Research Interests
The research programme in our laboratory has two strands:
1. Determination of the molecular structure and function of proteins, using the contractile proteins as a model system. Dynamic structural changes within proteins are investigated using the spectroscopic techniques of fluorescence circular dichroic and electron paramagnetic resonance (EPR) spectroscopy. We are collaborating on projects to determine the structure of proteins at atomic resolution using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Molecular biology techniques are being used to genetically engineer and express some of the muscle proteins we are interested in, to enable new spectroscopic probe sites to be incorporated into relevant proteins and to facilitate NMR and crystallographic studies.
2. Understanding cardiovascular disease at a molecular and cellular levels. Protein biochemical and structural techniques are being applied to understanding the protein defects that occur in disease processes such as familial hypertrophic cardiomyopathy and cardiac ischaemia. Additionally, we have developed tissue culture and animal models of atherosclerosis, which are being used to examine molecular and cellular changes during atherosgensis.
Personnel
Brett D Hambly, BSc(Med), MB BS, PhD, Dip Anat
Leena Singh BSc
Sanya Rushdi BSc
Cecily Oakley
Kim Tran
Adrian Tchen
Equipment
The laboratory has recently been refurbished and is fully equipped for protein biochemistry. Facilities include high and low speed centrifuges, spectrophotometer, chromatography equipment, electrophoresis and HPLC equipment. During refurbishment, a walk-in Cold Room was added to the laboratory. All relevant equipment is fully computer networked within the Department. Tissue culture/molecular biology equipment includes a laminar flow cabinet, incubators, shakers and DNA electrophoresis equipment. Access to spectroscopy facilities is provided by the Anatomy Department (steady state and lifetime fluorimeters), the Chemistry Department (EPR spectrometer) and the Biochemistry Department (NMR).
FUNDING
Dr BD Hambly - Senior Lecturer
Dr Hambly joined the Department of Pathology as a Lecturer in early 1992. The research of this laboratory is directed towards determining the molecular structure and function of proteins, primarily the contractile proteins.
Specific Projects
1. Molecular basis of force generation by contractile proteins.
One of the most fundamental questions in biology is the way in which motor proteins generate force within cells. A constant theme of my research over the last 22 years has been to address this issue, primarily by using spectroscopic techniques, especially electron paramagnetic resonance (EPR) spectroscopy. Over the last 6 years I have formed a strategic collaboration with Prof Piotr Fajer, a senior academic at the US National Science Foundation funded National High Magnet Field Laboratory, Florida State University, Tallahassee. The primary reason for this collaboration is access to the superior EPR instrumentation in Florida that is not available within this country.
Collaborator: Prof Piotr Fajer, National High Magnet Field Laboratory, Florida State University, Tallahassee
2. Molecular structure of filamentous proteins.
I continue an important collaboration with my former PhD supervisor Prof Cris dos Remedios on the basic biophysics of the filament forming protein actin. Our shared interest in the application of spectroscopic techniques to biophysical structural biology problems provides the ongoing stimulus to this work.
Collaborator: Prof Cris dos Remedios, Anatomy and Histology, University of Sydney
3. Functional effects of familial hypertrophic cardiomyopathy mutations in sarcomeric proteins.
Mutant human cardiac sarcomeric proteins are expressed in vitro using recombinant expression techniques. The altered function of these proteins is investigated using a range of spectroscopic techniques (fluorescence, circular dichroism, NMR) and functional assays (protein binding studies, incorporation into intact cardiac tissue). The major emphasis of this work has been on the study of cardiac myosin binding protein-C.
Collaborators: Prof Ron Trent, Dr Bing Yu, Department of Clinical and Molecular Genetics, RPA Hospital
Assoc Prof Richmond Jeremy and Assoc Prof David Richmond, Cardiology, RPA Hospital
Funding: National Heart Foundation
4. Protein degradation leading to cardiac stunning following myocardial ischaemia.
We are investigating proteolysis and chemical modification of cardiac contractile proteins (particularly the troponins) following transient myocardial ischaemia, which leads to the clinical phenomenon of myocardial stunning (temporarily hypokinetic areas of the ventricular wall without necrosis). Techniques involve the assessment of protein damage in an animal model of stunning, using Western blotting, protein sequencing and physiological assessment of reconstituted damaged proteins into cardiac tissue in vitro. More recently proteomics (protein profiling of tissues samples) has been successfully applied to stunning in an important collaboration with Dr Stewart Cordwell at Australian Proteomic Analysis Facility, Macquarie University.
Collaborator: Assoc Prof Richmond Jeremy, Department of Cardiology, RPA Hospital.
Dr Stewart Cordwell, Australian Proteomic Analysis Facility, Macquarie University.
