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R T Swank

Publications and source records attributed to R T Swank.

At least 91 records · Page 5Linked to original sources
PubMed1983

Subcellular redistribution of newly synthesized macrophage lysosomal enzymes. Correlation between delivery to the lysosomes and maturation.

Cultured mouse peritoneal macrophages were fractionated by two methods at various times after pulse labeling with [35S]methionine. The lysosomal enzymes beta-glucuronidase and beta-galactosidase were isolated from each fraction by immunoprecipitation and electrophoresis on sodium dodecyl sulfate-acrylamide gels. Two distinct peaks of label were obtained on Percoll density gradients. An early appearing peak of low density, containing the precursor forms of both enzymes, co-sedimented with markers for the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane. With time, immunoprecipitable label cosedimented with the bulk of the lysosomal enzyme activity at high density and corresponded to the mature forms of the lysosomal enzymes. By differential centrifugation, newly synthesized enzymes were found predominantly in small particle fractions, unlike the bulk of the lysosomal enzymic activity which was found in larger particle fractions. With increasing time, newly synthesized enzymes were transferred to assume a distribution similar to that of lysosomal enzymic activity. The results suggest that transport of newly synthesized enzymes to lysosomes and conversion to mature forms are closely linked events. Conversion of lysosomal precursors to mature forms occurs either in a prelysosomal vesicle or shortly after reaching the lysosome. The two enzymes follow similar subcellular pathways at similar rates. Also, the macrophage system appears suitable for direct analysis of newly synthesized lysosomal enzymes during subcellular transport.

Animals↗
PubMed1981

The mouse pale ear pigment mutant as a possible animal model for human platelet storage pool deficiency.

The mouse pigment mutant pale ear, ep/ep, which has a defect in kidney lysosomal enzyme secretion, had prolonged bleeding on experimental injury. Platelet counts and platelet protein did not differ from normal. There was, however, a deficiency in the platelet dense granule contents, serotonin, ATP, and ADP. Furthermore, a marked reduction of platelet dense granules was observed by electron microscopy. The results suggest that pale ear is a useful animal model in the study of platelet storage pool disease. Studies on this mutant and other pigment mutants have established that one gene can regulate at least three subcellular organelles, including the melanosome, the lysosome, and the platelet dense granule.

Adenosine Diphosphate↗
PubMed1980

Intracellular distribution of lysosomal enzymes in the mouse pigment mutants pale ear and pallid.

The size distribution of lysosomes was determined in kidney proximal tubule cells of two mouse pigment mutants, pale ear and pallid, which have an increase in kidney lysosomal enzyme content caused by a decreased rate of secretion of lysosomal enzymes into urine. Both mutations have larger lysosomes when compared with normal mice. However, neither mutant contains the giant lysosomes (up to 11 micron diameter) common to the well-characterized beige mutant, which has a kidney secretory defect similar to the pale ear and pallid mutants. Subcellular distribution studies, performed by the osmotic shock technique, likewise suggested differences among the pigment mutants. A very high content of soluble enzyme, indicative of lysosomal fragility during homogenization, was found in extracts from the beige mutation. By comparation, the percent of soluble enzyme became progressively lower in extracts of the pallid and pale ear mutants and was lowest in extracts from normal mice. All 3 pigment mutants had normal concentrations of osmotically resistant membrane-bound lysosomal enzymes. This indicates that the excess, non-secreted, lysosomal enzyme in all three pigment mutants likely is present in classical lysosomal organelles rather than in other non-lysosomal subcellular membrane fractions. The results also illustrate that mammalian mutants which exhibit lysosomal secretory rates can have strikingly different effects on morphology of lysosomes.

Animals↗
PubMed1980

Altered secretion of kidney lysosomal enzymes in the mouse pigment mutants ruby-eye, ruby-eye-2-J, and maroon.

Melanosomes and lysosomes share structural and biosynthetic properties. Three mouse pigment mutants, ruby-eye, ruby-eye-2-J, and maroon, have abnormally high concentrations of kidney lysosomal enzymes. concentrations of kidney nonlysosomal enzymes and of liver and serum lysosomal enzymes are normal. By light microscopy the mutants have normal kidney lysosome morphology. It does not appear that the mutant genes cause an increased rate of production of lysosomes since the increased kidney beta-glucuronidase concentration is not accompanied by a corresponding increase in rate of synthesis. The common defect in all mutants is a decreased rate of secretion of lysosomal enzymes from kidney into urine. Eight mouse pigment mutants are not known which affect both melanosome and lysosome function. They should serve as useful models for the study of the biogenesis, structure, and processing of these and other subcellular organelles.

