Role of p38γ MAPK in regulation of EMT and cancer stem cells

p38γ is a member of p38 MAPK family which contains four isoforms p38α, p38β, p38γ, and p38δ. p38γ MAPK has unique function and is less investigated. Recent studies revealed that p38γ MAPK may be involved in tumorigenesis and cancer aggressiveness. However, the underlying cellular/molecular mechanisms remain unclear. Epithelial-mesenchymal transition (EMT) is a process that epithelial cancer cells transform to facilitate the loss of epithelial features and gain of mesenchymal phenotype. EMT promotes cancer cell progression and metastasis, and is involved in the regulation of cancer stem cells (CSCs) which have self-renewal capacity and are resistant to chemotherapy and target therapy. We showed that p38γ MAPK significantly increased EMT in breast cancer cells; over-expression of p38γ MAPK enhanced EMT while its down-regulation inhibited EMT. Meanwhile, p38γ MAPK augmented CSC population while knock down of p38γ MAPK decreased CSC ratio in breast cancer cells. MicroRNA-200b (miR-200b) was down-stream of p38γ MAPK and inhibited by p38γ MAPK; miR-200b mimics blocked p38γ MAPK-induced EMT while miR-200b inhibitors promoted EMT. p38γ MAPK regulated miR-200b through inhibiting GATA3. p38γ MAPK induced GATA3 ubiquitination, leading to its proteasome-dependent degradation. Suz12, a Polycomb group protein, was down-stream of miR-200b and involved in miR-200b regulation of EMT. Thus, our study established an important role of p38γ MAPK in EMT and identified a novel signaling pathway for p38γ MAPK–mediated tumor promotion.     

涡鞭毛虫（甲藻）着丝粒／动粒蛋白的检查

利用ＡＣＡ血清、抗人着丝粒蛋白Ｂ的单抗和多抗、抗ＣＨＯ细胞动粒蛋白的单抗,对典型涡鞭毛虫隐沟虫（隐甲藻）（Ｃｒｙｐｔｈｅｃｏｄｉｎｉｕｍｃｏｈｎｉｉ）和特殊涡鞭毛虫尖尾虫（尖尾藻）（Ｏｘｙｒｒｈｉｓｍａｒｉｎａ）的着丝粒／动粒蛋白进行了检查。用ＡＣＡ血清作的荧光观察表明,隐沟虫的这些蛋白虽结合在核骨架上,但在间期时并不形成点状的前着丝粒。免疫印迹检查表明两种涡鞭毛虫的着丝粒蛋白Ｂ彼此一致,而且与四膜虫和眼虫的也高度一致。但用ＡＣＡ血清作免疫印迹检查时,尖尾虫的蛋白虽与四膜虫和眼虫的相近,与隐沟虫的却有极大的差异。以抗动粒蛋白的单抗作此种检查时,尖尾虫与眼虫的反应带相同,而隐沟虫则与源真核生物（Ａｒｃｈｅｚｏａ）贾第虫（Ｇｉａｒｄｉａｌａｍｂｌｉａ）的相同;而且隐沟虫和贾第虫都与几种原细菌有两条相同的反应带,其中５０ｋＤ的一条是尖尾虫和眼虫都没有的。上述发现不仅从一个新的方面支持了认为应把尖尾虫从典型涡鞭毛虫分出来独立为一个门的主张（李靖炎,１９９０）,而且指出典型涡鞭毛虫在后真核生物（Ｍｅｔａｋａｒｙｏｔａ）中间是非常原始的。     

