Phosphorylation of the triadin cytoplasmic domain by CaM protein kinase in rabbit fast-twitch muscle sarcoplasmic reticulum

Identification of estrogenresponsive genes is important to understand the molecular mechanisms of estrogen action. Suppression subtractive hybridization was employed to screen estrogenresponsive genes in chick liver. A single injection of estrogen into 6weekold chick induced upregulation of several known genes encoded for yolk proteins, such as Vitellogenin I and II and very low density lipoprotein II (apo-VLDL II). One novel sequence displayed a dramatic change (3fold increase) in response to estrogen treatment. This cDNA fragment was extended and the resultant sequence was analyzed. Translated amino acid sequence was 90, 88, 83 and 87% identical to the Larginine:glycine amidinotransferase of pig, rat, frog and human, respectively. The sequence has a conservative catalytic site of Larginine:glycine amidinotransferase. The expression pattern of this gene in organs is consistent with previous reports of Larginine:glycine amidinotransferase in chick. Thus, this clone represented the chicken Larginine:glycine amidinotransferase. It appeared that estrogeninduced alteration of arginine:glycine amidinotransferase was not dependent on protein synthesis, because concurrent administration of cycloheximide did not affect the estrogenmediated expression pattern. This is the first study demonstrating that Larginine:glycine amidinotransferase is a target of the estrogen receptor.     

Targeting of calsequestrin to the sarcoplasmic reticulum of skeletal muscle upon deletion of its glycosylation site

The glycoprotein calsequestrin (CS) is segregated to the junctional sarcoplasmic reticulum (jSR) and is responsible for intraluminal Ca(2+) binding. A chimeric CS-hemoagglutinin 1 (HA1), obtained by adding the nine amino acid viral epitope hemoagglutinin to the carboxy terminal of CS and shown to be correctly segregated to skeletal muscle jSR [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe (1997). Chimeric calsequestrin and its targeting to the junctional sarcoplasmic reticulum of skeletal muscle. Am. J. Physiol. 272, C1420-C1428] lends itself as a molecular tool to investigate the targeting domains of CS. A putative targeting mechanism of CS to jSR implies glycosylation-dependent steps in the endoplasmic reticulum (ER) and Golgi complex. To test this hypothesis, CS-HA1DeltaGly, a mutant in which the unique N-glycosylation site Asn316 was changed to Ile, was engineered by site-directed mutagenesis. The mutant cDNA was transiently transfected in either HeLa cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of CS-HA1DeltaGly was studied by double-labeling epifluorescence by means of antibodies against either CS, HA1, or the ryanodine receptor calcium release channel. CS-HA1DeltaGly was expressed and retained to ER and ER/sarcoplasmic reticulum of HeLa cells and myotubes, respectively, and expressed, sorted, and correctly segregated to jSR of regenerating soleus muscle fibers. Thus, the targeting mechanism of CS in vivo appears not to be affected by glycosylation-that is, the sorting, docking, and segregation of CS are independent of cotranslational and posttranslational glycosylation or glycosylations.     

On the Worrying Fate of Data Deficient Amphibians

The ‘Data Deficient’ (DD) category of the IUCN Red List assembles species that cannot be placed in another category due to insufficient information. This process generates uncertainty about whether these species are safe or actually in danger. Here, we give a global overview on the current situation of DD amphibian species (almost a quarter of living amphibians) considering land-use change through habitat modification, the degree of protection of each species and the socio-political context of each country harboring DD species. We found that DD amphibians have, on average, 81% of their ranges totally outside protected areas. Worryingly, more than half of DD species have less than 1% of their distribution represented in protected areas. Furthermore, the percentage of overlap between species’ range and human-modified landscapes is high, at approximately 58%. Many countries harboring a large number of DD species show a worrying socio-political trend illustrated by substantial, recent incremental increases in the Human Development Index and lower incremental increases in the establishment of protected areas. Most of these are African countries, which are located mainly in the central and southern regions of the continent. Other countries with similar socio-political trends are in southeastern Asia, Central America, and in the northern region of South America. This situation is concerning, but it also creates a huge opportunity for considering DD amphibians in future conservation assessments, planning, and policy at different levels of government administration.     

Effects of quercetin and menadione on intestinal calcium absorption and the underlying mechanisms

Quercetin (QT) could be considered as a potential therapeutic agent for different diseases due to its antioxidant, anti-inflammatory, antiviral and anticancer properties. This study was designed to investigate the ability of QT to protect the chick intestine against menadione (MEN) induced injury in vivo and in vitro. Four-week old chicks (Gallus gallus) were treated i.p. with 2.5 μmol of MEN/kg b.w. or with i.l. 50 μM QT or both. QT protected the intestinal Ca²⁺ absorption against the inhibition caused by MEN, but QT alone did not modify. Glutathione (GSH) depletion provoked by MEN in chick enterocytes was abolished by QT treatment, whereas QT alone did not modify the intestinal GSH content. The enhancement of GSH peroxidase activity produced by MEN was blocked by QT treatment. In contrast, superoxide dismutase activity remained high after simultaneous treatment of enterocytes with MEN and QT. The flavonol also avoided changes in the mitochondrial membrane permeability (swelling) produced by MEN. The FasL/Fas/caspase-3 pathway was activated by MEN, effect that was abrogated by QT. In conclusion, QT may be useful in preventing inhibition of chick intestinal Ca²⁺ absorption caused by MEN or other substances that deplete GSH, by blocking the oxidative stress and the FasL/Fas/caspase-3 pathway activation.     

