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Biomedical subjects

C A Miles

Publications and source records attributed to C A Miles.

At least 19 recordsLinked to original sources
PubMed2001

Thermal stability of collagen fibers in ethylene glycol.

The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.

Animals↗
PubMed2001

Thermally labile domains in the collagen molecule.

We have proposed that the denaturation kinetics of the characteristic sharp melting point of the collagen molecules is an irreversible rate rather than an equilibrium process as previously believed. This leads to the concept of domains of variable thermal stability along the length of the molecule. We have identified the major thermally labile domains from which the denaturation process is initiated as hydroxyproline deficient sequences of 65, 65 and 59 residues near the carboxy terminus in fibrillar collagen types I, II and III, respectively. These domains differ in that there is a single hydroxyproline in the type II domain and two hydroxyprolines in the type III domain. Similar sized domains are conserved in these collagen types across species including amphibians and invertebrates. The effective size of the domain is reduced in the fibrillar aggregates to 26 residues due to the interaction with adjacent molecules and because of the precise quarter-staggered alignment of the molecules the domains are located in the gap region. This spatial confinement within the lattice of the fibre leads to the significant increase in denaturation temperature of the fibre compared to the molecule. These labile domains have also been located in molecules that form the non-fibrillar type IV basement membrane collagens and the fibril-associated aggregates such as type IX. Based on the location of the different domains in type IX we have proposed a different arrangement of the type IX on the type II fibril. The model stresses the importance of hydroxyproline in stabilising the triple helix and supports the concept of hydrogen-bonded water-bridges originally proposed from X-ray diffraction studies in contrast to other studies indicating water-bridges do not play a role in stabilising the collagen molecule.

Amino Acid Sequence↗
PubMed2000

Identification of an intermediate state in the helix-coil degradation of collagen by ultraviolet light.

Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 degrees C following UV irradiation of collagen, compared with 39 degrees C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.

Animals↗
PubMed2000

Isinglass/collagen: denaturation and functionality.

Isinglass is widely used commercially to clarify alcoholic beverages by aggregation of the yeast and other insoluble particles. It is derived from swim bladders of tropical fish by solubilisation in organic acids and consists predominantly of the protein collagen. The low content of intermolecular cross-links allows ready dissolution of swim bladder compared to bovine hide which is cross-linked by a high proportion of stable bonds and requires enzymic digestion to solubilise. Isinglass is no longer effective as a clarifying agent if thermally denatured hence the collagenous triple helical structure must be maintained. Thermal denaturation of isinglass occurs at 29 degrees C, compared to 40-41 degrees C for mammalian collagens, primarily due to the lower hydroxyproline content. The hydroxyproline is essential for the formation of H-bonded water-bridges through the hydroxyl group and the peptide chain thereby stabilising the triple helix. Based on the lower enthalpy determined by differential scanning calorimetry we have calculated that the thermally labile domain of the isinglass molecule was 41 residues compared to 66 for mammalian collagen. The fining efficiency was unaffected by pH, chelating agents, detergents and removal of surface proteins from yeast cells. Studies on the mechanism of action of isinglass have shown that higher molecular weight aggregates that increase the length of the collagen molecules (trimers, tetramers, etc.) increase efficiency and that their surface charge are important in the clarification process. By chemical modification, we have shown that blocking positively charged groups had no effect on the fining process, whilst negative charges are clearly essential and that increasing the negative charge by succinylation increases its efficacy. Solutions of bovine hide collagen were shown to be equally effective in refining beers and standard yeast preparations. The higher thermal denaturation temperature, ready availability and reproducibility of bovine collagen preparations gives it considerable advantages over isinglass.

Air Sacs↗
PubMed1999

Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers.

