p-Cresol Affects Reactive Oxygen Species Generation,Cell Cycle Arrest,Cytotoxicity and Inflammation/Atherosclerosis-Related Modulators Production in Endothelial Cells and Mononuclear Cells

Aims

Cresols are present in antiseptics, coal tar, some resins, pesticides, and industrial solvents. Cresol intoxication leads to hepatic injury due to coagulopathy as well as disturbance of hepatic circulation in fatal cases. Patients with uremia suffer from cardiovascular complications, such as atherosclerosis, thrombosis, hemolysis, and bleeding, which may be partly due to p-cresol toxicity and its effects on vascular endothelial and mononuclear cells. Given the role of reactive oxygen species (ROS) and inflammation in vascular thrombosis, the objective of this study was to evaluate the effect of p-cresol on endothelial and mononuclear cells.

Methods

EA.hy926 (EAHY) endothelial cells and U937 cells were exposed to different concentrations of p-cresol. Cytotoxicity was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion technique, respectively. Cell cycle distribution was analyzed by propidium iodide flow cytometry. Endothelial cell migration was studied by wound closure assay. ROS level was measured by 2′,7′-dichlorofluorescein diacetate (DCF) fluorescence flow cytometry. Prostaglandin F_2α (PGF_2α), plasminogen activator inhibitor-1 (PAI-1), soluble urokinase plasminogen activator receptor (suPAR), and uPA production were determined by Enzyme-linked immunosorbant assay (ELISA).

Results

Exposure to 100–500 µM p-cresol decreased EAHY cell number by 30–61%. P-cresol also decreased the viability of U937 mononuclear cells. The inhibition of EAHY and U937 cell growth by p-cresol was related to induction of S-phase cell cycle arrest. Closure of endothelial wounds was inhibited by p-cresol (>100 µM). P-cresol (>50 µM) also stimulated ROS production in U937 cells and EAHY cells but to a lesser extent. Moreover, p-cresol markedly stimulated PAI-1 and suPAR, but not PGF_2α, and uPA production in EAHY cells.

Conclusions

p-Cresol may contribute to atherosclerosis and thrombosis in patients with uremia and cresol intoxication possibly due to induction of ROS, endothelial/mononuclear cell damage and production of inflammation/atherosclerosis-related molecules.     

The Eag Domain Regulates the Voltage-Dependent Inactivation of Rat Eag1 K+ Channels

Eag (Kv10) and Erg (Kv11) belong to two distinct subfamilies of the ether-à-go-go K⁺ channel family (KCNH). While Erg channels are characterized by an inward-rectifying current-voltage relationship that results from a C-type inactivation, mammalian Eag channels display little or no voltage-dependent inactivation. Although the amino (N)-terminal region such as the eag domain is not required for the C-type inactivation of Erg channels, an N-terminal deletion in mouse Eag1 has been shown to produce a voltage-dependent inactivation. To further discern the role of the eag domain in the inactivation of Eag1 channels, we generated N-terminal chimeras between rat Eag (rEag1) and human Erg (hERG1) channels that involved swapping the eag domain alone or the complete cytoplasmic N-terminal region. Functional analyses indicated that introduction of the homologous hERG1 eag domain led to both a fast phase and a slow phase of channel inactivation in the rEag1 chimeras. By contrast, the inactivation features were retained in the reverse hERG1 chimeras. Furthermore, an eag domain-lacking rEag1 deletion mutant also showed the fast phase of inactivation that was notably attenuated upon co-expression with the rEag1 eag domain fragment, but not with the hERG1 eag domain fragment. Additionally, we have identified a point mutation in the S4–S5 linker region of rEag1 that resulted in a similar inactivation phenotype. Biophysical analyses of these mutant constructs suggested that the inactivation gating of rEag1 was distinctly different from that of hERG1. Overall, our findings are consistent with the notion that the eag domain plays a critical role in regulating the inactivation gating of rEag1. We propose that the eag domain may destabilize or mask an inherent voltage-dependent inactivation of rEag1 K⁺ channels.     

