Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (kcat). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays.

Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5-1.7 µm/s; 25 °C) in the in vitro motility assay as well as maximum actin activated catalytic activity (kcat; 8-9 s-1) and actin concentration for half maximal activity (KATPase; 70-80 µM) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.

Expression and phosphorylation of eukaryotic translation initiation factor 4-gamma (eIF4G) in denervated atrophic and hypertrophic mouse skeletal muscle.

The eukaryotic translation initiation factor 4-gamma (eIF4G) is important for the initiation of protein synthesis and phosphorylation on S1108 regulates this function of eIF4G. Thus, increased phosphorylation has been reported in conditions associated with increased protein synthesis such as meal feeding and insulin/IGF-1 treatment whereas decreased phosphorylation occurs following starvation, dexamethasone treatment, in sepsis and in atrophic denervated hind-limb muscle. The aim of the present study was to test the hypothesis that S1108 phosphorylation of eIF4G is differentially affected in denervated atrophic hind-limb muscles and denervated hypertrophic hemidiaphragm muscle. Protein expression and phosphorylation in innervated and 6-days denervated atrophic hind-limb muscles (pooled gastrocnemius and soleus) and hypertrophic hemidiaphragms were studied semi-quantitatively using Western blots. Total expression of eIF4G did not change in denervated hind-limb muscles but increased about 77% in denervated hemidiaphragm. S1108 phosphorylated eIF4G decreased about 64% in denervated hind-limb muscles but increased about 1.3-fold in denervated hemidiaphragm. The ratio of S1108 phosphorylated eIF4G to total eIF4G decreased about 60% in denervated hind-limb muscles but no statistically significant change was observed in denervated hemidiaphragm. The differential effect of denervation on eIF4G expression and S1108 phosphorylation in hemidiaphragm (hypertrophic) and hind-limb muscle (atrophic) may represent a regulatory mechanism that helps clarify the differential response of these muscles following denervation.

Forkhead box O1 and muscle RING finger 1 protein expression in atrophic and hypertrophic denervated mouse skeletal muscle.

BACKGROUND: Forkhead box O (FoxO) transcription factors and E3 ubiquitin ligases such as Muscle RING finger 1 (MuRF1) are believed to participate in the regulation of skeletal muscle mass. The function of FoxO transcription factors is regulated by post-translational modifications such as phosphorylation and acetylation. In the present study FoxO1 protein expression, phosphorylation and acetylation as well as MuRF1 protein expression, were examined in atrophic and hypertrophic denervated skeletal muscle. METHODS: Protein expression, phosphorylation and acetylation were studied semi-quantitatively using Western blots. Muscles studied were 6-days denervated mouse hind-limb muscles (anterior tibial as well as pooled gastrocnemius and soleus muscles, all atrophic), 6-days denervated mouse hemidiaphragm muscles (hypertrophic) and innervated control muscles. Total muscle homogenates were used as well as separated nuclear and cytosolic fractions of innervated and 6-days denervated anterior tibial and hemidiaphragm muscles. RESULTS: Expression of FoxO1 and MuRF1 proteins increased 0.3-3.7-fold in all 6-days denervated muscles studied, atrophic as well as hypertrophic. Phosphorylation of FoxO1 at S256 increased about 0.8-1-fold after denervation in pooled gastrocnemius and soleus muscles and in hemidiaphragm but not in unfractionated anterior tibial muscle. A small (0.2-fold) but statistically significant increase in FoxO1 phosphorylation was, however, observed in cytosolic fractions of denervated anterior tibial muscle. A statistically significant increase in FoxO1 acetylation (0.8-fold) was observed only in denervated anterior tibial muscle. Increases in total FoxO1 and in phosphorylated FoxO1 were only seen in cytosolic fractions of denervated atrophic anterior tibial muscle whereas in denervated hypertrophic hemidiaphragm both total FoxO1 and phosphorylated FoxO1 increased in cytosolic as well as in nuclear fractions. MuRF1 protein expression increased in cytosolic as well as in nuclear fractions of both denervated atrophic anterior tibial muscle and denervated hypertrophic hemidiaphragm muscle. CONCLUSIONS: Increased expression of FoxO1 and MuRF1 in denervated muscles (atrophic as well as hypertrophic) suggests that these proteins participate in the tissue remodelling occurring after denervation. The effect of denervation on the level of phosphorylated and acetylated FoxO1 differed in the muscles studied and may be related to differences in fiber type composition of the muscles.

p38 mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 (MK2) signaling in atrophic and hypertrophic denervated mouse skeletal muscle.

