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Biomedical subjects

E Harms

Publications and source records attributed to E Harms.

At least 73 records · Page 4Linked to original sources
PubMed1996

Yeast artificial chromosome mapping of the cystinosis locus on chromosome 17p by fluorescence in situ hybridization.

The gene locus for cystinosis has been mapped between markers D17S1583 and D17S1584 on the short arm of chromosome 17. Using markers encompassing the cystinosis region, we assigned different yeast artificial chromosome (YAC) clones previously identified by sequence tagged site (STS) screening to 17p13.3. Three of the clones hybridized to the target 17p gene region; one of these was chimeric, hybridizing both to chromosomes 3p and 5q; two of the YACs did not contain sequences of 17p13.3. Our physical mapping has identified candidate YACs as a first step towards a positional cloning approach.

Amino Acid Sequence↗
PubMed1996

Effect of a reduced vitamin K supplementation on prothrombin time in prematures and high-risk neonates.

Recommendations for vitamin K supplementation were recently changed to 3 x 1 mg orally in healthy term neonates and 0.2 mg parenterally in prematures and high-risk neonates. Prothrombin times (PT) at reduced vitamin K doses (120 patients, 170 samples) during the first 6 weeks of life were below 40% in 6 patients only; all low PTs were unrelated to vitamin K. In the remaining patients, median PT was 100%; 79% of patients had values above 70%. PT was related to birthweight, gestational age and age. There was no decrease over the observation period. Following reduced oral and parenteral vitamin K regimens PTs were well within published reference values for neonates given larger amounts of vitamin K.

Administration, Oral↗
PubMed1996

The addition of aminoacids and phosphate to hemodiafiltration solutions in newborns with hyperammonemic coma.

A child with carbamyl-phosphate-synthetase defect who died after prolonged continuous hemodiafiltration in deep coma proved to have high aminoacid losses despite aminoacid infusion. We think that this results from high small-solute clearance during hemodialysis. In order to prevent these inevitable catabolic side-effects we decided to add aminoacids to the dialysate and substitution fluid in these children with metabolic diseases. Additionally we propose to add phosphate in order to avoid depletion. The aim is to achieve anabolic or at least non-catabolic hemodiafiltration.

Amino Acids↗
PubMed1995

Survival after early treatment for carbamyl phosphate synthetase (CPS) I deficiency associated with increase of intramitochondrial CPS I.

The basis for the benefit of early treatment in urea-cycle defects might be an increase in intramitochondrial mutant enzyme in hepatocytes in the postnatal period. In two siblings with carbamyl phosphate synthetase I (CPS I) deficiency, immunoreactive CPS I was greatly reduced in the liver and no residual enzyme activity was detectable. The elder child died at age 4 days, before the diagnosis of CPS I deficiency was established, but in the younger child, age 9 months, treatment was initiated on the 2nd day of life when ammonia concentration was moderately increased, and she has survived. Intramitochondrial CPS I was substantially higher in this sibling than in the elder sister. The different outcome in the younger patient was probably attributable to prompt treatment after early diagnosis.

Amino Acid Metabolism, Inborn Errors↗
PubMed1995

Fluorescence resolution of the intrinsic tryptophan residues of bovine protein tyrosyl phosphatase.

Fluorescence steady-state and lifetime measurements have been performed that permit the differentiation of the 2 intrinsic tryptophan residues in bovine low molecular weight phosphotyrosyl protein phosphatase (BPTP). Spectral information was obtained by use of two single-tryptophan mutant proteins, W39F and W49F, and the double mutant protein W39,49F. Fluorescence measurements show that Trp39 is characterized by a large blue shift, a low quantum yield, and a shorter mean lifetime compared to Trp49. Solute fluorescence quenching studies of W39F reveal that Trp49 is highly exposed to the aqueous environment. In contrast, Trp39 is situated within a hydrophobic core and is only partially accessible to quenching agents such as acrylamide, iodide ion, and cesium ion. The fluorescence contributions of Trp39 and Trp49 are additive, and their sum is equivalent to that observed for wild type BPTP. Calculated intramolecular distances between Trp39 or Trp49 and a 5-[[(acetylamino)-ethyl]amino]naphthalene-1- sulfonate group covalently bound at Cys12 or Cys17 of the respective protein mutants, place Trp49 within 10 A and Trp39 at least 20 A from the active site. The fluorescence decay of the single tryptophan mutants and, surprisingly, wild type BPTP were each adequately fitted as biexponentials. The latter is a consequence of the imprecision involved in determining actual minima in a three- and four-exponential fitting. Comparison of quenching results of wild type BPTP with those of the single tryptophan mutant proteins indicates that minor fluorescence components, easily resolved using a biexponential fitting for the mutant proteins, are unresolvable for wild type BPTP. These minor components skewed the weighted magnitudes and induced perturbations in lifetimes for the tryptophan fluorescence of wild type BPTP, which directly influenced the calculated values of Ksv and kq.

