Biomedical subjects

S Frutiger

Publications and source records attributed to S Frutiger.

At least 37 records · Page 2Linked to original sources

PubMed1995

Binding sites for hepatocyte nuclear factor 3 beta or 3 gamma and pancreas transcription factor 1 are required for efficient expression of the gene encoding pancreatic alpha-amylase.

Efficient expression of genes under the control of alpha-amylase 2 5'-flanking sequences in exocrine pancreatic cells requires, in addition to the pancreas transcription factor 1 binding site (M. Cockell, B.J. Stevenson, M. Strubin, O. Hagenbüchle, and P. K. Wellauer, Mol. Cell. Biol. 9:2464-2476, 1989), another cis-acting element at positions -60 to -86. This DNA element, which contains an AT-rich core, site for nuclear proteins present not only in the pancreas but also in other tissues and cell lines derived from the endoderm. Purification of binding activities from pancreatic cells by DNA affinity chromatography reveals several distinct proteins ranging in size from 45 to 54 kDa (p45, p47/48, and p54). All of these proteins interact with the specific DNA sequence upon renaturation in vitro. Protein sequencing, electrophoretic mobility shift assay, and immunoblot analyses identify p54 and p47/48 as members of the hepatocyte nuclear factor 3 (HNF3 [forkhead]) family of transcription factors. p54 belongs to the subfamily of HNF3 beta proteins, while p47/48 binding activity includes HNF3 gamma. The cDNAs for two HNF3 beta proteins differing only in N-terminal amino acid sequences were isolated from a pancreatic cDNA library. The mRNAs encoding the two protein species accumulate to different steady-state levels in poly(A)+ RNA of pancreatic cells. Our results support a model by which the pancreas-specific expression of the alpha-amylase gene is mediated by a combination of cell-specific and cell lineage-specific transcription factors.

Amino Acid Sequence↗

PubMed1994

A cyclin associated with the CDK-activating kinase MO15.

The eukaryotic cell cycle is regulated by the sequential activation of cyclin-dependent kinases (CDKs). CDK activation is dependent on cyclin binding and phosphorylation of a conserved threonine (T161 in Cdc2) mediated by the CDK-activating kinase CAK. A CDK-related kinase, MO15 (ref. 10), has been identified as the catalytic subunit of CAK (refs 11-13). Here we use a yeast two-hybrid screen to show that a new human cyclin (cyclin H) is a MO15-associated protein. Cyclin H is a major MO15 partner in vivo and enhances the kinase activity of MO15 towards Cdk2/cyclin A. These findings demonstrate that a cyclin/kinase complex can function as a regulator of other cyclin/kinase complexes, and suggest that cyclin/kinase cascades may exist.

Amino Acid Sequence↗

PubMed1994

Two-dimensional polyacrylamide gel electrophoresis analysis of cryoglobulins and identification of an IgM-associated peptide.

The clonality of immunoglobulins (Igs) in cryoprecipitates (n = 41) was studied by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Our series included 24 cryoglobulins characterized by immunofixation electrophoresis (IF), 12 'trace amount' cryoglobulins, defined by a protein content in the precipitate of less than 0.05 mg/ml of serum, and five cryoglobulins of undetermined protein composition by IF. 2-D PAGE analysis showed polyclonal IgG associated either with monoclonal Igs (type II cryoglobulins; n = 14) or with polyclonal IgM (type III cryoglobulins; n = 14). In ten cryoprecipitates (two 'trace amount' cryoglobulins as well as seven of 19 type II and as one of five type III cryoglobulins by IF) polyclonal IgG were associated with a mixture of polyclonal and monoclonal IgM. These cryoglobulins were tentatively named type II-III cryoglobulins. A monoclonal IgM was observed in one cryoprecipitate (type I cryoglobulins). Two cryoglobulins presented unexpected 2-D patterns, characterized by the presence of oligoclonal IgM, with trace amounts of Igs of different isotypes (tentatively named type II-III(variant) cryoglobulins). A peptide of 44 kDa with a pI of 5.45 was observed in all cryoglobulins containing IgM (n = 40). This peptide was also present in purified monoclonal or polyclonal IgM fractions. N-terminal microsequencing (12 amino acid residues) revealed that this IgM-associated peptide was an unknown protein. Our results highlight the role of 2-D PAGE as an aid in the analysis of cryoglobulins.

Amino Acid Sequence↗

PubMed1994

The yeast protein encoded by PUB1 binds T-rich single stranded DNA.

