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PubMed2002

Cytochrome P450 2B1 mediates oxidant injury in puromycin-induced nephrotic syndrome.

BACKGROUND: Reactive oxygen metabolites (ROM) are important mediators of puromycin aminonucleoside (PAN) induced minimal change nephrotic syndrome (NS) in rats. We have recently shown that cytochrome P450 (CYP) is a significant source of catalytic iron in this model of glomerular injury. The current study was designed to identify the CYP isozyme(s) in the rat glomeruli and explore the role of the specific isozyme(s) in PAN-induced minimal change NS. METHODS: NS was induced in rats by a single intravenous injection of PAN. Animals were sacrificed at different time points for variety of biochemical assays including Western blot, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Ultrastructural histochemistry was utilized to study hydrogen peroxide (H2O2) generation in the kidney. RESULTS: Several CYP isozymes were tested and CYP2B1 was localized exclusively in the rat glomeruli but not in the tubules. Treatment with PAN resulted in the generation of H2O2 in the glomerular basement membrane with significant loss of CYP2B1 content accompanied by a marked increase in the catalytic iron. CYP2B1 inhibitors cimetidine and piperine significantly reduced H2O2 generation, and prevented the loss of CYP2B1 content and the increase in the catalytic iron. CYP2B1 inhibitors also provided significant protection against PAN induced proteinuria. The induction of heme oxygenase and ferritin also was observed in the glomeruli in PAN-treated rats. Both cimetidine and piperine reduced the up-regulation of these proteins. CONCLUSION: Our data indicate that CYP2B1 plays an important role in PAN induced NS by serving as a site for ROM generation and a significant source of catalytic iron.

Animals↗
PubMed1969

Stereochemistry of nucleic acids and their constituents. IX. The conformation of the antibiotic puromycin dihydrochloride pentahydrate.

The elucidation of puromycin structure represents the first case where both a nucleic acid and a protein fragment are found together in a crystal. The molecule displays an elongated conformation with the nucleoside and the p-methoxy-L-phenylalanine in the preferred conformation. The purine and the tyrosine rings form alternating stacks with interplanar spacings of 3.4 A. The inhibitory effect of this antiobiotic may be due, in part, to its unique conformation and the ability of the amide proton on N(3') to hydrogen bond to the active site of the ribosomal enzyme, peptide synthetase.

Chemical Phenomena↗
PubMed1972

Enzymic removal and re-expression of a histocompatibility antigen, HL-A 2, at the surface of human peripheral lymphocytes ( 31 Cr-labeled cells-papain-puromycin-actinomycin D).

The removal by papain of a histocompatibility antigen, HL-A 2, from the surface of intact peripheral blood lymphocytes has been examined. Up to 80% of the antigenic determinant can be removed within 90 minutes. If the cells are reincubated in nutritive medium, re-expression of HL-A 2 is complete within 6 hours. Re-expression is reversibly inhibited by puromycin (50 mug/ml), but actinomycin D (6 mug/ml) only partially inhibited re-expression after 2 hours of prior incubation with the drug.

Antigens↗
PubMed1972

Peptidyl-puromycin synthesis on polyribosomes from Escherichia coli.

Peptide bond synthesis was studied with native polyribosomes of E. coli. With the use of this system for transpeptidation, it was possible to show that a single K(+) activates the ribosome monomers of polyribosomes; that protonation of a single group (probably imidazole or an N-terminal amino group) with a pK(a) equal to about 7.2 inactivates the transpeptidase complex; that Mn(++) can substitute for Mg(++), but that Ca(++), spermidine, and putrescine do so only very poorly; and that the K(m) for puromycin in this system is about 2.4 x 10(-6) M.

Ammonia↗
PubMed1973

Mechanism of puromycin action: fate of ribosomes after release of nascent protein chains from polysomes.

The exchange of ribosomal subunits during the release of growing polypeptide chains by puromycin has been investigated in a bacterial cell-free system engaged in protein synthesis. The addition of spermidine, used as a stabilizing agent of 70S monomers, caused a strong inhibition of the subunit exchange. This result led us to conclude that upon premature release of unfinished protein chains by the antibiotic, the ribosomes fall off mRNA as 70S particles. This behavior is different from that occurring during physiological termination of translation, where the ribosomes detach in a dissociated form. Some implications of the postulated mechanism are also discussed.

