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PubMed2001

A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus.

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 microM A23187 for 5 min were treated with 10 microg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.

Adenine↗
PubMed1990

Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences.

In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Coooperman, B. S. (1982) Biochemistry 21, 3809-3817] site specifically photoaffinity label protein L23 to the highest extent of any Escherichia coli ribosomal protein. In this work we demonstrate that L23 that has been photoaffinity labeled within a 70S ribosome by puromycin (puromycin-L23) can be separated from unmodified L23 by reverse-phase high-performance liquid chromatography (RP-HPLC) and further that puromycin-L23 can reconstitute into 50S subunits when added in place of unmodified L23 to a reconstitution mixture containing the other 50S components in unmodified form. We have achieved a maximum incorporation of 0.5 puromycin-L23 per reconstituted 50S subunit. As compared with reconstituted 50S subunits either containing unmodified L23 or lacking L23, reconstituted 50S subunits containing 0.4-0.5 puromycin-L23 retain virtually all (albeit low) peptidyl transferase activity but only 50-60% of mRNA-dependent tRNA binding stimulation activity. We conclude that although L23 is not directly at the peptidyl transferase center, it is sufficiently close that puromycin-L23 can interfere with tRNA binding. This conclusion is consistent with a number of other experiments placing L23 close to the peptidyl transferase center but is difficult to reconcile with immunoelectron microscopy results placing L23 near the base of the 50S subunit on the side facing away from the 30S subunit [Hackl, W., & Stöffler-Meilicke, M. (1988) Eur. J. Biochem. 174, 431-435].

Chromatography, High Pressure Liquid↗
PubMed2003

Hypercholesterolemia is a prerequisite for puromycin inducible damage in mouse kidney.

BACKGROUND: The mouse, as opposed to the rat, is relatively resistant to the experimental nephrosis induced by puromycin aminonucleoside. The reason for this species specificity is not known. Apolipoprotein E (apoE)-deficient mice were used to determine whether hypercholesterolemia plays a role in inducing proteinuria. METHODS: Thirty-two mice were divided into normal and high cholesterol diet groups and then divided further into four subgroups: puromycin, puromycin+probucol, probucol and control. Urinary albumin of these mice was analyzed by nephelometry. The lipid peroxidation (LPO) end products malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were detected by immunohistochemistry, and the expression level of the glomerular slit diaphragm protein, nephrin, was studied by immunohistochemistry and real time RT-PCR. RESULTS: Overt proteinuria was induced by puromycin only in the apoE knockout mice ingesting the high cholesterol diet. The staining intensities of MDA and 4-HNE were stronger in the glomeruli of proteinuric mice compared to glomeruli of non-proteinuric mice. When serum cholesterol levels were reduced by probucol, proteinuria decreased and fewer LPO end products were seen immunohistochemically. Three and eight days after puromycin injection the level of nephrin mRNA in the kidneys of proteinuric mice decreased in comparison to the controls. Puromycin-treated mice kidneys demonstrated a clearly reduced reactivity to the nephrin antibodies. CONCLUSIONS: Hypercholesterolemia, possibly via LPO, is a prerequisite for puromycin-inducible glomerular damage in the mouse. Furthermore, nephrin protein and mRNA levels appear to be candidate markers of glomerular damage in the mouse.

Animals↗
PubMed1971

Puromycin resistance in haploid and heteroploid frog cells: gene or membrane determined?

The frequency of colony formation in monolayers of cultured frog cell lines treated with puromycin was compared in (a) haploid and heteroploid lines and (b) mutagen-treated and nontreated haploid lines. Evidence that resistant colonies result from gene mutation was negative, since the colony frequency is independent of both ploidy and mutagen treatment. A study of five frog cell lines showed that colony formation in puromycin depends on (a) the concentration of puromycin, (b) preselection of the population with puromycin, and, particularly, (c) the capacity of the treated population to survive some exposure to puromycin. One haploid and one heteroploid strain showing stable resistance to puromycin have been isolated; comparison of those variants with sensitive populations has shown that resistance to puromycin is correlated with the cells' capacity to exclude the drug. The evidence for different levels of membrane permeability, combined with evidence for many degrees of resistance among and within cell populations, suggests a model of self-determining membrane units. The evolution of a resistant phenotype may result from changes in the proportion of specific units in the membrane population.

