Engineered elastin-like polypeptide improves the efficiency of adipose-derived stem cell-mediated cutaneous wound healing in type II diabetes mellitus.

Impaired cutaneous wound healing is a major complication in patients with diabetes mellitus (DM), leading to increased amputation and mortality rates in affected patients. Adipose-derived stem cells (ASCs) are widely used seed cells for promoted tissue regeneration to improve wound closure under diabetic conditions. However, ASCs-based therapies remain limited due to difficulties in maintaining cell quality during transplantation. To overcome this problem, extracellular matrix mimetic biomaterials have been developed for use in biomedical engineering field, including tissue engineering and regenerative medicine. Herein, a biosynthesized arginine-glycine-aspartate amino acid residues (RGD motif, known as a cell adhesion motif)-containing elastin-like polypeptides (REPs) improved the efficacy of ASCs in enhancing wound closure and skin elasticity in diabetic wounds by promoting the expression of angiogenic growth factors. Therefore, REPs can be used as potential supplements to stem cell-based therapeutic approach to accelerate diabetic wound repair.

Enhancing Viability of Human Embryonic Stem Cells during Cryopreservation via RGD-REP-Mediated Activation of FAK/AKT/FoxO3a Signaling Pathway.

BACKGROUND: Cryopreservation is a crucial method for long-term storage and stable allocation of human pluripotent stem cells (hPSCs), which are increasingly being used in various applications. However, preserving hPSCs in cryogenic conditions is challenging due to reduced recovery rates. METHODS: To address this issue, the Arginine-Glycine-Aspartate (RGD) motif was incorporated into a recombinant elastin-like peptide (REP). Human embryonic stem cells (hESCs) were treated with REP containing RGD motif (RGD-REP) during suspension and cryopreservation, and the survival rate was analyzed. The underlying mechanisms were also investigated. RESULTS: The addition of RGD-REP to the cryopreservation solution improved cell survival and pluripotency marker expression. The improvement was confirmed to be due to the activation of the FAK-AKT cascade by RGD-REP binding to hESC surface interin protein, and consequent inhibition of FoxO3a. The inactivation of FoxO3a reduced the expression of apoptosis-related genes, such as BIM, leading to increased survival of PSCs in a suspension state. CONCLUSION: RGD-REP, as a ligand for integrin protein, improves the survival and maintenance of hPSCs during cryopreservation by activating survival signals via the RGD motif. These results have potential implications for improving the efficiency of stem cell usage in both research and therapeutic applications.

Arg-Gly-Asp-modified elastin-like polypeptide regulates cell proliferation and cell cycle proteins via the phosphorylation of Erk and Akt in pancreatic ß-cell.

OBJECTIVE: Enhancement of ß-cell proliferation plays an important role in maintaining ß-cell mass and function, and in improving pancreatic ß-cell survival before transplantation. Extracellular matrix (ECM) components increase the adhesion and proliferation of ß-cells, and the RGD-modified elastin-like polypeptide (RGD-ELP, REP) has been described as a bioactive matrix. In this study, we investigated whether REP could enhance ß-cell adhesion and proliferation and elucidated the signaling pathways involved. METHODS: We investigated the effect of REP on cell adhesion, proliferation and insulin secretion via assays using Rin-m and rat islets. Crystal violet, CCK-8, and BrdU assay, FACS, western blot, real time q-PCR analyses and insulin ELISA were examined. To explain the associated mechanisms, phosphorylation of Akt and extracellular signal-regulated kinase (Erk) were measured. RESULTS: REP more increased the adhesion, proliferation and survival of Rin-m cells compared to elastin-like poly peptide (ELP) without RGD-motif. The enhancement of ß-cell proliferation by REP was associated with increased cyclin D1, cyclin D2 and cdk6, and decreased p27 levels. When ß-cells were cultured on REP, Erk and the phosphatidylinositol 3-kinase (PI3-kinase) downstream effector, Akt was stimulated. Treatment with the Erk pathway inhibitor and PI3-kinase inhibitor decreased REP-induced ß-cell adhesion and proliferation, and regulated REP-induced cell cycle proteins. Additionally, REP increased the mRNA and protein levels of insulin and its transcription factor, PDX-1, and insulin secretion. CONCLUSIONS: Our results demonstrate that the up-regulation of the PI3K/Akt and Erk signaling pathways and the regulation of cell cycle proteins by REP could serve as effective strategies for improving pancreatic ß-cell adhesion and proliferation.

