Two forms of elongation factor 1 alpha (EF-1 alpha O and 42Sp50), present in oocytes, but absent in somatic cells of Xenopus laevis.

We have purified and partially sequenced the EF-1 alpha protein from Xenopus laevis oocytes (EF-1 alpha O). We show that the two cDNA clones isolated by Coppared et al. (Coppard, N. J., K. Poulsen, H. O. Madsen, J. Frydenberg, and B. F. C. Clark. 1991. J. Cell Biol. 112:237-243) do not encode 42Sp50, as claimed by these authors, but two very similar forms of EF-1 alpha O (EF-1 alpha O and EF-1 alpha O1). 42Sp50 is the major protein component of a 42S nucleoprotein particle that is very abundant in previtellogenic oocytes of X. laevis, 42Sp50 differs from EF-1 alpha O not only by its amino acid sequence, but also by several properties already reported. In particular, 42Sp50 has a low EF-1 alpha activity. It is distributed uniformly in the cytoplasm of previtellogenic oocytes, in contrast to EF-1 alpha O which is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body.

Structure and transcription termination of a lysine tRNA gene from Xenopus laevis.

Termination of RNA polymerase III transcripts commonly occurs at clusters of T residues. A T4 tract located 72 base-pairs beyond a lysine tRNA gene from Xenopus laevis serves as an efficient termination site for the tRNA(Lys) precursors synthesized from this gene in homologous cell-free extracts. Nucleotides following this T tract influence the extent of read-through transcription in vitro, but in a way that differs from Xenopus 5 S RNA termination. Only approximately 50% of the transcripts initiated in vitro extend as far as this downstream T cluster. The remainder prematurely terminate at a second T4 tract located within the gene itself. The contrasting behaviour of these two T tracts in injected oocytes indicates that termination can be influenced by more than just RNA polymerase III alone, and that different components may contribute to, or hinder, termination at these sites. Prematurely terminated tRNA(Lys) transcripts are detectable in RNA from ovary tissue but not from a kidney cell line, suggesting that read-through transcription beyond intragenic T clusters can be modulated in vivo.

Differential expression of nucleoside diphosphate kinases (NDPK/NM23) during Xenopus early development.

In Xenopus laevis, three nucleoside diphosphate kinase (NDPK) monomers have been described (NDPK X1, X2 and X3) (Ouatas et al., 1997). In eucaryotes, this kinase is known as a hetero- or homohexamer. Here, we examine the distribution of the enzyme and its different subunit mRNAs during oogenesis and early embryogenesis of Xenopus laevis, respectively by immunohistofluorescence and whole-mount in situ hybridization. These analyses show that NDPKs and their mRNAs are differentially distributed throughout the oocyte and early embryos with a high level of transcription in somites and brain. We emphasize two points. First, each mRNA displays a distinct subcellular localization in somites, suggesting a complex regulation of NDPK genes both at the transcriptional and translational level and a possible involvement of NDPK X2 homohexamers in the dorsal muscle differentiation. Second, in oocytes and early embryos, the proteins are mainly localized in the nucleus, suggesting a new mechanism for their nuclear import, since they do not possess any known nuclear import sequences.

Thyroid hormone regulation of germ cell-specific EF-1 alpha expression during metamorphosis of Xenopus laevis.

In situ hybridization was used to follow the distribution of the mRNAs encoding the somatic form of elongation factor 1 alpha (EF-1 alpha S) and the germinal counterparts of this factor, thesaurin a and EF-1 alpha O, throughout metamorphosis in the gonads of Xenopus laevis tadpoles. EF-1 alpha S mRNA is detected before metamorphosis in both the somatic and germ cells of the gonads. In contrast, thesaurin a and EF-1 alpha O mRNAs are first detected in spermatogonia and oogonia at stages 60-62, corresponding to the climax of metamorphosis and to the peak of circulating thyroid hormone. To determine whether thyroid hormone, the instigator of metamorphosis, is involved in regulating the expression of the germinal gene EF-1 alpha O, Xenopus XTC cells were transfected with an EF-1 alpha O promoter sequence inserted in front of the luciferase reporter gene. Addition of T3 to the cell culture medium induced a dose-dependent increase in transcription from the EF-1 alpha O promoter. This effect was enhanced when the construct was cotransfected with an expression vector for a Xenopus thyroid hormone receptor. Our data show that germ cells switch from a somatic to a germ-cell specific mode of expression during metamorphosis. Furthermore, this switch appears to be induced by thyroid hormone.