Animals↗
PubMed1986

Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined.

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction'). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e. EF-G can already promote a translocation reaction at 0 degree C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[3H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe-tRNA per ribosome.

Binding Sites↗
PubMed1968

Puromycin: action on neuronal mitochondria.

Puromycin, in dosages that inhibit cerebral protein synthesis and expression of memory in mice, produces swelling of neuronal mitochondria. Acetoxycycloheximide, which inhibits cerebral protein synthesis to the same extent as puromycin, fails to produce swelling of neuronal mitochondria. Puromycin and heximide mixtures produce severe inhibition of protein synthesis, but result in a minimal swelling of neuronal mitochondria and in a decrease of peptidylpuromycin complexes to a level of 30 percent of that following the injection of puromycin alone. It is concluded that swelling of neuronal mitochondria in the presence of puromycin is not due to inhibition of cerebral protein synthesis per se, but is related to a specific action of puromycin on ribosomal protein synthesis. The findings are consistent with the hypothesis that peptidyl-puromycin complexes are responsible for mitochondrial swelling.

Animals↗
PubMed1978

Effects of puromycin and actinomycin D on fertilized rabbit eggs cultured in vitro.

Rabbit zygotes were cultured in media containing varying concentrations of puromycin and actinomycin D in order to determine what effect these agents have on events of fertilization and early development. Concentrations of puromycin greater than 10(-5) M inhibited mitosis, cytokinesis of fertilized eggs and leucine incorporation. In approximately 50% of the zygotes treated with puromycin (10(-5) M or greater) the female pronucleus remained at its site of formation, the cortex, and did not migrate centrad. In these instances the male pronucleus moved from its site of development to the female pronucleus. Concentrations of actinomycin D greater than 10(-7) M also inhibited mitosis and cytokinesis of fertilized eggs. Breakdown of the nuclear envelopes and association of the paternally- and maternally-derived chromatin did not occur in either puromycin- or actinomycin D-treated embryos. Actinomycin D at all of the concentrations employed (10(-9) to 10(-4) M) had little effect upon the structure of nucleolus-like bodies within male and female pronuclei. Male and female pronuclei of puromycin (10(-4) M) and actinomycin D (10(-5) M) treated zygotes, incubated with tritiated thymidine and prepared for scintillation counting and autoradiography, synthesized DNA at a reduced level when compared to control specimens. Although "side effects" of puromycin and actinomycin D treatment are not excluded, these results suggest that nascent proteins may be required for events of fertilization.

Animals↗
PubMed1993

Puromycin inhibits protein import into mitochondria by interfering with an intramitochondrial ATP-dependent reaction.

We have performed experiments which demonstrate that puromycin inhibits the import of proteins into mitochondria in in vitro reactions containing mitochondria isolated from the yeast Saccharomyces cerevisiae and precursor proteins synthesized in a nuclease-treated rabbit reticulocyte lysate. Puromycin inhibited the import of several precursor proteins including; a fusion protein consisting of the first 22 N-terminal residues of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase, both a destabilized and truncated form of this same fusion protein, the beta-subunit of the yeast mitochondrial F1-ATPase and yeast alcohol dehydrogenase III. The insertion of the yeast outer mitochondrial protein porin was not inhibited by puromycin. Puromycin-induced import inhibition could be overcome by adding additional ATP to the import reactions. However, if access of ATP to the mitochondrial matrix was prevented by blocking the adenine nucleotide translocase with carboxyatractyloside, ATP addition was unable to overcome the inhibitory effect of puromycin on protein import. Collectively, these results demonstrate that puromycin inhibits protein import into mitochondria by interfering with an ATP-dependent step in the import process and that the ATP-dependent component in the reaction is located inside the inner mitochondrial membrane. In addition to supporting the view that ATP is required in the matrix for efficient protein import, these results may provide a useful tool for identifying the ATP-binding components of the import apparatus.

Adenosine Triphosphate↗
PubMed1997

Differential activity of bcl-2 and ICE enzyme family protease inhibitors on Fas and puromycin-induced apoptosis of glioma cells.

