Hydrothermal conversion of mixed uranium(IV)-cerium(III) oxalates into U1-xCexO2+δ·nH2O solid solutions.

Uranium-cerium oxide solid solutions, U1-xCexO2+δ·nH2O, were prepared through hydrothermal conversion of mixed U(IV)-Ce(III) oxalate precursors, cerium being used as a surrogate for plutonium. Whatever the starting pH, the fluorite-type structure of AnO2 was obtained after heating at 250 °C for 24 h. The initial pH of the reaction media appeared to affect significantly the oxide morphology: for pH ≤ 2, the powder was found to be composed of microspheres, whereas for more alkaline pH values, agglomerates of nanocrystallites were found. Furthermore, a study of the hydrothermal treatment duration (T = 250 °C, pH = 8, t = 1-48 h) showed that fluorite-type mixed dioxides started to form after only 1 h, and then became single phase after 3 h. SEM and TEM/EDS analyses revealed that the cationic distribution narrowed with time to finally form highly homogeneous mixed oxides. Such a preparation route was then applied to various cerium incorporation rates and it was found that the formation of U1-xCexO2+δ·nH2O mixed oxides was possible for 0.1 ≤ x ≤ 0.75. In all the systems investigated, the speciation of uranium and cerium was questioned in both the solid and liquid phases. Thermodynamic calculations and evaluation of the O/M ratio in the final oxides led us to understand the complex redox behaviour of uranium and cerium in solution during hydrothermal processes and to propose a conversion mechanism.

The von Hippel-Lindau tumour suppressor gene: uncovering the expression of the pVHL172 isoform.

BACKGROUND: The von Hippel-Lindau (VHL) gene encodes two mRNA variants. Variant 1 encodes two protein isoforms, pVHL213 and pVHL160, that have been extensively documented in the literature. Variant 2 is produced by alternative splicing of exon 2 and encodes a pVHL isoform of 172 amino acids with a theoretical molecular weight of 19 kDa (pVHL172), the expression of which has never been demonstrated so far due to the absence of suitable antibodies. METHODS: We have generated an anti-pVHL monoclonal antibody (JD-1956) using pVHL172 recombinant protein. We tested the antibody against exogenous or endogenous expressed proteins in different cell lines. We identified the pVHL172 using a silencing RNA strategy. The epitope of the antibody was mapped using a peptide array. RESULTS: We efficiently detected the three different isoforms of pVHL in cell lines and tumorigenic tissues by western blotting and immunohistochemistry and confirmed for the first time the endogenous expression of pVHL172. CONCLUSIONS: The endogenous expression of the three isoforms and particularly the pVHL172 has never been shown before due to a lack of a highly specific antibody since none of the available commercial antibodies distinguish the three isoforms of pVHL in cells or in both normal and cancerous human tissues. Evidence of pVHL172 expression emphasises the need to further study its implication in renal tumorigenesis and VHL disease.

Overexpression of the polarity protein PAR-3 in clear cell renal cell carcinoma is associated with poor prognosis.

The partition-defective 3 (PAR-3) protein is implicated in the development and maintenance of cell polarity and is associated with proteins that mediate the changes in cytoskeleton organization required for cell polarity establishment. In this work, we used two original primary cell lines (R-180 and R-305) derived from clear cell Renal Cell Carcinoma (ccRCC) surgical specimens of a patient with unfavorable clinical course (R-180 cells) and a patient with favorable prognosis (R-305 cells) to identify genetic and molecular features that may explain the survival difference of the two patients. The cytogenetic analysis of these cell lines revealed that the PARD3 gene was amplified only in the R-180 cell line that was derived from an aggressive ccRCC. PARD3 gene amplification was associated with overexpression of the encoded protein and altered cytoskeleton organization. Consistently, PARD3 knockdown in R-180 cells restored the cytoskeleton organization and reduced cell migration in comparison to non-transfected cells. Immunohistochemical analysis of ccRCC samples from a cohort of 96 patients with a follow-up of 6 years revealed that PAR-3 overexpression was correlated with poor survival. Our results suggest that PAR-3 has a role in the clinical aggressiveness of ccRCC, possibly by promoting cell migration.

The protein phosphatase 2A B'-regulatory subunit par1p is implicated in regulation of the S. pombe septation initiation network.

