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T Funatsu

Publications and source records attributed to T Funatsu.

At least 19 recordsLinked to original sources
PubMed1999

Single-molecule imaging of RNA polymerase-DNA interactions in real time.

Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of lambda-phage DNA suspended in solution by optical traps. The interactions of RNA polymerase molecules were not homogeneous along DNA. They dissociated slowly from the positions of the promoters and sequences common to promoters at a rate of approximately 0.66 s-1, which was more than severalfold smaller than the rate at other positions. The association rate constant for the slow dissociation sites was 9.2 x 10(2) bp-1 M-1 s-1. The frequency of binding to the fast dissociation sites was dependent on the A-T composition; it was larger in the AT-rich regions than in the GC-rich regions. RNA polymerase molecules on the fast dissociation sites underwent linear diffusion (sliding) along DNA. The binding to the slow dissociation sites was greatly enhanced when DNA was released to a relaxed state, suggesting that the binding depended on the strain exerted on the DNA. The present method is potentially applicable to the examination of a wide variety of protein-nucleic acid interactions, especially those involved in the process of transcription.

Bacteriophage lambda↗
PubMed1998

Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin.

We have developed a technique that allows mechanical and ligand-binding events in a single myosin molecule to be monitored simultaneously. We describe how steps in the ATPase reaction are temporally related to mechanical events at the single molecule level. The results show that the force generation does not always coincide with the release of bound nucleotide, presumably ADP. Instead the myosin head produces force several hundreds of milliseconds after bound nucleotide is released. This finding does not support the widely accepted view that force generation is directly coupled to the release of bound ligands. It suggests that myosin has a hysteresis or memory state, which stores chemical energy from ATP hydrolysis.

Actin Cytoskeleton↗
PubMed1998

Contractile properties of thin (actin) filament-reconstituted muscle fibers.

Selective removal and reconstitution of the components of muscle fibers (fibrils) is a useful means of examining the molecular mechanism underlying the formation of the contractile apparatus. In addition, this approach is powerful for examining the structure-function relationship of a specific component of the contractile system. In previous studies, we have achieved the partial structural and functional reconstitution of thin filaments in the skeletal contractile apparatus and full reconstitution in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with plasma gelsolin, an actin filament-severing protein. Under these conditions, no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted by polymerization on the short actin fragments remaining at the Z line, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached as high as 30% of the original level in skeletal muscle, while it reached 140% in cardiac muscle. The augmentation of tension in cardiac muscle is mainly attributable to the elongation of reconstituted filaments, longer than the average length of thin filaments in an intact muscle. These results indicate that a muscle contractile apparatus with a high order structure and function can be constructed by the self-assembly of constituent proteins. Recently, we applied this reconstitution system to the study of the mechanism of spontaneous oscillatory contraction (SPOC) in thin (actin) filament-reconstituted cardiac muscle fibers. As a result, we found that SPOC occurs even in regulatory protein-free actin filament-reconstituted fibers (Fujita & Ishiwata, manuscript submitted), although the SPOC conditions were slightly different from the standard SPOC conditions. This result strongly suggests that spontaneous oscillation is intrinsic to actomyosin motors. We here summarize the contractile properties of the reconstitution system.

Actins↗
PubMed1998

YM866, a novel modified tissue-type plasminogen activator, affects left ventricular function and myocardial infarct development in dogs with coronary artery thrombi.

YM866 is a novel modified tissue-type plasminogen activator (t-PA). Its effects on left ventricular function and myocardial infarct development in dogs with copper coil-induced coronary artery thrombosis were compared with those of a native t-PA, alteplase. YM866 (bolus injection) and alteplase (bolus plus infusion) were administered 15 min after coronary artery occlusion. YM866 and alteplase produced reperfusion in all animals, with a median time to reperfusion of 10 min. In contrast, no reperfusion occurred in the vehicle control group. Left ventricular ejection fraction (LVEF) significantly decreased 15 min after coronary occlusion. YM866 and alteplase improved LVEF 3 hr and 4 hr after administration, respectively, while LVEF did not improve in the vehicle control group. Only slight myocardial infarct areas were observed in both YM866- and alteplase-administered groups, while the area in the vehicle control group accounted for 18.2% of left ventricular myocardial area. In conclusion, although both YM866 and alteplase reperfused occluded coronary arteries, inhibited myocardial infarct development and improved LVEF in dogs with coronary artery thrombi, only a single bolus injection of YM866 was necessary to achieve these improvements. Therefore, YM866 shows promise as an improved clinical agent in treating acute myocardial infarction.

Animals↗
PubMed1998

Sexual dimorphism in the tooth crown dimensions of the second deciduous and first permanent molars of Taiwan Chinese.

