In Vitro Evaluation of a Novel Osteo-Inductive Scaffold for Osteogenic Differentiation of Bone-Marrow Mesenchymal Stem Cells.

BACKGROUND: Demineralized bone matrices (DBMs) were demonstrated to be a promising candidate for bone regeneration by previous studies. However, the limited osteoinductivity of DBMs was insufficient for a better repairing of bone defect. Osteoblasts (OBs), the major cellular component of bone tissues, play an important role in the formation of new bone. The extracellular matrix (ECM) of OB is one of the main components of bone formation niche. OBJECTIVE: To combine the DBMs with the ECM of OBs to construct a novel scaffold that could be used for bone reconstruction. METHODS: In this study, OBs were cultured on the surface of DBMs for 10 days and removed by Triton X-100 and ammonium hydroxide to prepare the OBs-ECM-DBMs (OEDBMs). A series of material features such as residues of OBs and ECM, cytotoxity, and osteoinductive capability of OEDBMs were evaluated. RESULTS: Low cell residues and low content of DNA were observed in OEDBMs. Compared with DBMs, OEDBMs possessed more bone tissues organic matrix proteins, such as osteocalcin, osteopontin, and collagen I. Rat bone marrow mesenchymal stem cells (rBMSCs) presented a good viability when cultured on both 2 materials. The significant upregulations of osteogenic genes and proteins of rBMSCs were observed in OEDBMs group compared with DBMs group. CONCLUSION: Taken together, these findings suggested that the OB-secreted ECM may be qualified as an ideal modification method for enhancing the performance of engineered bone scaffold.

Dioxygen activation of a trinuclear Cu(I)Cu(I)Cu(I) cluster capable of mediating facile oxidation of organic substrates: competition between O-atom transfer and abortive intercomplex reduction.

The dioxygen activation of a series of Cu(I)Cu(I)Cu(I) complexes based on the ligands (L) 3,3'-(1,4-diazepane- 1,4-diyl)bis(1-{[2-(dimethylamino)ethyl](methyl)amino}propan-2-ol)(7-Me) or 3,3'-(1,4-diazepane-1,4-diyl)bis(1-{[2-(diethylamino)ethyl](ethyl)amino}propan-2-ol)(7-Et) forms an intermediate capable of mediating facile O-atom transfer to simple organic substrates at room temperature. To elucidate the dioxygen chemistry, we have examined the reactions of 7-Me, 7-Et, and 3,3'-(1,4-diazepane-1,4-diyl)bis[1-(4-methylpiperazin-1-yl)propan-2-ol] (7-N-Meppz) with dioxygen at -80, -55, and -35 °C in propionitrile (EtCN) by UV-visible, 77 K EPR, and X-ray absorption spectroscopy, and 7-N-Meppz and 7-Me with dioxygen at room temperature in acetonitrile (MeCN) by diode array spectrophotometry. At both -80 and -55 °C, the mixing of the starting [Cu(I)Cu(I)Cu(I)(L)](1+) complex (1) with O(2)-saturated propionitrile (EtCN) led to a bright green solution consisting of two paramagnetic species: the green dioxygen adduct [Cu(II)Cu(II)(µ-η(2):η(2)-peroxo)Cu(II)(L)](2+) (2) and the blue [Cu(II)Cu(II)(µ-O)Cu(II)(L)](2+) species (3). These observations are consistent with the initial formation of [Cu(II)Cu(II)(µ-O)(2)Cu(III)(L)](1+)(4), followed by rapid abortion of this highly reactive species by intercluster electron transfer from a second molecule of complex 1 to give the blue species 3 and subsequent oxygenation of the partially oxidized [Cu(II)Cu(I)Cu(I)(L)](2+)(5) to form the green dioxygen adduct 2. Assignment of 2 to [Cu(II)Cu(II)(µ-η(2):η(2)-peroxo)Cu(II)(L)](2+) is consistent with its reactivity with water to give H(2)O(2) and the blue species 3, as well as its propensity to be photoreduced in the X-ray beam during X-ray absorption experiments at room temperature. In light of these observations, the development of an oxidation catalyst based on the tricopper system requires consideration of the following design criteria: 1) rapid dioxygen chemistry; 2) facile O-atom transfer from the activated cluster to substrate; and 3) a suitable reductant to rapidly regenerate complex 1 to accomplish efficient catalytic turnover.