Amino Acids↗
PubMed1975

Neurohypophyseal hormones, analogs, and fragments: their effect on puromycin-induced amnesia.

Neurohypophyseal hormones and several of their analogs, as well as N-terminal and C-terminal fragments, have been studied for their ability to attenuate puromycin-induced amnesia in mice. [8-Lysine]vasopressin, [8-arginine]vasopressin, and the analogs des-9-glycinamide-[8-lysine]vasopressin, [1-beta-mercaptopropionic acid, 8-lysine]vasopressin, [1,6-aminosuberic acid, 8-lysine]vasopressin, [4-leucine, 8-lysine]vasopressin, glycyl-glycyl-glycyl-[8-lysine]vasopressin, [1-beta-mercaptopropionic acid, 8-D-arginine]vasopressin, and [1,6-aminosuberic acid, 8-arginine]vasopressin are active. [8-Arginine]oxytocin as well as oxytocin and all of its other analogs tested are inactive with the striking exception of glycyl-glycyl-glycyl-oxytocin. The structural aspects of the neurohypophyseal hormones which appear to be important for significant activity in memory consolidation include the combination of a cyclic moiety containing the Tyr and Phe residues along with a basic residue in position 8. Another series of active compounds comprises C-terminal neurohypophyseal peptides and analogs thereof, including the naturally occurring Pro-Leu-Gly-NH2 and, most surprisingly, Leu-Gly-NH2, as well as its derivatives D-Leu-Gly-NH2 and the diketopiperazine, cyclo(-Leu-Gly-).

Amnesia↗
PubMed2000

The role of protein kinase C in the increased generation in isolated rat hepatocytes of the hydroxyl radical by puromycin aminonucleoside.

Puromycin aminonucleoside (PAN) has been known to induce proteinuria. The increased generation of reactive oxygen species (ROS) has been implicated in this toxicity of PAN. We have reported that PAN increases the synthesis of methylguanidine (MG) and creatol which are the products of the reaction of creatinine and the hydroxyl radical in isolated rat hepatocytes. However, the mechanism for the increased ROS induced by PAN is still unclear. In this paper, we investigate the role of protein kinase C (PKC) on the PAN induced reactive oxygen generation in isolated rat hepatocytes. Isolated hepatocytes were incubated in Krebs-Henseleit bicarbonate buffer containing 3% BSA, 16.6 mM creatinine and tested reagents. MG and creatol were determined by high-performance liquid chromatography using 9,10-phenanthrenequinone for the post-labeling. PAN increased MG and creatol synthesis in isolated rat hepatocytes by 60%. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, at 10 and 100 microM significantly inhibited MG and creatol synthesis with or without PAN. The inhibition rate is dose dependent from 10 to 100 microM. H1004, a reagent used as control for H-7, did not affect (at 10 microM) or increased little (at 100 microM) the synthesis of MG and creatol. Ro31-8425, a potent PKC inhibitor, significantly inhibited (at 10 microM) MG synthesis in the presence of PAN. PKC in the membrane fraction, a marker of PKC activation, increased over the initial concentration by a factor of 1.65-fold at 60 min incubation and 2.16-fold at 120 min with PAN, while it changed little without PAN. These results indicate that PAN activates PKC resulting in increased hydroxyl radical generation in isolated rat hepatocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Synthesis of creatol, a hydroxyl radical adduct of creatinine and its increase by puromycin aminonucleoside in isolated rat hepatocytes.

Creatol is a hydroxyl radical adduct of creatinine and the precursor of methylguanidine (MG), a uremic toxin. We investigate the synthesis of creatol and MG from creatinine and the effect of substances that affect the hydroxyl radical in isolated rat hepatocytes. In the presence of increasing concentrations of creatinine, rising level of creatol were found after 2 h incubation in Krebs-Henseleit bicarbonate buffer. However, further increase of creatol was not observed after 4 and 6h incubations. On the other hand, MG after 2 h incubation achieved a level of about 50% that of creatol and increased depending on both the creatinine concentration and the incubation period. DMSO, a hydroxyl radical scavenger decreased the generation of creatol and MG by about 50% at 2.5 mM and the inhibition depended on DMSO concentration. Puromycin amino-nucleoside (PAN) increased both by about 170%. These findings demonstrated that hepatocytes synthesize creatol prior to MG and are inhibited by a hydroxyl] radical scavenger. They also show that PAN increased hydroxyl radical generation in tissue cells.