源真核生物贾第虫与原细菌着丝粒／动粒蛋白的免疫印迹检查

以抗人着丝粒蛋白Ｂ的单抗和多抗以及抗ＣＨＯ细胞动粒蛋白的单抗对源真核生物（ａｒｃｈｅｚｏａ）蓝氏贾第虫（Ｇｉａｒｄｉａｌａｍｂｌｉａ）和分别代表原细菌的３个枝的３种原细菌（Ｈａｌｏｂａｃｔｅｒｉｕｍ、Ｔｈｅｒｍｏｐｌａｓｍａ、Ｓｕｌｆｏｓｐｈａｅｒｅｌｌｕｓ）作了免疫电泳检查,并以小眼虫和大肠杆菌作为对照。结果表明,３种原细菌都呈阳性反应;而且贾第虫的反应情况显然比纤毛虫、眼虫、典型涡鞭毛虫、尖尾虫（Ｏｘｙｒｒｈｉｓ）等单细胞后真核生物的更接近于原细菌的情况。这不仅从一个新的方面为真核细胞起源于古代的原细菌的学说提供了新的佐证,而且从着丝粒／动粒蛋白方面证明了源真核生物贾第虫的原始性。本工作还为认识着丝粒蛋白Ｂ和动粒蛋白的起源和演化提供了线索。     

田菁茎瘤固氮根瘤菌对小麦叶组织的促生作用研究

利用GFP标记的田菁茎瘤固氮根瘤菌(Azorhizobium caulinodans ORS 571)侵染露白24h的小麦种子,分别在侵染后0、6、12、24、48、72和96h采样,利用实时荧光定量PCR方法检测小麦体内6条与促生作用相关miRNAs(miR156、miR159、miR160、miR167、miR168和miR403)的表达模式,检测其中3条miRNAs(miR159、miR167和miR168)的靶基因表达模式;以接菌8d的小麦样品做切片,利用激光共聚焦显微镜检测小麦叶部田菁茎瘤固氮根瘤菌的分布,并测定小麦的生理指标。结果显示:(1)田菁茎瘤固氮根瘤菌侵染小麦后能够在叶片边缘部位定殖。(2)小麦叶片中与促生作用相关的6条miRNAs出现了不同程度变化,在12~24h到达其峰值,随后逐渐下降,其中miR159在峰值时的表达量为初始表达量的2.88倍。(3)3条miRNAs的靶基因表达模式与相应miRNA表达模式相对应,但并不严格。(4)生理指标测定结果显示,接种田菁茎瘤固氮根瘤菌对小麦叶片产生明显的促生作用,其中叶鲜重在96h的变化与对照差异极显著。研究表明,接种的田菁茎瘤固氮根瘤菌能够到达小麦叶组织,对小麦叶片的生长产生明显的促生作用,其中miRNAs在促生过程中发挥重要作用。     

Mixed Compound of DCPTA and CCC Increases Maize Yield by Improving Plant Morphology and Up-Regulating Photosynthetic Capacity and Antioxidants

DCPTA (2-diethylaminoethyl-3, 4-dichlorophenylether) and CCC (2-chloroethyltrimethyl- ammonium chloride) have a great effect on maize growth, but applying DCPTA individually can promote the increase of plant height, resulting in the rise of lodging percent. Plant height and lodging percent decrease in CCC-treated plants, but the accumulation of biomass reduce, resulting in yield decrease. Based on the former experiments, the performance of a mixture which contained 40 mg DCPTA and 20 mg CCC as active ingredients per liter of solution, called PCH was tested with applying 40mg/L DCPTA and 20mg/L CCC individually. Grain yield, yield components, internode characters, leaf area per plant, plant height and lodging percent as well as chlorophyll content, chlorophyll fluorescence, enzymatic antioxidants, membranous peroxide and organic osmolyte were analyzed in two years (2011 and 2012), using maize hybrid, Zhengdan 958 (ZD 958) at density of 6.75 plants m^-2. CCC, DCPTA and PCH were sprayed on the whole plant leaves at 7 expanded leaves stage and water was used as control. Compared to control, PCH significantly increased grain yield (by 9.53% and 6.68%) from 2011 to 2012. CCC significantly decreased kernel number per ear (by 6.78% and 5.69%) and thousand kernel weight (TKW) (by 8.57% and 6.55%) from 2011 to 2012. Kernel number per ear and TKW increased in DCPTA-treated and PCH-treated plants, but showed no significant difference between them. In CCC-treated and PCH-treated plants, internode length and plant height decreased, internode diameter increased, resulting in the significant decline of lodging percent. With DCPTA application, internode diameter increased, but internode length and plant height increased at the same time, resulting in the augment of lodging percent. Bending strength and puncture strength were increased by applying different plant growth regulators (PGRs). In PCH-treated plants, bending strength and puncture strength were greater than other treatments. Compared to control, the bending strength of 3rd internode was increased by 14.47% in PCH-treated plants in 2011, increased by 18.40% in 2012, and the difference was significant. Puncture strength of 1st, 3rd and 5th internode was increased by 37.25%, 29.17% and 26.09% in 2011 and 34.04%, 25% and 23.68% in 2012, compared to control. Leaf area and dry weight per plant reduced significantly in CCC-treated plants, increased in DCPTA-treated and PCH-treated plants from 2011 to 2012. Chlorophyll content and chlorophyll fluorescence improved with CCC and DCPTA application. Due to the additive effect of DCPTA and CCC, PCH showed the significant effect on chlorophyll content and chlorophyll fluorescence. Compared to control, total enzyme activity (SOD, POD, CAT, APX and GR) and soluble protein content increased, malonaldehyde (MDA) and hydrogen peroxide (H₂O₂) content reduced in PCH-treated plants. The transportation of soluble sugar from leaf to kernel improved significantly at the late silking stage. The research provided the way for the further use of DCPTA and CCC into the production practice.     

