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T Yanagida

Publications and source records attributed to T Yanagida.

At least 91 records · Page 5Linked to original sources
PubMed1996

Torsional rigidity of single actin filaments and actin-actin bond breaking force under torsion measured directly by in vitro micromanipulation.

Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 +/- 1.2) x 10(-26) Nm2. This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90 degrees, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.

Actins↗
PubMed1996

Direct observation of single kinesin molecules moving along microtubules.

Kinesin is a two-headed motor protein that powers organelle transport along microtubules. Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule. Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead). The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a approximately 1% probability of detachment per mechanical cycle. Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl. Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism.

Amino Acid Sequence↗
PubMed1996

Accelerated generation of CD14+ monocyte-lineage cells from the bone marrow of rheumatoid arthritis patients.

OBJECTIVE: To examine the capacity of bone marrow progenitor cells to generate CD14+ cells, in order to assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA). METHODS: CD14- cells purified from bone marrow specimens of 11 patients with active RA and 8 control patients (osteoarthritis or trauma) were cultured in the presence or absence of granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 pg/ml). After incubation for various lengths of time, the cells were analyzed by flow cytometry for expression of CD14 and HLA-DR. RESULTS: The spontaneous generation of CD14+ cells from bone marrow CD14- progenitor cells was accelerated in RA patients compared with control patients. Moreover, the expression of HLA-DR on the bone marrow-derived CD14+ cells was also accelerated in RA patients compared with controls. GM-CSF significantly enhanced the generation of CD14+ cells, as well as the expression of HLA-DR, on CD14+ cells of control patients, but not those of RA patients. GM-CSF levels in the culture supernatants of bone marrow CD14- cells were not significantly different between RA patients and control patients (undetectable in most cases). CONCLUSION: These observations strongly support the hypothesis that the accelerated generation of CD14+ cells from bone marrow progenitor cells and the accelerated maturation of such CD14+ cells into HLA-DR+ cells play an important role in the pathogenesis of RA. Moreover, the data suggest a functional alteration of RA bone marrow CD14- cells in their responsiveness to GM-CSF.

Adult↗
PubMed1996

B7-1 synergizes with interleukin-12 in interleukin-2 receptor alpha expression by mouse T helper 1 clones.

Expression of interleukin-2 receptor alpha (IL-2R alpha) is critical to induce interleukin (IL)-2-dependent proliferation of T helper (Th)1 clones. The IL-2R alpha expression of Th1 clones is known to be up-regulated by IL-12. Co-stimulation via CD28/CTLA-4 is also known to be important for efficient activation of CD4+ T cells. In the present experiments, IL-12-induced enhancement of IL-2R alpha expression of Th1 clones stimulated with B cells as antigen-presenting cells (APC) is suppressed by the addition of anti-B7-1. To analyze the mechanism, Th1 clones were stimulated with immobilized anti-CD3 plus IL-12 in the presence or absence of Chinese hamster ovary cells that express mouse B7-1 (B7-1CHO) and the enhancement of IL-2R alpha expression induced by the co-stimulation was analyzed. The results of these experiments indicate that B7-1 synergizes with IL-12 in IL-2R alpha expression of the Th1 clone stimulated with anti-CD3, although B7-1CHO alone did not enhance IL-2R alpha expression of the clones. B7-1 stimulation is not mediated by the enhancement of IL-2 production: B7-1 enhancement of IL-2R alpha expression was FK506 resistant, while the inclusion of FK506 abrogated IL-2 production of the Th1 cells. B7-1 co-stimulation did not stabilize IL-2E alpha mRNA, but did synergize with IL-12 to enhance IL-2R alpha mRNA transcription.

Animals↗
PubMed1996

Modification of the bi-directional sliding movement of actin filaments along native thick filaments isolated from a clam.

