Triple Helix Structure Research Articles

Scurvy with gastrointestinal bleeding

A 48-year-old man was admitted to our hospital with 1-month history of petechiae and edema of the bilateral lower extremities, polyarthralgia, gait disturbance, and hematochezia. He also had 2-year history of anorexia and an unbalanced diet that did not include any fresh vegetables or fruits. On examination, his conjunctivae were pale and his hemoglobin was 5.7g/dL. He had leukopenia (2970/μL), thrombocytopenia (620000/μL), and hypoalbuminemia (2.9g/dL). The serum vitamin B1 level was 11.0ng/mL (24– 66ng/mL), the serum vitamin C level was <0.2μg/mL (5.5–16.8μg/mL), and the serum folate level was 2.8ng/mL (3.1–100,000ng/mL). Esophagogastroduodenoscopy (EGD) showedmultiple intramucosal hemorrhages and redness in the fornix (● Fig.1a), the upper gastric body, the antrum (● Fig.1b), and the descending portion of the duodenum (● Fig.1c). Colonoscopy showedmultiple intramucosal hemorrhages and redness extending from the ascending colon to the rectum (● Fig.2) and edema and erosions in the rectum. Histological examination of the biopsy specimens of the rectal lesions revealed fibrin exudation and mild infiltration of inflammatory cells (● Fig.3), consistent with hemorrhagic mucosa. The patient was diagnosed as having scurvy with gastrointestinal bleeding. Vitamin supplementation and adequate nutrition alleviated the symptoms. With the aid of rehabilitation management he was able to walk and was discharged. Ascorbic acid activates several hydrolases, one of which (prolyl hydroxylase) forms the hydroxyproline that stabilizes the collagen triple-helix structure. Thus, the present patient had probably become susceptible to bleeding from the capillaries because of defective collagen synthesis resulting in fragile capillary walls. Compared with earlier times, scurvy is now seen more rarely, most often occurring in elderly people, alcoholics, institutionalized patients, people on fad diets or those with malabsorption disorder [1]. People with scurvy have submucosal hemorrhages in the stomach, duodenum, and the colon [2–4]. It is important for gastroenterologists to include scurvy in the differential diagnosis of patients presenting with petechiae, polyarthralgia, and gastrointestinal bleeding.

Effect of pretreatment on enzymatic hydrolysis of bovine collagen and formation of ACE-inhibitory peptides

Bovine collagen was pre-treated (boiled or high pressure (HP)-treated) and then hydrolysed by 6 proteases. The degree of hydrolysis (DH) and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates were measured. All enzymes used were able to partly degrade collagen and release ACE-inhibitory peptides. The highest ACE-inhibitory activity was obtained with Alcalase. Pretreatment significantly influenced the DH and ACE-inhibition. For most enzymes, boiling for 5min resulted in a significantly higher DH and ACE-inhibitory activity. With Alcalase and collagenase, hydrolysis and release of ACE-inhibitory peptides occurred without any pretreatment, but HP-treatment significantly improved the DH and ACE-inhibitory activity. HP did not markedly affect the hydrolysis with the other enzymes. The major peptides obtained with Alcalase were identified; all were released from the triple helix structure of collagen. Many of these peptides had C-terminal sequences similar to known ACE-inhibitory peptides. The present results suggest that collagen-rich food materials are good substrates for the release of potent ACE-inhibitory peptides, when proper pre-treatment and enzymatic treatment is applied.

Hallopeau–Siemens dystrophic epidermolysis bullosa due to homozygous 5818delC mutation in the COL7A gene

Epidermolysis bullosa (EB) is a group of inherited mechanobullous skin disease. The dystrophic EB (DEB), one subtype of EB, is inherited in an autosomal dominant DEB or in an autosomal recessive (RDEB). DEB is caused by mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils. Over 300 pathogenic mutations have been detected within COL7A in DEB. Patients with the Hallopeau-Siemens type (HS-RDEB), most severe form of DEB, frequently have premature termination codon (PTC) mutations on both alleles. PTC mutations on both alleles result in depleted mRNA and α1 helix, and failure to form the triple helix structure characteristic of type VII collagen. As patients with HS-RDEB usually have a pair of heterozygous PTC mutations, there have been rarely reported homozygous ones in HS-RDEB. We report the first case of HS-RDEB homozygous PTC mutations of 5818delC in both COL7A1 alleles. This case report suggests the positional effect of PTC mutations and vigilance against early infantile death in EB including HS-RDEB.

