Biomedical subjects

J D Potter

Publications and source records attributed to J D Potter.

At least 37 records · Page 2Linked to original sources

PubMed2001

Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy.

Mutations in cardiac Troponin T (TnT) are responsible for approximately 15% of all cases of familial hypertrophic cardiomyopathy (FHC). This review summarizes recent data from in vitro assays, transgenic models and clinical studies on how TnT mutations alter the regulation of cardiac muscle contraction. Each TnT mutation has somewhat different effects on myofilament properties (increased myofilament Ca(2)+ sensitivity, decreased maximal force, decreased binding affinity to the thin filament, impaired pH-regulation). But when the in vitro data are correlated with the results from the transgenic models, essentially all mutations can be predicted to result in: (1) impaired relaxation, (2) reduced diastolic compliance, (3) reduced contractile reserve, (4) preserved systolic function under baseline conditions, and (5) cardiac dysfunction under inotropic stimulation. Thus, the alterations of myofilament function caused by TnT mutations likely play an important role in the pathogenesis of FHC.

Animals↗

PubMed2001

At the interfaces of epidemiology, genetics and genomics.

You come onto the court prepared for tennis but your partner seems to be ready for rugby. Neither of you is at all sure what it is that your opponent wants to play. The only recourse is to teach each other the rules of your own game and then decide whether you can collectively invent a new sport. Welcome to the dialogue at the intersections of epidemiology with genetics and genomics.

Alleles↗

PubMed2001

Invited Review: pathophysiology of cardiac muscle contraction and relaxation as a result of alterations in thin filament regulation.

Cardiac muscle contraction depends on the tightly regulated interactions of thin and thick filament proteins of the contractile apparatus. Mutations of thin filament proteins (actin, tropomyosin, and troponin), causing familial hypertrophic cardiomyopathy (FHC), occur predominantly in evolutionarily conserved regions and induce various functional defects that impair the normal contractile mechanism. Dysfunctional properties observed with the FHC mutants include altered Ca(2+) sensitivity, changes in ATPase activity, changes in the force and velocity of contraction, and destabilization of the contractile complex. One apparent tendency observed in these thin filament mutations is an increase in the Ca(2+) sensitivity of force development. This trend in Ca(2+) sensitivity is probably induced by altering the cross-bridge kinetics and the Ca(2+) affinity of troponin C. These in vitro defects lead to a wide variety of in vivo cardiac abnormalities and phenotypes, some more severe than others and some resulting in sudden cardiac death.

Actin Cytoskeleton↗

PubMed2001

Trans-fatty acids and colon cancer.

Trans-fatty acids have been hypothesized to be carcinogenic, although there are limited data in humans testing this hypothesis. In this study, we examine the association between trans-fatty acids and colon cancer using data from a case (n = 1,993)-control (n = 2,410) study conducted in Utah, Northern California, and Minnesota. Dietary data were collected using a detailed diet history questionnaire, and nutrient values were generated from the Nutrition Coordinating Center nutrient database. After adjustment for other variables, including age at diagnosis, body size, physical activity, aspirin and/or nonsteroidal anti-inflammatory drug (referred to collectively as NSAIDs) use, energy intake, and dietary fiber and calcium, we found a weak association in women [odds ratio (OR) = 1.5, 95% confidence interval (CI) = 1.1-2.0] but not in men (OR = 1.2, 95% CI = 0.9-1.7); no increased risk was observed for the cis form of the fatty acids. For men and women, slightly stronger associations were observed in those > or = 67 years of age (OR = 1.4, 95% CI = 0.9-2.1 for men; OR = 1.6, 95% CI = 1.0-2.4 for women). Those who did not use NSAIDs were at a 50% greater risk of developing colon cancer when they consumed high levels of trans-fatty acids. Women who were estrogen negative, i.e., postmenopausal not taking hormone replace therapy, had a twofold increase in risk from high levels of trans-fatty acids in the diet, while women who were estrogen positive did not experience an increased risk of colon cancer, regardless of level of trans-fatty acids consumed. We believe that these data have important public health implications. It seems prudent to avoid consuming partially hydrogenated fats, since no increased risk was observed for the cis form of fatty acids, while suggestions of increased risk from trans-fatty acids exist for subsets of the population.

