OBJECTIVE: Enzymatic fingerprinting of key enzymes of glucose metabolism is a valuable analysis tool in cell physiological phenotyping of plant samples. Yet, a similar approach for mammalian cell line samples is missing. In this study, we applied semi-high throughput enzyme activity assays that were originally designed for plant samples and tested their feasibility in extracts of six frequently used mammalian cell lines (Caco2, HaCaT, C2C12, HEK293, HepG2 and INS-1E). RESULTS: Enzyme activities for aldolase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, phosphofructokinase could be detected in samples of one or more mammalian cell lines. We characterized effects of sample dilution, assay temperature and repeated freeze-thaw cycles causing potential biases. After careful selection of experimental parameters, the presented semi-high throughput methods could be established as useful tool for physiological phenotyping of cultured mammalian cells.
- MeSH
- Fructose-Bisphosphate Aldolase metabolism MeSH
- Cell Line MeSH
- Hep G2 Cells MeSH
- Caco-2 Cells MeSH
- Enzyme Assays methods MeSH
- Phenotype MeSH
- Phosphofructokinases metabolism MeSH
- Phosphoglucomutase metabolism MeSH
- Phosphotransferases (Alcohol Group Acceptor) metabolism MeSH
- Glucosephosphate Dehydrogenase metabolism MeSH
- Glucose metabolism MeSH
- Glycolysis * MeSH
- HEK293 Cells MeSH
- Hexokinase metabolism MeSH
- Humans MeSH
- Carbohydrate Metabolism * MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Pilot Projects MeSH
- Feasibility Studies MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Phosphoglucomutase 1 (PGM1) encodes the metabolic enzyme that interconverts glucose-6-P and glucose-1-P. Mutations in PGM1 cause impairment in glycogen metabolism and glycosylation, the latter manifesting as a congenital disorder of glycosylation (CDG). This unique metabolic defect leads to abnormal N-glycan synthesis in the endoplasmic reticulum (ER) and the Golgi apparatus (GA). On the basis of the decreased galactosylation in glycan chains, galactose was administered to individuals with PGM1-CDG and was shown to markedly reverse most disease-related laboratory abnormalities. The disease and treatment mechanisms, however, have remained largely elusive. Here, we confirm the clinical benefit of galactose supplementation in PGM1-CDG-affected individuals and obtain significant insights into the functional and biochemical regulation of glycosylation. We report here that, by using tracer-based metabolomics, we found that galactose treatment of PGM1-CDG fibroblasts metabolically re-wires their sugar metabolism, and as such replenishes the depleted levels of galactose-1-P, as well as the levels of UDP-glucose and UDP-galactose, the nucleotide sugars that are required for ER- and GA-linked glycosylation, respectively. To this end, we further show that the galactose in UDP-galactose is incorporated into mature, de novo glycans. Our results also allude to the potential of monosaccharide therapy for several other CDG.
- MeSH
- Fibroblasts drug effects metabolism pathology MeSH
- Phosphoglucomutase deficiency MeSH
- Galactose administration & dosage MeSH
- Glycosylation MeSH
- Cohort Studies MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Uridine Diphosphate Galactose metabolism MeSH
- Uridine Diphosphate Glucose metabolism MeSH
- Congenital Disorders of Glycosylation drug therapy metabolism pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
PurposePhosphoglucomutase-1 deficiency is a subtype of congenital disorders of glycosylation (PGM1-CDG). Previous casereports in PGM1-CDG patients receiving oral D-galactose (D-gal) showed clinical improvement. So far no systematic in vitro and clinical studies have assessed safety and benefits of D-gal supplementation. In a prospective pilot study, we evaluated the effects of oral D-gal in nine patients.MethodsD-gal supplementation was increased to 1.5 g/kg/day (maximum 50 g/day) in three increments over 18 weeks. Laboratory studies were performed before and during treatment to monitor safety and effect on serum transferrin-glycosylation, coagulation, and liver and endocrine function. Additionally, the effect of D-gal on cellular glycosylation was characterized in vitro.ResultsEight patients were compliant with D-gal supplementation. No adverse effects were reported. Abnormal baseline results (alanine transaminase, aspartate transaminase, activated partial thromboplastin time) improved or normalized already using 1 g/kg/day D-gal. Antithrombin-III levels and transferrin-glycosylation showed significant improvement, and increase in galactosylation and whole glycan content. In vitro studies before treatment showed N-glycan hyposialylation, altered O-linked glycans, abnormal lipid-linked oligosaccharide profile, and abnormal nucleotide sugars in patient fibroblasts. Most cellular abnormalities improved or normalized following D-gal treatment. D-gal increased both UDP-Glc and UDP-Gal levels and improved lipid-linked oligosaccharide fractions in concert with improved glycosylation in PGM1-CDG.ConclusionOral D-gal supplementation is a safe and effective treatment for PGM1-CDG in this pilot study. Transferrin glycosylation and ATIII levels were useful trial end points. Larger, longer-duration trials are ongoing.
