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Biomedical subjects

J Prchal

Publications and source records attributed to J Prchal.

At least 19 recordsLinked to original sources
PubMed1999

Full-length human L1 insertions retain the capacity for high frequency retrotransposition in cultured cells.

Functional L1 elements are autonomous retrotransposons that can insert into human genes and cause disease. To date, 10 of 12 known L1 retrotranspositions into human genes have been found to be 5"-truncated and incapable of further retrotransposition. Here we report the nucleotide sequences of the two full-length L1 elements, L1beta-thal and L1RP, that have inserted into the beta-globin and retinitis pigmentosa-2 (RP2) genes, respectively. L1beta-thal is 99. 4% identical to a consensus sequence of active human L1s, while L1RP is 99.9% identical. Both elements retain impressive capacity for high frequency retrotransposition in cultured HeLa cells. Indeed, L1RP is the most active L1 isolated to date. Our data indicate that not all L1 insertions into human genes are 'dead on arrival'. Our findings also lend further credence to the concept of cis preference, that the proteins encoded by a particular L1 preferentially act upon their encoding RNA as opposed to other L1 RNAs.

Consensus Sequence↗
PubMed1995

Triosephosphate isomerase deficiency: repetitive occurrence of point mutation in amino acid 104 in multiple apparently unrelated families.

The molecular basis of triosephosphate isomerase (TPI) deficiency was studied in 3 patients from three separate families. In all 3 patients, genomic DNA directly sequenced after amplification by the polymerase chain reaction exhibited the point mutation TPI315C amino acid 104 Glu-->Asp. Although other mutations known to cause TPI deficiency have been restricted to single families, the amino acid 104 defect has now been described in nine apparently unrelated families throughout the world and is clearly the most frequently occurring form of the disorder. The basis of the repetitive occurrence of this mutation remains unexplained.

Anemia, Hemolytic↗
PubMed1993

Usefulness of HPLC methodology for the characterization of combinations of the common beta chain variants Hbs S, C, and O-Arab, and the alpha chain variant Hb G-Philadelphia.

We have analyzed the blood samples from seven members of two families that contained mixtures of different hemoglobin types due to the simultaneous presence of different beta chain variants and the alpha chain variant Hb G-Philadelphia. The methods used were isoelectrofocusing and cation exchange high performance liquid chromatography for the separation of the hemoglobin components, and reversed phase high performance liquid chromatography for that of the various alpha and beta chains; the chromatographic procedures also provided quantitative data. Application of these fast and simple high performance liquid chromatographic methods greatly aided in defining the different hemoglobinopathies that were present in these persons, including an additional alpha-thalassemia-2 heterozygosity or homozygosity.

Adult↗
PubMed1992

The origin of glucose-6-phosphate-dehydrogenase (G6PD) polymorphisms in African-Americans.

DNA samples from 54 male Afro-Americans were examined for glucose-6-phosphate dehydrogenase (G6PD) genotypes G6PD A(+)376G, G6PD A(-)202A/376G, and G6PD B and for polymorphisms in intron 5 (PvuII), at nucleotide 1311, and at nucleotide 1116 (PstI). In the G6PD B subjects, the nucleotide 1311 mutation and the PstI site appeared to be in linkage equilibrium. No PvuII+ G6PD men were encountered. The G6PD A(+) mutation was in disequilibrium with respect to both the nucleotide 1311 mutation and the PstI site. The G6PD A- nucleotide 202 mutation was in disequilibrium with all three polymorphic sites. No conclusion could be drawn with respect to the PvuII site, except that it preceded the nucleotide 202 (A-) mutation. We conclude from these and our previous studies that G6PD B is the most ancient genotype. The nucleotide 1311 mutation, with its worldwide distribution, probably occurred next. The PstI mutation, limited to Africans, probably arose next and is more ancient than the A(+) mutation, which occurred in a gene without either the PstI or the 1311 mutation. G6PD A-202A/376G is the most recent of these mutations and is still in linkage disequilibrium with all of the sites. Presumably it occurred in an individual with both the A(+) and PvuII mutations.