Animals↗
PubMed1995

Intact survival with transfusion-associated graft-versus-host disease proved by human leukocyte antigen typing of lymphocytes in skin biopsy specimens.

A transient transfusion-associated graft-versus-host disease occurred in a premature infant of 30 weeks of gestation. We demonstrated donor lymphocytes in a skin biopsy specimen with a two-step immunoperoxidase technique using monoclonal antibodies against human leukocyte antigen determinants specific for the donor. The girl survived and is immunocompetent.

Antibodies, Monoclonal↗
PubMed1995

Translocation of gliadin into HLA-DR antigen containing lysosomes in coeliac disease enterocytes.

Coeliac disease is triggered by ingestion of wheat gliadin and is probably immune mediated. There is evidence by light microscopy that expression of class II major histocompatibility complex (MHC) molecules is increased in the small intestinal epithelium of patients with untreated coeliac disease and that gliadin can be taken up by small intestinal enterocytes. The pathway by which gliadin is transported to class II MHC proteins has not been demonstrated. Using an immunogold technique and thin frozen sections of jejunal biopsy specimens, gliadin, HLA-DR antigens, and IgA were localised at an ultrastructural level in the jejunal epithelium of patients with both untreated and treated coeliac disease and controls. Cathepsin D was used as a marker for late endosomes or lysosomes. The results show that gliadin is translocated into vacuoles positive for HLA-DR antigens as well as cathepsin D in jejunal enterocytes of patients with untreated coeliac disease. Secretory IgA may have a role in this translocation of gliadin, which is a specific event that occurred only in jejunal enterocytes from patients with untreated coeliac disease but not in a patient maintained on a gluten free diet or in controls. These results support a central role for epithelial cells of the human intestinal mucosa in the transport of gliadin to an HLA-DR positive compartment which precedes antigen presentation of gliadin to antigen sensitive T lymphocytes.

Antigen Presentation↗
PubMed1995

Ultrastructural, immunocytochemical and stereological investigation of hepatocytes in a patient with the mutation of the ornithine transcarbamylase gene.

We studied a male newborn suffering from deficiency of ornithine transcarbamylase (OTC) that is due to a G-to-A substitution in codon 269 of the OTC gene. This study intends to define the cell biological mechanisms in this naturally occurring OTC mutation which may explain the mild clinical course in spite of the very low residual enzyme activity. Using immunogold labeling of thawed thin frozen sections of liver from this patient and a control liver, we analyzed the quantitative distribution of several mitochondrial proteins in the cytosol and the mitochondria of hepatocytes. In addition, the absolute volumes and surface densities of mitochondria and peroxisomes were determined. Our results show that the absolute volume of mitochondria in the patient's hepatocytes was increased to 141% (P < 0.001) without any change in the surface density indicating an increased number of mitochondria. In the patient's hepatocytes the peroxisomes were increased in size but not in number. The concentration of OTC was elevated in the cytosol (P < 0.001) and to a lesser extent in mitochondria (P < 0.01) of the patient's hepatocytes thus indicating a doubling of OTC relative to control liver cells. The quantity of OTC in mitochondria was 63% higher in diseased liver cells. By conventional thin section electron microscopy, mitochondria-like structures with poorly defined cristae and an electron-dense matrix were observed in the cytoplasm of the diseased hepatocytes. By immunoelectron microscopy, they contained the cytochrome c oxidase II subunit as well as DNA but lacked OTC, carbamylphosphate synthetase, F1-ATPase beta subunit and catalase. Thus it appears that these structures represent defective and probably degenerating mitochondria. Our data indicate that the reduced enzyme activity of the mutant OTC is partly compensated by an increased amount of enzyme molecules in the cytosol as well as mitochondria combined with an increase in the biogenesis of mitochondria.

Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzin↗
PubMed1995

Carbohydrate-deficient glycoprotein syndrome (CDGS)--glycosylation, folding and intracellular transport of newly synthesized glycoproteins.

Carbohydrate-deficient glycoprotein syndrome (CDGS) is a hereditary glycosylation disorder of unknown origin. In this study we used skin fibroblasts from patients with CDGS to study the glycosylation of three well characterized glycoproteins using gel mobility analysis, endoglycosidase treatments and protein folding studies. We show that glycoprotein transport along the secretory pathway was delayed. Dilation of the rough endoplasmic reticulum indicated a retention phenomenon for selected glycoproteins. However, for all examined glycoproteins cotranslational glycosylation in CDGS fibroblasts was normal.

Abnormalities, Multiple↗
PubMed1994

Asp129 of low molecular weight protein tyrosine phosphatase is involved in leaving group protonation.

Site-directed mutagenesis was used to explore the functions of a number of acidic residues of bovine low molecular weight protein tyrosine phosphatase. Residues Asp-129, Asp-56, and Asp-92 were mutated to Ala or Asn. The mutant enzymes D56A, D56N, and D92A showed no significant changes in Vmax values, although they did exhibit significantly altered Km values. In contrast, the D129A mutant enzyme exhibited a greater than 2000-fold reduction in Vmax, using p-nitrophenyl phosphate as a substrate. The Vmax values of D129A also exhibited a leaving group dependence, an altered solvent isotope effect of VmaxH/VmaxD of 0.78, and a lack of dependence on the presence of alternative phosphate acceptor alcohols, all properties that distinguish this mutant from wild type enzyme. The differences are due to a change of the rate-limiting step of the catalytic reaction. Asp-129 is concluded to be the proton donor to the leaving group in the phosphorylation step, and its mutation to alanine results in a reduced Vmax value and a change in the rate-limiting step of the catalysis from dephosphorylation to phosphorylation. Mechanistic considerations suggest that other phosphotyrosyl phosphatases having cysteine at the active site may be expected to have a similar requirement for a proton donor.

Animals↗
PubMed1994

Assessment of tubular reabsorption of sodium, glucose, phosphate and amino acids based on spot urine samples.

Reference values for tubular transport of sodium, phosphate, glucose and amino acids are generally based on inulin or creatinine short-term clearances, which are difficult to obtain in children. Hence, quantitative assessment of tubular transport capacities is rarely performed. For a simplified procedure, reference values for fractional sodium excretion, phosphate reabsorption related to glomerular filtration rate, percent glucose and percent amino acid reabsorption were established in 62 children from spot urine and simultaneously obtained blood samples. Sodium excretion, and glucose and amino acid reabsorption were significantly lower in infants than children, whereas phosphate reabsorption decreased during the first year of life. Results using the proposed protocol and those obtained from timed urine specimens correlated well; the phenomenon of renal adaptation during childhood could equally well be demonstrated. Renal tubular dysfunction can be diagnosed without timed urine specimens.

Absorption↗
PubMed1992

Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis.

Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding. The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis. Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.

Amino Acid Sequence↗
PubMed1992

Different concentrations of thyroid peroxidase and thyroglobulin in the nuclear envelope and the endoplasmic reticulum throughout the cytoplasm.

Thyroid peroxidase (TPO) and thyroglobulin (TG) represent two major glycoproteins of thyroid follicular cells performing biological functions such as iodination, transcytosis of thyroglobulin, and formation of thyroid hormones. They are involved in thyroid autoimmunity and thyroid inborn metabolic disorders. Studying these processes at a molecular level includes the determination of their precise intracellular distribution. An evaluation of the relative concentrations of TG and TPO in different subcellular compartments was carried out in stimulated human follicular cells using thin-frozen sections and the immunogold technique. It is documented that TG is transported from the endoplasmic reticulum and the Golgi apparatus to the follicular lumen by transport vesicles; most of it being present in the expanded endoplasmic reticulum throughout the cytoplasm. On the other hand, gold particles indicating TPO are adjacent to the membranes of the exocytotic pathway. They do not label the basolateral membrane but show the strongest density in the nuclear envelope and the apical membrane. The labeling density of TPO is about four times higher in the nuclear envelope than in the endoplasmic reticulum throughout the cytoplasm. In contrast, TG is concentrated three times higher in the rough endoplasmic reticulum throughout the cytoplasm than in the nuclear cisternae. Our results give the first quantitative evidence that TPO and TG are concentrated in different subcompartments of the endoplasmic reticulum. Because previous studies demonstrated the nuclear envelope as the site where the synthesis of endogenous peroxidase (Brökelmann, J., D. W. Fawcett, Biol. Reprod. 1, 59-71 (1969)) begins, we suggest that synthesis of these functionally related proteins happens in specialized parts of the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗
PubMed1991

A catalytic role for threonine-12 of E. coli asparaginase II as established by site-directed mutagenesis.

