Earticle

To investigate the effect of water extract of Saxifraga stolonifera MEERBURGH on skin care, we measured anti-oxidant and anti-aging activity. Saxifraga stolonifera MEERBURGH water extract itself had anti-oxidant activity in a dose-dependent manner in DPPH radical scavenging. Silica dose-dependently increased the intracellular ROS generation in RAW 264.7 cells. Saxifraga stolonifera MEERBURGH water extract inhibited silica-induced intracellular superoxide anion generation and H2O2 and hydroperoxide generation in RAW 264.7 cells. Saxifraga stolonifera MEERBURGH water extract significantly inhibited both hyaluronidase and elastase activity, also significantly inhibited MMP-1(collagenase) activity as well. In NIH 3T3 fibroblast cells, Saxifraga stolonifera MEERBURGH water extract significantly increased collagen-like polymer synthesis, which suggest that the Saxifraga stolonifera MEERBURGH water extract might be used as hydration and anti-wrinkle agents. From the above results, it is referred that Saxifraga stolonifera MEERBURGH water extract appears to have potent anti-oxidant and anti-aging activity. It is suggested that the main ingredients of stolonifera MEERBURGH water extract play an important role in anti-oxidant and anti-aging activity.

The efficient and safe gene delivery system using smart biomaterial has been a useful tool to enhance gene delivery for stem cell. We have demonstrated developed substrate-mediated gene delivery using Adeno-associated viral (AAV) vectors which have the capacity to mediate efficient and safe gene delivery and Elastin-like polypeptide (ELP) which has been recently employed as a macromolecular therapeutic delivery vehicle. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction toward stem cell, was non-specifically bound onto ELPs on a tissue culture polystyrene surface (TCPS). The cellular transduction was modulated by mechanical properties as the presence of different ELP quantities on the TCPS. Importantly, ELP-mediated AAV delivery system enhanced delivery efficiency with substantially reduced viral quantities compared to bolus delivery in fibroblasts and human neural stem cells. Therefore this system with capacity to enhance gene delivery efficiency has strong potential for use in tissue engineering applications and neurodegenerative disorder treatments.

Elastin-like polypeptides (ELPs) have the transition temperature (), so-called “lower critical solution temperature” (LCST), which leads to the phase transition; they are aggregated above like a gel and dissolved below like a sol.1) This thermal property of ELPs is applicable to the several fields such as inverse transition cycling (ITC) purification, dip-pen nanolithography (DPN), and drug delivery system (DDS), etc. Using the biological method, we synthesized genes of two different ELP sequences and combined two of these sequences to produce block copolymers. To study the change of thermal properties, two different sequences, and , were designed and combinatorial polypeptides were expressed in E.coli. The thermal properties of the expressed polypeptides are being investigated according to their chain length and sequence variation.

Elastin-like polypeptides (ELPs) consisting of the repeating pentapeptide of specific amino acids (Val-Pro-Gly-Xaa-Gly, where the “guest residue” Xaa is any amino except Proline) are thermally response polypeptides that undergo a reversible phase transition. In this study, we have analyzed the thermal behavior of ELPs by investigating the change of the transition temperature (Tt) according to their physical properties such as the ELP length or the amino acid composition of ELP as well as the external stimuli such as pH or ionic strength. This work would contribute to the various applications of ELPs including protein purification which are referred as inverse transition cycling (ITC), biosensors for heavy metals removal, and drug carrier scaffolds for tissue engineering, etc.

Elastin-like polypeptides (ELPs) have a characteristic thermal property, transition temperature (Tt), which leads to the phase transition. This thermal property depends on the ELPs molecular weight(MW), the ELPs concentration, the composition of the amino acids forming the ELPs, and the ionic strength of the aqueous solution. We have already obtained two different ELP sequences, (VPGVG)n and (VPAVG)n, and we combined two of them to study the change of thermal properties by the effect of ELP sequences. We also investigated the change of thermal properties when ELP was fused with a natural protein (thioredoxin). Our results of Tt variation according to the ELP length, sequence, and fusion protein are applicable to the diverse fields such as detection of heavy metal ions, protein purification by inverse transition temperature (ITC), etc.

