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Biomedical subjects

G Boileau

Publications and source records attributed to G Boileau.

At least 91 records · Page 5Linked to original sources
PubMed1989

Molecular heterogeneity of human motilinlike immunoreactivity explained by the processing of prepromotilin.

Results of studies on the molecular forms of canine motilin suggest that this hormone might be synthesized as a higher molecular weight precursor, as is the case for most other biologically active peptides. Chromatographic analysis of human duodenal mucosa extracts and of human serum, using motilin-specific antibodies, also shows the presence of multiple forms of motilinlike immunoreactivity. Cell free translation of human duodenal messenger ribonucleic acid (mRNA) and immunoprecipitation of nascent peptides with motilin antibodies confirm that motilin is synthesized as a 14-15-kilodalton polypeptide precursor. Sequence of the human intestinal motilin complementary deoxyribonucleic acid (cDNA) demonstrates that this precursor bears a 25-amino acid signal peptide, a single dibasic cleavage site immediately following the 22 amino acids of human motilin, and a 65-amino acid polypeptide (motilin-related peptide) following this dibasic processing site. Southern analysis of human genomic kilobases DNA demonstrates that only one motilin gene is contained within 3.5-4 of human genomic DNA. Northern analysis of human intestinal RNA reveals one species of motilin mRNA of an estimated 700 nucleotides in length. These results suggest that (a) only one gene encodes the synthesis of human motilin in the different tissues where this polypeptide is synthesized; (b) human intestinal motilin is translated from one mRNA species and the resulting precursor is processed within the duodenal mucosa by sequential proteolytic cleavage first at a site within the motilin-related peptide, which results in the liberation of a 6-kilodalton form of motilinlike immunoreactivity, and then at the dibasic amino acid cleavage site, thus freeing motilin 22 from its precursor.

Amino Acid Sequence↗
PubMed1988

Exploration of the catalytic site of endopeptidase 24.11 by site-directed mutagenesis. Histidine residues 583 and 587 are essential for catalysis.

Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His-142 and His-146) and a third histidine (His-231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His-583, His-587 and His-637) was explored by site-directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS-1 cells. Substitution of either His-583 or His-587 of NEP for Phe completely abolished the activity and Zn-directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His-142 and His-146 of thermolysin as zinc ligands. In contrast, substitution of His-637 for a phenylalanine residue was without effect on enzyme activity.

Amino Acid Sequence↗
PubMed1988

Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. Characterization of the recombinant enzyme and evidence for a glutamic acid residue at the active site.

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found in the plasma membrane of many cell types. The cDNA coding for the complete primary structure of neutral endopeptidase has recently been cloned and sequenced (Devault, A. Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317-1322). Comparison of the sequence of neutral endopeptidase with that of thermolysin, a bacterial Zn-metalloendopeptidase, suggests that Glu-584 in neutral endopeptidase probably corresponds to Glu-143 in thermolysin, which is an essential amino acid involved in catalysis. To test directly the importance of Glu-584 in the catalytic activity of neutral endopeptidase by site-directed metagenesis, we have constructed an expression vector in which the rabbit kidney cDNA encoding the entire neutral endopeptidase sequence is introduced downstream from the SV40 virus early promotor. After transfection in COS-1 monkey kidney cells, this vector was found to promote the expression of a protein with biochemical and catalytic properties identical to kidney neutral endopeptidase. Oligonucleotide-directed mutagenesis of Glu-584 to either valine or aspartic acid completely abolished the enzymatic activity of the recombinant protein without changing its affinity for the substrate-related tritiated inhibitor [3H]N-[(2R,2S)-3-hydroxyamino-carbonyl-2-benzyl-1-oxopropyl]-glycine. This observation clearly identifies Glu-584 as one of the important residues responsible for the catalytic activity of the enzyme.

Animals↗
PubMed1988

Higher order structures of the 5'-proximal region decrease the efficiency of translation of the porcine pro-opiomelanocortin mRNA.