Aged↗

PubMed2001

Breast cancer risk and "delayed" primary Epstein-Barr virus infection.

Parallel to its established causal association with both infectious mononucleosis (IM) and young adulthood Hodgkin disease (YAHD), we propose a hypothesis that "delayed" primary EBV infection (i.e., primary infection occurring during adolescence or adulthood) is associated with elevated breast cancer risk. We evaluated this hypothesis with two investigations, one descriptive and the other analytic. The descriptive study used international/United States cancer registry data to assess the association between incidence rates of breast cancer and those of YAHD. The incidence rates of the seemingly unrelated neoplasms were strongly correlated (correlation coefficients of 0.74 and 0.88 for international and United States data, respectively; these were higher than the correlation coefficients of YAHD with two other cancers that we considered). Populations with higher incidence rates corresponded to those with higher likelihood of delayed primary EBV infection. The analytical study was based on a population-based case-control study of breast cancer in middle-aged women. Age-adjusted odds ratios of breast cancer in women who reported a history of IM, relative to women who did not, increased monotonically from 0.55 [95% confidence interval (CI), 0.05-6.17] for women with 0-9 years of age at IM onset to 2.67 (CI, 1.04-6.89) for women with > or =25 years of age at IM onset (P = 0.016). An older age at tonsillectomy, another surrogate of delayed EBV exposure, was also associated with increased risk of breast cancer: odds ratios, 0.92 (CI, 0.57-1.48) and 1.76 (CI, 1.15-2.69) for women with tonsillectomy at 0-4 years of age and > or =15 years of age, respectively (P = 0.018). Adjusting for additional potential confounders did not modify the associations appreciably. The implications of the findings and a potential biological mechanism are presented.

Adolescent↗

PubMed2001

Standardization of cardiac troponin I assays: round Robin of ten candidate reference materials.

BACKGROUND: Cardiac troponin I (cTnI) results vary 100-fold among assays. As a step toward standardization, we examined the performance of 10 candidate reference materials (cRMs) in dilution studies with 13 cTnI measurement systems. METHODS: Solutions of 10 cTnI cRMs, each characterized by NIST, were shipped to the manufacturers of 13 cTnI measurement systems. Manufacturers used their respective diluents to prepare each cRM in cTnI concentrations of 1, 10, 25, and 50 microg/L. For the purpose of ranking the cRMs, the deviation of each cTnI measurement from the expected response was assessed after normalization with the 10 microg/L cTnI solution. Normalized deviations were examined in five formats. Parameters from linear regression analysis of the measured cTnI vs expected values were also used to rank performance of the cRMs. RESULTS: The three cRMs demonstrating the best overall rankings were complexes of troponins C, I, and T. The matrices for these three cRMs values differed; one was reconstituted directly from the lyophilized form submitted by the supplier; one was submitted in liquid form, lyophilized at NIST, and subsequently reconstituted; and the third was evaluated in the liquid form received from the supplier. The cRM demonstrating the fourth best performance was a binary complex of troponins C and I supplied in lyophilized form and reconstituted before distribution. CONCLUSIONS: The cRMs demonstrating the best performance characteristics in 13 cTnI analytical systems will be included in subsequent activities of the cTnI Standardization Committee of the AACC.

Algorithms↗

PubMed2001

Morphostats: a missing concept in cancer biology.