Animals↗
PubMed1979

Biosynthesis of two lysosomal enzymes in macrophages. Evidence for a precursor of beta-galactosidase.

We have purified beta-galactosidase and beta-glucuronidase from macrophages of thioglycollate-treated mice using concanavalin A chromatography and immunoprecipitation. The apparent molecular weight of the beta-galactosidase subunit, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, changed during a long term pulse-chase experiment. Following a 1-h pulse with [3H]leucine, radiolabel was present exclusively in an Mr = 82,000 form. However, after a 3-h chase in medium containing unlabeled leucine, most label migrated at Mr = 63,000, and at 24 h, all label was in the Mr = 63,000 form. Electrophoresis of peptides produced by cyanogen bromide cleavage of immunoprecipitates demonstrated structural similarities between precursor and mature forms. A mutation in the mouse, which is known to depress the rate of synthesis of beta-galactosidase in many cell types, proportionately decreased incorporation of [3H]leucine into both the Mr = 82,000 and 63,000 forms. Therefore, by kinetic, structural, and genetic evidence, the large molecular weight beta-galactosidase is a precursor of mature macrophage enzyme. No precursor of the Mr = 75,000 subunit of beta-glucuronidase was detected.

Animals↗
PubMed1979

Lysosomal dysfunctions associated with mutations at mouse pigment genes.

Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure of function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5-fold increased concentrations of kidney beta-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney beta-galactosidase and alpha-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysosomal enzyme concentrations.--A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney beta-glucuronidase and beta-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.--These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice.

Animals↗
PubMed1978

Effect of potassium deficiency on mouse kidney lysosomal enzymes.

Mice of inbred strains A/J, C57BL/6J and C57BL/6J beige were kept on a K+-deficient diet for up to 40 days to determine the magnitude and mechanism of changes in tissue lysosomal enzymes. From days 10 to 40 glucuronidase activity increased 3-fold in kidney of K+-deficient mice, but there was little effect on beta-galactosidase or acid phosphatase activity. Similar increases in kidney glucuronidase activity occurred in inbred strains known to have genetically altered control of the synthesis (A/J) and secretion (C57BL/6J beige) of glucuronidase in kidney proximal-tubule cells. Deprivation of K+ did not affect glucuronidase activity in liver, spleen, lung and brain, but there was a 2-3-FOld increase in glucuronidase activity in heart in the C57BL/6J and C57BL/6J beige strains. As shown by specific antibody titration, increased glucuronidase activity in kidney of K+-deficient mice was accompanied by accumulation of enzyme molecules. Likewise in kidney of deficient mice there was an increased rate of synthesis of glucuronidase as measured by incorporation of labelled leucine into immunoprecipitable glucuronidase. In kidney of K+-deficient mice the elevated glucuronidase activity was found in both collecting-tubule and interstitial cells of the medulla. It is probable therefore that a significant fraction of the increased kidney lysosomal synthesis and enzyme activity is due to infiltrating cells.

Animals↗
PubMed1978

Roles of growth hormone and testosterone in the synthesis of mouse kidney glucuronidase.

Mouse kidney beta-glucuronidase production is under multihormonal control. In normal mice, kidney glucuronidase is induced over 100-fold by testosterone. However, hypophysectomy reduces this induction to about 5% of normal. This loss in inducibility was in part restored by growth hormone. Simultaneous administration to hypophysectomized female mice of growth hormone and testosterone, but not of prolactin and testosterone, restored kidney glucuronidase concentration to half that found in testosterone-treated normal female mice. Growth hormone alone had no effect in hypophysectomized females nor did it enhance glucuronidase activity in testosterone-treated normal females. Radiolabeling experiments demonstrated that the enhancement by growth hormone of glucuronidase activity was accompanied by a corresponding increase in its rate of synthesis. Kidney hypertrophy and kidney glucuronidase production may be under common hormonal regulation. Testosterone or growth hormone treatment alone of hypophysectomized mice had little or no effect on either process, but combined treatment with the two hormones significantly enhanced both. The rate of synthesis of kidney glucuronidase is controlled by the Gur gene. Relative differences in kidney glucuronidase synthesis in mice of different Gur genotype were maintained in testosterone-treated hypophysectomized mice. This suggests that control of glucuronidase synthesis by the Gur locus is exerted by interaction with androgens rather than pituitary products.

