Puromycin Research Articles

Herpes simplex virus 1 infection is required to produce ICP27 immunoreactive triplet forms when ribosomal aminoacyl-tRNA translocation is blocked by cycloheximide

Infected cell protein (ICP) 27 is an essential herpes simplex virus type 1 (HSV-1) phosphoprotein required for optimal viral DNA and early or late gene synthesis. Three slow-migrating immunoreactive species were detected using multiple anti-ICP27 antibodies following HSV-1 infection of HEp-2 and Vero cells in the presence of cycloheximide (CHX). Generation of the protein triplet moieties required transcription of the α27 gene. These forms were observed following infection with a series of recombinant viruses that produce truncated ICP27 polypeptides, suggesting that alternative splicing is not involved in the process. These ICP27 species were not observed following translation inhibition by puromycin (PUR). Synthesis of the triplet occurred by 6 hpi and CHX addition as late as 3 hpi still enabled their production. That the ICP27 species were detected in uninfected ICP27-expressing cells without CHX, but not in its presence, suggests a mechanism in which virus infection is required to produce the forms when ribosomal aminoacyl-transfer RNA (tRNA) translocation is blocked.

The Destabilization of IL-2 mRNA by a Premature Stop Codon and Its Differential Stabilization byTrans-Acting Inhibitors of Protein Synthesis Do Not Support a Role for Active Translation in mRNA Stability

AbstractTo investigate the role that translation plays in the stabilization of the IL-2 mRNA, we inhibited protein synthesis in both cis and trans. To block translation in trans, we utilized the inhibitors puromycin (PUR) and cycloheximide (CHX), which differentially effect polysome structure. We found that CHX enhances the stability of IL-2 mRNA in cells stimulated with anti-TCR Ab alone, but it inhibits CD28-induced message stabilization in costimulated cells. In contrast, PUR had a minimal effect on IL-2 mRNA stability in either the presence or absence of costimulation. The differential effects of these two inhibitors suggest that: 1) CHX is unlikely to stabilize the IL-2 mRNA by inhibiting the expression of a labile RNase; 2) CD28-mediated IL-2 mRNA stabilization does not require translation; and 3) IL-2 mRNA decay is not coupled to translation. To block translation in cis, we generated sequence-tagged IL-2 genomic reporters that contain a premature termination codon (PTC). In both the presence and absence of costimulation, these PTC-containing mRNAs exhibit drastically diminished stability. Interestingly, the addition of CHX but not PUR completely restored CD28-mediated stabilization, suggesting that CHX can block the enhanced decay induced by a PTC. Finally, CHX was able to superinduce IL-2 mRNA levels in anti-TCR Ab-stimulated cells but not in CD28-costimulated cells, suggesting that CHX may also act by other mechanisms.

Roles for interleukin-1beta, phorbol ester and a post-transcriptional regulator in the control of bradykinin B1 receptor gene expression.

Bradykinin B1 receptor (BKB1R) is involved in a variety of pathophysiological processes, particularly those related to inflammation. The gene for this receptor is known to be upregulated by interleukin (IL)-1beta, a proinflammatory cytokine. However, the molecular mechanisms involved in the regulation of the BKB1R gene expression have not been defined. We demonstrated that IL-1beta induces a rapid increase in BKB1R mRNA level and the binding of desArg10-kallidin in human embryo lung fibroblasts (IMR90). This increase in BKB1R mRNA level is protein synthesis-independent as indicated by treatment of cells with cycloheximide (CHX) or puromycin (PUR). By testing the IL-1beta effect on BKB1R mRNA degradation, we showed that the IL-1beta upregulation of BKB1R expression is achieved through both transcriptional activation and post-transcriptional mRNA stabilization. In addition to the IL-1beta effects, translation inhibitors, CHX and PUR increase the steady state BKB1R mRNA level by inhibiting BKB1R mRNA degradation. Removal of the CHX block with subsequent resumption of protein synthesis results in a sizable increase of desArg10-kallidin binding. Using signalling pathway inhibitors, we show that IL-1beta functions through a protein tyrosine kinase, not protein kinase C or protein kinase A. However, activation of protein kinase C by phorbol 12-myristate 13-acetate increases the level of BKB1R mRNA and the binding of desArg10-kallidin. This increase is blocked by NF-kappaB activation inhibitors.

