Biomedical subjects

J M Tager

Publications and source records attributed to J M Tager.

At least 127 records · Page 7Linked to original sources

PubMed1987

Human peroxisomal 3-oxoacyl-coenzyme A thiolase deficiency.

We investigated the peroxisomal beta-oxidation system in liver from a patient with clinical features similar to those in the cerebrohepatorenal (Zellweger) syndrome and with elevated levels in body fluids of very-long-chain fatty acids and intermediates in the biosynthesis of bile acids. The peroxisomal beta-oxidation of fatty acids, measured as the cyanide-insensitive formation of [14C]acetyl units from [14C]palmitoyl-CoA, was very low in the patient (less than 10% of the values in control subjects). Immunoblotting experiments using antibodies to peroxisomal beta-oxidation enzymes indicated that peroxisomal 3-oxoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) was deficient. Addition of purified rat-liver peroxisomal 3-oxoacyl-CoA thiolase to a reaction mixture containing liver homogenate from the patient restored peroxisomal beta-oxidation. We conclude that the deficiency of peroxisomal 3-oxoacyl-CoA thiolase is responsible for the very low peroxisomal beta-oxidation activity and for the accumulation of very-long-chain fatty acids and intermediates in the biosynthesis of bile acids. Furthermore, the finding that both very-long-chain fatty acids and abnormal bile acids accumulate in this patient suggests that a single peroxisomal 3-oxoacyl-CoA thiolase is involved in the oxidative chain shortening of both very-long-chain fatty acids and the coprostanoic acids.

Acetyl-CoA C-Acyltransferase↗

PubMed1987

Direct evidence for a primary immune response of murine B-lymphocytes after in vitro immunization of dissociated splenocytes.

Monoclonal antibodies against human thyroglobulin were generated using splenocytes cultured in vitro with the antigen. When splenocytes from non-immunized mice were used, about 90% of the hybridomas obtained produced immunoglobulins of the IgM class. In contrast, when splenocytes from mice previously immunized in vivo with human thyroglobulin were cultured in vitro with the antigen about 85% of the hybridomas obtained produced immunoglobulins of the IgG class. The properties of the monoclonal antibody produced by hybridoma 3D12, obtained after culturing splenocytes from non-immunized mice with human thyroglobulin, were examined in detail. Monoclonal antibody 3D12 reacted only with human thyroglobulin and not with the murine homologue in an enzyme-linked immunosorbent assay, in immunoblotting experiments and in an immunohistochemical test. These results provide direct evidence that a primary response to an antigen can be elicited by adding the antigen to cultures of splenocytes from non-immunized mice.

Animals↗

PubMed1987

Characterization of five monoclonal antibodies obtained after immunization in vitro with a synthetic 19-amino acid peptide of thyroglobulin.

A 19-amino acid synthetic peptide representing the highly conserved amino terminus of thyroglobulin was used for the production of monoclonal antibodies after immunization of splenocytes in vitro. The properties of five of the antibodies were studied. One reacted only with the synthetic peptide. The others reacted with both the synthetic peptide and with thyroglobulin from all species tested so far, confirming that the amino terminus of thyroglobulin is highly conserved. Two of the five antibodies showed a positive reaction when tested on frozen sections of thyroid tissue, but with different reaction patterns. Monoclonal antibody F4 gave a positive reaction in the colloid, which contains mainly 19S thyroglobulin. In contrast, monoclonal antibody G4 gave a positive reaction only in the follicular cells. Monoclonal antibody G4 binds primarily to low molecular weight compounds in thyroglobulin preparations, possibly representing breakdown products of the protein.

Amino Acid Sequence↗

PubMed1987

Genetic diseases caused by peroxisomal dysfunction. New findings in clinical and biochemical studies.