- MeSH
- Administration, Oral MeSH
- Child MeSH
- Phosphoglucomutase metabolism MeSH
- Galactose administration & dosage adverse effects therapeutic use MeSH
- Glycogen Storage Disease drug therapy MeSH
- Glycoproteins metabolism MeSH
- Blood Coagulation MeSH
- Infant MeSH
- Creatine Kinase blood MeSH
- Blood Glucose metabolism MeSH
- Skin cytology metabolism MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Pilot Projects MeSH
- Child, Preschool MeSH
- Prospective Studies MeSH
- Transferrin metabolism MeSH
- Dose-Response Relationship, Drug MeSH
- Check Tag
- Child MeSH
- Infant MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Clinical Trial MeSH
OBJECTIVE: To define phenotypic groups and identify predictors of disease severity in patients with phosphoglucomutase-1 deficiency (PGM1-CDG). STUDY DESIGN: We evaluated 27 patients with PGM1-CDG who were divided into 3 phenotypic groups, and group assignment was validated by a scoring system, the Tulane PGM1-CDG Rating Scale (TPCRS). This scale evaluates measurable clinical features of PGM1-CDG. We examined the relationship between genotype, enzyme activity, and TPCRS score by using regression analysis. Associations between the most common clinical features and disease severity were evaluated by principal component analysis. RESULTS: We found a statistically significant stratification of the TPCRS scores among the phenotypic groups (P < .001). Regression analysis showed that there is no significant correlation between genotype, enzyme activity, and TPCRS score. Principal component analysis identified 5 variables that contributed to 54% variance in the cohort and are predictive of disease severity: congenital malformation, cardiac involvement, endocrine deficiency, myopathy, and growth. CONCLUSIONS: We established a scoring algorithm to reliably evaluate disease severity in patients with PGM1-CDG on the basis of their clinical history and presentation. We also identified 5 clinical features that are predictors of disease severity; 2 of these features can be evaluated by physical examination, without the need for specific diagnostic testing and thus allow for rapid assessment and initiation of therapy.
- MeSH
- Algorithms MeSH
- Principal Component Analysis MeSH
- Child MeSH
- Adult MeSH
- Phenotype * MeSH
- Phosphoglucomutase deficiency genetics MeSH
- Physical Examination MeSH
- Genetic Markers MeSH
- Genotype MeSH
- Glycogen Storage Disease diagnosis enzymology genetics MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Mutation MeSH
- Child, Preschool MeSH
- Regression Analysis MeSH
- Severity of Illness Index * MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
OBJECTIVES: A 10-year-old boy presented with cleft palate, hepatopathy, cholecystolithiasis, myopathy, coagulopathy, hyperlipidemia, hypoglycemia, hyperuricemia, short stature, obesity, hypothyroidism, microcephaly and mild intellectual disability. The multi-systemic manifestation involving certain distinct clinical features prompted us to search for a subtype of congenital disorders of glycosylation (CDG). METHODS: The patient was screened for CDG by examining the distribution of transferrin (TRF) and apolipoprotein C-III (ApoC-III) sialylated isoforms using isoelectric focusing of serum. This was followed by spectrophotometric measurement of phosphoglucomutase 1 (PGM1) activity in fibroblasts and molecular analysis including sequencing and PCR-RFLP of PGM1 gene. Selected bioinformatics tools were used to evaluate the data. RESULTS: Increased relative levels of di-, mono- and asialotransferrin reflected a defect of N-glycosylation in the patient. Markedly decreased activity of PGM1 corresponding to less than 5% of control´s was found. Sequencing of PGM1 gene revealed the presence of two heterozygous missense mutations c.1010C>T (p.T337M) and c.1508G>A (p.R503Q), whose pathogenicity was confirmed by in silico analysis. CONCLUSION: We report the first Czech patient with a glycosylation disorder due to PGM1 deficiency. Compared to the described cases, no dilated cardiomyopathy was noted in our patient. However, he suffered from a mild neurological impairment, which is an uncommon feature that extends the phenotype associated with PGM1-CDG. Lactose-rich diet, which was previously reported to have ameliorated the clinical symptoms in some PGM1-CDG patients, did not result in any improvement in our patient.
