High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles.

DNA restriction fragments and PCR products were separated by means of ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles with a mean diameter of 2.1 microns. Optimum resolution was obtained by using an acetonitrile gradient in 100 mM of triethylammonium acetate and a column temperature of 50 degrees C. This allowed the separation of DNA fragments differing in chain length by 1-5% up to a size of 500 base pairs. PCR products could be analyzed directly in less than two minutes with a concentration sensitivity of at least 300 ng/ml. Compared with anion-exchange chromatography or gel electrophoresis no desaltation of the purified DNA molecules is required because the volatile buffer system can be readily evaporated. Subsequently, the method was used for the semiquantitative evaluation of the expression of multidrug resistance genes in mononuclear white blood cells.     

Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.

Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316).     

Gel particles from spray-dried disordered polysaccharides

The formation of gel particles from alginate and ι-carrageenan was studied through a novel pathway of formation via an amorphous spray-dried intermediate. Dried biopolymer particles were suspended in solutions of different Ca²⁺ concentration. Particle size ranges and microscopic observation demonstrated that a range of swelling behaviour could be induced, with lower calcium concentrations resulting in more expanded particles, until a lower limit is reached below which particles initially dissolve. For the same calcium charge stoichiometry, larger swollen gel particles were obtained for alginate than for ι-carrageenan. The ability to produce a range of swollen biopolymer gel particle sizes, on the order of 1–600 μm, is attributed to the balance between gelation and dissolution kinetics, with fast gelation kinetics and slow dissolution promoting production of small gel particles whilst fast dissolution with slow gelation leads to larger gel particles. By controlling the solution environment in which rehydration is carried out, it is therefore possible to produce particles with desired degrees of swelling from a single starting material.     

Size-dependent, chromatographic separation of double-stranded DNA which is not based on gel permeation mode

A new procedure for size-dependent fractionation of DNA was investigated. DNA fragments ranging from 10 to 40 kbp were separated by using columns for high-performance gel permeation chromatography. However, the order of elution was opposite to that which would be expected for gel permeation chromatography, i.e., smaller fragments were eluted faster than larger fragments, though separation based on normal gel permeation chromatography was observed when smaller DNA fragments (less than 5 kbp) were applied. The size range of DNA which can be resolved by this new procedure was found to depend on both particle size and flow rate; the use of a column packed with smaller particles or the application of a faster flow rate enabled us to resolve smaller DNA fragments, but the pore size or chemical nature of the column packing had scarcely any effect on the resolution. This mode of separation was attained by using both silica and polymer packings. The results suggest that the separation is based on a hydrodynamic phenomenon.     

Effects of the micro-environment on the photophysical properties of hematoporphyrin

The photochemical degradation of histidine, cysteine and tyrosine with hematoporphyrin as sensitizer was potentiated by the presence of Sephadex, BioGel or Percoll particles. This effect could only partly be explained by binding of the sensitizer to the gel particles, leading a.o. ot monomerization of aggregated sensitizer molecules in the aquous environment. The hematoporphyrin triplet state life time increased from 250 μs in phosphate buffer to 1992 μs in the presence of 50% Percoll. Most likely the effect of the gel particles on the sensitizer triplet state is, at least partly, mediated by the solvent. A plausible explanation seems to be that the vicinal water structure at the particle interface stabilizes the sensitizer triplet.     

High-performance partition chromatography of peptides on a new cross-linked agarose

A new cross-linked agarose in bead form (20-40 microns in diameter) has been used for partition chromatography in two-phase solvent systems to give columns exhibiting efficiencies of several thousand theoretical plates. The efficiency of this high-performance partition column (70-80 plates per cm) far exceeds that of the most commonly used soft gel Sephadex G-50 (10 plates per cm). The column is operated under moderate pressure (5 p.s.i.) and can be regenerated. The new procedure appears capable of separating a peptide of moderate size, such as beta-endorphin (31 residues), from the majority of possible single-deletion sequences.     

Electronic particle counting for evaluating the quality of air in operating theatres: a potential basis for standards?

