Molecular mechanism regulating myosin and cardiac functions by ELC

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM^hVLC-1) or E56G-mutated hVLC-1 (hVLC-1^E56G; TgM^E56G). hVLC-1 or hVLC-1^E56G expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM^hVLC-1 (1.67 pN/nm and 2.3 μm/s, respectively) were significantly higher than myosin with hVLC-1^E56G prepared from TgM^E56G (1.25 pN/nm and 1.7 μm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 μm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM^hVLC-1 (80.0 mmHg) were significantly higher than hearts from TgM^E56G (66.2 mmHg) or C57/BL6 (59.3 ± 3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1 > hVLC-1^E56G ≈ mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations.     

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin

Background

Studies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin.

Methods

To measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (F_unb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin.

Results

F_unb from unregulated actin (0.10 ± 0.01 pN) was significantly increased in the presence of CaD (0.17 ± 0.02 pN), tropomyosin (0.17 ± 0.02 pN) or both regulatory proteins (0.18 ± 0.02 pN). ERK phosphorylation of CaD significantly reduced the F_unb (0.06 ± 0.01 pN). Inspection of the traces of the F_unb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment.

Conclusions

CaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels.

General significance

This study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.     

Regulation of myosin sliding alongChara actin bundles by native skeletal muscle tropomyosin

Summary Native tropomyosin from rabbit skeletal muscle introduced by intracellular perfusion intoChara cells inhibited the cytoplasmic streaming irrespective of the Ca²⁺ concentration. To find the action site of native tropomyosin inChara, the cytoplasmic streaming was reconstituted by introducing isolated endoplasm into actin donorChara cells from which native endoplasm had been removed. The reconstituted streaming was inhibited by pretreatment of the actin donor cells with native tropomyosin but not by that of the endoplasm, suggesting that the native tropomyosin inhibited the cytoplasmic streaming by binding toChara actin bundles. Staining of the actin bundles with FITC-labeled native tropomyosin also showed that the native tropomyosin could bind to the actin bundles. Streaming reconstituted fromChara actin bundles and skeletal muscle myosin was insensitive to Ca²⁺, but became sensitive on application of the native tropomyosin.Abbrevations APW artificial pond water - ATP adenosine 5-triphosphoric acid - BSA bovine serum albumin - EDTA ethylene diamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - FITC-NTM fluorescein isothiocyanate-labeled native tropomyosin - NTM native tropomyosin     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

Mechanism of inhibition of cytoplasmic streaming by a myosin inhibitor, 2,3-butanedione monoxime

Summary On the basis of the inhibition of myosin by 2,3-butanedione monoxime (BDM), the protein's involvement in various cell activities is discussed. However, it has not been established whether BDM inhibits plant myosin. In the present study, the effect of BDM on isolated plant myosin was analyzed in vitro. The sliding between myosin from lily (Lilium longiflorum) pollen tubes and actin filaments from skeletal muscle was inhibited to 25% at a concentration of 60 mM, indicating that BDM can be used as a myosin inhibitor for plant materials. Cytoplasmic streaming was completely inhibited by BDM at 30 mM in lily pollen tubes and at 70 mM in short root hair cells, and at 100 mM in long root hair cells ofHydrocharis dubia. However, BDM at high concentrations induced the disorganization of actin filament bundles in lily pollen tubes and short root hair cells. In addition, cortical microtubules were also fragmented in short root hair cells treated with BDM, suggesting a possible side effect of BDM.Abbreviations AF actin filament - BDM 2,3-butanedione monoxime - MT microtubule     

Mechanism of reducing knee adduction moment by shortening of the knee lever arm via medio-lateral manipulation of foot center of pressure: A pilot study

