姜黄素对血小板源性生长因子诱导的血管平滑肌细胞增殖及PTEN表达的影响

目的:研究在血小板源性生长因子（platelet-derived growth factor-BB,PDGF-BB）诱导下姜黄素对血管平滑肌细胞（vascular smooth muscle cell,VSMCs）增殖以及人第10号染色体缺失的磷酸酶及张力蛋白同源基因（phosphatase and tensin homology deletedon chromosome ten,PTEN）的影响，为侗族药物“姜黄”的临床应用提供更丰富的实验依据。方法:用20 ng/mL PDGF-BB孵育大鼠VSMCs 24 h，建立细胞增殖模型。MTT和CCK-8法观察姜黄素对VSMCs的细胞活性的影响，流式细胞术检测细胞周期，细胞划痕实验观察细胞迁移力，RT-PCR和Western blot观察PTEN mRNA、PTEN蛋白以及磷酸化PTEN（p-PTEN）蛋白的表达情况。结果:在20 ng/mL PDGF-BB的诱导下，10、30 μmol/L的姜黄素可显著抑制VSMCs增殖，以10 μmol/L姜黄素为最佳作用浓度，其不仅可以使细胞周期中G0/G1期比例明显升高，S期比例降低，还能显著抑制VSMCs迁移。同时可以上调PTEN mRNA和PTEN蛋白的表达，下调p-PTEN蛋白的表达。结论:姜黄素可以抑制PDGF-BB诱导的血管平滑肌细胞增殖与迁移，其对PTEN的影响，可能与上调PTEN mRNA和PTEN蛋白，下调p-PTEN蛋白相关。     

Biocompatibility of TiO2 nanotubes fabricated on Ti using different surfactant additives in electrolyte

This study examined the in vitro cell-material interactions on four different types of titanium surfaces: a polished Ti surface, TiO₂ nanotube surfaces fabricated in a fluorinated glycerol solution (TN), fluorinated glycerol solution with 1 wt% anionic surfactant sodium dodecyl sulphate (TN-SDS), and fluorinated glycerol solution with 1 wt% cationic surfactant cetyl trimethyl ammonium bromide (TN-CTAB), respectively. The surfaces exhibited distinct surface morphologies and geometrical features. Surface energy calculation shows that TN surface enhances the hydrophilic character by significantly increasing the surface energy. The osteoblast cell growth behavior on the four different surfaces was examined using the MC3T3-E1 cell line for 1 day. When the anodized surfaces were compared for the cell-materials interaction, each of the surfaces showed different properties that affected the cell–material interactions. Proliferation of the cells was noticed with distinctive cell-to-cell attachment on the TN surfaces. Good cellular adhesion with extracellular matrix extensions between the cells was noticed in the TN samples. The TiO₂ nanotubes grown in the surfactant-assisted fluorinated electrolyte did not show significant cell growth on the surface and some cell death was observed. The cell adhesion, differentiation and alkaline phosphatase activity were more pronounced on the TN surface. The MTT assays also revealed an increase in living cell density and proliferation on the TN surfaces. Overall, a rough surface morphology and surface energy are important factors for better cell material interactions.     

XIV. Yeast sequencing reports. A 43·5 kb segment of yeast chromosome XIV,which contains MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1, predicts an adenosine deaminase gene and 14 new open reading frames

A 43,481 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae was sequenced. A gene for tRNA^phe and 23 non-overlapping open reading frames (ORFs) were identified, seven of which correspond to known yeast genes: MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1. One ORF may correspond to the yet unindentified yeast adenosine deaminase gene. Among the 15 other ORFs, four exhibit known signatures, which include a protein tyrosine phosphatase, a cytoskeleton-associated protein and two ATP-binding proteins, four have similarities with putative proteins of yeast or proteins from other organisms and seven exibit no significant similarity with amino acid sequences described in data banks. One ORF is identical to yeast expressed sequence tags (EST) and therefore corresponds to an expressed gene. Six ORFs present similarities to human dbESTs, thus identifying motifs conserved during evolution. Nine ORFs are putative transmembrane proteins. In addition, one overlapping and three antisense ORFs, which are not likely to be functional, were detected. The sequence has been deposited in the EMBL data bank under Accession Number Z46843.     

Acclimation Temperature affects Activities of 5'-Nucleotidase and Acid Phosphatase and Lipid and Fatty Acid Composition in Carp Muscle Microsomes

The Km values of 5′-nucleotidase against IMP markedly increased below 6°C in the case of 32°C-acclimated carp muscle microsomes. This caused a break point at 6°C in the Arrhenius plot of 5′-nucleotidase activity against IMP at a low concentration. The discontinuity of temperature dependency was not found when a surfactant had been introduced. Muscle microsomes of carp acclimated at 32°C contained much more cholesterol and saturated fatty acid than those of 10°C-acclimated carp. Therefore, the difference of lipid composition seemed to be closely related to the change in kinetics of microsomal 5′-nucleotidase.     