Animals↗
PubMed1978

Turnover of kidney beta-glucuronidase in normal and Chédiak-Higashi (beige) mice.

Kidney beta-glucuronidase turnover has been examined by specific antibody methods in normal C57BL/6J mice and in coisogenic C57BL/6J beige mice, an animal model for the human Chédiak-Higashi syndrome. No effect of the beige gene on the rate of glucuronidase synthesis was detected in either untreated or testosterone-treated mice. Moreover, glucuronidase of beige mice decayed relatively slowly in pulse labeling and in hormone withdrawal experiments. Direct measurements of secretion confirmed that both in the presence of testosterone and following its withdrawal, there was a threefold lower rate of secretion of kidney glucuronidase in beige mice. Following hormone withdrawal, the loss of glucuronidase activity in beige mice was biphasic, with the second more slowly turning over component apparently lost by a nonsecretory mechanism. This persistent nonsecreted glucuronidase activity was specifically associated with giant lysosomes in kidney proximal tubule cells near the corticomedullary border. Thus, there are two major populations of lysosomes in proximal tubule cells of beige mice. Cells of the outer cortex contain mainly morphologically normal lysosomes, and their lysosomal enzymes are secreted at near normal rates. However, lysosomal enzymes derived from the giant lysosomes of cells near the corticomedullary border are secreted either very slowly or not at all. The altered secretion of lysosomal enzymes from specific kidney cells of beige mice may serve as a model system for study of defective fusion of lysosomes with phagocytosed bacteria in cells of Chédiak-Higashi patients.

Animals↗
PubMed1977

Differential effect of hypophysectomy on the synthesis of beta-glucuronidase and other androgen-inducible enzymes in mouse kidney.

The levels of several androgen responsive enzymes including beta-glucuronidase, alcohol dehydrogenase, D-amino acid oxidase and arginase, were compared in kidneys of normal and hypophysectomized female mice after treatment with testosterone. While hypophysectomy did not alter the basal level of glucuronidase, the androgen-mediated accumulation of kidney beta-glucuronidase was greatly decreased in hypophysectomized mice. Measurements of the rate of synthesis of glucuronidase showed that after androgen treatment the enzyme was synthesized in kidney of hypophysectomized mice at only 5% the normal rate. Glucuronidase activity in seven other organs was not appreciably affected by treatment with androgens or by hypophysectomy. Unlike the effect of hypophysectomy on kidney glucuronidase, there was no reduction in the accumulation of alcohol dehydrogenase or D-amino acid oxidase in kidney of hypophysectomized mice after androgen treatment. Hypophysectomy caused a large reduction in kidney arginase activity. However, subsequent administration of testosterone restored much of this activity. It is concluded that there are at least two mechanisms by which androgens increase enzyme activity in kidney. The normal increase in activity or rate of synthesis of beta-glucuronidase following androgen administration requires pituitary hormones and/or products of these hormones, while the increase in activity of enzymes like alcohol dehydrogenase and D-amino acid oxidase does not require pituitary hormones.

Adrenalectomy↗
PubMed1976

The Chediak-Higashi (beige) mutation in two mouse strains. Allelism and similarity in lysosomal dysfunction.

A mutation called beige, with a phenotype similar to that of the human Chediak-Higashi syndrome, has occurred independently in two inbred strains of mice. Beige-J (bgj) occurred as a spontaneous mutation in the C57B1/6J strain and beige (bg) was radiation-induced in mice of heterogenous background which were then inbred as strain SB/Le (bg/bg), the subject of the present study. As in the previously characterized C57Bl/6J beige-J mutant, there is a correlation between abnormal lysosome structure and defective lysosome function in SB/Le beige mice. They secrete much less than normal amounts of lysosomal enzymes from proximal tubule cells and, hence, have increased lysosomal enzyme activity in kidney. In addition, after treatment of either beige strain with androgen, numerous giant beta-glucuronidase-containing lysosomes are present in kidney proximal tubule cells near the corticomedullary border. By directly measuring the rate of beta-glucuronidase synthesis in androgen-treated SB/Le beige mouse kidney, it was shown that the greater accumulation of this lysosomal enzyme in proximal tubule cells was not due to an increase in its rate of synthesis. Genetic analysis of the beige mutations in the two mutant strains demonstrated that both mutant genes are recessive and, in fact, are allelic. The results suggest that both beige strains are defective in intracellular motility of lysosomes and/or their fusion with cellular membranes, and that both mutant strains are suitable experimental models for the human Chediak-Higashi syndrome.

