IR-inducible clusterin gene expression: a protein with potential roles in ionizing radiation-induced adaptive responses, genomic instability, and bystander effects

Clusterin (CLU) plays numerous roles in mammalian cells after stress. A review of the recent literature strongly suggests potential roles for CLU proteins in low dose ionizing radiation (IR)-inducible adaptive responses, bystander effects, and delayed death and genomic instability. Its most striking and evident feature is the inducibility of the CLU promoter after low, as well as high, doses of IR. Two major forms of CLU, secreted (sCLU) and nuclear (nCLU), possess opposite functions in cellular responses to IR: sCLU is cytoprotective, whereas nCLU (a byproduct of alternative splicing) is a pro-death factor. Recent studies from our laboratory and others demonstrated that down-regulation of sCLU by specific siRNA increased cytotoxic responses to chemotherapy and IR. sCLU was induced after low non-toxic doses of IR (0.02-0.5 Gy) in human cultured cells and in mice in vivo. The low dose inducibility of this survival protein suggests a possible role for sCLU in radiation adaptive responses, characterized by increased cell radioresistance after exposure to low adapting IR doses. Although it is still unclear whether the adaptive response is beneficial or not to cells, survival of damaged cells after IR may lead to genomic instability in the descendants of surviving cells. Recent studies indicate a link between sCLU accumulation and cancer incidence, as well as aging, supporting involvement of the protein in the development of genomic instability. Secreted after IR, sCLU may also alter intracellular communication due to its ability to bind cell surface receptors, such as the TGF-beta receptors (types I and II). This interference with signaling pathways may contribute to IR-induced bystander effects. We hypothesize that activation of the TGF-beta signaling pathway, which often occurs after IR exposure, can in turn activate the CLU promoter. TGF-beta and IR-inducible de novo synthesized sCLU may then bind the TGF-beta receptors and suppress downstream growth arrest signaling. This complicated negative feedback regulation most certainly depends on the cellular microenvironment, but undoubtedly represents a potential link between IR-induced adaptive responses, genomic instability and bystander effects. Further elucidation of clusterin protein functions in IR responses are clearly warranted.     

非天然氨基酸β-Homo天冬酰胺的合成研究

以α-氨基酸天冬酰胺为原料,经多步反应合成了β-氨基酸β-Homo天冬酰胺,中间产物和目的产物经熔点、红外、核磁、质谱以及元素分析,其结构得到证实。     

1型糖尿病葡萄糖代谢与胰岛素信号传导途径异常导致大鼠海马Alzheimer样tau蛋白过度磷酸化修饰

Tau蛋白过度磷酸化是Alzheimer病(Alzheimer disease, AD)的一个重要病理特征.采用 I 型糖尿病大鼠模型,研究胰岛素信号传导途径及葡萄糖代谢失调对tau蛋白过度磷酸化的形成机制进行探讨.以同龄Wistar大鼠做对照（CTL）,胰腺大部分切除造低胰岛素组（PX）,STZ较大剂量一次性注射造1型糖尿病模型即低胰岛素高血糖组（T1DM）.葡萄糖氧化酶法检测血浆血糖,放免法检测血浆胰岛素,蛋白质印迹分析海马内总tau蛋白及tau蛋白上部分位点（Ser199、Thr212、Ser214、Ser396及Ser422）的磷酸化及神经细胞膜上葡萄糖转运子3（Glucose transport 3,GLUT3）水平.γ-32P-ATP和特异性底物肽检测海马内胰岛素信号传导系统中的关键酶糖原合成酶激酶-3β（Glycogen synthase kinase-3β, GSK-3β）活性.发现3组大鼠海马回总tau蛋白水平无显著差异,但以高血糖、低胰岛素血症为特征的T1DM组在tau蛋白Ser199、Thr212、Ser214、Ser396及Ser422位点上,呈现过度磷酸化状态,以低胰岛素血症为特征而血糖正常的PX组在位点Ser199、Thr212及Ser396上磷酸化程度比CTL组显著上升, 在位点Ser214及 Ser422上的磷酸化程度的改变不显著;T1DM及PX组大鼠海马 GSK-3β活性显著高于CTL组, 而GLUT3水平在T1DM和PX组均降低, 尤以T1DM组降低更显著.研究结果显示,胰岛素水平低下可能通过激活GSK-3β和下调细胞内葡萄糖代谢的双重作用引起脑内tau蛋白过度磷酸化.     

Characteristics of stimulation of gonadotropin secretion by kisspeptin-10 in female goats

The aims of the present study were to clarify the effect of kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH), growth hormone (GH) and prolactin (PRL) in goats, and compare the characteristics of any response with those of the response to gonadotropin-releasing hormone (GnRH). The experiments were performed using four female goats (4–5 years old) in the luteal phase of estrous cycle. A single intravenous (i.v.) injection of 1, 5 and 10 μg/kg b.w. (0.77, 3.85 and 7.69 nmol/kg b.w.) of Kp10 stimulated the release of LH. Maximum values were observed 20–30 min after the injection. On the other hand, Kp10 did not alter plasma GH and PRL concentrations significantly. Three consecutive i.v. injections of Kp10 (5 μg/kg b.w.) or GnRH (5 μg/kg b.w.: 4.23 nmol/kg b.w.) at 2-h intervals increased both plasma LH and FSH levels after each injection (P < 0.05); however, the responses to Kp10 were different from a similar level of GnRH. The rate of decrease in LH and FSH levels following the peak was attenuated in Kp10-treated compared to GnRH-treated animals. These results show that Kp10 can stimulate the release of LH and FSH but not GH and PRL in female goats and suggest that the LH- and FSH-releasing effect of the i.v. injection of Kp10 is less potent than that of GnRH.     