The SP6 polymerase/promoter system was used to synthesize porcine pro-opiomelanocortin mRNAs with nucleotide sequence deletions in the 5'- as well as 3'-untranslated and coding regions. The translational efficiency of the mutant mRNAs was evaluated by cell-free translation or by monitoring the rate and extent of ribosome binding in the presence of sparsomycin. The results of these experiments indicate that specific nucleotide sequences in the 5'-untranslated and coding regions of the pro-opiomelanocortin mRNA decrease its rate of translation. Structure mapping of the mRNA with double-strand and single-strand specific nucleases suggests that these sequences can form stable secondary structures.

Animals↗
PubMed1988

Expression of porcine pro-opiomelanocortin cDNA in an established fibroblastic cell line: constitutive secretion of the precursor without proteolytic processing.

Pro-opiomelanocortin (POMC) is the common precursor of several pituitary hormones including alpha-melanotropic hormone, adrenocorticotropic hormone, beta-lipotropin and beta-endorphin. The porcine POMC cDNA was inserted downstream from the late promoter of an SV40-derived expression vector and co-transfected in NIH 3T3 cells with a marker plasmid carrying the neomycin resistance gene. Colonies resistant to the neomycin analog G418 were selected and analyzed for the production of POMC-related peptides by radioimmunoassay. Three clones were found to produce from 350 to 1750 pg of POMC-related peptides per 10(6) cells in 16 h and selected for further analysis. The number of POMC cDNA copies integrated in the host cell genome was determined and the levels of transcription were compared. POMC-related material released in the culture medium by the best producing clone (NJP 4-4) was further analyzed by gel filtration and reversed-phase high-pressure liquid chromatography combined with radioimmunoassays. POMC was found to be synthesized and secreted without further processing or degradation. Negligible amounts of POMC-immunoreactive species were found in cellular extracts indicating that the prohormone is secreted from the NIH 3T3 cells without storage, presumably through a constitutive pathway. Our results suggest that NIH 3T3 fibroblasts do not contain the enzymatic machinery to process complex precursors such as POMC.

Animals↗
PubMed1988

Double antibody radioimmunoassay for monitoring metastatic breast cancer.

We previously reported the production of a panel of murine monoclonal antibodies which recognize glycoproteins abnormally expressed in human breast tumours. Using two of these antibodies, a double antibody radioimmunoassay was designed to quantify levels of these breast tumour marker glycoproteins in serum. Marker levels greater than 28 units were considered abnormal. Using this criterion, 63% and 75% of patients with breast cancer stages I and II, respectively, and 88% of those with metastatic disease were found to have elevated marker levels. Thirteen percent of patients with non-malignant breast disease also had elevated marker levels. Elevated marker levels were also detected in patients with non breast neoplasms. One hundred and eleven women with metastatic disease were followed. Eighty-two percent of those with progressive disease and 73% of those where disease regressed had 20% changes in marker levels. These changes in marker levels preceded by up to 6 months changes in disease state. From these results we conclude that this assay may be useful for monitoring the course of disease in breast cancer patients.

Antibodies, Monoclonal↗
PubMed1987

Isolation and characterisation of a murine cDNA clone highly homologous to the yeast L29 ribosomal protein gene.

We report here the isolation of a murine cDNA clone (cCL3) which is homologous to the mRNA of the yeast ribosomal protein L29. Comparison of the deduced amino-acid composition of cCL3 to those known for rat ribosomal proteins indicates that this cDNA codes for mammalian ribosomal protein L27'. The gene corresponding to the cDNA is present at approximately 15 copies per genome, some of these probably representing processed pseudogenes. The cDNA hybridizes to an mRNA of 600 nucleotides from various mammals at high stringency, and to an avian transcript of the same size at low stringency. It has been suggested that L29 is involved in peptidyl transferase activity. The strong homology of mammalian L27' to yeast L29 suggests a function which has been conserved throughout evolution, and thus L27' may also be involved in peptidyl transferase activity.

