肝豆汤改良方对Wilson''s病模型TX乳鼠神经元内Cyt C/Caspase信号通路的分子调控机制

目的:探讨肝豆汤改良方调控TX乳鼠神经元内细胞色素C(Cyt C)/半胱氨酸天冬氨酸蛋白酶(Caspase)信号通路的分子靶点并观察其相应的调控机制。方法:本实验乳鼠神经元通过原代方法分离培养所得,分为正常组、模型组、肝豆汤改良方组、丁苯酞组,正常组为正常DL乳鼠神经元,用完全培养基培养,模型组为TX乳鼠神经元,用10%空白兔血清培养,肝豆汤改良方组为TX乳鼠神经元,加入含体积浓度(5%,10%,15%,20%)肝豆汤改良方兔血清的培养基继续培养,丁苯酞组为TX乳鼠神经元,用10%含丁苯酞兔血清培养。采用原子吸收分光光度法检测不同浓度含肝豆汤改良方兔血清作用24 h后,对TX乳鼠神经元内微量元素的影响;流式细胞仪检测经肝豆汤改良方兔血清作用后活性氧(ROS)释放量的变化;蛋白质免疫印迹(Western blot)法检测经含肝豆汤改良方兔血清作用后Cyt C,Caspase-9,Caspase-3蛋白的表达。结果:与模型组比较,含肝豆汤改良方兔血清可显著降低TX乳鼠神经元内铜、铁含量,增加锌含量(P0.01)。流式细胞仪检测发现,含肝豆汤改良方兔血清较模型组可显著降低TX乳鼠神经元内ROS的释放量(P0.01)。Western blot检测结果显示与模型组比较,含肝豆汤改良方兔血清可显著降低TX乳鼠神经元内Cyt C,Caspase-9,Caspase-3蛋白表达(P0.01)。结论:肝豆汤改良方可能是通过促进过量铜排出而抑制神经元内Cyt C,Caspase-9,Caspase-3表达,从而减轻高铜对神经元的损伤作用。肝豆汤改良方可通过减少脑内铜含量,进而调控Cyt C/Caspase信号通路达到减轻高铜诱导的神经元损伤的治疗效果。     

Folate deficiency is associated with the formation of complex nuclear anomalies in the cytokinesis‐block micronucleus cytome assay

Chromosomal instability (CIN) is an important hallmark to oncogenesis and can be diagnosed morphologically by the presence of nuclear anomalies such as micronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBuds). We have identified additional nuclear anomalies formed under folate‐deficient conditions, defined as “fused” nuclei (FUS), “circular” nuclei (CIR), and “horse‐shoe” nuclei (HS) and investigated their suitability for inclusion as additional CIN biomarkers in the lymphocyte cytokinesis‐block micronucleus cytome (CBMN‐Cyt) assay. Although the morphological appearance of FUS, CIR, and HS suggested an origin from multiple NPB in the fusion region between the two nuclei, the very low frequency of dicentric chromosomes in metaphase spreads from these cultures did not support this model. Fluorescence in situ hybridization (FISH) analysis of cytokinesis‐blocked binucleated (BN) cells with peptide nucleic acid probes for telomeres and centromeres (PNA–FISH) revealed a high proportion of fusion regions contained both centromeric and telomeric DNA. This suggests that folate deficiency may disrupt the process of sister chromatid separation and chromosome segregation during mitosis. It was concluded that the FUS, CIR, and HS morphologies represent promising biomarkers of CIN that are sensitive to folate deficiency, and further validation and investigation of the mechanisms responsible for their formation is warranted. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

A high isoflavone diet decreases 5′ adenosine monophosphate–activated protein kinase activation and does not correct selenium-induced elevations in fasting blood glucose in mice

