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T Funatsu

Publications and source records attributed to T Funatsu.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1996

[T cell lymphoma presenting as recurrent bilateral pulmonary infiltrates over five years].

A 52-year-old man presented with BOOP-like recurrent bilateral pulmonary infiltrates. In 1989 a chest X-ray film showed an infiltrative shadow in the right S6 region which disappeared after administration of prednisolone. Thereafter, bilateral patchy infiltrates recurred many times, and each time they resolved rapidly with steroid therapy. Skin eruptions on the face recurred. In October 1994 the patient underwent an open-lung biopsy of the infiltrate in the left S6 region. The pathological findings were consistent with BOOP, except for the moderate-to marked infiltration of lymphocytes. A lymphoproliferative disorder was suspected, and Southern blot analysis of the specimen revealed a rearrangement of the TCR-beta gene, which led to the diagnosis of T cell lymphoma. Ten months after the diagnosis, no recurrence of the lymphoma had been detected. In this case a gene analysis of the biopsy specimen was very useful for the diagnosis of T cell lymphoma.

Cryptogenic Organizing Pneumonia↗
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↗
PubMed1994

Single-molecule analysis of the actomyosin motor using nano-manipulation.

The elementary events in energy transduction by the actomyosin motor, driven by ATP hydrolysis, were directly recorded from multiple and single molecules using a recently developed technique for nano-manipulation of single actin filaments by a microneedle. In order to avoid the effects of random orientation of myosin and association of myosin with an artificial substrate in the surface motility assay, we used single myosin-rod cofilaments with various ratios. Distinct actomyosin attachment, force generation (the power stroke) and detachment events were detected at a very low myosin: rod ratio. At high load, one power stroke generated 5-6 pN peak force and 2.3 pN force averaged over the cycle, which were compatible with those deduced from noise analysis of force fluctuations caused by multiple molecules. As the load was reduced, the length of the power stroke increased. At near zero load, the length of a power stroke was approximately 17 nm. The results suggested that an ATPase cycle produces one power stroke at high load and many ones at low load.

Actins↗
PubMed1994

Structural and functional reconstitution of thin filaments in skeletal muscle.

Thin filaments were reconstituted by incorporating exogenous actin, tropomyosin and troponin into glycerinated skeletal muscle fibres or myofibrils. Firstly, thin filaments except short fragments at the Z line were selectively removed by treatment with plasma gelsolin, an actin severing protein. As a result, the fibres (or fibrils) lost the ability to generate active tension. Next, actin filaments were reconstituted by adding purified G-actin which polymerizes onto the actin fragments which remained at the Z line. Rhodamine phalloidin staining of myofibrils showed that exogenous actin was incorporated into the position where the intrinsic thin filaments located. Thin section electron micrographs of fibres showed that reconstituted actin filaments ran from the Z line to the inside of the A band, with some reaching the H zone. The number density of reconstituted actin filaments in the A band was about 20% of that found in intact fibres. The actin filament-reconstituted fibres (or fibrils) generated active tension in a Ca(2+)-insensitive manner and the tension was reversibly suppressed by 2,3-butanedione 2-monoxime. The recovered active tension was about 20% of tension developed by intact fibres. These results indicate that reconstituted actin filaments bear active tension similar to that borne by intact thin filaments. Thin filament-reconstituted fibres, which were prepared by adding purified tropomyosin-troponin complexes into actin filament-reconstituted fibres, showed Ca(2+)-sensitive tension generation. The maximum tension generated was not affected by the presence of tropomyosin and troponin. SDS-PAGE analysis showed that more than 25% of actin and 20% of tropomyosin and troponin was incorporated into the reconstituted fibres. These results indicate that the structure and function of thin filaments are substantially reconstituted by self-assembly of actin, tropomyosin and troponin. The reconstituted fibres and fibrils will be useful for studying the molecular mechanism of muscle contraction and its regulation.

Actin Cytoskeleton↗
PubMed1994

[A case of intractable pulmonary tuberculosis complicated by idiopathic thrombocytopenic purpura (ITP)].

The authors describe a case of pulmonary tuberculosis with multiple drug resistance and complicated by severe idiopathic thrombocytopenic purpura (ITP). The patient was initially treated with high-dose gamma globulin therapy and splenectomy. These procedures resulted in improvement of the patient's condition; the platelet count increased from 7,000/mm3 to 230,000/mm3 and the bleeding time fell from 15 min to 1 min 30 s. Given the significant improvement in the patient's condition, it was considered safe to carry out left pulmonary upper lobectomy and thoracoplasty. There have been no reports describing pulmonary resection performed for inflammatory lung disease in patients with ITP. High-dose gamma globulin therapy rapidly brings about an increase in the platelet count and a reduction of the bleeding time, and it thus extremely useful as preoperative treatment in surgical cases complicated by thrombocytopenia.

