Application of ultrasound treatment for improving the physicochemical, functional and rheological properties of myofibrillar proteins.

The aim of this study was to evaluate the impact of duration (10, 20 and 30min) and power (100 and 300W) of high-intensity ultrasound (20kHz) on physicochemical properties of beef myofibrillar proteins in order to investigate novel process for modification of its functional characteristics. Results showed that augmentation of duration and power of ultrasound led to enhance pH. Also, the water holding capacity and gel strength were improved by increasing pH. The highest value in pH, reactive sulfhydryl content, water holding capacity and gel strength was obtained in sample subjected to 30min of ultrasound at 300W. The particle size distribution of the proteins was decreased after ultrasound treatment because of the cavitation force of ultrasound waves. In this circumstance, an improvement of emulsifying properties can be obtained. Ultrasonic waves had significant effects on the rheological properties of myofibrillar proteins. Treated samples were more elastic and stiffer than control, although the inverse trend was observed after 30min treatment at each power. Finally, a reducing trend in viscosity was observed by increasing time and power of sonication. Ultrasonic treatment could successfully improve functional properties with effect on physicochemical properties of myofibrillar proteins.

Application of high temperature (14°C) aging of beef M. semimembranosus with low-dose electron beam and X-ray irradiation.

The effects of irradiation source (electron beam [EB] and X-ray [XR]), aging temperature (4°C and 14°C), and aging time (0, 3, 7, and 14days) were evaluated on microbial quality, physicochemical properties, and calpain-1 autolysis in beef M. semimembranosus. Regardless of irradiation source, irradiation prior to aging reduced the total number of aerobic bacteria in beef and this reduction was maintained during aging. Irradiation did not affect the pH, b⁎ value, shear force, or myofibrillar fragmentation index of beef at day 0. Degradation of sarcoplasmic and myofibrillar proteins was greater in beef aged at 14°C compared with beef aged at 4°C. EB- or XR-irradiated samples showed slower autolysis of calpain-1; however, beef tenderness was not affected. Therefore, EB or XR irradiation can be applied to beef prior to aging to control microbial growth during high temperature (14°C) aging, thus shortening the aging time without adversely affecting the physicochemical properties of beef.

A photoactivated artificial muscle model unit: reversible, photoinduced sliding of nanosheets.

A novel photoactivated artificial muscle model unit is reported. Here we show that organic/inorganic hybrid nanosheets reversibly slide horizontally on a giant scale and the interlayer spaces in the layered hybrid structure shrink and expand vertically by photoirradiation. The sliding movement of the system on a giant scale is the first example of an artificial muscle model unit having much similarity with that in natural muscle fibrils. In particular, our layered hybrid molecular system exhibits a macroscopic morphological change on a giant scale (~1500 nm) relative to the molecular size of ~1 nm by means of a reversible sliding mechanism.

Effects of low-intensity swimming on radiation-induced leg contracture in mice.

OBJECTIVE: To evaluate the effects of low-intensity swimming on radiation-induced leg contracture. METHODS: Forty mice were randomly and equally divided into four groups: 1) irradiation; 2) swimming before irradiation; 3) swimming after irradiation; 4) swimming after contracture, and their left hind legs were exposed to gamma irradiation of 60 Gy. The mice were allowed to swim freely for 10 minutes, three times per day. For group 2, the mice were allowed to swim for only 1 week before irradiation. For group 3, the mice were allowed to swim immediately after irradiation until day 130, post-irradiation. For group 4, the mice were allowed to swim after leg contracture happened (on day 30 post-irradiation) until day 130, post-irradiation. The leg lengths and knee joint angles were measured. Leg contracture was defined as the decrease in the hind leg lengths and the knee joint angles of each animal. The ultrastructural changes of gastrocnemius muscles were observed using transmission electron microscopy. RESULTS: The radiation could result in leg contracture and mitochondrial injury of the muscles. However, the group of swimming immediately after irradiation had less leg contracture and no vacuolar degeneration in the mitochondria, compared with the other groups. CONCLUSION: Low-intensity swimming that began immediately after the mice were irradiated could effectively prevent the irradiated legs from contracture. Patients with irradiated mastication muscles were recommended to begin mouth-opening exercises immediately after radiotherapy.

[The strategy of the "useful sun" improves physical endurance and structural adaptation in the myocardium].

The action of solar light transformed by special screens has been studied on CD-1 male mice. In the active control group, mice were irradiated through screens absorbing the UV-component. In the experimental group, screens transforming the UV-component into the orange-red light were used. In the active control, changes in the swimming activity, as compared to the same parameter before irradiation, were manifested much less than in animals of the experimental group. A morphological analysis showed changes in the structure of all cardiomyocyte organelles studied: the relative area of mitochondria in the experimental mice increased by more than 20% compared to intact animals (p < 0.05). A significant increase in the area of the sarcoplasmic reticulum, by 23.4% (p < 0.05), and in the volume of the myofibrillar apparatus, by 19.4% (p < 0.05), was detected. The results of our experiment show that the irradiation with using an additional orange-red component improves the physical endurance 1.5 times and initiates morphogenetic processes in cardiac muscle cells.

