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Studies on memory: evidence for a widespread memory trace in the neocortex after the suppression of recent memory by puromycin.

Intracerebral injections of puromycin in mice have provided evidence that the hippocampal area of the brain contains the memory trace of recently learned maze behavior and that, with time, the trace is also found in the neocortex. The present experiments were planned to determine whether this enlargement of locus would develop after the blockage of recent memory in the hippocampus by puromycin, i.e., whether the neocortical locus is established if hippocampal memory is suppressed. The experiments depend upon our earlier finding that the memory blockage caused by puromycin can be removed by small intracerebral injections of saline. Control experiments showed that saline injected into the frontal cortex three days after training did not release the puromycin blockage of memory in the hippocampal area. Injections of saline into the frontal cortex 6 and 11 days after training did, however, release memory in mice whose recent memory had been suppressed by puromycin; we interpret this result to mean that the memory trace appears widely in the cortex despite the suppression of recent memory in the hippocampus. The theoretical implications of this finding are briefly discussed.

Animals↗

Reversal of pactamycin inhibition of methionyl-puromycin synthesis and 80S initiation complex formation by a ribosomal joining factor.

A crude mixture of polypeptide chain initiation factors (0.5 M KCl ribosomal wash) from reticulocyte ribosomes was fractionated by DEAE-cellulose column chromatography. Among several initiation factors obtained from the column, one factor eluting at 0.22-0.25 M KCl showed a remarkable ability to overcome the inhibition of Met-puromycin and 80S initiation complex formation caused by the antibiotic, pactamycin. Earlier experiments had shown that pactamycin does not prevent the binding of Met-tRNA(f) to the small ribosomal subunit but does interfere with the joining of the 60S ribosomal subunit to form the 80S initiation complex. A Lineweaver-Burk plot of initial rates of Met-puromycin formation showed that the interaction of the factor and pactamycin was of a competitive type. In the absence of the factor, [(35)S]Met-puromycin was not synthesized and [(35)S]Met-tRNA(f) bound only to the small ribosomal subunit. The amount of [(35)S]Met-tRNA(f) bound to 80S ribosomes bearing endogenous mRNA and the amount of [(35)S]Met-puromycin formed were directly related to the amount of factor added. Thus, this factor can be termed a "joining factor," and a simple assay of its activity can be devised based on its ability to overcome the pactamycin inhibition of the puromycin reaction.

Antibiotics, Antineoplastic↗

The fine structure of puromycin-induced changes in mouse entorhinal cortex.

Bitemporal intracerebral injections of puromycin in mice suppress indefinitely expression of memory of avoidance-discrimination learning. Ultrastructural studies of the entorhinal cortex of puromycin-treated mice revealed the following: (a) Abnormalities were not observed in presynaptic terminals and synaptic clefts; many postsynaptic dendrites or somas contained swollen mitochondria. (b) Dispersion of polyribosomes into single units or condensation of ribosomes into irregular aggregates with loss of "distinctiveness" was noted in a few neurons 7-27 hr after puromycin treatment. (c) Cytoplasmic aggregates of granular or amorphous material were frequently noted within otherwise normal neuronal perikarya. (d) Mitochondria in many neuronal perikarya and dendrites were swollen. Mitochondria in axons, presynaptic terminals, and glial cells were unaltered. The relationships between these lesions and the effect of puromycin on protein synthesis and memory are examined. It is suggested that the disaggregation of polysomes is too limited to explain the effect of puromycin on memory. Special emphasis is given to the swelling of mitochondria. The possible mechanisms and the significance of this lesion are discussed.

Animals↗

A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin.

Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 microg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.

Animals↗

Two sensitivity levels of cattle oocytes to puromycin.

