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BACKGROUND: MicroRNA-210 (miR-210) is an oncogenic miRNA previously associated with prognosis in human gliomas, an incurable tumour type of the central nervous system. Here miR-210 was investigated as a potential serum biomarker in the diagnosis and prognosis of glioma. METHODS: Serum was immediately prepared from blood samples collected from patients with glioma grades I-IV at primary diagnosis (n=136) and healthy controls (n=50) from February 2007 to March 2014 in the Department of Neurosurgery of the First Affiliated Hospital of Wannan Medical College (Wuhu, China). Total RNA was isolated from serum. cDNA was synthesised with primers specific for miR-210 and miR-16-1 (internal control), and quantitative real-time RT-PCR was performed. Results were statistically analysed to determine the role of miR-210 in the diagnosis and prognosis of human glioma patients. RESULTS: An approximately seven-fold increase in miR-210 expression was detected in serum samples from glioblastoma patients relative to healthy controls. A threshold expression value (2.259) was chosen from receiver operator characteristic curves (ROC), and the low and high miR-210 expression groups were analysed by multivariate Cox proportional hazard regression and Kaplan-Meier analyses. Results revealed an association of high serum miR-210 expression with tumour grade and poor patient outcome (P-values <0.001). CONCLUSIONS: Serum miR-210 is a promising diagnostic and prognostic biomarker that can be detected in the peripheral blood of patients with glioma.

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Three new sesquiterpenoids were obtained from the leaves of Magnolia grandiflora L. Their structures were determined as 6 alpha,11-dihydroxy-12,13-diacetoxyelem-1,3-diene, 4 alpha,6 alpha,10 alpha-trihydroxy-13-acetoxyguaia-11-ene, and 12,13-diacetoxyguaia-4 alpha,6 alpha,10 alpha,11-tetraol on the basis of spectral evidence. In addition, the known sesquiterpenoid magnograndiolide was also obtained.

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Two new sesquiterpenoids, 4,5-epoxy-13-methoxy-1(10)-germacren-12,6-olide and 4,5-epoxy-13-acetoxy-1(10)-germacren-12,6-olide, were isolated from the leaves of Magnolia grandiflora, together with six known compounds, 2alpha-hydroxy-dihydroparthenolide, parthenolide, costunolide, syringaresinol, (+) medioresinol and 6,7-dimethoxycoumarin. The structures of the new compounds were elucidated on the basis of spectroscopic methods and X-ray diffraction.

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Four ent-pimarene diterpenoids. ent-18-acetoxy-8(14)-pimarene-15S, 16-diol, ent-18-acetoxy-16-hydroxy-8(14)-pimaren-15-one, ent-16,18-dihydroxy-8(14)-pimaren-15-one and ent-19-nor-4,16,18-trihydroxy-8(14)-pimaren-15-one, together with three known damarane triterpenoids, richenoic acid, eichleriainic acid and shoreic acid were isolated from the bark of Dysoxyhum hainanense Merr. Their structures were elucidated on the basis of spectroscopic techniques. The absolute configurations of four diterpenoids were assigned as ent-pimarene type by chemical transformation and by co-occurrence in the plant as well as by negative optical rotations for four compounds.

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Nitric oxide (NO) acts as a signal molecule in the nervous system, as a defense against infections, as a regulator of blood pressure, and as a gate keeper of blood flow to different organs. In vivo, it is thought to have a lifetime of a few seconds. Therefore, its direct detection at low concentrations is difficult. We report on a new type of hybrid, organic-semiconductor, electronic sensor that makes detection of nitric oxide in physiological solution possible. The mode of action of the device is described to explain how its electrical resistivity changes as a result of NO binding to a layer of native hemin molecules. These molecules are self-assembled on a GaAs surface to which they are attached through a carboxylate binding group. The new sensor provides a fast and simple method for directly detecting NO at concentrations down to 1 microM in physiological aqueous (pH=7.4) solution at room temperature.