Quantum biochemistry study of the T3-785 tropocollagen triple-helical structure

We estimate the residue–monomer and residue–residue interaction energies of the collagen-like peptide T3-785, whose triple helix structure is the sequence X–Y-glycine (X, Y are often the imino acids proline and hydroxyproline), considering its full X-ray diffraction crystal structure, including a hydratation layer of 111 water molecules. The computations are performed within the density functional theory (DFT) scope together with a Molecular Fractionation with Conjugate Caps (MFCC) approach. We found that the hydroxyproline and proline residues play a very important role in the stabilization of the T3-785 structure, with the arginine residue in a given peptide chain exhibiting the strongest residue–strand interaction.

Design of Structral Metal Sites in Heterospecific Collagen Peptides

Globular proteins commonly use structural metals to promote folding and enhance stability. There are no good examples of similar metal utilization in fibrillar proteins such as collagen. We hypothesized a metal binding site at the end of an A:B:C-type collagen triple helix could be designed to enhance folding and stability without compromising specificity. A heterospecific metal binding site was computationally designed by sampling backbone and sidechain conformations of C-terminal amino acids of the triple helix to optimize metal site geometry. Experimental characterization of the designed sequences confirms that zinc-binding enhances structure and thermal stability of the A:B:C heterotrimer peptides under physiological buffer conditions. By varying metal concentration, it is possible to study the relative contributions of electrostatic interactions and metal binding, to triple helix stability and structure. Metal-directed switching of triple helical structure has potential applications in self-assembly of higher order biomaterials, translational regenerative medicine and drug design.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Biomineralization on chemically synthesized collagen containing immobilized poly-γ-glutamic acid

Chemically synthesized collagen with a triple helix structure similar to that of natural collagen is attractive as a safe biomaterial. Hybrids of chemically synthesized collagen and apatite are proposed for novel bone substitutes. However their apatite-forming ability in simulated body fluid is still quite low. We examined acceleration of apatite formation on collagen by immobilized poly-γ-glutamic acid (PGA), which has excellent apatite-forming ability. Apatite was formed within 3 days when collagen was treated with PGA solutions containing an appropriate amount of CaCl2. A mixture of apatite and calcite was formed at high CaCl2 concentration. The present results indicate the possibility of preparing hybrid materials, with tailored mechanical and biological properties, based on chemically synthesized collagen.

Effect of crosslinking method on biological properties of grass carp skin collagen sponges

In this study,the effect of crosslinking method on biological properties of grass carp skin collagen sponge was discussed.Collagen was extracted from skin of grass carp(Ctenopharyngodon idellus)and collagen sponge was prepared from this collagen.Then,this collagen sponge was crosslinked with different methods,such as UV,dehydrothermal,EDC/NHS and glutaraldehyde crosslinking processes.At the same time,the biological and mechanical properties of those collagen sponges,including degree of crosslinking,denaturation temperature,tensile strength and enzymatic sensitivity in vitro,were evaluated and compared.Experiment results indicated that the grass carp skin collagen was type Ⅰ collagen.The degree of crosslinking of different crosslinking methods decreased in the order of glutaraldehyde(72.0%)EDC/NHS(32.5%)dehydrothermal(29.9%)UV(15.6%).Compared with control collagen sponges,the denaturation temperature(67.4 ℃),tensile strength(125.6 kPa)and enzymatic sensitivity in vitro of collagen sponges crosslinked by glutaraldehyde were significantly improved(P0.05);EDC/NHS crosslinking could lead to obvious increasing in denaturation enthalpy(6.86 J/g)and moderate improving on tensile strength(98.6 kPa)and enzymatic sensitivity for collagen sponge(P0.05).The changes of biological and mechanical properties of collagen sponges after being crosslinked by dehydrothermal and UV crosslinking processes were not notable.The results of FTIR showed that glutaraldehyde crosslinking could improve properties of collagen sponges by forming new covalent bond in triple helix structure of collagen,while EDC/NHS crosslinking do that by forming new hydrogen bonds among collagen molecules.This research shows that glutaraldehyde and EDC/NHS crosslinking could lead to obvious improvement in properties of collagen sponges but the influences on properties of collagen sponges after being crosslinked by dehydrothermal and UV crosslinking processes were limited.

