Structure of the C-terminal MA-3 domain of the tumour suppressor protein Pdcd4 and characterization of its interaction with eIF4A.

Programmed cell death protein 4 (Pdcd4) is a novel tumour suppressor protein, which is involved in the control of eukaryotic transcription and translation. The regulation of translation involves specific interactions with eukaryotic initiation factor (eIF)4A and eIF4G, which are mediated via the two tandem MA-3 domains. We have determined the structure of the C-terminal MA-3 domain of Pdcd4 (Pdcd4 MA-3(C)), characterized its interaction with eIF4A and compared the features of nuclear magnetic resonance (NMR) spectra obtained from the single domain and tandem MA-3 region. Pdcd4 MA-3(C) is composed of three layers of helix-turn-helix hairpins capped by a single helix and shows close structural homology to the atypical HEAT repeats found in many eIFs. The sequence conservation and NMR data strongly suggest that the tandem MA-3 region is composed of two equivalent domains connected by a somewhat flexible linker. Pdcd4 MA-3(C) was found to interact with the N-terminal domain of eIF4A through a conserved surface region encompassing the loop connecting alpha5 and alpha6 and the turn linking alpha3 and alpha4. This site is strongly conserved in other MA-3 domains known to interact with eIF4A, including the preceding domain of Pdcd4, suggesting a common mode of binding.

Differential association of transfer RNAs with the genomes of murine, feline and primate retroviruses.

The tRNAs that are bound to the genomic RNAs of several murine, feline, and primate retroviruses have been identified. Transfer RNAs were divided into those loosely bound and those tightly bound by stepwise thermal dissociation of the 70 S RNA. They were then identified and semiquantitated by aminoacylation. Proline tRNA is the most tenaciously bound tRNA in several strains of murine leukemia virus, two strains of feline leukemia virus, and the primate viruses simian sarcoma, baboon endogenous, and gibbon ape lymphoma. In the feline xenotropic virus, RD-114, tRNAGly is enriched in the most tightly bound fraction. In Mason-Pfizer monkey virus, as in the murine mammary tumor virus, tRNALys is the tRNA most tenaciously bound to its genomic RNA. Besides the most tightly associated tRNA, one or more different tRNAs are found in relatively large amounts in association with the 70 S RNA. (For convenience, we refer to the largest RNA ccomplex (50-70 S) isolated from any of the retroviruses studies as '70 S' RNA.) These tRNAs can be distinguished from the most tightly bound tRNA by the fact that they can be dissociated at lower temperatures. However, they occur in the same relative abundance as the tightly bound tRNA.

Studies on the function of the non-primer tRNAs associated with the 70 S RNA of avian myeloblastosis virus.

Significant amounts of three tRNAs are associated with the 70 S RNA of avian myeloblastosis virus (AMV). The temperatures at which they are half dissociated from the 70 S RNA in 50 mM NaCl and their respective quantities relative to 35 S RNA are: tRNAArg, 51 degree C, 1.6; tRNALys, 57 degree C, 0.7 and tRNATrp, 76 degree C, 1.0. Possible functions for the non-primer tRNAs (tRNAArg and tRNALys) were evaluated by determining the effect of their thermal dissociation on: (a) conversion of 70 S to 35 S RNA, (b) capacity of 70 S and/or 35 S RNA to be translated in vitro, and (c) capacity of 70 S and/or 35 S RNA to be reverse transcribed in vitro. Conversion of 70 S to 35 S RNA occurred with a tm of 56 degree C and is consistent with the hypothesis that tRNALys might be involved in joining two 35 S RNA subunits to form the 70 S RNA complex. There was no indication that the association of either tRNAArg or tRNALys influenced the rate or quality of translation of 70 S or 35 S RNA. A decrease in the rate at which 70 S RNA is transcribed occurs in parallel with the dissociation of tRNAArg and tRNALys.

Three second chromosome-linked clustered Cyp6 genes show differential constitutive and barbital-induced expression in DDT-resistant and susceptible strains of Drosophila melanogaster.