Prominent conservative treatment options for medial-compartment knee osteoarthritis include footwear that reduces knee adduction moment (KAM) correlated with detrimental loads in the medial compartment of the knee, thus providing clinical benefit. The proposed mechanism by which they reduce KAM is a lateral shift in foot center of pressure (COP) and a consequent shortening of the knee lever arm (KLA), thereby reducing KAM, which can be simply calculated as KLA multiplied by the frontal plane ground reaction force (FP-GRF). The present study investigated this mechanism for a unique biomechanical device capable of shifting COP by means of moveable convex elements attached to the shoe. Fourteen healthy young male subjects underwent gait analysis in two COP configurations of the device for comparison: (1) laterally and (2) medially deviated. Average midstance KLA and KAM were decreased by 8.2% and 8.7%, respectively, in the lateral COP compared to medial. Ground reaction force parameters, frontal plane knee angle (FP-KA), and spine lateral flexion angle (SLF) did not differ between COP configurations. No study parameters differed for terminal stance. Linear mixed effects models showed that COP and FP-GRF components, but not FP-KA and SLF, were significant predictors of KLA. In addition, KLA and FP-GRF were significant predictors of KAM; although, FP-GRF did not change significantly with medio-lateral COP shift, while KLA did. This suggests that the mechanism by which the study device reduces KAM is primarily through shortening of KLA brought on by a lateral shift in COP.     

Effects of cardiac myosin binding protein-C on the regulation of interaction of cardiac myosin with thin filament in an in vitro motility assay

Modulatory role of whole cardiac myosin binding protein-C (сMyBP-C) in regulation of cardiac muscle contractility was studied in the in vitro motility assay with rabbit cardiac myosin as a motor protein. The effects of cMyBP-C on the interaction of cardiac myosin with regulated thin filament were tested in both in vitro motility and ATPase assays. We demonstrate that the addition of cMyBP-C increases calcium regulated Mg-ATPase activity of cardiac myosin at submaximal calcium. The Hill coefficient for ‘pCa-velocity’ relation in the in vitro motility assay decreased and the calcium sensitivity increased when сMyBP-C was added. Results of our experiments testifies in favor of the hypothesis that сMyBP-C slows down cross-bridge kinetics when binding to actin.     

Depressed sliding velocity of isolated cardiac myosin from cardiomyopathic hamsters: Evidence for an alteration in mechanical interaction of actomyosin

We measured the relative sliding velocity of cardiomyopathic hamster cardiac myosin on actin cables by using anin vitro motility assay system. We also investigated the relationship between the velocity and both myosin isozyme content and ATPase activity. Cardiac myosin was obtained from cardiomyopathic hamsters (BIO 14.6;B) aged 3,6,9, and 18 months and age-matched controls (F1B;F). Long well-organized actin cables of an alga,Nitellopsis, wer used for the motility assay. Small latex beads (2 m in diameter) were coated with purified cardiac myosin. When myosin-coated beads were introduced into an algal cell in the presence of Mg-ATP, myosin interacted with actin and dragged the beads. Active movement of the beads along the actin cables was observed under a photomicroscope and the velocity was measured. The velocity was significantly lower in B than in F for each age group (0.47 vs. 0.71 m/s at the age of 3 months, p<0.05; 0.44 vs. 0.88 m/s at 6 months, p<0.01; 0.44 vs. 0.67 m/s at 9 months, p<0.01; 0.35 vs. 0.52 m/s at 18 months, p<0.05). Both Ca²⁺-activated ATPase activity and the percentage of -myosin heavy chain were also lower in B than in F for each age group. When examined for individual specimens, there was a positive correlation between the velocity and both myosin Ca²⁺-activated ATPase activity (r=0.84) and percentage of -myosin heavy chain (r=0.83). These data points of both control and cardiomyopathic hamsters were distributed near the regression line obtained from control and thyroxine-treated rabbits reported previously. The present results indicate that the difference in mechanical properties between control and cardiomyopathic cardiac myosin is attributed to isozyme redistribution and not to a qualitative change in each myosin molecule.     

A model of xylitol production by the yeast Candida mogii

Production of xylitol from xylose in batch fermentations of Candida mogii ATCC 18364 is discussed in the presence of glucose as the cosubstrate. Various initial ratios of glucose and xylose concentrations are assessed for their impact on yield and rate of production of xylitol. Supplementation with glucose at the beginning of the fermentation increased the specific growth rate, biomass yield and volumetric productivity of xylitol compared with fermentation that used xylose as the sole carbon source. A mathematical model is developed for eventual use in predicting the product formation rate and yield. The model parameters were estimated from experimental observations, using a genetic algorithm. Batch fermentations, which were carried out with xylose alone and a mixture of xylose and glucose, were used to validate the model. The model fitted well with the experimental data of cell growth, substrate consumption and xylitol production.     