Reduction of photobleaching and photodamage in single molecule detection: observing single actin monomer in skeletal myofibrils.

Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light.

Soft-X-ray damage to biological samples.

X-ray damage to biological samples was investigated in the wavelength region of 2.7-5 nm, which overlaps the so-called 'water-window', the wavelength range of 2.4-4.3 nm usually used in X-ray microscopy. Yeast cells and myofibrils were chosen as representatives of whole cell samples and motile protein systems, respectively. The samples were exposed to X-rays using an apparatus composed mainly of a laser-plasma X-ray source, a Wolter mirror condenser, and a sample cell. The yeast cells lost their dye exclusion ability when the X-ray flux was higher than 1 x 10(6) photons micron-2, while the myofibrils lost contractility when the X-ray flux was higher than 4 x 10(5) photons micron-2. These X-ray fluxes are lower than the flux required for the X-ray microscope observation of biological samples at a resolution higher than that of light microscopes.

A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis.

A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

Regeneration in denervated toad (Bufo viridis) gastrocnemius muscle and the promotion of the process by low energy laser irradiation.

BACKGROUND: It is known that while denervated skeletal muscles have the ability to regenerate, maturation of regenerated myofibres does not take place under these conditions. Denervation also causes elevation of "invasive" and satellite cells, but the role of these cells in the regeneration process after injury to the denervated muscle is still unknown. Low energy lasers have recently been found to modulate and accelerate physiological processes in cells. The aim of the present study was to compare regeneration in denervated and innervated amphibian muscles and to investigate whether this process in denervated muscles can be stimulated by low energy laser irradiation prior to injury in these muscles. METHODS: Denervated gastrocnemius muscles of toads were irradiated with He-Ne laser (6.0 mW, 31.2 J/cm2) 7 days postdenervation (control muscle received red light irradiation at the same wavelength). Nine days after denervation cold injury was performed on the site of irradiation of both groups of muscles. At 14 days postinjury all muscles were removed and processed for histology and histomorphometric analysis of mononucleated cells, myotubes, and young myofibres in the regenerated zone. RESULTS: The volume fraction (percent of total injured zone) of the various histological structures in the injured zones 14 days after cold injury in the denervated (9 days prior to injury) muscles did not differ from innervated injured muscles at the same time interval postinjury. The mononucleated cells and myotubes in the laser irradiated muscles comprised 49 +/- 4% and 6 +/- 1% of the injured area, respectively, which was significantly lower than their volume fraction (67 +/- 2% and 11 +/- 2%, respectively) in the control muscles. The young myofibres populated 34 +/- 4% of the total injured area in the denervated and laser irradiated muscles which was significantly higher than their volume fraction (12 +/- 2%) in control denervated muscles. CONCLUSIONS: It is concluded that initial stages of regeneration can also take place in skeletal denervated and injured muscles of amphibians. The kinetics of the regeneration process are identical in denervated and innervated muscles. The process of regeneration in denervated muscles can be markedly enhanced if the muscle is irradiated by low energy laser prior to injury, probably by activation (stimulation of proliferation and/or differentiation) cells in the muscles that are "recruited" and participate in the process of regeneration.

The effect of soft X-radiation on myofibrils.

Myofibrils, the contractile organelles from striated muscles, have been examined in the X-ray microscope to determine the effect of radiation on their function and structure. Using X-rays of energy 350-385 eV in the water window we find that after an exposure to 7.5 x 10(5) photons/micron2 (calculated to give an absorbed dose of 20,000 Gy) the myofibrils will no longer contract. The use of the free radical scavenging agent, DMSO, gives some protection to the fibrils. It has also been found that after this much irradiation the fibrils lose up to 20% of their mass. Further substantial mass loss occurs on subsequent irradiation. After 25 times the loss-of-function exposure only 30% of the mass remains. Analysis of a series of images of the same myofibril covering this range of exposures shows that the mass is preferentially lost in some areas of the structure and consequently significant structural changes occur.

Modification of myofibrils by fluorophore-induced photo-oxidation.

The excitation of fluorophores in the vicinity of a myofibril stops both shortening in the presence of ATP and Ca2+, and the extraction of the A-band by NaCl in the presence of Mg pyrophosphate. Shortening is more quickly affected than extraction. These effects can be induced by fluorescently-tagged antibodies bound in the A-band. Both depolymerization of the thick filament and the interaction between the myosin head and actin appear to be modified. Enzymatic lowering of the oxygen concentration in the bathing solution during excitation reduces these effects, indicating that they are due to photo-oxidation catalysed by excitation of the fluorophore. The results suggest that care needs to be exercised to minimize the consequences of these changes on the outcome of fluorescence-based assays of activity. Irradiated myofibrils that do not shorten, hydrolyse ATP at a rate comparable to those that contract, so they may be useful as a model system for the study of crossbridge activity in the ordered array of proteins of the myofibril.

