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1.
J Pept Sci ; 23(7-8): 610-617, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28370673

ABSTRACT

We recently identified a novel family of neutrophil-activating peptides including mitocryptide-1 and mitocryptide-2 (MCT-2) that are endogenously produced from various mitochondrial proteins. Among them, MCT-2 is an N-formylated pentadecapeptide derived from mitochondrial cytochrome b and is found to promote neutrophilic migration and phagocytosis efficiently. Signaling mechanisms of neutrophil activation by MCT-2 have been investigated at the cellular level, and MCT-2 has been demonstrated to be an endogenous specific ligand for formyl peptide receptor-2 (also referred to as formyl peptide receptor-like 1). It was also found that MCT-2 promoted neutrophilic functions via the activation of Gi2 proteins and phosphorylation of ERK1/2 consecutively. However, the physiological production, distribution, and functions of MCT-2 are not yet elucidated. Here, to investigate the roles of MCT-2 in vivo, we generated monoclonal antibodies (mAbs) against human MCT-2 (hMCT-2) that have two different characteristics. One mAb, NhM2A1, not only bound to the region of positions 10-15 of hMCT-2 but also recognized its C-terminal cleavage site that is presumably produced upon enzymatic hydrolysis of cytochrome b, indicating that NhM2A1 specifically interacts with hMCT-2 but not its parent protein. Moreover, we succeeded in acquiring a specific neutralizing mAb, NhM2A5, which blocks the bioactivities of hMCT-2. Specifically, NhM2A5 inhibited hMCT-2-induced ß-hexosaminidase release in neutrophilic/granulocytic differentiated HL-60 cells by binding to the region of positions 5-12 of hMCT-2. Functional analysis using obtained mAbs that specifically recognize hMCT-2 but not its parent protein, cytochrome b, and that neutralize bioactivities of hMCT-2 is expected to reveal the physiological roles of MCT-2, which are presently very difficult to investigate. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.


Subject(s)
Antibodies, Monoclonal/immunology , Mitochondrial Proteins/immunology , Animals , Cytochromes b/metabolism , Enzyme-Linked Immunosorbent Assay , HL-60 Cells , Humans , Mice , Mitochondrial Proteins/metabolism , Peptides/immunology , Peptides/metabolism , Swine
2.
Biophys Physicobiol ; 14: 207-220, 2017.
Article in English | MEDLINE | ID: mdl-29362706

ABSTRACT

We have studied the stiffness of myofilament lattice in sarcomeres in the pre-force generating state, which was realized by a relaxing reagent, BDM (butane dione monoxime). First, the radial stiffness for the overlap regions of sarcomeres of isolated single myofibrils was estimated from the resulting decreases in diameter by osmotic pressure applied with the addition of Dextran. Then, the radial stiffness was also estimated from force-distance curve measurements with AFM technology. The radial stiffness for the overlap regions thus obtained was composed of a soft and a rigid component. The soft component visco-elastically changed in a characteristic fashion depending on the physiological conditions of myofibrils, suggesting that it comes from cross-bridge structures. BDM treatments significantly affected the soft radial component of contracting myofibrils depending on the approach velocity of cantilever: It was nearly equal to that in the contracting state at high approach velocity, whereas as low as that in the relaxing state at low approach velocity. However, comparable BDM treatments greatly suppressed the force production and the axial stiffness in contracting glycerinated muscle fibers and also the sliding velocity of actin filaments in the in vitro motility assay. Considering that BDM shifts the cross-bridge population from force generating to pre-force generating states in contracting muscle, the obtained results strongly suggest that cross-bridges in the pre-force generating state are visco-elastically attached to the thin filaments in such a binding manner that the axial stiffness is low but the radial stiffness significantly high similar to that in force generating state.

3.
J Pharmacol Sci ; 123(3): 279-83, 2013.
Article in English | MEDLINE | ID: mdl-24152962

ABSTRACT

Stimulation of ß-adrenergic receptors in cardiac myocytes activates cyclic AMP-dependent protein kinase A (PKA). PKA-mediated phosphorylation of myofibrils decreases their longitudinal stiffness, but its effect on transverse stiffness is not fully understood. We thus examined the effects of PKA treatment on the transverse stiffness of cardiac myofibrils by atomic force microscopy and determined the phosphorylation levels of myofibril components by SDS-PAGE. Transverse stiffness was significantly decreased by PKA treatment concomitantly with increased phosphorylation of troponin I, myosin-binding protein C, and titin (also called connectin). Subsequent treatment with protein phosphatase 1 abrogated these PKA-mediated effects.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/pharmacology , Elasticity/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/physiology , Myofibrils/physiology , Carrier Proteins/metabolism , Cells, Cultured , Connectin/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/physiology , Electrophoresis, Polyacrylamide Gel , Humans , Microscopy, Atomic Force , Myocytes, Cardiac/ultrastructure , Myofibrils/metabolism , Myofibrils/ultrastructure , Phosphorylation , Protein Phosphatase 1/pharmacology , Receptors, Adrenergic, beta , Troponin I/metabolism
4.
J Physiol Sci ; 63(4): 299-310, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23690090

