Intra-specific hybridization: generator of genetic diversification and heterosis in Andrographis paniculata Nees. A bridge from extinction to survival

Andrographis paniculata (AP) has been stated as a low-diverse, endangered and red-listed plant species. Self-pollinated mating system, being an introduced species and experiencing a bottleneck as well as over exploitation cause such a consequence. Inter and intra-specific hybridizations have been suggested as essential techniques for generating genetic diversity. To test the effect of intra-specific hybridization on diversification and heterosis of AP, seven accessions were outcrossed manually in all 21 possible combinations. Three types of markers including morphological, phytochemical and RAPD markers were employed to evaluate the mentioned hypothesis. The results revealed that hybridization acted as a powerful engine for diversification of AP as it caused heterotic expression of the studied traits, simultaneously. Initially, it seems that additive and non-additive gene effects both can be considered as the genetic basis of heterosis in AP for the investigated traits. Agronomic and morphological traits were differentiated from each other, while positive heterosis was recorded mainly for agronomic traits but not for the morphological traits. Intra-specific hybridization increased the genetic diversity in AP population. Nevertheless, a part of this variation could also be attributed to the negative heterosis. The current exploration demonstrated the first ever conducted manual intra-specific hybridization among AP accessions in a mass scale. However, the 17 RAPD primers produced a monomorph pattern, but perhaps increasing the number of markers can feature a new genetic profile in this plant.     

黄芪多糖对缺血性脑损伤大鼠的神经保护作用及其机制研究

目的:探究缺血性脑损伤后,黄芪多糖(AG)对海马CA1区神经元重塑中粘附分子(NCAM)以及c-fos表达的影响。方法:取Wistar雄性大鼠100只,随机分成假手术组(SOG)、模型组(MG-1d,3d,7d),低剂量黄芪多糖治疗组(L-AGTG-1d,3d,7d),高剂量黄芪多糖治疗组(H-AGTG-1d,3d,7d),每组10只。所有MG和AGTG组颈部切开阻断右侧大脑中动脉,造成缺血性脑损伤后,AGTG组腹腔注射AG(5 mg/kg和15 mg/kg)。于1 d,3 d和7 d分别脑血流再灌注,随即评分神经功能缺损情况后取材,测算神经元凋亡数,免疫组织化学法和RT-PCR法半定量分析检测海马CA1区神经元NCAM和c-fos的表达。结果:L-AGTG和H-AGTG的神经功能缺损评分和海马神经元凋亡数显著低于MG(P<0.05或P<0.01),海马CA1区NCAM和c-fos的表达显著高于MG(P<0.05或P<0.01)。结论:黄芪多糖改善缺血性脑损伤大鼠的神经功能,可能与促进海马NCAM和c-fos表达,而阻止或逆转海马神经元凋亡有关。     

AG1478抑制剂对白鲫体色发育的影响

鱼类的体色由基本色素细胞相互配合而成,色素细胞主要有4种类型:黑色素细胞、红色素细胞、黄色素细胞和虹彩细胞。AG1478是针对酪氨酸激酶(Tyr)的一种新型抑制剂。Tyr是黑色素生成过程中的关键酶,在黑色素细胞中呈特异性表达。本文主要阐述了白鲫四种色素细胞以及两种不同形态黑色素细胞的超微结构观察,探讨了AG1478抑制剂对白鲫早期体色发育的影响,实验结果表明抑制剂对白鲫早期体色发育没有明显影响。     

Nitrosative stress in plants

Nitrosative stress has become a usual term in the physiology of nitric oxide in mammalian systems. However, in plants there is much less information on this type of stress. Using olive leaves as experimental model, the effect of salinity on the potential induction of nitrosative stress was studied. The enzymatic l-arginine-dependent production of nitric oxide (NOS activity) was measured by ozone chemiluminiscence. The specific activity of NOS in olive leaves was 0.280nmol NOmg(-1) proteinmin(-1), and was dependent on l-arginine, NADPH and calcium. Salt stress (200mM NaCl) caused an increase of the l-arginine-dependent production of nitric oxide (NO), total S-nitrosothiols (RSNO) and number of proteins that underwent tyrosine nitration. Confocal laser scanning microscopy analysis using either specific fluorescent probes for NO and RSNO or antibodies to S-nitrosoglutathione and 3-nitrotyrosine, showed also a general increase of these reactive nitrogen species (RNS) mainly in the vascular tissue. Taken together, these findings show that in olive leaves salinity induces nitrosative stress, and vascular tissues could play an important role in the redistribution of NO-derived molecules during nitrosative stress.     

