Gilbert's Syndrome: Analytical Subcellular Fractionation of Liver Biopsy Specimens. Enzyme Activities, Organelle Pathology and Evidence for Subpopulations of the Syndrome

1979 ◽  
Vol 57 (6) ◽  
pp. 491-497 ◽  
Author(s):  
J. Dawson ◽  
C. A. Seymour ◽  
T. J. Peters
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Liver Biopsy ◽  
Enzyme Activities ◽  
Subcellular Fractionation ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Gilbert’S Syndrome ◽  
Gilbert's Syndrome ◽  
Glutamyl Transferase

1. The hepatic organelle pathology of 12 patients with Gilbert's syndrome was studied by analytical subcellular fractionation in combination with enzymic microanalysis of liver biopsy material. 2. All patients showed increased activities of the endoplasmic reticulum marker enzyme, neutral α-glucosidase. Seven patients showed a decrease in the modal density of the endoplasmic reticulum, from 1·20 to 1·15 g/ml. These patients also showed striking hypertrophy of the hepatocyte smooth endoplasmic reticulum on electron microscopy. The remaining five patients showed normal endoplasmic reticulum density distribution with a peak at 1·20 g/ml, and had normal appearance of the endoplasmic reticulum on electron microscopy. 3. All patients showed increased activity of three lysosomal marker enzymes: N-acetyl-β-glucosaminidase, acid phosphatase and β-glucuronidase. The distribution of these enzymes in the sucrose gradients showed less enzyme in the high-density region of the gradients, indicating a reduced equilibrium density of the lysosomes. Assays of latent and sedimentable N-acetyl-β-glucosaminidase, a measure of lysosomal integrity, were normal. 4. Marker enzyme activities and density gradient distribution of other organelles, including plasma membrane (5′-nucleotidase), mitochondria (malate dehydrogenase), biliary canaliculi (γ-glutamyl transferase) and cytosol (lactate dehydrogenase) were normal. Increased catalase activities were noted.

Subcellular Fractionation Studies of the Intestinal Mucosa in Congenital Sucrase—Isomaltase Deficiency

1979 ◽  
Vol 57 (2) ◽  
pp. 181-185 ◽  
Author(s):  
B. T. Cooper ◽  
D. C. A. Candy ◽  
J. T. Harries ◽  
T. J. Peters
Keyword(s):  
Electron Microscopy ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Enzyme Activities ◽  
Subcellular Fractionation ◽  
The Other ◽  
Marker Enzyme ◽  
Jejunal Biopsy ◽  
Biopsy Specimens ◽  
Highly Sensitive

1. Jejunal biopsy specimens from three children with congenital sucrase—isomaltase deficiency were assayed for disaccharidase activity and were subjected to analytical subcellular fractionation with enzymic microanalysis. 2. By use of the highly sensitive fluorigenic modification of the disaccharidase assay, brush-border sucrase and isomaltase activities were depressed but nevertheless detectable in each child. 3. Apart from the expected decrease in brush-border α-glucosidase activity, the other enterocyte marker-enzyme activities were normal. 4. There were no abnormalities in the enterocytes of any child on analytical subcellular fractionation or on electron microscopy.

Sequential Enzyme and Subcellular Fractionation Studies on Jejunal Biopsy Specimens from Patients with Post-Infective Tropical Malabsorption

1979 ◽  
Vol 56 (5) ◽  
pp. 479-486 ◽  
Author(s):  
T. J. Peters ◽  
P. E. Jones ◽  
G. Wells ◽  
G. G. Cook
Keyword(s):  
Brush Border ◽  
Giardia Lamblia ◽  
Gradient Centrifugation ◽  
Selective Reduction ◽  
Subcellular Fractionation ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Jejunal Biopsy ◽  
Biopsy Specimens ◽  
Glutamyl Transferase

