Lower Denaturation Temperature Research Articles

Aging of a Regenerative Biologic Scaffold (AlloDerm Native Tissue Matrix) During Storage at Elevated Humidity and Temperature

Tissue products will age upon storage. Although a number of tissue products have been used for tissue repair and regeneration for more than a decade, no study has been published on the aging of tissue products and its potential effect on product function. This study investigated aging-caused changes in a regenerative biologic scaffold (AlloDerm native tissue matrix) upon storage at accelerated conditions. Tissue matrix was stored at elevated humidity (33%, 75%, and 85% relative humidity [RH]) and temperature (40 +/- 2 degrees C). The study measured the accumulation of advanced glycation end-products (Maillard products), and the changes of tissue structure, tissue stability, and mechanical properties as well as in vitro fibroblast repopulation. Tissue products stored at 75% RH and 85% RH changed significantly, including collagen condensation and cross-linking, increased breaking strength, and decreased elasticity. The aged products became less stable, as demonstrated by lower denaturation temperature, lower denaturation enthalpy, and higher susceptibility to nonspecific proteolytic action. In comparison, changes were nondetectable in control products stored at 2 degrees C to 8 degrees C, or very small in tissue products stored at 33% RH and 40 degrees C. Changes of tissue structure and stability were correlated highly with the formation of Maillard products, suggesting a role of Maillard reactions in tissue aging during storage. Calorimetric analysis revealed that tissue products stored at 2 degrees C to 8 degrees C and at 33% RH and 40 degrees C were in the glassy state, whereas the products stored at 75% RH, 85% RH, and 40 degrees C were not in the glassy state, suggesting a role of the glassy state in preserving tissue products during storage. Aging did not affect in vitro fibroblast proliferation on tissue matrix, and further tests are needed to investigate how aging may affect in vivo performance of the tissue product.

Electro-spinning of pure collagen nano-fibres – Just an expensive way to make gelatin?

Scaffolds manufactured from biological materials promise better clinical functionality, providing that characteristic features are preserved. Collagen, a prominent biopolymer, is used extensively for tissue engineering applications, because its signature biological and physico-chemical properties are retained in in vitro preparations. We show here for the first time that the very properties that have established collagen as the leading natural biomaterial are lost when it is electro-spun into nano-fibres out of fluoroalcohols such as 1,1,1,3,3,3-hexafluoro-2-propanol or 2,2,2-trifluoroethanol. We further identify the use of fluoroalcohols as the major culprit in the process. The resultant nano-scaffolds lack the unique ultra-structural axial periodicity that confirms quarter-staggered supramolecular assemblies and the capacity to generate second harmonic signals, representing the typical crystalline triple-helical structure. They were also characterised by low denaturation temperatures, similar to those obtained from gelatin preparations (p>0.05). Likewise, circular dichroism spectra revealed extensive denaturation of the electro-spun collagen. Using pepsin digestion in combination with quantitative SDS-PAGE, we corroborate great losses of up to 99% of triple-helical collagen. In conclusion, electro-spinning of collagen out of fluoroalcohols effectively denatures this biopolymer, and thus appears to defeat its purpose, namely to create biomimetic scaffolds emulating the collagen structure and function of the extracellular matrix.

Isolation and characterisation of collagens from the skin, scale and bone of deep-sea redfish (Sebastes mentella)

To make more effective use of underutilised resources, collagens from skin, scale and bone (SKC, SCC and BOC) of deep-sea redfish were isolated with acetic acid and characterised for their potential in commercial applications. The abundant ash and fat in the materials could be removed effectively by EDTA and hexane treatment in 24h, with high recoveries of protein. The yield of SKC (47.5%) was significantly higher than that of SCC and BOC (6.8% and 10.3%, respectively). The denaturation temperatures of SKC, SCC and BOC were 16.1°C, 17.7°C and 17.5°C, respectively, which were lower than those of most other fish species. The amino acid profiles of these collagens were similar with a low imino acid content, which might be the reason for the low denaturation temperature. All the collagens were type I mainly and maintained their triple helical structures well with slight molecular structure differences. SKC possessed a higher degree of intermolecular cross-linking and molecular order, but the extent of peptide chain unwinding was also higher, due to the existence of fewer hydrogen bonds, compared to SCC and BOC.