Optimized liposomes with transactivator of transcription peptide and anti-apoptotic drugs to target hippocampal neurons and prevent tau-hyperphosphorylated neurodegeneration.

Liposomes (lip) carrying pharmaceuticals have shown promise in their ability to advance the therapy for neurodegenerative diseases. However, the low nerve-targeting capacity and poor penetration rate of lip through the blood-brain barrier (BBB) are major hurdles to achieving successful treatment. Herein, we developed lip incorporating cardiolipin (CL) and phosphatidic acid (PA) to promote their capability against hyperphosphorylation of tau protein, and a transactivator of transcription (TAT) peptide to permeate the BBB for delivering nerve growth factor (NGF), rosmarinic acid (RA), curcumin (CURC) and quercetin (QU). We derived an optimization method to assess a better composition of phospholipids in the lip loaded with the four medicines. Experimental results revealed that this optimized lip increased the viability of SK-N-MC cells insulted with ß-amyloid peptide (Aß) fibrils and prevented Wistar rat brain from producing hyperphosphorylated tau. CL and PA and the grafted TAT peptide on the carrier surface improved the rescue efficiency by inhibiting Aß deposition and reducing the expressions of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), c-Jun N-terminal protein kinase, p38, tau at serine 202 and caspase-3. The lip also enhanced the expressions of p-ERK5 and p-cyclic adenosine monophosphate response element-binding protein. The amalgamated activity of NGF, RA, CURC and QU, and the effect of charged CL/PA on Aß deposits supported the therapeutic efficacy of lip. The optimized TAT-NGF-RA-CURC-QU-CL/PA-lip can be a capable drug delivery system to cross the BBB and protect Alzheimer's disease brains from tau hyperphosphorylation. STATEMENTS OF SIGNIFICANCE: The therapeutic efficiency of liposomes (lip) against neurodegenerative disorder depends on their nerve-targeting capacity and ability to permeate the blood-brain barrier (BBB). Lip was developed incorporating cardiolipin (CL) and phosphatidic acid (PA) to promote their target specificity against hyperphosphorylation of tau protein, and a transactivator of transcription (TAT) peptide to permeate the BBB. We have successfully derived an optimization method using a new mathematical expression for the first time to assess a better composition of phospholipids in lip loaded with nerve growth factor (NGF), rosmarinic acid (RA), curcumin (CURC) and quercetin (QU). The optimized TAT-NGF-RA-CURC-QU-CL/PA-lip efficaciously down-regulated the expressions of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), c-Jun N-terminal protein kinase, p38, tau at serine 202 and caspase-3, and up-regulated the expressions of p-ERK5 and p-cyclic adenosine monophosphate response element-binding protein in Alzheimer's disease Wistar rat model.

Preparation and characterization of a (Cu,Zn)-pMMO from Methylococcus capsulatus (Bath).

We report the preparation of a (Cu,Zn)-particulate methane monooxygenase (pMMO) in which the bulk of the copper ions of the electron-transfer clusters (E-clusters) has been replaced by divalent Zn ions. The Cu and Zn contents in the (Cu,Zn)-pMMO were determined by both inductively coupled plasma mass spectroscopy (ICP-MS) and X-ray absorption K-edge spectroscopy. Further characterization of the (Cu,Zn)-pMMO was provided by pMMO-activity assays as well as low-temperature electron paramagnetic resonance (EPR) spectroscopy following reductive titration and incubation in air or air/propylene mixtures. The pMMO-activity assays indicated that the (Cu,Zn)-pMMO was no longer capable of supporting catalytic turnover of hydrocarbon substrates. However, the EPR studies revealed that the catalytic cluster (C-cluster) copper ions in the (Cu,Zn)-pMMO were still capable of supporting the activation of dioxygen when reduced, and that the 14N-superhyperfine features associated with one of the type 2 Cu(II) centers in the hydroxylation C-cluster remained unperturbed. The replacement of the E-cluster copper ions by Zn ions did compromise the ability of the protein to mediate the transfer of reducing equivalents from exogenous reductants to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles for the E-cluster and C-cluster copper ions, respectively.

