空心莲子草响应南方菟丝子寄生的生长-防御权衡

为探讨全寄生植物南方菟丝子(Cuscuta australis)防治入侵植物空心莲子草(Alternanthera philoxeroides)的可行性,以二者野外天然生长的种群为研究对象,分析南方菟丝子寄生对空心莲子草生长及防御的影响,阐明空心莲子草在受到寄生胁迫时如何权衡自身生长与防御的关系,进而发展出一套应对南方菟丝子寄生的生长-防御策略。结果显示:(1)南方菟丝子寄生显著改变空心莲子草茎的形态,茎直径和平均节间长均增加,茎直径变化极显著(P0.01);(2)南方菟丝子寄生显著减少空心莲子草叶片数,但同时显著增加后者茎的分枝数,而茎上的节是潜在的无性繁殖体,故有利于空心莲子草的克隆繁殖;此外,南方菟丝子寄生显著降低了空心莲子草的根、茎、叶生物量和总生物量,抑制空心莲子草的生长;(3)南方菟丝子寄生显著增加空心莲子草茎的单宁、总酚、三萜皂苷含量,增强其防御能力;(4)南方菟丝子寄生的空心莲子草的生物量与茎部木质素、三萜皂苷、单宁和总酚含量均呈现显著负相关性(P0.01),对照组则不存在相关性;且寄生组较对照组相比,生物量的相对百分比显著低于对照组(P0.01),而用于防御的次生代谢产物总含量的相对百分比显著高于对照组(P0.01)。以上结果表明,受到南方菟丝子寄生胁迫后,空心莲子草改变自身的生长-防御策略,减少营养生长投入而将更多的资源投向克隆繁殖,同时增强对"防御"物质的投入,增强其防御能力,以利于后代生存和繁衍。     

Multiple Sequence Motifs in the Rubisco Small Subunit Transit Peptide Independently Contribute to Toc159-Dependent Import of Proteins into Chloroplasts

