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J M Tager

Publications and source records attributed to J M Tager.

At least 19 recordsLinked to original sources
PubMed1998

Lipid metabolism in peroxisomes in relation to human disease.

Peroxisomes were long believed to play only a minor role in cellular metabolism but it is now clear that they catalyze a number of important functions. The importance of peroxisomes in humans is stressed by the existence of a group of genetic diseases in man in which one or more peroxisomal functions are impaired. Most of the functions known to take place in peroxisomes have to do with lipids. Indeed, peroxisomes are capable of 1. fatty acid beta-oxidation 2. fatty acid alpha-oxidation 3. synthesis of cholesterol and other isoprenoids 4. ether-phospholipid synthesis and 5. biosynthesis of polyunsaturated fatty acids. In Chapters 2-6 we will discuss the functional organization and enzymology of these pathways in detail. Furthermore, attention is paid to the permeability properties of peroxisomes with special emphasis on recent studies which suggest that peroxisomes are closed structures containing specific membrane proteins for transport of metabolites. Finally, the disorders of peroxisomal lipid metabolism will be discussed.

Animals↗
PubMed1996

Control analysis of biliary lipid secretion.

UNLABELLED: Biliary lipid secretion is a complex process involving a multitude of metabolic pathways. It has always been assumed that bile salt secretion (BSec) fully controls this process. Recently we have demonstrated, that mdr2 P-glycoprotein (P-gp) is an important controlling step as well. In this study we have analysed the control structure of this pathway with Metabolic Control Analysis. METHODS: FVB mice homozygous (+/+) or heterozygous (+/-) for mdr2 P-glycoprotein were infused via the tail vein with tauroursodeoxycholate in stepwise increasing concentrations. Bile was collected and biliary lipids were determined by standard techniques. RESULTS: To simplify the pathway we have lumped all reactions involved in BSsec into bile in one step. Since this step is not controlled by the canalicular BS concentration, the FCC of BS secretion on phospholipid secretion (PLsec) could be calculated from a plot between BS and PL secretion. The FCC of BSsec varied from 80% at low flux to a value of 90% at maximal BS output. The FCC of mdr2 P-gp was determined by varying the gene dose of mdr2 P-gp. Since PLsec showed linear kinetics towards canalicular BS the FCC could be calculated via the Deviation index. The values for the FCC of mdr2 P-gp in (+/+) mice vary from 80% at low flux to 125% at maximal BS output. CONCLUSIONS: Both BS secretion and mdr2 P-gp strongly control biliary phospholipid secretion. The sum of the FCCs of both steps is always much higher than 100% implicating the presence of step(s) which exert negative control. We hypothesize that steps controlling biliary water transport account for the negative control.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1994

Signals on proteins, intracellular targeting and inborn errors of organellar metabolism.

Newly synthesized polypeptides contain signals that direct them to the appropriate intracellular organelles and the organelles contain receptors that recognize the signals. Protein synthesis occurs either on free ribosomes or on ribosomes bound to the endoplasmic reticulum. The proteins synthesized on bound ribosomes are co-translationally translocated into the lumen of the endoplasmic reticulum and contain or acquire targeting information for retention in the endoplasmic reticulum or for sorting to lysosomes and other compartments of the secretory and endocytic pathways. Proteins synthesized on free ribosomes remain in the cytosol or contain signals for import into the nucleus, mitochondria or peroxisomes. The nature of the targeting signals and the mechanisms of import are discussed briefly. Examples are given of inborn errors of metabolism caused by incorrect or impaired incorporation of proteins into mitochondria, lysosomes or peroxisomes.

Biological Transport↗
PubMed1994

Clinical and biochemical characteristics of peroxisomal disorders: an update.

Peroxisomal disorders represent a recently recognized group of inherited diseases in man, now comprising 14 different disorders. If discussion is restricted to those peroxisomal disorders in which there is neurological involvement (thereby excluding hyperoxaluria and acatalasaemia), results over the least few years have shown that analysis of very-long-chain fatty acids (VLCFAs) is a highly reliable initial test to establish whether or not one is dealing with a peroxisomal disorder. Rhizomelic chondrodysplasia punctata, its recently identified variant form and glutaryl-CoA oxidase deficiency will show no abnormalities and must be identified by other means. Recently we have found a few clinically proven cases of adrenoleukodystrophy showing normal VLCFA in plasma but clearly abnormal values in fibroblasts. This suggests that great care is warranted in interpreting plasma VLCFA analyses. Furthermore, plasma bile acids, phytanic acid, pristanic acid and pipecolic acid should be analysed in any patient with clinical symptoms suggestive for a peroxisomal disorder but normal plasma VLCFAs.