Three different genes encode NM23/nucleoside diphosphate kinases in Xenopus laevis.

Nucleoside diphosphate kinases (NDPKs) catalyse the phosphorylation of nucleoside diphosphates. In mammals, the functional enzyme is a hexamer composed of different amounts of two homologous acidic (A) and basic (B) subunits encoded by separate genes. In prokaryotes and invertebrate eukaryotes, only one cytoplasmic enzyme has been isolated. Other genes encoding chloroplastic and mitochondrial forms as well as related proteins have been cloned. Here, we show that in Xenopus laevis, as in mammals, the cytoplasmic NDPK is encoded by several homologous genes. With Xenopus laevis being a pseudotetraploid species, each monomer is encoded by two genes. The amino acid sequences are very similar, and all the differences concern amino acids located at the outer surface of the hexameric enzyme. The Xenopus genes share 82-87% identity with their human counterparts. Interestingly, in vitro, the Xenopus X1 enzyme binds to a specific nuclease hypersensitive element (NHE) of the human c-myc promoter, as does its human counterpart. X1 also binds to a single-stranded (CT)(n) dinucleotide repeat. The NHE is present in the coding strand of a pyrimidine-rich region of the 3' non-coding sequence of the Xenopus NDPK genes. We propose that NDPK is indeed able to bind to its own mRNA and prevent polyadenylation at the normal position. This could provide an autoregulatory translation mechanism. A phylogenetic tree of the vertebrate NDPK sequences supports the idea that in amphibians, as in mammals, gene duplication has resulted in functional diversification.

Analysis of the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene sequence.

A basis for the intrinsic resistance of some Plasmodium vivax isolates to pyrimethamine is suggested following the isolation of the bifunctional gene encoding dihydrofolate reductase-thymidylate synthase (DHFR-TS) of this human malaria parasite. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to an inhibition of DNA replication. The gene was isolated by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotides designed to hybridize on the highly conserved regions of the sequence. The nucleotide sequence was completed by screening P. vivax genomic bank. Sequence analysis revealed an open reading frame (ORF) of 1872 nucleotides encoding a deduced protein of 623 amino acids (aa). Alignment with other malarial DHFR-TS genes showed that a 237-residue DHFR domain and a 286-residue TS domain were separated by a 100-aa linker region. Comparison with other malarial species showed low and essentially no isology in the DHFR and junctional domains, respectively, whereas an extensive isology was observed in the TS domain. The characteristic features of the P. vivax DHFR-TS gene sequence include an insertion of a short repetitive tandem array within the DHFR domain that is absent in another human malaria parasite, P. falciparum, and a GC-biased aa composition, giving rise to highly GC-rich DHFR (50.8%), junctional (58.7%), and TS (40.5%) domains, as compared with other malaria parasites. Analysis of the 5' noncoding region revealed the presence of a putative TATA box at 116 nucleotides upstream of the ATG start codon as well as a putative GC box at -636. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed two residue changes: Ser Arg-58 and Ser Asn-117. These aa residues correspond to codons 59 and 108 in the P. falciparum DHFR active site in which similar aa substitutions (Cys Arg-59 and Ser Asn-108) are associated with pyrimethamine resistance. These findings may explain the intrinsic resistance of some P. vivax isolates to pyrimethamine.

Thesaurin a, the major protein of Xenopus laevis previtellogenic oocytes, present in the 42 S particles, is homologous to elongation factor EF-1 alpha.

We have purified in SDS X.laevis thesaurin a (Mr 50,000) which is part of the 42 S storage particles. Its N-terminal amino acid is blocked and several peptides obtained by V8 protease treatment were purified and sequenced. As expected from one of the functional roles of the 42 S particles (tRNA binding, protection against deacylation and exchange with the ribosome), the amino acid sequence of thesaurin a was found to be closely related to that of the elongation factor EF-1 alpha. We suggest that all three proteins involved in 5 S RNA and tRNA storage in previtellogenic oocytes, TFIIIA, thesaurin a and thesaurin b, have a dual function: storage and a role in transcription or in protein synthesis.

The nucleotide sequence of phenylalanine tRNA of Xenopus laevis.