We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only after fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with apparent mobility of 60kDa (ACBP-60). This protein co-fractionates with nuclei, is present at several thousand copies per cell and has a Kd for the T-rich single strand of the ARS consensus between 10(-9) and 10(-10) M. Competition studies with simple nucleic acid polymers show that ACBP-60 has marginally higher affinity for poly dT30 than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximately 10 fold higher affinity for poly rU. Internal sequence information of purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The second binding activity, ACBP-67, also binds specifically to the T-rich single strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is identical to the major polyA binding protein in yeast, PAB1.

Base Composition↗

PubMed1993

Human alpha and beta parvalbumins. Structure and tissue-specific expression.

alpha and beta parvalbumins are Ca(2+)-binding proteins of the EF-hand type. We determined the protein sequence of human brain alpha parvalbumin by mass spectrometry and cloned human beta parvalbumin (or oncomodulin) from genomic DNA and preterm placental cDNA. beta parvalbumin differs in 54 positions from alpha parvalbumin and lacks the C-terminal amino acid 109. From MS analyses of alpha and beta parvalbumins we conclude that parvalbumins generally lack posttranslational modifications. alpha and beta parvalbumins were differently expressed in human tissues when analyzed by immunoblotting and polymerase-chain-reaction techniques. Whereas alpha parvalbumin was found in a number of adult human tissues, beta parvalbumin was restricted to preterm placenta. The pattern of alpha parvalbumin expression also differs in man compared to other vertebrates. For example, in rat, alpha parvalbumin was found in extrafusal and intrafusal skeletal-muscle fibres whereas, in man, alpha parvalbumin was restricted to the muscle spindles. Different functions for alpha and beta parvalbumins are discussed.

Amino Acid Sequence↗

PubMed1993

Transcytosis of the polymeric Ig receptor requires phosphorylation of serine 664 in the absence but not the presence of dimeric IgA.

MDCK cells expressing the polymeric immunoglobulin (poly-Ig) receptor, cocultured with IgA-producing hybridoma cells, transported dimeric IgA (dIgA) from the basolateral into the lumenal compartment, where it was recovered as secretory component-dIgA complexes. The tail of the receptor was phosphorylated on serines 664 and 726. Each serine was mutated to alanine. Appearance of A726 receptor at the basolateral surface was reduced approximately 5-fold. This was accompanied by a approximately 5-fold reduction in dIgA transcytosis. Basolateral delivery of receptor was not affected by mutation A664, and in the absence of dIgA, the receptor accumulated in recycling basolateral endosomes. In coculture, however, dIgA transcytosis by A664 receptor was normal. Thus, entry of receptor into the transcytotic pathway requires Ser-664 phosphorylation only in the absence of dIgA.

Amino Acid Sequence↗

PubMed1993

The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences.

The focusing positions in narrow range immobilized pH gradients of 29 polypeptides of known amino acid sequence were determined under denaturing conditions. The isoelectric points of the proteins calculated from their amino acid sequences matched with good accuracy the experimentally determined pI values. We show the advantages of being able to predict the position of a protein of known structure within a two-dimensional gel.

Amino Acid Sequence↗

PubMed1993

Human liver protein map: update 1993.

This publication updates the reference human liver protein map. By microsequencing, 27 spots or 34 polypeptide chains were identified. The most abundant polypeptides detected on the silver stained liver map were key elements in major hepatic biochemical pathways. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing the 1993 reference human liver SWISS-2DPAGE database. SWISS-2DPAGE and the SWISS-PROT protein sequence databases are closely linked together through the use of common accession numbers.

Amino Acid Sequence↗

PubMed1993

Plasma and red blood cell protein maps: update 1993.

This publication updates the reference plasma and red blood cell protein maps obtained with immobilized pH gradients. Seventeen polypeptide spots or chains were partially characterized by direct N-terminal sequencing or by sequencing of peptides obtained from enzymatic digestion. Additional new polypeptides and previously known proteins are listed in a table and/or labeled on the protein maps, thus providing the 1993 update of the human plasma and red blood cell two-dimensional gel SWISS-2DPAGE database. SWISS-2DPAGE and the SWISS-PROT protein sequence databases are closely linked together through the use of common accession numbers.

Amino Acid Sequence↗

PubMed1993

Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins.

