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Biomedical subjects

A Horvath

Publications and source records attributed to A Horvath.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of the glycine-to-arginine substitution G2043R in type VII collagen in a family with dominant dystrophic epidermolysis bullosa from Hungary.

Epidermolysis bullosa (EB) represents a group of genodermatoses characterized by fragility and easy blistering of the skin. In the dystrophic forms of EB (DEB), blisters occur below the basement membrane, at the level of the anchoring fibrils. In the dominantly inherited forms (DDEB), the predominant type of mutation detected thus far is the substitution of a glycine residue which occurs within the collagenous domain of the molecule characterized by the repeating Gly-X-Y amino acid sequence. In this study, we searched for mutations in DDEB in a family from Hungary, by PCR amplification of segments of COL7A1, followed by heteroduplex analysis. Examination of the PCR fragment corresponding to exon 73 revealed a heteroduplex in affected individuals from the family. Sequence analysis revealed a G-to-A transition at nucleotide 6127 in the triple-helical domain of COL7A1, which converted a glycine residue at amino acid position 2043 to an arginine. This report represents the second incidence of this mutation, G2043R, described first in a family with DDEB from Italy.

Alleles↗
PubMed1995

The yeast spt14 gene is homologous to the human PIG-A gene and is required for GPI anchor synthesis.

The protein encoded by the yeast gene SPT14 shows high sequence similarity to the human protein, PIG-A, whose loss of activity is at the origin of the disease paroxysmal nocturnal hemoglobinuria. The symptoms of this disease are apparently due to a loss of cell surface, glycosylphosphatidylinositol (GPI)-anchored proteins. Like PIG-A mutant cells, spt14 mutant cells are defective in GPI anchoring due to a defect in the synthesis of GlcNAc-PI, the first step of GPI synthesis. The spt14 mutant causes several other abnormalities including transcriptional defects and a downregulation of inositolphosphoceramide synthesis. We suggest that these defects are indirect results of the loss of GPI anchoring.

Acetylglucosamine↗
PubMed1994

Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast.

Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide-containing, mild base-resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI-anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI-anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI-anchored proteins.

Acyltransferases↗
PubMed1994

Rapid protein extraction from Saccharomyces cerevisiae.

We have developed and evaluated an easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate (SDS)-gel electrophoresis and Western blotting. The procedure comprises a centrifugation step to harvest the cells, addition of a sample buffer and heating, then another centrifugation step before applying the extracted proteins found in the supernatant to an SDS gel. It is applicable to the study of large numbers of samples in 1 day. This procedure is easier, quicker, and as efficient as procedures using base and 2-mercaptoethanol, but somewhat less efficient than lysis with glass beads under certain conditions.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Immunocytochemical detection of cyclin A and cyclin D in formalin-fixed, paraffin-embedded tissues: novel, pertinent markers of cell proliferation.

Cyclin proteins in association with cyclin-dependent protein kinase subunits represent a new class of potentially oncogenic serine/threonine protein kinases that function to execute critical cell cycle transitions in all eukaryotic cells. Characterized by dramatic fluctuations in abundance, which occur in accordance with the periodicity of the cell cycle, the expression patterns of specific cyclins provide a unique and relevant indicator of cellular activation and cell cycle progression. In this study, we introduce a series of monospecific antibodies that are selective for human cyclin A and cyclin D, respectively, and we assess the feasibility of utilizing these reagents for immunocytochemical analyses. Conditions were optimized for detecting cyclin A and cyclin D in formalin-fixed, paraffin-embedded sections of the postnatal human palatine tonsil, in which normal cell proliferation is well characterized. Subsequent studies demonstrated the performance of these antibodies in the examination of pediatric bone tumors, in which decalcification methods are additionally performed. In both cases, the proliferative status of individual cells was monitored with an exceedingly high degree of resolution. Taken together with the available biochemical data, the results of these studies reveal a novel means of characterizing the proliferative status of normal as well as neoplastic tissues. The demonstrated utility of these immunochemical reagents will potentially facilitate retrospective studies aimed at examining cell proliferation in a wide variety of archival histopathologic specimens.

Antibodies↗
PubMed1994

Intracellular transport of GPI-anchored proteins by yeast.

We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphatidylinositol (GPI)-anchoring and intracellular transport of the yeast Gas1 protein. No effect on anchor attachment was demonstrable, but a selective delay in transport from the endoplasmic reticulum to the Golgi complex was observed. The compound also blocked remodeling of GPI-anchors from their base-sensitive to base-resistant forms. A recessive mutation was found that caused resistance to the drug, restored transport of Gas1p, but did not restore ceramide biosynthesis in the presence of the inhibitor. Our results suggest that intracellular transport of GPI-anchored proteins is stimulated by new ceramide synthesis. The role of ceramide may be direct or may be through its use in the remodeling of GPI-anchored proteins other than Gas1p. The need for ceramide can be overcome in the mutant strain.

Ceramides↗
PubMed1993

Analysis of the sequence requirements for glycosylphosphatidylinositol anchoring of Saccharomyces cerevisiae Gas1 protein.

The Saccharomyces cerevisiae Gas1 protein is synthesized as a precursor with a hydrophobic extension at the carboxyl terminus which is removed and replaced with an inositol containing glycolipid that anchors the protein to the plasma membrane. We performed saturation mutagenesis on the anchor attachment site (Asn506) and showed that only a subset of amino acids with small side chains could act as substrates for peptide cleavage and glycolipid addition. After Asn, which is the most efficient anchor attachment site, Ser, Gly, Ala, Asp, and Cys function with decreasing effectiveness. Mutational analysis also revealed that the 2 adjacent amino acids to the carboxyl side of the anchor attachment site are important for efficient anchoring. These two amino acids should have relatively short side chains with the second position being more critical. Analysis of the region between the anchor attachment site and the carboxyl-terminal hydrophobic region indicated that this region may not simply perform a spacer function.