Purification and characterization of a puromycin-hydrolyzing enzyme from blasticidin S-producing Streptomyces morookaensis.

Blasticidin S-producing Streptomyces morookaensis JCM4673 produces an enzyme which inactivates puromycin (PM) by hydrolyzing an amide linkage between its aminonucleoside and O-methyl-L-tyrosine moieties [Nishimura et al. (1995) FEMS Microbiol. Lett. 132, 95-100]. In this study, we purified to homogeneity the enzyme from the cell-free extracts of S. morookaensis. The molecular weight of PM-hydrolyzing enzyme, estimated by SDS-PAGE and gel filtration, was 68 and 66 kDa, respectively, suggesting that this protein is monomeric. The PM-hydrolyzing activity was strongly inhibited by Zn2+, Fe2+, Cu2+, Hg2+, and N-bromosuccinimide, but was stimulated by DTT. The optimum pH and temperature for PM-hydrolyzing activity were 8.0 and 45 degrees C, respectively. Several L-aminoacyl-beta-naphthylamides were good substrates for the enzyme, suggesting that the PM-inactivating enzyme has an aminopeptidase activity. The N-terminal sequence of the first 14 amino acids (Val-Ser-Thr-Ala-Pro-Tyr-Gly-Ala-Trp-Gln-Ser-Pro-Ile-Asp) of the enzyme showed no significant homology with any published hydrolase sequences.

Induction of P‐Glycorprotein mRNA by protein synthesis inhibition is not controlled by a transcriptional repressor protein in rat and human liver cells

Recent studies have suggested that a labile transcriptional repressor protein is important in the regulation of pgp mRNA expression. However, cycloheximide (CHX) the protein synthesis inhibitor used, can increase mRNAs by either stabilizing the mRNA transcript or directly activating gene transcription. To determine whether CHX posttranscriptionally increased pgp mRNA, we compared the effect of CHX, which inhibits protein synthesis by stabilizing polysomes, with puromycin (PURO), which inhibits protein synthesis by polysome destabilization. In rat hepatocytes, CHX induced pgp2 mRNA, and the increase was proportional to the degree of protein synthesis inhibition. In contrast, despite almost complete inhibition of protein synthesis, PURO did not induce pgp2 mRNA. Further studies demonstrated that PURO pretreatment could block pgp2 mRNA induction by CHX. Likewise, in cultures of primary human hepatocytes CHX, but not PURO, induced MDR1 mRNA. A polymerase chain reaction assay was developed to assess whether CHX treatment altered the length of the 3'-untranslated region (UTR) of pgp2. CHX treatment time dependently increased the length of the pgp2 3'-UTR. To determine whether CHX acts as a transcriptional agonist, we performed nuclear run-off analysis and found no increase in pgp2 gene transcription compared to untreated control. Further, transcription studies were performed by transiently transfecting HepG2 cells with plasmids containing 5' segments of human MDR1 fused with the reporter chloramphenicol acetyltransferase (CAT). These plasmids were not transcriptionally activated by CHX. In summary, our results cast doubt on the existence of a labile transcriptional repressor protein for pgp. Furthermore, these are the first studies to demonstrate that polysomal destabilization by PURO can block CHX induction of pgp.

Stabilization of methionine enkephalin in various rabbit mucosal extracts by enzyme inhibitors