Collagen molecules in solution unfold close to the maximum body temperature of the species of animal from which the molecules are extracted. It is therefore vital that collagen is stabilized during fiber formation. In this paper, our concept that the collagen molecule is thermally stabilized by loss of configurational entropy of the molecule in the fiber lattice, is refined by examining the process theoretically. Combining an equation for the entropy of a polymer-in-a-box with our previously published rate theory analysis of collagen denaturation, we have derived a hyperbolic relationship between the denaturation temperature, Tm, and the volume fraction, epsilon, of water in the fiber. DSC data were consistent with the model for water volume fractions greater than 0.2. At a water volume fraction of about 0.2, there was an abrupt change in the slope of the linear relationship between 1/Tm and epsilon. This may have been caused by a collapse of the gap-overlap fiber structure at low hydrations. At more than 6 moles water per tripeptide, the enthalpy of denaturation on a dry tendon basis was independent of hydration at 58.55 +/- 0.59 J g-1. Between about 6 and 1 moles water per tripeptide, dehydration caused a substantial loss of enthalpy of denaturation, caused by a loss of water bridges from the hydration network surrounding the triple helix. At very low hydrations (less than 1 mole of water per tripeptide), where there was not enough water to form bridges and only sufficient to hydrogen bond to primary binding sites on the peptide chains, the enthalpy was approximately constant at 11.6 +/- 0.69 J g-1. This was assigned mainly to the breaking of the direct hydrogen bonds between the alpha chains.

Animals↗
PubMed1998

Differences between the thermal stabilities of the three triple-helical domains of type IX collagen.

Fibre-forming collagens in dilute solution show highly co-operative helix-coil transitions at temperatures that are remarkably close to the body temperature of the animal from which the collagen was extracted. This close correlation holds across animal Phyla and the transition temperatures, which range from 5 degrees C to 40 degrees C, are adjusted to suit by changing the primary structure, especially the concentration of the water-bridge-enhancing hydroxyproline residue. Fibril-forming collagens are thermally stabilised by fibrillogenesis, which causes a loss of random coil configurational entropy by intermolecular and intramolecular cross-linking and by spacial confinement of the molecule within the lattice of the fibre. But this mechanism cannot apply to the full length of the type IX collagen molecule, since its COL3 arm, according to current models, projects out from the stabilising influence of the type II fibre. In this paper we examine the thermal stability of the type IX collagen molecule and its three triple-helical domains, thereby demonstrating that the COL3 arm is much more stable than the rest of the molecule. At a scanning rate of 60 deg. C/h COL3 exhibited an unfolding endotherm with a tmax at 49.0 degrees C, well above body temperature. Corresponding peak maxima for COL1 and COL2 were seen at 40.6 degrees C and 39.6 degrees C, respectively. The sizes of the thermally labile units of COL1, COL2 and COL3, calculated from the measured activation enthalpies, were 24, 28 and 28 residues, respectively, much smaller than type I (65 residues) because of the relatively short lengths of triple helix to be unfolded. However, unlike type I collagen, no regions of the required size were found completely devoid of hydroxyproline. Consequently, the intrinsic stabilities of these thermally labile units were higher than that of type I with DeltaH updownarrow DeltaS updownarrow for COL1, COL2 and COL3 being, respectively, 385 K, 371 K and 384 K, contrasting with the much lower 349 K of type I collagen. We therefore speculate that the increased thermal stability of the thermally labile units was caused by the presence of the water-bridge-enhancing residue, hydroxyproline. Finally the stabilisation of type IX collagen tissue is considered and an alternative structural organisation of the type IX molecule on the type II fibre is proposed.

Animals↗
PubMed1998

Survival and biological activity of heat damaged DNA.

The thermal degradation of plasmid pUC18 held at temperatures between 100 and 135 degrees C was examined by measuring the ability of heat-treated plasmid preparations to transform Escherichia coli to ampicillin resistance using electroporation. Substantial protection against loss of transforming ability during heating was provided by concentrations of NaCl between 0.25 and 2.0 mol l-1. For example, the addition of 1.0 mol l-1 NaCl to samples heated at 100 degrees C for 15 min increased transformation frequency about 200-fold compared with samples heated without NaCl. In the presence of 0.5-2.0 mol l-1 NaCl, transforming capacity was not destroyed even by heating at 121 degrees C for 15 min, i.e. after a typical sterilization treatment. These findings may have implications for the safe disposal of genetically modified micro-organisms and recombinant DNA preparations.