Acetylglucosaminidase↗
PubMed1976

Eosinophil and neutrophil granulocyte exudation in the Chediak-Higashi (beige) mouse.

Humans with Chediak-Higashi syndrome (CHS) and mice carrying the beige mutation have a heritable defect which results in the presence of giant inclusion granules in the cytoplasm in a wide variety of cells and a markedly increased susceptibility to infections. To test whether this increased susceptibility to infection might be a consequence of impaired accumulation of granulocytes at sites of inflammatory-immune stimulation, we quantitated the exudation of granulocytes into the peritoneum in response to secondary tetanus toxoid challenge in normal mice and in two inbred mouse strains with the beige mutation. Neutrophil and eosinophil granulocyte responses in the peritoneal cavity were not diminished in the beige mice as compared to normal mice when previously sensitized animals were challenged intraperitoneally with tetanus toxoid. Since accumulation of cells at the in vivo site of inflammatory immune stimulation did not seem impaired in the mutant beige mice, it would appear that their increased susceptibility to infections is not due to impairment of cellular exudative responses, including the immune components of the eosinophil response.

Animals↗
PubMed1975

The molecular genetics of mammalian glucuronidase.

The genetic factors known to be involved in the final realization of beta-glucuronidase activity in mice are considered from the standpoint of structural genes determining the catalytic activity of enzyme molecules as well as the recognition features of enzyme molecules that identify them for subsequent processing by the cell; processing genes determining the cellular apparatus involved with the conjugation, intracellular localization and eventual degradation of enzyme molecules; regulatory genes determining rates of enzyme synthesis, especially in response to physiological signals such as hormones; and temporal genes determining the developmental programs for expression of these classes during growth and differentiation. The properties of genetic variants of beta-glucuronidase falling into each of these classes are described. When those results are considered in concert with the properties of genetic variants known for other mammalian enzymes several generalizations emerge. Structural genes of enzymes are not usually linked to the processing genes determining the post-assembly events in the life of that enzyme. In contrast, all of the regulatory and temporal gene sites so far identified are in close proximity to the structural genes they modulate. Regulatory and temporal sites appear to act in a cis fashion to control the amount of enzyme synthesized from the adjacent structural allele on the same chromosome.

Animals↗
PubMed1975

Defective lysosomal enzyme secretion in kidneys of Chediak-Higashi (beige) mice.

The beige mouse is an animal model for the human Chediak-Higashi syndrome, a disease characterized by giant lysosomes in most cell types. In mice, treatment with androgenic hormones causes a 20-50-fold elevation in at least one kidney lysosomal enzyme, beta-glucuronidase. Beige mice treated with androgen had significantly higher kidney beta-glucuronidase, beta-galactosidase, and N-acetyl-beta-D-glucosaminidase (hexosaminidase) levels than normal mice. Other androgen-inducible enzymes and enzyme markers for the cytosol, mitochondria, and peroxisomes were not increased in kidney of beige mice. No significant lysosomal enzyme elevation was observed in five other organs of beige mice with or without androgen treatment, nor in kidneys of beige females not treated with androgen. Histochemical staining for glucuronidase together with subcellular fractionation showed that the higher glucuronidase content of beige mouse kidney is caused by a striking accumulation of giant glucuronidase-containing lysosomes in tubule cells near the corticomedullary boundary. In normal mice lysosomal enzymes are coordinately released into the lumen of the kidney tubules and appreciable amounts of lysosomal enzymes are present in the urine. Levels of urinary lysosomal enzymes are much lower in beige mice than in normal mice. It appears that lysosomes may accumulate in beige mice because of defective exocytosis resulting either from decreased intracellular motility of lysosomes or from their improper fusion with the plasma membrane. A similar defect could account for characteristics of the Chediak-Higashi syndrome.

Alcohol Oxidoreductases↗