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Papers by Brett Hambly

Research paper thumbnail of Fluorescence energy transfer between Cys-10 residues in F-actin filaments

Biochemistry international

Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-... more Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-[2-((iodoacetyl)amino)-ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) as a fluorescence energy donor and 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) as the acceptor. Both labels were covalently attached to Cys-10 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Cys-10 was calculated to be 23 A. This corresponds to a distance between adjacent sites along the long pitch helix of 56.1 A and along the genetic helix of 53.3 A.

Research paper thumbnail of Identification of the nucleotide and phalloidin binding sites on actin

Research paper thumbnail of Structural and functional domains on actin

Research paper thumbnail of Localization of the phalloidin and nucleotide-binding sites on actin

European Journal of Biochemistry

Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide... more Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy-transfer spectroscopy. They are separated by less than 1 .0 nm. Added confirmation of the close proximity of phalloidin to nucleotide was obtained by extracting a small peptide-ATP complex from an actin digest. The peptide comprises residues 114-118, whch'are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815-28181. The results suggest that phalloidin has two major effects. It traps actin monomers in a conformation which appears to be distinct from G-actin and it stabilizes the structure of F-actin, an event accompanied by the trapping of ADP.

Research paper thumbnail of Fang Y, Gong SJ, Xu YH, Hambly BD, Bao SImpaired cutaneous wound healing in granulocyte/macrophage colony-stimulating factor knockout mice. Br J Dermatol 157:458-465

British Journal of Dermatology

Wound healing involves various cells and cytokines, resulting in the regular progression of remod... more Wound healing involves various cells and cytokines, resulting in the regular progression of remodelling events. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a multifunctional pleiotropic cytokine and is known to facilitate wound healing, although the precise molecular and cellular mechanisms remain to be explored. To use GM-CSF gene knockout (GM-CSF KO) mice to investigate the role of GM-CSF in cutaneous wound healing following full-thickness skin injury. Full-thickness skin wounds were made in GM-CSF KO and wild-type mice. The wound closure, leucocyte infiltration, vascularization and extent of cytokine production were determined. Wound healing was significantly delayed in GM-CSF KO mice, accompanied by reduced cytokine production (interleukin-6, monocyte chemoattractant protein-1 and macrophage inflammatory protein-2), and platelet-endothelial cell adhesion molecule-1 expression. Consequently there was reduced recruitment of neutrophils and macrophages and reduced vascularization in the wounds of GM-CSF KO mice. Although collagen deposition was delayed, it was significantly increased in the wounds of the GM-CSF KO mice in the later stages of wound healing. We conclude that GM-CSF plays an important role in the complex network of effector molecules that regulate keratinocyte proliferation and the inflammatory response. These data have important implications for further development of the therapeutic manipulation of wound healing using GM-CSF.

Research paper thumbnail of A proposed mechanism for the binding of phalloidin to actin and the relationship between the phalloidin and nucleotide binding sites

Molecular and Cellular Biology

Research paper thumbnail of Granulocyte-macrophage colony-stimulating factor enhances wound healing in diabetes via upregulation of proinflammatory cytokines

British Journal of Dermatology

Chronic ulceration, especially in diabetes, remains a substantial clinical problem. Exogenous gra... more Chronic ulceration, especially in diabetes, remains a substantial clinical problem. Exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) is efficacious in the treatment of chronic wound healing in both animal models and patients, but its role in diabetic wounds remains to be explored. Objectives Using a diabetic mouse model, to investigate the role of GM-CSF in wound healing. Clinical observation, histopathology, immunohistochemistry and cytokine assays. There was a significant reduction (50%) in GM-CSF production in the wounds of the diabetics compared with nondiabetics. Exogenous GM-CSF substantially enhanced the wound healing in diabetic mice, accompanied by increased interleukin-6 and monocyte chemoattractant protein-1 production. The elevated cytokines correlated with increased neovascularization, and infiltration of macrophages and neutrophils. GM-CSF showed no beneficial effects in nondiabetic wound healing. Our results provide useful guidelines for the clinica...

Research paper thumbnail of Relationship Between Heart Rate Variability and Angiotensinogen Gene Polymorphism in Diabetic and Control Individuals

Heart Rate Variability (HRV) is extensively used to investigate general Autonomic Nervous System ... more Heart Rate Variability (HRV) is extensively used to investigate general Autonomic Nervous System (ANS) func-tion and is affected by many factors including age, gender, pathology such as diabetes and genetic polymorphisms. One of these genetic polymorphisms is the Angiotensin Converting Enzyme (ACE) polymorphism corresponding to insertion (I) or deletion (D) of a 287-base pair sequence of DNA in intron 16 of the ACE gene (rs4340). Some studies have addressed the relationship between HRV and D/D, I/D and I/I ACE polymorphism while others combined I/D and I/I ACE groups. In this study HRV is determined for diabetic and control individuals with different ACE polymorphism considering either separate or combined I/D and I/I genotypes. Linear time domain parameters, entropy, low frequency and total power of HRV were found to be significantly different between diabetic and control individuals with combined I/I and I/D ACE polymorphism, while only entropy was different for diabetic and contr...

