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Biomedical subjects

D B Kell

Publications and source records attributed to D B Kell.

At least 73 records · Page 4Linked to original sources
PubMed1994

Dielectric properties of human blood and erythrocytes at radio frequencies (0.2-10 MHz); dependence on cell volume fraction and medium composition.

The dielectric properties of human erythrocytes (red blood cells) suspended in whole blood and in isotonic media at various volume fractions (haematocrits) have been studied in the frequency range 0.2-10 MHz, in which the so-called beta-dispersion due to the Maxwell-Wagner effect is known to occur. The capacitance and conductance at 25 degrees C were measured by an instrument interfaced to a computer. The rectangular sample cavity (1 ml volume) contained four pure gold electrode pins, and the sample could be circulated by a roller pump. The frequency-dependence of the permittivity and conductivity were fitted by non-linear least squares regression. Corrections were applied for non-linearity in the dielectric increment at high haematocrit, and for electrode polarisation when diluting the blood in saline. Data were interpreted in terms of a simple equivalent resistor-capacitor circuit. From the measured haematological values the specific membrane capacitance (Cm) and the conductivities internal and external to the cells (sigma i' and sigma o' respectively) were estimated. The conductivities behaved in a predictable manner with a mean of 0.458 S.m-1 (s.d. +/- 0.044) for sigma i', whereas the value of Cm (and indeed the actual capacitance of the suspension) was dependent on the amount of plasma present. Hence, in stationary normal (anticoagulated) whole blood samples, Cm was as high as 2.98 mu F.cm-2 (s.d. +/- 0.40), in contrast to about 0.9 mu F.cm-2 in blood diluted more than two-fold (to less than 20% hct) in isotonic media. The high value remained when the diluent was plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Cells↗
PubMed1994

Rapid identification using pyrolysis mass spectrometry and artificial neural networks of Propionibacterium acnes isolated from dogs.

Curie-point pyrolysis mass spectra were obtained from reference Propionibacterium strains and canine isolates. Artificial neural networks (ANNs) were trained by supervised learning (with the back-propagation algorithm) to recognize these strains from their pyrolysis mass spectra; all the strains isolated from dogs were identified as human wild type P. acnes. This is an important nosological discovery, and demonstrates that the combination of pyrolysis mass spectrometry and ANNs provides an objective, rapid and accurate identification technique. Bacteria isolated from different biopsy specimens from the same dog were found to be separate strains of P. acnes, demonstrating a within-animal variation in microflora. The classification of the canine isolates by Kohonen artificial neural networks (KANNs) was compared with the classical multivariate techniques of canonical variates analysis and hierarchical cluster analysis, and found to give similar results. This is the first demonstration, within microbiology, of KANNs as an unsupervised clustering technique which has the potential to group pyrolysis mass spectra both automatically and relatively objectively.

Animals↗
PubMed1994

Influence of Viable Cells on the Resuscitation of Dormant Cells in Micrococcus luteus Cultures Held in an Extended Stationary Phase: the Population Effect.

A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). In the present work, such cultures were studied by both flow cytometry and conventional microbiological methods and were found to contain various numbers of viable cells. Pretreatment of such cultures with penicillin G, and subsequent dilution, was used to vary this number. When the initial number of colony-forming cells per 30-ml flask was approximately nine (+/-five) or more, resuscitation of 10 to 40% of the cells, and thus culture growth, was observed. The lag period before the appearance of a population of cells showing significant accumulation of the fluorescent dye rhodamine 123 (i.e., of cells with measurable membrane energization) decreased from 70 to 27 h when the number of viable cells was increased from 30 to 10 per flask, while the lag period before an observable increase in the number of colony-forming cells occurred was almost constant (at some 20 h). Provided there were more than nine (+/-five) initially viable cells per flask, the number of initially viable cells did not affect the final percentage of resuscitable cells in the culture. The lag period could be ascribed in part to the time taken to restore the membrane permeability barrier of starved cells during resuscitation, as revealed by flow cytometric assessment of the uptake of the normally membrane-impermeant fluorescent DNA stain PO-PRO-3 {4-[3-methyl-2, 3-dihydro-(benzo-1, 3-oxazole)-2-methylidene]-1-(3'-trimethylammonium propyl)-pyridinium diiodide}. Although cell populations which contained fewer than nine +/-five viable cells per flask failed to grow, 4 to 20% of the cells (of 1.2 X 10) were able to accumulate rhodamine 123 after 80 to 100 h of incubation, showing the ability of a significant number of the cells in the population at least to display "metabolic resuscitation." Resuscitation and cell growth under such conditions were favored by the use of a 1:1 mixture of fresh lactate medium and supernatant from late-logarithmic-phase M. luteus cultures as the resuscitation medium. We conclude that the presence of a small fraction of viable cells at the onset of resuscitation facilitates the recovery of the majority of the remaining (dormant) cells. The cell density dependence of the kinetics, or population effect, suggests that this recovery is due to the excretion of some factor(s) which promoted the transition of cells from a state in which they are incapable of growth and division to one in which they are capable of colony formation.