ABSTRACT

The radial stability of the actomyosin filament lattice in skeletal myofibrils was examined by using atomic force microscopy. The diameter and the radial stiffness of the A-band region were examined based on force-distance curves obtained for single myofibrils adsorbed onto cover slips and compressed with the tip of a cantilever and with the Dextran treatment. The results obtained indicated that the A-band is composed of a couple of stiffness components having a rigid core-like component. It was further clarified that these radial components changed the thickness as well as the stiffness depending on the physiological condition of myofibrils. Notably, by decreasing the ionic strength, the diameter of the A-band region became greatly shrunken, but the rigid core-like component thickened, indicating that the electrostatic force distinctly affects the radial structure of actomyosin filament components. The results obtained were analyzed based on the elementary structures of the filament lattice composed of cross-bridges, thin filaments and thick filament backbones. It was clarified that the actomyosin filament lattice is radially deformable greatly and that (1), under mild compression, the filament lattice is stabilized primarily by the interactions of myosin heads with thin filaments and thick filament backbones, and (2), under severe compression, the electrostatic repulsive interactions between thin filaments and thick filament backbones became predominant.


Subject(s)
Actomyosin/ultrastructure , Myofibrils/ultrastructure , Animals , Dextrans/pharmacology , Microscopy, Atomic Force , Muscle Contraction/physiology , Myofibrils/drug effects , Myofibrils/physiology , Psoas Muscles/ultrastructure , Rabbits
5.
J Physiol Sci ; 59(6): 439-46, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19669390

ABSTRACT

The effects of adrenaline (also called epinephrine) on glycogenolysis in living anaerobic muscles were examined based on time-dependent changes of (31)P-NMR spectra of resting frog skeletal muscles with and without iodoacetate treatments. The phosphate-metabolite concentration and the intracellular pH determined from the NMR spectra changed with time, reflecting the advancement of various phosphate metabolic reactions coupled with residual ATPase reactions to keep the ATP concentration constant. The results could be explained semi-qualitatively as the ATP regenerative reactions, creatine kinase reaction and glycogenolysis, advanced with time showing the characteristic two phases. Thus, it was clarified for living muscles that adrenaline activates the phosphorylase step of glycogenolysis, and the adrenaline-activated glycogenolysis is further regulated at the phosphofructokinase step by PCr and also possibly by AMP. Associated with the adrenaline-activated glycogenolysis in the examined muscles, the P(i) concentration and the intracellular pH, factors affecting the muscle force, changed significantly, suggesting complicated effects of adrenaline on the muscle contractility.


Subject(s)
Epinephrine/pharmacology , Glycogen/metabolism , Muscle Contraction/physiology , Muscle, Skeletal/metabolism , Anaerobiosis , Animals , Cyanides/pharmacology , Epinephrine/metabolism , Hydrogen-Ion Concentration , Iodoacetates/pharmacology , Magnetic Resonance Spectroscopy , Muscle Contraction/drug effects , Muscle, Skeletal/drug effects , Phosphates/metabolism , Radioisotopes/metabolism , Rana catesbeiana
6.
Cell Struct Funct ; 31(2): 135-43, 2006.
Article in English | MEDLINE | ID: mdl-17110784

ABSTRACT

The mechanical strength of sarcomere structures of skeletal muscle was studied by rupturing single myofibrils of rabbit psoas muscle by submicromanipulation techniques. Microbeads coated with alpha-actinin were attached to the surface of myofibrils immobilized to coverslip. By use of either optical tweezers or atomic force microscope, the attached beads were captured and detached from the myofibrils. During the detachment of the beads, the actin filaments bound specifically to the beads were peeled off from the bulk structures of myofibrils, thus rupturing the peripheral components of the myofibrils bound to the actin filaments. By analyzing the ruptures thus produced in various myofibril preparations, it was found that the sarcomere structure of myofibrils is maintained by numerous molecular components having the mechanical strength sufficient to sustain the contractile force produced by the actomyosin system. The present techniques could be applied to study the mechanical strength of cellular organelles containing actin filaments as their component.


