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Biomedical subjects

C Richaud

Publications and source records attributed to C Richaud.

At least 19 recordsLinked to original sources
PubMed1992

Cloning of dapD, aroD and asd of Leptospira interrogans serovar icterohaemorrhagiae, and nucleotide sequence of the asd gene.

Metabolites such as diaminopimelate and some aromatic derivatives, not synthesized in mammalian cells, are essential for growth of bacteria. As a first step towards the design of a new human live vaccine that uses attenuated strains of Leptospira interrogans, the asd, aroD and dapD genes, encoding aspartate beta-semialdehyde dehydrogenase, 3-dehydroquinase and tetrahydrodipicolinate N-succinyltransferase, respectively, were cloned by complementation of Escherichia coli mutants. The complete nucleotide sequence of the asd gene was determined and found to contain an open reading frame capable of encoding a protein of 349 amino acids with a calculated Mr of 38,007. Comparison of this deduced L. interrogans aspartate beta-semialdehyde dehydrogenase amino acid sequence with those of the same enzyme from Saccharomyces cerevisiae and Corynebacterium glutamicum revealed 46% and 36% identity, respectively. By contrast, the identity between the L. interrogans enzyme and the Streptococcus mutans or E. coli enzymes was less than 31%. Highly conserved sequences within aspartate semialdehyde dehydrogenase from the five organisms were observed at the amino and carboxyl termini, and around the cysteine of the active site.

Acyltransferases↗
PubMed1992

Cloning, characterization, and expression of the dapE gene of Escherichia coli.

The dapE gene of Escherichia coli encodes N-succinyl-L-diaminopimelic acid desuccinylase, an enzyme that catalyzes the synthesis of LL-diaminopimelic acid, one of the last steps in the diaminopimelic acid-lysine pathway. The dapE gene region was previously purified from a lambda bacteriophage transducing the neighboring purC gene (J. Parker, J. Bacteriol. 157:712-717, 1984). Various subcloning steps led to the identification of a 2.3-kb fragment that complemented several dapE mutants and allowed more than 400-fold overexpression of N-succinyl-L-diaminopimelic acid desuccinylase. Sequencing of this fragment revealed the presence of two closely linked open reading frames. The second one encodes a 375-residue, 41,129-M(r) polypeptide that was identified as N-succinyl-L-diaminopimelic acid desuccinylase. The first one encodes a 118-residue polypeptide that is not required for diaminopimelic acid biosynthesis, as judged by the wild-type phenotype of a strain in which this gene was disrupted. Expression of the dapE gene was studied by monitoring amylomaltase activity in strains in which the malPQ operon was under the control of various fragments located upstream of the dapE gene. The major promoter governing dapE transcription was found to be located in the adjacent orf118 gene, while a minor promoter allowed the transcription of both orf118 and dapE. Neither of these two promoters is regulated by the lysine concentration in the growth medium.

Amidohydrolases↗
PubMed1992

Scattering of the rRNA genes on the physical map of the circular chromosome of Leptospira interrogans serovar icterohaemorrhagiae.

Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.

Base Sequence↗
PubMed1991

Overproduction and characterization of the iclR gene product of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2.

The iclR gene of Escherichia coli K-12, which encodes a regulatory protein (repressor) for the aceBAK operon, is located between that operon and metH in the 91-min region of the chromosome. The iclR gene was cloned and expressed in a coupled T7 RNA polymerase/promoter system and the gene product was identified by specific binding to a fragment containing the aceBAK operator region. The iclR gene product is a polypeptide of 274 amino acids (aa) with a calculated Mr of 29,741. Comparison of the deduced IclR aa sequence to that of Salmonella typhimurium revealed that the two IclR repressors exhibit 89% identity. A possible helix-turn-helix motif characteristic of DNA-binding proteins was found within the IclR sequence. A search in protein data banks revealed that IclR has a score of similarity of 43.7% with GylR, a transcriptional regulator of the glycerol operon of Streptomyces coelicolor.