The development of environmentally responsive drug carriers requires a new method for assembling stimuli-responsive hydrogel. Recent progress suggests that short peptide motifs can be engineered into biopolymers with specific temperture-dependent behavior.Protein polymers derived from thermally responsive elastin-mimetic peptide can be synthesized with near-absolute control of macromolecular architecture using genetic engineering techniques. Elastin-mimetic triblock copolymers contain hydrophobic and hydrophilic segments and display different phase behavior in aqueous solution by changing the copolymer's length or sequence. The selective collapse of the hydrophobic blocks above the transition temperature can cause 3 dimensional protein-based nanoparticles or nano-textured hydrogels. We designed triblock peptide copolymers with various length and are investigating their expression in E. coli.

Elastin-like polypeptides (ELPs) are a family of polypeptides derived from a pentapeptide Val-Pro-Gly-Xaa-Gly repeat in the amino acid sequence of mammalian elastin. ELPs undergo a thermodynamically driven phase transition, characterized by intramolecular coacervation at the inverse transition temperature (Tt). Because of their specific thermal behavior, ELPs have been applied to the various fields including protein purification (ITC), biosensors for heavy metals removal, and drug carrier scaffolds. Despite the extensive application and valuable usefulness of these materials, it has some challenges to synthesize the long ELPs in vivo. First of all, repetitive units in ELPs gene are easily eliminated in the E.coli cells. In addition, owing to slipped strand mispairing, insufficiency of tRNA for translation, causative mutation in the translation process can occur frequently. Thus the productivity of the long ELPs are much lower than those of the short ELPs in the same condition. To break through these shortcomings, we used cell-free protein synthesis system. By selective addition of specific amino acids constituting ELPs, we improved the productivity of the long ELPs. It is thought that this technique can be more effective in producing longer repetitive polypeptides or in preventing mutation caused by the insufficiency of specific amino acids.

Accumulation of heavy metals is one of the major concerns for the environmental safety because of their toxicity and non-degradability. In particular, cadmium and several cadmium-containing compounds are known as carcinogens and can induce many types of cancer. Among promising strategies of cadmium removal, one emerging technology is the use of tunable, metal binding biopolymers based on elastin-like polypeptides (ELPs). An ELP consisting of the repeating pentapeptide of specific amino acids, Val-Pro-Gly-Xaa-Gly (where the “guest residue” Xaa is any amino except Proline), undergoes a reversible phase transition at a specific temperature (transition temperature, Tt). For effective elimination of cadmium, synthetic phytochelatin (EC), which is a metal-binding protein and has a repetitive motif (Glu-Cys)nGly, was fused with the ELP. We expected that a fusion protein constituting ELP and EC can detect and remove cadmium without any complex process. In this study, a fusion protein was expressed in E.coli and metal binding ability of EC to cadmium was examined quantitatively. In addition, transition temperature variation when the fusion protein binds cadmium was analyzed.

The elastin-like polypeptides (ELPs) are artificial polypeptides based on the amino acid sequence of elastin. ELPs have a distinctive thermal property which leads to reversible phase transition within a very narrow temperature. At a temperature below transition temperature (Tt), ELPs are highly soluble in aqueous solutions. However, upon a temperature shift to above Tt, ELPs become insoluble and form an aggregate of polypeptides. The epoxide hydrolases (EHs) catalyze the addition of water molecule to an epoxide to form the corresponding vicinal diol. A marine epoxide hydrolase from Mugil cephalus (McEH) has enantioselective hydrolysis activity toward (R)-enantiomer of racemic epoxide, resulting in the preparation of enantiopure (S)-enantiomer. McEH showed higher enantioselectivity and stability at lower temperature. In order to develop a novel EH biocatalyst system that catalyzes enantioselective hydrolysis reaction at low temperature and can be recycled by thermo-responsive reversible aggregation at high temperature, ELP-tagged McEH was developed. The recombinant McEH fused with elastin-like polypeptides was cloned and characterized at the molecular level.

We have examined 24 human aortas aged $46{\sim}90$ years obtained from autopsies. Most exhibited gross lesions of some degree on the lumenal surface. Using hot alkaline treatment (0.1 N NaOH) at $70{\sim}75^{\circ}C$ for 5 hours, we extracted and quantitated elastin portions from the aortic wall in 3 different segments (UTA=upper thoracic aorta, LTA=lower thoracic aorta, AA=abdominal aorta). We have found UTA had $70.6%{\pm}1.39$ (SE), LTA $61.6%{\pm}1.94$ (SE), AA $49.2%{\pm}1.84$ (SE) elastin respectively based on wet weight. The differences between segments are statistically significant (p＜0.05, 0.025). However, there is no significant correlation between the age of the patients and the relative amounts of elastin in each segment. We have also observed the structure of elastin in the internal elastic lamina (IEL) and tunica media (TM) with SEM (scanning electron microscopy), and discovered that the IEL shows various forms of elastolysis- broken sheets, discontinuity, various sizes of lumps, vesicles, and possible newly formed elastin in the aortic lesions (Song and Roach submitted to YMJ). From these studies we conclude that elastin in the aortic wall remains well balanced quantitatively with age in spite of evidence suggesting vigorous degeneration and regeneration in the atherosclerotic lesions.