A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry

An efficient system to regenerate shoots on excised leaves of greenhouse-grown wild lowbush blueberry (Vaccinium angustifolium Ait.) was developed in vitro. The effect of thidiazuron (TDZ) on adventitious bud and shoot formation from apical, medial, and basal segments of the leaves was tested. Leaf cultures produced multiple buds and shoots with or without an intermediary callus phase on 2.3–4.5 μM TDZ within 6 wk of culture initiation. The greatest shoot regeneration came from young expanding basal leaf segments positioned with the adaxial side touching the culture medium and maintained for 2 wk in darkness. Callus development and shoot regeneration depended not only on the polarity of the explants but also on the genotype of the clone that supplied the explant material. TDZ-initiated cultures were transferred to medium containing 2.3–4.6 μM zeatin and produced usable shoots after one additional subculture. Elongated shoots were dipped in 39.4 mM indole-3-butyric acid powder and planted on a peat:perlite soilless medium at a ratio of 3:2 (v/v), which yielded an 80–90% rooting efficiency. The plantlets were acclimatized and eventually established in the greenhouse with 75–85% survival.     

In vitro test system for compounds affecting cholesterol pathway: Studies in primary rat liver cell cultures

Rat liver cells derived from male and female animals in primary monolayer cultures were investigated for suitability as a test system for xenobiotics affecting the cholesterol pathway. An appropriate mode of extraction and separation of newly formed cholesterol and precursors is described. This system can be widely applied.Rat liver cells from females in oestrus cycle had a higher synthesis rate of cholesterol than those from males. The disadvantages related to the cycle phases make male rats more appropriate donor animals for the test system developed. The altered in vitro cholesterol synthesis is relevant to that in vivo.The extraction of newly synthesized cholesterol and its precursors by means of columns packed with large-pore kieselgur is precise and time saving. The modified separation by thin-layer chromatography on silica gel layers impregnated with silver nitrate enables direct separation from the extract and is sufficient to recognize cholesterol and its precursors.The method in this form is suitable for processing a large number of specimens e.g. for screening.Dedicated to Prof. F.H. Schmidt on the occasion of his 60th birthday     

Lactic acid production by batch fermentation of whey permeate: A mathematical model

Summary The batch fermentation of whey permeate to lactic acid was improved by supplementing the broth with enzyme-hydrolyzed whey protein. A mathematical model based on laboratory results predicts to a 99% confidence limit the kinetics of this fermentation. Cell growth, acid production and protein and sugar use rates are defined in quantifiable terms related to the state of cell metabolism. The model shows that the constants of the Leudeking-Piret model are not true constants, but must vary with the medium composition, and especially the peptide average molecular weight. The kinetic mechanism on which the model is based also is presented.Nomenclature K _i lactic acid inhibition constant (g/l) - K _pr protein saturation constant during cell growth (g/l) - K _pr protein saturation constant during maintenance (g/l) - K _s lactose saturation constant (g/l) - [LA] lactic acid concentration (g/l) - [PR] protein concentration (g/l) - [S] lactose concentration (g/l) - t time (h) - [X] cell mass concentration (g/l) - , fermentation constants of Leudeking and Piret - specific growth rate (l/h) - Y _{g, LA/S} acid yield during cell growth (g acid/g sugar) - Y _{m, LA/S} acid yield during maintenance (g acid/g sugar) - Y _x/pr yield (g cells/g protein) - specific sugar use rate during cell growth (g sugar/h·g cell) - specific sugar use rate during maintenance (g sugar/h·cell)     

Negative gravitropism in Chara protonemata: A model integrating the opposite gravitropic responses of protonemata and rhizoids

