Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

A proteolytic activity has been shown to be associated with the heat shock protein 70 (hsp70). In order to study this, I have constructed RNA transcribing vectors with the coding sequences of the D. melanogaster (pBUG7) and the human (pMAN70) genes coding hsp70, and with an internal deletion (pBUG301) in D. melanogaster. Proteins from 37 kDa to 70 kDa were translated in a rabbit reticulocyte lysate in the presence of 35S-methionine from RNA synthesized in vitro off the full length templates (pBUG7, and pMAN70), or altered templates. Restriction digestion of pBUG7 with BamH I and Nar I yields templates that produce carboxy-terminal truncated proteins of 37 kDa and 61 kDa respectively. The full length and the truncated proteins contain a proteolytic activity when assayed by SDS/PAGE in two dimensions. The internally deleted protein does not maintain the proteolytic activity. The proteolytic activity was shown not to be the result of non-enzymatic cleavage. A general serine proteinase inhibitor eliminates the proteolytic activity of the full length human and D. melanogaster hsp70. This evidence shows that the proteolytic activity is directly connected to hsp70.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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Ho, Sin Chu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 112-135). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.ii / Acknowledgement --- p.iii / Table of Contents --- p.iv / List of Abbreviations --- p.vii / List of Figures --- p.x / List of Tables --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Test Organisms: Daphnia magna and Moina macrocopa --- p.1 / Chapter 1.2 --- Biomarkers --- p.2 / Chapter 1.3 --- Heat shock protein (HSP) --- p.4 / Chapter 1.3.1 --- Regulation of hsp70 gene expression --- p.5 / Chapter 1.3.2 --- Review of studies on hsp70 of various species --- p.6 / Chapter 1.3.3 --- Review of studies using molecular methods in Daphnia and Moina --- p.7 / Chapter 1.4 --- Environmental stress studied --- p.9 / Chapter 1.5 --- Objectives --- p.11 / Chapter Chapter 2 --- Materials and Methods --- p.12 / Chapter 2.1 --- Experimental organisms --- p.12 / Chapter 2.2 --- "The effects of temperature, metals and nutritional stress on cladocerans" --- p.13 / Chapter 2.3 --- Demographic measurement --- p.15 / Chapter 2.4 --- Cloning and sequencing of D. magna and M. macrocopa hsp70 --- p.17 / Chapter 2.4.1 --- Acute heat shock --- p.17 / Chapter 2.4.2 --- Total RNA extraction --- p.17 / Chapter 2.4.3 --- Reverse transcription and polymerase chain reaction (PCR) --- p.18 / Chapter 2.4.4 --- Degenerative PCR --- p.21 / Chapter 2.4.5 --- Cloning and sequencing of hsp70 partial sequence --- p.25 / Chapter 2.4.6 --- Rapid amplification of cDNA ends (RACE) --- p.28 / Chapter 2.4.7 --- Confirmation of D. magna and M. macrocopa hsp70 sequences --- p.30 / Chapter 2.5 --- Molecular measurements - heat shock protein 70 gene expression --- p.31 / Chapter 2.5.1 --- Sample collection --- p.31 / Chapter 2.5.2 --- Quantification of cDNA level by real-time PCR --- p.32 / Chapter 2.5.2.1 --- Primer design --- p.32 / Chapter 2.5.2.2 --- Validation of real-time PCR conditions --- p.32 / Chapter 2.5.2.3 --- Determination of hsp70 gene expression levels --- p.35 / Chapter 2.6 --- Statistical analysis --- p.36 / Chapter Chapter 3 --- Results --- p.37 / Chapter 3.1 --- Demographic measurements --- p.37 / Chapter 3.1.1 --- Effects of temperature on life history parameters of M macrocopa --- p.37 / Chapter 3.1.2 --- Effects of temperature on life history parameters of D. magna --- p.40 / Chapter 3.1.3 --- Effects of Metals on life history parameters of M. macrocopa --- p.42 / Chapter 3.1.4 --- Effects of Anabaena on life history parameters of M. macrocopa --- p.47 / Chapter 3.2 --- Cloning and sequencing of hsp70 --- p.50 / Chapter 3.2.1 --- Partial sequences of D. magna and M. macrocopa hsp70 --- p.50 / Chapter 3.2.2 --- Sequences of 3´ة and 5´ة RACE amplified products --- p.53 / Chapter 3.2.3 --- Full sequences of D. magna and M. macrocopa hsp70 --- p.53 / Chapter 3.2.4 --- Phylogenetic relationships of the hsp70 gene --- p.60 / Chapter 3.3 --- Molecular measurements - heat shock protein 70 gene expression --- p.71 / Chapter 3.3.1 --- Validation of real-time PCR conditions --- p.71 / Chapter 3.3.2 --- Effect of temperature stress on hsp70 gene expression levels --- p.76 / Chapter 3.3.3 --- Effect of metals on hsp70 gene expression levels --- p.78 / Chapter 3.3.4 --- Effect of Anabaena on Hsp70 Gene Expression Levels --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Demographic responses of M. macrocopa and D. magna by different stress --- p.85 / Chapter 4.1.1 --- Effects of temperature on life history parameters --- p.85 / Chapter 4.1.2 --- Effects of metal stress on life history parameters --- p.87 / Chapter 4.1.3 --- Effects of Anabaena on life history parameters --- p.89 / Chapter 4.2 --- Characterization of hsp70 of M. macrocopa and D. magna --- p.92 / Chapter 4.3 --- Hsp70 gene expression in response to different stress --- p.94 / Chapter 4.3.1 --- Effect of temperature stress on hsp70 gene expression levels --- p.94 / Chapter 4.3.2 --- Effect of metals on Hsp70 gene expression levels --- p.97 / Chapter 4.3.3 --- Effect of Anabaena on hsp70 gene expression levels --- p.101 / Chapter 4.4 --- Comparison of demographic and molecular measurements --- p.102 / Chapter 4.5 --- Performance of hsp70 gene expression as a biomarker of environmental stress --- p.106 / Chapter 4.6 --- Conclusion --- p.111 / References --- p.112 / Appendix --- p.136

