Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 8 uppsatser.

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination and post-germination. Here I test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating and post-germination seeds. It is demonstrated that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean seed germination and post-germination. Immunogold electron microscopy (EM) with VSR antibodies demonstrates that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in Day-1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at Day-3 post-germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating and post-germination seeds. Further immunogold EM studies demonstrate that VSR and aleurain colocalize to MVBs, as well as PSVs in germinating and post-germination seeds. Thus, MVBs in germinating and post-germination seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed, and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination and post-germination. / Storage proteins synthesized during seed development are transported to PSVs for storage. However, relatively little is known about the mechanisms of storage protein transport. A putative VSR-interacting protein termed S2 was identified as mung bean 8S globulin. Thus, I test the hypothesis that VSR proteins may be involved in storage protein transport to PSVs in developing mung bean seeds. Immunogold EM with 52 (8S globulin) antibody demonstrates that transport of 8S globulin to PSVs is Golgi-mediated, involving dense vesicle (DV) and a novel prevacuolar compartment (PVC). The novel PVC consists of storage protein aggregates and small internal vesicles. Immunogold EM with S2 (8S globulin) antibody demonstrates that MVBs contain 8S globulin at early stage of seed development. Further immunogold EM studies demonstrate that VSR and 8S globulin colocalize to DVs and the novel PVCs. In vitro binding study demonstrates that calcium ion can stabilize interaction between VSRs and 8S globulin. Thus, VSR proteins may mediate storage protein transport to PSVs via a novel PVC. / Wang, Junqi. / "March 2007." / Adviser: Jiang Liwen. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0052. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 120-131). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

No description available.

Graduation date: 1996

Iron is one of the essential elements involved in various fundamental biological activities. However, excess iron may bypass the negative feedback regulatory systems, leading to the formation of iron overload. The increase of iron deposition generates cellular toxicity and subsequently damages vital organs. Primary and secondary iron overload are affecting patients worldwide. Iron overload cardiomyopathy is the primary cause of cardiac dysfunction and cardiovascular mortality in β-thalassaemia major patients. Current effective therapy includes chelation treatment with conventional and new iron chelators, while potential new therapies are currently under development. The pathophysiology of iron overload cardiomyopathy remains unclear. Controversial findings on the mechanism of excessive iron entry into cardiomyocytes exist. Using novel real-time approach to trace iron entry into HL-1 cardiomyocytes, the only beating cardiac cell line with mature cardiac phenotype available currently, we visualized the patterns of iron entry following ferric iron incubation with and without ascorbate. Iron entry could be partly blocked by pretreatment with L-type calcium channel blockers but not T-type calcium channel blocker. Such blockage effect by L-type calcium channel blockers occurred in ferric iron overload. This finding suggested a role of L-type calcium channels for ferric iron uptake into cardiomyocytes under iron overload condition. For the pathophysiology of iron cardiac toxicity, we assessed the iron overload induced apoptosis using both in vitro and in vivo approaches. The results demonstrated that iron-overloaded mouse HL-1 atrial cardiomyocytes and human embryonic stem cell derived ventricular cardiomyocytes underwent apoptosis via the mitochondria-mediated caspase-3 dependent pathway. Supportive data was found in iron-overloaded mouse myocardium by an increase in DNA fragmentation. However, despite the blockage of iron entry, L-type calcium channel blockers did not significantly prevent iron induced apoptosis in vitro. The mechanism of cardiac contractile dysfunction caused by iron overload on cardiomyopathy has not yet been fully characterized. Given the central role of titin, the giant myofilament protein, as the main determinant in myocardial passive tension, stiffness, diastolic and systolic cardiac function, as well as myocardial twisting and untwisting motion, we investigated its expression in iron-overloaded cardiomyocytes in vitro and in vivo. Our results indicated that significant degradation of cardiomyocytes titin was induced by iron overload. This was associated with the cleavage at the elastic domain. Its potential upstream protease, calpain, was further identified to be activated under iron overload. The specific role of titin proteolysis in iron-overloaded cardiomyocytes merited further investigation. The findings in this project provided new insights to the pathophysiology of iron overload cardiomyopathy, in terms of the route for iron entry, iron induced cardiac apoptosis, and titin proteolysis. Novel therapeutic approaches for prevention and treatment of iron overload cardiomyopathy can focus on inhibiting excessive iron uptake, as well as by targeting pathways involved in cardiac apoptosis and titin proteolysis. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Not available / text

No description available.

No description available.

No description available.

The endolysosomal system is a multifunctional system and is involved in catabolism, antigen presentation and regulation of hormones. The descriptions of, and functions assigned to organelles within the system are often applied using different criteria. Further, the properties of the hydrolases within the system, and the organelles that house them are usually studied in isolation from one another. Considering that the endolysosomal system may be extremely dynamic, an understanding of the system as an integrated whole is a necessary first step. Thus, a review of the endolysosomal system was undertaken. It was determined that the enzymes within the endolysosomal system are probably subject to 'organelle-dependent' regulation, i.e. the organelles create the appropriate luminal conditions for these enzymes to work. It is also likely that the effectors of these luminal conditions are regulated in a manner that is related to GTPase networks that control much of the cell's functions. The organisation of the endolysosomal system may be hierarchical, with proteases being downstream effectors of a system that is regulated at the whole body level. The main endoprotease class within the endolysosomal system are cysteine proteases. A literature review suggested that these enzymes may not be redox regulated within the endolysosomal system. However, the lysosomal endoprotease cathepsin B has been implicated in many pathologies where it is operating in pH and redox conditions different from the endolysosomal system. To study this, cathepsin B was isolated from bovine livers using a novel procedure that exploits the ability of tertiary butanol to temporarily inhibit protease interactions in tissue homogenates. This would prevent artefactual, as well as protease-inhibitor interactions. This novel procedure resulted in increased yields of cathepsin B. Cathepsin D, an aspartic protease, was isolated using established methods. In order to test the hypothesis that cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine and/or cystine-containing redox buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0 - 7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.