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PubMed1982

Gene technology, characterization of insulin gene and the relationship to diabetes research.

The development and introduction of modern molecular biological methods was a prerequisite to the characterization of the insulin gene. The transfer of chemically synthesized genes for human insulin A and B chains as well as the naturally occurring rat insulin gene into bacteria opened possibilities to produce insulin in bacterial systems. Moreover, the structure of rat I and rat II insulin genes as well as human insulin gene were determined. Structural analysis of human insulin genes isolated from a human chromosomal gene library led to the identification of allelic variants of a single human insulin gene. The human insulin gene is located on the short arm of chromosome 11. The hypothesis that structurally abnormal insulin could play a role in the development of diabetes mellitus could be proved in clinical practice. Human insulin isolated from a diabetic patient showed an abnormality in the amino acid sequence and a decreased biological activity of the hormone. This finding demonstrates that in certain cases of diabetes mellitus the synthesis of structurally abnormal insulin due to a mutation in the insulin gene can be the cause for the development of relative insulin deficiency and hyperglycaemia.

Amino Acid Sequence↗

PubMed1981

Detection of inborn errors of metabolism in 1,117 patients studied because of suspected inherited disease.

Results of screening tests for the detection of inborn errors of metabolism in 1,117 consecutive patients are reported in this work; patients came for a second consultation to the Departamento de Genética, Centro Médico de Occidente, IMSS. Simple qualitative test were made that revealed the presence of abnormal metabolites (amino-acids, sugars, organic acids and mucopolysaccharides) in urine and blood as well as identification tests for variant proteins. Results were positive in 138 patients and test for confirmation and/or specificity were made in all patients; in 35 the following diagnosis and incidence were established: classic galactosemia, 2; glucose-6-phosphate dehydrogenase deficiency, 20; essential pentosuria, 1; hyperphenylalaninemia, 5; blue diaper syndrome, 1; cistinuria 1, and type 1 mucopolysaccharidosis, 5.

Amino Acid Metabolism, Inborn Errors↗

PubMed1995

Localized glenoid hypoplasia. An anatomic variation of possible clinical significance.

Examinations were done on 1150 scapular bone specimens from museum collections. A localized hypoplasia of the posteroinferior glenoid was found in significant numbers (20%-35%) depending on the population group studied. Criteria were established for the identification of this variant in computed tomography and magnetic resonance studies. Localized hypoplasia of the posteroinferior glenoid was detected as an incidental finding in 54 (18%) of 300 such studies. Using these same imaging criteria in a prospective manner, 9 of 12 patients seen for voluntary multidirectional instability of the shoulder displayed a localized posteroinferior glenoid hypoplasia. This phenomenon also may add to the picture of extreme posterior glenoid wear that sometimes is seen in association with osteoarthritis of the shoulder.

Adolescent↗

PubMed1993

Introduction of magnetic beads in diagnosis: a simple and rapid method to detect mutations of beta globin gene, directly amplified from blood.

In this communication we present the results obtained by the use of magnetic beads in diagnosis, for the identification of genetic variants at the molecular level by sequencing, in comparison with the more laborious method of the production of ssDNA with asymmetric PCR. We compared the two techniques studying variants of beta globin gene: Hb Abruzzo [beta 143 (H21) His -> Arg] and Hb D Los Angeles [beta 121 (GH4) Glu -> Gln].

Base Sequence↗

PubMed1993

Separation and tryptic digest mapping of normal and variant haemoglobins by capillary electrophoresis.

