Mechanical stretch inhibits oxidized low density lipoprotein-induced apoptosis in vascular smooth muscle cells by up-regulating integrin alphavbeta3 and stablization of PINCH-1

To determine the mechanisms involved in regulating the balance between apoptosis and survival in vascular smooth muscle cells (VSMC), we studied anti-apoptotic stimuli that can counteract pro-apoptotic events in the process of early atherosclerotic lesions formation. Such a process involves VSMC accumulation even in the presence of oxidized low density lipoprotein (Ox-LDL). In the arch of the aorta, we find that integrin beta3 is higher than in descending arteries. In the advanced atherosclerosis lesion, we found an inverse correlation between the level of integrin beta3 and apoptosis (deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive). We also found an increase in integrin alphaVbeta3 (but not integrin beta1) expression in VSMC that are subjected to cyclic stretch. VSMC subjected to stretch as well as VSMC with forced expression of alphaVbeta3 were demonstrated to be resistant to Ox-LDL-induced cytoskeleton disruption and apoptosis. The anti-apoptotic effect of stretch was abolished by treatment of VSMC with small interfering RNA against integrin beta3 as well as VSMC isolated from integrin beta3 knock-out mice. Disruption of the cytoskeleton abolished the protective effect of stretch or alphaVbeta3 overexpression on Ox-LDL-induced activation of Bax and apoptosis. We also demonstrated that stretch-mediated protection of Ox-LDL-induced apoptosis involved stabilization of PINCH-1; Ox-LDL decreased the level of PINCH-1, but the application of mechanical stretch or overexpression of either integrin beta1 or integrin beta3 prevented its down-regulation. In the arteries of integrin beta3 null mice, there were lower levels of PINCH-1 and ILK-1. Moreover, deletion of integrin beta3 in VSMC abolished the stretch protective effect on PINCH-1. Small interfering RNA-mediated knockdown of PINCH-1 disrupted the cytoskeleton and caused apoptosis of VSMC. These findings provided experimental evidence that mechanical stretch acted as a survival factor in the arches of aortas. Furthermore, mechanical stretch prevented VSMC from apoptosis via a mechanism that involves alphaVbeta3 integrin expression, stabilization of PINCH-1, and remodeling of the cytoskeleton.     

出、入肺血中ET-1、VEGF含量与慢性低氧性肺动脉高压的相关性研究

目的：观察间断性减压低氧大鼠出、入肺血血浆、肺组织匀浆和体循环动脉匀浆中内皮素-1（ET-1）的含量变化,以及出、入肺血血清中血管内皮生长因子（VEGF）的含量变化及其与低氧性肺动脉高压的关系。方法：建立间断性减压低氧模型,采用插管法分别测定左侧颈总动脉压力（CAP）、右心室平均压分别代表体循环压力和平均肺动脉压力（mPAP）,放射免疫测定法测定ET-1含量,双抗体夹心ABC—ELISA法测定VEGF含量。结果：①低氧2周组（H2）、低氧3周组（H3）大鼠平均肺动脉压力、右心肥厚指数（RV／LV＋S）均分别显著高于对照组,而各组平均颈动脉压力之间无明显变化（P〉0．05）。②对照组大鼠出肺血中ET含量比入肺血中低（P〈0．05）。H2、H3组大鼠出、入肺血中ET-1含量较对照组均明显增高（P〈0．01）,且H2、H3组大鼠出肺血中ET-1含量均高于入肺血中ET-1含量（P〈0．05）,刚好与对照组相反。H2、H3组大鼠肺组织匀浆中ET—1含量与对照组肺组织和低氧组体循环动脉组织匀浆比较,均明显增高（P〈0.01）,而各组体循环动脉组织匀浆中ET-1含量间无明显差异（P〉0．05）：③对照组大鼠出、入肺血中VEGF含量无明显差异（P〉0．05）,H2、H3组大鼠出肺血血清中VEGF含量较对照组明显增高（P〈0.01）。结论：在慢性低氧可使肺组织产生ET-1和VEGF增多,这可能是慢性肺动脉高压发生、发展的重要机制之一。肺动脉局部缩血管物质ET-1增高,而体循环动脉局部ET-1不增高。可能是长期慢性低氧可导致肺动脉高压的形成,却不发生高血压的机制之一。     

