Genome-Wide Analyses of Recombination Suggest That Giardia intestinalis Assemblages Represent Different Species

Giardia intestinalis is a major cause of waterborne enteric disease in humans. The species is divided into eight assemblages suggested to represent separate Giardia species based on host specificities and the genetic divergence of marker genes. We have investigated whether genome-wide recombination occurs between assemblages using the three available G. intestinalis genomes. First, the relative nonsynonymous substitution rates of the homologs were compared for 4,009 positional homologs. The vast majority of these comparisons indicate genetic isolation without interassemblage recombinations. Only a region of 6 kbp suggests genetic exchange between assemblages A and E, followed by gene conversion events. Second, recombination-detecting software fails to identify within-gene recombination between the different assemblages for most of the homologs. Our results indicate very low frequency of recombination between the syntenic core genes, suggesting that G. intestinalis assemblages are genetically isolated lineages and thus should be viewed as separated Giardia species.     

Host defense peptides in skin secretions of Odorrana tiannanensis: Proof for other survival strategy of the frog than merely anti-microbial

Genus Odorrana, among all amphibians studied, is generally reported to have the most abundant and diversified anti-microbial peptides even from a single individual frog. In our previous work, 46 cDNA sequences encoding precursors of 22 different anti-microbial peptides (AMPs) were characterized from the skin of frog, Odorrana tiannanensis. In this work, we reported the purification of three AMPs from skin secretions of O. tiannanensis. Their amino acid sequences matched well with the sequences deduced from cDNAs and they were designated as Odorranain-C7HSa, Brevinin-1-OT2 and Odorranain-G-OT, respectively. Furthermore, we selected to analyze the four most structurally diversified sequences among the 22 AMPs that are significantly different from all reported AMPs. By structural characterization, three of them were designated as pleurain-E-OT, odorranain-G-OT, odorranain-A-OT, belonging to AMP families already identified. The forth one with a unique 14-mer sequence of AILTTLANWARKFLa and C-terminal amidation represents the prototypes of a new class of amphibian AMP, and thereby named tiannanensin. Such broad diversity in sequences and structures are consistent with other species in Genus Odorrana. Multi-functions of the synthesized four special AMPs were screened, including anti-microbial, antioxidant, cytotoxic and hemolytic activities. The results suggest that these AMPs may employ sophisticated mechanisms of action in host defense in addition to anti-microbial, although their precise contribution to host defense still seems unclear.     

培养时间对蛹拟青霉(Paecilomyces militaris)菌丝体中代谢产物的影响

[目的]通过测定不同培养时间蛹拟青霉(Paecilomyces militaris)菌丝体中次级代谢产物的变化,分析蛹拟青霉次级代谢产物与培养时间之间的关系.[方法]液体种子接入SDAY固体培养基 ;培养温度为25℃,培养周期为9d,从第2天开始每天取样 ;用甲醇与乙酸乙酯分别提取菌丝体中次级代谢产物,离心、过滤后合并提取液,用液质联用仪进行分析,用MetaboAnalyst software软件进行数据采集分析.[结果]主成分分析结果表明供试菌株在不同培养时间内其菌丝中次级代谢产物差异显著.聚类分析结果显示,供试拟青霉对生物碱、肽类和核苷等易形成阳离子类物质的代谢物可分为前中后三个阶段.供试拟青霉对糖类和有机酸等易形成阴离子类物质的代谢主要分为前后两个阶段.差异代谢物及热图分析结果表明,在培养的第2和第3天含量显著增加的代谢产物种类较多,主要有酯类及其水解产物、细胞破坏素B和拟青霉素,以及多种尚未鉴定的含氮化合物等 ;在培养第4和第5天含量显著增加的差异代谢物质种类较少,主要有细胞破坏素A和团囊虫草素等肽类抗菌杀虫物质 ;在培养第6天至第9天含量显著增加的代谢物种类较多,除多种白僵菌交酯和细胞破坏素等肽类抗生素外,显著增加的还有多种脂肪酸、氨基酸、鼠李糖、海藻糖、脑苷脂类化合物和核黄素等物质.[结论]培养时间对蛹拟青霉菌丝中次级代谢产物的产生有显著影响.在培养初期,菌体中酯类及细胞破坏素B和拟青霉素等含氮化合物的合成旺盛.在培养中期,次生代谢物明显减少,但细胞破坏素A2和团囊虫草素等肽类抗生素的合成却仍显著增多.在培养后期,供试蛹拟青霉除代谢出多种白僵菌交酯等肽类抗生素外,还大量产生有机酸、氨基酸和海藻糖等物质.虽然在整个培养过程中都有肽类抗生素产生,但这些抗生素并不相同,同时后一阶段新的抗生素的产生常常伴随着前一阶段的抗生素减少,因此前期的抗生素可能是后期产生的抗生素的前体.     

