Centriole duplication, centrosome maturation and spindle assembly in lysates of Spisula solidissima oocytes.
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Biomedical subjects
Publications and source records attributed to W Steffen.
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Vibrational modes in suspensions of soft colloids in a fluid can be detected experimentally by Brillouin light scattering. Besides the usual acoustic mode, being essentially the longitudinal phonon of the liquid matrix, an "opticlike" mode is observed in giant starlike micelles at low volume fractions. We propose that this opticlike mode is due mainly to the internal vibration of each hairy particle.
Motivated by the rapid increase in atmospheric CO2 due to human activities since the Industrial Revolution, several international scientific research programs have analyzed the role of individual components of the Earth system in the global carbon cycle. Our knowledge of the carbon cycle within the oceans, terrestrial ecosystems, and the atmosphere is sufficiently extensive to permit us to conclude that although natural processes can potentially slow the rate of increase in atmospheric CO2, there is no natural "savior" waiting to assimilate all the anthropogenically produced CO2 in the coming century. Our knowledge is insufficient to describe the interactions between the components of the Earth system and the relationship between the carbon cycle and other biogeochemical and climatological processes. Overcoming this limitation requires a systems approach.
The structure factor of a fragile glass-forming liquid orthoterphenyl was measured in the previously inaccessible intermediate q range between the conventional light scattering (LS) and small-angle x-ray scattering (SAXS) q ranges using the low-angle scattering beam line at the European Synchrotron Radiation Facility. At low q the structure factor exhibits an excess scattering and matches well the LS data. This excess scattering is due to long-range density fluctuations also observed in the isotropic component of scattered light. At high q the structure factor decays to a plateau corresponding to the isothermal compressibility in agreement with the conventional SAXS data. In the intermediate q range, the structure factor exhibits a power law q dependence which indicates that the excess scattering is due to fractal aggregates of denser domains.
Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161-164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis.
The dynamics of the fragile glass-forming liquid diglycidyl ether of bisphenol-A was studied by depolarized Rayleigh-Brillouin light-scattering and photon correlation spectroscopy above the glass transition, in the temperature range from 261 to 473 K and in the frequency range from 1 Hz to 300 GHz. The structural (alpha-) relaxation process was revealed and no signature of the secondary relaxation previously evidenced by dielectric spectroscopy at about 0.1 GHz was observed. The characteristic time of the alpha process differs from that determined by dielectric spectroscopy of an amount, which increases with increasing temperature. The relaxation times were compared with viscosity data to test the predictions of the classic Stokes-Einstein-Debye model. The tau proportional, variant eta behavior was verified for dielectric data, while a fractional power law of viscosity tau proportional, variant eta(0.89) was obtained for light-scattering relaxation times, extending over more than seven decades in viscosity and time. This deviation of light scattering from viscosity data could be interpreted in terms of cooperative motion in the supercooled liquid with a characteristic length xi(a) proportional, variant(T-T0)(-v) where T(0)=229 K is the Vogel temperature and v is close to 2 / 3 which is consistent with the prediction of the fluctuation theory of glass transition.
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Meiosis I spindle assembly is induced in lysate-extract mixtures prepared from clam (Spisula solidissima) oocytes. Unactivated lysate prepared from unactivated oocytes contain nuclei (germinal vesicles, GVs) which house condensed chromosomes. Treatment of unactivated lysate with clarified activated extract prepared from oocytes induced to complete meiosis by treatment with KCl induces GV breakdown (GVBD) and assembly of monopolar, bipolar, and multipolar aster-chromosome complexes. The process of in vitro meiosis I spindle assembly involves the assembly of microtubule asters and the association of these asters with the surfaces of the GVs, followed by GVBD and spindle assembly. Monoclonal antibody m74-1, known to react specifically with the N terminus of the intermediate chain of cytoplasmic dynein, recognizes Spisula oocyte dynein and inhibits in vitro meiosis I spindle assembly. Control antibody has no affect on spindle assembly. A similar inhibitory effect on spindle assembly was observed in the presence of orthovanadate, a known inhibitor of dynein ATPase activity. Neither m74-1 nor orthovanadate has any obvious affect on GVBD or aster formation. We propose that dynein function is required for the association of chromosomes with astral microtubules during in vitro meiosis I spindle assembly in these lysate-extract mixtures. However, we conclude that dynein function is not required for centrosome assembly and maturation or for centrosome-dependent aster formation.
Catheter-delivered, therapeutic ultrasound was shown to effectively dissolve thrombus in vitro and in vivo. This first study in 14 patients with acute myocardial infarctions demonstrates that it is a safe and effective treatment alternative that deserves further clinical evaluation.
