Expression of kinesin in Escherichia coli.
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Ammonium Sulfate↗
Biomedical subjects
M F Stock
Publications and source records attributed to M F Stock.
Kinesin's IAK tail domain inhibits initial microtubule-stimulated ADP release.
Kinesin undergoes a global folding conformational change from an extended active conformation at high ionic concentrations to a compact inhibited conformation at physiological ionic concentrations. Here we show that much of the observed ATPase activity of folded kinesin is due to contamination with proteolysis fragments that can still fold, but retain an activated ATPase function. In contrast, kinesin that contains an intact IAK-homology region exhibits pronounced inhibition of its ATPase activity (140-fold in 50 mM KCl) and weak net affinity for microtubules in the presence of ATP, resulting from selective inhibition of the release of ADP upon initial interaction with a microtubule. Subsequent processive cycling is only partially inhibited. Fusion proteins containing residues 883-937 of the kinesin alpha-chain bind tightly to microtubules; exposure of this microtubule-binding site in proteolysed species is probably responsible for their activated ATPase activities at low microtubule concentrations.
Adenosine Diphosphate↗
Formation of the compact confomer of kinesin requires a COOH-terminal heavy chain domain and inhibits microtubule-stimulated ATPase activity.
Full-length Drosophila kinesin heavy chain from position 1 to 975 was expressed in Escherichia coil (DKH975) and is a dimer. The sedimentation coefficient of DKH975 shifts from 5.4 S at 1 M NaCl to approximately 6.9 S at <0.2 M NaCl. This transition of DKH975 between extended and compact conformations is essentially identical to that for the heavy chain dimer of bovine kinesin (Hackney, D. D., Levitt, J. D., and Suhan, J. (1992) J. Biol. Chem. 267, 8696-8701). Thus the capacity for undergoing the 7 S/5 S transition is an intrinsic property of the heavy chains and requires neither light chains nor eukaryotic post-translational modification. DKH960 undergoes a similar transition, indicating that the extreme COOH-terminal region is not required. More extensive deletions from the COOH-terminal (DKH945 and DKH937) result in a shift in the midpoint for the transition to lower salt concentrations. DKH927 and shorter constructs remaining extended even in the absence of added salt. Thus the COOH-terminal approximately 50 amino acids are required for the formation of the compact conformation. Separately expressed COOH-terminal tail segments and NH2-terminal head/neck segments interact in a salt-dependent manner that is consistent with the compact conformer being produced by the interaction of domains from these regions of the heavy chain dimer. The microtubule-stimulated ATPase rate of DKH975 in the compact conformer is strongly inhibited compared with the rate of extended DKH894 (4 s-1 and 35 s-1, respectively, for kcat at saturating microtubules).
Adenosine Triphosphatases↗
Influence of the kinesin neck domain on dimerization and ATPase kinetics.
Motor domains of kinesin were expressed that extend from the N terminus to positions 346, 357, 365, 381, and 405 (designated DKH346-DKH405) to determine if the kinetic differences observed between monomeric DKH340 and dimeric DKH392 (Hackney, D. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6865-6869) were specific to these constructs or due to their oligomeric state. Sedimentation analysis indicated that DKH346, DKH357, and DKH365 are predominantly monomeric and that DKH381 and DKH405 are predominantly dimeric at 0. 01-0.03 microM, the concentrations used for ATPase assays. In buffer with 25 mM KCl, all have high kcat values of 38-96 s-1 at saturating microtubule (MT) levels. Monomeric DKH346, DKH357, and DKH365 have K0.5(MT) values of 17, 9, and 1.4 microM, respectively, but the K0. 5(MT) values for the dimeric species are significantly lower, with 0. 02 and 0.14 microM for DKH381 and DKH405, respectively. The three new monomers release all of their ADP on association with microtubules, whereas the two new dimers retain approximately half of their ADP, consistent with the half-site reactivity observed previously with dimeric DKH392. Both the kbi(ATPase) (=kcat/K0. 5(MT)) values for stimulation of ATPase by MTs and the kbi(ADP) for stimulation of ADP release by MTs were determined in buffer containing 120 mM potassium acetate. The ratio of these rate constants (kbi(ratio) = kbi(ATPase)/kbi(ADP)) is 60-100 for the dimers, indicating hydrolysis of many ATP molecules per productive encounter with a MT as observed previously for DKH392 (Hackney, D. D. (1995) Nature 377, 448-450). For the monomers, kbi(ratio) values of approximately 4 indicate that they also may hydrolyze more than one ATP molecule per encounter with a MT and that the mechanism of hydrolysis is therefore fundamentally different from that of actomyosin. DKH340 is an exception to this pattern and may undergo uncoupled ATP hydrolysis.