Macrophage-stimulated cardiac fibroblast production of IL-6 is essential for TGF β/Smad activation and cardiac fibrosis induced by angiotensin II

Interleukin-6 (IL-6) is an important cytokine participating in multiple biologic activities in immune regulation and inflammation. IL-6 has been associated with cardiovascular remodeling. However, the mechanism of IL-6 in hypertensive cardiac fibrosis is still unclear. Angiotensin II (Ang II) infusion in mice increased IL-6 expression in the heart. IL-6 knockout (IL-6-/-) reduced Ang II-induced cardiac fibrosis: 1) Masson trichrome staining showed that Ang II infusion significantly increased fibrotic areas of the wild-type mouse heart, which was greatly suppressed in IL-6-/- mice and 2) immunohistochemistry staining showed decreased expression of α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1) and collagen I in IL-6-/- mouse heart. The baseline mRNA expression of IL-6 in cardiac fibroblasts was low and was absent in cardiomyocytes or macrophages; however, co-culture of cardiac fibroblasts with macrophages significantly increased IL-6 production and expression of α-SMA and collagen I in fibroblasts. Moreover, TGF-β1 expression and phosphorylation of TGF-β downstream signal Smad3 was stimulated by co-culture of macrophages with cardiac fibroblasts, while IL-6 neutralizing antibody decreased TGF-β1 expression and Smad3 phosphorylation in co-culture of macrophage and fibroblast. Taken together, our results indicate that macrophages stimulate cardiac fibroblasts to produce IL-6, which leads to TGF-β1 production and Smad3 phosphorylation in cardiac fibroblasts and thus stimulates cardiac fibrosis.     

Macrophages Facilitate Coal Tar Pitch Extract-Induced Tumorigenic Transformation of Human Bronchial Epithelial Cells Mediated by NF-κB

Objective

Chronic respiratory inflammation has been associated with lung cancer. Tumor-associated macrophages (TAMs) play a critical role in the formation of inflammation microenvironment. We sought to characterize the role of TAMs in coal tar pitch extract (CTPE)-induced tumorigenic transformation of human bronchial epithelial cells and the underlying mechanisms.

Methods

The expression of TAMs-specific CD68 in lung cancer tissues and paired adjacent tissues from cancer patients was determined using immunostaining. Co-culture of human bronchial epithelial cells (BEAS-2B) and macrophage-like THP-1 cells were conducted to evaluate the promotive effect of macrophages on CTPE-induced tumorigenic transformation of BEAS-2B cells. BEAS-2B cells were first treated with 2.4 µg/mL CTPE for 72 hours. After removal of CTPE, the cells were continuously cultured either with or without THP-1 cells and passaged using trypsin-EDTA. Alterations of cell cycle, karyotype, colony formation in soft agar and tumor xenograft growth in nude mice of BEAS-2B cells at passages 10, 20 and 30, indicative of tumorigenecity, were determined, respectively. In addition, mRNA and protein levels of NF-κB in BEAS-2B cells were measured with RT-PCR and western blot, respectively. B(a)P was used as the positive control.

Results

The over-expression of TAMs-specific CD68 around lung tumor tissues was detected and associated with lung cancer progression. The tumorigenic alterations of BEAS-2B cells including increase in cell growth rate, number of cells with aneuploidy, clonogenicity in soft agar, and tumor size in nude mice in vivo occurred at passage 10, becoming significant at passages 20 and 30 of the co-culture following CTPE removal in compared to BEAS-2B cells alone. In addition, the expression levels of NF-κB in BEAS-2B cells were positively correlated to the malignancy of BEAS-2B cells under different conditions of treatment.

Conclusion

The presence of macrophages facilitated CTPE-induced tumorigenic transformation of BEAS-2B cells, which may be mediated by NF-κB.     

纳豆激酶液态发酵条件的研究

考察纳豆杆菌在7.5 L发酵罐中分批发酵产纳豆激酶的条件,纳豆激酶酶活采用四肽底物测定。结果表明:纳豆杆菌生长和产酶的适宜条件不一致。发酵过程中发酵罐搅拌转速控制为500 r/min不变,0～12 h时控制pH为8.0、温度为37℃;12～36 h调整pH为7.0、温度为30℃;16 h时补加外源C源,连续发酵36 h。该过程中发酵液中比酶活最高达到3 232 U/mL,与摇瓶发酵相比,比酶活提高了58%。     