Several factors been reported to influence the mobility of polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) including the brand of SDS. Using microtubule proteins from axonemes of Lytechinus pictus and Spisula solidissima sperm and meiotic spindles of Spisula solidissima we demonstrate that the change in mobility was caused by sodium tetradecyl sulfate (STS), a major contaminant of many commercial SDS brands. We also examined the use of sodium tetradecyl sulfate and different SDS brands as a tool in extracting more information from immunoblot studies. Commercial SDS containing contaminants other than sodium tetradecyl sulfate reduced or eliminated the immunosignal from certain polypeptides and the loss of antigenicity could not even be recovered by immunoblot under "renaturing" conditions. It can thus be concluded that STS can be useful in separating and identifying comigrating polypeptides and in detecting additional immunobands in immunoblots.
Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74-1, m74-2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 micrograms/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74-1 and m74-2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC-p150Glued interaction. Thus these findings demonstrate that direct IC-p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.
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UNLABELLED: BASIS AND AIM OF STUDY: Low-frequency, high-intensity ultrasound has been shown, both in vivo and in vitro, selectively to remove arteriosclerotic plaques and thrombi. This study was undertaken to investigate the safety and effectiveness of intracoronary ultrasound angioplasty. PATIENTS AND METHODS: Ultrasound coronary angioplasty (UCA) with highly flexible ultrasound catheters (1.2 mm or 1.7 mm probe tip) was performed in 50 patients (36 men, 14 women; mean age 64.7 [33-79] years) with coronary heart disease involving one (n = 26 or several (n = 24) vessels. Indications for treatment were exercise-induced (n = 35) or unstable (n = 14) angina or acute myocardial infarction (n = 11). Treated lesions were in the anterior interventricular branch (n = 25), circumflex branch of the left coronary artery (n = 3) and right coronary artery (n = 22). 19 vessels were occluded, 24 lesions were partially thrombosed, 19 were calcified. 22 stenoses were longer than 20 mm. According to AHA/ACC criteria, 10 type A, 17 type B1, 6 type B2 and 17 type C lesions were treated. RESULTS: Total ultrasound time was 336 +/- 308 s; mean passing time through the stenosis was 233 +/- 289 (10-556) s. 17 of 19 occlusions were recanalised after 285 +/- 224 s. Percutaneous transluminal angioplasty (PTCA) was subsequently performed in 49 patients. The mean stenosis grade was reduced by UCA from initially 82 +/- 3 to 64 +/- 2% and by subsequent PTCA to 38 +/- 1%. Average flow grade rose from 1.5 to 1.9 after UCA and to 2.8 after PTCA. No vasospasm, atrioventricular block or perforation was caused by UCA. Angiography demonstrated dissection after PTCA in seven patients, treated in two with a stent. Both UCA and PTCA failed to achieve recanalization in one patient with a thrombotic occluded coronary artery after acute myocardial infarction. In all other patients there were no complications. CONCLUSION: The results show UCA to be a safe method for removing high-degree coronary artery stenosis or recanalize thrombotic occlusions.
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OBJECTIVES: This study assessed the efficacy of a new high intensity, low frequency therapeutic coronary ultrasound catheter for thrombus dissolution in vitro and in vivo in canine coronary arteries. BACKGROUND: Therapeutic ultrasound has been shown to dissolve thrombi in vitro and in peripheral arteries in vivo. There have been no previous studies on in vivo coronary thrombus dissolution by ultrasound. METHODS: In vitro, we exposed 1- to 4-h old human blood clots for 3 min to pulsed-wave ultrasound. Clot dissolution under various conditions was evaluated. In vivo occlusive coronary thrombi were induced in 18 dogs. RESULTS: In vitro irrigation alone (10 ml/min of normal saline solution) and ultrasound alone each contributed to a reduction of clot weight by 47.1 +/- 11.4 mg and 84.6 +/- 25.6 mg, respectively, after 3 min (p < 0.001). Ultrasound plus irrigation resulted in a reduction of clot weight by 216.5 +/- 31.5 mg after 3 min (p < 0.001). The magnitude of clot dissolution was considerably amplified when ultrasound energy was combined with irrigation, probably because of cavitational effects. In vivo, in three dogs mechanical passage of the unactivated probe failed to recanalize the artery, and the arteries remained thrombotically occluded. After passage of the activated ultrasound probe, angiography revealed widely patent coronary arteries in 13 of 15 dogs and partial recanalization with filling defects indicative of residual thrombus in 2 of 15 dogs. Three of 15 coronary arteries were histologically free of residual thrombi. Mural thrombi extending to < or = 10% of the vessel circumference were seen in 10 of 15 dogs. Residual thrombi > or = 50% of the vessel circumference were found in two cases. There was no histologic evidence of ultrasound-mediated vessel damage. CONCLUSIONS: Catheter-delivered therapeutic ultrasound effectively dissolves clots in vitro and in canine coronary arteries in vivo. Thus, therapeutic catheter-delivered ultrasound has the potential to serve as an adjunct or alternative treatment for thrombus-mediated coronary ischemic syndromes or myocardial infarction.
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