rs9939609 FTO genotype associations with FTO methylation level influences body mass and telomere length in an Australian rural population.

BACKGROUND: The fat mass- and obesity-associated (FTO) gene influences energy homeostasis in humans. Although the obesity-related variant, rs9939609 has been replicated across a number of cohort studies, there remains significant variance and a low to modest association. Telomere length is another commonly reported obesity risk factor. We hypothesize understanding the associations between FTO rs9939609 with FTO methylation and telomere length will provide a more accurate assessment of obesity risk. METHODS: Overall, 942 participants free of diabetes or pre-diabetes were included in the retrospective study. Leukocyte genomic DNA was analyzed for rs9939609 genotyping, FTO gene methylation and leukocyte telomere length (LTL) measurement. RESULTS: In general linear models, rs9939609 AA genotypes were associated with increased fat percentage (3.15%, P=0.001), fat mass (4.16 kg, P=0.001), body mass index (BMI) (1.38, P=0.006) and waist circumference (3.35 cm, P=0.006), but not with FTO methylation or LTL in this overall population. However, when participants were stratified into higher and lower FTO methylation groups, the AA genotype possesses a 2.04-fold increased obesity risk in comparison to TT genotype (95%CI, 1.07-3.89, P=0.031) in participants with a higher FTO methylation level, but this association was absent in the lower FTO methylation sub-group. Moreover, AT and AA genotype carriers were associated with shorter LTL compared to TT carriers (P=0.020 and P=0.111, respectively) in the higher FTO methylation level group. However, this association was absent in the lower methylation group. Furthermore, FTO gene methylation level was significantly associated with LTL in the 942 samples (P=0.017). CONCLUSIONS: FTO rs9939609 is associated with obesity risk and LTL in this study, where this association is only observed at higher, but not lower, FTO methylation levels of participants. Our data suggest association of multiple factors, including FTO methylation level, may be involved in one of several mechanisms underlying the commonly reported obesity risk of this FTO polymorphism.

TRPV2 in the development of experimental colitis.

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.

Spinodal decomposition and the emergence of dissipative transient periodic spatio-temporal patterns in acentrosomal microtubule multitudes of different morphology.

We have studied a spontaneous self-organization dynamics in a closed, dissipative (in terms of guansine 5'-triphosphate energy dissipation), reaction-diffusion system of acentrosomal microtubules (those nucleated and organized in the absence of a microtubule-organizing centre) multitude constituted of straight and curved acentrosomal microtubules, in highly crowded conditions, in vitro. Our data give experimental evidence that cross-diffusion in conjunction with excluded volume is the underlying mechanism on basis of which acentrosomal microtubule multitudes of different morphologies (straight and curved) undergo a spatial-temporal demix. Demix is constituted of a bifurcation process, manifested as a slow isothermal spinodal decomposition, and a dissipative process of transient periodic spatio-temporal pattern formation. While spinodal decomposition is an energy independent process, transient periodic spatio-temporal pattern formation is accompanied by energy dissipative process. Accordingly, we have determined that the critical threshold for slow, isothermal spinodal decomposition is 1.0 ± 0.05 mg/ml of microtubule protein concentration. We also found that periodic spacing of transient periodic spatio-temporal patterns was, in the overall, increasing versus time. For illustration, we found that a periodic spacing of the same pattern was 0.375 ± 0.036 mm, at 36 °C, at 155th min, while it was 0.540 ± 0.041 mm at 31 °C, and at 275th min after microtubule assembly started. The lifetime of transient periodic spatio-temporal patterns spans from half an hour to two hours approximately. The emergence of conditions of macroscopic symmetry breaking (that occur due to cross-diffusion in conjunction with excluded volume) may have more general but critical importance in morphological pattern development in complex, dissipative, but open cellular systems.

RUNX1T1 rs34269950 is associated with obesity and metabolic syndrome.

