The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Delta11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Delta11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small alpha,beta-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.     

Angiotensin converting enzyme-2 confers endothelial protection and attenuates atherosclerosis

The endothelium plays a central role in the maintenance of vascular homeostasis. One of the main effectors of endothelial dysfunction is ANG II, and pharmacological approaches to limit ANG II bioactivity remain the cornerstone of cardiovascular therapeutics. Angiotensin converting enzyme-2 (ACE2) has been identified as a critical negative modulator of ANG II bioactivity, counterbalancing the effects of ACE in determining net tissue ANG II levels; however, the role of ACE2 in the vasculature remains unknown. In the present study, we hypothesized that ACE2 is a novel target to limit endothelial dysfunction and atherosclerosis. To this aim, we performed in vitro gain and loss of function experiments in endothelial cells and evaluated in vivo angiogenesis and atherosclerosis in apolipoprotein E-knockout mice treated with AdACE2. ACE2-deficient mice exhibited impaired endothelium-dependent relaxation. Overexpression of ACE2 in human endothelial cells stimulated endothelial cell migration and tube formation, and limited monocyte and cellular adhesion molecule expression; effects that were reversed in ACE2 gene silenced and endothelial cells isolated from ACE2-deficient animals. ACE2 attenuated ANG II-induced reactive oxygen species production in part through decreasing the expression of p22phox. The effects of ACE2 on endothelial activation were attenuated by pharmacological blockade of ANG-(1-7) with A779. ACE2 promoted capillary formation and neovessel maturation in vivo and reduced atherosclerosis in apolipoprotein E-knockout mice These data indicate that ACE2, in an ANG-(1-7)-dependent fashion, functions to improve endothelial homeostasis via a mechanism that may involve attenuation of NADPHox-induced reactive oxygen species production. ACE2-based treatment approaches may be a novel approach to limit aberrant vascular responses and atherothrombosis.     

Leporipoxvirus Cu,Zn-superoxide dismutase (SOD) homologs are catalytically inert decoy proteins that bind copper chaperone for SOD

Many Chordopoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD). The biological function of these proteins is unknown, although the proteins encoded by Leporipoxviruses have been shown to promote a slow decline in the level of superoxide dismutase activity in virus-infected cells. To gain more insights into their function, we have further characterized the enzymatic and biochemical properties of a SOD homolog encoded by Shope fibroma virus. Shope fibroma virus SOD has retained the zinc binding properties of its cellular homolog, but cannot bind copper. Site-directed mutagenesis showed that it requires at least four amino acid substitutions to partially restore copper binding activity, but even these changes still did not restore catalytic activity. Reciprocal co-immunoprecipitation experiments showed that recombinant Shope fibroma virus SOD forms very stable complexes with cellular copper chaperones for SOD and these observations were confirmed using glutathione-S-transferase tagged proteins. Similar viral SOD/chaperone complexes were formed in cells infected with a closely related myxoma virus, where we also noted that some of the SOD antigen co-localizes with mitochondrial markers using confocal fluorescence microscopy. About 2% of the viral SOD was subsequently detected in gradient-purified mitochondria extracted from virus-infected cells. These poxviral SOD homologs do not form stable complexes with cellular Cu,Zn-SOD or affect its concentration. We suggest that Leporipoxvirus SOD homologs are catalytically inert decoy proteins that are designed to interfere in the proper metallation and activation of cellular Cu,Zn-SOD. This reaction might be advantageous for tumorigenic poxviruses, since higher levels of superoxide have been proposed to have anti-apoptotic and tumorigenic activity.     

Multi-axial spine biomechanical testing system with speckle displacement instrumentation

This paper reports on the design and development of a multi-axis (up to 6 axes) mechanical tester for spinal research and testing. The developed spine tester allowed true motion to be simulated on a specimen in pure or combined modes. To demonstrate the capability of the new tester flexural stiffness properties of sheep lumbar motion segments were evaluated together wiith a non-contact speckle displacement measurement system. The flexural stiffness of the specimens was measured and compared under constrained and non-constrained testing conditions; with relieving of shear forces (non-constrained), it was found that the specimen behaved in a 'stiffer' manner.     

Acetylcholine-mediated relaxation in rat thoracic aorta is enhanced following acute exposure to physiological concentrations of 17β-estradiol

We investigated the effects of short-term exposure to low concentrations of 17-estradiol on vasorelaxation using an in vitro rat thoracic aortic ring preparation. Supraphysiological levels of 17-estradiol directly relaxed phenylephrine-contracted rings. Although acute incubation (20 min) with 1-100 nM of the female sex hormone did not have any significant effect on phenylephrine-contracted rings, relaxation evoked by acetylcholine was significantly potentiated. In contrast, calcium ionophore A23187-elicited endothelium-dependent relaxation as well as cromakalim- and sodium nitroprusside-mediated endothelium-independent relaxation was unchanged following the same regime with 17-estradiol. These results demonstrate that short-term treatment with physiologically relevant levels of 17-estradiol, which on their own have no effect, enhances endothelium-dependent relaxation by acetylcholine. The vascular effects observed herein may partly account for some of the improved acute vasodilatory responses reported with 17-estradiol on blood flow in humans.     

Adiponectin and cardiovascular disease: state of the art?

The cardiometabolic syndrome, associated with increased cardiovascular disease risk in the industrialized world, is estimated to affect one in four adults. Although the mechanisms linking obesity and cardiovascular disease remain unclear, research continues to unravel the molecular pathways behind this pandemic. Adipose tissue has emerged as a metabolically active participant in mediating vascular complications, serving as an active endocrine and paracrine organ secreting adipokines, which participate in diverse metabolic processes. Among these adipokines is adiponectin, which seems to possess antiatherogenic and anti-inflammatory effects and may be protective against cardiovascular disease development. The current review describes the pathophysiology of adiponectin in atherosclerotic disease.     

Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics

The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC₅₀ values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.     

Optimal Designs of the Median Run Length Based Double Sampling X? Chart for Minimizing the Average Sample Size

Designs of the double sampling (DS) chart are traditionally based on the average run length (ARL) criterion. However, the shape of the run length distribution changes with the process mean shifts, ranging from highly skewed when the process is in-control to almost symmetric when the mean shift is large. Therefore, we show that the ARL is a complicated performance measure and that the median run length (MRL) is a more meaningful measure to depend on. This is because the MRL provides an intuitive and a fair representation of the central tendency, especially for the rightly skewed run length distribution. Since the DS chart can effectively reduce the sample size without reducing the statistical efficiency, this paper proposes two optimal designs of the MRL-based DS chart, for minimizing (i) the in-control average sample size (ASS) and (ii) both the in-control and out-of-control ASSs. Comparisons with the optimal MRL-based EWMA and Shewhart charts demonstrate the superiority of the proposed optimal MRL-based DS chart, as the latter requires a smaller sample size on the average while maintaining the same detection speed as the two former charts. An example involving the added potassium sorbate in a yoghurt manufacturing process is used to illustrate the effectiveness of the proposed MRL-based DS chart in reducing the sample size needed.