氧化葡萄糖酸杆菌生物催化1,3-丙二醇合成3-羟基丙酸

3-羟基丙酸是一种潜在的重要化工产品,可作为中间体合成多种有经济价值的工业用化合物。文中利用氧化葡萄糖酸杆菌生物催化1,3-丙二醇合成3-羟基丙酸。首先在50 mL摇瓶中(转化体系为10 mL)考察细胞加入量、底物和产物浓度等对催化反应的影响。在此基础上,在2 L鼓泡塔中(转化体系为1 L),采取适当的补料方式和生物转化与分离相耦合的手段解除抑制,以提高目标产物终浓度。结果表明:高底物和产物浓度通过降低反应初速度抑制转化的进行,并确定了最佳催化反应条件为6 g/L菌体量,pH 5.5。利用流加补料方式维持反应体系中底物浓度在15~20 g/L,经过60 h的反应,3-羟基丙酸的浓度达到60.8 g/L,生产强度为1.0g/(L.h),转化率为84.3%。采用生物转化与分离相耦合的方法,经过50 h的转化反应,3-羟基丙酸的总产量达76.3 g/L,生产强度为1.5 g/(L.h),转化率83.7%。研究结果对利用氧化葡萄糖酸杆菌的不完全氧化醇类化合物特性实现其在工业生物催化中的应用具有一定的指导意义。     

Post-endocytic fates of delta-opioid receptor are regulated by GRK2-mediated receptor phosphorylation and distinct beta-arrestin isoforms

Once internalized, some G protein-coupled receptors (GPCRs) can recycle back to the cell surface, while some of them are delivered to lysosomes for degradation. Because recycling and degradation represent two opposing receptor fates, understanding the mechanisms that determine post-endocytic fate of GPCRs is of great importance. Our recent work has verified that agonist-induced internalization of delta-opioid receptor (DOR) employs both phosphorylation-dependent and -independent mechanisms in HEK293 cells. To investigate whether these two internalization mechanisms work differently in receptor regulation, we monitored receptor post-endocytic fates using flow cytometry, surface receptor biotinylation and radioligand binding assays. Results showed that the internalized wild type DOR could either recycle to the cell surface or be degraded. Mutant DOR M4/5/6, which lacks all three G protein-coupled receptor kinase 2 (GRK2) phosphorylation sites, could also internalize upon agonist challenge although in a reduced level as compared with the wild type counterpart. However, the internalized mutant DOR could not recycle back to the cell surface and all mutant DOR was degraded after internalization. Inhibition of GRK2 expression by GRK2 RNAi also strongly attenuated recycling of DOR. Furthermore, overexpression of GRK2, which significantly increased receptor phosphorylation and internalization, also targeted more internalized receptors to the recycling pathway. These data suggest that GRK2-catalyzed receptor phosphorylation is critically involved in DOR internalization and recycling, and the phosphorylation-independent internalization leads to receptor degradation. Data obtained from beta-arrestin1 and beta-arrestin2 RNAi experiments indicated that both beta-arrestin1 and beta-arrestin2 participate in phosphorylation-dependent internalization and the subsequent recycling of DOR. However, phosphorylation-independent internalization and degradation of DOR were strongly blocked by beta-arrestin2 RNAi, but not beta-arrestin1 RNAi. Taken together, these data demonstrate for the first time that GRK2 phosphorylation-dependent internalization mediated by both beta-arrestin1 and beta-arrestin2 leads DOR to recycle, whereas GRK2-independent internalization mediated by beta-arrestin2 alone leads to receptor degradation. Thus, the post-endocytic fate of internalized DOR can be regulated by GRK2-catalyzed receptor phosphorylation as well as distinct beta-arrestin isoforms.     

Liquid chromatography with multi-channel electrochemical detection for the determination of epigallocatechin gallate in rat plasma utilizing an automated blood sampling device

A liquid chromatography method with multi-channel electrochemical detection was developed for the determination of epigallocatechin gallate (EGCG) in rat plasma. After administration of EGCG, blood samples were periodically collected by Culex (an automated blood sampling robot). EGCG was extracted from 50 μl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 10 min using a C₈ (150×4.6 mm) 5 μm column with a mobile phase containing 20 mM sodium monochloroacetate, pH 2.8 and 12% acetonitrile at a flow-rate of 1.2 ml/min. A four-channel detector with glassy carbon electrodes was used with applied potentials of +700, 600, 500, 400 mV vs. Ag/AgCl. The limit of detection was 2 ng/ml at a signal-to-noise ratio of 3:1 and the limit of quantitation was 5 ng/ml. The calibration curve was linear over the range of 5–800 ng/ml. The intra- and inter-assay precisions were in the range of 1.3–4.5% and 2.2–4.4%, respectively. Using this method it was possible to determine plasma concentration following a single dose of EGCG to rats with good accuracy and precision. Thus the pharmacokinetic properties of EGCG in rats can be examined for intravenous, intraperitoneal and oral dosing.