Effects of sampling method on foliar δ13C of Leymus chinensis at different scales

Stable carbon isotope composition (δ¹³C) usually shows a negative relationship with precipitation at a large scale. We hypothesized that sampling method affects foliar δ¹³C and its response pattern to precipitation. We selected 11 sites along a precipitation gradient in Inner Mongolia and collected leaves of Leymus chinensis with five or six replications repeatedly in each site from 2009 to 2011. Additionally, we collected leaves of L. chinensis separately from two types of grassland (grazed and fenced) in 2011. Foliar δ¹³C values of all samples were measured. We compared the patterns that foliar δ¹³C to precipitation among different years or different sample sizes, the differences of foliar δ¹³C between grazed and fenced grassland. Whether actual annual precipitation (AAP) or mean annual precipitation (MAP), it was strongly correlated with foliar δ¹³C every year. Significant difference was found between the slopes of foliar δ¹³C to AAP and MAP every year, among the slopes of foliar δ¹³C to AAP from 2009 to 2011. The more samples used at each site the lower and convergent P‐values of the linear regression test between foliar δ¹³C and precipitation. Furthermore, there was significant lower foliar δ¹³C value in presence of grazed type than fenced type grassland. These findings provide evidence that there is significant effect of sampling method to foliar δ¹³C and its response pattern to precipitation of L. chinensis. Our results have valuable implications in methodology for future field sampling studies.     

B3GNT3 Expression Is a Novel Marker Correlated with Pelvic Lymph Node Metastasis and Poor Clinical Outcome in Early-Stage Cervical Cancer

Background

The β1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) encodes a member of the B3GNT family that functions as the backbone structure of dimeric sialyl-Lewis A and is involved in L-selectin ligand biosynthesis, lymphocyte homing and lymphocyte trafficking. B3GNT3 has been implicated as an important element in the development of certain cancers. However, the characteristics of B3GNT3 in the development and progression of cancer remain largely unknown. Thus, our study aimed to investigate the expression pattern and the prognostic value of B3GNT3 in patients with early-stage cervical cancer.

Methods

The mRNA and protein levels of B3GNT3 expression were examined in eight cervical cancer cell lines and ten paired cervical cancer tumors, using real-time PCR and western blotting, respectively. Immunohistochemistry (IHC) was used to analyze B3GNT3 protein expression in paraffin-embedded tissues from 196 early-stage cervical cancer patients. Statistical analyses were applied to evaluate the association between B3GNT3 expression scores and clinical parameters, as well as patient survival.

Results

B3GNT3 expression was significantly upregulated in cervical cancer cell lines and lesions compared with normal cells and adjacent noncancerous cervical tissues. In the 196 cases of tested early-stage cervical cancer samples, the B3GNT3 protein level was positively correlated with high risk TYPES of human papillomavirus (HPV) infection (P = 0.026), FIGO stage (P < 0.001), tumor size (P = 0.025), tumor recurrence (P = 0.004), vital status (P < 0.001), concurrent chemotherapy and radiotherapy (P = 0.016), lymphovascular space involvement (P = 0.003) and most importantly, lymph node metastasis (P = 0.003). Patients with high B3GNT3 expression had a shorter overall survival (OS) and disease-free survival (DFS) compared with those with low expression of this protein. Multivariate analysis suggested that B3GNT3 expression is an independent prognostic indicator for cervical cancer patients.

Conclusions

Our study demonstrated that elevated B3GNT3 expression is associated with pelvic lymph node metastasis and poor outcome in early-stage cervical cancer patients. B3GNT3 may be a novel prognostic marker and therapeutic target for the treatment of cervical cancer.     

Up-Regulation of MiR-300 Promotes Proliferation and Invasion of Osteosarcoma by Targeting BRD7

Increasing reports suggest that deregulated microRNAs (miRNAs) might provide novel therapeutic targets for cancers. However, the expression and function of miR-300 in osteosarcoma is still unknown. In our study, we found that the expression of miR-300 was up-regulated in osteosarcoma tissues and cells compared with paired adjacent non-tumor bone tissues and osteoblastic cells using RT-qPCR. The enforced expression of miR-300 could promote cell proliferation, invasion and epithelial-mesenchymal transition (EMT). Moreover, we identified that bromodomain-containing protein 7 (BRD7), a new tumor suppressor gene, was a direct target of miR-300. Ectopic expression of BRD7 could significantly inhibit miR-300-promoted proliferation, invasion and EMT. Therefore, our results identify an important role for miR-300 in osteosarcoma through regulating BRD7 expression.     

