树鼩IL-2全长编码序列的克隆及分子特征分析

树鼩作为多种人类疾病模型已受到广泛关注,而免疫因子对于树鼩模型评价至关重要,但目前对其白细胞介素-2(IL-2)的研究鲜有报道。该实验以经ConA(concanavalin)诱导培养的树鼩淋巴细胞总RNA为模板,RT-PCR克隆出465bp的树鼩IL-2全长编码序列,并采用ClustalW软件分析其序列和分子特征。结果表明树鼩IL-2cDNA编码一个由154个氨基酸组成的蛋白质,其cDNA及氨基酸序列与人的同源性分别为93%及80%,且其整体结构与人IL-2相似。MEGA5.0软件构建的进化树表明,树鼩与人及恒河猴的亲缘关系较近。Pymol软件对树鼩和人IL-2氨基酸序列进行的三维结构模建表明,两者的IL-2分子三维空间结构基本相似,表面大部分区域所带电荷相同,但在某些区域差异较大,且树鼩多出一个糖基化位点,这些差异对抗体的结合可能存在影响。该研究为今后树鼩IL-2单克隆抗体的制备及功能研究奠定了基础。     

Involvement of 15-lipoxygenase in the inflammatory arthritis

15-Lipoxygenase (15-LOX) is involved in many pathological processes. The aim of this study is to examine the role of 15-LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. It was found that treatment of 15-LOX downstream product of 15-(S)-HETE (15-S-hydroxyeicosatetraenoic acid) increased the mRNA and protein levels of MMP-2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15-(S)-HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF-κB inhibitor). Treatment of 15-(S)-HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF-α and IL-1β are the key cytokines involved in arthritis and also increase the activity of MMP-2 in RASF, which was antagonized by pretreatment with 15-LOX inhibitor PD146176 or knockdown of 15-LOX. It was also found that these two cytokines increased the expression of 15-LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15-(S)-HETE-induced expression of MMP-2. In comparison with wild-type mice, adjuvant-induced arthritis and MMP-2 expression in synovial membrane were markedly inhibited in 15-LOX knockout (KO) mice. These results indicate that 15-LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF-α and IL-1β. 15-LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis.     

Skin Autofluorescence Is Associated with Endothelial Dysfunction in Uremic Subjects on Hemodialysis

Background

Elevated levels of advanced glycation end products (AGEs) within tissues may contribute to endothelial dysfunction, an early indicator of atherosclerosis. We aimed to investigate whether levels of skin AGEs could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.

Methods and Results

One hundred and nineteen uremic patients on hemodialysis and 57 control subjects with moderate-to-high cardiovascular risk factors and without chronic kidney disease (CKD) were enrolled. We used ultrasound to measure flow-mediated vasodilation (FMD). An AGE reader measured skin autoflurorescence (AF). We then compared differences in FMD and skin AF values between the two groups. The uremic subjects had significantly higher levels of skin AF (3.47±0.76 AU vs. 2.21±0.45 arbitrary units; P<0.01) and significantly lower levels of FMD (4.79%±1.88% vs. 7.19%±2.17%; P<0.01) than the non-CKD subjects. After adjusting for all potential covariates, we found that skin AF level independently predicted FMD in both the hemodialysis and the non-CKD groups. In the hemodialysis group, skin AF ≥ 3.05 arbitrary units predicted abnormal FMD at a sensitivity of 87.9% and a specificity of 78.6% (P<0.01).

Conclusions

Skin AF could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.     

A Report on the Chromosome Numbers of Chinese Umbelliferae

Chromosome numbers are reported for 26 species and varieties of Umbelliferae which belong to 3 subfamilies and 19 genera in this paper. Of these, 13 counts are new records and some problems about chromosome numbers of Umbelliferae are simplydiscussed.     

