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Biomedical subjects

L Lemieux

Publications and source records attributed to L Lemieux.

At least 19 recordsLinked to original sources
PubMed1992

Demonstration by FTIR that the bo-type ubiquinol oxidase of Escherichia coli contains a heme-copper binuclear center similar to that in cytochrome c oxidase and that proper assembly of the binuclear center requires the cyoE gene product.

Amino acid sequence data have revealed that the bo-type ubiquinol oxidase from Escherichia coli is closely related to the eukaryotic aa3-type cytochrome c oxidases. In the cytochrome c oxidases, the reduction of oxygen to water occurs at a binuclear center comprised of heme a3 and Cu(B). In this paper, Fourier transform infrared (FTIR) spectroscopy of CO bound to the enzyme is used to directly demonstrate that the E. coli bo-type ubiquinol oxidase also contains a heme-copper binuclear center. Photolysis of CO ligated to heme o at low temperatures (e.g., 30 K) results in formation of a CO-Cu complex, showing that there is a heme-Cu(B) binuclear center similar to that formed by heme a3 and Cu(B) in the eukaryotic oxidase. It is further demonstrated that the cyoE gene product is required for the correct assembly of this binuclear center, although this polypeptide is not required as a component of the active enzyme in vitro. The cyoE gene product is homologous to COX10, a nuclear gene product from Saccharomyces cerevisiae, which is required for the assembly of yeast cytochrome c oxidase. Deletion of the cyoE gene results in an inactive quinol oxidase that is, however, assembled in the membrane. FTIR analysis of bound CO shows that Cu(B) is present in this mutant but that the heme-Cu(B) binuclear center is abnormal. Analysis of the heme content of the membrane suggests that the cyoE deletion results in the insertion of heme B (protoheme IX) in the binuclear center, rather than heme O.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkyl and Aryl Transferases↗
PubMed1992

Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.

The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.

Amino Acid Sequence↗
PubMed1992

Multimodality imaging and intracranial EEG display for stereotactic surgery planning in epilepsy.

We describe a computer program for stereotactic surgery planning based on multimodality imaging and the display of intracranial EEG data in relation to anatomical data. The program is primarily designed to be used by surgeons to assist them in localizing brain structures and plan the safe and accurate insertion of surgical tools and depth electrodes. The mathematics underlying these concepts are briefly described. Estimates of the accuracy of the procedures are calculated and made available to the user. An extension of the program was developed to integrate intracranial EEG data with conventional images in order to help the neurologist and neurosurgeon in visualizing the EEG data in relation to the anatomy. It can be used to visualize the measured EEG voltages, or processed signals such as the application of the tetrahedral field calculation, which provides an extension of conventional bipolar EEG to 3 dimensions.

Brain↗
PubMed1992

Equivalent source estimation based on the calculation of the electric field from depth EEG data.

This paper describes a method for the estimation of equivalent source position and strength based on the estimation of the electric field from depth electroencephalographic (EEG) data. The calculation method for the electric field is based on a tetrahedral geometry. The proposed approach for source parametrization is twofold. Firstly, the distribution of electrical energy by the squared norm of the electric field vector can give an estimate of the source position, without having to assume a dipole source. Secondly, the average electric field can be related to the dipole magnitude and orientation of the equivalent source. Simulation results demonstrate the potential usefulness of the method. The effects of noise and sampling, and the geometry of the measurement system (i.e., implanted electrodes) relative to the source are also investigated through simulations.

Artifacts↗
PubMed1990

High-performance liquid chromatography of casein hydrolysates phosphorylated and dephosphorylated. I. Peptide mapping.

A mixture of small peptides of molecular weight averaging 1000 daltons, obtained by controlled hydrolysis of casein with proteases, chymotrypsin and trypsin, was separated by size-exclusion and reversed-phase high-performance liquid chromatography. Peptides were identified and located in the known casein structures from their amino acid content and their N- and C-terminal amino acid analyses. The primary structure of peptides identified from casein hydrolysate phosphorylated and casein hydrolysate dephosphorylated is presented.

Amino Acid Sequence↗
PubMed1990

Recent studies of the cytochrome o terminal oxidase complex of Escherichia coli.