PKCε regulates contraction‐stimulated GLUT4 traffic in skeletal muscle cells

The signaling pathways that stimulate glucose uptake in response to muscle contraction are not well defined. Recently, we showed that carbachol, an acetylcholine analog, stimulates contraction of C2C12 myotube cultures and the rapid arrival of myc‐epitope tagged GLUT4 glucose transporters at the cell surface. Here, we explore a role for protein kinase C (PKC) in regulating GLUT4 traffic. Cell surface carbachol‐induced GLUT4myc levels were partly inhibited by the conventional/novel PKC inhibitors GF‐109203X, Gö6983, and Ro‐31‐8425 but not by the conventional PKC inhibitor Gö6976. C2C12 myotubes expressed several novel isoforms of PKC mRNA with PKCδ and PKCε in greater abundance. Carbachol stimulated phosphorylation of PKC isoforms and translocation of PKCδ and PKCε to membranes within 5 min. However, only a peptidic inhibitor of PKCε translocation (myristoylated‐EAVSLKPT), but not one of PKCδ (myristoylated‐SFNSYELGSL), prevented the GLUT4myc response to carbachol. Significant participation of PKCε in the carbachol‐induced gain of GLUT4myc at the surface of C2C12 myotubes was further supported through siRNA‐mediated PKCε protein knockdown. These findings support a role for novel PKC isoforms, especially PKCε, in contraction‐stimulated GLUT4 traffic in muscle cells. J. Cell. Physiol. 226: 173–180, 2010. © 2010 Wiley‐Liss, Inc.     

SSR-based linkage map of flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and mapping of QTLs underlying fatty acid composition traits

Flax (Linum usitatissimum L.) seeds contain nearly 50% oil which is high in linolenic acid (an omega-3 fatty acid). In this study, a genetic linkage map was constructed based on 114 expressed sequence tag-derived simple sequence repeat (SSR) markers in addition to five single nucleotide polymorphism markers, five genes (fad2A, fad2B, fad3A, fad3B and dgat1) and one phenotypic trait (seed coat color), using a doubled haploid (DH) population of 78 individuals generated from a cross between SP2047 (a yellow-seeded Solin™ line with 2–4% linolenic acid) and UGG5-5 (a brown-seeded flax line with 63–66% linolenic acid). This map consists of 24 linkage groups with 113 markers spanning ~833.8 cM. Quantitative trait locus (QTL) analysis detected two major QTLs each for linoleic acid (LIO, QLio.crc-LG7, QLio.crc-LG16), linolenic acid (LIN, QLin.crc-LG7, QLin.crc-LG16) and iodine value (IOD, QIod.crc-LG7, QIod.crc-LG16), and one major QTL for palmitic acid (PAL, QPal.crc-LG9). The mutant allele of fad3A, mapped to the chromosomal segment inherited from the parent SP2047, underlies the QTL on linkage group 7 and was positively associated with high LIO content but negatively associated with LIN and IOD. This fad3A locus accounted for approximately 34, 25 and 29% of the phenotypic variation observed in this DH population for these three traits, respectively. The QTL localized on linkage group 16 explained approximately 20, 25 and 13% of the phenotypic variation for these same traits, respectively. For palmitic acid, QPal.crc-LG9 accounted for ~42% of the phenotypic variation. This first SSR-based linkage map in flax will serve as a resource for mapping additional markers, genes and traits, in map-based cloning and in marker-assisted selection.     

Separation and purification of a keratinase as pesticide against root-knot nematodes

A new alkaline keratinase, which could kill Meloidogyne incognita (a root-knot nematode) was separated and purified from Bacillus sp. 50-3 in this study. The solid ammonium sulfate was selected to precipitate the enzyme and its proper adding mass was also determined. After solid ammonium sulfate precipitation and liquid chromatography on DEAE-Sephadex-A50 column, there was 17.7-fold purification with a yield of 46.5%, as determined by azokeratin as substrate. The purification effect was determined through SDS-PAGE and the molecular weight of the enzyme was found to be 27,423 Da by the MALDI-TOF-MS. When the second-stage juveniles of Meloidogyne incognita were exposed to 50 μg/ml of keratinase solution, 98.5% of Meloidogyne incognita mortality rates were obtained compared to control after 24 h. Its simple purification step and high yield from the cheap medium affords this keratinase great biotechnological potential, especially in controlling root-knot nematodes such as Meloidogyne incognita. To the best of our knowledge, this study is the first report that uses keratinase as a pesticide.     