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A new triterpenoid, 1 alpha,7 alpha-diacetoxyapotirucall-14-en e-3 alpha, 21,22,24,25-pentaol (1), and the two known compounds odoratone (2) and 2 beta,3 beta,4 beta-trihydroxypregnan-16-o ne (3) were isolated from a methanolic extract of the seed kernels of Azadirachta indica. Their structures were elucidated on the basis of spectral methods.

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Four tirucallane derivatives, 3beta,22S-dihydroxy-tirucalla-7,24-dien-23-one, 22,23-epoxy-tirucalla-7-ene-3beta,24,25-triol, 3beta,25-dihydroxy-tirucalla-7,23-diene, 23,26-dihydroxy-tirucalla-7,24-dien-3-one, together with two known triterpenoids, 24,25-epoxy-3beta,23-dihydroxy-7-tirucallene, tirucalla-7,24-diene-3beta,23-diol, were isolated from Dysoxylum hainanense. Their structures were elucidated on the basis of spectroscopic evidence.

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Five new tetranortriterpenoids-walsurin (1), isowalsuranolide (2), walsuranolide (3), 11beta-acetoxywalsuranolide (4), and 20, 22-dihydro-22,23-epoxywalsuranolide (5)-and three new natural tetranortriterpenoids-11beta-hydroxydihydrocedrelone (6), 11beta-acetoxydihydrocedrelone (7), and 11beta-hydroxycedrelone (8)-together with a known tetranortriterpenoid, cedrelone (9), were isolated from the bark of Walsura yunnanensis. The structures of 1-8 were determined on the basis of spectral evidence.

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A novel monoterpenoid-derived metabolite, paeonilide, was isolated from the roots of Paeonia delavayi. Its structure was established by a combination of spectroscopic and X-ray crystallographic analyses. It showed an anti-PAF effect with an IC50 value of ca. 8 microg/ml.

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Two novel secoergosterols, 3beta-hydroxy-8alpha,9alpha-oxido-8, 9-secoergosta-7,9(11),22-triene (tylopiol A) (1) and 3beta-hydroxy-8alpha,9alpha-oxido-8,9-secoergosta-7,22 -dien-12-one (tylopiol B) (2), were isolated from the fresh fruit bodies of Tylopilus plumbeoviolaceus, along with three known compounds, ergosta-7,22-dien-3beta-ol, uridine, and allitol. Their structures were elucidated by NMR techniques, including (1)H NMR, (13)C NMR, HMQC, HMBC, and MS. The structure and stereochemistry of compound 1 were demonstrated by X-ray crystallography.

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Four compounds were isolated from dry seeds of Cipadessa baccifera (Roth) Miq. along with the known 2beta,3beta,4beta-trihydroxypregnan-16-one, febrifugin, and khaysin T. Their structures were elucidated on the basis of spectral analysis to be cipadesin, 17alpha,20R-dihydroxypregnan-3,16-dione, 1,4-epoxy-16-hydroxyheneicos-1,3,12,14,18-pentaene and 1,4-epoxy-16-hydroxyheneicos-1,3,12,14-tetraene.

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Two new compounds, cedrellin (1) and 2,6,10,15-phytatetraene-14-ol (2), together with five known compounds, 7 alpha-obacunyl acetate, 6-acetoxyobacunol acetate, 7 alpha-acetoxydihydronomilin, 2,6,10-phytatriene-1,14,15-triol and phytol were isolated from leaves of Cedrela sinensis. Their structures were elucidated on the basis of combined one- and two-dimensional spectral techniques.

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In order to study the relationship between the structure of A-nor-5 alpha-androstane derivatives and their antifertility activity, we designed and synthesized 16 A-nor-5 alpha-androstane compounds through several reaction steps with dehydroepiandrosterone acetate as a starting material. Their structures were confirmed by IR, 1HNMR, MS, elemental analyses, etc. Preliminary pharmacological tests showed that compounds 8, 9, 10 and 16 possess antiimplantation activity to some extent (2.5 mg.kg-1, administered po, gave 67-75% antiimplantation rate). Other compounds showed low activity. The possible relationship between compound structures and their activities is analysed briefly.

