Puromycin Research Articles

General properties of 14C-L-Valine-binding to human brain tissue.

L-Valine (VAL) binding is saturable, reversible, linear within a protein concentration of 0.2 to 0.8 mg prot./ml, pH dependent (pH-optimum 7.1 to 7.4), temperature dependent with extremely low binding at 0-4 degrees C; the assay runs at 37 degrees C; it is ion dependent (Bmax:CaCl2 (5mM): + 100%; NaCl (5 mM): -50%); very sensitive to TRITON X-100 (Bmax at 0.01%; -70%); N-ethylmaleimide (0.5 mM): Competitive inhibition; no change in non-specific binding; there are no effects by puromycin plus GTP; VAL binds to synaptic membranes of human frontal cortex with KD-values several fold lower than that observed with homogenates, the distribution of VAL-binding is different in various human brain areas and can be inhibited by L-leucine, L-isoleucine, L-serine, DL-threonine, glycine and D-valine, while aromatic amino acids, beta-alanine, GABA, taurine, glutamate, proline and gamma-glutamyltaurine have no effects of 10(-3) M.

The preparation of puromycin antibody and its use in enzyme immunoassay for the quantification using beta-D-galactosidase as a label.

An antibody specific for puromycin (PU) was prepared by immunization of rabbits with a PU conjugate of bovine serum albumin, which was newly synthesized by coupling PU to mercaptosuccinylated bovine serum albumin via a cross-linker, N-(gamma-maleimidobutyryloxy)succinimide. Enzyme labeling of PU was performed using beta-D-galactosidase [EC 3.2.1.23] via N-(m-maleimidobenzoyloxy) succinimide. An ultrasensitive and specific enzyme immunoassay for PU was developed utilizing these reagents by a double antibody technique. The standard curve of the assay was linear in the range of 2 pg to 100 pg, and the lower limit of detection was 28.2 pm (2 pg/tube); so the enzyme immunoassay was found to be approximately 326,000 times more sensitive than a microbiological assay. Further, the enzyme immunoassay is free from interference by any purine or pyrimidine analogs, or by other drugs commonly used for the inhibition of protein synthesis. Using this assay, drug levels were easily determined in rat tissue following PU administration. Since PU is extensively available as an inhibitor of protein synthesis, the enzyme immunoassay should provide useful tool for developing biochemical and toxicity studies of PU.

Effects of cycloheximide and puromycin on learning and retention in the cockroach, P. americana

A new one-session T-maze training procedure for cockroaches, in which animals were trained to turn right or left to avoid shock, is described. This paradigm was utilized to investigate effects of protein synthesis inhibiting drugs on learning and retention. Cycloheximide (CXM), which inhibited protein synthesis by over 90% during the training period, did not impair acquisition and did not produce retention deficits at any interval up to 1 day after training. Puromycin (PURO), which inhibited protein synthesis by about 70% during the training period, produced amnesia 5 hr after training, while acquisition was not affected. Thus invertebrates, as well as vertebrates, are susceptible to amnesic effects of puromycin. Although PURO-injected animals showed retention deficits as measured by the number of correct turns, no retention deficit occurred for the behavioral modification consisting of an increase in runway time during the training period. Therefore, PURO appears to show specificity for the different types of longer-term memories that are formed in a training situation.

Inhibition of protein synthesis antagonizes induction of sister-chromatid exchanges by exogenous agents

Cycloheximide (CH) and puromycin (PM) strongly antagonize induction of sister-chromatid exchanges (SCEs) by exogenous agents regardless of the mechanisms for initiating damage. 5-Bromodeoxyuridine (BUdR) substitution was used to monitor SCEs, but the background level of BUdR-induced SCEs was unaffected by the presence of protein inhibitors. Antagonism between DNA-damaging agents and protein inhibitors was strongest in euchromatic regions. Possible relationships between SCE formation and the mechanism of antagonism by protein inhibitors are discussed.

