Thermostable bFGF Improves Cell Lifespan by Enhancing Cell Activity in the Long-Term Culture of Human Orbicularis Oculi Stem Cells.

Stem cells derived from human orbicularis oculi muscle (hOOM) are a valuable resource for cell therapy. However, when stem cells are continuously cultured, their abilities tend to deteriorate over time. One method to address this issue is to use basic fibroblast growth factor (bFGF) to maintain the stem cell functionality.

Thermostable Basic Fibroblast Growth Factor Enhances the Production and Activity of Human Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Wharton's jelly-derived mesenchymal stem cell (WJ-MSC)-derived exosomes contain a diverse cargo and exhibit remarkable biological activity, rendering them suitable for regenerative and immune-modulating functions. However, the quantity of secretion is insufficient. A large body of prior work has investigated the use of various growth factors to enhance MSC-derived exosome production.

Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can differentiate into various tissues and are an essential source of various disease models and therapeutics. Various growth factors are required in order to culture pluripotent stem cells, among which basic fibroblast growth factor (bFGF) is essential for maintaining stem cell ability. However, bFGF has a short half-life (8 h) under normal mammalian cell culture conditions, and its activity decreases after 72 h, posing a serious problem in the production of high-quality stem cells.

Development of stabilized dual growth factor-loaded hyaluronate collagen dressing matrix.

Patients with diabetes experience impaired growth factor production such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), and they are reportedly involved in wound healing processes. Here, we report dual growth factor-loaded hyaluronate collagen dressing (Dual-HCD) matrix, using different ratios of the concentration of stabilized growth factors-stabilized-EGF (S-EGF) and stabilized-bFGF (S-bFGF). At first, the optimal concentration ratio of S-EGF to S-bFGF in the Dual-HCD matrix is determined to be 1:2 in type I diabetic mice.

Target Proteins of Phloretin for Its Anti-Inflammatory and Antibacterial Activities Against -Induced Skin Infection.

Phloretin is a natural chalcone with antibacterial and anti-inflammatory effects. This study investigated the anti-acne activity of phloretin against -induced skin infection and the potential target proteins of its anti-inflammatory and antibacterial effects. Phloretin potently inhibited the growth of and -induced Toll-like receptor (TLR) 2-mediated inflammatory signaling in human keratinocytes.

Effects of structurally stabilized EGF and bFGF on wound healing in type I and type II diabetic mice.

Unlabelled: Diabetes mellitus comprises a multiple metabolic disorder that affects millions of people worldwide and consequentially poses challenges for clinical treatment. Among the various complications, diabetic ulcer constitutes the most prevalent associated disorder and leads to delayed wound healing. To enhance wound healing capacity, we developed structurally stabilized epidermal growth factor (ST-EGF) and basic fibroblast growth factor (ST-bFGF) to overcome limitations of commercially available EGF (CA-EGF) and bFGF (CA-bFGF), such as short half-life and loss of activity after loading onto a matrix.

Protein Loop Structure Prediction Using Conformational Space Annealing.

We have developed a protein loop structure prediction method by combining a new energy function, which we call E (energy for protein loop modeling), with the conformational space annealing (CSA) global optimization algorithm. The energy function includes stereochemistry, dynamic fragment assembly, distance-scaled finite ideal gas reference (DFIRE), and generalized orientation- and distance-dependent terms. For the conformational search of loop structures, we used the CSA algorithm, which has been quite successful in dealing with various hard global optimization problems.

Development of Stabilized Growth Factor-Loaded Hyaluronate- Collagen Dressing (HCD) matrix for impaired wound healing.

Background: Diabetes mellitus is a disease lack of insulin, which has severely delayed and impaired wound healing capacity. In the previous studies, various types of scaffolds and growth factors were used in impaired wound healing. However, there were several limitations to use them such as short half-life of growth factors in vivo and inadequate experimental conditions of wound-dressing material.

Temperature-dependent structural change of D-penicillamine-capped chiral gold nanoparticles investigated by infrared spectroscopy.

The structure and stability of D-penicillamine-capped gold nanoparticles (d-Pen Au NPs) were studied using spectroscopic tools. The synthesis of d-Pen Au NPs was examined using high-resolution transmission electron microscopy (HR-TEM), UV-vis absorption spectroscopy, and circular dichroism (CD). Temperature-dependent reversible structural changes of d-Pen Au NPs were observed using infrared spectroscopic tools.

Design and efficient production of bovine enterokinase light chain with higher specificity in E. coli.

Enterokinase light chain (EKL) is a serine protease that recognizes Asp-Asp-Asp-Asp-Lys (D(4)K) sequence and cleaves the C-terminal peptide bond of the lysine residue. The utility of EKL as a site-specific cleavage enzyme is hampered by sporadic cleavage at other sites than the canonical D(4)K recognition sequence. In order to produce more site-specific EKL, we have generated several EKL mutants in E.

Role of the surface loop on the structure and biological activity of angiogenin.

Angiogenin is a member of the ribonuclease superfamily that induces the formation of new blood vessels. It has been suggested that the surface loop of angiogenin defined by residues 59-71 plays a special role in angiogenic function (1); however, the mechanism of action is not clearly defined. To elucidate the role of the surface loop on the structure, function and stability of angiogenin, three surface loop mutants were produced in which 14 amino acids in the surface loop of RNase A were substituted for the 13 amino acids in the corresponding loop of angiogenin.

Development of human tumor necrosis factor-alpha muteins with improved therapeutic potential.