Airborne particle counting in eight size ranges (0.5- greater than 20 microns), by computerized electronic equipment, was compared with the numbers of bacteria-carrying particles (BCP) assessed by slit sampling in ultra-clean and turbulently ventilated operating theatres. In the ultra-clean theatre the number of particles of 5-7 microns size range correlated with BCP while peaks in the numbers of particles less than 3 microns and greater than 15 microns corresponded with activity. Comparative relationships also occurred in the turbulently ventilated theatre but the use of this equipment in that environment cannot yet replace counts of airborne bacteria. We consider that electronic particle counting in the 0-20 microns size range may be used to judge the performance of a clean air operating theatre distribution system, including efficiency and integrity of the filter/seal systems and the presence or absence of entrainment of bacteria and other particles. The sampling techniques and analysis of particle concentration results described here may be a suitable basis for standards.     

Effects of osmotic stress on mast cell vesicles of the beige mouse

The large size of the vesicles of beige mouse peritoneal mast cells (4 microns in diameter) facilitated the direct observation of the individual osmotic behavior of vesicles. The vesicle diameter increased as much as 73% when intact cells were perfused with a 10 mM pH buffer solution; the swelling of the vesicle membranes exceeded that of the insoluble vesicle gel matrix, which resulted in the formation of a clear space between the optically dense gel matrix and the vesicle membrane. Hypertonic solutions shrank intact vesicles of lysed cells in a nonideal manner, suggesting a limit to the compressibility of the gel matrix. The nonideality at high osmotic strengths can be adequately explained as the consequence of an excluded volume and/or a three-dimensional gel-matrix spring. The observed osmotic activity of the vesicles implies that the great majority of the histamine known to be present is reversibly bound to the gel matrix. This binding allows vesicles to store a large quantity of transmitter without doing osmotic work. The large size of the vesicles also facilitated the measurement of the kinetics of release as a collection of individual fusion events. Capacitance measurements in beige mast cells revealed little difference in the kinetics of release in hypotonic, isotonic, and hypertonic solutions, thus eliminating certain classes of models based on the osmotic theory of exocytosis for mast cells.     

Efficiency of particle retention and clearance rate in the polychaete Sabellaria alveolata L

The development of Sabellaria alveolata, a gregarious reef-building polychaete species, is maximal in Mont-Saint-Michel Bay (France), where trophic capacity is now threatened by increasing shellfish farming. As no data are available concerning the ecophysiological response of this species, the purpose of the present study was to obtain clearance rate and retention efficiency values to provide a first order of magnitude for the trophic role of this species. Data were obtained using a flow-through system with novel troughs suitable for 225 cm2 reef blocks containing a mean number of 940 +/- 102 (S.E.) individuals. The experimental diet used consisted of a mixture of two live microalgae, Skeletonema costatum (3800 cell ml-1) and Isochrysis galbana (23,700 cell ml-1), chosen to cover a broad size range (2 to 16 microns equivalent spherical diameter, ESD), as determined by a particle counter. On the basis of a mean clearance rate of 0.7 lh-1 obtained with reef blocks, the mean rate for an individual was estimated at 7.5 x 10(-4) L h-1. Particles larger than 6 microns ESD were cleared with 100% efficiency, but S. alveolata was unable to retain particles smaller than 2 microns ESD. The results are compared with data obtained for other polychaete species, and clearance rate values are extrapolated to an entire reef.     

The effect of size of polystyrene particles on their retention within the rat peritoneal compartment, and on their interaction with rat peritoneal macrophages in vitro

Polystyrene particles (size range 300 nm-3 microns diameter) were radioiodinated and their capture by rat peritoneal macrophages measured in vitro. For unmodified particles, most efficient accumulation was observed using a diameter of 600 nm (Endocytic Index (E.I.) = 16.4 +/- 2.9 microliters/10(6) cells/h). Particles (3 microns diameter) which had been modified to become more hydrophilic by hydroxymethylation showed an increased rate of capture (E.I. = 136.6 +/- 91.2 microliters/10(6) cells/h). Following intraperitoneal administration to rats, unmodified 3 micron particles showed selective accumulation in the omentum (18.4% injected dose/g), and this was increased for the hydroxymethylated bead (35.3% dose/g). The smaller (800 nm) particles were better able to leave the peritoneal compartment. Radiolabelled particles isolated from a peritoneal wash after 5 h were mostly cell-associated (72-86%, depending on the type of particle).     

Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. I. Measurement of concentration and size of single platelets and aggregates.