The nucleotide sequence of Xenopus laevis phenylalanine tRNA extracted from oocytes was determined to be: pGCCGAAAUAm2GCUCm1AG DDGGGAGAGCm22 G psi psi AGACmUGmAAYA psi C UAAAGm7GDCm5CCUGGT psi CGm1AUCCCGG GUUUCGGCACCAoH. This result was achieved by analysing, with classical procedures [6], the oligonucleotides obtained after digestion by T1 or pancreatic ribonuclease. This sequence is identical to the mammalian sequence. It has been entirely conserved during 10(8) years, the time lapse between the divergence of amphibians and mammals in evolution. In contrast to 5S RNA, no important heterogeneity has been found in the oocyte sequence, suggesting that there is only a single sequence for tRNAphe in X. laevis. Small differences are seen in the elution pattern from RPC-5 columns for immature oocyte and somatic tRNAphe. They are probably due to a submodification of methyl-5-cytidine residues, which appear to be about half methylated in tRNAphe as well as in total tRNA from immature oocytes.

Sequence variations in the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene and their relationship with pyrimethamine resistance.

The gene encoding dihydrofolate reductase-thymidylate synthase of the human malaria parasite, Plasmodium vivax, was isolated by polymerase chain reaction from genomic DNA and cloned. The sequences of the dihydrofolate reductase domain of 30 clinical isolates originating from various geographic areas were compared. Interstrain analysis revealed several genotypic variations, including short tandem repeat arrays which produced length polymorphism between different parasite isolates and point mutations in the putative dihydrofolate reductase active site cavity corresponding to those associated with pyrimethamine resistance in P. falciparum and rodent malaria parasites. Amino acid substitutions Ser-->Asn-117 and Ser-->Arg-58 were associated with decreased level of in vitro pyrimethamine sensitivity. These findings suggest that the P. vivax dihydrofolate reductase domain is characterized by polymorphism that has not been observed in P. falciparum and may explain the resistance of some P. vivax isolates to pyrimethamine. Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB databases under the accession numbers X98123 (isolate ARI/Pakistan), AJ003050 (isolate CNC/Thailand), AJ003051 (isolate COU/unknown geographic origin), AJ003052 (isolate DUF/French Guiana), AJ003053 (isolate GRO/Madagascar), AJ003054 (isolate HRT/Comoros Islands), AJ003071 (isolate LFT/Cambodia), AJ003072 (isolate LGF/'India), AJ003073 (isolate MAN/Comoros Islands), AJ003074 (isolate MAT/Surinam), AJ003075 (isolate PHI/Djibouti), AJ003076 (isolate PIT/Madagascar), AJ003077 (isolate YTZ/Indonesia), AJ222630 (isolate Burma-1), AJ222631 (isolate Burma-151), AJ222632 (isolate Burma-5), AJ222633 (isolate Burma-6), AJ222634 (isolate Burma-98).

Kinetic properties of dihydrofolate reductase from wild-type and mutant Plasmodium vivax expressed in Escherichia coli.

Antifolate drugs inhibit malarial dihydrofolate reductase (DHFR). In Plasmodium falciparum, antifolate resistance has been associated with point mutations in the gene encoding DHFR. Recently, mutations at homologous positions have been observed in the P. vivax gene. Since P. vivax cannot be propagated in a continuous in vitro culture for drug sensitivity assays, the kinetic properties of DHFR were studied by expression of the DHFR domain in Escherichia coli. Induced expression yielded a protein product that precipitated as an inclusion body in E. coli. The soluble, active DHFR recovered after denaturation and renaturation was purified to homogeneity by affinity chromatography. Kinetic properties of the recombinant P. vivax DHFR showed that the wild-type DHFR (Ser-58 and Ser-117) and double mutant DHFR (Arg-58 and Asn-117) have similar K(m) values for dihydrofolate and NADPH. Antifolate drugs (pyrimethamine, cycloguanil, trimethoprim, and methotrexate), but not proguanil (parent compound of cycloguanil) inhibit DHFR activity, as expected. The kinetics of enzyme inhibition indicated that point mutations (Ser58Arg and Ser117Asn) are associated with lower affinity between the mutant enzyme and pyrimethamine and cycloguanil, which may be the origin of antifolate resistance.

A clinicopathologic review of 25 cases of chordoma (a pleomorphic and metastasizing neoplasm).

