人胰高血糖素样肽1在转基因番茄中的表达

人胰高血糖素样肽1 (GLP1)是一种短肽激素,对Ⅱ型糖尿病具有很好的疗效.本研究在设计合成GLP1基因并构建植物表达载体的基础上,通过农杆菌介导将GLP1基因导入番茄基因组中,经过PCR扩增和Southern Blot分析,证实GLP1基因已整合进入9个株系的番茄基因组中.Western Blot检测表明,其中4个株系转基因番茄的叶片能够检测到hGLP1融合蛋白的表达.通过Ni-NTA亲和层析分离纯化转基因番茄表达的hGLP1融合蛋白,动物实验表明该融合蛋白具有显著的降血糖生物活性.本研究结果将为转基因番茄作为生物反应器表达药用蛋白提供重要的理论和技术支持,并将为培育具有糖尿病治疗功能的番茄新品种奠定基础.     

Application of plasma lipidomics in studying the response of patients with essential hypertension to antihypertensive drug therapy

Hypertension is a key risk factor in the progression of cardiovascular disease (CVD). Dyslipidemia, a strong predictor of CVD, frequently coexists with hypertension. Therefore, the control of hypertension and dyslipidemia may help reduce CVD morbidity and mortality. In the present study, the therapeutic effects of antihypertensive agents on blood pressure control and plasma lipid metabolism were evaluated. The plasma lipid profiles of patients with treated (n = 25) or untreated (n = 30) essential hypertension as well as of subjects with normotension (n = 28) were analyzed using liquid chromatography mass spectrometry. Principal component analysis of the lipidomics data revealed distinct clusters among studied subjects across three human populations. Phosphatidylcholines and triacylglycerols (TG) dominated the pattern of hypertension-influenced plasma lipid metabolism. Discriminatory lipid metabolites were analyzed using one-way analysis of variance followed by a post hoc multiple comparison correction. TG lipid class was significantly increased by 49.0% (p < 0.001) in hypertensive vs. normotensive groups while tended to decrease (-21.2%, p = 0.054) in hypertensive patients after treatment. Total cholesteryl esters were significantly decreased by -16.9% (p < 0.001) in hypertensive patients after treatment. In particular, a large number of individual neutral lipid species were significantly elevated in hypertensive subjects but significantly decreased after treatment with antihypertensive agents. The present study applied, for the first time, a systems biology based lipidomics approach to investigate differentiation among plasma lipid metabolism of patients with treated/untreated essential hypertension and subjects with normotension. Our results demonstrate that antihypertensive medications to lower blood pressure of hypertensive patients to target levels produced moderate plasma lipid metabolism improvement of patients with hypertension.     

1H, 13C and 15N resonance assignments of rhodanese GlpE from Escherichia coli

Rhodanese catalyzes the sulfur-transfer reaction in which a sulfur atom is transferred from thiosulfate to cyanide by a double-displacement mechanism. During the reaction, a persulfide-intermediate form of rhodanese is generated by the reaction of a conserved active cysteine residue with thiosulfate. Escherichia coli GlpE is a prototype for the single-domain rhodanese superfamily. Though there are some studies on rhodaneses, the molecular mechanism of the catalytic activity of rhodaneses is still unclear. Herein, we report the resonance assignments of (1)H, (13)C and (15)N atoms of E. coli GlpE, which provides the basis for further structural, dynamic and functional studies of rhodaneses using NMR technique.     

Lentiviral gene transfer into the dorsal root ganglion of adult rats

Background

Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K⁺ channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K_2P channels after peripheral axotomy in mammals.

Results

Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured in vivo. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo.

Conclusions

In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability.     

Enhanced oxidative stress impairs cAMP-mediated dilation by reducing Kv channel function in small coronary arteries of diabetic rats

We have shown that short-term exposure of rat small coronary arteries (RSCAs) to high glucose enhances superoxide (O2-*) formation and impairs cAMP-mediated dilation by reducing voltage-gated K+ (Kv) channel function. However, it is not clear whether the impairment also occurs in diabetes mellitus (DM), where alternate mechanisms could mask or aggravate vasodilator dysfunction. RSCAs were isolated from control and streptozotocin-induced diabetic rats. Reduced constriction to 4-aminopyridine (4-AP) was observed in RSCAs from DM rats, indicating Kv channel impairment. Forskolin increased 4-AP-inhibitable K+ channel open-state probability and whole cell K+ current density in coronary myocytes from non-DM rats but had little effect on K+ current density in cells from DM rats. Diminished dilation to 8-bromo-cAMP, forskolin, or isoproterenol was observed in DM RSCAs. The attenuated dilation to forskolin or isoproterenol in DM RSCAs was partially restored by application of the superoxide dismutase mimetic manganese[III] tetrakis (4-benzoic acid) porphyrin. Histofluorescence studies using hydroethidine revealed a blockage of O2-* generation by the NADPH oxidase inhibitor apocynin in DM RSCAs. Sepiapterin, a precursor of tetrahydrobiopterin, had little effect on hyperglycemia-induced O2-* formation. Consistent with the findings from the concurrent fluorescence study, apocynin also partially restored the reduced dilator response to forskolin in DM RSCAs. Forskolin-induced cAMP production was unaltered in DM. We conclude that in diabetes, enhanced O2-* formation by activation of NADPH oxidase impairs cAMP-medicated dilation in RSCAs by inhibiting Kv channel activity.     