1. Jejunal biopsy specimens were obtained from seven patients with post-infective tropical malabsorption, four of whom were infected with Giardia lamblia. 2. Enzymic analysis of the biopsy homogenates revealed a selective reduction of certain enzymes (alkaline phosphatase, γ-glutamyl transferase) localized at least in part to the brush border. Other enzymes (leucyl-2-naphthylamidase, α-glucosidase) were unaffected. 3. Density gradient centrifugation confirmed the selective decrease in the brush-border activity but the equilibrium density of the organelle was normal. Homogenization studies revealed that the brush border was unusually fragile. 4. Apart from reduced activities of the basal—lateral marker enzyme 5′-nucleotidase and enhanced lysosomal fragility, other organelles showed normal properties. There were no significant differences in any of these parameters in the patients with or without giardiasis. 5. The patients were restudied after their jejunal morphology and clinical features had returned to normal. All these parameters, apart from a persistently reduced activity of 5′-nucleotidase, returned to normal. 6. It is suggested that the principal lesion in patients with post-infective tropical malabsorption affects the cell membrane, specifically affecting the absorptive—digestive capacity of the enterocyte. These findings suggest that there is direct brush-border damage rather than mucosal invasion by bacteria, viruses or Giardia lamblia.

scholarly journals Analytical subcellular fractionation of needle-biopsy specimens from human liver

1978 ◽  
Vol 174 (2) ◽  
pp. 435-446 ◽  
Author(s):  
T J Peters ◽  
C A Seymour
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Density Gradient ◽  
Needle Biopsy ◽  
Human Liver ◽  
Resolving Power ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Acid Hydrolases ◽  
Biopsy Specimens

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5′-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5′-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5′-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.

Formation of chlamydospores in Gilbertella persicaria

1981 ◽  
Vol 59 (5) ◽  
pp. 908-928 ◽  
Author(s):  
Martha J. Powell ◽  
Charles E. Bracker ◽  
David J. Sternshein
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Light And Electron Microscopy ◽  
Multivesicular Bodies ◽  
Marker Enzyme ◽  
Transparent Layer ◽  
Thiamine Pyrophosphatase ◽  
Gilbertella Persicaria

The cytological events involved in the transformation of vegetative hyphae of the zygomycete Gilbertella persicaria (Eddy) Hesseltine into chlamydospores were studied with light and electron microscopy. Thirty hours after sporangiospores were inoculated into YPG broth, swellings appeared along the aseptate hyphae. Later, septa, traversed by plasmodesmata, delimited each end of the hyphal swellings and compartmentalized these hyphal regions as they differentiated into chlamydospores. Nonswollen regions adjacent to chlamydospores remained as isthmuses. Two additional wall layers appeared within the vegetative wall of the developing chlamydospores. An alveolate, electron-dense wall formed first, and then an electron-transparent layer containing concentrically oriented fibers formed between this layer and the plasma membrane. Rather than a mere condensation of cytoplasm, development and maturation of the multinucleate chlamydospores involved extensive cytoplasmic changes such as an increase in reserve products, lipid and glycogen, an increase and then disappearance of vacuoles, and the breakdown of many mitochondria. Underlying the plasma membrane during chlamydospore wall formation were endoplasmic reticulum, multivesicular bodies, vesicles with fibrillar contents, vesicles with electron-transparent contents, and cisternal rings containing the Golgi apparatus marker enzyme, thiamine pyrophosphatase. Acid phosphatase activity was localized cytochemically in a cisterna which enclosed mitochondria and in vacuoles which contained membrane fragments. Tightly packed membrane whorls and single membrane bounded sacs with finely granular matrices surrounding vacuoles were unique during chlamydospore development. Microbodies were rare in the mature chlamydospore, but endoplasmic reticulum was closely associated with lipid globules. As chlamydospores developed, the cytoplasm in the isthmus became highly vacuolated, lipid globules were closely associated with vacuoles, mitochondria were broken down in vacuoles, unusual membrane configurations appeared, and eventually the membranes degenerated. Unlike chlamydospores, walls of the isthmus did not thicken, but irregularly shaped appositions containing numerous channels formed at intervals on the inside of these walls. The pattern of cytoplasmic transformations during chlamydospore development is similar to events leading to the formation of zygospores and sporangiospores.

scholarly journals Integrated stereological and biochemical studies on hepatocytic membranes. I.V. Heterogeneous distribution of marker enzymes on endoplasmic reticulum membranes in fractions.