The role of specific morphogens is well established in the determination of body plans in development. A variety of morphogens have been identified; others are suspected. Pathways have been delineated. In complex tissues, the ability to maintain fidelity of microarchitectural structure is crucial. Microarchitecture is a consequence of relationships among cells, not a function of single cells. Epithelial layers, in particular, are able to maintain their microarchitecture with remarkable accuracy over many decades despite recurrent damage, regular cell turnover, and complexity of structure. Nonetheless, metaplasia and transdifferentiation (change in tissue structure without cell dysplasia) do occur, suggesting that there is the possibility of loss of control or change of control of the microarchitecture. A strong inference to be derived from the above is that there are control systems and molecules and that these are derived from cells that are outside, but plausibly adjacent to, the respective epithelia. It is postulated is that there are morphogen-like controller molecules with morphogen-like functions in adult epithelial tissues. These are responsible for the maintenance of normal tissue microarchitecture. Because the function of these putative molecules is maintenance of tissue structure, I have chosen to call them morphostats by analogy with morphogens. It seems plausible that morphostats and morphogens may constitute overlapping families of molecules. Evidence for the existence of morphostats can be derived from a variety of in vivo and in vitro data and from studies of normal tissue, precancer, and cancer, including: (a) the existence but rarity of metaplasia and transdifferentiation; (b) the fact that metaplasias are multicentric and are only one step from normal but do not show any consistent epithelial mutation; (c) the genesis of animal cancers by simple transplantation of tissues into the wrong environment and the evidence that epithelial mutation is not a feature of such transplantation carcinogenesis; (d) the fact that carcinogenesis occurs frequently at the junctions of different epithelial types, e.g., squamocolumnar junctions in gastrointestinal and genital tracts; (e) the fact that cancer-associated fibroblasts can stimulate proliferation in transformed cells but not influence normal cells; and (f) the failure to grow most epithelial organs in a fully differentiated structural pattern in monolayer culture. It is suggested that morphostats may function like morphogens inasmuch as they may act via a diffusion gradient from source mesenchymal cells and provide architectural instruction for complex adult epithelia. Morphostats may influence architecture via control of cell adhesion, apoptosis, and proliferation. Some specific predictions follow from this hypothesis, most notably, a new two-hit model of cancer: one mutation in an epithelial cell resulting in disruption of cell function and structure (e.g., dysplasia); and the other in a mesenchymal or other supporting cell resulting in disruption of tissue microarchitecture. The corollary of this is that there will be mesenchymal mutations producing microarchitectural abnormalities without epithelial dysplasia and vice versa. Disruption of the functions of morphostats may result in a variety of abnormalities. Such disruption may be a key event in carcinogenesis.

Animals↗

PubMed2001

Soyfood intake during adolescence and subsequent risk of breast cancer among Chinese women.

Many experimental but few epidemiological studies have suggested that soyfoods and their constituents have cancer-inhibitory effects on breast cancer. No epidemiological study has evaluated the association of adolescent soyfood intake with the risk of breast cancer. To evaluate the effect of soyfood intake during adolescence, one of the periods that breast tissue is most sensitive to environmental stimuli, on subsequent risk of breast cancer, we analyzed data from a population-based case-control of 1459 breast cancer cases and 1556 age-matched controls (respective response rates were 91.1% and 90.3%). Information on dietary intake from ages 13-15 years was obtained by interview from all study participants and, in addition, from mothers of subjects less than 45 years of age (296 cases and 359 controls). Odds ratios (ORs) and 95% confidence intervals (CIs) derived from unconditional logistic models were used to measure soyfood intake and breast cancer risk. After adjustment for a variety of other risk factors, adolescent soyfood intake was inversely associated with risk, with ORs of 1.0 (reference), 0.75 (95% CI, 0.60-0.93), 0.69 (95% CI, 0.55-0.87), 0.69 (95% CI, 0.55-0.86), and 0.51 (95% CI, 0.40-0.65), respectively, for the lowest to highest quintiles of total soyfood intake (trend test, P < 0.001). The inverse association was observed for each of the soyfoods examined and existed for both pre- and postmenopausal women. Adolescent soyfood intakes reported by participants' mothers were also inversely associated with breast cancer risk (P for trend < 0.001), with an OR of 0.35 (95% CI, 0.21-0.60) for women in the highest soyfood intake group. Adjustment for rice and wheat products, the major energy source in the study population, and usual adult soyfood intake did not change the soyfood associations. Our study suggests that high soy intake during adolescence may reduce the risk of breast cancer in later life.

Adolescent↗

PubMed2001

Vitamin D receptor polymorphism and the risk of colorectal adenomas: evidence of interaction with dietary vitamin D and calcium.

