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Biomedical subjects

D Faucher

Publications and source records attributed to D Faucher.

At least 19 recordsLinked to original sources
PubMed1995

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.

Amino Acid Sequence↗
PubMed1995

Identification of the cytoskeletal protein alpha-actinin as a platelet thrombospondin-binding protein.

Binding of the alpha-granular thrombospondin (TSP) to the plasma membrane of activated platelets has long been documented, yet the molecular mechanism involved in its secretion and surface expression have not been elucidated. Using a ligand blot binding assay where electrophoretically separated platelet proteins were incubated with purified 125I-labeled TSP, we observed a strong interaction of [125I]TSP with a 100 kDa single chain protein. On performing a platelet subfractionation, the 100 kDa protein was predominantly localized in the cytosol from which it was purified by preparative electrophoresis and was identified by amino acid sequencing to the cytoskeletal protein, alpha-actinin. We further demonstrated that [125I]TSP interacts with alpha-actinin in a specific manner and with a high affinity (Kd = 6.6 nM) in a solid-phase binding assay.

Actinin↗
PubMed1995

Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis.

High levels of conversion of 14C-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PIIB was an intermediate on the pathway to PIIA. In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PIIB to PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), and molecular oxygen. Two enzymes were shown to be necessary to catalyze this reaction. Both were purified to homogeneity from S. pristinaespiralis by a coupled enzyme assay based on the formation of PIIA and by requiring addition of the complementing enzyme. One enzyme was purified about 3,000-fold by a procedure including a decisive affinity chromatography step on FMN-agarose. It was shown to be a NADH:FMN oxidoreductase (E.C. 1.6.8.1.) (hereafter called FMN reductase), providing reduced FMN (FMNH2) to the more abundant second enzyme. The latter was purified only 160-fold and was called PIIA synthase. Our data strongly suggest that this enzyme catalyzes a transient hydroxylation of PIIB by molecular oxygen immediately followed by a dehydration leading to PIIA. The native PIIA synthase consists of two different subunits with Mrs of around 50,000 and 35,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the FMN reductase seems to be a monomer with a Mr of around 28,000 and containing one molecule of tightly bound FMN. Stepwise Edman degradation of the entire polypeptides or some of their trypsin-digested fragments provided amino acid sequences for the two isolated proteins.

Amino Acid Sequence↗
PubMed1994

Primary structure control of recombinant proteins using high-performance liquid chromatography, mass spectrometry and microsequencing.

The conformity of two recombinant proteins (a von Willbrand factor fragment and human serum albumin, consisting of respectively 289 and 585 amino acids) has been examined by HPLC combined with mass spectrometry and microsequencing, on both intact material and fragment peptides obtained by proteolytic cleavage. These studies confirmed that the primary structure of the recombinant proteins corresponds to that predicted from their gene, particularly the integrity of their N and C termini, and, in the case of albumin, the agreement between the observed disulfide bond pattern and the published model. Furthermore, the structure of an albumin-related compound could be elucidated. Application of LC-MS for batch-to-batch quality control is also under discussion.

Amino Acid Sequence↗
PubMed1994

Solution structure of RP 71955, a new 21 amino acid tricyclic peptide active against HIV-1 virus.

The structure of RP 71955, a new tricyclic 21 amino acid peptide active against human immunodeficiency virus 1, was determined. Its amino acid composition was inferred from the results of fast atom bombardment mass spectrometry, nuclear magnetic resonance, Raman spectroscopy, and amino acid analysis. Its sequence could not be determined classically, using Edman degradation, given the lack of a free terminal NH2. It was deduced from the interpretation of interresidue nuclear Overhauser effects and confirmed by the sequencing of peptides obtained by limited chemical hydrolysis. It was found to be CLGIGSCNDFAGCGYAVVCFW. An internal amide bond between the NH2 of C1 and the gamma-COOH of D9 was observed, as well as two disulfide bridges, one between C1 and C13 and one between C7 and C19. The three-dimensional structure of RP 71955 was determined from nuclear magnetic resonance derived constraints using distance geometry, restrained molecular dynamics, nuclear Overhauser effect back calculation, and an iterative refinement using a full relaxation matrix approach. Analogies between the structure of RP 71955 and some functional domains of gp41, the transmembrane protein of human immunodeficiency virus 1, suggest hypotheses concerning the mode of action of RP 71955.

Amino Acid Sequence↗
PubMed1994

The major biotinyl protein from Pisum sativum seeds covalently binds biotin at a novel site.

Seeds of Pisum sativum contain a biotinyl polypeptide called SBP65 that behaves as a putative sink for the free vitamin, representing more than 90% of the total protein-bound biotin in mature seeds. A cDNA encoding SBP65 was cloned and sequenced. The deduced primary structure of the protein was confirmed by protein sequencing. Peptide sequencing also indicated binding of the biotin to lysine 103. The biotinylation domain of SBP65 differs markedly from that of presently known biotin enzymes. Molecular analysis of the protein sequence reveals an extremely hydrophilic protein containing several repeated motifs. These properties, as well as the temporal and spatial patterns of expression of this protein, suggest that SBP65 belongs to the LEA (late embryogenesis-abundant) group of proteins.

