PubMed Health⌕ Search

Biomedical subjects

P Amiguet

Publications and source records attributed to P Amiguet.

14 recordsLinked to original sources
PubMed1993

Paralytic tremor (pt) rabbit: a sex-linked mutation affecting proteolipid protein-gene expression.

Paralytic tremor (pt) is a neurological sex-linked recessive mutation in rabbits which is characterized by a coarse body tremor and limb paresis. Morphological studies showed that this mutation affects CNS myelination. Although the number of oligodendrocytes is not reduced, myelination is slower, irregular and defective. We have made a biochemical and molecular analysis of 4-wk-old mutant and normal rabbits. The amount of myelin in the mutant represents only approximately 25% of the normal level. Radioimmunoassay for myelin basic protein showed a reduction to approximately 40% in pt whole-brain homogenate but the difference was not significant in purified myelin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of brain homogenates followed by immunoblotting showed that all major myelin proteins are affected by the pt mutation, although to different degrees. While most of the myelin proteins are reduced to approximately 60-80% of the normal level, an important reduction to approximately 30%, was measured for the proteolipid protein (PLP). In purified myelin, the difference in PLP concentration was significant while the other specific proteins were less affected. A similar reduction in myelin-protein gene expression was detected at the mRNA level. Sex-linked transmission, low concentrations of PLP and its specific mRNA in the CNS indicate that the pt mutation primarily affects the expression of the Plp gene.

Animals↗
PubMed1992

Myelin/oligodendrocyte glycoprotein is a unique member of the immunoglobulin superfamily.

Myelin/oligodendrocyte glycoprotein (MOG) is a primary target autoantigen in experimental autoimmune encephalomyelitis, a widely used animal model for autoimmune demyelinating diseases such as multiple sclerosis. We have isolated several rat MOG cDNAs and confirmed their identity by comparison with MOG N-terminal peptide sequence. As expected, MOG mRNA expression is CNS-specific and peaks during active myelination. Our studies show that full length MOG mRNA is approximately 1.6 kb and encodes a signal peptide of 27 amino acids, followed by 218 residues for mature MOG (24,962 MW). A single site for N-glycosylation is found at Asn-31. Rather than the ubiquitous AAUAAA polyadenylation signal, a series of three overlapping, rare poly A signals were identified. The N-terminal half of mature MOG shares 52% identity with bovine butyrophilin, a possible lipid receptor. This same region has 39% identity with chicken B-G antigen, a major histocompatibility complex antigen involved in B cell selection and immune repertoire development. We show that both MOG and butyrophilin, each exhibiting a single Ig-like variable region domain, meet criteria for inclusion in the immunoglobulin superfamily. Moreover, MOG appears to represent a unique member of this superfamily in that it possesses two potential transmembrane domains, in contrast to a single membrane-spanning domain or glycophospholipid anchor found in all other members of Ig superfamily members.

Animals↗
PubMed1992

Purification and partial structural and functional characterization of mouse myelin/oligodendrocyte glycoprotein.

The myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8-18C5 identifies MOG. Several studies have shown that anti-MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25-kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that there are no or few O-linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N-terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal-transducing GTP-binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.

Amino Acid Sequence↗
PubMed1991

Structural features of the human C3 gene: intron/exon organization, transcriptional start site, and promoter region sequence.

The third component of human complement (C3) is a key molecule in the activation of the complement cascade. C3 cDNA fragments were used to identify seven cosmid clones that covered all but 1 kilobase pair (kb) of the C3 gene. The remainder of the gene was cloned by using the polymerase chain reaction. These clones were used to identify the intron/exon boundaries and to map the gene. The C3 gene is 42 kb in length and comprises 41 exons ranging in size from 52 to 213 base pairs (bp). The transcription start site was identified by primer extension, and approximately 1 kb of DNA upstream of this site was sequenced. Putative TATA and CAAT boxes were identified along with a number of regions that shared homology with known regulatory sequences. These include responsive elements for interferon-gamma, interleukin-6, nuclear factor kappa B, estrogen, glucocorticoids and thyroid hormone. Several of these agents have been shown to affect C3 synthesis and mRNA levels. The sizes of the exons in C3 were compared to those of C4 and alpha 2-macroglobulin (alpha 2M). Thirty-nine of 41 exons in C4 were found to be of similar size to the analogous ones in C3, and two-thirds of those in alpha 2M were also similarly sized, supporting the hypothesis that these genes arose from a common ancestor.

Amino Acid Sequence↗
PubMed1990

Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene.

p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been determined from analysis of cloned cDNA. The protein contains 338 amino acids, has an approximate molecular weight of 39,000, and is comprised of several distinct domains, including four 75 amino acid repeats. From two overlapping cosmid clones isolated from different mouse genomic liver libraries, the complete intron/exon structure of the p36 gene was determined and the 5' and 3' noncoding regions of the gene were analyzed. The coding and 3' untranslated region of the p36 gene contains 12 exons which range in size from 48 to 322 base pairs (bp) with an average size of 107 bp. The repeat structures found at the protein level are not delineated by single exons, but the N-terminal p11-binding domain is encoded by a single exon. Structural mapping of the gene demonstrated that the lengths of the first two introns in the coding region are together approximately 6 kilobases (kb), while the other introns range in size from 600 to 3600 bp with an average size of 1650 bp. The p36 gene is at least 22 kb in length and has a coding sequence of approximately 1 kb, representing only 4.5% of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Localization and molecular modelling of the membrane-inserted domain of the ninth component of human complement and perforin.

Upon interaction with the membrane-bound C5b-8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazirine (125I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125I-TID-labeled C9, were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292-308 and 313-333) separated by a turn (309-312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perforin/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.