Combinatorial methods for refined neuronal gene targeting

Methods for the selective and reproducible expression of genetically encoded tools in targeted subsets of cells are required to facilitate studies of neuronal development, connectivity, and function in living animals. In the absence of techniques for synthesizing promoters that target defined cell groups, current methods exploit the regulatory elements of endogenous genes to achieve specificity of transgene expression. However, single promoters often have expression patterns too broad to pinpoint the functional roles of specific neurons. In this review, we describe emerging combinatorial techniques that make transgene expression contingent not upon a single promoter, but upon two or more promoters. Although only a few such techniques are currently available, recent developments promise rapid growth in this area in the coming years.     

Sinomenine inhibits primary CD4+ T-cell proliferation via apoptosis

Sinomenine is an active component isolated from Sinomenium acutum and is widely used as an immunosuppressive drug for treating autoimmune diseases. CD4(+) T-cell population plays a key role in adaptive immune response and is related to some autoimmune diseases. In this study, we investigated the possible immunosuppressive effect of sinomenine on CD4(+) T cells and its underlying mechanism. Our data demonstrated that sinomenine remarkably suppressed the proliferation of CD4(+) T cells, blocked the cell cycle progression from G0/G1 phase to S plusG2/M phases. Finally, the immunosuppressive activity elicited by sinomenine in CD4(+) primary lymphocytes was found to be largely accounted for by caspase 3-dependent cells apoptosis. Sinomenine did not significantly alter the expression of bcl-2 in activated CD4(+) primary T cells, suggesting that bcl-2 might not be involved in sinomenine-induced T cells apoptosis. In sum, this study proposes a novel mechanism for the immunosuppressive function of sinomenine on primary mouse CD4(+) T cells.     

TNFα-initiated oxidative/nitrative stress mediates cardiomyocyte apoptosis in traumatic animals

Whole body non-penetrating trauma causes myocardial infarction in humans and mechanical trauma (MT) results in cardiac dysfunction in animals. Our recent study demonstrated that incubation of cardiomyocytes with plasma isolated from MT animals causes significant cardiomyocyte apoptosis that can be blocked by neutralization of TNFα. The present study attempted to obtain direct in vivo evidence to support that overproduction of TNFα plays a causative role in trauma-induced cardiomyocyte apoptosis. Non-lethal MT caused significant TNFα overproduction (2.4-fold at 1.5 h after MT) and increased cardiomyocyte apoptosis (starting 3 h and peaking 12 h after MT). Pharmacological inhibition of TNFα with etanercept or TNFα gene deletion reduced post-trauma myocyte apoptosis (P < 0.01). Expression of iNOS and NADPH oxidase, overproduction of NO and , and excessive protein nitration in the MT heart were all significantly reduced in etanercept-treated or TNFα^−/− mice, suggesting that oxidative/nitrative stress may contribute to TNFα-initiated myocyte apoptosis in MT hearts. Additional experiments demonstrated that inhibiting iNOS (1400W) or NADPH oxidase (apocynin), or scavenging peroxynitrite (FP15) significantly reduced myocyte apoptosis in MT animals (P < 0.01). Collectively, these data demonstrated that non-lethal mechanical trauma caused significant TNFα production that in turn stimulated myocardial apoptosis via oxidative/nitrative stress.     