Peroxisomes play an essential role in human cellular metabolism. Peroxisomal disorders, a group of genetic diseases caused by peroxisomal dysfunction, can be classified in three groups namely a group of disorders with a general peroxisomal dysfunction (Zellweger syndrome; infantile type of Refsum's disease; neonatal adrenoleukodystrophy, hyperpipecolic acidemia), a group with an impairment of some, but not all peroxisomal functions (rhizomelic chondrodysplasia punctata) and a group with impairment of only a single peroxisomal function (acatalasemia, X-linked adrenoleukodystrophy/adrenomyeloneuropathy; adult type of Refsum's disease; peroxisomal thiolase deficiency; peroxisomal acyl-CoA oxidase deficiency; hyperoxaluria type I). In this paper we report the typical findings in ophthalmological examinations of patients suspected of Zellweger syndrome contributing to the clinical diagnosis of this disorder. In biochemical studies using a rapid gaschromatographic detection method for plasmalogens we confirmed that plasmalogens are severely deficient in all tissues of Zellweger patients studied. Moreover, using a recently developed radiochemical method, de novo plasmalogen biosynthesis was found to be impaired in fibroblasts from patients with Zellweger syndrome, infantile Refsum's disease, neonatal adrenoleukodystrophy or rhizomelic chondrodysplasia punctata, this in contrast to X-linked chondrodysplasia in which a normal plasmalogen biosynthesis was found. From the literature it is known that peroxisomal beta-oxidation with both long-chain (C16:0) and very long-chain (C24:0; C26:0) fatty acids is deficient in Zellweger syndrome, infantile Refsum's disease and neonatal adrenoleukodystrophy. In contrast, in X-linked adrenoleukodystrophy only the peroxisomal beta-oxidation of the very long chain fatty acids is impaired. As a result very long-chain fatty acids accumulate in tissues, plasma, fibroblasts and amniotic fluid cells from patients with Zellweger syndrome, infantile Refsum's disease, neonatal and X-linked adrenoleukodystrophy, but not in rhizomelic chondrodysplasia punctata or X-linked chondrodysplasia. Finally we confirmed that the peroxisomal enzyme alanine glyoxylate aminotransferase is severely deficient in liver from a patient that died because of the neonatal type of hyperoxaluria type I, but not in liver from Zellweger patients.

Chromatography, Gas↗

PubMed1987

alpha-Glucosidase deficiency (Pompe's disease).

alpha-Glucosidase is deficient (less than 30% of control) in Pompe's disease, but the extent of the deficiency does not always correlate with the severity of the clinical symptoms. The defects that lead to a deficiency of alpha-glucosidase include synthesis of catalytically inactive protein, absence of mRNA for the enzyme, decreased synthesis of the precursor, lack of phosphorylation of the precursor, impaired conversion of the precursor to the mature enzyme and synthesis of unstable precursor. A single type of defect can lead to different clinical phenotypes. The precursor of alpha-glucosidase is present in the brush border of the polarized epithelial cells of small intestine and kidney and is secreted into urine.

Biological Transport↗

PubMed1987

Clinical diversity in glycogenosis type II. Biosynthesis and in situ localization of acid alpha-glucosidase in mutant fibroblasts.

The molecular basis of clinical diversity in glycogenosis type II (Pompe's disease) was investigated by comparing the nature of acid alpha-glucosidase deficiency in cultured fibroblasts from 30 patients. Biosynthetic forms of acid alpha-glucosidase with different molecular mass were separated electrophoretically and identified by immunoblotting. Immuno-electron microscopy was employed to determine the intracellular localization of mutant enzyme. Our studies illustrate that maturation of acid alpha-glucosidase is associated with transport to the lysosomes. Deficiency of catalytically active mature enzyme in lysosomes is common to all clinical phenotypes but, in the majority of cases, is more profound in early onset than in late onset forms of the disease. Thus, the results suggest that the clinical course of glycogenosis type II is primarily determined by the amount of functional acid alpha-glucosidase. The role of secondary factors can, however, not be excluded because three adult patients were identified with very low activity and little enzyme in the lysosomes.

Adult↗

PubMed1987

Peroxisomal fatty acid beta-oxidation in relation to the accumulation of very long chain fatty acids in cultured skin fibroblasts from patients with Zellweger syndrome and other peroxisomal disorders.