Molecular cytotoxic mechanisms of catecholic polychlorinated biphenyl metabolites in isolated rat hepatocytes

Polychlorinated biphenyl (PCB) and PCB metabolites are highly lipophilic and accumulate easily in the lipid bilayer and fat deposits of the body. The molecular cytotoxic mechanisms of these metabolites are still not understood. The aim of the present study was to compare the cytotoxicity and toxicological properties of six dihydroxylated metabolites using isolated rat hepatocytes. All of the metabolites were more cytotoxic than 4-chlorobiphenyl (4-ClBP) and less cytotoxic than phenyl hydroquinone (PHQ). The order of cytotoxic effectiveness of catecholic metabolites expressed as LC(50) (2h) was 3',4'-diCl-2,3-diOH-biphenyl>PHQ>4'-Cl-2,5-diOH-biphenyl, 4'-Cl-2,3-diOH-biphenyl>2',5'-diCl-3,4-diOH-biphenyl>2',3'-diCl-3,4-diOH-biphenyl>3',4'-diCl-3,4-diOH-biphenyl>4'Cl-3,4-diOH-biphenyl>4'-Cl-biphenyl; showing that the positions of hydroxyl and chlorine groups were important for their hepatotoxicity and that the two 2,3-diOH congeners were the most cytotoxic. Cytotoxicity for 3,4-diOH metabolites correlated with the number and position of chlorine atoms with the more chlorine atoms being more cytotoxic. The cytotoxic order of metabolites with two chlorine atoms being 2',5'>2',3'>3',4'. Borneol, an uridine diphosphate glucuronosyltransferases (UGT) inhibitor, increased the cytotoxicity of all tested metabolites; suggesting that glucuronidation was a major mechanism of elimination of these compounds. On the other hand entacapone, a catechol-O-methyl transferase (COMT) inhibitor, only increased the cytotoxicity of 3',4'-diCl-3,4-diOH-biphenyl, 3',4'-diCl-2,3-diOH-biphenyl and 4'-Cl-2,3-diOH-biphenyl. Hepatocyte GSH was depleted (oxidized and conjugated) by these metabolites before cytotoxicity ensued in a similar order of effectiveness to their cytotoxicity with PHQ being the most effective. Hepatocyte mitochondrial membrane potential also decreased before cytotoxicity ensued with a similar order of effectiveness as their cytotoxicity. These results suggest that catecholic cytotoxicity can be attributed to mitochondrial toxicity and oxidative stress. Semiquinone or benzoquinone species were also important in the cytotoxicity of catecholic metabolites.     

Carbachol induces p70S6K1 activation through an ERK-dependent but Akt-independent pathway in human colonic epithelial cells

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser⁴⁷³ induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr³⁸⁹ in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells.     

Carbohydrate structural analysis of wheat flour arabinogalactan protein

The water-extractable arabinogalactan protein (AGP) was isolated from bread wheat flour (Triticum aestivum L. variety Cadenza) and the structure of the arabinogalactan (AG) carbohydrate component was studied. Oligosaccharides, released by hydrolysis of the AG with a range of AGP-specific enzymes, were characterised by Matrix Assisted Laser Desorption Ionisation (MALDI)-Time of Flight (ToF)-Mass Spectrometry (MS), MALDI-ToF/ToF high energy collision induced dissociation (CID) and Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE). The AG is composed of a β-(1→3)-d-galactan backbone with β-(1→6)-d-galactan side chains. These side chains are highly variable in length, from one to at least 20 Gal residues and are highly substituted with α-l-Araf. Single GlcA residues are also present at the non-reducing termini of some short β-(1→6)-galactan side chains. In addition, the β-(1→6)-galactan side chains are also substituted with β-l-Arap. We propose a polysaccharide structure of the wheat flour AGP that is substantially revised from earlier models.     