群落构建研究的新进展:进化和生态相结合的群落谱系结构研究

群落如何构建足群落生态学中的重要问题.群落谱系结构研究将物种间的亲缘进化关系运用到群落生态学研究中,利用物种的系统发育状况推测历史因素对现有群落的影响,为推断影响群落组成的生态学机制提供了有效方法.群落谱系结构的研究方法是首先建立可代表群落物种库的超级系统进化树,然后计算群落内物种间的谱系距离,最后通过统计方法检测其与随机模型下的谱系距离是否有显著差异来获得谱系结构(如谱系聚集、谱系发散),从而揭示群落构建中的关键生态过程(如生境过滤、竞争作用).群落谱系结构与空间尺度、分类群尺度、时间尺度等不同研究尺度有关.在小的空间尺度下,随着分类群尺度降低、树木年龄级增大,群落谱系结构从聚集逐渐转为发散;而随群落空间尺度的增大,谱系趋向于聚集.谱系结构受到环境因素影响,因此分析集合群落下的谱系可以揭示区域生态过程的影响.另外,群落谱系结构研究还有助于探讨中性理论、密度制约假说等生态学理论,并预测干扰作用下的群落演化趋势.在利用谱系结构深入探讨群落构建成因时,需要基于生态特征和环境变量共同分析,同时考虑小尺度局域过程(群落的微环境或群落内种间相互作用等)和大尺度区域过程(地史过程和物种形成等),并可结合生态控制实验,以确认群落构建的关键因素.在研究方法和手段上,今后需要注重通过选择合适的基因片段建立系统树,然后通过生态特征来加以校正,以更准确地反映物种间的亲缘距离.另外,获得谱系树后还需要寻找更加合理的统计模型和指数,增加统计分析和解决问题的能力.     

Seed-to-seed potential allelopathic effects between <Emphasis Type="Italic">Ligularia virgaurea</Emphasis> and native grass species of Tibetan alpine grasslands

Allelopathy is an important process in plant communities, but the role of seed allelopathy in natural ecosystems remains poorly understood. In the present study, we examined the potential allelopathic effects of Ligularia virgaurea (a dominant species in degraded Tibetan grasslands) seeds on the germination of four native grass species (Festuca sinensis, Agrostis gigantean, Bromus inermis, and Elymus nutans). The results showed that L. virgaurea seeds can have potential allelopathic effects on seed germination, mean time to germination and root growth rates of native grass species. We further demonstrate that these effects are driven by a water-soluble seed leachate. Species with smaller seeds were generally more sensitive than larger seeded species. The results suggest that seed-to-seed allelopathic potential may be an important mechanism driving the dominance of L. virgaurea in degraded alpine grasslands on the Tibetan Plateau. Further studies are required to demonstrate effects of seed-to-seed potential allelopathy in a field setting.     

假单胞菌YZ-26来源环酰亚胺水解酶中半胱氨酸残基的反应性能

【目的】研究环酰亚胺水解酶(Imidase,CIH)中的两个半胱氨酸残基的反应性及功能。【方法】设计了3个半胱氨酸突变酶:CIH7,108、CIH7、CIH108。将天然酶以及突变酶基因分别与麦芽糖结合蛋白(MBP)基因在大肠杆菌(Escherichia coli)中进行融合表达,融合蛋白经纯化后得到了电泳纯的样品。使用5,5’-二硫代双(2-硝基苯甲酸)(DTNB)对天然酶CIH的巯基基团进行修饰,并分析了DTT对分子状态的影响。进一步研究了经H2O2处理后CIH及其突变酶的锌离子结合能力及分子状态。【结果】酶活测定表明CIH7,108和CIH7的活力基本丧失,而CIH108仍保持了72%的酶活性。CIH中的两个半胱氨酸残基以游离形式存在,不形成链内或链间二硫键。CIH与CIH108为四聚体结构且具有一定的锌离子结合能力,CIH7,108为多聚体,CIH7为单体及多体的混合物且都不具备锌离子结合能力,随着H2O2浓度的增加,CIH中的链内二硫键及CIH108中的链间二硫键逐渐增加。【结论】说明Cys7是结合锌离子和稳定CIH分子结构的必要残基。     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.