Funding: NHMRC
5. Electrical instability in revascularised myocardium following myocardial infarction.
An rabbit model of reperfusion following myocardial infarction has been developed. Electrical instability and cardiac remodeling during myocardial repair has been studied using electrophysiological techniques and histopathological morphometric analysis (and immunohistochemistry).
Collaborator: Dr Mark McGuire, Department of Cardiology, RPA Hospital
Funding: National Heart Foundation
6. Regulation of arterial smooth muscle proliferation during atherogenesis.
We have developed several animal models and tissue culture techniques to investigate neointimal proliferation of vascular smooth muscle cells. In particular the development of our unique mouse model of endothelial damage/neointimal proliferation has allowed us to access gene knockout mice to selectively examine the effects of specific proteins in neointimal proliferation. Currently we are examining the effects of interferon gamma.
Collaborator: Dr Bob Bao, Department of Pathology, University of Sydney
Funding: Industry sources
Recent Publications
1. Hambly, B., Franks, K. & Cooke, R. (1991) Orientation of spin-labeled myosin light chain-2 exchanged onto cross-bridges in glycerinated muscle fibers. Biophys. J. 59: 127-138.
2. O'Donoghue, S., Hambly, B. & dos Remedios, C. (1992) Models of the actin monomer and filament from fluorescence resonance-energy transfer. Eur. J. Biochem. 205: 591-601.
3. Hambly, B., Franks, K. & Cooke, R. (1992) Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers. Biophys. J. 63: 1306-1313.
4. dos Remedios, C. G., Kiesling, P. & Hambly, B. D. (1994) DNase I binding induces a conformational change in the actin monomer. In: "Synchotron Radiation in the Biosciences." (Ed. Chance, Sakabe & Sasaki), pp. 418-425, Clarendon Press, Oxford.
5. Hambly, B. D., Kiessling, P. & dos Remedios, C. G. (1994) Evidence for an F-actin like conformation in the actin: DNase 1 complex. Adv. Exp. Med. Biol. 358: 25-34.
6. Boey, W., Huang, B., Bennetts, J., Sparrow, J., dos Remedios, C. & Hambly, B.D. (1994) Fluorescence resonance energy transfer within the regulatory light chain of myosin. Eur. J. Biochem. 219: 603-610.
7. Dingley, A.J., Mackay, J.P., Chapman, B.E., Morris, M.B., Kuchel, P.W., Hambly, B.D. & King, G.F. (1995) Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: Application to myosin light chain 2. J. Biomol. NMR 6: 321-328.
8. Kekic, M., Huang, W., Moens, P., Hambly, B.D. & dos Remedios, C.G. (1996) Distance measurements near the myosin head-rod junction using fluorescence spectroscopy. Biophys. J. 71: 40-47.
9. Watts, A.D., Hunt, N.H., Hambly, B.D. & Chaudhri, G. (1997) Separation of tumour necrosis factor a isoforms by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 18: 1086-1091.
10. Dingley, A.J., Mackay, J.P., Shaw, G.L., Hambly, B.D. & King, G.F. (1997) Measuring macromolecular diffusion using heteronuclear multiple-quantum pulsed-field-gradient NMR. J. Biomol. NMR. 10: 1-8.
11. Yu, B., French, J., Carrier, L., Jeremy, R., McTaggart, D., Nicholson, M., Hambly, B., Semsarian, C., Richmond, D., Schwartz, K & Trent, R. (1998) Molecular pathology of familial hypertrophic cardiomyopathy caused by mutations in the cardiac myosin binding protein C gene. J. Med. Genet. 35: 205-210.
12. May, J., White, G.H., Yu, W, Huang, W., Hambly, B. and Little, D. The pathology of healing and changes in morphology of abdominal aortic aneurysms treated by endoluminal prostheses. In: "Endoluminal Vascular Prostheses." (Second Edition) (Ed. T.A.M. Chuter, C.E. Donayre and R.A. White), Futura Publication Company, New York (in press).
13. Brown, L., Yin, J.L. & Hambly, B.D. (1998) Direct cloning of PCR products into the Pinpoint Xa1-T vector protein expression system. Electrophoresis 19: 860-866
14. Gayagay, G., Yu, B., Hambly, B., Boston, T., Hahn, A., Celermajer, D. & Trent, R.J. (1998) Elite endurance athletes and the ACE I allele - role of genes in athletic performance. Hum. Genet. 103: 48-50.
15. Huang, W., Wilson, G., Brown, L., Lam, H. & Hambly, B.D. (1998) EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine. Eur. J. Biochem. 257: 457-465.
16. Chaufour, X., White, G.H., Hambly, B.D., May, J., Yu, W., Harris, J.P. & Stephen, M.S. (1998) Evaluation of the risks of using an oversized balloon catheter in the human infrarenal abdominal aorta. Eur. J. Vasc. Endovasc. Surg. 16: 142-147.