Acetates↗
PubMed1991

[Björk-Shiley-Monostrut prosthesis of the aortic valve. Mid-term development].

From July 84 to June 88, 100 patients underwent an isolated aortic valve replacement by a Monostrut-Björk-Shiley prosthesis. Fifty-one had calcified aortic stenosis, 24% aortic insufficiency, 25% mixed aortic lesions. Pre-operatively, their mean age was 57 years, 68% were male, 46% were in NYHA class II or IV, 43% had angina, all were in sinus rhythm. Operative mortality was 4%. All the survivors were followed-up for a mean period of 22 months (6 to 58) with a cumulative follow-up of 183 patients-years. All patients were placed on a long-term regimen of anticoagulant therapy. Two late deaths occurred (1 myocardial infarction, 1 cerebral hemorrhage). The 4 years survival rate was 94%. Postoperative functional results were excellent. Nearly all patients were asymptomatic. Concerning valve related complications: the 4 years actuarial rate of patients free from thromboembolism, hemorrhage, valve thrombosis, periprosthetic leakage and endocarditis were respectively 97%, 97%, 100%, 100% and 100%. No patient were reoperated on. Valvular function was evaluated by mean transprothetic gradient on echo-doppler (61 cases) and by the calculation of the valvular area with transseptal catheterization (21 cases). For small sizes prosthesis (annulus diameter of 19 or 21 mm), medium size prosthesis (23 or 25 mm), large size prosthesis (27 to 29 mm), mean transprothetic gradient were respectively 16, 10 and 6.9 mmHg and valvular aortic area were respectively 1.5, 1.9 and 2.4 cm2. Mid term results of the Monostrut-Björk-Shiley prosthesis on aortic position are good with a low rate of valve related complications and good hemodynamic results, even with the small valve size.

Actuarial Analysis↗
PubMed1990

Two genomic species in Borrelia burgdorferi.

A total of 13 Borrelia burgdorferi strains (responsible for Lyme borreliosis) and representatives of 3 other Borrelia species (B. hermsii, B. parkeri, B. turicatae) associated with relapsing fever were studied by DNA/DNA hybridization and rRNA gene-restriction patterns. Two genomic DNA hybridization groups were observed which could be differentiated by rRNA gene-restriction patterns. Moreover, the number and size of restriction fragments suggest the existence of a single set of 16 and 23 S rRNA genes in Borrelia.

Borrelia burgdorferi Group↗
PubMed1990

Cloning of genes required for amino acid biosynthesis from Leptospira interrogans serovar icterohaemorrhagiae.

Leptospira interrogans belongs to a large family of important pathogens, which is part of the order Spirochaetales, a distinct group of eubacteria. In order to obtain a better understanding of the genetic organization of this species, we have constructed a DNA library of the serovar icterohaemorrhagiae, using the Escherichia coli vector pUC13. We have isolated Leptospira DNA fragments containing the genetic information required to complement strains of E. coli with defects in proline and leucine biosynthesis. While a 3.9 kb fragment which complemented proA also complemented proB, a 15 kb fragment complementing leuB could not complement other leu mutations. The L. interrogans origin of the cloned DNA fragments was confirmed by DNA-DNA hybridization. The hydridization was specific to the pathogenic species and was not seen with the saprophytic species L. biflexa.

Amino Acids↗
PubMed1990

[Study of the etiologic mechanism of valvular regurgitation using Doppler echography].

Doppler echocardiography is currently the method of choice for diagnosing and determining the mechanism and etiology of valvular regurgitation. The recent introduction of transesophageal echo coupled with color Doppler has increased the value of these ultrasonic methods. The analysis of the valvular lesion should be particularly precise and accurate in severe mitral insufficiency because of the possibility of surgical valvuloplasty, the indications of which are now much broader than was the case at the beginning of the nineteen eighties.

Aortic Valve Insufficiency↗
PubMed1990

[Long-term evaluation of the treatment results of male urethral stenosis].

