Genetic Structure of Wild Rice Oryza Glumaepatula Populations in Three Brazilian Biomes Using Microsatellite Markers

The existence of Oryza glumaepatula is threatened by devastation and, thus, the implementation of conservation strategies is extremely relevant. This study aimed to characterize the genetic variability and estimate population parameters of 30 O. glumaepatula populations from three Brazilian biomes using 10 microsatellite markers. The levels of allelic variability for the SSR loci presented a mean of 10.3 alleles per locus and a value of 0.10 for the average allelic frequency value. The expected total heterozygosity (H_e) ranged from 0.63 to 0.86. For the 30 populations tested, the mean observed (H_o) and expected heterozygosities (H_e) were 0.03 and 0.11within population, respectively, indicating an excess of homozygotes resulting from the preferentially self-pollinating reproduction habit. The estimated fixation index ( _IS ) was 0.79 that differed significantly from zero, indicating high inbreeding within each O. glumaepatula population. The total inbreeding of the species (_IT ) was 0.98 and the genetic diversity indexes among populations, _ST and _ST, were 0.85 and 0.90, respectively, indicating high genetic variability among them. Thus, especially for populations located in regions threatened with devastation, it is urgent that in situ preservation conditions should be created or that collections be made for ex situ preservation to prevent loss of the species genetic variability.     

The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway

The atypical protein kinase C (aPKC)-interacting protein, p62, has previously been shown to interact with RIP, linking these kinases to NF-kappaB activation by tumor necrosis factor alpha (TNFalpha). The aPKCs have been implicated in the activation of IKKbeta in TNFalpha-stimulated cells and have been shown to be activated in response to interleukin-1 (IL-1). Here we demonstrate that the inhibition of the aPKCs or the down-regulation of p62 severely abrogates NF-kappaB activation by IL-1 and TRAF6, suggesting that both proteins are critical intermediaries in this pathway. Consistent with this we show that p62 selectively interacts with the TRAF domain of TRAF6 but not that of TRAF5 or TRAF2 in co-transfection experiments. The binding of endogenous p62 to TRAF6 is stimulus dependent, reinforcing the notion that this is a physiologically relevant interaction. Furthermore, we demonstrate that the N-terminal domain of TRAF6, which is required for signaling, interacts with zetaPKC in a dimerization-dependent manner. Together, these results indicate that p62 is an important intermediary not only in TNFalpha but also in IL-1 signaling to NF-kappaB through the specific adapters RIP and TRAF6.     

Pattern Dynamics in Adaxial-Abaxial Specific Gene Expression Are Modulated by a Plastid Retrograde Signal during Arabidopsis thaliana Leaf Development

The maintenance and reformation of gene expression domains are the basis for the morphogenic processes of multicellular systems. In a leaf primordium of Arabidopsis thaliana, the expression of FILAMENTOUS FLOWER (FIL) and the activity of the microRNA miR165/166 are specific to the abaxial side. This miR165/166 activity restricts the target gene expression to the adaxial side. The adaxial and abaxial specific gene expressions are crucial for the wide expansion of leaf lamina. The FIL-expression and the miR165/166-free domains are almost mutually exclusive, and they have been considered to be maintained during leaf development. However, we found here that the position of the boundary between the two domains gradually shifts from the adaxial side to the abaxial side. The cell lineage analysis revealed that this boundary shifting was associated with a sequential gene expression switch from the FIL-expressing (miR165/166 active) to the miR165/166-free (non-FIL-expressing) states. Our genetic analyses using the enlarged fil expression domain2 (enf2) mutant and chemical treatment experiments revealed that impairment in the plastid (chloroplast) gene expression machinery retards this boundary shifting and inhibits the lamina expansion. Furthermore, these developmental effects caused by the abnormal plastids were not observed in the genomes uncoupled1 (gun1) mutant background. This study characterizes the dynamic nature of the adaxial-abaxial specification process in leaf primordia and reveals that the dynamic process is affected by the GUN1-dependent retrograde signal in response to the failure of plastid gene expression. These findings advance our understanding on the molecular mechanism linking the plastid function to the leaf morphogenic processes.     

