Biomedical subjects
Publications and source records attributed to J L Couderc.
Ceftriaxone penetrates into the aqueous humour in man. The interaction of indomethacin on the disposition of ceftriaxone appeared to be time-dependent. Indomethacin delayed ceftriaxone penetration into, but enhanced its persistence in, the aqueous humour, permitting a period of antibioprophylaxis covering the required time for surgical treatment.
The expression of 56D and 60C beta-tubulin genes has been examined in Drosophila melanogaster Kc cells in response to the insect moulting hormone, 20-hydroxyecdysone (20-OH-E). Northern blots probed with beta-tubulin subclones show that the 56D beta-tubulin gene encodes a 1.8 kb mRNA whose abundance is not affected by 20-OH-E. The 60C gene probe detects two mRNAs: one of 1.8 kb present in untreated and 20-OH-E-treated cells, and one of 2.6 kb present only in 20-OH-E-treated cells; using a 60C 3'-specific probe, only the 2.6 kb is revealed. Hybrid selection translation experiment demonstrates that a 20-OH-E-inducible mRNA homologous to the 60C gene encodes a beta-tubulin subunit (P4); this subunit is the so-called beta 3-tubulin. Translation of size-fractionated mRNA shows that the 20-OH-E-induced beta 3-tubulin subunit is encoded, in treated cells, by the 2.6 kb mRNA.
Used at a physiological concentration, the steroid hormone 20-hydroxyecdysone (20-OHE) induces, in Kc cultured Drosophila melanogaster cells, important and specific changes. Modifications occur at morphological and enzymatical levels. Variations in specific protein synthesis are observed. At the molecular level, 20-OHE particularly induces a decrease in expression of the mobile dispersed genetic element 412. This repeated element which belongs to the "copia-like" family is more widely represented in Kc cells (80 fold) compared to fly cells (25 fold). 412 transcripts are heterogeneous in size, essentially polyadenylated and restricted to the nucleus. A minimal concentration of 10(-8) M and a time treatment of 16 hours are necessary to obtain a strong decrease in 412 expression. The decrease is at least an effect on these sequences at the transcriptional level. Structural similarities between the 412 element and the proviral forms of vertebrate retroviruses are strengthened by the characterization of extrachromosomal circular DNA forms revealed by the 412 probe. Quantifying experiments have shown that the steady state level of such forms is not affected by the steroid treatment.
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The steroid hormone 20-hydroxyecdysone (20-OHE) induces, in Kc cultured Drosophila melanogaster cells, important morphological transformations and specific changes of enzymatic activities and of protein synthesis. These changes are accompanied by an increase of synthesis and an accumulation of actin. Specific probes were used to reveal transcripts of each actin gene in mRNA populations isolated from cells at various times of 20-OHE treatment. Only the two cytoplasmic actin genes 5C and 42A are expressed in Kc cells and the hormone induces the accumulation of transcripts of these two genes. We have also taken advantage of S1 mapping and extension procedures to identify the 5' ends of the actin mRNAs from these two genes and to compare their respective levels of expression. The 5C gene is more expressed than the 42A one in untreated and in hormone treated cells. The 5C gene encodes three RNAs that differ in their 3' end. The two genes are interrupted by an intervening sequence immediately upstream of ATG initiation codon but not at the same position. The transcription rate for the two genes is increased up to five fold upon 20-OHE treatment, demonstrating a direct effect of the steroid hormone at the transcriptional level for these genes.
One polypeptide, P4, of molecular weight 55000 daltons and pHi, 5,1, is synthesized in Drosophila melanogaster Kc 0% cells only when they were treated by the insect moulting hormone, 20 hydroxyecdysone (20-HE). P4 is precipitated with vinblastine sulfate and migrates as a tubulin subunit. Immunoblot experiment confirms that P4 is a beta subunit of tubulin. This beta tubulin is recovered by in vitro translation only when mRNAs are extracted from treated cells. Thus, regulation of expression of this polypeptide is at the level of transcripts and is under a steroid hormone control. The 20-HE induced beta tubulin comigrates with the beta 3 subunit and like the latter, is specific to the mid period of embryogenesis.
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A procedure for extracting RNA-free plasmid DNA from bacterial cells is described. The method is simple and rapid enough to obtain pure plasmid DNA in 8 to 10 h after plasmid amplification. The protocol uses the alkaline extraction procedure described by Birnboim and Doly (1979, Nucl. Acid Res. 7, 1513-1523). Plasmid DNA is then separated from high-molecular-weight RNA by ammonium acetate precipitation and from low-molecular-weight RNA contaminants by Ultrogel A2 column chromatography. The plasmid DNA obtained by this inexpensive technique is sufficiently pure to be used for restriction endonuclease analysis, 5'-end labeling, S1 mapping, DNA sequencing, and colony hydridization.
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The steroid hormone ecdysterone induced characteristic and specific changes of morphology, enzymatic activities and protein synthesis in a Kc 0% Drosophila melanogaster cell line. To study the ecdysterone action at a molecular level, a Drosophila genomic library was screened by differential hybridization to poly(A)+ RNA from control and ecdysterone-treated cells. Two recombinant phages were selected for hybridizing very intensively with poly(A)+ RNA of ecdysterone-treated cells and very weakly with poly(A)+ RNA of untreated ones. These two clones (lambda Dm 1632 and lambda Dm A5A1) mapped at the 5 C locus on polytene chromosomes; they overlap for a 9000 base-pair sequence that contains an abundantly transcribed region in ecdysterone-treated cells of about 2000 base-pairs. This region permits the selection of mRNA that gives, after translation in vitro, two polypeptides identified as cytoplasmic actin II and III. We demonstrated that these two recombinant phages, hybridizing preferentially with poly(A)+ RNA of ecdysterone-treated cells, contain the 5 C actin gene. Poly(A)+ RNA prepared from various times of treatment of cells were electrophoresed on agarose gels, transferred to nitrocellulose paper and then hybridized with the cloned actin probe. Results of these experiments indicate that there is a sharp increase in the level of RNA coding for actin after ecdysterone treatment of the cell, and that there are two forms of actin-specific RNA in the D. melanogaster cells. Using genomic blots with specific probes derived from lambda Dm 1632, we show that there are six actin genes per haploid Drosophila cell genome contained on six EcoRI fragments, as in Drosophila embryos, indicating that there is no rearrangement of these sequences in cultured cells. Our results suggest that the expression of actin genes in D. melanogaster Kc 0% cells is modulated by ecdysterone.
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