RGD-containing elastin-like polypeptide improves islet transplantation outcomes in diabetic mice.

Successful islet transplantation critically depends on the isolation of healthy islets. However, the islet isolation procedure itself contributes to islet death due to the destruction of intra- and peri-islet extracellular matrices (ECMs) during digestion. We investigated whether an RGD-containing elastin-like polypeptide (REP) could function as a self-assembling matrix to replenish ECMs and protects islets from cell death. Immediately following isolation, islets were coated with REP coacervate particles via isothermal adsorption of an REP solution followed by thermal gelation. REP-coated islets displayed increased viability and insulin secretory capacity in pretransplant culture compared to untreated islets. Co-transplantation of REP-treated islets and REP beneath the renal sub-capsule in streptozotocin-induced diabetic mice restored normoglycemia and serum insulin levels. Mice that received co-transplants maintained normoglycemia for a longer period of time than those receiving untreated islets without REP. Moreover, co-transplantation sites exhibited enhanced ß-cell proliferation and vascularization. Thus, the REP-based coacervation strategy improve the survival, function and therapeutic potential of transplanted islets. STATEMENT OF SIGNIFICANCE: 1). An artificial matrix polypeptide comprised of thermoresponsive elastin-like peptides and integrin-stimulatory RGD ligands (REP) to reconstitute damaged or lost matrices. 2). Through body temperature-induced coacervation, REP reconstitutes intra-islet environment and enhances islet viability and insulin secretion by activating the pro-survival and insulin signaling pathways. 3). REP-coated islets were transplanted together with the matrix polypeptide under the kidney sub-capsule of mice, it develops a new peri-insular environment, which protects the islet grafts from immune rejection thus extending islet longevity. 4). Our data suggest that in situ self-assembly of biomimetic islet environments become a new platform allowing for improved islet transplantation at extrahepatic sites.

Thermo-sensitive assembly of the biomaterial REP reduces hematoma volume following collagenase-induced intracerebral hemorrhage in rats.

Intracerebral hemorrhage (ICH) frequently results in severe disabilities and high mortality. RGD-containing elastin-like polypeptide (REP), a modified elastin-like polypeptide (ELP), is a thermally responsive biopolymer. REP has high affinity for cells and is known to show non-immunotoxicity, -cytotoxicity, and -inflammatory responses. Here we found that administration of REP in the acute phase of the ICH rat model reduced the hematoma volume, decreased the number of activated microglia, attenuated the expression of von Willebrand Factor (vWF), and prevented the leakage of immunoglobulin G (IgG) into the cerebral parenchyma without any occlusion of intact microvessels. Therefore, the present data suggest that REP treatment could be a novel therapeutic strategy for attenuating the acute phase of ICH.

Integrin-binding elastin-like polypeptide as an in situ gelling delivery matrix enhances the therapeutic efficacy of adipose stem cells in healing full-thickness cutaneous wounds.

One crucial issue in stem cell therapy used for tissue repair is often the lack of selective carriers to deliver stem cells to the site of injury where the native extracellular matrix is pathologically damaged or lost. Therefore, it is necessary to develop a biomaterial that is permissive to stem cells and is suitable to replace injured or missing matrix. The major aim of this study is to investigate the potential of an RGD-containing elastin-like polypeptide (REP) with the structure TGPG[VGRGD(VGVPG)6]20WPC to engraft adipose stem cells (ASC) to full-thickness excisional wounds in mice. We implanted REP into the wound defects via body temperature-induced in situ aggregation. Engrafted REP exhibited a half-life of 2.6days in the wounds and did not elicit any pathological immune responses. REP itself significantly accelerated wound closure and reepithelialization and upregulated the expression of dermal tissue components. A combined administration of REP and ASC formed a hydrogel-like ASC/REP composite, which provided better neovascularization than the use of ASCs alone and increased the viability of transplanted ASC, improving overall wound healing. In vitro and in vivo mechanistic investigations suggested that REP enhances ASC survival at least in part via the Fak/Src adhesion-induced upregulation of Mek/Erk and PI3K/Akt survival pathways. We conclude that REP is a promising therapeutic agent for the improvement of stem cell-based therapy for enhanced tissue regeneration and repair.

