Oscillatory work and the step that generates force in single myofibrils from rabbit psoas.

The elementary molecular step that generates force by cross-bridges (CBs) in active muscles has been under intense investigation in the field of muscle biophysics. It is known that an increase in the phosphate (Pi) concentration diminishes isometric force in active fibers, indicating a tight coupling between the force generation step and the Pi release step. The question asked here is whether the force generation occurs before Pi release or after release. We investigated the effect of Pi on oscillatory work production in single myofibrils and found that Pi-attached state(s) to CBs is essential for its production. Oscillatory work is the mechanism that allows an insect to fly by beating its wings, and it also has been observed in skeletal and cardiac muscle fibers, implying that it is an essential feature of all striated muscle types. With our studies, oscillatory work disappears in the absence of Pi in experiments using myofibrils. This suggests that force is generated during a transition between steps of oscillatory work production, and that the states involved in force production must have Pi attached. With sinusoidal analysis, we obtained the kinetic constants around the Pi release steps, established a CB scheme, and evaluated force generated (and supported) by each CB state. Our results demonstrate that force is generated before Pi is released, and the same force is maintained after Pi is released. Stretch activation and/or delayed tension can also be explained with this CB scheme and forms the basis of force generation and oscillatory work production.

Biomechanical evaluation of flash-frozen and cryo-sectioned papillary muscle samples by using sinusoidal analysis: cross-bridge kinetics and the effect of partial Ca2+ activation.

We examined the integrity of flash-frozen and cryo-sectioned cardiac muscle preparations (introduced by Feng and Jin, 2020) by assessing tension transients in response to sinusoidal length changes at varying frequencies (1-100 Hz) at 25 °C. Using 70-µm-thick sections, we isolated fiber preparations to study cross-bridge (CB) kinetics: preparations were activated by saturating Ca2+ as well as varying concentrations of ATP and phosphate (Pi). Our results showed that, compared to ordinary skinned fibers, in-series stiffness decreased to 1/2, which resulted in a decrease of isometric tension to 62%, but CB kinetics and Ca2+ sensitivity were little affected. The pCa study demonstrated that the rate constant of the force generation step (2πb) is proportionate to [Ca2+] at < 5 µM, suggesting that the activation mechanism can be described by a simple second order reaction. We also found that tension, stiffness, and magnitude parameters are related to [Ca2+] by the Hill equation, with a cooperativity coefficient of 4-5, which is consistent with the fact that Ca2+ activation mechanisms involve cooperative multimolecular interactions. Our results support the long-held hypothesis that Process C (Phase 2) represents the CB detachment step, and Process B (Phase 3) represents the force generation step. Moreover, we discovered that constant H may represent the work-performing step in cardiac preparations. Our experiments demonstrate excellent CB kinetics with two well-defined exponentials that can be more distinguished than those found using ordinary skinned fibers. Flash-frozen and cryo-sectioned preparations are especially suitable for multi-institutional collaborations nationally and internationally because of their ease of transportation.

Investigating the role of core needle biopsy in evaluating tumor-stroma ratio (TSR) of invasive breast cancer: a retrospective study.

PURPOSE: Tumor-stroma ratio (TSR) of invasive breast carcinoma has gained attention in recent years due to its prognostic significance. Previous studies showed TSR is a potential biomarker for indicating the tumor response to neoadjuvant chemotherapy. However, it is not clear how well TSR evaluation in biopsy specimens might reflect the TSR in resection specimens. We conducted a study to investigate whether biopsy evaluation of TSR can be an alternative method. METHOD: We collected cases with invasive breast carcinoma of no special type (IBC-NST) from University of Yamanashi hospital between 2011 and 2017 whose biopsy and resection specimens both had a pathologically diagnosis of IBC-NST (n = 146). We conceptualized a method for evaluating TSR in biopsy specimens within a preliminary cohort (n = 50). Within the studied cohort (n = 96), biopsy-based TSR (b-TSR) and resection-based TSR (r-TSR) were scored by two pathologists. We then evaluated our method's validity and performance by measuring interobserver variability between the two pathologists, Spearman's correlation between b-TSR and r-TSR, and the receiver operating characteristics (ROC) analysis for defining stroma-rich and stroma-poor tumors. RESULTS: Intra-class coefficient between the two pathologists was 0.59. The correlation coefficients between b-TSR and r-TSR in the two pathologists were 0.45 and 0.37. The ROC areas under the curve were 0.7 and 0.67. By considering an r-TSR of < 50% as stroma-rich, the sensitivity and specificity of detecting stroma-rich tumors were 64.1% and 66.7%, respectively, when b-TSR was < 40%. CONCLUSION: Our current b-TSR evaluation method can provide information about r-TSR and facilitate pre-treatment therapy follow-up.