Journal Article↗
PubMed1993

On the role of enzyme kinetic parameters in determining the effectiveness with which channelling can decrease the size of a metabolite pool.

Recently, it has been argued that the phenomenon of direct transfer of intermediate metabolites between adjacent enzymes, also known as metabolic channelling, would not decrease the concentration of those intermediates in the 'bulk' solution. However, this conclusion has been drawn by extrapolation from the results of simulations with a rather restricted set of parameters. We show that, for a number of kinetic cases, the existence of metabolic channelling can decrease the size of the soluble pool of intermediates. When the enzyme(s) 'downstream' of the channel have a catalytic capacity that is large relative to the enzymes 'upstream' of the channel, the decrease of concentration can be substantial (3 orders of magnitude).

Enzymes↗
PubMed1993

Dormancy in Stationary-Phase Cultures of Micrococcus luteus: Flow Cytometric Analysis of Starvation and Resuscitation.

Cultures of the copiotrophic bacterium Micrococcus luteus were stored in spent growth medium for an extended period of time following batch culture. After an initial decrease, the total cell counts remained constant at approximately 60 to 70% of the counts at the beginning of storage. The level of viability, as judged by plate counts, decreased to less than 0.05%, while respiration and the ability to accumulate the lipophilic cation rhodamine 123 decreased to undetectable levels. However, using penicillin pretreatment (to remove viable cells) and flow cytometry and by monitoring both the total and viable counts, we found that at least 50% of the cells in populations of 75-day-old cultures were not dead but were dormant. Resuscitation in liquid medium was accompanied by the appearance of a population of larger cells, which could accumulate rhodamine 123 and reduce the dye 5-cyano-2,3-ditolyl tetrazolium chloride to a fluorescent formazan, while a similar fraction of the population was converted to colony-forming, viable cells. We surmise that dormancy may be far more common than death in starving microbial cultures.

Journal Article↗
PubMed1992

Channelling can decrease pool size.

It is widely considered that a possible advantage of metabolite channelling, in which a product of an enzyme is transferred to the next enzyme in a metabolic pathway without being released to the 'bulk' solution, is that channelling can decrease the steady-state concentrations of 'pool' intermediates. This then spares the limited solvent capacity of the cell, and reduces the loss of pathway flux due to leakage or instability of the free intermediate. Recently, however, based on simulations of a particular model of a 'dynamic' channel, Cornish-Bowden ["Failure of channelling to maintain low concentrations of metabolic intermediates" (1991) Eur. J. Biochem. 195, 103-108] has argued that this is not in fact the case; his simulations indicated that the channel was rather ineffective at decreasing the concentration of the pool intermediate, and in some cases actually increased it. However, although his simulations were restricted to very specific thermodynamic and kinetic parameters, he generalised his conclusions, arguing that "channelling has no effect on the free concentration of a channelled intermediate in a pathway". By showing that, for a number of kinetic cases, the concentration of the pool intermediate did decrease substantially with increased channelling, we demonstrate here that the conclusion of Cornish-Bowden is not correct. In particular, if the reaction catalysed by the enzymes forming the channel has an equilibrium constant K higher than 1, and if the enzyme removing the product of the channel reaction is kinetically competent, channelling in the model system studied by Cornish-Bowden (1991) can decrease the steady-state concentration of the pool by a factor of 1000, independently of the mechanism of the terminal reaction and under conditions of essentially constant overall flux. If the channel is a 'static' channel, the decrease in the pool can be to arbitrarily low levels. This conclusion also holds for a system in which other reactions may consume the pool intermediate. Thus, channelling can maintain metabolite concentrations at low levels.

Catalysis↗
PubMed1992

New materials and technology for cell immobilization.

The choice of the most effective manner in which to operate an immobilized cell system is both complicated and, to some extent, a matter of guesswork. There is increasing awareness of the factors affecting reactor choice, and present work is aimed at making reactor performance more predictable.

Animals↗
PubMed1991

Confirmation by using mutant strains that the membrane-bound H(+)-ATPase is the major source of non-linear dielectricity in Saccharomyces cerevisiae.