BACKGROUND: p38 mitogen-activated protein kinase has been implicated in both skeletal muscle atrophy and hypertrophy. T317 phosphorylation of the p38 substrate mitogen-activated protein kinase-activated protein kinase 2 (MK2) correlates with muscle weight in atrophic and hypertrophic denervated muscle and may influence the nuclear and cytoplasmic distribution of p38 and/or MK2. The present study investigates expression and phosphorylation of p38, MK2 and related proteins in cytosolic and nuclear fractions from atrophic and hypertrophic 6-days denervated skeletal muscles compared to innervated controls. METHODS: Expression and phosphorylation of p38, MK2, Hsp25 (heat shock protein25rodent/27human, Hsp25/27) and Hsp70 protein expression were studied semi-quantitatively using Western blots with separated nuclear and cytosolic fractions from innervated and denervated hypertrophic hemidiaphragm and atrophic anterior tibial muscles. Unfractionated innervated and denervated atrophic pooled gastrocnemius and soleus muscles were also studied. RESULTS: No support was obtained for a differential nuclear/cytosolic localization of p38 or MK2 in denervated hypertrophic and atrophic muscle. The differential effect of denervation on T317 phosphorylation of MK2 in denervated hypertrophic and atrophic muscle was not reflected in p38 phosphorylation nor in the phosphorylation of the MK2 substrate Hsp25. Hsp25 phosphorylation increased 3-30-fold in all denervated muscles studied. The expression of Hsp70 increased 3-5-fold only in denervated hypertrophic muscles. CONCLUSIONS: The study confirms a differential response of MK2 T317 phosphorylation in denervated hypertrophic and atrophic muscles and suggests that Hsp70 may be important for this. Increased Hsp25 phosphorylation in all denervated muscles studied indicates a role for factors other than MK2 in the regulation of this phosphorylation.

Factors influencing the prescription of drugs of different price levels.

PURPOSE: Socioeconomic factors have been suggested to influence the prescribing of newer and more expensive drugs. In the present study, individual and health care provider factors were studied in relation to the prevalence of differently priced drugs. METHODS: Register data for dispensed drugs were retrieved for 18 486 individuals in a county council in Sweden. The prevalence of dispensed drugs was combined with data for the individual's gender, age, education, income, foreign background, and type of caregiver. For each of the diagnostic groups (chronic obstructive pulmonary disease [COPD], depression, diabetes, and osteoporosis), selected drugs were dichotomized into cost categories, lower and higher price levels. Univariate and multivariate logistic regressions were performed using cost category as the dependent variable and the individual and provider factors as independent variables. RESULTS: In all four diagnostic groups, differences were observed in the prescription of drugs of lower and higher price levels with regard to the different factors studied. Age and gender affected the prescription of drugs of lower and higher price levels more generally, except for gender in the osteoporosis group. Income, education, foreign background, and type of caregiver affected prescribing patterns but in different ways for the different diagnostic groups. CONCLUSIONS: Certain individual and provider factors appear to influence the prescribing of drugs of different price levels. Because the average price for the cheaper drugs versus more costly drugs in each diagnostic group was between 19% and 69%, there is a risk that factors other than medical needs are influencing the choice of drug.

Awareness among nurses about reporting of adverse drug reactions in Sweden.

BACKGROUND: The purpose of this study was to investigate awareness among nurses regarding their new role as reporters of adverse drug reactions in Sweden and factors that may influence reporting by nurses. METHODS: In 2007, all nurses were included in the adverse drug reaction reporting scheme in Sweden. A questionnaire was sent to 753 randomly selected nurses in September 2010. RESULTS: Of the 453 (60%) responding nurses, 265 (58%) were aware that nurses were included in the reporting of adverse drug reactions. Sixty-one nurses (14%) stated that they had reported an adverse drug reaction. Fifteen percent (n = 70) of the respondents had received training about reporting of adverse drug reactions. Almost one third of these (n = 21, 30%) had reported an adverse drug reaction on at least one occasion. Among nurses without training, a smaller proportion (n = 40, 11%, P < 0.05) had reported an adverse drug reaction on at least one occasion. The two factors considered most important by nurses for reporting were the severity of the adverse drug reaction and if the reaction was to a newly approved drug. A majority of the nurses (n = 397, 88%) were interested in a training course in pharmacology as part of their ongoing professional development. One third (32%) of all nurses stated that one reason for not reporting a suspected adverse drug reaction was that the physician responsible did not regard the reaction necessary to report. CONCLUSION: We found that more than half of the study population of nurses in Sweden were aware of their new role as reporters of adverse drug reactions, but few of the responding nurses had reported an adverse drug reaction. Given that training seems to be associated with high reporting frequency, we suggest more training in pharmacovigilance for nurses.