Tumor necrosis factor-alpha (TNF-alpha) exhibits cytotoxicity towards various tumor cells in vitro and induces apoptotic necrosis in transplanted tumors in vivo. It also shows severe toxicity when used systemically for the treatment of cancer patients, hampering the development of TNF-alpha as a potential anticancer drug. In order to understand the structure-function relation of TNF-alpha with respect to receptor binding, we selected four regions on the bottom of the TNF-alpha trimer that are in close contact with the receptor and carried out mutagenesis studies and computational modeling.

Protein solubility and folding enhancement by interaction with RNA.

While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding.

Oxidative folding and N-terminal cyclization of onconase.

Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein.

Correlation of folding kinetics with the number and isomerization states of prolines in three homologous proteins of the RNase family.

Several studies attribute the slower phases in protein folding to prolyl isomerizations, and several others do not. A correlation exists between the number of prolines in a protein and the complexity of the mechanism with which it folds. In this study, we have demonstrated a direct correlation between the number of cis-prolyl bonds in a native protein and the complexity with which it folds via slower phases by studying the folding of three structurally homologous proteins of the ribonuclease family, namely RNase A, onconase and angiogenin, which differ in the number and isomerization states of their proline residues.

Analysis of the thermostability determinants of hyperthermophilic esterase EstE1 based on its predicted three-dimensional structure.

The three-dimensional (3D) structure of the hyperthermophilic esterase EstE1 was constructed by homology modeling using Archaeoglobus fulgidus esterase as a reference, and the thermostability-structure relationship was analyzed. Our results verified the predicted 3D structure of EstE1 and identified the ion pair networks and hydrophobic interactions that are critical determinants for the thermostability of EstE1.

Mutational analysis of human tumor necrosis factor-alpha.

To understand the structure-function relationship of human tumor necrosis factor-alpha (TNF-alpha), mutational analysis was carried out on the lower regions (regions 1-6) of the molecule. The muteins were prepared as a soluble form by using a chaperonin co-expression system and the cytotoxic activities of the purified muteins were evaluated on TNF-sensitive murine fibrosarcoma L929 cells. Three regions (regions 1, 2 & 4) were found where mutations significantly influenced the bioactivity.

High level production of bovine angiogenin in E. coli by an efficient refolding procedure.

Recombinant bovine angiogenin (rbAng) was expressed in E. coli at up to 30% of total cell proteins but was produced as inclusion bodies. By investigating the effect of various factors on the refolding yield, we obtained about 60% refolding.

Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris.

A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin clots. The cDNA encoding batroxobin was cloned, expressed in Pichia pastoris and the molecular function of purified recombinant protein was also characterized. The recombinant batroxobin had an apparent molecular weight of 33 kDa by SDS-PAGE analysis and biochemical activities similar to those of native batroxobin.

Formation of an alpha-helix in human tumor necrosis factor-alpha by guanidine hydrochloride-induced unfolding.

Human tumor necrosis factor-alpha (TNF-alpha) is a trimeric protein consisting primarily of beta-sheet. GdnHCl-induced unfolding of TNF-alpha was investigated at room temperature by circular dichroism (CD) and size exclusion chromatography. The secondary and tertiary structure of TNF-alpha persisted up to 0.

Role of disulfide bonds in the structure and activity of human insulin.

Insulin contains two inter-chain disulfide bonds between the A and B chains (A7-B7 and A20-B19), and one intra-chain linkage in the A chain (A6-A11). To investigate the role of each disulfide bond in the structure, function and stability of the molecule, three des mutants of human insulin, each lacking one of the three disulfide bonds, were prepared by enzymatic conversion of refolded mini-proinsulins. Structural and biological studies of the three des mutants revealed that all three disulfide bonds are essential for the receptor binding activity of insulin, whereas the different disulfide bonds make different contributions to the overall structure of insulin.

Role of the [65-72] disulfide bond in oxidative folding of bovine pancreatic ribonuclease A.

To assess the role of the [65-72] disulfide bond in the oxidative folding of RNase A, use has been made of [C65S, C72S], a three-disulfide-containing mutant of RNase A which regenerates from its two-disulfide precursor in an oxidation and conformational folding-coupled rate-determining step. The distribution of disulfide bonds in the one-disulfide-containing ensemble of this mutant has been characterized. In general, the disulfide-bond distribution in its 1S ensemble agrees relatively well with the corresponding distribution in wt-RNase A and with distributions based on calculations of loop entropy, except for the absence of the [65-72] disulfide bond.

Effect of protein disulfide isomerase on the rate-determining steps of the folding of bovine pancreatic ribonuclease A.

The effects of protein disulfide isomerase (PDI) on the four structured des species that accumulate in the rate-determining steps of ribonuclease A folding were investigated at pH 8.0 and 15 degrees C. The results indicate that PDI catalyzes the conversion of the kinetically trapped intermediates, des-[26-84] and des-[58-110], by reshuffling them into the on-pathway intermediate, des-[40-95], and the formation of native protein, by acting as both a chaperone and an oxidase on this on-pathway intermediate.

Oxidative folding of bovine pancreatic ribonuclease A: insight into the overall catalysis of the refolding pathway by phosphate.

The effects of the strong stabilizing anion, phosphate, on the oxidative folding of bovine pancreatic ribonuclease A were examined. Phosphate was found to catalyze several steps involved in the oxidative folding process at pH 8.0 and 25 degrees C, resulting in an increase in the rate of pre-equilibration of unstructured species on the folding pathway.