Thermodynamic and structural characteristics of partially denatured collagen

Thermodynamic and structural parameters of partially denaturated collagen which had undergone denaturation of different degrees are measured. On the basis of comparative analysis of these data it is established that denaturation enthalpy and secondary structure degree are linearly linked. These investigations made it possible to determine special features of heat absorption curves as well. It is concluded that heat absorption at collagen denaturation must be followed by corresponding conformational alteration.     

Calorimetric study of the glass transition of denatured collagen

Temperature dependence of heat capacity of native and denatured collagen samples with different content of bound water (6 divided by 27%) has been studied by DSC method in the temperature range from -50 to 150 degrees C. Heat capacity of denatured samples demonstrates a jump of 0.50 J/g.grad. at temperature Tg, which depends on humidity of the sample. It has been shown that Tg value also depends on the heating rate and thermal history. Annealing at the temperature below Tg produces an additional maximum in the temperature dependence on heat capacity. The magnitude of this maximum, as well as the Tg value increase with the annealing time. It is concluded that these properties of heat capacity reflect glass transition in the denatured collagen.     

Agarose gel electrophoresis of denatured RNA with silver staining

This paper describes agarose gel electrophoresis and silver staining of denatured RNAs. Glyoxal- or formaldehyde-denatured RNAs are electrophoresed in an agarose gel cast on a plastic support using an inert, low conductivity buffer. Following electrophoresis, the gel is stained with a sensitive silver stain. The method produces sharp, well-resolved bands and yields accurate RNA size estimates. Because of its sensitivity and simplicity, it is suitable for routine laboratory use.     

The effects of low temperature on Lymnaea truncatula.

A group of 20 young and another of 20 adult Lymnaea truncatula were abundantly supplied with food and kept continuously under cold conditions (5 degrees C) in the laboratory for 3 months and the effects of low temperature on their behavior, growth and reproduction were studied. The results indicate that at low temperature the activity of L. truncatula was markedly reduced but complete hibernation did not occur. The snails seem to be unaffected by the low temperature itself since none (both young and adult) died during the 3 months that they were kept at 5 degrees C. Reduced feeding, even in the presence of abundant food, during the cold conditions caused an almost total inhibiton of growth. Of even more significance was the suppression of reproduction which was connected with the metabolic rate of adult snails kept at low temperature. Young snails seem to profit by exposure to low temperature. On the return to normal laboratory temperature (16-22 degrees C) the young snails became very active, fed voraciously, grew rapidly, tended to live longer and produced more offspring than the controls. Low temperature, however, appears to have an adverse after-effect on the growth and reproduction of mature snails. Relatively, fewer eggs were deposited in this case. The results indicate that under natural field conditions in England, where temperature fluctuations during the usually mild winter months are common, the greater burden of increasing the population in overwintered snails must rest on the younger members of the community.     

Influence of chondroitin sulfate on collagen gel structure and mechanical properties at physiologically relevant levels

The ability to alter collagen organization could lead to more physiologically relevant scaffolds for tissue engineering. This study examined collagen organization in the presence of polysaccharide and the resulting effects on viscoelastic properties. Fibrillogenesis in the presence of chondroitin sulfate (CS) resulted in changes in the collagen network organization with an increase in void space present. The increased void space caused by CS addition correlated with a decreased stiffness of the collagen gel. These changes occurred with physiologically relevant ratios of collagen to CS, at physiological pH and ionic strength, and without a decrease in the amount of collagen incorporated into fibrils. The addition of dextran, an uncharged polysaccharide, yielded no change in network void space or mechanical properties. Changes in fibril diameter caused by CS or dextran were not correlated with mechanical properties. The results of this study demonstrate that collagen organization can be modified by the addition of GAG, leading to altered matrix mechanical properties. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 841-851, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Digestion of native collagen, denatured collagen, and collagen fragments by extracts of rat liver lysosomes.