Research paper thumbnail of TRPV2 in the development of experimental colitis

Scandinavian journal of immunology, 2014

Colitis is still a significant disease challenge in humans, but its underlying mechanism remains ... more Colitis is still a significant disease challenge in humans, but its underlying mechanism remains to be fully elucidated. The transient receptor potential vanilloid (TRPV) ion channel plays an important pathological role in host immunity, as deficiency of TRPV compromises host defence in vivo and in vitro. Using a DSS-induced colitis mouse model, the function of TRPV2 in the development of colitis was investigated, utilizing TRPV2(-/-) and Wt mice. Less severe colitis was observed in TRPV2(-/-) , compared to that of Wt mice, at the clinical, histopathological and immunohistochemical levels. Compared to Wt mice, reduced severity of colitis in TRPV2(-/-) mice may be due to less intestinal inflammation via reduced recruitment of macrophages. The TRPV2 pathway contributes to the development of colitis. These data provide useful information for potential therapeutic intervention in colitis patients.

Research paper thumbnail of How can food extracts consumed in the Mediterranean and East Asia suppress prostate cancer proliferation?

The British journal of nutrition, 2012

We have developed a blend of food extracts commonly consumed in the Mediterranean and East Asia, ... more We have developed a blend of food extracts commonly consumed in the Mediterranean and East Asia, named blueberry punch (BBP), with the ultimate aim to formulate a chemoprevention strategy to inhibit prostate cancer progression in men on active surveillance protocol. We demonstrated previously that BBP inhibited prostate cancer cell proliferation in vitro and in vivo. The purpose of this study was to determine the molecular mechanism responsible for the suppression of prostate cancer cell proliferation by BBP. Treatment of lymph node-metastasised prostate cancer cells (LNCaP) and bone-metastasised prostate cancer cells (PC-3 and MDA-PCa-2b) with BBP (up to 0·8 %) for 72 h increased the percentage of cells at the G0/G1 phase and decreased those at the S and G2/M phases. The finding was supported by the reduction in the percentage of Ki-67-positive cells and of DNA synthesis measured by the incorporation of 5-ethynyl-2'-deoxyuridine. Concomitantly, BBP treatment decreased the prote...

Research paper thumbnail of Evidence for an F-actin like conformation in the actin:DNase I complex

Advances in experimental medicine and biology, 1994

We demonstrate that a ribose modified analogue of ATP, TNP-ATP, can exchange with a resident nucl... more We demonstrate that a ribose modified analogue of ATP, TNP-ATP, can exchange with a resident nucleotide in F-actin, but fails to bind to G-actin. TNP-ATP is also able to bind to actin in the actin:DNase I complex, suggesting that the nucleotide binding site in the actin:DNase I complex adopts a conformation similar to that found in F-actin. This result is consistent with the hypothesis that the two major domains of actin on either side of the cleft are able to "flex" or move relative to each other in G-actin, but that this flexing motion is limited as a consequence of either polymerisation or DNase I binding. F-actin, in which approximately 80% of the bound nucleotide is TNP-ADP, appears to be functionally similar to native ADP-F-actin. It can superprecipitate with myosin and, following regulation with troponin-tropomyosin, exhibits a Ca(2+)-sensitivity during superprecipitation. Sonication induced nucleotide exchange in regulated F-actin was not sensitive to the presence ...

Research paper thumbnail of Measuring macromolecular diffusion using heteronuclear multiple-quantum pulsed-field-gradient NMR

