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PubMed2004

Antifibrotic effect of the Chinese herbs, Astragalus mongholicus and Angelica sinensis, in a rat model of chronic puromycin aminonucleoside nephrosis.

UNLABELLED: Nephrotic syndrome has long been treated in China with two herbs, Astragalus mongholicus and Angelica sinensis, which may have antifibrotic effects. METHODS: Rats with chronic puromycin-induced nephrosis were treated with Astragalus and Angelica 3 mL/d (n = 7) or enalapril 10 mg/kg/d (n = 7). Normal control rats (n = 7) received saline rather than puromycin, and an untreated control group (n = 7) received puromycin but no treatment. After 12 weeks, stained sections of the glomerulus and tubulointerstitium were evaluated for injury. Immunohistochemistry staining measured extracellular matrix components, transforming growth factor-beta1 (TGFbeta1), osteopontin, ED-1-positive cells, and alpha-actin. TGFbeta1 mRNA was assessed by in situ hybridization. Renin, ACE activity, angiotensin, and aldosterone were measured by radioimmunoassay or colorimetry. In the untreated rats, chronic renal injury progressed to marked fibrosis at 12 weeks. Astragalus and Angelica significantly reduced deterioration of renal function and histologic damage. Expressions of type III and IV collagen, fibronectin, and laminin also decreased significantly. This anti-fibrotic effect was similar to that of enalapril. The herbs had no effect on the renin-angiotensin system but did reduce the number of ED-1-positive, and alpha-actin positive cells and expression of osteopontin compared to untreated controls. The combination of Astragalus and Angelica retarded the progression of renal fibrosis and deterioration of renal function with comparable effects of enalapril. These effects were not caused by blocking the intrarenal renin-angiotensin system, but associated with suppression of the overexpression of TGFbeta1 and osteopontin, reduction of infiltrating macrophages, and less activation of renal intrinsic cells [corrected].

Angelica sinensis↗
PubMed1991

Effects of YM264, a novel PAF antagonist, on puromycin aminonucleoside-induced nephropathy in the rat.

We investigated the effects of YM264, WEB2086, methylprednisolone and ticlopidine on puromycin-induced nephropathy in the rat. Puromycin produces marked proteinuria, hypercholesterolemia, and hypoalbuminemia. The structurally differing PAF antagonists YM264 and WEB2086 inhibited proteinuria and improved hypercholesterolemia and hypoalbuminemia. Methylprednisolone also exhibited a beneficial effect on these variables. However, ticlopidine, a platelet inhibitor, showed no inhibitory effect on nephropathy. These results indicate that PAF may play a major role in puromycin-induced nephropathy in the rat, and that PAF antagonists may prove of therapeutic value in the treatment of nephropathy in humans.

Animals↗
PubMed1966

Life cycle analysis of mammalian cells. 3. The inhibition of division in Chinese hamster cells by puromycin and actinomycin.

Analysis of the effects of actinomycin and puromycin on the G(2) and mitotic parts of the life cycle in Chinese hamster ovary cells grown in suspension and synchronized by thymidine treatment has been carried out. Rates of division of partially synchronized cell populations were measured in the presence and absence of the drugs, and various controls were performed to test for absence of complex side effects. Actinomycin produces a block 1.9 hr before completion of division, while puromycin produces a block almost coinciding with the initiation of mitosis. Evidence is presented that the puromycin block may be a double one, inhibiting one kind of protein synthesis that virtually coincides with the beginning of mitosis and another that occurs about 8 min earlier. The data are interpreted in terms of the time interval between messenger formation and its associated protein synthesis in this region of the life cycle. The various events studied have been provisionally mapped in the G(2) and mitotic periods of the life cycle.

Animals↗
PubMed2001

Effect of activation with Ca ionophore A23187 and puromycin on the development of human oocytes that failed to fertilize after intracytoplasmic sperm injection.