Adenosine Diphosphate↗
An attempt to define a just detectable effect for airborne chemicals on the respiratory tract in mice.
We have attempted to define a just detectable effect (JDE) for three different types of reactions along the respiratory tract: (a) sensory irritation of the upper airways (S), (b) airflow limitation along the conducting airways (A), and (c) pulmonary irritation at the alveolar level (P1 or P). Each type of reaction, S, A, P1 or P, was recognized by analyzing the breathing pattern of unanesthetized mice held in body plethysmographs. A rule-based computer program analyzed each breath during a period of 3.75 h and classified each breath as normal (N) or falling in any of the above categories (i.e., S, A, P1 or P). Eight groups of four mice were used for sham exposures: exposed to water vapor. These data sets were used, as sham exposure data, to define the variation which can occur with time in order to define an expected range of normal variation. Once this range was established, we defined JDE values for each type of effect and used such values to evaluate the results obtained in exposed animals. Eight groups of four mice were exposed to a mixture of airborne chemicals, machining fluid G (MFG), at concentrations from 0.17 to 55 mg/m3. Data sets for individual animals and for each group of animals exposed to MFG were analyzed to determine if and when a particular effect occurred. It was possible to recognize the effects of low exposure concentrations on groups of exposed animals or individual animals within each group. This procedure will be valuable when investigating the effect of airborne chemicals and when it is impossible to generate high exposure concentrations to define concentration-response relationships.
Air Pollutants↗
Distribution and reactivity of inhaled 14C-labeled toluene diisocyanate (TDI) in rats.
Inhalation exposure to toluene diisocyanate (TDI) can result in a variety of airway diseases. Concern has been expressed that a putative carcinogenic potential of TDI exists as a result of the formation of toluenediamine (TDA) by hydrolysis of the isocyanate in the body. Results from long-term bioassays (TDI inhalation versus gavage in rats and mice) are contradictory and discrepancies do exist concerning the interpretation of adverse effects. This study was performed to analyze the distribution and reactivity of radioactively-labeled TDI using vapor exposure in a rat model system. Rats were exposed to 14C-TDI vapors at concentrations ranging from 0.026 to 0.821 ppm for 4 h. All tissues examined showed detectable quantities of radioactivity, with the airways, gastrointestinal system and blood having the highest levels which increased with exposure concentration. The concentration of radioactivity in the bloodstream after exposure was linear with respect to dose. The majority (74-87%) of the label associated with the blood was recovered in the plasma, and of this, 97-100% of the 14C existed in the form of biomolecular conjugates. Analysis of stomach contents shows that the majority of the label is also associated with high (> 10 kDa) molecular weight species. While a larger percentage (28%) of the label is found in the low molecular weight fraction relative to blood, this low molecular weight labeled material represents at least eight different components. Thus, over the vapor exposure concentrations and time tested, it appears that conjugation is the predominant reaction and that free TDA is not a primary in vivo reaction product under the conditions tested.
Administration, Inhalation↗
Computer assisted recognition and quantitation of the effects of airborne chemicals acting at different areas of the respiratory tract in mice.
The pattern and timing of a normal breath in unanesthetized mice was analyzed from measurement of inspiratory and expiratory airflows (VI and VE). Airflow was measured via a differential pressure transducer, attached to a pneumotachograph, which itself was attached to a body plethysmograph into which a mouse was placed. The analog voltage from the differential pressure transducer was digitized and stored for analysis on a microcomputer. Criteria were developed to classify each breath as normal (N) or belonging into one of seven abnormal categories. The abnormal categories were arrived at by computer analysis, recognizing specific modifications of the normal pattern into patterns of: sensory irritation of the upper respiratory tract (S), airflow limitation within the conducting airways of the lungs (A) or pulmonary irritation at the alveolar level (P). Combinations of these effects, i.e., S+A, P+A, P+S and P+S+A were also recognized. Computer analysis of each breath also permitted quantitative evaluation of the degree of S, A or P abnormalities. To induce each type of effect we used inhalation exposures to 2-chlorobenzylchloride, carbamylcholine or propranolol. We propose that this approach will permit rapid evaluation of the possible effects of airborne chemicals at three levels of the respiratory tract, with the classification of the type of effect easily obtained in an objective way using well defined criteria, followed by quantitation of the degree of each effect.