Akt (protein kinase B) isoform phosphorylation and signaling downstream of mTOR (mammalian target of rapamycin) in denervated atrophic and hypertrophic mouse skeletal muscle.

BACKGROUND: The present study examines the hypothesis that Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signaling is increased in hypertrophic and decreased in atrophic denervated muscle. Protein expression and phosphorylation of Akt1, Akt2, glycogen synthase kinase-3beta (GSK-3beta), eukaryotic initiation factor 4E binding protein 1 (4EBP1), 70 kD ribosomal protein S6 kinase (p70S6K1) and ribosomal protein S6 (rpS6) were examined in six-days denervated mouse anterior tibial (atrophic) and hemidiaphragm (hypertrophic) muscles. RESULTS: In denervated hypertrophic muscle expression of total Akt1, Akt2, GSK-3beta, p70S6K1 and rpS6 proteins increased 2-10 fold whereas total 4EBP1 protein remained unaltered. In denervated atrophic muscle Akt1 and Akt2 total protein increased 2-16 fold. A small increase in expression of total rpS6 protein was also observed with no apparent changes in levels of total GSK-3beta, 4EBP1 or p70S6K1 proteins. The level of phosphorylated proteins increased 3-13 fold for all the proteins in hypertrophic denervated muscle. No significant changes in phosphorylated Akt1 or GSK-3beta were detected in atrophic denervated muscle. The phosphorylation levels of Akt2, 4EBP1, p70S6K1 and rpS6 were increased 2-18 fold in atrophic denervated muscle. CONCLUSIONS: The results are consistent with increased Akt/mTOR signaling in hypertrophic skeletal muscle. Decreased levels of phosphorylated Akt (S473/S474) were not observed in denervated atrophic muscle and results downstream of mTOR indicate increased protein synthesis in denervated atrophic anterior tibial muscle as well as in denervated hypertrophic hemidiaphragm muscle. Increased protein degradation, rather than decreased protein synthesis, is likely to be responsible for the loss of muscle mass in denervated atrophic muscles.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) in skeletal muscle atrophy and hypertrophy.

Skeletal muscle is a highly plastic tissue. Overall muscle growth (hypertrophy) or muscle wasting (atrophy) results from alterations in intracellular signaling pathways with important regulatory steps occurring in the nucleus as well as in the cytoplasm. Previous studies have identified components of the Akt/mTor pathway as well as the p38 MAPK pathway as important for skeletal muscle hypertrophy and/or atrophy. The present study tests the hypothesis that MK2, a substrate of p38 which following phosphorylation, can be exported from the nucleus in a complex with p38, may be important for skeletal muscle growth. The expression of MK2 was examined in denervated mouse hind-limb (atrophic) and hemidiaphragm (transiently hypertrophic) muscles. MK2 mRNA expression decreased after denervation in both atrophic (48% of innervated controls, P < 0.001) and hypertrophic muscle (34% of innervated controls, P < 0.01) but MK2 protein expression decreased only in atrophic muscle (32% of innervated controls, P < 0.01). The level of T205 phosphorylated MK2 increased after denervation in both atrophic (fourfold increase, P < 0.01) and hypertrophic muscles (almost sevenfold increase, P < 0.001) whereas the level of T317 phosphorylated MK2 (necessary for nuclear export) increased after denervation in hypertrophic muscle (nearly threefold increase, P < 0.001) but not in atrophic muscle. Logarithmically transformed relative changes in MK2 phosphorylated at T317 correlated well (r(2) = 0.7737) with relative changes in muscle weight. The results suggest a role for MK2 in the regulation of muscle mass, a role which, at least in part, may be related to determining the subcellular localization of p38 in muscle fibers.

Galectin-1 expression in innervated and denervated skeletal muscle.

