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PubMed2002

Renal structural abnormalities following recovery from acute puromycin nephrosis.

BACKGROUND: Rats that recover from acute puromycin nephrosis later develop widespread glomerular and tubulointerstitial injury. The current study sought to identify structural changes present in the recovery phase that could precipitate progressive renal disease. METHODS: Stereologic studies were performed 10 weeks after administration of puromycin (PAN) or saline (Cont). Serial sections were examined to assess glomerular structure. RESULTS: Rats receiving puromycin developed heavy proteinuria that returned nearly to control levels at 10 weeks. Kidneys in these animals were moderately enlarged and exhibited expansion of the interstitium (PAN, 254 +/- 47 mm3; Cont, 152 +/- 23 mm3; P < 0.05). The average glomerular volume was not different from control (PAN, 1.90 +/- 0.38 x 10(6) microm3; Cont, 2.07 +/- 0.47 x 10(6) microm3), but a subpopulation of glomeruli of about half normal size was found in PAN rats. Serial sections revealed that most of these glomeruli were not connected to normal tubule segments. Serial sections also revealed that more than 90% of glomeruli in rats recovering from nephrosis had synechias joining the tuft to Bowman's capsule. Synechias occupied an average of 8 +/- 11% of the Bowman's capsule surface in PAN animals versus less than 1% of the surface in controls. The appearance of synechias was not associated with a reduction in the mean number of visceral or parietal epithelial cells per glomerulus. CONCLUSIONS: Acute puromycin nephrosis does not cause a notable reduction in visceral epithelial cell number. However, widespread glomerular injury characterized by synechia between the tuft and Bowman's capsule is present following remission of proteinuria. Progression of this residual glomerular injury could contribute to the late development of glomerular segmental sclerosis following recovery from acute nephrosis.

Acute Disease↗
PubMed1971

Studies on memory: distribution of peptidyl-puromycin in subcellular fractions of mouse brain.

Previous experience has led us to suggest that sustained blockage of memory in mice upon administration of puromycin is caused by the persistence of peptidyl-puromycin at synapses. The block can be removed and memory restored by intracerebral injections of water and by subcutaneous or intraperitoneal injections of several psychotropic drugs. The present experiments provide evidence that peptidyl-puromycin persists for long periods in subcellular fractions of synaptosomes (nerve endings), and that the peptide is lost from synaptosomal, but not from mitochondrial, fractions when memory is restored. These findings strengthen our interpretation of the mode of action of puromycin on memory.

Acetylcholinesterase↗
PubMed1981

Localization of the puromycin binding site on the large ribosomal subunit of Escherichia coli by immunoelectron microscopy.

By using immunoelectron microscopy, we have localized the binding site on 50S Escherichia coli ribosomal subunits for puromycin, an antibiotic that interacts with the ribosomal peptidyltransferase center. This was achieved by affinity-labeling 50S subunits with N-bromoacetyl puromycin and treating the labeled subunits with an antibody specific for the N6,N6-dimethyladenosine moiety of puromycin. The position of the puromycin binding site was then revealed by localizing the attachment sites of the IgG molecules on the surfaces of the 50S subunits under the electron microscope: it was located at the interface between the subunits, on and around the wider lateral protuberance of the 50S subunit. This localizes directly the peptidyl transferase center on the surface of the large ribosomal subunit.

Binding Sites↗
PubMed1968

Antibody production in vitro. II. Effects of actinomycin D and puromycin on the secondary response to sheep erythrocytes.