Black People↗
PubMed1991

Four different mutations in codon 28 of alpha spectrin are associated with structurally and functionally abnormal spectrin alpha I/74 in hereditary elliptocytosis.

Hereditary elliptocytosis (HE) Sp alpha I/74 is a disorder associated with defective spectrin (Sp) heterodimer self-association and an abnormal tryptic cleavage of the 80-kD alpha I domain of Sp resulting in increased amounts of a 74-kD peptide. The molecular basis of this disorder is heterogeneous and mutations in codons 28, 46, 48, and 49 (codons 22, 40, 42, and 43 in the previous nomenclature which did not include the six NH2-terminal amino acids) have been reported. In this study we present data on seven unrelated HE Sp alpha I/74 kindred from diverse racial backgrounds in whom we identified four different mutations all occurring in exon 2 of alpha Sp at codon 28. Utilizing the polymerase chain reaction we established a CGT----CTT; Arg----Leu 28 mutation in one kindred of Arab/Druze origin. In two unrelated white kindred of English/European origin the substitution is CGT----AGT; Arg----Ser 28 and in two apparently unrelated white kindred from New Zealand, the mutation is CGT----TGT; Arg----Cys 28. Finally, in one American black kindred and in a black kindred from Ghana the mutation involves CGT----CAT; Arg----His 28. Allele specific oligonucleotide hybridization confirmed that the probands are heterozygous for the respective mutant alleles. All four point mutations abolished an Aha II restriction enzyme site which allowed verification of linkage of the mutation with HE Sp alpha I/74. Our results imply that codon 28 of alpha Sp is a "hot spot" for mutations and also indicate that Arg 28 is critical for the conformational stability and functional self association of Sp heterodimers.

Base Sequence↗
PubMed1986

Lack of association between glutathione content and development of thermal tolerance in human fibroblasts.

Thermal tolerance is a transient state of heat resistance occurring in cells and tissues after exposure to sublethal heat or certain chemicals. Although the mechanism of such resistance is unknown, it has been recently shown that preceding its development, cellular glutathione (GSH) levels rise. We have used a glutathione synthetase-deficient [GSH(-)] human fibroblast line to study the relationship between glutathione content and thermal tolerance. The GSH(-) cells had approximately 6% as much GSH as normal fibroblasts. Normal and GSH(-) fibroblasts showed similar survival after exposure to 45 degrees C. Exposure of normal fibroblasts to heat (45 degrees C for 15 min) led to a prompt rise in cellular GSH content as well as development of transient thermal tolerance. Similar treatment of GSH(-) fibroblasts produced no change in the very low GSH levels but was associated with a degree of thermal tolerance similar to that of normal cells. Thermal tolerance decayed more rapidly in GSH(-) cells than in normal fibroblasts. We conclude that the development of thermal tolerance in human fibroblasts is independent of GSH content.

Child, Preschool↗
PubMed1985

Decreased population of Leu-7+ natural killer cells in lymph nodes of homosexual men with AIDS-related persistent lymphadenopathy.

Natural killer (NK) cells were studied in the lymph nodes of homosexual men with the persistent lymphadenopathy syndrome (PLS) and other signs of the disease complex related to the acquired immune deficiency syndrome (AIDS). The NK cells were identified by their Leu-7+ phenotype and enumerated in frozen sections of lymph nodes in conjunction with the quantification of T-lymphocyte subsets. Lymph nodes from patients with AIDS-related PLS contained 91% and 81% fewer NK cells than normal lymph nodes and lymph nodes from patients with non-AIDS-related hyperplastic lymphadenopathy respectively. This decrease in NK cells in PLS is consistent with the immune dysregulation leading to persistent infection and neoplasia in AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Uncertainty in identification of blood group A subtypes by agglutination test.

Identification of the blood group A subtypes, i.e. A1, A2, and A1-A2 intermediate (Aint), by agglutination test, particularly in AB red cells, is ambiguous. The expressions of A subtypes in red blood cells are the consequences of diverse formations of the A substances by the action of three types of blood group N-acetyl-galactosaminyl-transferases controlled by A1, A2, and Aint genes. Therefore, the A subtypes are more directly identified by examining the kinetic characters of A-enzymes existing in plasma. Several Black AB subjects classified as non-A1 by the agglutination test were identified as A1B and AintB on the enzyme basis. A subject serologically classified as A1 had A2-enzyme in her plasma, i.e. she is genetically A2O or A2A2. The present and previous studies indicate that red cell A2 status is occasionally expressed as a result of the combination of Aint and B, and of A1 and superactive B. The imbalance between A1/A2 and A1B/A2B observed in some Black populations could be attributed to high frequencies of the Aint and B. sup. genes in Blacks.