BACKGROUND: Runt-related transcription factor 1 (RUNX1T1) isoforms are involved in adipogenesis. RUNX1T1 is mediated by the fat mass and obesity-associated (FTO). However, the extent to which RUNX1T1 single-nucleotide polymorphisms (SNPs) are associated with obesity risk or metabolic abnormalities in a community population basis is unknown. METHODS: Samples were obtained from the Australian Crossroads study bio-bank. SNPs located in the coding region and 3'untranslated regions of RUNX1T1 with minor allele frequency ≥0.05 were analysed using Taqman genotyping assays. RESULTS: Eight candidate SNPs were genotyped successfully in 1440 participants. Of these SNPs only rs34269950 located in the 'RRACH' motif, the most common N6-methyladenosine (m6A) methylation modification site (recognized by FTO), was significantly associated with obesity risk and metabolic abnormalities. Specifically, compared to AA genotype, rs34269950 del/del genotype was associated with a 1.47 [95% confidence interval (CI): 1.01-2.14, P = 0.042] fold higher rate of obesity risk. Additionally, the del/del genotype was associated with a 60% increased risk of metabolic syndrome (MetS) [odds ratio (OR) = 1.60, 95% CI: 1.10-2.32, P = 0.015], in comparison to the AA genotype. Finally, rs34269950 del/del increased the risk of a larger waist circumference (OR = 1.65, 95% CI: 1.15-2.36, P = 0.007), but not other components of MetS. CONCLUSION: Our study demonstrates that RUNX1T1 rs34269950, located in a potential FTO recognition motif, is significantly associated with waist circumference. This provides novel evidence to suggest SNPs located in RRACH motif may be involved in RNA m6A modification and mechanistic pathways that influence abdominal obesity.

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

BACKGROUND: 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. METHODS: Clinical observation, histopathology, immunohistochemistry and cytokine assays. RESULTS: 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. CONCLUSIONS: Our results provide useful guidelines for the clinical management of chronic ulceration in diabetes.

Sex-specific educational attainment is associated with telomere length in an Australian rural population.

BACKGROUND: There is limited understanding on whether and how socioeconomic status (SES), particularly educational attainment and household income, impacts on telomere length in an Australian rural context. Additionally, it is unknown whether access to health services via the Australian public or private health system influences telomere length. AIM: This study investigates whether there is a relationship between telomere length and SES indicators (income, education) as well as health insurance status in a rural Australian population. METHODS: Samples were drawn from the Australian Rural Victoria cross-sectional Crossroads Study. Leucocyte telomere length (LTL) was measured using a multiplex quantitative polymerase chain reaction method. RESULTS: Among 1424 participants, we did not find a significant main effect association with LTL across education, income level and health insurance. An exploratory finding was sex may influence the relationship between educational attainment and LTL (P = 0.021). In males, but not females, higher education was associated with longer LTL by 0.033 [95% confidence interval (CI) 0.002-0.063, P = 0.035]; in those with low education attainment, male participants had shorter LTL by 0.058 (95% CI -0.086 to -0.029) than female participants (P < 0.0001). CONCLUSION: Being male and having lower education attainment was associated with shorter telomere length in our rural population. Evidence from our study supports the importance of education on LTL in males in rural Australia. Our studies also support previous findings that LTL in later life may not be closely associated with indicators of SES.

Fluorescence energy transfer between nucleotide binding sites in an F-actin filament.

Fluorescence energy transfer between nucleotide binding sites in an F-actin filament was measured using 1-N6-ethenoadenosine diphosphate (epsilon-ADP) as a fluorescent donor and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) as an acceptor, both of which were bound to F-actin. Taking into consideration the helical structure of the F-actin filament, the radial coordinate of the nucleotide binding site was calculated to be 25 A, which corresponds to a distance between these sites along the long-pitch helix of 56.3 A and along the genetic helix of 56.7 A.

Evidence for an F-actin like conformation in the actin:DNase I complex.