1980 ◽  
Vol 85 (3) ◽  
pp. 577-586 ◽  
Author(s):  
R P Bolender ◽  
D Paumgartner ◽  
D Muellener ◽  
G Losa ◽  
E R Weibel
Keyword(s):  
Endoplasmic Reticulum ◽  
Surface Area ◽  
Enzyme Activities ◽  
Membrane Surface ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Heterogeneous Distribution ◽  
Membrane Surface Area ◽  
Total Membrane ◽  
Nadph Cytochrome C Reductase

The purpose of the study was to consider quantitatively the relationships between the surface area of the endoplasmic reticulum (ER) and constituent marker enzyme activities, as they occur in fractions collected from rat liver homogenates. The ER surface area was estimated in five membrane-containing fractions by use of a combined cytochemical-stereological technique (5), while, at the same time, ER marker enzymes were assayed biochemically. Fraction/homogenate recoveries for the ER enzymes averaged 100%, total membrane surface area 98%, and ER surface area 96%. Relative specific activities, which compare the relative amounts of ER marker enzyme activities to the relative ER surface area in the membrane-containing fractions, indicate variable distributions for glucose-6-phosphatase and NADPH cytochrome c reductase, but not for esterase.

Analytical Subcellular Fractionation of Jejunal Biopsy Specimens: Enzyme Activities, Organelle Pathology and Response to Gluten Withdrawal in Patients with Coeliac Disease

1978 ◽  
Vol 55 (3) ◽  
pp. 285-292 ◽  
Author(s):  
T. J. Peters ◽  
P. E. Jones ◽  
G. Wells
Keyword(s):  
Endoplasmic Reticulum ◽  
Coeliac Disease ◽  
Brush Border ◽  
Enzyme Activities ◽  
Subcellular Fractionation ◽  
Density Gradients ◽  
Marker Enzymes ◽  
Jejunal Biopsy ◽  
Essentially Normal ◽  
Biopsy Specimens

1. Portions of jejunal biopsy specimens from patients with coeliac disease were homogenized in isotonic sucrose solution and subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation. 2. The activities and distribution of marker enzymes for the principal subcellular organelles were determined by highly sensitive enzymic micro-assays employing fluorigenic and radiolabelled substrates. 3. In untreated coeliac disease the brush border showed decreased activities of five marker enzymes, which were reduced to up to one-eighth of control values. On sucrose density gradients the distinct brush-border peak at density 1.21 was replaced by a broad peak at density 1.16. 4. Two out of three lysosomal enzymes showed increased activities and there was evidence of enhanced fragility of both lysosomes and peroxisomes. 5. There were reduced activities of both mitochondrial and cytosol malate dehydrogenase, with increased activities of cytosol lactate dehydrogenase. 6. The endoplasmic reticulum and basal-lateral membranes were relatively unaffected except that the equilibrium densities were slightly increased and decreased respectively. 7. The patients were treated by gluten withdrawal and subsequent biopsies examined as above. Biopsies from patients treated for 2–4 weeks and who had not yet achieved a morphological improvement showed a partial return to normal of the pathological organelles. 8. Treated patients with partial villus atrophy showed progressively improved enzyme activities and a partial reappearance of the brush-border peak on the sucrose density gradients. 9. Patients whose intestinal mucosa had returned to morphological normality after prolonged gluten withdrawal showed a persistent defect in the brush-border enzyme activities. This was particularly striking for β-glucosidase, which was persistently deficient from the brush-border although approximately one-third of the total activity of this enzyme is normally found in this organelle. 10. The properties and enzyme activities of lysosomes, peroxisomes, endoplasmic reticulum, mitochondria and cytosol were essentially normal in the biopsies from fully treated patients.