A double infusion flow system and particle sizing technique were developed to study the effect of time and shear rate on adenosine diphosphate-induced platelet aggregation in Poiseuille flow. Citrated platelet-rich plasma, PRP, and 2 microM ADP were simultaneously infused into a 40-microliters cylindrical mixing chamber at a fixed flow ratio, PRP/ADP = 9:1. After rapid mixing by a rotating magnetic stirbar, the platelet suspension flowed through 1.19 or 0.76 mm i.d. polyethylene tubing for mean transit times, t, from 0.1 to 86 s, over a range of mean tube shear rate, G, from 41.9 to 1,000 s-1. Known volumes of suspension were collected into 0.5% buffered glutaraldehyde, and all particles in the volume range 1-10(5) microns 3 were counted and sized using a model ZM particle counter (Coulter Electronics Inc., Hialeah, FL) and a logarithmic amplifier. The decrease in the single platelet concentration served as an overall index of aggregation. The decrease in the total particle concentration was used to calculate the collision capture efficiency during the early stages of aggregation, and aggregate growth was followed by changes in the volume fraction of particles of successively increasing size. Preliminary results demonstrate that both collision efficiency and particle volume fraction reveal important aspects of the aggregation process not indicated by changes in the single platelet concentration alone.     

Thrombogenicity of prothrombin complex concentrates]

PCC's were isolated either by adsorption to DEAE-Sephadex A-50 (particle size 40-120 microns; Pharmacia Uppsala) or by adsorption to Molselect DEAE-50 (particle size 100-320 microns; Reanal Budapest) from human citrated plasma after separation of factor VIII by cryoprecipitation. The two products obtained (without heparin) were both compared by in vitro (NAPTT, TGt50) and in vivo (venous stasis) model in rats acc. to WESSLER) tests. The agent prepared by adsorption to DEAE-Sephadex A-50 showed a significantly higher thrombogenicity than that prepared by Molselect DEAE-50. ED50 values for thrombus formation to amounted 15 Factor IX U/kg for the first product and to 210 factor IX U/kg for the second.     

Effect of processing on the recovery of recombinant beta-glucuronidase (rGUS) from transgenic canola

This study addresses the processing of transgenic canola seed for production of recombinant proteins by using beta-glucuronidase (rGUS) as a model protein. The major processing steps that were investigated included dry and wet grinding of the seed, solvent extraction of canola oil, and protein extraction. rGUS in canola seed was stable for at least 2 weeks of incubation at 38 degrees C and for more than 5 months at 10 degrees C. At 70 degrees C, the residual activity changed inversely to the initial moisture content of the seed. The comparison of wet and dry processing revealed no significant differences in protein recovery. rGUS was stable during the defatting of transgenic canola flakes with hexane at 66 degrees C, whereas 2-propanol extraction at the same temperature reduced the extractable enzyme activity by almost 50%. The particle size of the ground seed was important for the extraction efficiency. A faster extraction and greater protein yield was achieved by extracting particles with an average diameter equal to or smaller than 255 microm. More than 80% rGUS was extracted in one stage with sodium phosphate buffer of pH 7.5.     

Optical plankton analyser: a flow cytometer for plankton analysis, II: Specifications

An analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton analysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 microns in width and over 1,000 microns in length can be analysed. Sample flow rates of up to 55 microliters/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 microns. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aeruginosa colonies, whereas one sample showed a dynamic range of 10(6). A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.     

High-performance liquid chromatography using novel square tile-shaped hydroxyapatite crystals as adsorbent

High-performance liquid chromatography using, as adsorbent, novel square tile-shaped hydroxyapatite crystals (with thicknesses of about 2 microns and diameters of 3-7 microns) has been developed. The chromatographic efficiencies of the novel hydroxyapatite packed columns are almost equal to those of the previously developed spherical hydroxyapatite packed columns; high chromatographic resolutions can be obtained by using extremely reduced column lengths of 0.5-3 cm. Since both the square and the spherical hydroxyapatite have roughly the same particle size of some micrometers, the chromatographic efficiency can be deduced to be determined mainly by the particle size rather than the particle shape.     