Objective : We present the difference of histopathologic changes of the internal elastic lamina (IEL) and collagen III in the superficial temporal artery (STA) between aneurysmal patients and non-aneurysmal patients. Also, the pathologic data with clinical features by comparative methods to validate the risk factor of the intracranial aneurysm are presented. Methods : Samples of the STA were harvested form 38 patients including aneurysmal and non-aneurysmal patients undergoing craniotomy. Paraffin-embedded sections were examined, using hematoxylin and eosin, van Giebson and mouse anti-collagen III staining techniques. Histopathological observations were ana lysed and correlated with clinical features such as presence of aneurysm, hypertension, age, and sex. Results : Twenty-seven patients had the intracranial aneurysm. Of these 24 patients were 50 years old or older. Nineteen patients had a history of hypertension. Twenty patients were female. Histopathological study demostrated the derangement of IEL and the deficiency of type III collagen were prominent in aneurysmal patients (p < 0.05). Fifty years old or older patients did not show correlation with the deficiency of type III collagen, but with the derangement of IEL (p < 0.05). The female sex was not correlated with the derangement of IEL but with the deficiency of type III collagen (p < 0.05). However, Hypertension was not correlated with these pathologic data. Conclusion : Patients with intracranial aneurysms have severe histopathologic changes of the arterial wall showing the derangement of IEL and the deficiency of type III collagen. In the clinico-pathologic study, the advanced age and female sex were considered as risk factors of the intracranial aneurysm.

Purpose: Elastin is a major arterial structural protein, and elastin-derived peptides are related to arterial change. We previously reported on a novel assay developed using aortic elastin peptides; however, its clinical implications remain unclear. In this study, we assessed whether anti-elastin antibody titers reflect the risk of coronary artery disease (CAD) or its characteristics. Materials and Methods: We included 174 CAD patients and 171 age- and sex-matched controls. Anti-elastin antibody titers were quantified by enzyme-linked immunosorbent assay. Parameters of arterial stiffness, including the augmentation index (AI) and heart-to-femoral pulse wave velocity (hfPWV), were measured non-invasively. The clinical and angiographic characteristics of CAD patients were also evaluated. Associations between anti-elastin levels and vascular characteristics were examined by linear regression analysis. Results: The median blood level of anti-elastin was significantly lower in the CAD group than in the controls [197 arbitrary unit (a.u.) vs. 63 a.u., p<0.001]. Levels of anti-elastin were significantly lower in men and in subjects with hypertension, diabetes mellitus,hyperlipidemia, or high hfPWV. Nevertheless, anti-elastin levels were not dependent on atherothrombotic events or the angiographicseverity of CAD. In a multivariate analysis, male sex (β=-0.38, p<0.001), diabetes mellitus (β=-0.62, p<0.001), hyperlipidemia(β=-0.29, p<0.001), and AI (β=-0.006, p=0.02) were ultimately identified as determinants of anti-elastin levels. Conclusion: Lower levels of anti-elastin are related to CAD. The association between antibody titers and CAD is linked to arterial stiffness rather than the advancement of atherosclerosis.

Pluripotent stem cells (PSCs) have lots of potential in biomedical sciences owing to its potential to differentiate into any kind of cells in the body. However, it is still a challenge to culture PSCs on a large scale for application to regenerative medicine. Herein, we introduce a synthetic polymer that enables large-scale suspension culture of human PSCs. By employing suspension culture, it became unnecessary to use conventional substrata such as mouse embryonic fibroblast (MEF) or Matrigel$^{TM}$, which are believed to be main causative sources of xenogeneic contamination in cultured human PSCs in vitro. Human PSCs were cultured in the medium in which elastin-like polypeptide (ELP) dissolved. The ELP in the medium became harden as temperature increases by transforming the medium into a semi-solid gel that supported growth of human PSCs in suspension. Gel-sol transition temperature of ELP can be adjusted by modifying the peptide sequence in which 5 amino acids, Val-Pro-Gly-Xaa-Gly, repeated sequentially. We constructed 3D suspension media having transition temperature around $33{\sim}35^{\circ}C$ using an ELP consisted of 40, 60, or 80 repeats of a monomer, which was Val-Pro-Gly-Val-Gly. Among the ELPs, ELP80 was chosen as the best ELP to support growth of human PSCs in suspension culture. This result suggests that the ELP80 can be a medium component for culturing human PSCs in large-scale.

