Dynamics of structural parameters of mast cells and accumulation of collagen fibers during radiation-induced pneumosclerosis in rats (a quantitative study)

The methods of stereometry were used to study dynamics of accumulation of collagen fibres and alteration of the number, sizes and state of mast cells of rat lung during the period of 12 months after single local X-irradiation with doses of 10, 14.3 and 20 Gy. A statistically significant correlation between the number of mast cells and spatial density of collagen fibres in the exposed pulmonary tissue was shown. Severity of changes in the structural characteristics of mast cells and the degree of collagen fibres accumulation were a function of radiation dose.     

Formation of collagen type I fibrils in vitro

The assembly of collagen fibrils as a function of temperature and collagen concentration was studied. It was shown that temperature increases from 25 to 35 degrees C, the degree of ordering of collagen fibrils increases 1.5-fold at collagen concentration above 1 mg/ml and 2-fold at low collagen concentration. A maximum ordering of fibril structure occurs under conditions close to physiological (T approximately 35 degrees C and collagen concentration 1.2 mg/ml). As temperature is elevated from 30 to 35 degrees C, the packing of collagen molecules in fibrils becomes more ordered: the values of enthalpy and entropy of the transition of fibrils from the native to a disordered state decrease at all collagen concentrations used. At high collagen concentration, the dimensions of cooperative blocks in fibrils formed at 25 and 30 degrees C coincide with those of cooperative blocks of monomeric collagen in solution. Upon increasing the temperature to 35 degrees C, the dimensions of cooperative blocks increase.     

Temperature dependence of the gel electrophoretic mobility of superhelical DNA.

We have determined the gel electrophoretic behavior of closed circular plasmid pSM1 DNA (5420 bp) as a function of both temperature and of linking number (Lk). At temperatures below 37 degrees, the electrophoretic mobility first increases, then becomes constant as Lk is decreased below that of the relaxed closed DNA. As the temperature is increased above 37 degrees the electrophoretic mobility first increases as Lk decreases and then varies in a cyclic manner with further decreases in Lk. As the temperature is increased over the range 37 degrees - 65 degrees the cyclic behavior is manifested at progressively smaller decreases in Lk and the amplitude of the cycles increases. We interpret the results in terms of the early melting of superhelical DNA, in which the free energy associated with superhelix formation is progressively transferred to local denaturation. Using a two state approximation, we estimate the free energy change in the first cyclic transition to be 35 Kcal/mole DNA at 37 degrees and to decrease linearly with temperature. The free energy becomes equal to zero at a temperature of 71.6 degrees, which lies within 3 degrees of the melting temperature for the corresponding nicked circular DNA. From the slope of this relationship we estimate the apparent entropy and enthalpy of the first mobility transition to be 6.0 Kcal/mole base pair and 17.3 cal/mole base pair/degree, values consistent with duplex melting.     

The increase in denaturation temperature following cross-linking of collagen is caused by dehydration of the fibres

Differential scanning calorimetry (DSC) was used to study the thermal stability of native and synthetically cross-linked rat-tail tendon at different levels of hydration, and the results compared with native rat-tail tendon. Three cross-linking agents of different length between functional groups were used: malondialdehyde (MDA), glutaraldehyde and hexamethylene diisocyanate (HMDC). Each yielded the same linear relation between the reciprocal of the denaturation temperature in Kelvin, T(max), and the water volume fraction, epsilon (1/T(max)=0.000731epsilon+0.002451) up to a critical hydration level, the volume fraction of water in the fully hydrated fibre. Thereafter, water was in excess, T(max) was constant and the fibre remained unchanged, no matter how much excess water was added. This T(max) value and the corresponding intrafibrillar volume fraction of water were as follows: 84.1 degrees C and 0.48 for glutaraldehyde treated fibres, 74.1 degrees C and 0.59 for HMDC treated fibres, 69.3 degrees C and 0.64 for MDA treated fibres, and 65.1 degrees C and 0.69 for untreated native fibres. Borohydride reduction of the native enzymic aldimines did not increase the denaturation temperature of the fibres. As all samples yielded the same temperature at the same hydration, the temperature could not be affected by the nature of the cross-link other than through its effect on hydration. Cross-linking therefore caused dehydration of the fibres by drawing the collagen molecules closer together and it was the reduced hydration that caused the increased temperature stability. The cross-linking studied here only reduced the quantity of water between the molecules and did not affect the water in intimate contact with, or bound to, the molecule itself. The enthalpy of denaturation was therefore unaffected by cross-linking. Thus, the "polymer-in-a-box" mechanism of stabilization, previously proposed to explain the effect of dehydration on the thermal properties of native tendon, explained the new data also. In this mechanism, the configurational entropy of the unfolding molecule is reduced by its confinement in the fibre lattice, which shrinks on cross-linking.     