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 of Ca2+ which argues against a significant conformational change in the vicinity of the nucleotide binding site during Ca(2+)-sensitive thin filament regulation.

Triamcinolone acetonide inhibits p38MAPK activation and neuronal apoptosis in early diabetic retinopathy.

OBJECTIVE: Intravitreal glucocorticoids and anti-vascular endothelial growth factor (VEGF) therapies are novel strategies for the treatment of advanced diabetic retinopathy, a condition with inflammatory and neuropathic elements. In contrast with anti-VEGF therapy, glucocorticoids may also exert neuroprotective effects. How glucocorticoids protect retinal neurons is unknown. The aims of the study are to investigate the anti-apoptotic actions of glucocorticoids on diabetic retinal neurons, and characterize the signalling pathways involved. RESEARCH DESIGN AND METHODS: The regulation of gene expression of the four p38 mitogen-activated protein kinase (MAPK) isoforms (α, ß, δ and γ) and the glucocorticoid receptor (GR) in the retinas was evaluated using quantitative RT-PCR, Western blot and immunohistochemistry. Phosphorylation of all isoforms p38MAPK (Thr180/Tyr182) and GR (S-211) was further evaluated. Apoptosis was confirmed by immunolocalization of active CASPASE-3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP) following intravitreal injection of triamcinolone acetonide (IVTA), in an early diabetic rat model (26 days after induction of diabetes. RESULTS: IVTA significantly down-regulated mRNA expression of Caspase 3. Activation of CASPASE-3, the subsequent cleavage of PARP-1 and phosphorylation of p38MAPK induced by diabetes were attenuated by IVTA treatment, concomitantly with activation by phosphorylation of the glucocorticoid receptor (GR S-211). CONCLUSIONS: IVTA activates the GR and exerts neural protective effects on retinal neurons. Inhibition of the p38MAPK pathway and activation of GR play a critical anti-apoptotic role in retinal neurons of diabetes following IVTA treatment. Both the anti-inflammatory and anti-apoptotic effects of glucocorticoids may be mediated through inhibition of the p38MAPK pathway in diabetic retinopathy.

Chronic NG-nitro-l-arginine methyl ester (L-NAME) administration in C57BL/6J mice induces a sustained decrease in c-kit positive cells during development of cardiac hypertrophy.

Chronic NG-nitro-l-arginine methyl ester (L-NAME) administration induces cardiac hypertrophy in rodent models. Our aims is to determine the role of c-kit expression in L-NAME induced cardiac hypertrophy. 12-20 week old C57BL/6J mice (5 per group) were administered L-NAME (0.325mg/ml) in the drinking water. Hearts were excised at 1-day, 2-days, 5-days, 2-weeks or 6-weeks; or controls which received no L-NAME. Ventricular cross-sectional wall thickness and individual cardiac myocytes cross-sectional area and cardiomyocyte/nuclear ratio to determine cardiac hypertrophy. Immuno-histochemical staining for c-kit, sca-1 and BCRP undertaken. Six weeks L-NAME administration induced significant cardiac hypertrophy compared to control hearts, evidenced by an increase in the thickness of the cross-sectional free ventricular wall (p<0.05) and an increase in mean individual cross-sectional area of cardiac myocytes in the LV wall (p<0.007). We observed c-kit(+) cells (predominately non-mast cell sub-types) in both healthy mice and in the L-NAME treated mice. C-kit staining in the left ventricular cross sections following L-NAME remained stable at 1 and 2 days compared to controls (p=NS). After 5 days of L-NAME we observed c-kit expression to decrease below control levels (p<0.05) and these lower levels were sustained at 2 and 6 weeks. C-kit expression does not decrease during two days of L-NAME administration, suggesting, firstly, that the later decrease in c-kit is not due to NOS inhibition directly and, secondly, there is the possibility for c-kit(+) cell differentiation into other cell types, possibly inducing myocardial cellular hyperplasia, without significant replacement of the original pool of c-kit(+) cells.