Journal of Biomolecular Nmr, 1997

We have previously shown that (1)H pulsed-field-gradient(PFG) NMR spectroscopy provides a facile ... more We have previously shown that (1)H pulsed-field-gradient(PFG) NMR spectroscopy provides a facile method for monitoring proteinself-association and can be used, albeit with some caveats, to measure theapparent molecular mass of the diffusant [Dingley et al. (1995) J. Biomol.NMR, 6, 321-328]. In this paper we show that, for(15)N-labelled proteins, selection of(1)H-(15)N multiple-quantum (MQ) coherences in PFGdiffusion experiments provides several advantages over monitoring(1)H single-quantum (SQ) magnetization. First, the use of agradient-selected MQ filter provides a convenient means of suppressingresonances from both the solvent and unlabelled solutes. Second,(1)H-(15)N zero-quantum coherence dephases morerapidly than (1)H SQ coherence under the influence of a PFG.This allows the diffusion coefficients of larger proteins to be measuredmore readily. Alternatively, the gradient length and/or the diffusion delaymay be decreased, thereby reducing signal losses from relaxation. In orderto extend the size of macromolecules to which these experiments can beapplied, we have developed a new MQ PFG diffusion experiment in which themagnetization is stored as longitudinal two-spin order for most of thediffusion period, thus minimizing sensitivity losses due to transverserelaxation and J-coupling evolution.

Research paper thumbnail of Actin and Its Binding Proteins in Heart Failure

... Maurizio Stefani, Masako Tsubakihara, Brett D. Hambly, Choon C. Liew, Paul D. Allen, Peter S.... more ... Maurizio Stefani, Masako Tsubakihara, Brett D. Hambly, Choon C. Liew, Paul D. Allen, Peter S. Macdonald, and Cristobal G. dos Remedios ... Mogensen, J., Klausen, IC, Pedersen, AK, Egeblad, H., Bross, P., Kruse, TA, Gregersen, N., Hansen, PS, Baandrup, U. and Borglum, AD ...

Research paper thumbnail of Intrinsic synergistic-topological mechanism versus synergistic-topological matrix in microtubule self-organization

EPJ Nonlinear Biomedical Physics, 2014

Background: In this body of work we investigate the synergistic-topological relationship during s... more Background: In this body of work we investigate the synergistic-topological relationship during self-organization of the microtubule fiber in vitro, which is composed of straight, axially shifted and non-shifted, acentrosomal microtubules under crowded conditions. Methods: We used electron microscopy to observe morphological details of ordered straight microtubules. This included the observation of the differences in length distribution between microtubules in ordered and non-ordered phases followed by the observation of the formation of interface gaps between axially shifted and ordered microtubules. We performed calculations to confirm that the principle of summation of pairwise electrostatic forces act between neighboring microtubules all their entire length.

Research paper thumbnail of Molecular mechanisms of inherited thoracic aortic disease – from gene variant to surgical aneurysm

Biophysical Reviews, 2014

ABSTRACT Aortic dissection is a catastrophic event that has a high mortality rate. Thoracic aorti... more ABSTRACT Aortic dissection is a catastrophic event that has a high mortality rate. Thoracic aortic aneurysms are the clinically silent precursor that confers an increased risk of acute aortic dissection. There are several gene mutations that have been identified in key structural and regulatory proteins within the aortic wall that predispose to thoracic aneurysm formation. The most common and well characterised of these is the FBN1 gene mutation that is known to cause Marfan syndrome. Others less well-known mutations include TGF-β1 and TGF-β2 receptor mutations that cause Loeys–Dietz syndrome, Col3A1 mutations causing Ehlers–Danlos Type 4 syndrome and Smad3 and-4, ACTA2 and MYHII mutations that cause familial thoracic aortic aneurysm and dissection. Despite the variation in the proteins affected by these genetic mutations, there is a unifying pathological end point of medial degeneration within the wall of the aorta characterised by vascular smooth muscle cell loss, fragmentation and loss of elastic fibers, and accumulation of proteoglycans and glycosaminoglycans within vascular smooth muscle cell-depleted areas of the aortic media. Our understanding of these mutations and their post-translational effects has led to a greater understanding of the pathophysiology that underlies thoracic aortic aneurysm formation. Despite this, there are still many unanswered questions regarding the molecular mechanisms. Further elucidation of the signalling pathways will help us identify targets that may be suitable modifiers to enhance treatment of this often fatal condition.

Research paper thumbnail of Vascular endothelial growth factor-A: A multifunctional molecular player in diabetic retinopathy

The International Journal of Biochemistry & Cell Biology, 2009

Vascular endothelial growth factor-A (VEGF-A), first described as &am... more Vascular endothelial growth factor-A (VEGF-A), first described as "vascular permeability factor", is a critical molecule in the pathogenesis of diabetic retinopathy at several levels. Previous studies have outlined the importance of VEGF-A in mediating vascular pathology in both experimental models and clinical diabetic retinopathy, which are characterized by retinal vascular leakage, preretinal neovascularisation and neuronal degeneration. Paradoxically, recent reports have emphasized the potential neurotrophic effects of VEGF-A on the quiescent vasculature, as well as its direct and indirect protective effects on retinal neurons. VEGF-A has also been identified as an important signalling regulator in the normal central nervous system. Consequently, anti-VEGF therapy for diabetic retinopathy has become a controversal issue. This review outlines recently developed concepts relating to the role of VEGF-A in the pathogenesis of diabetic retinopathy, with particular emphasis on its implications for clinical practice.