Non-linear dielectric spectroscopy is a novel technique for determining the activity of (predominantly) membranous enzymes as their ability to generate harmonics when excited with a sinusoidal electrical field. In washed suspensions of yeast cells, the ability to generate harmonics is inhibited by low concentrations of sodium vanadate, suggesting that the vanadate-sensitive H(+)-ATPase is the major source of the non-linear dielectricity. This conclusion is greatly strengthened by the demonstration herein that the generation of harmonics by a strain containing a vandate-resistant H(+)-ATPase is also highly resistant to sodium metavanadate.

Cell Membrane↗
PubMed1991

New materials and technology for cell immobilization.

The choice of support materials for immobilizing cells is rapidly expanding. The literature that has appeared over the past year suggests that hydrogels will remain the first choice for the forseeable future, even though they are associated with many widely recognized problems. There is increasing interest in the use of tougher polymeric materials, and especially of inorganic ceramic supports. However, the most suitable cell support can be selected only after the process or form of reactor in which it is to be used has been assessed.

Biotechnology↗
PubMed1990

Hydrodynamic deposition: a novel method of cell immobilization.

A novel method of cell immobilization is described. The cell support consists of ceramic microspheres of approximately 50-75 microns diameter. The spheres are hollow, having a wall thickness of 10-15 microns and one entrance (ca. 20 microns diameter). The walls are porous with a mean pore size of approximately 90 nm. When a cell suspension (of S. cerevisiae) is passed through a column of such particles, cells are immobilized. Conditions are devised such that the overwhelming majority of cells are held in the central cavity of the support and not between the particles. Provided turbulence is avoided, the distribution of cells along the column length in the steady state is rather homogeneous. The facts that (a) essentially all particles, regardless of orientation, entrap cells, and (b) nonporous particles also entrap cells with high efficiency, indicate that filtration effects are irrelevant and that heretofore unrecognized hydrodynamic forces are alone responsible for the cell immobilization. Cells can be immobilized to high biomass densities, while the hydrodynamic properties of columns containing such immobilized cells are excellent. We describe an on-line electronic method for the real-time measurement of immobilized cellular biomass. Cell growth (so recorded) and metabolism continue to occur in such particles at high rates. Using the glycolytic production of ethanol by S. cerevisiae as a model reaction, volumetric productivities as great as any published are obtained. Thus the "lobster-pot effect" or "hydrodynamic deposition" represents a novel, promising, and generally applicable method of cell immobilization.

Biotechnology↗
PubMed1987

The roles of osmotic stress and water activity in the inhibition of the growth, glycolysis and glucose phosphotransferase system of Clostridium pasteurianum.

Growth of Clostridium pasteurianum was inhibited in media of high solute content. At equal osmolarities, 'permeant' solutes (glycerol and acetamide) were much less growth-inhibitory than 'non-permeant' solutes (KC1 and xylitol). Glycolysis by washed cell suspensions was inhibited by these solutes in parallel with growth. However, in their inhibition of glucose 6-phosphate dissimilation by permeabilized cells the distinction between 'permeant' and 'impermeant' solutes was significantly less marked. The glucose phosphotransferase system (PTS) of intact cells was much more strongly inhibited by 'non-permeant' than by 'permeant' solutes. It was concluded that the predominant inhibitory effects on this organism of media of high solute content are due not to the low water activity of such media per se, but to the creation of an osmotic pressure across the bacterial cytoplasmic membrane, which acts to inhibit the glucose PTS by which the organism effects glucose uptake. Parallel measurements of the effects of xylitol on both glycolysis and the activity of the glucose PTS suggested that despite this correlation between the osmotic inhibition of growth, glycolysis and the PTS, the flux-control coefficient of the PTS on glycolysis did not exceed 0.2 under the conditions used.

Acetamides↗
PubMed1987

The growth and nutrition of Clostridium sporogenes NCIB 8053 in defined media.

Various defined and minimal media are described for the growth of Clostridium sporogenes NCIB 8053. The organism requires 10 amino acids and one vitamin for growth, whilst three other vitamins are growth stimulatory. L-alpha-hydroxy acid analogues can replace eight, and fatty acid analogues four, of these amino acids. The organism may generate free energy by a variety of Stickland reactions. Most Stickland acceptors, but not glycine, stimulate the growth of this organism on glucose. Nonetheless, cells grown in the presence of glycine will reductively deaminate it. The media described support the growth of several other strains of this species. The simplified growth media which we have developed permit quantitative studies of the physiology of this organism.

Acetates↗