Administration, Inhalation↗
Characteristic modifications of the breathing pattern of mice to evaluate the effects of airborne chemicals on the respiratory tract.
A system was developed for exposure of unanesthetized mice to airborne chemicals and for continuous measurement of their breathing pattern prior to, during and following exposure. By measuring inspiratory and expiratory airflows (VI and VE), and integration with time to yield tidal volume (VT), we obtained characteristic modifications to the normal breathing pattern. These permitted recognition that a specific portion of the respiratory tract was affected by the selected airborne chemicals. Following recognition, we also quantitated the degree of effect using one specific measurement in each case. An effect on the upper respiratory tract, induced by the sensory irritant, 2-chlorobenzylchloride, was quantitated by measuring a decrease in respiratory frequency. An effect on the conducting airways, induced by the airway constrictor, carbamylcholine, was quantitated by a decrease in VE at the mid-point of VT. An effect at the alveolar level, induced either by the vagal nerve ending stimulant, propranolol, or by the pulmonary irritant, machining fluid G, was quantitated by an increase in the length of a pause induced at the end of expiration. The system is easy to construct and operate and can be used to rapidly evaluate the effects of airborne chemicals on the respiratory tract.
Aerosols↗
Uptake and distribution of 14C during and following inhalation exposure to radioactive toluene diisocyanate.
Inhalation of toluene diisocyanate (TDI) results in toxic responses ranging from pulmonary irritation to immunological sensitization. The use of radioactively labeled isocyanate has made it possible to follow the initial uptake of the compound into the bloodstream independent of the final fate of the isocyanate. This study shows that the rate of uptake into the blood is linear during exposure to concentrations ranging from 0.00005 to 0.146 ppm and that the uptake continues to increase slightly postexposure. It also demonstrates that the radioactivity clears from the bloodstream to a level corresponding to approximately a 100 nM concentration of tolyl group after 72 hr and persists at a nanomolar level even 2 weeks following the exposure. This is similar to the response previously reported by this group for radioactively labeled methyl isocyanate. The initial rate of 14C uptake is also a linear function of the concentration of TDI when expressed either as concentration (ppm) or as concentration multiplied by duration of exposure (ppm.hr). This is discussed in comparison with the toxic responses as a function of both ppm and ppm.hr. Finally, the inclusion of the data on methyl isocyanate indicates that the uptake into arterial blood is a function of exposure concentration, independent of isocyanate structure.
Administration, Inhalation↗
Effects of inhaled municipal refuse incinerator fly ash in the guinea pig.
Fly ash was collected from two municipal refuse incinerators. It was analyzed for heavy metals, elements, and a wide range of toxic organics. It was resuspended in air for inhalation exposure of guinea pigs. These animals were exposed at high concentrations of each ash 6 h/d for 5 d, and tissues were taken 45 d after the exposure. Following the first exposure and after each daily exposure the ventilatory response of these animals upon challenge with CO2 was found to be depressed. Recovery occurred following exposure. Heavy metals, cadmium, lead, zinc, and mercury were elevated in the lungs of these animals. Histologic evaluation of pulmonary tissue revealed multifocal pneumoconiosis. Interstitial infiltration by macrophages and smooth muscle hypertrophy of blood vessels and bronchioles were also observed. There was no evidence of a dioxinlike toxic effect following inhalation of these ashes.
Air Pollutants↗
Uptake and distribution of 14C during and following exposure to [14C]methyl isocyanate.
