~(125)Ⅰ标记单抗LC-I与肺腺癌细胞体内外结合特性的研究

本文用~(125)Ⅰ标记LC-1进行了一些体内外实验。实验结果表明:LC-1单抗的结合常数为4.8×10~8M~(-1),LC-1针对的SPC-A_1细胞表面抗原的位点数为7.2×10~4/细胞;LC-1与LAC-122两单抗针对的抗原决定簇没有交叉;用蛋白酶和过碘酸钠处理SPC-A_1细胞,前者对LC-1的结合抑制39%,后者抑制66%;LC- 1不但有较强的体外结合靶细胞的能力,从LC-1在荷瘤裸鼠中的组织器官分布来看,LC-1与肿瘤有较高的体内亲和性,并且是特异性的结合。     

Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, skeletal myocyte differentiation, and cardiac hypertrophy. We previously showed that active MEKK3 is capable of stimulating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes through phosphorylation of modulatory calcineurin-interacting protein 1 (MCIP1). However, the protein kinases that function downstream of MEKK3 to mediate MCIP1 phosphorylation and the mechanism of MCIP1-mediated calcineurin regulation have not been defined. Here, we show that MEK5 and big MAP kinase 1 (BMK1) function downstream of MEKK3 in a signaling cascade that induces calcineurin activity through phosphorylation of MCIP1. Genetic studies showed that BMK1-deficient mouse lung fibroblasts failed to mediate MCIP1 phosphorylation and activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring BMK1 to the deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. Thus, using BMK1-deficient mouse lung fibroblast cells, we provided the genetic evidence that BMK1 is required for angiotensin II-mediated calcineurin/NFAT activation through MICP1 phosphorylation. Finally, we discovered that phosphorylated MCIP1 dissociates from calcineurin and binds with 14-3-3, thereby relieving its inhibitory effect on calcineurin activity. In summary, our findings reveal a previously unrecognized essential regulatory role of mitogen-activated protein kinase signaling in calcineurin activation through the reversible phosphorylation of a calcineurin-interacting protein, MCIP1.     

Effects of hypomagnetic field on noradrenergic activities in the brainstem of golden hamster

Previous studies found that elimination of the geomagnetic field (GMF) interferes with the normal brain functions, but the underlying mechanism remains unknown. The present study examined the effects of long-term exposures to a near-zero magnetic environment on the noradrenergic activities in the brainstem of golden hamsters. Both the content of norepinephrine (NE) and the density of NE-immunopositive neurons in the tissue decreased significantly after the treatment, and the effects could be progressive with time. These variations may substantially contribute to behavioral and mood disorders reported in other studies when animals are shielded from the GMF.     

PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex

PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Furthermore, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5, and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1, and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a potential role of PTIP in the regulation of gene expression.     

Cytoplasm-localized SIRT1 enhances apoptosis

In general, SIRT1 is localized in nuclei. Here, we showed that endogenous and exogenous SIRT1 were both able to partially localize in cytoplasm in certain cell lines, and cytoplasm-localized SIRT1 was associated with apoptosis and led to increased sensitivity to apoptosis. Furthermore, we demonstrated that translocation of nucleus-localized SIRT1 from nuclei to cytoplasm was the main pathway leading to localization of SIRT1 in cytoplasm. In HeLa cells, wild type SIRT1 was completely localized in nuclei. By truncation of two predicted nuclear localization signals or fusion with an exogenous nuclear export signal, SIRT1 was partially localized in cytoplasm of HeLa cells and resulted in increased sensitivity to apoptosis. The apoptosis enhanced by cytoplasm-localized SIRT1 was independent of its deacetylase activity, but dependent on caspases. SIRT1 was distributed in cytoplasm at metaphase during mitosis, and overexpression of SIRT1 significantly augmented apoptosis for cells at metaphase. In summary, we found SIRT1 is able to localize in cytoplasm, and cytoplasm-localized SIRT1 enhances apoptosis.     