The level of expression of the Cyp6a2 gene is much higher in the DDT-resistant 91-R strain than in the susceptible 91-C strain of Drosophila melanogaster (Waters et al. (1992b) Proc. Natl. Acad. Sci. USA, 89, 4855-4859). To understand the role of Cyp6a2 and related genes in insecticide resistance, we have isolated and characterized two new Cyp6 genes from the 91-R strain. The polypeptides encoded by these two genes, Cyp6a8 and Cyp6a9, show 77 and 75% amino acid sequence similarity, and 60 and 55% identity with Cyp6a2 of D. melanogaster, respectively. In the genome, Cyp6a8 and Cyp6a9 genes are closely clustered within 4 kb and map at region 51C of the second chromosome. In between them another Cyp gene is present which is more related to Cyp6a9 than to Cyp6a8. The Cyp6a8 gene which is transcriptionally highly active in 91-R, moderately active in ry506 and silent in the 91-C strain hybridizes with 2.0- and 1.8-kb RNAs. Two different-sized RNAs, 2.1 and 1.8 kb, also hybridize with the Cyp6a9 and/or Cyp6a9-related genes. While the level of 2.1-kb RNA is similar in all three strains, the level of 1.8-kb RNA is highest in the 91-R strain and barely detectable in 91-C strain. Transgenic experiments showed that a 8.3-kb BamHI fragment contains the cis-regulatory elements for the expression of both Cyp6a8 and Cyp6a9-related genes. Barbital induces all these genes in all three strains and increases the levels of the two Cyp6a8 transcripts and the 1.8-kb RNA produced by the Cyp6a9 and/or Cyp6a9-related genes. Expression of the Cyp6a8 gene is down-regulated in the hybrids of 91-R and 91-C strains despite the fact that the hybrids carry one copy of the highly active allele of the Cyp6a8 gene of the 91-R strain. Based on these results we propose that the Cyp6a8 gene in 91-C strain may be turned off by an active repressor which might be inhibited by barbital treatment. In the 91-R strain, the putative repressor may be defective, allowing high level of constitutive expression of the Cyp6a8 gene.

Factors on the third chromosome affect the level of cyp6a2 and cyp6a8 expression in Drosophila melanogaster.

The expression of two second chromosome-linked cytochrome P450 genes, Cyp6a2 and Cyp6a8, of Drosophila melanogaster was measured in various strains. Six different strains, including ry(506) and 91-C, showed low or undetectable levels of CYP6A2 and CYP6A8 mRNAs, suggesting that low expression is the wild-type phenotype of Cyp6a2 and Cyp6a8 genes. In the 91-R and MHIII-D23 strains, however, both these genes are overexpressed. In order to examine the genetic basis of Cyp6a2 and Cyp6a8 expression, CYP6A2 and CYP6A8 RNA levels were measured in the F1 hybrids of overproducer (91-R and MHIII-D23) and underproducer (ry(506) and 91-C) strains. Results showed that the total amounts of CYP6A2 and CYP6A8 mRNAs in the F1 hybrids were lower than half the amounts of these RNAs found in the overproducer parental strains. This suggested that the underproducer strains carry loci which downregulate Cyp6a2 and Cyp6a8 gene expression. To determine the chromosome linkage of these loci, several stocks homozygous for the second chromosome of overproducer 91-R strain and, therefore, homozygous for the Cyp6a2-91R and Cyp6a8-91R alleles were synthesized. The third chromosomes in all these stocks were from the underproducer ry(506) strain. The levels of expression of both Cyp6a2-91R and Cyp6a8-91R genes in these three stocks were significantly lower than that observed in the 91-R strain. One of these stocks, named iso-2, showing reduced expression, was used to synthesize two new isogenic stocks by resubstituting the third chromosome of ry(506) origin with third chromosomes of the 91-R strain. Expression of both Cyp6a2-91R and Cyp6a8-91R alleles was found to be much higher in these two resubstituted isogenic stocks than in the progenitor iso-2 stock. Taken together, these results suggest that the second chromosome-linked Cyp6a2 and Cypa8 genes are regulated by loci present on the third chromosome, and the wild-type function of these loci is to repress these two Cyp genes. The data also suggest that Cyp6a2 and Cyp6a8 overexpression in the 91-R and MHIII-D23 strains is more likely due to mutation in the repressor locus (or loci) rather than in the cis-regulatory sequences of the Cyp6a2 and Cyp6a8 genes.

