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71.
Sharpe PL Dicosimo D Bosak MD Knoke K Tao L Cheng Q Ye RW 《Applied and environmental microbiology》2007,73(6):1721-1728
The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C(40) carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations-the fliCS, hsdM, ccp-3, cysH, and nirS regions-were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes. 相似文献
72.
Su H Sun X Ma H Zhang HF Yu QJ Huang C Wang XM Luan RH Jia GL Wang HC Gao F 《American journal of physiology. Endocrinology and metabolism》2007,293(3):E629-E635
There is a close association between hyperglycemia and increased risk of mortality after acute myocardial infarction (AMI). However, whether acute hyperglycemia exacerbates myocardial ischemia/reperfusion (MI/R) injury remains unclear. We observed the effects of acute hyperglycemia on MI/R injury and on the cardioprotective effect of glucose-insulin-potassium (GIK). Male rats were subjected to 30 min of myocardial ischemia and 6 h of reperfusion. Rats were randomly received one of the following treatments (at 4 ml.kg(-1).h(-1) iv): Vehicle, GIK (GIK during reperfusion; glucose: 200g/l, insulin: 60 U/l, KCL: 60 mmol/l), HG (high glucose during ischemia; glucose:500 g/l), GIK + HG (HG during I and GIK during R) or GIK + wortmannin (GIK during R and wortmannin 15 min before R). Blood glucose, plasma insulin concentration and left ventricular pressure (LVP) were monitored throughout the experiments. Hyperglycemia during ischemia not only significantly increased myocardial apoptosis (23.6 +/- 1.7% vs. 18.8 +/- 1.4%, P < 0.05 vs. vehicle), increased infarct size (IS) (45.6 +/- 3.0% vs. 37.6 +/- 2.0%, P < 0.05 vs. vehicle), decreased Akt and GSK-3beta phosphorylations (0.5 +/- 0.2 and 0.6 +/- 0.1% fold of vehicle, respectively, P < 0.05 vs. vehicle) following MI/R, but almost completely blocked the cardioprotective effect afforded by GIK, as evidenced by significantly increased apoptotic index (19.1 +/- 2.0 vs. 10.3 +/- 1.2%, P < 0.01 vs. GIK), increased myocardial IS (39.2 +/- 2.8 vs. 27.2 +/- 2.1%, P < 0.01 vs. GIK), decreased Akt phosphorylation (1.1 +/- 0.1 vs. 1.7 +/- 0.2%, P < 0.01 vs. GIK) and GSK-3beta phosphorylation (1.4 +/- 0.2 vs. 2.3 +/- 0.2%, P < 0.05 vs. GIK). Hyperglycemia significantly exacerbates MI/R injury and blocks the cardioprotective effect afforded by GIK, which is, at least in part, due to hyperglycemia-induced decrease of myocardial Akt activation. 相似文献
73.
Cheng J Zhang J Merched A Zhang L Zhang P Truong L Boriek AM Du J 《The Journal of biological chemistry》2007,282(47):34268-34275
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. 相似文献
74.
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 总被引:1,自引:0,他引:1
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。 相似文献
75.
Combinatorial methods for refined neuronal gene targeting 总被引:1,自引:0,他引:1
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. 相似文献
76.
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. 相似文献
77.
Li S Tao L Jiao X Liu H Cao Y Lopez B Luan RH Christopher T Ma XL 《Apoptosis : an international journal on programmed cell death》2007,12(10):1795-1802
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. 相似文献
78.
Cytogenetic studies on two F1 hybrids of autotetraploid rice varieties showing extremely high level of heterosis 总被引:1,自引:0,他引:1
L. Luan S. B. Tu W. B. Long X. Wang Y. H. Liu F. L. Kong T. He W. G. Yan M. Q. Yu 《Plant Systematics and Evolution》2007,267(1-4):205-213
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. 相似文献
79.
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. 相似文献80.
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. 相似文献