Biomedical subjects

C Cagnon

Publications and source records attributed to C Cagnon.

8 recordsLinked to original sources

PubMed1997

Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization.

Specific molecular interactions involved in catalysis by antibody 6D9 were investigated by site-directed mutagenesis. The catalytic antibody 6D9, which was generated against a transition state analog (III), hydrolyzes a non-bioactive chloramphenicol monoester derivative (I) to produce chloramphenicol (II). Construction of a three-dimensional molecular model of 6D9 and sequence comparison within a panel of related antibodies suggested candidates for catalytic residues, His (L27d), Tyr (L32), Tyr (H58) and Arg (H100b); these were targeted for the site-directed mutagenesis study. The Y-H58-F and R-H100b-A mutants possessed catalytic activities comparable to that of the wild-type, and the Y-H58-H and Y-L32-F mutant displayed an approximately fivefold decrease in k(cat)/Km. In the transition state analysis, the plots of logK(TSA) versus log(k(cat)/Km) for the mutants are linear, with a slope of approximately 1.0, indicating that the entire hapten-binding energy in the mutants is also utilized to bind the transition state and to accelerate the catalysis. In addition, a dramatic change in the catalytic activity was observed when the histidine residue (27d) in the CDR1 light chain was replaced with alanine. The H-L27d-A mutant had no detectable catalytic activity. This mutation led to a large, 40-fold reduction in transition state binding, with no change in substrate binding. Coupled with the previous kinetic studies and chemical modifications of the intact 6D9 antibody, this mutagenesis study has demonstrated that His L27d plays an essential role in stabilization of the transition state, the mechanism of catalysis by the 6D9 antibody.

Amino Acid Sequence↗

PubMed1997

Proline-dependent oligomerization with arm exchange.

BACKGROUND: Oligomerization is often necessary for protein activity or regulation and its efficiency is fundamental for the cell. The quaternary structure of a large number of oligomers consists of protomers tightly anchored to each other by exchanged arms or swapped domains. However, nothing is known about how the arms can be kept in a favourable conformation before such an oligomerization. RESULTS: Upon examination of such quaternary structures, we observe an extremely frequent occurrence of proline residues at the point where the arm leaves the protomer. Sequence alignment and site-directed mutagenesis confirm the importance of these prolines. The conservation of these residues at the hinge regions can be explained by the constraints that they impose on polypeptide conformation and dynamics: by rigidifying the mainchain, prolines favour extended conformations of arms thus favouring oligomerization, and may prevent interaction of the arms with the core of the protomer. CONCLUSIONS: Hinge prolines can be considered as 'quaternary structure helpers'. The presence of a proline should be considered when searching for a determinant of oligomerization with arm exchange and could be used to engineer synthetic oligomers or to displace a monomers to oligomers equilibrium by mutation of this proline residue.

Acetyltransferases↗

PubMed1997

Mammography film processor replenishment rate: bromide level monitoring.

The effects of the mammography film processing replenishment rate on contrast and speed are studied sensitometrically. Two experiments studied decreasing replenishment rates in the Kodak RP developer and quantified changes in the developer by measuring bromide ion concentrations. First, values of NaBr concentration from 1.7 to 8.4 g/L, achieved by reducing the replenishment rate, were tested with sensitometry strips. Second, the developer replenishment rate of a high volume dedicated mammography processor was reduced by one-third, to 20 cm3/1560 cm2, so that the NaBr concentration rose from 2.0 to 12.36. Sensitometric results for four film types and patient films were tested for changes from standard values as NaBr concentration was restored to 3.31 g/L. Fifty-five clinical images obtained at 7.3-9.3 NaBr g/L were compared to their matching previous films, with NaBr levels of 2-3 g/L, for contrast and visibility of the skin line. For the range of the NaBr ion from 1.7 to 7 g/L, no significant sensitometric differences were found. Above 7 g/L, different film types had different sensitometric results. From 7.3 to 9.3 NaBr g/L, 47.5% of the clinical films reviewed by four radiologists had less contrast compared to previous films. Dedicated mammography processors with high film volume (i.e., those that do not have excessive oxidation or foreign dye problems) can operate at lower replenishment rates than are currently employed. All common mammography film types are stable at these lower replenishment rates up to 7.0 NaBr g/L.

Biophysical Phenomena↗

PubMed1997

CM-gag, a transposable-like element reiterated in the genome of Culex pipiens mosquitoes, contains only a gag gene.