Fas ligand is a potent inducer of apoptosis in human glioma cells by the Fas/Fas ligand pathway. With comparable efficiency, metalloprotease inhibitors including puromycin and bestatin induce apoptosis in glioma cells. To evaluate the involvement of potential components involved in Fas ligand- and metalloprotease inhibitor-induced apoptosis, we investigated the effect of anti human Fas antibody, soluble Fas ligand and puromycin on cultures of human malignant glioma cell lines (LN-18, LN-229, T98G). Stimulation with Fas ligand lead to apoptotic cell death within 16 h. Costimulation with the translational inhibitor cycloheximide and the transcription blocker actinomycin D did not reduce Fas ligand toxicity. In contrast, apoptosis induced by puromycin was blocked by cycloheximide and decreased by subtoxic doses of actinomycin D in all three gliomas. Whereas inhibition of caspase activity with the general inhibitor zVAD-fmk resulted in a complete block of Fas ligand-induced cell death, puromycin-mediated apoptosis was found to be unaffected by zVAD-fmk as well as by more specific inhibitors for caspase-1 (Interleukin-1 beta converting enzyme) and caspase-3 (CPP32/Yama). Other prominent components involved in many apoptotic pathways as bcl-2 and reactive oxygen intermediates were also examined. Bcl-2 which protects glioma cells from Fas ligand-induced cell death, was shown to have only a small protective effect on puromycin-induced apoptosis. The tested radical scavengers did not reduce Fas- or puromycin-mediated killing of human glioma cells.

Amino Acid Chloromethyl Ketones↗
PubMed1985

Puromycin photoaffinity labels small- and large-subunit proteins at the A site of the Drosophila ribosome.

[3H]Puromycin covalently incorporates into the protein and to a much lesser extent into the RNA components of Drosophila ribosomes in the presence of 254-nm light. The photoincorporation reaction takes place with a small number of large- (L2 and L17) and small- (S8 and S22) subunit proteins as determined by two-dimensional gel analysis. More quantitative one-dimensional gel results show that puromycin reacts with each of these proteins in a functional site specific manner. The small percentage of the total labeling that occurs with rRNA also appears to be site specific. The rRNA labeling arises from a puromycin-mediated cross-linking of ribosomal protein and rRNA. Ionic conditions shift the pattern of puromycin-labeled ribosomal proteins. These results suggest that puromycin can occupy two distinct sites on Drosophila 80S ribosomes. The pattern of ribosomal proteins labeled by puromycin is affected by the presence of other antibiotics such as emetine, anisomycin, and trichodermin.

Affinity Labels↗
PubMed1986

Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding.

In previous work we have shown that puromycin photoaffinity labels two proteins, L23 and S14, from separate sites of high affinity on Escherichia coli ribosomes [Jaynes, E. N., Jr., Grant, P. G., Giangrande, G., Wieder, R., & Cooperman, B. S. (1978) Biochemistry 17, 561-569; Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274], that puromycin-modified S14 is separable from native S14 by reverse-phase high-performance liquid chromatography (RP-HPLC), and that ribosomal proteins prepared by RP-HPLC can be reconstituted into active 30S subunits [Kerlavage, A. R., Weitzmann, C. J., & Cooperman, B. S. (1984) J. Chromatogr. 317, 201-212]. In this work we definitively identify puromycin-modified S14 by tryptic fingerprinting, an analysis that also provides evidence that the single tryptophan-containing peptide in S14 is the site of puromycin photoincorporation. We show that reconstituted 30S subunits, in which all of the S14 present is stoichiometrically modified with puromycin and all other ribosomal components are present in unmodified form, lack Phe-tRNAPhe binding activity and further that 70S ribosomes containing such reconstituted 30S subunits have substantially diminished binding activity to both the A and P sites, as differentiated through use of tetracycline. Suitable control experiments strongly indicate that this loss of activity is a direct consequence of puromycin photoincorporation.

Affinity Labels↗
PubMed1986

Carbocyclic puromycin: synthesis and inhibition of protein biosynthesis.