Impaired cutaneous wound healing in granulocyte/macrophage colony-stimulating factor knockout mice.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Interaction of phalloidin with chemically modified actin.

Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report [Lehrer, S. S. and Elzinga, M. (1972) Fed. Proc. 31, 502]. 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, 67-72], 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. Modification of Tyr-53 with 5-diazonium-(1H)tetrazole blocks actin polymerization [Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G. and Volk, K. E. (1976) Eur. J. Biochem. 64, 215-218]. Phalloidin is unable to induce the polymerization of this modified actin nor does it bind to it. Phalloidin does not induce the polymerization of the trypsin-digested actin core. These results indicate that the site at which phalloidin binds is involved in polymerization and the probable conformational change involved in polymerization may be modulated through this site.

Extraction of myosin light chains and actin from bovine cardiac muscle acetone powder.

A joint preparation of the two myosin light chains and actin from bovine cardiac muscle acetone powder is described. There is a significant improvement in the ease of purification, while the yield of the myosin light chains equals the best yields obtained from the use of established techniques. The actin yield greatly exceeds that obtained in an earlier published report.

Localization of the phalloidin and nucleotide-binding sites on actin.

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, which are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815-2818]. 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.

Fluorescence energy transfer between Cys-10 residues in F-actin filaments.

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.

Models of the actin monomer and filament from fluorescence resonance-energy transfer.

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 mirror-image 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. in Heidelberg [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. & Holmes, K. C. (1990) Nature 347, 37-44]. The root-mean-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 Cys10 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 Cys10 FRET data have probably located Cys10 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 Heidelberg group [Holmes, K., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44-49], largely because of the discrepancies in the Cys10 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 Cys10 data.

Separation of tumor necrosis factor alpha isoforms by two-dimensional polyacrylamide gel electrophoresis.

The mouse macrophage cell-line RAW264.7, stimulated with lipopolysaccharide, was used as a model for the study of the production of tumor necrosis factor (TNF) isoforms. TNF is synthesised initially as a 26 kDa transmembrane precursor, which is then processed enzymatically by a protease to release a mature molecule of 17 kDa. Dose-dependent production of transmembrane TNF was assessed by fractionation of cell membranes and Western blot analysis followed by autoradiography and densitometry. Isoforms of both the precursor and mature molecules were separated using two-dimensional (2-D) electrophoresis with immobilised pH gradient 3-10 linear gels as the first dimension. After radiolabelling of cells with 35S, both cell-associated and supernate-associated TNF isoforms were immunoprecipitated. A large number of protein spots were visualised on the 2-D gel map, for both the transmembrane and mature TNF species, more than have been detected previously using one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The likelihood that these putative isoforms were the result of differential glycosylation was tested by preincubating the cells with tunicamycin. This had the effect of reducing the number of protein spots, notably the higher molecular weight species. There were a number of precursor TNF isoforms that were unchanged upon tunicamycin treatment and these presumably reflect protein modifications other than glycosylation.

Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.

We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region.

Monoclonal antibodies directed against skeletal muscle actin.

The development of a monoclonal antibody directed against rabbit skeletal muscle monomeric actin is described. The production of the monoclonal antibody followed a standard hybridoma technique, the antibody being purified by affinity chromatography. It was found to be of the IgM class. Antibody specificity for rabbit skeletal actin was demonstrated by radioimmunoassay. The antibody failed to bind to actin in Western Blot experiments, presumably due to modification of the antigenic determinant on actin during the Western Blot procedure. The antibody was also shown to bind to two other isotypes of actin, i.e. actin from squid mantle muscle and bovine myocardium.

EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine.

The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half-maximal effects at TFP concentrations of approximately 14-50 microM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407-424]. EPR spectroscopy provided structural information from paramagnetic probes on C-terminal domain surfaces. In the absence of TFP, tauR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (approximately 10 ns). TFP progressively recruited the probes into populations with tauR sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of approximately 370-800 microM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to 'specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.