Foraging behavior of Adélie penguins during incubation period in Lützow-Holm Bay

The diving behavior of Adélie penguins Pygoscelis adeliae was investigated using time–depth recorders during the incubation period in the fast sea-ice area of Lützow-Holm Bay, Antarctica. Dive profiles and activity/time allocation suggested that penguins were obligated to walk on the fast-ice for 90–100 km until a polynya, which they used as an access to the pack-ice zone. Dive depth did not differ between males and females, though males’ dive duration was longer than that of females. Dive depth was slightly shallower and dive duration was shorter during the incubation than during the chick-rearing phase. Birds dove throughout the day, although less frequently around midnight, and there was no clear diel change in dive depth. This daily dive pattern during incubation period was similar to that previously observed during the chick-rearing period in a fast sea-ice area, but differed from that observed in sea-ice-free area. Variations in diving behavior resulted from different environmental conditions, such as foraging area with different sea-ice condition, as well as from different life history strategies.     

Identification of cis-localization elements of the maize 10-kDa δ-zein and their use in targeting RNAs to specific cortical endoplasmic reticulum subdomains

The RNAs for the storage proteins of rice ( Oryza sativa ), prolamines and glutelins, which are stored as inclusions in the lumen of the endoplasmic reticulum (ER) and storage vacuoles, respectively, are targeted by specific cis -localization elements to distinct subdomains of the cortical ER. Glutelin RNA has one or more cis -localization elements (zip codes) at the 3' end of the RNA, whereas prolamine has two cis -elements; one located in the 5' end of the coding sequence and a second residing in the 3'-untranslated region (UTR). We had earlier demonstrated that the RNAs for the maize zeins ('prolamine' class) are localized to the spherical protein body ER (PB-ER) in developing maize endosperm. As the PB-ER localization of the 10-kDa δ-zein RNA is maintained in developing rice seeds, we determined the number and proximate location of their cis -localization elements by expressing GFP fusions containing various zein RNA sequences in transgenic rice and analyzing their spatial distribution on the cortical ER by in situ RT-PCR and confocal microscopy. Four putative cis -localization elements were identified; three in the coding sequences and one in the 3'-UTR. Two of these zip codes are required for restricted localization to the PB-ER. Using RNA targeting determinants we show, by mis-targeting the storage protein RNAs from their normal destination on the cortical ER, that the coded proteins are redirected from their normal site of deposition. Targeting of RNA to distinct cortical ER subdomains may be the underlying basis for the variable use of the ER lumen or storage vacuole as the final storage deposition site of storage proteins among flowering plant species.     

Cooperative alkylation of double-strand human telomere repeat sequences by PI polyamides with 11-base-pair recognition based on a heterotrimeric design

We designed and synthesized alkylating conjugates 5–7 and their partner N-methylpyrrole-N-methylimidazole (PI) polyamides 8, 9. The DNA alkylating activities of conjugates 5–7 were evaluated by high-resolution denaturing polyacrylamide gel electrophoresis with a 219 base pair (bp) DNA fragment containing the human telomere repeat sequence. Conjugate 5 efficiently alkylated the sequence, 5′-GGTTAGGGTTA-3′, in the presence of partner PI polyamide 8 or distamycin A (Dist). In contrast, the heterodimer system of 5 with 9 showed very weak alkylating activity. Accordingly, this heterotrimeric system of 5 with two short partners is an expedient way to attain improved precision and extension of the recognition of DNA sequences.     

Age-related myelin dynamics revealed by increased oligodendrogenesis and short internodes

Aging is associated with many functional and morphological central nervous system changes. It is important to distinguish between changes created by normal aging and those caused by disease. In the present study we characterized myelin changes within the murine rubrospinal tract and found that internode lengths significantly decrease as a function of age which suggests active remyelination. We also analyzed the proliferation, distribution and phenotypic fate of dividing cells with Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). The data reveal a decrease in glial cell proliferation from 1 to 6, 14 and 21 months of age in gray matter 4 weeks post-BrdU injections. However, we found an increase in gliogenesis at 21st  month in white matter of the spinal cord. Half of newly generated cells expressed NG2. Most cells were positive for the early oligodendrocyte marker Olig2 and a few also expressed CC1. Very few cells ever became positive for the astrocytic markers S100β or GFAP. These data demonstrate ongoing oligodendrogenesis and myelinogenesis as a function of age in the spinal cord.     

GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers

GAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice.     

Proteomic analysis of laser-microdissected paraffin-embedded tissues: (2) MRM assay for stage-related proteins upon non-metastatic lung adenocarcinoma

A preceding paper suggested 81 candidates of stage-specifically expressed proteins for either stage IA or IIIA by global shotgun proteomics and spectral counting. Six proteins, a subset of these proteins, were chosen for a further verification study since they are potentially soluble and/or secretory, which nature is convenient for detecting them in blood in clinical practice. The multiple-reaction monitoring (MRM) quantitative analysis suggested that napsin-A and anterior gradient protein 2 homolog (hAG-2) out of the 6 candidates would be useful for determining stage IA or IIIA and are related to metastasis. In the study we noted that stage IIIA patients with better outcome showed napsin-A profiles similar to that of stage IA patients. We therefore examined 14 additional patients for analysis, which contained the IA-stage patients of poorer outcome and the IIIA-stage patients of better outcome. The MRM analysis of napsin-A for all patients suggests that napsin-A contents correlate with better outcome in stage IA. This and discovery studies demonstrate that direct isolation of tumor cells alone by laser microdissection (LMD) greatly reduces complexity on comprehensive analyses, and that MRM mass spectrometry using the endogenous internal standard is a feasible technology for quantitative verification of target proteins in formalin-fixed paraffin embedded (FFPE) tissues.     