Selenium (Se) has been implicated as a micronutrient that decreases adenosine monophosphate–activated protein kinase (AMPK) signaling and may increase diabetes risk by reducing insulin sensitivity. Soy isoflavones (IF) are estrogen-like compounds that have been shown to attenuate insulin resistance, hyperglycemia, adiposity, and increased AMPK activation. We hypothesized that a high IF (HIF) diet would prevent the poor metabolic profile associated with high Se intake. The purpose of this study was to examine changes in basal glucose metabolism and AMPK signaling in response to an HIF diet and/or supplemental Se in a mouse model. Male FVB mice were divided into groups receiving either a control diet with minimal IF (low IF) or an HIF diet. Each dietary group was further subdivided into groups receiving either water or Se at a dose of 3 mg Se/kg body weight daily, as Se-methylselenocysteine (SMSC). After 5 months, mice receiving SMSC had elevated fasting glucose (P < .05) and a tendency for glucose intolerance (P = .08). The increase in dietary IF did not result in improved fasting blood glucose. Interestingly, after 6 months, HIF-fed mice had decreased basal AMPK activation in liver and skeletal muscle tissue (P < .05). Basal glucose metabolism was changed by SMSC supplementation as evidenced by increased fasting blood glucose and glucose intolerance. High dietary IF levels did not protect against aberrant blood glucose. In FVB mice, decreased basal AMPK activation is not the mechanism through which Se exerts its effect. These results suggest that more research must be done to elucidate the role of Se and IF in glucose metabolism.     

深低温停循环后大鼠海马线粒体通透性转换孔开闭情况研究

目的通过对深低温停循环大鼠海马线粒体通透性转换孔(MPTP)开闭情况的研究,从亚细胞水平探讨深低温脑保护的机制。方法取成年SD大鼠33只,3只作为供血动物,剩余30只随机分为深低温停循环组(n=10)、常温停循环组(n=10)、正常对照组(n=10)。采用闭胸式体外插管法建立大鼠体外循环模型。其后采用断头取脑法迅速取出大鼠双侧海马组织,提取线粒体后应用蛋白印记法测量线粒体内细胞色素C(CytC)研究MPTP的开闭情况。结果常温停循环组的MPTP较正常对照组、深低温停循环组的开放明显增加(P<0.05);正常对照组的MPTP开放较深低温停循环组的开放少(P<0.05)。结论缺血缺氧可导致MPTP开放;深低温可抑制MPTP开放,保证了细胞的正常代谢活动从而起到脑保护的作用。     

Assessment of oxidative stress responses and the cytotoxic and genotoxic potential of the herbicide tembotrione in HepG2 cells

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus “cytome” assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.     

Immune recognition of cytochrome C. I. Investigation by synthesis whether antigenic sites of polymeric cytochrome coincide with locations of sequence differences between the immunizing and host cytochromes

Previously, we had shown that the locations of the antigenic sites of myoglobin, lysozyme, hemoglobin and serum albumin are independent of the host species and that their antigenicity is inherent in their structural locations. However, studies of others on cytochrome c (Cyt c) had been consistently analyzed on the basis of the assumption that protein antigenic sites are localized at locations of amino acid differences between the injected protein and its counterpart in the host. The intriguing possibility that immune recognition of Cyt c may proceed via a unique mechanism was tested here using beef Cyt c as the antigen in rabbits. Regions around the four locations of amino acid differences between beef Cyt c and rabbit Cyt c (positions 44, 62, 89 and 92) were synthesized, and their ability to bind ¹²⁵I-labelled antibodies against beef Cyt c was determined by immunoadsorbent titration studies. Of these peptides, only the peptide around the substitution at position 44 (i.e. peptide 42–50) had antibody binding activity, which was almost equal to that of the homologous antigen and which also fully accounted for an autoreactivity of the antibodies with rabbit Cyt c. Since, of the four positions of amino acid differences, only one was localized in an antigenic site, it was concluded that this occurence was a chance happening. The antigenicity (and autoreactivity) of the region 42–50 was most likely inherent in its conformational location and not related to sequence differences between beef Cyt c and rabbit Cyt c.     

Signaling of endothelial cytoprotection in transplantation

A better knowledge of the processes by which endothelium can resist to cell death and adapt to injury by specific intracellular signaling pathways and dedicated protein regulation is a key step to understand how vascular inflammation/injury develops and how it is regulated. This review focuses on signaling pathways and molecular effectors that trigger the balance between endothelial cell activation and dysfunction. In addition to the canonical nuclear factor-κB (NF-κB), phosphatidyl inositol 3-kinase (PI-3K) and mitogen-activated protein kinases (MAPK) that orchestrated the inflammatory response and its termination we report here additive pathways such as Notch pathway and protein C/protease activated receptor (PAR) pathway that have been also reported to play a role in the control of EC activation and apoptosis. This review also provides an update of the characteristics of some established and novel protective molecules for the endothelium, identified in transplantation.     