Alleles↗
PubMed1991

Mesocestoides corti: a GC/MS study of the effects of oxaloacetate and iodoacetate on the isotope ratios in succinate and lactate excreted in the presence of NaH13CO3.

The current study was undertaken to gain further insight into the carbohydrate metabolism of the proliferating larvae of Mesocestoides corti by studying the effect of the metabolic inhibitors oxaloacetate and iodoacetate on the incorporation of label from NaH13CO3 into the excretory products lactate and succinate. These products were converted to their methyl derivatives and analyzed by gas chromatography/mass spectrometry (GC/MS) to determine the isotope ratios. The percentage of [1-13C]lactate, [1-13C]succinate, and [1,4-13C2]succinate decreased in the presence of oxaloacetate; iodoacetate did not significantly influence the relative amount of the former, but markedly increased percentages of the latter two. The data provide information on the relative rates of the pathways and steps within them and the influence of the inhibitors on these pathways.

Animals↗
PubMed1991

The reaction of reducing sugars with histones.

The epsilon amino groups of histone proteins were eliminated by condensation with glucose, fructose or mannose. The trichloroacetic acid extracted, DNA negative nuclei treated with reducing sugars, stained easily with basic dyes.

Carbohydrates↗
PubMed1990

Both ongoing suppression and clonal elimination contribute to graft-host tolerance after transplantation of HLA mismatched T cell-depleted marrow for severe combined immunodeficiency.

Lymphocytes from children with severe combined immunodeficiency who had been immunologically reconstituted with haploidentical T cell-depleted bone marrow were analyzed with regard to their immunologic recognition of donor, host, or third party alloantigens. When compared with freshly isolated donor lymphocytes, the engrafted donor cells exhibited markedly reduced to absent responses toward host Ag in primary or secondary MLC and cell-mediated lympholysis assays. However, under limiting dilution conditions, cytotoxic responses to host Ag could be demonstrated, indicating that small numbers of host reactive cells were present, although down-regulated at high responder cell doses. These results are consistent with prior observations in limiting dilution cultures that indicate that cells with the potential to lyse autologous target cells exist in the peripheral blood of all normal individuals. The number of host reactive cells present in these patients is significantly less than that present in cells isolated directly from the marrow donors, and is also less than the number of autocytotoxic cells normally seen in peripheral blood. Together, these observations indicate that two mechanisms contribute to donor host tolerance in these patients. The majority of host reactive cells appear to have undergone clonal deletion or inactivation, whereas the small residual host-reactive population appears to be under ongoing immunoregulatory control.

Bone Marrow Transplantation↗
PubMed1990

O-phosphohydroxylysinuria: a new inborn error of metabolism?

An abnormal ninhydrin positive compound was observed in the urine of two unrelated patients with neurological abnormalities. The compound was isolated by cation exchange followed by preparative paper chromatography and finally purified via cation exchange column chromatography. Its identification as O-phosphohydroxylysine resulted from FAB mass spectrometry and NMR spectroscopy. Chemical synthesis confirmed the structure. It was tentatively postulated that these patients had a defect of the metabolism of hydroxylysine, viz., a deficiency of the enzyme O-phosphohydroxylysine phospholyase.

Adolescent↗
PubMed1990

Evidence for presence of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in human brain cortex.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was previously isolated from bovine hypothalamus. We have now purified it from the parietal cortex of human brain tissue by gel filtration chromatography and four subsequent high performance liquid chromatographic steps. During isolation, the peptide content was followed by radioimmunoassay and compared with the elution of synthetic Tyr-MIF-1 in identical chromatographic systems. This extends evidence for the presence of Tyr-MIF-1 from bovine to human brain tissue and from hypothalamus to cortex.

Amino Acid Sequence↗
PubMed1990

Immunoreactive erythropoietin concentration and haemoglobin type in the perinatal period in sheep of various haemoglobin genotypes.

This study examines the hypotheses that (i) erythropoietin (the hormone responsible for red blood cell production) is higher in the fetus (at low PO2) than in the neonate (at high PO2); and (ii) that the level of erythropoietin in the neonate is influenced by the presence of high oxygen affinity haemoglobin. Haematocrit (PCV), PO2 and plasma immunoreactive erythropoietin were measured in 4 chronically cannulated fetal sheep (120 days to birth, n = 22) and in 7 neonatal lambs until 233 days post-conception (75-85 days after birth, n = 83). The percentage of globin chains (alpha, gamma, beta A, beta B, beta C) was quantitated by gel electrophoresis. Plasma erythropoietin values, in 4 fetuses were 9.8 +/- 1.3 mU/ml at 120-132 days of gestation, declined significantly (P less than 0.01) to 5.2 +/- 0.4 at 133 days until birth, then increased significantly (P less than 0.001) to 24.2 +/- 5.5(5), 26.3 +/- 7.3 (6), and 24.8 +/- 8.5 (6), respectively in weeks 1, 2 and 3 of postnatal life. By weeks 5-8 the erythropoietin was 13 +/- 0.6 (4) mU/ml. PO2 was 17.4 +/- 0.9 mmHg before birth and 88.0 +/- 10.7 in the first week after birth. PCV was constant until three weeks after birth and then declined. Fetal haemoglobin had virtually disappeared from the circulation by 166 days (3 weeks after birth); in the 4 heterozygotes (beta A beta B) beta C was expressed transiently, with a maximum value of 4%, whilst in the homozygote lamb (beta A beta A) the maximum beta C was 12%.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