Cytogenetic studies on two F1 hybrids of autotetraploid rice varieties showing extremely high level of heterosis

Mechanisms of two F1 hybrids (D46A × DTP-4 and D46A × Dminghui63) of autotetraploid rice (2n = 4x = 48) showing extremely high pollen fertility 87.40% and 85.97%, respectively, seed set 82.00% and 79.00%, respectively and extremely high level of heterosis were analyzed cytologically. The chromosome pairing of D46A × DTP-4 and D46A × Dminghui63 was normal at metaphase I(MI), and had almost no I or III, with an average of 0.020I +14.36 II 6.44rod+7.91ring) +0.01III + 4.80 IV + 0.01VIII and 0.06 I + 17.67 II (11.01rod + 6.67ring)] + 0.06 III +3.10IV+0.01VI, respectively. The most frequent chromosome configurations were 10II+7IV and 12II+bIV. The bivalent frequency was less frequent in hybrids than that in restoring parents, and the same results were gained from univalents, trivalent and multivalents. However, the quadrivalent frequency was significantly higher in hybrids than that in restoring parents at MI. The other meiotic phases progressed normally, except for low percentages of PMCs with lagging chromosomes at AI and low percentages of PMCs with micronuclei at telophaseI (TI) and telophaseII (TII). PMCs with lagging chromosomes at AI and PMCs with micronuclei at TI and TII showed negative correlation between pollen fertility and seed set. Above 90% of the PMCs could form normal microspores, which resulted in the production of viable pollen grains, abnormal microspores were observed including penta-fission and hexa-fission. Based on these results we suggest that the two F1 hybrids had better behaviors of chromosome pairing and genetic stability than autotetraploid rice and other autotetraploid plants ever studied.     

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins

Background  

Expression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as these domains/fragments. Furthermore, a HTP cloning pipeline incorporated with bioinformatics domain/fragment selection methods will be beneficial to studies of structure and function genomics/proteomics.     

Selection of Stable Reference Genes for Quantitative Real-Time PCR on <i>Paeonia ostii</i> T. Hong et J. X. Zhang Leaves Exposed to Different Drought Stress Conditions

The definition of relatively stable expressed internal reference genes is essential in both traditional blotting quantification and as a modern data quantitative strategy. Appropriate internal reference genes can accurately standardize the expression abundance of target genes to avoid serious experimental errors. In this study, the expression profiles of ten candidate genes, ACT1, ACT2, GAPDH, eIF1, eIF2, α-TUB, β-TUB, TBP, RNA Pol II and RP II, were calculated for a suitable reference gene selection in Paeonia ostii T. Hong et J. X. Zhang leaves under various drought stress conditions. Data were processed by the four regularly used evaluation software. A comprehensive analysis revealed that RNA Pol II was the most stable gene and eIF2 was the least stable one. In addition, the geNorm program provided the optimal choice of two reference gene combination, RNA Pol II and β-TUB, for qRT-PCR normalization in P. ostii subjected to different drought stress levels. Our research provided convenience for gene expression analysis in P. ostii under drought stress and promoted research of effective methods to alleviate P. ostii drought stress in the future.     

FERONIA receptor kinase interacts with S‐adenosylmethionine synthetase and suppresses S‐adenosylmethionine production and ethylene biosynthesis in Arabidopsis

Environmental inputs such as stress can modulate plant cell metabolism, but the detailed mechanism remains unclear. We report here that FERONIA (FER), a plasma membrane receptor‐like kinase, may negatively regulate the S‐adenosylmethionine (SAM) synthesis by interacting with two S‐adenosylmethionine synthases (SAM1 and SAM2). SAM participates in ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for protein and DNA methylation reactions. The Arabidopsis fer mutants contained a higher level of SAM and ethylene in plant tissues and displayed a dwarf phenotype. Such phenotype in the fer mutants was mimicked by over‐expressing the S‐adenosylmethionine synthetase in transgenic plants, whereas sam1/2 double mutant showed an opposite phenotype. We propose that FER receptor kinase, in response to environmental stress and plant hormones such as auxin and BR, interacts with SAM synthases and down‐regulates ethylene biosynthesis.