The properties of bi-directional sliding of F-actin prepared from rabbit skeletal muscle moving along clam thick filaments have been characterized in the presence of agents known to modify unloaded shortening velocity in muscle to determine if the sliding characteristics of actin are similar in the two directions of movement. Actin filaments moved at a fast velocity towards the central bare zone (11.1 +/- 0.2 microns s-1) and at a slower velocity away from the bare zone (3.9 +/- 0.3 microns s-1). Movement of filaments at the slow sliding velocity is thought to be sustained by a change in orientation of the myosin head. The Michaelis Menten constant (Km values) of approximately 0.3 mM in the presence of MgATP concentrations of 0.01-2.0 mM at an ionic strength of 43.5 mM were reduced to approximately 0.1 mM at low ionic strength (18.5 mM) although the Km values at the fast and slow sliding velocities at each ionic strength were similar. In the presence of constant concentrations of MgATP, increasing the MgADP concentrations from 0.5 to 2mM, decreased the bi-directional sliding velocity of actin. The data were well fitted with an equation described by Michaelis Menten kinetics yielding mean absolute Km and Ki values of 0.41 +/- 0.01 and 0.44 +/- 0.05 mM for the fast velocity and 0.29 +/- 0.07 and 0.45 +/- 0.02 mM for the slow velocity of sliding, respectively. The Km and Ki values were not significantly different from each other at either the fast or slow sliding velocities. The actin filament sliding velocity appeared to be controlled through the thick filament as actin was devoid of regulatory proteins and the presence of Ca2+ modified the MgATP dependent movement of actin. The pCa value for half maximal sliding velocity was 7.0 for both fast and slow velocities. The Km and Ki values and the Ca2+ sensitivity of the actin movement at the fast and slow sliding velocity are similar suggesting that no major biochemical changes have occurred in the myosin head as a result of a change in orientation.

Actins↗
PubMed1996

Direct inhibition of the actomyosin motility by local anesthetics in vitro.

Using a recently developed in vitro motility assay, we have demonstrated that local anesthetics directly inhibit myosin-based movement of single actin filaments in a reversible dose-dependent manner. This is the first reported account of the actions of local anesthetics on purified proteins at the molecular level. In this study, two tertiary amine local anesthetics, lidocaine and tetracaine, were used. The inhibitory action of the local anesthetics on actomyosin sliding movement was pH dependent; the anesthetics were more potent at higher pH values, and this reaction was accompanied by an increased proportion of the uncharged form of the anesthetics. QX-314, a permanently charged derivative of lidocaine, had no effect on actomyosin sliding movement. These results indicate that the uncharged form of local anesthetics is predominantly responsible for the inhibition of actomyosin sliding movement. The local anesthetics inhibited sliding movement but hardly interfered with the binding of actin filaments to myosin on the surface or with actomyosin ATPase activity at low ionic strength. To characterize the actomyosin interaction in the presence of anesthetics, we measured the binding and breaking force of the actomyosin complex. The binding of actin filaments to myosin on the surface was not affected by lidocaine at low ionic strength. The breaking force, measured using optical tweezers, was approximately 1.5 pN per micron of an actin filament, which was much smaller than in rigor and isometric force. The binding and breaking force greatly decreased with increasing ionic strength, indicating that the remaining interaction is ionic in nature. The result suggests that the binding and ATPase of actomyosin are governed predominantly by ionic interaction, which is hardly affected by anesthetics; whereas the force generation requires hydrophobic interaction, which plays a major part of the strong binding and is blocked by anesthetics, in addition to the ionic interaction.

Actins↗
PubMed1996

Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: unitary steps and forces.

We have developed a new technique for measurements of piconewton forces and nanometer displacements in the millisecond time range caused by actin-myosin interaction in vitro by manipulating single actin filaments with a glass microneedle. Here, we describe in full the details of this method. Using this method, the elementary events in energy transduction by the actomyosin motor, driven by ATP hydrolysis, were directly recorded from multiple and single molecules. We found that not only the velocity but also the force greatly depended on the orientations of myosin relative to the actin filament axis. Therefore, to avoid the effects of random orientation of myosin and association of myosin with an artificial substrate in the surface motility assay, we measured forces and displacements by myosin molecules correctly oriented in single synthetic myosin rod cofilaments. At a high myosin-to-rod ratio, large force fluctuations were observed when the actin filament interacted in the correct orientation with a cofilament. The noise analysis of the force fluctuations caused by a small number of heads showed that the myosin head generated a force of 5.9 +/- 0.8 pN at peak and 2.1 +/- 0.4 pN on average over the whole ATPase cycle. The rate constants for transitions into (k+) and out of (k-) the force generation state and the duty ratio were 12 +/- 2 s-1, and 22 +/- 4 s-1, and 0.36 +/- 0.07, respectively. The stiffness was 0.14 pN nm-1 head-1 for slow length change (100 Hz), which would be approximately 0.28 pN nm-1 head-1 for rapid length change or in rigor. At a very low myosin-to-rod ratio, distinct actomyosin attachment, force generation (the power stroke), and detachment events were directly detected. At high load, one power stroke generated a force spike with a peak value of 5-6 pN and a duration of 50 ms (k(-)-1), which were compatible with those of individual myosin heads deduced from the force fluctuations. As the load was reduced, the force of the power stroke decreased and the needle displacement increased. At near zero load, the mean size of single displacement spikes, i.e., the unitary steps caused by correctly oriented myosin, which were corrected for the stiffness of the needle-to-myosin linkage and the randomizing effect by the thermal vibration of the needle, was approximately 20 nm.