Research paper thumbnail of Myosin binding protein—C: Enigmatic regulator of cardiac contraction

The International Journal of Biochemistry & Cell Biology, 2007

Myosin binding protein C (MyBPC) is a sarcomeric protein whose role in sarcomere structure and re... more Myosin binding protein C (MyBPC) is a sarcomeric protein whose role in sarcomere structure and regulation of contraction is currently under investigation. It is a member of the immunoglobulin superfamily and is found in the C-zone of the A-band of the sarcomere. The elongated structure of MyBPC is composed of a series of immunoglobulin and fibronectin domains, with the C-terminal domains binding to the myosin thick filament and the N-terminal domains interacting with the myosin subfragment-2 (S2) neck region and possibly the actin thin filament. The functions of MyBPC are to stabilise the sarcomere structure and to regulate contraction. When phosphorylated near its N-terminus, MyBPC no longer binds myosin-S2, causing an increase in the ordering of the myosin heads, ATPase activity, F(max) and Ca(2+) sensitivity of contraction. Mutations in MyBPC have been found to cause familial hypertrophic cardiomyopathy (FHC) and changes in MyBPC phosphorylation have been linked to cardiac ischaemia-reperfusion injury.

Research paper thumbnail of Localization of the phalloidin and nucleotide-binding sites on actin

European Journal of Biochemistry, 1987

Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide... more Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy-transfer spectroscopy. They are separated by less than 1 .0 nm. Added confirmation of the close proximity of phalloidin to nucleotide was obtained by extracting a small peptide-ATP complex from an actin digest. The peptide comprises residues 114-118, whch'are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815-28181. The results suggest that phalloidin has two major effects. It traps actin monomers in a conformation which appears to be distinct from G-actin and it stabilizes the structure of F-actin, an event accompanied by the trapping of ADP.

Research paper thumbnail of Interaction of phalloidin with chemically modified actin

European Journal of Biochemistry, 1987

Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance... more Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report Fed. Proc. 31, 5021. Phalloidin induces this chemically modified actin to form the same characteristic helical thread-like structure as normal F-actin. The filaments bind myosin heads and activate the myosin ATPase activity as effectively as normal F-actin. When a dansyl group is introduced at the same point [Chantler, P. D. and Gratzer, W. B. (1975) Eur. J. Biochem. 60,67-721, phalloidin still induces the polymerization. The filaments bind myosin heads and activate the myosin ATPase activity. These results indicate that Tyr-69 is not directly involved in either an actin-actin binding site or the myosin binding site on actin. Moreover, the results suggest that phalloidin binds to actin monomer in the presence of salt and its binding induces a conformational change in actin which is essential for polymerization, or that actin monomer fluctuates between in unpolymerizable and polymerizable form while phalloidin binds to actin only in the polymerizable form and its binding locks the conformation which causes the irreversible polymerization of actin.

Research paper thumbnail of Models of the actin monomer and filament from fluorescence resonance-energy transfer

European Journal of Biochemistry, 1992

We have developed algorithms for combining fluorescence resonance-energy transfer (FRET) efficien... more We have developed algorithms for combining fluorescence resonance-energy transfer (FRET) efficiency measurements into structural models which predict the relative positions of the chemical groups used in FRET. We used these algorithms to construct models of the actin monomer and filament derived solely from FRET measurements based on seven distinct loci. We found a mirrorimage pair of monomer models which best fit the FRET data. One of these models agrees well with the atomic-resolution crystal structure recently published by Kabsch et al. ]. The rootmean-square deviation between this FRET model and the crystal structure was about 0.9 nm. Other macromolecular models assembled from FRET measurements are likely to have a similar resolution. The largest discrepancy was for the CyslO locus which deviated 1.44 nm from the crystal position. We discuss the limitations of the FRET method that may have contributed to this discrepancy, and conclude that the CyslO FRET data have probably located CyslO incorrectly in the FRET monomer model. Using the FRET monomer models, we found three orientations in the filament which best fit the intermonomer FRET data. These orientations differ substantially from the atomic-resolution filament model proposed by the ], largely because of the discrepancies in the CyslO data. These data should probably be excluded from the analysis; however, this would leave too few measurements to assemble a filament model. In the near future, we hope to obtain additional FRET measurements to other actin loci so that the filament modelling can be done without the CyslO data.

