Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells

Leptospermum flavescens Sm. (Myrtaceae), locally known as ‘Senna makki’ is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC₅₀ values of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml respectively. LF1 treated cells showed a sub-G₁ region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treatment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death.     

Influence of borate complexation on the electrophoretic behavior of 2-AA derivatized saccharides in capillary electrophoresis

A complete separation with baseline resolution of the 2-AA derivatized saccharides, including mono-, di-, and oligosaccharides, was achieved using 50 mM sodium phosphate-150 mM borate solution, pH 7.0 as running buffer by capillary electrophoresis. It was thought to be a result of the inclusion of 150 mM borate in the running electrolyte solution. The formation of borate complexes was observed by means of ¹¹B and ¹³C NMR spectroscopy and the electrophoretic mobilities of the various derivatives were calculated. It was found that steric factors play an important role in the stability of the formed borate complexes, which depends strongly on the configuration of the three vicinal hydroxyl groups at C-2, C-3, and C-4. 2-AA-Glc mainly forms stable 1,2-diester complexes with borate and 2-AA-Mal can form stable 1,2-monoesters. In turn, for 2-AA-Rib the formation of complexes is difficult to take place. The results implied that the configurational difference between the hydroxyl groups could cause the difference in formation of borate complexes leading to significant difference among saccharide molecules in their migration time on CE analysis.     

Rapid biochemical synthesis of 11C-labeled single chain variable fragment antibody for immuno-PET by cell-free protein synthesis

Immuno-PET is a promising approach for improved cancer diagnosis, by taking advantage of the high specificity of antibodies. Here, we present a novel cell-free protein synthesis method for preparing a positron emitter labeled-antibody. Functional anti-human EGFRvIII single chain Fv, MR1-1, was successfully labeled with carbon-11 (half-life = 20.4 min) in 5 min (36% yield) by the direct incorporation of the clinical PET tracer, l-[¹¹C]methionine. The product [¹¹C]MR1-1 was easily and rapidly isolated with high radiochemical purity (>95%) from the reaction solution, by affinity purification. This method would be widely applicable to the preparation of radiolabeled antibodies for PET imaging.     

Cell death of Nicotiana benthamiana is induced by secreted protein NIS1 of colletotrichum orbiculare and is suppressed by a homologue of CgDN3

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein-based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.     

Sulfation of the galactose residues in the glycosaminoglycan-protein linkage region by recombinant human chondroitin 6-O-sulfotransferase-1

6-O-Sulfated galactose residues have been demonstrated in the glycosaminoglycan-protein linkage region GlcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser isolated from shark cartilage chondroitin 6-sulfate (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027-6035). In this study, we investigated whether a recombinant human chondroitin 6-sulfotransferase-1 (C6ST-1) catalyzes the sulfation of C6 on both galactose residues in the linkage region using structurally defined acceptor substrates. The C6ST-1 was expressed as a soluble protein A chimeric form in COS-1 cells and purified using IgG-Sepharose. The purified C6ST-1 utilized the linkage tri-, tetra-, penta-, and hexasaccharide-serines and hexasaccharide alditols, including GlcUAbeta1-3GalNAc(4-O-sulfate)beta1-4GlcUAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O-Ser and DeltaGlcUAbeta1-3GalNAc(6-O-sulfate)beta1-4GlcUAbeta1-3Galbeta1-3Gal(6-O-sulfate)beta1-4Xyl-ol. Identification of the reaction products obtained with the linkage tetra-, penta-, and hexasaccharide-serines revealed that the C6ST-1 catalyzed the sulfation of C6 on both galactose residues in the linkage region. Notably, the linkage tetrasaccharide-peptide GlcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-(Gly)Ser-(Gly-Glu) was a good acceptor substrate for the C6ST-1, suggesting that the sulfation of the galactose residues can occur before the transfer of the first N-acetylhexosamine residue to the linkage tetrasaccharide. In contrast, no incorporation was observed into DeltaGlcUAbeta1-3GalNAc(4-O-sulfate)beta1-4GlcUAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xyl-ol, indicating that an intact xylose is necessary for the transfer of a sulfate to the second sugar residue Gal from the reducing end. These findings clearly demonstrated that the recombinant C6ST-1 catalyzes the sulfation of C6 on both galactose residues in the linkage region in vitro. This is the first identification of the sulfotransferase responsible for the sulfation of galactose residues in the glycosaminoglycan-protein linkage region.     

