Growth control via TOR kinase signaling,an intracellular sensor of amino acid and energy availability,with crosstalk potential to proline metabolism

The TOR (Target of Rapamycin) protein kinase pathway plays a central role in sensing and responding to nutrients, stress, and intracellular energy state. TOR complex 1 (TORC1) is comprised of TOR, Raptor, and Lst8 and its activity is sensitive to inhibition by the macrolide antibiotic rapamycin. TORC1 regulates protein synthesis, ribosome biogenesis, autophagy, and ultimately cell growth through the phosphorylation of S6 K, 4E-BP, and other substrates. As TORC1 activity is positively or negatively modulated in response to upstream regulators, cellular growth rate is, respectively, enhanced or suppressed. A separate multiprotein TOR complex, TORC2, is insensitive to direct inhibition by rapamycin and does not regulate growth patterns directly; TORC2 can, however, impact certain aspects of TORC1 signaling and cell survival. TOR signaling is an ancient pathway, conserved among the yeasts, Dictyostelium, C. elegans, Drosophila, mammals, and Arabidopsis. This review will focus on the regulation of TORC1 in mammalian cells in the context of amino acid sensing/regulation and intracellular ATP homeostasis, but will also include comparisons among other organisms.     

A novel nitrite biosensor based on the direct electron transfer of hemoglobin immobilized on CdS hollow nanospheres

A novel nitrite biosensor based on the direct electron transfer of hemoglobin (Hb) immobilized on CdS hollow nanospheres (HS-CdS) modified glassy carbon electrode was constructed. The direct electron transfer of Hb showed a pair of redox peaks with a formal potential of -286 mV (vs. SCE) in 0.1M pH 7.0 phosphate buffer solution. It was a surface-controlled electrode process involving a single proton transfer coupled with a reversible one-electron transfer for each heme group of Hb. HS-CdS had a large specific surface area and good biocompatibility and had a better electrochemical response than that of solid spherical CdS. The immobilized Hb on HS-CdS displayed an excellent response to NO(2)(-) with one irreversible electrode process for NO reduction. Under optimal conditions, the biosensor could be used for the determination of NO(2)(-) with a linear range from 0.3 to 182 microM and a detection limit of 0.08 microM at 3 sigma based on the irreversible reduction of NO. HS-CdS provided a good matrix for protein immobilization and had a promising application in constructing sensors.     

Identification of the Xenopus DNA2 protein as a major nuclease for the 5'->3' strand-specific processing of DNA ends

The first step of homology-dependent DNA double-strand break (DSB) repair is the 5′ strand-specific processing of DNA ends to generate 3′ single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5′ strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5′→3′ degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the purification and identification of the Xenopus DNA2 (xDNA2) as one of the nucleases responsible for the 5′→3′ degradation of single-strand tails. Immunodepletion of xDNA2 resulted in a significant reduction in end processing and homology-dependent DSB repair. These results provide strong evidence that xDNA2 is a major nuclease for the resection of DNA ends for homology-dependent DSB repair in eukaryotes.     