The peroxisomal oxidation of the long chain fatty acid palmitate (C16:0) and the very long chain fatty acids lignocerate (C24:0) and cerotate (C26:0) was studied in freshly prepared homogenates of cultured skin fibroblasts from control individuals and patients with peroxisomal disorders. The peroxisomal oxidation of the fatty acids is almost completely dependent on the addition of ATP, coenzyme A (CoA), Mg2+ and NAD+. However, the dependency of the oxidation of palmitate on the concentration of the cofactors differs markedly from that of the oxidation of lignocerate and cerotate. The peroxisomal oxidation of all three fatty acid substrates is markedly deficient in fibroblasts from patients with the Zellweger syndrome, the neonatal form of adrenoleukodystrophy and the infantile form of Refsum disease, in accordance with the deficiency of peroxisomes in these patients. In fibroblasts from patients with X-linked adrenoleukodystrophy the peroxisomal oxidation of lignocerate and cerotate is impaired, but not that of palmitate. Competition experiments indicate that in fibroblasts, as in rat liver, distinct enzyme systems are responsible for the oxidation of palmitate on the one hand and lignocerate and cerotate on the other hand. Fractionation studies indicate that in rat liver activation of cerotate and lignocerate to cerotoyl-CoA and lignoceroyl-CoA, respectively, occurs in two subcellular fractions, the endoplasmic reticulum and the peroxisomes but not in the mitochondria. In homogenates of fibroblasts from patients lacking peroxisomes there is a small (25%) but significant deficiency of the ability to activate very long chain fatty acids. This deficient activity of very long chain fatty acyl-CoA synthetase is also observed in fibroblast homogenates from patients with X-linked adrenoleukodystrophy. We conclude that X-linked adrenoleukodystrophy is caused by a deficiency of peroxisomal very long chain fatty acyl-CoA synthetase.

Facial Bones↗

PubMed1986

Efficient routing of glucocerebrosidase to lysosomes requires complex oligosaccharide chain formation.

The biosynthesis and intracellular transport of the membrane-associated lysosomal enzyme glucocerebrosidase was studied in the monoblast cell line U937. Addition to the cultures of the oligosaccharide trimming inhibitors swainsonine or deoxymannojirimycin led to an increased intracellular activity of glucocerebrosidase. This was due to prevention of the lysosomal degradation of the enzyme. When homogenates of control cells were fractionated on Percoll gradients glucocerebrosidase, like beta-hexosaminidase, was distributed in two peaks, one at low density and one at high density. When homogenates of cells cultured in the presence of oligosaccharide trimming inhibitors were fractionated beta-hexosaminidase was still distributed in two peaks but glucocerebrosidase was found mainly in low density fractions also containing galactosyltransferase activity. It is concluded that complex type oligosaccharide chain formation is required for efficient routing of glucocerebrosidase to the lysosomes in U937 cells.

1-Deoxynojirimycin↗

PubMed1986

Acyl-CoA:dihydroxyacetone phosphate acyltransferase in human skin fibroblasts: study of its properties using a new assay method.

In relation to the finding that human skin fibroblasts are capable of de novo either phospholipid biosynthesis, we have studied the properties of acyl-CoA:dihydroxyacetone phosphate acyltransferase in fibroblast homogenates using a new assay method. The results indicate that the acylation of dihydroxyacetone phosphate shows an optimum at pH 5.5 with a broad shoulder of activity up to pH 6.4 and a decline in activity up to pH 8.2. At pH 5.5 the acyltransferase accepts dihydroxyacetone phosphate, but not glycerol 3-phosphate as a substrate. Furthermore, the transferase activity was found to be membrane-bound and inactivated by Triton X-100 at concentrations above 0.025% (w/v). Similar properties have been described for the enzyme as present in rat-liver and guinea-pig liver peroxisomes. These data, together with the finding that acyl-CoA:dihydroxyacetone phosphate acyltransferase is deficient in cultured skin fibroblasts from patients without peroxisomes (Zellweger syndrome), suggest that in cultured skin fibroblasts the enzyme is primarily located in peroxisomes.

Acyltransferases↗

PubMed1986

Comparison of the properties of a soluble form of glucocerebrosidase from human urine with those of the membrane-associated tissue enzyme.