The effect of gender and obesity in modulating cross-bridge function in cardiac muscle fibers.

The effect of obesity on cross-bridge (CB) function was investigated in mice lacking functional Melanocortin-4 Receptor (MC4R-/-), the loss of which causes dilated cardiomyopathy (DCM) in humans and mice. Skinned cardiac muscle fibers from male and female mice were used, and activated in the presence of Ca2+. To characterize CB kinetics, we changed the length of fibers in sinewaves (15 frequencies: 1‒187 Hz) at a small amplitude (0.2%L0), studied concomitant tension transients, and deduced the kinetic constants of the CB cycle from the ATP and Pi effects. In males, active tension and stiffness during full activation and rigor were ~ 1.5X in WT compared to MC4R-/- mice. This effect was not observed in females. We also observed that ATP binding and subsequent CB detachment steps were not altered by the mutation/gender. The equilibrium constant of the force generation step (K4) and Pi release step (association constant: K5) were not affected by the mutation, but there was a gender difference in WT mice: K4 and K5 were ~ 2.2X in males than in females. Concomitantly, the forward rate constant (r4) and backward rate constant (r-4) of the force generation step were 1.5-2.5X in muscles from female MC4R-/- mice relative to male MC4R-/- mice. However, these effects did not cause a significant difference in CB distributions among six CB states. In both genders, Ca2+ sensitivity decreased slightly (0.12 pCa unit) in mutants. We conclude that the CB functions are differentially affected both by obesity induced in the absence of functional MC4R-/- and gender.

Mechanisms of Frank-Starling law of the heart and stretch activation in striated muscles may have a common molecular origin.

Vertebrate cardiac muscle generates progressively larger systolic force when the end diastolic chamber volume is increased, a property called the "Frank-Starling Law", or "length dependent activation (LDA)". In this mechanism a larger force develops when the sarcomere length (SL) increased, and the overlap between thick and thin filament decreases, indicating increased production of force per unit length of the overlap. To account for this phenomenon at the molecular level, we examined several hypotheses: as the muscle length is increased, (1) lattice spacing decreases, (2) Ca2+ sensitivity increases, (3) titin mediated rearrangement of myosin heads to facilitate actomyosin interaction, (4) increased SL activates cross-bridges (CBs) in the super relaxed state, (5) increased series stiffness at longer SL promotes larger elementary force/CB to account for LDA, and (6) stretch activation (SA) observed in insect muscles and LDA in vertebrate muscles may have similar mechanisms. SA is also known as delayed tension or oscillatory work, and universally observed among insect flight muscles, as well as in vertebrate skeletal and cardiac muscles. The sarcomere stiffness observed in relaxed muscles may significantly contributes to the mechanisms of LDA. In vertebrate striated muscles, the sarcomere stiffness is mainly caused by titin, a single filamentary protein spanning from Z-line to M-line and tightly associated with the myosin thick filament. In insect flight muscles, kettin connects Z-line and the thick filament to stabilize the sarcomere structure. In vertebrate cardiac muscles, titin plays a similar role, and may account for LDA and may constitute a molecular mechanism of Frank-Starling response.

Lesion-targeted cytology to improve cytological sampling for atypical polypoid adenomyomas of the uterus: A case series and review of the literature.