Fabrication of Composites from Apatite and Chemically Synthesized Collagen

Chemically synthesized collagen with triple helix structure similar to natural collagen has been developed as a safe biomaterial. If the chemically synthesized collagen is deposited with apatite, they are expected for novel bone substitutes having bioactivity and bioresorbability. Although apatite formation on the chemically synthesized collagen has been examined, highly supersaturated condition such as 1.5SBF with ion concentration 1.5 times those of simulated body fluid (SBF) is needed to achieve apatite formation. In the present study, we intended acceleration on the apatite formation on the chemically synthesized collagen by immobilization with polyglutamic acid (PGA). PGA is known as biodegradable and biocompatible polypeptide having excellent apatite-forming ability. We examined effects of the immobilization procedure on mineralization behavior in SBF. At first, PGA was immobilized on porous sponges of chemically synthesized collagen in aqueous solutions containing PGA and CaCl2. As a result, not only apatite but also calcite-type CaCO3 was deposited on the specimens in SBF. The calcite formation was occurred during the treatment with PGA solution. pH of the solution was adjusted to 7 by NaOH solution in order to avoid dissolution of the collagen. During this procedure, Ca (OH)2 would be precipitated by locally increase in pH of the solution and converted into the calcite. When the PGA solution treatment was shortened so as to prevent the calcite formation, single phase of the apatite was formed in SBF. The present results indicate that crystalline phase deposited on the chemically synthesized collagen can be controlled by fabrication procedure, and provide fundamental design of composites containing apatite and chemically synthesized collagen useful for bone regeneration.

Preparation and physicochemical properties of digested collagen fragments with varying molecular weights

The porcine dermal collagen molecules were successfully digested by clostridium histolyticun collagenases (CHC) and then ultra-filtrated using varying grades of ultra-filtration membranes. Gel permeation analyses revealed that collagen fragments with varying molecular weights were successfully segregated using ultra-filtration membranes with varying grades of pore sizes. Fourier transform infra-red analyses suggest that digested collagen fragments and digested collagen fragments prepared after ultra-filtration still preserve certain percentages of triple helix structures of collagen molecules, although the percentages of preserved triple helix structures present in digested and ultra-filtrated collagen fragments reduce significantly as their Mw values reduce. Thermal and denaturation temperature analysis suggest that denaturation temperature and thermal degradation temperature values of digested collagen fragments and ultra-filtrated collagen fragments decrease significantly as their Mw values reduce. The absorbed/desorbed amounts of digested and ultra-filtrated collagen fragments in PA6/PP flocking specimens are significantly higher than those of the original collagens, and increase significantly as their Mw values reduce. Possible reasons accounting for the above degradation, ultra-filtration physicochemical, absorbing and desorbing properties of original, digested and ultra-filtrated digested collagen molecules are reported.

Ultrasensitive, high temperature and ionic strength variation-tolerant Cu2+ fluorescent sensor based on reconstructed Cu2+-dependent DNAzyme/substratecomplex

A previously reported Cu2+-dependent DNAzyme/substrate complex was reconstructed in this work, which makes possible the use of an intramolecular stem–loop structure and is, therefore, a good choice for the design of Cu2+ sensors. To demonstrate this, a fluorescent sensor was designed on the basis of the reconstructed complex. In this sensor, the fluorophore/quencher pair was caged tightly in an intramolecular double-helix structure; thus, the background signal was greatly suppressed. Cu2+-dependent cleavage of the complex could cause the release of the fluorophore, leading to restoration of the fluorescence signal. High quenching efficiency provides the sensor with three important characteristics: high sensitivity, high temperature variation tolerance and high ionic strength tolerance. The proposed sensor allows specific detection of aqueous Cu2+ down to a limit of 0.6nM, and the performance is independent of temperature and ionic strength in the range of 4–40°C and 0.8–3.0M NaCl, respectively. This work identifies a good choice for sensor design on the basis of DNAzymes containing triple-helix structures.