Human urine contains a soluble form of glucocerebrosidase, an enzyme associated with the lysosomal membrane in cells and tissues. Urinary glucocerebrosidase is identical to the enzyme extracted from tissues with respect to the following parameters: Km for natural and artificial substrates, inhibition by conduritol B-epoxide, and stimulation by taurocholate. The enzyme is greater than 90% precipitable by polyclonal anti-(placental glucocerebrosidase) antiserum. Upon isoelectric focussing of urinary glucocerebrosidase multiple peaks of activity were observed. Partial deglycosylation (removal of sialic acid, N-acetylglucosamine and galactose) of the urinary enzyme increased the isoelectric point to a value identical to that of the main form found after partial deglycosylation of the placental enzyme. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by immunoblotting, the immunopurified urinary enzyme shows the same molecular mass forms as the enzyme immunopurified from brain and kidney. In placenta the apparent molecular mass is somewhat higher but upon removal of sialic acid, N-acetylglucosamine and galactose the urinary and the placental enzyme show identical molecular masses of 57 kDa. We conclude that the enzymes extracted from urine and tissue are identical and that differences in apparent molecular mass and isoelectric point are probably due to heterogeneity in the oligosaccharide moieties of the molecules.

Cell Membrane↗

PubMed1986

Genetic relationship between lysosomal and lamellar body-specific alpha-glucosidases in human lung.

In lamellar bodies isolated from adult human lung at least two acid alpha-glucosidases are present: one similar to the lung lysosomal alpha-glucosidase, and another lamellar body-specific isoenzyme. In the present study we measured the activity of this lamellar body-specific alpha-glucosidase and of lysosomal alpha-glucosidase in a patient with an inherited deficiency of lysosomal alpha-glucosidase. The activity of the lamellar body-specific alpha-glucosidase was not affected in the patient, whereas the lysosomal alpha-glucosidase activity was strongly depressed. The results strongly suggest that the lysosomal alpha-glucosidase and the lamellar body-specific alpha-glucosidase are different gene products.

Concanavalin A↗

PubMed1986

Peroxisomal beta-oxidation of palmitoyl-CoA in human liver homogenates and its deficiency in the cerebro-hepato-renal (Zellweger) syndrome.

The presence of a beta-oxidation system in peroxisomes has been well documented. Rather than a duplicate of the mitochondrial beta-oxidation system, peroxisomes seem specially equipped to initiate the oxidation of very-long-chain fatty acids. Thus, the accumulation of very-long-chain fatty acids in tissues and body fluids from patients with a limited (X-linked adrenoleukodystrophy) or generalized (cerebro-hepato-renal (Zellweger) syndrome, infantile Refsum disease, neonatal adrenoleukodystrophy) peroxisomal dysfunction probably results from an impairment in the peroxisomal beta-oxidation system. In order to study this, we have developed an original assay which allows measurement of the overall peroxisomal beta-oxidation activity in human liver homogenates. Compared to controls, a strong deficiency of this activity was detected in liver from Zellweger patients using palmitoyl-CoA as a substrate.

Acyl Coenzyme A↗

PubMed1986

Deficient activity of glucocerebrosidase in urine from patients with type 1 Gaucher disease.

Glucocerebrosidase is present in considerable amounts in human urine. The enzyme is stable in concentrated urine for several days when stored at 0 degrees C. Like tissue glucocerebrosidase, the urinary enzyme is inhibited by conduritol B-epoxide and hydrolyses not only glucocerebroside but also the synthetic substrate 4-methyl-umbelliferyl-beta-D-glucoside. The enzyme is deficient in urine from patients with Gaucher disease (type 1). It is possible to discriminate completely between patients with type 1 Gaucher disease and control subjects by measuring the ratio glucocerebrosidase/beta-hexosaminidase in urine. The value of this ratio (mean +/- SE) with the synthetic substrates 4-methylumbelliferyl-beta-glucoside and p-nitrophenyl-beta-N-acetylglucosaminide, respectively, was 34.2 +/- 3.7 (n = 24) in the controls and 2.1 +/- 0.9 (n = 21) in the patients.

