miR-27b attenuates dexamethasone-inhibited proliferation and osteoblastic differentiation in MC3T3-E1 cells by targeting PPARγ2.

Osteoporosis is a metabolic bone illness characterized by low bone density and a high risk of fracture. It is estimated that there are >60 million individuals in China suffering from this disease, which highlights an urgent requirement for the development of novel and safe drugs for the long-term treatment of osteoporosis. MicroRNAs (miRNAs/miRs) have previously been identified as critical regulators in the progression of osteoporosis. As an intronic miRNA, miR-27b enhances the osteoblastic differentiation of stem cells from the bone marrow and the maxillary sinus membrane. However, the mechanism underlying miR-27b in osteoporosis remains to be elucidated. In the present study, MC3T3-E1 pre-osteoblasts were treated with dexamethasone (DEX) to establish an in vitro model of osteoporosis. The results of the present study demonstrated that DEX treatment markedly inhibited the viability of MC3T3-E1 cells, and downregulated the expression level of miR-27b. The results of reverse transcription-quantitative PCR, western blotting and dual-luciferase assays revealed that miR-27b directly regulated and suppressed the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) in MC3T3-E1 cells. Furthermore, overexpression of miR-27b by transfection of cells with miR-27b mimic attenuated DEX-mediated inhibition of cell viability, alkaline phosphatase (ALP) activity and the expression levels of bone morphogenetic protein-2 (BMP2), runt-related protein 2 (Runx2) and osteocalcin (OCN). The results of the present study indicated that miR-27b alleviated DEX-inhibited proliferation and osteoblastic differentiation. Moreover, miR-27b knockdown repressed MC3T3-E1 cell viability, ALP activity and protein levels of BMP2, Runx2 and OCN. However, these effects were abrogated by small interfering RNA-mediated PPARγ2 silencing. In conclusion, the results of the present study demonstrated that miR-27b attenuated DEX-inhibited proliferation and osteoblastic differentiation in MC3T3-E1 pre-osteoblasts by targeting PPARγ2.

Identification of epigenetically altered genes and potential gene targets in melanoma using bioinformatic methods.

This study aimed to analyze epigenetically and genetically altered genes in melanoma to get a better understanding of the molecular circuitry of melanoma and identify potential gene targets for the treatment of melanoma. The microarray data of GSE31879, including mRNA expression profiles (seven melanoma and four melanocyte samples) and DNA methylation profiles (seven melanoma and five melanocyte samples), were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated positions (DMPs) were screened using the linear models for microarray data (limma) package in melanoma compared with melanocyte samples. Gene ontology (GO) and pathway enrichment analysis of the DEGs were carried out using the Database for Annotation, Visualization, and Integrated Discovery. Moreover, differentially methylated genes (DMGs) were identified, and a transcriptional regulatory network was constructed using the University of California Santa Cruz genome browser database. A total of 1,215 DEGs (199 upregulated and 1,016 downregulated) and 14,094 DMPs (10,450 upregulated and 3,644 downregulated) were identified in melanoma compared with melanocyte samples. Additionally, the upregulated and downregulated DEGs were significantly associated with different GO terms and pathways, such as pigment cell differentiation, biosynthesis, and metabolism. Furthermore, the transcriptional regulatory network showed that DMGs such as Aristaless-related homeobox (ARX), damage-specific DNA binding protein 2 (DDB2), and myelin basic protein (MBP) had higher node degrees. Our results showed that several methylated genes (ARX, DDB2, and MBP) may be involved in melanoma progression.

2,6-Bis(1H-benzimidazol-2-yl)pyridine butyric acid monosolvate dihydrate.

In the title compound, C(19)H(13)N(5)·C(4)H(8)O(2)·2H(2)O, the mol-ecular skeleton of the 2,6-bis-(benzimidazol-2-yl)pyridine (bbip) mol-ecule is essentially planar (r.m.s. deviation = 0.023 Å). An extensive three-dimensional network of inter-molecular N-H⋯O, O-H⋯O and O-H⋯N hydrogen bonds consolidates the crystal packing, which also exhibits π-π inter-actions between the five- and six-membered rings from neighbouring bbip mol-ecules.

Glutaric acid-2-(pyridin-4-yl)-1H-benzimidazole (1/1).