Humans↗
PubMed1994

[Diagnosis of mediastinal lymph node metastasis in primary lung cancer: comparing CT scan and mediastinoscopy].

In 105 patients with primary lung cancer who were operated on, the rate of diagnosis of mediastinal lymph node metastasis was determined, and the results of CT scanning and mediastinoscopy were compared. The rate of accuracy of mediastinoscopy was 93.8%, considerably higher than that of CT, which was less than 85%. For the lymph nodes other than #7, which indicate contralateral metastasis, the rate of accuracy of mediastinoscopy was even better and reached 96.3%. In the 9 patients who were proved by mediastinoscopy to have contralateral lymph node metastasis, the rate of accuracy of CT was only 44%. Mediastinoscopy was very effective in the diagnosis of mediastinal lymph node metastases from primary lung cancers.

Humans↗
PubMed1994

Preoperative mediastinoscopic assessment of N factors and the need for mediastinal lymph node dissection in T1 lung cancer.

The significance of preoperative N factor assessment in T1 lung cancer and the need for mediastinal node dissection in T1 N0 M0 cases were investigated. The results of mediastinoscopy were evaluated in patients with T1 adenocarcinoma or squamous cell carcinoma who underwent preoperative mediastinoscopy and thoracotomy from 1971 to 1991 (n = 164). Mediastinoscopy gave true negative results in 90% of patients, false negative in 1%, and true positive in 9%. The 5-year survival was 90% for patients with T1 N0 M0 disease who underwent nonradical dissection (n = 64) and 70% for those who underwent radical dissection (n = 61), indicating that the prognosis was significantly better (p < 0.05) with nonradical dissection. Distant metastasis was a common cause of death, and no death was related to local recurrence, whether nonradical or radical dissection had been performed. The results strongly suggest that preoperative mediastinoscopy and intraoperative node sampling are sufficient for assessment of N factors in T1 lung cancer. The possible relation between immunologic functional changes associated with mediastinal lymph node dissection and the prognosis in patients without evidence of positive lymph nodes should be clarified by a prospective randomized study.

Adenocarcinoma↗
PubMed1994

[A case of solitary plasmacytoma of the chest wall].

A 58-year-old man with a solitary plasmacytoma of the chest wall is reported. He was admitted because of a painless tumor on the right lateral chest wall. The chest CT scan showed a chest wall tumor surrounding the 7th and 8th ribs without rib destruction. A transcutaneous needle biopsy of the tumor with a Sure-Cut-Needle revealed a plasmacytoma. The bone marrow biopsy findings were normal. Under a diagnosis of solitary plasmacytoma, the chest wall tumor was resected. The tumor was an extramedullary plasmacytoma. Southern blot analysis of the immunoglobulin light chain gene of the tumor cells detected a rearrangement of genes in the lambda chain, while no rearrangements of immunoglobulin genes were detected in the bone marrow specimen. Seven months later another solitary plasmacytoma was found at the left radius, and was resected. Southern blot analysis of the immunoglobulin light chain gene was useful in the differential diagnosis from systemic myeloma and in determining the monoclonality of the tumor.

Blotting, Southern↗
PubMed1994

[Preoperative staging in T1 and T2 lung cancers by mediastinoscopy and mediastinal lymph node dissection].

Between 1975 and 1992, mediastinoscopy and thoracotomy were performed on 184 T1 and 271 T2 lung cancer cases consisting of adenocarcinoma and squamous cell carcinoma. Mediastinoscopy gave true negative findings in 90.8% of the T1 patients, true positive findings in 7.6% and false negative findings in 1.6%. The comparable rates were 76.7%, 17.0% and 6.3% in T2 patients. The 5-year survival rate was 91.3% for T1N0M0 patients (n = 64) who underwent non-radical dissection (= NRD), and 69.4% for those (n = 70) who underwent radical dissection (= RD). The rate with NRD was significantly better (p < 0.006). The 5-year survival rate was 63.2% for T2N0M0 patients (n = 62) undergoing NRD, and 49.8% for those (n = 72) undergoing RD, but the difference was not significantly. Distant metastasis was a common cause of death, whereas there were no deaths due to local recurrence in the T1N0M0 patients, whether NRD or RD was performed. These results support our opinions that preoperative mediastinoscopy and intraoperative node staging are sufficient for assessment of the N factor in T1 and T2 lung cancer, and that mediastinal node dissection should not be performed in T1N0M0 patients.

Adenocarcinoma↗