Characterization of haemoglobin (Hb) through whole protein separations and tryptic digest mapping allows the identification of structural Hb variants which result in haemoglobinopathies. Tryptic digest mapping by conventional two-dimensional paper chromatograph-electrophoresis provides high resolution but requires 48 h, while gradient elution reversed-phase high-performance liquid chromatography (HPLC) is faster (1.5 h), there is decreased resolution. CE analysis provides a fast separation with high resolution. We have used CE to optimise the tryptic digestion of globin purified from normal human haemoglobin A and to analyze tryptic digests from normal Hb. The separations were optimised and peak identification performed using UV scanning detection. In the optimised tryptic digest separation up to 28 peaks could be resolved in < 20 min. These peaks were identified as far as possible and a high-resolution map of the digest was constructed. The optimised analytical conditions were used to observe the separation pattern obtained from normal adult haemoglobin (HbA), common variant haemoglobins and some rarer haemoglobin variants.

Adult↗

PubMed2003

Identification of a novel gene and a common variant associated with uric acid nephrolithiasis in a Sardinian genetic isolate.

Uric acid nephrolithiasis (UAN) is a common disease with an established genetic component that presents a complex mode of inheritance. While studying an ancient founder population in Talana, a village in Sardinia, we recently identified a susceptibility locus of approximately 2.5 cM for UAN on 10q21-q22 in a relatively small sample that was carefully selected through genealogical information. To refine the critical region and to identify the susceptibility gene, we extended our analysis to severely affected subjects from the same village. We confirm the involvement of this region in UAN through identical-by-descent sharing and autozygosity mapping, and we refine the critical region to an interval of approximately 67 kb associated with UAN by linkage-disequilibrium mapping. After inspecting the genomic sequences available in public databases, we determined that a novel gene overlaps this interval. This gene is divided into 15 exons, spanning a region of approximately 300 kb and generating at least four different proteins (407, 333, 462, and 216 amino acids). Interestingly, the last isoform was completely included in the 67-kb associated interval. Computer-assisted analysis of this isoform revealed at least one membrane-spanning domain and several N- and O-glycosylation consensus sites at N-termini, suggesting that it could be an integral membrane protein. Mutational analysis shows that a coding nucleotide variant (Ala62Thr), causing a missense in exon 12, is in strong association with UAN (P=.0051). Moreover, Ala62Thr modifies predicted protein secondary structure, suggesting that it may have a role in UAN etiology. The present study underscores the value of our small, genealogically well-characterized, isolated population as a model for the identification of susceptibility genes underlying complex diseases. Indeed, using a relatively small sample of affected and unaffected subjects, we identified a candidate gene for multifactorial UAN.

Amino Acid Sequence↗

PubMed1993

Novel rat calpastatin mRNA variants.

Rat calpastatin cDNAs obtained by RT-PCR method were isolated and directly sequenced; this allowed the identification of two new variants both characterized by a 23 amino acids deletion at the end of the unique N-terminal domain L, increasing the number of functional isoforms. The deletion shows high homology to the amino acid sequence coded by exon 8 of human calpastatin gene and properly conserved splicing consensus sequences, suggesting exon skipping in domain L. Furthermore, three point mutations scattered along the coding region were found.

Amino Acid Sequence↗

PubMed2003

Adoptive transfer of an anti-MART-1(27-35)-specific CD8+ T cell clone leads to immunoselection of human melanoma antigen-loss variants in SCID mice.

The identification of appropriate mouse models could be useful in carefully evaluating the actual role of the in vivo development of antigen-loss variants during antigen-specific vaccine therapy of human tumors. In this study we investigated the level of efficacy of a MART-1/Melan-A-specific CD8+ T cell clone against its autologous melanoma in a severe combined immunodeficiency (SCID) mouse model, in which the tumor cells expressed in vivo heterogeneous and suboptimal levels of MART-1. The subcutaneous co-injection of the MART-1/Melan-A-reactive T cell clone A42 with MART-1/Melan-A+ autologous human melanoma cells into SCID mice caused a total inhibition of tumor growth. However, the systemic treatment with A42 clone lymphocytes resulted in only 50-60% inhibition of tumor growth, although the T cell clone targeted the tumors and the MART-1+ cells virtually disappeared from the tumors. This study suggests that an immunotherapy based on the expansion of an antigen-specific T cell clone generated in vitro is highly efficient in abolishing tumor growth when the target antigen is fully expressed, but leads to in vivo immunoselection of antigen-loss variants in the presence of suboptimal levels of antigen expression. Furthermore, this work shows that human tumors/SCID mouse models may be useful in evaluating the in vivo efficacy of adoptive immunotherapies.