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli $DH5{\alpha}$ at $37^{\circ}C$ for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at $20^{\circ}C$ in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature ($T_t$) of levansucrase-ELP[V-40] was $17^{\circ}C$ with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.

Skin aging appears to be the result of overlapping intrinsic (including genetic and hormonal factors) and extrinsic (external environment including chronic light exposure, chemicals, and toxins) processes. These factors cause decreases in the synthesis of collagen type I and elastin in fibroblasts and increases in the melanin in melanocytes. Collagen Type I is the most abundant type of collagen and is a major structural protein in human body tissues. In previous studies, many products containing collagen derived from land and marine animals as well as other sources have been used for a wide range of purposes in cosmetics and food. However, to our knowledge, the effects of human collagen-derived peptides on improvements in skin condition have not been investigated. Here we isolate and identify the domain of a human COL1A2-derived protein which promotes fibroblast cell proliferation and collagen type I synthesis. This human COL 1A2-derived peptide enhances wound healing and elastin production. Finally, the human collagen alpha-2 type I-derived peptide (SMM) ameliorates collagen type I synthesis, cell proliferation, cell migration, and elastin synthesis, supporting a significant anti-wrinkle effect. Collectively, these results demonstrate that human collagen alpha-2 type I-derived peptides is practically accessible in both cosmetics and food, with the goal of improving skin condition.

A series of elastin-like proteins, $SKGPG[V(VKG)_3VKVPG]_n$-(ELP1-90)WP (n = 1, 2, 3, and 4), were biosynthesized based on the hydrophobic and lysine linkage domains of tropoelastin. The formation of self-assembled hydrophobic aggregates was monitored in order to determine the influence of cationic segments on phase transition properties as well as the sensitivity to changes in salt and pH. The thermal transition profiles of the proteins fused with only one or two cationic blocks (n = 1 or 2) were similar to that of the counterpart ELP1-90. In contrast, diblock proteins that contain 3 and 4 cationic blocks displayed a triphasic profile and no transition, respectively. Upon increasing the salt concentration and pH, a stimulus-induced phase transition from a soluble conformation to an insoluble aggregate was observed. The effects of cationic segments on the stimuli sensitivity of cationic bimodal ELPs were interpreted in terms of their structural and molecular characteristics.

The pancreatic and neutrophil elastases are associated with several illnesses including lung and vascular diseases, various cancers, and pancreatitis. The development of a potent and specific inhibitor to the elastases could lead to new therapies. In this study, an agar diffusion method was modified to include a substrate-dye conjugate (Elastin-Congo red) as a substrate of elastase and an indicator of elastase inhibitory activity. The Elastin-Congo red agar plates consisted of 0.1 % Elastin-Congo red and 2.5% agar. The elastase and elastase inhibitors were simultaneously loaded into wells, ultimately resulting in halo formations in which the halo diameter decreased as the concentration of elastase inhibitor increased. The concentration of elastase inhibitor in the samples, therefore, was inversely proportional to the halo diameters. This simplified method provided an excellent correlation with the standard microplate technique, which uses a chromogenic substrate. The concentration of elastase inhibitor obtained from the culture supernatant of a recombinant elastase inhibitor produced by the yeast Pichia pastoris was easily determined. This study has established a simple modified and inexpensive agar diffusion method that is potentially useful for the identification, quantification, and screening of new elastase inhibitors.

Williams-Beuren syndrome (WS) is a rare genetic disorder that results from microdeletion at chromosome 7, which harbors the elastin gene. Clinical findings include arteriopathy, aortic stenosis, hypertension, and laxities and contractures in different joints throughout the body. While many components of the temporomandibular joint (TMJ) normally contain elastin, there are few reports on TMJ manifestations of WS. This study reports a TMJ ankylosis case in a WS patient and shares insight on a possible link between development of TMJ ankylosis and elastin deficiency in WS patients. A WS patient presented with bilateral TMJ ankylosis and was successfully treated with TMJ gap arthroplasty. Hypermobility of TMJ and lack of elastin in retrodiscal tissue can induce anterior disc displacement without reduction. Due to lack of elastin, which has a significant role in the compensatory and reparatory mechanism of TMJ, WS patients might be prone to TMJ ankylosis.