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H Denis

Publications and source records attributed to H Denis.

At least 37 records · Page 2Linked to original sources
PubMed1989

Immunotoxicotherapy: present status and future trends.

Immunotoxicotherapy (ITT) is currently used in humans for the treatment of snake venom and cardiac glycoside poisoning. Other toxins have been studied in animals or in vitro to assess their suitability as candidates for detoxification by specific antibodies. Testing conditions are often empirical suggesting that numerous improvements need to be introduced in ITT. Basic mechanisms in ITT include three phases: sequestration, extraction and elimination. The pharmacokinetics of these three phases depend on the type of antidotal binding site (ABS). IgG or its Fab2, Fab or Fv fragment are the possible choices. The Fab fragment is the most frequently used ABS because of its diffusion properties in the peripheral compartments and its renal excretion by glomerular filtration. Toxicokinetic and pharmacokinetic considerations indicate that the dosage cannot be satisfactorily calculated from stoichiometric principles. Study of the toxin dose-lethality curves shows that ABS dosage can be lowered. Moreover, clinical data reveal that some FAb fragments are directly eliminated without acting on toxin molecules. In order to counteract these drawbacks, a compromise between dosage and duration of infusion is suggested. Other improvements will stem from advances in immunologic methodology. Monoclonal and chimeric antibodies are new tools that will help resolve the clinical problems of immunogenicity and adverse reactions associated with polyclonal ABS.

Animals↗
PubMed1988

Characterization by human antibodies of two HeLa cell proteins which are related to Xenopus laevis transcription factor TFIIIA.

The sera of two patients with autoimmune disorders recognize in HeLa cell extracts two proteins with apparent molecular masses of 37,000 (p37) daltons and 32,000 daltons (p32). These proteins are non covalently associated with 5S RNA and sediment as 7-10 S particles in sucrose density gradients. Both proteins are antigenetically related to TFIIIA, a previously described protein of Xenopus laevis, which is known as a 5S RNA transcription factor and occurs in oocytes as a noncovalent complex with 5S RNA. Like TFIIIA, HeLa cell proteins p37 binds in vitro to 5S RNA and to cloned 5S RNA genes. These results suggest that protein p37 fulfils in HeLa cells a function similar to that of TFIIIA in amphibian oocytes, ie control of 5S RNA transcription.

Animals↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

Biochemical research on oogenesis. Binding of tRNA to the nucleoprotein particles of Xenopus laevis previtellogenic oocytes.

In previtellogenic oocytes of Xenopus laevis, nearly all tRNA is included in nucleoprotein particles (thesaurisomes) sedimenting at 42 S. We evaluate the possibility of a tRNA exchange between the particles and the ribosomes during protein synthesis. We find that the particles take up tRNA after a very short incubation in vitro. In the absence of ATP, the particles preferentially bind charged tRNA. In the presence of ATP, more tRNA binds to the particles, and the sedimentation coefficient of the integrated tRNA is displaced to 45 S. When added to nonfractionated homogenates of oocytes together with ATP, poly(U) strongly stimulates the incorporation of radioactive phenylalanine into tRNA and protein. The labeled protein (polyphenylalanine) cosediments with the ribosomes, whereas most of phenylalanyl tRNA cosediments with the thesaurisomes. These data suggest that the thesaurisomes participate to some extent in protein synthesis. They release charged tRNA, thereby supplying the ribosomes with activated amino acids. Discharged tRNA is then taken up, reacylated, and stored in the particles until the next round of peptide bond formation. The aminoacylation and storage functions are probably carried out by two very unequal populations of particles. The main subclass of particles (42 S) binds and stores tRNA in an ATP-independent manner. A much smaller subclass of particles (45 S) is responsible for reacylation of discharged tRNA.

Animals↗
PubMed1987

Biochemical research on oogenesis: protein synthesis in whole cells and in cell-free extracts of Xenopus laevis immature ovaries.

