Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.     

Influence of puromycin aminonucleoside on photoreactivation and pyrimidine dimer repair

We have measured the effect of puromycin aminonucleoside (PAN) on photoreactivation of mutations and loss of pyrimidine dimers in structural gene Strep A in ultraviolet (UV)-irradiatedEscherichia coli B/r T-cells. Photoreactivating illumination between 3100 and 4000 Å wavelengths for 45 min on brain-heart infusion (BHI) of B/r T-eliminated 55% of the pyrimidine dimers and 75% of the mutations in gene Strep A. When BHI-PAN medium was employed, there was a 45% loss of dimers and 73% reduction in mutations. Incubation for 3.5 on BHI or BHI-PAN prior to such treatment results in no loss of mutations. These results suggest that the photoreactivity of mutations in gene Strep A is related to the ability of the cells to repair pyrimidine dimers. However, no direct correlation has been made between the observed repair of mutations and repair of pyrimidine dimers. Further experiments on the kinetics of the repair process, designed to elucidate the mechanism of PAN action, show that slope increases with increasing concentration of the drug that follows an enzyme-like pattern.     

Physiological and genetic effects of puromycin aminonucleoside on ultraviolet-irradiated Escherichia coli strains.

Puromycin aminonucleoside (PAN) increased significantly the mutation rate of Escherichia coli B/r strains when used in conjunction with certain ultraviolet dosages. PAN (2.5 mM) when added to the post-irradiation medium of hcr+ cells slowed down RNA synthesis to 65%, protein to 76% and DNA to 48% of the control rate. Purine ribosides such as adenosine decreased the inhibitory action of PAN on DNA, RNA and protein synthesis. Quantitatively quite different results were obtained with the hcr- strains. PAN did not increase killing of UV, but decreased the frequency of UV-induced mutations. Antimutagenic purine ribosides decreased the synergistic mutagenic activity of PAN. Increases in DNA synthesis in the presence of antimutagens correspond to reductions in the rate of mutation to streptomycin resistance. The excision of UV-induced pyrimidine dimers was investigated in the presence and absence of PAN. The pattern of repair-inhibition reversion of pre-mutagenic lesions by adenosine suggests that PAN behaves as a feedback inhibitor of purine biosynthesis in UV-irradiated cells. It is probable that this inhibition results in an impairment of repair which produces the increase in mutant numbers.     

In vitro effects of puromycin aminonucleoside on the ultrastructure of rat glomerular podocytes

Summary Puromycin aminonucleoside (PAN)-induced nephrosis in rats provides a model for studying the pathogenesis of severe proteinuric conditions, such as minimal change disease. The present study used scanning (SEM) and transmission (TEM) electron microscopy to investigate the in vitro effects of PAN on rat glomerular podocytes. Slices of rat kidney were incubated for up to 3 days in Medium 199 with Hanks' salts (control) or in medium with PAN. Semiquantitative SEM analysis of glomeruli on the upper surface of kidney slices indicated that incubation with PAN (100 g/ml and 500 g/ml) decreased the number of microvilli on podocyte cell bodies (days 1, 2 and 3), increased the number of glomeruli showing flattening of podocyte cell bodies and major processes (days 2 and 3), and increased the number of glomeruli showing surface membrane blebbing on podocyte foot processes (day 3) (p<0.001 in all cases). TEM morphometry revealed that incubation with 500 g/ml PAN retarded significantly (p<0.001 at days 2 and 3) the loss of podocyte foot processes observed in control cultures. Whilst the SEM changes to podocyte ultrastructure largely mimic those seen in PAN nephrosis in vivo, the retardation of foot process loss runs counter to the major TEM change observed in vivo.     

Gluconeogenesis in the kidney cortex. Effects of d-malate and amino-oxyacetate

1. Rat kidney-cortex slices incubated with d-malate alone formed very little glucose. d-Malate, however, augmented gluconeogenesis from l-lactate and inhibited gluconeogenesis from pyruvate and l-malate. 2. d-Malate had little effect on the rate of the tricarboxylic acid cycle with or without other substrates added. 3. d-Malate inhibited the activity of the l-malate dehydrogenase in a high-speed-supernatant fraction from kidney cortex. 4. It was concluded that d-malate inhibited either the operation of the cytoplasmic l-malate dehydrogenase or malate outflow from the mitochondria in the intact kidney-cortex cell. This supports the hypothesis of Lardy, Paetkau & Walter (1965) and Krebs, Gascoyne & Notton (1967) on the role of malate as carrier for carbon and reducing equivalents in gluconeogenesis. 5. Gluconeogenesis from l-lactate in kidney-cortex slices was strongly inhibited by a low concentration (0.1mm) of amino-oxyacetate, whereas glucose formation from pyruvate, malate, aspartate and several other compounds was only slightly affected. 6. High concentrations of l-aspartate largely reversed the inhibition of gluconeogenesis from l-lactate caused by amino-oxyacetate. 7. Amino-oxyacetate inhibited strongly the glutamate-oxaloacetate transaminase in the 30000g supernatant fraction of a kidney-cortex homogenate. The presence of l-aspartate decreased the inhibition of the transaminase by amino-oxyacetate. 8. Detritiation of l-[2-(3)H]aspartate was inhibited by 90% during an incubation of kidney-cortex slices with l-lactate and amino-oxyacetate. 9. Low concentrations (10mum) of artificial electron acceptors such as Methylene Blue and phenazine methosulphate abolished most of the inhibition of gluconeogenesis from l-lactate by amino-oxyacetate. This is interpreted as an activation of net malate outflow from the mitochondria by-passing the inhibited transfer of oxaloacetate. 10. These findings support the concept that transamination to aspartate is involved in the transfer of oxaloacetate from mitochondria to cytosol required in gluconeogenesis from l-lactate.     