Galectin-1 is a soluble carbohydrate-binding protein with a particularly high expression in skeletal muscle. Galectin-1 has been implicated in skeletal muscle development and in adult muscle regeneration, but also in the degeneration of neuronal processes and/or in peripheral nerve regeneration. Exogenously supplied oxidized galectin-1, which lacks carbohydrate-binding properties, has been shown to promote neurite outgrowth after sciatic nerve sectioning. In this study, we compared the expression of galectin-1 mRNA and immunoreactivity in innervated and denervated mouse and rat hind-limb and hemidiaphragm muscles. The results show that galectin-1 mRNA expression and immunoreactivity are up-regulated following denervation. The galectin-1 mRNA is expressed in the extrasynaptic and perisynaptic regions of the muscle, and its immunoreactivity can be detected in both regions by Western blot analysis. The results are compatible with a role for galectin-1 in facilitating reinnervation of denervated skeletal muscle.

The effects of transdermal scopolamine on simulated ship navigation and attention/reaction time.

Transdermal scopolamine is commonly used by mariners to prevent or treat seasickness. Most studies indicate that scopolamine administered transdermally via an adhesive patch does not impair performance of skills required to navigate a vessel, but trials have not been conducted testing navigation and ship handling under realistic conditions. The aim of this study was to test the effects of transdermal scopolamine on performance using training simulators to assess complex vessel navigation and rough-weather ship handling abilities. A randomized double-blind crossover study assessed 32 Swedish maritime cadets under transdermal scopolamine and placebo conditions on simulated navigation and ship handling performance, sleepiness, and subjective measures of fitness and performance. There were no significant differences on occupational outcomes by medication condition, but sustained reaction time was significantly increased under transdermal scopolamine, relative to placebo. We conclude that the transdermal scopolamine patch does not impair simulated ship handling.

Diffusion dynamics of motor-driven transport: gradient production and self-organization of surfaces.

The interaction between cytoskeletal filaments (e.g., actin filaments) and molecular motors (e.g., myosin) is the basis for many aspects of cell motility and organization of the cell interior. In the in vitro motility assay (IVMA), cytoskeletal filaments are observed while being propelled by molecular motors adsorbed to artificial surfaces (e.g., in studies of motor function). Here we integrate ideas that cytoskeletal filaments may be used as nanoscale templates in nanopatterning with a novel approach for the production of surface gradients of biomolecules and nanoscale topographical features. The production of such gradients is challenging but of increasing interest (e.g., in cell biology). First, we show that myosin-induced actin filament sliding in the IVMA can be approximately described as persistent random motion with a diffusion coefficient (D) given by a relationship analogous to the Einstein equation (D = kT/gamma). In this relationship, the thermal energy (kT) and the drag coefficient (gamma) are substituted by a parameter related to the free-energy transduction by actomyosin and the actomyosin dissociation rate constant, respectively. We then demonstrate how the persistent random motion of actin filaments can be exploited in conceptually novel methods for the production of actin filament density gradients of predictable shapes. Because of regularly spaced binding sites (e.g., lysines and cysteines) the actin filaments act as suitable nanoscale scaffolds for other biomolecules (tested for fibronectin) or nanoparticles. This forms the basis for secondary chemical and topographical gradients with implications for cell biological studies and biosensing.

Secreted frizzled related protein 1 (Sfrp1) and Wnt signaling in innervated and denervated skeletal muscle.

Wnts are secreted proteins with functions in differentiation, development and cell proliferation. Wnt signaling has also been implicated in neuromuscular junction formation and may function in synaptic plasticity in the adult as well. Secreted frizzled-related proteins (Sfrps) such as Sfrp1 can function as inhibitors of Wnt signaling. In the present study a potential role of Wnt signaling in denervation was examined by comparing the expression levels of Sfrp1 and key proteins in the canonical Wnt pathway, Dishevelled, glycogen synthase kinase 3beta and beta-catenin, in innervated and denervated rodent skeletal muscle. Sfrp1 mRNA and immunoreactivity were found to be up-regulated in mouse hemidiaphragm muscle following denervation. Immunoreactivity, detected by Western blots, and mRNA, detected by Northern blots, were both expressed in extrasynaptic as well as perisynaptic parts of the denervated muscle. Immunoreactivity on tissue sections was, however, found to be concentrated postsynaptically at neuromuscular junctions. Using beta-catenin levels as a readout for canonical Wnt signaling no evidence for decreased canonical Wnt signaling was obtained in denervated muscle. A role for Sfrp1 in denervated muscle, other than interfering with canonical Wnt signaling, is discussed.

