Potential of the bean α‐amylase inhibitor αAI‐1 to inhibit α‐amylase activity in true bugs (Hemiptera)

True bugs (Hemiptera) are an important pest complex not controlled by Bt‐transgenic crops. An alternative source of resistance includes inhibitors of digestive enzymes, such as protease or amylase inhibitors. αAI‐1, an α‐amylase inhibitor from the common bean, inhibits gut‐associated α‐amylases of bruchid pests of grain legumes. Here we quantify the in vitro activity of α‐amylases of 12 hemipteran species from different taxonomic and functional groups and the in vitro inhibition of those α‐amylases by αAI‐1. α‐Amylase activity was detected in all species tested. However, susceptibility to αAI‐1 varied among the different groups. α‐Amylases of species in the Lygaeidae, Miridae and Nabidae were highly susceptible, whereas those in the Auchenorrhyncha (Cicadellidae, Membracidae) had a moderate susceptibility, and those in the Pentatomidae seemed to be tolerant to αAI‐1. The species with αAI‐1 susceptible α‐amylases represented families which include both important pest species but also predatory species. These findings suggest that αAI‐1‐expressing crops have potential to control true bugs in vivo.     

Bruchid resistance of transgenic azuki bean expressing seed α-amylase inhibitor of common bean

Various species of bruchid beetles including Callosobruchus chinensis, C. maculatus and C. analis cause postharvest damage of azuki bean seeds, an important East Asian grain legume. The -amylase in the midguts of these insects is inhibited by the -amylase inhibitor (AI) present in common bean seeds. Transformation of azuki bean with the AI gene driven by the promoter of phytohemagglutinin results in high levels of AI in the seeds and the complete block of bruchid development on the seeds. Zabrotes subfasciatus, a South and Central American bruchid that is a storage pest of common bean, develops normally on the transgenic azuki bean.     

A novel wheat α‐amylase inhibitor gene,TaHPS, significantly improves the salt and drought tolerance of transgenic Arabidopsis

On the basis of microarray analyses of the salt‐tolerant wheat mutant RH8706‐49, a previously unreported salt‐induced gene, designated as TaHPS [Triticum aestivum hypothetical (HPS)‐like protein], was cloned. Real‐time quantitative polymerase chain reaction analyses showed that expression of the gene was induced by abscisic acid, salt and drought. The encoded protein was found to be localized mainly in the plasma membranes. Transgenic Arabidopsis plants overexpressing TaHPS were more tolerant to salt and drought stresses than non‐transgenic wild‐type (WT) plants. Under salt stress, the root cells of the transgenic plants secreted more Na⁺ and guard cells took up more Ca²⁺ ions. Compared with wild‐type plants, TaHPS‐expressing transgenic plants showed significantly lower amylase activity and glucose and malic acid levels. Our results showed that the expression of TaHPS inhibited amylase activity, which subsequently led to a closure of stomatal apertures and thus improved plant tolerance to salt and drought.     

Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle α‐actin gene

Smooth muscle α actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells (vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full‐length SMA promoter and intronic sequences. We determined that the founders and F1 progeny of five independent lines contain 1–3 copies of the mCherry‐substituted BAC vector. Furthermore, we characterized the expression of SMA‐mCherry in relation to endogenous SMA in the embryo and in adult tissues, and found that the transgenic reporter in each line recapitulated endogenous SMA expression at all time points. We were also able to isolate SMA expressing cells from embryonic tissues using fluorescence‐activated cell sorting (FACS). We demonstrated that this marker can be combined with other vital fluorescent reporters and it can be used for live imaging of embryonic cardiodynamics. Therefore, these transgenic mice will be useful for isolating live SMA‐expressing cells via FACS and for studying the emergence, behavior, and regulation of SMA‐expressing cells, including vascular smooth muscle cells and pericytes throughout embryonic and postnatal development. genesis 48:457–463, 2010. © 2010 Wiley‐Liss, Inc.     

Plasma and liver proteomic analysis of 3Z‐3‐[(1H‐pyrrol‐2‐yl)‐methylidene]‐1‐(1‐piperidinylmethyl)‐1,3‐2H‐indol‐2‐one‐induced hepatotoxicity in Wistar rats

3Z‐3‐[(1H‐pyrrol‐2‐yl)‐methylidene]‐1‐(1‐piperidinylmethyl)‐1,3‐2H‐indol‐2‐one (Z24), a synthetic anti‐angiogenic compound, inhibits the growth and metastasis of certain tumors. Previous works have shown that Z24 induces hepatotoxicity in rodents. We examined the hepatotoxic mechanism of Z24 at the protein level and looked for potential biomarkers. We used 2‐DE and MALDI‐TOF/TOF MS to analyze alternatively expressed proteins in rat liver and plasma after Z24 administration. We also examined apoptosis in rat liver and measured levels of intramitochondrial ROS and NAD(P)H redox in liver cells. We found that 22 nonredundant proteins in the liver and 11 in the plasma were differentially expressed. These proteins were involved in several important metabolic pathways, including carbohydrate, lipid, amino acid, and energy metabolism, biotransformation, apoptosis, etc. Apoptosis in rat liver was confirmed with the terminal deoxynucleotidyl transferase dUTP‐nick end labeling assay. In mitochondria, Z24 increased the ROS and decreased the NAD(P)H levels. Thus, inhibition of carbohydrate aerobic oxidation, fatty acid β‐oxidation, and oxidative phosphorylation is a potential mechanism of Z24‐induced hepatotoxicity, resulting in mitochondrial dysfunction and apoptosis‐mediated cell death. In addition, fetub protein and argininosuccinate synthase in plasma may be potential biomarkers of Z24‐induced hepatotoxicity.     