Germinal vesicle breakdown (GVBD) in cumulus-enclosed and denuded cattle oocytes was sensitive to puromycin at concentrations at or above 50 micrograms/ml. Media supplemented with 5-25 micrograms/ml of puromycin did not significantly reduce either rate or sequence of GVBD after 8 h of culture (82-96% GVBD). In concentrations of 50, 75, and 100 micrograms/ml, GVBD occurred in 15, 4, and 2% of oocytes, respectively. However, 50 micrograms puromycin/ml did postpone the time sequence of GVBD, since all treated oocytes underwent GVBD after 20 h of culture. Oocytes arrested in the germinal vesicle (GV) stage possessed GV filled with highly condensed bivalents. The puromycin block (100 micrograms/ml) was fully reversible, and the time sequence of GVBD was two times faster than in control medium. Proteins important for GVBD were synthesized during the first 4 h of culture, and 81% of oocytes underwent GVBD when puromycin (100 micrograms/ml) was added after 4 h of preincubation in control medium. The first polar body (I PB) expulsion was more sensitive to inhibition of protein synthesis, as shown by the observation that 2.5 and 5 micrograms puromycin/ml significantly (69 and 61%) reduced the incidence of Metaphase II, and 10 micrograms/ml highly significantly (31%) reduced it. The I PB expulsion in concentrations of 25 and 37 micrograms puromycin/ml was less than 5%. The subsequent culture in puromycin (8 h) and 6-dimethylaminopurine (8 h) proved that nuclear membrane breakdown is less sensitive to inhibition of protein phosphorylation than the process of chromatin condensation.

Adenine↗

Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties.

One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 microg/mL puromycin for the first 2 days of culture or 3 microg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 x 10(-6) cm/s) and a high electrical resistance (up to 500 Omega x cm(2)). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for cellular and molecular biology studies as well as pharmacological investigations.

Animals↗

Puromycin effect on memory may be due to occult seizures.

Intracerebral injections of puromycin, which have been shown to impair memory 3 hours after training, increase the susceptibility of mice to seizures after administration of normally subconvulsive doses of pentylenetetrazol. Cycloheximide, which antagonizes the puromycin-induced amnesia 3 hours after training, also antagonizes the puromycin effect on susceptibility to seizure. The anticonvulsant diphenylhydantoin antagonizes the puromycin effect on memory. The puromycin effect on memory may be due to occult seizures.

Amnesia↗

Chloramphenicol binding site with analogues of chloramphenicol and puromycin.

The effect of a series of puromycin analogues and aminoacyl chloramphenicol derivatives on poly(U,C)-directed polyphenylalanine synthesis in an Escherichia coli cell-free system was examined. A comparison between the structures and activities of the puromycin and chloramphenicol analogues was made to examine the proposal that ribosomal binding sites for both antibiotics overlap. Our results suggest that the dichloroacetamido group in the chloramphenicol molecule does not correspond to the role of the aminoacyl moieties of either puromycin or aminoacyl transfer ribonucleic acid. These results comparing the structures and activities of puromycin and chloramphenicol analogues also seem inconsistent with a common binding site for the p-substituted phenyl moieties of the two antibiotics. Previous data have indicated that both sites are mutually affected by the prior binding of either antibiotic. Although it is possible that chloramphenicol and puromycin may have overlapping bindings sites, no common structural features between the two antibiotics are supported by our data.

Binding Sites↗

Puromycin effect on amino acid transport: differential rates of carrier protein turnover.

In fetal rat calvaria, puromycin selectively inhibited the uptake of certain groups of amino acids. Puromycin treatment decreased the uptake of glycine, L-proline, and alpha-aminoisobutyric acid but was without effect on the active uptake of all other amino acids tested. In studies of alpha-aminoisobutyric acid uptake, puromycin decreased the maximal transport velocity by 70% but had no effect on the affinity of the transport system for the amino acid. With puromycin treatment, the fall-off in rates of alpha-aminoisobutyric acid uptake was first order with a half-life of 68 min. Insulin treatment increased this half-life to 118 min. These findings suggest that protein components of specific transport systems are degraded at varying rates after puromycin blockade of protein synthesis. Hormones that stimulate amino acid transport (e.g., insulin) may decrease the rate of degradation of these protein components.

Amino Acids↗

[Sex chromosome analysis and IGF-II expression on activated human unfertilized oocytes after ICSI with calcium ionophore A23187 and puromycin].