Actomyosin↗
PubMed1996

Role of endothelin as a mitogen in experimental glomerulonephritis in rats.

Recent studies have revealed that endothelin-1 (ET-1) is a potent mitogen for mesangial cells in vitro. To determine whether ET-1 exerts the mitogenic action on mesangial cells in vivo, we examined the glomerular expression of ET-1 and its receptors in a rat model of mesangial proliferative glomerulonephritis and assessed the effect of a specific endothelin A (ET(A)) receptor antagonist, FR139317, on mesangial cell proliferation in this model. The levels of preproET-1 mRNA expression and ET-1 protein production in glomeruli increased markedly on days 4 and 7 after disease induction, and the levels changed in concordance with the glomerular cell proliferation. In contrast, the level of ET(A) receptor mRNA initially decreased on day 1, and thereafter increased on days 4 and 7. Administration of FR139317 to rats with experimental glomerulonephritis induced a significant reduction in mesangial cell proliferation. In addition, in situ hybridization of preproET-1 mRNA and double-immunolabeling of ED-1 and OX-7 in a mirror image section revealed that the principal cell expressing ET-1 in glomeruli were infiltrating macrophages on day 1, and they were replaced by mesangial cells on day 4. These findings indicate that ET-1 functions as a potent mitogen for mesangial cells in vivo in an autocrine or paracrine fashion.

Animals↗
PubMed1996

Transforming growth factor-beta 1 in hypertensive renal injury in Dahl salt-sensitive rats.

The expression of transforming growth factor-beta 1 (TGF-beta 1) for hypertensive renal injury was investigated in Dahl salt-sensitive (Dahl-S) rats fed a high-salt (HS; 8% NaCl) diet or a low-salt (LS; 0.3% NaCl) diet for 4 wk. The HS rats developed severe hypertension and renal damage, including glomerulosclerosis and arteriosclerosis. TGF-beta biosynthesis by isolated glomeruli, the TGF-beta localization, and the gene expression of TGF-beta 1, latent TGF-beta binding protein (LTBP), and TGF-beta receptors (Types I, II, and III) were compared between the HS rats and LS rats. A TGF-beta bioassay revealed that the isolated glomeruli from the HS rats secreted a larger amount of latent TGF-beta than those from the LS rats. Northern blotting analysis demonstrated that the HS diet led to the increases in cortical gene expression of TGF-beta 1, LTBP, and TGF-beta receptors, compared with the LS diet. The glomerular biosynthesis of fibronectin and plasminogen activator inhibitor-1 (PAI-1), and cortical mRNA expression for fibronectin, collagen I, and PAI-1 (which may be affected by TGF-beta) in the HS rats were elevated, compared with the LS rats. The latent TGF-beta immunostained by anti-LTBP antibody was localized on the sclerosing glomeruli and vascular walls. Furthermore, fibronectin, collagen I, and PAI-1 were also localized in the sclerotic area. The TGF-beta 1-positive cells, immunostained by antibody for latency-associated peptide of TGF-beta 1, increased in the glomeruli and vascular walls in the HS rats. These results thus suggested that TGF-beta 1 may be related to hypertensive renal injury in this model.

Animals↗
PubMed1996

[Growth patterns of colorectal carcinoma and mucin property of transitional mucosa].

To examine the relationship of growth pattern of colorectal carcinoma with mucin property and cellular proliferation of transitional mucosa (TM), 49 lesions of colorectal carcinomas invading to the muscularis propria were studied. Growth patterns were classified as polypoid growth (PG), non-polypoid growth (NPG), or intermediate type basically according to Shimoda's classification. Mucin property of TM was examined using HID-AB staining. Cellular proliferation was assessed from PCNA staining. NPG carcinomas were smaller in size, found in younger age, and more often to have lymph node metastasis than PG carcinoma. Mucin property of TM significantly correlated with growth pattern of carcinoma; sulphomucin dominance in TM was found in 5 out of 12 PG carcinomas where as it was found in only 1 out of 22 NPG carcinomas. In addition, none of the 8 carcinomas whose TM showed a sulphomucin dominance had lymph node metastasis. However cellular proliferation in TM did not differ by the growth pattern of carcinoma or the mucin property of TM. Preoperative assessment of the growth pattern of lesions and the mucin property of TM appears to be useful for predicting lymph node metastasis of colorectal carcinomas invading to the muscularis propria.

Aged↗
PubMed1995

Inhibition of expression of cyclin A in human B cells by an immunosuppressant mizoribine.