- MeSH
- Central Nervous System physiopathology MeSH
- Child MeSH
- Phenotype MeSH
- Phosphoglucomutase genetics MeSH
- Glycogen Storage Disease complications genetics MeSH
- Humans MeSH
- Intellectual Disability complications diagnosis genetics MeSH
- Microcephaly complications diagnosis genetics MeSH
- Mutation, Missense MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
- Research Support, Non-U.S. Gov't MeSH
We tested the virulence of 15 strains of Entamoeba histolytica, imported to Czechoslovakia, by intracaecal inoculation of laboratory rats. According to the scoring system of Neal, none of the 15 strains possessed the virulence index greater than 2. This indicates that all the organisms tested should be classified as avirulent. However, it should be noted that all the strains produced infection of the caecum and thus should be considered infective for rat. For 7 strains, isoenzyme patterns were determined for PGM, HK and ME. One imported strain, obtained from student from Congo, demonstrated isoenzyme pattern for PGM and HK indicated that the strain was virulent. This organisms had the index of virulence 1.8 (avirulent) in animal experiment; it was isolated from cysts of clinically asymptomatic patient. Examination of the rectal mucosa of the donor of the strain indicated typical chronic catarrhal proctitis of mild degree. Examination of the patient's serum demonstrated the presence of anti E. histolytica antibodies by CIEP, while the ELISA test was negative. Twenty-one cyst carriers were examined by rectoscopy. Pathologic changes were observed in 20 of these, as follows: altered vascular structure (13X), roughened mucosa (12X), mucosal reddening (10X), decreased glistening (7X), mucus in mucosa (5X), inflammatory pseudopolypes (2X), ulcers (2X), enanthema (1X). Histological biopsies were obtained in 15 cases. One was considered normal. Remaining 14 biopsies exhibited following morphological changes: increased mucus secretion (8X), edema (7X), lymphocytic and plasmocytic infiltration (6X), lymphocytic and plasmocytic infiltration in addition to the presence of eosinophilic granulocytes (6X), presence of mucophages (5X), haemorrhages (4X), increased vascularity (3X), lymphocytic and plasmocytic infiltration with presence of extremely abundant eosinophilic granulocytes (1X), erosive-ulcerative changes of mucosa (1X). The changes observed indicated chronic catarrhal proctitis with expression to greater or less degree of signs of chronic catarrhal inflammation.
- MeSH
- Amebiasis parasitology MeSH
- Electrophoresis, Starch Gel MeSH
- Entamoebiasis parasitology MeSH
- Entamoeba histolytica enzymology pathogenicity MeSH
- Phosphoglucomutase analysis MeSH
- Hexokinase analysis MeSH
- Isoenzymes analysis MeSH
- Rats MeSH
- Malate Dehydrogenase analysis MeSH
- Virulence MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
About 3,500 subjects from Italy and Czechoslovakia have been analyzed by acid starch gel electrophoresis for the subtyping of PGM1 polymorphism. The Italian sample included three different subgroups, from Northern, Central and Southern Italy. The allele frequencies found in the three groups do not differ significantly from each other; the observed values in the pooled sample are: PGM1S1 = 0.594, PGM1F1 = 0.118, PGM2S1 = 0.231, PGM2F1 = 0.057. In the Czechoslovakian group, which differs significantly from the Italian population, the following allele frequencies were found: PGM1S1 = 0.639, PGM1F1 = 0.118, PGM2S1 = 0.180, PGM2F1 = 0.063. The analysis of 217 families did not show any exception to Mendelian inheritance of the patterns.
- MeSH
- Phosphoglucomutase genetics MeSH
- Gene Frequency MeSH
- Isoenzymes genetics MeSH
- Humans MeSH
- Polymorphism, Genetic MeSH
- Pedigree MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czechoslovakia MeSH
- Italy MeSH
The electrophoretic and thermostability polymorphisms of the PGM1 locus were examined in about 700 Czechoslovakians (Prague) and 3000 Italians. The Italian sample consisted of individuals from Pavia (Northern Italy), Viareggio and Rome (Central Italy) and Naples (Southern Italy). The eight PGM1 alleles, PGM1Str1, PGM1Sts1, PGM1Ftr1, PGM1Fts1, PGM2Str1, PGM2Sts1, PGM2Ftr1, PGM2Fts1, have been considered as combinations of mutations at three different sites, 1/2, S/F and tr/ts, within the PGM1 gene and their frequencies discussed in terms of linkage disequilibrium between these sites. All pairwise differences between the samples were significant except for Pavia-Viareggio and Viareggio-Rome. The frequencies of the PGMts1 alleles have been found to range from 0.0981 (Prague) to 0.0546 (Naples) and can be ordered according to a North-South cline.
- MeSH
- Alleles MeSH
- Adult MeSH
- Phenotype MeSH
- Phosphoglucomutase genetics MeSH
- Gene Frequency * MeSH
- Isoenzymes genetics MeSH
- Humans MeSH
- Infant, Newborn MeSH
- Polymorphism, Genetic * MeSH
- Temperature MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Infant, Newborn MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czechoslovakia MeSH
- Italy MeSH
