Studies were conducted to determine the post-mortem stability of lysosomes in the white muscle of rainbow trout (Salmo gairdneri) and to determine whether lysosomal stability was related to rigor mortis and its subsequent dissolution. In addition, isolated lysosomes were subjected to different environments in an attempt to detect some of the factors that might influence their stability. After slaughter, fish were aged at either 4 or 15. Muscle samples were excised from each fish 1 hr post-mortem, during rigor mortis, soon after the dissolution of rigor mortis, and after a period of aging. Lysosonnes were extracted by blending the muscle in 0. 25 M sucrose-0.175 M KC1-1 mM EDTA in a solution:muscle ratio of 4:1. After blending, the mixture was centrifuged at 1000 x G to sediment the nuclear-debris pellet, followed by centrifugation of the supernatant at 27,000 x G to obtain a lysosomal pellet and a supernatant containing the soluble enzymes. The distribution of the lysosomal enzymes, cathepsin and α-glucosidase, was measured in each of the above fractions at each sampling interval. The stability of lysosomes was thus followed by monitoring the release of these lysosomal enzymes with time. The lysosomal pellet extracted at 1 hr post-naortem contained 23% of the catheptic activity of the muscle. This percentage decreased to approximately 15% by the end of rigor mortis with small changes noted thereafter. The nuclear-debris pellet contained a relatively constant amount of catheptic activity, generally 55-65%, while the soluble activity increased with decreasing lysosomal activity. Approximately one-third of the extractable lysosomes appeared to rupture and release catheptic enzymes before or during rigor mortis. Freezing and thawing the muscle four times reduced the catheptic activity in the lysosomal pellet to 8% of the total activity and increased the soluble activity to 51%. Isolated lysosomes rapidly released their enzymes in response to environmental conditions, but once adjusted they were relatively stable. Seventy-five percent of the cathepsins from lysosomes suspended in 0.25 M sucrose-0.005 M phosphate buffer (pH 6.5) at 4° remained sedimentable for 105 hr. Increasing the sucrose concentration toward 0.5 M and decreasing the incubation temperature increased the stability. The minimum release of the enzymes occurred between pH 6 and 7. Addition of salts (NaCl, KCl, CaCl₂) decreased the initial solubilization of the enzymes, but appeared to increase solubilization over a 70 hr incubation. Ca⁺² was more effective in producing this pattern than Na⁺. Fish saline had little protective effect relative to distilled water. The detergent Triton X-100 caused nearly complete solubilization of the enzyme. The solubilization of α-glucosidase in response to incubation conditions paralleled that of cathepsin but was invariably greater. This suggested that the enzymes have different binding affinities with the membrane surface. However, when the changing distribution of the two enzynnes was determined in aging muscle, an identical pattern resulted. / Graduation date: 1974

Bibliography: leaves 165-174. / vi, 174 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the synthesis and kinetic studies of substrate analogues for the human lysosomal enzyme, N-acetylgalactosamine-4-sulfatase. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry 1998

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No description available.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Bibliographies. Also available via the Internet.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 227-233).