An activated medium with high durability and low nonspecific adsorption: application to protein A chromatography

Activated media allow the user to easily synthesize a variety of affinity media. We have developed a novel activated medium based on porous silica modified with phosphorylcholine (PC) and N-hydroxysuccinimide (NHS) groups for the purpose of high-throughput purification and reducing nonspecific protein adsorption. The PC groups function as suppressors of nonspecific protein adsorption, whereas the NHS groups are able to covalently bind to the primary amino groups of ligands. Because protein A affinity medium is the most frequently used affinity medium, we prepared protein A media in which a recombinant protein A was bound to the NHS groups of the activated media and evaluated its utility. After optimizing various factors in the synthetic process, the resultant protein A medium showed improved durability at a high flow rate over 300 purification cycles and reduced nonspecific protein adsorption compared with commercially available protein A media.     

Megalin is downregulated via LPS-TNF-α-ERK1/2 signaling pathway in proximal tubule cells

Expression and function of megalin, an endocytic receptor in proximal tubule cells (PTCs), are reduced in diabetic nephropathy, involved in the development of proteinuria/albuminuria. Lipopolysaccharide (LPS) is chronically increased in diabetic sera, by the mechanism called metabolic endotoxemia. We investigated low-level LPS-mediated signaling that regulates megalin expression in immortalized rat PTCs (IRPTCs). Incubation of the cells with LPS (10 ng/ml) for 48 h suppressed megalin protein expression and its endocytic function. TNF-α mRNA expression was increased by LPS treatment, and knockdown of the mRNA with siRNA inhibited LPS-mediated downregulation of megalin mRNA expression at the 24-h time point. Incubation of IRPTCs with exogenous TNF-α also suppressed megalin mRNA and protein expression at the 24- and 48-h time points, respectively. MEK1 inhibitor PD98059 competed partially but significantly TNF-α-mediated downregulation of megalin mRNA expression. Collectively, low-level LPS-mediated TNF-α-ERK1/2 signaling pathway is involved in downregulation of megalin expression in IRPTCs.     

Chimeric microbial rhodopsins containing the third cytoplasmic loop of bovine rhodopsin

G-protein-coupled receptors transmit stimuli (light, taste, hormone, neurotransmitter, etc.) to the intracellular signaling systems, and rhodopsin (Rh) is the most-studied G-protein-coupled receptor. Rh possesses an 11-cis retinal as the chromophore, and 11-cis to all-trans photoisomerization leads to the protein structural changes in the cytoplasmic loops to activate G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. Microbial rhodopsins possess an all-trans retinal, and all-trans to 13-cis photoisomerization triggers protein structural changes for each function. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. However, it was reported that bacteriorhodopsin (BR) chimeras containing the third cytoplasmic loop of bovine Rh are able to activate G-protein, suggesting a common mechanism of protein structural changes. Here we design chimeric proteins for Natronomonas pharaonis sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin), which has a two-orders-of-magnitude slower photocycle than BR. Light-dependent transducin activation was observed for most of the nine SRII chimeras containing the third cytoplasmic loop of bovine Rh (from Y223, G224, Q225 to T251, R252, and M253), but the activation level was 30,000–140,000 times lower than that of bovine Rh. The BR chimera, BR/Rh223-253, activates a G-protein transducin, whereas the activation level was 37,000 times lower than that of bovine Rh. We interpret the low activation by the chimeric proteins as reasonable, because bovine Rh must have been optimized for activating a G-protein transducin during its evolution. On the other hand, similar activation level of the SRII and BR chimeras suggests that the lifetime of the M intermediates is not the simple determinant of activation, because SRII chimeras have two-orders-of-magnitude's slower photocycle than the BR chimera. Activation mechanism of visual and microbial rhodopsins is discussed on the basis of these results.     

Dietary adaptations of temperate primates: comparisons of Japanese and Barbary macaques

Habitat, diet and leaf chemistry are compared between Japanese and Barbary macaques to reveal the similarities and differences in dietary adaptations of temperate primates living at the eastern and western extremes of the genus Macaca. Tree species diversity and proportion of fleshy-fruited species are much higher in Japan than in North Africa. Both species spend considerable annual feeding time on leaves. Japanese macaques prefer fruits and seeds over leaves, and Barbary macaques prefer seeds. These characteristics are adaptive in temperate regions where fruit availability varies considerably with season, since animals can survive during the lean period by relying on leaf and other vegetative foods. The two species are different with respect to the higher consumption of herbs by Barbary macaques, and the leaves consumed contain high condensed and hydrolysable tannin for Barbary but not for Japanese macaques. Barbary macaques supplement less diverse tree foods with herbs. Because of the low species diversity and high tannin content of the dominant tree species, Barbary macaques may have developed the capacity to cope with tannin. This supports the idea that digestion of leaves is indispensable to survive in temperate regions where fruit and seed foods are not available for a prolonged period during each year.