The non-receptor tyrosine kinase c-Src mediates the PDGF-induced association between Furin and pro-MT1-MMP in HPAC pancreatic cells

Furin is a member of the proprotein convertase family, which is capable of cleaving the precursors of a wide variety of substrates including membrane-type 1 matrix metalloproteinase (MT1-MMP) proenzyme. c-Src is activated by growth factors, and has been linked with a poor prognosis in pancreatic cancer (PCa). Both c-Src and Furin play crucial roles in tumorigenesis, and the mechanism controlling their association is not understood. Modulation of the association between Furin and pro-MT1-MMP by c-Src inhibitor PP2 was evaluated by western blotting, assay of in vitro enzyme, co-immunoprecipitation (co-IP), and confocal immunofluorescence microscopy. Human platelet-derived growth factor BB (PDGF-BB) activated c-Src and induced c-Src-dependent association of Furin with pro-MT1-MMP in HPAC pancreatic cancer cells. Co-IP and confocal immunofluorescence assays revealed that c-Src interacts with Furin in vivo. The SH2 domain appeared to be important for c-Src interaction with Furin. In addition, we showed that Furin protein is tyrosine phosphorylated. Association between Furin and MT1-MMP is regulated by the tyrosine kinase c-Src.     

拒盐型盐生植物小花碱茅肌动蛋白基因片段的克隆及序列分析

本研究根据其它植物Actin基因的保守序列设计一对简并性引物,以拒盐型盐生植物小花碱茅根部总RNA为模板,采用RT-PCR的方法扩增出Actin基因片段并克隆到PUCm-T载体,阳性克隆经PCR检测后进行测序,在GenBank中注册;序列分析结果表明:该片段长约600 bp,编码198个氨基酸;所得序列与GenBank中注册的其它植物Actin基因序列同源性均在84%以上,与其它肌动蛋白的氨基酸序列同源性达94%以上.     

Prediction of Catalytic Residues Using the Variation of Stereochemical Properties

In this paper, we investigate a simple protein sequence conservation measure which takes amino acid similarity into account. Instead of grouping 20 amino acids into disjoint sets in previous methods, we consider ten overlapping classes. The method is based on the assumption that a column in a multiple sequence alignment is evolved from an identical column in the evolutionary history. Two ten-dimensional vectors are constructed for each position to denote frequencies of ten classes in a column and the corresponding hypothetical identical column. Then the cosine function of the angle between these two vectors is considered as a measure of divergence of stereochemical properties at this position. This divergence, combining with other conservation scores, is used as conservation measure of the column. Finally, we evaluate our methods by identifying catalytic sites, using rank analysis criterion and receiver operator characteristic analysis criterion.     