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The subfamily of guanine nucleotide-binding regulatory (G proteins) designated Gq has been shown to regulate the activity of phospholipase C by reconstitution. However, the role of these proteins in hormonal regulation of this activity has not been demonstrated. Two antisera were used in attempts to interrupt this pathway. Antiserum W082, developed against a peptide representing an internal sequence in alpha q, was specific for alpha q by immunoblots but did not recognize the native protein. Antiserum X384 was developed against a peptide representing the 12 amino acids of the common carboxyl termini of alpha q and alpha 11. It had a broader specificity for this subfamily of G protein alpha subunits and recognized the native proteins. Antiserum X384 specifically immunoprecipitated alpha q and its homologs from purified preparations and detergent extracts of membranes. Affinity-purified antibodies attenuated stimulation of phosphatidylinositide 4,5-bisphosphate hydrolysis by bradykinin, angiotensin, and histamine in membranes derived from NG108-15 cells, rat liver, and 1321N1 cells, respectively. Activation of the phospholipase C activity by guanosine 5'-3-O-(thio)triphosphate alone was also inhibited. Inclusion of the peptide to which the antisera were raised blocked the effect of the antibody. In contrast, affinity-purified W082, which did not recognize native proteins, did not alter regulation of phospholipase C. This indicates that the Gq family of signaling proteins can couple to several receptors and is responsible for the hormonal regulation of phospholipase C in these diverse systems. The further generality of this regulatory pathway remains to be established.

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Bacteriophage T4 gene 17 amplification mutants (Hp17) selected by growth of gene 17 amber mutants on ochre suppressor strains of Escherichia coli carry two to more than sixfold tandem head-to-tail repeats of the gene 17-18 region (Wu & Black, 1987). We characterized the structures of Hp17 isolates by restriction enzyme mapping and Southern blot analysis. The left and right boundaries of the amplified sequences were mapped within genes 16 and gene 18 or 19, respectively. The TaqI-restriction fragments containing the novel junctions arising from fusion of the amplified gene were then cloned and sequenced. Three Hp17 mutants arose from rearrangement in one five base-pair (bp) block within a G + C-rich region of partial homology (24 bp with 4 mismatches) between genes 16 and 19. Moreover, an oligonucleotide probe showed that 190/191 mutants isolated had recombined within the 5 bp block, and other rearrangements within this 24 bp region were not detected. Only one anomalous Hp mutant rearranged elsewhere between genes 16 and 18 in a 14 bp homology region with one mismatch. Elimination of gene alt of phage T4 is required for isolation of Hp17 mutants, apparently because more DNA can be packaged into alt- heads. Requirements for the dispensable replication and recombination genes of T4 were probed; T4 topoisomerase (39, 52, 60), primase (58/61), and uvsX are required, whereas the host recA gene and T4 denV gene do not appear to be required for isolation of the Hp17 mutants. The evidence suggests an initiating sequence-specific rearrangement leads to the T4 Hp17 amplification mutants.

