1.90-kDa heat shock protein (HSP90) in cardiac sudden death

Molecular and biochemical role of heat shock protein 90 in cardiac sudden death with HCM

     The proposal bases on the results of previous study of hypertrophic cardiomyopathy (HCM) to continue the investigation of the cause-and-effect relationship between HSP90 metabolism and animal cardiac sudden death. We will focus the methodology on transgenic mice and gene knock-out mice in the future years. Wild type and mutated hsp90 from porcine or human will be inserted into expression vectors. Before conducting oocyte microinjection, the expression activity of the transgenes will be assessed in the cellular level. The transgenic mice will be identified by regular PCR and RT-PCR for verifying gene insertion and transcript expression, respectively. Stress test and/or aorta banding of the transgenic animals with their controls will be performed to evaluate the protective effect of HSP90 to the animal and cardiac sudden death. In contrast, we will microinject a hsp90 gene knock-out plasmid into mouse embryonic stem cells. The positive clone will be injected to mouse embryos to produce gene knock-out mice. By comparing the HSP90 overexpression and deficient mice, the effects of HSP90 will be realized. Taken together the above observations, we will understand the regulation mechanism of HSP90 in more detail and this may be helpful for clinician to design the prognosis and therapeutic protocols for cardiovascular diseases.
 
 
 

2.HSP70 in thermoresistant animals including mice and pigs

Study of thermoresistance in transgenic mice overexpressing procine inducible stress protein 70

      Living cells or organisms respond to heat or other enviromental insults by inducing expression of the so-called heat shock proteins (HSPs). Thermoresistance (TR) is the acquired ability to survive at a lethal temperature of the cells that have preexposed to a sublethal elevated temperature. TR can also manifestated its effect in an organism level. There have several lines of evidence demonstrated the close relathionship between HSPs and TR. Firstly, there exists a parallel trend between development and decay of TR and synthesis and degradation of HSPs, respectively. Secondly, thermosensitivity can be increased by injecting anti-HSP70 monoclonal antibody and by blocking transcription of the HSP70. In contrast, the transfected cells that overexpressed HSP70 confer the cells with TR. Thirdly, the levels of HSP70-like protein in the lizard is strongly correleted with the mean temperature surrounding the ecological area; and transcriptional regulation of this genetic basis may be responsible for the animals' stress-resistance. Lastly, the ability of inducing HSP70 synthesis in skin samples from tropical desert Tuckman is significantly higher than that samples from temperate Russian. The above observations indicate that the amount of HSP70 plays a very important role in acquired or genetically transmittable TR of a living organism.
     The ultimate goal of this two years-period proposal is to demonstrate that overexpressing of HSP70 can increase the stress resistance of the transgenic mice insulted by a lethal hyperthermia. In the first year, we will conduct the experiments of molecular and cellular biology and get the constructs including the transgene hsp70i(inducible form of hsp70) from porcine cDNA and/or genomic DNA libraries. The expression of these constructs with different promoters in mammalian cells will be monitored by RT-PCR, indirect immunofluorescence microscopy, and immunoblotting. The capable constructs will be microinjected into the oocytes and transgenic mice will be produced in the transgenic lab. at Pig Research Institute, Taiwan (PRIT). Overexpression of porcine hsp70i(phsp70i) will be assessed by techniques including RT-PCR, Southern hybridization , DNA sequencing, and/or immunoblotting. The ability against heat insults of transgenic mice overexpressing phsp70i will then be measured by following-up the survival rate of the animlas after 7 days. Once the TR evidence raised, we may have the oppertunity and may get more confidence to work on the studies about the TR of transgenic pigs overexpressing hsp70 and the affected performance such as growth rate, fat deposition, and pork quality. For a long-term view, this model may be benificial to the cardiovascular research for heart protection.
 
 

Topic II. Acute Phase Responses

3. Kawasaki Disease: early diagnosis ans effective treatment