The influence of specific binding of collagen–silk chimeras to silk biomaterials on hMSC behavior

Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in Escherichia coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen–silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen–silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen–silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications.

An Innovation Strategy to End the Second Great Depression

This paper provides insights into a science-based strategy aimed to address the Great Depression of the 1930s and examines its relevance to policies addressing the contemporary economic crisis. This early science-based strategy was not accepted at the national level at the time, but was enacted at the regional level in New England and became the basis of an innovation response to the Second World War emergency. A few key individuals, like Massachusetts Institute of Technology's Karl Compton, President, and Vannevar Bush, dean of engineering, led this effort. The resulting innovation system, which had a significant triple-helix structure, was taken apart after the war, but key elements persisted, under a banner of support for basic research, health and military innovation, and provided the foundation of the contemporary US innovation system. We argue that a dual strategy to address the contemporary economic crisis should be pursued, combining Keynesian stimulation of the “old economy” with coherent support to the “new economy” through advanced technology development.

A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation

Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.

Human collagen isolated from adipose tissue

Collagen, the most abundant protein in vertebrates, is a useful biomaterial in pharmaceutical and medical industries. So far, most collagen has been extracted from animals and cadavers. Herein, we suggest human adipose tissue, which is routinely abandoned after liposuction, as a plentiful source of human collagen. In this study, human collagen was obtained from adipose tissue through two successive major steps: (i) extraction of the extracellular matrix (ECM) by pulverization, centrifugation, alkaline, and alcohol treatment; (ii) isolation of collagen from ECM by pepsin treatment in dilute acetic acid. The purified human adipose-derived collagen was characterized by Fourier transform infrared spectroscopy, polyacrylamide gel electrophoresis, amino acid analysis, and circular dichroism spectroscopy. The extracted collagen showed a typical triple helix structure, good thermal stability due to abundant imino acids, and high solubility at acidic pH. The collagen greatly facilitated the adhesion and proliferation of human adipose-derived stem cells and normal human dermal fibroblasts on polystyrene plates. These results suggest that human adipose tissue obtained by liposuction can provide human collagen for use in cosmetics, pharmaceutics, and medicine.

Influence of palygorskite on the structure and thermal stability of collagen

We investigated the influence of palygorskite on the conformation and thermal stability of type I collagen. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FTIR) studies demonstrated that the interaction between collagen and purified palygorskite (PAL) led to the contraction and aggregation of collagen molecules, but did not destroy the triple helix backbone of collagen. Due to the structure of collagen and PAL, the interaction mainly involved hydrogen bonding and electrostatic forces. Atomic force microscopy (AFM) observations clearly displayed the nanorod morphology of PAL, and further revealed the fibril aggregation of collagen in the presence of PAL. Differential scanning calorimetry (DSC) measurements indicated that the PAL-collagen nanocomposites improved the thermal stability in comparison with pure collagen. The present study showed that PAL could modify collagen as a reinforcing agent and preserve the triple helix structure of collagen.

Energetic and conformational changes upon complexation of gelatin with sodium dodecyl sulfate

The interaction between gelatin and sodium dodecyl sulfate (SDS) is investigated by calorimetry and photometric spectrum. The conformational change in gelatin chains upon binding of SDS, the morphology development of gelatin–SDS complex, and the nature of the interaction are studied by isothermal titration calorimetry (ITC), circular dichroism (CD), fluorescence, and freeze-fracture transmission electronic microscope micrograph (FF-TEM). The results show that the exothermic binding process is driven by both electrostatic and hydrophobic interaction. The triple helix structure of gelatin is decomposed to random coil upon the binding of SDS. The morphology of complex transfers from branch-like to “necklace-and-bead”. The interaction can be controlled by the concentration of SDS, the pH value of the solution, and the ionic strength of the system.