The carbocyclic analogue of puromycin was prepared by the coupling of N-(benzyloxycarbonyl)-p-methoxy-L-phenylalanine to the racemic aminonucleoside (+/-)-9-[3 beta-amino-2 beta-hydroxy-4 alpha-(hydroxymethyl)cyclopent-1 alpha-yl]-6-(dimethylamino)purine, followed by separation of the diastereomers and subsequent removal of the Cbz blocking group. Kinetic studies indicate that carbocyclic puromycin is an excellent substrate for the peptidyltransferase reaction with both prokaryotic and eukaryotic ribosomes. A comparison of carbocyclic puromycin with previously synthesized analogues indicate that the furanosyl ring oxygen and the hydroxymethyl group of puromycin do not contribute to ribosomal binding, but both moieties contribute to the rate of product formation from the enzyme-substrate complex. Carbocyclic puromycin was equal to puromycin when evaluated for cytotoxicity using P-388 mouse lymphoid leukemia cells in culture.

Animals↗
PubMed1995

Puromycin aminonucleoside inhibits mesangial cell-induced contraction of collagen gels by stimulating production of reactive oxygen species.

Glomerular epithelial cell injury is thought to be the primary reason for the development of proteinuria in puromycin aminonucleoside nephrosis (PAN), the rat model of nephrotic syndrome. By comparison mesangial cells are considered resistant to the effects of puromycin. The purpose of the present study was to investigate whether puromycin in non cytotoxic concentrations caused mesangial cell dysfunction, with particular reference to cell-extracellular matrix interactions. Mesangial cells, when embedded in collagen gels, contact after exposure to minimal essential medium (MEM) containing fetal bovine serum (FBS). This contractility, measured by determining changes in area of the collagen gel, is inhibited by puromycin in a dose dependent manner from 2.5 micrograms/ml to 160 micrograms/ml. At these concentrations there is no alteration of cell viability as measured by the tetrazolium salt (MTT) method and trypan blue exclusion. Immunocytochemistry with rhodamine phalloidin reveals that actin filaments are not disrupted. The antioxidants, superoxide dismutase (SOD) and catalase as well as diphenylene iodonium (DPI), a flavoprotein inhibitor, not only counteracted the effect of puromycin on gel contraction, but also enhanced gel contraction when added to mesangial cells on their own. Aminotriazole, an inhibitor of endogenous catalase, inhibited mesangial cell-induced gel contraction in a dose dependent manner (5 mM to 40 mM), and this effect was completely reversed by addition of catalase. Mesangial cells preloaded with dihydrorhodamine and exposed to puromycin (5 micrograms/ml to 160 micrograms/ml) exhibited a dose dependent increase in rhodamine 123 fluorescence, indicating production of reactive oxygen species (ROS). This effect was blocked by the addition of DPI.(ABSTRACT TRUNCATED AT 250 WORDS)

Amitrole↗
PubMed1980

Immunoelectron microscopic localization of the site of photo-induced affinity labeling of the small ribosomal subunit with puromycin.

Ribosomes from Escherichia coli strain TPR201 (which lack N6,N6-dimethyladenosine) have been photoaffinity labeled with [3H]puromycin in the presence of chloramphenicol. Puromycin-modified 30S ribosomal subunits appear to be identical to untreated subunits in electron micrographs and are efficiently precipitated by antibodies to the puromycin analog N6,N6-dimethyladenosine. Electron micrographs of subunit-antibody complexes show ribosomal subunits to which an individual antibody molecule is bound and pairs of 30S subunits which appear to be crosslinked by a single IgG molecule. A predominant site of puromycin photoaffinity labeling has been identified from the apparent point of contact of antibody and ribosomal subunit. The puromycin site is localized to the small upper portion of the particle on the side opposite to the subunit platform. This location is close to that reported for ribosomal protein S14, the major puromycin-labeled protein in the small ribosomal subunit.

Affinity Labels↗
PubMed2000

Puromycin-induced lipid peroxidation in the cochlea of ApoE knockout mice.