Subject(s)
Micromanipulation/methods , Myofibrils/physiology , Sarcomeres/physiology , Actin Cytoskeleton/physiology , Actin Cytoskeleton/ultrastructure , Actinin , Animals , Biomechanical Phenomena , Electrophoresis, Polyacrylamide Gel , Microscopy, Atomic Force , Microspheres , Muscle Contraction/physiology , Myofibrils/ultrastructure , Psoas Muscles/physiology , Psoas Muscles/ultrastructure , Rabbits , Sarcomeres/ultrastructure
7.
J Physiol Sci ; 56(2): 145-51, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16839448

ABSTRACT

The transverse stiffness of single myofibrils of skeletal and cardiac muscles was examined by atomic force microscopy. The microscopic images of both skeletal and cardiac myofibrils in a rigor state showed periodical striation patterns separated by Z-bands, which is characteristic of striated muscle fibers. However, sarcomere patterns were hardly distinguishable in the stiffness distributions of the relaxed myofibrils of skeletal and cardiac muscles. Myofibrils in a rigor state were significantly stiff compared with those in a relaxed state, and in each state, cardiac myofibrils were significantly stiffer compared with skeletal myofibrils. By proteolytic digestions of sarcomere components of myofibrils, it was suggested that cardiac myofibrils are laterally stiffer than skeletal myofibrils because Z-bands, connectin (titin) filament networks, and other components of sarcomere structures for the former myofibrils are stronger than those for the latter.


Subject(s)
Microscopy, Atomic Force , Muscle Fibers, Skeletal/physiology , Myocytes, Cardiac/physiology , Myofibrils/diagnostic imaging , Myofibrils/physiology , Animals , Calpain/pharmacology , Electrophoresis, Polyacrylamide Gel , Muscle Contraction , Muscle Fibers, Skeletal/ultrastructure , Muscle Proteins/metabolism , Myocardial Contraction , Myocytes, Cardiac/ultrastructure , Myofibrils/drug effects , Rats , Sarcomeres/physiology , Sarcomeres/ultrastructure , Trypsin/pharmacology , Ultrasonography
8.
Biophys J ; 86(1 Pt 1): 346-52, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14695276

ABSTRACT

The 9 + 2 configuration of axonemes is one of the most conserved structures of eukaryotic organelles. Evidence so far has confirmed that bending of cilia and flagella is the result of active sliding of microtubules induced by dynein arms. If the conformational change of dynein motors, which would be a key step of force generation, is occurring in a three-dimensional manner, we can easily expect that the microtubule sliding should contain some transverse component, i.e., a motion in a direction at a right angle to the longitudinal axis of axonemes. Using a modified technique of atomic force microscopy, we found such transverse motion is actually occurring in an oscillatory manner when the axonemes of sea-urchin sperm flagella were adhered onto glass substrates. The motion was adenosine triphosphate-dependent and the observed frequency of oscillation was similar to that of oscillatory sliding of microtubules that had been shown to reflect the physiological activity of dynein arms (S. Kamimura and R. Kamiya. 1989. Nature: 340:476-478; 1992. J. Cell Biol. 116:1443-1454). Maximal amplitude of the diameter oscillation was around 10 nm, which was within a range of morphological change observed with electron microscopy (F. D. Warner. 1978. J. Cell Biol. 77:R19-R26; N. C. Zanetti, D. R. Mitchell, and F. D. Warner. 1979. J. Cell Biol. 80:573-588).


Subject(s)
Adenosine Triphosphate/metabolism , Biological Clocks/physiology , Molecular Motor Proteins/physiology , Sea Urchins/physiology , Sperm Motility/physiology , Sperm Tail/physiology , Animals , Male , Microscopy, Atomic Force/methods , Sperm Tail/ultrastructure
9.
Biophys J ; 85(3): 1741-53, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12944289

ABSTRACT

Isometric skinned muscle fibers were activated by the photogeneration of a substoichiometric amount of ATP and their cross-bridge configurations examined during the development of the rigor force by x-ray diffraction and electron microscopy. By the photogeneration of approximately 100 microM ATP, approximately 2/3 of the concentration of the myosin heads in a muscle fiber, muscle fibers originally in the rigor state showed a transient drop of the force and then produced a long-lasting rigor force (approximately 50% of the maximal active force), which gradually recovered to the original force level with a time constant of approximately 4 s. Associated with the photoactivation, muscle fibers revealed small but distinct changes in the equatorial x-ray diffraction that run ahead of the development of force. After reaching a plateau of force, long-lasting intensity changes in the x-ray diffraction pattern developed in parallel with the force decline. Two-dimensional x-ray diffraction patterns and electron micrographs of the sectioned muscle fibers taken during the period of 1-1.9 s after the photoactivation were basically similar to those from rigor preparations but also contained features characteristic of fully activated fibers. In photoactivated muscle fibers, some cross-bridges bound photogenerated ATP and underwent an ATP hydrolysis cycle whereas a significant population of the cross-bridges remained attached to the thin actin filaments with no available ATP to bind. Analysis of the results obtained indicates that, during the ATP hydrolysis reaction, the cross-bridges detached from actin filaments and reattached either to the same original actin monomers or to neighboring actin monomers. The latter cross-bridges contribute to produce the rigor force by interacting with the actin filaments, first producing the active force and then being locked in a noncycling state(s), transforming their configuration on the actin filaments to stably sustain the produced force as a passive rigor force.