Animals↗
PubMed2006

A simple and efficient synthesis of puromycin, 2,2'-anhydro-pyrimidine nucleosides, cytidines and 2',3'-anhydroadenosine from 3',5'-O-sulfinyl xylo-nucleosides.

Synthesis of antibiotics, puromycin and 3'-amino-3'-deoxy-N6,N6-dimethyladenosine 11 was achieved by utilizing the cyclic sulfite 6a of the xylo-3',5'-dihydroxy group as a new protective group. The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization of 2-alpha-carbamate 7. In a similar manner 2,2'-anhydro-pyrimidine nucleosides 15, ribo-cytidines 17 and 2',3'-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4, 5, and 6b, respectively.

Adenosine↗
PubMed2000

Antiproteinuric effect of calcium antagonists on puromycin-induced experimental nephrosis.

Calcium antagonists have a potential for beneficial effects on kidney function unrelated to their antihypertensive action. In this study we have investigated the efficacy of calcium antagonists compounds (verapamil, nifedipine and diltiazem) on reversible acute renal insufficiency, proteinuria and interstitial nephritis induced by the puromycin ammonucleoside (PAN). An increase in blood pressure (BP) was detected on day 14, with no statistical differences in the response to calcium antagonists. Serum creatinine concentration increased to 1.2 mg/dL on day 7 after PAN and decreased to 0.7 mg/dL at 14 days, calcium antagonists shortened the time required to reach baseline or control levels. Calcium antagonists also reduced proteinuria in the PAN-treated animals, in both day 7 and day 14. Differential effects of the antagonists were observed. Verapamil caused a greater reduction (p < 0.01) in proteinuria than nifedipine or diltiazem in day 7. Moreover, verapamil (p < 0.01) and nifedipine (p < 0.01) reduced the total number of interstitial infiltrating leukocytes from 690 to 120 and 425 positive cells/20 high power fields (x63) respectively, by contrast, diltiazem had no effect. We conclude that in this model of PAN nephropathy verapamil is more effective in reducing both proteinuria and the severity of acute interstitial nephritis than either nifedipine or diltiazem. The possible clinical implications of these results remain to be elucidated.

Animals↗
PubMed2000

Caffeine augments proteinuria in puromycin-aminonucleoside nephrotic rats.

Several studies indicate that increased intrarenal adenosine concentrations may attenuate puromycin-aminonucleoside (PAN)-induced nephropathy in rats. The purpose of this study was to investigate the chronic effects of caffeine, a nonselective adenosine receptor antagonist, on renal function and structure in PAN-induced nephropathy. Animals were randomized to receive drinking water or 0.1% caffeine solution. PAN was administered in two doses to a subset from each group at 1 week (100 mg/kg, s.c.; Purom-1) and 15 wks (80 mg/kg, s.c.; Purom-2) after initiating caffeine treatment (PAN and CAFF-PAN groups). The remaining animals served as time controls (CON and CAFF groups). Renal excretory function was followed for 23 wks. Caffeine consumption significantly augmented PAN-induced proteinuria after both PAN injections (Purom-1 and Purom-2, p<0.05 and p<0.001 respectively; CAFF-PAN vs. PAN). In addition, caffeine potentiated the transient reduction in creatinine clearance (CrCl) induced by PAN. Caffeine consumption for 23 wks significantly reduced CrCl in conscious nephrotic animals (4.76 +/- 0.98 vs. 8.51 +/- 1.55 L/kg/day, CAFF-PAN vs. PAN). Seven days after both PAN injections, increased plasma renin activity was detected in animals that were consuming caffeine as compared with corresponding control groups (CAFF and CAFF + PAN vs CON and PAN, respectively). Eight weeks after the second injection of PAN, acute measures of renal hemodynamic and excretory function were compared in anesthetized animals and renal samples were analyzed for histological changes. In PAN-rats, caffeine treatment for 23 weeks significantly reduced inulin clearance (0.28 +/- 0.09 vs. 0.57 +/- 0.12 mL/min/gr kidney. CAFF-PAN vs PAN, p<0.05), tended to increase renal vascular resistance (59.0 +/- 9.5 vs. 42.9 +/- 5.5 mmHg/mL/min/gr kidney, CAFF-PAN vs. PAN, p < 0.06), potentiated the development of more severe tubulointerstitial damage (tubular atrophy, presence of proteinaceous material, tubular dilatation, interstitial inflammation, interstitial fibrosis), and tended to increase glomerulosclerosis. In conclusion, this study indicates that caffeine adversely affects renal function in PAN-nephrotic rats, and that this effect may be due, in part, to increased activity of the renin angiotensin system.

