Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes

Endocytosis and intracellular sorting of transforming growth factor-β (TGF-β) receptors play an important regulatory role in TGF-β signaling. Two major endocytic pathways, clathrin- and caveolae-mediated endocytosis, have been reported to independently mediate the internalization of TGF-β receptors. In this study, we demonstrate that the clathrin- and caveolae-mediated endocytic pathways can converge during TGF-β receptor endocytic trafficking. By tracking the intracellular dynamics of fluorescently-labeled TGF-β type I receptor (TβRI), we found that after mediating TβRI internalization, certain clathrin-coated vesicles and caveolar vesicles are fused underneath the plasma membrane, forming a novel type of caveolin-1 and clathrin double-positive vesicles. Under the regulation of Rab5, the fused vesicles are targeted to early endosomes and thus deliver the internalized TβRI to the caveolin-1 and EEA1 double-positive early endosomes (caveolin-1-positive early endosomes). We further showed that the caveolin-1-positive early endosomes are positive for Smad3/SARA, Rab11 and Smad7/Smurf2, and may act as a multifunctional device for TGF-β signaling and TGF-β receptor recycling and degradation. Therefore, these findings uncover a novel scenario of endocytosis, the direct fusion of clathrin-coated and caveolae vesicles during TGF-β receptor endocytic trafficking, which leads to the formation of the multifunctional sorting device, caveolin-1-positive early endosomes, for TGF-β receptors.     

Alleviation of Salt Stress by Enterobacter sp. EJ01 in Tomato and Arabidopsis Is Accompanied by Up-Regulation of Conserved Salinity Responsive Factors in Plants

Microbiota in the niches of the rhizosphere zones can affect plant growth and responses to environmental stress conditions via mutualistic interactions with host plants. Specifically, some beneficial bacteria, collectively referred to as Plant Growth Promoting Rhizobacteria (PGPRs), increase plant biomass and innate immunity potential. Here, we report that Enterobacter sp. EJ01, a bacterium isolated from sea china pink (Dianthus japonicus thunb) in reclaimed land of Gyehwa-do in Korea, improved the vegetative growth and alleviated salt stress in tomato and Arabidopsis. EJ01 was capable of producing 1-aminocy-clopropane-1-carboxylate (ACC) deaminase and also exhibited indole-3-acetic acid (IAA) production. The isolate EJ01 conferred increases in fresh weight, dry weight, and plant height of tomato and Arabidopsis under both normal and high salinity conditions. At the molecular level, short-term treatment with EJ01 increased the expression of salt stress responsive genes such as DREB2b, RD29A, RD29B, and RAB18 in Arabidopsis. The expression of proline biosynthetic genes (i.e. P5CS1 and P5CS2) and of genes related to priming processes (i.e. MPK3 and MPK6) were also up-regulated. In addition, reactive oxygen species scavenging activities were enhanced in tomatoes treated with EJ01 in stressed conditions. GFP-tagged EJ01 displayed colonization in the rhizosphere and endosphere in the roots of Arabidopsis. In conclusion, the newly isolated Enterobacter sp. EJ01 is a likely PGPR and alleviates salt stress in host plants through multiple mechanisms, including the rapid up-regulation of conserved plant salt stress responsive signaling pathways.     

Leukocyte-mediated cell dissemination and metastasis: findings from multiple types of human tumors

Our previous studies revealed that leukocyte infiltration could trigger human breast and prostate tumor invasion through focal disruptions of the tumor capsule, which selectively favors monoclonal proliferation of tumor progenitors or a biologically more aggressive cell clone overlying the focal disruptions. Our current study, involving multiple types of human tumors, further shows that leukocyte infiltration could also trigger tumor metastasis through the following pathways: [1] more leukocytes migrate to focally disrupted tumor capsules, which forms leukocyte aggregates surrounding newly formed tumor cell clusters, [2] the physical movement of leukocytes into proliferating tumor cells disrupts the intercellular junctions and cell-surface adhesion molecules, causing the disassociation of tumor cells from the tumor core, [3] leukocytes are conjoined with some of these tumor cells through plasma membrane fusion, creating tumor cell-leukocyte chimeras (TLCs), and [4] the leukocyte of TLCs impart migratory capacity to associated tumor cell partners, physically dragging them to different tissue sites. Our findings suggest a novel pathway for tumor cell dissemination from the primary sites and the subsequent journey to new sites. Our findings also provide a unique explanation for the cellular mechanism of leukocytes on tumor invasion and metastasis. If confirmed, our hypothesis and technical approach may significantly facilitate early detection and intervention of tumor invasion and metastasis.     

Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

Molecular cloning, heterologous expression, and phylogenetic analysis of a novel y-type HMW glutenin subunit gene from the G genome of Triticum timopheevi.