Laboratory studies and epidemiological investigations suggest that vitamin D plays a role in the etiology of colorectal adenomas, possibly through a mechanism mediated by the vitamin D receptor (VDR). We conducted a clinic-based case-control study to examine the association between VDR polymorphisms and colorectal adenomas. We selectively identified a random subset of 393 cases of colorectal adenomas and 406 colonoscopy-negative controls from a clinic-based case-control study conducted in the metropolitan Minneapolis/St. Paul area during 1991-1994. A self-administered questionnaire was used to collect data on dietary and supplement intake of vitamin D and calcium, as well as on demographics, physical activity, medical information, lifestyle factors, reproductive history, and anthropometry. DNA was extracted from whole blood and assayed for the BsmI VDR polymorphism using an ABI 7700 TaqMan assay. Adjusted odds ratios (OR) and 95% confidence intervals (CIs) were evaluated using logistic regression. Compared with the bb genotype (33% of controls), neither the Bb (48.8% of controls) nor the BB (18.2% of controls) genotypes was strongly associated with risk of colorectal adenomas (OR = 0.86, CI = 0.63-1.19 and OR = 0.77, CI = 0.50-1.18, respectively). However, those with the lowest tertile of vitamin D intake and the BB genotype had a lower risk of colorectal adenoma (OR = 0.24, CI = 0.08-0.76) than those with the highest tertile of intake and the bb genotype. Similarly, those with the lowest tertile of calcium intake and the BB genotype had a reduced risk of colorectal adenoma (OR = 0.34, CI = 0.11-1.06). Although it has generally been shown that higher calcium and vitamin D intake are associated with a modestly reduced risk of colorectal neoplasia, our data suggest that those with the BB BsmI VDR genotype may be at reduced risk of colorectal adenoma in the presence of lower calcium and vitamin D intake.

Adenoma↗

PubMed2001

Epoxide hydrolase Tyr113His polymorphism is associated with elevated risk of colorectal polyps in the presence of smoking and high meat intake.

Microsomal epoxide hydrolase (mEH) metabolizes polycyclic aromatic hydrocarbons, carcinogens found in cigarette smoke and cooked meat. Polymorphisms in exon 3 and exon 4 of the mEH gene have been found to alter mEH activity. We investigated the association between these polymorphisms and colorectal polyps within the Minnesota Cancer Prevention Research Unit case-control study. Cases were diagnosed with colonoscopically confirmed adenomas (n = 530) or hyperplastic polyps (n = 202); controls (n = 649) were polyp-free at colonoscopy. Smoking history and meat consumption were obtained from self-administered questionnaires before colonoscopy. mEH genotypes were determined by PCR/RFLP or oligonucleotide ligation assay. The overall risks associated with exon 3 or exon 4 polymorphisms for both adenomas and hyperplastic polyps were not statistically different from 1.0. Compared with exon 3 Tyr/Tyr, 0 pack-years, risk was highest among those with the exon 3 His/His genotype and >25 pack-years of smoking [adenoma, odds ratio (OR) = 4.9 (1.9-12.8); hyperplastic, OR = 7.7 (2.5-24.0)]. Risks were not elevated among exon 4 homozygous variants, even in the presence of heavy smoking. Fried, baked, or broiled meat intake of > or =two servings/week (high) compared with < or =one serving/week was associated with a 2-fold increase in risk of adenoma. The highest risks were seen for those with the exon 3 His/His genotype and high cooked meat intake [OR = 3.3 (1.4-7.9); reference group: Tyr/Tyr, < or = 1 serving/week). Although mEH polymorphisms are not associated with an increased risk of colorectal polyps overall, genotypes that produce a slow phenotype appear to be associated with an increased risk in the presence of smoking and high intakes of cooked meat.

Adult↗

PubMed2000

Inotropic stimulation induces cardiac dysfunction in transgenic mice expressing a troponin T (I79N) mutation linked to familial hypertrophic cardiomyopathy.

The cardiac troponin T (TnT) I79N mutation has been linked to familial hypertrophic cardiomyopathy and a high incidence of sudden death, despite causing little or no cardiac hypertrophy. In skinned fibers, I79N increased myofilamental calcium sensitivity (Miller, T., Szczesna, D., Housmans, P. R., Zhao, J., deFreitas, F., Gomes, A. V., Culbreath, L., McCue, J., Wang, Y., Xu, Y., Kerrick, W. G., and Potter, J. D. (2001) J. Biol. Chem. 276, 3743-3755). To further study the functional consequences of this mutation, we compared the cardiac performance of transgenic mice expressing either human TnT-I79N or human wild-type TnT. In isolated hearts, cardiac function was different depending on the Ca(2+) concentration of the perfusate; systolic function was significantly increased in Tg-I79N hearts at 0.5 and 1 mmol/liter. At higher Ca(2+) concentrations, systolic function was not different, but diastolic dysfunction became manifest as increased end-diastolic pressure and time to 90% relaxation. In vivo measurements by echocardiography and Doppler confirmed that base-line systolic function was significantly higher in Tg-I79N mice without evidence for diastolic dysfunction. Inotropic stimulation with isoproterenol resulted only in a modest contractile response but caused significant mortality in Tg-I79N mice. Doppler studies ruled out aortic outflow obstruction and were consistent with increased chamber stiffness. We conclude that in vivo, the increased myofilament Ca(2+) sensitivity due to the I79N mutation enhances base-line contractility but leads to cardiac dysfunction during inotropic stimulation.