OBJECTIVE: To determine the effect of sequential oocyte activation with calcium ionophore A23187 (A23187) and puromycin on oocytes that were unfertilized after intracytoplasmic sperm injection (ICSI). DESIGN: Laboratory examination. SETTING: The in vitro fertilization/embryo transfer unit in Tokushima University Hospital. PATIENT(S): Discarded unfertilized oocytes following ICSI. INTERVENTION(S): All 172 oocytes that showed no evidence of normal fertilization 18 hours after ICSI were exposed to A23187 (5 microM) for 5 minutes and consequently were treated with puromycin (10 microg/mL) for 5 hours. MAIN OUTCOME MEASURE(S): The activation rate, proportion of oocytes that showed two pronuclei with the second polar body (2PN2PB), and cleavage rate were calculated. Chromosomal analysis of the oocytes with 2PN2PB was also performed. RESULT(S): The treatment activated 146 out of 172 oocytes (84.9%) and 30.1% of the activated oocytes showed 2PN2PB. Sixteen of 25 oocytes with 2PN2PB (64.0%) cleaved. There was no difference in the activation rate, proportion of activated oocytes with 2PN2PB, or cleavage rate between oocytes that were injected with a motile spermatozoon and those injected with an immotile spermatozoon. Chromosomal analysis showed a normal diploid set of chromosomes (n = 46) in four of five analyzable oocytes. CONCLUSION(S): The sequential treatment of calcium ionophore A23187 and puromycin activates unfertilized oocytes after ICSI. The resultant oocytes with 2PN2PB can cleave.

Calcimycin↗
PubMed2000

Puromycin-sensitive alanyl aminopeptidase from human liver cytosol: purification and characterization.

A cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human liver cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of liver cells and renal tubules, and was ubiquitously localized in various human tissues.

Aminopeptidases↗
PubMed2006

Degradation of tau protein by puromycin-sensitive aminopeptidase in vitro.

Tau, a microtubule associated protein, aggregates into intracellular paired helical filaments (PHFs) by an unknown mechanism in Alzheimer's disease (AD) and other tauopathies. A contributing factor may be a failure to metabolize free cytosolic tau within the neuron. The buildup of tau may then drive the aggregation process through mass action. Therefore, proteases that normally degrade tau are of great interest. A recent genetic screen identified puromycin-sensitive aminopeptidase (PSA) as a potent modifier of tau-induced pathology and suggested PSA as a possible tau-degrading enzyme. Here we have extended these observations using human recombinant PSA purified from Escherichia coli. The enzymatic activity and characteristics of the purified PSA were verified using chromogenic substrates, metal ions, and several specific and nonspecific protease inhibitors, including puromycin. PSA was shown to digest recombinant human full-length tau in vitro, and this activity was hindered by puromycin. The mechanism of amino terminal degradation of tau was confirmed using a novel N-terminal cleavage-specific tau antibody (Tau-C6g, specific for cleavage between residues 13-14) and a C-terminal cleavage-specific tau antibody (Tau-C3). Additionally, PSA was able to digest soluble tau purified from normal human brain to a greater extent than either soluble or PHF tau purified from AD brain, indicating that post-translational modifications and/or polymerization of tau may affect its digestion by PSA. These results are consistent with observations that PSA modulates tau levels in vivo and suggest that this enzyme may be involved in tau degradation in human brain.

Alzheimer Disease↗
PubMed2001

Syntheses of puromycin from adenosine and 7-deazapuromycin from tubercidin, and biological comparisons of the 7-aza/deaza pair.

Protection (O5') of 2',3'-anhydroadenosine with tert-butyldiphenylsilyl chloride and epoxide opening with dimethylboron bromide gave the 3'-bromo-3'-deoxy xylo isomer which was treated with benzylisocyanate to give the 2'-O-(N-benzylcarbamoyl) derivative. Ring closure gave the oxazolidinone, and successive deprotection concluded an efficient route to 3'-amino-3'-deoxyadenosine. Analogous treatment of the antibiotic tubercidin [7-deazaadenosine; 4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine] gave 3'-amino-3'-deoxytubercidin. Trifluoroacetylation of the 3'-amino function, elaboration of the heterocyclic amino group into a (1,2,4-triazol-4-yl) ring with N,N'-bis[(dimethylamino)methylene]hydrazine, and nucleophilic aromatic substitution with dimethylamine gave puromycin aminonucleoside [9-(3-amino-3-deoxy-beta-D-ribofuranosyl)-6-(dimethylamino)purine] and its 7-deaza analogue. Aminoacylation [BOC-(4-methoxy-L-phenylalanine)] and deprotection gave puromycin and 7-deazapuromycin. Most reactions gave high yields at or below ambient temperature. Equivalent inhibition of protein biosynthesis in a rabbit reticulocyte system and parallel growth inhibition of several bacteria were observed with the 7-aza/deaza pair. Replacement of N7 in the purine ring of puromycin by "CH" has no apparent effect on biological activity.