CM-gag elements constitute an homogeneous family of sequences that are reiterated in the genome of Culex pipiens strains from different continents. Apparently complete 1.75 kb CM-gag copies are flanked by target-site duplications and have a polyadenylation signal near their 3' end. They potentially contain a unique gene encoding a putative protein that displays homologies with nucleic acid binding proteins and the gag polypeptide of retroviruses and retrotransposons, but that does not encode a reverse transcriptase. CM-gag elements are similar in their genetic organization to the telomeric transposable sequences Het-A from Drosophila melanogaster, but Southern-hybridization patterns indicate that the former are more probably dispersed in various areas of the mosquito genome. The homogeneity of CM-gag copies that are distributed worldwide suggests that they have most probably been amplified recently. Furthermore, selective constraints against amino acid changes have been acting on these sequences, suggesting that they need to encode the gag-like protein to be incorporated into the chromosomes.

Amino Acid Sequence↗

PubMed1995

Characterization of the unlinked 16S rDNA and 23S-5S rRNA operon of Wolbachia pipientis, a prokaryotic parasite of insect gonads.

The rRNA-encoding genes (rDNAs) have been cloned and characterized from Wolbachia pipientis (Wp), the gonadial bacteria-like parasite of the mosquito Culex pipiens (Cp) and the moth Ephestia cautella (Ec). In Wp from both insect species the rDNAs are organized in a way which appears to be very unusual. The rRNAs are encoded by two unlinked transcription units, each present in a single copy per genome. One contains the 16S rDNA only, while the other is an operon encoding both the 23S and 5S rDNAs. Each transcription unit contains two putative upstream promoters, and downstream a Rho-independent terminator. The 16S rDNA, as well as the 23S-5S rRNA operon are not linked to any tRNA-encoding sequence and lack the antitermination boxes which are usually present immediately downstream from eubacterial promoters of rDNAs. Wp infecting Ec and Cp are highly similar taking as criteria the rDNAs and their flanking sequences. However, it clearly appears that each insect species harbours a different and specific Wp strain, or even subspecies. Phylogenetic relationships deduced from the complete sequences of their rDNAs undoubtedly confirm that Wp from Cp and Ec belong to the alpha-group of Proteobacteria, and are closely related to the Rickettsia.

Animals↗

PubMed1994

Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering.

The antibiotic bleomycin, a strong DNA cutting agent, is naturally produced by actinomycetes which have developed a resistance mechanism against such a lethal compound. The crystal structure, at 2.3 A resolution, of a bleomycin resistance protein of 14 kDa reveals a structure in two halves with the same alpha/beta fold despite no sequence similarity. The crystal packing shows compact dimers with a hydrophobic interface and involved in mutual chain exchange. Two independent solution studies (analytical centrifugation and light scattering) showed that this dimeric form is not a packing artefact but is indeed the functional one. Furthermore, light scattering also showed that one dimer binds two antibiotic molecules as expected. A crevice located at the dimer interface, as well as the results of a site-directed mutagenesis study, led to a model wherein two bleomycin molecules are completely sequestered by one dimer. This provides a novel insight into antibiotic resistance due to drug sequestering, and probably also into drug transport and excretion.

Acetyltransferases↗

PubMed1991

A new family of sugar-inducible expression vectors for Escherichia coli.

A set of 11 expression vectors was constructed, each of them harbouring a cloning cassette under the control of the araB promoter. Some of these vectors enable expression of foreign proteins in the cytoplasm, while others include a synthetic sequence coding for a very efficient secretion signal sequence. Other features are an f1 origin of replication (in plus or minus orientation) and a promoter(up) mutation that enhances the already very high level of expression from these vectors. With such a versatile vector family, cloning, sequencing and site-directed mutagenesis can be performed on the same vector, and the level of expression can be defined according to the specific constraints of a given protein.

Amino Acid Sequence↗

PubMed1989

Crystallization and preliminary X-ray data of a phleomycin-binding protein from Streptoalloteichus hindustanus.

Large single crystals of a glycopeptide resistance protein encoded by the SH-ble gene from Streptoalloteichus hindustanus have been grown using vapor diffusion techniques with ammonium sulfate as the precipitant. The diffraction pattern extends to 2.0 A resolution. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have unit cell dimensions of a = 48.8 A and c = 112.5 A.

Actinomycetales↗