The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of Escherichia coli when the bacteria are grown under conditions of high aeration. The oxidase is a ubiquinol oxidase and reduces molecular oxygen to water. Electron transport through the enzyme is coupled to the generation of a protonmotive force. The purified cytochrome o complex contains four or five subunits, two protoheme IX (heme b) prosthetic groups, plus at least one Cu. The subunits are all encoded by the cyo operon. Sequence comparisons show that the cytochrome o complex is closely related to the aa3-type cytochrome c oxidase family. Gene fusions have been used to define the topology of each of the gene products. Subunits I, II, III and IV are proposed to have 15, 2, 5 and 3 transmembrane spans, respectively. The fifth gene product (cyoE) encodes a protein with 7 membrane spanning segments, and this may also be a subunit of this enzyme. Fourier transform infrared spectroscopy has been used to monitor CO bound in the active site where oxygen is reduced. These data provide definitive proof that the cytochrome o complex has a heme-copper binuclear center, similar to that present in the aa3-type cytochrome c oxidases. Site-directed mutagenesis is being utilized to define which amino acids are ligands to the heme iron and copper prosthetic groups.

Alkaline Phosphatase↗
PubMed1990

The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa3-type family of cytochrome c oxidases.

The cytochrome o complex is one of two ubiquinol oxidases in the aerobic respiratory system of Escherichia coli. This enzyme catalyzes the two-electron oxidation of ubiquinol-8 which is located in the cytoplasmic membrane, and the four-electron reduction of molecular oxygen to water. The purified oxidase contains at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and has been shown to couple electron flux to the generation of a proton motive force across the membrane. In this paper, the DNA sequence of the cyo operon, containing the structural genes for the oxidase, is reported. This operon is shown to encode five open reading frames, cyoABCDE. The gene products of three of these, cyoA, cyoB, and cyoC, are clearly related to subunits II, I, and III, respectively, of the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. This family of cytochrome c oxidases contain heme a and copper as prosthetic groups, whereas the E. coli enzyme contains heme b (protoheme IX) and copper. The most striking sequence similarities relate the large subunits (I) of both the E. coli quinol oxidase and the cytochrome c oxidases. It is likely that the sequence similarities reflect a common molecular architecture of the two heme binding sites and of a copper binding site in these enzymes. In addition, the cyoE open reading frame is closely related to a gene denoted ORF1 from Paracoccus dentrificans which is located in between the genes encoding subunits II and III of the cytochrome c oxidase of this organism. The function of the ORF1 gene product is not known. These sequence relationships define a superfamily of membrane-bound respiratory oxidases which share structural features but which have different functions. The E. coli cytochrome o complex oxidizes ubiquinol but has no ability to catalyze the oxidation of reduced cytochrome c. Nevertheless, it is clear that the E. coli oxidase and the aa3-type cytochrome c oxidases must have very similar structures, at least in the vicinity of the catalytic centers, and they are very likely to have similar mechanisms for bioenergetic coupling (proton pumping).

Amino Acid Sequence↗
PubMed1990

Expression of cyoA and cyoB demonstrates that the CO-binding heme component of the Escherichia coli cytochrome o complex is in subunit I.

The cytochrome o complex of the Escherichia coli aerobic respiratory chain is a ubiquinol oxidase. The enzyme consists of at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and contains two heme b prosthetic groups (b555 and b562) plus copper. The sequence of the cyo operon, encoding the subunits of the oxidase, reveals five open reading frames, cyoABCDE. This paper describes results obtained by expressing independently cyoA and cyoB in the absence of the other subunits of the complex. Polyclonal antibodies which react with subunits I and II of the purified oxidase demonstrate that cyoA and cyoB correspond to subunit II and subunit I, respectively, of the complex. These subunits are stably inserted into the membrane when expressed. Furthermore, expression of cyoB (subunit I) results in elevated heme levels in the membrane. Reduced-minus-oxidized spectra suggest that the cytochrome b555 component is present but that the cytochrome b562 component is not. This heme component is shown to bind to CO, as it does in the intact enzyme. Hence, subunit I alone is sufficient for the assembly of the stable CO-binding heme component of this oxidase.

Blotting, Western↗
PubMed1990

Characterization of amplification core and esterase B1 gene responsible for insecticide resistance in Culex.

