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Biomedical subjects

R B Kemp

Publications and source records attributed to R B Kemp.

At least 19 recordsLinked to original sources
PubMed2006

Pycnometric, viscometric and calorimetric studies of the process to release the double-stranded DNA from the Un bacteriophage.

Knowledge of both the packaging of the linear, double-stranded (ds)DNA in bacteriophages and its subsequent release into the bacterial host is vital to our understanding of phage infection. There is now strong evidence that packaging requires a powerful rotary motor fuelled by ATP. From thermodynamic studies, however, it has been proposed that, at least for those viruses with a contractile tail, the dsDNA ejection from the phage head is a relatively simple physical process that does not require cellular energy and is facilitated by the difference in the conditions of the medium in the environments inside and outside the head. In this case, there should be no enthalpic effects associated with the dehiscence of the capsid and no destruction of it or the other structural elements of the phage. For the present study of temperature-induced phage dehiscence, we used a newly discovered phage with a contractile tail, named the Un (unknown) bacteriophage. Evidence is given of its characteristics in terms of ultrastructural morphology, serological parameters, host range and interaction with host cell. These show that, although it has similarities with the T-even phages and, in particular, the DDVI phage, it appears to be a new type. Earlier viscometric studies with it had shown that the temperature-induced release of the capsid dsDNA was completed at 70 degrees C. In the present investigation, a concentrated suspension of purified phage was subjected to pycnometric analysis through the temperature range of 30 to 70 degrees C. This showed that a significant and abrupt increase in the phage partial volume takes place, which remarkably is in the order of threefold. Viscometric measurements over time at 72 degrees C gave a kinetic curve from which evidence it was suggested that the temperature-induced DNA release is similar to a second order phase transition. At the same time, data from differential scanning calorimetry over the same temperature range showed no enthalpic effect. Our results indicate that the ejection of DNA from the capsid tail is driven by an entropy change.

Bacteriophages↗
PubMed1999

Detection of the changing substrate requirements of cultured animal cells by stoichiometric growth equations validated by enthalpy balances.

As part of an overall aim to base the feeding of substrates to cultured animal cells on their actual metabolic needs, we have developed a stoichiometric approach centred on the macronutrients in the medium. Heat flux records the overall metabolic activity and therefore was the sensitive indicator of changing metabolic requirement. Analyses were made of the experimental measurements on two engineered cell lines in batch culture, the 2C11-12 macrophage hybridoma cell capable of the respiratory burst and the CHO320 constitutively producing human interferon-gamma. The crux was to construct simplified stoichiometric equations for the growth reactions to represent metabolic activity as it changed with time. Beforehand, it was essential to select the appropriate components for the equations. The choice was then justified by constructing enthalpy balances in which the ratio of heat flux to enthalpy flux must be close to unity for validation. By combining the stoichiometric approach with heat flow measurements, it was shown both theoretically and experimentally that the set of stoichiometric coefficients constituting a validated growth equation has a one-to-one corresponding relationship to the metabolic activity of the average cell population. Thus, a strategy was established for feeding the cells at any one time with the correct ratio of the major substrates, glucose and glutamine, in response to metabolic requirements that change with time.

Animals↗
PubMed1998

Specific heat flow rate: an on-line monitor and potential control variable of specific metabolic rate in animal cell culture that combines microcalorimetry with dielectric spectroscopy.

One of the requirements for enhanced productivity by the animal culture systems used in biotechnology is the direct assessment of the metabolic rate by on-line biosensors. Based on the fact that cell growth is associated with an enthalpy change, it is shown that the specific heat flow rate is stoichiometrically related to the net specific rates of substrates, products, and indeed to specific growth rate, and therefore a direct reflection of metabolic rate. Heat flow rate measured by conduction calorimetry has a technical advantage over estimates for many material flows which require assays at a minimum of two discrete times to give the rate. In order to make heat flow rate specific to the amount of the living cellular system, it would be advantageous to divide it by viable biomass. This requirement has been fulfilled by combining a continuous flow microcalorimeter ex situ with a dielectric spectroscope in situ, the latter measuring the viable cell mass volume fraction. The quality of the resulting biosensor for specific heat flow rate was illustrated using batch cultures of Chinese hamster ovary cells (CHO 320) producing recombinant human interferon-gamma (IFN-gamma) during growth in a stirred tank bioreactor under fully aerobic conditions. The measuring scatter of the probe was decreased significantly by applying the moving average technique to the two participant signals. It was demonstrated that the total metabolic rate of the cells, as indicated by the specific heat flow rate sensor, decreased with increasing time in batch culture, coincident with the decline in the two major substrates, glucose and glutamine, and the accumulation of the by-products, ammonia and lactate. Furthermore, the specific heat flow rate was an earlier indicator of substrate depletion than the flow rate alone. The calorimetric-respirometric ratio showed the intensive participation of anaerobic processes during growth and the related IFN-gamma production. Specific heat flow rate was monotonically related to specific cell growth rate and associated with specific IFN-gamma production. Specific heat flow rate is potentially a valid control variable for the growth of genetically engineered cell lines producing target proteins.