The crystal structure of the title co-crystal, C(12)H(9)N(3)·C(5)H(8)O(4), N-H⋯O and O-H⋯N hydrogen bonds link the components. There are also π-π stacking inter-actions between the imidazole rings, between the imidazole and pyridine rings and between the pyridine and benzene rings [centroid-centroid distances = 3.643 (2), 3.573 (2) and 3.740 (1)Å, respectively].

[Bleaching of non-vital discolored tooth: clinical analysis of 30 cases].

PURPOSE: The purpose of this study was to evaluate the bleaching efficacy of non-vital discolored tooth. METHODS: Thirty-three non-vital discolored anterior teeth with intact crowns from 30 patients were included in this clinical study. Bleaching treatment was performed using a combination of intracoronal and extracoronal applications of 15% carbamide peroxide. Intracoronal bleaching with 30% hydrogen peroxide was used as control group. The effective cases of the experimental group were reexamined one year later. Statistical analysis of all data was performed with SPSS 17.0 software package. RESULTS: Combined therapy with intracoronal and extracoronal applications of 15% carbamide peroxide presented higher bleaching efficacy than intracoronal applications of 30% hydrogen peroxide alone, the difference was significant. But the color shade change one year later was not significant. CONCLUSION: A combination of intracoronal and extracoronal applications of 15% carbamide peroxide was an effective bleaching technique for non-vital discolored teeth.Supported by Key Research Project of Science and Technology Bureau of Hefei City (Grant No.2007-1016).

NFATc1: functions in osteoclasts.

NFATc1 is an important transcription factor which is critical for lineage selection in T-cell differentiation, cardiac valve morphogenesis, lymphatic endothelial development, osteoblast differentiation and osteoclastogenesis. Especially, it is a master regulator of RANKL-induced osteoclast differentiation and plays a pivotal role in osteoclast fusion and osteoclast activation via up-regulation of various genes responsible for osteoclast adhesion, migration, acidification, degradation of inorganic and organic bone matrix. In the present review, some of the known features of NFATc1 such as structure, function and its roles in physiological or pathophysiological processes are highlighted.

CLC-7: a potential therapeutic target for the treatment of osteoporosis and neurodegeneration.

CLC-7 is a member of the voltage-gated chloride channels family. It resides mainly in the late endosomes, lysosomes and the ruffled membrane of osteoclasts. Mice deficient in the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. In the present review, some of the known features of CLC-7 such as structure, function and its roles in physiological or pathophysiological processes are highlighted.

Nontemplate-dependent polymerization processes: polyhydroxyalkanoate synthases as a paradigm.

This review focuses on nontemplate-dependent polymerases that use water-soluble substrates and convert them into water-insoluble polymers that form granules or inclusions within the cell. The initial part of the review summarizes briefly the current knowledge of polymer formation catalyzed by starch and glycogen synthases, polyphosphate kinase (a polymerase), cyanophycin synthetases, and rubber synthases. Specifically, our current understanding of their mechanisms of initiation, elongation (including granule formation), termination, remodeling, and polymer reutilization will be presented. General underlying principles that govern these types of polymerization reactions will be enumerated as a paradigm for all nontemplate-dependent polymerizations. The bulk of the review then focuses on polyhydroxyalkanoate (PHA) synthases that generate polyoxoesters. These enzymes are of interest as they generate biodegradable polymers. Our current knowledge of PHA production and utilization in vitro and in vivo as well as the contribution of many proteins to these processes will be reviewed.

Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation.

Poly-(R)-3-hydroxybutyrate (PHB) homeostasis in Ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic PHB granule. PHB synthesis and degradation are therefore intimately linked to the process of granule assembly and breakdown. Unraveling this time-dependent three-dimensional process requires an understanding of the kinetics of synthesis of relevant proteins. Reverse transcriptase quantitative PCR and quantitative Western blotting were carried out on batch cultures of R. eutropha H16 in order to gain insight into how expression of the PHB-related genes phaA, phaB, phaC, phaP, phaR, phaZ1a, phaZ1b, and phaZ1c changed during a cell growth phase, a PHB production phase, and a PHB utilization phase. phaA, phaB, phaC, phaR, and phaZ1a were transcribed throughout cell growth, PHB production, and PHB degradation. PHB-mediated induction of PhaP expression was shown to occur at the transcriptional level, with transcript levels increasing during PHB production and decreasing during PHB utilization. Levels of PhaP correlated strongly with levels of PHB. Levels of phaZ1b transcript and protein increased sharply during production and decreased during degradation, but transcript accumulation did not depend on PHB production as in the case of phaP. No evidence of phaZ1c expression was found under the experimental conditions used in this study.