Regulatory effects of estrogen on acute lung inflammation in mice

Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency. mitochondria; oxidative phosphorylation; rhodamine 123; fibroblast     

Properties of isonucleotide-incorporated oligodeoxynucleotides and inhibition of the expression of spike protein of SARS-CoV

Antisense oligonucleotides are recognized to be very efficient tools for the inhibition of gene expression in a sequence specific way. For the discovery of a novel efficient way to modify oligonucleotides, a series of single isonucleotide-incorporated antisense oligodeoxynucleotides have been synthesized, in which an isonucleotide was introduced at different positions of the sequences. The binding behaviors of modified oligodeoxynucleotides to the complementary sequence were studied by UV, CD, and molecular dynamics simulation. The results showed that although the incorporated isonucleotides at certain positions of the sequence interfere with the binding ability to a different extent, B-form duplexes were maintained and the binding abilities of the 3'-end-modified duplexes were better than the corresponding mismatched duplexes. The digestion of modified oligodeoxynucleotides by snake venom phosphodiesterase showed that an isonucleotide strongly antagonizes hydrolysis. The DNA/RNA hybrid formed by a modified oligodeoxynucleotide and its target RNA could activate RNase H. The 3'-end-modified antisense oligodeoxynucelotides inhibited S-glycoprotein expression of SARS-CoV at the mRNA levels in insect Sf9 cells. This study indicated the possibility of designing a novel and effective antisense oligodeoxynucleotide by incorporating an isonucleotide at the 3'-end of the sequence.     

Use of Ssp dnaB derived mini-intein as a fusion partner for production of recombinant human brain natriuretic peptide in Escherichia coli

To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, a modified Ssp dnaB mini-intein linked with a chitin binding domain tag was used as a fusion partner for production of human brain natriuretic peptide (hBNP), a hormone for the treatment of congestive heart failure. The fusion protein was expressed as an inclusion body in Escherichia coli. After refolding, the fusion protein was purified with a chitin affinity column, and dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH in the chitin column to 7.0 at 25 degrees C for 16 h, which led to the release and separation of hBNP from its fusion partner. The hBNP sample was further purified with reverse phase HPLC and its biological activity was assayed in vitro. It was found that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC(50) of (1.24+/-0.32)x10(-6)mg/ml, which was similar to that of the synthetic BNP standard. The expression strategy described here promises to produce small peptides without use of proteolytic or chemical reagents.     

Kinetic resolution of ibuprofen catalyzed by Candida rugosa lipase in ionic liquids

Candida rugosa lipase-catalyzed esterification of ibuprofen with 1-propanol was conducted in seven ionic liquids and the results were compared with those in isooctane. Although the enzyme showed comparable or higher activity in some ionic liquids compared to that in isooctane, only in the case of [BMIM]PF6 was the enantioselectivity (E = 24.1) almost twice that (E = 13.0) of isooctane. In another six ionic liquids the enzyme enantioselectivity was much poorer (E = 1.1-6.4). At the same conversion of 30%, E of [BMIM]PF6 is more than triple that of isooctane. The lipase stability in [BMIM]PF6 was improved by 25% of that in isooctane. It was concluded that [BMIM]PF6 could be applied to substitute the conventional organic solvent (isooctane) in the esterification of ibuprofen.     

Third-order NLO properties of the DMF solutions of 3-D network and 2-D layered organic-metal polymers with (4, 4) grid units

Reactions of 1,1′-(1,4-butanediyl)bis-1H-benzimidazole (bbbm) with Cd(II), Ni(II), or Co(II) afford three organic-metal polymers: {[Cd(bbbm)(SO₄)(H₂O)₂]CH₃OH}_n (1), {[Ni(bbbm)₂(H₂O)₂](NO₃)₂ · 2CH₃OH · 6H₂O}_n (2) and {[Co(bbbm)₂(H₂O)₂](NO₃)₂ · 2CH₃OH · 6H₂O}_n (3). In 1, bbbm and coordinate to Cd(II) simultaneously leading to a 3-D structure. In 2 and 3, each bridging bbbm ligand links two Ni(II) or Co(II) ions forming the 2-D layered structure with (4, 4) grid units. Each (4, 4) grid unit is a 44-membered ring and constructed by four bbbm ligands acting as four sides and four Ni(II) or Co(II) ions representing four corners. Their third-order nonlinear optical (NLO) properties in DMF solution have been studied by Z-scan technique. The results show that the DMF solution of 2 possesses both the large third-order NLO absorptive and strong refractive behaviors; and 3’s shows large NLO absorptive effect and weak NLO refractive behavior. A reasonably good fit between the experimental data and the theoretical curve suggests that the experimentally obtained NLO effects are effective third-order in nature.