Cloning and characterization of a human cDNA ACAD10 mapped to chromosome 12q24.1

Mitochondrial fatty acid -oxidation is an important energy resource for many mammal tissues. Acyl-CoA dehydrogenases (ACADs) are a family of flavoproteins that are involved in the -oxidation of the fatty acyl-CoA derivatives. Deficiency of these ACADs can cause metabolic disorders including muscle fatigue, hypoglycaemia, hepatic lipidosis and so on. By large scale sequencing, we identified a cDNA sequence of 3960 base pairs with a typical acyl-CoA dehydrogenase function domain. RT-PCR result shows that it is widely expressed in human tissues, especially high in liver, kidney, pancreas and spleen. It is hypothesized that this is a novel member of ACADs family. Abbreviations: ACADs – acyl-CoA dehydrogenases, FAD – flavinadenine dinucleotide, SCAD – short-chain acyl-CoA dehydrogenase,MCAD – medium-chain acyl-CoA dehydrogenase, LCAD – long-chain acyl-CoAdehydrogenase, VLCAD – very long- chain acyl-CoA dehydrogenase, IVD –isocalery-CoA dehydrogenase, SBCAD – short/branched chain acyl-CoAdehydrogenase, GCD – glutaryl- CoA dehydrogenase, ETF – electron transferflavoprotein, ACAD8 – acyl-CoA dehydrogenase 8, ACAD9 – acyl-CoAdehydrogenase 9, ACAD10 – acyl-CoA dehydrogenase 10.     

Isolating high-quality RNA from mangroves without liquid nitrogen

Mangroves form unique communities in tropical coastal regions and tidal lowlands. Isolating RNA from mangrove leaves is difficult because of high amounts of secondary metabolites and polysaccharides. Conventional extraction methods produce poor-quality mangrove RNA. We present a simple, fast, and convenient protocol for isolating RNA from 5 mangrove species:Aegiceras corniculatum, Bruguiera gymnorrhiza, Ceriops tagal, Kandelia candel, andSonneratia apetala. Isolating RNA from other mangrove species is also possible. Obtained RNA was of high quality and used in an RT-PCR reaction that amplified 0.6 kb of theA. corniculatum CPI-1 gene.     

P2X7 receptor-mediated apoptosis of human cervical epithelial cells

Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X₇ receptor analog 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 µM in hECE cells and 1 µM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-, it had only a mild effect on caspase-8. Both BzATP and TNF- activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was 0.5 µM, which is in the range of values that suffice to activate the P2X₇ receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X₇ receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway. cervix; epithelium; ATP; 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate     

Synthesis of polyhydroxysterols (IV): synthesis of 24-methylene-cholesta-3beta,5alpha,6beta,19-tetrol, a cytotoxic natural hydroxylated sterol

The cytotoxic, polyhydroxylated sterol 24-methylene-cholesta-3beta,5alpha,6beta,19-tetrol (1), previously isolated from the soft corals Nephthea albida and N. tiexieral verseveldt, was synthesized using stigmasterol as the starting material by 10 steps in 9% overall yield. The spectral data and physical constants of 1 were identical with those of the natural product. This is the first report of the synthesis of 1.     

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors

Inhibitors of receptor tyrosine kinases are implicated as therapeutic agents for the treatment of many human diseases including cancer, inflammation and diabetes. Cell-based assays to examine inhibition of receptor tyrosine kinase mediated intracellular signaling are often laborious and not amenable to high-throughput cell-based screening of compound libraries. Here we describe the development of a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the activation and inhibition of ligand-induced phosphorylation of the colony-stimulating factor-1 receptor (CSF-1R) in 96-well microtiter plate format. The assay involves the capture of the Triton X-100 solubilized human CSF-1R, from HEK293E cells overexpressing histidine epitope-tagged CSF-1R (CSF-1R/HEK293E), with immobilized CSF-1R antibody and detection of phosphosphorylation of the activated receptor with a phosphotyrosine specific antibody. The assay exhibited a 5-fold increase in phosphorylated CSF-1R signal from CSF-1R/HEK293E cells treated with colony-stimulating factor (CSF-1) relative to treated vector control cells. Additionally, using a histidine epitope-specific capture antibody, this method can also be adapted to quantify the phosphorylation state of any recombinantly expressed, histidine-tagged receptor tyrosine kinase. This method is a substantial improvement in throughput and quantitation of CSF-1R phosphorylation over conventional immunoblotting techniques.     

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1

In plants, Glycoside Hydrolase (GH) Family 1 -glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thalianagenome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron–exon organizations. The forty-eighth member of this family (At3g06510; sfr2) is a -glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44-encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for -mannosides including 1,4--D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm -mannosidase and barley seed -glucosidase/-mannosidase BGQ60.     

Millimeter wave exposure reverses TPA suppression of gap junction intercellular communication in HaCaT human keratinocytes

The effect of 30.16 GHz millimeter wave (MMW) exposure at 1.0 and 3.5 mW/cm2 on gap junction intercellular communication (GJIC) was studied in cultured HaCaT keratinocytes, using the fluorescence recovery after photobleaching (FRAP) technique and laser confocal scanning microscopy to follow the intracellular movement of 5,6-carboxyfluorescein diacetate dye. While MMW exposure alone for 1 h at either 1.0 or 3.5 mW/cm2 did not affect GJIC, MMW exposure in combination with 5 ng/ml TPA treatment reversed TPA induced suppression of GJIC. Exposure at 1.0 mW/cm2 resulted in a partial reversal, and exposure at 3.5 mW/cm2 resulted in essentially full reversal of the TPA suppression.