ABO Blood-Group System↗
PubMed1984

A molecular defect of spectrin in a subset of patients with hereditary elliptocytosis. Alterations in the alpha-subunit domain involved in spectrin self-association.

Hereditary elliptocytosis (HE) is a clinically and biochemically heterogenous group of diseases characterized by elliptically shaped erythrocytes and an autosomal dominant mode of inheritance. Whereas the self-association of spectrin heterodimers to tetramers is defective in a subpopulation of HE patients, designated HE[SpD-SpD], it is normal in others. We have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from patients with HE[SpD-SpD] to determine if the functional defects in spectrin self-association could be correlated with structural changes in the spectrin molecule. Although the peptide pattern produced by limited tryptic digestion of spectrin extracts from those HE patients with normal spectrin self-association was indistinguishable from the pattern from control normal volunteers, digestion of the spectrin extracts from the HE[SpD-SpD] patients showed a reproducible diminution in the 80,000-D domain of the alpha-subunit, which is involved in spectrin dimer self-association. The decrease in the 80,000-D fragment was associated with an increase in a 74,000-D fragment in eight of nine families, or, in one family, with an increase of fragments at 46,000 and 17,000 D. These atypical peptide patterns were similar to those previously reported in two variants of hereditary pyropoikilocytosis (HPP), which also had defective self-association of spectrin. These data indicate that two distinct structural variants of spectrin alpha-subunit are associated with the defective spectrin heterodimer self-association in a subpopulation of HE patients.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Gas chromatographic/mass spectrometric evidence for the identification of a heptitol and an octitol in human and Octodon degu eye lenses.

Gas chromatographic analysis of alditols obtained from post-mortem human and South American rodent Octodon degu eye lenses indicated the presence of two high molecular weight, late eluting components which could not be identified. Thus, the samples were subjected to gas chromatographic/mass spectrometric analysis under both electron impact and chemical ionization conditions. The data obtained from these analyses indicate that both human and Octodon degu lenses contain a heptitol and an octitol, sugars which have not previously been reported to be present in any animal tissue.

Animals↗
PubMed1983

Molecular heterogeneity of hereditary pyropoikilocytosis: identification of a second variant of the spectrin alpha-subunit.

In hereditary pyropoikilocytosis (HPP), the red cell membrane skeletons exhibit a mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To determine the underlying molecular defect, we have subjected HPP spectrin to limited tryptic digestion, followed by one- and two-dimensional separations of the peptides. Two of the HPP kindreds exhibited a marked decrease in 80,000-dalton peptide (previously identified as the spectrin dimer-dimer contact domain of the alpha-subunit) and a concomitant increase of the 74,000-dalton polypeptide (presumably derived from the 80,000-dalton domain) and a decrease in a 22,000-dalton polypeptide. We now report tryptic digests of two other HPP kindred that are characterized by a decrease or complete absence of the 80,000-dalton tryptic fragment, with a concomitant increase in fragments at 46,000 and 17,000 daltons. The 46,000-dalton fragment separated into multiple spots on isoelectric focusing, ranging in isoelectric point from 5.25 to 5.35, and the 17,000-dalton fragment focused to a single spot at 5.4. Minor fragments at 56,000 and 22,000 daltons were also decreased, while a 38,000-dalton fragment increased. Limited tryptic digestion of the separated alpha- and beta-subunits revealed that the 74,000-dalton fragment in the first group of patients and the 46,000-dalton fragment in the second group of patients were derived from the alpha-subunit. Both subtypes exhibited a similar defect of spectrin self-association, with 30%-38% of spectrin dimers in O degrees C extracts. The results indicate that at least two distinct forms of structurally defective spectrin may give rise to the clinical presentation of HPP.