Animals↗
PubMed2000

Does captopril change oxidative stress in puromycin aminonucleoside nephropathy?

Puromycin aminonucleoside (PAN) nephropathy in rats has been induced by the intraperitoneal injections of PAN. One group of animals which received PAN has been treated simultaneously with captopril (angiotensine converting enzyme-ACE-inhibitor) with the aim to test whether continuing treatment with captopril along with PAN injections would be able to modulate the toxic effects of PAN. The third group of rats was given only captopril. Morphological changes in the kidney were evaluated by scanning electron microscopy that showed the loss of podocyte foot processes in the kidney of PAN treated animals but also in the kidney of captopril treated ones as well as in the animals treated with both drugs simultaneously. Reduced glutathione content, catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), xanthine oxidase activities as well as lipid peroxides were investigated in rat blood and kidney. Captopril given alone produced a significant decrease of plasma lipid peroxides, but it did not show any significant effect on investigated antioxidative factor levels neither in blood nor in the kidney. PAN given alone produced a significant depletion of plasma lipid peroxides, kidney catalase and erythrocyte GSH-Px activity as well as a significant increase of plasma catalase and erythrocyte SOD activity. Treatment of animals with both drugs simultaneously resulted in a significant increase of erythrocyte SOD activity and a significant decrease of plasma lipid peroxides, erythrocyte GSH-Px and kidney SOD activities. Kidney xanthine oxidase activity showed a significant increase in both PAN and PAN plus captopril treated animals in comparison with the values of captopril treated rats. These data suggest that PAN changes the antioxidative factor pattern in rat blood and kidney. Contrary to our expectations that captopril may protect the toxic effects of PAN it only to a certain extent modifies these effects showing protective effect only on tissue catalase activity.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1966

Studies on the mechanism of hormonal induction of alkaline phosphatase in human cell cultures. I. Effects of puromycin and actinomycin D.

Increased alkaline phosphatase activity is induced in certain epithelial cell cultures by hormones with adrenal glucocorticoid activity or their analogues such as prednisolone (Delta(I)-hydrocortisone). Enzyme induction occurs in two distinct phases. During the first 12 hr after the addition of prednisolone, there is a small increase in alkaline phosphatase levels. After 15 to 24 hr, the enzyme activity shows a sudden, marked linear rise, reaching a maximum at 60 to 80 hr. Puromycin blocks enzyme induction immediately, even when added during the period of rapid increase of enzyme. Actinomycin D blocks induction when added no later than 8 hr after the addition of prednisolone. On the other hand, Actinomycin D added during the phase of rapid enzyme induction has no effect for at least 12 hr. These findings suggest that de novo protein synthesis is involved in prednisolone induction of alkaline phosphatase and that the RNA messenger for this enzyme is relatively stable.

Cell Division↗
PubMed1966

Relationship between RNA synthesis, cell division, and morphology of mammalian cells. I. Puromycin aminonucleoside as an inhibitor of RNA synthesis and division in HeLa cells.

Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P(32) and H(3)-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division within the period studied (3 generation times). The cell content of RNA returned to normal when the inhibitor was removed.

Cell Division↗
PubMed1973

Synthesis of Paramecium surface proteins. I. Puromycin insensitive amino acid incorporation.

The synthesis of a surface protein has been studied in Paramecium through double-labeling experiments using [(14)C]- and [(3)H]leucine-labeled bacteria as the source of radioactive amino acid. Over a 4-5 h incubation period, the turnover rate was found to be higher than that of overall cell protein. In addition, the initial label is apparently utilized during the chase period, being incorporated into protein via a puromycin insensitive pathway.