Constitutive and barbital-induced expression of the Cyp6a2 allele of a high producer strain of CYP6A2 in the genetic background of a low producer strain.

The levels of one or more cytochrome P450 (CYP) enzymes and the respective mRNAs are found to be higher in insecticide-resistant insects than in susceptible insects. To understand better how insects regulate the levels of CYPs, we examined the expression of the Cyp6a2 gene in various strains of Drosophila melanogaster. We also took a transgenic approach to understand the molecular mechanisms that are involved in strain variation of Cyp6a2 expression. RNA blot analysis showed that the constitutive expression of Cyp6a2 varies from strain to strain; the level of CYP6A2 mRNA is barely detectable in the underproducer ry506 strain, whereas it is very high in the overproducer 91-R and MHIII-D23 strains. The long terminal repeat (LTR) of mobile element 17.6 that is found in the 3' untranslated region (UTR) of the Cyp6a2 gene of some strains does not appear to have any role on the steady-state CYP6A2 mRNA level. We also found that the Cyp6a2 gene is inducible by barbital in 91-R, ry506 as well as 91-C, which carries an LTR insertion. To examine the genetic background of the underproducer ry506 strain with respect to Cyp6a2 expression, we transformed the ry506 strain with the Cyp6a2 allele of the overproducer 91-R strain (Cyp6a2-91 R) and measured the constitutive and barbital-induced expression of the Cyp6a2-91 R transgene in the transformed flies. The Cyp6a2-91 R transgene carrying 129 bp of DNA upstream of the ATG codon did not show any constitutive or barbital-induced expression in the ry506 host genome. However, transgenes with 1331 and 985 bp upstream DNA showed similar levels of constitutive expression that were higher than that of the endogenous Cyp6a2 gene of the ry506 host strain, but lower than the expression of the same gene in the 91-R strain. Both these transgenes, with 1331 and 985 bp upstream DNA, also showed induction with 0.1 M barbital. DNA sequence analysis revealed that in both 91-R and ry506, the upstream DNA between +1 and -985 bp contains a distal and a proximal group of three potential barbie boxes, i.e. cis-elements that are thought to be involved in barbiturate-mediated induction of CYP genes. Except for four bases located near the distal cluster of barbie boxes and two other bases, the base sequence of the upstream DNA is identical in ry506 and 91-R strains. These results suggest that the underproducer ry506 strain has the trans-regulatory factors to support constitutive and induced expression of the Cyp6a2-91 R allele carrying DNA between -129 and -1331 bp regions. Possible reasons for low constitutive expression of the endogenous Cyp6a2 gene and moderate level of expression of the Cyp6a2-91 R allele in the ry506 genetic background are discussed.

The sources, fate, and toxicity of chemical warfare agent degradation products.

We include in this review an assessment of the formation, environmental fate, and mammalian and ecotoxicity of CW agent degradation products relevant to environmental and occupational health. These parent CW agents include several vesicants: sulfur mustards [undistilled sulfur mustard (H), sulfur mustard (HD), and an HD/agent T mixture (HT)]; nitrogen mustards [ethylbis(2-chloroethyl)amine (HN1), methylbis(2-chloroethyl)amine (HN2), tris(2-chloroethyl)amine (HN3)], and Lewisite; four nerve agents (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX), tabun (GA), sarin (GB), and soman (GD)); and the blood agent cyanogen chloride. The degradation processes considered here include hydrolysis, microbial degradation, oxidation, and photolysis. We also briefly address decontamination but not combustion processes. Because CW agents are generally not considered very persistent, certain degradation products of significant persistence, even those that are not particularly toxic, may indicate previous CW agent presence or that degradation has occurred. Of those products for which there are data on both environmental fate and toxicity, only a few are both environmentally persistent and highly toxic. Major degradation products estimated to be of significant persistence (weeks to years) include thiodiglycol for HD; Lewisite oxide for Lewisite; and ethyl methyl phosphonic acid, methyl phosphonic acid, and possibly S-(2-diisopropylaminoethyl) methylphosphonothioic acid (EA 2192) for VX. Methyl phosphonic acid is also the ultimate hydrolysis product of both GB and GD. The GB product, isopropyl methylphosphonic acid, and a closely related contaminant of GB, diisopropyl methylphosphonate, are also persistent. Of all of these compounds, only Lewisite oxide and EA 2192 possess high mammalian toxicity. Unlike other CW agents, sulfur mustard agents (e.g., HD) are somewhat persistent; therefore, sites or conditions involving potential HD contamination should include an evaluation of both the agent and thiodiglycol.