287 urethral strictures were treated between 1974 and 1984. Two-stage urethroplasty provided 47.6% of good results at 5 years, although there was a possibility of very delayed recurrences. Endoscopic urethrotomy only gave 26% of good results at 5 years, while 30% of good results were obtained after dilatation with Olbert's sound. Endourethral techniques do not compete with two-stage urethroplasty, provided they are performed repeatedly.

Catheterization↗
PubMed1989

Expression of recombinant diaminopimelate epimerase in Escherichia coli. Isolation and inhibition with an irreversible inhibitor.

Recombinant diaminopimelate epimerase is overproduced to give 1% of soluble protein when grown under the appropriate conditions in Escherichia coli. This compares with 0.02% of the constitutive level of wild-type enzyme. A new purification procedure now yields milligram quantities of homogeneous enzyme of high specific activity (192 U/mg). This has enabled sufficient amounts of enzyme both to compare with wild-type enzyme and to enable active site modification studies to be performed. Incubation of the enzyme with 2-(4-amino-4-carboxybutyl)-2-aziridine-carboxylic acid (AZIDAP), results in time-dependent irreversible inhibition. Tryptic digestion of the inactivated enzyme and peptide-mapping show that AZIDAP is specifically and covalently bound to the enzyme at a unique peptide. Determination of the amino acid sequence of this peptide and comparison with the sequence deduced from the DNA sequence of the dapF gene shows that Cys73 is labelled. Finally based on limited sequence similarities around this cysteine and active-site cysteines of proline racemase and 1-hydroxyproline 2-epimerase, together with mechanistic considerations, we propose that all three non-pyridoxal-phosphate-containing racemases/epimerases derive from a common evolutionary origin.

Amino Acid Isomerases↗
PubMed1989

Linear chromosome of Borrelia burgdorferi.

The DNA organization of several European and American isolates of Borrelia burgdorferi, the aetiological agent of Lyme disease, was analysed in pulse-field agarose gel electrophoresis. The results of in situ cell lysis in agarose plugs demonstrated a unique arrangement for the DNA of this spirochete. The chromosome of Borrelia behaved as a eukaryotic linear chromosome with a size of around 1,000 kb. The genome also comprised several circular and linear plasmids which varied in size from 15 to 60 kb.

Borrelia burgdorferi Group↗
PubMed1989

Does medical treatment of lymphedema act by increasing lymph flow?

The lymphatic origin of chronic edema of the lower limbs was identified by lymphoscintigraphic exploration. Patients underwent therapy involving 8 days of manual lymph drainage combined with elevation of the limbs during rest periods and double compression bandaging. Manual drainage increased lymph flow in 16 limbs, implying that the edema resulted from a functional lymphatic anomaly. In contrast, manual drainage did not increase lymph flow in 9 limbs, suggesting a structural anomaly of the lymphatics. Hence, the same clinical picture corresponded to two different lymphatic anomalies, distinguished by lymphoscintigraphy. However, the therapeutic results were independent of the lymphoscintigraphic results. Increased lymph flow is therefore not the only explanation for the decrease in edema during therapy.

Adolescent↗
PubMed1988

Incorporation of LL-diaminopimelic acid into peptidoglycan of Escherichia coli mutants lacking diaminopimelate epimerase encoded by dapF.