OBJECTIVE: Atypical polypoid adenomyomas (APAs) are uncommon tumours consisting of atypical endometrioid glands and fibromyomatous stroma. Identifying the biphasic nature of atypical glandular components and spindle mesenchymal components without atypia is crucial for the cytological diagnosis of APA. We investigated the utility of lesion-targeted cytology (LTC) to directly collect firm spindle components. METHODS: We recruited seven consecutive surgical patients who underwent cytological examinations before surgery and were diagnosed with APA on postoperative histological examinations. Cytological smears were obtained by routine sampling in five cases and by targeted sampling using transvaginal ultrasonography, that is, LTC, in two cases. We retrospectively analysed the cytological findings from our cases and compared them to those of APA cases previously reported in the English literature. RESULTS: Among 5/7 cases that involved routine cytological sampling, normal cytological findings were found in 2 and atypical glandular cells were found in 3, but spindle cells from mesenchymal components were not detected. In contrast, among 2/7 cases in which sampling involved LTC, spindle cells without atypia, in addition to atypical glandular cells were found. CONCLUSIONS: Lesion-targeted cytology is useful to assess mesenchymal components of APAs and may improve the cytological diagnosis of APA.

High expression of CD10 in anaplastic thyroid carcinomas.

AIMS: CD10 is an endopeptidase that degrades various bioactive peptides in the extracellular matrix. In addition to enzymatic degradation, it affects multiple intracellular signal transduction pathways. CD10 expression has been extensively studied in human epithelial cancers of numerous organs and sites. However, its presence in thyroid carcinomas, especially in anaplastic thyroid carcinoma (ATC), has not been fully determined. An actual CD10 expression in thyroid lesions including a large series of ATC was evaluated. METHODS AND RESULTS: We examined CD10 by immunohistochemistry (IHC) in 152 thyroid lesions: nine adenomatous goitres (AGs) and 143 tumours, including 47 anaplastic carcinomas. IHC showed diffuse and strong positivity for CD10 in the epithelial components of almost all ATCs. However, epithelia with squamous metaplasia and oncocytic change from AGs, follicular adenomas and differentiated carcinomas had focal CD10 reactivity. Some papillary thyroid carcinomas (PTCs), along with the PTC components of some ATCs, showed CD10 positivity in fibroblast-like stromal cells and fibrous material. CONCLUSION: Our results imply that the CD10 expression pattern depended on the histotypes of thyroid lesions. When possible metastatic tumours and non-epithelial tumours are excluded, high CD10 expression may be useful in determining whether a primary thyroid carcinoma includes an anaplastic component.

Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model.

The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2+-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca2+-sensitivity and a decrease (0.8×) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5×), reversal step (k-4) increased (2×), and the phosphate-release step (1/K5) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics-possibly caused by changes in the α-myosin heavy chain phosphorylation profile-as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.

Development of apical hypertrophic cardiomyopathy with age in a transgenic mouse model carrying the cardiac actin E99K mutation.

In both humans and mice, the Glu-99-Lys (E99K) mutation in the cardiac actin gene (ACTC) results in little understood apical hypertrophic cardiomyopathy (AHCM). To determine how cross-bridge kinetics change with AHCM development, we applied sinusoidal length perturbations to skinned papillary muscle fibres from 2- and 5-month old E99K transgenic (Tg) and non-transgenic (NTg) mice, and studied tension and its transients. These age groups were chosen because our preliminary studies indicated that AHCM develops with age. Fibres from 5-month old E99K mice showed significant decreases in tension, stiffness, the rate of the medium-speed exponential process and its magnitude compared to non-transgenic control. The nucleotide association constants increased with age, and they were significantly larger in E99K compared to NTg. However, there were no large differences in the rates of the cross-bridge detachment step, the rates of the force generation step, or the phosphate association constant. Our result on force/cross-bridge demonstrates that the decreased active tension of E99K fibres was caused by a decreased amount of force generated per each cross-bridge. The effects were generally less or insignificant at 2 months. A pCa-tension study showed increased Ca2+-sensitivity (pCa50) with age in both the E99K and NTg sample groups, and pCa50 was significantly larger (but only for 0.05-0.06 pCa units) in E99K than in NTg groups. A significant decrease in cooperativity (nH) was observed only in 5-month old E99K mice. We conclude that the AHCM-causing ACTC E99K mutation is associated with progressive alterations in biomechanical parameters, with changes smaller at 2 months but larger at 5 months, correlating with the development of AHCM.