Animals↗
PubMed1990

Anaerobic metabolism in aerobic mammalian cells: information from the ratio of calorimetric heat flux and respirometric oxygen flux.

Calorimetric and respirometric studies of cultured cells show that both neoplastic and non-neoplastic cell types maintain an anaerobic contribution to their total heat flux. In many mammalian cells this can be explained quantitatively by lactate production observed under fully aerobic conditions. Uncoupling and enhanced futile substrate cycling increase the ratio of heat flux to oxygen flux, the calorimetric-respirometric (CR) ratio. The interpretation of calorimetric and respirometric measurements requires an energy balance approach in which experimentally measured CR ratios are compared with thermochemically derived oxycaloric equivalents. The oxycaloric equivalent is the enthalpy change per mole of oxygen consumed, and equals -470 kJ/mol O2 in the aerobic catabolism of glucose, assuming that catabolism is 100% dissipative (the net efficiency of metabolic heat transformation is zero). CR ratios more negative than -470 kJ/mol O2 have been reported in well-oxygenated cell cultures and are discussed in terms of integrated aerobic and anaerobic metabolism.

Aerobiosis↗
PubMed1989

Changes in the functions of the intestinal brush border membrane during the development of the ruminant habit in lambs.

1. Brush border membrane vesicles were prepared from lamb enterocytes. These were used to study the changes in the enzyme contents and the transport capacities which occur during the change from a milk to a roughage diet. 2. Na+-dependent transport of D-glucose was present in all regions of the small intestine of pre-ruminant lambs and absent in ruminants. 3. Na+-dependent transport of L-proline was present in all regions of the small intestine irrespective of the age of the animal. 4. Phosphate transport was seen only in the presence of a transmembrane pH gradient (acid outside). The transport was not stimulated by either Na+ or K+. The transport capacity increases 2-fold as the animal becomes ruminant. 5. The activities of lactase and maltase diminished with age. Alkaline phosphatase and aminopeptidase N activities remain constant. Sucrase activity cannot be detected in lambs of any age.

Alkaline Phosphatase↗
PubMed1988

Comparison of cell death and adenosine triphosphate content as indicators of acute toxicity in vitro.

1. Anchorage-independent LS cells, derived from L929 mouse fibroblasts, were used as an in vitro alternative to animals for the assessment of acute toxicity. The two end points were cell death, indicated by fluorescein diacetate and ethidium bromide, and intracellular adenosine triphosphate (ATP) content. 2. Concentrations of 20 test compounds which produced a 50% decrease in the ATP contents of control cells (ATP50) ranged from 17 micrograms ml-1 for diethylstilboestrol to 7.0 mg ml-1 for sodium chloride. 3. The concentrations which caused 50% cell death ranged from 16 micrograms ml-1 for diethylstilboestrol to 8.0 mg ml-1 for paracetamol. 4. There was a good numerical correlation (r = 0.99) between the ranks of ATP50 and CD50 end-points, there being only minor changes in order between the ranks. 5. The slopes of the dose-response plots for individual chemicals were markedly different.

Acetaminophen↗
PubMed1985

Toxicity of commercial products on cells in suspension culture: a possible screen for the Draize eye irritation test.

Incubation of LS cells in suspension for 4 hr in the presence of water-soluble commercial products with known Draize rabbit-eye irritation potential has shown that viability, as indicated by fluorescent dyes, is a credible screen for irritation. For product formulations containing immiscible and/or insoluble substances, there are difficulties that have been reduced by extraction of the soluble components of the formulations into the culture medium. A more sensitive and automated, though more expensive, technique based on the estimation of intracellular ATP levels has been developed and the ATP50 values (concentration that reduces intracellular ATP by 50%) for several substances indicate that this may be another suitable endpoint in cytotoxicity screening for animal irritation studies.

Adenosine Triphosphate↗
PubMed1985

Estimation of heat production by cultured cells in suspension using semi-automated flow microcalorimetry.

Modifications to a heat conduction flow microcalorimeter are described which allow registration of heat production by cells cultured in suspension. LS cells produced 34 +/- 3 pW per cell. Over an 8.5 h period, cell numbers increased by 9% and heat production per cell by 18%. Oxygen consumption per cell was 0.244 +/- 0.02 mumol min-1 per 10(8) cells and the enthalpy change was -836 kJ/mol O2. An automated pumping system allowed sequential registration of heat production by untreated cells and those exposed to a metabolic inhibitor. The results showed that 0.1 mM 2,4-dinitrophenol caused a greater increase in power (+65% at 1.5 h) than in oxygen consumption (+36%). The opposite occurred in the case of cells treated with 1 mM potassium cyanide, heat dissipation being depressed (-48%) slightly less than oxygen uptake (-52%). The results illustrate the potential of careful calorimetric determinations in studying metabolic events in the growth and division of cells in culture.

2,4-Dinitrophenol↗
PubMed1983

A rapid cell culture technique for assessing the toxicity of detergent-based products in vitro as a possible screen for eye irritancy in vivo.