The immunogenicity and protective efficacy of a DNA vaccine encoding a truncated groEL heat shock gene (pcDNA3/groEL) from Burkholderia pseudomallei was evaluated in vaccinated BALB/c mice infected with B. pseudomallei or B. cenocepacia. After vaccination, the levels of anti-GroEL total IgG and IgG2a were increased in mouse sera. The clonal expansion of the spleen cells increased, and the GroEL protein induced IFN-£^ production by spleen cells. The anti-GroEL antibody-mediated opsonic killing effect was not able to eliminate the growth of B. pseudomallei but was able to eliminate the growth of B. cenocepacia. After intravenous challenge of the vaccinated Balb/c mice with B. pseudomallei, the number of bacteria colonizing the in liver and/or spleen was not reduced. Over 50% of vaccinated mice infected with B. pseudomallei died within 7 days post-infection. By contrast, the bacterial loads in organs were significantly reduced if the vaccinated mice were infected with B. cenocepacia. All of vaccinated mice were alive 7 days post-infection. Liver damage, as assessed by histological observation, and abnormalities in the levels of liver enzymes rapidly resolved in vaccinated mice. We suggest that B. pseudomallei groEL plasmid DNA immunization of Balb/c mice induces a Th1-type immune response and provides cross-protection against B. cenocepacia but not against B. pseudomallei infection.

­^ ¤å ºK ­n We investigated the role of heat shock proteins (HSPs), particularly HSP60, HSP70 or HSP90 in E. coli lipopolysaccharide (LPS)-induced naïve pheochrommocytoma cell (PC12) death. PC12 cells seeded at a density of 1x105 cells per poly-L-lysine-coated 3.5 cm diameter polystyrene dish were incubated with LPS (1 mg/ml; serotype O55:B5) for 3, 6, 12, or 24 hr. Cell viability was measured by trypan blue test, and expression of HSP60, HSP70, and HSP90 were detected by Western blot analysis. We found that the viability of PC12 cell decreased significantly after treatment with LPS for 12 hr, and viability was only 30% at 24 hr post-treatment. Western blot analysis revealed that LPS-induced PC12 cell death was associated with an increase in HSP70 or HSP60. HSP70 was markedly up-regulation at 12 hr; and both HSP70 and HSP60 increased significantly by over 1000% and 200%, respectively, 24 hr after administration of LPS. There was no significant change in HSP90 level 3, 6, 12, or 24 hr after LPS treatment. To further investigate the role of HSP70, 60, or HSP90 in LPS-induced PC12 cell death, we treated PC12 cells with hsps antisense oligonucleotide (AODN) for 24 hr. The effects of LPS on cell viability and HSP60, HSP70, or HSP90 expression were again tested. We found that suppression of HSP70 or HSP60 expression accelerated the process of LPS-induced cell death. A reduction in HSP90 level, however, had little effect. The study revealed that HSP70 and HSP60 played an anti-death role during LPS-induced PC12 cell death, and HSP90 did not appear to be involved.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 174-188).

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