Measurement of the ratio sigma (W-->e nu )/ sigma (Z-->ee) in p-barp collisions at sqrt s =1.8 TeV.
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Journal Article↗
Biomedical subjects
D Crane
Publications and source records attributed to D Crane.
Isozymes and the micro organization of organellar structure and function.
One of the most valuable of the wide-ranging attributes of isozymes is the novel insight they allow into some of the most complex aspects of cell biology. The microorganization of subcellular structure and function is one such area, and the present study utilizes the peroxisome as an example of these applications. The biological characteristics of the major enzymic component of this organelle, catalase, have been detailed in mammalian tissues, where the enzyme exhibits a complex heterogeneity, which is due to multiple types of epigenetic modification. The major native multiplicity, however, has been demonstrated as attributable to sialic acid attachment to the enzyme. In studying the compartmentalization of these isozymes within liver cells, the technique of differential extraction with digitonin was employed, and evidence provided which supports the presence of an appreciable proportion of catalase activity in the cytoplasmic compartment. The source of these cytoplasmic isozymes was traced to release from the peroxisome, and the mechanism of this release identified with variations in the content of lysophosphatidyl choline in the peroxisomal membrane. Studies of the association of catalase with the subcellular membranes indicated an appreciable ionic interaction which varied with membrane type and isozyme status. Activity was enhanced in the bound form, providing support for the interpretation of a general protective role of this enzyme against oxidation of membrane components within the cell. Overall, these studies are considered to contribute significantly to current knowledge of the biological role and subcellular localization of catalase in relation to organellar structure and function.
Animals↗
Measurement of the mass and width of the Z0 boson at the Fermilab Tevatron.
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Journal Article↗
On the interactions of catalase with subcellular structure.
The interaction of mouse liver catalase with subcellular membranes was studied, and an ionic interaction with a variety of membranes, including those derived from the microsomes, was observed. The interaction with microsomal membranes was found to be abolished by pre-treatment of catalase with neuraminidase, indicating a functional significance for catalase-bound sialic acid. Catalase activity was found to be enhanced when bound to membranes, and evidence for a weak association of catalase with peroxisomal structure in mouse liver was also obtained. It is concluded that mouse liver catalase has a capacity to bind to a variety of subcellular membranes in vivo and that this interaction may be consistent with a general protective role for the enzyme, as well as being compatible with a model of peroxisomal biogenesis which involves the interaction of catalase with microsomal membranes.
Animals↗
On the compartmentalization of catalase, fatty acyl-CoA oxidase and urate oxidase in mammalian livers, and the influence of clofibrate treatment on this microlocalization.
The compartmentalization of catalase, fatty acyl-CoA oxidase and urate oxidase was examined in the livers of mice, rats and guinea pigs, using the technique of digitonin extraction in order to avoid the trauma associated with centrifugation procedures. The results are interpreted as indicating that an appreciable proportion of catalase activity occurs in the cytoplasmic compartment of these cells. Following treatment of the animals with clofibrate, the specific activity in both peroxisomal and cytoplasmic compartments was increased, with a higher proportion of cytoplasmic catalase being evident in mice. The results for catalase were compared with those for fatty acyl-CoA oxidase and urate oxidase both of which were indicated as showing a closer association with the peroxisomal compartment than was the case for catalase. These data have been discussed in relation to their significance on present understanding of peroxisomal structure and function.
Acyl-CoA Oxidase↗
On the multiplicity of the enzyme catalase in mammalian liver.
The literature on the complex multiplicity of mammalian catalase and the nature of the epigenetic modifications undergone by this enzyme has been reviewed, along with relevant comment on the subcellular localization and biological role of the enzyme. The epigenetic causations of multiplicity are established as being multifactorial and include oxidoreductive conversions of sulphydryl groups, the covalent attachment of carbohydrate, and partial proteolysis of the enzyme. Each of these epigenetic transformations may give rise to sets of multiple forms, and overlaps between these separate sets may give rise to extremely complex multiplicity patterns. It is concluded that any interpretation of catalase multiplicity which places emphasis on a single epigenetic causation is not compatible with the scope and variety of the available data on this enzyme. Instead, a holistic approach is urged - one giving due emphasis to the multiple causation of catalase multiplicity, and the interrelationships of these causations in the cellular situation. Rather than viewing the multiplicity of this enzyme as merely a series of interesting chemical modifications, emphasis is directed towards the fact that catalase heterogeneity provides a sensitive indication of the functional variations which occur within separate compartments of the subcellular structure, and hence becomes an essential element in any satisfactory understanding of the role of this enzyme in cellular processes.