人肝癌细胞系HepG2中survivin-3鉴定及其真核载体构建

目的:鉴定肝癌细胞系HepG2中survivin异构体(survivin variant,SVV variant)并构建其真核表达栽体.方法:提取HepG2细胞总RNA,根据Gen-Bank内survivin 3个异构体核苷酸序列设计3条引物对其进行鉴定;设计含有BamH I和Xho I双酶切位点的SVV-3引物,逆转录聚合酶链反应(RT-PCR)扩增SVV-3完整编码区,扩增产物用BamHI和XhoI双酶切后定向克隆到真核细胞表达栽体pcDNA3.1中,序列测定进行鉴定.结果:HepG2细胞表达SVV-3、1,SVV-3表达尤为丰富.对SVV-3克隆测序,与Gen-Bank报道完全一致.结论:成功鉴定出HepG2表达SVV-3、1,构建了SVV-3真核表达载体.     

Testing the responses of four wheat crop models to heat stress at anthesis and grain filling

Higher temperatures caused by future climate change will bring more frequent heat stress events and pose an increasing risk to global wheat production. Crop models have been widely used to simulate future crop productivity but are rarely tested with observed heat stress experimental datasets. Four wheat models (DSSAT‐CERES‐Wheat, DSSAT‐Nwheat, APSIM‐Wheat, and WheatGrow) were evaluated with 4 years of environment‐controlled phytotron experimental datasets with two wheat cultivars under heat stress at anthesis and grain filling stages. Heat stress at anthesis reduced observed grain numbers per unit area and individual grain size, while heat stress during grain filling mainly decreased the size of the individual grains. The observed impact of heat stress on grain filling duration, total aboveground biomass, grain yield, and grain protein concentration (GPC) varied depending on cultivar and accumulated heat stress. For every unit increase of heat degree days (HDD, degree days over 30 °C), grain filling duration was reduced by 0.30–0.60%, total aboveground biomass was reduced by 0.37–0.43%, and grain yield was reduced by 1.0–1.6%, but GPC was increased by 0.50% for cv Yangmai16 and 0.80% for cv Xumai30. The tested crop simulation models could reproduce some of the observed reductions in grain filling duration, final total aboveground biomass, and grain yield, as well as the observed increase in GPC due to heat stress. Most of the crop models tended to reproduce heat stress impacts better during grain filling than at anthesis. Some of the tested models require improvements in the response to heat stress during grain filling, but all models need improvements in simulating heat stress effects on grain set during anthesis. The observed significant genetic variability in the response of wheat to heat stress needs to be considered through cultivar parameters in future simulation studies.     

Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.     

Homodimeric PHD Domain-containing Rco1 Subunit Constitutes a Critical Interaction Hub within the Rpd3S Histone Deacetylase Complex

Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way.     

辽宁碱蓬SlCMO基因盐胁迫表达分析及盐诱导启动子鉴定

胆碱单加氧酶（choline monooxygenase, CMO）是合成甜菜碱的关键酶,甜菜碱在植物抵抗渗透胁迫中起着重要的作用。本研究室前期克隆了盐生植物辽宁碱蓬CMO（Suaeda liaotungensis CMO）基因及启动子。本研究对SlCMO基因在盐胁迫下的表达及盐诱导启动子进行分析。qRT-PCR分析SlCMO基因在辽宁碱蓬不同器官及盐胁迫下的表达,结果表明,SlCMO基因在根、茎、叶中均有表达,其中茎、叶中的表达量较高,SlCMO基因在根、茎、叶中的表达均受盐胁迫诱导。5′端缺失分析SlCMO启动子的盐诱导区段,结果表明,pC5（-267~+128 bp）是SlCMO启动子的盐诱导功能区段,推测pC5调控SlCMO 基因的盐诱导表达。本研究为SlCMO 基因表达调控研究奠定基础,也为植物抗盐基因工程提供可用的启动子。