A large number of plastid proteins encoded by the nuclear genome are posttranslationally imported into plastids by at least two distinct mechanisms: the Toc159-dependent and Toc132/Toc120-dependent pathways. Light-induced photosynthetic proteins are imported through the Toc159-dependent pathway, whereas constitutive housekeeping plastid proteins are imported into plastids through the Toc132/Toc120 pathway. However, it remains unknown which features of the plastid protein transit peptide (TP) determine the import pathway. We have discovered sequence elements of the Rubisco small subunit TP (RbcS-tp) that play a role in determining import through the Toc159-dependent pathway in vivo. We generated multiple hybrid mutants using the RbcS-tp and the E1α-subunit of pyruvate dehydrogenase TP (E1α-tp) as representative peptides mediating import through the Toc159-dependent and Toc159-independent pathways, respectively. Import experiments using these hybrid mutants in wild-type and ppi2 mutant protoplasts revealed that multiple sequence motifs in the RbcS-tp independently contribute to Toc159-dependent protein import into chloroplasts. One of these motifs is the group of serine residues located in the N-terminal 12-amino acid segment and the other is the C-terminal T5 region of the RbcS-tp ranging from amino acid positions 41 to 49. Based on these findings, we propose that multiple sequence elements in the RbcS-tp contribute independently to Toc159-dependent import of proteins into chloroplasts.The plastid is a crucial organelle in plant cells. It plays a role in critical cellular processes such as photosynthesis, ATP generation, amino acid metabolism, and synthesis of fatty acids and lipid components. Accordingly, a large number of proteins are required for all these activities in plastids. Some of these proteins are encoded by the chloroplast genetic system and are translated in the plastids. However, most plastid proteins (over 90%) are encoded by the nuclear genome and are imported into plastids from the cytosol posttranslationally (Kessler and Schnell, 2006; Jarvis, 2008).Most plastid interior proteins that undergo posttranslational import from the cytosol contain a cleavable N-terminal targeting signal, a transit peptide (TP), of 50 to 70 amino acid residues (Jarvis, 2008; Lee et al., 2008). However, recently, some plastid interior proteins have been identified that do not have the N-terminal canonical TP (Miras et al., 2002, 2007; Nada and Soll, 2004). The long TP consists of multiple domains or motifs that encode information for preprotein import into plastids (von Heijne et al., 1989; Pilon et al., 1995; Rensink et al., 2000; Lee et al., 2006, 2008). The preproteins transit through the cytosol as unfolded protein. During passage through the cytosol, they may form a complex with heat shock proteins, such as Hsp70 and Hsp90, and guidance factors such as 14-3-3 (May and Soll, 2000; Qbadou et al., 2006). However, 14-3-3 may not be essential for the targeting of these proteins to chloroplasts (Lee et al., 2002, 2006; Nakrieko et al., 2004). To cross the two envelope membranes, the TP interacts with components of the Toc and Tic complexes located at the outer and inner envelopes of chloroplasts, respectively (Jarvis, 2008). These include members of the Toc159 family, Toc33/Toc34, Toc75, and Tic20. At the late stage or after translocation, the TP is recognized and cleaved off by stromal processing peptidases (Richter and Lamppa, 1999; Chen and Li, 2007).Despite extensive study of the TPs, it is not fully understood how the information encoded in these peptides is decoded by the plastid protein import machinery. TPs display some degree of similarity in their amino acid composition, including a higher content of Ala, Gly, and the hydroxylated amino acids Ser and Thr, and a lack of acidic amino acids (von Heijne et al., 1989; Bruce, 2001; Zhang and Glaser, 2002). However, it is clear that the entire family of TPs, termed the transit peptidome, cannot be represented by a single consensus sequence. Growing evidence has pointed to a functional classification of TPs. The first indication is that the transit peptidome may be classified into two groups: Toc159-dependent and Toc159-independent TPs (Ivanova et al., 2004; Kubis et al., 2004; Smith et al., 2004). The TPs that confer Toc159 dependence in protein import are typically used by light-induced photosynthetic proteins, whereas Toc159-independent TPs are used by nonphotosynthetic and housekeeping proteins (Kessler and Schnell, 2006). This was clearly demonstrated in the ppi2 mutant that has a T-DNA insertion in atTOC159 (Smith et al., 2004). In accord with this observation, the expression of atTOC159 is high in young and photosynthetic tissues whereas atTOC132 and atTOC120 are expressed uniformly in all plant tissues at low levels (Kubis et al., 2004). In addition, in nonphotosynthetic tissues, such as roots, the mRNA level of atTOC132 or atTOC120 is much higher than that of atTOC159. These results are consistent with the hypothesis that TPs may contain sequence motifs that determine the targeting pathway. However, the sequence information that confers Toc159 dependence or Toc132/120 dependence on these proteins during protein import remains unknown. In addition, Lee et al. (2008) recently demonstrated that the transit peptidome may be divided into several groups based on critical sequence motifs present in the TP. However, the role of the sequence motifs embedded in the TPs is not entirely clear yet with respect to translocation through the envelope membranes and also to the molecular machinery that recognizes these sequence motifs. Furthermore, the sequence information that confers Toc159 dependence or Toc132/120 dependence in protein import on these proteins remains unknown.The Rubisco small subunit (RbcS) and E1α TPs (RbcS-tp and E1α-tp) confer Toc159 dependence and Toc159 independence in protein import into chloroplasts, respectively (Smith et al., 2004). In this study, using these two TPs, we have determined the RbcS-tp sequence motifs that confer Toc159 dependence. Here, we have demonstrated that Toc159-dependent protein import is mediated independently by multiple sequence motifs: one of them is the group of Ser residues located in the N-terminal 12-amino acid segment and the other is in the C-terminal region ranging from amino acid positions 41 to 49.     

The Hepatitis E Virus Open Reading Frame 3 Product Interacts with Microtubules and Interferes with Their Dynamics