Adrenoleukodystrophy↗
PubMed1993

Topology of catalase assembly in human skin fibroblasts.

The biogenesis, assembly and import of the peroxisomal enzyme catalase was studied in human skin fibroblasts from control persons and from patients with the Zellweger syndrome. For this purpose, two monoclonal antibodies were generated which are able to discriminate between the monomeric or dimeric form and the tetrameric, enzymically active conformation of the enzyme. Metabolic labelling studies showed that catalase is assembled to the tetrameric conformation within one hour after its synthesis, while it is still in the cytosol of the cell. Subsequently, the enzyme becomes particle-bound in the control cells, a process that is retarded by addition of the catalase inhibitor 3-amino-1,2,4-triazole. However, the tetramer remains in the cytosol in cells from Zellweger patients. It is concluded that newly synthesized catalase can be assembled to a tetramer in the cytosol in human skin fibroblasts. Unfolding of this tetramer prior to import into peroxisomes is indicated.

Antibodies, Monoclonal↗
PubMed1993

Demonstration of the existence of a second, non-lysosomal glucocerebrosidase that is not deficient in Gaucher disease.

In addition to the lysosomal glucocerebrosidase, a distinct beta-glucosidase that is also active towards glucosylceramide could be demonstrated in various human tissues and cell types. Subcellular fractionation analysis revealed that the hitherto undescribed glucocerebrosidase is not located in lysosomes but in compartments with a considerably lower density. The non-lysosomal glucocerebrosidase differed in several respects from lysosomal glucocerebrosidase. The non-lysosomal isoenzyme proved to be tightly membrane-bound, whereas lysosomal glucocerebrosidase is weakly membrane-associated. The pH optimum of the non-lysosomal isoenzyme is less acidic than that of lysosomal glucocerebrosidase. Non-lysosomal glucocerebrosidase, in contrast to the lysosomal isoenzyme, was not inhibited by low concentrations of conduritol B-epoxide, was markedly inhibited by taurocholate, was not stimulated in activity by the lysosomal activator protein saposin C, and was not deficient in patients with Gaucher disease. Non-lysosomal glucocerebrosidase proved to be less sensitive to inhibition by castanospermine or deoxynojirimycin but more sensitive to inhibition by D-gluconolactone than the lysosomal glucocerebrosidase. The physiological function of this second, non-lysosomal, glucocerebrosidase is as yet unknown.

Cells, Cultured↗
PubMed1993

An efficient procedure for the generation of human monoclonal antibodies based on activation of human B lymphocytes by a murine thymoma cell line.

A new, efficient procedure for the generation of human monoclonal antibodies has been developed. The procedure is based on the activation of human B cells in microwells by murine thymoma EL4B5 cells. This mode of B cell stimulation leads to proliferation of at least one per eight of human B cells and to a high rate of antibody production. Subsequently, supernatants of the microwells are screened by ELISA for the presence of antibody of the desired specificity and B cells from selected wells are hybridized by electroporation. To optimize the procedure, the kinetics of the B cell expansion induced by EL4B5 cells were analysed. Counting and phenotyping of cultured cells at different time points indicated that the peak of B cell expansion occurred at day 5 for tonsil B cells (16-fold increase) and at day 7 for peripheral blood B cells (20-fold increase). The B cells did not merely proliferate but also differentiated, as indicated by loss of CD20 expression and increase of CD38 expression. At the peak of B cell expansion, B cells could be hybridized efficiently with myeloma cells. The majority of the resultant hybridomas secreted human immunoglobulin. The efficiency of the procedure is exemplified by the generation of hybridomas secreting human IgG against Haemophilus influenzae from limited numbers of either human tonsil B lymphocytes or peripheral blood B lymphocytes.