DDBJ Read Annotation Pipeline: A Cloud Computing-Based Pipeline for High-Throughput Analysis of Next-Generation Sequencing Data

High-performance next-generation sequencing (NGS) technologies are advancing genomics and molecular biological research. However, the immense amount of sequence data requires computational skills and suitable hardware resources that are a challenge to molecular biologists. The DNA Data Bank of Japan (DDBJ) of the National Institute of Genetics (NIG) has initiated a cloud computing-based analytical pipeline, the DDBJ Read Annotation Pipeline (DDBJ Pipeline), for a high-throughput annotation of NGS reads. The DDBJ Pipeline offers a user-friendly graphical web interface and processes massive NGS datasets using decentralized processing by NIG supercomputers currently free of charge. The proposed pipeline consists of two analysis components: basic analysis for reference genome mapping and de novo assembly and subsequent high-level analysis of structural and functional annotations. Users may smoothly switch between the two components in the pipeline, facilitating web-based operations on a supercomputer for high-throughput data analysis. Moreover, public NGS reads of the DDBJ Sequence Read Archive located on the same supercomputer can be imported into the pipeline through the input of only an accession number. This proposed pipeline will facilitate research by utilizing unified analytical workflows applied to the NGS data. The DDBJ Pipeline is accessible at http://p.ddbj.nig.ac.jp/.     

Positive autoregulation of a KNOX gene is essential for shoot apical meristem maintenance in rice

Self-maintenance of the shoot apical meristem (SAM), from which aerial organs are formed throughout the life cycle, is crucial in plant development. Class I Knotted1-like homeobox (KNOX) genes restrict cell differentiation and play an indispensable role in maintaining the SAM. However, the mechanism that positively regulates their expression is unknown. Here, we show that expression of a rice (Oryza sativa) KNOX gene, Oryza sativa homeobox1 (OSH1), is positively regulated by direct autoregulation. Interestingly, loss-of-function mutants of OSH1 lose the SAM just after germination but can be rescued to grow until reproductive development when they are regenerated from callus. Double mutants of osh1 and d6, a loss-of-function mutant of OSH15, fail to establish the SAM both in embryogenesis and regeneration. Expression analyses in these mutants reveal that KNOX gene expression is positively regulated by the phytohormone cytokinin and by KNOX genes themselves. We demonstrate that OSH1 directly binds to five KNOX loci, including OSH1 and OSH15, through evolutionarily conserved cis-elements and that the positive autoregulation of OSH1 is indispensable for its own expression and SAM maintenance. Thus, the maintenance of the indeterminate state mediated by positive autoregulation of a KNOX gene is an indispensable mechanism of self-maintenance of the SAM.     

Direct examination of chromosomal clustering of organ-specific genes in the chordate Ciona intestinalis

One of challenges in the field of developmental biology is to understand how spatially and/or temporally coordinated expression of genes is controlled at the chromosomal level. It remains controversial whether genes expressed in a given tissue are randomly distributed throughout a given animal genome, or instead resolve into clusters. Here we used microarray analysis to identify more than 1,700 genes that are expressed preferentially in each of 11 organs of the chordate Ciona intestinalis adult, and determined the location of these genes on the 14 pairs of Ciona chromosomes. In spite of extensive mapped gene analysis, we only confirmed small clusters containing two or three genes. Our result indicates that organ-specific genes are distributed rather evenly all over chromosomes, suggesting that the notion of clustering of organ-specific genes in animal genomes is not generally applicable to this chordate.     

Intestinal Na(+)/Ca(2+) exchanger protein and gene expression are regulated by 1,25(OH)(2)D(3) in vitamin D-deficient chicks

The role of 1,25(OH)(2)D(3) on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)(2)D(3) were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)(2)D(3) simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)(2)D(3). NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)(2)D(3). Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)(2)D(3) treatment. Rapid effects of 1,25(OH)(2)D(3) on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)(2)D(3) on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)(2)D(3) enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.     

Global trends of habitat destruction and consequences for parrot conservation

Human advance on natural habitats is a major cause of biodiversity loss. This transformation process represents a profound change in wooded environments, disrupting original communities of flora and fauna. Many species are highly dependent on forests, especially parrots (Psittaciformes) with almost a third of their species threatened by extinction. Most parrot species occur in tropical and subtropical forests, and given the forest dependence of most species, this is the main reason why habitat loss has been highlighted as the main threat for the group. Such habitat loss acts in synergy with other important threats (e.g., logging and poaching), which become especially problematic in certain developing countries along tropical latitudes. In this study, we used available information on parrot distributions, species traits, IUCN assessment, habitat loss and timber extraction for different periods, and distribution of protected areas, to determine conservation hotspots for the group, and analyze potential changes in the conservation status of these species. We detected four conservation hotspots for parrots: two in the Neotropics and two in Oceania, all of them facing different degrees of threat in regard of current habitat loss and agricultural trends. Our results suggest that the future of the group is subject to policymaking in specific regions, especially in the northeastern Andes and the Atlantic Forest. In addition, we predicted that agricultural expansion will have a further negative effect on the conservation status of parrots, pushing many parrot species to the edge of extinction in the near future. Our results have conservation implications by recommending protected areas in specific parrot conservation hotspots. Our recommendations to mitigate conservation risks to this group of umbrella species would also benefit many other coexisting species as well.