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J C Merchuk

Publications and source records attributed to J C Merchuk.

12 recordsLinked to original sources
PubMed2002

Phase separation rates of aqueous two-phase systems: correlation with system properties.

The kinetics of phase separation in aqueous two-phase systems have been investigated as a function of the physical properties of the system. Two distinct situations for the settling velocities were found, one in which the light, organic-rich (PEG) phase is continuous and the other in which the heavier, salt-rich (phosphate) phase is continuous. The settling rate of a particular system is a crucial parameter for equipment design, and it was studied as a function of measured viscosity and density of each of the phases as well as the interfacial tension between the phases. Interfacial tension increases with increasing tie line length. A correlation that describes the rate of phase separation was investigated. This correlation, which is a function of the system parameters mentioned above, described the behavior of the system successfully. Different values of the parameters in the correlation were fitted for bottom-phase-continuous and top-phase-continuous systems. These parameters showed that density and viscosity play a role in the rate of separation in both top continuous- and bottom continuous-phase regions but are more dominant in the continuous top-phase region. The composition of the two-phase system was characterized by the tie line length. The rate of separation increased with increasing tie line length in both cases but at a faster rate when the bottom (less viscous) phase was the continuous phase. These results show that working in a continuous bottom-phase region is advantageous to ensure fast separation.

Models, Chemical↗
PubMed2000

RHIZOSCAN: A semiautomatic image processing system for characterization of the morphology and secondary metabolite concentration in hairy root cultures.

We describe the application of the newly developed RHIZOSCAN software for accurate morphological analysis and determination of overall and local secondary metabolite concentrations in two clones of Beta vulgaris throughout the growth period. Local secondary metabolite concentrations may be determined at any point in the root, and pigment gradients in each lateral root can be followed during culture and saved to the computer. Throughout the entire analysis process, an image appears in a graphical result window on the screen, which enables visual evaluation of the numerical output at each stage of the analysis. Biosynthetic data on concentrations obtained by image analysis were validated by spectrophotometric analysis. The importance of determining appropriate scanning and analysis conditions (scanning resolution, background color, threshold value, segmentation plane in the hue-saturation-intensity color system and pruning length) for obtaining accurate morphological measurements is examined and the means of fixing these parameters is described. Our results show that, using RHIZOSCAN, detailed and accurate information on root architecture and secondary metabolite concentrations can be obtained in a short time.

Automation↗
PubMed2000

Determination of the dissociation constant of valine from acetohydroxy acid synthase by equilibrium partition in an aqueous two-phase system.

An aqueous polyethylene glycol/salt two-phase system was used to estimate the dissociation constant, K(dis), of the Escherichia coli isoenzyme AHAS III regulatory subunit, ilvH protein, from the feedback inhibitor valine. The amounts of the bound and free radioactive valine in the system were determined. A Scatchard plot of the data revealed a 1:1 valine-protein binding ratio and K(dis) of 133+/-14 microM. The protein did not bind leucine, and the ilvH protein isolated from a valine resistant mutant showed no valine binding. This method is very simple, rapid and requires only a small amounts of protein compared to the presently used equilibrium dialysis method.

Acetolactate Synthase↗
PubMed2000

Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system.

Extraction in a polyethylene glycol (PEG)-phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E. coli extract. Extraction optimization was achieved by varying the system parameters. Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)-phosphate (6%) and PEG-4000 (14%)-phosphate (5.5%)-KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system. Significant purification was achieved by a single extraction step with 70% recovery of the enzyme. After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.

Acetolactate Synthase↗
PubMed2000

Hepatocyte behavior within three-dimensional porous alginate scaffolds.

A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100-150 microm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 microm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 microg albumin/10(6) cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 microg/10(6) cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation.

Albumins↗
PubMed1998

Aqueous two-phase systems for protein separation. Studies on phase inversion.

Phase equilibrium studies were done with the PEG 400-phosphate system, obtaining equilibrium binodal lines, tie lines and phase inversion points. A method of calculation of the critical point on the binodal curve is described. The influence of the presence of NaCl in solution was studied, and the comparative results are presented. It was found that in some range of concentration the shift produced in the binodal line can be important. The rate of phase separation can be used as an indication of which of the phases is continuous. Using this method the phase inversion point can be determined in the system for each tie line. A range of ambiguity was found, where the continuity of the phases is affected not only by the composition of the mixture, but also by the fluid dynamics. Within this range, gentle agitation produces a bottom-continuous suspension. while strong agitation produces a top-continuous suspension. Two inversion points exist therefore on each tie line, delimiting on the phase equilibrium plane a region where the phase continuity depends on fluid dynamics. The convergence of this region towards the critical point can be used to control of the consistency of the experimental data.