The effects of actinomycin D and puromycin on spleen cell suspensions from rabbits immunized to SRC's were studied. These inhibitors, in high concentration, suppressed PFC's when added initially to recently isolated cells. When such cells were incubated for several days in the presence of both antigen and inhibitor, both actinomycin D and puromycin produced an increase in PFC's after the initial suppression. This recovery effect was best seen with cells from rabbits killed 3 days after boosting with SRC's, and was usually absent when cells were taken from rabbits killed 2 days after boosting. When actinomycin D or puromycin was added after several days in culture in the presence of SRC's, surviving PFC's were found to be not only resistant to these inhibitors, but there was also an increased number of PFC's compared to similar cultures incubated without these agents. Radioautographic studies showed that PFC's stimulated by the presence of actinomycin D or puromycin were not incorporating precursors for RNA or protein synthesis. In view of the known mode of action of these inhibitors, it was postulated that they were stimulating antibody production by PFC's in vitro either by interfering with represser mechanisms or stimulating the completion of antibody molecules, perhaps by causing the release of preformed antibody chains from ribosomes. Since the presence of specific antigens in vitro were necessary for these observed stimulatory effects on PFC's, and since antigens were producing an effect on antibody production on cells which were being suppressed by these inhibitors, added initially, it was further suggested that one role of antigen in the immune response was concerned with the completion of antibody synthesis on the ribosomes, perhaps by acting as an inducer as has been suggested previously (1).

Animals↗
PubMed1981

Necessity of detergent for efficient puromycin-mediated release of nascent peptides from rat liver ribosomes.

Puromycin-mediated in vitro release of nascent peptides from rat liver ribosomes was significantly stimulated by the presence of low concentrations of a detergent, and the stimulation was much more marked with bound ribosomes than with free ribosomes. The release of nascent peptides from ribosomes could be carried out in two steps, first with puromycin in the absence of a detergent and then with a detergent, to give two separate nascent peptide fractions S1 and S2, respectively. Although S1 and S2 fractions were not significantly different in hydrophobicity and in the size of the puromycin-conjugated peptides when examined by alkyl-Sepharose column chromatography and SDS-polyacrylamide gel electrophoresis, the fractionation of the released peptides by immunoprecipitation showed significant difference in the distribution of the nascent peptides of two specific proteins, serum albumin and NADPH-cytochrome c reductase, between these two fractions. The nascent peptides of serum albumin were found mainly in fraction S1 obtained from bound ribosomes. On the other hand, a larger portion of the nascent peptides of NADPH-cytochrome c reductase was detected in fraction S2 from free ribosomes than in other fractions. The presence of a detergent is indispensable for efficient in vitro release of nascent peptides from ribosomes by puromycin and this finding may be important in studying the synthesis of specific proteins in mammalian cells.

Animals↗
PubMed1991

Protein modification by phosphorylation during the process of nuclear membrane dissolution in puromycin-treated mouse oocytes.

The present study was undertaken to elucidate the mechanism of nuclear membrane dissolution (NMD) in puromycin-treated mouse oocytes. Treatment of germinal vesicle breakdown (GVBD) oocytes with puromycin (50 micrograms/ml) induced chromosome decondensation with formation of a polar body; these are designated nuclear membrane (NM) oocytes. After withdrawal of puromycin, NM oocytes underwent NMD (approximately 70%) during a 12-h culture period. Either dibutyryl cyclic AMP (dbcAMP, 25-100 micrograms/ml) or isobutylmethylxanthine (IBMX, 0.1-1.0 mM) inhibited the process of NMD in a dose-dependent manner, suggesting the involvement of cAMP in the process of NMD. To determine which protein(s) participated in the transition from interphase to metaphase II during NMD, NM oocytes were labeled with [35S]methionine, and one- and two-dimensional gel electrophoresis were performed. Although the synthesis of stage-specific proteins during NMD was not found, two specific proteins of Mr 27,000 and 46,000, which were synthesized at interphase following removal of puromycin, were modified during NMD. Phosphatase treatment and 32PO4-labeling experiments indicated that phosphorylation was responsible for these modifications, which were inhibited by either dbcAMP or IBMX. Therefore, it appears that phosphorylation of specific proteins may play an important role in the transition from interphase to metaphase II.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Mechanism of self-protection in a puromycin-producing micro-organism.

Puromycin is a potent inhibitor of bacterial protein synthesis, but puromycin-producing Streptomyces alboniger KCC S-0309 is tolerant to the antibiotic in vivo. Puromycin bound to both 30S and 50S ribosomal subunits from S. alboniger and inhibited polyuridylate-directed polyphenylalanine synthesis by the ribosomes. However, the organism possessed a novel puromycin-inactivating enzyme which acetylated the antibiotic at the 2''-NH2 group of the O-methyltyrosine moiety.