Nearly all tRNA molecules in previtellogenic oocytes of Xenopus laevis are included in nucleoprotein particles sedimenting at 42S. The tRNA-binding sites of these particles have several properties in common with those of the ribosomes. This suggests that the 42S particles might behave like unprogrammed ribosomes and be the site of a template-independent polymerization of amino acids. We expected this reaction to be insensitive to protein synthesis inhibitors, such as cycloheximide and puromycin. We found that these antibiotics almost completely inhibit the incorporation of labeled amino acids into protein, when added to the incubation medium of whole ovaries or free oocytes. In cell-free extracts of ovaries, the incorporation of amino acids is partially insensitive to cycloheximide and puromycin. When such extracts are fractionated by sucrose density centrifugation and incubated with ATP, a major peak of amino acid incorporation can be detected, which nearly coincides with the 42S particle peak.

Adenosine Triphosphate↗
PubMed1987

Biochemical research on oogenesis: protein synthesis by purified 42S particles from Xenopus laevis and Tinca tinca previtellogenic oocytes.

When incubated with ATP and a labeled amino acid, the 42S particles from early oocytes of Xenopus laevis and Tinca tinca incorporate radioactivity into tRNA and into a high molecular mass material which can be identified as protein. This incorporation is totally independent of ribosomes of cytosolic, mitochondrial or bacterial origin. The incorporated amino acids are linked to a broad spectrum of proteins by covalent bonds. Simple treatments such as incubation in buffer or addition of synthetic polyribonucleotides can inhibit the protein-labeling activity of the particles without affecting their tRNA aminoacylation activity. The former activity corresponds either to an amino acid polymerization reaction or to a protein-modifying reaction of a novel type. No involvement of mRNA in this process has been demonstrated. The alleged amino acid polymerization activity of the 42S particles could be a consequence of the conditions provided to aminoacyl tRNA by the tRNA-binding sites of the particles. These conditions are likely to allow the peptidyl transfer reaction to take place, although at a much lower rate than in the ribosome.

Adenosine Triphosphate↗
PubMed1987

Biochemical research on oogenesis: distribution of tRNA-linked peptides and proteins in previtellogenic oocytes of Xenopus laevis.

Peptides and proteins were detected in the deacylation products of tRNA purified from the 42S particles and from the messenger ribonucleoprotein particles (mRNPs) present in the previtellogenic oocytes of Xenopus laevis. Only a small fraction of particle tRNA carries a peptide or protein chain. The bulk of particle tRNA is simply aminoacylated. The tRNA-linked peptide chains of the particles appear to turn over more slowly in vivo than aminoacyl tRNA. These chains could arise in the particles by a peptidyl transfer reaction similar to that carried out by the ribosome.

Acylation↗
PubMed1987

[A variety of human autoantibodies recognizes in HeLa cells 2 proteins related to the TFIIIA factor of Xenopus laevis which regularizes the transcription of ribosomal 5S RNA].

Using the sera from two patients with autoimmune disorders, we have identified by immunoprecipitation of HeLa cell extracts two proteins with apparent molecular masses of 37 kDa (p 37) and 32 kDa (p 32). These proteins are associated with 5 S RNA. They are antigenetically related to Xenopus laevis 5 S RNA transcription factor TFIIIA, which is very abundant in early oocytes of this species. In contrast to what is observed in X. laevis oocytes, the TFIIIA-related proteins of HeLa cells are present in very small amounts. Our data suggest that proteins p 37 and p 32 are involved in the control of 5 S RNA transcription.

Animals↗
PubMed1986

Enzyme-linked immunosorbent assay for amitriptyline and other antidepressants using a monoclonal antibody.

We describe and evaluate a method to measure amitriptyline and other tricyclic antidepressants by enzyme linked immunosorbent assay, using monoclonal antibody. In this assay, biological samples were first incubated with the antibody; in a second step, free remaining antibody was allowed to bind to lysozyme-nortriptyline coated immunotitration plates. The bound fraction of the monoclonal antibody was revealed with rabbit anti-mouse serum coupled to horseradish peroxidase. The optical density of the reaction product was measured with a colorimeter at 410 nm. Specificity of the antibody was investigated by means of a Farr test showing interferences in therapeutic ranges only for chlorpromazine and phenytoine. Means of intra- and inter-assay variations were 10 and 13%, respectively. The results when compared to those obtained by gas chromatography with a selective nitrogen detector gave a correlation coefficient of 0.897. Finally, the great reliability of the monoclonal antibody, the advantages of a decreased analysis time, low cost and high capacity of the procedure contribute to make this immunoassay most suitable for clinical monitoring and pharmacokinetic studies of tricyclic antidepressants.