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An epidermal growth factor (EGF) receptor monoclonal antibody (mAb), mAb LA22, was used to analyze the covalent coupling of human EGF receptors to mouse EGF by the amine-reactive cross-linking agent disuccinimidyl suberate. A soluble Mr 105,000 truncated form of the receptor secreted by A-431 epidermoid carcinoma cells and consisting of the ligand-binding extracellular domain was cross-linked to 125I-labeled EGF. Digestion of this complex with an endoproteinase that specifically cleaves at the COOH side of glutamyl residue released a single radiolabeled glycosylated fragment of Mr 18,000 that reacted with mAb LA22. As the epitope for mAb LA22 resided between Ala-351 and Asp-364 of the mature receptor, this result localized the cross-linked receptor residue(s) to the 47-amino acid interval from Phe-321 to Glu-367. The receptor residue(s) involved in the covalent coupling of rat 125I-labeled transforming growth factor alpha was similarly localized to this region of the receptor. This receptor interval, which included two glycosylated asparaginyl residues at positions 328 and 337, contained but three amino acid residues that were potentially reactive with disuccinimidyl suberate: Lys-332, Lys-333, and Lys-336. Characterization of mAb LA22-reactive 125I-EGF-labeled receptor fragments generated by an endoproteinase specific for the COOH side of lysyl residue placed the NH2 termini of the two smallest fragments between the glycosylated residues Asn-328 and Asn-337. These results indicated that disuccinimidyl suberate cross-linked the NH2 group of EGF residue Asn-1 to the human EGF receptor residue Lys-336. Our results further suggest that EGF and transforming growth factor alpha, two members of the EGF family of peptide growth factors, interact with closely apposed or identical features of the receptor.

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Epitopes recognized by three epidermal growth factor (EGF) competitive monoclonal antibodies, LA22, LA58, and LA90, have been localized to a 14-amino acid region in the extracellular domain of the human EGF receptor. The binding of each of these mutually competitive antibodies to A431 epidermoid carcinoma cells was inhibited up to 87% by EGF. Furthermore, binding to A431 cells was inhibited 100% by the EGF competitive monoclonal antibody 528 IgG. The EGF receptor monoclonal antibody 455 IgG, which recognizes a blood group A-related carbohydrate modification of A431 receptors and does not inhibit EGF binding, did not inhibit the binding of these three antibodies to A431 cells. Antibodies LA22, LA58, and LA90 were unusual in that they bound to recognized denatured and endoglycosidase F-treated antigenic determinants in Western blots. This suggested that the antibodies recognized continuous peptide epitopes. The epitopes for these antibodies were first localized in cyanogen bromide- and V8 protease-generated fragments of a truncated form of the EGF receptor secreted by A431 cells. In experiments with synthetic peptides, all three antibodies were found to bind to the 14 amino acids from Ala-351 to Asp-364 of the mature human EGF receptor. These amino acids are located between the two Cys-rich regions of the extracellular domain of the receptor, and they include an Arg-Gly-Asp-Ser recognition site for adhesion molecule receptors. The homologous sequence in the chicken EGF receptor, which binds mouse EGF with a 100-fold lower affinity than the human EGF receptor, contains four amino acid differences including two in the Arg-Gly-Asp-Ser tetramer. The mutually competitive binding of EGF and antibodies LA22, LA58, and LA90 implied that the amino acids between Ala-351 and Asp-364 participated in the formation of the EGF-binding site of the human EGF receptor.

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Bacteriophage T4 mutants hyperproducing gene 17 protein (Hp17) have been isolated at high frequency by growing gene 17 amber mutants on ochre suppressor strains of Escherichia coli. Most mutants showed the co-hyperproduction of gene 18 protein, although one anomalous mutant hyperproduced a 60,000 Mr partial polypeptide of gene 18. Hybridization of T4 late RNAs to cloned plasmid DNA containing genes 17, 18 or control T4 genes revealed that approximately five times more gene 17 mRNA and two to three times more gene 18 mRNA were synthesized in the Hp17 mutant infections. DNA-DNA hybridizations showed that Hp17 mutant DNA contained two to three times more copies of genes 17 and 18 than wild-type DNA. Southern blot analysis suggested that Hp17 mutants carry a direct tandem repeat of the gene 17-18 region, with variable copy number from one to at least six copies. Hyperproduction of gene 17 and 18 proteins appears therefore to result from gene amplification specific to the gene 17-18 region. In contrast to gene duplications reported in lambda and T4 phage, and numerous cases of gene amplification in bacteria, a similar or identical novel junctional fragment created by the amplification event was observed among 28 independent T4 Hp17 isolates; therefore, the mechanism giving gise to amplified sequences may involve specific sequences in this region of the T4 genome.

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