Species Displacement Between an Introduced and A ‘vulnerable’ Crayfish: The Role of Aggressive Interactions and Shelter Competition

In eastern Hokkaidô, Japan, the native crayfish Cambaroides japonicus de Haan has been declining rapidly while the exotic crayfish Pacifastacus leniusculus Dana has been expanding its range. We speculated that the observed displacement of Cambaroides by Pacifastacus is partly due to an interference interaction for shelter between the two species. We studied the impacts of Pacifastacus on Cambaroides by testing experimentally whether intensity of interference, the outcome of dominance order and shelter occupancy differed between single- and mixed-species combinations of the two species. Pacifastacus showed less defensive behaviours towards Cambaroides than to conspecifics. In contrast, Cambaroides exhibited more defensive behaviours towards Pacifastacus than to conspecifics. Pairwise comparison of agonistic behaviours in mixed species groups revealed that Pacifastacus exhibiting frequently more aggressive attacks while Camaboides showing more defensive behaviours to their respective counterparts. Dominance in aggressive encounter generally dictated shelter occupancy in mixed-species and Pacifastacus pairs but not in Cambaroides pairs. Because shelter utilization, when these are not limited in supply, was much more frequent in Cambaroides than Pacifastacus, Cambaroides suffered interference from both conspecifics and heterospecifics when they competed for a single shelter. Pacifastacus, however, did not show any change in shelter occupancy in the presence or absence of conspecics or heterospecifics. Inferiority in aggressive interactions and shelter occupancy can therefore be a critical disadvantage for Cambaroides if shelters are limited in natural situations.     

Evaluation of accumulation of hepatitis C virus mutations in a chronically infected chimpanzee: comparison of the core, E1, HVR1, and NS5b regions

Four hepatitis C virus genome regions (the core, E1, HVR1, and NS5b) were amplified and sequenced from yearly samples obtained from a chronically infected chimpanzee over a 12-year span. Nucleotide substitutions were found to accumulate in the core, E1, and HVR1 regions during the course of chronic infection; substitutions within the NS5b region were not detected for the first 8 years and were found to be minimal during the last 4 years. The rate of accumulation of mutations in the core and E1 regions, based on a direct comparison between the first 1979 sequence and the last 1990 sequence, was 1.120 x 10(-3), while phylogenetic ancestral comparison using the 12 yearly sequences showed a rate of 0.816 x 10(-3) bases per site per year. Temporal evaluation of the sequences revealed that there appeared to be periods in which substitutions accumulated and became fixed, followed by periods with relative stasis or random substitutions that did not persist. Synonymous and nonsynonymous substitutions within the core, E1, and HVR1 regions were also analyzed. In the core and E1 regions, synonymous substitutions predominated and gradually increased over time. However, within the HVR1 region, nonsynonymous substitutions predominated but gradually decreased over time.     

Identification and characterization of a novel protein inhibitor of type 1 protein phosphatase

