Effect of obesity and troglitazone on expression of two glutathione peroxidases: Cellular and extracellular types in serum,kidney and adipose tissue

To determine the effect of obesity on expression of cellular- (C-) and extracellular (EC-) glutathione peroxidase (GPX) in serum, kidney and adipose tissue, we measured GPX in serum, kidneys and adipose tissue of the obese Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat and its lean counterpart (LETO). We also investigated the effect of troglitazone. Five each of OLETF and LETO rats were fed diet with or without 0.2% troglitazone for 10 days. Final body weight, kidney weight, blood glucose and serum tumor necrosis factor-α (TNF-α) level were higher in OLETF rats than in LETO rats. Serum and kidney GPX activities were higher, but adipose tissue GPX activity was lower, in OLETF rats than in LETO rats. Troglitazone treatment decreased adipose tissue GPX activity and abolished overproduction of TNF-α in OLETF rats. Immunoblot analysis, for the first time, revealed that both obesity and troglitazone suppressed the protein signals for C-GPX and EC-GPX in adipose tissue. Serum protein carbonyl groups were increased in OLETF rats and troglitazone completely blocked this increase. Increased serum GPX activity in obese rat was due to the increased secretion of EC-GPX from the kidney. Troglitazone protected against the enhanced oxidative stress induced by obesity independently of the serum GPX concentration.     

A combination of Flt3 ligand cDNA and CpG oligodeoxynucleotide as nasal adjuvant elicits protective secretory-IgA immunity to Streptococcus pneumoniae in aged mice

Our previous study showed that a combination of a plasmid-expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotides (CpG ODN) as a combined nasal adjuvant elicited mucosal immune responses in aged (2-y-old) mice. In this study, we investigated whether a combination of pFL and CpG ODN as a nasal adjuvant for a pneumococcal surface protein A (PspA) would enhance PspA-specific secretory-IgA Ab responses, which could provide protective mucosal immunity against Streptococcus pneumoniae infection in aged mice. Nasal immunization with PspA plus a combination of pFL and CpG ODN elicited elevated levels of PspA-specific secretory-IgA Ab responses in external secretions and plasma in both young adult and aged mice. Significant levels of PspA-specific CD4(+) T cell proliferative and PspA-induced Th1- and Th2- type cytokine responses were noted in nasopharyngeal-associated lymphoreticular tissue, cervical lymph nodes, and spleen of aged mice, which were equivalent to those in young adult mice. Additionally, increased numbers of mature-type CD8, CD11b-expressing dendritic cells were detected in mucosal inductive and effector lymphoid tissues of aged mice. Importantly, aged mice given PspA plus a combination of pFL and CpG ODN showed protective immunity against nasal S. pneumoniae colonization. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for protection against S. pneumoniae in the elderly.     

Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry‐induced increase of phosphorylation of eukaryotic elongation factor‐2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2‐mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore‐assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin‐dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)‐evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP‐induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Localized role of CRMP1 and CRMP2 in neurite outgrowth and growth cone steering

Collapsin response mediator protein 1 (CRMP1) and CRMP2 have been known as mediators of extracellular guidance cues such as semaphorin 3A and contribute to cytoskeletal reorganization in the axonal pathfinding process. To date, how CRMP1 and CRMP2 focally regulate axonal pathfinding in the growth cone has not been elucidated. To delineate the local functions of these CRMPs, we carried out microscale‐chromophore‐assisted light inactivation (micro‐CALI), which enables investigation of localized molecular functions with highly spatial and temporal resolutions. Inactivation of either CRMP1 or CRMP2 in the neurite shaft led to arrested neurite outgrowth. Micro‐CALI of CRMP2 in the central domain of the growth cones consistently arrested neurite outgrowth, whereas micro‐CALI of CRMP1 in the same region caused significant lamellipodial retraction, followed by retardation of neurite outgrowth. Focal inactivation of CRMP1 in its half region of the growth cone resulted in the growth cone turning away from the irradiated site. Conversely, focal inactivation of CRMP2 resulted in the growth cone turning toward the irradiated site. These findings suggest different functions for CRMP1 and CRMP2 in growth cone behavior and neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Cross-linked protein complex exhibiting asymmetric oxidation activities in the absence of added cofactor

A protein complex (PC) suspension exhibits asymmetric biooxidation activities in the absence of any added cofactor such as NAD(P)⁺ or FAD. It can be extracted from pea protein (PP)‐gel (PP encapsulated with Ca²⁺ alginate gel and aerated in air for several hours) using hot water by rotary shaking and powdered by the following three steps: (1) forming precipitates from the suspension using 30% (w/v) aqueous (NH₄)₂SO₄, (2) crosslinking the precipitates with 0.25% (v/v) GA, and (3) preparing the cross‐linked powder by freeze‐drying. The cross‐linked PC (CLPC) performed asymmetric oxidation of the toward (R)‐isomers of rac‐ 1 and rac ‐2 in 50 mM glycine–NaOH (pH 9.0) buffer/DMSO cosolvent [2.07% (v/v)] with high enantioselectivity; thus, the (S)‐isomers can be obtained in greater than 99% ee from the corresponding rac‐p‐substituted naphthyl methyl carbinol (rac‐ 1 and rac ‐2 ). The CLPC activity was not only competitively inhibited by addition of either 1.0 mM ZnCl₂ or a chelating agent such as 1.0 mM EDTA but also denatured by pretreatments: autoclaving at 121°C (20 min) or using 6.0 M guanidine–HCl containing 50 mM DTT. These results indicated that the PC catalytic process may utilize an electron transfer system incorporating a redox cation (e.g., Fe²⁺ ? Fe³⁺ or Zn). Therefore, the newly introduced CLPC can asymmetrically oxidize the substrates without the addition of any cofactor resulting in a low‐cost organic method. Overall, our results show that the CLPC is an easily prepared, low‐cost reagent that can function under mild conditions and afford stereoselectivity, regioselectivity, and substrate specificity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 953–961, 2012     

Novel Genetic Resources in the Genus Vigna Unveiled from Gene Bank Accessions

The genus Vigna (Fabaceae) consists of five subgenera, and includes more than 100 wild species. In Vigna, 10 crops have been domesticated from three subgenera, Vigna, Plectrotropis, and Ceratotropis. The habitats of wild Vigna species are so diverse that their genomes could harbor various genes responsible for environmental stress adaptation, which could lead to innovations in agriculture. Since some of the gene bank Vigna accessions were unidentified and they seemed to be novel genetic resources, these accessions were identified based on morphological traits. The phylogenetic positions were estimated based on the DNA sequences of nuclear rDNA-ITS and chloroplast atpB-rbcL spacer regions. Based on the results, the potential usefulness of the recently described species V. indica and V. sahyadriana, and some wild Vigna species, i.e., V. aconitifolia, V. dalzelliana, V. khandalensis, V. marina var. oblonga, and V. vexillata, was discussed.