Animals↗

PubMed1984

Hb F-La Grange or alpha 2 gamma 2 101(G3)Glu----Lys; 75Ile; 136Gly: a high oxygen affinity fetal hemoglobin variant observed in a Caucasian newborn.

The identification of a newly discovered gamma-chain variant is reported. This abnormal fetal hemoglobin is characterized by a Glu----Lys substitution at position 101(G3) of its gamma chain and was observed in a Caucasian baby girl. Because glutamic acid residue in position gamma 101 is involved in the alpha 1-gamma 2 chain contact, its replacement by a lysine residue results in changes in physicochemical and functional properties. The variant readily forms hybrid hemoglobins at room temperature, is mildly unstable at higher temperature, and has an increased oxygen affinity with a somewhat lower heme-heme interaction.

Adolescent↗

PubMed1986

Biochemical characterization for identification of ovine sarcosporidia.

Electrophoretic variants of enzymes from 100 individual macroscopic sarcocysts from sheep carcasses were examined to see if they could serve as genetic markers for the identification of ovine sarcosporidia. Of the 31 enzymes screened, 12 that represented single genetic loci were tested. There were two patterns of isoenzyme mobility, which differed at 7 out of 12 of the loci being studied, and corresponded to enzymes from either 'fat' or 'thin' cysts, indicating that these two sarcocyst types may be considered quite different species. In a parallel study, extracts of microscopic sarcocysts digested from sheep heart muscle were compared with those from macroscopic sarcocysts and found to have a distinct electrophoretic isoenzyme profile for each of four loci.

Animals↗

PubMed1977

Application of isoelectric focusing in alpha1-antitrypsin phenotyping.

alpha1-Antitrypsin, the major protease inhibitor in human serum, occurs in a considerable number of variant forms, some of which are associated with lung and liver diseases. The identification of these genetic variants, generally called Pi-types, by means of isoelectric focusing is described, as well as investigations concerning the practical application of isoelectric focusing as a routine procedure for typing the variants of alpha1-antitrypsin. Finally, isoelectric focusing is compared with the most widely used Pi-phenotyping technique, namely acid starch-gel electrophoresis followed by immunoelectrophoresis in antibody-containing agarose gel.

Genetic Variation↗

PubMed2001

Could capillary zone electrophoresis of tryptic peptides be used for the characterization of hemoglobin variants?

The present article describes a simple and rapid new peptide mapping procedure that could be used to assist identification of rare hemoglobin variants in clinical laboratories. Four hemoglobin variants were taken as example, namely Hb D-Ouled Rabah, Hb Marseille, Hb G-Philadelphia, and Hb Ube-2, and isolated by electrophoresis at alkaline pH. The globin chains were aminoethylated and, after tryptic digestion, the peptides were separated by a capillary zone electrophoresis method. Highly reproducible migration times of the peptides were obtained with intra-assay and inter-assay coefficients of variation of less than 1 and 2%, respectively.

Amino Acid Sequence↗

PubMed1975

A new beta-chain variant in human milk.

Investigation on the genetic polymorphism of human casein at the population level resulted in the identification of a new variant within the beta-casein system. This discovery throws new light on the type of genetic control applicable to beta-casein.

Caseins↗

PubMed2004

Identification and functional analysis of two rare allelic variants of the thiopurine S-methyltransferase gene, TPMT16 and TPMT19.

