Distribution of bacteria in the velocity gradient centrifuge.

Cells in different parts of the cell cycle can be separated by brief centrifugation in a density stabilized gradient: the Mitchison-Vincent technique. The position of a cell in the tube depends upon its size, shape, and density, upon the gradients of density, viscosity, and centrifugal force through which it sediments, and upon time. A program to compute the velocities and integrate the velocity profile for particles of a particular size class is presented. Because enteric bacteria are a form intermediate between right cylinders and prolate ellipsoids of revolution, the program uses values for the frictional coefficient intermediate between those calculated for ellipsoids and for cylinders. The formula f=6pietab(a/b)1/2 possesses this property and because of its simplicity greatly speeds the calculations. A second program computes the distribution of masses and then of sedimentation constants for a bacterial population, expressed either as a frequency distribution or as total mass per s-class. The effect of the known variation in cell size at division is included in these calculations, which apply to organisms undergoing balanced, asynchronous growth in which mass increase is proportional to cell size. The two programs in conjunction compute the mass or cell-number profile in an arbitrary gradient. The programs have been used to design gradients to maximize the resolution of the technique.     

Ideal and non-ideal concentration gradient propagation in chemotaxis studies

The shape and spatio-temporal development of a concentration gradient depend on the method and medium of its generation. Model gradients of low molecular weight (MW) dyes develop with predictable characteristics in 1% agarose. In contrast, any method of delivering dyes into free solution, e.g., via capillary pipets, produces anomalous gradients, unless the chambers are extremely shallow (less than 1 mm deep). Thus, most methods of chemo-attractant delivery which are popular in chemotaxis studies are incapable of propagating gradients by molecular diffusion; rather, they tend to be dominated by the effects of wholesale flow of the chemo-attractant and convection and turbulence of the free solution. This may have serious consequences for the interpretation of some chemotaxis experiments, since several gradient qualities of chemo-attractants (and probably morphogens) could influence cell behaviour. The possible effects on cells of the motion and development of gradients are also discussed.     

Prediction of a protein band profile in preparative reversed-phase gradient elution chromatography

The overloaded band profiles of lysozyme in reversedphase preparative chromatography were recorded on a C18 chemically bonded silica column, with acetonitrile/water as the mobile phase. These experiments were carried out under isocratic conditions at 31.6, 31.9, and 32.2% acetonitrile (ACN) for loading factors up to 43% of the column saturation capacity and under linear-solvent-strength gradientelution with gradient slopes of 0.5 and 1% ACN/min, for loading factors up to 11.3%. The adsorption isotherms of lysozyme were measured for the same solvent compositions and found to be accurately accounted for by a bi-Langmuir isotherm model.With the use of a Craig model implementation of the equilibrium-dispersive model of chromatography, the band profiles of lysozyme were calculated. An excellent agreement was observed between these calculated profiles and the experimental profiles recorded at loading factors below 5%. By contrast, band profiles calculated using a Langmuir isotherm failed to describe the experimental bands. At column loadings exceeding 8%, a slight but systematic deviation takes place between calculated and experimental profiles. It is most probably explained by the considerable concentration effect of the gradient, making the band experience phase equilibrium in a concentration range that exceeds largely the one where the isotherm data have been measured.     

The development of the International Space Station centrifuge.

Gravitational biology research facility "Centrifuge" is currently under development for the International Space Station. Research in the Complex Organism Biology, indispensable to the progress in Health Science, is only possible in the Centrifuge aboard the station. So, on-orbit 1 G controls for various specimens including small mammals, fish, and higher plants will be rigorously done in the Centrifuge. This facility is also capable of providing "reduced gravity" likely on the Moon or on Mars. Thus, it will play a key role in creating knowledge of space fundamental biology. As part of the offset of NASA's Shuttle launch services for the Japanese Experiment Module, JAXA is developing the Centrifuge Rotor (CR), the Life Sciences Glovebox (LSG) and the Centrifuge Accommodation Module (CAM). Critical Design Review (CDR) of LSG was conducted on July 2004, while the system CDRs of the CAM and CR are scheduled for December 2004 and August 2005, respectively. Their launch schedules are under review.     

Quasi-elastic light scattering from migrating chemotactic bands of Escherichia coli. III. Studies of band formation propagation and motility in oxygen and serine substrates.

A series of light scattering experiments have been performed to study both macroscopic aspects of band formation and propagation and microscopic motility parameters of Escherichia coli in the combined substrate gradients of oxygen and serine. From the band formation experiment the conclusion is drawn that a minimum threshold gradient of the substrate is required for bacteria to form a band. From the band propagation experiment in the serine substrate the motility coefficient mu and chemotactic coefficient delta are determined. A separate quasi-elastic scattering experiment has been made with a propagating band to obtain three microscopic motility parameters: mean twiddle time tau 1, mean run time tau 2, and mean run speed V2. Finally, a scaling argument is made to connect the macroscopic parameters mu and delta with the microscopic parameters tau 1, tau 2, and V2, thus achieving a unified understanding of macroscopic and microscopic aspect of chemotaxis.     

