Protein Phosphatase Type 1-Interacting Protein Ysw1 Is Involved in Proper Septin Organization and Prospore Membrane Formation during Sporulation

Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of YSW1 partially suppresses the sporulation defect of a temperature-sensitive allele of gip1. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in ysw1Δ mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in ysw1Δ cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.Diploid cells of Saccharomyces cerevisiae subjected to nitrogen limitation in the presence of a nonfermentable carbon source undergo the developmental process of sporulation (14, 23, 35). Four nuclei produced by two rounds of nuclear division, meiosis I and II, are encapsulated by newly formed double-membrane structures, called prospore membranes, and are finally packaged into spores covered with layered spore walls (35).In this process, prospore membrane formation is one of the most dynamic events. Early in meiosis II, the cytoplasmic surface of the meiotic spindle pole body (SPB) is modified by the recruitment of sporulation-specific protein complex that acts as a site of vesicle recruitment (2, 22, 39). Post-Golgi secretory vesicles dock to the surface of the SPBs and fuse with each other, generating prospore membranes (33, 34). The prospore membranes then grow to engulf daughter nuclei through a series of stages that are categorized by the membranes'' appearance in the fluorescence microscope (12). Initially, the membranes appear as small horseshoes that enlarge to become small round membrane structures. The prospore membranes then extend into a tube-like shape, engulfing the nucleus, as well as some cytosol and organelles (12). After this extension, prospore membrane undergoes a rapid change to a mature round form. This rounding of the membrane is coordinated with membrane closure (12). Spore wall materials are then deposited into the luminal space created by closure of the prospore membrane (9).In addition to the meiotic plaque of the SPB, two protein complexes are associated with the prospore membrane as it forms. One is the leading edge protein complex, which exists at the lip of the prospore membranes and consists of three components: Ssp1, Ady3, and Don1 (27, 30, 38). Ssp1 is the most important of the three and is required for proper extension of the prospore membrane (30). The second complex is a sporulation-specific septin structure. The septins are a family of cytoskeletal proteins, which form filaments (18, 50). Septins are conserved from yeast to mammals. They were originally found and have been extensively studied in S. cerevisiae. In vegetatively growing S. cerevisiae cells, five septin proteins—Cdc3, Cdc10, Cdc11, Cdc12, and Shs1—form a ring at the bud neck that serves as a scaffold for many additional proteins, as well as a barrier to diffusion of proteins between the mother and the bud (19, 29, 50). In sporulating cells, the set of septin proteins is changed. Cdc3 and Cdc10, along with two sporulation-specific septins, Spr3 and Spr28, form a pair of parallel bars or sheets associated with each prospore membrane (11, 15, 29). Although deletion of sporulation-specific septins has only modest effects on sporulation (11, 15), their specific localization suggests that they have some function during prospore membrane formation. Septin organization in vegetatively growing cells is regulated by phosphorylation and dephosphorylation of septin components and septin-associated proteins (29). In sporulating cells, a sporulation-specific protein phosphatase type 1 (PP1) complex Gip1-Glc7 is required for the formation of septin structures (46), although whether this phosphatase acts directly on the septin proteins is unknown.The PP1 catalytic subunit is highly conserved in eukaryotes and is involved in a variety of cellular processes (8, 44). In S. cerevisiae it is encoded by an essential gene, GLC7, and functions in glycogen synthesis, glucose repression, chromosome segregation, cell wall organization, endocytosis, mating, and sporulation (3, 17, 24, 42, 44, 47, 53). The specificity of this enzyme is determined by targeting subunits. GIP1 was originally isolated in a two-hybrid screen by using GLC7 as a bait, and this interaction was confirmed by coimmunoprecipitation of the two proteins (48). GIP1 is a sporulation-specific gene required for sporulation. Further analysis revealed that Gip1 and Glc7 colocalize with septins during sporulation and are required for both septin organization and spore wall formation (46). The specific targets or cofactors of this PP1 complex are unknown.To elucidate the role of Gip1-Glc7 phosphatase, we screened for high-copy suppressors of a temperature-sensitive allele of gip1 and isolated YSW1. Ysw1 interacts with Gip1 and colocalizes with septins similar to Gip1. Furthermore, a ysw1Δ mutant displays aberrant septin structures and prospore membrane extension. These results suggest that Ysw1 may function with Gip1-Glc7 to regulate proper septin organization and prospore membrane formation.     