Guinea pigs were exposed to [14C]methyl isocyanate (14CH3-NCO, 14C MIC) for periods of 1 to 6 hr at concentrations of 0.5 to 15 ppm. Arterial blood samples taken during exposure revealed immediate and rapid uptake of 14C. Clearance of 14C was then gradual over a period of 3 days. Similarly 14C was present in urine and bile immediately following exposure, and clearance paralleled that observed in blood. Guinea pigs fitted with a tracheal cannula and exposed while under anesthesia showed a reduced 14C uptake in blood indicating that most of the 14C MIC uptake in normal guinea pigs occurred from retention of this agent in the upper respiratory tract passages. In exposed guinea pigs 14C was distributed to all examined tissues. In pregnant female mice similarly exposed to 14C MIC, 14C was observed in all tissues examined following exposure including the uterus, placenta, and fetus. While the form of 14C distributed in blood and tissues has not yet been identified, these findings may help to explain the toxicity of MIC or MIC reaction products on organs other than the respiratory tract, as noted by several investigators.
Animals↗
Arterial blood gas measurements in guinea pigs and inspired CO2 concentrations for ventilatory performance challenges.
Guinea pigs were fitted with a carotid cannula under anesthesia and used 2 days later for exposure to increasing CO2 concentrations to evaluate their ventilatory response (tidal volume and respiratory frequency) while blood samples were taken for measurement of O2, CO2, and pH. The same measurements were made on another group of animals while changing O2 concentrations. The ventilatory response increased with increasing CO2 concentrations up to 10% while there was little change induced by low oxygen. Decreasing O2 during challenge of animals with 10% CO2 had no effect on the ventilatory response induced by 10% CO2 alone. The ventilatory response of guinea pigs is greater than for other small laboratory animals tested under similar conditions but is not as pronounced as in humans. Challenging guinea pigs with 10% CO2 has revealed that their ventilatory response can be abnormal following pneumotoxicity induced by a variety of agents and blood gas measurements as described here may add to our understanding of such abnormal responses.
Animals↗
Performance evaluation under intoxicating atmospheres.
A new behavioral model has been developed and used to assess the performance of mice during exposure to carbon monoxide, hydrogen chloride, or subambient levels of oxygen. The apparatus is a ventilated 150-ft series of glass tubes forming an S-shaped exposure system. Performance evaluation was obtained for two sublethal responses: distance traveled/time and incapacitation. Performance of normal mice (Type I) or mice previously fitted with a tracheal cannula (Type II) was very reproducible and similar. Concentration-response relationships were obtained showing the deterioration of performance with exposures to CO from 2500 ppm, HCl from 1095 ppm, and below 8.8% ambient O2 level. This model is likely to be sensitive to other asphyxiants and irritants. It includes both distance traveled and time of performance prior to incapacitation. Both are critical parameters to be included in escape hazard analysis in fire situations and possibly in other accidents involving chemical spills.
Animals↗
Sensory and pulmonary irritation of methyl isocyanate in mice and pulmonary irritation and possible cyanidelike effects of methyl isocyanate in guinea pigs.
Methyl isocyanate (MIC) was evaluated for sensory and pulmonary irritation in mice. MIC was found to be both a potent sensory and pulmonary irritant in this species. From these results, a safe level of exposure for a period of 8 hr was estimated to be about 0.02 ppm for humans. Guinea pigs were also exposed to MIC for a single 3-hr exposure at a concentration of 37 ppm. During exposure to MIC, coughing was observed in all animals. Pulmonary function was evaluated immediately following exposure and intermittently on the next 35 days using CO2 challenges and flow-volume loops. Highly abnormal responses to CO2 were observed immediately after exposure in all animals. Six of the eight animals exposed to MIC died. In the two survivors, an apparent recovery was seen during the 5 days following exposure, but a worsening effect was observed at days 21 and 28, with a partial recovery at day 35. The data clearly demonstrated that the primary pulmonary effect of MIC was one of airways obstruction. Oxygen uptake and carbon dioxide output were also measured in the guinea pigs following exposure to MIC. No evidence of a cyanidelike effect was observed, in contrast to a severe depression of oxygen uptake following exposure to hydrogen cyanide.
Airway Obstruction↗
Evaluation of sister chromatid exchange and cytotoxicity in murine tissues in vivo and lymphocytes in vitro following methyl isocyanate exposure.
