PubMed Health⌕ Search

Biomedical subjects

M Stoppini

Publications and source records attributed to M Stoppini.

35 records · Page 2Linked to original sources
PubMed1993

Application of monoclonal anti-idiotypes in the study of AL amyloidosis: therapeutic implications.

A monoclonal anti-idiotyped antibody (IgG1k MAb 3B11D4) has been raised against the lambda-chain dimers isolated from the urine of a patient (DEP) with AL amyloidosis. This antibody binds a conformational idiotope present on the monoclonal DEP IgA, but does not recognize the reduced and alkylated lambda-chain monomers, nor the 15- to 17-kDa light chain fragments obtained from the amyloid fibrils, which have the same N-terminal sequence as the urinary light chains. The nonreactivity of this MAb with amyloid fibrils was confirmed by immunohistochemical examination of cryostatic sections of an amyloidoma surgically removed from the patient's subcutaneous tissue. Our data demonstrate that the deletion of about 70 amino acid residues of the C-terminus of the lambda chain prevents the formation of the self-limiting dimer and may facilitate the deposition of fragments into amyloid fibrils. With regard to the amyloidogenic clone, MAb 3B11D4 recognizes the plasma cell clone in bone marrow and 9% of circulating B lymphocytes. Panning and cytotoxicity experiments demonstrate that this antibody has the capability of selectively eliminating the idiotype-positive cells from peripheral blood. Antibodies with these properties could find application in a new therapeutic strategy which provides high-dose chemotherapy, total body irradiation, and rescue with circulating stem cells. These antibodies could be used in two distinct phases: first, in the purging of the stem cells to be infused from the amyloidogenic clone and, secondly, in an attempt to eliminate residual disease by intravenous infusion.

Amyloid↗
PubMed1992

The primary structure of piscine (Oncorhynchus mykiss) retinol-binding protein and a comparison with the three-dimensional structure of mammalian retinol-binding protein.

1. The primary structures of two variants of rainbow trout (Oncorhynchus mykiss) plasma retinol-binding protein (RBP) were determined and found to be approximately 60% identical with those of both human and Xenopus laevis RBPs. The comparable sequence similarities that we have found agree with the estimate of similar divergence times between bony fishes and mammals and between bony fishes and amphibians. The two piscine RBP variants differ by six amino acid substitutions at positions that are not crucial for the interaction with retinol, on the basis of the human RBP three-dimensional structure [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1990) Proteins Struct. Func. Genet. 8, 44-61]. 2. Models were developed for the three-dimensional structures of rainbow trout and X. laevis RBPs, based on that of human RBP. The overall three-dimensional structure appears to be very well preserved for RBPs isolated from vertebrate species for which the divergence time is 350-400 million years. At variance with an almost absolute conservation for the residues that participate in the formation of the retinol binding site in mammalian RBPs, several amino acid replacements are found for this part of the RBP molecule when the comparison is extended to piscine and amphibian RBPs. However, the only allowed amino acid replacements are either conservative or more than 0.4 nm distant from retinol. Besides the retinol binding site, a few regions at the protein surface appear to be rather conserved during phylogenetic development of vertebrates and, therefore, might be involved in molecular interactions.

Amino Acid Sequence↗
PubMed1992

Two alloalbumins with identical electrophoretic mobility are produced by differently charged amino acid substitutions.

We describe the amino acid substitutions of albumins Sondrio and Paris 2, two slow moving variants of human serum albumin, which show an identical electrophoretic mobility on cellulose acetate at three different pH values. These variants have been found in several instances in a wide geographic area including Northern Italy and France. Both alloalbumins were isolated from the sera of heterozygous subjects. Isoelectric focusing analysis of CNBr fragments from the purified variants allowed us to localize the mutation of albumin Sondrio in fragment CNBr V (residues 330-446) and that of albumin Paris 2 in CNBr VII (residues 549-585). Sequential analysis of the variant CNBr VII established the molecular defect of albumin Paris 2 as 563 Asp----Asn. Fragments CNBr V from normal and Sondrio albumins were isolated on a preparative scale and subjected to tryptic and V8 proteinase digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution of glutamic acid 333 by lysine. Thus, a +1 change in the C-terminal region of the albumin molecule produces a variant with the same electrophoretic mobility as an alloalbumin with a +2 substitution in the central domain, suggesting a higher degree of exposure to the solvent of the C-terminal tailpiece. Both amino acid substitutions are consistent with a G----A transition in the first position of the corresponding codon in the structural gene.