Animals↗

PubMed2000

Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca2+ binding, and phosphorylation.

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.

Amino Acid Sequence↗

PubMed2000

Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation.

This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine alpha-myosin heavy chain promoter. Extensive characterization of the transgenic I79N lines compared with wild type and/or nontransgenic mice demonstrated: 1) normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca(2+) sensitivity of ATPase activity and force in skinned fibers; 3) a substantial increase in the rate of force activation and an increase in the rate of force relaxation; 4) lower maximal force/cross-sectional area and ATPase activity; 5) loss of sensitivity to pH-induced shifts in the Ca(2+) dependence of force; and 6) computer simulations that reproduced experimental observations and suggested that the I79N mutation decreases the apparent off rate of Ca(2+) from troponin C and increases cross-bridge detachment rate g. Simulations for intact living fibers predict a higher basal contractility, a faster rate of force development, slower relaxation, and increased resting tension in transgenic I79N myocardium compared with transgenic wild type. These mechanisms may contribute to mortality in humans, especially in stimulated contractile states.

Animals↗

PubMed2000

Interplay between dietary inducers of GST and the GSTM-1 genotype in colon cancer.

The purpose of this study is to determine if cruciferous vegetables and coffee, two dietary inducers of glutatione-S-transferases, interact with GSTM-1 genotype to alter risk of colon cancer. Data were available on 1579 incident cases of adenocarcinoma of the colon and 1898 population-based controls. Intake of cruciferous vegetables, specific types of cruciferous vegetable, and coffee were not associated with colon cancer; GSTM-1 genotype did not modify these associations. However, age at diagnosis and cigarette smoking appeared to be important effect modifiers of the associations between GSTM-1, cruciferous vegetables and colon cancer. Among GSTM-1 null individuals, <55 years at diagnosis, we observed an inverse association between colon cancer and high levels of cruciferous vegetable intake relative to people who did not eat cruciferous vegetables (ORs 0.23 95% CI 0.10-0.54); broccoli was the cruciferous vegetable associated with the strongest inverse association (OR 0.30 95% CI 0.13-0.70). Among younger individuals who were GSTM-1 present (relative to those with GSTM-1 null), we observed an inverse association with colon cancer regardless of level of cruciferous vegetable intake (OR 0.74 95% CI 0.30-1.79 for no intake; OR 0.44 95% CI 0.21-0.92 for <4 servings/week; and OR 0. 44 95% CI 0.19-0.99 for >/=4 servings/week). These associations were further modified by cigarette smoking. People <65 years of age who smoked had a greater reduction in risk of colon cancer from consumption of cruciferous vegetables than non-smokers at the same age. In summary, although cruciferous vegetables do not appear to modify colon cancer risk in the total population, there are subgroups of the population for whom these vegetables may be important. These subgroups are defined mostly by age and smoking status.

Adenocarcinoma↗

PubMed2000

Association of menstrual and reproductive factors with breast cancer risk: results from the Shanghai Breast Cancer Study.

The incidence of breast cancer among women in Shanghai, a traditionally low-risk population, has increased substantially over the past 20 years. To evaluate the association of menstrual and reproductive factors with breast cancer risk and the influence of these factors on the temporal trend of breast cancer incidence, we analyzed data from the Shanghai Breast Cancer Study, a population-based case-control study of breast cancer recently completed among Chinese women in urban Shanghai. In-person interviews were completed for 1,459 women newly diagnosed with breast cancer between ages 25 and 64 and for 1,556 controls frequency-matched to cases by age. Unconditional logistic regression was employed to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs) related to menstrual and reproductive factors. Earlier menarcheal age, nulliparity, and later age at first live birth were associated with increased risk of breast cancer among both pre- and post-menopausal women, while never having breast-fed and later age at menopause were associated with elevated risk only among post-menopausal women. Among controls, 32% of younger women (</=40 years) and 24% of older women (>40 years) reported starting menarche at age of 13 or younger, and this factor contributed to 44% of cases diagnosed among younger women and 26% to 28% of cases in older women. Older age at first live birth or at menopause explained a considerable portion of cases diagnosed in older, but not younger, women. Our study suggests that the changes in menstrual and reproductive patterns among women in Shanghai have contributed to the recent increase in breast cancer incidence, particularly among younger women.