The unicellular protonema of Chara fragilis Desv. was investigated in order to establish a reaction chain for negative gravitropism in tip-growing cells. The time course of gravitropic bending after stimulation at angles of 45 degrees or 90 degrees showed three distinct phases of graviresponse. During the first hour after onset of stimulation a strong upward shift of the tip took place. This initial response was followed by an interval of almost straight growth. Complete reorientation was achieved in a third phase with very low bending rates. Gravitropic reorientation could be completely abolished by basipetal centrifugation of the cells, which lastingly removed conspicuous dark organelles from the protonema tip, thus identifying them as statoliths. Within minutes after onset of gravistimulation most or all statoliths were transported acropetally from their resting position 20-100 micrometers from the cell apex to the lower side of the apical dome. This transport is actin-dependent since it could be inhibited with cytochalasin B. Within minutes after arrival of the statoliths, the apical dome flattened on its lower side and bulged on the upper one. After this massive initial response the statoliths remained firmly sedimented, but the distance between this sedimented complex and the cell vertex increased from 7 micrometers to 22 micrometers during the first hour of stimulation and bending rates sharply declined. From this it is concluded that only statoliths inside the apical dome convey information about the spatial orientation of the cell in the gravitropic reaction chain. After inversion of the protonema the statoliths transiently arranged into a disk-shaped complex about 8 micrometers above the vertex. When this statolith complex tilted towards one side of the apical dome, growth was shifted in the opposite direction and bending started. It is argued that the statoliths intruding into the apical dome may displace a growth-organizing structure from its symmetrical position in the apex and may thus cause bending by bulging. In the positively gravitropic Chara rhizoids only a more stable anchorage of the growth-organizing structure is required. As a consequence, sedimented statoliths cannot dislocate this structure from the vertex. Instead they obstruct a symmetrical distribution of cell-wall-forming vesicles around the structure and thus cause bending by bowing.     

Physarum myosin light chain one: A potential regulatory factor in cytoplasmic streaming

Summary Physarum myosin is composed of a heavy chain of about 225,000 daltons and two small polypeptides of 17,700 and 16,100 daltons, called light chain one (LC 1) and two (LC 2). Light chain one is shown to belong to the general class of regulating light chains by two independent criteria. After denaturation, purification and renaturation of thePhysarum light chains only LC 1 will combine with scallop myofibrils in which one myosin regulatory light chain has been removed. This LC 1 can restore inhibition of the ATPase activity of the myofibrils at 10^–8 M Ca⁺⁺ just as well as light chains from rabbit skeletal myosin. Secondly, this LC 1 is the only component of the myosin that is significantly phosphorylated by an endogenous kinase present in crude actomyosin. An active phosphatase is also present. Preliminary results could not detect calcium sensitivity for either kinase or phosphatase, nevertheless the importance of phosphorylation in affecting activity of biological systems suggests that LC 1 may serve some regulating function for plasmodial actomyosin.     

A dynamic in vitro model for evaluating antimicrobial activity against bacterial biofilms using a new device and clinical-used catheters

The activity of daptomycin compared to vancomycin against Staphylococcus epidermidis-biofilms on intravascular catheters has been evaluated using the new Sevilla device that enables to use medical grade-catheters, in an in vitro model that simulates the in vivo conditions. S. epidermidis-biofilms were obtained on polyurethane catheter segments using the Sevilla device linked to a continuous culture system for 24 h. To assess the antimicrobial activity, at this time the continuous culture system was changed to therapeutic antimicrobial concentration solutions for 48 h. At each 24 h interval time, catheter segments were taken out, washed and sonicated. Viable adherent bacteria were determined by agar plating. Data of surviving bacteria numbers attached to the catheter surface obtained with the Sevilla device showed a very good reproducibility. Daptomycin showed a good activity against S. epidermidis-biofilm on polyurethane catheter surface. After 48 h exposure to daptomycin, surviving adherent bacteria were reduced by 4 log compared to the control with no antimicrobial. Using the same model, vancomycin reduced bacterial survival by only 1.3 log. The Sevilla device enables antimicrobial agent activity against bacterial biofilms grown on the external surface of catheters used in clinical practice to be evaluated. The model used replicates as closely as possible the biofilm formed in a highly standardized way. Using this model, daptomycin demonstrates potent in vitro activity against S. epidermidis-biofilm on a polyurethane catheter; this activity was greater than that showed by vancomycin.     

A quick and precise technique for identifying ectomycorrhizas by PCR

A rapid procedure was developed to amplify ITS fragments directly from Tuber ectomycorrhizas either synthesized in a greenhouse or collected from the field. The addition of Bovine Serum Albumin (BSA) to the reaction mixtures overcame the presence of reaction inhibitors present in fungal and root cells, and enabled the amplification of the ITS regions directly from ectomycorrhizal tissues. This method is cheaper and less time-consuming than conventional procedures, and reduces the time required from 1-4 h to a few minutes. It is also much more sensitive, allowing the identification of just a small fragment of a mycorrhizal root tip. Because of this it is possible to select only the target fungal tissue and hence minimise the risk of contamination by saprobic or other mycorrhizal species. The method also avoids the use of toxic or hazardous substances. This method could have a wider application in other areas of applied mycology.     