Amino Acid Sequence↗
PubMed1992

The piscine plasma retinol-binding protein. Purification, partial amino acid sequence and interaction with mammalian transthyretin of rainbow trout (Oncorhynchus mykiss) retinol-binding protein.

1. Retinol-binding protein (RBP) has been isolated from the pooled plasma or rainbow trouts (Oncorhinchus mykiss) by gel filtration, hydrophobic interaction chromatography and ion-exchange chromatography. By this procedure two forms of the protein, both with a molecular mass (approximately 20 kDa) similar to that of mammalian RBP, were purified to homogeneity. Five amino acid substitutions have been found in the partial (about 60%) sequences of the two forms of trout RBP, which are presumably acetylated at their N terminus. The apparent participation of six conserved cysteines in the formation of disulphide bridges, as in human RBP, and the similarity (about 60%) of the amino acid sequence of trout and mammalian RBPs, indicate the existence of a similar overall structure organization in evolutionary distant RBPs. 2. Although the two forms of trout RBP are not physiologically involved in the formation of any protein--protein complex in plasma, they are capable of interacting with mammalian transthyretin, albeit with a binding affinity (K'd = 15-40 microM) considerably lower than that of mammalian RBP. Our data indicate that the two forms of trout RBP also possess the region that in mammalian RBP has the functional role of binding transthyretin. It is suggested that transthyretin (or a homologous protein) was modified, during phylogenetic development of the non mammalian vertebrates, to acquire a binding site for such a region of the RBP molecule.

Amino Acid Sequence↗
PubMed1992

Use of an anti-idiotypic monoclonal antibody in studying amyloidogenic light chains in cells, urine and fibrils: pathophysiology and clinical implications.

A monoclonal anti-idiotype antibody (IgG1k MoAb 3B11D4) raised against the amyloidogenic DEP lambda chain dimer binds a conformational idiotope also present on the monoclonal DEP IgA immunoglobulin. MoAb 3B11D4 does not recognize the reduced and alkylated lambda chain monomers, nor the 15-17-kDa fibrillar light chain fragments which have the same N-terminal sequence of the urinary light chains. The lack of about 70 amino acid residues of the C terminal of the protein prevents the formation of the self-limiting dimer and may facilitate the deposition of the fragments into amyloid fibrils. MoAb 3B11D4 recognizes the plasma cell clone in bone marrow and 9% of circulating B lymphocytes. Panning experiments demonstrate that this antibody has the capability to selectively eliminate the idiotype positive cells from peripheral blood. Antibodies with these characteristics could become a useful tool for better understanding the pathogenesis of the disease and for new therapeutic options.

Amino Acid Sequence↗
PubMed1991

Amino acid sequence of k Sci, the Bence Jones protein isolated from a patient with light chain deposition disease.

Light chain Sci was isolated from the urine of a patient affected by light chain deposition disease with an apparent exclusive localization to the kidney. Sci protein is an intact light chain: it consists of 214 amino acid residues and has an Mr of 23.65. Its complete primary structure has been determined by sequence analysis of the corresponding tryptic peptides and by partially sequencing the intact protein. Sequence comparison shows that Sci protein is strictly related to the light chains of kIIIa family (88% structural identity) which are usually expressed in autoimmune rheumatoid syndromes. Computer graphics model suggests a perturbation in k Sci three-dimensional structure due to the unusual replacement of residues 53 and 77.

Aged↗
PubMed1991

Bacterial pyruvate kinases have a shorter N-terminal domain.

The N-terminal portions of the two forms of pyruvate kinase (EC2.7.1.40) from Escherichia coli have been sequenced up the 48th and 43rd residue, respectively. Comparison with the known primary structures shows that bacterial enzymes lack a substantial portion of the N-terminal sequence with respect to pyruvate kinases from vertebrates. This makes the suggested functional role of the N-terminal domain unlikely [Muirhead, H. (1990) Biochem. Soc. Trans. 18, 193-196] although an elongation of this domain with evolution is apparent.

