Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands

The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.     

Differential localization of P-selectin and von Willebrand factor during megakaryocyte maturation

Abstract

An important step in megakaryocyte maturation is the appropriate assembly of at least two distinct subsets of α-granules. The mechanism that sorts the α-granule components into distinct structures and mediates their release in response to specific stimuli is now emerging. P-selectin and von Willebrand factor are two proteins present in the α-granules that recognize P-selectin glycoprotein ligand on neutrophils and collagen in the subendothelial matrix. These proteins may play an important role in determining the differential release of the α-granule contents in response to external stimuli. If P-selectin and von Willebrand factor are localized in the same or different α-granules is not known. To clarify this question, we analyzed by immunoelectron microscopy the localization of von Willebrand factor and P-selectin during the maturation of wild-type and Gata1^low megakaryocytes induced in vivo by treating animals with thrombopoietin. Gata1^low is a hypomorphic mutation that blocks megakaryocyte maturation, reduces the levels of von Willebrand factor expression and displaces P-selectin on the demarcation membrane system. The maturation block induced by this mutation is partially rescued by treatment in vivo with thrombopoietin. In immature megakaryocytes, both wild-type and Gata1^low, the two receptors were co-localized in the same cytoplasmic structures. By contrast, the two proteins were segregated to separate α-granule subsets as the megakaryocytes matured. These observations support the hypothesis that P-selectin and von Willebrand factor may ensure differential release of the α-granule content in response to external stimuli.     

植物转录因子最新研究方法

转录因子可以调控众多下游基因的表达,在植物的生长发育、代谢及对外界环境的反应中起着重要作用。我们结合近年来植物转录因子的研究进展,归纳分析了高等植物转录因子研究的主要策略和最新的技术方法,并从生物信息学分析、瞬间转化技术的应用、突变体表型分析及调控网络等几个方面进行了全面阐述,为植物转录因子的预测、功能鉴定及靶基因分析等相关研究提供理论和方法的参考。     

五个WRKY转录因子在水稻叶片生长和抗病反应中的表达研究

WRKY是植物基因组中最大的转录因子家族之一,它们在抗病及其他信号转导途径中发挥着重要的调控作用．为了解水稻WRKY的功能,我们选择了5个WRKY转录因子,用免疫印迹技术调查了它们在水稻叶片生长和在Xa21介导的白叶枯病抗性反应中的表达丰度变化．结果表明,OsWRKY13、23和71在叶片中表达,且随叶片生长而逐步增加,至成熟期略有下降,但在叶片中检测不到OsWRKY45和OsWRKY53的表达信号．在Xa21介导的白叶枯病抗性反应中,OsWRKY45、53和71均受诱导表达,而OsWRKY13和 OsWRKY23蛋白质的表达没有可见的变化．进一步比较OsWRKY45、OsWRKY53和OsWRKY71在抗、感和对照(Mock)反应中的表达,发现它们在抗、感反应中均发生相似变化．上述结果说明,OsWRKY13和OsWRKY23可能在叶片正常生长过程中发挥作用,OsWRKY45和OsWRKY53可能在水稻-白叶枯病菌互作过程中发挥作用,而OsWRKY71在二种条件下均有功能．     

基于林业清查资料的桂西北植被碳空间分布及其变化特征

基于2005-2010年林业资源清查数据,采用材积源生物量法,运用地理信息系统技术,估算和分析了桂西北植被碳密度及其储量的空间分布及其变化。结果显示:(1) 研究区域从2005年到2010年呈现碳汇变化趋势,植被碳储量由4.19×10⁴t增加到4.27×10⁴t(增幅为1.84%),植被碳密度从29.04t/hm²增加到29.57 t/hm²。(2) 从治理措施、林种起源方式及林种类型来看,自然保护区的植被碳密度最大,超过40 t/hm²。2005-2010年,人工植苗、直播、飞播和萌生方式植被碳密度增加,退耕还林工程的植被碳密度均呈明显增长(增加3.00 t/hm²),所有林种碳密度都呈不同程度的增长。 (3)植被碳密度空间分布上,大致表现为西部高、中东部低,北部高、南部低。西部区植被碳密度均值超过40 t/hm²,中东部区植被碳密度均值低于25 t/hm²。植被碳密度变化在空间分布上表现为无论是非喀斯特区还是喀斯特区的植被碳密度都有增长趋势,其中有7个县市植被碳密度升级为更高等级。研究表明,随着退耕还林、生态移民等治理措施的实施,区域植被碳密度显著增加,生态环境好转。     