Comparison of elementary steps of the cross-bridge cycle in rat papillary muscle fibers expressing α- and ß-myosin heavy chain with sinusoidal analysis.

In mammalian ventricles, two myosin heavy chain (MHC) isoforms have been identified. Small animals express α-MHC, whereas large animals express ß-MHC, which contribute to a large difference in the heart rate. Sprague-Dawley rats possessing ~99% α-MHC were treated with propylthiouracil to result in 100% ß-MHC. Papillary muscles were skinned, dissected into small fibers, and used for experiments. To understand the functional difference between α-MHC and ß-MHC, skinned-fibers were activated under the intracellular ionic conditions: 5 mM MgATP, 1 mM Mg2+, 8 mM Pi, 200 mM ionic strength, pH 7.00 at 25 °C. Small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100 Hz (corresponding time domain: 1.6-1200 ms), and effects of Ca2+, Pi, and ATP were studied. The results show that Ca2+ sensitivity was slightly less (10-15%) in ß-MHC than α-MHC containing fibers. Sinusoidal analysis at pCa 4.66 (full Ca2+ activation) demonstrated that, the apparent rate constants were 2-4× faster in α-MHC containing fibers. The ATP study demonstrated that, in ß-MHC containing fibers, K 1 (ATP association constant) was greater (1.7×), k 2 and k -2 (cross-bridge detachment and its reversal rate constants) were smaller (×0.6). The Pi study demonstrated that, in ß-MHC containing fibers, k 4 (rate constant of the force-generation step) and k -4 were smaller (0.75× and 0.25×, respectively), resulting in greater K 4 (3×). There were no differences in active tension, rigor stiffness, or K 2 (equilibrium constant of the cross-bridge detachment step). Our study further demonstrated that there were no differences in parameters between fibers obtained from left and right ventricles, but with an exception in K 5 (Pi association constant).

A qPCR assay that specifically quantifies Tricholoma matsutake biomass in natural soil.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, "matsutake" mushrooms along densely developed mycelia, called "shiro," in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A0 horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.

The immediate effect of HCM causing actin mutants E99K and A230V on actin-Tm-myosin interaction in thin-filament reconstituted myocardium.

Human cardiac actin mutants E99K and A230V were expressed with baculovirus/insect cells and used to reconstitute the thin-filament of bovine cardiac (BVC) muscle fibers, together with tropomyosin (Tm) and troponin (Tn) purified from bovine ventricles. Effects of [Ca(2+)], [ATP], and [phosphate] on tension and its transients were studied at 25°C. In the absence of Tm/Tn, both mutants significantly decreased the tension of actin filament reconstituted fibers (WT: 0.75±0.06 T0, E99K: 0.58±0.04 T0, A230V: 0.58±0.03 T0), where T0 is active tension of native fibers (T0=26.9±1.1kPa, N=41), indicating diminished actin-myosin interactions. However, in the presence of Tm and Tn, WT, E99K, and A230V recovered tension (0.85±0.06 T0, 0.89±0.06 T0, and 0.85±0.05 T0, respectively), demonstrating the compensatory effect of Tm/Tn. Ca(2+) sensitivity (pCa50) increased (5.59±0.02, 5.80±0.03, 5.77±0.03, respectively) and cooperativity (nH) decreased (2.6±0.3, 1.87±0.21, 1.60±0.11, respectively). The kinetic constants of the cross-bridge cycle were deduced using sinusoidal analysis. E99K did not show any significant changes in any of the kinetic constants compared to those of WT. A230V caused a decrease in K1 (ATP association constant), k2 and k-2 (rate constants of the cross-bridge detachment step). The cross-bridge distribution was similar among WT, E99K, and A230V. In conclusion, our experiments demonstrate that the first step of HCM pathogenesis with E99K is increased pCa50 and decreased nH, which result in larger tension during partial activation to cause a diastolic problem. The effect on nH is more severe with A230V. In addition, A230V has a problem of decreased cross-bridge kinetics, which affects the normal functions of the cross-bridge cycle and may contribute to the first step of the HCM pathogenesis.