Adolescent↗
PubMed1982

Molecular defect of spectrin in hereditary pyropoikilocytosis. Alterations in the trypsin-resistant domain involved in spectrin self-association.

In hereditary pyropoikilocytosis (HPP) the erythrocyte membrane skeleton exhibits mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To detect structural changes in the functional domains of HPP spectrin we have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from two families that contain three HPP patients. Limited tryptic digestion of all three HPP patients revealed a similar and reproducible decrease in the staining intensity of an 80,000-, and 22,000-, and an 88,000-dalton polypeptide with a concomitant increase in a 74,000- and a 90,000-dalton polypeptide as compared with controls. Only changes in the 80,000-, and 74,000-, and 22,000-dalton polypeptides could be correlated to defective spectrin self-association and the amount of spectrin dimers in 0 degrees C extracts of the HPP patients and their affected kindred. Similar results were obtained when the tryptic digests were analyzed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the affected 74,000- and 80,000-dalton polypeptides focusing into multiple spots ranging in isoelectric point from 5.3-5.4. When HPP spectrin dimers and tetramers were separated and subjected to trypsin digestion, changes in the 80,000-, 74,000-, and 22,000-dalton polypeptides were found predominantly in the spectrin dimer pool. Similar results were obtained for spectrin from two of the probands' mother, whom we have identified as an HPP carrier. We conclude that these HPP patients contain a population of normal, (principally tetrameric) and mutant (principally dimeric) spectrin. The latter is characterized by a defective spectrin dimer self-association due to conformational changes that affect the 80,000-dalton domain.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Two siblings with unusually mild homozygous beta-thalassemia: a didactic example of the effect of a nonallelic modifier gene of the expressivity of a monogenic disorder.

Two adult Black sibs with homozygous beta-thalassemia had severe deficiency of beta-chain production but an unusually mild clinical course and almost normal hematocrit values. Their father had the typical hematological findings of beta-thalassemia trait but the mother, in addition to the beta-thalassemia trait, had elevated F cells (42.2%). Elevated F cells were also present in a hematologically normal sister of the affected providing evidence that a gene for heterocellular hereditary persistence of fetal hemoglobulin (HPFH) was also running in this family. The unusually mild clinical course of the two homozygotes is attributed to their inheritance from their mother of the heterocellular HPFH gene. The gene for heterocellular HPFH is nonallelic to the beta-globin locus and apparently acted as a modifier of the homozygous beta-thalassemia phenotype by facilitating F-cell production and thus diminishing the pathophysiological consequences of the beta-thalassemia defect. The effect of heterocellular HPFH on the expression of homozygous beta-thalassemia or Hb S genotypes is an impressive example of the pathophysiological basis of changes in the expressivity of monogenic disorders due to action of nonallelic modifiers.

Adult↗
PubMed1981

Altered spectrin dimer-dimer association and instability of erythrocyte membrane skeletons in hereditary pyropoikilocytosis.

Hereditary pyropoikilocytosis (HPP) is a hemolytic anemia characterized by microspherocytosis, poikilocytosis, and an unusual thermal sensitivity of erythrocytes. We have investigated the contribution of abnormal membrane skeletal assembly to these abnormal HPP erythrocyte properties. Skeletons prepared from fresh HPP ghosts with Triton X-100 were considerably more fragile than skeletons from control erythrocytes. Spectrin, the major skeleton component, extracted at 0 degrees C from normal erythrocytes, was present primarily as tetramers and high molecular weight complexes. In contrast, spectrin extracted from HPP erythrocytes under identical conditions contained a significant amount of dimers with a concomitant decrease of tetramers. Furthermore, spectrin dimers from HPP erythrocytes differed from normal spectrin dimers in their failure to reassociate into tetramers both in solution and in the membrane. Presumptive HPP carriers (asymptomatic mothers of the two patients) exhibited a mild but reproducible increase of spectrin dimers in 0 degrees C extracts and a defective reassociation of spectrin dimers of tetramers both in solution and in the membrane. We conclude that in HPP, self-association of spectrin dimers into tetramers is defective, which accounts for the instability of membrane skeletons.

Cytoplasm↗