Puromycin-treated apolipoprotein E (ApoE)-deficient mice were used to study lipid peroxidation (LPO) in the cochlea. Puromycin causes accelerated peroxidation of lipids and induces both inner ear and renal lesions in experimental animals presenting with abnormally high serum cholesterol. To prevent LPO, we used probucol, an effective inhibitor of LPO, and, simultaneously, also a lipid-lowering drug. The mice were given a single injection of the aminonucleoside of puromycin (25 mg/100 g). Polyclonal malondialdehyde and 4-hydroxynonenal antibodies were used to localize the LPO products. LPO products were mainly found in the stria vascularis of puromycin-treated mice. No LPO products were observed in the hair cells. LPO product immunoreactivity was clearly diminished in the animal group treated with both puromycin and probucol. In the cochlea of the ApoE-deficient mouse, puromycin affects mainly the stria vascularis due to the accelerated peroxidation of structural lipids. Probucol treatment prevented the formation of LPO products.

Animals↗
PubMed1980

Effect of puromycin on guinea pig lymphotoxin release and lectin-induced cellular cytotoxicity.

In order to confirm the role of guinea pig lymphotoxin (GLT) in lectin-induced cellular cytotoxicity (LICC), the effect of puromycin, a potent enhancer of GLT activity, on the LICC to target L . P3 cells induced by phytohemagglutinin (PHA) was investigated under serum-free conditions. LICC was completely inhibited by puromycin, when it was added at the initiation of LICC culture, because of the inhibition of the release of GLT from the effector lymph node cells. However, LICC was markedly enhanced when puromycin was added several hours after the initiation of LICC culture. The interpretation of these facts is that GLT release can be inhibited by puromycin, but that the GLT already released exerts an enhanced cytotoxic effect on the target cells in the presence of puromycin. Enhancement of the cytotoxicity by the addition of puromycin several hours after the initiation of LICC culture was observed even after the removal of the GLT present in the supernatant, suggesting that the morphologically intact target cells were already affected by GLT in the early stages of LICC culture.

Animals↗
PubMed2006

Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction.

In vitro blood-brain barrier (BBB) models using primary rat brain microvessel endothelial cells (BMEC) are often hampered by a lack of culture purity and poor barrier properties. To address these problems, the translation inhibitor puromycin was used to purify rat BMEC cultures. BMEC purities of 99.8% were routinely attained using puromycin treatment, and this technique proved to be far superior to other purification methods of similar difficulty. In contrast to cultures without puromycin treatment, purity of puromycin-treated cultures was unaffected by initial seeding density. Next, rat BMEC monolayer transendothelial electrical resistance (TEER) was increased by glucocorticoid treatment with either corticosterone (CORT) or hydrocortisone (HC), and a corresponding decrease in monolayer permeability to small molecules was observed. Importantly, cultures treated with both puromycin and glucocorticoid attained significantly higher TEER values (CORT 168 +/- 13 Omega x cm2; HC 218 +/- 66 Omega x cm2) than those treated by the glucocorticoid alone (CORT 57 +/- 5 Omega x cm2; HC 70 +/- 2 Omega x cm2). Glucocorticoid induction resulted in BMEC morphological changes that accompanied the increases in TEER, and BMEC tight junctions exhibited improved integrity as visualized by the localization of tight junction proteins zonula occluden-1, occludin and claudin-5. The combined use of puromycin and glucocorticoid therefore provides an in vitro system that is well suited for molecular level BBB investigations.

Animals↗
PubMed1985

Measurement of protein synthetic activity by determination of peptidyl[3H]puromycin formation in liver slices after ethanol administration.

We investigated the utility of [3H]puromycin as an alternate and adjunct precursor to amino acids for measuring protein synthetic activity in rat liver slices. Slices were incubated in the presence of either [3H]puromycin or radiolabeled valine to compare the incorporation of these isotopic precursors into nascent hepatocellular proteins. Compared to liver slices from controls, comparable decreases in the incorporation of both [3H]puromycin and labeled valine were observed in experiments using slices from fasted rats and in slices preincubated with 25 mM ethanol. Radiolabeling of nascent polypeptides with either [3H]puromycin or labeled valine in liver slices from rats fed a liquid diet containing ethanol was also decreased compared to slices from pair-fed control and chow-fed animals. Our results demonstrated the validity of using [3H]puromycin to detect changes in protein synthetic activity under these conditions. The potential advantage of using [3H]puromycin for in vivo studies is discussed.

Alcoholism↗