Ampicillin↗
PubMed1996

Factors affecting the ultrasonic properties of equine digital flexor tendons.

The velocity, attenuation and apparent backscattering coefficient of 6-11-MHz ultrasound were measured in three orthogonal directions in equine deep digital flexor (DDF) and superficial digital flexor (SDF) tendons at 0 degree C. Ultrasonic measurements were examined for correlation with tendon water, collagen, DNA and glycosaminoglycans contents, determined by chemical analyses and with structure observed by scanning electron microscopy. The SDF tendon contained more water, more DNA (i.e., more cells), less collagen and less glycosaminoglycans and exhibited lower velocities and attenuations than the DDF tendon. Velocities were governed primarily by the adiabatic bulk modulus and density, perturbed by a highly direction-dependent rigidity. Ultrasound propagating across tendon generated frequency-independent backscattering which appeared to derive from the large interfaces between the fascicles, while along the fibres backscattering varied as f3.62 +/- 0.88 and appeared to derive from small structures such as collagen fibres. The mechanisms by which ultrasound is attenuated by tendon remain unknown.

Animals↗
PubMed1996

Ultrasonic properties of tendon: velocity, attenuation, and backscattering in equine digital flexor tendons.

Ultrasound velocity, attenuation, and backscattering were measured in vitro in samples of equine digital flexor tendon sandwiched between plane, parallel rexolite buffer rods. The buffer rods were coupled to transmitting and receiving transducers (nominally 10 MHz) mounted in-line and facing one another on the jaws of a digital caliper. Six superficial digital flexor (SDF) tendons and six deep digital flexor (DDF) tendons were measured in three orthogonal directions: along the long axis of the tendon (D), and across the tendon in the dorsal-volar (C), and lateral (L) directions. Substantial anisotropy was apparent in all the measured properties. The velocity data, which in both tendons showed a higher velocity along the fibers than across (e.g., in the DDF tendon at 0 degrees C: 1713 +/- 9 m/s in the D direction compared with 1650 +/- 5 m/s in the C direction), were consistent with a composite comprising stiff fibers embedded in a less stiff medium of lower speed. The apparent backscattering coefficient adjusted for the tissue's frequency-dependent attenuation (e.g., in the C direction of the DDF tendon at 0 degrees C: 7.4 x 10(-3) cm-1 sr-1), was independent of frequency in both transverse directions and larger than that measured along the long axis of the tendon (e.g., in DDF tendon at 0 degrees C: 1.2 x 10(-3) cm-1 sr-1 at 7 MHz) in which direction the apparent backscattering coefficient increased with frequency as f4.0 +/- 1.2. The frequency-independent backscattering was thought to be due to specular reflection from the boundaries between the fascicles, i.e., the bundles of fibers making up the tendon, while backscattering along the axis was due to structures of unknown origin, but of a size much smaller than 45 microns. Attenuation of ultrasound directed along the fibers was higher than that across (at 7 MHz in DDF tendon at 0 degrees C: 58 dB/cm in the D direction compared with 11.3 dB/cm in the C direction). Calculations indicated that the attenuation was primarily caused by absorption rather than scattering.

Animals↗
PubMed1995

The kinetics of the thermal denaturation of collagen in unrestrained rat tail tendon determined by differential scanning calorimetry.