Semaphorin 6C expression in innervated and denervated skeletal muscle.

Semaphorins are secreted or transmembrane proteins important for axonal guidance and for the structuring of neuronal systems. Semaphorin 6C, a transmembrane Semaphorin, has growth cone collapsing activity and is expressed in adult skeletal muscle. In the present study the expression of Semaphorin 6C mRNA and immunoreactivity has been compared in innervated and denervated mouse hind-limb and hemidiaphragm muscles. Microscopic localization of immunoreactivity was studied in innervated and denervated rat skeletal muscle. The results show that Semaphorin 6C mRNA expression and immunoreactivity on Western blots are down-regulated following denervation. The mRNA of Semaphorin 6C as well as immunoreactivity determined by Western blots are expressed in extrasynaptic as well as perisynaptic regions of muscle. Immunohistochemical studies, however, show Semaphorin 6C-like immunoreactivity to be concentrated at neuromuscular junctions. The results suggest a role for Semaphorin 6C in neuromuscular communication.

Contact angle measurements by confocal microscopy for non-destructive microscale surface characterization.

Contact angle measurements are of great importance in surface characterization but the practical use has often been limited to macroscopic dimensions (millimeters). Therefore, we have developed a confocal microscopy method that allows non-destructive measurements of both low (<30 degrees ) and high (30 degrees -90 degrees ) contact angles. Low contact angles were measured by reconstructing the drop profile from the interference patterns in droplets condensed from atmospheric humidity. At higher contact angles water droplets with a small amount of fluorescein were sprayed onto the surfaces and 3D-image stacks were recorded and used to extract the contact angle. Suitable drop sizes were between a few up to about 50 mum radius, using a 40x magnification objective. Using drops >10 micrometers radius for microcontact angle measurements a good correlation was obtained between measured micro- and macrocontact angles. After microcontact angle measurements the surfaces were rinsed and heavy meromyosin motor fragments were adsorbed to the surface. Importantly, the sensitive actin propelling function of these motor proteins was not affected by the previous contact angle measurements using fluorescent droplets. This suggests that the methodology should be suitable for non-destructive characterization of different parts of micropatterned surfaces being developed for biological assays.

Actin filament guidance on a chip: toward high-throughput assays and lab-on-a-chip applications.

Biological molecular motors that are constrained so that function is effectively limited to predefined nanosized tracks may be used as molecular shuttles in nanotechnological applications. For these applications and in high-throughput functional assays (e.g., drug screening), it is important that the motors propel their cytoskeletal filaments unidirectionally along the tracks with a minimal number of escape events. We here analyze the requirements for achieving this for actin filaments that are propelled by myosin II motor fragments (heavy meromyosin; HMM). First, we tested the guidance of HMM-propelled actin filaments along chemically defined borders. Here, trimethylchlorosilane (TMCS)-derivatized areas with high-quality HMM function were surrounded by SiO(2) domains where HMM did not bind actin. Guidance along the TMCS-SiO(2) border was almost 100% for filament approach angles between 0 and 20 degrees but only about 10% at approach angles near 90 degrees . A model (Clemmens, J.; Hess, H.; Lipscomb, R.; Hanein, Y.; Bohringer, K. F.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Langmuir 2003, 19, 10967-10974) accounted for essential aspects of the data and also correctly predicted a more efficient guidance of actin filaments than previously shown for kinesin-propelled microtubules. Despite the efficient guidance at low approach angles, nanosized (<700 nm wide) TMCS tracks surrounded by SiO(2) were not effective in guiding actin filaments. Neither was there complete guidance along nanosized tracks that were surrounded by topographical barriers (walls and roof partially covering the track) unless there was also chemically based selectivity between the tracks and surroundings. In the latter case, with dually defined tracks, there was close to 100% guidance. A combined experimental and theoretical analysis, using tracks of the latter type, suggested that a track width of less than about 200-300 nm is sufficient at a high HMM surface density to achieve unidirectional sliding of actin filaments. In accord with these results, we demonstrate the long-term trapping of actin filaments on a closed-loop track (width < 250 nm). The results are discussed in relation to lab-on-a-chip applications and nanotechnology-assisted assays of actomyosin function.