Amino Acid Sequence↗
PubMed1987

Expression of porcine pro-opiomelanocortin cDNA in heterologous monkey kidney cells. Biosynthesis and secretion of the prohormone without processing.

Pro-opiomelanocortin is the common precursor to several pituitary hormones. These include alpha-melanotropic hormone, adrenocorticotropic hormone, beta-lipotropic hormone, and beta-endorphin. The porcine pro-opiomelanocortin cDNA was inserted downstream from the early promoter of a SV40-derived expression vector and transfected into the monkey kidney COS-1 cells. Transient expression of the pro-opiomelanocortin cDNA was observed between 48 and 70 h after transfection. Analysis of pro-opiomelanocortin-related material in COS-1 cell extracts and culture medium revealed that these cells synthesize and secrete constitutively pro-opiomelanocortin without further processing into its mature hormones. Our results suggest that COS-1 cells do not contain the necessary enzymatic machinery to process complex precursors such as pro-opiomelanocortin.

Amino Acid Sequence↗
PubMed1987

Reassembly of active 30S ribosomal subunits with an unmethylated in vitro transcribed 16S rRNA.

A plasmid has been constructed, which contains the 16S ribosomal RNA gene of Escherichia coli immediately downstream from a phage T7 promoter. In vitro transcription of this gene by RNA polymerase of the T7 phage yielded an unmethylated 16S rRNA that could be used for the assembly of functional 30S subunits. These subunits when assayed in a poly(U)-directed translation assay, were as active as 30S subunits reconstructed with native 16S rRNA. This system opens the possibility of investigating the role of the methylations of the rRNA and the functional consequences of site-directed mutagenesis in a rRNA gene. An application of this system was provided by generating a 16S rRNA transcript shortened by about 30 nucleotides at the 3' end. This truncated rRNA could be reassembled into 30S subunits, about 70% as active as 30S subunits reconstructed with the full-length 16S rRNA transcript.

Base Sequence↗
PubMed1986

Ambulatory hepatic artery infusion chemotherapy for cancer of the liver.

We have used continuous hepatic arterial infusion chemotherapy to treat 105 patients with cancer of the liver originating from colorectal, other gastrointestinal, and nongastrointestinal sites. The response rate seen in colorectal metastases was two to three times that expected for systemic chemotherapy. The median survival of responders of 16 months was significantly better then for nonresponders (6 months). The median duration of response was 9 months. The results for patients with other tumor types were less encouraging. Although minor problems developed in about 30 percent of the patients, major complications requiring removal of the catheter were not common. Expertise derived from managing many patients and a team approach, with a defined protocol for catheter care and follow-up, contributed to the success of the ambulatory program. However, the role of hepatic arterial infusion chemotherapy remains under debate. At the root of the controversy is the lack of randomized, controlled trials supporting the superiority of hepatic arterial infusion over systemic chemotherapy in the treatment of colorectal liver metastases. This and other issues, including the current liberal use of implanted infusion pumps, should be studied.

Adult↗
PubMed1984

Cross-linking of streptomycin to the 30S subunit of Escherichia coli with phenyldiglyoxal.

[3H]Dihydrostreptomycin was covalently linked to the 30S subunit of Escherichia coli K12A19 with the bifunctional cross-linking reagent phenyldiglyoxal. The cross-linking was abolished under conditions that prevent the binding of streptomycin, which indicates that the cross-linking occurs at the specific binding site of streptomycin. The cross-linking involved 16S RNA and the ribosomal proteins S1, S5, S11, and S13. This suggests that the streptomycin binding site is located in the upper part of the 30S subunit, facing the 50S subunit. Unexpectedly, the same extent and pattern of cross-linking were observed with the 30S subunits from a streptomycin-resistant mutant. We have shown previously that streptomycin induces conformational changes in the ribosomes from sensitive bacteria but not from streptomycin-resistant mutants. From this and from the results in the present study, it is suggested that the binding of streptomycin to streptomycin-sensitive ribosomes is a two-step reaction wherein an initial loose interaction at the antibiotic binding site is followed by a conformational rearrangement of the ribosomal particle. The second step would tighten the association with streptomycin and cause interference with protein synthesis. That step would be lacking in streptomycin-resistant mutants.

