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D Faucher

Publications and source records attributed to D Faucher.

16 recordsLinked to original sources
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↗
PubMed1989

[Natural inhibitor of metalloproteinases: structural and functional study].

A potent collagenase inhibitor was purified from cells of calf aorta medial tissue maintained in culture. This molecule was characterized and identified as TIMP (Tissue Inhibitor of Metalloproteinases). Formation of a TIMP--collagenase complex was demonstrated chromatographically using pure TIMP and pure pig synovial cell collagenase. The N-terminal aminoacid sequence of TIMP was determined and, using appropriate oligonucleotide probes the human genes was cloned from a human cDNA bank. This gene was expressed in E. coli, and fully active TIMP was obtained after a denaturation renaturation process. The interest of TIMP as a model for the design of novel collagenase inhibitors is discussed.

Amino Acid Sequence↗
PubMed1988

Two rat homologues of human cystatin C.

Two immunochemically related forms of cystatin C-like inhibitors which differ in their Mr app and isoelectric point have been found both in urine and seminal vesicles of rats. Amino-terminal sequences of these two cystatins are identical within the same fluid and exhibit a high degree of homology with that of human cystatin C. However, cystatins C purified from urine lack eight residues at their amino-terminal end when compared to those of seminal vesicles. The occurrence of two cystatin C-like components in rat fluids has been found to be due to the presence of a glycosylated form reported here as cystatin Cg which specifically binds concanavalin A and is susceptible to endo-beta-N-acetylglucosaminidase treatment.

Amino Acid Sequence↗
PubMed1985

[Orbital apex syndrome in a child with Wegener's granulomatosis].

The authors report the case of a 10 year-old child presenting with orbital apex syndrome as the initial manifestation of Wegener's granulomatosis. Orbital apex syndrome is characterized by complete or partial paralysis of the third, fourth and sixth cranial nerves with involvement of the trigeminal nerve's ophthalmic branch. The different mechanisms responsible for ocular complications in Wegener's granulomatosis and the usefulness of computer assisted tomography in this clinical situation are discussed.

Child↗
PubMed1976

[Non-specific effects of a drug study in hypertensive patients].

During the first phase of a drug study, 33 hypertensive patients received 2 placebo tablets daily for 2 weeks as the sole treatment. Analysis of the blood pressure readings during this placebo phase shows that the study in itself creates a psychological climate which tends to decrease blood pressure. This effect should be taken into account when analyzing results of drug studies; only in studies with a double blind design is drug effect adequately judged.

Adult↗