Research paper thumbnail of Fluorescence energy transfer between Cys-10 residues in F-actin filaments

Biochemistry international

Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-... more Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-[2-((iodoacetyl)amino)-ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) as a fluorescence energy donor and 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) as the acceptor. Both labels were covalently attached to Cys-10 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Cys-10 was calculated to be 23 A. This corresponds to a distance between adjacent sites along the long pitch helix of 56.1 A and along the genetic helix of 53.3 A.

Research paper thumbnail of Identification of the nucleotide and phalloidin binding sites on actin

Research paper thumbnail of Structural and functional domains on actin

Research paper thumbnail of Localization of the phalloidin and nucleotide-binding sites on actin

European Journal of Biochemistry

Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide... more Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy-transfer spectroscopy. They are separated by less than 1 .0 nm. Added confirmation of the close proximity of phalloidin to nucleotide was obtained by extracting a small peptide-ATP complex from an actin digest. The peptide comprises residues 114-118, whch'are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815-28181. The results suggest that phalloidin has two major effects. It traps actin monomers in a conformation which appears to be distinct from G-actin and it stabilizes the structure of F-actin, an event accompanied by the trapping of ADP.

Research paper thumbnail of Fang Y, Gong SJ, Xu YH, Hambly BD, Bao SImpaired cutaneous wound healing in granulocyte/macrophage colony-stimulating factor knockout mice. Br J Dermatol 157:458-465

British Journal of Dermatology

Wound healing involves various cells and cytokines, resulting in the regular progression of remod... more Wound healing involves various cells and cytokines, resulting in the regular progression of remodelling events. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a multifunctional pleiotropic cytokine and is known to facilitate wound healing, although the precise molecular and cellular mechanisms remain to be explored. To use GM-CSF gene knockout (GM-CSF KO) mice to investigate the role of GM-CSF in cutaneous wound healing following full-thickness skin injury. Full-thickness skin wounds were made in GM-CSF KO and wild-type mice. The wound closure, leucocyte infiltration, vascularization and extent of cytokine production were determined. Wound healing was significantly delayed in GM-CSF KO mice, accompanied by reduced cytokine production (interleukin-6, monocyte chemoattractant protein-1 and macrophage inflammatory protein-2), and platelet-endothelial cell adhesion molecule-1 expression. Consequently there was reduced recruitment of neutrophils and macrophages and reduced vascularization in the wounds of GM-CSF KO mice. Although collagen deposition was delayed, it was significantly increased in the wounds of the GM-CSF KO mice in the later stages of wound healing. We conclude that GM-CSF plays an important role in the complex network of effector molecules that regulate keratinocyte proliferation and the inflammatory response. These data have important implications for further development of the therapeutic manipulation of wound healing using GM-CSF.

Research paper thumbnail of A proposed mechanism for the binding of phalloidin to actin and the relationship between the phalloidin and nucleotide binding sites

Molecular and Cellular Biology

Research paper thumbnail of Granulocyte-macrophage colony-stimulating factor enhances wound healing in diabetes via upregulation of proinflammatory cytokines

British Journal of Dermatology

Chronic ulceration, especially in diabetes, remains a substantial clinical problem. Exogenous gra... more Chronic ulceration, especially in diabetes, remains a substantial clinical problem. Exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) is efficacious in the treatment of chronic wound healing in both animal models and patients, but its role in diabetic wounds remains to be explored. Objectives Using a diabetic mouse model, to investigate the role of GM-CSF in wound healing. Clinical observation, histopathology, immunohistochemistry and cytokine assays. There was a significant reduction (50%) in GM-CSF production in the wounds of the diabetics compared with nondiabetics. Exogenous GM-CSF substantially enhanced the wound healing in diabetic mice, accompanied by increased interleukin-6 and monocyte chemoattractant protein-1 production. The elevated cytokines correlated with increased neovascularization, and infiltration of macrophages and neutrophils. GM-CSF showed no beneficial effects in nondiabetic wound healing. Our results provide useful guidelines for the clinica...

Research paper thumbnail of Relationship Between Heart Rate Variability and Angiotensinogen Gene Polymorphism in Diabetic and Control Individuals

Heart Rate Variability (HRV) is extensively used to investigate general Autonomic Nervous System ... more Heart Rate Variability (HRV) is extensively used to investigate general Autonomic Nervous System (ANS) func-tion and is affected by many factors including age, gender, pathology such as diabetes and genetic polymorphisms. One of these genetic polymorphisms is the Angiotensin Converting Enzyme (ACE) polymorphism corresponding to insertion (I) or deletion (D) of a 287-base pair sequence of DNA in intron 16 of the ACE gene (rs4340). Some studies have addressed the relationship between HRV and D/D, I/D and I/I ACE polymorphism while others combined I/D and I/I ACE groups. In this study HRV is determined for diabetic and control individuals with different ACE polymorphism considering either separate or combined I/D and I/I genotypes. Linear time domain parameters, entropy, low frequency and total power of HRV were found to be significantly different between diabetic and control individuals with combined I/I and I/D ACE polymorphism, while only entropy was different for diabetic and contr...