Increased Crystalline Cellulose Activity via Combinations of Amino Acid Changes in the Family 9 Catalytic Domain and Family 3c Cellulose Binding Module of Thermobifida fusca Cel9A

Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.Cellulases catalyze the breakdown of cellulose into simple sugars that can be fermented to ethanol. The large amount of natural cellulose available is an exciting potential source of fuels and chemicals. However, the detailed molecular mechanisms of crystalline cellulose degradation by glycoside hydrolases are still not well understood and their low efficiency is a major barrier to cellulosic ethanol production.Thermobifida fusca is a filamentous soil bacterium that grows at 50°C in defined medium and can utilize cellulose as its sole carbon source. It is a major degrader of plant cell walls in heated organic materials such as compost piles and rotting hay and produces a set of enzymes that includes six different cellulases, three xylanases, a xyloglucanase, and two CBM33 binding proteins (12). Among them are three endocellulases, Cel9B, Cel6A, and Cel5A (7, 8), two exocellulases, Cel48A and Cel6B (6, 19), and a processive endocellulase, Cel9A (5, 7).T. fusca Cel9A-90 (Uniprot {"type":"entrez-protein","attrs":{"text":"P26221","term_id":"2506384","term_text":"P26221"}}P26221 and {"type":"entrez-protein","attrs":{"text":"YP_290232","term_id":"72162575","term_text":"YP_290232"}}YP_290232) is a multidomain enzyme consisting of a family 9 catalytic domain (CD) rigidly attached by a short linker to a family 3c cellulose binding module (CBM3c), followed by a fibronectin III-like domain and a family 2 CBM (CBM2). Cel9A-68 consists of the family 9 CD and CBM3c. The crystal structure of this species (Fig. ​(Fig.1)1) was determined by X-ray crystallography at 1.9 Å resolution (Protein Data Bank [PDB] code 4tf4) (15). Previous work has shown that E424 is the catalytic acid and D58 is the catalytic base (11, 20). H125 and Y206 were shown to play an important role in activity by forming a hydrogen bonding network with D58, an important supporting residue, D55, and Glc(−1)O1. Several enzymes with amino acid changes in subsites Glc(−1) to Glc(−4) had less than 20% activity on bacterial cellulose (BC) and markedly reduced processivity. It was proposed that these modifications disturb the coordination between product release and the subsequent binding of a cellulose chain into subsites Glc(−1) to Glc(−4) (11). Another variant enzyme with a deletion of a group of amino acids forming a block at the end of the catalytic cleft, Cel9A-68 Δ(T245-L251)R252K (DEL), showed slightly improved filter paper (FP) activity and binding to BC (20).Open in a separate windowFIG. 1.Crystal structure of Cel9A-68 (PDB code 4tf4) showing the locations of the variant residues, catalytic acid E424, catalytic base D58, hydrogen bonding network residues D55, H125, and Y206, and six glucose residues, Glc(−4) to Glc(+2). Part of the linker is visible in dark blue.The CBM3c domain is critical for hydrolysis and processivity. Cel9A-51, an enzyme with the family 9 CD and the linker but without CBM3c, had low activity on carboxymethyl cellulose (CMC), BC, and swollen cellulose (SWC) and showed no processivity (4). The role of CBM3c was investigated by mutagenesis, and one modified enzyme, R557A/E559A, had impaired activity on all of these substrates but normal binding and processivity (11). Variants with changes at five other CBM3c residues were found to slightly lower the activity of the modified enzymes, while Cel9A-68 enzymes containing either F476A, D513A, or I514H were found to have slightly increased binding and processivity (11) (see Table ​Table1).1). In the present work, CBM3c has been investigated more extensively to identify residues involved in substrate binding and processivity, understand the role of CBM3c more clearly, and study the coordination between the CD and CBM3c. An additional goal was to combine amino acid variants showing increased crystalline cellulose activity to see if this further increased activity. Finally, we have investigated whether the changes that improved the activity of Cel9A-68 also enhanced the activity of intact Cel9A-90.

TABLE 1.

Activities of Cel9A-68 CBM3c variant enzymes and CD variant enzymes used to create the double variants