We have isolated human cDNA for a novel type 1 protein phosphatase (PP1) inhibitory protein, named inhibitor-4 (I-4), from a cDNA library of germ cell tumors. I-4, composed of 202 amino acids, is 44% identical to a PP1 inhibitor, inhibitor-2 (I-2). I-4 conserves functionally important structure of I-2 and exhibited similar biochemical properties. I-4 inhibited activity of the catalytic subunit of PP1 (PP1C), specifically with an IC(50) of 0.2 nM, more potently than I-2 with an IC(50) of 2 nM. I-4 weakly inhibited the activity of myosin-associated phosphates (PP1M). However, the level of inhibition of PP1M was increased during preincubation of PP1M with I-4, suggesting that the inhibition is caused by interaction of I-4 with PP1C in such a manner that it competes with the M subunit of PP1M. Gel overlay experiments showed that I-4 binds PP1C directly. Three I-4 peptides containing the N-terminal residues 1-123, 1-131, and 1-142 all showed strong binding ability to PP1C but did not show PP1 inhibitory activity, whereas an I-2 peptide (residues 1-134), lacking the corresponding C-terminal residues, potently inhibited PP1C activity as previously reported. Removal of the 18 N-terminal amino acid residues from I-4 dramatically reduced the PP1 binding activity with a correlated loss of inhibitory activity, whereas removal of the 10 N-terminal residues had only a little effect. The two peptides GST-I-4(19-131) and GST-I-4(132-202) showed ability to bind to PP1C, albeit very weakly. These results strongly suggest a multiple-point interaction between I-4 and PP1C, which is thought to cause the inhibition of I-4 which is stronger than the inhibition of I-2.     

Purification and partial amino acid sequence analysis of the larval settlement-inducing pheromone from adult extracts of the barnacle,Balanus amphitrite (=Amphibalanus amphitrite)

A previously undescribed larval settlement-inducing protein was purified from adult extracts of the barnacle, Balanus amphitrite (=Amphibalanus amphitrite). Results of SDS-PAGE indicated that the relative molecular mass of the protein in reduced and denatured form is 31,600 ± 500 kDa, and that it is distinct from the Settlement Inducing Protein Complex (SIPC) which has previously been determined as a larval settlement-inducing pheromone. The N-terminal 33-residue sequence of the intact protein showed no similarity with previously reported proteins in the EMBL/Genbank/DDBJ databases. The purified protein at a concentration of 10 μg ml^?1 induced approximately four times more larval settlement than the control (filtered natural seawater). In addition, results of the assay using both 24-well polystyrene plates and agarose gels indicated that this protein is probably released into seawater and attracts cypris larvae. These results suggest that the purified protein is a waterborne type pheromone which induces settlement of larvae of B. amphitrite.     

Adventitious shoot regeneration from cultured petal explants of carnation

Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 M BA with or without 5 M NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N-phenylurea and N-1,2,3-thiadiazol-5-yl-N-N-phenylurea were more effective than BA, kinetin, N⁶-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - GA₃ gibberellic acid - 2iP N⁶-2-isopentenyl adenine - KT-30 N-2-chloro-4-pyridyl-N-phenylurea (also called 4PU) - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Plasmin decreases the BH3-only protein BimEL via the ERK1/2 signaling pathway in hepatocytes

Since the signal transduction mechanisms responsible for liver regeneration mediated by the plasminogen/plasmin system remain largely undetermined, we have investigated whether plasmin regulates the pro-apoptotic protein Bim(EL) in primary hepatocytes. Plasmin bound to hepatocytes in part via its lysine binding sites (LBS). Plasmin also triggered phosphorylation of ERK1/2 without cell detachment. The plasmin-induced phosphorylation of ERK1/2 was inhibited by the LBS inhibitor epsilon-aminocaproic acid (EACA), the serine protease inhibitor aprotinin, and the MEK inhibitor PD98059. DFP-inactivated plasmin failed to phosphorylate ERK1/2. Plasmin temporally decreased the starvation-induced expression of Bim(EL) and activation of caspase-3 via the ERK1/2 signaling pathway, resulting in an enhancement of cell survival. The amount of mRNA for Bim increased 1 day after the injection of CCl(4) in livers of plasminogen knockout (Plg-KO) and the wild-type (WT) mice. The increase in Bim(EL) protein persisted for at least 7 days post-injection in livers of Plg-KO mice, whereas WT mice showed an increase in Bim(EL) protein 1 day after the injection. Plg-KO and WT mice showed notable phosphorylation of ERK1/2 7 and 3 days after the injection of CCl(4), respectively. Our data suggest that the plasminogen/plasmin system could decrease Bim(EL) expression via the ERK1/2 signaling pathway during liver regeneration.