The inhibition of the enzymatic degradation of methionine enkephalin (Met-Enk) was investigated kinetically in nasal, rectal, and vaginal extracts of rabbits with and without inhibitors, such as puromycin (PM), amastatin (AM), thiorphan (TP), Na 2EDTA, and thimerosal (TM), alone or in combination, by analyzing the parent peptide and its hydrolytic fragments by HPLC. The effects of variation of pH in the nasal extracts, and the addition of 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CyD) on the stabilization of Met-Enk were also studied. The degradation of Met-Enk was found to be fastest at around pH 7, indicating that the activity of enkephalin-degrading enzymes is optimal at this pH. Addition of 2-HP-β-CyD (10%) to the nasal, rectal, and vaginal extracts was noted to reduce the first-order degradation rate constants for Met-Enk by 2.5-2.8-fold, compared to the control. AM alone inhibited the enzymatic degradation of Met-Enk with IC 50 values of 3.5 and 0.22 μM for the rectal and vaginal extracts, respectively, whereas PM was found to be approx. 14.2- and 26.8-fold less potent than AM, respectively. The effects of both aminopeptidase inhibitors in the nasal extracts were smaller. Even at 50 μM, TP (a potent enkephalinase A inhibitor) alone revealed only a small increase of Met-Enk stability in the various mucosal extracts, however, EDTA (5 μM) was observed to inhibit enzymatic hydrolysis considerably by blocking both enkephalinase A and B and, to some extent, aminopeptidase. On the other hand, TM (0.05%) was found to be a new and potent inhibitor for enkephalinase B and aminopeptidases, which was more potent than AM (50 μM) in inhibiting the degradation of Met-Enk in various mucosal extracts. Furthermore, the addition of TM (0.01%) to a combination of AM (50 μM) and EDTA (5 μM) was observed to protect Met-Enk from enzymatic degradation in nasal, rectal, and vaginal extracts by more than 90%, after 24 h of incubation, by inhibiting almost completely all the enkephalin-degrading enzymes present in the incubation mixtures.

Low-protein diet attenuates increased gene expression of platelet-derived growth factor and transforming growth factor-beta in experimental glomerular sclerosis.

The present study was designed to assess whether platelet-derived growth factor (PDGF)-A and -B chain and transforming growth factor-beta (TGF-beta) mRNA expression in glomeruli are affected by a low-protein (6%) diet during the course of focal glomerulosclerosis (FGS). Puromycin aminonucleoside (PAN) was injected intraperitoneally in rats, and the right kidney was removed on day 22. The nephrotic rats received successive intraperitoneal injections of PAN on days 27, 34, and 41. Control rats were subjected to a sham operation on day 22. The PAN-injected rats were divided into two groups. Group 1 rats were fed a standard diet containing 22% protein, whereas group 2 rats were fed a low-protein diet containing 6% protein, starting on the same day as the first PAN injection. Rats were killed on days 0, 48, 60, and 80 after the initial PAN injection. The percentage of sclerotic glomeruli in group 1 rats increased markedly with time, reaching 73% on day 80. The PDGF-A and -B chain and TGF-beta mRNA levels increased significantly as glomerulosclerosis progressed. A positive correlation was noted between the PDGF and TGF-beta mRNA levels and the incidence of glomerular sclerosis. The low-protein diet reduced the prevalence of glomerular sclerosis (10% on day 80) and attenuated the abnormally high expression of PDGF-A and -B chain and TGF-beta genes in FGS glomeruli. These findings suggest that PDGF and TGF contribute to glomerulosclerosis and that a low-protein diet attenuates markedly the increased glomerular expression of the PDGF and TGF-beta genes in glomerular sclerosis.

Studies on the glomerular permeability with human liver ferritin.

To clarify the roles of the glomerular basement membrane (GBM) in filtration mechanisms, human liver ferritin was used for the first time as a tracer. Urinary excretion of human liver ferritin was measured and the injected ferritin was tracked under electron microscopy. Puromycin aminonucleoside (PAN) nephrosis was induced in Sprague Dawley rats and normal saline was injected into control rats. Monomeric and polymeric human ferritin were isolated from post mortem samples. Both kinds of human liver ferritin were injected into the experimental and control rats and urine samples were examined for human ferritin by radioimmunoassay. Rats with PAN nephrosis excreted approximately 33 times more monomeric ferritin than the controls. Appreciably more monomeric ferritin was excreted than polymeric ferritin. In control rats, monomeric ferritin particles were restricted in the lamina rara interna and inner aspect of the glomerular basement membrane 30 min after injection. On the other hand, in rats with PAN nephrosis, monomeric ferritin particles were seen throughout the width of the GBM and in the epithelial cells. With human liver ferritin, we were able to demonstrate the escape of the ferritin into the urine in addition to conducting the conventional electron microscopic tracer study of the glomerular capillary wall. Human liver ferritin shows potential as a useful tracer in the study of glomerular permselectivity.