Structural Alignment of RNA with Triple Helix Structure

Structural alignment is useful in identifying members of ncRNAs. Existing tools are all based on the secondary structures of the molecules. There is evidence showing that tertiary interactions (the interaction between a single-stranded nucleotide and a base-pair) in triple helix structures are critical in some functions of ncRNAs. In this article, we address the problem of structural alignment of RNAs with the triple helix. We provide a formal definition to capture a simplified model of a triple helix structure, then develop an algorithm of O(mn(3)) time to align a query sequence (of length m) with known triple helix structure with a target sequence (of length n) with an unknown structure. The resulting algorithm is shown to be useful in identifying ncRNA members in a simulated genome.

Collagen characteristics of farmed Atlantic salmon with firm and soft fillet texture

The possible role of collagen in texture variations among Atlantic salmon (Salmo salar L.) grown under commercial conditions at a Norwegian farm was studied. The texture was determined instrumentally as the breaking strength, and collagen and its salt (SSC), acid (ASC), pepsin (PSC) and insoluble (IF) fractions were analysed in order to determine the collagen aggregation degree. The collagen solubility and its overall amino acid (AA) composition showed no correlation to the breaking strength, but a positive correlation was observed between the breaking strength and glycine (r=0.74) and alanine (r=0.87) contents of the PSC fraction. Salmon with high breaking strength had higher T(peak) (temperature of transition) and ΔH (enthalpy of transition), and the collagen seemed to have triple helix structures mainly stabilized by covalent associations as compared to salmon with low breaking strength. The glycosylation degree was also positively correlated to the breaking strength (r=0.88, p ≤ 0.05). It is concluded that firmness of salmon muscle was not related to the total amount of collagen in the muscle, but rather higher collagen stability.

Protein secondary structure and solvent accessibility of proteins in decellularized heart valve scaffolds

Decellularized heart valve scaffolds can be used for pulmonary heart valve replacements in heart surgery. In order to assess quality parameters of heart valves prior to implantation into patients various techniques can be applied, including ultrastructural evaluation and biomechanical testing. Fourier transform infrared spectroscopy (FTIR) was used here to study protein secondary structure and solvent accessibility in decellularized heart valve matrices. FTIR was used to study proteins in the three main types of structures in decellularized heart valves: leaflet, pulmonary artery wall and heart muscle. The amide-I band region was used to reveal differences in the overall protein secondary structure amongst tissues. Leaflet material contains a relatively high contribution of α-helical structures, whereas artery matrix contains a relatively high content of triple-helix and β-sheet structures. Solvent accessibility of tissue proteins was studied by exposing material to D2O. The exchange of hydrogen of the NH amide-II bond for deuterium originating from D2O was visible as an increase in the area of the amide-II band (1500– 1400 cm−1). The amide-I band also depicted changes in shape and position during incubation in D2O: a low frequency band at around 1625 cm−1 increased as a function of time. Remarkable differences in hydrogen-to-deuterium exchange kinetics and protein solvent accessibility were observed among the three types of heart valve matrices: proteins in artery matrix are highly accessible to solvent, whereas proteins in leaflet and heart muscle structures are relatively inaccessible to solvent and show slow hydrogen/deuterium exchange. The differences in protein solvent accessibility in the different types of matrices likely reflect differences in scaffold porosity.

Design of Aptamer-Based Sensing Platform Using Triple-Helix Molecular Switch

For successful assay development of an aptamer-based biosensor, various design principles and strategies, including a highly selective molecular recognition element and a novel signal transduction mechanism, have to be engineered together. Herein, we report a new type of aptamer-based sensing platform which is based on a triple-helix molecular switch (THMS). The THMS consists of a central, target specific aptamer sequence flanked by two arm segments and a dual-labeled oligonucleotide serving as a signal transduction probe (STP). The STP is doubly labeled with pyrene at the 5'- and 3'-end, respectively, and initially designed as a hairpin-shaped structure, thus, bringing the two pyrenes into spacer proximity. Bindings of two arm segments of the aptamer with the loop sequence of STP enforce the STP to form an "open" configuration. Formation of aptamer/target complex releases the STP, leading to new signal readout. To demonstrate the feasibility and universality of our design, three aptamers which bind to human α-thrombin (Tmb), adenosine triphosphate (ATP), and L-argininamide (L-Arm), respectively, were selected as models. The universality of the approach is achieved by virtue of altering the aptamer sequence without change of the triple-helix structure.