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D L Stokes

Publications and source records attributed to D L Stokes.

43 records · Page 3Linked to original sources
PubMed1990

Structure of CaATPase: electron microscopy of frozen-hydrated crystals at 6 A resolution in projection.

Thin, three-dimensional crystals of CaATPase have been studied at high resolution by electron crystallography. These crystals were grown by adding purified CaATPase to appropriate concentrations of lipid, detergent and calcium. A thin film of crystals was then rapidly frozen and maintained in the frozen-hydrated state during electron microscopy. The resulting electron diffraction patterns extend to 4.1 A resolution and images contain phase data to 6 A resolution. By combining Fourier amplitudes from electron diffraction patterns with phases from images, a density map has been calculated in projection. Comparison of this map from unstained crystals with a previously determined map from negatively stained crystals reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and of the packing of molecules in the crystal, we have proposed a specific arrangement for the ten alpha-helices that have been suggested as spanning the bilayer.

Animals↗
PubMed1990

Neurobehavioral and immunological effects of prenatal cocaine exposure in rat.

Time-pregnant Sprague-Dawley rats were injected subcutaneously with 20 mg/kg of cocaine HCl or 0.9% saline daily from gestation days 15 through 21. Maternal plasma levels of approximately 720 ng/ml of cocaine did not alter maternal weight gain during treatment, duration of pregnancy, any of the litter variables or several indices of maternal behavior. Offsprings' body weight from birth to 30 days of age and physical maturation were not generally affected by prenatal cocaine exposure. While the development of surface righting, cliff avoidance, and the startle response was accelerated in cocaine-exposed offspring, acquisition of a preference for a social odor was unaltered. Prenatal cocaine also attenuated the locomotor response of the offspring to d-amphetamine and cocaine at PND 15; at PND 30 both of these catecholaminergic agonists increased activity in prenatal saline and prenatal cocaine offspring. However, the difference in plasma levels of cocaine at PND 30 suggests a possible down-regulation of adrenergic receptors following prenatal cocaine exposure. Decreased thymus/body weight ratios and splenomegaly were observed in prenatal cocaine animals at 55 days of age. Although complete neutralization of herpes simplex virus-type 1 was not observed, sera from prenatal cocaine offspring showed an increased rate of appearance of cytopathic effect, while sera from animals given cocaine postnatally showed a reduction in the rate at which viral infectivity was expressed in culture. These results indicate that prenatal cocaine exposure can alter neurobehavioral ontogeny and humoral immune responsitivity in the offspring.

Animals↗
PubMed1990

Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing.

Structural studies of CaATPase from sarcoplasmic reticulum have so far been restricted to low resolution due to the poor order of two-dimensional crystal forms. However, we report that three-dimensional microcrystals of detergent-solubilized CaATPase diffract to 7.2 A in x-ray powder patterns and may therefore provide an opportunity to study CaATPase structure at higher resolutions. In the present study, we have characterized the symmetry and molecular packing of negatively stained crystals by electron microscopy (em). By altering the detergent-to-lipid ratio, different sized crystals were produced, which adhere to an em grid in different orientations. Thus, we obtained micrographs of three different projections and from these determined unit cell dimensions to be 151 X 51 X 158 A and the three-dimensional space group to be C2 with an angle beta very close to 90 degrees; x-ray powder patterns of hydrated, unstained crystals yielded dimensions of 166 X 58 X 164 A. Micrographs from each of two principal projections were averaged to produce two-dimensional density maps. Based on these maps and on the previously determined low-resolution structure of CaATPase, a packing diagram for these three-dimensional crystals is presented and major intermolecular contacts are proposed.

Animals↗
PubMed1987

The variable twist of actin and its modulation by actin-binding proteins.

Previous studies demonstrated that actin filaments have variable twist in which the intersubunit angles vary by approximately +/- 10 degrees within a filament. In this work we show that this variability was unchanged when different methods were used to prepare filaments for electron microscopy. We also show that actin-binding proteins can modulate the variability in twist. Three preparations of actin filaments were photographed in the electron microscope: negatively stained filaments, replicas of rapidly frozen, etched filaments, and frozen hydrated filaments. In addition, micrographs of actin + tropomyosin + troponin (thin filaments), of actin + myosin S1 (decorated filaments), and of filaments frayed from the acrosomal process of Limulus sperm (Limulus filaments) were obtained. We used two independent methods to measure variable twist based on Fourier transforms of single filaments. The first involved measuring layer line intensity versus filament length and the second involved measuring layer line position. We measured a variability in the intersubunit angle of actin filaments of approximately 12 degrees independent of the method of preparation or of measurement. Thin filaments have 15 degrees of variability, but the increase over pure actin is not statistically significant. Decorated filaments and Limulus filaments, however, have significantly less variability (approximately 2 and 1 degree, respectively), indicating a torsional stiffening relative to actin. The results from actin alone using different preparative methods are evidence that variable twist is a property of actin in solution. The results from actin filaments in the presence of actin-binding proteins suggest that the angular variability can be modulated, depending on the biological function.

Actin Cytoskeleton↗
PubMed1981

Nonlinear increases in diffusing capacity during exercise by seated and supine subjects.

To study the effects of exercise on pulmonary diffusing capacity, we measured the lungs' diffusing capacity for carbon monoxide (DLCO) during exhalation from 30 to 45% exhaled vital capacity in eight healthy subjects at rest and during exercise while both sitting and supine. We found that DLCO at these lung volumes in resting subjects was 26.3 +/- 3.2% (mean +/- SE) higher in the supine than in the sitting position (P less than 0.001). We also found that, in both positions, DLCO at these lung volumes increased significantly (P less than 0.001) with increasing exercise and approached similar values at maximal exercise. The pattern of increase in DLCO with an increase in oxygen consumption in both positions was curvilinear in that the rate of increase in DLCO during mild exercise was greater than the rate of increase in DLCO during heavy exercise (P = 0.02). Furthermore, in the supine position during exercise, it appeared that DLCO reached a physiological maximum.

Adult↗
PubMedDate not supplied

Development of a new capillary electrophoresis-based fibre optic sensor.

A new fluorescence-based fibre optic sensor is described which combines the sensitivity offered by laser-induced fluorescence with the selectivity offered by capillary electrophoresis (CE). A single optical fibre directly probes the terminus of a 5-8 cm separation capillary. The linear geometry associated with this sensor necessitates a 'single reservoir' design, thus presenting major challenges to overcome in comparison to the conventional two-reservoir configuration common to a typical laboratory setup. Some of the challenges confronted by the design features presented in this work include the reduction of gravity-driven hydrostatic flow, the ejection of electrolytic gases evolved at the detection-side electrode and the establishment a suitable compromise between detectability and separation performance. The success of such design features demonstrates the feasibility of a CE-based sensor which offers several amenities particularly useful for in situ sensing. Such attributes include selectivity, diminutive size, flexibility, reusability, high sensitivity, speed, and remote control. Detailed descriptions of sensor fabrication are included, including two variations on a general design concept. In addition, the single-fibre optical detection system is described. Separation characteristics of the new CE-based sensor are presented, highlighted by an observed separation efficiency of up to 8000 theoretical plates (for a 5 cm capillary). The separation of a three-component mixture of the laser dyes, Rhodamine 6G, fluorescein isothyocyanate and sodium fluorescein, is demonstrated.

Biosensing Techniques↗