IQ technology: an automated, high-throughput screening assay for kinase, phosphatase, and protease targets

IQ^® Technology, a homogeneous, universal-detection platform, originally designed for high-throughput screening (HTS) of kinases and phosphatases, has now been applied to protease screening. Representative enzymes from the major classes of proteases have been assayed in the IQ^® format. Enzyme activity and compound inhibition data are presented for such enzymes as Trypsin, Matrix Metalloproteinase 3 (MMP-3) and Calpain 1. The technology has been tested in 96- to 384- to 1536-well microplate formats and is universally suited for automated screening. IQ^® Technology is a direct, noncompetitive assay that does not require antibodies or radioisotopes. Fluorophore-labeled peptides are used as enzyme substrates. Kinase or phosphatase activity is quantified by direct measurement of the phosphorylation state of the substrate. For protease activity, cleavage is quantified with a peptide substrate containing a phospho-residue distal to the fluorphore. Cleavage of the substrate liberates the fluorphore-labeled terminus from the terminus containing the phospho-residue. Protease activity is measured by the change in fluorescence intensity that occurs when a proprietary compound binds specifically to phosphoryl groups on peptides and quenches the fluorescence. IQ^® Technology can be used with any peptide sequence and is insensitive to high concentrations of ATP and substrate. The IQ^® Technology has been validated against a large number of detergents, organics, and other reagents found in reaction mixtures and has been optimized for HTS applications exhibiting representative Z' values of 0.7.     

3-hydroxybenzene 1,2,4-trisphosphate, a novel second messenger mimic and unusual substrate for type-I myo-inositol 1,4,5-trisphosphate 5-phosphatase: Synthesis and physicochemistry

3-Hydroxybenzene 1,2,4-trisphosphate 4 is a new myo-inositol 1,4,5-trisphosphate analogue based on the core structure of benzene 1,2,4-trisphosphate 2 with an additional hydroxyl group at position-3, and is the first noninositol based compound to be a substrate for inositol 1,4,5-trisphosphate 5-phosphatase. In physicochemical studies on 2, when three equivalents of protons were added, the (31)P NMR spectrum displayed monophasic behaviour in which phosphate-1 and phosphate-2 behaved independently in most of the studied pH range. For compound 4, phosphate-2 and phosphate-4 interacted with the 3-OH group, which does not titrate at physiological pH, displaying complex biphasic behaviour which demonstrated co-operativity between these groups. Phosphate-1 and phosphate-2 strongly interacted with each other and phosphate-4 experienced repulsion because of the interaction of the 3-OH group. Benzene 1,2,4-trisphosphate 2 is resistant to inositol 1,4,5-trisphosphate type I 5-phosphatase catalysed dephosphorylation. However, surprisingly, 3-hydroxybenzene 1,2,4-trisphosphate 4 was dephosphorylated by this 5-phosphatase to give the symmetrical 2,3-dihydroxybenzene 1,4-bisphosphate 16. The extra hydroxyl group is shown to form a hydrogen bond with the vicinal phosphate groups at -15 degrees C, and (1)H NMR titration of the ring and hydroxyl protons in 4 shows the OH proton to be strongly stabilized as soon as the phosphate groups are deprotonated. The effect of the phenolic 3-OH group in compound 4 confirms a critical role for the 6-OH group of the natural messenger in the dephosphorylation mechanism that persists even in radically modified analogues.     

Functional characterization of the PP2C phosphatase CaPtc2p in the human fungal pathogen Candida albicans

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ion. There are seven PP2C‐like genes in Candida albicans. In this study, we demonstrate that CaPtc2p is a PP2C phosphatase. Surprisingly, in addition to the cytoplasmic localization, CaPtc2p is partially associated with mitochondria in yeast‐form and filamentous cells of C. albicans. Expression of CaPTC2 is developmentally regulated during the serum‐induced filamentation. Deletion of CaPTC2 renders C. albicans cells sensitive to SDS and azole antifungals, as well as the DNA methylation agent methylmethane sulphonate and the DNA synthesis inhibitor hydroxyurea. Therefore, CaPtc2p might fulfil multiple functions, including the regulation of mitochondrial physiology and checkpoint recovery from DNA damage in C. albicans cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of the disector method in the light microscopic quantification of type II pneumocytes in control and ozone-exposed rats

Alveolar type II cells in control and ozone-exposed rat lungs were counted at the light microscopical level with the ‘disector method’. The type II cells were unequivocally marked by histochemical staining for alkaline phosphatase activity in 2 μm plastic sections. By this counting method, the mean number of type II cells per lung in control rats was of the same magnitude as those reported in the literature, using point counting methods. After exposure of rats to 1.6 mg ozone/m³ for 7 days, a 50% increase in the mean number of type II cells was observed. The use of the disector method at the light microscopical level offers some advantages above a quantification at the electron microscopical level. The procedure is less time-consuming, larger areas can be screened, two parallel countings can be performed in one set of sections and there is no need for an exact knowledge about the diameter of the measured particle.     

DOGR1 and DOGR2: Two genes from Saccharomyces cerevisiae that confer 2-deoxyglucose resistance when overexpressed

Saccharomyces cerevisiae contains two genes (DOG^R1 and DOG^R2) that are able to confer 2-deoxyglucose resistance when they are overexpressed. These genes are very similar, sharing 92% identity at the protein level. They code for two isoenzymes with 2-deoxyglucose-6 phosphate (2-DOG-6P) phosphatase activity. These enzymes have been purified and characterized. Dog^R1p shows an optimum pH of 6, an optimum temperature of 30°C and a K_M on 2-DOG-6P of 17 mM. Dog^R2p shows a similar optimum pH, but the optimum temperature is 40°C and it exhibits a K_M on 2-DOG-6P of 41 mM. Both enzymes require 10 mM-MgCl₂ for maximal activity and they are inhibited by inorganic phosphate.