Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not on NADPH oxidase

Calcium ions (Ca(2+)) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca(2+) in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca(2+) induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death.     

Development of a novel transgenic rice with hypocholesterolemic activity via high-level accumulation of the α′ subunit of soybean β-conglycinin

Soybean 7S globulin, known as β-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α′ subunit of β-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α′ subunit of β-conglycinin in rice seeds could lead to the production of new functional rice that could promote human health. Herein, we used the low-glutelin rice mutant ‘Koshihikari’ (var. a123) and suppressed its glutelins and prolamins, the major seed storage proteins of rice, by RNA interference. The accumulation levels of the α′ subunit in the lines with suppressed glutelin and prolamin levels were >20 mg in 1 g of rice seeds, which is considerably higher than those in previous studies. Oral administration of the transgenic rice containing the α′ subunit exhibited a hypocholesterolemic activity in rats; the serum total cholesterol and LDL cholesterol levels were significantly reduced when compared to those of the control rice (var. a123). The cholesterol-lowering action by transgenic rice accumulating the α′ subunit induces a significant increase in fecal bile acid excretion and a tendency to increase in fecal cholesterol excretion. This is the first report that transgenic rice exhibits a hypocholesterolemic activity in rats in vivo by using the β-conglycinin α′ subunit.     

N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications.     

An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.     

Linkages among trait-mediated indirect effects: a new framework for the indirect interaction web

Plants have diverse ways of responding to damage by herbivores, such as changes in allelochemistry, physiology, morphology, growth, and phenology. These responses form the mechanistic basis for trait-mediated indirect interactions (TMIIs) between organisms on the plants. There is a growing appreciation that such TMIIs form complex networks (i.e., indirect interaction webs) in terrestrial plant-associated arthropod communities. Almost all previous studies have had the same framework: examining trait-mediated indirect effects within a single interactive unit consisting of one initiator of herbivore, a host plant as a mediator, and one receiver [trait-mediated indirect interaction unit (TMIU)]. However, this framework is too simple to understand the dynamics of the indirect interaction web. Recent studies suggest that there is a wide variety of interactions among TMIUs within a community, which may largely affect the outcomes of indirect effects in each unit. Here, we review recent advance in studies of trait-mediated indirect effects in plant-associated arthropod communities and explore the mechanisms of linkages among TMIUs. Then, we argue the importance of examining linkages among TMIUs as a new framework for future studies on the indirect interaction web. Finally, we propose the hypothesis that linkages among TMIUs contribute to the maintenance of biodiversity.     

Community structure of insect herbivores on introduced and native Solidago plants in Japan

We compared community composition, density, and species richness of herbivorous insects on the introduced plant Solidago altissima L. (Asteraceae) and the related native species Solidago virgaurea L. in Japan. We found large differences in community composition on the two Solidago species. Five hemipteran sap feeders were found only on S. altissima. Two of them, the aphid Uroleucon nigrotuberculatum Olive (Hemiptera: Aphididae) and the scale insect Parasaissetia nigra Nietner (Hemiptera: Coccidae), were exotic species, accounting for 62% of the total individuals on S. altissima. These exotic sap feeders mostly determined the difference of community composition on the two plant species. In contrast, the herbivore community on S. virgaurea consisted predominately of five native insects: two lepidopteran leaf chewers and three dipteran leaf miners. Overall species richness did not differ between the plants because the increased species richness of sap feeders was offset by the decreased richness of leaf chewers and leaf miners on S. altissima. The overall density of herbivorous insects was higher on S. altissima than on S. virgaurea, because of the high density of the two exotic sap feeding species on S. altissima. We discuss the importance of analyzing community composition in terms of feeding guilds of insect herbivores for understanding how communities of insect herbivores are organized on introduced plants in novel habitats.     

An EF-hands protein, centrin-1, is an EGTA-sensitive SUMO-interacting protein in mouse testis

A multifunctional calcium‐binding protein, centrin‐1, is specifically expressed in male germ cells, certain neurons and ciliated cells. We identified centrin‐1 as a protein interacting with SUMO‐2/3 using yeast two‐hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction between centrin‐1 and SUMO‐2/3 was reduced in the presence of EGTA and facilitated by the addition of CaCl_2. immunostaining of seminiferous tubules in 35‐day‐old mouse testes revealed that cells in the layer containing spermatogonia showed colocalization of SUMO‐2/3 with centrin‐1 in cytoplasmic spots. Identification of centrin‐1 as the EGTA‐sensitive SUMO‐2/3‐interacting protein indicates the possible role of calcium in modulating the centrin‐1–SUMO‐2/3 interaction and suggests the importance of this interaction in mouse testis. Copyright © 2010 John Wiley & Sons, Ltd.     

Expression of receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin in the inner blood-retinal barrier of rats

The retina is protected from somatic circulation by the blood-retinal barrrier (BRB) composed of tight junctions between retinal vascular endothelial cells (the inner BRB) and those between retinal pigment epithelial cells (the outer BRB). Our recent studies showed that glial cell line-derived neurotrophic factor (GDNF) secreted from astrocytes regulates the permeability of the BBB. In the present study, we immunohistochemically examined the expression of GDNF, neurturin (NTN) and their receptors, GFRalpha1 for GDNF and GFRalpha2 for NTN, because the capillaries of the inner BRB show specialization very similar to the blood-brain barrier (BBB). GDNF and NTN were detected in glial fibrillary acidic protein (GFAP)-positive cells, including Müller cells. GFRalpha1 and GFRalpha2 were localized in von Willebrand factor-positive cells. GDNF and NTN enhanced the barrier function of endothelial cells derived from porcine brain cortex. These results strongly suggest that the barrier function of the BRB is regulated by GDNF and NTN secreted from glial cells, like the BBB.