门诊患者医疗服务感受度影响因素研究

??????? 目的 了解门诊患者对就医服务感受度影响因素,以期提高患者满意度。 方法 现场问卷调查法、专家访谈法。 结果 患者在门诊过程中最为关注的就诊流程、等候时间、就医环境、医德医风四个因素,非常看重医护人员服务规范,服务流程便捷流畅。病情不同患者对等候时间要求不同,对医院熟悉程度与就诊环境感受度不同。结论 在门诊服务中为患者提供便捷流畅、规范化的服务,同时应注重患者的不同服务需求。     

Significance of the anti-aging protein Klotho

Abstract

The Klotho gene was identified as an ‘aging suppressor' in mice. Overexpression of the Klotho gene extends lifespan and defective Klotho results in rapid aging and early death. Both the membrane and secreted forms of Klotho have biological activity that include regulatory effects on general metabolism and a more specific effect on mineral metabolism that correlates with its effect on aging. Klotho serves as a co-receptor for fibroblast growth factor (FGF), but it also functions as a humoral factor that regulates cell survival and proliferation, vitamin D metabolism, and calcium and phosphate homeostasis and may serve as a potential tumor suppressor. Moreover, Klotho protects against several pathogenic processes in a FGF23-independent manner. These processes include cancer metastasis, vascular calcification, and renal fibrosis. This review covers the recent advances in Klotho research and discusses novel Klotho-dependent mechanisms that are clinically relevant in aging and age-related diseases.     

NF-κB p65的敲减加重结肠上皮HCT116细胞氧化损伤

炎症细胞诱导的活性氧类生成和肠道氧化应激与慢性炎症性肠疾病以及结直肠肿瘤的发病密切相关。NF-κB信号通路参与氧化应激反应以及在结直肠炎症和肿瘤发生中的作用还并不完全清楚。本研究将化学合成一对编码小干扰RNA 序列、靶向人NF-κB 基因的长60 bp寡核苷酸链定向克隆至pSUPER小干扰RNA表达载体中,通过单酶切、双酶切及测序证实重组RNA干扰载体构建成功. 将构建成功的质粒转染至结肠上皮细胞HCT116中敲减p65,分别采用Western blot方法检测NF-κB p65蛋白表达水平,（3-(4,5-二甲基噻唑-2)-3,5-二苯基四氮唑溴盐（MTT）方法检测细胞存活情况. 结果显示,pSUPER-NF-κB p65载体可特异性下调NF-κB p65蛋白表达;下调p65表达可导致过氧化氢诱导的HCT116内活性氧类物质生成增高,存活细胞数目显著减少,氧化损伤加重。研究表明,在人结肠上皮细胞内NF-κB p65通路的抑制显著加重了结肠上皮细胞氧化损伤情况.     

米诺环素对不完全脊髓损伤大鼠脊髓BDNF和NT-3表达的影响

目的观察腹腔注射米诺环素对改良Allen’s法造成的不完全脊髓损伤大鼠脊髓中脑源性神经营养因子以及神经营养因子3表达的影响,探讨米诺环素治疗脊髓损伤的作用机制。方法成年雌性Sprague-Dawley(SD)大鼠54只,改良Allen’s法造成不完全脊髓损伤,根据实验需要可以分为3组,空白组,只打开脊柱椎板,不损伤;治疗组,大鼠脊髓损伤,并腹腔注射米诺环素;损伤组,大鼠脊髓损伤,腹腔注射等剂量的生理盐水。观察各组大鼠的后肢能力Basso-Beattie-Bresnahan评分,并于不同时段(3d、7d,14d)取大鼠脊髓T8-9段采用逆转录PCR,以及免疫化学组织染色法测定脑源性神经营养因子以及神经营养因子3的表达。结果米诺环素能够明显改善不完全脊髓损伤大鼠的功能,逆转录PCR和脊髓组织冰冻切片免疫组织化学染色DAB都能证实米诺环素治疗组脑源性神经营养因子以及神经营养因子3表达显著增多。结论米诺环素在治疗不完全脊髓损伤大鼠的机制还应与其上调了大鼠体内的脑源性神经营养因子以及神经营养因子3表达有关。