Critical role of angiopoietins/Tie-2 in hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are the two ligands of the Tie-2 receptor, a receptor tyrosine kinase that is expressed on the endothelium. A balanced angiopoietin/Tie-2 system is critical for the maintenance of vascular integrity. We investigated the potential role of a disrupted angiopoietin/Tie-2 system on hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Using streptozotocin (STZ) mice subjected to myocardial ischemia, we examined the effects of shifting the Ang-2-to-Ang-1 ratio on myocardial infarction size, apoptosis, bone marrow (BM) cell-endothelial progenitor cell (EPC) differentiation, and angiogenesis. In control mice, myocardial ischemia increased expression of both Ang-2 and Tie-2. In STZ mice, Ang-2 expression was elevated, whereas Tie-2 expression was reduced, and neither was significantly altered by ischemia. Myocardial infarct size and apoptosis were increased in STZ compared with control mice. Using in vivo administration of an adenovirus containing Ang-1 or Ang-2, we found that shifting the Ang-2-to-Ang-1 ratio to favor Ang-1 reduced myocardial apoptosis and infarct size in STZ mice, while shifting the Ang-2-to-Ang-1 ratio to favor Ang-2 resulted in a significant increase in myocardial infarct size and apoptosis in control mice. Myocardial ischemia-stimulated BM cell-EPC differentiation was inhibited and myocardial angiogenesis was reduced in STZ mice. Systemic administration of Ad-Ang-1 restored BM cell-EPC differentiation and increased myocardial VEGF expression and angiogenesis in STZ mice. Our data demonstrate that disturbed angiopoietin/Tie-2 signaling contributes to the hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Restoration of the Ang-2-to-Ang-1 ratio may be a novel therapeutic strategy for the treatment of diabetic myocardial ischemic diseases.     

Prevention of aortic fibrosis by N-acetyl-seryl-aspartyl-lysyl-proline in angiotensin II-induced hypertension

Fibrosis is an important component of large conduit artery disease in hypertension. The endogenous tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has anti-inflammatory and antifibrotic effects in the heart and kidney. However, it is not known whether Ac-SDKP has an anti-inflammatory and antifibrotic effect on conduit arteries such as the aorta. We hypothesize that in ANG II-induced hypertension Ac-SDKP prevents aortic fibrosis and that this effect is associated with decreased protein kinase C (PKC) activation, leading to reduced oxidative stress and inflammation and a decrease in the profibrotic cytokine transforming growth factor-beta1 (TGF-beta1) and phosphorylation of its second messenger Smad2. To test this hypothesis we used rats with ANG II-induced hypertension and treated them with either vehicle or Ac-SDKP. In this hypertensive model we found an increased collagen deposition and collagen type I and III mRNA expression in the aorta. These changes were associated with increased PKC activation, oxidative stress, intercellular adhesion molecule (ICAM)-1 mRNA expression, and macrophage infiltration. TGF-beta1 expression and Smad2 phosphorylation also increased. Ac-SDKP prevented these effects without decreasing blood pressure or aortic hypertrophy. Ac-SDKP also enhanced expression of inhibitory Smad7. These data indicate that in ANG II-induced hypertension Ac-SDKP has an aortic antifibrotic effect. This effect may be due in part to inhibition of PKC activation, which in turn could reduce oxidative stress, ICAM-1 expression, and macrophage infiltration. Part of the effect of Ac-SDKP could also be due to reduced expression of the profibrotic cytokine TGF-beta1 and inhibition of Smad2 phosphorylation.     

Octylphenol stimulates resistin gene expression in 3T3-L1 adipocytes via the estrogen receptor and extracellular signal-regulated kinase pathways

Resistin is known as an adipocyte-specific secretory hormone that can cause insulin resistance and decrease adipocyte differentiation. It can be regulated by sexual hormones. Whether environmental estrogens regulate the production of resistin is still not clear. Using 3T3-L1 adipocytes, we found that octylphenol upregulated resistin mRNA expression in dose- and time-dependent manners. The concentration of octylphenol that increased resistin mRNA levels by 50% was approximately 100 nM within 6 h of treatment. The basal half-life of resistin mRNA induced by actinomycin D was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin mRNA degradation. In addition, octylphenol stimulated resistin protein expression and release. The basal half-life of resistin protein induced by cycloheximide was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin protein degradation. While octylphenol was shown to increase activities of the estrogen receptor (ER) and MEK1, signaling was demonstrated to be blocked by pretreatment with either ICI-182780 (an ERalpha antagonist) or U-0126 (a MEK1 inhibitor), in which both inhibitors prevented octylphenol-stimulated phosphorylation of ERK. These results imply that ERalpha and ERK are necessary for the octylphenol stimulation of resistin mRNA expression. Moreover, U-0126 antagonized the octylphenol-increased resistin protein expression and release. These data suggest that the way octylphenol signaling increases resistin protein levels is similar to that by which it increases resistin mRNA levels; it is likely mediated through an ERK-dependent pathway. In vivo, octylphenol increased adipose resistin mRNA expression and serum resistin and glucose levels, supporting its in vitro effect.     