Gaucher disease is the most prevalent lysosomal lipid storage disease caused by deficient glucocerebrosidase activity. It is transmitted as an autosomal recessive trait. Three clinical forms of Gaucher disease have been described: type 1, non-neuronopathic; type 2, acute neuronopathic; and type 3, subacute neuronopathic. It has been known that in most cases, the deficient glucocerebrosidase activity is due to mutations in the glucocerebrosidase gene. However, some mutant alleles remain unidentified. In this study, we performed DNA sequence analysis of 12 mutant alleles from 6 unrelated type 1 and type 2 Gaucher patients. Two novel mutations (649T and 1366G) from one type 1 and one type 2 Gaucher patient, and two rare mutations (48IT and 1604A) from two type 1 Gaucher patients were identified. To demonstrate that these mutations are deleterious and not neutral mutations, we inserted the full-length normal and mutant glucocerebrosidase cDNA into the genome of baculovirus AcUW1.lacZ and expressed the recombinant enzyme in Spodoptera frugiperda cells (Sf9). The levels of glucocerebrosidase, activities from crude extracts of transfected Sf9 cells with the Gaucher 649T, 1366G, 48IT, and 1604A alleles are 2.8%, 2.9%, 17.3% and 6.9% of that expressed by the normal allele [normal = 352.0 nmol/hr/mg protein, using a fluorogenic substrate 4-methylumbeffifery 1-β-D-glucopyranoside (4MUGP)]. The results demonstrated that the two novel mutations (1604A and 1366G) and the two rare mutations (481T and 1604A) are deleterious, resulting in profoundly deficient glucocerebrosidase activity and subsequent Gaucher disease. To explore the feasibility of the heterologous expression of the recombinant glucocerebrosidase in the yeast Pichia pastoris, we cloned the glucocerebrosidase cDNA into transformation vectors pPIC9K and pPlCαZ downstream of the AOX1 promoter, and integrated into yeast hosts KM71 and SMD 1168 of Pichia pastoris. The recombinant glucocerebrosidase was expressed and secreted into the induced culture medium when the native targeting signal of glucocerebrosidase cDNA was replaced by an α-factor secretion signal of Saccharomyces cerevisiae. The maximum expression level under flask culture conditions reached the specific activity of 494 nmol/hr/mg protein on a natural substrate (N-palmitoyl dihydroglucocerebroside). The secreted form of recombinant glucocerebrosidase was determined to have a molecular weight of 66 KDa. After deglycosylation, the peptide backbone has a molecular weight of 58 kDa. The recombinant enzyme exhibits similar kinetic properties to that of native glucocerebrosidase. A successive two-step chromatography procedure was developed to purify the recombinant enzyme to apparent homogeneity. / Graduate

Chemotherapy frequently fails to cure cancer patients due to toxicity or resistance to treatment. Variability in toxicity and resistance is influenced by polymorphisms and mutations in the individual's genome (Pharmacogenetics). Zebrafish has extensive evolutionary conservation with human, its genetics is a powerful gene discovery tool, and it has been described as a suitable model in cancer research. To study chemotherapy resistance, we used ENU-mutagenised zebrafish in a forward genetic screen to identify genes that modify responses to cancer chemotherapy. Zebrafish larvae were challenged with two chemotherapeutic drugs and stained with acridine orange (AO) to detect apoptosis and reveal hypo- or hyper-responders to chemotherapy. A mutation, conferring an increased uptake of AO, was identified by genetic mapping as a premature stop codon truncating the ribonuclease T2 (rnaset2) gene. Human RNASET2 encodes a putative lysosomal RNase. Lysosomal storage disorders, due to deficiencies in lysosomal hydrolases and resultant accumulation of macromolecules within lysosomes, are collectively among the commonest genetic diseases. RNASET2 deficiency in man results in a static encephalopathy arising in infancy and characterized by multifocal bilateral white matter lesions, subcortical cysts and focal enlargement of the anterior inferior horn. This doctoral thesis demonstrates that rnaset2 deficient zebrafish embryos suffer from a lysosomal storage disorder accumulating undigested ribosomal RNA (rRNA) in enlarged lysosomes within neurons of the brain. Moreover, high-field intensity μMRI revealed white matter lesions in the brain of adult rnase2 mutant animals. Thus, this zebrafish mutant can be used as a model to study the abnormalities observed in RNASET2 deficient individuals. This model suggests that the leukoencephalopathy results from a lysosomal storage disorder and provides a preclinical model for further elaborating disease mechanisms and evaluating candidate therapeutics.RNASET2 has also been advanced as a candidate tumour suppressor in several solid tumours. Recombinant rnaset2 protein has been tested in the clinic as an anti-cancer cytotoxic agent, with anti-angiogenic properties. By combining the rnaset2 mutant presented here with a transgenic melanoma model developed in the laboratory, the tumour suppressor and angiogenic role for rnaset2 was refuted.

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No description available.