Mizoribine has been used to prevent rejection of organ allografts in humans and in animal models. Recent clinical trials have demonstrated its efficacy in rheumatoid arthritis and lupus nephritis, in which abnormalities of B cell functions are also involved. We therefore examined the effects of mizoribine on the in vitro function of human B cells. IgM production was induced from highly purified B cells obtained from healthy donors by stimulation with Staphylococcus aureus Cowan I (SA) plus IL-2. Mizoribine suppressed the production of IgM at its pharmacologically attainable concentrations (1 to 3 micrograms/ml) in a dose-dependent manner. Mizoribine had to be present within the first 96 h after the initiation of cultures to exert its suppressive effects on B cell responses. Cell cycle analysis disclosed that mizoribine significantly decreased the numbers of B cells in S + G2 + M phases. Mizoribine did not decrease GTP levels in SA-stimulated B cells, whereas mizoribine led to a decrease in GTP levels in activated T cells, which was reversed by addition of GMP. Consistently, the suppressive effects of mizoribine on the IgM production of SA-stimulated B cells was not reversed by the addition of GMP as much as 40 microM, which overcame the inhibitory effects of mizoribine on the proliferation of anti-CD3-stimulated T cells. Although mizoribine did not suppress the expression of CD25 and cdc2 kinase, mizoribine markedly suppressed the expression of cyclin A in SA-activated B cells. These results indicate that mizoribine directly suppresses the function of human B cells without interfering with the initial phase of activation. Moreover, the data demonstrate that mizoribine interferes with the cell cycle progression of activated B cells by suppressing the expression of cyclin A by a mechanism distinct from guanine ribonucleotide depletion.

Adult↗
PubMed1995

Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution.

Visualization of single actin filaments by fluorescence microscopy led to the development of new in vitro assays for analysing actomyosin-based motility at the molecular level. The ability to manipulate actin filaments with a microneedle or an optical trap combined with position-sensitive detectors has enabled direct measurements of nanometre displacements and piconewton forces exerted by individual myosin molecules. To elucidate how myosin generates movement, it is necessary to understand how ATP hydrolysis is coupled to mechanical work at the level of the single molecule. But the most sensitive microscopic ATPase assay available still requires over 1,000 myosins. To enhance the sensitivity of such assays, we have refined epifluorescence and total internal reflection microscopies to visualize single fluorescent dye molecules. We report here that this approach can be used directly to image single fluorescently labelled myosin molecules and detect individual ATP turnover reactions. In contrast to previously reported single fluorescent molecule imaging methods, which used specimens immobilized on an air-dried surface, our method allows video-rate imaging of single molecules in aqueous solution, and hence can be applied to the study of many types of enzymes and biomolecules.

Adenosine Triphosphate↗
PubMed1995

Selective induction of IgM rheumatoid factors by CD14+ monocyte-lineage cells generated from bone marrow of patients with rheumatoid arthritis.

OBJECTIVE: To determine the capacity of CD14+ monocyte-lineage cells induced from bone marrow of rheumatoid arthritis (RA) patients to stimulate the production of IgM rheumatoid factor (IgM-RF), in order to explore the functional abnormalities of CD14+ cells and gain insight into the mechanism of selective synthesis of IgM-RF in RA. METHODS: CD14+ cells were induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of CD14- cells purified from bone marrow cells obtained from 6 RA patients and 6 osteoarthritis (OA) patients. The production of IgM and IgM-RF was induced by stimulating B cells from normal healthy individuals with immobilized anti-CD3-activated autologous CD4+ T cells. The effects of CD14+ cells on the proportion of IgM-RF to total IgM produced by the normal B cells were assessed. RESULTS: CD14+ cells induced by GM-CSF stimulation of bone marrow CD14- cells from the 6 RA patients significantly enhanced the proportion of IgM-RF to total IgM produced by anti-CD3-activated CD4+ T cell-stimulated normal B cells (P < 0.05), whereas GM-CSF-induced CD14+ cells from the bone marrow of the 6 OA patients did not significantly affect IgM-RF production. CD14+ cells induced by GM-CSF obtained from different sites in the same RA patient on different occasions consistently enhanced the proportion of IgM-RF to IgM produced by B cells from different normal subjects. CONCLUSION: These results indicate that abnormal CD14+ monocytes stimulate RF-producing B cells to be ready to be activated by the signals delivered through noncognate T-B interactions with anti-CD3-activated T helper cells. Moreover, the data suggest that the accelerated generation of such functionally abnormal CD14+ cells from bone marrow precursors might play an important role in the pathogenesis of RA.

Aged↗