This paper shows that the position and shape of the denaturation endothem of collagen fibrils are governed by the kinetics of an irreversible rate process. This was proved by measuring the rate of denaturation in rat tail tendons held isothermally at different temperatures, thereby determining rate constant characteristics such as the activation enthalpy and entropy and predicting endotherm position and shape therefrom. Comparison with actual scanning results showed good correspondence. Isothermal measurements of the rate of collagen denaturation, measured continuously using a calorimetric method, were used to determine rate constants for collagen denaturation in tendons immersed in water and 0.5 M acetic acid. The temperature dependence of the rate constants were fitted to the three rate process models, previously examined theoretically: the D and z formulation, the Arrhenius equation and the absolute rate theory. For example, in water the activation enthalpy was 0.518 (+/- 0.016) Mj mol-1 and the activation entropy 1.485 (+/- 0.049) kj mol-1 K-1, while in acetic acid the corresponding figures were 1.306 (+/- 0.099) Mj mol-1 and 4.142 (+/- 0.323) kj mol-1 K-1. These characteristics are discussed in terms of the thermal activation of a region of the molecule, the co-operative unit. The ratio of the activation enthalpy to the calorimetry enthalpy of denaturation indicated a co-operative unit that was 66 (+/- 5) residues long when fibrils were swollen in acetic and the collagen molecules acted essentially independently. On the other hand the intact fibrils in water gave a co-operative unit of 26 (+/- 1) residues long. The reason for the reduction in size of the co-operative unit is that it is surrounded, and therefore stabilized by other molecules in the fibre. It is interesting to note that the suggested co-operative unit lies almost entirely within the "gap" zone of the collagen fibril in its quarter-staggered arrangement of molecules. We believe that the co-operative unit would be represented by a domain that is free of stabilising hydroxyproline residues. Indeed such a domain exists near the C terminus of the triple helix from Gly877 to Pro941, i.e. 65 residues. In acetic acid, activation is similar to that of collagen molecules in solution. All the inter alpha-chain hydrogen bonds in the co-operative unit are broken and the separate chains in this short region are free to flail around under the action of thermal collisions relatively unimpeded by intermolecular interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

A mathematical analysis of microbial inactivation at linearly rising temperatures: calculation of the temperature rise needed to kill Listeria monocytogenes in different foods and methods for dynamic measurements of D and z values.

From a theoretical analysis of the inactivation of microbes heated at linearly rising temperatures an equation was derived for predicting the linear temperature rise needed to reduce viable numbers of microbes by any chosen factor. This equation is used to predict the temperatures needed to inactivate Listeria monocytogenes in different foods based on published D and z values. Two novel mathematical methods for deriving D and z values from viable counts obtained at linearly rising temperatures are also presented.

Animals↗
PubMed1994

Differential scanning calorimetric studies of superficial digital flexor tendon degeneration in the horse.

Differential scanning calorimetry (DSC) of equine superficial digital flexor tendons revealed the presence of a small exothermic peak at 23 degrees C of unknown origin, and a large endothermic peak at 70 degrees C due to denaturation of cross-linked collagen fibres. In the central degenerated core of damaged tendons the denaturation temperature remained at 70 degrees C but the enthalpy decreased in relation to the extent of degeneration of the tendon. We suggest that this reduction in enthalpy is due to depolymerisation and denaturation of the collagen fibres. This contention is supported by the observed increased activity of the degradative enzyme cathepsin B secreted by the fibroblasts. DSC analysis of cultured porcine tendon fibroblasts revealed a multicomponent endotherm, denaturation beginning at 46 degrees C, a temperature capable of being achieved within the tendon during intensive exercise. DSC clearly has considerable potential in complementing morphological and biochemical studies to determine the aetiology and progress of equine tendon degeneration.

Animals↗
PubMed1993

Chemistry of collagen cross-links: glucose-mediated covalent cross-linking of type-IV collagen in lens capsules.

The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or 'strength') was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.

Animals↗
PubMed1993

Kinetics of collagen denaturation in mammalian lens capsules studied by differential scanning calorimetry.

Calorimetry was used to measure the rates of irreversible denaturation of collagen in intact mammalian lens capsules, isothermally stored in water at different temperatures, and the kinetic characteristics of the process determined. At 47.4 degrees C, the number of native collagen molecules declined by a factor of 10 in 21.4 +/- 2.9 min (this is called the D-value) and increasing the storage temperature by 2.32 +/- 0.12 degrees C reduced the D-value by a factor of 10. The activation energy of the process was 860 +/- 44 kJ mol-1, and the activation enthalpy and entropy were respectively 858 +/- 45 kJ mol-1 and 2.38 +/- 0.14 kJ mol-1 K-1. Using equations derived in the paper, these kinetic characteristics were used to predict the position and shape of the denaturation endotherms over a range of scanning rates. Comparison of the predicted and actual positions of peak maxima showed a close correspondence, demonstrating that the principal phenomenon determining the position of the peak maxima was an irreversible rate process. While there was fairly good agreement between the predicted and actual shape of the endotherm at low scanning rates, measurements at high rates were distorted by the finite response time of the calorimeter, and the agreement was poor.