Human thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. During routine genotyping of patients with Crohn's disease, one novel missense mutation, 365A>C (TPMT*19, Lys(122)Thr), and a recently described missense mutation, 488G>A (TPMT*16, Arg(163)His), were identified in a Caucasian and a Moroccan patient, respectively. Using a heterologous yeast expression system, kinetic parameters (K(m) and V(max)) of the two variants with respect to 6-thioguanine S-methylation were determined and compared with those obtained with the wild-type enzyme. The Lys(122)Thr exchange did not significantly decrease the intrinsic clearance value (V(max)/K(m)) of the variant enzyme. In contrast, the Arg(163)His substitution significantly decreased the intrinsic clearance value by three-fold. The Arg(163) is located in a highly conserved region of the human TPMT protein and, as such, the Arg(163)His substitution is expected to result in a marked reduction of enzyme activity, as confirmed by the in vitro data. Phenotyping by measurement of red blood cell TPMT activity indicated that the patient heterozygous for the Lys(122)Thr mutation had normal TPMT activity, whereas the patient heterozygous for the Arg(163)His mutation was an intermediate methylator, which demonstrated a positive correlation between TPMT phenotyping and the in vitro data. The identification of a novel non-functional allele of the TPMT gene improves our knowledge of the genetic basis of interindividual variability in TPMT activity. These data further enhance the efficiency of genotyping methods to predict patients at risk of an inadequate response to thiopurine therapy.

Alleles↗

PubMed1984

Multiple isoelectric and molecular weight variants of choline acetyltransferase. Artifact or real?

The existence of isoelectric variants of bovine brain and human placental choline acetyltransferase was confirmed by chromatofocusing. The identification of molecular weight variants (bovine brain, Mr = 68,000 and 63,000; human placental, Mr = 66,000 and 64,000) was also demonstrated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by "Western blotting" and immunochemical visualization of choline acetyltransferase with monoclonal antibodies. No correlation between the isoelectric variants and molecular weight variants could be observed; however, in the case of the bovine brain enzyme the more alkaline isoelectric variant was enriched in the higher molecular weight form of the enzyme. Treatment of the bovine brain enzyme with Staphylococcus aureus V8 protease resulted in the conversion of the Mr = 68,000 form of the enzyme to the Mr = 63,000 form. During this conversion no change in the enzyme activity was observed demonstrating that the Mr = 63,000 form of the enzyme and probably also the Mr = 68,000 form of the enzyme are active. Preparation of the bovine brain enzyme in the presence of proteolytic enzyme inhibitors yielded a new higher molecular weight form of the enzyme, Mr = 73,000, which was enzymatically active. The Mr = 73,000 form of the enzyme exhibited a single isoelectric form when analyzed by chromatofocusing. These results suggest that the Mr = 73,000 form of the enzyme represents the native form, and that other molecular weight and isoelectric variants may arise by proteolysis.

Animals↗

PubMed1995

Three-dimensional solution structure of the cyanide adduct of a Met80Ala variant of Saccharomyces cerevisiae iso-1-cytochrome c. Identification of ligand-residue interactions in the distal heme cavity.

The 1H NMR spectrum of the the cyanide adduct of a triply mutated Saccharomyces cerevisiae iso-1-cytochrome c (His39Gln/Met80Ala/Cys102Ser) in the oxidized form has been assigned through 1D NOE and 2D COSY, TOCSY, NOESY, and NOE-NOESY experiments; 562 protons out of a total of 683 have been assigned. The solution structure, the first of a paramagnetic heme protein, was determined using 1426 meaningful NOE constraints out of a total of 1842 measured NOEs. The RMSD values at the stage of restrained energy minimization of 17 structures obtained from distance geometry calculations are 0.68 +/- 0.11 and 1.32 +/- 0.14 A for the backbone and all heavy atoms, respectively. The quality, in terms of RMSD, of the present structure is the same as that obtained for the solution structure of the diamagnetic horse heart ferrocytochrome c [Qi, P. X., et al. (1994) Biochemistry 33, 6408-6419]. The secondary structure elements and the overall folding in the variant are observed to be the same as those of the wild-type protein for which the X-ray structure is available. However, the replacement of the methionine axial ligand with an alanine residue creates a ligand-binding "distal cavity". The properties of the distal cavity seen in this solution structure are compared to those of other heme proteins.