A clinicopathologic study of 25 cases of chordoma revealed that this tumor occurs principally in (68%), with a predominance for the sixth decade of life (seven patients--28%), and shows a predilection for the sacrococcygeal region (52%). The symptomatology was intimately related to the location of the tumor. Histologically, chordoma showed an extremely wide range in its cellular composition and pattern, not only from tumor to tumor, but also often in different portions of the same tumor. In addition to the large physaliferous cells in a lobular arrangement, large cells with apparently degenerating nuclei (ghost cells) were commonly seen; cells arranged in concentric spherical formations were observed in two cases, whereas small, round cells predominated in another case. A sarcomatous pattern was prominent in two cases. Large pink cells were frequently seen and in one case were arranged in epithelial-like columns. Whether these neoplastic components can be related to different degrees of tumor differentiation is difficult to establish. Histologic features of five cases in which metastasis occurred were compared to previously described metastasizing cases. These appear to be few reliable features helpful in suggesting the metastatic potential of this neoplasm.

Immunoblastic lymphoma following Hodgkin's disease: a case report with translocation t(8;14) in tumoral cells and sporadic t(7;14) in peripheral lymphocytes.

A non-Hodgkin's lymphoma was observed in a patient who had been treated for Hodgkin's disease (HD). The initial treatment consisted of radiotherapy alone, but following three subsequent relapses, both chemotherapy and radiotherapy were administered several times. Twenty years later, the biopsy of an isolated cervical lymph node revealed a non-Hodgkin's lymphoma. The histologic subtype was immunoblastic. Cytogenetic studies of the tumoral cells revealed a t(8;14)(q24;q32) translocation. At the same time, multiple chromosomal rearrangements were observed in peripheral blood lymphocytes, especially t(7;14)(q35;q12), which was noted in 6 of 53 mitoses. This anomaly, frequently observed in patients with ataxia telangiectasia or severe immunodeficiency, has not previously been described in such circumstances.

Translocation involving chromosome 22 in Ewing's sarcoma. A cytogenetic study of four fresh tumors.

Ewing's sarcoma was described in 1921 by James Ewing as a diffuse endothelioma of bone and, for some time, was believed to be an undifferentiated type of Parker's sarcoma. At present, these two entities are thought to be distinct, the macroscopic and microscopic aspects of Ewing's sarcoma being very characteristic, although the exact cell type of this tumor remains unknown. This has lead many workers to study this sarcoma in order to recognize its origin. We thought it of interest to carry out cytogenetic investigations of our cases of Ewing's sarcoma, since very few chromosomal data on this malignancy exist in the literature [1-3].

Desmoid tumors in adults: the role of radiotherapy in their management.

Twenty-six adult patients with the pathologic diagnosis of desmoid tumor were treated between 1964 and 1983 at the Institut Curie in Paris with megavoltage irradiation. Twenty of these patients (76 percent) had extraabdominal tumors. Definitive surgical resection was performed on nine patients (one received preoperative radiotherapy). At last follow-up 1 1/2 to 10 years after treatment, all of the patients had no evidence of disease. Seven of the nine had follow-up examinations from 5 to 10 years after treatment. Seven patients had postoperative radiotherapy with doses from 4,700 to 6,500 rads (47 to 65 Gy) for either microscopic (three patients) or gross (four patients) residual disease. All but one patient had no evidence of disease from 2 to 8 years after treatment. Nine patients had radiotherapy for recurrent inoperable tumors and six had no evidence of disease from 3 to 20 years after treatment. Recurrences developed in three patients; outside the treatment portal in one, and the other two had received less than 5,000 rads (50 Gy). Clinical regression of tumors after treatment was slow, with complete regression taking up to 2 years. Postoperative radiotherapy with doses of at least 5,000 to 6,000 rads (50 to 60 Gy) was effective in achieving local control of inoperable or incompletely resected tumors, thus the need for repeated resections was avoided. Computerized tomography has greatly improved the assessment of tumor extension and should be used routinely before either operation or radiotherapy to obtain adequate margins and minimize the chance of missing disease.

Ewing's sarcoma: transplantation in nude rat.