Adenosine↗
PubMed1968

Biosynthesis of intestinal mucins. Effect of puromycin on mucoprotein biosynthesis by sheep colonic mucosal tissue.

1. Surviving sheep colonic mucosal tissue incorporated l-[U-(14)C]threonine when incubated in Krebs medium III at 37 degrees in an atmosphere of oxygen, into a well-characterized mucoprotein fraction, isolated by papain digestion of the incubated scrapings. 2. Acidic hydrolysis and chromatography of the labelled mucoprotein showed that threonine was the only constituent to become labelled. In the presence of puromycin the incorporation of l-[U-(14)C]threonine was considerably diminished (6.7 and 18.5% of control in duplicate experiments). Furthermore, puromycin also decreased incorporation of radioactivity from d-[U-(14)C]-glucose (48.0 and 31.6% of control) and (35)SO(4) (2-) (21.2 and 23.6% of control) into the mucoprotein fraction. 3. In a puromycin-inhibited system, with d-[U-(14)C]-glucose, where the overall specific radioactivity of the mucoprotein was 48% of control, the labelling of the individual monosaccharide constituents (as% of control) was: N-acetylneuraminic acid, 44%; N-glycollylneuraminic acid, 61%; hexosamines, 46%; fucose, 68%; galactose, 34%.

Animals↗
PubMed1970

Adrenalectomy and the suppression of memory by puromycin.

It has previously been shown that expression of memory of maze-learning in mice is blocked by puromycin injected intracerebrally one or more days after the training experience. Bilateral adrenalectomy before training has now been found to protect memory against the effects of puromycin. This protection is absent when adrenalectomy follows training. In view of control experiments, we conclude that adrenalectomy before training modifies factors necessary for the expression of memory and that this alteration makes puromycin ineffective in blocking memory.

Adrenal Glands↗
PubMed1995

Puromycin-sensitive aminopeptidase. Sequence analysis, expression, and functional characterization.

Among the molecular mechanisms that control the cell division cycle, proteolysis has emerged as a key regulatory process enabling cells to pass critical check points. Such proteolysis involves a cascade of enzymes including a multisubunit complex termed 26S proteasome. Here we report on the analysis of a novel mouse cDNA encoding the puromycin-sensitive aminopeptidase (PSA) and on its expression in COS cells and 3T3 fibroblasts. PSA is 27-40% homologous to several known Zn(2+)-binding aminopeptidases including aminopeptidase N. Immunohistochemical analysis revealed that PSA is localized to the cytoplasm and to the nucleus and associates with microtubules of the spindle apparatus during mitosis. Furthermore, puromycin and bestatin both arrested the cell cycle, leading to an accumulation of cells in G2/M phase, and ultimately induced cells to undergo apoptosis at concentrations that inhibit PSA. Control experiments including cycloheximide further suggested that the induction of apoptosis by puromycin was not attributable to inhibition of protein synthesis. Taken together, these data favor the novel idea that PSA participates in proteolytic events essential for cell growth and viability.

3T3 Cells↗
PubMed1966

Puromycin-induced necrosis of crypt cells of the small intestine of mouse.

Chemical and radioautographic analysis of the small intestine of mice injected intraperitoneally with puromycin revealed an immediate decrease of precursor incorporation into DNA and protein and a delayed decrease of precursor incorporation into RNA. In addition to this decrease of precursor incorporation, damage to the crypt cells, but not to the cells of the villus of the small intestine, was observed. Further examination of other dividing cells (spleen) and nondividing cells (liver and heart) of these mice showed again that only cells of actively dividing tissues were damaged. The metabolic inhibitors actinomycin D, cytosine arabinoside, actidione, and puromycin aminonucleoside were used in an attempt to clarify the mechanism of cell damage by puromycin. The results showed that there was no clear correlation between cell necrosis and the pattern of inhibition of synthesis of DNA, RNA, or protein.