Acetylation↗
PubMed1984

Effect of P and A site substrates on the binding of a macrolide to ribosomes. Analysis of the puromycin-induced stimulation.

The puromycin-induced stimulation of [3H]dihydrorosaramicin binding is due to a twofold increase in affinity of the macrolide antibiotic, with no change in the number of binding sites. Conversely, the binding of [3H]puromycin (A site) is stimulated by rosaramicin. The synergistic effect observed between the two antibiotics can be explained by a conformational change with positive effect, which occurs at the level of their binding sites. Various effectors of [3H]dihydrorosaramicin binding have been tested. Adenosine and dimethyladenosine stimulate the binding; phenylalanine, uridine and gougerotin (A site) have no effect whereas AMP, ADP, ATP, GTP, puromycin 5'-phosphate and lincomycin (P site) are inhibitors. These results point to the importance of the purine moiety in the stimulatory effect and of the phosphate function in reversing this effect. It is concluded that rosaramicin binds to the ribosomal P site and that the synergism observed between rosaramicin and puromycin may be related to interactions between the A and P sites.

Adenosine Triphosphate↗
PubMed1993

The pur8 gene from the pur cluster of Streptomyces alboniger encodes a highly hydrophobic polypeptide which confers resistance to puromycin.

A novel puromycin-resistance determinant (pur8) was isolated from one end of the pur cluster that encodes the puromycin biosynthetic pathway from Streptomyces alboniger and expressed in Streptomyces lividans. The gene pur8 induced antibiotic resistance that was highly specific for puromycin. The nucleotide sequence of pur8 contains an open reading frame of 1512 bp whose deduced amino acid sequence encodes a polypeptide (Pur8) with 14 possible transmembrane-spanning segments. It shows significant similarities to other known or putative transmembrane proteins, including a number which confer drug resistance in a variety of antibiotic-producing Streptomyces, Gram-positive and Gram-negative bacteria, and some solute transporters of prokaryotic and eukaryotic origin. As is probably the case for most of these proteins, Pur8 may be involved in active puromycin efflux energized by a proton-dependent electrochemical gradient. In addition, it could be implicated in secreting N-acetylpuromycin, the last intermediate of the biosynthesis pathway, to the environment.

Amino Acid Sequence↗
PubMed1994

Lysosomal iron accumulation and tubular damage in rat puromycin nephrosis and ageing.

1. Energy dispersive X-ray spectrometry was used to examine the relationship between proteinuria and increased urinary iron excretion, and structural and functional damage in puromycin nephrosis. 2. After 11-12 days rats treated with puromycin (10 mg/100 g, i.v.i.) had greater proteinuria (211.6 +/- 35.7 mg/day, mean +/- s.e.m.) and urinary iron excretion (15.4 +/- 2.2 micrograms/day) than saline-treated controls (14.5 +/- 1.4 mg/day and 1.1 +/- 0.2 micrograms/day, respectively, both P < 0.001). 3. On day 13, mean lysosomal iron concentration of proximal tubular cells (306.6 +/- 64.5 vs 11.9 +/- 8.6 mg%, P < 0.001), and proximal tubular cell damage assessed semi-quantitatively (1.17 +/- 0.10 vs 0.62 +/- 0.10, P < 0.001) were higher and creatinine clearance (0.15 +/- 0.01 vs 0.29 +/- 0.02 mL/min per g kidney weight, P < 0.001) lower than in control rats. 4. At days 35, 60 and 360 there were no differences in any of the measured parameters between rats treated with puromycin or saline, and in both groups proteinuria, tissue damage and lysosomal iron concentration increased with time. 5. Lysosomal iron accumulation was the only independent predictor of both functional and structural damage. 6. In conclusion, the apparent association between proteinuria and tubulo-interstitial damage in puromycin nephrosis, and with ageing, is best explained by factors associated with accumulation of iron within lysosomes of proximal tubule cells.

Aging↗
PubMed1966

Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells.

