The novel neutrophil differentiation marker phosphatidylglucoside mediates neutrophil apoptosis.

A new type of glycolipid, phosphatidylglucoside (PtdGlc), was identified as a component of raft-like membrane domains of the human leukemia cell line HL-60. In this study, we show that PtdGlc forms functional domains that are different from those produced by lactosylceramide (LacCer)-enriched lipid rafts. These rafts initiate neutrophil apoptosis. Neutrophils are the only type of human peripheral blood leukocyte or monocyte-derived dendritic cell to express large amounts of PtdGlc on their cell surfaces. PtdGlc was not colocalized with LacCer. Anti-PtdGlc IgM DIM21 did not induce neutrophil chemotaxis or superoxide generation, whereas anti-LacCer IgM T5A7 induced these activities. DIM21, but not T5A7, significantly induced neutrophil apoptosis. DIM21-induced apoptosis was inhibited by specific inhibitors of cysteine-containing aspartate-specific proteases (caspases)-8, -9, and -3 but not by the Src family kinase inhibitor PP1, PIP(3) kinase inhibitor LY294002, NADPH oxidase inhibitor diphenyleneiodonium, superoxide dismutase, or catalase. PtdGlc was colocalized with Fas on the neutrophil plasma membrane. DIM21 and the agonist anti-Fas Ab DX2 induced the formation of large Fas-colocalized clusters of PtdGlc on the plasma membrane. Furthermore, the antagonistic anti-Fas Ab ZB4 significantly inhibited DIM21-induced neutrophil apoptosis. These results suggest that PtdGlc is specifically expressed on neutrophils and mediates apoptosis of these cells, and that the Fas-associated death signal may be involved in PtdGlc-mediated apoptosis.

Involvement of cholesterol-enriched microdomains in class A scavenger receptor-mediated responses in human macrophages.

OBJECTIVE: Lipid rafts are cholesterol-enriched microdomains on cell membranes. We hypothesized that these microdomains could involve modified low-density lipoprotein (LDL) uptake. METHODS AND RESULTS: Co-localizations of cholesterol-enriched microdomains and CD204 during the uptake of acetyl LDL (AcLDL) and oxidized LDL were observed using Alexa488-labeled polyethylene glycol cholesteryl ester, which is a sensitive probe used to analyze the dynamics of cholesterol-rich lipid microdomains in living cells. The lipid raft disruptors, methyl-ß cyclodextrin and filipin, inhibited the uptake of AcLDL. CD204 siRNA treatments significantly reduced AcLDL uptake by 80%. We also demonstrated the presence of CD204 in the detergent-resistant membrane fraction (DRM) by immunoblotting analysis. The ratio of CD204/flotillin-1 in DRM was increased 11.5-fold by modified LDL administration. The PI3 kinase inhibitor LY294002, but not the Src kinase inhibitor PP1 or the Gαi/o inhibitor pertussis toxin, inhibited modified LDL uptake. The production of interleukin (IL)-8, but not CCL2, CXCL2, CXCL3, IL-6 or tumor necrosis factor-α was increased by AcLDL administration. The AcLDL-induced IL-8 production was inhibited by LY294002 and filipin. CONCLUSIONS: These data firstly demonstrated that PI3 kinase-associated cholesterol-enriched microdomains are involved in CD204-mediated modified LDL uptake in human macrophages. Cholesterol-enriched microdomains may play a critical role in inflammatory processes.

Antibody-specific aptamer-based PCR analysis for sensitive protein detection.

Nucleic acid amplification techniques were applied to the enzyme linked immunosorbent assay (ELISA) with an antibody-specific aptamer, R18. This novel detection system is a modification of the original immuno-polymerase chain reaction (immuno-PCR), but oligonucleotide-labeled antibodies are not required in the assay. This method is performed with the usual ELISA protocol, using an RNA aptamer for rabbit IgG instead of the conventional secondary antibody. After the assay plate was washed, quantitative reverse transcription (RT)-PCR was performed. Ribonuclease (RNase) inhibitors are not needed for this method. The detection limit of the quantitative RT-PCR is over 100 times more sensitive than the original ELISA method, even with the same sandwich-antibody combination. Only 1 mg of aptamer is sufficient for more than 10 million assays. This aptamer-based quantitative PCR successfully detected 16 attomoles (16 x 10(-18)) of vascular endothelial growth factor (VEGF). This is a cost-effective and easy method to increase the sensitivity of the rabbit antibody-based ELISA systems. The new method is referred to as immuno-aptamer PCR (iaPCR), to distinguish it from the original immuno-PCR.

Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica.

We cloned two forms of the secreted and thermostable luciferase genes, MpLuc1 and MpLuc2, from the marine copepod, Metridia pacifica. The 840-bp MpLuc1 cDNA comprised a 630-bp open reading frame encoding a 210-amino acid polypeptide (22.7 kDa). MpLuc1 had the closest homology with Metridia longa luciferase. The 753-bp MpLuc2 cDNA consisted of a 567-bp open reading frame (20.3 kDa), and it had the closest homology with Gaussia princeps luciferase. Single-specimen genomic PCR confirmed the presence of two luciferase genes in M. pacifica, and single-specimen RT-PCR revealed that both luciferase mRNAs were expressed. Both MpLuc1 and MpLuc2 (MpLucs) specifically reacted with the substrate coelenterazine producing identical bioluminescent spectra (lambdamax, 485 nm), but with different kinetics. Adding salt such as MgCl2 and CaCl2 to the reaction mixture significantly enhanced MpLuc1 and MpLuc2 activities. Wild-type MpLucs were remarkably thermostable; MpLuc1 retained about 60% of the original activity even after incubation at 90 degrees C for 30 min. MpLucs expressed in NIH-3T3 and HeLa cells were largely secreted into the culture medium. Continuous monitoring of secreted MpLuc1 driven by the c-fos promoter demonstrated the potential usefulness of MpLuc1 in nondisruptive reporter assays.

A novel yellowish-green fluorescent protein from the marine copepod, Chiridius poppei, and its use as a reporter protein in HeLa cells.

A crustacean gene, encoding for a new class of GFP-like protein, has been isolated from a cDNA library of the deep-sea (benthic) copepod crustacean, Chiridius poppei, by expression cloning. The cDNA library was constructed in a pBluescript II vector and screened using a non-UV transilluminator, obtaining a positive clone. The clone consisted of a 781-bp fragment of cDNA with a 660-bp open reading frame, which encoded for a 219-amino acid polypeptide with a calculated molecular mass of 24.7 kDa. The protein was overexpressed in Escherichia coli, purified to homogeneity by anion-exchange and size-exclusion chromatographies. The protein, CpYGFP, had excitation and emission maxima at 507 and 517 nm, respectively. CpYGFP existed as a dimer in solution and could be expressed either alone or as a fusion protein in HeLa cells. Dual labeling experiments carried out with CpYGFP-actin and DsRed2-Nuc demonstrated the usefulness of CpYGFP as a reporter in the subcellular localization of actin.

Structural characterization and artificial fiber formation of Bombyx mori silk fibroin in hexafluoro-iso-propanol solvent system.

High-resolution solution (13)C-NMR and CD studies of Bombyx mori silk fibroin revealed the presence of an ordered secondary structure 3(10)-helix, in hexafluoro-iso-propanol (HFIP). The solid-state structure of the silk fibroin film prepared by drying it gently from the HFIP solution still keep the structure, 3(10)-helix, which was studied with high-resolution solid state (13)C-NMR. The structural transition from the 3(10)-helix to silk II structure, heterogeneous structure including antiparallel beta-sheet, occurred during the artificial spinning from the HFIP solution. The wide-angle x-ray diffraction and differential scanning calorimetry thermograms of the artificial spinning fiber after postspinning treatments were observed together with the stress-strain curves. The results emphasize that the molecular structures, controlled morphology, and mechanical properties of the protein-based synthetic polymers can be modulated for enhancing biocompatibility.

PPARgamma activation and adipocyte differentiation induced by AS-6, a prenyl-phenol antidiabetic antibiotic.

The prenyl-phenol antibiotics ascochlorin-related compounds, are known to reduce serum cholesterol and triglyceride, suppress hypertension, and ameliorate types-I and II diabetes. However, little is known about the molecular mechanism for these physiological effects. Here we report that the ascochlorin derivative, 4-O-carboxymethyl ascochlorin (AS-6) acts as a potent activator of the nuclear hormone receptor, PPARgamma, although it does not activate the related receptors, PPARalpha, PPARdelta or RARalpha. AS-6 interacts directly with the PPARgamma molecule in vitro, and induces differentiation of the mouse preadipocyte cell line 3T3-L1. Our results suggest that AS-6 is a partial agonist for PPARgamma with a novel chemical structure.