Extracts of highly purified lysosomes from rat liver were examined for their ability to degrade native collagen and thermally denatured collagen at pH values between 3.5 and 7.0. After a 24-h digestion at 36 degrees with the lysosomal extract at a pH of 5.5 or lower (collagen/lysosomal protein; 2/1 or 8/1), both native and denatured collagen were degraded to an extent equivalent to 60 to 70% of that observed upon total acid hydrolysis in 6 N HCl as measured by the ninhydrin reaction (570 nm). At a pH of 6.0, native collagen and denatured collagen were degraded by the mixture of lysosomal proteinases to 11% and 40% of total acid hydrolysis, respectively. At pH 6.5 AND 7.0, the corresponding values were 3% versus 33% and 0.3% versus 11%, respectively. Fragments of collagen (TCA and TCB) are produced when mammalian collagenase degrades native collagen at 25 degrees. These fragments were degraded by the lysosomal extract at 36 degrees to an extent equivalent to 28% and 8% of total acid hydrolysis at pH 6.5 and 7.0, respectively. The experiments at pH 6.5 and 7.0 were done using a collagen/lysosomal protein ratio of 2/1. At pH 5.0 (a pH which is found within secondary lysosomes), the lysosomal extracts degraded collagen to a mixture of free amino acids and small peptides. Amino acid analysis established that approximately 30% of the amino acid residues of the collagen appeared in the lysosomal hydrolysate as free amino acids. Hydroxyproline and perhaps hydroxylysine were the only amino acids found in collagen which did not appear at least to some extent as the free amino acid in this hydrolysate.     

Two stress proteins of the endoplasmic reticulum bind denatured collagen

A differentiation-related gelatin-binding 46 kilodalton (kDa) glycoprotein in myoblasts (GP46, colligin) shares several properties with the 78-kDa glucose-regulated protein (GRP78), including location in the endoplasmic reticulum and related C-terminal sequences. These similarities extend to stress inducibility, since we find that GP46 is a heat-shock protein; its synthesis is elevated at 42 degrees C, resulting in a two- to three-fold increase in protein level. Further, GRP78 is a gelatin-binding protein; together with GP46 it is retained on gelatin-Sepharose beads. GRP78 and GP46 do not interact; each protein can be individually eluted, GP46 at low pH and GRP78 by ATP. These results suggest that the proteins have distinct roles in the synthesis of collagen and point to a simple method for purification.     

Scaling behavior and structure of denatured proteins

An ensemble of random-coil conformations with no persistent structures has long been accepted as the classical model of denatured proteins due to its consistency with the experimentally determined scaling of protein sizes. However, recent NMR spectroscopy studies on proteins at high chemical denaturant concentrations suggest the presence of significant amounts of native-like structures, in contrast to the classical random-coil picture. To reconcile these seemingly controversial observations, we examine thermally denatured states of experimentally characterized proteins by using molecular dynamics simulations. For all studied proteins, we find that denatured states indeed have strong local conformational bias toward native states while a random-coil power law scaling of protein sizes is preserved. In addition, we explain why experimentally determined size of the protein creatine kinase does not follow general scaling. In simulations, we observe that this protein exhibits a stable intermediate state, the size of which is consistent with the reported experimental observation.     

Effect of low temperature on chloroplast structure in cultivars of rice

The chloroplasts in cold-susceptive indica varieties and indica? japonica lines swelled and then accumulated excess starchunder low temperature. Disorganization of the thylakoids anddiscoloration of the leaf were also observed. ¹ Home address: Kongju Teacher's College, Choong-Chung Nam Province,301 Korea. (Received April 7, 1979; )     

Water-silica gel interactions,X-ray diffraction study at room and low temperature

X-ray diffraction experiments on water confined in silica gel powder hydrated at about 20% are presented and analyzed at room temperature and down to 77 K. The structural modification of confined water observed at second neighbors is due to the competition between the confinement effect and the water-silica interaction.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Direct effects of altered temperature on renal structure and function

Although marked alterations in temperature often accompany ischemic, acute renal failure (ARF), the effects of altered temperature on renal structure and function have received little attention. In the present investigation, isolated rat kidneys perfused at 41 degrees C had extensive tubular damage and decreased function compared to kidneys perfused at 37 degrees C. In contrast, kidneys perfused at 30 degrees C had less tubular damage, and better function, than kidneys perfused at 37 degrees C. Increased temperature caused a 50% reduction in renal ATP (0.46 +/- 0.04 microM/100 mg tissue protein. 37 degrees C vs. 0.26 +/- 0.03 microM/100 mg tissue protein, 41 degrees C; p less than 0.05). The decreased ATP occurred despite reduced sodium reabsorption (129 +/- 8 microM/min/g, 37 degrees C vs. 65 +/- 12 microM/min/g, 41 degrees C, p less than 0.05) and normal renal oxygen consumption (QO2). These results suggest that increased temperature may cause an uncoupling of QO2 and sodium chloride transport, and an increase in nontransport mediated, basal metabolic rate may result in depleted cellular ATP levels and renal tubular cell death.     