Chemistry Techniques, Analytical↗
PubMed1998

On the kinetics of phase separation in aqueous two-phase systems.

The effect of the tie-line location (phase volume ratio) on the kinetics of phase separation in batch PEG/salt aqueous two-phase systems (ATPS) has been investigated. PEG/sulphate systems with a stability ratio (sr) of 0.34 and 0.37 and relative tie-line lengths in the range 0.1 to 0.6 for a continuous top phase and in the range 0.03 to 0.15 for a continuous bottom phase were used in the batch studies. A continuous settler was designed with three different inlet geometries. Phase separation is much faster when the bottom phase is continuous and in this case the location on the tie-line and the presence or absence of Bacillus subtilis extract makes little difference. When the top phase is continuous the relative sizes of the phases (phase ratio, R. relative distance on tie-line, rd) has an important effect, the larger the top phase (larger R and rd) the slower the phase separation. The presence of Bacillus extract also makes the operation slower which is more marked at the largest values of R (and rd). At the largest volume ratios (R or rd) three different settling regions have been recognised, a region of coalescence, a region of drops moving to the interphase and a region where drops queue at the interphase to coalesce into the large phase. A modified correlation that takes into account the location on the tie-line and thus volume ratio (R) and relative distance (rd) has been proposed and successfully tested. The behavior of batch and continuous systems in the presence and absence of Bacillus subtilis extract in systems with continuous bottom phase was also studied. The settling velocity was lower in the continuous than in the batch systems, and in both cases the initial rate was lower in the presence of Bacillus extract.

Bacillus subtilis↗
PubMed1998

Polysaccharide production by plant cells in suspension: experiments and mathematical modeling.

Symphytum officinale L cells were grown in Erlenmeyer flasks at four different temperatures: 15, 20, 25, and 30 degrees C. A mathematical model of the culture growth is presented. The intracellular and extracellular products are considered in separate equations. An interrelation between fresh weight, dry weight, and viability is considered in the balances. The model includes a description of the changes in time of wet and dry biomass, cell viability, substrate concentration and polysaccharide concentration, both intra- and extracellular. The model was tested by fitting the numerical results to the data obtained.

Biomass↗
PubMed1991

Shear effects on suspended cells.

Shear has been mainly considered in the technical literature as a destructive element, when applied to microorganisms and cells. Indeed, most of the research work addressing the subject aims at the identification of damaging levels of shear on a given culture. The present work is focused on the effects of shear on suspended cultures before the damaging levels are attained. Inspection of the literature reveals that shear may influence growth rate, cellular volume, metabolite production rate and distribution, and membrane permeabilities. Available devices for study and evaluation of shear effects on suspended cultures are described and critically reviewed. The review reveals the possibility of an influence of the liquid dynamics on the kinetics of the biochemical process. This is relevant for bioreactor design and scale up, and stresses the importance of using structural bioreactor models in order to describe the hydrodynamics of the system.

Animals↗
PubMed1987

Evaluation of a new type of membrane for blood oxygenation.

Results of tests on oxygen transfer rates and on a series of membranes, both in saline solutions and blood are reported. A series of hematological tests was carried out during the tests with blood, in order to assess the damage caused to the blood by contact with the membrane. All the results were compared with results obtained with the same equipment using Celgard membranes. A series of measurements at high liquid flow rate was carried out both with Celgard and the MFT membrane in order to eliminate the liquid-side resistance and evaluate the permeability of the membranes. The results of oxygen transfer rate into blood, when extrapolated by the "constant slope method" showed excellent agreement with the data obtained with saline solutions, justifying the use of oxygen absorption into saline during the process of screening membranes for use in blood oxygenators.

Absorption↗
PubMed1981

Performance of a new type of blood oxygenator.

A new type of direct-contact oxygenator has been tested satisfactorily on dogs in nine experiments. It is based on the principle of the "packed column" and has the advantage of a small hold-up (meaning a small priming volume), no formation of foam, and gentle flowing of the blood. The apparatus is simple enough to be commercially available at a low price and discarded after use. Both PO2 increase and PCO2 decrease were satisfactory and plasma hemoglobin values remained low after 75 minutes of perfusion. Preliminary in vitro experiments on ox and human blood showed a low degree of blood damage.

Animals↗