High ionic strength depresses muscle contractility by decreasing both force per cross-bridge and the number of strongly attached cross-bridges.

An increase in ionic strength (IS) lowers Ca(2+) activated tension in muscle fibres, however, its molecular mechanism is not well understood. In this study, we used single rabbit psoas fibres to perform sinusoidal analyses. During Ca(2+) activation, the effects of ligands (ATP, Pi, and ADP) at IS ranging 150-300 mM were studied on three rate constants to characterize elementary steps of the cross-bridge cycle. The IS effects were studied because a change in IS modifies the inter- and intra-molecular interactions, hence they may shed light on the molecular mechanisms of force generation. Both the ATP binding affinity (K1) and the ADP binding affinity (K 0) increased to 2-3x, and the Pi binding affinity (K5) decreased to 1/2, when IS was raised from 150 to 300 mM. The effect on ATP/ADP can be explained by stereospecific and hydrophobic interaction, and the effect on Pi can be explained by the electrostatic interaction with myosin. The increase in IS increased cross-bridge detachment steps (k2 and k-4), indicating that electrostatic repulsion promotes these steps. However, IS did not affect attachment steps (k-2 and k4). Consequently, the equilibrium constant of the detachment step (K2) increased by ~100%, and the force generation step (K4) decreased by ~30%. These effects together diminished the number of force-generating cross-bridges by 11%. Force/cross-bridge (T56) decreased by 26%, which correlates well with a decrease in the Debye length that limits the ionic atmosphere where ionic interactions take place. We conclude that the major effect of IS is a decrease in force/cross-bridge, but a decrease in the number of force generating cross-bridge also takes place. The stiffness during rigor induction did not change with IS, demonstrating that in-series compliance is not much affected by IS.

Using baculovirus/insect cell expressed recombinant actin to study the molecular pathogenesis of HCM caused by actin mutation A331P.

Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca(2+)], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25°C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75±0.06 T0, N=11; BVC: 0.73±0.07 T0, N=12, where T0 is the tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85±0.06 T0 (N=11) compared to 0.98±0.04 T0 (N=12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca(2+) sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69±0.01, N=10; WT: 5.69±0.02, N=11; BVC: 5.68±0.02, N=12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55±0.03 T0, N=13) and in their presence (0.65±0.02 T0, N=13) compared to those of the WT (0.75±0.06 T0 and 0.85±0.06 T0, respectively, N=11). A331P also showed decreased pCa50 (5.57±0.03, N=13) compared to that of WT (5.69±0.02, N=11). The cross-bridge kinetics and its distribution were similar between WT and A331P actin reconstituted fibers, indicating that force/cross-bridge was decreased by A331P. In conclusion, A331P causes a weakened cross-bridge force, which leads to a decreased active tension, reduces left-ventricular ejection fraction, and eventually results in the HCM phenotype.

The host ranges of conifer-associated Tricholoma matsutake, Fagaceae-associated T. bakamatsutake and T. fulvocastaneum are wider in vitro than in nature.

Tricholoma matsutake is the most commercially important edible mushroom in pine forests in Japan. Tricholoma bakamatsutake and T. fulvocastaneum, species closely related to T. matsutake, occur in Fagaceae forests. We examined ectomycorrhizal (EM) formation by these Tricholoma species by in vitro synthesis among seven strains (two of T. matsutake, four of T. bakamatsutake, one of T. fulvocastaneum) and axenic plants of pine (Pinus densiflora) and oak (Quercus serrata, Q. phillyraeoides). All strains, except for one of T. matsutake, formed EM associations with both pine and oak. Plant growth and mycelial development were differently affected by EM formation depending on the plant-fungus combination.