Animals↗
PubMed1993

Postnatal diagnosis of peroxisomal disorders: a biochemical approach.

In recent years an increasing number of inherited decreases in man has been identified in which there is an impairment of one or more peroxisomal functions. Sofar 15 different peroxisomal disorders have been identified which can be subdivided into three distinct groups depending upon whether there is a generalized (group A), multiple (group B) or single (group C) loss of peroxisomal functions. In this paper we will briefly describe the functions of peroxisomes in man which are of direct relevance for the peroxisomal disorders known up to now. Based upon the biochemical characteristics of the different peroxisomal disorders, we well describe a straightforward approach for the postnatal identification of patients suspected to suffer from a peroxisomal disorder. Furthermore, a detailed analysis of the biochemical procedures which should be used preferably, is given.

Adrenoleukodystrophy↗
PubMed1993

Direct evidence that human follicular dendritic cells (FDC) rescue germinal centre B cells from death by apoptosis.

It is supposed that FDC have a pivotal role in the rescue of germinal centre (GC) B lymphocytes from apoptosis. However, formal proof for this hypothesis has not as yet been presented. In the present study FDC and GC B cells were isolated from human tonsils and cultured. When brought into culture FDC and B cells rapidly formed spherical clusters. T cells were not observed inside these clusters. At different time points cultures of FDC and B cells were supravitally stained with Hoechst 33258 or acridine orange and examined by direct observation using fluorescence microscopy. Viable B cells appeared to be profoundly restricted to clusters, whereas cells not taking part in clusters all had an apoptotic appearance. The formation of clusters could be prevented by addition of MoAbs against CD11a (LFA-1 alpha) or CD49d (VLA-4 alpha), resulting in an apoptotic appearance of virtually all B lymphocytes. The present data demonstrate that a physical interaction between FDC and germinal centre B lymphocytes is able to rescue the latter from apoptotic cell death.

Apoptosis↗
PubMed1993

Role of pH in determining the cell-type-specific residual activity of glucocerebrosidase in type 1 Gaucher disease.

The properties of control and 370Asn-->Ser glucocerebrosidase, the frequently encountered mutated form of the enzyme in type 1 Gaucher disease, were studied in vitro as well as in situ. The catalytic properties of purified 370Asn-->Ser glucocerebrosidase were highly dependent on the assay conditions. The enzyme was deficient in activity towards substrate and in reactivity with the irreversible inhibitor conduritol B-epoxide (CBE) when activated by the bile salt taurocholate. In the presence of more physiological activators, the lysosomal activator protein saposin C and phosphatidylserine, the 370Asn-->Ser enzyme was near normal in kinetic properties at pH values approximately 5, but not at higher pH. In intact fibroblasts, the enzymic activity of the 370Asn-->Ser glucocerebrosidase and its reactivity with CBE were found to be clearly deficient. However, in intact lymphoblasts from the same patients, the behavior of the mutant enzyme was near normal. The catalytic efficiency of 370Asn-->Ser glucocerebrosidase in situ was also found to be highly pH dependent. When intact lymphoblasts were cultured in the presence of permeant weak bases, which increase the pH of acidic intracellular compartments, the catalytic efficiency of the mutant enzyme, as assessed by its reactivity with CBE, became markedly impaired. Our findings indicate that the intralysosomal pH in the intact cell can be expected to have a critical influence on the activation state of 370Asn-->Ser glucocerebrosidase and its ability to hydrolyse substrate. This phenomenon may partly underly the marked heterogeneity in clinical manifestation of Gaucher disease among patients with this mutated form of glucocerebrosidase.

Amino Acid Sequence↗
PubMed1993

CD40 plays an essential role in the activation of human B cells by murine EL4B5 cells.