Reproductive death of chinese hamster V79 cells after exposure to chemical inhibitors of DNA synthesis

1. 1. The results of this study have contributed to the definition of three categories of chemical inhibitors of DNA replication in mammalian cells. 2. 2. Inhibitors of replicon cluster initiation [4-nitroquinoline-. N-oxide (4-NQO), etoposide (VP-16), teniposide (VM-26), amsacrine ( m-AMSA), N-methyl- N′-nitro- N-nitrozoguanidine (MNNG), cis-Pt(II)diammine diehloride ( cis-Pdd)], which needed similar doses to produce a slow and persistent (up to 4 hr) inhibition of DNA synthesis, followed by significant cell killing. 3. 3. Inhibitors of DNA replication by indirect action [3-aminobezamide (3-AB), cycloheximide (CHX), puromycin (PRC), bisbenzimide Hoechst No. 33258 (H-33258)], that showed reduced cytotoxic effects, and caused a slow (60 min) and reversible inhibition of DNA synthesis. 4. 4. Inhibitors of formation and/or polymerization of deoxyribonucleotides [5-aminouracil (5-AU), bisbenzimide Hoechst No. 33342 (H-33342)], which induced a fast (20 min) and reversible suppression of DNA replication, associated with limited cell killing.

Protein synthesis inhibitors prevent both spontaneous and hormone‐dependent maturation of isolated mouse oocytes

The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors [CX, emetine (EM), and puromycin (PUR)] each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of low protein diet in puromycin aminonucleoside-induced nephrotic rats.

Wistar rats were injected with puromycin aminonucleoside (PAN) to induce massive proteinuria and were raised on a low protein diet to determine whether the urinary protein excretion might be reduced. Although PAN nephrotic rats fed with a normal protein diet containing 20% protein (group A) did not show any reduction of their urinary protein excretion, PAN rats fed with a low protein diet containing 6% protein (group B) revealed a marked reduction at day 9 of the present study in comparison with group A. The total serum protein levels were low in groups A and B as compared to group C, the non-treated control. However, group A recovered to normal levels at day 13. In terms of the absolute amount of oral protein intake, marked differences were observed between groups B and C, but little difference was observed between groups A and C. The body weight in all three groups was decreased at 8 days after the start of this study. The serum levels of creatinine were elevated to 1.1 +/- 0.3 mg/dl and 1.0 +/- 0.2 mg/dl in groups A and B, respectively, on day 7, but abruptly recovered to normal levels by day 12. These data indicate that the decreased urinary protein excretion in group B might be dependent on the total protein volume intake, and not on the renal function nor serum levels of total protein.

Study of anionic sites on the mesangium in aminonucleoside nephrosis.

In recent years, various reports have appeared concerning anionic sites in glomerular lesions. However, no detailed results for anionic sites in the mesangium have been reported. The authors therefore prepared nephrotic rats by the administration of puromycin aminonucleoside (PA) and investigated the anionic sites in various mesangial regions, using polyethyleneimine (PEI) as a cationic probe. In the mesangium of the control group as well as the experimental groups, PEI particles were most numerous in the subendothelial regions, followed by the central region and paramesangial region, in that order. In the group treated with PA, the numbers of PEI particles in each mesangial region decreased as compared with those in the control group. Among the three mesangial regions considered, this decrease was most pronounced in the paramesangial region. In the group which received elastase, no significant decrease in numbers of PEI particles was observed in subgroups I and II, indicating maintenance of the anionic sites. The results obtained suggested that regional investigations of the mesangial matrix may be of considerable significance for elucidating the etiologic factors in glomerular sclerosis, and that negative charge impairment in the mesangial matrix plays an important role in the development of glomerular sclerotic lesions.

The Effect of Puromycin and Cycloheximide on the Infection of Algae‐Free Paramecium bursaria by Symbiotic Chlorellae1

ABSTRACTIt has been suggested that the infection of algae‐free Paramecium bursaria by symbiotic algae involves an induction in the ciliate. Such a process suggests a need for the synthesis of specific proteins. Therefore, an attempt was made to determine the role of protein synthesis during the initial phases of host‐symbiont interaction by examining the capacity of the ciliate to form a stable association with algae when the ciliate is exposed to puromycin (PURO) or cycloheximide (CYC) during the first 1–3 h of algal insestion. Cycloheximide (100 μg/ml) blocked algal but not ciliate growth and protein synthesis while PURO (250 μg/ml) appeared to inhibit these processes in both Puromycin significantly inhibited the infection when presented to the ciliate during the first hour of algal exposure and had little effect when added after that period. Inhibition of ciliate, as compared to the alga, protein synthesis appears to be significant in relationship to those processes leading to infection, as CYC when presented during the first hour of algae‐ciliate exposure has no inhibitory effects. Experiments on algal sugar secretion and ciliate ingestion of algae indicated that neither process was significantly affected by these inhibitors. These results point to a need for host protein synthesis during the initial phase of ingestion of algae which appears to be important to establishment of the symbiotic association.