Animals↗
Confirmation that catalase is a glycoprotein.
Catalases which had been purified from the livers of mouse, rat and guinea pig were subjected to mild periodate oxidation followed by reduction with sodium boro[3H]hydride in order to test for the presence of sialic acid. A radioactively labelled moiety resulted, which behaved as a derivative of N-acetyl neuraminic acid during mild acid hydrolysis, neuraminidase treatment, ion exchange chromatography and paper chromatography. It is concluded that mammalian catalases are glycoproteins, and possess variable amounts of N-acetyl neuraminic acid in their carbohydrate moiety.
Animals↗
In vitro bioassay for dioxinlike activity based on alterations in epithelial cell proliferation and morphology.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to induce changes in morphology and proliferation characteristics of a nonkeratinizing derivative (XBF) of a keratinizing epithelial cell line (XB), cloned from a mouse teratoma, when cocultured with irradiated feeder cells. Polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), polynuclear aromatic hydrocarbons (PAHs), and pesticides (24 compounds in total) were tested for their ability to induce these effects. The results indicated that, for the representative compounds tested, these changes are relatively specific for-and that the XBF cells are extremely sensitive to--PCDDs and PCDFs. TCDD was the most potent congener tested, capable of inducing the effects at a concentration as low as 10(-11) M. The activities of other tested PCDDs and PCDFs ranged from 10(-1) to 10(-3) of TCDD activity. The PCBs, PAHs, and pesticides had lower activities ranging from 10(-3) to 10(-6) that of TCDD. This assay system using XBF cells cocultured with irradiated 3T3 fibroblast feeder cells was examined as a possible in vitro screening assay for dioxinlike activity by testing benzene extracts of soot from a fire involving a PCB-containing transformer. The results were compared to a high-resolution gas chromatographic/mass spectrometric analysis for total PCDFs in the same samples. This comparison showed a good correlation, suggesting that the XBF-3T3 system has potential for use as a semiquantitative assay for dioxinlike activity.
Animals↗
The influence of insulin on the flux of lipid metabolism in vivo.
The effects of insulin on the balance of lipid metabolism have been investigated by measuring the relative in vivo incorporation of 3H- and 14C-labelled glycerol into the major tissues and lipid classes of diabetic mice over a three day period. Several significant alterations in the relative uptake of label were caused by the insulin treatment. Generally, there were indications of decreased synthesis and degradation of lipids in most tissues, but with an increased synthesis of hepatic triacylglycerol being a notable exception to these trends. These data indicate that insulin treatment produces widespread changes in the relative emphasis of lipid metabolism, and allow a detailed description of these responses in relation to the individual tissues and lipid classes of the living animal. The implications of these metabolic changes have been discussed in relation to hormonal effects, and tissue specific and whole body aspects of the regulation of lipid metabolism.
Animals↗
Reversible inhibition of in vitro epithelial cell proliferation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Subconfluent cultures of a mouse epithelial cell line, which after prolonged subculturing exhibited an elevated saturation density as compared to the original cell line, were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cultures of cells with or without TCDD grew at equal rates until confluency was reached. At confluency, cultures treated with as little as 10(-11) M TCDD showed a decline in cell proliferation relative to controls as demonstrated by cell enumeration and supported by reduced [3H]thymidine incorporation (both by liquid scintillation spectrometry of whole culture and autoradiography of individual cells). After 14 days of exposure, the saturation density of the treated culture was about 50% of the control culture. This TCDD-induced, increased sensitivity to density-dependent inhibition of replication ( DDIR ) was accompanied by a change from a fusiform morphology in the high-saturation-density control cells to a flat cobblestone appearance in the treated low-saturation-density cells. The nondividing cultures treated for 14 days with 10(-11) M TCDD had the same viability as control cultures. Upon trypsin suspension and reseeding , these formerly quiescent cultures were again capable of growing to high cell density and of again showing susceptibility to TCDD-induced changes in cell growth and morphology. Evidence is presented to suggest that this reversible increase in sensitivity to DDIR and the morphological change are not a consequence of cell growth inhibition. This system may provide the basis for an in vitro model to study the effect of TCDD on the control of replication of these cells.