OBJECTIVE: To investigate the sex chromosomes and the expression of insulin-like growth factor-II (IGF-II) on activated human unfertilized oocytes after intracytoplasmic sperm injection(ICSI) with calcium ionophore A23187 and puromycin. METHODS: All 95 discarded oocyes that showed no evidence of fertilization at 16-18 h after in vitro maturation and intracytoplasmic sperm injection cycles (IVM-ICSI)/conventional ICSI were exposed to calcium ionophore A23187 (5 micromol/L) for 5 min and then were incubated with puromycin (10 microg/mL) for 4 h. After activation, the oocytes were cultured in vitro for 3-5 days. The sex chromosome analysis was performed by dual color fluorescence in situ hybridization. The expression of IGF-II on the activated embryos, normal embryos, and parthenotes was examined. RESULTS: The combination of calcium ionophore A23187 with puromycin could activate the unfertilized oocytes 22 h after ICSI. The activated rate, cleavage rate, and quality of activated embryos of the IVM-ICSI group were similar to those of ICSI group, respectively. Sex chromosome analysis indicated that 8 male and 5 female embryos had been derived from two pronucleus and a second polar body. The expression of IGF-II on activated embryos and normal embryos was high and similar, which was much stronger than that of parthenotes. CONCLUSION: The combination of calcium ionophore A23187 with puromycin could effectively activate unfertilized oocytes 22 h after ICSI. Moreover, the unfertilized oocytes activated by calcium ionophore A23187 and puromycin had normal sex chromosomes and expression of IGF-II like the normal embryos. These suggest that oocyte activation may be considered as a remedial measure in the presence of total or nearly total fertilization failure in ICSI.

Calcimycin↗

Enkephalin degradation in the guinea-pig ileum: effect of aminopeptidase inhibitors, puromycin and bestatin.

Degradation of enkephalin by aminopeptidases has been established as an important functional mechanism that terminates the pharmacological action of enkephalins in the guinea-pig ileum. Aminopeptidases are a family of enzymes and little is known regarding the specificity of individual enzymes with respect to the degradation of enkephalins. Puromycin is a general inhibitor of aminopeptidases and bestatin is a more selective inhibitor of Leu-aminopeptidases and aminopeptidase B. Both agents are capable of inhibiting enkephalin degradation in broken cell preparations from brain. However, only bestatin enhanced the pharmacological response to enkephalin in the guinea-pig ileum and ileal longitudinal muscle. Bestatin enhanced the response to enkephalin in a concentration-dependent fashion. Furthermore, bestatin also decreased the formation of [3H]Tyr and increased [3H]Leu-enkephalin content after incubation of the guinea-pig ileum with [3H]Leu-enkephalin. In contrast, puromycin did not shift the concentration-response curve to Met-enkephalin in either the intact guinea-pig ileum or the ileal longitudinal muscle and, likewise, no alteration in the degradation of [3H]Leu- or Met-enkephalin occurred with puromycin. A small enhancing effect of puromycin on the duration of the inhibitory effect of enkephalin was observed only in the guinea-pig longitudinal muscle. This enhancement cannot be explained by an effect on enkephalin degradation, but may be related to some other action of puromycin. These data support the importance of aminopeptidase activity to the degradation of enkephalin and indicate that enzymes which have properties in common with Leu-aminopeptidases rather than arylamidases may be the primary aminopeptidases responsible for terminating the pharmacological actions of enkephalins in intact guinea-pig ileal preparations.

Aminopeptidases↗

Differential transport of cholesterol and oleic acid in lymph lipoproteins: sex differences in puromycin sensitivity.

Adult rats of both sexes were prepared with indwelling drainage catheters in the left thoracic lymphatic duct, and with duodenal infusion catheters. Control and puromycin-treated animals were administered an aqueous test emulsion containing [7alpha-(3)H]cholesterol and [1-(14)C]oleic acid, followed two hours later, by a tracer dose of [1-(14)C]-leucine. Successive 2-hr lymph samples were subjected to ultracentrifugal separations of the major lipoprotein classes. These were specifically extracted for lipids, and for DNA- and lipid-free protein. In both sexes, oleic acid absorption was largely associated with the d < 1.006 g/ml chylomicron fraction throughout the 6-hr experimental period. Small but consistent levels of labeled fatty acid appeared in the 1.006 < d < 1.019 g/ml VLDL fraction. However, with both sexes 25-35% of the absorbed cholesterol appearing in lymph was recovered in the VLDL fraction. Furthermore, there were statistically greater levels of cholesterol in this lymph fraction in females than in males. Cumulative protein levels and leucine incorporation into chylomicron proteins was comparable in both sexes. However, VLDL protein in the female was significantly greater than in the male and this difference was mimicked by the greater incorporation of leucine into VLDL proteins in the female. In males, there were no significant effects of puromycin on cholesterol or oleic acid absorption, despite a marked inhibition in chylomicron protein levels and leucine incorporation into this fraction. There was also no effect of the inhibitor on VLDL protein levels or on leucine incorporation into VLDL peptides. Cholesterol but not oleic acid absorption in females was significantly depressed by administration of puromycin, and this was largely attributed to a decrease in VLDL transport of the sterol. Also, unlike males, leucine incorporation into VLDL peptides was inhibited by 75% by puromycin administration. These results emphasize the importance of non-chylomicron transport of cholesterol during absorption and suggest a hormonal influence on intestinal VLDL synthesis in female rats.-Vahouny, G. V., E. M. Blendermann, L. L. Gallo, and C. R. Treadwell. Differential transport of cholesterol and oleic acid in lymph lipoproteins: sex differences in puromycin sensitivity.