Amino Acid Sequence↗
PubMed1990

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP): amino acid sequence of the subunits from isoenzyme I and structural relationship with isoenzyme II.

The structural relationship between isoenzymes I and II of chloroplast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating) EC 1.2.1.13) has been established at the protein level. The complete primary structure of subunits A and B of glyceraldehyde-3-phosphate dehydrogenase I from Spinacia oleracea has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide and Staphylococcus proteinase V8 digestions and by partially sequencing each intact subunit. Subunit A has an Mr of 36,225 and consists of 337 amino acid residues, whilst subunit B (Mr 39,355) consists of 368 residues. The amino acid sequence of subunit B, as determined through direct analysis of the protein, is identical to that recently deduced at cDNA level (Brinkmann et al. (1989) Plant Mol. Biol. 13, 81-94). The two subunits share a common portion of amino acid sequence which differs by 66 amino acid residues. Subunit B has an extra C-terminal sequence of 31 amino acid residues. Chloroplast glyceraldehyde-3-phosphate dehydrogenase II was partially characterized by sequencing the N-terminal portion of the intact protein and some of its tryptic peptides. The sequences of all the examined fragments fit precisely that of the corresponding regions of subunit A from glyceraldehyde-3-phosphate dehydrogenase I.

Amino Acid Sequence↗
PubMed1990

The bovine plasma retinol-binding protein. Amino acid sequence, interaction with transthyretin, crystallization and preliminary X-ray data.

1. The primary structure of bovine plasma retinol-binding protein (RBP) has been determined and found to be more than 90% identical to human and rabbit RBPs, and more than 80% identical to rat RBP. Main changes in amino acid sequence are observed in two regions on the surface of the protein molecule (residues 138-148 and 169-183). 2. The interactions of bovine RBP with bovine and human transthyretins were investigated using the technique of fluorescence polarization. Bovine RBP was able to form high affinity complexes (K'd = 0.34 +/- 0.02 microM) with both bovine and human transthyretins, displaying a stoichiometry of approximately 2 molecules RBP/molecule transthyretin in both cases. The sites that participate in protein-protein interactions are thus very similar, and this tends to exclude the involvement of the superficial regions more significantly substituted in mammalian RBPs (residues 138-151 and 167-183) in the protein-protein recognition. 3. Bovine RBP has been crystallized (space group P2(1)2(1)2(1), with a = 4.61 nm, b = 4.91 nm, c = 7.61 nm) and the crystals are suitable for high-resolution X-ray diffraction studies.

Amino Acid Sequence↗
PubMed1989

A new proalbumin variant: albumin Jaffna (-1 Arg----Leu).

Albumin Jaffna is an electrophoretically slowly moving genetic variant of human serum albumin found in two members of a Tamil family from Jaffna (Northern Sri Lanka), both heterozygous for the abnormal protein. Sequential analysis of albumin Jaffna, purified from serum by ion exchange chromatography on DEAE Sephadex and Mono Q columns, revealed that this variant is a new abnormal proalbumin, arising from a -1 Arg----Leu substitution, which prevents the proteolytic removal of the N-terminal hexapeptide and allows the mutated proalbumin to enter the circulation. The presence of two additional positive charges is in keeping with the decreased electrophoretic mobility of albumin Jaffna, as well as with its isoelectric point of 5.01, determined by chromatofocusing on a Mono P column. The variant is selectively cleaved by trypsin in vitro, leaving leucin -1 as N-terminal residue.

Arginine↗
PubMed1989

Structural characterization of kappa II Inc, a new amyloid immunoglobulin.

Light chain Inc, obtained from a patient with amyloid arthropathy, has an Mr of 23,550 and consists of 219 amino acid residues. The complete primary structure of its variable domain has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide digestion, and by partially sequencing the intact protein. Although closely related to protein of the V kappa II subgroup, light chain Inc differs from its counterpart by the replacement of some invariant residues in its variable domain. By comparing its sequence with that of the nonamyloid kappa II Nim, a different distribution of some polar and apolar amino acid residues through the molecule is evidenced. A computer graphic analysis shows that some of the replaced amino acid residues cannot be readily accommodated in the known three-dimensional structure of the immunoglobulin light chains.