复合稻田生态系统温室气体交换及其综合增温潜势

研究稻田CO2、CH4、N2O等温室气体的综合增温潜势,有助于科学评价复合稻田生态系统在减少温室气体排放和减缓全球变暖方面的作用,为稻鸭、稻鱼复合种养模式的发展提供依据。2006年采用静态箱法研究了养鸭稻田（RD）、养鱼稻田（RF）和常规淹水稻田（CK）的CH4、N2O的排放量。水稻整个生育期间,RD、CK和RF的CH4排放量分别是19.11、26.71g/m^2和25.01g/m^2;N2O排放量分别是0.237、0.229、0.237g/m^2。采用干物质积累法测得,水稻整个生长期内RD处理地上稻株对CO2的净固定量为2766.4g/m2,RF为2759.59g/m^2,CK为2533.9g/m^2。采用土壤有机碳库的变化估算土壤CO2净交换通量,水稻整个生育期间,三类稻田土壤亚系统均表现为对CO2的净固定,相当于固定CO2量分别为RD675.55g/m^2、CK575.43g/m^2、RF562.62g/m^2。三类稻田温室气体的交换均表现为CO2的净吸收、CH4、N2O的净排放,综合增温潜势以RD为最低。稻田养鸭能显著减少甲烷排放,降低增温潜势,其减缓综合温室效应的潜力是常规淹水稻田的1.6倍左右。     

Genetically engineered biosynthesis of macrolide derivatives including 4-amino-4,6-dideoxy-L-glucose from Streptomyces venezuelae YJ003-OTBP3

Two sugar biosynthetic cassette plasmids were used to direct the biosynthesis of a deoxyaminosugar. The pOTBP1 plasmid containing TDP-glucose synthase (desIII), TDP-glucose-4,6-dehydratase (desIV), and glycosyltransferase (desVII/desVIII) was constructed and transformed into S. venezuelae YJ003, a strain in which the entire gene cluster of desosamine biosynthesis is deleted. The expression plasmid pOTBP3 containing 4-aminotransferase (gerB) and 3,5-epimerase (orf9) was transformed again into S. venezuelae YJ003- OTBP1 to obtain S. venezuelae YJ003-OTBP3 for the production of 4-amino-4,6-dideoxy-L-glucose derivatives. The crude extracts obtained from S. venezuelae ATCC 15439, S. venezuelae YJ003, and S. venezuelae YJ003-OTBP3 were further analyzed by TLC, bioassay, HPLC, ESI/MS, LC/MS, and MS/MS. The results of our study clearly shows that S. venezuelae YJ003-OTBP3 constructs other new hybrid macrolide derivatives including 4-amino-4,6-dideoxy-L-glycosylated YC-17 (3, [M+ Na+] m/z=464.5), methymycin (4, m/z=480.5), novamethymycin (6, m/z=496.5), and pikromycin (5, m/z=536.5) from a 12- membered ring aglycon (10-deoxymethynolide, 1) and 14-membered ring aglycon (narbonolide, 2). These results suggest a successful engineering of a deoxysugar pathway to generate novel hybrid macrolide derivatives, including deoxyaminosugar.     

MT-4 Suppresses Resistant Ovarian Cancer Growth through Targeting Tubulin and HSP27

Objective

In this study, the anticancer mechanisms of MT-4 were examined in A2780 and multidrug-resistant NCI-ADR/res human ovarian cancer cell lines.

Methods

To evaluate the activity of MT-4, we performed in vitro cell viability and cell cycle assays and in vivo xenograft assays. Immunoblotting analysis was carried out to evaluate the effect of MT-4 on ovarian cancer. Tubulin polymerization was determined using a tubulin binding assay.

Results

MT-4 (2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-ethyl]-phenol), a derivative of moscatilin, can inhibit both sensitive A2780 and multidrug-resistant NCI-ADR/res cell growth and viability. MT-4 inhibited tubulin polymerization to induce G2/M arrest followed by caspase-mediated apoptosis. Further studies indicated that MT-4 is not a substrate of P-glycoprotein (p-gp). MT-4 also caused G2/M cell cycle arrest, accompanied by the upregulation of cyclin B, p-Thr161 Cdc2/p34, polo-like kinase 1 (PLK1), Aurora kinase B, and phospho-Ser10-histone H3 protein levels. In addition, we found that p38 MAPK pathway activation was involved in MT-4-induced apoptosis. Most importantly, MT-4 also decreased heat shock protein 27 expression and reduced its interaction with caspase-3, which inured cancer cells to chemotherapy resistance. Treatment of cells with SB203580 or overexpression of dominant negative (DN)-p38 or wild-type HSP27 reduced PARP cleavage caused by MT-4. MT-4 induced apoptosis through regulation of p38 and HSP27. Our xenograft models also show the in vivo efficacy of MT-4. MT-4 inhibited both A2780 and NCI-ADR/res cell growth in vitro and in vivo.