X-ray diffraction study into the effects of liming on the structure of collagen

The manufacture of parchment from animal skin involves processes that remove hair, fats, and other macromolecules. Although it is well understood that the collagen fibers "open up" during processing, this study uses small and wide-angle X-ray diffraction to measure quantitatively the changes induced at the nanoscopic and microscopic levels. The axial rise per residue distance within the collagen molecules is unaffected by salt and lime treatments. Salting of the hides appears to remove noncollagenous materials. The intermolecular lateral packing distance between the hydrated collagen molecules (1.4 nm) increases after salting ( approximately 1.5 nm) and liming ( approximately 1.55 nm); drying is responsible for a reduction to approximately 1.2 nm in all samples. The axial staggered array (d spacing) is reduced by 1 nm after liming and is unaffected by drying. The average fibril diameter increases from 103.2 to 114.5 nm following liming, and the fibril-to-fibril distance increases from 122.6 to 136.1 nm.     

Mechanically overloading collagen fibrils uncoils collagen molecules,placing them in a stable,denatured state

Due to the high occurrence rate of overextension injuries to tendons and ligaments, it is important to understand the fundamental mechanisms of damage to these tissues' primary load-bearing elements: collagen fibrils and their constituent molecules. Based on our recent observations of a new subrupture, overload-induced mode of fibril disruption that we call discrete plasticity, we have sought in the current study to re-explore whether the tensile overload of collagen fibrils can alter the helical conformation of collagen molecules. In order to accomplish this, we have analyzed the conformation of collagen molecules within repeatedly overloaded tendons in relation to their undamaged matched-pair controls using both differential scanning calorimetry and variable temperature trypsin digestion susceptibility. We find that tensile overload reduces the specific enthalpy of denaturation of tendons, and increases their susceptibility to trypsin digestion, even when the digestion is carried out at temperatures as low as 4 °C. Our results indicate that the tensile overload of collagen fibrils can uncoil the helix of collagen molecules, placing them in a stable, denatured state.     

Growth of human renal cortical tissue on collagen gel

Summary A model system for 3-dimensional “native-state” culture of tissues on collagen gels (Proc. Natl. Acad. Sci. USA 86:2013–2017; 1989) has been applied in this study to histologically normal human renal cortical tissue from 11 patients undergoing nephrectomy for renal cell carcinoma elsewhere in the kidney. Microbial contamination occurred in 12/90 cultures, the rest (78) were studied by visual inspection, histology, immunohistochemical analysis for pankeratin (epithelial cell origin), vimentin (mesenchymal cell origin), andp-glycoprotein (associated with proximal tubules), transmission electron microscopy (EM), incorporation of tritiated thymidine (³HTdR). In the first 10 days, explants showed³HTdR-labeled cells in tubule structures. The surrounding gel was invaded by cells forming tubule structures, sometimes with basement membrane. Some of these cells showed labeling by³HTdR and immunostaining positive for pankeratin andp-glycoprotein. EM showed well-polarized epithelial cells in tubule structures with tight junctions, interdigitating lateral processes, and microvilli characteristic of proximal and distal convoluted tubules.³HTdR-labeled cells in tubule structures were observed even 2 mo. after Passage 1, 6 mo. after the initial explantation. Tubule growth was most active and fibroblast proliferation was negligible from 2 to 4 wk postexplantation. The proliferation of tubulelike cells and formation of tubulelike structures in this system represents an opportunity to study human renal cortical tissue in vitro, under conditions more closely resembling in vivo circumstances than are present in other in vitro systems suitable for long-term study. This model has potential use for in vitro toxicology studies and studies of renal physiology.     

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~ 10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.