"Reduced" analogs of puromycin. Synthesis of 3'-O-(L-2-amino-3-phenylpropyl)-N6,N6-dimethyladenosine and the corresponding 2' isomer

"Reduced" analogs of puromycin. Synthesis of 3'-O-(L-2-amino-3-phenylpropyl)-N6,N6-dimethyladenosine and the corresponding 2' isomer

Studies on the validity of urinary enzyme assay in the diagnosis of drug-induced renal lesions in rats

The validity of urinary enzyme assays in the diagnosis of renal lesions was studied in rats for evaluation in comparison with other tests such as urinalysis and serum biochemical assay. Tubular damages of slight or severe degree were induced by daily intramuscular administration of 500 mg/kg or 1,000 mg/kg of kanamycin (KM) for 7 and 6 days respectively. Glomerular lesions were induced subcutaneous injection of 30 mg/kg of puromycin (PM) for 6 days. The enzymes, assayed daily before and after the onset of administration, were lactic dehydrogenase (LDH), aspartate aminotransferase (GOT), alkaline phosphatase (AI-P), acid phosphatase (Ac-P), leucin aminopeptidase (LAP) and γ-glutamyl transpeptidase (γ-GT). The distribution of these enzymic activities in the kidneys was histochemically examined. Assays of LDH and GOT on the kidney tissue homogenate were also conducted. Histological alterations of the kidneys were examined in the rats sacrificed after termination of administration. Significant elevation of the LDH and GOT urine levels were observed within 24 hours in rats treated with 500 mg/kg of KM with the high enzyme levels being maintained throughout the administration period, while the other enzymes remained within pretreatment levels or were only slightly elevated. Serum urea nitrogen (BUN) and creatinine levels remained unchanged with no abnormality being found in the urinalysis of this group. In the group treated with 1,000 mg/ kg of KM, concomitant rising of all urine enzyme levels was observed, with elevation of the LDH and GOT levels being extreme while the BUN and serum creatinine levels rose only at the termination of the administration period. The BUN level rose earlier than did most of the urinary enzymes in puromycin-treated rats. Depletion of LDH activity was histochemically demonstrated in the kidneys where no histological alteration was observable, while the activities of AI-P, γ-GT, and LAP showed no distinct changes until the tabular destruction became severe. Upon termination of both KM and PM administration, the depletion of LDH and GOT activities was noted in a kidney homogenate assay for the enzymes. These results clearly showed that among the enzymes studied, the LDH and GOT urine levels are the most sensitive indicators for detecting proximal tubular lesions induced by KM. It was further demonstrated that the urinary enzyme assay in combination with serum BUN measurement is an effective examination for distinguishing tubular from glomerular lesions.

Puromycin: two distinct behavioral effects with different temporal parameters in the pigeon.

Pigeons were injected intracerebrally with either puromycin (PM) or control saline solution following training for one 12-min session on a visual discrimination. Injections were made either immediately following training, 1 hr later or 24 hr later. Retention testing 3 days after training showed that PM produced marked amnesia in the first two groups, but had no effect in the 24 hr condition. However, all PM groups were retarded subsequently in the number of days required to reach a 90% discimination criterion. This differentiation of two separate behavioral effects with different temporal gradients suggests that PM may be working through two distinct physiological mechanisms.

Effect of antibiotics on retention of visual discrimination training and on protein synthesis in the pigeon

Forty-three pigeons were trained for one day on a visual discrimination (horizontal vs. vertical stripes) and then immediately injected with either puromycin (PM), cycloheximide (CXM), control saline, or combined PM and CXM solution. PM produced a marked amnesic effect, CXM a weaker effect. PM injected animals (but not CXM's) also took significantly more than 1 day longer than controls to reach criterion levels, indicating an effect on continued acquisition of the discrimination beyond the amnesia for Day 1. Combination with CXM did not attenuate the PM effects. Protein inhibition profiles showed maximum PM activity of 90% inhibition, whereas CXM's maximum was 98% and was distributed more widely throughout different brain regions. In a second experiment, PM (N = 8), CXM (N = 8) or saline (N = 8) were again injected immediately after training, but the S+ was reversed on all postinjection training trials. In this case, PM animals were superior in percentage of S+ pecks on Day 2 to controls and CXM's. Further, the continued acquisition deficit was absent in the PM group, indicating that this effect is highly specific to those behavioral circuits active at the time of injection.

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

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

180) - Puromycin blockage of anti-Inflammatory of glucocorticoid

180) - Puromycin blockage of anti-Inflammatory of glucocorticoid

Correlation between proton extrusion and stimulation of cell enlargement effects of fusicoccin and of cytokinins on leaf fragments and isolated cotyledons

Fusicoccin (FC), a diterpene glucoside which stimulates growth of pea internode segments and of leaf fragments, is shown to induce in isolated cotyledons from germinating squash and radish seeds, an effect on cell enlargement markedly greater than that of benzyladenine (BA) and kinetin. The stimulation of cell enlargement induced in leaf fragments by FC, and in cotyledons by FC or by the cytokinins, is accompanied by a proportional rapid decrease in the pH of the medium, similar to the one previously observed when the pea internode segments are treated with auxin or with FC. This apparent proton extrusion accompanying growth stimulation by auxins, cytokinins and FC, is markedly reduced by the phosphorylation uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP) and by the protein synthesis inhibitors cycloheximide (CH) and puromycin (PUR). A comparison of these results with those of a parallel investigation shows that the effects of growth stimulators and of the inhibitors on cell enlargement and on proton extrusion are accompanied by corresponding changes in the negative transmembrane electric potential. A model is proposed for the interpretation of the interrelationship between these effects.