Amino Acid Sequence↗
PubMed1993

Topical anesthesia during circumcision in newborn infants.

OBJECTIVE: To determine the efficacy of topical anesthetic cream, eutectic mixture of local anesthetics (EMLA), in alleviating pain from neonatal circumcision. DESIGN: Randomized, controlled trial. SETTING: Normal newborn nursery in a university teaching hospital. PATIENTS: Twenty-seven newborn, full-term male neonates, aged 1 to 3 days. OUTCOME MEASURES: Heart rate, transcutaneous oxygen saturation, facial activity, and crying. RESULTS: Compared with baseline, all newborns experienced pain as evidenced by increased heart rate of an average of 40 beats per minute (F [8, 25] = 14.12; P < .0001), decreased oxygen saturation of 3% (F [8, 25] = 15.02; P < .0001), and more facial actions indicative of pain (F [8, 25] = 5.25; P < .002) during all phases of the procedure. Compared with placebo, EMLA significantly attenuated the pain response as shown by lower heart rate of an average of 25 beats per minute (F [1, 25] = 14.92; P < .001), higher oxygen saturation of 5%, particularly during the clamping and lysis phases (F [1, 25] = 19.83; P < .0001), 20% less facial activity (F [1, 25] = 12.01; P < .002), and 15% less crying during the clamping, Gomco clamp application, and incision of the foreskin. There were no differences between groups in the spectral crying parameters. CONCLUSIONS: Circumcision procedure produces pain responses that EMLA diminishes. Thus, EMLA may be a useful agent for pain management in neonatal circumcision.

Administration, Topical↗
PubMed1993

Stable periplasmic secretion intermediate in the general secretory pathway of Escherichia coli.

The secretion of the Klebsiella oxytoca cell surface lipoprotein pullulanase involves translocation across the cytoplasmic and outer membranes of the Gram-negative bacterial cell envelope. A variant of pullulanase was created by fusing the signal peptide-encoding 5' region of the Escherichia coli gene for periplasmic MalE protein to the 3' end of the pulA gene encoding almost the entire mature part of pullulanase. When produced in E. coli carrying the malE-pulA gene fusion on a high copy number plasmid and the complete set of genes specifically required for pullulanase secretion on a second plasmid, the hybrid protein differed from wild-type pullulanase as follows: (i) it was not fatty-acylated; (ii) it was apparently processed by LepB signal peptidase rather than by LspA lipoprotein signal peptidase; (iii) it was released into the periplasm and was only slowly transported across the outer membrane, and (iv) it was released directly into the medium rather than via the usual surface-anchored intermediate. The hybrid protein was secreted more rapidly when malE-pulA was expressed from a low copy number plasmid. The two steps in the secretion pathway could be totally uncoupled by expressing first the malE-pulA gene fusion and then the cognate secretion genes. These results show that fatty-acylation of wild-type PulA is not essential for secretion but may improve its efficiency when large amounts of the protein are produced, that the two steps in secretion can occur quite independently and that the periplasmic intermediate can persist for long periods under certain circumstances.

ATP-Binding Cassette Transporters↗
PubMed1991

High-level secretion of correctly processed recombinant human interleukin-1 beta in Kluyveromyces lactis.

The lactose-assimilating yeast, Kluyveromyces lactis, has been developed as a microbial host for the synthesis and secretion of human proteins. Here, we report the use of multi-copy vectors based on the 2 mu-like plasmid pKD1 from Kluyveromyces drosophilarum [Chen et al., Nucleic Acids Res. 14 (1986) 4471-4481] for the secretion of recombinant human interleukin-1 beta (reIL-1 beta). High levels of reIL-1 beta were secreted into the growth medium when the structural gene was fused in-frame to a synthetic secretion signal derived from the 'pre'-region of the K. lactis killer toxin. N-terminal sequencing of the excreted protein showed highly efficient (greater than 95%) maturation of the signal sequence. Synthesis as prepro-IL-1 beta, the 'pro'-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1 beta. Cytoplasmic production of Met-IL-1 beta, without a secretion signal, was found to be toxic to K. lactis. As in Saccharomyces cerevisiae [Baldari et al., EMBO J. 6 (1987) 229-234], but unlike native human IL-1 beta, K. lactis reIL-1 beta is glycosylated. This glycosylation led to a 95% loss of its biological activity. Removal of the carbohydrate chains by endo-beta-N-acetyl-glucosamidase H treatment fully restored the biological activity. A modified form of IL-1 beta (Asn7----Gln7), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1 beta. The level of secretion of mature recombinant IL-1 beta ws glycosylation-independent.

Amino Acid Sequence↗
PubMed1991

Outer membrane translocation of the extracellular enzyme pullulanase in Escherichia coli K12 does not require a fatty acylated N-terminal cysteine.