Animals↗
PubMed1991

Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry.

Thermograms of whole cells of Escherichia coli obtained by differential scanning calorimetry contained ten main peaks (denoted f, l, m1, m2, m3, n, p, q, r and s) occurring at temperatures of approximately 25, 54, 61, 71, 76, 81, 95, 105, 118 and 124 degrees C, respectively. After cooling to 5 degrees C and reheating, peaks denoted fr, mr and pr were observed at 23, 73 and 94 degrees C, respectively. By examining thermograms of different cell fractions we have identified the following thermal denaturation events. During primary heating there is a broad endotherm (f) beginning below 20 degrees C and extending to just above 40 degrees C that is caused by melting of membrane lipids. Superimposed on this is an exothermic process associated with a change of state of the peptidoglycan. The first irreversible denaturation event occurs just above 47 degrees C, associated with the onset of denaturation of the 30S ribosomal subunit and soluble cytoplasmic proteins. Ribosome melting is a complex process occurring between 47 and 85 degrees C and is characterized by peaks m1, m2 and n. Peak m3 at 75-76 degrees C is of unknown identity but may possibly represent melting of tRNA. Peak p at 95 degrees C results from melting of a portion of the cellular DNA combined with denaturation of a cell wall component. Peak q at 105 degrees C is multicomponent and may be caused by melting of a different region of DNA together with denaturation of another cell wall component. The complex events denoted r and s at 118 and 125 degrees C, respectively, are associated with denaturation of a component of the cell envelope, and possibly also of DNA. Following cooling and reheating there is a broad endotherm with a maximum at 23 degrees C caused by remelting of membrane lipid and a very broad endotherm extending between 40 and 100 degrees C caused by the remelting of ribosomal RNA. Peak pr at 94 degrees C is caused by the melting of reannealed DNA. Additional features not appearing in whole cells were evident in some cell fractions. These observations should allow us to distinguish events that may lead to loss of viability from those that do not.

Bacterial Proteins↗
PubMed1988

Attenuation of ultrasound in suspensions of bovine muscle myofibrils and myosin.

The attenuation of 1.5-7 MHz ultrasound was measured over the pH range 3-7 in 100 mM KCl suspensions of bovine M. semitendinosus myofibrils, precipitated myosin and the residue of myofibrils after partial extraction of myosin. In all fractions attenuation showed a similar dependence on pH over the range 3-7, with a broad, substantial maximum in the region of pH 4.5-pH 5.5 and similar mass attenuation coefficients (per g protein). At pH 7 and 7 MHz these were 3.49 +/- 0.20 cm2 g-1 in the myofibrils, 3.26 +/- 0.31 cm2 g-1 in the myofibrilar residue and 2.83 +/- 0.68 cm2 g-1 in the precipitated myosin. Measurements at 5.3 MHz of precipitated myosin over a wider pH range revealed an attenuation titration curve similar to that previously observed in homogenates of muscle and muscle myofibrils, with substantial peaks at about pH 5 and 11.5, and a shoulder perhaps indicating a small underlying peak at about pH 8-9. Myosin dissolved in 800 mM KCl gave attenuation levels that were typically 50% lower than precipitated myosin e.g. at pH 7 and 7 MHz: 2.83 +/- 0.68 cm2 g-1 in the precipitated form, 1.29 +/- 0.10 cm2 g-1 in solution. These results indicated that: (a) attenuation by myosin filaments contributed substantially to the total attenuation in suspensions of myofibrils and (b) the peak in the myofibrilar attenuation is caused, or substantially contributed to, by a process taking place in the myosin component.

Animals↗