A mutant subclone of the murine thymoma EL-4, known as EL4B5, can strongly activate human B cells to proliferate and differentiate in a cell-cell contact-dependent manner. We have investigated whether interaction via CD40 plays a role in this helper activity. For this purpose, three newly generated anti-CD40 monoclonal antibodies (mAb) were used. In contrast with other anti-CD40 mAb described in the literature, these mAb did not co-stimulate proliferation of human B cells. On the other hand, these novel mAb could inhibit the co-stimulatory effect of the previously described anti-CD40 mAb S2C6 on anti-IgM-induced human B-cell proliferation. It was found that addition of these non-stimulatory anti-CD40 mAb could completely inhibit EL4B5-induced human B-cell proliferation. Maximal inhibition occurred already at a mAb concentration of 10 ng/ml. Similarly, a fusion protein, consisting of the extracellular portion of CD40 and human IgM constant domains CH2, CH3 and CH4, could completely inhibit EL4B5-induced human B-cell proliferation. Induction of human B-cell proliferation by EL4B5 cells was also inhibited by anti-CD40 mAb S2C6 and G28.5, but less effectively. In contrast, mAb against B-cell surface antigens CD20 or B7 had no inhibitory effects. It is concluded that interaction via CD40 is essential for the induction of human B-cell proliferation by EL4B5 cells.

Animals↗
PubMed1992

Production and characterisation of monoclonal antibodies against native and disassembled human catalase.

Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either monomer/dimer catalase or native, tetrameric enzyme. Three stable hybridoma clones were selected and the characteristics of the antibodies produced were investigated by ELISA, immunofluorescence, immunoprecipitation and immunoblotting experiments. One monoclonal antibody (17E10) was shown to interact with both native tetramer catalase and--to a lesser extent--with monomer/dimer catalase. Two monoclonal antibodies (10B12H9, 13A10) were found to react only with completely denatured catalase or with monomer/dimer catalase but not with native catalase.

Antibodies, Monoclonal↗
PubMed1992

A procedure for selecting mammalian cells with an impairment in oxidative phosphorylation.

The mitochondrial encephalomyopathies in man are characterized by heterogeneous defects leading to an impairment in the pathway of aerobic energy production. As a means of investigating the molecular and genetic mechanisms underlying these disorders we have developed a procedure for selecting mammalian cell lines with features resembling the human pathological phenotypes. The principle of the selection is the use of a fluorescent amphiphilic dye, 2,4-(dimethylamino)-1-styrylmethylpyridiniumiodine, a cation showing two main features. Firstly, it is accumulated by mitochondria to an extent correlated with the magnitude of the electrochemical gradient of protons across the mitochondrial inner membrane. Secondly, upon irradiation with UV light, it gives rise to formation of free radicals, which inflict damage to the cell. Mutant cells with an impairment in oxidative phosphorylation will have more chance to survive than wild type cells. The selection procedure was applied to a stock of mutagenized Chinese hamster ovary cells. After subcloning of the cells which survived the selection procedure, twenty-six independent clones were isolated. Eighteen of the clones had a partial deficiency of cytochrome c oxidase ranging from 30 to 60% of the activity in control cells. The properties of two of the clones are described. One clone has been cultured under non-selective conditions for at least 12 months with retention of the partial deficiency of cytochrome c oxidase.

Animals↗
PubMed1992

Subcellular localisation and processing of non-specific lipid transfer protein are not aberrant in Rhizomelic Chondrodysplasia Punctata fibroblasts.

The import into peroxisomes and maturation of peroxisomal 3-oxoacyl-CoA thiolase are impaired in patients with the Rhizomelic form of Chondrodysplasia Punctata (RCDP). Here we show by means of immunoblotting and subcellular fractionation that non-specific lipid transfer protein (nsLTP), another peroxisomal protein synthesised as a larger precursor, is localised in peroxisomes and is present as the mature protein in RCDP fibroblasts. Thus the component of the import machinery defective in RCDP is not required for the import of nsLTP into peroxisomes.

Acetyl-CoA C-Acetyltransferase↗
PubMed1992

Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata--a complementation study.

The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: dihydroxyacetone phosphate acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3-oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.

Acetyl-CoA C-Acyltransferase↗
PubMed1992

X-linked adrenoleukodystrophy: biochemical diagnosis and enzyme defect.

The adrenoleukodystrophies refer to three genetically distinct disorders all characterized by the accumulation of very long-chain fatty acids. In this paper we will review the biochemical aspects of these leukodystrophies with particular emphasis on the methods used to measure very long-chain fatty acid levels in plasma and their reliability. Furthermore, we will concentrate on the primary defect in the X-linked form of adrenoleukodystrophy.

Adrenoleukodystrophy↗