Imatinib mesylate induction of ROS-dependent apoptosis in melanoma B16F0 cells

Background

Imatinib mesylate (STI571), a protein tyrosine kinase inhibitor, was shown to reduce the viability of several cancer cell lines via apoptosis induction; however, the role of reactive oxygen species (ROS) in STI571-induced melanoma cell apoptosis is still undefined.

Objective

In this study, we investigated the contribution of ROS to STI571-induced apoptosis in melanoma B16F0 cells, and the apoptotic mechanism elicited by STI571 was illustrated.

Methods

Using an in vitro cell culture system, the effects of STI571 on ROS production, cell cycle progression, caspase activation, and mitochondrial functions were examined via Western blotting, a flow cytometric analysis, an enzyme activity assay, and a DNA integrity assay. In pharmacological studies, the ROS scavenger, N-acetyl cysteine (NAC), the NADPH oxidase inhibitor, dipheylene iodide (DPI), and mitogen-activated protein kinase (MAPK) inhibitors (PD98059, SP600125, and SB203580) were applied to investigate the mechanism.

Results

STI571 reduced the viability of melanoma cells B16F0, but not human skin fibroblasts WS1, via apoptosis induction. Besides, apoptosis induced by STI571 was inhibited by the addition of NAC and DPI, and an increase in the intracellular peroxide level by STI571 was identified in melanoma B16F0 cells. Activation of caspases 3 and 9 enzyme activities accompanied by disrupting the mitochondria membrane potential in according with stimulating JNK and p38 protein phosphorylation was identified in STI571-treated B16F0 cells. STI571-mediated a ROS-dependent apoptosis potentiated by JNK inhibitor SP600125 was first identified in melanoma B16F0 cells.

Conclusion

Our results support the idea that ROS-dependent apoptosis in STI571-treated melanoma cells B16F0. The combination of a JNK inhibitor with STI571 for treating melanomas is suggested for further in vivo studies.     

Phylogenetic and developmental study of CD4, CD8 α and β T cell co-receptor homologs in two amphibian species, Xenopus tropicalis and Xenopus laevis

CD4 and CD8 co-receptors play critical roles in T cell development and activation by interacting both with T cell receptors and MHC molecules. Although homologs of these genes have been identified in many jawed vertebrates, there are still unresolved gaps concerning their evolution and specialization in MHC interaction and T cell function. Using experimental and computational procedures we identified CD4, CD8α and CD8β gene homologs both in Xenopus tropicalis, whose full genome has been sequenced, and its sister species Xenopus laevis. Multiple alignments of deduced amino acid sequences reveal a poor conservation of the residues involved in binding of CD4 to MHC class II, and CD8α to class I in non-mammalian species, presumably related to the co-evolutionary pressure of MHC I and II genes. Phylogenetic study suggests that Xenopodinae co-receptor genes are more closely related to their homologs in other tetrapods than those of bony fish. Furthermore, the developmental and cell-specific expression patterns of these genes in X. laevis are very similar to that of mammals. X. laevis CD4 is mainly expressed by peripheral non-CD8 T cells and detected in the thymus as early as four days post-fertilization (dpf) at the onset of thymic organogenesis. CD8β expression is specific to adult surface CD8⁺ T cells and thymocytes, and is first detected in the thymus at 5 dpf in parallel with productive TCRγ transrcipts, whereas productive TCRβ and α rearrangements are not detected before 7-9 dpf.     

Effects on Fertility of Rabbits After Active Immunization With Rat Testicular Cytochrome ct

ABSTRACT: A Male rabbit was immunized with rat testicular cytochrome (Cyt) c_t and mated with normal, unimmunized females. The matings resulted in abnormal pregnancies: no offspring or stillborn or undersized liveborn offspring weighing 25–30 gm each. Another unusual observation was that fur-pulling behavior, normally exhibited by pregnant female rabbits at the end of the gestational period, was absent in all of these pregnancies. Therefore, immunization of a normal rabbit with testicular cyt c_t appeared to interfere with physiological and behavioral aspects of pregnancy in normal female rabbits. The immunological basis of these findings remains to be determined.