Pds1 phosphorylation in response to DNA damage is essential for its DNA damage checkpoint function

In Saccharomyces cerevisiae, Pds1 is an anaphase inhibitor and plays an essential role in DNA damage and spindle checkpoint pathways. Pds1 is phosphorylated in response to DNA damage but not spindle disruption, indicating distinct mechanisms delaying anaphase entry. Phosphorylation of Pds1 is Mec1 and Chk1 dependent in vivo. Here, we show that Pds1 is phosphorylated at multiple sites in vivo in response to DNA damage by Chk1. Mutation of the Chk1 phosphorylation sites on Pds1 abolished most of its DNA damage-inducible phosphorylation and its checkpoint function, whereas its anaphase inhibitor functions and spindle checkpoint functions remain intact. Loss of Pds1 phosphorylation correlates with APC-dependent Pds1 destruction in response to DNA damage. We also show that APC(Cdc20) is active in preanaphase arrested cells after DNA damage. This suggests that Pds1 is stabilized by phosphorylation in response to DNA damage, but APC(Cdc20) activity is not altered. Our results indicate that phosphorylation of Pds1 by Chk1 is the key function of Chk1 required to prevent anaphase entry.     

Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells

Toll-like receptors (TLR) are sentinel receptors capable of recognizing pathogen-associated molecule patterns (PAMP) such as lipopolysaccharide (LPS) and CpG-containing oligonucleotides (CpG ODN). TLR2 and TLR4 are major receptors for Gram-positive and Gram-negative bacterial cell wall components, respectively. TLR9 is necessary for CpG signalling. LPS or CpG ODN can activate immature dendritic cells (DC) and induce DC maturation characterized by production of cytokines, up-regulation of co-stimulatory molecules, and increased ability to activate T cells. However, little is known regarding the regulation of TLR gene expression in mouse DC. In this study, we investigated the regulation of TLR2, TLR4 and TLR9 gene expression by LPS in murine immature DC. TLR2, TLR4 and TLR9 mRNA were up-regulated following LPS stimulation. The up-regulation of TLR9 expression coincided with significantly increased production of tumour necrosis factor-alpha induced by LPS plus CpG ODN. While inhibition of extracellular signal-related kinase and NF-kappaB activation suppressed the up-regulation of the expression of TLR2, TLR4 and TLR9 mRNA, inhibition of p38 kinase prevented the up-regulation of TLR2 and TLR4 mRNA expression but enhanced the up-regulation of TLR9 expression. These results demonstrated that TLR2, TLR4 and TLR9 gene expression was differently regulated by LPS in mouse immature DC. Up-regulation of TLR2, TLR4 and TLR9 expression by LPS might promote the overall responses of DC to bacteria and help to explain the synergy between LPS and other bacterial products in the induction of cytokine production.     

平行平板流动腔的合理设计和使用

高度远小于横向和纵向几何尺寸的平行平板流动腔，是当前用以体外研究细胞力学行为，特别是细胞粘附特性的主要工具之一。本文从目前常用的平行平板流动腔内流体定常流动时的切应力表达式出发，指出该切应力表达式仅适用于小雷诺数条件，由此确定平行平板流动腔几何尺寸的合理选择；通过对小孔入流和出流的平行平板流动腔流场的分析，指出该切应力表达式只在远离孔口边界一定距离，即达到均匀流动后成立。     

Urothelial carcinoma of the urinary bladder with a component of acinar/tubular type differentiation simulating prostatic adenocarcinoma

We report a case of an 83-year-old man with a high-grade carcinoma of the urinary bladder who underwent cystoprostatectomy. The invasive carcinoma showed mixed, morphologically distinct patterns consisting of conventional high-grade urothelial carcinoma, glandular differentiation resembling enteric type adenocarcinoma, and acinar/tubular type differentiation, morphologically similar to Gleason grade 3 prostatic adenocarcinoma. Immunohistochemical studies revealed the acinar/tubular component of the tumor to be negative for prostate-specific antigen and prostatic acid phosphatase, but positive for cytokeratin 7, cytokeratin 20, high molecular weight cytokeratin (34 beta E12), and thrombomodulin, consistent with origin from the bladder rather than the prostate. Although bladder carcinomas composed of mixed morphologic patterns are not uncommon, to our knowledge, the presence of acinar/tubular type features simulating prostatic adenocarcinoma in such tumors has not been described elsewhere.     

酵母双杂合系统AD端阴离子交换蛋白C-末端表达质粒的构建

利用PCR方法，从阴离子交换蛋白1（AE1）全长cDNA中扩增出约350bp c末端cDNA片段，测序后将其克隆至pGADT7载体上，用醋酸锂法构建好的pADT7-AE1-c末端转染酵母菌HA109，观察其在选择性培养基上的表达情况。结果表明，获得了530bp AE1c－末端cDNA,pGADT7-AE1-c末端对酵母无毒性，不能激活检测基因，可作为酵母双杂合系统中的靶基因。     

Shear Rate Dependence of Ultrasound Backscattering from Blood Samples Characterized by Different Levels of Erythrocyte Aggregation