Subcellular Fractionation of Bone Marrow-Derived Macrophages: Localization of Phospholipase A1 and A2 and Acyl-CoA:1-Acylglycero-3-phosphorylcholine-0-acyltransferase

1982 ◽  
Vol 37 (3-4) ◽  
pp. 314-320 ◽  
Author(s):  
Ellen E. Krüner ◽  
Herbert Fischer ◽  
Ernst Ferber
Keyword(s):  
Bone Marrow ◽  
Endoplasmic Reticulum ◽  
Subcellular Fraction ◽  
Subcellular Fractionation ◽  
Enrichment Factors ◽  
Phospholipase A ◽  
Subcellular Localisation ◽  
Marker Enzyme ◽  
Localization Analysis ◽  
Special Respect

Abstract Phospholipase A1 and A2, Lysolecithin-Acyltransferase, Macrophage, Subcellular Localization Analysis of the subcellular distribution of lipid-metabolizing enzymes was carried out in bone marrow-derived macrophages with special respect to a comparison of the subcellular localisation of phospholipase A1 and A2 and to acyl-CoA: 1-acylglycero-3-phosphorylcholine-0-acyltransferase.After cell disruption differential centrifugation was followed by additional sucrose gradient purification of three main fractions.Satisfactory enrichment factors were obtained by this method for the following marker enzymes. The plasma-membrane enzyme alkaline phosphodiesterase I was enriched up to 25-fold and the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase was enriched up to 30-fold. The marker enzyme for the endoplasmic reticulum, NADPH-cytochrome c reductase showed a similar enrichment and distribution as the acyltransferase. Therefore it was concluded that the acyl-CoA:1-acylglycero-3-phosphorylcholine-O-acyltransferase of bone marrow-derived macrophages is mainly located in the endoplasmic reticulum. Phospholipase A1 and A2 occurred in a high proportion together with the lysosomal marker enzyme N-acetyl-β-glucosaminidase in the soluble supernatant and in the gradient fractions. In the endoplasmic reticulum phospholipase A2 occurred only in trace activities whereas phospholipase A 1 was maximally enriched in this subcellular fraction. No subcellular fraction could be obtained where phospholipase A2 was enriched exclusively. However, it can be concluded that the two enzymes which are responsible for the balance of fatty acid liberation and re-acylation are located in two different cellular compartments. Furthermore it can be claimed that in the cell there has to exist an exchange of substrates and products between these compartments to achieve a complete metabolic cycle of the de-and re-acylation reaction of phospholipids in bone marrow-derived macrophages.

Changes in Hepatic Enzymes and Organelles in Alcoholic Liver Disease

1978 ◽  
Vol 55 (4) ◽  
pp. 383-389 ◽  
Author(s):  
Carol A. Seymour ◽  
T. J. Peters
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Liver Disease ◽  
Fatty Liver ◽  
Density Gradient ◽  
Alcoholic Liver Disease ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Marker Enzyme

1. Liver biopsy specimens obtained from patients with alcoholic liver disease of varying severity were assayed for lysosomal and microsomal enzyme activities, the results being compared with values previously obtained in control subjects. 2. Analytical subcellular fractionation by sucrose-density-gradient centrifugation was performed on extracts of the biopsies and the properties of the lysosomes, plasma membrane, biliary canaliculi and endoplasmic reticulum membranes were determined. Increased activities of plasma membrane marker enzymes, particularly γ-glutamyl transpeptidase believed to be localized to the biliary canalicular membrane, were demonstrated. These findings were most marked in alcoholic cirrhosis. The centrifugation studies revealed no abnormalities in the properties of these membranes. 3. Although the total activities of the endoplasmic reticulum marker enzyme neutral α-glucosidase were unaltered in alcoholic liver disease, centrifugation studies showed a decrease in the density distribution of the membrane-bound enzyme in cirrhosis indicating an increase in the proportion of smooth endoplasmic reticulum membranes. 4. Apart from a small decrease in activity of certain acid hydrolases in fatty liver and in cirrhosis the activities of the lysosomal enzymes were unaffected by alcoholic liver disease. 5. Measurements of lysosomal integrity and density-gradient-centrifugation studies revealed no significant abnormalities in the various patient groups apart from increased stability and reduced equilibrium density of certain lysosomes in fatty liver. It is concluded that lysosomal disruption is not implicated in the pathogenesis of alcoholic liver disease.

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