Analysis of transient polyhydroxybutyrate production in Wautersia eutropha H16 by quantitative Western analysis and transmission electron microscopy.

Polyhydroxybutyrates (PHBs) are polyoxoesters generated from (R)3-hydroxybutyryl coenzyme A by PHB synthase. During the polymerization reaction, the polymers undergo a phase transition and generate granules. Wautersia eutropha can transiently accumulate PHB when it is grown in a nutrient-rich medium (up to 23% of the cell dry weight in dextrose-free tryptic soy broth [TSB]). PHB homeostasis under these growth conditions was examined by quantitative Western analysis to monitor the proteins present, their levels, and changes in their levels over a 48-h growth period. The proteins examined include PhaC (the synthase), PhaP (a phasin), PhaR (a transcription factor), and PhaZ1(a), PhaZ1(b), and PhaZ1(c) (putative intracellular depolymerases), as well as PhaZ2 (a hydroxybutyrate oligomer hydrolase). The results show that PhaC and PhaZ1(a) were present simultaneously. No PhaZ1(b) or PhaZ1(c) was detected at any time throughout growth. PhaZ2 was observed and exhibited an expression pattern different from that of PhaZ1(a). The levels of PhaP changed dramatically and corresponded kinetically to the levels of PHB. Transmission electron microscopy (TEM) provided the dimensions of the average cell and the average granule at 4 h and 24 h of growth (J. Tian, A. J. Sinskey, and J. Stubbe, J. Bacteriol. 187:3814-3824, 2005). This information allowed us to calculate the amount of each protein and number of granules per cell and the granule surface coverage by proteins. The molecular mass of PHB (10(6) Da) was determined by dynamic light scattering at 4 h, the time of maximum PHB accumulation. At this time, the surface area of the granules was maximally covered with PhaP (27 to 54%), and there were one or two PhaP molecules/PHB chain. The ratio of PHB chains to PhaC was approximately 60, which required reinitiation of polymer formation on PhaC. The TEM studies of wild-type and deltaphaR strains in TSB provided further support for an alternative mechanism of granule formation.

Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I.

The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass of 24,563Da. The uncommon start codon TTG was identified by matching the N-terminal amino acid sequence of purified Nocardia GCH with the deduced amino acid sequence. A likely ribosomal binding site was identified 9bp upstream of the translational start site. The 3' end flank region encodes a peptide that shares high homology with dihydropteroate synthases. Nocardia GCH has 73 and 60% identity to the proteins encoded by the putative gch of Mycobacterium tuberculosis and Streptomyces coelicolor, respectively. Nocardia GCH was highly expressed in Escherichia coli cells carrying a pHAT10 based expression vector, and moderately expressed in Mycobacterium smegmatis cells carrying a pSMT3 based expression vector. Enterokinase digestion of recombinant Nocardia GCH, and in-gel digestion of Nocardia GCH and recombinant GCH followed by MALDI-TOF-MS analysis, confirmed that the actual subunit size of the enzyme was 24.5kDa. Thus, we conclude that the active form of native Nocardia GCH is a decamer. Our earlier incorrect conclusion was that the native enzyme was an octamer derived from the anomalous SDS-PAGE migration of the subunit.

Nocardia sp. carboxylic acid reductase: cloning, expression, and characterization of a new aldehyde oxidoreductase family.

We have cloned, sequenced, and expressed the gene for a unique ATP- and NADPH-dependent carboxylic acid reductase (CAR) from a Nocardia species that reduces carboxylic acids to their corresponding aldehydes. Recombinant CAR containing an N-terminal histidine affinity tag had K(m) values for benzoate, ATP, and NADPH that were similar to those for natural CAR, and recombinant CAR reduced benzoic, vanillic, and ferulic acids to their corresponding aldehydes. car is the first example of a new gene family encoding oxidoreductases with remote acyl adenylation and reductase sites.

GTP cyclohydrolase I: purification, characterization, and effects of inhibition on nitric oxide synthase in nocardia species.

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH4), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a Km for GTP of 6.5 micromoles. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56 degrees C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH4 inhibited enzyme activity by 25% at a concentration of 100 micromoles. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a Ki of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO2- plus NO3-. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH4.