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated α-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.The hepatitis E virus (HEV), the sole member of the genus Hepevirus, is a single-strand positive-sense RNA virus that is the causative agent in endemics and epidemics of acute human hepatitis in many parts of the world (5). Transmitted mainly from contaminated water through the fecal-oral route, HEV infection causes a fulminant form of hepatitis that has a mortality rate of up to 20% in pregnant women (28). HEV infection is considered zoonotic. Swine and chicken HEV strains have been found in the United States (11, 23). A swine strain can infect chimpanzees under experimental conditions, and a human strain that is genetically similar to the swine strain can experimentally infect pigs (22). Direct evidence of the zoonotic nature of HEV infection has been provided in reports of a series of cases of HEV infection in people who ate undercooked deer meat 6 to 7 weeks before the onset of the disease (19, 33, 39). HEV RNA recovered from the leftover deer meat was found to be identical in nucleotide sequence to the HEV RNA recovered from the individuals who became ill (31).The HEV genome is approximately 7.2 kb in length and consists of three open reading frames (ORFs) (32). ORF1 encodes a nonstructural polyprotein that includes the RNA-dependent RNA polymerase. ORF2 encodes the capsid protein, the major structural protein in virion. ORF3 encodes a phosphoprotein that was found to be essential for establishing an HEV infection in macaques and pigs under experimental conditions (9, 12). It has been reported that ORF3 translation initiates at the third in-frame AUG codon, which lies 23 bases downstream of the ORF1 termination codon (10, 12). Propagation of HEV and studies of virus replication still rely upon nonhuman primates due to the lack of an effective cell culture system. As a result, functional study of the ORF3 product in HEV biology and infection is limited.The phosphoprotein encoded by HEV ORF3 has a molecular mass of approximately 13 kDa (hereinafter called vp13) (32). The exact functions of vp13 in HEV infection remain unknown although the findings of a number of studies have shown that it plays a role in cellular signaling pathways (13, 17, 24, 34-36, 40). During subcellular fractionation of COS-7 cells transfected with a vp13-expressing plasmid, vp13 was found to partition with the cytoskeletal fraction (40). Deletion of the N-terminal hydrophobic domain of vp13 abolished the association with the cytoskeleton fraction. The vp13-binding proteins in the cytoskeleton and the nature of this interaction are not known.In this study, we found that the HEV ORF3 product localizes to microtubules and interferes with their dynamics. The filamentous pattern of vp13 distribution in the cell was abolished by a microtubule-destabilizing drug. vp13 led to elevation of acetylated α-tubulin. These results suggested that vp13 interaction with the microtubules might facilitate HEV infection. We further studied the nature of the vp13-microtubule interaction.     

Organic Matter Stable Isotope (δ13C, δ15N) Response to Historical Eutrophication of Lake Taihu, China

We explored the use of carbon and nitrogen isotope ratios (δ¹³C, δ¹⁵N) in sediment organic matter as proxy indicators of historical changes in the trophic state of Lake Taihu, the third largest freshwater lake in China. Stable isotope signatures in four sediment cores spanning the 20th century were compared with instrumental records of lake-water trophic state. The comparative study shows that, between ∼ ∼1950 and 1990 AD, the δ¹³C and δ¹⁵N of sediment organic matter throughout Lake Taihu increased along the trophic gradient from oligotrophy to eutrophy due to biological isotopic fractionation. However, in the 1990s, the trophic state of Lake Taihu diverged into two different trophic systems, a hypereutrophic western Lake Taihu dominated by blue-green algae and a mesoeutrophic eastern Lake Taihu dominated by vascular aquatic plants. During the post-1990 AD shift from mesoeutrophic to hypereutrophic state in western Lake Taihu, organic matter δ¹³C and δ¹⁵N decreased sharply in response to pronounced shifts in the aquatic ecosystem. The results indicate that ¹³C-depleted phytoplankton replaced macrophytes in western Lake Taihu. δ¹⁵N values in western Lake Taihu also decreased because of N₂ fixation by cyanobacteria in this highly productive ecosystem. By contrast, in eastern Lake Taihu, organic matter δ¹³C and δ¹⁵N values show a post-1990 AD trend towards slightly lower values, but they remain higher than the long-term average. This recent ¹³C–enrichment of organic matter indicates that periods of high productivity in the restricted eastern sub-basin of Lake Taihu limited aqueous CO₂ availability, causing a decrease in isotopic discrimination during photosynthesis. After ∼ ∼1990 AD, organic matter δ¹⁵N values for eastern Lake Taihu only dropped slightly, suggesting that the contribution of phytoplankton to the sediment organic matter increased slightly. Taken together, the results indicate that nitrogen-fixing cyanobacteria probably played a much smaller role in primary productivity in this part of eastern Lake Taihu, compared with western Lake Taihu. Despite the complexity of carbon and nitrogen cycles in lakes, the agreement between the stable isotope signatures and instrumental records for Lake Taihu suggests that δ¹³C and δ¹⁵N in sediment organic matter are capable of recording important shifts in the spatial and temporal evolution of lake-water trophic state.     