Metagenomic analysis reveals the contribution of anaerobic methanotroph-1b in the oxidation of methane at the Ulleung Basin,East Sea of Korea

We have previously identified a sulfate methane transition zone (SMTZ) within the methane hydrate-bearing sediment in the Ulleung Basin, East Sea of Korea, and the presence of ANME-1b group in the sediment has been shown by phylogenetic analysis of a 16S rRNA gene. Herein, we describe taxonomic and functional profiling in the SMTZ sample by metagenomic analysis, comparing with that of surface sediment. Metagenomic sequences of 115 Mbp and 252 Mbp were obtained from SMTZ and surface sediments, respectively. The taxonomic profiling using BLASTX against the SEED within MG-RAST showed the prevalence of methanogens (19.1%), such as Methanosarcinales (12.0%) and Methanomicrobiales (4.1%) predominated within the SMTZ metagenome. A number of 185,200 SMTZ reads (38.9%) and 438,484 surface reads (62.5%) were assigned to functional categories, and methanogenesis-related reads were statistically significantly overrepresented in the SMTZ metagenome. However, the mapping analysis of metagenome reads to the reference genomes, most of the sequences of the SMTZ metagenome were mapped to ANME-1 draft genomes, rather than those of methanogens. Furthermore, the two copies of the methyl-coenzyme M reductase gene (mcrA) segments of the SMTZ metagenome were clustered with ANME-1b in the phylogenetic cluster. These results indicate that ANME-1b reads were miss-annotated to methanogens due to limitation of database. Many of key genes necessary for reverse methanogenesis were present in the SMTZ metagenome, except for N⁵,N¹⁰-methenyl-H₄MPT reductase (mer) and CoB-CoM heterodisulfide reductase subunits D and E (hdrDE). These data suggest that the ANME-1b represents the primary player the anaerobic methane oxidation in the SMTZ, of the methane hydrate-bearing sediment at the Ulleung Basin, East Sea of Korea.     

Involvement of Mst1 in tumor necrosis factor-alpha-induced apoptosis of endothelial cells

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-α-induced apoptosis of ECs. Western blot analysis revealed that TNF-α induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-α-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting of propidium iodide-stained cells showed that TNF-α induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-α-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-α-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-α-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.     

Fertile Arabidopsis cyp704b1 mutant,defective in sporopollenin biosynthesis,has a normal pollen coat and lipidic organelles in the tapetum

The exine acts as a protectant of the pollen from environmental stresses, and the pollen coat plays an important role in the attachment and recognition of the pollen to the stigma. The pollen coat is made of lipidic organelles in the tapetum. The pollen coat is necessary for fertility, as pollen coat-less mutants, such as those deficient in sterol biosynthesis, show severe male sterility. In contrast, the exine is made of sporopollenin precursors that are biosynthesized in the tapetum. Some mutants involved in sporopollenin biosynthesis lose the exine but show the fertile phenotype. One of these mutants, cyp704b1, was reported to lose not only the exine but also the pollen coat. To identify the cause of the fertile phenotype of the cyp704b1 mutant, the detailed structures of the tapetum tissue and pollen surface in the mutant were analyzed. As a result, the cyp704b1 mutant completely lost the normal exine but had high-electron-density granules localized where the exine should be present. Furthermore, normal lipidic organelles in the tapetum and pollen coat embedded between high-electron-density granules on the pollen surface were observed, unlike in a previous report, and the pollen coat was attached to the stigma. Therefore, the pollen coat is necessary for fertility, and the structure that functions like the exine, such as high-electron-density granules, on the pollen surface may play important roles in retaining the pollen coat in the cyp704b1 mutant.