A novel y-type high molecular weight (HMW) glutenin subunit gene from the G genome of Triticum timopheevi (2n=4x=28, AAGG) was isolated and characterized. Genomic DNA from accession CWI17006 was amplified and a 2200 bp fragment was obtained. Sequence analysis revealed a complete open reading frame including N- and C-terminal ends and a central repetitive domain encoding 565 amino acid residues. The molecular weight of the deduced subunit was 77,031, close to that of the x-type glutenin subunits. Its mature protein structure, however, demonstrated that it was a typical y-type HMW subunit. To our knowledge, this is the largest y-type subunit gene among Triticum genomes. The molecular structure and phylogenetic analysis assigned it to the G genome and it is the first characterized y-type HMW glutenin subunit gene from T. timopheevi. Comparative analysis and secondary structure prediction showed that the subunit possessed some unique characters, especially 2 large insertions of 45 (6 hexapeptides and a nonapeptide) and 12 (2 hexapeptides) amino acid residues that mainly contributed to its higher molecular weight and allowed more coils to be formed in its tertiary structure. Additionally, more alpha-helixes in the repeat domain of the subunit were found when compared with 3 other y-type subunits. We speculate that these structural characteristics improve the formation of gluten polymer. The novel subunit, expressed as a fusion protein in E. coli, moved more slowly in SDS-PAGE than the subunit Bx7, so it was designated Gy7*. As indicated in previous studies, increased size and more numerous coils and alpha-helixes of the repetitive domain might enhance the functional properties of HMW glutenins. Consequently, the novel Gy7* gene could have greater potential for improving wheat quality.     

PA2800 Plays an Important Role in Both Antibiotic Susceptibility and Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important human pathogen which causes a variety of infections. P. aeruginosa infections are often difficult to treat due to the pathogen’s resistance to many antibiotics. Previously, it has been reported that a transposon insertion mutant in gene PA2800 of P. aeruginosa PAO1 was more sensitive to tetracycline and ciprofloxacin. Further characterization of this gene, a vacJ homolog, in this study indicated that this gene plays an important role in both antibiotic susceptibility and virulence in P. aeruginosa. The role of PA2800 in antibiotic susceptibility probably signifies its involvement in maintaining outer membrane stability, similar to the role of vacJ in E. coli and Shigella flexneri. However, in contrast to vacJ in other bacteria, PA2800 also affects antibiotic susceptibility by affecting the expression of oprH in P. aeruginosa. As shown by in vivo studies using a Drosophila melanogaster infection model, significantly increased virulence was observed in the PA2800 mutant when compared to the wild type, and such a difference is likely a result of disrupted outer membrane stability and altered expression of znuA in the mutant. The role of PA2800 or vacJ in antibiotic susceptibility and pathogenicity seems to be unique in P. aeruginosa in which it affects both outer membrane stability as well as gene expression.     

A simple and sensitive method for lipoprotein and lipids profiles analysis of individual micro-liter scale serum samples

A simple and sensitive method to determine lipoprotein and lipids profiles in micro-liter scale individual serum sample is not presently available. Traditional lipoprotein separation techniques either by ultra-centrifugation or by liquid chromatography methods have their disadvantages in both lipoprotein separation and lipids component quantification. In this study we used small volume needing size-exclusion fast protein liquid chromatography to separate different lipoprotein subclasses in 50μL serum. And lipids contents, such as cholesterol, cholesterol ester and triacylglycerol, were measured by using two different fluorescence-based lipid detection methods. With this method, very low density lipoprotein, low density lipoprotein and high density lipoprotein could be easily separated, and follow-up lipid detection was completed by simple kinds of reactions. Serum lipoprotein and lipids profiling from C57BL/6 mice (n=5) and human (n=5) were analyzed. The elution profiles of five individuals were highly reproducible, and there were lipoprotein and lipids distribution variations between C57BL/6 mice and human beings. In conclusion, this method which combined small volume needing size-exclusion fast protein liquid chromatography and fluorescence-based lipids measurement, provided a simple, efficient, integrity and reproducible procedure for determining serum lipoprotein and lipids profiles in micro-liter scale levels. It becomes possible that determination of lipoprotein profiles and gaining information of lipids in different lipoproteins can be accomplished simultaneously.     

EP300 contributes to high-altitude adaptation in Tibetans by regulating nitric oxide production

The genetic adaptation of Tibetans to high altitude hypoxia likely involves a group of genes in the hypoxic pathway,as suggested by earlier studies.To test the adaptive role of the previously reported candidate gene EP300 (histone acetyltransferase p300),we conducted resequencing of a 108.9 kb gene region of EP300 in 80 unrelated Tibetans.The allele-frequency and haplotype-based neutrality tests detected signals of positive Darwinian selection on EP300 in Tibetans,with a group of variants showing allelic divergence between Tibetans and lowland reference populations,including Han Chinese,Europeans,and Africans.Functional prediction suggested the involvement of multiple EP300 variants in gene expression regulation.More importantly,genetic association tests in 226 Tibetans indicated significant correlation of the adaptive EP300 variants with blood nitric oxide (NO) concentration.Collectively,we propose that EP300 harbors adaptive variants in Tibetans,which might contribute to high-altitude adaptation through regulating NO production.     

铜绿假单胞菌多重耐药基因的筛选及鉴定

[目的]研究铜绿假单胞菌中与耐药性相关的基因.[方法]筛选转座突变体文库中对多种抗菌药物敏感的突变体,通过随机PCR、核苷酸测序及序列比对确定突变体中转座子的插入位点及其破坏的基因.[结果]筛选得到2株对多种抗菌药物敏感的突变体,其中被破坏的基因分别为功能未知的新基因PA2580和PA2800.[结论]PA2580和PA2800可能分别通过参与细胞氧化还原作用和细胞壁合成进而与铜绿假单胞菌耐药性相关.