Amino Acid Sequence↗
PubMed1989

Study of three patients with monoclonal gammopathies and 'lupus-like' anticoagulants.

In three patients with monoclonal gammopathies: a case of multiple myeloma, a case of monoclonal gammopathy of uncertain significance (MGUS) and a case of monoclonal gammopathy associated with lymphocytic lymphoma, we found the presence of a circulating lupus-like anticoagulant. Coagulative studies showed that the paraproteins: an IgG3k, an IgG1k and an IgMlambda, were responsible for the anticoagulant activity by interacting with the thromboplastin phospholipids. Using isoelectrofocusing we demonstrated that the three monoclonal immunoglobulins had a strong basic charge which may have contributed to determining their interaction with the acidic thromboplastin phospholipids. The binding of various phospholipids to the monoclonal proteins was assessed by the fluorescence quenching method which showed heterogeneous specificity. In order to establish whether the electrical charge is also relevant in cases with polyclonal lupus anticoagulant, the polyclonal immunoglobulins were fractionated according to their charge. The strongest inhibitor activity was found in the most basic immunoglobulins. Monoclonal lupus-like anticoagulants represent useful tools for investigating the heterogeneous world of polyclonal lupus-like anticoagulants.

Aged↗
PubMed1989

Primary structure of three peptides at the catalytic and allosteric sites of the fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli.

Three peptides containing 6-pyridoxyllysine have been isolated from the tryptic digest of the allosteric fructose-1,6-bisphosphate-dependent pyruvate kinase from Escherichia coli, which had been almost completely inactivated with pyridoxal 5'-phosphate. The labelled peptides have been sequenced. The comparison of their sequences with the primary structure of the cat muscle pyruvate kinase allowed to state that peptide I fits the region spanning residues 423-438 (53% identity), peptide II corresponds to residues 442-457 (44% identity) and peptide III encompasses residues 342-368 (70% identity). These findings are discussed in connection with our previous results on the involvement of the three peptides in the catalytic and regulatory properties of the enzyme (Valentini, G., Speranza, M.L., Iadarola, P., Ferri, G. & Malcovati, M. (1988) Biol. Chem. Hoppe-Seyler 369, 1219-1226) and in connection with their location in the three-dimensional structure of the cat muscle pyruvate kinase (Muirhead, H., Clayden, D.A., Lorimer, C.G., Fothergill-Gilmore, L.A., Schiltz, E. & Schmitt, W. (1986) EMBO J. 5, 475-481).

Amino Acid Sequence↗
PubMed1988

High-performance liquid chromatography of complex mixtures of cyanogen bromide-produced peptides from different proteins.

A variety of approaches have been required in order to achieve the resolution of large fragments from cyanogen bromide (CNBr) digests of Inc k chain (an immunoglobulin light chain), human serum albumin (HSA) and four of its mutants. Reversed-phase high-performance liquid chromatography (RP-HPLC) under different conditions failed to resolve the Inc k chain digest; the three CNBr fragments (3.1, 6.7 and 13.7 kDa) were separated in a homogeneous form by gel HPLC. Five of the seven CNBr fragments (ranging from 3.4 to 20.0 kDa) obtained from CNBr cleavage of HSA can be resolved by a single reversed-phase HPLC step; separation of the other two requires modification of the eluent composition. Some structural features of the peptides seem to influence their chromatographic behaviour; by examining the elution patterns from albumin mutants, the sequence-related contribution of single amino acid residues is apparent.

Albumins↗
PubMed1987

Structural characterization of two genetic variants of human serum albumin.

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.

Amino Acids↗
PubMed1986

The molecular defect of albumin Tagliacozzo: 313 Lys----Asn.

Albumin Tagliacozzo is a fast-moving genetic variant of human serum albumin found in 19 unrelated families. The protein was isolated from the serum of a heterozygous healthy subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to CNBr fragment IV (residues 299-329). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by RP-HPLC, revealed the variant was caused by 313 Lys----Asn substitution, probably due to a point mutation in the structural gene. The lack of a lysine residue accounts for the electrophoretic behavior of albumin Tagliacozzo.

Amino Acid Sequence↗