Affinity Labels↗
PubMed1990

Myelin/oligodendrocyte glycoprotein expression during development in normal and myelin-deficient mice.

The myelin/oligodendrocyte glycoprotein (MOG) is identified by monoclonal antibody 8-18C5. MOG is localized on the surface of myelin and oligodendrocyte processes. Recently, several studies have shown that MOG plays an important role as a target for antibody-induced demyelination. In the present study, we investigated MOG expression in the brains of normal and myelin-deficient (mld) mutant mice during development. By gel electrophoresis and immunoblotting, we observed the developmental pattern of two closely migrating bands, with apparent molecular masses of 26 and 28 kilodaltons. Their concentrations increased coordinately during the most active phase of myelin and myelin basic protein (MBP) synthesis. Between 20 and 25 days of age, the MOG developmental pattern superimposed that of MBP as well as myelin yields. In mld mutant mice, which are affected by a severe deficit of MBP synthesis, MOG was present at reduced levels (40% of controls at 60 days of age). At 85 days of age, mld mice exhibited increased concentrations of MBP, and myelin was better compacted. At this age, MOG concentrations increased and reached 70% of controls. These results suggest that MOG could play a role in the maintenance or completion of the myelin sheath. Its expression level may be modulated by the presence of compact myelin and/or MBP in the myelin sheath.

Animals↗
PubMed1989

Complete intron/exon organization of DNA encoding the alpha' chain of human C3.

The third component of human complement (C3), a central molecule in both the classical and alternative pathways of complement, is comprised of two polypeptides, termed the alpha and beta chains. Activation of C3 cleaves the alpha chain into two fragments, C3a, an inflammatory peptide, and the alpha' chain which remains covalently linked to the beta chain. Proteolytic fragments derived from the alpha' chain during activation and regulation of complement play a significant role in host defense and regulation of the immune response. Two cosmid clones covering the alpha' chain region were used to characterize the structure of this portion of the C3 gene. The alpha' chain is encoded by 24 exons, which range in size from 52 to 213 base pairs (bp) with an average size of 115 bp. The splice donor sequence at the beginning of intron 12 has a rare sequence variant of GC instead of the usual GT sequence. Ten introns have been completely sequenced and were surprisingly short, ranging in size from 85 to 242 bp with an average of 140 bp. Other introns range in size from 250 bp to over 4 kilobases in length. The gene size for this portion of C3 is estimated to be 23-24 kilobases. Comparison of exon structure with protein domains and with peptide mapping studies demonstrates that several binding sites on C3 are encoded by single exons. These data support the hypothesis that individual exons can code for functional protein domains.

Base Sequence↗
PubMed1986

Increased hemolytic activity of the trypsin-cleaved ninth component of complement.

Human C9 treated with trypsin is initially cleaved into two fragments with relative mol. wts of 53,000 and 20,000. This limited cleavage of C9 induces a 2.4-times increase in the hemolytic activity of C9 when compared to untreated C9. This difference diminishes when C9 activity is tested in an assay using a prolonged incubation time of C9 with C5b-8-bearing red blood cells. Trypsinization of C9 also promotes spontaneous C9 polymerization. SDS-resistant tubular C9 complexes are formed at a C9 concn of 1 mg/ml within 8 hr at 37 degrees C. Our data indicate that specific limited proteolysis of C9 not only induces spontaneous C9 polymerization but also increases the hemolytic activity of C9, suggesting that a similar molecular mechanism is involved in both processes.

Biopolymers↗
PubMed1985

The membrane attack complex of complement: lipid insertion of tubular and nontubular polymerized C9.

The membrane-restricted photoactivatable carbene generator 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)diazirine [Brunner, J., & Semenza, G. (1981) Biochemistry 20, 7174-7182] was used to label the subunits of the membrane attack complex of complement (C5b-9). C5b-9 complexes either were assembled from serum on erythrocyte membranes or were reconstituted from purified components on liposomes. After irradiation, most of the probe is bound to C9 independent of the membrane system used, indicating that the wall of the transmembrane channel is predominantly composed of C9. No difference was observed whether polymerized C9 was in the tubular or nontubular form [Podack, E. R., & Tschopp, J. (1983) J. Biol. Chem. 257, 15204-15212], showing that tubule closure is not essential for successful lipid insertion. The same label distribution between the two forms of polymerized C9 was obtained by analyzing zinc-polymerized C9 in the absence of C5b-8. Since the photoreactive probe reacted with at least two distinct polypeptide segments within C9, lipid interaction does not occur via a single segment of hydrophobic amino acids.

Animals↗
PubMed1983

Comparison of the interactions of soya bean protease inhibitors with rat pancreatic enzymes and human trypsin.

The present work describes the effect of two soya bean protease inhibitors: Kunitz type (SBTI) and Bowman-Birk type (BBTI) on rat trypsin I (TrI), trypsin II (TrII) and chymotrypsin (Chtr) and on human cationic trypsin (hTr). The inhibition spectra show that: (1) SBTI inhibits TrI, TrII, Chtr and hTr esterase activities by 80, 80, 83 and 45%, respectively, at inhibitor-to-enzyme molar ratios of one-to-one, and (2) BBTI inhibits TrI, TrII, Chtr, and hTr esterase activities by 50, 65, 75 and 30%, respectively, at an inhibitor-to-enzyme molar ratio of two-to-one. A similar inhibition pattern was obtained by testing proteolytic activities. It would appear that hTr is less sensitive to soya bean protease inhibitors than each of the rat proteases investigated. This difference in inhibition should be considered when a rat is used as a model to predict the effects of dietary soya bean protease inhibitors on humans.

Animals↗
PubMed1976

Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase.

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.

Alcohol Oxidoreductases↗