土壤水分和供硼状况对不同硼效率油菜苗期生长、水分含量和硼形态的影响

通过土培试验,研究了土壤水分和供硼状况对不同硼效率油菜苗期生长、叶片水分含量和硼形态的影响.结果表明:低水分胁迫条件下,硼高效甘蓝品种和硼低效甘蓝品种在高供硼水平下的单株鲜质量比低供硼水平分别增加了43.1%和31.7%,但品种间没有显著差异;硼高效品种在两种供硼水平下的束缚态水分含量分别比低效品种高11.4%和1.7%,半束缚态硼分配比例分别比低效品种高6.9%和23.8%.正常水分条件下,硼高效甘蓝品种和硼低效甘蓝品种在高供硼水平下的单株鲜质量比低供硼水平分别增加了11.1%和27.4%;硼高效品种在两种供硼水平下的自由态水分含量较硼低效品种多,自由态硼累积量为低效品种的2倍多,这有利于硼在高效植物体内的移动运输.     

Organogenesis of Phaseolus angularis L.: high efficiency of adventitious shoot regeneration from etiolated seedlings in the presence of N6-benzylaminopurine and thidiazuron

A step-wise procedure for the regeneration of fertile plants by organogenesis from cultures of the economically important Phaseolus angularis L., cultivars: KS-6, KS-7 and KS-8 using etiolated seedlings was established. Pre-culture of 5-day old seedling explants with MS (Murashige and Skoog (1962) Physiol Plant 15:473–493) + B₅-vitamins (Gamborg et al. (1968) Exp Cell Res 50:151–158) liquid medium containing either 5.0 μM TDZ or 5.0 μM BAP under dark condition was essential for organogenesis. Bud growth and shoot multiplication were stimulated by reducing the BAP concentrations from 5.0 to 2.5 μM after 3 weeks. The maximum frequency of shoot induction was 65.2% (33.8 ± 2.54 shoots/explant) in cultivar KS-8 followed by KS-7 34.6% (23.4 ± 1.91 shoots/explant) and KS-6 30.6% (21.2 ± 2.28 shoots/explant). The multiplied buds elongated after transferring to solid MSB₅ medium supplemented with 4.0 μM GA₃, 12.5 μM AgNO₃ and 0.4 μM IBA. Up to 98% rooting efficiency of was obtained when the shoots were pulse-treated with liquid medium containing 4.5 μM IBA for 10 min. The rooted plantlets were transferred to pots in the greenhouse, where they grew, mature, flowered and bared pod normally. The efficient shoot bud induction capability was found to be cultivar dependent. All the three cultivars tested formed multiple shoots. This efficient and rapid regeneration system may also be helpful for Agrobacterium- or particle gun-mediated transformation for this important legume crop.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

干旱耐逆基因（HS1）转化甘薯获得转基因植株

以甘薯（1pomoeabatatas（L.）Lam．）品种栗子香的胚性悬浮细胞为受体材料,用根癌农杆菌介导法,获得了表达除草剂抗性基因bar基因的转HSl基因甘薯植株。共计380个遗传转化的胚性细胞团,在添加2mg／L2．4-D、100mg／L Carb和10mg／L Glu（glufosinate）的固体Ms培养基上选择培养9周后,得到了12个Glu抗性愈伤组织。将这些抗性愈伤组织转移到添加1mg／L ABA、100mg／L羧苄青霉素和10mg／L Glu的固体MS培养基上,其中的3个抗性愈伤组织再生出拟转基因植株。PCR鉴定它们为转基因植株。Southern blot分析表明,HS1基因已整合到基因组中。转基因植株具有稳定的除草剂抗性。结薯观察实验结果表明,转基因植株结薯正常。     