Exploring differential scanning calorimetry as a tool for evaluating freezing stress sensitivity in Baker's yeasts

Thermal transitions were analyzed after freezing and thawing of two commercial baker's yeast cells of Saccharomyces cerevisiae having high and low tolerance to sugar. Yeast in stationary phase of growth were exposed to two cycles of freezing (20 h and 4 h) at −20 °C after being exposed to various physical and chemical mild stresses and differential scanning calorimetry (DSC) runs were performed at 10 °C/min from 20 °C to 100 °C. Thermograms showed two peaks that were attributed to low and high protein onset denaturation temperatures: 47–58 °C and 67–77 °C, respectively. Apparent enthalpy changes showed that the sugar tolerant strain had a relatively higher resistance to freezing and thawing than the non-tolerant one. On the other hand, some pre-stress treatments ( e.g., temperature, ethanol and NaCl) increased both enthalpy values and survival after freezing and thawing. The analysis of data clearly showed that DSC can be a very useful tool to analyze the behavior of yeast cells after freezing and thawing stress.

Site-specific Glycosylation at Asn-292 in Ovalbumin is Essential to Efficient Secretion in Yeast

Chicken ovalbumin (OVA) exists as mono-N-glycosylated form with a carbohydrate chain on Asn-292 in egg white, despite the possession of two potential N-glycosylation sites. To investigate the roles of N-glycosylation of OVA, we constructed a series of N-glycosylation mutants deleted N-glycosylation site and compared the secretion level of the mutants in Pichia pastoris. N292Q and N292/311Q mutants resulted in greater lowering of the secretion level as compared with wild-type, whereas N311Q mutant was secreted in approximately equal amounts to wild-type. However, secretion of wild-type and N311Q mutant was inhibited completely by tunicamycin treatment. All the N-glycosylation mutants have been expressed in the cells, as well as wild-type. Circular dichroism and fluorescence spectra of secreted N311Q mutant were almost identical to those of wild-type, while those of N292Q and N292/311Q mutants were different from wild-type; and, N292Q and N292/311Q mutants showed considerably lower denaturation temperature than wild-type. The results indicate that N-glycosylation at Asn-292 of OVA is required for the folding and secretion.

Evaluation of a PCR Melting Profile Technique for Bacterial Strain Differentiation

In search of an effective DNA typing technique for hospital epidemiology use, the performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation-mediated PCR (LM PCR) of bacterial DNA was tested. A number of Escherichia coli isolates from patients of the Clinical Hospital in Gdańsk, Poland, were examined. We found that the PCR MP technique is a rapid method that offers good discriminatory power and excellent reproducibility and may be applied for epidemiological studies. The usefulness of the PCR MP for molecular typing was compared with the pulsed-field gel electrophoresis method, which is currently considered the gold standard for epidemiological studies of isolates recovered from patients and the environment. Clustering of PCR MP fingerprinting data matched pulsed-field gel electrophoresis data. The features of the PCR MP technique are discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning E. coli that may occur in a hospital.