Immunologic competence of alveolar cells. II. Modification of the plaque-forming response by inhibitors, tolerance, and chronic stimulation.

SUMMARy _ After intranasal immunization of rabbits with either sheep erythrocytes or human serum albumin, plaque-forming cells were detected among cells collected by alveolar lavage. Direct plaque formation was inhibited by 10 JLg of actinomycin D per ml, 10 JLg of puromycin per ml, 0.02 M 2-mercapto­ ethanol, guinea pig anti-rabbit immunoglobulin ~I serum, and antilymphocyte serum. Thus, direct plaque formation required protein synthesis and was mediated by immunoglobulin M antibody. During a primary response to human serum albumin, 75 per cent of the plaque-forming cells could be detected with the direct plaque assay and 25 per cent required anti-rabbit immunoglobulin G serum. After prolonged immunization, two thirds of the plaque-forming cells required the facili­ tating anti-immunoglobulin G serum. Intraperitoneal injection of sheep erythrocytes into newborn rabbits produced a state of tolerance accompanied by inhibition of plaque formation after subse­ quent intranasal immunization. Alveolar exudates contain cells that synthesize immunoglobulin G and immunoglobulinM anti­ bodies. These cells are a likely source of some of the immunoglobulins in respiratory secretions.

Properties of phage T4 messenger RNA synthesized in the absence of protein synthesis

Phage T4 specific messenger RNA (mRNA) synthesized by Escherichia coli B cells infected in the absence of protein synthesis was studied. Protein synthesis was inhibited by treatment of bacterial cells with the antibiotics chloramphenicol (CM), puromycin (PM), or amicetin (AMI) or by K + depletion of the K + accumulation defective mutant strain B207. As shown by hybridization competition experiments only “immediate early” mRNA was made in the presence of CM or in the absence of K +. When inhibition of protein synthesis was released and further RNA synthesis was blocked with rifampicin (RIF), the immediate early mRNA accumulated either in the presence of CM, PM, or AMI or in the absence of K + could serve as messenger for the synthesis of the protein necessary for resistance to lysis from without, but could not serve as messenger for the synthesis of two early phage enzymes, deoxynucleotide kinase (TMP kinase) and deoxycytidine triphosphatase (dCTPase). RNA synthesized in the presence of CM could serve as mRNA for the synthesis of the early enzyme deoxycytidylate hydroxymethylase (HMase). However, RNA synthesized in the absence of K + or in the presence of PM or AMI could not direct the synthesis of this enzyme. The results indicate that although phage mRNA is made in the absence of concomitant protein synthesis, it contains only a very restricted portion of the RNA made during a normal infection. Furthermore, the RNA made in the presence of CM is not identical with that synthesized during inhibition of protein synthesis by the other methods employed.

Fragmentation and Nondisjunction of Barley Chromosomes After the Treatment of Chloramphenicol and Cycloheximide

Barley root meristematic cells were treated with three antibiotics known as the inhibitors of protein synthesis, i.e., chloramphenicol (CAP), cycloheximide (CH) and puromycin (PU), at each stage of the interphase in first cell cycle. The results indicated that both CAP and CH induced the chromosome fragments of dot type after the treatment in early S, late S or early G2 cells, but PU produced no chromosomal aberrations. These chromosome fragments seemed to be produced by a 1-hit event.On the other hand, S cells treated with CAP or CH markedly reduced the number of chromosomes in a cell. The chromosome reduction is likely due to the nondisjunction of chromosomes.Moreover, CH inhibited the protein synthesis as revealed by the incorporation of 3H-lysine, but CAP did not depress at the concentrations used. It was discussed that chromosome fragments are attributable to the chromosome nondisjunction.

Depressant effect of puromycin on the sympathetic ganglionic function: A study of the possible site and mechanism of action

The possible site and mechanism of action of puromycin (PU) on sympathetic ganglion isolated in vitro are investigated. We have observed that: (1) the depressant effect of PU on functional activity is higher on active than on resting ganglia; (2) PU interference with the divalent cations of the medium is negligible; (3) PU depresses the post-ganglionic action potentials to a higher extent than those recorded from preganglionic fibres; (4) synaptic potentials (recorded from curarised ganglia) are depressed to the same extent as post-ganglionic action potentials; (5) PU depresses the postganglionic response evoked by chemical stimulation with acetylcholine; (6) in the presence of PU, ganglia incubated with tritiated choline under sustained activity have a greater content of labelled acetylcholine. It is therefore suggested that PU exerts its action at both a pre- and a post-synaptic level.