利用DNA池技术研究猪GH基因启动子序列的多态性

目的:分析猪GH基因启动子区序列的多态性,期望筛选出对猪生长性状有显著影响的SNP位点,为地方猪种的选育及选种提供一定的理论依据.方法:以大约克、可乐猪、香猪和黔北黑猪为试验对象,构建品种DNA池,采用PCR产物直接测序法对猪GH基因启动子区-856～+171片段共1 027bp进行单核苷酸多态性检测.结果:除香猪外,在其他3个猪种5'-端侧翼序列发现5个SNP位点:C22T、A- 26T、G- 219A、T- 385A和C-391T,并且在大约克GH基因启动子区-640处发现了一个12bp碱基序列(GGCAAAGTGTAG)的缺失.结论:DNA池结合PCR产物直接测序技术能够很好的筛选SNP位点,本研究采用该技术在猪GH基因启动子区- 856～+171片段检测到了5个SNP位点.     

梁子湖湿地植物的氮磷积累特征

采用野外测定结合室内分析的方法,探讨了梁子湖湿地植物对氮磷的积累特征。结果表明,湿地不同植物种类对氮磷的积累特征明显不同。浮游植物积累各种营养物质量最多,其体内全氮量平均为29.07g.kg-1,全磷量平均为9.53g.kg-1。梁子湖湿地各类植物氮磷含量的大小依次为浮游植物>浮叶植物>沉水植物>水稻>挺水植物。各类植物中氮磷积累的大小依次为浮叶植物>挺水植物>沉水植物>沼泽植物>水稻。梁子湖湿地水生植物氮磷营养元素的吸收系数大于湿生植物,各类植物氮元素的吸收系数均大于磷。     

Transforming growth factor-β1 suppresses hepatitis B virus replication by the reduction of hepatocyte nuclear factor-4α expression

Several studies have demonstrated that cytokine-mediated noncytopathic suppression of hepatitis B virus (HBV) replication may provide an alternative therapeutic strategy for the treatment of chronic hepatitis B infection. In our previous study, we showed that transforming growth factor-beta1 (TGF-β1) could effectively suppress HBV replication at physiological concentrations. Here, we provide more evidence that TGF-β1 specifically diminishes HBV core promoter activity, which subsequently results in a reduction in the level of viral pregenomic RNA (pgRNA), core protein (HBc), nucleocapsid, and consequently suppresses HBV replication. The hepatocyte nuclear factor 4alpha (HNF-4α) binding element(s) within the HBV core promoter region was characterized to be responsive for the inhibitory effect of TGF-β1 on HBV regulation. Furthermore, we found that TGF-β1 treatment significantly repressed HNF-4α expression at both mRNA and protein levels. We demonstrated that RNAi-mediated depletion of HNF-4α was sufficient to reduce HBc synthesis as TGF-β1 did. Prevention of HNF-4α degradation by treating with proteasome inhibitor MG132 also prevented the inhibitory effect of TGF-β1. Finally, we confirmed that HBV replication could be rescued by ectopic expression of HNF-4α in TGF-β1-treated cells. Our data clarify the mechanism by which TGF-β1 suppresses HBV replication, primarily through modulating the expression of HNF-4α gene.     

Modular synthesis of Petri nets for modeling flexible manufacturing systems

This paper proposes a modular Petri net synthesis method for modeling flexible manufacturing systems based on synchronization among control processes of the manufacturing resources (such as robots and machines). In the method, the target system is modeled in a bottom-up and uniform manner by first describing the system's control processes using strongly connected state machines (SCSMs) as the basic modules. Each SCSM may contain multiple tokens to represent resources from the same type such as spaces in a buffer. Next, the common transitions and common transition subnets of the modules are merged to represent their synchronization. The system model constructed is proven to be conservative and thus bounded. Moreover, a restricted class of merged nets is proven to be live and reversible. For general classes of merged nets, this paper shows theorems that easily calculateP-invariants of the final net without solving the linear system equations. TheseP-invariants can be used to help in verifying the model's qualitative properties such as liveness.