Animals↗

Decrease of glomerular disialogangliosides in puromycin nephrosis of the rat.

Puromycin aminonucleoside nephrosis (PAN) is a model for human minimal change nephropathy induced in rats by injection of puromycin. In PAN, defective sialylation of a major sialoprotein of podocytes, podocalyxin, has been demonstrated and the consequent decrease of anionic charge suggested as a causative factor for increased glomerular permeability and proteinuria. Whether defective sialylation is a general feature of PAN affecting also glomerular glycosphingolipids is not known. We have shown that rat glomeruli are rich in disialogangliosides GD3 and O-acetyl GD3, the functions of which are not known. Here, we made a sequential analysis of the glomerular gangliosides, especially of GD3 and its O-acetyl derivative in acute PAN using immunohistochemical and biochemical techniques and compared the results with another rat model of glomerular disease, Heymann nephritis. The prominent immunohistochemical finding was the almost total disappearance of glomerular O-acetyl GD3 and a substantial decrease of its precursor GD3 peaking at 10 days after injection of puromycin. Segmental areas lacking these gangliosides remained in glomeruli still at 30 days after injection. The response was dose dependent. Semiquantitative analysis by thin layer chromatograms showed that O-acetyl GD3 was decreased by 41% already at 3 days and by 60% at 10 days after injection of puromycin. Also GD3, the immediate precursor of O-acetyl GD3, was decreased by 20 and 19%, respectively, at 3 and 10 days after injection. At 3 days after injection, overt proteinuria had not started. At these times, no other changes were observed in the glomerular gangliosides. The decrease of glomerular GD3 and O-acetyl GD3 indicates a decrease of GD3 synthase activity and perhaps of O-acetyltransferase activity in PAN nephrosis. As these changes preceded the overt proteinuria, they may have a causal relationship to it. In the glomeruli of Heymann nephritic rats, no similar changes were seen, suggesting that the sialylation defect is not due to proteinuria but is a consequence of targeted puromycin action on cells.

Albuminuria↗

The pur6 gene of the puromycin biosynthetic gene cluster from Streptomyces alboniger encodes a tyrosinyl-aminonucleoside synthetase.

The pur6 gene of the puromycin biosynthetic gene (pur) cluster from Streptomyces alboniger is shown to be essential for puromycin biosynthesis. Cell lysates from this mycelial bacterium were active in linking L-tyrosine to both 3'-amino-3'-deoxyadenosine and N6,N6-dimethyl-3'-amino-3'-deoxyadenosine with a peptide-like bond. Identical reactions were performed by cell lysates from Streptomyces lividans or Escherichia coli transformants that expressed pur6 from a variety of plasmid constructs. Physicochemical and biochemical analyses suggested that their products were tridemethyl puromycin and O-demethylpuromycin, respectively. Therefore, it appears that Pur6 is the tyrosinyl-aminonucleoside synthetase of the puromycin biosynthetic pathway.

Chemistry, Physical↗

INHIBITION OF RIBONUCLEIC ACID SYNTHESIS IN NEWCASTLE DISEASE VIRUS-INFECTED CELLS BY PUROMYCIN AND 6-AZAURIDINE.