Cell‐specific effects on surface α7 nicotinic receptor expression revealed by over‐expression and knockdown of rat RIC3 protein

We tested whether surface α7 nicotinic acetylcholine receptor expression is dependent on an endogenous chaperone named Resistance to Inhibitors of Cholinesterase 3 (RIC3) by comparing RIC3 protein in rat GH4C1 and human SH‐EP1 cells, which express strikingly different surface receptor levels following α7 transfection. Cloned rat RIC3 exists in at least two isoforms because of an ambiguous splice site between exons 4 and 5. Both rat isoforms permit surface α7 expression in SH‐EP1 and human embryonic kidney (HEK) cells measured by α‐bungarotoxin binding. Contrary to expectations, endogenous RIC3 protein expression determined by immunoblots did not differ between untransfected GH4C1 or SH‐EP1 cells. siRNA against rat RIC3 exon 4 and shRNA against exons 2, 5 and 6 knocked down transfected rat RIC3 expression in SH‐EP1 cells and simultaneously blocked toxin binding. However, no RNAi construct blocked binding when co‐transfected with α7 into GH4C1 cells. shRNA against rat exons 2 and 5 knocked down rat RIC3 protein transfected into GH4C1 cells with a time course suggesting a protein half‐life of a few days. These results suggest GH4C1 cells may possess unknown chaperone(s) allowing high surface α7 expression in the absence of known RIC3 splice variants.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Adaptation to β‐myrcene catabolism in Pseudomonas sp. M1: An expression proteomics analysis

β‐Myrcene, a monoterpene widely used as a fragrance and flavoring additive, also possesses analgesic, anti‐mutagenic, and tyrosinase inhibitory properties. In order to get insights into the molecular mechanisms underlying the ability of Pseudomonas sp. M1 to catabolize β‐myrcene, an expression proteomics approach was used in this study. Results indicate that the catabolic enzyme machinery for β‐myrcene utilization (MyrB, MyrC, and MyrD and other uncharacterized proteins) is strongly induced when β‐myrcene is present in the growth medium. Since an M1 mutant, lacking a functional 2‐methylisocitrate dehydratase, is not able to grow in mineral medium with β‐myrcene or propionic acid as the sole C‐source, and also based on the expression proteomic analysis carried out in this study, it is suggested that the β‐myrcene catabolic intermediate propionyl‐CoA is channeled into the central metabolism via the 2‐methylcitrate cycle. Results also suggest that the major alteration occurring in the central carbon metabolism of cells growing in β‐myrcene‐containing media is related with the redistribution of the metabolic fluxes leading to increased oxaloacetate production. Other up‐regulated proteins are believed to prevent protein misfolding and aggregation or to play important structural roles, contributing to the adaptive alteration of cell wall and membrane organization and integrity, which are essential features to allow the bacterium to cope with the highly lipophilic β‐myrcene as C‐source.     

Phospholipase Dα1‐mediated phosphatidic acid change is a key determinant of desiccation‐induced viability loss in seeds

High sensitivity of seeds to water loss is a widespread phenomenon in the world's plant species. The molecular basis of this trait is poorly understood but thought to be associated with critical changes in membrane function. We profiled membrane lipids of seeds in eight species with varying levels of desiccation tolerance and found a close association between reducing seed viability and increasing phosphatidic acid (PA). We applied hydration–dehydration cycles to Arabidopsis seeds, which are normally desiccation tolerant, to mimic the onset of desiccation sensitivity with progression towards germination and examined the role of phospholipase D (PLD) in desiccation stress‐induced production of PA. We found that PLDα1 became more abundant and migrated from the cytosol to the membrane during desiccation, whereas PLDδ did not change, and that all desiccation‐induced PA was derived from PLDα1 hydrolysis. When PLDα1 was suppressed, the germination level after each hydration–dehydration cycle improved significantly. We further demonstrated that PLDα1‐mediated PA formation modulates desiccation sensitivity as applying its inhibitor improved seed desiccation tolerance and its suppression in protoplasts enhanced survival under dehydration. The insights provided by comparative lipidomics enable us to propose a new membrane‐based model for seed desiccation stress and survival.     

Amyloid precursor protein expression is induced by tumor necrosis factor α in 3T3‐L1 adipocytes

Amyloid precursor protein (APP) has been characterized as an adipocyte‐secreted protein that might contribute to obesity‐related insulin resistance, inflammation, and dementia. In the current study, regulation of APP by the proinflammatory and insulin resistance‐inducing cytokine tumor necrosis factor (TNF) α was determined in 3T3‐L1 adipocytes. Interestingly, APP protein synthesis and mRNA expression were significantly increased by TNFα in a time‐dependent manner with maximal induction observed after 24 h of treatment. Furthermore, TNFα induced APP mRNA expression dose‐dependently with maximal 6.4‐fold upregulation seen at 100 ng/ml effector. Moreover, inhibitor experiments suggested that TNFα‐induced APP expression was mediated by nuclear factor κ B. Taken together, we show for the first time a potent upregulation of APP by TNFα suggesting a potential role of this adipocyte‐secreted protein in TNFα‐induced insulin resistance and inflammatory disease. J. Cell. Biochem. 108: 1418–1422, 2009. © 2009 Wiley‐Liss, Inc.