Heat-Stable Phytases in Transgenic Wheat (Triticum aestivumL.): Deposition Pattern, Thermostability, and Phytate Hydrolysis

The present paper addresses the question of thermotolerance of in planta synthesized heterologous enzymes using phytase as a model. Two individual transgenic wheat materials expressing an Aspergillus fumigatus phytase with a low denaturation temperature (62.5 degrees C) but a high refolding capacity, and a rationally designed consensus phytase engineered to a high denaturation temperature (89.3 degrees C), were evaluated. High levels of endosperm specific expression were ensured by the wheat high molecular weight glutenin 1DX5 promoter. Immunodetection at the light and electron microscopical level shows unequivocally that the heterologous phytase is deposited in the vacuole, albeit that the transformation constructs were designed for secretion to the apoplast. Evaluation of heat stability properties and kinetic properties unraveled that, under these deposition conditions, heat stability based on high unfolding temperature is superior to high refolding capacity and represents a realistic strategy for improving phosphate and mineral bioavailability in cereal-based feed and food.

Molecular basis of cellulose biosynthesis disappearance in submerged culture of Acetobacter xylinum.

Acetobacter xylinum strains are known as very efficient producers of bacterial cellulose which, due to its unique properties, has great application potential. One of the most important problems faced during cellulose synthesis by these bacteria is generation of cellulose non-producing cells, which can appear under submerged culture conditions. The reasons of this remain unknown. These studies have been undertaken to compare at the molecular level wild-type, cellulose producing (Cel(+)) A. xylinum strains with Cel(-) forms of cellulose-negative phenotype. Comparison of protein profiles of both forms of A. xylinum by 2D electrophoresis allowed for the isolation of proteins which were produced exclusively by either Cel+ or Cel- cells. Sequences of peptides derived from these proteins were aligned with those of proteins deposited in databases. This analysis revealed that Cel(-) cells lacked two enzymes: phosphoglucomutase and glucose-1-phosphate uridylyltransferase, which generates UDP-glucose being the substrate for cellulose synthase. DNA was analyzed by ligation-mediated PCR carried out at low denaturation temperature (PCR-MP). Two DNA fragments of different thermal stability (218 and 217 bp) were obtained from the DNA of Cel(+) and Cel(-) forms, respectively. The only difference between these Cel(-) and Cel(+) DNA fragments is deletion of one T residue. Alignment of those two sequences with those deposited in the GenBank database revealed that similar fragments are present in the genomes of some bacterial cellulose producers and are located downstream from open reading frames (ORF) encoding phosphoglucomutase. The meaning of this observation is discussed.

Highly Stable Mutants of Human Fibroblast Growth Factor-1 Exhibit Prolonged Biological Action

Fibroblast growth factor 1 (FGF-1) shows strong angiogenic, osteogenic and tissue-injury repair properties that might be relevant to medical applications. Since FGF-1 is partially unfolded at physiological temperature we decided to increase significantly its conformational stability and test how such an improvement will affect its biological function. Using an homology approach and rational strategy we designed two new single FGF-1 mutations: Q40P and S47I that appeared to be the most strongly stabilizing substitutions among those reported so far, increasing the denaturation temperature by 7.8 deg. C and 9.0 deg. C, respectively. As our goal was to produce highly stable variants of the growth factor, we combined these two mutations with five previously described stabilizing substitutions. The multiple mutants showed denaturation temperatures up to 27 deg. C higher than the wild-type and exhibited full additivity of the mutational effects. All those mutants were biologically competent in several cell culture assays, maintaining typical FGF-1 activities, such as binding to specific cell surface receptors and activation of downstream signaling pathways. Thus, we demonstrate that the low denaturation temperature of wild-type FGF-1 is not related to its fundamental cellular functions, and that FGF-1 action is not affected by its stability. A more detailed analysis of the biological behavior of stable FGF-1 mutants revealed that, compared with the wild-type, their mitogenic properties, as probed by the DNA synthesis assay, were significantly increased in the absence of heparin, and that their half-lives were extensively prolonged. We found that the biological action of the mutants was dictated by their susceptibility to proteases, which strongly correlated with the stability. Mutants which were much more resistant to proteolytic degradation always displayed a significant improvement in the half-life and mitogenesis. Our results show that engineered stable growth factor variants exhibit enhanced and prolonged activity, which can be advantageous in terms of the potential therapeutic applications of FGF-1.