The role of the alpha2 chain in the stabilization of the collagen type I heterotrimer: a study of the type I homotrimer in oim mouse tissues

We have previously reported that the fragility of skin, tendon and bone from the oim mouse is related to a significant reduction in the intermolecular cross-linking. The oim mutation is unlikely to affect the efficacy of the lysyl oxidase, suggesting that the defect is in the molecule and fibre. We have therefore investigated the integrity of both the oim collagen molecules and the fibre by differential scanning calorimetry.The denaturation temperature of the oim molecule in solution and the fibre from tail tendon were found to be higher than the wild-type by 2.6deg.C and 1.9deg.C, respectively. With the loss of the alpha2 chain, the hydroxyproline content of the homotrimer is higher than the heterotrimer, which may account for the increase.There is a small decrease in the enthalpy of the oim fibres but it is not significant, suggesting that the amount of disorder of the triple-helical molecules and of the fibres is small and involves only a small part of the total bond energy holding the helical structure together. The difference in denaturation temperature of the skin collagen molecules (t(m)) and fibres (t(d)) is significantly lower for the oim tissues, 19.9deg.C against 23.1deg.C, indicating reduced molecular interactions and hence packing of the molecules in the fibre. Computation of the volume fraction of the water revealed that the interaxial separation of the oim fibres was indeed greater, increasing from 19.6A to 21.0A. This difference of 1.4A, equivalent to a C-C bond, would certainly decrease the ability of the telopeptide aldehyde to interact with the epsilon -amino group from an adjacent molecule and form a cross-link. We suggest, therefore, that the reduction of the cross-linking is due to increased water content of the fibre rather than a distortion of the molecular structure.The higher hydrophobicity of the alpha2 chain appears to play a role in the stabilisation of heterotrimeric type I collagen, possibly by increasing the hydrophobic interactions between the heterotrimeric molecules, thereby reducing the water content and increasing the binding of the molecules in the fibre.     

On the application of polyelectrolyte “limiting laws” to the helix-coil transition of DNA. I. Excess univalent cations

A general theory of polyelectrolyte solutions is here used to calculate the differences in Gibbs free energy, enthalpy, and entropy between the coil and helix forms of DNA at any temperature and salt concentration. The salt has univalent cations and is assumed present in excess over the base concentration. The results are restricted to sufficiently dilute solutions. It is shown that the salt concentrations effect is entirely entropic in origin. When applied to the melting temperature, the calculations yield a relation between the enthalpy difference at the melting temperature and the slope of the plot of melting temperature vs. the logarithm of the salt concentration. In accord with observation, both the Gibbs free energy difference at any fixed temperature and the melting temperature are predicted to be linear functions of the log of the salt concentration. However, the theory is not in quantitative agreement with enthalpy data. Data on various colligative and transport properties of both helix and coil forms are reviewed in the text and in Appendix B, and good agreement is found with theory for both forms. No attempt is made to explain why the theory is quantitative for these properties but not for heat measurements. Finally, in Appendix A, an approximate calculation is made of the free energy contributions due to ionic effects not associated with the salt concentration.     