The objectives were (1) to determine the effect of the erythrocyte aggregation level (wide range of aggregation) and shear rate (which also affects aggregation) on the ultrasound backscattered power, and (2) to evaluate the reproducibility of the ultrasound method. Experiments were performed under steady flow (100–1250 ml/min) in a 12.7 mm diameter vertical tube. Doppler ultrasound at 10 MHz was used to measure simultaneously the velocity and the backscattered power across the tube. For each radial position, the shear rate was computed from the derivative of the velocity profile. The backscattered power decayed exponentially as a function of the shear rate, and for a given shear rate, the power increased monotonically with the level of aggregation measured by laser reflectometry. Using blood samples simulating hypo-, normal, and hyperaggregating erythrocytes, the power of the ultrasound signal varied respectively by –7.8, –13.2, and –16.1 dB as a function of the shear rate (from 0.4 to 50 s^–1). The reproducibility of the backscattered power was 5.5 dB, which is less than the variations observed as a function of the shear rate. In conclusion, ultrasound backscattering is sensitive to the level of erythrocyte aggregation. At a first glance, ultrasound seems less accurate when compared to laser reflectometry but it is suggested that this is because ultrasound backscattering may be sensitive to structural aggregate changes that are not detected by the laser method. © 2000 Biomedical Engineering Society. PAC00: 8718-h, 8719Tt, 8750Kk     

Injection of substance P (SP) N-terminal fragment SP(1-7) into the ventral tegmental area modulates the levels of nucleus accumbens dopamine and dihydroxyphenylacetic acid in male rats during morphine withdrawal

The biologically active substance P (SP) N-terminal metabolite SP_1–7 has been reported to modulate several neural processes such as learning, locomotor activity and reaction to opioid withdrawal. Although all these processes are believed to be associated with dopaminergic transmission no evidence of an interaction between SP_1–7 and dopamine in the case of morphine withdrawal has so far been reported. Therefore, in this work we applied in vivo microdialysis to investigate the effect of SP_1–7 injection into the ventral tegmental area on dopamine release in nucleus accumbens of male rats during naloxone precipitated morphine withdrawal. The result showed that the heptapeptide enhances dopamine release and also elevates the level of the dopamine metabolite dihydroxyphenylacetic acid in this brain area. It was suggested that the observed action of the SP fragment on the dopamine system represents the underlying mechanism for a previously observed ability of SP_1–7 to counteract the aversion response to morphine withdrawal.     

Defective Treg response in acute kidney injury was caused by a reduction in TIM-3+ Treg cells

Background: Despite years of research, the treatment of acute kidney injury (AKI) remains a significant challenge. Animal studies presented causal links between elevated regulatory T cell (Treg) response and better prognosis in AKI. Previous studies in mice and humans showed that TIM-3+ Treg cells were more potent than TIM-3- Treg cells. In this study, we investigated the role of TIM-3 in Treg in AKI patients.

Methods: Peripheral blood from AKI patients and healthy controls were gathered, and TIM-3+ Treg subset was examined.

Results: Compared to healthy controls, the AKI patients presented a significant upregulation in the frequency of circulating CD4+CD25+ T cells; however, the majority of this increase was from the CD4+CD25+TIM-3- subset, and the frequency of CD4+CD25+TIM-3+ T cells was downregulated in AKI patients. In both healthy controls and AKI patients, the CD4+CD25+TIM-3+ T cells expressed higher levels of Foxp3, and were more potent at expressing LFA-1, LAG-3, CTLA-4, IL-10 and TGF-β. In addition, the CD4+CD25+TIM-3+ T cells from both healthy controls and AKI patients presented higher capacity to suppress CD4+CD25- T cell proliferation than the CD4+CD25+TIM-3- T cells. Interestingly, the total CD4+CD25+ T cells from AKI patients presented significantly lower inhibitory capacity than those from healthy controls, indicating that the low frequency of CD4+CD25+TIM-3+ T cells was restricting the efficacy of the Treg responses in AKI patients.

Conclusions: We demonstrated that TIM-3 downregulation impaired the function of Treg cells in AKI. The therapeutic potential of CD4+CD25+TIM-3+ T cells in AKI should be investigated in future studies.     

非悬滴开放式培养法在鸡胚背根节体外培养中的应用

本研究针对悬滴培养法在操作和应用上存在的问题和局限性，改用操作简便、适用范围广的非悬滴开放式培养法培养鸡胚背根节。将数个鸡胚背根节按一定间隔种植在内置生长基质盖玻片的35mm培养皿中，加人适量培养液，置于CO2。孵箱中进行培养。结果显示.从培养24h至60h各时期，培养皿中背根节生长状况均良好，神经突起明显增长，表明用非悬滴开放式培养法培养鸡胚背根节是可行且可靠的。     

IV型II类反式激活因子基因新变异体的克隆、鉴定和功能测定

为获取有功能的IV型II类反式激活因子基因 (CIITA IV ) ,诱导肿瘤细胞表达MHCII类分子 ,从IFN γ刺激的THP 1细胞中以RT PCR获得CIITA IV ,将其连接到pGEMT easy载体。对所构建的pcDNA3 1 CIITA IV型表达载体进行反复测序后发现 ,所获得的CIITA IV基因存在结构变异 ,在 2 87位插入了 3个核苷酸TAG ,使 2 86 2 88位的AAG改变成为ATAGAG(2 86 2 90 ) ,并引起其他 8个座位核苷酸 (及推导的氨基酸残基 )发生改变。将表达载体转入原先不表达MHCII类分子的HeLa细胞中 ,检测到所获得的IV型CIITA变异体具有诱导人II类分子HLA DR表达的能力。空载体和CIITA IV基因导入的HeLa细胞中 ,DR阳性细胞百分率分别为 0 0 1 %和 37 6 4 %。该基因已从GenBank得到登录号 ,表明这是一个具有诱导HLA DR分子表达功能的IV型CIITA新基因。