Cooperation between osteoblastic cells and endothelial cells enhances their phenotypic responses and improves osteoblast function

Osteogenesis requires close co-operation with angiogenesis to create vascularized bone tissue. In this study, an indirect co-culture model using osteoblasts (OBs), primary endothelial cells (ECs) and Matrigel interlayer was established to understand the impact of each cell type on the other. ECs synergistically enhanced osteoblastic gene expression by OBs, while OBs were capable of supporting tubule-like structures formed by ECs on Matrigel, enhancing mean tubule length from 146.5 ± 23.5 μm in ECs alone to 192 ± 28.6 μm in co-culture (p < 0.05). Similar improvements were noted in terms of tubule number. An applicability study of the co-culture model to bone tissue engineering, performed on a biopolymer fibrous membrane, showed substantially enhanced deposition of calcified nodules. These results demonstrate the efficacy of co-culture with ECs to improve osteogenesis for bone tissue engineering.     

High dose of glucose promotes chondrogenesis via PKCα and MAPK signaling pathways in chick mesenchymal cells

We investigated the molecular mechanism of the glucose effect on the regulation of chondrogenesis. Exposure of chick wing bud mesenchymal cells to high concentrations of glucose stimulated chondrogenesis 2–fold to 2.5-fold without affecting cell proliferation. Glucose increased protein levels and the membrane translocation of protein kinase C alpha (PKC), leading to a reduction of extracellular signal-regulated kinase (ERK) phosphorylation. Phosphorylation of p38 was also increased in a PKC-independent manner by glucose treatment. Glucose also increased cell adhesion molecules such as fibronectin, integrin 1, and N-cadherin at early stages and then decreased these adhesion molecules at later stages of chondrogenesis. These alterations in protein level of adhesion molecules and in the phosphorylation of mitogen-activated protein kinases by glucose were blocked by inhibition of PKC or p38 but were synergistically increased by the inhibition of ERK. Therefore, high doses of glucose induce the down-regulation of ERK activity via PKC and the up-regulation of p38 and result in the stimulation of chondrogenesis of chick mesenchymal cells through modulating the expression of adhesion molecules.This work was supported by the Korea Research Foundation (KRF-2000-DP0352)     

Styrylheterocycles as a novel class inhibitor of cyclooxygenase-2-mediated prostaglandin E(2) production

The inhibitory effects of a series of styrylheterocycles on the production of cyclooxygenase-2-mediated prostaglandin E(2) (PGE(2)) were evaluated in lipopolysaccharide-stimulated RAW264.7 murine macrophages. A new series of potential inhibitors, including 3-[2-(4-methoxy-phenyl)-vinyl]-thiophene, have been identified, thus providing novel chemical leads for the further development of potential inhibitors in this capacity. The suppression of COX-2 mRNA expression by the active styrylheterocycles, in part, was involved in the inhibitory activity against the overproduction of PGE(2).     

Crystal structure of (R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha

(R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha H16 (RePhaB) is an enzyme that catalyzes the NADPH-dependent reduction of acetoacetyl-CoA, an intermediate of polyhydroxyalkanoates (PHA) synthetic pathways. Polymeric PHA is used to make bioplastics, implant biomaterials, and biofuels. Here, we report the crystal structures of RePhaB apoenzyme and in complex with either NADP⁺ or acetoacetyl-CoA, which provide the catalytic mechanism of the protein. RePhaB contains a Rossmann fold and a Clamp domain for binding of NADP⁺ and acetoacetyl-CoA, respectively. The NADP⁺-bound form of RePhaB structure reveals that the protein has a unique cofactor binding mode. Interestingly, in the RePhaB structure in complex with acetoacetyl-CoA, the conformation of the Clamp domain, especially the Clamp-lid, undergoes a large structural change about 4.6 Å leading to formation of the substrate pocket. These structural observations, along with the biochemical experiments, suggest that movement of the Clamp-lid enables the substrate binding and ensures the acetoacetyl moiety is located near to the nicotinamide ring of NADP⁺.     

Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K_m, V_max, and K_cat values of 11.58 μM, 1.5 μmol/min, and 102.18 s⁻¹, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.     

Sulfonamide derivatives of styrylheterocycles as a potent inhibitor of COX-2-mediated prostaglandin E2 production

The overproduction of prostaglandin E(2) (PGE(2)) plays an important role in a variety of pathophysiological processes including inflammation and carcinogenesis. Therefore, the modulation of PGE(2) production is a promising target in the design of chemotherapeutic agents. In the present study, the inhibitory effects of a series of styrylheterocycles having either a p-SO(2)NH(2) or p-SO(2)Me group on the production of cyclooxygenase-2-mediated PGE(2) were evaluated in lipopolysaccharide-stimulated RAW264.7 murine macrophages. Among the series of styrylheterocycle derivatives, (E)-4-(2-(thiophen-3-yl)vinyl)benzenesulfonamide exhibited a potent inhibitory activity, with an IC(50) value of 0.013 μM. The inhibitory activity against the overproduction of PGE(2) by the active compound was found to be due in part to the suppression of COX-2 mRNA expression.     

Molecular cloning of Plasmodium vivax calcium-dependent protein kinase 4

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.