Animals↗
PubMed1972

Effects of puromycin on the structure of rat intestinal epithelial cells during fat absorption.

This report provides information on the morphology of rat intestinal epithelial cells during fat absorption. In addition, the role of protein metabolism in this process has been evaluated by blocking its synthesis with puromycin and studying the fine structure of mucosal cells from rats at various times after fat intubation. The results indicate that SER-derived vesicles, containing fat droplets, migrate from the apical cytoplasm of the absorptive cell and fuse with saccules or vacuoles of the Golgi complex. Arguments are made that the Golgi complex is important in completing chylomicron formation and in providing appropriate enveloping membranes for the chylomicron. Such membranes may be necessary for Golgi vacuoles to fuse with the lateral cell membranes and release chylomicra. Puromycin treatment causes the absorptive cell to accumulate increased quantities of lipid that are devoid of membrane during fat absorption. In addition, puromycin-treated cells contain much less RER and Golgi membranes are strikingly decreased in number. In this paper we discuss the consequences of these abnormalities and suggest that continued protein synthesis by the RER is required in order to generate Golgi membranes. If such membranes are absent the cell's ability to discarge chylomicra is impaired and lipid accumulates.

Animals↗
PubMed1967

The circadian rhythm in photosynthesis in Acetabularia in the presence of actinomycin D, puromycin, and chloramphenicol.

Anucleate Acetabularia crenulata shows a circadian rhythm in photosynthesis. In this study, an oxygen electrode was employed to measure this photosynthetic rhythm in the presence and absence of the inhibitors, actinomycin D, chloramphenicol, and puromycin. High concentrations of the inhibitors were used: actinomycin D, 20-40 micrograms ml-1; puromycin, 30 and 100 micrograms ml-1; and chloramphenicol, 250 micrograms ml-1. The effectiveness of these inhibitors on protein synthesis was also measured under the same conditions used for the determination of rhythmicity. In spite of large effects of all three inhibitors on the incorporation of 14C leucine, no effect on the period or the phase of the photosynthetic rhythm was observed. The higher concentration of puromycin and chloramphenicol produced toxic effects which were expressed as a reduction in the amount of photosynthesis, but rhythmicity was still apparent. After 3 or 4 days' exposure to actinomycin, Acetabularia became resistant to its effect. Recovery was also observed in the ability to incorporate leucine. The implications of these results for theories of the basic oscillator responsible for circadian rhythmicity are discussed.

Acetabularia↗
PubMed1998

Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: a transient gene-integration marker for ES cells.

Gene targeting in embryonic stem (ES) cells is a powerful tool for generating mice carrying specifically designed mutations in the germline. Puromycin can completely kill ES cells within 24 to 48 h whereas G418 and hygromycin cannot. We have, therefore, proposed that the puromycin N-acetyltransferase ( pac ) gene, may be utilized as a transient gene-integration marker. Using a circular expression vector of cre and pac genes, Cre-mediated mutant cells were effectively enriched by pulse treatment of puromycin without stable integration of their genes. We have thus demonstrated the first application of pac as a transient gene-integration marker for ES cells.

Acetyltransferases↗
PubMed2004

Melatonin decreases apoptosis and expression of apoptosis-associated proteins in acute puromycin aminonucleoside nephrosis.