Conclusion

These findings indicate that MT-4 could be a potential lead compound for the treatment of multidrug-resistant ovarian cancer.     

平滑肌肌球蛋白轻链激酶的非激酶作用及对ATP 酶活性的调节

肌球蛋白轻链激酶（myosin light chain kinase, MLCK）具有激酶活性和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为探寻MLCK的非激酶活性区域对MLCK活性的影响,以进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制.采用PCR技术构建MLCK部分氨基酸缺失的重组表达载体pGEX-F6-5/D,经大肠杆菌表达得到可溶性GST融合蛋白,利用SDS-PAGE及Western 印迹鉴定表达的MLCK在细胞中的分布,结果还显示,提取液的上清和沉淀中均有MLCK片段的表达.运用亲和层析技术分离并纯化删除前、后表达的MLCK片段（F6.5和F6-5/D）,经谷胱甘肽琼脂糖凝胶 4B 纯化,SDS-PAGE鉴定显示为单一表达条带.应用EnzChek磷分析试剂盒和孔雀绿两种方法分别测定不同浓度的MLCK对非磷酸化肌球蛋白Mg²⁺-ATP酶活性的影响.两种MLCK的片段均具有激活ATP酶活性的作用,并随MLCK浓度的增加,酶的活性增加.比较删除前后不同MLCK片段对ATP酶活性的影响结果显示,删除MLCK片段1002位丙氨酸至1019位亮氨酸后,对ATP酶的激活作用较删除前明显降低,表明删除的部分氨基酸序列为MLCK非激酶活性所必需的区域.利用电镜技术观察到MLCK片段（F6.5）使非磷酸化肌球蛋白构象发生明显的变化.加入MLCK片段后肌球蛋白的构象由非活性型转化为活性型,并且MLCK片段还具有促进肌球蛋白单体形成肌丝的作用.     

Efficient transient expression of human GM-CSF protein in Nicotiana benthamiana using potato virus X vector

The human granulocyte macrophage colony-stimulating factor (GM-CSF) is a glycoprotein with important clinical applications for the treatment of neutropenia and aplastic anemia and reducing infections associated with bone marrow transplants. We evaluated the potential for using a potato virus X (PVX) viral vector system for efficient expression of the biologically functional GM-CSF protein in Nicotiana benthamiana leaves. The GM-CSF gene was cloned into PVX viral expression vector, driven with the CaMV 35S promoter. Gene transfer was accomplished by inoculating N. benthamiana leaves with the plasmid DNA of PVX vector containing the GM-CSF gene. The expression level of the recombinant GM-CSF protein was determined with ELISA and its size was confirmed by Western blot analysis. The results showed that: (1) leaf age significantly affects GM-CSF protein concentration with younger leaves accumulating 19.8 mg g⁻¹ soluble protein which is 2.6 times the concentration in older leaves, (2) recombinant protein accumulation within a given leaf declined slightly over time but was not significantly different between 7 and 11 days post-inoculation (dpi), and (3) the two leaves immediately above the inoculated leaves play an important role for GM-CSF accumulation in the younger leaves. Protein extracts of infected N. benthamiana leaves contained recombinant human GM-CSF protein in concentrations of up to 2% of total soluble protein, but only when the pair of leaves immediately above the inoculated leaves remained intact. The recombinant protein actively stimulated the growth of human TF-1 cells suggesting that the recombinant human GM-CSF expressed via PVX viral vector was biologically active.