In order to identify regulators of the Schizosaccharomyces pombe septation initiation network (SIN), which signals the onset of cell division, we have isolated extragenic suppressors of mutations in the GTPase spg1p, which is a central element in this pathway. One of these encodes the protein phosphatase 2A (PP2A) B'-regulatory subunit par1p. Loss of par1p function rescues mutants in cdc11, cdc7, and spg1, but no other SIN mutants. Our data suggest that PP2A-par1p acts as a negative regulator of SIN signalling.

The S. pombe rlc1 gene encodes a putative myosin regulatory light chain that binds the type II myosins myo3p and myo2p.

In order to identify additional components important for cell division in the fission yeast Schizosaccharomyces pombe we have screened a bank of conditional cold-sensitive mutants for cytokinesis defects. One of these mutants showed a delay in cell cleavage, and strong genetic interactions with other genes implicated in medial ring formation. Cloning of the corresponding gene indicates that it encodes a protein with significant homology to the regulatory light chain of non-muscle myosins. We have named the gene rlc1 (regulatory light chain 1). The gene is not essential for division, but null mutants display a cell cleavage defect and form an aberrant F-actin ring. Two myosin-II heavy chains have been identified in fission yeast: Co-immunoprecipitation experiments indicate that rlc1p associates more strongly with myo3p than myo2p.

Analysis of the cps1 gene provides evidence for a septation checkpoint in Schizosaccharomyces pombe.

The fission yeast gene cps1, which encodes the catalytic subunit of beta-glucan synthase, was isolated in a screen for mutants that show an increase in ploidy at the restrictive temperature. cps1 mutants display defects in both polarity and septation at the permissive temperature, and become swollen and multinucleate at the restrictive temperature. Analysis of the interaction of cps1 with other mutations suggests the existence of a septation checkpoint, which requires the activity of the protein kinase weel for function.

Controlling septation in fission yeast: finding the middle, and timing it right.

The fission yeast Schizosaccharomyces pombe provides a simple eukaryotic model for the study of cytokinesis. S. pombe cells are rod-shaped, grow mainly by elongation at their tips, and divide by binary fission after forming a centrally placed division septum. Analysis of mutants has begun to shed light upon how septum formation and cytokinesis are regulated both spatially and temporally. Some of the proteins involved in these events have been functionally conserved throughout eukaryotic evolution, suggesting that aspects of this control will be common to all eukaryotic cells.

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.

Overexpression of human release factor 1 alone has an antisuppressor effect in human cells.

Two eukaryotic proteins involved in translation termination have recently been characterized in in vitro experiments. Eukaryotic release factor 1 (eRF1) catalyzes the release of the polypeptide chain without any stop codon specificity. The GTP-binding protein eRF3 confers GTP dependence to the termination process and stimulates eRF1 activity. We used tRNA-mediated nonsense suppression at different stop codons in a cat reporter gene to analyze the polypeptide chain release factor activities of the human eRF1 and eRF3 proteins overexpressed in human cells. In a chloramphenicol acetyltransferase assay, we measured the competition between the suppressor tRNA and the human release factors when a stop codon was present in the ribosomal A site. Whatever the stop codon (UAA, UAG, or UGA) present in the cat open reading frame, the overexpression of human eRF1 alone markedly decreased translational readthrough by suppressor tRNA. Thus, like the procaryotic release factors RF1 and RF2 in Escherichia coli, eRF1 seems to have an intrinsic antisuppressor activity in human cells. Levels of antisuppression of overexpression of both eRF3 and eRF1 were almost the same as those of overexpression of eRF1 alone, suggesting that eRF1-eRF3 complex-mediated termination may be controlled by the expression level of eRF1. Surprisingly, when overexpressed alone, eRF3 had an inhibitory effect on cat gene expression. The results of cat mRNA stability studies suggest that eRF3 inhibits gene expression at the transcriptional level. This indicates that in vivo, eRF3 may perform other functions, including the stimulation of eRF1 activity.

Expression of the release factor eRF1 (Sup45p) gene of higher eukaryotes in yeast and mammalian tissues.