The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type.

The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

Decrease of reactive oxygen species-related biomarkers in the tissue-mimic 3D spheroid culture of human lung cells exposed to zinc oxide nanoparticles.

Common 2-dimensional (2D) cell cultures do not adequately represent cell-cell and cell-matrix signaling and substantially different diffusion/transport pathways. To obtain tissue-mimic information on nanoparticle toxicity from in vitro cell tests, we used a 3-dimensional (3D) culture of human lung cells (A549) prepared with elastin-like peptides modified with an arginine-glycine-aspartate motif. The 3D cells showed different cellular phenotypes, gene expression profiles, and functionalities compared to the 2D cultured cells. In gene array analysis, 3D cells displayed the induced extracellular matrix (ECM)-related biological functions such as cell-to-cell signaling and interaction, cellular function and maintenance, connective tissue development and function, molecular transport, and tissue morphology. Additionally, the expression of ECM-related molecules, such as laminin, fibronectin, and insulin-like growth factor binding protein 3 (IGFBP3), was simultaneously induced at both mRNA and protein levels. When 0.08-50 microg/ml zinc oxide nanoparticles (ZnO-NPs) were administered to 2D and 3D cells, the cell proliferation was not significantly changed. The level of molecular markers for oxidative stress, such as superoxide dismutase (SOD), Bcl-2, ATP synthase, and Complex IV (cytochrome C oxidase), was significantly reduced in 2D culture when exposed to 10 microg/ml ZnO-NPs, but no significant decrease was detected in 3D culture when exposed to the same concentration of ZnO-NPs. In conclusion, the tissue-mimic phenotype and functionality of 3D cells could be achieved through the elevated expression of ECM components. The 3D cells were expected to help to better predict the nanotoxicity of ZnO-NPs at tissue-level by increased cell-cell and cell-ECM adhesion and signaling. The tissue-mimic morphology would also be useful to simulate the diffusion/transport of the nanoparticles in vitro.

Construction and application of elastin like polypeptide containing IL-4 receptor targeting peptide.

Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP) polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R). The fluorescently labeled [AP1-V12]6, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V12]6 to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V12]6 demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V12]6 to tumor tissue. These results indicate that designed [AP1-V12]6 can serve as a novel carrier for selective delivery of therapeutic drugs to tumors.

Cytotoxicity and inhibition of intercellular interaction in N2a neurospheroids by perfluorooctanoic acid and perfluorooctanesulfonic acid.

Effects of perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) on the neuronal lineage marker expression, cell-cell interaction, caspase-3 mRNA transcription and reactive oxygen species production by N2a neuronal cells were assesses in 3-dimensional (3D) spheroid cultures, and the cytotoxicity were thoroughly compared with those of a conventional 2D monolayer-based toxicity assay. Increasing concentrations of PFOA or PFOS resulted in an increase in cell death. The half maximal inhibitory concentrations measured with spheroids were approximately one and a half times greater than the respective values for monolayer cells. Necrosis was prevalent in spheroids regardless of the dose, whereas the major injury mechanism in monolayers was dependent on compound concentration. Both PFOA and PFOS inhibited neuronal, astrocyte and oligodendrocyte marker gene expression by monolayers and spheroids grown under undifferentiated and all-trans-retinoic acid-induced differentiating conditions. In the presence of PFOA or PFOS, expression levels of E-cadherin and connexin-43 mRNAs were significantly downregulated, and spheroids were dissociated into single cell populations, indicating that the compounds affect the synthesis of E-cadherin and connexin-43 at the transcriptional level. Results from 3D cultures may provide an insight into potential inhibitory mode of action on gap junctional intercellular communication.