The purpose of this study was to assess sister chromatid exchange (SCE) levels and cell cycle kinetics in various murine tissues following MIC exposure. Following exposure of mice to MIC, these parameters were measured in bone marrow and alveolar macrophages labeled with BrdUrd in vivo and in peripheral blood and spleen lymphocytes cultured in the presence of BrdUrd in vitro. Target concentrations of MIC were 2, 15, and 30 ppm (3 hr). Neither elevated SCE frequencies nor inhibition of cell cycling were evident in lipopolysaccharide (LPS)- or concanavalin A (ConA)-stimulated spleen lymphocytes, or in LPS-stimulated peripheral blood lymphocyte (PBL) cultures from mice exposed for 3 hr to MIC concentrations as high as 30.5 ppm. Inhibition of cell cycling and poor culture success rates were apparent in ConA-stimulated PBLs following MIC exposures as low as 2.3 +/- 0.4 ppm for 3 hr. At the lowest MIC dose employed, the cycling characteristics of bone marrow and alveolar macrophages were not altered, and SCE frequencies were at control levels. However, severe cell cycle inhibition was observed in these tissues at MIC concentrations of 15 ppm or greater. A marker of cytotoxicity at this dose was a high frequency (approximately 33-90%) of occurrence of first division cells containing a late-replicating Y chromosome. Despite its apparent cellular toxicity, MIC is not genotoxic as measured by SCE analysis in the tissues examined in this study.
Animals↗
Sensory and pulmonary irritation with exposure to methyl isocyanate.
Methyl isocyanate (MIC) was tested for its potency as a sensory irritant and as a pulmonary irritant in mice. To evaluate sensory irritation, animals were exposed to MIC at concentrations between 0.5 and 7.6 ppm for a period of 90 min. A characteristic reflex decrease in respiratory rate indicating sensory irritation was observed. The concentration evoking a 50% decrease in respiratory rate (RD50) was found to be 1.3 ppm. To evaluate pulmonary irritation, animals were first anesthetized and fitted with a tracheal cannula. Following recovery from anesthesia, they were exposed to MIC at concentrations between 0.4 and 7.3 ppm for a period of 90 min. A characteristic decrease in respiratory rate indicating pulmonary irritation in tracheally cannulated (TC) mice was observed. The concentration evoking a 50% decrease in respiratory rate (RD50TC) was found to be 1.9 ppm. Thus, MIC was found to be a potent sensory and pulmonary irritant.
Animals↗
Evaluation of the pulmonary effects of wood smoke in guinea pigs by repeated CO2 challenges.
Male, English smooth haired guinea pigs were exposed to thermal decomposition products, i.e., smoke, generated by heating Douglas fir in an open system. Various amounts of Douglas fir were placed in a furnace, at room temperature, and heated at a rate of 11 degrees C/min until completely decomposed. Major decomposition occurred between 160 and 490 degrees C, and the animals were exposed during this time for a period of 30 min. Immediately before exposure and at various times after exposure, each animal was evaluated by whole-body plethysmography to measure tidal volume and respiratory frequency during air breathing as well as during challenge with 10% CO2. Exposure to smoke from Douglas fir resulted in a diminished ventilatory response to 10% CO2. Comparing the effect of wood smoke to the effect of smoke from polyvinylchloride from previous experiments wood smoke was found to be 10 times less potent than smoke from polyvinylchloride and animals recovered much more rapidly than with smoke from polyvinylchloride.
Animals↗
Evaluation of the pulmonary toxicity of plasticized polyvinyl chloride thermal decomposition products in guinea pigs by repeated CO2 challenges.
Male guinea pigs were exposed to thermal decomposition products of plasticized polyvinyl chloride (PVC-A) at different concentrations up to levels inducing acute lethality. Several groups exposed at sublethal levels were then evaluated for pulmonary performance for a period of 57 days following exposure. Pulmonary performance was evaluated by challenging each animal with a mixture containing 10% CO2, 20% O2, and 70% N2. In control animals, this mixture induced an increase in both tidal volume and respiratory frequency. This hyperventilatory response was greatly depressed during the first 3 days following exposure and gradually returned to normal during the following weeks with the exception of the highest exposure group which still showed a diminished response 57 days after exposure. The pulmonary toxicity induced by thermal decomposition products of PVC-A is probably related to the very large amount of HCl released during thermal decomposition. The CO2 response test, a nonintrusive and noninvasive method to evaluate pulmonary performance in guinea pigs, is easily performed and appears to be a very promising type of pulmonary function test for toxicological evaluations.
Animals↗