Glycoprotein synthesis by the trachea.

Slices of rat trachea incubated with d-glucosamine-1-14C incorporated radioactivity into protein, that is, material insoluble in cold ethanol, cold and hot trichloroacetic acid and not extracted by lipid solvents. Incorporation was dose-dependent, occurred at an optimal pH of 8 and was decreased in the presence of glucose. A small amount of d-glucosamine-14C was metabolized to 14CO2. Compared to l-leucine-14C, the curve of glucosamine incorporation rose less steeply initially, and the ascending limb assumed a sigmoid shape. Puromycin inhibited incorporation of radioactivity from glucosamine by 44 per cent to 72 per cent and from leucine by more than 95 per cent. The microsomal fraction of tracheal homogenate had the highest specific activity of the subcellular fractions throughout a four-hour incubation. The alcohol-insoluble radioactive product was mostly resistant to hyaluronidase but was made soluble by Pronase®; treatment with neuraminidase, and acid hydrolysis yielded radioactive materials having the...

Rat glomerular basement membrane composition and metabolism in aminonucleoside nephrosis.

Abstract Rats treated with aminonucleoside of puromycin had a significant increase in incorporation of 3 H-proline and conversion to 3 H-hydroxyproline in glomeruli and glomerular basement membrane with the onset of proteinuria. There were no significant changes in the specific activity of hydroxyproline in bone or urine at any time during the course of the disease. The half-life of glomerular hydroxyproline measured 48 to 192 hours after the administration of 3 H-proline was 15 days. Two per cent of glomerular basement membrane hydroxyproline was solubilized by dilute acid and in this fraction the specific activity of hydroxyproline was higher than whole glomerular basement membrane. No changes in dilute acid solubility or amino acid composition of glomerular basement membrane were seen in aminonucleoside-treated animals with proteinuria.

Effects of antibiotics on androgenization of the neonatal female rat.

In order to elucidate the possible mechanisms of action of neonatally administered androgen in inducing permanent sterility, actinomycin D (Act-D) or puromycin (PM), inhibitors of DNA-dependent RNA and protein synthesis, were injected sc into 5-day-old female rats simultaneously with 30 μg testosterone propionate (TP), or 2,4 or 6 hr before, or 2,4,6 or 12 hr after TP injection. Act-D inhibited the effect of TP when the antibiotic was administered 6 or 4 hr before the injection of TP. When Act-D was given simultaneously with, or 2 hr before the injection of TP, however, it had no effect on the sterilizing action of TP. On the other hand, Act-D administered 2 or 4 hr after TP injection markedly inhibited the action of TP. PM significantly inhibited the action of TP only when injected 4 hr after the androgen; its inhibitory effect was much less than that of Act-D at the dose used in this experiment. At 90 days of age, however, none of these groups showed an incidence of sterility significantly different fro...

Effects of Antibiotics, Inhibitors against Protein Synthesis, on Tobacco Mosaic Virus Multiplication and the Host Metabolism

Blasticidin S (BcS) and puromycin (PM), inhibitors against protein synthesis in fungi and higher organisms, inhibited tobacco mosaic virus (TMV) multiplication in tobacco leaf-discs, whereas chloramphenicol (CM), inhibitor against bacterial protein synthesis, did not inhibit. TMV produced by antibiotics treatment had the same infectivity as that produced by untreatment. BcS and PM inhibited 14C-leucine incorporation into the cytoplasmic protein of tobacco leaf-discs, but CM did not. CM reduced more rapidly the chlorophyll content in leaf-discs than BcS and PM. 14CO2-incorporation into infected leaf-discs was strongly inhibited by CM, but not by BcS and PM. Although, under BcS and PM treatments, the rate of 14C-amino acids and 14CO2-incorporations into TMV was in proportion to the amount of TMV produced, the rate of the former incorporation rather increased by CM under both light and dark conditions and that of 14CO2-incorporation was decreased, whereas the amount of TMV was not altered by CM. Most of 14CO2 incorporated into TMV located in TMV protein. Site of TMV synthesis in tobacco leaf-discs and the role of photosynthetic activity of chloroplasts are discussed.

Restoration of expression of memory lost after treatment with puromycin.

Restoration of expression of memory lost after treatment with puromycin.