Adolescent↗

PubMed1986

Production of murine monoclonal antibodies against human thyroglobulin using an in vitro immunization procedure in serum-free medium.

A serum-free in vitro immunization method for the generation of hybridomas producing specific antibodies to an antigen is described. The method was tested with human thyroglobulin as antigen. The serum-free medium used (Yssel et al., 1984) consisted of Iscove's modification of Dulbecco's modified Eagle's medium, supplemented with albumin, transferrin, insulin, ethanolamine and linoleic, oleic and palmitic acids. An optimal response was obtained when splenocytes from BALB/c mice were cultured for 3 days in the presence of 1.5 nM thyroglobulin and thymocyte-conditioned medium prior to fusion with SP2/0 myeloma cells and seeding of the fused cells in microtitre plates. The frequency of positive wells, defined as the number of wells secreting anti-(thyroglobulin) antibodies/number of viable cells used for the fusion, was 1.6 X 10(-6) +/- 0.25 X 10(-6) (mean +/- SD; n = 4). Eight stable clones producing anti-(thyroglobulin) antibodies were isolated. One clone (3D12) produced antibodies reacting only with human thyroglobulin. The antibodies produced by the other clones reacted with human, murine and porcine thyroglobulins. Seven of the clones produced antibodies of the IgM class and one clone produced IgG. The specificity of 3D12 (IgM) for human thyroglobulin and the absence of any reactivity with murine thyroglobulin provides evidence for a primary response of splenocytes in culture to the presence of an antigen.

Antibodies, Monoclonal↗

PubMed1986

Control of gluconeogenesis in rat liver cells. Flux control coefficients of the enzymes in the gluconeogenic pathway in the absence and presence of glucagon.

We have used control analysis to quantify the distribution of control in the gluconeogenic pathway in liver cells from starved rats. Lactate and pyruvate were used as gluconeogenic substrates. The flux control coefficients of the various enzymes in the gluconeogenic pathway were calculated from the elasticity coefficients of the enzymes towards their substrates and products and the fluxes through the different branches in the pathway. The elasticity coefficients were either calculated from gamma/Keq. ratios (where gamma is the mass-action ratio and Keq. is the equilibrium constant) and enzyme-kinetic data or measured experimentally. It is concluded that the gluconeogenic enzyme pyruvate carboxylase and the glycolytic enzyme pyruvate kinase play a central role in control of gluconeogenesis. If pyruvate kinase is inactive, gluconeogenic flux from lactate is largely controlled by pyruvate carboxylase. The low elasticity coefficient of pyruvate carboxylase towards its product oxaloacetate minimizes control by steps in the gluconeogenic pathway located after pyruvate carboxylase. This situation occurs when maximal gluconeogenic flux is required, i.e. in the presence of glucagon. In the absence of the hormone, when pyruvate kinase is active, control of gluconeogenesis is distributed among many steps, including pyruvate carboxylase, pyruvate kinase, fructose-1,6-bisphosphatase and also steps outside the classic gluconeogenic pathway such as the adenine-nucleotide translocator.

Animals↗

PubMed1986

Transport and processing of endocytosed lysosomal alpha-glucosidase in cultured human skin fibroblasts.

In the present study we have characterised the molecular products that arise from processing of a precursor form of alpha-glucosidase isolated from urine after endocytosis at 37 degrees C by cultured human skin fibroblasts. The urinary precursor (Mr 110 000) was processed to forms with Mr of 100 000, 80 000 and 74 000. These forms were approximately 4000 Da larger than the corresponding forms of endogenously synthesized alpha-glucosidase. Digestion of the different forms of the enzyme with endoglycosidase F showed that the differences in apparent molecular mass between the exogenous and corresponding endogenous forms were due to difference in glycosylation. Intracellular transport of endocytosed alpha-glucosidase was followed by incubating fibroblast homogenates with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap), which leads to specific lysis of lysosomes. Transport to the lysosomes was a fast process: within 45 min after endocytosis more than 50% of the enzyme was present in the lysosome. The first step in the processing of endocytosed alpha-glucosidase started in a Gly-Phe-NH-Nap-insensitive (prelysosomal) compartment, but further processing of the enzyme to lower-Mr forms was coupled to transport to the lysosomes. Processing of alpha-glucosidase after uptake at 20 degrees C was also studied. At this temperature the enzyme accumulated in an organelle with a low buoyant density, presumably the endosome; this compartment appeared to be heterogeneous, ranging in density from 1.04 g/ml to 1.08 g/ml. Under these conditions only the first step in the processing of the enzyme occurred. It is concluded that endocytosed enzyme is processed more rapidly than endogenously synthesized enzyme owing to the fact that endocytosed enzyme is transported more rapidly to the lysosomes. Furthermore, processing may start in a prelysosomal organelle.