Termination of tyrphostin AG1478 application results in different recovery of EGF receptor tyrosine residues 1045 and 1173 phosphorylation in A431 cells

Tyrphostin AG1478 is known as a specific and reversible inhibitor of TK (tyrosine kinase) activity of the EGFR [EGF (epidermal growth factor) receptor]. It is attractive as an anticancer agent for cancers with elevated EGFR TK levels. However, post‐application effects of AG1478 are not well studied. We have analysed EGFR phosphorylation after termination of AG1478 application using human epidermoid carcinoma A431 cells. It was found that AG1478 inhibitory action is fast, but not fully reversible: removal of tyrphostin resulted in incomplete restoration of the overall EGFR phosphorylation. Analysing the state of two individual autophosphorylation sites of internalized EGFR, Tyr¹⁰⁴⁵ and Tyr¹¹⁷³, we demonstrated that phosphorylation of Tyr¹¹⁷³ involved in stimulation of the MAPK (mitogen‐activated protein kinase) cascade was restored much more efficiently than that in position 1045, which binds the ubiquitin ligase c‐Cbl and is necessary for targeting the receptor for lysosomal degradation. c‐Cbl association with EGFR abolished by AG1478 was not reestablished after tyrphostin cessation. As a consequence, ubiquitination‐dependent EGFR delivery to lysosomes was blocked, while phosphorylation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) was even increased. Thus, after termination of AG1478, the intracellular level of the inhibitor can be reached at which mitogenic signalling will be restored, whereas the EGFR negative regulation due to lysosomal degradation will not.     

表皮生长因子对精原干细胞的作用机理

为了探究表皮生长因子(EGF)对体外培养的精原干细胞增殖的调控作用及其作用机制.应用不连续Percoll梯度液和选择性贴壁法分离纯化精原干细胞,c-kit细胞免疫组化鉴定细胞,MTT法研究EGF对精原干细胞增殖的效应,再加入JAK-STAT信号通路特异性抑制剂AG490,探究EGF对精原干细胞增殖作用的可能机制.c-kit细胞免疫组化结果显示分离得到细胞为精原干细胞;MTT结果显示各实验组比对照组细胞数量均有显著增多(P0.01);与对照组相比,加入AG490组的活细胞数有显著下降(P0.01).实验结果表明EGF能够促进精原干细胞的增殖,并且可以通过JAK-STAT信号通路起作用.     

Somatic cell mutation. Detection and quantification of x-ray-induced mutation in cultured, diploid human fibroblasts

We describe a system for detecting somatic cell mutation to 8-azaguanine (8AG) resistance in cultured, diploid human fibroblasts. Hypoxanthine-guanine phosphoribosyltransferase (HG-PRT)-deficient, AG-resistant fibroblasts from boys with the X-chromosomal, Lesch-Nyhan (L-N) mutation served as one type of prototype mutant cells. Both spontaneous and X-ray-induced mutation were studied. Recovery of L-N cells was a function both of density of normal cells and of the AG concentration used for selection. Optimum recovery was achieved at an initial inoculum of 2·10⁴ normal cells per 60 mm diameter culture dish and an AG concentration of 8·10^?6M. Efficiency of recovery was between 39 and 90% and controls to determine this efficiency were included in mutagenesis experiments.Attempts to free normal cell populations of pre-existing AG-resistant mutant cells by pregrowth in HAT medium failed because, unlike L-N mutants, most spontaneous AG-resistant mutants can grow in HAT medium. Although pre-existing mutants probably caused overestimation, the average spontaneous mutation rate derived from our experiments was 4.5·10^?6 per cell generation. Eliminating one large-yieldv experiment reduced this estimate to 1.9·10^?6. Clonal survival of cultured human fibroblasts as a function of X-ray dose was studied. X-Irradiation increased the mutation rate above spontaneous background. Minimum estimates of the increases were 1.13·10^?9 per R per cell at 75 R, 7.49·10^?8 per R per cell at 125 R, 6.87·10^?8 per R per cell at 150 R and 2.16·10^?7 per R per cell at 250 R. The total mutagenic effect and the induced mutation rate appeared to be dose-dependent. Normal parental cell strains and their derived AG-resistant mutants had similar X-ray sensitivities indicating that X-rays induced mutations rather than selected for pre-existing mutants.Because of the realism of the cultured diploid, human fibroblast model vis-a-vis in vivohuman cellular events, the mutation detection system described herein is proposed as being potentially useful for environmental monitoring.