Ingrid Willers

Two-dimensional electrophoresis of soluble and structure-bound proteins from cultured human fibroblasts and hair root cells: Qualitative and quantitative variation

SummaryProteins from cultured human fibroblasts and native human hair root cells were investigated using the twodimensional electrophoresis (2DE) technique. Cell material from 35 different healthy persons was examined. Proteins of different sources were separated: total proteins of fibroblasts (12 cell lines), soluble proteins of fibroblasts (12 cell lines), structurebound proteins of fibroblasts (eight cell lines) and soluble proteins of hair root cells (12 subjects). The protein samples of different individuals were run in pairs through the electrophoresis procedure and the two patterns of each pair were compared. All changes in the electrophoretic mobility of polypeptide spots (qualitative variants) and all clearly visible differences in the staining intensity of the spots (quantitative variants) were scored.Less than 1% of the qualitative variants per pattern was found in total cell proteins and this percentage was not increased in soluble proteins. No qualitative variation was detected in structure-bound proteins. Quantitative variation occurred to a considerably higher degree in the 2DE patterns than qualitative changes. The incidence of quantitative variants was about three times higher in soluble proteins (11%) than in structure-bound proteins (3.5%); in the total cell proteins it lay in between (7%). Cultured cells (fibroblasts) and native cells (hair root cells) showed a similar degree of variation. A comparison of the data shown here with data obtained by an investigation on inbred strains of the mouse suggest that the major part of the quantitative variants observed in the 2DE patterns of proteins were genetically determined.The results presented here and the mouse data mentioned above lead us to the conclusion that the genetic variability of proteins may be characterized by quantitative changes rather than by qualitative changes, and that the genetic variability occurs to quite different degrees in different classes of proteins: structure-bound proteins

5-Ethyl-2'-deoxyuridine: absence of effects on the chromosomes of human lymphocytes and fibroblasts in culture.

SummaryIn vitro studies have been performed with 5-ethyl-2′-deoxyuridine (EDU) concerning its incorporation into the cell nuclear material and its effects on the chromosome morphology of cultivated human lyphocytes and skin fibroblasts. This compound is presumably incorporated in the DNA without visible chromosome aberrations as is seen with many other pyrimidine analogues. A slight inhibition of the cell growth was noted at high concentration (120μg/ml) of EDU. A similar degree of cell growth inhibition was found with corresponding doses of deoxythymidine.ZusammenfassungEs wurden in vitro-Untersuchungen hinsichtlich des Äthyldeoxyuridineinbaus (ÄDU) und dessen Einfluß auf menschliche Lymphocyten- und Fibroblastenchromosomen durchgeführt. Diese Substanz wird in die DNS eingebaut und führt nicht zu sichtbaren Chromosomenaberrationen wie bei einigen anderen Pyrimidinanaloga. Eine leichte Hemmung des Zellwachstums wurde mit 120 μg/ml ÄDU festgestellt. Eine ähnliche Hemmung des Zellwachstums ließ sich auch mit gleicher Deoxythymidinkonzentration nachweisen.

Heterogeneity in maple syrup urine disease: aspects of cofactor requirement and complementation in cultured fibroblasts.

Fibroblast strains derived from six patients with maple syrup urine disease have been investigated for their requirements of the cofactors NAD, CoASH, Mg++ and TPP in comparison with 10 normal control strains. The reconstitution of the decarboxylase function of branched chain α‐keto acid (BCKA) dehydrogenase complex in lysed cells was studied with respect to the substrates u‐keto‐isocaproic acid, α‐keto‐isovaleric acid, and α‐keto‐β‐methylvaleric acid (KIC, KIVA, MEVA). The enzyme activity of all normal control strains for the substrates KIC and KIVA was not reconstituted by TPP + Mg++ alone, but CoASH + NAD could reconstitute the enzyme activity with KIC and KIVA in different degrees. Only two control strains were tested with MEVA as substrate, and these showed in contrast that TPP + Mg++ could partly reconstitute the enzyme activity. In contrast to the relative homogeneiy in the reconstitution profiles of normal strains, the five classical and one intermittent MSUD strains showed heterogeneity in cofactor requirements.

High-resolution protein mapping of human fibroblasts and hair root cells: a standardized reproducible procedure considering the effect of cell culture parameters.

The effect of different cell culture parameters on two-dimensional polypeptide maps of human fibroblasts is considered. Improved culture methods are introduced to get better resolution and reproducibility. Based on these investigations, highly standardized techniques for the protein mapping of this tissue and also of hair root cell lysates are presented. These methods seem to be quite suitable for analysis of cellular synthesized proteins and study of variability in normal subjects and in patients with genetic disorders.