Settling of fixed erythrocyte suspension droplets

The fractionation of micron-size particles according to physical properties of size, density and surface characteristics by centrifugation and electrophoresis is hindered when the particles behave collectively rather than individually. The formation and sedimentation of droplets containing particles is an extreme example of collective behavior and a major problem for these separation methods when large quantities of particles need to be fractionated. In this paper, experiments that measured droplet sizes and settling rates for a variety of particles and droplets are described. Expressions are developed relating the particle concentration in a drop to measurable quantities of the fluids and particles. The number of particles in each droplet was then estimated along with the effective droplet density and certain trends are noted. Since a major application of this work is the purification of biological cells in the range of 10 microns, for which monodisperse inert particles are not available, red blood cells from different animals fixed in glutaraldehyde provided model particle groups with the necessary size range, visibility and stability for these fluid dynamical studies.     

Gel matrices for scanning gel chromatography

I have examined the light-scattering behavior of a number of gel matrices used in gel filtration chromatography. The angular dependence of light scattering by Sephadexes is consistent with treatment of the particles as large scattering particles with a low refractive index increment (mu). Such particles scatter light almost exclusively in the forward direction, permitting their use in direct scanning gel chromatography systems without corrections for multiple scattering and the consequent variation in pathlength through the column. Any matrix material with an appropriate combination of these two properties (large effective size and low mu relative to solvent) will perform reasonably well in direct scanning systems while any material with very small effective particle size (agaroses) or high mu (glass beads) may be expected to perform poorly. Agaroses may be acceptable for work in the visible region of the spectrum while glass beads are not.     

Optimization of the production of virus-like particles in insect cells

In this work the maximal operational hydrodynamic conditions (agitation and aeration rate) that cause no adverse effect in Sf-9 cells growth in SF900II serum-free medium were determined. Shear stresses higher than 1 N m-2 and aeration rates higher than 0.04 vvm affect cell growth and when these conditions increase to 1.5 N m-2 and 0.11 vvm, cell growth is completely inhibited with significant cell morphology changes and a strong decrease in viability. Although the pO2 did not show a significant effect upon cell growth in the range from 10 to 50%, cell infection and specific productivity were dramatically affected. The production was optimal at a pO2 of 25% with decreases higher than 50% being observed when the pO2 decreased to 10 or increased to 50%. The maximum product quality, i.e., the percentage of product in the form of high molecular weight particles, is not coincident with maximum product titer. Although the highest Pr55gag particle titer was obtained at 96 hours post infection (hpi) and at pO2 of 25%, the best product quality (defined by gel filtration chromatography and Western immunoblot) was obtained at 48 hpi, independently of the pO2 used. The effect of overcritical conditions upon productivity was also studied. As obtained for cell growth, cell infection is affected by shear stresses above 1 N m-2 and by aeration rates higher than 0.04 vvm, with decreases in Pr55gag particle titer higher than 70%, even when the overcritical values are still far from the limit at which cell death occurs. The results obtained and the optimization strategy used allowed the maximization of the oxygen supply without damaging the cells, with important consequences on the scale-up of a production process involving this insect cell/baculovirus expression system.     

Physico-chemical and functional characteristics of a subfraction of low-density lipoproteins isolated by ion-exchange column chromatography

Fractionation of human blood plasma low density lipoproteins (LDL) was performed by ion-exchange chromatography, using a linear NaCl gradient. It was shown that the binding of LDL subfractions eluted with a low ionic strength buffer (i.e., containing the particles with a lower negative charge) to B, E-receptors of fibroblasts was more effective than that of subfractions eluted with a high ionic strength buffer (i.e., containing the particles with a higher negative charge). The LDL particles with a lower negative charge had lower values of flotation coefficients (according to analytical ultracentrifugation data), smaller dimensions (according to gradient gel electrophoresis data) and a lower phospholipid/protein ratio (w/w). The experimental results suggest that LDL subfractions having different electrical parameters of the particle surface also differ in other physicochemical properties and seem to play a different role in atherogenesis.     

Changes in physical and chemical properties of pretreated wheat straw during hydrolysis with cellulase

Summary After 5 days of enzymatic hydrolysis, the average wheat straw particle size has decreased by 62% and there is a dramatic shift to particle sizes less than 25 microns in size. Before hydrolysis, all particle size fractions have the same composition. After hydrolysis, the cellulose fraction is reduced in all particles and the decrease is greater for smaller particles.