Detection of the edible ectomycorrhizal fungus Lyophyllum shimeji colonising seedlings of cultivated conifer species in New Zealand.

Lyophyllum shimeji is an edible ectomycorrhizal fungus that is widely distributed in East Asia and also present in the northern regions of Europe. In Japan, L. shimeji is a culinary delicacy, considered amongst all edible mushrooms to have the best taste and to be second only to Tricholoma matsutake in price. Traditionally, fruiting bodies of L. shimeji have been collected from the wild but fruiting of L. shimeji is now relatively uncommon and cannot keep up with increasing consumer demand. As a result, methods for its cultivation are being developed for commercial production in Japan and other countries. In this work, techniques were developed to cultivate L. shimeji on coniferous seedlings using a pure culture inoculum. They resulted in successful mycorrhization of Pinus pinaster and Picea abies in only 8 to 10 months. As ectomycorrhizae of L. shimeji are difficult to identify morphologically, mycorrhization was confirmed using an L. shimeji-specific PCR diagnostic, which was designed following a phylogenetic analysis of the Lyophyllum section Difformia using DNA sequences of the internal transcribed spacer (ITS), intergenic spacer (IGS) and elongation factor 1-α (EF1-α) gene. L. shimeji is a member of the Lyophyllum decastes complex in section Difformia, which also includes Lyophyllum fumosum and L. decastes. This analysis confirmed the separation of L. shimeji from closely related Lyophyllum spp. and enabled its unambiguous detection using an IGS-based PCR diagnostic. This is the first report of successful mycorrhization of L. shimeji on P. pinaster and P. abies and provides an opportunity for its commercial cultivation on conifers in New Zealand.

Analysis of the molecular pathogenesis of cardiomyopathy-causing cTnT mutants I79N, ΔE96, and ΔK210.

Three troponin T (TnT) mutants that cause hypertrophic, restrictive, and dilated cardiomyopathy (I79N, ΔE96, and ΔK210, respectively), were examined using the thin-filament extraction/reconstitution technique. Effects of Ca(2+), ATP, phosphate, and ADP concentrations on force and its transients were studied at 25°C. Maximal Ca(2+) tension (THC) and Ca(2+)-activatable tension (Tact), respectively, were similar among I79N, ΔE96, and WT, whereas ΔK210 led to a significantly lower THC (∼20% less) and Tact (∼25% less) than did WT. In pCa solution containing 8 mM Pi and ionic strength adjusted to 200 mM, the Ca(2+) sensitivity (pCa50) of I79N (5.63 ± 0.02) and ΔE96 (5.60 ± 0.03) was significantly greater than that of WT (5.45 ± 0.04), but the pCa50 of ΔK210 (5.54 ± 0.04) remained similar to that of WT. Five equilibrium constants were deduced using sinusoidal analysis. All three mutants showed significantly lower K0 (ADP association constant) and larger K4 (equilibrium constant of force generation step) relative to the corresponding values for WT. I79N and ΔK210 were associated with a K2 (equilibrium constant of cross-bridge detachment step) significantly lower than that of ΔE96 and WT. These results demonstrated that at pCa 4.66, the force/cross-bridge is ∼18% less in I79N and ∼41% less in ΔK210 than that in WT. These results indicate that the molecular pathogenesis of the cardiac TnT mutation-related cardiomyopathies is different for each mutation.

Diversity and similarity of motor function and cross-bridge kinetics in papillary muscles of transgenic mice carrying myosin regulatory light chain mutations D166V and R58Q.