Improved neural progenitor cell proliferation and differentiation on poly(lactide-co-glycolide) scaffolds coated with elastin-like polypeptide.

Poly(lactide-co-glycolide) (PLGA) and elastin-like polypeptide (ELP) have been widely used as a biodegradable scaffold and thermoresponsive matrix, respectively. However, little attention has focused on the combinatorial use of these biomaterials for tissue engineering applications. An ELP matrix TGPG[VGRGD(VGVPG)6]20WPC (referred to as REP) contains multiple Arg-Gly-Asp motifs. This study fabricated porous PLGA scaffolds coated with various concentration of matrix via thermally induced phase transition to improve adhesion-mediated proliferation and differentiation of neural progenitor cells. Matrix-coated scaffolds were characterized by FTIR, SEM, and hematoxylin and eosin staining with respect to coating efficiency, porosity, and pore size and shape. On the matrix-coated scaffolds, cells grew as a single cell or associated each other to form a multicellular layer or cluster. In biological evaluations, cell adhesion and proliferation were significantly promoted in a matrix concentration-dependent manner. More importantly, in combination with retinoic acid, the differentiation of progenitor cells into neuronal and astroglial lineages was highly stimulated in the cells cultured on matrix-coated scaffolds than on untreated controls. Taken together, our results indicated that the REP matrix-functionalized PLGA scaffolds are suitable for improving neuronal cell functions, and thus applicable for neural tissue engineering.

Morphogenetic and neuronal characterization of human neuroblastoma multicellular spheroids cultured under undifferentiated and all-trans-retinoic acid-differentiated conditions.

In this study, we aimed to compare the morphogenetic and neuronal characteristics between monolayer cells and spheroids. For this purpose, we established spheroid formation by growing SH-SY5Y cells on the hydrophobic surfaces of thermally-collapsed elastin-like polypeptide. After 4 days of culture, the relative proliferation of the cells within spheroids was approximately 92% of the values for monolayer cultures. As measured by quantitative assays for mRNA and protein expressions, the production of synaptophysin and neuronspecific enolase (NSE) as well as the contents of cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins are much higher in spheroids than in monolayer cells. Under the all-trans-retinoic acid (RA)-induced differentiation condition, spheroids extended neurites and further up-regulated the expression of synaptophysin, NSE, CAMs, and ECM proteins. Our data indicate that RA-differentiated SH-SY5Y neurospheroids are functionally matured neuronal architectures.

Stem cell-based tissue engineering approaches for musculoskeletal regeneration.

The field of regenerative medicine and tissue engineering is an ever evolving field that holds promise in treating numerous musculoskeletal diseases and injuries. An important impetus in the development of the field was the discovery and implementation of stem cells. The utilization of mesenchymal stem cells, and later embryonic and induced pluripotent stem cells, opens new arenas for tissue engineering and presents the potential of developing stem cell-based therapies for disease treatment. Multipotent and pluripotent stem cells can produce various lineage tissues, and allow for derivation of a tissue that may be comprised of multiple cell types. As the field grows, the combination of biomaterial scaffolds and bioreactors provides methods to create an environment for stem cells that better represent their microenvironment for new tissue formation. As technologies for the fabrication of biomaterial scaffolds advance, the ability of scaffolds to modulate stem cell behavior advances as well. The composition of scaffolds could be of natural or synthetic materials and could be tailored to enhance cell self-renewal and/or direct cell fates. In addition to biomaterial scaffolds, studies of tissue development and cellular microenvironments have determined other factors, such as growth factors and oxygen tension, that are crucial to the regulation of stem cell activity. The overarching goal of stem cell-based tissue engineering research is to precisely control differentiation of stem cells in culture. In this article, we review current developments in tissue engineering, focusing on several stem cell sources, induction factors including growth factors, oxygen tension, biomaterials, and mechanical stimulation, and the internal and external regulatory mechanisms that govern proliferation and differentiation.