It is generally accepted that the Draize rabbit eye irritation test has several disadvantages and inadequacies when used as an indicator for potential irritancy of detergent-based commercial products. In the search for a possible in vitro screen, it was decided to use mouse LS cells in suspension, taking 50% cell death (CD50) after exposure to the product for 4 h as the endpoint. This result for eleven samples was compared with rabbit eye irritation data ranked on an arbitrary classification of mild, moderate and severe. All samples with a CD50 less than 0.5 mg ml-1 were severe eye irritants while, with one exception, all those with a CD50 greater than 1.0 mg ml-1 were mild irritants. Samples between these two cytotoxicity levels were, in general, moderately irritant to the rabbit eye. It would appear that the present in vitro test is a possible screen for the irritancy of detergent-based products.

Animals↗
PubMed1978

The similarity of the two high-molecular-weight polypeptides of erythrocyte spectrin.

1. The two major polypeptides (P1 and P2) of erythrocyte-membrane spectrin were isolated by preparative polyacrylamide-gel electrophoresis. 2. The two polypeptides were shown to possess similar amino acid compositions, both with the characteristically high glutamate and leucine contents of the parent spectrin. 3. The tryptic-peptide 'maps' of the two polypeptides were prepared by a combination of t.l.c. and electrophoresis. 4. Radioactive peptides were prepared by [14C]carboxymethylation and chloramine-T-catalysed [125I]iodination. 5. 'Maps' of both sets of peptides demonstrate a marked similarity between the two polypeptides. 6. These new data confirm earlier evidence for the similarity of the two chains. 7. The number of peptides in the 'maps' of carboxymethylated peptides suggest that polypeptides P1 and P2 are not aggregates.

Amino Acids↗
PubMed1976

Specific binding of anti-myosin and -actin gamma-globulins to the surface of trypsin-dissociated embryonic chick cells.

125I-labelled sheep anti-rabbit gamma-globulin antibodies were used to locate rabbit antibodies to smooth- and striated-muscle actomyosins at the surface of trypsin-dissociated embryonic chick cells. Statistical analysis of electron microscope autoradiographs revealed that the plasma membrane of these cells was significantly labelled with both antibodies. Further tests revealed that there were a significantly greater number of antigenic sites present on the cell surface for the gizzard smooth-muscle antibodies than for those against pectoralis straited-muscle actomyosin. It was further shown that both the rate and extent of binding of the 125I-labelled smooth-muscle actomyosin antibodies to the cells were greater than for anti-straited-muscle gamma-globulins. Binding of the former was reduced to a level similar to that of 125I-NIS conjugate by preincubation of the gamma-globulins with smooth-muscle heavy meromyosin, while a simiar reduction was observed when anti-pectoralis actomyosin was treated with actin. It was concluded that actin- and myosin-like proteins must now be considered as integral components of the plasma membrane.

Actins↗
PubMed1976

Anti-actomyosin. Influence on adhesive behaviour of eukaryotic cells and of Cuvierian tubules.

The effect of antisera against chicken gizzard smooth-muscle actomyosin and against pectoralis striated-muscle actomyosin on adhesive behaviour of eukaryotic cells (from sea urchin embryos and from a silicious sponge) and of Cuvierian tubules has been studied. The results with sea urchin cells, which require divalent cations for aggregation, showed that antiserum to chicken gizzard smooth-muscle actomyosin inhibited reaggreagation of trypsin-treated cells better than mechanically dissociated cells, while anti-chicken pectoralis striated-muscle had no effect. Primary reaggreagation of trypsin-dissociated sponge cells, in the presence of calcium and magnesium, is also inhibitable by anti-gizzard smooth-muscle but not by anti-pectoralis straited muscle. Anti-gizzard smooth-muscle had no effect on secondary reaggregation of sponge cells mediated by a soluble aggregation factor. Anti-gizzard smooth-muscle inhibited Cuvierian tubule adhesion.

Actomyosin↗
PubMed1975

(Ca-2+ + Mg-2+)-Activated ATPase in the plasma membrane mouse liver cells.

1. Purified plasma membranes from dissociated adult mouse liver cells posses a (Ca-2+ + Mg-2+)-stimulated ATPase (EC 3.6.1.3) activity. 2. Enzyme activity is at a maximum with the addition of 0.3 mM Ca-2+ and 3 mM Mg-2+. 3. Using medium devoid of alkali metal ions (Ca-2+ + Mg-2+)-ATPase enzyme activity was observed with Km1 = 0.35 - 10-3 M at a substrate concentration of 1 mM or less and an apparent Km2 = 0.88 - 10-3 M at higher substrate concentrations. 4. In the presence of Na+ and 4 mM ATP, an increase in activity was seen, suggesting the presence of a (Ca-2+ + Mg-2+ + Na+)-activated ATPase. 5. In the presence of both Na+ and K+ the (Ca-2+ + Mg-2+)-dependent enzyme activity was further increased, indicating that a (Ca-2+ +Mg-2+ + K+)-stimulated ATPase may also be present.

Adenosine Triphosphate↗