Polypeptide chain termination in eukaryotic cells is mediated in part by the release factor eRF1 (Sup45p). We have isolated and characterised cDNAs encoding this translation factor from Syrian hamster (Mesocricetus auratus) and human (Homo sapiens) Daudi cells. Comparison of the deduced amino acid sequence of these new eRF1 (Sup45p) sequences with those published for Saccharomyces cerevisiae, Arabidopsis thaliana, Xenopus laevis and human indicates a high degree of amino acid identity across a broad evolutionary range of species. Both the 5' and 3' UTRs of the mammalian eRF1 (Sup45p)-encoding cDNAs show an unusually high degree of conservation for non-coding regions. In addition, the presence of two different lengths of 3' UTR sequences in the mammalian eRF1 (Sup45p) cDNAs indicated that alternative polyadenylation sites might be used in vivo. Northern blot analysis demonstrated that eRF1 (Sup45p) transcripts of differing length, consistent with the use of alternative polyadenylation sites, were detectable in a wide range of mammalian tissues. The Xenopus, human and Syrian hamster eRF1 (Sup45p) cDNAs were shown to support the viability of a strain of S cerevisiae carrying an otherwise lethal sup45::HIS3 gene disruption indicating evolutionary conservation of function. However, the yeast strains expressing the heterogenous eRF1 (Sup45p) showed a defect in translation termination as defined by an enhancement of nonsense suppressor tRNA activity in vivo. Western blot analysis confirmed that Xenopus eRF1 (Sup45p) was primarily ribosome-associated when expressed in yeast indicating that the ribosome-binding domain of eRF1 (Sup45p) is also conserved.

Is there a human [psi]?

The yeast Sup35p protein which is responsible for the [psi] phenotype, is a GTP-binding protein involved in translation termination. It was suggested recently that the [psi] determinant has prion-like properties that were localized in the 114 N-terminal amino acids of the protein. In this study, we show that the 5' end of the human SUP35 gene open reading frame is longer than previously reported by 138 codons. This N-terminal sequence presents similarities with the N-terminus of S. cerevisiae Sup35p protein, involved in [psi] maintenance. By transfection of human cells and Western blotting, we demonstrate that translation is initiated at the first AUG encountered at the 5' end of the human SUP35 gene. The longest form of the protein, which contains the N-terminal extension, is the major form of Sup35p protein in non transfected cells. Moreover, an analog of the long form of Sup35p protein is found in various mouse tissues. We suggest that the protein encoded by SUP35 gene could have, at least in human, the properties described for the yeast [psi] element.

Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase.

Termination of translation in eukaryotes is governed by two polypeptide chain release factors, eRF1 and eRF3 on the ribosome. eRF1 promotes stop-codon-dependent hydrolysis of peptidyl-tRNA, and eRF3 interacts with eRF1 and stimulates eRF1 activity in the presence of GTP. Here, we have demonstrated that eRF3 is a GTP-binding protein endowed with a negligible, if any, intrinsic GTPase activity that is profoundly stimulated by the joint action of eRF1 and the ribosome. Separately, neither eRF1 nor the ribosome display this effect. Thus, eRF3 functions as a GTPase in the quaternary complex with ribosome, eRF1, and GTP. From the in vitro uncoupling of the peptidyl-tRNA and GTP hydrolyses achieved in this work, we conclude that in ribosomes both hydrolytic reactions are mediated by the formation of the ternary eRF1-eRF3-GTP complex. eRF1 and the ribosome form a composite GTPase-activating protein (GAP) as described for other G proteins. A dual role for the revealed GTPase complex is proposed: in " GTP state," it controls the positioning of eRF1 toward stop codon and peptidyl-tRNA, whereas in "GDP state," it promotes release of eRFs from the ribosome. The initiation, elongation, and termination steps of protein synthesis seem to be similar with respect to GTPase cycles.

Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.

Termination of translation in higher organisms is a GTP-dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35-like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C-domain of the X.laevis eRF3 protein is highly conserved with other Sup35-like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.

A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor.

The termination of protein synthesis in ribosomes is governed by termination (stop) codons in messenger RNAs and by polypeptide chain release factors (RFs). Although the primary structure of prokaryotic RFs and yeast mitochrondrial RF is established, that of the only known eukaryotic RF (eRF) remains obscure. Here we report the assignment of a family of tightly related proteins (designated eRF1) from lower and higher eukaryotes which are structurally and functionally similar to rabbit eRF. Two of these proteins, one from human and the other from Xenopus laevis, have been expressed in yeast and Escherichia coli, respectively, purified and shown to be active in the in vitro RF assay. The other protein of this family, sup45 (sup1) of Saccharomyces cerevisiae, is involved in omnipotent suppression during translation. The amino-acid sequence of the eRF1 family is highly conserved. We conclude that the eRF1 proteins are directly implicated in the termination of translation in eukaryotes.