Animals↗
On the role of catalase in the oxidation of tissue fatty acids.
The role of catalase in lipid metabolism has been studied by means of a comparison of the turnover characteristics of the major lipid classes in the normal mouse with those of animals in which the catalase activity had been inhibited and blocked by aminotriazole and allylisopropylacetamide. Double isotope ratios were determined in the lipid fractions of several tissues following the injection of labeled glycerol, and a number of significant differences were identified between these treatments. Since catalase is recognized as an integral component of the peroxisomal pathway of fatty acid oxidation, these results may be taken as indicating that interruption of the process of peroxisomal beta-oxidation in this manner cause extensive perturbations of lipid metabolism in the living animal, and these perturbations extend well beyond those tissues where the predominant localization of these organelles occurs. The concept which derives from these data--that of a significant regulatory role of peroxisomes in relation to the overall balance of lipid metabolism in the animal body--is described and discussed.
Adipose Tissue↗
On the role of peroxisomes in the metabolism of lipids--evidence from studies on mammalian tissues in vivo.
Recent investigations into the role of peroxisomes in mammalian lipid metabolism have employed double isotope methodologies to examine the influence of peroxisomal agents on lipid turnover in the liver and extra hepatic tissues of the living animal. The action of these agents, all of which caused extensive changes in the flux of lipid metabolism in the treated animals, may best be viewed in relation to their effects on the common pathway of fatty acid oxidation in peroxisomes. Clofibrate, for example, acts through induction of peroxisomal oxidases and catalase; glycolate and ethanol through activation of this pathway; and aminotriazole and allylisopropylacetamide through inhibition of the catalase step in the sequence. The data from these studies provide support for the concept of an important contributory and regulatory role of peroxisomes in relation to the overall balance of lipid metabolism, and emphasize that these organelles play a significant role in the oxidation of common fatty acids, as well as a potential for the elimination of fatty acids that are poorly oxidized by mitochondria. Additionally, the data raise intriguing questions on the extension of peroxisomal influence to include phospholipid metabolism and the substantial degree of inter-tissue communication which is involved in the balance of lipid metabolism in the whole animal.
Adipose Tissue↗
Sequential alterations in the micro-localization of catalase in mouse liver after treatment with hypolipidemic drugs.
A comparative study has been carried out on the micro-localization of catalase in mouse tissues subsequent to treatment with a representative range of hypolipidemic drugs. A commonality of effect was shown by clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate), Wy-14,643 (4-chloro-6-[2,3 xylidino)-2-pyrimidinylthio] acetic acid), RMI-15,414 (5-tetradecyloxy-2-furancarboxylic acid) and aspirin (acetyl salicylic acid), in that treatments with each of these drugs was associated with the release of peroxisomal catalase into the cytoplasmic compartment of liver and kidney. It was also noticeable that this increased cytosolic activity was characterized by the presence of an 'aged' form of the enzyme with different mobility and activity characteristics to that of the peroxisomal enzyme. Possible molecular bases for these effects and their relationship to peroxisomal biogenesis are discussed.
Animals↗
Application of an in vitro keratinization assay to extracts of soot from a fire in a polychlorinated biphenyl-containing transformer.
A fire in the State Office Building in Binghamton, New York, involving a polychlorinated biphenyl-containing electrical transformer, resulted in contamination of the structure with soot containing 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran. Benzene extracts of soot collected from various areas of the building were tested for in vitro keratinization-inducing activity by the method of J. C. Knutson and A. Poland (Cell 22, 27-36, 1980). The results, in terms of relative keratinization-inducing activity, are compared to a high-resolution gas chromatographic/mass spectrometric analysis for total polychlorinated dibenzofurans in the same samples. This comparison showed a good correlation and suggests that the in vitro keratinization model has potential for use as a semiquantitative assay for dioxinlike activity.