BACKGROUND: The anti-apoptotic properties of melatonin have been demonstrated previously in several in vivo and in vitro studies. Previous reports have shown increased apoptosis during puromycin aminonucleoside nephrosis (PAN). The aim of this study was to determine if melatonin (MEL) can prevent apoptosis and modify oxidative stress, an apoptosis inducer, in this experimental model. METHODS: Rats were injected intraperitoneally with puromycin aminonucleoside. In addition, by the intragastric route they received 1 mg/kg/day of MEL or vehicle 3 days before puromycin injection and throughout the experiment. Animals were sacrificed at weeks 1 and 2 of nephrosis and frozen renal sections were studied for apoptosis by TUNEL, for apoptosis-associated proteins by monoclonal and polyclonal antibodies, and for superoxide anion (O(2)(-)) by a histochemical method. Nitric oxide (NO), malondialdehyde (MDA) and reduced glutathione (GSH), and the activities of superoxide dismutase (SOD) and catalase were measured in homogenized kidney tissue by appropriate biochemical and enzymatic methods. RESULTS: Increases in apoptosis, p53, Fas and Fas-ligand were observed in nephrotic animals. MEL treatment decreased apoptosis at weeks 1 and 2 in the glomerular, interstitial and tubular compartments. This was accompanied by decreased expression of p53 (glomerulus, week 1; tubules, weeks 1 and 2), Fas (glomerulus and interstitium, week 2; tubules, weeks 1 and 2) and Fas-ligand (interstitum and tubules, week 2). Increased expression of Bcl-2-positive cells was observed at week 2 in all renal compartments in MEL-treated animals. High levels of O(2)(-) and NO generation and lipid peroxidation (MDA) were found in nephrotic animals. SOD and GSH remained unchanged, and only decreased catalase activity (week 1) was observed in PAN animals. Tendencies toward decreased values of O(2)(-) and MDA content along with recovery of catalase activity (week 1) were observed in MEL-treated nephrotic animals, but were insignificant in magnitude. MEL, however, did significantly downregulate pro-apoptotic genes and upregulated anti-apoptotic genes. CONCLUSIONS: The data demonstrate that, in PAN, melatonin has anti-apoptotic effects, which might in part be independent of the modulation of the oxidative status.

Animals↗
PubMed1980

Time-dependency of neurohypophyseal peptide attenuation of puromycin amnesia in mice.

[Ile3, Arg8]vasopressin (arginine-vasotocin), as well as the C-terminal tripeptides of the neurohypophyseal hormones arginine and lysine vasopressin, Pro-Arg-Gly-NH2 and Pro-Lys-Gly-NH2, were protective against puromycin-induced amnesia in mice when administered 24h before training. The N-protected tripeptide derivative, Z-Pro-Lys-Gly-NH2, was effective when given 5 days before training. The effectiveness of all peptides to attenuate puromycin-induced amnesia decreased as the interval between training and peptide treatment increased, indicating that the peptides influence memory processes, rather than general arousal. Z-Pro-Lys-Gly-NH2 was active at 24h after training, when the other peptides were no longer effective. Although it seems clear that neurohypophyseal hormones per se can attenuate puromycin-induced amnesia, these results are in line with the possibility that some portion of hormone action may be mediated via formation of longer-lived hormone fragments in the CNS.

Amnesia↗
PubMed1980

Effects of puromycin on the differentiation of the freshwater sponge: Ephydatia fluviatilis.

As could be expected, the most general effect of puromycin on the hatching and differentiation of freshwater sponges from their gemmules is complete inhibition. However, when gemmules are treated with 7-microgram/ml puromycin from the beginning of incubation, hatching does occur, and the escaping archaeocytes secrete an unusual number of spicules, while failing to differentiate in any other way or to undertake any morphogenesis. It is suggested that puromycin-treated archaeocytes could provide a suitable material for the study of spicule formation.

Animals↗
PubMed1995

Podocytic degeneration and regeneration in puromycin aminonucleoside nephropathy in the rat.

In various types of chronic glomerulonephritis, proliferation of mesangial and endothelial cells is often seen together with extracellular matrix accumulation. In contrast, the proliferation of visceral glomerular epithelial cells (podocytes) has not been detected in any disease in which these cells are the principal target of injury. To examine whether proliferation of podocytes occurs, puromycin aminonucleoside nephropathy was induced in Wistar rats by intraperitoneal injection of puromycin aminonucleoside (15 mg/100 g of bodyweight). In this nephropathy model of nephrotic syndrome with minimal change, the main lesions consisted of vacuolation and flattened foot processes of podocytes, which are the primary target of puromycin aminonucleoside. Urine protein excretion and proliferating cell nuclear antigen (PCNA) positive cells were examined. The PCNA+ podocytic count reached its maximum on day 3, indicating that the most marked podocytic proliferation occurs just after the injection of PAN. Twenty-four hour urinary excretion in this nephropathy model supported the hypothesis that mitosis of podocytes occurs in association with recovery on day 13.

Animals↗