Amino Acids↗
PubMed1977

Binding of viral glycoprotein mRNA to endoplasmic reticulum membranes is disrupted by puromycin.

Previous studies showed that the glycoprotein (G) of vesicular stomatitis virus is synthesized in association with the endoplasmic reticulum (ER) membrane and that all G mRNA co-fractionates with ER membrane. Here, we show that treatment of infected cells with puromycin results in dissociation of G mRNA, and presumably the associated ribosomes, from the ER membrane. Even it extracts from treated cells are kept at low ionic strength (0.01 M KCl), over 80% of G mRNA is found unattached to membranes. There is no evidence for direct interaction of GmRNA with membranes; rather, the linkage apparently is mediated by the nascent G polypeptide.

Animals↗
PubMed1976

Phase shifting the circadian rhythm of neuronal activity in the isolated Aplysia eye with puromycin and cycloheximide. Electrophysiological and biochemical studies.

The effects of pulse application of puromycin (PURO) or cycloheximide (CHX) were tested on the circadian rhythm (CR) of spontaneous compound action potential (CAP) activity in the isolated Aplysia eye. CAP activity was recorded from the optic nerve in constant darkness at 15degreesC. PURO pulses (6, 12 h; 12--134 mug/ml) and CHX pulses (12 h, 500--2,000 mug/ml) caused dose-dependent phase delays in the CR when administered during projected night. PURO pulses (6 h, 125 mug/ml) caused phase advances when given during projected day and caused phase delays when given during projected night. In biochemical experiments PURO (12 h, 20 mug/ml) and CHX (12 h, 500 mug/ml) inhibited leucine incorporation into the eye by about 50%. PURO (12 h; 50, 125 mug/ml) also changed the molecular weight distribution of proteins synthesized by the eye during the pulse. The effect of PURO (12 h, 125 mug/ml) on the level of incorporation was almost completely reversible within the next 12 h but the phase-shifted eye showed an latered spectrum of proteins for up to 28 h after the pulse. In electrophysiological experiments spontaneous CAP activity and responses to light were measured before, during, and after drug treatments. In all, eight parameters in three periods were analyzed quantitatively. Of these 24 indices, only 3 showed significant changes. PURO increased spontaneous CAP frequency by 67% 0-7 h after the drug pulse and increased the CAP amplitude of the tonic light response by 23% greater than 7 h after the pulse. CHX increased the intraburst spontaneous CAP frequency by 33% during the pulse and CAP frequency of the tonic light response by 32% 0-7 h after the pulse. The above data indicate that phase-shifting doses of PURO and CHX inhibit protein synthesis in the eye without causing adverse electrophysiological effects, and suggest that protein synthesis is involved in the production of the CR of the isolated Aplysia eye.

Action Potentials↗
PubMed1992

Mechanisms involved in the progression to glomerular sclerosis induced by systemic hypertension during mild puromycin aminonucleoside nephrosis.

To evaluate the contribution of systemic hypertension in the progression of nephropathies to glomerular sclerosis, a mild form of puromycin aminonucleoside (PAN) nephrosis was associated with Goldblatt hypertension and studied after 18 weeks. We studied four groups: Group I, controls; Group II, Goldblatt hypertension; Group III, PAN nephrosis; and Group IV, both conditions. Systolic blood pressure, 24-h proteinuria, serum cholesterol, triglycerides, glomerular hemodynamics, and histological studies were compared among the groups. Rats in groups II and IV developed systemic hypertension, but only group IV rats showed persistent proteinuria. No alterations in lipid metabolism were present in any of the groups. The most striking findings in the micropuncture studies were a significant increase of glomerular capillary pressure in group IV rats (63.15 +/- 1.34 mm Hg) as compared to controls (48.74 +/- 0.97 mm Hg) and to groups II and III (55.31 +/- 2.11 and 48.17 +/- 1.23 mm Hg, respectively), and a marked fall in Kf in groups III and IV. Only group IV showed significant histological alterations such as glomerular sclerosis, interstitial damage, and increased glomerular area. These results suggest that, in the presence of an underlying nephropathy, a greater fraction of systemic pressure is transmitted to the glomerular capillaries when systemic hypertension is present; the resulting elevation in glomerular pressure and proteinuria seems to be responsible for the progression to glomerular sclerosis.

Animals↗