Selective spatial localization of actomyosin motor function by chemical surface patterning.

We have previously described the efficient guidance and unidirectional sliding of actin filaments along nanosized tracks with adsorbed heavy meromyosin (HMM; myosin II motor fragment). In those experiments, the tracks were functionalized with trimethylchlorosilane (TMCS) by chemical vapor deposition (CVD) and surrounded by hydrophilic areas. Here we first show, using in vitro motility assays on nonpatterned and micropatterned surfaces, that the quality of HMM function on CVD-TMCS is equivalent to that on standard nitrocellulose substrates. We further examine the influences of physical properties of different surfaces (glass, SiO(2), and TMCS) and chemical properties of the buffer solution on motility. With the presence of methylcellulose in the assay solution, there was HMM-induced actin filament sliding on both glass/SiO(2) and on TMCS, but the velocity was higher on TMCS. This difference in velocity increased with decreasing contact angles of the glass and SiO(2) surfaces in the range of 20-67 degrees (advancing contact angles for water droplets). The corresponding contact angle of CVD-TMCS was 81 degrees. In the absence of methylcellulose, there was high-quality motility on TMCS but no motility on glass/SiO(2). This observation was independent of the contact angle of the glass/SiO(2) surfaces and of HMM incubation concentrations (30-150 microg mL(-)(1)) and ionic strengths of the assay solution (20-50 mM). Complete motility selectivity between TMCS and SiO(2) was observed for both nonpatterned and for micro- and nanopatterned surfaces. Spectrophotometric analysis of HMM depletion during incubation, K/EDTA ATPase measurements, and total internal reflection fluorescence spectroscopy of HMM binding showed only minor differences in HMM surface densities between TMCS and SiO(2)/glass. Thus, the motility contrast between the two surface chemistries seems to be attributable to different modes of HMM binding with the hindrance of actin binding on SiO(2)/glass.

Denervation-induced alterations in gene expression in mouse skeletal muscle.

Motoneurons are important for regulating the function and properties of skeletal muscle. In the present study high-density oligonucleotide arrays have been used to compare gene expression in innervated and six-days denervated NMRI mouse skeletal muscle. To avoid looking at genes mainly participating in the process of atrophy, both hind-limb muscles (atrophic after denervation) and hemidiaphragm muscle (transiently hypertrophic after denervation) were used. Only genes previously not known to respond to denervation and with potential roles in DNA/RNA interactions/transcription and/or cellular communication/signalling are presented. Data for additional genes are provided as supplementary material. Thirty-two genes, up-regulated by a factor of two or down-regulated to the same extent after denervation, are presented. These include genes that may act through chromatin remodelling and/or as transcription factors/regulators (Cdkn1a, Cdr2, Hrmt1l2, Idb2, Myc/c-myc, L-myc1, Rb1, Sap30 and Tgif), genes possibly involved in the regulation of muscle membrane properties and/or excitation-contraction coupling (Cacng1, Camk2d, Hrmt1l2, Kcnj12, Kcna7 and Rrad) and genes potentially involved in neuromuscular interactions and/or receptor signalling (Acvr2b, Adam19, D0H4S114, Kai1, Maged1, Mt2, Prkcabp, Ptp4a3, Ramp1, Rras, Timp1, Vegfa and Zfp145). A set of five genes with altered expression after denervation (Fzd9, Nr4a1, Frat2, Ctgf and Cyr61) indicate that Wnt signalling may be reduced in denervated skeletal muscle.

In vitro sliding of actin filaments labelled with single quantum dots.

We recently refined the in vitro motility assay for studies of actomyosin function to achieve rectified myosin induced sliding of actin filaments. This paves the way, both for detailed functional studies of actomyosin and for nanotechnological applications. In the latter applications it would be desirable to use actin filaments for transportation of cargoes (e.g., enzymes) between different predetermined locations on a chip. We here describe how single quantum dot labelling of isolated actin filaments simultaneously provides handles for cargo attachment and bright and photostable fluorescence labels facilitating cargo detection and filament tracking. Labelling was achieved with preserved actomyosin function using streptavidin-coated CdSe quantum dots (Qdots). These nanocrystals have several unique physical properties and the present work describes their first use for functional studies of isolated proteins outside the cell. The results, in addition to the nanotechnology developments, open for new types of in vitro assays of isolated biomolecules.