Size-separation of yeast mitochondria in the zonal centrifuge

Mitochondria, released from yeast spheroplasts and subjected to rate separation through sorbitol gradients in the zonal centrifuge, migrated in a wide symmetrical zone. Electron micrographs showed that the mitochondria had been resolved within the zone according to size. The mean mitochondrial diameter at the leading edge was approximately twice that at the trailing edge of the particle zone. Activities of the enzymes cytochrome oxidase, malate dehydrogenase, and reduced nicotinamide adenine dinucleotide- and d-lactate cytochrome c reductases were essentially uniform throughout the mitochondrial zone. Mitochondria from a vegetative-petite mutant had almost the same size distribution as the isogenic wild type, but with somewhat larger mean diameter and either absent or markedly reduced enzyme activities. Mixtures of wild-type and petite mitochondria produced sedimentation profiles showing overlap of particle populations with respect to mean sedimentation rates and mitochondrial diameters, as well as intermediate levels of enzyme activities. Both cristate and noncristate organelles were present throughout the mitochondrial zone from these mixtures. Mitochondria centrifuged in sorbitol density gradients were well-preserved and yielded consistent sedimentation profiles, whereas particles in sucrose density gradients migrated more slowly, produced varied sedimentation profiles, and often showed spurious peaks, presumably due to particle aggregations.     

The total containment of a batch-type zonal centrifuge.

A batch-type zonal centrifuge has been modified and totally contained for use with biologically hazardous materials. A sealed cabinet encloses the centrifuge and the ancilliary equipment. It is operated with a flow of filtered air when the zonal system is on, decontaminated with ethylene oxide, and maintained at a negative pressure throughout. The centrifuge subsystems can be drained, flushed, and decontaminated with ethylene oxide before an engineer services the machine. The sample handling system within the cabinet is remotely controlled.     

A single band does not always represent single bacterial strains in denaturing gradient gel electrophoresis analysis

DNA in a denaturing gradient gel electrophoresis (DGGE) band that could not be sequenced after recovery from the gel was cloned into a TA cloning vector and a library was constructed and then 13 clones randomly picked up from the library was sequenced. Although the excised DNA from the DGGE gel showed a single band, the library consisted of several different sequences phylogenetically. This phenomenon was also observed in several other DGGE bands. Therefore, this suggests that a single DGGE band does not always represent a single bacterial strain and a new bias for quantitative analyses based on band intensities has been identified.     

Purification and properties of human erythrocyte band 4.2. Association with the cytoplasmic domain of band 3

We have purified the human erythrocyte membrane protein band 4.2 to greater than 85% homogeneity. The protein was extracted from spectrin-actin-depleted inside-out vesicles in a pH 11 medium and purified by gel filtration in the presence of 1 M KI. The purified protein was heterogeneous and had an average S20,w of 5.5 and an average Stokes radius of 82 A. By electron microscopy, the protein appeared heterogeneous in size and shape, having a diameter ranging from 80 to 150 A. The protein bound saturably to band 4.2-depleted red cell inside-out vesicles, and the binding exhibited a concave Scatchard plot. Binding was reduced greater than 90% by proteolytic digestion of membranes. Digestion studies suggested that there are two classes of binding sites for band 4.2 on the cytoplasmic aspect of red cell membranes, one of which is likely to be band 3. The purified 43-kDa cytoplasmic domain of band 3 competed for band 4.2 binding to red cell membranes and could completely abolish binding when added at a concentration of greater than 200 micrograms/ml. The purification of band 4.2 and the characterization of its association with red cell membranes should facilitate the discovery of the function of this major red cell membrane protein.     

Critical band in auditory lateralization.

A new and powerful procedure for determining frequency analysis in the auditory system, as evidence by the critical band, is described. The onset time difference, delta T, needed to lateralize 30-msec tone bursts toward the leading ear was measured as a function of the frequency difference, delta F, between the brust in one ear and the burst in the other ear. When delta F was less than the critical band, threshold delta T was constant at 100 mu sec or less, depending on center frequency; beyond the critical band, delta T increased with delta F. These dichotically measured critical bandwidths increased from 110 Hz at a center frequency of 500 Hz to 1100 Hz at a center frequency of 6000 Hz. They were unaffected by varying signal level from 25 to 80 dB or signal duration from 10 to 300 msec. The sam e critical-band values have been measured with monaural stimuli in loudness summation, maskin, detection, phase perception, consonance, and so forth.     

Both ankyrin and band 4.1 are required to restrict the rotational mobility of band 3 in the human erythrocyte membrane.

A population of band 3 proteins in the human erythrocyte membrane is known to have restricted rotational mobility due to interaction with cytoskeletal proteins. We have further investigated the cause of this restriction by measuring the effects on band 3 rotational mobility of rebinding ankyrin and band 4.1 to ghosts stripped of these proteins as well as spectrin and actin. Rebinding either ankyrin or 4.1 alone has no detectable effect on band 3 mobility. Rebinding both these proteins together does, however, reimpose a restriction on band 3 rotation. The effect on band 3 rotational mobility of rebinding ankyrin and 4.1 are similar irrespective of whether or not band 4.2 is removed from the membrane. We suggest that ankyrin and 4.1 together promote the formation of slowly rotating clusters of band 3.