Development and characterization of eleven polymorphic microsatellite loci from South China field mouse (Apodemus draco)

One population distributed in Yunnan of China was regarded as a new species based on mitochondrial cytochrome b sequences. However, the usefulness of mitochondrial sequence data in determining species boundaries is not universally agreed upon, the frequency data from multiple nuclear gene loci is necessary in determining species boundaries. So, we describe in this paper the isolation and characterization of eleven microsatellite loci in the South China field mouse from genomic DNA-enriched libraries. The eleven loci were tested in 24 individuals from two populations in Southwest China. These loci were highly polymorphic with numbers of alleles per locus ranging from 9 to 24 and expected heterozygosities from 0.898 to 0.967. Eight loci followed Hardy–Weinberg expectations after Bonferroni correction for multiple comparisons. No significant linkage association was found among all these loci. The eleven polymorphic microsatellite loci will be useful in determining species boundaries of the South China field mouse.     

Distribution of ground-dwelling beetle assemblages (Coleoptera) across ecotones between natural oak forests and mature pine plantations in North China

This paper studied edge effects resulting from logging to reforestation on the distribution of ground-dwelling beetles (Coleoptera) across ecotones between natural oak forests and mature pine plantations established after harvesting of natural forests. Using pitfall traps, ground-dwelling beetles were investigated at three replicated plots (ecotones) with three sampling positions of slope (lower, middle and upper) for each plot. Rarefaction estimates of species richness indicated that traps on natural forests and transition zones had more species than mature plantations did, and traps on the middle slope had more species than on the lower and upper slopes did. Results of an ANOVA analysis, which used forest type and slope position as factors and number of species and individuals as the response variables, showed a significant effect of forest type and slope position, and a significant interaction between forest type and slope position. Multivariate analyses (DCA and CCA) showed that beetles of transition zones were more similar to those of natural forests than to those of mature plantations, and that some environmental characteristics, i.e., proportion of broad-leaved trees, canopy cover and elevation (slope position), significantly affected species abundances. We conclude that the logging of natural oak forests and the reforestation of pine plantations can result in subtle variation in the composition and distribution of beetle assemblages at a local scale and such variation should be taken into account when conservation issues are involved.     

三峡水库及香溪河库湾理化特征的比较研究

根据2003年6月三峡水库初期蓄水后对香溪河库湾的常规监测,对该水域的理化特征及其动态进行了分析,并与三峡水库库首的数据进行了比较研究。结果显示库湾TN、NO3-N浓度要显著低于库首,前者两周年平均值为1.29mg/L,0.88mg/L,后者两周年平均值为1.62mg/L,1.22mg/L。而PO4-P则是库湾显著高于库首,并且在7—9月库首的TP/PO4-P有显著提高。结果同时表明库首的水土流失较严重,而库湾则有较好的水土保持。最后对TSIM的计算结果表明,由于TP、TN都处于高水平,库首呈现中营养化(TSIM>37),而库湾则呈现严重富营养化(TSIM>53)。     

苍耳柄锈菌三裂叶豚草专化型冬孢子基因组DNA快速提取方法的比较研究

以苍耳柄锈菌三裂叶豚草专化型Puccinia xanthii f. sp. ambrosiae-trifidae为研究试材,比较研究了其冬孢子DNA提取的9种方法,其中CTAB-钢珠法、改良的微型电钻法以及EZ-Kit改良法获得的基因组DNA经检测质量较好。在此基础上,利用ITS-PCR和ISSR引物UBC#835将待用DNA进行PCR扩增检测。结果表明,上述3种方法提取的DNA适合于ISSR反应。研究结果为专性寄生锈菌分子遗传变异的研究提供了保障。     

鸡舍环境中镰孢菌的种类分布及潜在产单端孢霉烯族毒素菌株的检测

采用AGI-30生物采样器收集鸡舍空气样本,同时采集鸡舍中饲料、积尘、土壤和饮用水在内的环境基质样品。采用形态学方法对分离获得的镰孢菌菌株进行鉴定,利用tri5-PCR技术对镰孢菌菌株中产单端孢霉烯族毒素的菌株进行检测,目的是探明鸡舍环境中镰孢菌种类的分布特征和产毒菌株。结果表明,从采集的50份样品中分离获得139个镰孢菌菌株,鸡舍空气和基质中的优势菌株均为Fusarium verticillioides;在各基质中,土壤中镰孢菌总浓度最高,为4×102–1.35×104CFU/g,其次为饲料和饮用水;采用tri5-PCR技术筛选到42株tri5阳性镰孢菌菌株,其中以F. graminearum所占比例最高。研究明确鸡舍中镰孢菌种类及其分布特征对鸡只疾病控制及保障人类和动物的健康具有重要意义。     