Research paper thumbnail of TRPV2 in the development of experimental colitis

Scandinavian journal of immunology, 2014

Colitis is still a significant disease challenge in humans, but its underlying mechanism remains ... more Colitis is still a significant disease challenge in humans, but its underlying mechanism remains to be fully elucidated. The transient receptor potential vanilloid (TRPV) ion channel plays an important pathological role in host immunity, as deficiency of TRPV compromises host defence in vivo and in vitro. Using a DSS-induced colitis mouse model, the function of TRPV2 in the development of colitis was investigated, utilizing TRPV2(-/-) and Wt mice. Less severe colitis was observed in TRPV2(-/-) , compared to that of Wt mice, at the clinical, histopathological and immunohistochemical levels. Compared to Wt mice, reduced severity of colitis in TRPV2(-/-) mice may be due to less intestinal inflammation via reduced recruitment of macrophages. The TRPV2 pathway contributes to the development of colitis. These data provide useful information for potential therapeutic intervention in colitis patients.

Research paper thumbnail of How can food extracts consumed in the Mediterranean and East Asia suppress prostate cancer proliferation?

The British journal of nutrition, 2012

We have developed a blend of food extracts commonly consumed in the Mediterranean and East Asia, ... more We have developed a blend of food extracts commonly consumed in the Mediterranean and East Asia, named blueberry punch (BBP), with the ultimate aim to formulate a chemoprevention strategy to inhibit prostate cancer progression in men on active surveillance protocol. We demonstrated previously that BBP inhibited prostate cancer cell proliferation in vitro and in vivo. The purpose of this study was to determine the molecular mechanism responsible for the suppression of prostate cancer cell proliferation by BBP. Treatment of lymph node-metastasised prostate cancer cells (LNCaP) and bone-metastasised prostate cancer cells (PC-3 and MDA-PCa-2b) with BBP (up to 0·8 %) for 72 h increased the percentage of cells at the G0/G1 phase and decreased those at the S and G2/M phases. The finding was supported by the reduction in the percentage of Ki-67-positive cells and of DNA synthesis measured by the incorporation of 5-ethynyl-2'-deoxyuridine. Concomitantly, BBP treatment decreased the prote...

Research paper thumbnail of Evidence for an F-actin like conformation in the actin:DNase I complex

Advances in experimental medicine and biology, 1994

We demonstrate that a ribose modified analogue of ATP, TNP-ATP, can exchange with a resident nucl... more We demonstrate that a ribose modified analogue of ATP, TNP-ATP, can exchange with a resident nucleotide in F-actin, but fails to bind to G-actin. TNP-ATP is also able to bind to actin in the actin:DNase I complex, suggesting that the nucleotide binding site in the actin:DNase I complex adopts a conformation similar to that found in F-actin. This result is consistent with the hypothesis that the two major domains of actin on either side of the cleft are able to "flex" or move relative to each other in G-actin, but that this flexing motion is limited as a consequence of either polymerisation or DNase I binding. F-actin, in which approximately 80% of the bound nucleotide is TNP-ADP, appears to be functionally similar to native ADP-F-actin. It can superprecipitate with myosin and, following regulation with troponin-tropomyosin, exhibits a Ca(2+)-sensitivity during superprecipitation. Sonication induced nucleotide exchange in regulated F-actin was not sensitive to the presence ...

Research paper thumbnail of Measuring macromolecular diffusion using heteronuclear multiple-quantum pulsed-field-gradient NMR