Mutations induced by ionizing radiation in a plasmid replicated in human cells. I. Similar, nonrandom distribution of mutations in unirradiated and X-irradiated DNA.

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations.

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.

Biotesting of wastewater: a comparative study using the Salmonella and CHO assay systems.

Means to assess the toxicity of wastewaters are essential to implementing the Federal Clean Water Act. Health risk assessment based on single chemicals is limited by the number of chemicals that can be identified and to those chemicals for which toxicity data are available. Long-term whole animal tests on large numbers of wastewater samples are not practical. In this study, two short-term tests, the Salmonella mutagenicity assay and the Chinese hamster ovary (CHO) cell assay for mutagenicity and cytotoxicity, were evaluated as potentially useful biomonitors of wastewaters. Standard assay protocols were modified to allow testing of up to 2.5 and 3.4 ml of unconcentrated water in the bacterial and mammalian cell tests, respectively. Cytotoxicity and mutagenicity were detected in some unconcentrated wastewater samples using these modifications. Data on eight wastewater samples, representing five different sites, indicated that the Salmonella test is the more sensitive indicator of mutagenic activity in those samples, whereas the CHO test is a sensitive indicator of the presence of cytotoxic components. Wastewater concentrates, prepared by adsorption onto XAD-2 and "blue cotton," were compared in the two bioassays. In a single concentrate, the two short-term tests detected distinctly different mutagens. Advantages of using the CHO-AS52 cell line instead of the CHO-K1BH4 line for detecting wastewater mutagens were indicated. This study illustrates the complementary use of multiple bioassays and concentration methods to detect and characterize toxic components in wastewater.

Studies on the relationship between dimethylnitrosamine-demethylase activity and dimethylnitrosamine-dependent mutagenesis in Drosophila melanogaster.

The relationship between dimethylnitrosamine (DMN) demethylase activity and DMN-induced mutagenesis was investigated in Drosophila melanogaster. The activity of DMN-demethylase was at least 10-fold greater in the Hikone-R strain than in three other Drosophila strains. However, the sex-linked recessive lethal (SLRL) mutations induced by DMN in the four strains differed by less than 2-fold. Several possibilities to explain the lack of correlation between DMN-demethylase activity and DMN-induced mutations were tested and eliminated. They include: (i) the presence of inhibitors of DMN-demethylase in extracts of low-activity strains, (ii) a sex bias in the Hikone-R strain in which the enzyme activity is confined to the females, (iii) the possibility that DMN treatment induces DMN-demethylase activity in the low-activity strains and (iv) the possibility that Hikone-R has a much more efficient DNA repair system than the other strains. The results are discussed in terms of what is known about the role of DMN-demethylase in the metabolic activation of DMN in other systems.

Effects of genotype and age on mixed-function oxidase activities in adult Drosophila melanogaster.

The mixed-function oxidases that metabolize dimethylnitrosamine, aminopyrine, benzphetamine, 7-ethoxycoumarin and benzo[alpha]pyrene were measured in adults of the Canton-S, Oregon-R and Hikone-R strains of Drosophila melanogaster. The expression of these activities is both genotype and age dependent.