Overexpression of PRE1 and its homologous genes activates Gibberellin-dependent responses in Arabidopsis thaliana

Gibberellins control various aspects of growth and development. Here, we identified a gene, designated paclobutrazol resistance1 (PRE1), by screening Arabidopsis activation-tagged lines. PRE1 encodes a helix-loop-helix protein and belongs to a small gene family. Physiological and genetic analysis indicated that overexpression of PRE1 altered various aspects of gibberellin-dependent responses such as germination, elongation of hypocotyl/petiole, floral induction and fruit development, and suppressed gibberellin-deficient phenotypes of the ga2 mutant. Expression of some gibberellin-responsive genes was also affected by PRE1. Expression of PRE1 was shown to be early gibberellin inducible in the wild-type plants and under control of SPY and GAI, upstream negative regulators of gibberellin signaling. The shortened hypocotyl length phenotype of the gai-1 mutant was suppressed by PRE1 overexpression. Ectopic overexpression of each of the four PRE1-related genes conferred pleiotropic phenotypes similar to PRE1 overexpression, indicative of overlapping functions among the PRE gene family. Our results of gain-of-function studies suggest that PRE genes may have a regulatory role in gibberellin-dependent development in Arabidopsis thaliana.     

Nuclear Multidrug-Resistance Related Protein 1 Contributes to Multidrug-Resistance of Mucoepidermoid Carcinoma Mainly via Regulating Multidrug-Resistance Protein 1: A Human Mucoepidermoid Carcinoma Cells Model and Spearman's Rank Correlation Analysis

Background

Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells.

Methods

Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman''s rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter.

Results

MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues.

Conclusions

Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors.     

大肠杆菌 LT B亚基基因表达载体对犬细小病毒VP2 DNA疫苗免疫应答的增强作用

【目的】通过融合基因表达载体和共免疫基因表达载体研究大肠杆菌不耐热肠毒素(LT)B亚基基因对犬细小病毒VP2DNA疫苗免疫应答的影响。【方法】提取大肠杆菌44815菌株基因组DNA,通过PCR方法从基因组DNA中扩增LTB基因,同时采用PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2的主要抗原表位基因(VP2-70,编码70个氨基酸)。将上述基因分别连接到含有人CD5信号肽序列的载体pcDNA-CD5sp上,分别构建成它们的分泌型真核表达载体,pcDNA-CD5sp-LTB和pcDNA-CD5sp-VP2-70。再利用酶切连接的方法构建LTB与VP2-70融合的真核表达载体pcDNACD5sp-LTB-VP2-70。然后用pcDNACD5sp-VP2-70(VP2-70组)、pcDNACD5sp-LTB-VP2-70(VP2-LTB融合组)、pcDNA-CD5sp-LTB/pcDNACD5sp-VP2-70(VP2-LTB共免疫组)和pcDNA3.1A(空载体对照组)分别免疫小鼠。免疫后用间接ELISA检测不同时间小鼠血清的抗体水平,用MTT方法检测小鼠免疫5周后脾脏淋巴细胞的增殖活性。【结果】经过测序表明本研究扩增的LTB和VP2基因序列和构建的相关表达载体结构正确。通过Western-blot检测证明构建的表达载体均能介导相应基因在真核细胞进行分泌表达。ELISA检测结果表明,3组实验组小鼠接受VP2DNA疫苗免疫后均能产生特异的体液免疫应答反应,特别是VP2-LTB基因融合组小鼠的抗体水平在第5周时高达1:5120,明显高于其它两组(P<0.01)。3组免疫小鼠抗体的亚型均表现IgG1抗体水平明显高于IgG2a抗体水平(P<0.01)。淋巴细胞增殖实验结果表明,在ConA的刺激下,3组免疫小鼠的淋巴细胞刺激指数均明显高于对照组(P<0.01),说明VP2DNA疫苗能够引起淋巴细胞的增殖。但3组免疫小鼠之间的刺激指数没有明显差异(P>0.05)。【结论】在小鼠体内,LTB基因表达载体可明显提高CPVVP2DNA疫苗的体液免疫应答水平。     

Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD(+). LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD(+). However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD(+) redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.