Dextran causes aggregation of mitochondria and influences their oxidoreductase activities and light scattering

It has been reported that dextrans diminish the intermembrane space of mitochondria, increase the number of contact sites between the inner and the outer mitochondrial membranes, decrease the outer membrane permeability to adenosine 5(')-diphosphate, and change the kinetic properties of mitochondrial kinases. In the present work the influence of dextran M40 (5% w/v) on the oxidoreductase activities of the inner and outer membranes of mitochondria, the interaction of cytochrome c with mitochondrial membranes, and the light scattering by rat liver mitochondria were studied. No influence of dextran on the release of cytochrome c from mitochondria or its interaction with mitochondrial membranes was observed. Decreases in the NADH-oxidase (to 80+/-2% of the control), NADH-cytochrome c reductase (to 26+/-2%), succinate-cytochrome c reductase (to 70+/-5%), and NADH-ferricyanide reductase (to 75+/-3%) activities induced by dextran, which may be due to the mitochondrial aggregation, were observed. The formation of aggregates was registered by light scattering, confirmed by light microscopy, and explained within the framework of the Gouy-Chapman theory of the electrical double layer. The observed mitochondrial aggregation seems to be useful also for understanding the mechanisms of mitochondrial condensation and perinuclear clustering during apoptosis.     

Change of serum L-tryptophan levels following the development and recovery of acute puromycin aminonucleoside nephrosis in rats

Summary It is known that total L-tryptophan (Trp) levels decrease with a decrease in albumin-bound Trp levels and an increase in free Trp levels in the plasma or serum of nephrotic children. We, therefore, examined the change of serum Trp levels following the development and recovery of acute nephrosis in 6-week-old male Wistar rats injected once with puromycin aminonucleoside (100mg/kg body weight) and checked the levels of 16 amino acids including Trp in the serum and the levels of Trp in the liver, kidney, and urine under nephrotic conditions. In this study, the development and recovery of nephrosis were checked by the changes of levels of urinary protein and serum protein and albumin. Total serum Trp and albumin-bound serum Trp levels decreased with the development of nephrosis and these decreased levels returned to the normal level with its recovery. In contrast, free serum Trp levels increased with the development of nephrosis and this increased level returned to the normal level with its recovery. In the serum of nephrotic rats, the decrease of albumin-bound Trp levels and the increase of free Trp levels were well consistent with a decrease in albumin levels and an increase in the level of non-esterified fatty acids which are known to weaken the binding of Trp to albumin and among 16 amino acids studied, only Trp showed a significant change in its levels. Trp levels increased in the liver and kidney but not in the urine under nephrotic conditions. These results indicate that the change of serum Trp levels should be closely related to the condition of nephrosis and that although serum Trp is lost under nephrotic conditions, the lost serum Trp is accumulated in the liver and kidney.     

Plasma and urinary lipids and lipoproteins during the development of nephrotic syndrome induced in the rat by puromycin aminonucleoside

This study was undertaken to ascertain whether the alterations of plasma lipoproteins found in nephrotic syndrome induced by puromycin aminonucleoside were due to nephrotic syndrome per se, or, at least in part, to the aminonucleoside. The purpose of the present study was to investigate the changes in plasma and urinary lipoproteins during the administration of puromycin aminonucleoside (20 mg/kg for 7 days) and the subsequent development of nephrotic syndrome. Since massive albuminuria occurred after 6 days of treatment, the time-course study was divided into two stages: pre-nephrotic stage (day 1–5) and nephrotic stage (day 6–11). In pre-nephrotic stage the plasma level of fatty acids, triacylglycerol and VLDL decreased while that of phospholipid, cholesteryl esters and HDL remained constant. Plasma apolipoprotein A-I tended to increase (40% increase at day 5). At the beginning of nephrotic stage (day 6) the concentration of plasma albumin dropped to a very low level, while that of apolipoprotein A-I increased abruptly (4-fold increase) and continued to rise, although less steeply, in the following days. The plasma concentration of HDL followed the same pattern. Plasma VLDL and LDL increased at a later stage (day 9). Plasma apolipoprotein A-I was found not only in HDL (1.063–1.210 g/ml) but also in the LDL density class (1.025–1.050 g/ml). In the pre-nephrotic stage lipoproteinuria was negligible, while in the early nephrotic stage the urinary loss of plasma lipoproteins consisted mainly of HDL. These observations indicate that puromycin aminonucleoside alters plasma lipoproteins by lowering VLDL and increasing HDL. It is likely that the early and striking increase of plasma HDL found in nephrotic rats is related to a direct effect of the drug on HDL metabolism.