Real-time treatment of dairy manure: implications of oxidation reduction potential regimes to nutrient management strategies

A pilot-scale sequencing batch reactor (SBR) was operated at a dairy farm to test real-time based control in winter operation conditions. A combination of high loading and low oxidation reduction potential (ORP) conditions in the aerobic stage of SBR treatment (an end value of -50 to -150 mV) inhibited nitrification while maintaining carbon removal. After a period of over-aeration over several cycles, the ORP at the end of the aerobic stage increased to values of 50-75 mV. Subsequently, nitrification was observed, accompanied by higher total cycle times. Significant increase in removal efficiencies of ammonical nitrogen (alpha<0.0001) and chemical oxygen demand (alpha<0.001) were observed for the high ORP phase. It is postulated that higher ORP regimes are needed for nitrification. In low ORP regimes, nitrification is absent or occurs at an extremely low rate. It is also noted that nitrifying systems treating high strength animal manure can possibly lead to unacceptably high levels of effluent nitrate+nitrite nitrogen (NO(x)-N). Two manure management schemes are proposed that give the farmer an option to either retain the nutrients, or remove them from the wastewater. Some advantages and disadvantages of the schemes are also discussed.     

Haemagglutinating and antibiotic activities of freshwater microalgae

We analysed the haemagglutinating activity of algal extracts from 44 species of freshwater microalgae against native and trypsin/papain-treated cow, pig, sheep, and human A-, B-, and O-type erythrocytes. Algal extracts obtained with aqueous ethanol exhibited higher haemagglutinating activity than those obtained with aqueous acetone. Most of the algal extracts agglutinated at least one of the erythrocyte types analysed. Human erythrocytes were the most sensitive of the cell types analysed. In the other species, the sensitivity of algal haemagglutinating activity for erythrocytes was pig > sheep > cow. Pre-treating erythrocytes with trypsin and papain improved the detection of most algal agglutinins and increased the haemagglutination titre; pre-treatment with papain was most effective for pig erythrocytes. Algal extracts stored at –20 °C for 4 months lost their haemagglutinating activity. Algal extracts also exhibited strong antibiotic activity against food pathogenic bacteria, especially against Bacillus. Our numerical taxonomy data showed that these microalgae might be grouped into several clusters according to their haemagglutinating activity. The detection of haemagglutinating activity may provide an efficient biochemical or physiological character to classify and differentiate microalgae. Our results suggest that freshwater microalgae might provide a potent source of haemagglutinins and antibacterial compounds for biochemical and medical studies and applications.     

A new geranylgeranyl diphosphate synthase gene from Ginkgo biloba, which intermediates the biosynthesis of the key precursor for ginkgolides.

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for ginkgolide biosynthesis. Here we reported for the first time the cloning of a new full-length cDNA encoding GGPPS from the living fossil plant Ginkgo biloba. The full-length cDNA encoding G. biloba GGPPS (designated as GbGGPPS) was 1657bp long and contained a 1176bp open reading frame encoding a 391 amino acid protein. Comparative analysis showed that GbGGPPS possessed a 79 amino acid transit peptide at its N-terminal, which directed GbGGPPS to target to the plastids. Bioinformatic analysis revealed that GbGGPPS was a member of polyprenyltransferases with two highly conserved aspartate-rich motifs like other plant GGPPSs. Phylogenetic tree analysis indicated that plant GGPPSs could be classified into two groups, angiosperm and gymnosperm GGPPSs, while GbGGPPS had closer relationship with gymnosperm plant GGPPSs.