A study of NO trafficking from dinitrosyl-iron complexes to the recombinant E. coli transcriptional factor SoxR.

SoxR is a transcriptional factor in Escherichia coli that induces the expression of SoxS to initiate the production of enzymes in response to oxidative stress. In addition to superoxide, SoxR is also sensitive to cellular NO to produce a protein-bound dinitrosyl-iron complex (DNIC) with a characteristic electron paramagnetic resonance (EPR) signal at g(av)=2.03. Toward developing a strategy for NO sensing based on this property of SoxR, we have overexpressed and purified the recombinant His-tagged SoxR protein. Upon treatment of the purified protein under anaerobic conditions with (1) NO solution, (2) S-nitrosothiol (RSNO), and (3) chemically synthesized low molecular weight DNICs (LMW-DNICs), we have observed enhancement of the EPR signal at g(av)=2.03 from the protein-bound DNICs over time, reflecting the redistribution of NO from the NO solution, RSNO and LMW-DNICs to the SoxR. We have exploited this NO exchange to investigate the kinetics and mechanisms of release and delivery of NO from various LMW-DNICs to an isopropyl-beta-D-thiogalactopyranoside-dependent SoxR expressed in E. coli cells. These experiments revealed that the NO from RSNO and LMW-DNICs could cross the biological membrane and enter the cytoplasm of the cell to form the SoxR protein-bound DNIC complex. For comparison, we have also studied the direct NO transfer from the LMW-DNICs to the SoxR protein in buffer. The NO transfer was found to be rapid. From the kinetic data derived, we showed that LMW-DNICs with bidentate thiolate ligands displayed greater stability in aqueous solution but exhibited more facile NO delivery to cytoplasmic SoxR in whole cells.

Sequential electrochemical oxidation of alternating benzene-furan oligomers.

Electrochemical oxidation of pentaaryl 2 containing two furan moieties occurs sequentially to give diketone 8 after two-electron transfer. Further oxidation with another two-electron transfer gives the corresponding tetraketone 9. Radical cation intermediate is detected by absorption spectroscopy. The radical intermediates of different regiochemistry have been shown to exhibit different oxidation potentials as revealed by the differential pulse voltammetry.

Toward delineating the structure and function of the particulate methane monooxygenase from methanotrophic bacteria.

The particulate methane monooxygenase (pMMO) is a complex membrane protein complex that has been difficult to isolate and purify for biochemical and biophysical characterization because of its instability in detergents used to solubilize the enzyme. In this perspective, we summarize the progress recently made toward obtaining a purified pMMO-detergent complex and characterizing the enzyme in pMMO-enriched membranes. The purified pMMO is a multi-copper protein, with ca. 15 copper ions sequestered into five trinuclear copper clusters: two for dioxygen chemistry and alkane hydroxylation (catalytic or C-clusters) and three to provide a buffer of reducing equivalents to re-reduce the C-clusters following turnover (electron transfer or E-clusters). The enzyme is functional when all the copper ions are reduced. When the protein is purified under ambient aerobic conditions in the absence of a hydrocarbon substrate, only the C-clusters are oxidized; there is an apparent kinetic barrier for electron transfer from the E-cluster copper ions to the C-clusters under these conditions. Evidence is provided in support of both C-clusters participating in the dioxygen chemistry, but only one C-cluster supporting alkane hydroxylation. Acetylene modification of the latter C-cluster in the hydrophobic pocket of the active site lowers or removes the kinetic barrier for electron transfer from the E-clusters to the C-clusters so that all the copper ions could be fully oxidized by dioxygen. A model for the hydroxylation chemistry when a hydrocarbon substrate is bound to the active site of the hydroxylation C-cluster is presented. Unlike soluble methane monooxygenase (sMMO), pMMO exhibits limited substrate specificity, but the hydroxylation chemistry is highly regioselective and stereoselective. In addition, the hydroxylation occurs with total retention of configuration of the carbon center that is oxidized. These results are consistent with a concerted mechanism involving direct side-on insertion of an active singlet "oxene" from the activated copper cluster across the "C-H" bond in the active site. Finally, in our hands, both the purified pMMO-detergent complex and pMMO-enriched membranes exhibit high NADH-sensitive as well as duroquinol-sensitive specific activity. A possible role for the two reductants in the turnover of the enzyme is proposed.