Journal of Biomolecular Nmr, 1997

We have previously shown that (1)H pulsed-field-gradient(PFG) NMR spectroscopy provides a facile ... more We have previously shown that (1)H pulsed-field-gradient(PFG) NMR spectroscopy provides a facile method for monitoring proteinself-association and can be used, albeit with some caveats, to measure theapparent molecular mass of the diffusant [Dingley et al. (1995) J. Biomol.NMR, 6, 321-328]. In this paper we show that, for(15)N-labelled proteins, selection of(1)H-(15)N multiple-quantum (MQ) coherences in PFGdiffusion experiments provides several advantages over monitoring(1)H single-quantum (SQ) magnetization. First, the use of agradient-selected MQ filter provides a convenient means of suppressingresonances from both the solvent and unlabelled solutes. Second,(1)H-(15)N zero-quantum coherence dephases morerapidly than (1)H SQ coherence under the influence of a PFG.This allows the diffusion coefficients of larger proteins to be measuredmore readily. Alternatively, the gradient length and/or the diffusion delaymay be decreased, thereby reducing signal losses from relaxation. In orderto extend the size of macromolecules to which these experiments can beapplied, we have developed a new MQ PFG diffusion experiment in which themagnetization is stored as longitudinal two-spin order for most of thediffusion period, thus minimizing sensitivity losses due to transverserelaxation and J-coupling evolution.

Research paper thumbnail of Actin and Its Binding Proteins in Heart Failure

... Maurizio Stefani, Masako Tsubakihara, Brett D. Hambly, Choon C. Liew, Paul D. Allen, Peter S.... more ... Maurizio Stefani, Masako Tsubakihara, Brett D. Hambly, Choon C. Liew, Paul D. Allen, Peter S. Macdonald, and Cristobal G. dos Remedios ... Mogensen, J., Klausen, IC, Pedersen, AK, Egeblad, H., Bross, P., Kruse, TA, Gregersen, N., Hansen, PS, Baandrup, U. and Borglum, AD ...

Research paper thumbnail of Intrinsic synergistic-topological mechanism versus synergistic-topological matrix in microtubule self-organization

EPJ Nonlinear Biomedical Physics, 2014

Background: In this body of work we investigate the synergistic-topological relationship during s... more Background: In this body of work we investigate the synergistic-topological relationship during self-organization of the microtubule fiber in vitro, which is composed of straight, axially shifted and non-shifted, acentrosomal microtubules under crowded conditions. Methods: We used electron microscopy to observe morphological details of ordered straight microtubules. This included the observation of the differences in length distribution between microtubules in ordered and non-ordered phases followed by the observation of the formation of interface gaps between axially shifted and ordered microtubules. We performed calculations to confirm that the principle of summation of pairwise electrostatic forces act between neighboring microtubules all their entire length.

Research paper thumbnail of Molecular mechanisms of inherited thoracic aortic disease – from gene variant to surgical aneurysm

Biophysical Reviews, 2014

ABSTRACT Aortic dissection is a catastrophic event that has a high mortality rate. Thoracic aorti... more ABSTRACT Aortic dissection is a catastrophic event that has a high mortality rate. Thoracic aortic aneurysms are the clinically silent precursor that confers an increased risk of acute aortic dissection. There are several gene mutations that have been identified in key structural and regulatory proteins within the aortic wall that predispose to thoracic aneurysm formation. The most common and well characterised of these is the FBN1 gene mutation that is known to cause Marfan syndrome. Others less well-known mutations include TGF-β1 and TGF-β2 receptor mutations that cause Loeys–Dietz syndrome, Col3A1 mutations causing Ehlers–Danlos Type 4 syndrome and Smad3 and-4, ACTA2 and MYHII mutations that cause familial thoracic aortic aneurysm and dissection. Despite the variation in the proteins affected by these genetic mutations, there is a unifying pathological end point of medial degeneration within the wall of the aorta characterised by vascular smooth muscle cell loss, fragmentation and loss of elastic fibers, and accumulation of proteoglycans and glycosaminoglycans within vascular smooth muscle cell-depleted areas of the aortic media. Our understanding of these mutations and their post-translational effects has led to a greater understanding of the pathophysiology that underlies thoracic aortic aneurysm formation. Despite this, there are still many unanswered questions regarding the molecular mechanisms. Further elucidation of the signalling pathways will help us identify targets that may be suitable modifiers to enhance treatment of this often fatal condition.

Research paper thumbnail of Vascular endothelial growth factor-A: A multifunctional molecular player in diabetic retinopathy

The International Journal of Biochemistry & Cell Biology, 2009

Vascular endothelial growth factor-A (VEGF-A), first described as &am... more Vascular endothelial growth factor-A (VEGF-A), first described as "vascular permeability factor", is a critical molecule in the pathogenesis of diabetic retinopathy at several levels. Previous studies have outlined the importance of VEGF-A in mediating vascular pathology in both experimental models and clinical diabetic retinopathy, which are characterized by retinal vascular leakage, preretinal neovascularisation and neuronal degeneration. Paradoxically, recent reports have emphasized the potential neurotrophic effects of VEGF-A on the quiescent vasculature, as well as its direct and indirect protective effects on retinal neurons. VEGF-A has also been identified as an important signalling regulator in the normal central nervous system. Consequently, anti-VEGF therapy for diabetic retinopathy has become a controversal issue. This review outlines recently developed concepts relating to the role of VEGF-A in the pathogenesis of diabetic retinopathy, with particular emphasis on its implications for clinical practice.