Alanine↗

PubMed1989

Identification and biologic characterization of an acutely lethal variant of simian immunodeficiency virus from sooty mangabeys (SIV/SMM).

A virus pool isolated from lymphoid tissue of a macaque (PBj) infected for 14 months with SIV/SMM was found to be associated with acute disease and death. Six of six pig-tailed macaques, one of three rhesus macaques, and three of four SIV/SMM-seronegative mangabeys developed acute disease within 5 days and died from 7 to 13 days postinoculation; however, neither of two SIV/SMM-infected mangabeys died or developed disease. The virus associated with acute disease and death was shown by electron microscopy to be a lentivirus and was serologically indistinguishable from SIV/SMM by immunofluorescence and radioimmunoprecipitation assays. A biologic clone generated from lymphoid tissue of an animal that died 7 days after inoculation of the lethal pool resulted in death within 8 days of three of three pig-tailed macaques. Comparison of the lethal virus, designated SIV/SMM(PBj14), with the parent virus, SIV/SMM-9 (the isolate with which macaque PBj was originally inoculated), showed that although the kinetics of replication in peripheral blood mononuclear cells (PBMC) from pig-tailed macaques and mangabey monkeys were similar, SIV/SMM(PBj14) replicated more efficiently than SIV/SMM-9 in human PBMC and also replicated in chimpanzee PBMC whereas SIV/SMM and other SIV isolates did not. In addition, the variant was shown to replicate efficiently in some established cell lines whereas replication of SIV/SMM-9 in cell lines could be demonstrated only occasionally. That parental SIV/SMM-9, but not SIV/SMM(PBj14), was neutralized by serum from macaque PBj suggests that the variant may have been generated by immune selection. Comparison of sequential virus isolates from macaque PBj for host range and the ability to be neutralized and of sequential serum samples for neutralization activity indicated that changes in biologic properties were detected in virus isolates and serum obtained at 6 months after infection and later. Normal macaque PBMC infected in vitro with SIV/SMM(PBj14), but not with SIV/SMM-9 or other virus pools from PBj, formed syncytia with Sup-T1 cells, whereas all isolates formed syncytia with MOLT-4 clone 8 cells. These data suggest that, relative to SIV/SMM-9, SIV/SMM(PBj14) acquired multiple mutations, at least one (or more) of which is in the gene coding for the envelope glycoprotein. Continued analysis of this series of SIV/SMM isolates with diverse properties may lead to the identification of specific regions of the viral genome that influence defined biologic properties. Furthermore, the availability of a strain of SIV that induces rapid onset of disease and death may facilitate screening of drugs for antiviral activity against lentiviruses.

Animals↗

PubMed2001

Phosphorylation of the oncofetal variant of the human bile salt-dependent lipase. identification of phosphorylation site and relation with secretion process.

In this paper, we report, for the first time, the localization of the phosphorylation site of the fetoacinar pancreatic protein (FAPP), which is an oncofetal variant of the pancreatic bile salt-dependent lipase. Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radiolabeled the enzyme with (32)P, and then the protein was purified by affinity chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis. Analysis of peptides by high pressure liquid chromatography, associated with the radioactivity profile, revealed that the phosphorylation site is located at threonine 340. Site-specific mutagenesis experiments, in which the threonine was replaced by an alanine residue, were used to invalidate the phosphorylation of FAPP and to study the influence of the modification on the activity and secretion of the enzyme. These studies showed that CHO cells, transfected with the mutated cDNA of FAPP, kept all of their ability to synthesize the protein, but the loss of the phosphorylation motif prevented the release of the protein in the extracellular compartment. However, the mutated enzyme, which was sequestrated in the transfected CHO cells, remains active on bile salt-dependent lipase substrates.

Animals↗