17. Thomas Palm, Ken Sale, Louise Brown, Huichun Li, Brett Hambly and Peter Fajer (1999) Intradomain Distances in Regulatory Domain of Myosin Head in Prepower and Postpower Stroke States: Fluorescence Energy Transfer. Biochemistry 38: 13026-34
18. Craig S. McLachlan, Jianlin Yin, Catherine Driussi, Adam J. Landau, Xavier Chaufour, Geoffery White, Brett Hambly and Mark A. McGuire. (1999) Delay in opening the infarct related coronary artery increases plasma Atrial Natriuretic Peptide levels. Eur. J. Pharmacol. 379: R3-4
19. Worthington, R.A., Brown, L., Chang, A.C.M., Reddel, R.R., Hambly, B.D. & Barden, J.A. (1999) Expression and localisation of stanniocalcin 1 receptors in rat bladder, kidney and ovary. Electrophoresis 20: 2071-7076
20. Jianlin Yin, Xavier Chaufour, Craig McLachlan, Mark McGuire, Geoffrey White, Nicholas King and Brett Hambly (2000) Apoptosis of vascular smooth muscle cells induced by cholesterol and its oxides in vitro and in vivo. Atherosclerosis 148: 365-374
21. C McLachlan, H Jelinek, S Kummerfeld, N Rummery, P Jusuf, B Hambly and M McGuire (2000) Cross-sectional infarct edge jaggedness does not influence ventricular electrical stability in a rabbit model of late myocardial infarct healing. Redox Report (in press)
22. C McLachlan, J Yin, P Jusuf, C Driussi, B Hambly and M McGuire. (2000) Validation of a semi-quantitative PCR method for the detection of apoptotic DNA fragmentation in a rabbit heart failure model. Jap J Physiol (in press).
23. Jianlin Yin, Craig McLachlan, Xavier Chaufour, , Mark McGuire, Geoffrey White, Virginia Turner, Nicholas King and Brett Hambly. (2000) Growth arrest-specific gene 6 expression in proliferating vascular smooth muscle cells in vitro and in vivo. Electrophoresis (in press).
24. Hambly, B.D., Walsh, B.J. and dos Remedios, C.G. (2000) Electrophoresis - a multidisciplinary and unifying technology. Electrophoresis 21: 3781-3783.
25. Pulvirenti, T.J., Yin, J.L., Chaufour, X., McLachlan, C., Hambly, B.D., Bennett, M.R. & Barden, J.A. (2000) P2X (purinergic) receptor redistribution in rabbit aorta following injury to endothelial cells and cholesterol feeding. J. Neurocytol. 29: 623-631.
26. Bruce A. J. Baumann, Brett D. Hambly, Kalman Hideg and Peter G. Fajer. (2001) The Regulatory Domain of the Myosin Head Behaves as a Rigid Lever. Biochemistry 40: 7868-7873.
27. Louise J. Brown, Nicholas Klonis, William H. Sawyer, Peter G. Fajer and Brett D. Hambly. (2001) Independent movement of the regulatory and catalytic domains of myosin heads revealed by phosphorescence anisotropy. Biochemistry 40: 8283-8291.
28. Jian L Yin, Helen Pimore, Yi Q Yan, Geoffrey McCaughan, G Alex Bishop, Brett D Hambly, Josette M Eris. (2002) Expression of growth arrest-specific gene 6 and its receptors in a rat model of chronic renal transplant rejection. Transplantation 73: 657-60.
29. Irina Dedova, Vadim Dedov, Neil Nosworthy, Brett D. Hambly, and Cris G. dos Remedios. (2002) Cofilin and DNase I Affect the Conformation of the Small Domain of Actin. Biophys. J. 82: (in press).
30. Ananth M Prasan, Hugh C K McCarron, Brett D Hambly, Gary G Fermanis, David R Sullivan, Richmond W Jeremy. (2002) Effect of treatment on ventricular function and troponin I proteolysis in reperfused myocardium. J. Mol. Cell. Cardiol. 34: 401–411.
31. Ananth M Prasan, Hugh C K McCarron, Melanie Y White, Susan V McLennan, Adrian S Tchen, Brett D Hambly, Richmond W Jeremy. (2002) Duration of ischaemia determines matrix metalloproteinase-2 activation in the reperfused rabbit heart. Proteomics (in press)
32. Louise J. Brown, Leena Singh, Kenneth L. Sale, Bing Yu, Ronald Trent, Peter G. Fajer and Brett D. Hambly. (2002) Functional and spectroscopic studies of a Familial Hypertrophic Cardiomyopathy mutation in Motif X of cardiac Myosin Binding Protein-C. Eur. Biophys. J. (in press).