山西文峪河上游河岸林群落稳定性评价

稳定性是植物群落结构与功能的综合特征。该文运用模糊综合评判理论评价了山西文峪河上游13种河岸林群落的稳定性。基于群落整体稳定性和结构稳定性的考虑, 选取乔木层优势树种更新潜力、物种多样性、Godron指数、立地质量和保护程度等5项特征指标, 通过计算各群落5项指标隶属度的平均值来评价群落稳定性。研究结果表明, 多数群落的稳定性隶属度介于0.40-0.60, 属于低山森林演替系列的青杨辽东栎(Populus cathayana + Quercus wutaishanica)混交林和油松白桦(Pinus tabulaeformis + Betula platyphylla)混交林的稳定性居中; 中高山森林演替系列中, 群落稳定性随着演替的进展而增加, 青杨(Populus cathayana)林结构简单, 稳定性最低, 青杄(Picea wilsonii)林接近演替顶极, 稳定性最高; 但并非只有近演替顶极的群落是稳定的, 客观存在的各种林冠干扰和河岸生境的高度异质性也使得白杄杨桦(Picea meyeri + Populus cathayana + Betula platyphylla)混交林、落叶松白杄(Larix principis-rupprechtii + Picea meyeri)混交林和落叶松青杄(Larix principis- rupprechtii + Picea wilsonii)混交林有较高的稳定性。     

施氮量和种植密度对东北特早熟棉区棉铃生物量和氮素累积的影响

以辽棉19号和美棉33B为材料,研究了不同施氮量(0、240、480 kg ·hm-2)和不同种植密度(75000、97500、120000 plants·hm-2)对东北特早熟棉区棉花棉铃生物量和氮素累积特征的影响.结果表明： 棉花单铃、棉籽和纤维的生物量及其氮素累积随棉花生育进程的动态变化均符合“S”型曲线,种植密度和施氮量可以显著影响棉铃各部分生物量和氮素累积的动态特征,以及棉花产量与品质;在施氮量240 kg·hm-2和种植密度97500 plants·hm-2处理下,单铃、棉籽和纤维的生物量均达到最大,生物量和氮素累积的快速累积起始时间和终止时间较早但持续时间较短,生物量快速累积速率最大,生物量和氮素在铃壳中的分配系数最低,在棉籽和纤维中分配系数最高.     

Targeting IFN-γ-inducible lysosomal thiol reductase overcomes chemoresistance in AML through regulating the ROS-mediated mitochondrial damage

The persistence of leukemia stem cells (LSCs) is one of the leading causes of chemoresistance in acute myeloid leukemia (AML). To explore the factors important in LSC-mediated resistance, we use mass spectrometry to screen the factors related to LSC chemoresistance and defined IFN-γ-inducible lysosomal thiol reductase (GILT) as a candidate. We found that the GILT expression was upregulated in chemoresistant CD34+ AML cells. Loss of function studies demonstrated that silencing of GILT in AML cells sensitized them to Ara-C treatment both in vitro and in vivo. Further mechanistic findings revealed that the ROS-mediated mitochondrial damage plays a pivotal role in inducing apoptosis of GILT-inhibited AML cells after Ara-C treatment. The inactivation of PI3K/Akt/ nuclear factor erythroid 2-related factor 2 (NRF2) pathway, causing reduced generation of antioxidants such as SOD2 and leading to a shifted ratio of GSH/GSSG to the oxidized form, contributed to the over-physiological oxidative status in the absence of GILT. The prognostic value of GILT was also validated in AML patients. Taken together, our work demonstrated that the inhibition of GILT increases AML chemo-sensitivity through elevating ROS level and induce oxidative mitochondrial damage-mediated apoptosis, and inhibition of the PI3K/Akt/NRF2 pathway enhances the intracellular oxidative state by disrupting redox homeostasis, providing a potentially effective way to overcome chemoresistance of AML.     

The cardioprotective effect of fluvastatin on ischemic injury via down-regulation of toll-like receptor 4

To determine whether the cardioprotection effect of fluvastatin mediates by toll-like receptor 4 (TLR4) signaling pathway, fifty Sprague–Dawley rats were randomly divided into five groups: sham operation group, ischemia/reperfusion (I/R) group, fluvastatin groups (high-dosage, medium-dosage, low-dosage, n = 10 in each group). Except sham operation group, the rest four groups of rats were artificially afflicted with coronary occlusion for 30 min, then reperfusion 2 h. Light microscope and transmission electronic microscope were used to observe structural changes of myocardium. RT–PCR was used to measure TLR4 mRNA expression level, TLR4 protein expression was detected by immunohistochemistry. Western blot was used to measure myocardial NF-κB protein level; ELISA was used to measure the level of TNF-α in myocardium. The results demonstrated that fluvastatin treatment markedly decreased ischemic injury caused by ischemia/reperfusion, and inhibited the expression levels of TLR4, TNF-α and NF-κB, all of which up-regulated by ischemia/reperfusion. Taken together, our results suggest that proper dosage of fluvastatin may have protective effect on the ischemic injury mediated by ischemia/reperfusion in the hearts, which might be associated with inhibition of TLR4 signaling pathway and inflammatory response during ischemia/reperfusion.