Recently a dapF mutant of Escherichia coli lacking the diaminopimelate epimerase was found to have an unusual large LL-diaminopimelic acid (LL-DAP) pool as compared with that of meso-DAP (C. Richaud, W. Higgins, D. Mengin-Lecreulx, and P. Stragier, J. Bacteriol. 169:1454-1459, 1987). In this report, the consequences of high cellular LL-DAP/meso-DAP ratios on the structure and metabolism of peptidoglycan were investigated. For this purpose new efficient high-pressure liquid chromatography techniques for the separation of the DAP isomers were developed. Sacculi from dapF mutants contained a high proportion of LL-DAP that varied greatly with growth conditions. The same was observed with the two DAP-containing precursors, UDP-N-acetylmuramyl-tripeptide and UDP-N-acetylmuramyl-pentapeptide. The limiting steps for the incorporation of LL-DAP into peptidoglycan were found to be its addition to UDP-N-acetylmuramyl-L-alanyl-D-glutamate and the formation of the D-alanyl-DAP cross-bridges. The Km value of the DAP-adding enzyme for LL-DAP was 3.6 x 10(-2) M as compared with 1.1 x 10(-5) M for meso-DAP. When isolated sacculi were treated with Chalaropsis N-acetylmuramidase and the resulting soluble products were analyzed by high-pressure liquid chromatography, the proportion of the main peptidoglycan dimer was lower in the dapF mutant than in the parental strain. Moreover, the proportion of LL-DAP was higher in the main monomer than in the main dimer, where it was almost exclusively located in the donor unit. There are thus very few D-alanyl-LL-DAP cross-bridges, if any. We also observed that large amounts of LL-DAP and N-succinyl-LL-DAP were excreted in the growth medium by the dapF mutant.

Amino Acid Isomerases↗
PubMed1988

[Diagnostic and therapeutic problems posed by renal or para-renal masses of cystic appearances].

The authors report some forty cases of renal masses, cystic in appearance and having raised a certain number of diagnostic or therapeutic problems. These masses included 7 cystic carcinomas, 3 benign tumors of cystic appearance and 28 cysts including 2 infected and 2 hydatid cysts. No paraclinical investigation is infallible in confirming the nature of the mass in doubtful cases, and pre-operative diagnosis is more easily obtained by investigation including the majority of standard imaging methods often completed by puncture. Amongst clinical factors which raise doubts as to the benign nature of the cystic mass seen, hematuria remains the most important, other symptoms and signs being of little significance. With regard to imaging, septate calcified masses with echoes within them, with a thick wall or high CT scan density are theoretically suspect. The same applies to masses having rapidly increased at two successive investigations or where puncture fluid is bloody. In all cases if proof of the benign nature cannot be obtained, surgical exploration would appear to be required.

Calcinosis↗
PubMed1987

Purification and characterization of RNA polymerase from the cyanobacterium Anabaena 7120.

A procedure for the purification of RNA polymerase from vegetative cells of the filamentous cyanobacterium Anabaena 7120 is described. Polyethyleneimine precipitation followed by gel filtration and affinity chromatography steps results in greater than 99% purification with 46% yield. The enzyme has a novel core component of Mr = 66,000, designated gamma, in addition to the typical prokaryotic beta'beta alpha 2 core enzyme. The sigma subunit has been identified by reconstitution of specific transcriptional activity from core enzyme and gel-purified sigma. In transcription assays, this RNA polymerase initiates at a number of Anabaena vegetative cell promoters, as well as from a bacteriophage T4 early promoter, but does not initiate at nitrogen fixation (nif) promoters used in heterocysts. The promoter specificity of Anabaena RNA polymerase is compared with that of Escherichia coli RNA polymerase.

Base Sequence↗
PubMed1987

Molecular cloning, characterization, and chromosomal localization of dapF, the Escherichia coli gene for diaminopimelate epimerase.

The Escherichia coli dapF gene was isolated from a cosmid library as a result of screening for clones overproducing diaminopimelate epimerase. Insertional mutagenesis was performed on the cloned dapF gene with a mini-Mu transposon, leading to chloramphenicol resistance. One of these insertions was transferred onto the chromosome by a double-recombination event, allowing us to obtain a dapF mutant. This mutant accumulated large amounts of LL-diaminopimelate, confirming the blockage in the step catalyzed by the dapF product, but did not require meso-diaminopimelate for growth. The dapF gene was localized in the 85-min region of the E. coli chromosome between cya and uvrD.

Amino Acid Isomerases↗