Stabilization of Low Denaturation Temperature Collagen from Fish by Physical Cross-Linking Methods

Collagen matrices were prepared from atelo salmon collagen (SC). SC has a lower denaturation temperature (19°C) than mammalian collagen. SC matrices were successfully stabilized by ultraviolet irradiation and dehydrothermal treatment, and their optimum conditions were determined. By sponging at 37°C, partial denaturation of the collagen molecules resulted in shrinkage of the matrices.

Ligation mediated PCR performed at low denaturation temperatures--PCR melting profiles.

We show that using low denaturation temperatures (80-88 degrees C) during ligation mediated PCR (LM PCR) of bacterial DNA leads to the amplification of limited sets of the less stable DNA fragments. A set of electrophoretic patterns of such fragments obtained at different denaturation temperatures forms the PCR melting profile (PCR MP). A single pattern obtained for a given temperature and a set of patterns arising after application of several denaturation temperatures (PCR MP) are very specific for the given bacterial genome and may be used for strain characterisation and differentiation. The method may also be used for amplification and isolation of the less stable DNA fragments in a genome.

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.

Dual effect of high electric field in capillary electrophoresis study of the conformational stability of Bungarus fasciatus acetylcholinesterase

The effect of high electric field in capillary zone electrophoresis (CZE) was evaluated for the study of the thermally induced unfolding of Bungarus fasciatus acetylcholinesterase. This monomer enzyme is characterised by two interdependent uncommon structural features, the asymmetrical distribution of charged residues and a relatively low thermal denaturation temperature. Both traits were presumed to interfere in the thermal unfolding of this enzyme as investigated by CZE. This paper analyses the effect of high electric field on the behaviour of the enzyme native state. It is shown that increasing the applied field causes denaturation-like transition of the enzyme at a current power which does not induce excessive Joule heating in the capillary. The susceptibility to electric field of proteins like cholinesterases, with charge distribution anisotropy, large permanent dipole moment and notable molecular flexibility associated with moderate thermal stability, was subsequently discussed.

Thermal Stability of Onconase and Some Mutant Forms

Onconase is a small globular protein of the pancreatic ribonuclease superfamily. It is an important molecule because it possesses a selective antitumor activity. The interesting finding is that onconase has a high thermal stability, with a denaturation temperature close to 90d`C at pH 6.0. A detailed comparison between the tertiary structures of onconase and bovine pancreatic ribonuclease has been accomplished in order to identify the molecular determinants of the high stability. The results of differential scanning calorimetry measurements confirm that the mutant forms of onconase, designed to be less stable than the parent enzyme, exhibit lower denaturation temperatures. In particular, the disulfide bridge at the C-terminus of onconase seems to play a pivotal role in stability.

Thermal and pH-induced conformational changes of a beta-sheet protein monitored by infrared spectroscopy.

The stability of a lentil lectin, an all-beta protein, has been perturbed by changes in pH and temperature. In the pH interval 5.0 --> 10.0, the overall secondary structure does not undergo significant changes. However, if the individual components of the infrared amide I band are considered, changes in band components attributed to variations in beta-sheet and beta-turns cross-interactions are detected. The combined effects of pH and temperature reveal that the protein is more compact at pH 7.5 with lower denaturation temperatures at pH 5.0 or 10.0, indicating a less stable protein under those conditions. According to our results, the structural stability of the beta-sheet would depend not only on the intermolecular interactions among the strands but also on the conformation of the segments connecting these strands. The protein infrared band assignment has also been examined since the three-dimensional structure of the lentil lectin protein is known from X-ray diffraction studies. Two of the bands observed are attributed to beta-sheet. The one at 1620 cm-1, not affected if the medium is deuterated, is assigned to hairpins composed by two strands connected by a rigid turn whereas that located at 1633 cm-1 corresponds to strands associated by more flexible segments. The band appearing at 1645 cm-1 in H2O corresponds to the open, flexible loops that are connecting the beta-strands. The simplest assumption of the various secondary structure components having identical IR extinction coefficients is enough to provide IR-derived data that are in good agreement with the structure solved by X-ray diffraction.