Unloading-induced remodeling in the normal and hypertrophic left ventricle

To date, no study has assessed the degree of similarity between left ventricular (LV) reverse remodeling and atrophic remodeling. Stable LV hypertrophy was induced by creation of an arteriovenous fistula (AVF) in Lewis rats (32 days). LV unloading was induced by heterotopic transplantation of normal (NL-HT) and/or hypertrophic (AVF-HT) hearts (7 days). We compared indexes of remodeling in AVF, NL-HT, and AVF-HT groups with those of normal controls. LV unloading induced decreases in cardiomyocyte size in NL-HT and AVF-HT hearts. NL-HT and AVF-HT LV were both characterized by relative increases in collagen concentration that were largely a reflection of decreases in myocyte volume. NL-HT and AVF-HT LV were associated with similar increases in matrix metalloproteinase (MMP-2 and -9) zymographic activity, without change in the abundance of the tissue inhibitors of the MMPs. In contrast, AVF-HT, but not NL-HT, was associated with a dramatic increase in collagen cross-linking. Our findings suggest an overall similarity in the response of the normal and hypertrophic LV to surgical unloading. However, the dramatic increase in collagen cross-linking after just 1 wk of unloading suggests a potential difference in the dynamics of collagen metabolism between the two models. Further studies will be required to determine the precise molecular mechanisms responsible for these differences in extracellular matrix regulation. However, with respect to these and related issues, heterotopic transplantation of hypertrophied hearts will be a useful small animal model for defining mechanisms of myocyte-matrix interactions during decreased loading conditions.     

X-ray diffraction evidence of collagen molecular packing and cross-linking in fibrils of rat tendon observed by synchrotron radiation.

X-ray diffraction patterns of fibres from 90 day (mature) rat-tail tendons were investigated using synchrotron radiation. The specimens were kept isometric at their corresponding in vitro rest length, and effects of pH and ionic strength were studied during short X-ray exposures. The results indicate that fibrils, equilibrated in physiological Ringer prior to exposure, have segregated lateral regions of well ordered collagen molecular packing. Lowering the ionic strength or the pH to 4.0 causes an order/disorder transition during which the fibril crystallinity decreases. At pH 3.5 a dramatic increase in the lateral swelling was observed. This effect was absent for fibres pretreated with sodium borohydride. The results are interpreted on the basis of cross-linking phenomena whereby the aldimine cross-link seems to be a controlling component of the lateral packing arrangement of collagen molecules.     

Comparative study of the stability of poplar plastocyanin isoforms

The stability of the two isoforms of poplar plastocyanin (PCa and PCb) was studied with differential scanning calorimetry (DSC) technique. It was shown that the thermal unfolding of both isoforms is an irreversible process with two endothermic and one exothermic peaks. The melting temperature of PCb was found to be 1.3+/-0.2 K degrees higher than of PCa, which indicates that PCb is more stable. The enthalpy of unfolding was estimated from the heat capacity curves and was found to be significantly higher for PCb at salt concentration I=0.1 M. In addition, PCb unfolding enthalpy and melting temperature are much more sensitive to the changes in the salt concentration as found in the experiments done at different ionic strength. The experiments were complemented with numerical calculations. The salt effect on the stability was modeled using the X-ray structure of PCa and a homology modeled structure of PCb. It was found, in agreement with the experimental data, that the stability of PCb changes by 4.7 kJ more than PCa, as the salt concentration increases from zero to 0.1 M. Thus, the differences in only 12 amino acid positions between "a" and "b" isoforms result in a measurable difference in the folding enthalpy and a significant difference in the salt dependence. The optimization of the electrostatic energies of PCa and PCb were studied and it was shown that PCb is better electrostatically optimized.     

Calorimetric study of the complexes between polyuridylic acid and adenylic nucleotides.

Soluble complexes of poly (U) and adenylic nucleotides in NaCl solutions were studied by scanning microcalorimetry. The melting enthalpies, delta Hm, of poly (U) complexes with adenosine, 2',3' -cAMP, 2'(3')-AMP, 5-AMP, ADP, ATP in 1 M NaCl are 50.5; 45.0; 42.9; 28.6; 26.1 and 25.6 kJ/mole triplets, respectively. Delta Hm is independent of the complex melting temperature, Tm. The calorimetric enthalpies are considerably lower than the apparent delta Hv.H. obtained from Tm dependence on free monomer concentration. The enthalpy of complex formation in 1 M NaCl depends neither ob the number nor on the degree of ionization of the phosphate groups but is essentially determined by their 5' - or 2'(3')-position. In contrast to 2'(3')- AMP. 2 poly (U), delta Hm of 5'AMP. 2 poly (U) increases considerably at lowering Na+ concentration. The enthalpy of poly (U) double helix melting in 1 M NaCl is 8.8 kJ/mole pairs which is 2.5 times lower than that in MgCl2 solutions.     

Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy

Multiphoton microscopy of collagen hydrogels produces second harmonic generation (SHG) and two-photon fluorescence (TPF) images, which can be used to noninvasively study gel microstructure at depth (~1 mm). The microstructure is also a primary determinate of the mechanical properties of the gel; thus, we hypothesized that bulk optical properties (i.e., SHG and TPF) could be used to predict bulk mechanical properties of collagen hydrogels. We utilized polymerization temperature (4–37°C) and glutaraldehyde to manipulate collagen hydrogel fiber diameter, space-filling properties, and cross-link density. Multiphoton microscopy and scanning electron microscopy reveal that as polymerization temperature decreases (37–4°C) fiber diameter and pore size increase, whereas hydrogel storage modulus (G′, from 23 ± 3 Pa to 0.28 ± 0.16 Pa, respectively, mean ± SE) and mean SHG decrease (minimal change in TPF). In contrast, glutaraldehyde significantly increases the mean TPF signal (without impacting the SHG signal) and the storage modulus (16 ± 3.5 Pa before to 138 ± 40 Pa after cross-linking, mean ± SD). We conclude that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties. Thus, optical imaging may be a useful noninvasive tool to assess tissue mechanics.     

Thermodynamic characteristics of collagen fibrils reconstructed in vitro at different temperatures and concentrations

The results of a calorimetric study of type I collagen fibrillogenesis were analyzed. The dependence of the half-width of the temperature transition of a collagen solution on the concentration and temperature of collagen formation was studied. It was demonstrated that, by varying temperature and collagen concentration, one can regulate the density of packing and dimensions of cooperative fibril blocks. At temperatures below the physiological level (25 degrees C and 30 degrees C), and a relatively low concentration of collagen (0.3 mg/ml), fibrils with the lowest density of packing are formed. The degree of order does not change as the collagen concentration increases twofold but grows as the concentration increases fourfold. It was shown that, at the physiological temperature (35 degrees C), fibrils with a dense packing of molecules are formed at all collagen concentrations studied. The value of fibril formation enthalpy is minimal at a temperature of 35 degrees C, pH 7.2, an ionic strength of 0.17 M and a concentration of 1.2 mg/ml. Based on the results obtained, a conclusion was made that the packing density of fibrils formed at physiological temperature does not depend on collagen concentration over the concentration range of 0.3 - 1.2 mg/ml.     

Role of collagen content and cross-linking in large pulmonary arterial stiffening after chronic hypoxia

Chronic hypoxic pulmonary hypertension (HPH) is associated with large pulmonary artery (PA) stiffening, which is correlated with collagen accumulation. However, the mechanisms by which collagen contributes to PA stiffening remain largely unexplored. Moreover, HPH may alter mechanical properties other than stiffness, such as pulse damping capacity, which also affects ventricular workload but is rarely quantified. We hypothesized that collagen content and cross-linking differentially regulate the stiffness and damping capacity of large PAs during HPH progression. The hypothesis was tested with transgenic mice that synthesize collagen type I resistant to collagenase degradation (Col1a1(R/R)). These mice and littermate controls (Col1a1(+/+)) were exposed to hypoxia for 10 days; some were treated with β-aminopropionitrile (BAPN), which prevents new cross-link formation. Isolated PA dynamic mechanical tests were performed, and collagen content and cross-linking were measured. In Col1a1(+/+) mice, HPH increased both collagen content and cross-linking, and BAPN treatment prevented these increases. Similar trends were observed in Col1a1(R/R) mice except that collagen content further increased with BAPN treatment. Mechanical tests showed that in Col1a1(+/+) mice, HPH increased PA stiffness and damping capacity, and these increases were impeded by BAPN treatment. In Col1a1(R/R) mice, HPH led to a smaller but significant increase in PA stiffness and a decrease in damping capacity. These mechanical changes were not affected by BAPN treatment. Vessel-specific correlations for each strain showed that the stiffness and damping capacity were correlated with the total content rather than cross-linking of collagen. Our results suggest that collagen total content is critical to extralobar PA stiffening during HPH.     