Organophosphorus insecticide (OP) resistance in several Culex species is associated with increased esterase activity resulting from amplification of the corresponding structural gene. In Culex pipiens quinquefasciatus, high levels of OP resistance (approximately 800 times) are due to the esterase B1 gene, which is amplified at least 250-fold. This gene has now been sequenced, and the structure of the amplification unit (amplicon) encompassing the structural gene has been partially characterized. The inferred amino acid sequence of the enzyme revealed regions of strong homology with other eukaryotic serine-esterases, such as cholinesterases, which are the target of OPs. The amplicon covers at least 30 kilobases and contains a constant and highly conserved "core" of 25 kilobases. This core carries a single copy of the esterase gene (2.8 kilobases) as well as other sequences that are present as single or low number copies in the genomes of mosquitoes lacking overproduction of the esterase B1 protein. In the amplicon, the esterase gene is framed by two DNA sequences that are repeated in other parts of the genome of resistant mosquitoes and found in the genome of susceptible mosquitoes but not near the esterase B1 gene. It is suggested that these repetitive sequences may have a role in the amplification process.

Amino Acid Sequence↗
PubMed1986

Serum folates in gestating swine after folic acid addition to diet.

Folic acid was added to the diet as a simple means to increase serum folates in gestating sows. At weaning, 95 multiparous sows were randomly assigned to five treatments. Of these sows, 67 farrowed and were used for this trial. Three supplementation levels of folic acid added to a commercial diet at 3, 9 and 27 mg per kg were studied. A commercial diet without any supplementation of folic acid was used as a control treatment. A fifth treatment consisted of eight im injections of 15 mg of folic acid each, according to a predetermined schedule that was previously effective in improving the reproductive performance of sows when combined with flushing. Each sow was kept in an individual cage and received 2 kg of feed daily. Serum folates were measured at weaning, mating and on d 14, 28, 42 and 56 after mating. The time-response curve of serum folates in sows injected with folic acid was higher than that of sows fed the unsupplemented diet (P = .057). Adding folic acid to diet may be as efficient as folic acid injections to elevate serum folates when compared with sows fed the control diet. The mean supplementary level of folic acid sufficient to maintain the serum folate concentration at approximately the same levels as those observed in sows injected with folic acid was estimated to be near 4.3 mg per kg of feed.

Animals↗
PubMed1985

Myeloperoxidase activity of polymorphonuclear leukocytes in iron deficiency anemia and anemia of chronic disorders.

Myeloperoxidase (MPO) activity was studied in adults with iron deficiency anemia (IDA) or anemia of chronic disorders (ACD). MPO activity (biochemical quantitation) was found to be decreased in IDA when compared to the control group (p less than 0.05); ACD subjects also had lower values although the difference was not significant (p less than 0.05). MPO scores (MPO staining) were significantly lower in IDA and ACD subjects than in the healthy control group (p less than 0.05). A significant positive correlation was noted between ferritin (R = 0.40) and percent transferrin saturation (R = 0.37) and MPO activity (p less than 0.001) in IDA and for the healthy controls.

Adult↗
PubMed1984

Biological activities of v-myc and rearranged c-myc oncogenes in rat fibroblast cells in culture.

Two distinct forms of the myc oncogene were assayed for their ability to induce, in cultured rat fibroblast cells, the alterations of cellular growth controls observed upon transfer of the gene of polyoma virus encoding only the large T protein (plt). Both of these rearranged myc genes and the plt gene had been previously shown to cooperate with ras oncogenes for transformation of rat embryo fibroblasts (REF) and were thought to induce the same early step ("immortalization") of the tumoral transformation pathway. We now report that these two different oncogenes elicite the same response in the following biological assays: (i) reduction of the requirements in serum factors for growth in culture of cells of the established FR3T3 line; (ii) expression of transformed properties in low serum medium after transfer into FR3T3 cells expressing only the middle T protein of polyoma virus (MTT lines); (iii) conferring on REF cells the ability to grow as clonal colonies after seeding at low cell density; (iv) conferring on REF cells the ability to grow continuously in cell culture. These congruent phenotypes suggest that the activities of the large T and myc proteins result in the induction of the same molecular events. These results also provide simple biological assays and selective systems for oncogenes of the myc class.

Animals↗