Gilden, R. V. (Wistar Institute, Philadelphia, Pa.), and R. I. Carp. Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells. J. Bacteriol. 91:1295-1297. 1966.-Synthesis of the simian virus 40 (SV40) T antigen in primary African green monkey kidney cells was abolished when cycloheximide was added up to 10 hr postinfection. In contrast, puromycin, another inhibitor of protein synthesis, did not suppress antigen production. The basis of this differential effect was the inability of puromycin to inhibit protein synthesis in the cells used. This was shown by the failure of the drug to depress the incorporation of labeled amino acid into protein and also failure to inhibit poliovirus synthesis. The puromycin preparation used was very effective in inhibiting poliovirus synthesis in HeLa cells. Thus, appearance of the SV40 T antigen is dependent on protein synthesis in infected cells.

Animals↗
PubMed1975

Recovery of pancreas from mild puromycin-induced injury. A histologic and ultrastructure study in rats.

Pancreatic acinar cells undergo degeneration or necrosis following injection of puromycin intraperitoneally in rats. The purpose of this study was to characterize recovery following injection of four hourly doses of puromycin, 40mg/kg of body weight, examining the pancreas histologically and by electron microscopy. The number of dividing acinar cells increased following injury. By 12 to 24 hours following treatment, electron microscopy showed numerous autophagic vacuoles and intracisternal granules in the cells. By 48 hours, these were largely cleared from surviving cells although the intracisternal granules persisted in isolated acinar cells as long as 144 hours. At 24 hours, there was debris in the acinar lumens and interstitial space. We conclude that some acinar cells injured by puromycin may survive and be restored to normal structure; that surviving acinar cells can extrude autophagic vacuoles; and that necrotic acinar cells are replaced by regeneration following puromycin-induced injury in rats.

Animals↗
PubMed2006

[Oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that fail to fertilize after intracytoplasmic sperm injection].

OBJECTIVE: To investigate the effect of assisted oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that fail to fertilize after intracytoplasmic sperm injection (ICSI). METHODS: All 113 discarded oocytes that showed no evidence of fertilization at 16 - 18 hours after in vitro maturation (IVM)-ICSI cycles and conventional ICSI were assigned to four groups according to the time after ICSI: IVM-ICSI 22-hour group (n = 33), IVM-ICSI 44-hour group (n = 18), ICSI 44-hour group (n = 37) and ICSI 68-hour group (n = 25). All unfertilized oocytes were exposed to calcium ionophore A23187 (5 micromol/L) for 5 minutes and subsequently incubated with puromycin (10 microg/ml) for 4 hours. After incubation, the oocytes were cultured in vitro for 3 - 5 days. The activation rate, proportion of oocytes that showed pronucleus formation and cleavage rate were calculated after activation. Sex chromosomal analysis was performed by dual color fluorescence in situ hybridization (FISH) on the embryos that displayed two pronuclei and a second polar body. RESULTS: The combination of calcium ionophore A23187 with puromycin could activate the unfertilized oocytes 22 - 68 hours after ICSI. Best results were achieved in IVM-ICSI 22-hour group, which elicited 88% (29/33) of activation rate, 62% (18/29) of cleavage rate and 28% (5/18) of 4-cell embryos. One embryo in this group developed to the morular stage. The activation rate and developmental potential of the activated embryos in IVM-ICSI 44-hour group, ICSI 44-hour group and ICSI 68-hour group decreased. FISH analysis showed 4 embryos with XX and 9 embryos with XY in 16 embryos. CONCLUSIONS: The combination of calcium ionophore A23187 with puromycin could effectively activate unfertilized oocytes 22 - 68 hours after ICSI. The cultured time of unfertilized oocytes after ICSI affects activation efficiency and developmental potential of the activated embryos. The activated zygotes that display two pronuclei and a second polar body can develop normally.

Calcimycin↗
PubMed1979

Photoaffinity labeling of rat liver ribosomes by N-(2-nitro-4-azidobenzoyl)puromycin.