TGF-β/Smad通路对造血干细胞的调控作用

造血干细胞(hematopoietic stem cells,HSCs)是典型的成体干细胞,造血系统的稳定依靠造血干细胞正确的自我更新、增殖和分化。TGF-β超家族包括TGF-β、骨生成蛋白(BMP)和激活素,可通过Smad蛋白对造血干细胞进行调节。TGF-β/Smad通路可通过降低CDK4的表达、增加p21蛋白表达和改变p27分布,将造血干细胞阻断于G1期;通过上调CD34表达,抑制造血干细胞的分化。但也有不同的观点,认为TGF-β对HSCs的调节与Smads无关,TGF-β并非通过调控p21和p27抑制HSCs的增殖,TGF-β/Smad通路对维持HSCs静止状态无关。     

褪黑素对海人酸致痫大鼠海马内TGF-β3水平的影响

目的探讨褪黑素(me1atonin,MT)对海人酸(kainic acid,KA)致痫大鼠海马内TGF-β3的影响,进一步明确其在中枢内的作用。方法将实验大鼠随机分为3组:生理盐水对照组(NS组)、海人酸组(KA组)、褪黑素+海人酸组(MT+KA组)。各组大鼠给予相应试剂处理后观察并记录大鼠行为学改变,用免疫组织化学方法、RT-PCR检测大鼠海马内TGF-β3(transforming growth factor-β3)的表达情况及其mRNA变化。结果动物行为学观察显示,NS组无癫痫发作,KA组发作程度为Ⅲ-V级,MT+KA组为0-Ⅲ级;免疫组织化学结果显示,TGF-β3在3组大鼠海马内均有表达,其中KA组、MT+KA组较NS组表达增强,MT+KA组较KA组增强,差异具有显著性意义(P0.05);RT-PCR结果显示,与NS组相比较,KA组、MT+KA组大鼠海马内TGF-β3 mRNA含量均升高;但MT+KA组升高较KA组多,差异具有显著性意义(P0.05)。结论褪黑素能明显改善海人酸诱发的大鼠癫痫,增强海马内TGF-β3的表达,减轻海马神经元损伤,发挥中枢保护作用。     

The RING Domain of TRAF2 Plays an Essential Role in the Inhibition of TNFα-Induced Cell Death but Not in the Activation of NF-κB

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of κB kinase (IKK), as well as in inhibiting apoptosis induced by TNFα. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFα-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-ΔR) has been widely used as a dominant negative in transient overexpression systems to block TNFα-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-ΔR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFα-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells efficiently inhibited the TNFα-induced later phase of prolonged JNK activation, yet failed to inhibit TNFα-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFα and is essential for suppressing the noncanonical nuclear factor κB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFα-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.     

Prediction of the parallel/antiparallel orientation of beta-strands using amino acid pairing preferences and support vector machines

In principle, structural information of protein sequences with no detectable homology to a protein of known structure could be obtained by predicting the arrangement of their secondary structural elements. Although some ab initio methods for protein structure prediction have been reported, the long-range interactions required to accurately predict tertiary structures of β-sheet containing proteins are still difficult to simulate. To remedy this problem and facilitate de novo prediction of β-sheet containing protein structures, we developed a support vector machine (SVM) approach that classified parallel and antiparallel orientation of β-strands by using the information of interstrand amino acid pairing preferences. Based on a second-order statistics on the relative frequencies of each possible interstrand amino acid pair, we defined an average amino acid pairing encoding matrix (APEM) for encoding β-strands as input in the prediction model. As a result, a prediction accuracy of 86.89% and a Matthew's correlation coefficient value of 0.71 have been achieved through 7-fold cross-validation on a non-redundant protein dataset from PISCES. Although several issues still remain to be studied, the method presented here to some extent could indicate the important contribution of the amino acid pairs to the β-strand orientation, and provide a possible way to further be combined with other algorithms making a full ‘identification’ of β-strands.     

Intramolecular Movements in EF-G, Trapped at Different Stages in Its GTP Hydrolytic Cycle, Probed by FRET

Elongation factor G (EF-G) is one of several GTP hydrolytic proteins (GTPases) that cycles repeatedly on and off the ribosome during protein synthesis in bacterial cells. In the functional cycle of EF-G, hydrolysis of guanosine 5′-triphosphate (GTP) is coupled to tRNA-mRNA translocation in ribosomes. GTP hydrolysis induces conformational rearrangements in two switch elements in the G domain of EF-G and other GTPases. These switch elements are thought to initiate the cascade of events that lead to translocation and EF-G cycling between ribosomes. To further define the coupling mechanism, we developed a new fluorescent approach that can detect intramolecular movements in EF-G. We attached a fluorescent probe to the switch I element (sw1) of Escherichia coli EF-G. We monitored the position of the sw1 probe, relative to another fluorescent probe anchored to the GTP substrate or product, by measuring the distance-dependent, Förster resonance energy transfer between the two probes. By analyzing EF-G trapped at five different functional states in its cycle, we could infer the cyclical movements of sw1 within EF-G. Our results provide evidence for conformational changes in sw1, which help to drive the unidirectional EF-G cycle during protein synthesis. More generally, our approach might also serve to define the conformational dynamics of other GTPases with their cellular receptors.