Protection of Chinese Hamster Ovary Cells from Heat Killing by Treatment with Cycloheximide or Puromycin: Involvement of HSPs?

Cycloheximide (CHM) or puromycin (PUR) added for 2 h before heating at 43 degrees C followed by either PUR or CHM during heat greatly protected cells from heat killing. This protection increased with inhibition of protein synthesis. Since treatment with a drug both before and during heating was required for heat protection, and since one drug could be exchanged for the other after the 2-h pretreatment without affecting the heat protection, a common mode of action involving inhibition of protein synthesis is suggested for the two drugs. Drug treatment reduced the synthesis of heat-shock proteins (HSPs) as studied by one-dimensional gel electrophoresis by 80-98% relative to 37 degrees C untreated controls. Synthesis of large molecules (greater than 30 kDa) was preferentially inhibited by PUR but not by CHM. Also for CHM, but not for PUR treatment, a 42 kDa band appeared along with a great reduction in the 43 kDa actin band during CHM treatment at both 37 and 43 degrees C. Furthermore, during CHM or PUR treatment, incorporation of [35S]methionine into HSP families 70, 87, or 110 was not increased relative to incorporation into total protein. However, synthesis of the 70 kDa HSP family was selectively suppressed when cells were incubated at 37 degrees C after CHM treatment, but when cells were incubated at 37 degrees C after treatment at 43 degrees C with CHM, synthesis of the 70 kDa HSP family resumed. When cells were labeled for 3 days, there was no preferential accumulation or turnover of HSP families during heating with or without CHM. Therefore, heat protection caused by treatment with CHM or PUR apparently involves a common mode of action not associated with changes in either total levels or synthesis of HSP families during drug treatment before and during heating. The significance of the changes observed in the synthesis of the HSP 70 family after heat is unknown. As thermotolerance developed during 5 h at 42 degrees C without drugs, synthesis of HSP families 70, 87, and 110, as studied with one-dimensional gels, increased 1.4-fold relative to synthesis of total protein, but compared to HSP families in cells labeled for 5 h at 37 degrees C incorporation was reduced by 40%. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater.(ABSTRACT TRUNCATED AT 400 WORDS)

Protection of Chinese Hamster Ovary Cells from Hyperthermic Killing by Cycloheximide or Puromycin

Cycloheximide (CHM) and puromycin (PUR) were used at various concentrations up to maxima of 10 micrograms/ml and 100 micrograms/ml, respectively, which inhibited protein synthesis by 95% without any cytotoxicity. The drugs were added to the cells for a maximum period of 7 h, with various combinations for treatment before, during, and after heating. Maximum protection, i.e., a 10,000-fold increase in survival from 5 X 10(-6) to 5 X 10(-2) after 4 h at 43 degrees C, required both 1-2 h of treatment before heating and 1-2 h of treatment during heating. For treatments at 45.5 degrees C, the protection was less, i.e., a 100-fold increase in survival from 10(-5) to 10(-3). Little or no protection was observed if after treatment, the drug was removed before heating, or if the drug was added at the start of heating and left on for 5 min to 3 h after heating. For both drugs, the amount of protection increased as inhibition of protein synthesis increased. However, the amount of protection from the drugs was the same only at about 95% inhibition; at 60-85% inhibition, CHM afforded more protection than PUR. Therefore, the modes of action of the drugs might be common at high drug concentrations, but different when intermediate concentrations are used.