Eight Ewing's sarcoma, primary tumor or metastasis, have been transplanted in Nude Rats. These tumors grow slowly and only in female rats. One of them has been maintained for 13 months with 5 passages. It has conserved all the characteristics of the primary tumor, histologic and ultramicroscopic morphology, glycogen secretion and cytogenetic modification (11.22 translocation). The graft of Ewing's sarcoma to Nu/Nu rats is a valuable system to get more material in good condition to study the nature and the origin of Ewing's cells, to test the new chemotherapy trials and to prepare and test the monoclonal antibodies.

[Experimental production of bone sarcomas in the rabbit by a single local injection of beryllium]. / Production expérimentale de sarcomes osseux chez le lapin par injection unique locale de Béryllium

The local intra-osseous injection of double zinc beryllium silicate into the tibial or femoral epiphysis of a rabbit causes an osteogenic sarcoma in 70 p. 100 of cases. These experimental conditions make it possible to reveal early non specific radiological alterations, later on secondary alterations corresponding to the development of the sarcoma and finally to follow the spontaneous evolution of the tumor. Moreover, this experimental process of induction of an osteogenic sarcoma by means of a local intra-osseous injection is vastly better than an intra-venous injection which causes straight-away multiple visceral lesions.

[A case of epidermoid carcinoma developing from contact with an articular hip prosthesis]. / Un cas de carcinome épidermoïde développé au contact d'une prothèse articulaire de hanche.

Several cases of sarcomas which developed in contact with joint prosthetic material were reported previously. They were mostly malignant fibrous histiocytoma, but to our knowledge, no case of epidermoid carcinoma was reported. We report here one case of epidermoid carcinoma which developed in contact with a hip prosthesis. Surgery was complicated by local infection and necessitated multiple reoperations, which may be responsible for the implantation of skin cells. It is generally admitted that multiple factors contribute to the development of a tumor clone. In our patient we must take into consideration such factors as chronic infection with fistula and prosthetic material. However carcinomas, even rare, which form on fistulas appear only after many years of suppuration. The short delay between the prosthesis setting and the carcinoma onset in our patients, suggests that a cofactor may have speeded up tumor induction. This cofactor could be the platelet derived growth factor (PDGF) in the case of sarcomas appearing on bone infarcts without foreign body introduction, and possibly other tumors.

[Sarcomatoid spindle-cell epithelioma. Anatomicoclinical study of 15 cases]. / Le problème des "épithéliomas fusocellulaires sarcomatoïdes". Etude anatomoclinique de 15 cas.

Sarcomatoid spindle cell carcinoma, which is a rather rare cancer, is of epithelial origin, but histologically resembles a connective spindle cell sarcoma. It remains subject to controversy, especially concerning the reality of epitheliomesenchymatous transformation of the neoplastic cells which, indeed, take on not only the shape but also the behaviour of connective cells. The debate is of both a dogmatic and practical nature, dogmatic in that it has bearing on the question of cellular specificity, and practical in that histological transmutation has repercussions on the macroscopic aspect of the tumor, its clinical evolution and even its behaviour vis-a-vis radiation therapy. The preferred, but not the only, sites of such tumors are the upper respiratory and digestive tracts. In many cases, they occur in subjects who many years previously had undergone radiological treatment, with the tumor appearing in the irradiated area. The problems raised by these unusual cancers explain and seem to justify the publication over the last few years of isolated cases or series of reports made in various centers. The present work concerns 15 anatomoclinical cases observed at the Institut Curie.

[Reflections on the classification validation of the most common forms of osteosarcomas by functional isotopic methods]. / Réflexions sur la validation de la classification des formes les plus fréquentes des sarcomes ostéogènes par des méthodes fonctionnelles isotopiques.

After noting the limits of a purely morphological approach, either the descriptive methods for classifying certain osteosarcomas, or the techniques connected with these limitations such as particularly limited or non significant sampling, the authors consider the possible contribution of new methods such as immunohistochemistry or in vivo radioisotope tests to improve osseous tumor classification. The latter methods may be particularly useful in clarifying a significant number of difficult diagnoses or in providing information on tumor location, to be combined with morphological observations regarding osteosarcomas.

[Pathological anatomy of sarcomas of the bone and soft tissue]. / Anatomie pathologique des sarcomes osseux et des tissus mous.

The different pathologic aspects of bone and soft-tissue sarcomas are discussed: usefulness of special techniques, particularly immunohistochemistry, histologic classification and grading, diagnosis of frozen sections and assessment of treatment effects, particularly of postoperative chemotherapy.