Growth-related changes in the mechanical properties of collagen fascicles from rabbit patellar tendons

Growth-related changes in the mechanical properties of collagen fascicles (approximately 300 microm in diameter) were studied using patellar tendons obtained from skeletally immature 1 and 2 months old and matured 6 months old rabbits. Tensile properties were determined using a specially designed micro-tensile tester. In each age group, there were no significant differences in the properties among cross-sectional locations in the tendon. Tangent modulus and tensile strength significantly increased with age; the rates of their increases between 1 and 2 months were higher than those between 2 and 6 months. The tangent modulus and tensile strength were positively correlated with the body weight of animals. However, growth-related changes in the mechanical properties were different between collagen fascicles and bulk patellar tendons, which may be attributable to such non-collagenous components as ground substances and also to mechanical interactions between collagen fascicles.     

Maintenance of bactericidal activity of pectin stabilized aqueous solutions]

Maintenance of the bactericidal activity and organoleptic properties of 3% and 5% stabilized aqueous solutions of pectin (Pepidol PEG, 3% and Pepidol PEG, 5%) stored for prolonged periods and at various temperatures was studied. Pepidol PEG was shown to preserve its bactericidal activity and organoleptic properties for 28 months when stored at rather wide ranges of the environmental temperature. The drug did not lose its properties after freezing and subsequent melting. The results of the study allowed to increase the period of Pepidol PEG use up to 18 months and the storage temperature ranges up to -30 degrees to +30 degrees C.     

Cross-Linking in Collagen by Nonenzymatic Glycation Increases the Matrix Stiffness in Rabbit Achilles Tendon

Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174–180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendon. The glycation process was initiated by incubating the Achilles tendons (n = 6) in phosphate-buffered saline containing ribose, whereas control tendons (n = 6) were incubated in phosphate-buffered saline without ribose. Eight weeks following glycation, the biomechanical attributes as well as the degree of collagen cross-linking were determined to examine the potential associations between matrix stiffness and molecular properties of collagen. Compared to nonglycated tendons, the glycated tendons showed increased maximum load, stress, strain, Young''s modulus of elasticity, and toughness indicating that glycation increases the matrix stiffness in the tendons. Glycation of tendons resulted in a considerable decrease in soluble collagen content and a significant increase in insoluble collagen and pentosidine. Analysis of potential associations between the matrix stiffness and degree of collagen cross-linking showed that both insoluble collagen and pentosidine exhibited a significant positive correlation with the maximum load, stress, and strain, Young''s modulus of elasticity, and toughness (r values ranging from .61 to .94) in the Achilles tendons. However, the soluble collagen content present in neutral salt buffer, acetate buffer, and acetate buffer containing pepsin showed an inverse relation with the various biomechanical attributes tested (r values ranging from .22 to .84) in the Achilles tendons. The results of the study demonstrate that glycation-induced collagen cross-linking is directly associated with the increased matrix stiffness and other mechanical attributes of the tendon.     