Site-directed mutagenesis was used to construct three mutant derivatives of the extracellular, cell surface lipoprotein pullulanase (PulA) in which the normally fatty acylated cysteine of the signal peptide-bearing precursor was replaced by other amino acids. When produced in Escherichia coli expressing all genes required for pullulanase secretion, approximately 90% of the PulA derivatives persisted as cell-associated precursors, indicating inefficient signal peptide processing. Processed (intermediate-sized) forms of the two derivatives that were studied in detail were found to result from proteolytic cleavage at different sites within the signal peptide. Both were further processed to smaller polypeptides by cleavage at an undetermined site that is presumably close to their C termini. The intermediate-sized pullulanase derived from prepullulanase in which Cys+1 had been replaced by Leu and Gly-1 by Glu (PulA:C1L/G-1E) appeared rapidly, was apparently entirely extracellular, and accounted for approximately 10% of synthesized PulA. Prolonged incubation did not result in further conversion of the precursor to the intermediate form, and the precursor remained anchored to the cytoplasmic membrane. The smaller processed form was also found extracellularly. The active form of the extracellular enzyme was monomeric, which is again in contrast to the fatty acylated, wild-type enzyme. Taken together, these results indicate that replacement of Cys+1 of prePulA eliminates processing by lipoprotein signal peptidase and does not permit processing by leader peptidase, but allows inefficient, aberrant processing by an unknown peptidase and immediate secretion of the resulting polypeptide, which retains most of its signal peptide. Processing and secretion only occur when the pullulanase secretion functions are expressed.

Acylation↗
PubMed1991

Identification of intact neurotensin in the substantia nigra after its retrograde axonal transport in dopaminergic neurons.

Two hours after the injection of (3-[125I]iodotyrosyl3) neurotensin into the striatum, a labeling was observed in the ipsilateral substantia nigra. In the present study, we demonstrated by HPLC that this radioactivity corresponded to intact neurotensin and to degradation products of this peptide. This finding provides the first clearcut evidence that a neuropeptide can be internalized and retrogradely transported in brain neurons. Therefore, the fact that intact neurotensin can be seen to exist over a long period of time in the cell body suggests that the retrograde transport process could perhaps be involved in the long-term effects of neuropeptides.

Animals↗
PubMed1991

Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii.

An S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity has been identified in Methanobacterium ivanovii and was purified 4,500-fold to homogeneity with a 38% yield. The enzyme had an apparent molecular weight of 58,200 by gel filtration and consisted of two identical subunits of Mr 29,000, as estimated by gel electrophoresis under denaturing conditions. The Km value for uroporphyrinogen III was 52 nM. The enzyme catalyzed the two C-2 and C-7 methylation reactions converting uroporphyrinogen III into precorrin-2. Unlike Pseudomonas denitrificans SUMT, the only SUMT characterized to date (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet and B. Cameron, J. Bacteriol. 171:4222-4231, 1989), M. ivanovii SUMT did not show substrate inhibition at uroporphyrinogen III concentrations of up to 20 microM. Oligonucleotide probes from limited peptide sequence information were used to clone the corresponding gene. The encoded polypeptide showed more than 40% strict homology with P. denitrificans SUMT. The M. ivanovii SUMT structural gene is likely to be, as is P. denitrificans cobA, involved in corrinoid synthesis.

Amino Acid Sequence↗
PubMed1990

Low molecular weight, sequence based, collagenase inhibitors selectively block the interaction between collagenase and TIMP (tissue inhibitor of metalloproteinases).

Sequence-based inhibitors of collagenase bearing an hydroxamate group capable of chelating the active site zinc atom were synthesized and tested. The effect of one of these molecules (RP 59794; Ki about 10(-8) M) on the formation of the TIMP: collagenase complex was also tested. RP 59794 blocks complex formation and can partially dissociate established TIMP: collagenase complexes. It exhibits the same stereospecificity in this activity as in its inhibition of collagenase suggesting that TIMP and RP 59794 both interact with the active site region of collagenase.

Amino Acid Sequence↗
PubMed1990

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase.

An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 mumol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30 degrees C under the control of an E. coli promoter and ribosome-binding site.

Amides↗
PubMed1989

Limited proteolysis of T-kininogen (thiostatin). Release of comparable fragments by different endopeptidases.

Limited proteolysis of T-kininogen by heterologous and homologous endopeptidases (bovine trypsin, human leukocyte elastase, rat submaxillary gland endopeptidase k, and rat mast cell chymase) produced similar fragmentation. Amino-terminal sequence analysis of whole T-kininogen lysates and purified proteolytic fragments identified four susceptible regions which contained all the preferential cleavage sites for these proteinases. Two of these susceptible regions were close to the junction between heavy chain cystatin-like domains, the third was in the kinin-containing region, and the fourth was close to the carboxyl terminus of the T-kininogen light chain. There was only one primary site for each proteinase in the kinin-containing region, which explains why catalytic amounts of these proteinases did not release immunoreactive kinin from this kininogen. However, preferential cleavage of T-kininogen close to the junction between cystatin-like domains released fragments which, provided they included cystatin-like domains 2 and/or 3, strongly inhibited papain and cathepsin L. The fragments were inhibitory even when parts of the amino-terminal ends of the domains were lacking. The highly conserved glycyl residue, thought to be involved in the inhibitory reactive site of cystatin-like inhibitors, was not required in purified domain 3 for inhibition of cathepsin L.

Amino Acid Sequence↗