Contrasting effects of RNA and protein synthesis blocking on natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

In this study, earlier observations concerning the independence of both natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) from DNA synthesis have been confirmed. In addition, blocking of RNA synthesis by actinomycin D and of protein synthesis, reversibly by puromycin (PM) and irreversibly by emetine (EM) had different effects on NCMC and LDCC against 3H-thymidine-prelabeled HEp-2 target cells. Similarly to the Con A-induced proliferation of lymphocytes, LDCC activity was also inhibited by blocking of RNA and protein synthesis. NCMC to HEp-2 target cells was not affected by blocking of RNA synthesis, while both PM and EM strongly enhanced NCMC activity.

New evidence for the inhibition by harringtonine in the formation of the first peptide bond in protein synthesis.

New evidence for the inhibition by harringtonine in the formation of the first peptide bond in protein synthesis was obtained by means of experiments in the cell-free system. The antitumour drug strongly blocked dipeptide synthesis in the system without elongation factor G and GTP. Furthermore, it inhibited N-Acetyl-phenylalanyl-puromycin formation from N-Acetyl-phenylalanyl-tRNA, puromycin, and ribosomes.

Expression of a herpes simplex virus glycoprotein in two clonal lines of hamster cells transformed by herpes simplex virus type 2.

Expression of herpes simplex virus type 2 (HSV-2) glycoproteins on the cell surface of two clones of a hamster cell line transformed by HSV-2 was examined by immunofluorescence tests with hamster antisera prepared against three size-class glycoprotein fractions obtained from hamster embryo fibroblast cells infected with HSV-2. Enhanced expression of one of the three size-class glycoprotein fractions (molecular weight, 82,000-94,000) was found in one clone (155-4-213) after replating of the cells, and in the other (155-4-03) after actinomycin D treatment. Enhanced expression of the antigen in clone 155-4-213 was inhibited by 2-deoxy-D-glucose, but not by puromycin or protease inhibitors, antipain and p-nitrophenyl-p'-guanidinobenzoate. On the other hand, the antigen expression induced by actinomycin D in clone 155-4-03 was inhibited by these four drugs. These results indicate that the expressions of the antigen in the two clones are enhanced by different mechanisms.

Enhanced release of a chemoattractant for alveolar macrophages after asbestos inhalation.

Alveolar macrophage supernatants from 2 groups of asbestos-exposed rats and a group of sham-exposed rats were tested for chemoattractant activity towards rat alveolar macrophages. Enhanced chemotaxin release was observed in culture supernatants from both crocidolite and chrysotile asbestos-exposed rats when compared with supernatants from sham-exposed rats. These between-group differences persisted for as long as 15 months after exposure had ceased. Chemotactic factor release was maximal after 24 h of culture in all animal groups. Partial characterization of the chemoattractant from each of the 3 rat groups revealed that it was thermolabile, nondialyzable, and trypsin-sensitive. Separation on SDS-polyacrylamide gel electrophoresis revealed 3 major peaks of activity. The production of the chemotaxin in supernatants from asbestos-exposed rats was partially inhibited by both actinomycin D and puromycin. These agents had no appreciable effect on the production of chemoattractant in cultures from sham-exposed animals. The enhanced release of an alveolar macrophage chemoattractant after asbestos inhalation may explain why macrophages accumulate at sites of asbestos deposition in the lungs.

Temporal and pharmacological parameters of puromycin-induced amnesia

Mice were trained in step-down passive avoidance behavior. Bitemporal injections of puromycin (PM) were given either immediately or delayed until 24 hrs after training. PM produced a marked amnesia in both cases during retention testing 3 days later. The amnesia persisted during a second retention test 6 days after training. Of all the antibiotics, only PM is effective as an amnestic agent when injections are delayed 24 or more hours after training. Cycloheximide (CXM) was also injected bitemporally immediately after training. However, CXM produced a weaker amnestic effect even though it produced a much greater inhibition of cerebral protein synthesis, more rapidly, and of longer duration. In an effort to attenuate the amnesia produced by PM, in separate experiments, the mice were injected with combined injections of PM and CXM (bitemporally); mice were also given combined injections of PM (bitemporally) and amphetamine (subcutaneously). The amnesia produced by immediate injections of PM was not attenuated by either CXM or amphetamine. However, the amnesia produced by delayed injections of PM was attenuated by both CXM and amphetamine. These results suggest that delayed injections of PM (24 hours after training) block the e expression or retrieval of memory. This study also supports the contention that puromycin has two separate effects on memory with different temporal parameters depending on when the drug is injected relative to initial training.