Rapid determination of hypoxanthine-guanine-phosphoribosyl transferase in human fibroblasts and amniotic cells

SummaryA micromodification of the method of HGPRT and APRT assay is described, which measures the incorporation of 14C hypoxanthine and 14C adenine into cultured skin fibroblasts and amniotic cells grown on microtiter plates. Only about 10 000 cells are needed per assay. By this method HGPRT deficient cells can be easily distinguished from normal cells. Investigations with respect to the effect of substrate concentrations and time of incubation have been carried out on some normal fibroblast cell lines, amniotic cell lines and 3 Lesch-Nyhan cell lines.Another modified method is described for quantitative determination of HGPRT activity by means of radio thin-layer chromatography.

A rapid micromethod for prenatal diagnosis of Lesch Nyhan syndrome

A method is described which enables prenatal diagnosis of Lesch Nyhan Syndrome (HGPRT deficiency) to be made within 7–10 days. The procedure is based on the direct cultivation of amniotic cells on microtest II plates; the HGPRT reaction is performed in individual wells containing between 500 to 10,000 cells, and is followed by separation of the radioactive reaction products by means of microchromatography on 3 cm × 5 cm PEI plates. This method permits determination of the actual HGPRT enzyme activity of the cell lines.

Lesch-Nyhan Syndrome and HPRT Variants: Study of Heterogeneity at the Gene Level

The purine salvage enzyme hypoxanthine-guanine phospho-ribosyltransferase (HPRT: EC 2.4.2.8) catalyses the conversion hypoxanthine and guanine to their respective nucleotides, IMP and GMP. A partial deficiency of HPRT is associated with overproduction of uric acid which results in the development of severe form of Gout and nephrolithiasis.(Kelly et al. 1967). An almost complete deficiency of HPRT leads to an Xlinked disorder Lesch-Nyhan Syndrome, which in its classical form shows clinical symptoms of hyperuricemia, choreoathetosis, mental retardation and compulsive self mutilation. A large number of patients with different severity of these symptoms and levels of rest activity of HPRT have been reported and attempts have been made to classify them into various catagories on the basis of enzyme kinetics or behaviour of the cultured fibroblasts or lyphoblasts in various selection media (Page et al.1983., Singh et al., 1986). By sequence analysis of tryptic peptides, several HPRT variants have been chracterised to be due to single amino acid substitution(Wilson et al. 1983). Two of these have been recently shown to be due to point mutation of a single base by employing the strategy of cDNA cloning or genomic amplification of a specific region of the gene and followed by nucleotide sequencing (Fujimori et al., 1988; Cariello et al., 1988). Some of them have also been detected as gross rearrangements of the Gene (Yang et al., 1984.; Wilson et al. 1986). By employing the ribonuclease cleavage anlysis technique, also known as RNA mapping, Gibbs and Casky (1987) have been successful in localising a number of mutations of one to five nucleotides in five patients.

Growth Studies on Fibroblasts of Patients with Autosomal Recessive Friedreich’s Ataxia

Fibroblasts derived from patients with undisputed autosomal recessive Friedreichs ataxia were examined for their growth characteristics including plating efficiency, proliferation curves and cumulative number of population doublings. In comparison to cells of healthy controls, the cells from ataxia patients showed lower plating efficiency, growth rate and number of cumulative population doublings in these parameters. Cells of heterozygotes showed clonal growth and growth curves in the range of the healthy controls.

Genetic heterogeneity of hypoxanthine-phosphoribosyl transferase in human fibroblasts of 3 families.

Incorporation of hypoxanthine, resistance to 8‐azaguanine and activation by lyophilisation have been studied in cultured human fibroblasts. Cells from one family where there was a boy with Lesch‐Nyhan syndrome, from two families with variant H‐PRT mutations and three cell strains from patients with the Lesch‐Nyhan syndrome were investigated. Cells from patients with the Lesch‐Nyhan syndrome showed almost no hypoxanthine incorporation and resistance to concentrations of 8‐azaguanine up to 10‐3 M, whereas cells of patients with partial H‐PRT deficiency demonstrated variant patterns of hypoxanthine uptake and partial resistance to 8‐azaguanine. Lyophilisation of fibroblast sediment from patients with the Lesch‐Nyhan syndrome and patients with variant H‐PRT mutations showed activation of the deficient or partially deficient H‐PRT enzyme. No such activation was observed in healthy controls. Activation of lyophilised fibroblast extract from patients and controls was not obtained. These results suggest that H‐PRT could be associated with the cell membranes.

Purine Metabolism in Fibroblasts of Patients with Duchenne’s Muscular Dystrophy

Fibroblasts derived from patients with Duchenne’s muscular dystrophy have been investigated for incorporation of adenine and AMP and the enzyme activities in comparison with normal control strains. Cells of patients and controls showed an overlap in regard to adenine and AMP uptake and conversion to nucleotides. Activity values of HPRT and APRT of these patients were in the normal range. The activity of 5’-nucleotidase in different cell fractions showed a broad variability and no clear differences between Duchenne’s muscular dystrophy patients and normal controls.