Enthalpies of DNA melting in the presence of osmolytes

The melting of DNA in the presence of osmolytes has been studied with the intention of obtaining information about how base pair stability is affected by changes in solution conditions. In previous investigations, the melting enthalpies were assumed to be constant as osmolalities change, but no systematic evaluation of whether this condition is true has been offered. This paper presents calorimetric data on the melting of two synthetic DNA samples in the presence of a number of common osmolytes. Poly(dAdT)*poly(dTdA) and poly(dGdC)*poly(dCdG) melting have been examined by differential scanning calorimetry in solutions containing ethylene glycol, glycerol, sucrose, urea, betaine, PEG 200 and PEG 1450 at increasing osmolalities. The results show small, but significant changes in the enthalpy of melting of the two polynucleotides that are different, depending on the structure of the cosolvent. The polyols, ethylene glycol, glycerol, PEG 200 and also urea all show decreases in melting enthalpy, while betaine and sucrose display increases with increasing concentration of cosolvent. The large stabilizing PEG 1450 shows no change within the experimental errors. Using concepts relating to preferential interactions of the cosolvents with the DNA base pairs, it is possible to interpret some of the observed changes in the thermodynamic properties of melting. The results indicate that there is strong entropy-enthalpy compensation upon melting base pairs, but entropy increases dominate to cause the decreases in stability with increased cosolvent concentration. Excess hydration parameters are evaluated and their magnitudes discussed in terms of changes in cosolvent interactions with the DNA base pairs.     

The effect of plant hormone abscisic acid on model membranes - differential scanning calorimetry and planar bilayer membranes investigations

The effect of abscisic acid on the thermotropic properties of dipalmitoylphosphatidylcholine (DPPC) and on phosphatidylethanolamines (natural (PE) and dipalmitoylphosphatidylethanolamine (DPPE)) bilayers was investigated by differential scanning calorimetry (DSC). Abscisic acid eliminates the pretransition of DPPC, causes a downward shift of its temperature of melting (T_m) and broadens the melting peak without changing the enthalpy of melting. In natural PE bilayers interacting with abscisic acid a small decrease in the enthalpy of melting almost without change of T_m was detected, whereas in synthetic DPPE abscisic acid caused a small shift of T_m and small broadening of the melting peak without changing the enthalpy of melting. Abscisic acid increases the conductance to Na⁺ or K⁺ by three orders of magnitude in planar lipid membranes formed from PE monolayers and by less than two orders of magnitude in membranes formed from PC monolayers.     

热变性前后水合醛缩酶中水状态的热力学研究

 用差示扫描量热计,测定了不同水合态(水含量h从0.08g/g至2.50g /g)冻结的水合醛缩酶样品中冰的熔化温度和熔化焓,并计算出熔化熵。实验结果表明,变性前,水合醛缩酶中的水转化成四种状状:(1)不冻结水,(2)熔化焓和熔化温度均低了普通水的可冻结水,(3)熔化焓与普通水相同而熔化温度低于普通水的可冻结水,(4)熔化焓和熔化温度均与普通水相同的容积水。变性后,水合醛缩酶中的水可处于五种状态,即不冻结水,熔化焓低于普通水而熔化温度与普通水相同的可冻结水,以及熔化温度均低于普通水且彼此不相同的三种状态的可冻结水。实验还观察到,水合醛缩酶在热变性前后,水在这些态中的量是不同的。     

Heat capacity of hydrogen-bonded networks: an alternative view of protein folding thermodynamics

Large changes in heat capacity (deltaCp) have long been regarded as the characteristic thermodynamic signature of hydrophobic interactions. However, similar effects arise quite generally in order-disorder transitions in homogeneous systems, particularly those comprising hydrogen-bonded networks, and this may have significance for our understanding of protein folding and other biomolecular processes. The positive deltaCp associated with unfolding of globular proteins in water, thought to be due to hydrophobic interactions, is also typical of the values found for the melting of crystalline solids, where the effect is greatest for the melting of polar compounds, including pure water. This suggests an alternative model of protein folding based on the thermodynamics of phase transitions in hydrogen-bonded networks. Folded proteins may be viewed as islands of cooperatively-ordered hydrogen-bonded structure, floating in an aqueous network of less-well-ordered H-bonds in which the degree of hydrogen bonding decreases with increasing temperature. The enthalpy of melting of the protein consequently increases with temperature. A simple algebraic model, based on the overall number of protein and solvent hydrogen bonds in folded and unfolded states, shows how deltaCp from this source could match the hydrophobic contribution. This confirms the growing view that the thermodynamics of protein folding, and other interactions in aqueous systems, are best described in terms of a mixture of polar and non-polar effects in which no one contribution is necessarily dominant.