Research paper thumbnail of Myosin binding protein—C: Enigmatic regulator of cardiac contraction

The International Journal of Biochemistry & Cell Biology, 2007

Myosin binding protein C (MyBPC) is a sarcomeric protein whose role in sarcomere structure and re... more Myosin binding protein C (MyBPC) is a sarcomeric protein whose role in sarcomere structure and regulation of contraction is currently under investigation. It is a member of the immunoglobulin superfamily and is found in the C-zone of the A-band of the sarcomere. The elongated structure of MyBPC is composed of a series of immunoglobulin and fibronectin domains, with the C-terminal domains binding to the myosin thick filament and the N-terminal domains interacting with the myosin subfragment-2 (S2) neck region and possibly the actin thin filament. The functions of MyBPC are to stabilise the sarcomere structure and to regulate contraction. When phosphorylated near its N-terminus, MyBPC no longer binds myosin-S2, causing an increase in the ordering of the myosin heads, ATPase activity, F(max) and Ca(2+) sensitivity of contraction. Mutations in MyBPC have been found to cause familial hypertrophic cardiomyopathy (FHC) and changes in MyBPC phosphorylation have been linked to cardiac ischaemia-reperfusion injury.

Research paper thumbnail of Localization of the phalloidin and nucleotide-binding sites on actin

European Journal of Biochemistry, 1987

Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide... more Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy-transfer spectroscopy. They are separated by less than 1 .0 nm. Added confirmation of the close proximity of phalloidin to nucleotide was obtained by extracting a small peptide-ATP complex from an actin digest. The peptide comprises residues 114-118, whch'are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815-28181. The results suggest that phalloidin has two major effects. It traps actin monomers in a conformation which appears to be distinct from G-actin and it stabilizes the structure of F-actin, an event accompanied by the trapping of ADP.

Research paper thumbnail of Interaction of phalloidin with chemically modified actin

European Journal of Biochemistry, 1987

Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance... more Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report Fed. Proc. 31, 5021. Phalloidin induces this chemically modified actin to form the same characteristic helical thread-like structure as normal F-actin. The filaments bind myosin heads and activate the myosin ATPase activity as effectively as normal F-actin. When a dansyl group is introduced at the same point [Chantler, P. D. and Gratzer, W. B. (1975) Eur. J. Biochem. 60,67-721, phalloidin still induces the polymerization. The filaments bind myosin heads and activate the myosin ATPase activity. These results indicate that Tyr-69 is not directly involved in either an actin-actin binding site or the myosin binding site on actin. Moreover, the results suggest that phalloidin binds to actin monomer in the presence of salt and its binding induces a conformational change in actin which is essential for polymerization, or that actin monomer fluctuates between in unpolymerizable and polymerizable form while phalloidin binds to actin only in the polymerizable form and its binding locks the conformation which causes the irreversible polymerization of actin.

Research paper thumbnail of Models of the actin monomer and filament from fluorescence resonance-energy transfer

European Journal of Biochemistry, 1992

We have developed algorithms for combining fluorescence resonance-energy transfer (FRET) efficien... more We have developed algorithms for combining fluorescence resonance-energy transfer (FRET) efficiency measurements into structural models which predict the relative positions of the chemical groups used in FRET. We used these algorithms to construct models of the actin monomer and filament derived solely from FRET measurements based on seven distinct loci. We found a mirrorimage pair of monomer models which best fit the FRET data. One of these models agrees well with the atomic-resolution crystal structure recently published by Kabsch et al. ]. The rootmean-square deviation between this FRET model and the crystal structure was about 0.9 nm. Other macromolecular models assembled from FRET measurements are likely to have a similar resolution. The largest discrepancy was for the CyslO locus which deviated 1.44 nm from the crystal position. We discuss the limitations of the FRET method that may have contributed to this discrepancy, and conclude that the CyslO FRET data have probably located CyslO incorrectly in the FRET monomer model. Using the FRET monomer models, we found three orientations in the filament which best fit the intermonomer FRET data. These orientations differ substantially from the atomic-resolution filament model proposed by the ], largely because of the discrepancies in the CyslO data. These data should probably be excluded from the analysis; however, this would leave too few measurements to assemble a filament model. In the near future, we hope to obtain additional FRET measurements to other actin loci so that the filament modelling can be done without the CyslO data.