Wilson, D. E. (Rensselaer Polytechnic Institute, Troy, N.Y.), and P. LoGerfo. Inhibition of ribonucleic acid synthesis in Newcastle disease virus-infected cells by puromycin and 6-azauridine. J. Bacteriol. 88:1550-1555. 1964.-Puromycin and 6-azauridine were used to inhibit protein and ribonucleic acid (RNA) synthesis during Newcastle disease virus replication. Viral RNA, measured by the incorporation of uridine-2-C(14), increased rapidly 6 to 7 hr after infection and reached a peak at 12 hr. Viral RNA synthesis was inhibited by puromycin added at the time of infection or at any time up to 4 hr after infection, but not thereafter. 6-Azauridine (3 mg/ml) inhibited over 90% of the viral RNA synthesis and inhibited protein synthesis to a small extent. Infected cells synthesized more viral RNA than did untreated controls when exposed to azauridine alone for 5 hr, followed by puromycin alone for 9 hr. It was concluded that immediately after virus infection, virus-specific proteins, which are necessary for viral RNA synthesis, were synthesized. Virus-specific protein synthesis may occur even when viral RNA synthesis is greatly inhibited. After virus-specific protein synthesis has taken place, viral RNA synthesis may take place when protein synthesis is almost totally inhibited.

Animals↗

Biochemical studies of the isolated rat glomerulus and the effects of puromycin aminonucleoside administration.

Using analytical subcellular fractionation techniques in combination with enzymic microanalysis, studies have been performed on the enzymology and organelle pathology of isolated glomeruli from normal rats and from animals with puromycin aminonucleoside nephrosis. There was a marked decrease in 5'-nucleotidase (plasma membrane) activity 2 days after puromycin aminonucleoside administration. In contrast, catalase (peroxisomes) and N-acetyl-beta-glucosaminidase (lysosomes) showed a significant rise during the first 6 days of treatment. By 10 days most marker enzymes showed a significant decrease in activity. The glomeruli were homogenized in isotonic sucrose and extracts were fractionated by isopycnic centrifugation on linear sucrose gradients in the Beaufay automatic small-volume zonal centrifuge. The properties of the principal organelles were characterized. ed. After 10 days of puromycin aminonucleoside treatment there were no marked differences in the centrifugal properties of these organelles. Morphological correlations with the biochemical changes are discussed and it is suggested that the plasma membrane changes represent the primary site of puromycin aminonucleoside damage.

Acetylglucosaminidase↗

Effect of puromycin on the ultrastructure of keratohyalin granules.

Keratohyalin granules (KHG) from control and puromycin-treated hamster cheek pouch epithelium were studied by electron microscopy. The animals were killed 1-4 hours after injection with 75-100 microgram puromycin per gram body weight; controls were injected with saline. The KHG from control animals were round or oval and consisted of a finely granular background matrix containing many small, round, homogeneous electron-dense deposits. The KHG from puromycin-treated animals showed marked variation in morphology and size. They consisted of a coarse granular or speckled matrix and round, club-shaped or comma-shaped electron-dense deposits. Since puromycin is known to inhibit protein synthesis, alterations in KHG structure support previous reports suggesting the protein nature of KHG.

Animals↗

Morphological aspects of type II alveolar pneumocytes following treatment with puromycin in vivo.

Ultrastructural modifications of type II pneumocytes (PNM-II) in mice were analysed 125 and 155 minutes after puromycin treatment (12 mg/100 gm at 0, 30, 60 and 90 minutes). A quantitative evaluation of the cell compartments was carried out and the inhibition of protein synthesis in PNM-II was monitored by light microscopic radioautography, following 3H-leucine injection. In electron micrographs, following a 125-minute puromycin treatment, the number and size of lamellar bodies, the precursors of lung surfactant material appeared markedly reduced. The multivesicular bodies (MVB), which are normally very frequent in PNM-II, had almost completely disappeared, as had composite bodies. Golgi saccules were dilated, while the area occupied by Golgi vesicles was enlarged. Observations following the 155-minute puromycin treatment showed a strong enhancement of these modifications. Smooth and coated vesicles of the Golgi area, as well as peroxisomes, did not appear modified by puromycin. Elongated zones of autophagy were more prevalent after 125-minute treatment than after the 155-minute one. Small bodies were frequently observed in the cytoplasm, near the Golgi zone. They were bounded by a smooth membrane and contained tiny vesicles and/or electron-dense lamellae similar to those present within the lamellar bodies. Parallel membranes formed folds, some of them in continuity with lamellar bodies, thus encircling portions of cytoplasm. These structures, which were few in number in controls, were very frequently observed in treated cells, mainly after the 125-minute treatment. These extensive alterations of PNM-II morphology appeared to be related to a disturbed production of pulmonary surfactant.

Animals↗