Adult↗

PubMed2000

Structure-fluorescence correlations in a single tryptophan mutant of carp parvalbumin: solution structure, backbone and side-chain dynamics.

Heterogeneous fluorescence intensity decays of tryptophan in proteins are often rationalized using a model which proposes that different rotameric states of the indole alanyl side-chain are responsible for the observed fluorescence lifetime heterogeneity. We present here the study of a mutant of carp parvalbumin bearing a single tryptophan residue at position 102 (F102W) whose fluorescence intensity decay is heterogeneous and assess the applicability of a rotamer model to describe the fluorescence decay data. We have determined the solution structure of F102W in the calcium ligated state using multi-dimensional nuclear magnetic resonance (NMR) and have used the minimum perturbation mapping technique to explore the possible existence of multiple conformations of the indole moiety of Trp102 of F102W and, for comparison, Trp48 of holo-azurin. The maps for parvalbumin suggest two potential conformations of the indole side-chain. The high energy barrier for rotational isomerization between these conformers implies that interwell rotation would occur on time-scales of milliseconds or greater and suggests a rotamer basis for the heterogeneous fluorescence. However, the absence of alternate Trp102 conformers in the NMR data (to within 3 % of the dominant species) suggests that the heterogeneous fluorescence of Trp102 may arise from mechanisms independent of rotameric states of the Trp side-chain. The map for holo-azurin has only one conformation, and suggests a rotamer model may not be required to explain its heterogeneous fluorescence intensity decay. The backbone and Trp102 side-chain dynamics at 30 degrees C of F102W has been characterized based on an analysis of (15)N NMR relaxation data which we have interpreted using the Lipari-Szabo formalism. High order parameter (S(2)) values were obtained for both the helical and loop regions. Additionally, the S(2) values imply that the calcium binding CD and EF loops are not strictly equivalent. The S(2) value for the indole side-chain of Trp102 obtained from the fluorescence, NMR relaxation and minimum perturbation data are consistent with a Trp moiety whose motion is restricted.

Amino Acid Substitution↗

PubMed2000

Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy.

To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G(1) --> A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca(2+) sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca(2+) sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca(2+) sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca(2+) regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.

Amino Acid Sequence↗

PubMed2000

Investigating the role of Ca2+-binding site IV in barnacle troponin C.

Two genetically engineered, recombinant versions of native barnacle troponin C (TnC) (BTnC,) were created from the bacterially expressed, recombinant, wild-type BTnC (BTnCWT) to investigate the role of the Ca(2+)-specific sites in force regulation. The mutant BTnC4- contains a single amino acid mutation in site IV which results in the inactivation of site IV Ca2+ binding; the mutant BTnCTrunc lacks the last II amino acids of the C-terminal, and hence most of site IV. Both mutant proteins, which retain an active site II, bind to native TnC-depleted myofibrillar bundles and restore approximately 40% of the tension-generating capacity, about half that seen with purified native BTnC1 or BTnC2. This observation implies that the Mg(2+)-dependent interaction with troponin I (TnI) is at a location on TnC other than the C-terminal Ca(2+)-binding sites of BTnC2. Replacement with BTnCTrunc increases the sensitivity of the myofibrillar bundle to changes in ionic strength. Decreasing the ionic strength from 0.15 to 0.075 M increased force by 34%, a value much greater that the 8% increase seen in control bundles or bundles substituted with BTnC4-. These findings implicate TnC in determining this fibre characteristic, although this cannot be simply due to the alteration in the numbers of Ca2+ ions bound by the troponin complex since both BTnC4- and BTnCTrunc bind only 1 mol Ca2+/mol protein.

Amino Acid Sequence↗