Effects of Arg-Gly-Asp-modified elastin-like polypeptide on pseudoislet formation via up-regulation of cell adhesion molecules and extracellular matrix proteins.

Extracellular matrix (ECM) plays an important role in controlling the ß-cell morphology, survival and insulin secretary functions. An RGD-modified elastin-like polypeptide (RGD-ELP), TGPG[VGRGD(VGVPG)(6)](20)WPC, has been reported previously as a bioactive matrix. In this study, to investigate whether RGD-ELP affects ß-cell growth characteristics and insulin secretion, ß-TC6 cells were cultured on the RGD-ELP coatings prepared via thermally induced phase transition. On RGD-ELP, ß-TC6 cells clustered into an islet-like architecture with high cell viability. Throughout 7days' culture, the proliferation rate of the cells within a pseudoislet was similar to that of monolayer culture. Under high glucose (25mM), ß-TC6 pseudoislets showed up-regulated insulin gene expression and exhibited glucose-stimulated insulin secretion. Importantly, the mRNA and protein abundances of cell adhesion molecules (CAM) E-cadherin and connexin-36 were much higher in pseudoislets than in monolayer cells. The siRNA-mediated inhibition of E-cadherin or connexin-36 expression severely limited pseudoislet formation. In addition, the mRNA levels of collagen types I and IV, fibronectin and laminin were significantly elevated in pseudoislets. The results suggest that RGD-ELP promotes pseudoislet formation via up-regulation of the CAM and ECM components. The functional roles of RGD-ELP are discussed in respect of its molecular composition.

Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides.

BACKGROUND: Integrin-mediated interaction of neuronal cells with extracellular matrix (ECM) is important for the control of cell adhesion, morphology, motility, and differentiation in both in vitro and in vivo systems. Arg-Gly-Asp (RGD) sequence is one of the most potent integrin-binding ligand found in many native ECM proteins. An elastin-mimetic recombinant protein, TGPG[VGRGD(VGVPG)6]20WPC, referred to as [RGD-V6]20, contains multiple RGD motifs to bind cell-surface integrins. This study aimed to investigate how surface-adsorbed recombinant protein can be used to modulate the behaviors and differentiation of neuronal cells in vitro. For this purpose, biomimetic ECM surfaces were prepared by isothermal adsorption of [RGD-V6]20 onto the tissue culture polystyrene (TCPS), and the effects of protein-coated surfaces on neuronal cell adhesion, spreading, migration, and differentiation were quantitatively measured using N2a neuroblastoma cells. RESULTS: The [RGD-V6]20 was expressed in E. coli and purified by thermally-induced phase transition. N2a cell attachment to either [RGD-V6]20 or fibronectin followed hyperbolic binding kinetics saturating around 2 µM protein concentration. The apparent maximum cell binding to [RGD-V6]20 was approximately 96% of fibronectin, with half-maximal adhesion on [RGD-V6]20 and fibronectin occurring at a coating concentration of 2.4 × 10-7 and 1.4 × 10-7 M, respectively. The percentage of spreading cells was in the following order of proteins: fibronectin (84.3% ± 6.9%) > [RGD-V6]20 (42.9% ± 6.5%) > [V7]20 (15.5% ± 3.2%) > TCPS (less than 10%). The migration speed of N2a cells on [RGD-V6]20 was similar to that of cells on fibronectin. The expression of neuronal marker proteins Tuj1, MAP2, and GFAP was approximately 1.5-fold up-regulated by [RGD-V6]20 relative to TCPS. Moreover, by the presence of both [RGD-V6]20 and RA, the expression levels of NSE, TuJ1, NF68, MAP2, and GFAP were significantly elevated. CONCLUSION: We have shown that an elastin-mimetic protein consisting of alternating tropoelastin structural domains and cell-binding RGD motifs is able to stimulate neuronal cell behaviors and differentiation. In particular, adhesion-induced neural differentiation is highly desirable for neural development and nerve repair. In this context, our data emphasize that the combination of biomimetically engineered recombinant protein and isothermal adsorption approach allows for the facile preparation of bioactive matrix or coating for neural tissue regeneration.