A threonine-12 to alanine mutant of E. coli asparaginase II (EC 3.5.1.1) has less than 0.01% of the activity of wild-type enzyme. Both tertiary and quaternary structure of the enzyme are essentially unaffected by the mutation; thus the activity loss seems to be the result of a direct impairment of catalytic function. As aspartate is still bound by the mutant enzyme, Thr-12 appears not be involved in substrate binding.

Asparaginase↗
PubMed1991

Mutations replacing the leucine codons or altering the length of the amino acid-coding portion of the ilvGMEDA leader region of Escherichia coli.

The specificity of regulation by attenuation of the ilvGMEDA operon of Escherichia coli was examined by making alterations in the peptide-coding portion of the leader region. The effects of the alterations on attenuation control were monitored by operon fusions with the lacZ or cat gene. Substitution of the tandem leucine codons with arginine codons did not result in arginine control of attenuation even though the altered leader transcripts contained three consecutive arginine codons. Substitution of the single leucine codon with a proline codon at position 10 of the putative peptide, which had been shown to be important in the regulation of the Serratia marcescens ilv operon, did not result in control of attenuation by proline. Since the formation of neither proline nor arginine biosynthetic enzymes is regulated by attenuation control, the effect of tandem phenylalanine codons in place of the tandem leucine codons was examined and found not to result in control by phenylalanine supply. The latter failure may have been due to a configuration in the secondary structure of the protector stem of the leader transcript different from that of the wild-type transcript. The results of the study favored the idea that the lead ribosome does not initiate translation of the leader transcript until after the RNA polymerase has reached the pause site (117 bases into the leader region).

Amino Acids, Branched-Chain↗
PubMed1989

Ganglioside GM3 sialidase activity in fibroblasts of normal individuals and of patients with sialidosis and mucolipidosis IV. Subcellular distribution and and some properties.

Sensitive assays for the determination of the ganglioside sialidase activity of fibroblast homogenates were established using ganglioside GM3, 3H-labelled in the sphingosine moiety, as a substrate. Ganglioside GM3 sialidase activity was greatly stimulated by the presence of the non-ionic detergent Triton X-100 and was further enhanced by salts such as NaCl; the optimal pH was 4.5. The subcellular localization of this activity was determined by fractionation using free-flow electrophoresis and found to be exclusively associated with the marker for the plasma membrane, but not with that for lysosomes. This Triton-stimulated ganglioside sialidase activity was selectively inhibited by preincubating intact cells in the presence of millimolar concentrations of Cu2+, suggesting that the activity resides on the external surface of the plasma membrane. In normal fibroblasts homogenates, ganglioside GM3 sialidase was also greatly stimulated by sodium cholate. In contrast to the Triton X-100-activated reaction, however, it was not diminished by prior incubation of intact cells in the presence of Cu2+. Only after cell lysis was Cu2+ inhibitory. the cholate-stimulated ganglioside sialidase activity thus paralleled the behaviour of the lysosomal 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4-MU-NeuAc) sialidase. In fibroblasts from sialidosis patients, the cholate-stimulated ganglioside GM3 sialidase activity, but not that of the Triton-activated enzyme, was profoundly diminished. In fibroblasts from patients with mucolipidosis IV (ML IV), both the Triton X-100- and the cholate-stimulated ganglioside GM3 sialidase activities were in the range of normal controls. The Triton-activated enzyme was associated with the plasma membrane in the same manner as in normal cells. Our findings suggest that, in human fibroblasts, there exist two sialidases that degrade ganglioside GM3: one on the external surface of the plasma membrane, and another that is localized in lysosomes and seems identical with the activity that acts on sialyloligosaccharides and 4-MU-NeuAc. As neither activity was found to be deficient in ML IV fibroblasts, our results argue against the hypothesis of a primary involvement of a ganglioside GM3 sialidase in the pathogenesis of ML IV.

Cell Membrane↗