Air Pollutants↗
Relationships between the flux of lipid metabolism in vivo and the uncoupling of respiratory control.
The influence of dinitrophenol treatment on the balance of lipid metabolism in mouse tissues has been studied by measuring the incorporation in vivo of 3H- and 14C-labelled glycerol. Several significant alterations in the relative utilization of these labels were observed in treated animals, with the greatest influence being evident in liver lipids, and diverse responses occurring in kidney, heart, muscle and adipose tissue. These data establish that dinitrophenol perturbs lipid metabolism in all the major tissues of the animal, and point to the detailed nature of this response in the individual classes of neutral and phospholipids. The implications of individual changes have been discussed in relation to the relative roles of mitochondria and peroxisomes in the regulation of mammalian lipid metabolism.
Adipose Tissue↗
On the flux of lipid metabolism in diabetic animals.
The influence of diabetes on the balance of lipid metabolism has been studied by measuring the simultaneous incorporation in vivo of 3H- and 14C-labelled glycerol into all the major tissues and lipid classes of diabetic mice. The induction of the diabetic condition caused significant alterations in the relative utilization of these labels, with double isotope ratios (3H/14C) being increased in the total lipid fractions of all tissues, but most extensively in muscle and adipose tissue. A degree of individuality was evident in relation to the response of tissue phospholipids, particularly in regard to the incorporation of tritium label into phosphatidyl choline fractions. These data establish that diabetes causes widespread and major perturbations of lipid metabolism in these animals, and indicate the detailed nature of the responses in relation to individual tissues and lipid classes. The implications of these alterations have been discussed with respect to tissue-specific and whole body aspects of the regulation of lipid metabolism.
Adipose Tissue↗
Decreased flux through pyruvate dehydrogenase during calcium ion movements induced by vasopressin, alpha-adrenergic agonists and the ionophore A23187 in perfused rat liver.
Vasopressin or alpha-adrenergic agents such as phenylephrine or adrenaline, but not glucagon, elicited an initial decrease in flux through pyruvate dehydrogenase assayed by 14CO2 production from [1-14C]pyruvate in perfused rat liver. This rapid decrease in 14CO2 production was maximal within 1-2 min of exposure, concomitant with a rise in effluent pyruvate concentration: a subsequent return towards initial values in both parameters was completed well before 5 min. This time course was superposed with Ca2+ efflux from perfused liver, maximal (at 116 nmol/min per g wet wt. of liver) at 1-2 min of exposure. The percentage of the active (dephospho) form of pyruvate dehydrogenase was not decreased at 2 min of exposure. The effect on flux through pyruvate dehydrogenase by phenylephrine was abolished by prazosine, phentolamine or phenoxybenzamine. Ionophore A23187 also caused a depression in 14CO2 production from [1-14C]pyruvate and a rise in effluent pyruvate concentration, but this effect was stable for longer times, and it was delayed when Ca2+ was omitted from the perfusion medium. Responses of phenylephrine and A23187 were not additive. The results demonstrate that under the experimental conditions employed in intact perfused liver, the mitochondrial multienzyme system of pyruvate dehydrogenase is sensitive to vasopressin, alpha-adrenergic agents and A23187. The similar time course in Ca2+ efflux may be indicative of the involvement of Ca2+ in mediating this effect.
Adrenergic alpha-Agonists↗
On the synthesis and incorporation of catalase and urate oxidase into the peroxisomes of mouse liver.
The processes associated with the biogenesis of peroxisomes in mouse liver have been studied by following the incorporation of radiolabelled leucine into major enzymic components of this organelle. Maximal incorporation of label into peroxisomal catalase and urate oxidase occurred within 2 hr, with the urate oxidase being labelled before catalase, but subsequent to the incorporation of phospholipid into this organelle. Subsequently, immunoprecipitation of catalase from the large granular fraction of mouse liver was shown to result in the isolation of a catalase molecule which had lost a peptide of approx. 2000 dalton from each subunit by comparison with the newly-synthesized enzyme. It was observed that the modification of catalase was obviated by the presence of leupeptin and iodoacetamide and this information has enabled the purification of both modified and unmodified forms of the enzyme. The possible significance of these data has been discussed and the major features incorporated into a working model of peroxisomal biogenesis.
Animals↗