Coptisine inhibits RANKL-induced NF-κB phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells.

Excessive receptor activator of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1α,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1α,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-κB p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 µM coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction.

Palmatine attenuates osteoclast differentiation and function through inhibition of receptor activator of nuclear factor-κb ligand expression in osteoblast cells.

Osteoclasts are the only cell type capable of resorbing mineralized bone, and they act under the control of numerous cytokines produced by supporting cells such as osteoblasts and stromal cells. Among cytokines, receptor activator of nuclear factor-κB ligand (RANKL) was found to be a key osteoclastogenetic molecule that directly binds to its cognate receptor, RANK, on osteoclast precursor cells. In turn, RANKL, which is an essential factor for differentiation and activation of osteoclasts, is one of the major targets of anti-resorptive agents. In this study, we found that palmatine, an isoquinoline alkaloid originally isolated from Coptis chinensis, had an inhibitory effect on osteoclast differentiation and function in vitro. Palmatine inhibited osteoclast formation in the co-culture system with mouse bone marrow cells (BMC) and osteoblasts in the presence of 10 nM 1α,25-(OH)(2)D(3). Palmatine did not affect osteoclast formation induced by RANKL in the BMC cultures. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that palmatine significantly inhibited the expression of 1α,25-(OH)(2)D(3)-induced expression of RANKL mRNAs in stromal cells without loss of cell viability. Moreover, palmatine suppressed resorption pit formation by mature osteoclasts on dentin slices and induced disruption of actin ring formation in mature osteoclasts with an impact on cell viability. Taken together, these results suggest that palmatine attenuates osteoclast differentiation through inhibition of RANKL expression in osteoblast cells, and its inhibitory effect on bone resorption is due to its disruptive effect on actin rings in mature osteoclasts. Therefore, palmatine might be an ideal candidate as an anti-resorptive agent for the prevention and treatment of bone disorders such as osteoporosis.

Retrospective analyses of the bottleneck in purification of eukaryotic proteins from Escherichia coli as affected by molecular weight, cysteine content and isoelectric point.

Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value.

Alisol-B, a novel phyto-steroid, suppresses the RANKL-induced osteoclast formation and prevents bone loss in mice.

Osteoclasts, bone-resorbing multinucleated cells, are differentiated from hemopoietic progenitors of the monocyte/macrophage lineage. Bone resorption by osteoclasts is considered a potential therapeutic target to the treatment of erosive bone diseases, including osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we found that alisol-B, a phyto-steroid from Alisma orientale Juzepczuk, exhibited inhibitory effects on osteoclastogenesis both in vitro and in vivo. Although RT-PCR analysis showed that alisol-B did not affect the 1alpha,25(OH)(2)D(3)-induced expressions of RANKL, OPG and M-CSF mRNAs in osteoblasts, addition of alisol-B to co-cultures of mouse bone marrow cells and primary osteoblasts with 10(-8)M 1alpha,25(OH)(2)D(3) caused significant inhibition of osteoclastogenesis. We further examined the direct effects of alisol-B on osteoclast precursors. Alisol-B strongly inhibited RANKL-induced osteoclast formation when added during the early stage of cultures, suggesting that alisol-B acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, alisol-B inhibited the phosphorylation of JNK, which are upregulated in response to RANKL in bone marrow macrophages, alisol-B also inhibited RANKL-induced expression of NFATc1 and c-Fos, which are key transcription factors for osteoclastogenesis. In addition, alisol-B suppressed the pit-forming activity and disrupted the actin ring formation of mature osteoclasts. In a hypercalcemic mouse model induced by 2-methylene-19-nor-(20S)-1alpha,25(OH)(2)D(3) (2MD), an analog of 1alpha,25(OH)(2)D(3), administration of alisol-B significantly suppressed 2MD-induced hypercalcemia as resulting from the inhibition of osteoclastogenesis. Taken together, these findings suggest that alisol-B may be a potential novel therapeutic molecule for bone disorders by targeting the differentiation of osteoclasts as well as their functions.

New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster.

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO2. A unique [NiFe4S4] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH*Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe3S4] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step.