Aldehydes↗
PubMed1983

Complete structure of the porcine pro-opiomelanocortin mRNA derived from the nucleotide sequence of cloned cDNA.

Polyadenylated RNA isolated from porcine pituitary neurointermediate lobes was used to construct a cDNA library. The library was screened with a rat genomic DNA fragment specific for pro-opiomelanocortin sequences. Two positive clones, pJA-19 and pJA-20, containing respectively 850 bp and 550 bp were characterized. Sequence analysis of the cDNA inserts revealed the complete structure of the porcine pro-opiomelanocortin mRNA. This mRNA would include 129 5'-untranslated nucleotides, 801 nucleotides coding for the 267 amino acids precursor and 162 3'-untranslated nucleotides. Comparison with pro-opiomelanocortin mRNA sequences from other species shows regions of high homology not only in the coding sequences but also in the 5'untranslated region where the first 50 nucleotides are over 80% purines.

Adrenocorticotropic Hormone↗
PubMed1983

Cross-links between ribosomal proteins of 30S subunits in 70S tight couples and in 30S subunits.

Ribosome 70S tight couples and 30S subunits derived from them were modified with 2-iminothiolane under conditions where about two sulfhydryl groups per protein were added to the ribosomal particles. The 70S and 30S particles were not treated with elevated concentrations of NH4Cl, in contrast to those used in earlier studies. The modified particles were oxidized to promote disulfide bond formation. Proteins were extracted from the cross-linked particles by using conditions to preclude disulfide interchange. Disulfide-linked protein complexes were fractionated on the basis of charge by electrophoresis in polyacrylamide/urea gels at pH 5.5. The proteins from sequential slices of the urea gels were analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Final identification of proteins in cross-linked complexes was made by radioiodination of the proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis. Attention was focused on cross-links between 30S proteins. We report the identification of 27 cross-linked dimers and 2 trimers of 30S proteins, all but one of which were found in both 70S ribosomes and free 30S subunits in similar yield. Seven of the cross-links, S3-S13, S13-S21, S14-S19, S7-S12, S9-S13, S11-S21, and S6-S18-S21, have not been reported previously when 2-iminothiolane was used. Cross-links S3-S13, S13-S21, S7-S12, S11-S21, and S6-S18-S21 are reported for the first time. The identification of the seven new cross-links is illustrated and discussed in detail. Ten of the dimers reported in the earlier studies of Sommer & Traut (1976) [Sommer, A., & Traut, R. R. (1976) J. Mol. Biol. 106, 995-1015], using 30S subunits treated with high salt concentrations, were not found in the experiments reported here.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Direct cross-links between initiation factors 1, 2, and 3 and ribosomal proteins promoted by 2-iminothiolane.

Complexes were prepared containing 30S ribosomal subunits from Escherichia coli and the three initiation factors IF1, IF2, and IF3. In different experiments, each of the factors was radiolabeled with the others unlabeled. The complexes were allowed to react with 2-iminothiolane and then oxidized to promote the formation of intermolecular disulfide bonds, some of which were between factors and ribosomal proteins. Each of the labeled factors becomes covalently cross-linked to the complex as judged by its failure to dissociate when centrifuged in a sucrose gradient containing a high salt concentration. Proteins from the complexes were extracted and analyzed on two-dimensional polyacrylamide gels by nonequilibrium isoelectric focusing and sodium dodecyl sulfate gel electrophoresis. Spots corresponding to cross-linked dimers that contained initiation factors, as indicated on autoradiographs, were cut out and analyzed further. The following cross-linked dimers between factors and ribosomal proteins were identified: IF1-S12, IF1-S18, IF2-S13, IF3-S7, IF3-S11, IF3-S13, and IF3-S19. Cross-links between factors IF1-IF2 and IF3-IF2 were also identified. A model integrating these findings with others on the protein topography of the ribosome is presented.