N-(2-Nitro-4-azidobenzoyl)-[3H]puromycin (NAB-puromycin) was synthesized as a photoreactive derivative of puromycin in order to detect ribosomal proteins located near the peptidyltransferase centre of rat liver ribosomes. Irradiation of ribosome-NAB-puromycin complexes leads to covalent attachment of the affinity label to proteins of the large ribosomal subunit, in particular to proteins L28/29, and, to a somewhat lower extent, to proteins L4, L6, L10 and L24. The results are discussed in the light of earlier studies performed with other affinity labels that attacked the peptidyltransferase region of rat liver ribosomes.

Affinity Labels↗
PubMed1986

Puromycin aminonucleoside nephropathy: ultrastructure, glomerular polyanion, and cell surface markers.

Puromycin aminonucleoside nephropathy with heavy proteinuria and oedema was induced in rats by 10 consecutive daily subcutaneous injections of aminonucleoside (1.67 mg/100 g of body weight). The main ultrastructural lesions were vacuolation of podocytes and total fusion of foot processes with loss of colloidal iron-reactive polyanion layer on the epithelial surface adjacent to the basement membrane. On the other hand the outer surface of podocytes and intravacuolar granular substance stained with colloidal iron. In scanning electron microscopy of freeze-fractured tissue the swollen podocytes and the urinary spaces displayed granular and filamentous precipitates. Seven cell surface antigens were examined by indirect enzyme immunohistochemistry with a series of MRC OX monoclonal antibodies. Glomeruli of control rats exhibited rare isolated Ia- positive endocapillary cells, possibly monocytes; these elements were significantly reduced in puromycin aminonucleoside nephropathy but there was an increase in Ia- positive cells in the cortical interstitium. Control kidneys harboured scanty interstitial T lymphocytes. These latter, especially the T8- positive cytotoxic/suppressor subpopulation, were markedly augmented in puromycin aminonucleoside nephropathy. The expression of class I histocompatibility antigens and of differentiation antigens (Thy 1) was not altered by aminonucleoside.

Animals↗
PubMed1987

The role of superoxide anion and hydrogen peroxide in glomerular injury induced by puromycin aminonucleoside in rats.

1. The nephrotic syndrome was induced in inbred female Wistar rats by the intravenous injection of puromycin aminonucleoside (PA) (5 mg/100 g body weight). 2. One group (n = 12) received superoxide dismutase (SOD) (15 mg/kg body weight), a second group (n = 12) received polyethylene glycol coupled catalase (PEG-catalase) (5000 i.u./kg body weight) and the third (n = 9) saline (150 mmol/l NaCl) via the intraperitoneal route, in addition to the PA. 3. SOD and PEG-catalase reduced the 24 h urine protein on days 8 and 15 compared with unmodified puromycin treated animals and this difference was significant on day 15 for SOD (P less than 0.05) and for PEG-catalase (P less than 0.01). Glomerular filtration rate, as measured by the creatinine clearance, was lower in the PEG-catalase group but did not differ significantly from the saline treated group. 4. These data suggest that superoxide anion and hydrogen peroxide, or their reaction products, are involved in the glomerular injury of puromycin nephropathy.

Animals↗
PubMed1984

Self-association of puromycin as studied by proton magnetic resonance spectroscopy.

The self-association of puromycin has been studied using proton magnetic resonance spectroscopy. The concentration, temperature and pH dependence studies of the proton chemical shifts of the adenine protons indicate that puromycin in aqueous solution at pD 7.4 self associates predominantly through adenine-adenine interaction. At this pD, the amino group of the aminoacyl segment of puromycin has been demonstrated to exist in a equilibrium blend of protonated and non-protonated forms. At pD 2.6, PM is found to exist predominantly in the monomeric from in which the methyl groups of the 6N-dimethyladenine are found to be non-equivalent due to hindered rotation about the C6-N6 bond.

Hydrogen-Ion Concentration↗
PubMed1964

PUROMYCIN EFFECT ON MEMORY FIXATION IN THE GOLDFISH.

Puromycin injected intracranially into the goldfish produces impairment of memory for a shock-avoidance response. Intracranial injection of puromycin aminonucleoside, or of saline has no effect. Puromycin does not afject performance in naive or overtrained goldfish.

Animals↗