We describe some simple modifications to the micropreparative two-dimensional (2-D) electrophoresis procedure using immobilized pH gradients in the first dimension and sodium dodecyl sulfate-electrophoresis in the second dimension. The geometry of the immobilized pH gradient strips has been changed to allow the use of large sample application cups that can accommodate greater sample volumes. The use of narrow range immobilized pH gradients with a large sample loading volume allows an efficient resolubilization of polypeptides after the first dimension. As a result, the vertical streaking caused by too high a protein concentration is eliminated in the second dimension. Protein identification by N-terminal sequencing is facilitated by the large protein load (1-15 mg) which can be employed using this modification. Spots not normally detectable on conventional analytical 2-D maps, even with sensitive silver staining, are observed. Results for plasma and liver proteins are shown.

Blood Proteins↗

PubMed1992

Disulfide bond assignment in human J chain and its covalent pairing with immunoglobulin M.

The assignment of disulfide bonds in human J chain and its covalent pairing with immunoglobulin M was determined under conditions which minimize disulfide bond interchange. We show that in J chain the three intradisulfide bridges are formed between Cys 12 and 100, Cys 71 and 91, and Cys 108 and 133. Previous reports [reviewed by Koshland, M. E. (1985) Annu. Rev. Immunol. 3, 425-453] have proposed that cysteines 12, 14, or 68 were linked to the penultimate cysteine 575 of two mu chain tails. In this work, we demonstrate that cysteines 14 and 68 are disulfide-bridged to mu chains. A revised, albeit putative, model of J chain folding is presented which takes into account the correct disulfide pairing and the predictive secondary structure assignment.

Amino Acid Sequence↗

PubMed1992

Polypeptide marker and disease patterns found while mapping proteins in ascitis.

To assess the protein composition of ascitis, 28 samples of ascitic fluid were obtained from patients admitted to Geneva University Hospital. The samples were analysed randomly and blindly by high-resolution two-dimensional polyacrylamide gel electrophoresis. The final visual evaluation was compared with the discharged summary and diagnosis. The protein pattern of ascitis was, as expected, very similar to normal or diseased plasma, with the exception of two spots which were present in ascitic fluids but not in the 200 plasma samples analyzed in parallel. After microsequencing, they proved to be beta-fibrinogen fragments. Several diseases showed distinct patterns, especially acute pancreatitis. A group of intense spots with an apparent molecular mass between those of alpha 1-antitrypsin and beta-haptoglobin were found in all ascitic fluid from pancreatitis cases (six patients). These spots had isoelectric points similar to those of alpha 1-antitrypsin and beta-haptoglobin and microsequencing revealed that they were three different fragments of alpha 1-antitrypsin.

Acute Disease↗

PubMed1992

Structural and electron-microscopic studies of jacalin from jackfruit (Artocarpus integrifolia) show that this lectin is a 65 kDa tetramer.

The 133-amino-acid sequences of the alpha-subunit of jacalin (a lectin from Artocarpus integrifolia) and of the slightly larger alpha'-subunit were determined. The alpha'- and alpha-subunits, in the approximate ratio of 1:3, were found to be virtually identical in their primary structures, except for one valine for isoleucine substitution at position 113. Although both alpha'- and alpha-chains were glycosylated, the extent of glycosylation in the alpha'-chain was much greater than that in the alpha-subunit. In the alpha'-polypeptide, all molecules contained an N-linked oligosaccharide at position 74 and some contained sugar at position 43. The alpha- and alpha'-subunits were found to be strongly non-covalently associated with three distinct beta-subunits containing 20 amino acids each. Electron-microscopic visualization of native jacalin disclosed a structure composed of four alpha-type subunits with a clear-cut 4-fold symmetry. Analytical-ultracentrifugation studies of jacalin revealed an average molecular mass of 65 kDa, a value compatible with a tetrameric structure of the alpha(alpha')-subunits. The recalculated number of sugar-binding sites per jacalin molecule, given a molecular mass of 65 kDa, would yield 0.8 sites per alpha(alpha')-promoter, i.e. about twice the value previously determined [Appukutan & Basu (1985) FEBS Lett. 180, 331-334; Ahmed & Chatterjee (1989) J. Biol. Chem. 264, 9365-9372].

Amino Acid Sequence↗

PubMed1992

Human liver protein map: a reference database established by microsequencing and gel comparison.

This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.

Amino Acid Sequence↗

PubMed1991

A nodulin specifically expressed in senescent nodules of winged bean is a protease inhibitor.

Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspective of improving the host-bacteroid interaction. In winged bean nodules, a 21-kilodalton protein is specifically expressed when senescence begins. Using subcellular fractionation, we observed that this plant protein interacts with the bacteroids. Microsequencing of the protein allowed us to obtain a specific oligonucleotide that was used to isolate the corresponding nodule cDNA. Sequence analysis of this cDNA revealed that the 21-kilodalton protein has all of the features of a legume Kunitz protease inhibitor. Subsequent analysis confirmed that this nodulin is indeed a protease inhibitor. Immunocytochemical study showed that the protease inhibitor is exclusively localized in infected senescent cells of the nodule, particularly in disorganized bacteroids, the peribacteroid membrane, vacuole membranes, and in the vacuole fluid. The specific expression of a protease inhibitor at senescence may be of particular interest if the targeted proteolytic activity is important for the symbiotic relationship. This point is discussed in relation to the known nodule proteases.

Aging↗

PubMed1990

The amino acid sequence of rabbit J chain in secretory immunoglobulin A.

The primary structure of rabbit J chain, which occurs covalently bound to secretory IgA, was determined. J chain was isolated in its S-carboxymethylated form, in one step, by SDS/PAGE followed by electro-elution; 5 nmol of protein (approx. 75 micrograms), in all, was necessary for the determination of the complete sequence by the 'shot-gun' microsquencing technique; with the use of several site-specific endoproteinases, the various digests of S-carboxymethylated J chain were separated by micro-bore reverse-phase h.p.l.c. and the partial N-terminal sequences of all peptides were analysed. From the sequence alignment, gaps were filled by further extensive sequencing of the relevant overlapping fragments isolated from selected digests. Rabbit J chain comprises 136 amino acid residues, out of which eight are conserved cysteine residues, and is more closely similar to the human sequence (73.5% identify) than to the mouse sequence (68% identity). There is one unique glycosylation site at asparagine-48.

Amino Acid Sequence↗

PubMed1990

Immunological heterogeneity of autoreactive T lymphocytes against the nicotinic acetylcholine receptor in myasthenic patients.

The response of human T lymphocytes against the nicotinic acetylcholine receptor (AChR) was studied in five patients with myasthenia gravis (MG) and in six healthy donors using either native Torpedo AChR or recombinant protein derived from the mammalian AChR alpha subunit (X4, residues 6-216 of mouse AChR alpha subunit). The present study demonstrates that (a) AChR-specific T helper cell lines can be generated from MG patients [either from peripheral blood lymphocytes (PBL) or from thymocytes] as well as from PBL of normal controls, (b) lymphocytes from MG patients, but not from controls, recognize the mammalian AChR but not the Torpedo receptor, (c) in humans, the HLA-DR2-associated T cell epitope is probably located in the region of residues 162-216 of the AChR alpha subunit and (d) there is a considerable heterogeneity of autoreactive T cell responses: (i) T cell lines from different HLA-type donors have distinct epitope profiles; (ii) the epitope specificity of the PBL-derived T cell line is different from that of the thymocyte-derived line; (iii) the epitope specificities of patient-derived T cell lines are different from those generated from normal controls who share the same HLA phenotype.

Animals↗

PubMed1990

Chemical and immunochemical identification of the second major rabbit immunoglobulin lambda chain polypeptide bearing c7 epitopes.

The purification of lambda chain-containing IgG fraction from pooled sera of Basilea rabbits, which were bred and selected for the expression of a high level of lambda chains positive for c7 but negative for c21, was carried out. On the basis of specific binding to anti-c7 antiserum, the c7 lambda chain fraction in serum IgG was shown to account for up to 70% of the total immunoglobulin light chains, the remaining 30%, bearing the expected k2 bas isotype. Peptide mapping of the mixed light chains (lambda + k2 bas) followed by microsequencing of the constant region fragments indicated that the C lambda region originated from the cDNA sequence derived from a c7+,c21- Basilea rabbit (i.e. identical to gene C lambda 6), as described in the preceding report (Hayzer, D. J. et al., Eur. J. Immunol. 1990. 20: 2707). In addition, a synthetic peptide encompassing residues 139-159 of the constant region derived from the predicted sequence of gene C lambda 6 was shown to partially inhibit the c7-anti-c7 binding reaction in a sensitive enzyme-linked immunosorbent assay. Taken together, the chemical and immunochemical data clearly demonstrate that gene C lambda 6 indeed encodes c7 epitopes.

Amino Acid Sequence↗