Binding Sites↗
PubMed1983

Levels of ribosomal protein S1 and elongation factor G in the growth cycle of Escherichia coli.

The relative levels of ribosomes, ribosomal protein S1, and elongation factor G in the growth cycle of Escherichia coli were examined with two-dimensional polyacrylamide gel electrophoresis. Nonequilibrium pH gradient polyacrylamide gel electrophoresis was used in the first dimension, and polyacrylamide gradient-sodium dodecyl sulfate gel electrophoresis was used in the second dimension. The identities of protein spots containing S1 and elongation factor G were confirmed by radioiodination of the proteins and peptide mapping of the radiolabeled peptides. The levels of ribosomes and ribosomal protein S1 were coordinately reduced during transition from exponential phase to stationary phase. There was no accumulation of S1 in the stationary phase. In marked contrast, the level of elongation factor G showed no significant change from exponential phase to stationary phase. The relative level of elongation factor G compared with ribosomes or S1 increased by about 2.5-fold during transition from exponential phase to stationary phase. The results show that there are differences between the regulation of the levels of elongation factor G and of ribosomal proteins, including S1, apparent during the transition from exponential to stationary phase.

Bacterial Proteins↗
PubMed1983

Cell-free synthesis of porcine proopiomelanocortin: two distinct primary translation products.

Polyadenylated RNA prepared from porcine pituitary intermediate lobes was translated in the cell-free rabbit reticulocyte system. Two translation products with apparent relative masses (Mrs) of 37 500 and 35 000 on polyacrylamide-sodium dodecyl sulfate gel electrophoresis were immunoprecipitated with a beta-melanophore-stimulating hormone (beta-MSH) antiserum. Tryptic digestions after citraconylation of the lysine residues and purification of the peptides by high performance liquid chromatography demonstrated that both polypeptides contained authentic beta-endorphin sequences. N-terminus microsequencing of the translation products labeled with [3H]leucine revealed the following partial amino acid sequence for the polypeptides with Mrs of 37 500 and 35 000: Leu 8, 11, 12, 13, 15, 16, 17, 20, 28, and 36 and Leu 3, 11, 12, 13, 15, 16, 17, 28, and 36, respectively. These results taken together prove the existence of two distinct mRNAs coding for proopiomelanocortin in the porcine pituitary intermediate lobe.

Animals↗
PubMed1982

Characterization of multiple forms of porcine anterior pituitary proopiomelanocortin amino-terminal glycopeptide.

Chromatography on a molecular sieve column of a preparation of porcine proopiomelanocortin N-terminal glycopeptide purified from anterior pituitary resulted in the isolation of three forms of the peptide with respective apparent Mr 21 000, 17 500, and 13 500 on polyacrylamide/sodium dodecyl sulfate gel. Determination of the amino acid composition of each peptide revealed that the form with a molecular weight of 17 500 corresponds to the 80 amino acid residue porcine N-terminal glycopeptide (PNT 1-80) previously characterized [Larivière, N., Seidah, N.G., & Chrétien, M. (1981) Int. J. Pept. Protein Res. 18, 487-491]. The forms with molecular weight of 21 000 and 13 500 correspond respectively to longer and shorter forms of the N-terminal glycopeptide. The high molecular weight form contains 107 amino acid residues. Sequencing of the fragments obtained after cleavage of the molecule with cyanogen bromide and Myxobacter Lys-C protease indicated that an extension of 27 amino acid residues is linked to PNT 1-80 through a -Lys-Arg-sequence. The sequence of the extension is reported. The low molecular weight form corresponds to the first 61 residues of PNT 1-80. Pronase digestion of the peptide and dansylation of the digest revealed the presence of a residue of phenylalanine amide at position 61. A general model for the maturation of the N-terminal glycopeptide of proopiomelanocortin in porcine anterior pituitary is presented.

Amino Acid Sequence↗