Biological Transport↗

PubMed1986

A procedure for the rapid purification in high yield of human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibodies.

A novel chromatographic immunoaffinity procedure is described for the purification of Form I glucocerebrosidase (see J. M. F. G. Aerts, W. E. Donker-Koopman, M. K. Van der Vliet, L. M. V. Jonsson, E. I. Ginns, G. J. Murray, J. A. Barranger, J. M. Tager, and A. W. Schram, 1985, Eur. J. Biochem. 150, 565-574) from extracts of human tissues. The affinity support consists of two monoclonal anti-(glucocerebrosidase) antibodies immobilized by covalent coupling to CNBr-activated Sepharose 4B. After adsorption of the enzyme from a crude detergent extract, the column is washed successively with 30% ethylene glycol in citrate buffer (pH 6), 1% Triton X-100 in citrate phosphate buffer (pH 5.2), and 50% ethylene glycol in citrate buffer. The enzyme is eluted with 90% ethylene glycol in citrate buffer. After dilution to 30% ethylene glycol, the immunoaffinity purification is repeated. The procedure can be completed within less than 18 h. The final preparations have a high specific activity (50 U/mg protein (n = 4) for the placental enzyme) and contain no detectable impurities after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The yield is high (81 +/- 8% for the placental enzyme). The immunoaffinity column has a high capacity, can be regenerated easily, and can be utilized repeatedly without loss of activity.

Antibodies, Monoclonal↗

PubMed1986

The malate/aspartate shuttle and pyruvate kinase as targets involved in the stimulation of gluconeogenesis by phenylephrine.

The mechanisms responsible for the stimulation by phenylephrine of gluconeogenesis from dihydroxyacetone and glycerol were studied in perifused rat hepatocytes. The stimulation by phenylephrine of glucose formation from dihydroxyacetone was biphasic. Transient stimulation of about 25% after 3 min was followed by a stable stimulation of about 15% 7 min later. Concomitantly there was a transient inhibition by phenylephrine of pyruvate kinase flux during the first few minutes followed by a more stable inhibition after about 10 min. The stable inhibition could quantitatively account for the stable stimulation of gluconeogenesis by the hormone when dihydroxyacetone was the gluconeogenic substrate. The effects of phenylephrine were independent of cAMP. With the reduced gluconeogenic substrate, glycerol, a transient aminooxyacetate-sensitive stimulation of gluconeogenesis occurs, followed by a stable stimulation of smaller magnitude [Leverve, X. M., Groen, A. K., Verhoeven, A. J. and Tager, J. M. (1985) FEBS Lett. 181, 43-46]. This transient phase of stimulation was abolished by addition of low concentrations of pyruvate, in accordance with our previous proposal that phenylephrine transiently accelerates the mitochondrial oxidation of cytosolic NADH via the malate/aspartate shuttle. The transient stimulation of gluconeogenesis from glycerol was accompanied by a transient increase in the cytosolic/mitochondrial aspartate concentration gradient. Phenylephrine caused a transient stimulation of mitochondrial aspartate efflux in two other systems dependent on the operation of the malate/aspartate shuttle, i.e. gluconeogenesis from lactate and the oxidation of ethanol. It is proposed that addition of phenylephrine to hepatocytes brings about a transient increase in the mitochondrial membrane potential and hence an increased rate of transport of aspartate from the mitochondria to the cytosol.

Animals↗