Mechanical properties of skinned papillary muscle fibers from transgenic mice expressing familial hypertrophic cardiomyopathy associated mutations D166V and R58Q in myosin regulatory light chain were investigated. Elementary steps and the apparent rate constants of the cross-bridge cycle were characterized from the tension transients induced by sinusoidal length changes during maximal Ca(2+) activation, together with ATP, ADP, and Pi studies. The tension-pCa relation was also tested in two sets of solutions with differing Pi and ionic strength. Our results showed that in both mutants the fast apparent rate constant 2πc and the rate constants of the cross-bridge detachment step (k2) were smaller than those of wild type (WT), demonstrating the slower cross-bridge kinetics. D166V showed significantly smaller ATP (K1) and ADP (K0) association constants than WT, displaying weaker ATP binding and easier ADP release, whereas those of R58Q were not significantly different from WT. In tension-pCa study, both D166V and R58Q mutations exhibited increased Ca(2+) sensitivity and less cooperativity. We conclude that, while the two FHC mutations have similar clinical manifestations and prognosis, some of the mechanical parameters of cross-bridges (K0, K1) are differently modified, whereas some others (Ca(2+)-sensitivity, cooperativity, k2) are similarly modified by these two FHC associated mutations.

A re-interpretation of the rate of tension redevelopment (k(TR)) in active muscle.

A slackening to zero tension by large length release (~20%) and a restretch of active muscle fibres cause a fall and a redevelopment in tension. According to the model of Brenner (Proc Natl Acad Sci USA 85(9):3265-3269, 1988), the rate constant of tension redevelopment (k TR) is the sum of attachment and detachment rate constants, hence is limited by the fast reaction. Here we propose a model in which, after restretch, cross-bridges cycle many times by stretching series elastic elements, hence k(TR) is limited by a slow reaction. To set up this model, we made an assumption that the stepping rate (v) decreases linearly with tension (F), which is consistent with the Fenn effect. The distance traveled by a cross-bridge stretches series elastic elements with stiffness σ. With these assumptions, we set up a first order differential equation, which results in an exponential time course with the rate constant k(TR) = ση(0)ν(0)(1 - λ)/F(1), where λ = ν(1)/ν(0), η = step size, the subscript 0 indicates unloaded condition, and the subscript 1 indicate isometric condition. We demonstrate that the ATP hydrolysis rate (=[myosin head]/ν(0)) is proportionate to k(TR) as the ambient temperature is changed, and that the published data fit to this relationship well if λ = 0.28. We conclude that k(TR) is limited by the cross-bridge turnover rate; hence it represents the rate constant of the slowest reaction of the cross-bridge cycle, i.e. the ADP isomerization step before ADP is released.

A study of tropomyosin's role in cardiac function and disease using thin-filament reconstituted myocardium.

Tropomyosin (Tm) is the key regulatory component of the thin-filament and plays a central role in the cardiac muscle's cooperative activation mechanism. Many mutations of cardiac Tm are related to hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and left ventricular noncompaction (LVNC). Using the thin-filament extraction/reconstitution technique, we are able to incorporate various Tm mutants and protein isoforms into a muscle fiber environment to study their roles in Ca(2+) regulation, cross-bridge kinetics, and force generation. The thin-filament reconstitution technique poses several advantages compared to other in vitro and in vivo methods: (1) Tm mutants and isoforms are placed into the real muscle fiber environment to exhibit their effect on a level much higher than simple protein complexes; (2) only the primary and immediate effects of Tm mutants are studied in the thin-filament reconstituted myocardium; (3) lethal mutants of Tm can be studied without causing a problem; and (4) inexpensive. In transgenic models, various secondary effects (myocyte disarray, ECM fibrosis, altered protein phosphorylation levels, etc.) also affect the performance of the myocardium, making it very difficult to isolate the primary effect of the mutation. Our studies on Tm have demonstrated that: (1) Tm positively enhances the hydrophobic interaction between actin and myosin in the "closed state", which in turn enhances the isometric tension; (2) Tm's seven periodical repeats carry distinct functions, with the 3rd period being essential for the tension enhancement; (3) Tm mutants lead to HCM by impairing the relaxation on one hand, and lead to DCM by over inhibition of the AM interaction on the other hand. Ca(2+) sensitivity is affected by inorganic phosphate, ionic strength, and phosphorylation of constituent proteins; hence it may not be the primary cause of the pathogenesis. Here, we review our current knowledge regarding Tm's effect on the actomyosin interaction and the early molecular pathogenesis of Tm mutation related to HCM, DCM, and LVNC.