Tumor suppressor protein Pdcd4 interacts with Daxx and modulates the stability of Daxx and the Hipk2-dependent phosphorylation of p53 at serine 46.

The tumor suppressor protein Pdcd4 is a nuclear/cytoplasmic shuttling protein that has been implicated in the development of several types of human cancer. In the nucleus, Pdcd4 affects the transcription of specific genes by modulating the activity of several transcription factors. We have identified the Daxx protein as a novel interaction partner of Pdcd4. Daxx is a scaffold protein with roles in diverse processes, including transcriptional regulation, DNA-damage signaling, apoptosis and chromatin remodeling. We show that the interaction of both proteins is mediated by the N-terminal domain of Pdcd4 and the central part of Daxx, and that binding to Pdcd4 stimulates the degradation of Daxx, presumably by disrupting the interaction of Daxx with the de-ubiquitinylating enzyme Hausp. Daxx has previously been shown to serve as a scaffold for protein kinase Hipk2 and tumor suppressor protein p53 and to stimulate the phosphorylation of p53 at serine 46 (Ser-46) in response to genotoxic stress. We show that Pdcd4 also disrupts the Daxx-Hipk2 interaction and inhibits the phosphorylation of p53. We also show that ultraviolet irradiation decreases the expression of Pdcd4. Taken together, our results support a model in which Pdcd4 serves to suppress the phosphorylation of p53 in the absence of DNA damage, while the suppressive effect of Pdcd4 is abrogated after DNA damage owing to the decrease of Pdcd4. Overall, our data demonstrate that Pdcd4 is a novel modulator of Daxx function and provide evidence for a role of Pdcd4 in restraining p53 activity in unstressed cells.

Pdcd4 directly binds the coding region of c-myb mRNA and suppresses its translation.

Pdcd4 is a novel tumor suppressor protein that functions in the nucleus and the cytoplasm, and appears to be involved in the regulation of transcription and translation. In the cytoplasm, Pdcd4 has been implicated in the suppression of translation of mRNAs containing structured 5'-untranslated regions; however, the mechanisms that recruit Pdcd4 to specific target mRNAs and the identities of these mRNAs are mostly unknown. In this study, we have identified c-myb mRNA as the first natural translational target mRNA of Pdcd4. We have found that translational suppression of c-myb mRNA by Pdcd4 is dependent on sequences located within the c-myb-coding region. Furthermore, we have found that the N-terminal domain of Pdcd4 has an important role in targeting Pdcd4 to c-myb RNA by mediating preferential RNA binding to the Pdcd4-responsive region of c-myb mRNA. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA and provides the first evidence for a key role of the RNA-binding domain in targeting Pdcd4 to a specific mRNA.

Laboratory identification of Rothia dentocariosa and its occurrence in human clinical materials.

Fifty isolates of Rothia dentocariosa from diverse clinical sources were characterized by 28 separate tests. An attempt was made to select practical tests that could be completed in a minimal length of time. Rothia is also compared with Actinomyces and Nocardia with which it is often confused. Of the isolates 100% were positive in the following reactions: catalase production, nitrate and nitrite reduction, esculin hydrolysis, and acid production from glucose, sucrose, maltose, salicin, and glycerol. The importance of recognizing this organism is based on the fact that it is frequently isolated from human clinical materials and must be differentiated from morphologically similar organisms of the genera Actinomyces and Nocardia, which contain pathogenic members.

Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

Significant purification of the ubiquitous cytochrome P-450-A and the strain-specific P-450-B from Drosophila melanogaster has been achieved by sequential chromatography on octylamino-agarose, DEAE-cellulose and hydroxyapatite. Preparations of P-450-A (specific contents of 7-9 nmol/mg) were homogeneous as determined by SDS/polyacrylamide-gel electrophoresis (PAGE) analysis. Preparations enriched for P-450-B (specific contents of 4-7 nmol/mg) contained significant amounts of P-450-A but were essentially free of other proteins as judged by SDS/PAGE. Partial reconstitution of 7-ethoxycoumarin de-ethylase activity was achieved using rabbit NADPH: cytochrome P450 reductase and purified preparations containing P450-B.