Collagen-Carboxymethyl Dextran Conjugate without Platelet Aggregation

Pepsin solubilized collagen-carboxymethyl dextran (PSC-CMD) conjugate was prepared to improve the function of collagen by using cyanogen bromide. The conjugation between PSC and CMD was confirmed by SDS-PAGE, isoelcctric focusing and size-exclusion chromatography. The molar ratio of the conjugate was PSC:CMD=1:2.3. The isoelectric point was shifted from 9.0 to 5.5 by conjugation. Denaturation temperature of the conjugate molecule was 42.2°C, which is 1.9°C higher than that of PSC. The PSC-CMD conjugate maintained self-assembling ability but showed a very slow aggregation as compared with that of PSC. Reconstructed matrix of the conjugate had lower denaturation temperature and random molecular rearrangement structure as compared with PSC matrix. The platelet aggregation inducing ability, analyzed by spectroscopy disappeared.

Effect of dynamic glutaraldehyde fixation on the viscoelastic properties of bovine pericardial tissue

We have previously proposed dynamic fixation as an alternative method to fix a porcine aortic heart valve xenograft with better tissue fixation and better preservation of its natural biomechanical properties. Bovine pericardium was fixed under dynamic conditions, low pressures (<4 mmHg) and low vibration rate (1.2 Hz) in a 0.5% glutaraldehyde phosphate buffer (pH 7.4, 0.2 M). After fixation, tensile testing (i.e. relaxation and stress–strain curves) was performed at low and high extension rates (3 and 30 mm s -1) and tissue denaturation temperatures were determined by the hydrothermal isometric tension method. Conventional fresh and statically fixed pericardium were used as controls. In this instance, we found no significant biomechanical differences between the dynamically and statically fixed pericardial tissue (e.g. moduli and stress relaxation). However, differences in tissue extensibility were delineated, since the extensibility of the dynamically fixed tissue was closer to that of the fresh tissue compared to that of the statically fixed tissue. The final relaxation rate of the dynamically fixed tissue (−3.5±1.0% of stress remaining per log(second)) was similar to that of the statically fixed tissue (−3.2±0.6% log(s -1)) and significantly lower than the fresh tissue (−9.5±1.2% log(s -1)). The denaturation temperatures of the dynamically fixed pericardial tissue (mean±SD) (86.0±1.2°C) and the statically fixed (85.2±1.6°C) were similar but significantly higher than that of the untreated (fresh) valves (69.3±0.4°C). The results suggest a similar degree of internal cross-linking for both statically and dynamically fixed pericardium. Although fundamental structural differences exist between both porcine and bovine xenograft tissue, how these differences contribute to biomechanical differences in the effects of dynamic versus static fixation remain to be explained.

Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction.

In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 10(7) to 10(3) cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 10(5) genomes (i.e., amplifiable target sequences) per g of soil.

Short PCR product yields improved by lower denaturation temperatures

Short PCR product yields improved by lower denaturation temperatures

Phenotypic variability and abnormal type I collagen unstable at body temperature in a family with mild dominant osteogenesis imperfecta

Autosomal dominant inheritance of a mild form of osteogenesis imperfecta (osteogenesis imperfecta type I) with different phenotypic expression was found in a family. Phenotypic expression was different for the affected mother and son, in the presence of the same biochemical results. Dermal fibroblast cultures synthesized normal and mutant type I collagen alpha chains. Collagen heterotrimers containing abnormal chains were overmodified along the entire triple helical domain and showed an unusually low denaturation temperature, so far found only in lethal cases. The mild phenotype in the family is probably due to the fact that abnormal type I collagen molecules are more likely to be degraded than utilized in the extracellular matrix.