Subject(s)
Adenosine Triphosphate/chemistry , Muscle, Skeletal/pathology , Adenosine Triphosphate/pharmacology , Animals , Calcium/chemistry , Light , Microscopy, Electron , Muscle Contraction , Muscle Fibers, Skeletal/chemistry , Muscle Rigidity , Muscles/metabolism , Muscles/pathology , Potassium/chemistry , Rabbits , Stress, Mechanical , Synchrotrons , Time Factors , X-Ray Diffraction
10.
Neuroreport ; 14(4): 645-9, 2003 Mar 24.
Article in English | MEDLINE | ID: mdl-12657904

ABSTRACT

An error signal from the anterior forebrain pathway (AFP) in the songbird brain is necessary for juvenile song learning and adult song maintenance. It induces the expression of protein kinase C (PKC) which is related to the plasticity in the robust nucleus of the archistriatum (RA), one of the song control nuclei in the forebrain. The glutamatergic inputs from the AFP activate mainly the NMDA receptors of the RA neurons. In order to clarify the molecular mechanism of error signal-induced PKC activation, two experiments were carried out. First, Ca2+ concentration was measured in a brain slice preparation from zebra finches using the fluorescent Ca2+ indicator Fura 2-AM. Glutamate increased the intracellular Ca2+ concentration ([Ca2+](i)) in RA neurons. This increase was inhibited by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (AP5). Second, we examined the expression of PKC in the RA slice preparation after stimulation with glutamate for 10 min using PKCbeta1 fluorescence immunohistochemistry. Glutamate induced the activation of PKC as the translocation from the cytosol to the cell membrane, and the translocation was inhibited by AP5. These results indicate that the translocation of the PKC caused by the [Ca2+](i) elevation through NMDA receptors is concerned with the initial stage of error signal-induced plasticity in the RA.


Subject(s)
Basal Ganglia/physiology , Prosencephalon/physiology , Protein Kinase C/metabolism , Vocalization, Animal/physiology , 2-Amino-5-phosphonovalerate/pharmacology , Animals , Calcium/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Excitatory Amino Acid Agents/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Fluorescent Antibody Technique/methods , Fura-2/metabolism , Glutamic Acid/pharmacology , In Vitro Techniques , Male , Medial Forebrain Bundle/cytology , Medial Forebrain Bundle/metabolism , Protein Kinase C/classification , Signal Transduction/physiology , Songbirds , Time Factors
11.
Adv Exp Med Biol ; 538: 285-94; discussion 294, 2003.
Article in English | MEDLINE | ID: mdl-15098676

ABSTRACT

By applying AFM technology, we studied mechanical characteristics of myofibrils of skeletal muscle. The obtained results indicate that (1) the Z-band is the most rigid sarcomere component stabilizing the myofibril structures, (2) various filamentous components are inter-connected in sarcomere with sufficient mechanical strength to support the contractile force, and (3) the molecular structure of the overlap region between actin and myosin filaments is anisotropic. In any case the present studies clearly indicate that the AFM technique is a powerful tool to investigate the mechanical characteristics of sarcomere structure of muscle fiber.


Subject(s)
Microscopy, Atomic Force/methods , Muscle, Skeletal/metabolism , Myofibrils/chemistry , Actins/chemistry , Animals , Anisotropy , Cells, Cultured , Myofibrils/metabolism , Myosins/chemistry , Psoas Muscles/cytology , Rabbits , Sarcomeres/metabolism , Software
12.
Biochim Biophys Acta ; 1573(1): 93-9, 2002 Oct 10.
Article in English | MEDLINE | ID: mdl-12383947

ABSTRACT

The motions of myosin filaments actively sliding along suspended actin filaments were studied. By manipulating a double-beam laser tweezers, single actin filaments were suspended between immobilized microbeads. When another beads coated with myosin filaments were dragged to suspended actin filaments, the beads instantly and unidirectionally slid along the actin filaments. The video image analysis showed that the beads slid at a velocity of ca. 3-5 microm/s accompanied with zigzag motions. When beads were densely coated with myosin filaments, the sliding motions became straight and smooth. The obtained results indicate that (1) during the sliding motions, the interaction between myosin heads and actin filaments is weak and susceptible to random thermal agitations, (2) the effects of thermal agitations to the sliding motions of myofilaments are readily suppressed by mechanical constraints imposed to the filaments, and (3) the active sliding force is produced almost in parallel to the filaments axis.


Subject(s)
Actin Cytoskeleton/physiology , Actins/physiology , Myosins/physiology , Animals , Lasers , Microspheres , Muscle Contraction , Rabbits , Video Recording
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