Amitriptyline↗
PubMed1985

Biochemical research on oogenesis. Aminoacyl tRNA turns over in the 42-S particles of Xenopus laevis oocytes, but its ester bond is protected against hydrolysis.

The ester bond aminoacyl tRNA is protected against hydrolysis in the 42-S particles (thesaurisomes) present in Xenopus laevis previtellogenic oocytes. Deacylation of tRNA is very slow in vitro, unless ATP is present. ATP causes a partial turnover of aminoacyl tRNA in vitro, with no detectable decrease in the overall aminoacylation level of tRNA, which remains close to 100%. tRNA in the particles turns over rapidly in vivo. Since the ester bond of aminoacyl tRNA is stabilized inside the 42-S particles, this turnover cannot be a consequence of spontaneous deacylation of tRNA, followed by reacylation by the aminoacyl-tRNA synthetases associated with the particles. We rather consider this turnover as reflecting a true metabolic activity of the particles, and a direct or indirect involvement of these particles in the oocyte's protein-synthesizing system.

Adenosine Triphosphate↗
PubMed1980

Biochemical Research on oogenesis. Composition of the 42-S storage particles of Xenopus laevix oocytes.

Previtellogenic oocytes of Xenopus laevis contain far more 5-S RNA and tRNA than 28-S + 18-S RNA. tRNA and 5-S RNA are storage products that will be used in later oogenesis for protein synthesis and ribosome assembly. Stored tRNA and 5-S RNA are not free in the cell sap but belong to nucleoprotein particles of various sizes. There are two prominent kinds of storage particles in previtellogenic oocytes of X. laevis. The smaller ones (7-S) contain about half of the cell's 5-S RNA. The larger ones (42-S) contain the remainder of the 5-S RNA and 90% of the tRNA. The 7-S particles consist of one molecule of 5-S RNA and one molecule of protein. In this paper we describe the biochemical and physical properties of the 42-S particles. The 42-S particle contains four main components: tRNA, 5-S RNA, a 50000-Mr protein (a) and a 40000-Mr protein (b) in the following molar ratios: 3/1/2/1. We propose a 28-component model for the 42-S particles. This model is consistent with all the biochemical and physical data that we report here. A 42-S particle is made up of four subunits, each of which contains three molecules of tRNA, one molecule of 5-S RNA, two molecules of protein a, and one molecule of protein b. Protein b from the 42-S particles and the 7-S particle protein are indistinguishable by all tests that we have tried. We present evidence showing that protein a binds tRNA whereas protein b binds 5-S RNA in the 42-S particles as well as in the 7-S particles.

Animals↗
PubMed1979

Biochemical research on oogenesis. Transfer RNA is fully charged in the 42-S storage particles of Xenopus laevis oocytes.

1. Transfer RNA makes up 30-40% of total RNA in previtellogenic oocytes of Xenopus laevis. The bulk of tRNA is associated with 5-S RNA and two proteins in a high-molecular-weight complex sedimenting at 42S. 2. We show here that all kinds of tRNA are present in the 42-S particles and all of them sediment coincidently. Particle tRNA is fully charged in vivo. During purification of the 42-S particles tRNA becomes partially uncharged. When purified particles are incubated in vitro with amino acids and ATP a charging reaction occurs without disruption of the nucleoprotein complex. Many aminoacyl-tRNA synthetases can be shown to co-sediment with the 42-S particles. We conclude that complete aminoacylation of tRNA within the storage particles results from the activity of particle-bound aminoacyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Evolution of the 5 S RNA genes in vertebrates.

We have built the phylogenetic tree of Vertebrate 5S RNA using the sequence data of thirteen species belonging to six groups. Evolution of the 5S genes has been very slow in Vertebrates since 90 residues are identical in all 5S RNAs which are presently sequenced. In Amphibians and Teleosts different 5S genes are active in oocytes and in somatic cells. This dual gene system has probably been acquired independently by Amphibians and Teleosts. In Amphibians, the oocyte-type 5S genes have evolved much faster than the somatic-type genes. This is not true in all species since the oocyte-type genes of one Teleost (Tinca tinca) have evolved more slowly than the somatic-type genes. There are in all Vertebrate 5S RNAs five complementary regions which can be base-paired. The sequence data are compatible with the three secondary-structure models that have been proposed for 5S RNA.

Animals↗