Sexual dimorphism in the crown components in the second deciduous molar (dm2) and the first permanent molar (M1) of the dental casts taken from Chinese living in Kaohsiung (Taiwan) was investigated. Mesiodistal and buccolingual crown diameters, and 4 main-cusp sizes in the maxillary molars and mesiodistal diameters of the trigonid and talonid in the mandibular molars were measured using a digital caliper (0.01 mm). Percentage sexual differences were calculated. With the exception of trigonid mesiodistal diameters, the mean values of males were larger than females. In both of dm2 and M1 the mean values of the trigonid mesiodistal diameters were slightly larger in females than in males, but the differences were not significant. Percentage sexual dimorphism was smaller in the mesiodistal diameters than in the buccolingual in both dm2 and M1. The crown components showed larger sexual difference in dm2 than in M1, while in the maxilla the external crown size showed larger sexual difference in M1 than in dm2. The buccal cusps showed larger sexual difference than the lingual in the maxillary molars and the talonid showed larger sexual difference than the trigonid in the mandibular. Sexual dimorphism of dm2 and M1 in their crown components displayed similar pattern, although the different degree was noted. This result is thought to relate to the fact these molars belong to the first dentition embryologically.

China↗
PubMed1998

[Limited operation for stage I lung cancer: a retrospective study].

Although lobectomy is standard therapy for Stage I non-small cell lung cancer, it often cannot be performed in poor-risk patients. In this report, we describe the results of a retrospective study to assess the usefulness of limited operation for stage I lung cancer. Over a 21-year period, 1,286 lung cancers were resected at our center. Among the 497 patients with stage I lung cancer, 36 sublobar resections were performed. There was only one surgery-related death, and the 5-year survival rate was 46% for all patients. At 5 years, survival was 69.2% for patients with squamous cell carcinoma and 33.7% for patients with adenocarcinoma. Survival rates were higher in patients who underwent mediastinoscopy than those who did not, and depended on histological findings and accurate pathological staging.

Adenocarcinoma↗
PubMed1998

[A pulmonary tumorlet with caseous granuloma associated with atypical mycobacterium].

We encountered a case of pulmonary tumorlet with caseous granuloma associated with atypical mycobacterium. A 73-year-old woman was admitted to the hospital because a chest x-ray film showed enlargement of an abnormal shadow in the middle lobe of the right lung. Primary lung cancer was suspected and right middle lobectomy was performed. Acid-fast bacilli (Gaffky 1) were found in a caseous lesion and examination of intraoperatively obtained frozen specimens showed caseous granulomas. The bacilli were later identified as Mycobacterium avium complex. The permanent specimen showed a minute lesion consisting of small clusters of epithelial cells resembling carcinoid tumor in contact with granulomatous tissue. Histopathological examination revealed argyrophilia on Grimelius stain and immunoreactivity to chromogranin-A in the clusters of epithelial cells. Although these results are consistent with small cell carcinoma or peripheral carcinoid tumor, pulmonary tumorlet was diagnosed because of the lesion's small and minimal cytologic atypia, and because of chronic pulmonary damage around the lesion. Pulmonary tumorlets are minute, usually microscopic, tumor-like lesions mostly found in damaged lung tissue obtained at autopsy or during surgery. Morphological diagnosis is sometimes very difficult, but recently these lesions have been regarded as hyperplastic lesions arising in pulmonary neuroendocrine cells (Kultschitzky cells) and caused by chronic pulmonary damage, such as hypoxia and inflammation. Pulmonary tumorlets must be considered in the differential diagnosis of minute lesions suspected to be small cell carcinoma or peripheral carcinoid tumor.

Aged↗
PubMed1997

Single molecular assay of individual ATP turnover by a myosin-GFP fusion protein expressed in vitro.

Fusion proteins of a truncated mutant of myosin subfragment-1 (S1dC) and green fluorescent protein (GFP) were expressed in vitro by T7 RNA polymerase and rabbit reticulocyte lysate. Single S1dC-GFP fusion proteins were clearly seen and their individual ATP turnovers were directly monitored using low background total internal reflection fluorescence microscopy (LBTIRFM), recently developed by our laboratory. LBTIRFM using GFP as a fluorescent tag allowed us to assay functions of single protein molecules expressed in vitro. Thus, the results suggested that this method may be particularly useful to analyze functions of proteins that cannot be produced in an active form and/or in large quantities in conventional heterologous expression systems.

Adenosine Triphosphate↗
PubMed1997

Interhead distances in myosin attached to F-actin estimated by fluorescence energy transfer spectroscopy.

Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine distances between probes attached to the most reactive sulfhydryl (SH1) group on individual myosin "heads." We measured intramolecular and intermolecular interhead distances as well as the distance between one head of heavy meromyosin (HMM) mixed with subfragment-1 (S1) heads attached to F-actin under rigor conditions. The SH1 cysteine was specifically labeled with either a donor (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid) or an acceptor probe (5-iodoacetamidofluorescein). In free solution, the distance between these probes was too large to allow significant FRET, but in the rigor complex with F-actin, intermolecular interhead distances between S1 molecules, HMM molecules, or S1 and HMM were determined to be 6.0-6.3 nm. The radial coordinate of the labels relative to F-actin was 5.0-6.4 nm. However, the intramolecular interhead distance in HMMs in which the two heads were labeled with D and A probes was estimated to be larger. The binding affinity of the second head of HMM(D/A) to F-actin may be reduced because of heterogeneous modification of the SH1 groups, such that the probability of single-head binding is increased.

Actins↗
PubMed1997

Imaging and nano-manipulation of single biomolecules.

We have developed a new technique for imaging single fluorescent dye molecules by refining epifluorescence and total internal reflection fluorescence microscopies. In contrast to previously reported single fluorescent molecule imaging methods, in which specimens were immobilized on an air-dried surface, our method enables video-rate imaging of single molecules in aqueous solution. This approach enabled us to directly image the processive movement of individual fluorescently labeled kinesin molecules along a microtubule. This method was also used to visualize individual ATP turnover reactions of single myosin molecules. The method can be combined with molecular manipulation using an optical trap. A single kinesin molecule attached to a polystyrene bead was brought into contact with a microtubule adsorbed onto the glass surface. The lifetime of bound Cy3-nucleotide in the absence or presence of the microtubule was 10 s or 0.08 s, respectively, showing that ATPase activity of the kinesin is strongly activated by microtubules. As the present system is equipped with a nanometer sensor, elemental steps of a single kinesin molecule can also be measured. By simultaneously measuring the individual ATP turnovers and elementary mechanical events of a single kinesin molecule, we will be able to obtain a clear answer to the fundamental problem of how the mechanical events are coupled to the ATPase reaction.

Adenosine Triphosphate↗
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

Structural and functional reconstitution of thin filaments in the contractile apparatus of cardiac muscle.

The muscle contractile apparatus has a highly ordered liquid crystalline structure. The molecular mechanism underlying the formation of this apparatus remains, however, to be elucidated. Selective removal and reconstitution of the components are useful means of examining this mechanism. In addition, this approach is a powerful technique for examining the structure and function of a specific component of the contractile system. In this study we have achieved the structural and functional reconstitution of thin filaments in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with gelsolin. Under these conditions no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached 135 +/- 64% of the original level. The augmentation of tension was attributable to the elongation of reconstituted filaments. As another possibility for augmented tension generation, we suggest the presence of an inhibitory system that was not reconstituted. In any case, the thin filaments of the cardiac contractile apparatus are considered to be assembled so as not to develop the highest degree of tension. Incorporation of the tropomyosin-troponin complex fully restored Ca2+ sensitivity without affecting maximum tension. The present results indicate that a muscle contractile apparatus with a higher order structure and function can be constructed by the self-assembly of constituent proteins.

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

[Chest wall reconstruction using polyester mesh].

From January 1987 through December 1994, we performed chest wall reconstruction using the polyester mesh in 15 patients with lung cancer, 11 with empyema after open drainage, 8 with chest wall tumor and 1 with radiation dermatitis and costal chondritis. Twenty five patients were resected 3 or more ribs. Chest wall defects were reconstructed with the polyester mesh covered with Gore-Tex soft tissue patch. Twenty two cases passed more than a year without signs of infection and follow-up averaged 27.6 months. Polyester mesh was removed due to bronchial fistula (3 cases), deformities (3 cases) and abscess formation (1 case). In these cases, polyester mesh was well incorporated and had no foreign body change. In conclusion, the polyester mesh seems to be a dependable prosthetic material for chest wall reconstruction.

Adult↗
PubMed1996

[Flow cytometric analysis of the DNA content of resected non-small cell lung cancer with reference to long-term follow-up].

We measured the cellular DNA content of paraffin-embedded tumor specimens by flow cytometry from 340 cases of resected non-small cell lung cancer, and investigated the correlation of DNA content and prognosis of these cases with long-term follow-up. These 340 cases were divided into some populations according to pathological stage, histologic type, surgical curativity and N factor, and we compared the prognosis of DNA diploidy cases and DNA aneuploidy cases in each population. DNA aneuploidy cases had a significantly less favorable prognosis than DNA diploidy cases in population of stage I adenocarcinoma, stage IIIA non-small cell lung cancer and N2 cases among stage IIIA non-small cell lung cancer, all after curative operation. But in other populations, there was no significant difference in prognosis between DNA diploidy cases and DNA aneuploidy cases. In conclusion, DNA ploidy pattern is a prognostic factor for survival in patients with stage I adenocarcinoma and N2 cases of stage IIIA non-small cell lung cancer.

Adenocarcinoma↗