A cellular model for drug interactions on hematopoiesis: The use of human umbilical cord blood progenitors as a model for the study of drug-related myelosuppression of normal hematopoiesis

A cellular model of hematopoiesis which would be more convenient than bone marrow (BM) progenitors and directly relevant to human pathology is needed in order to investigate xenobiotic toxicity. Human umbilical cord blood (HCB), previously shown to be able to repopulate BM, provides a powerful in vitro model of normal human hematopoiesis. In order to validate the use of normal HCB progenitors as targets for dose-related myelosuppression, we used clonogenic assays and expansion in a liquid culture of progenitor-enriched cell suspensions from HCB. A series of 8 reference molecules, doxorubicin, cytosine-arabinoside, 5-fluorouracil, 3-azido-3-deoxythymidine, acetylsalicyclic acid, sodium valproate and two cephalosporin antibiotics, were tested. In vitro 50% inhibition concentrations (IC₅₀) were compared to those observed or reported with BM progenitors, and to the values of plasma concentrations from treated patients. HCB progenitors as in vitro targets for cytotoxic molecules were easy to access and handle, and their use was sensitive, specific and reproducible. They gave results similar to BM progenitors and allowed a qualitative approach to cellular metabolism and toxicity using morphological, flow cytometric and chromatographic methods.Abbreviations ARA-CC cytosine arabinoside - AS acetylsalicylic acid - AZTT 3-azido-3-deoxythymidine - BFUU burst-forming units - BM bone marrow - CFU colony-forming units - DOXX doxorubicin - FU 5-fluorouracil - glyAA glyAcophorin A - HCB human umbilical cord blood - IU international units - PCMEM human placenta-conditioned medium - VA sodium valproate     

A simple structured model for recombinant IDShr protein production in Pichia pastoris

A simple structured model is proposed for the methanol production phase of the iduronate 2-sulphate sulfatase recombinant enzyme (IDShr) in Pichia patoris Mut(+). The model is mainly focused in oxidative stress phenomenon due to methanol consumption and based on extracellular experimental information and the basic knowledge of methanol metabolism in Pichia pastoris yeast (P. pastoris). The model's prediction shows a reasonable accuracy as compared with the experimental data. Likewise, it was proved that this model is able to simulate the production of other recombinant protein in P. pastoris.     

Improvement in the in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor

In this study, we examined the effects of inhibitors of mitochondrial permeability transition (MPT), caspase activity, intracellular Ca²⁺ chelator and mitochondrial Ca²⁺ uniporter on survival assessed by morphological observation and in vitro maturation (IVM) of porcine vitrified germinal vesicle (GV) oocytes. When vitrified GV oocytes were matured only present in the IVM medium with an MPT inhibitor, cyclosporin A (CsA), the survival and IVM rates (36.1% and 26.8%, respectively) were significantly higher (P < 0.05) than those in the other vitrified groups (10.3–12.3% and 6.2–10.3%, respectively). However, Z-VAD-fmk (Z-VAD), a caspase inhibitor, did not improve the survival and IVM rates (11.7–21.6% and 8.5–155%, respectively). When BAPTA-AM, an intracellular Ca²⁺ chelator, was present in the IVM medium, the survival and IVM rates of vitrified GV oocytes (34.5–36.2% and 25.0–26.9%, respectively) were significantly higher (P < 0.05) than those in the absent vitrified groups (17.2–24.2% and 12.9–19.3%, respectively). When ruthenium red (RR), an inhibitor of mitochondrial Ca²⁺ uniporter, was present only in the IVM medium, the survival and IVM rates (54.5% and 39.4%, respectively) were significantly higher than those in the other vitrified groups (25.8–38.4% and 14.4–24.2%, respectively). Furthermore, blastocysts were successfully produced using porcine vitrified GV oocytes matured in the IVM medium with RR after in vitro fertilization.These results suggested that CsA, BAPTA-AM and RR but not Z-VAD have improved the survival and IVM rates of porcine vitrified GV oocytes.