Irradiations of rabbit myofibrils with an ultraviolet microbeam. I. Effects of ultraviolet light on the myofibril components necessary for contraction.

Glycerinated rabbit psoas myofibrils, F-actin, and myofibril ghosts were irradiated with ultraviolet light (UV) to investigate how UV blocks myofibril contraction. Myofibril contraction is most sensitive to 270- and 290-nm wavelength light. We irradiated I and A bands separately with 270- and 290-nm wavelength light using a UV microbeam and constructed dose-response curves for blocking sarcomere contraction. For both wavelengths, irradiations of A bands required less energy per area to block contraction than did irradiations of I bands, suggesting that the primary effects of both 270- and 290-nm wavelength light in stopping myofibril contraction are on myosin. We investigated whether the primary effect of UV in blocking I-band contraction is the depolymerization of actin by comparing the relative sensitivities of I-band contraction, F-actin depolymerization, and thin filament depolymerization to 270- and 290-nm light. We also compared the dose of UV required to depolymerize F-actin in solution with the dose needed to block I-band contraction and the dose required to alter thin filament structure in myofibril ghosts. The results confirm that UV blocks I-band contraction by depolymerizing actin. We discuss how the results might be relevant to the hypothesis that an actomyosin-based system is involved in chromosome movement.

Irradiations of rabbit myofibrils with an ultraviolet microbeam. II. Phalloidin protects actin in solution but not in myofibrils from depolymerization by ultraviolet light.

We tested whether phalloidin protects actin in myofibrils from depolymerization by ultraviolet light (UV). I bands in glycerinated rabbit psoas myofibrils were irradiated with a UV microbeam in the presence and absence of phalloidin. We used the retention of contractility of the irradiated I band as the assay for protection of actin by phalloidin, since previous experiments indicated that UV blocks contraction of an irradiated I band by depolymerizing the thin filaments. The I bands of myofibrils incubated in phalloidin were as sensitive to UV as control I bands, indicating that phalloidin did not protect the thin filaments. However, phalloidin did protect F-actin in solution from depolymerization by UV. This apparent contradiction between F-actin in myofibrils and F-actin in solution was resolved by observing unirradiated myofibrils that were stained with rhodamine-phalloidin. It was found that phalloidin does not bind uniformly to the thin filaments, though as the fluorescence image is observed over time the staining pattern changes until it does appear to bind uniformly. We conclude that phalloidin does not protect F-actin in myofibrils from depolymerization by UV because it does not bind uniformly to the filaments.

Analysis of chromosome movement in crane fly spermatocytes by ultraviolet microbeam irradiation of individual chromosomal spindle fibres. II. Action spectra for stopping chromosome movement and for blocking ciliary beating and myofibril contractions.

Chromosome-to-pole movement in crane fly spermatocytes was temporarily blocked by ultraviolet light focussed to a 4-micrometer-diameter spot on single chromosomal spindle fibres. Since similar irradiation of the interzonal region did not alter chromosome-to-pole movement, this effect was specific to spindle fibres. The action spectrum for blocking chromosome movement in this specific way had two peaks, one at 270 nm and one at 290 nm. To block movement, irradiations with 280-nm-wavelength light required two to four times more energy than irradiations with 270- or 290-nm-wavelength light. Action spectra were obtained for blocking ciliary beating and for blocking myofibril contraction. The action spectrum for blocking ciliary beating had a broad peak, between 260 nm and 280 nm, whilst that for blocking myofibril contraction had two peaks, at 270 and 290 nm, just like that for blocking chromosome movement. We discuss the similarities and differences in the various action spectra, and we compare the action spectra to absorption spectra of spindle components and to other action spectra (e.g., that for depolymerizing actin-containing filaments). Absorption spectra were obtained for ultraviolet light passing through spindle fibres as well as for ultraviolet light passing through the interzone.

[Ultrastructure of skeletal muscle tissue of microwave damaged chick embryos]. / Ul'trastruktura skeletnoi myshechnoi tkani kurinykh émbrionov pri mikrovolnovom porazhenii.

The effect of ultrahigh-frequency energy of electromagnetic field of low intensity on certain morphometric indices of intracellular organoids and on cellular ultrastructure were studied in the developing skeletal muscular tissue of chick embryos. In the skeletal muscles of irradiated embryos a limited cellular destruction, structural disorders in myonic organoids were revealed. Reactive-recovery processes manifested themselves in hyperplasy and hypertrophy of organoids, in activation of protein synthesis, in increasing amount of myosatellites. At early stages after irradiation peripheral mitochondria are subjected to greater changes. After hatching, central mitochondria in myons suffer more. Quantitative changes in myonic organoids and degree of their destruction seem to depend on.a peculiar differentiation of the muscular tissue, blood supply and innervation of the muscle as an organ.