated metanephric mesenchymes. Whole kidney rudiments developed into advanced stages, expressed the segment-specific antigenic markers of tubules and the polyanionic coat of the glomeruli. In contrast to development in vitro, these grafts regularly showed glomeruli with an endothelial component and a basement membrane expressing type IV collagen and laminin. The glomerular endothelial cells in these grafts were shown to carry the nuclear structure of the host. This confirms the outside origin of these cells and the true hybrid nature of the glomeruli. When in vitro induced mesenchymes were grafted on chorioallantoic membranes, abundant vascular invasion was regularly found but properly vascularized glomeruli were exceptional. Uninduced, similarly grafted mesenchymal explants remained avascular as did the undifferentiated portions of partially induced mesenchymal blastemas. It is concluded that the stimulation of the host endothelial cells to invade into the differentiating mesenchyme requires the morphogenetic tissue interaction between the ureter bud and the mesenchyme. The induced metanephric cells presumably start to produce chemoattractants for endothelial cells at an early stage of differentiation. Kidney development thus seems to require an orderly, synchronized development of the three cell lineages: the branching ureter, the induced, tubule-forming mesenchyme, and the invading endothelial cells of outside origin.     

A paired-tracer dilution method for characterizing membrane transport in the perfused rat hindlimb. Effects of insulin, feeding and fasting on the kinetics of sugar transport.

We have applied the paired-tracer dilution method to the study of transport processes in a mixed mammalian muscle preparation, the perfused rat hindlimb. The method is suitable for the characterization of the kinetic parameters of sugar and amino acid transport and its regulation by hormones, contractile activity, hypoxia, etc. Insulin stimulates sugar transport by increasing the Vmax. of the process 2-3 fold, but its affinity is unchanged. Starvation increases the affinity of sugar transport in perfused skeletal muscle.     

The inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.

The activity of glycogen synthase phosphatase in rat liver stems from the co-operation of two proteins, a cytosolic S-component and a glycogen-bound G-component. It is shown that both components possess synthase phosphatase activity. The G-component was partially purified from the enzyme-glycogen complex. Dissociative treatments, which increase the activity of phosphorylase phosphatase manyfold, substantially decrease the synthase phosphatase activity of the purified G-component. The specific inhibition of glycogen synthase phosphatase by phosphorylase a, originally observed in crude liver extracts, was investigated with purified liver synthase b and purified phosphorylase a. Synthase phosphatase is strongly inhibited, whether present in a dilute liver extract, in an isolated enzyme-glycogen complex, or as G-component purified therefrom. In contrast, the cytosolic S-component is insensitive to phosphorylase a. The activation of glycogen synthase in crude extracts of skeletal muscle is not affected by phosphorylase a from muscle or liver. Consequently we have studied the dephosphorylation of purified muscle glycogen synthase, previously phosphorylated with any of three protein kinases. Phosphorylase a strongly inhibits the dephosphorylation by the hepatic G-component, but not by the hepatic S-component or by a muscle extract. These observations show that the inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.     

Bidirectional transport of glutamine across the cell membrane in rat liver.

Hepatocytes isolated from fed rats were used to investigate glutamine transport. Glutamine transport appears as a composite process involving at least two saturable components. The Na+-dependent component probably represents the entry through the N system. The Na+-independent component was also inhibited by histidine and exhibited trans-stimulation, suggestive of a facilitated diffusion process. Kinetic parameters for both systems suggest that facilitated diffusion only plays a minor role in glutamine influx. In contrast, the Km for glutamine efflux was consistent with a physiological role of the facilitated-diffusion component in glutamine release. In Na+ medium, relatively constant distribution ratios (about 8) between intra- and extra-cellular concentrations were observed, with external glutamine ranging from 0.5 to 5 mM. The present observations suggest that glutamine influx might largely be mediated by the N system, whereas facilitated diffusion allows hepatocytes to release glutamine when intracellular concentrations are elevated. The physiological consequences of this bidirectional transfer of glutamine across the liver cell membrane is discussed.     

Regulation of lipogenesis in vivo by glucose availability and insulin secretion in maternal and foetal tissues during late gestation in the rat. Effect of glucose intubation, streptozotocin-induced diabetes and starvation.

Administration of an oral load of glucose did not change the rate of lipogenesis in maternal liver during late gestation. However, streptozotocin-induced diabetes or starvation decreased maternal liver lipogenesis at 20-22 days of gestation. Glucose intubation, on the other hand, increased foetal lipogenesis at 21-22 days. In addition, maternal starvation decreased foetal lipogenesis and plasma insulin concentration. However, chronic hyperglycaemia induced by streptozotocin administration to the mother did not change foetal liver lipogenesis.