Overexpression of constitutively active Arabidopsis RabG3b promotes xylem development in transgenic poplars

An Arabidopsis small GTPase, RabG3b, was previously characterized as a component of autophagy and as a positive regulator for xylem development in Arabidopsis. In this work, we assessed whether RabG3b modulates xylem-associated traits in poplar in a similar way as in Arabidopsis. We generated transgenic poplars (Populus alba × Populus tremula var. glandulosa) overexpressing a constitutively active form of RabG3b (RabG3bCA) and performed a range of morphological, histochemical and molecular analyses to examine xylogenesis. RabG3bCA transgenic poplars showed increased stem growth due to enhanced xylem development. Autophagic structures were observed in differentiating xyelm cells undergoing programmed cell death (PCD) in wild-type poplar, and were more abundant in RabG3bCA transgenic poplar plants and cultured cells. Xylogenic activation was also accompanied by the expression of secondary wall-, PCD- and autophagy-related genes. Collectively, our results suggest that Arabidopsis RabG3b functions to regulate xylem growth through the activation of autophagy during wood formation in Populus, as does the same in Arabidopsis.     

Population genetic structure of wild and hatchery black rockfish Sebastes inermis in Korea, assessed using cross-species microsatellite markers

The population structure of the black rockfish, Sebastes inermis (Sebastidae), was estimated using 10 microsatellite loci developed for S. schlegeli on samples of 174 individuals collected from three wild and three hatchery populations in Korea. Reduced genetic variation was detected in hatchery strains [overall number of alleles (N(A)) = 8.07; allelic richness (A(R)) = 7.37; observed heterozygosity (H(O)) = 0.641] compared with the wild samples (overall N(A) = 8.43; A(R) = 7.83; H(O) = 0.670), but the difference was not significant. Genetic differentiation among the populations was significant (overall F(ST) = 0.0237, P < 0.05). Pairwise F(ST) tests, neighbor-joining tree, and principal component analyses showed significant genetic heterogeneity among the hatchery strains and between wild and hatchery strains, but not among the wild populations, indicating high levels of gene flow along the southern coast of Korea, even though the black rockfish is a benthic, non-migratory marine species. Genetic differentiation among the hatchery strains could reflect genetic drift due to intensive breeding practices. Thus, in the interests of optimal resource management, genetic variation should be monitored and inbreeding controlled within stocks in commercial breeding programs. Information on genetic population structure based on cross-species microsatellite markers can aid in the proper management of S. inermis populations.     

Prevention of salt-induced renal injury by activation of NAD(P)H:quinone oxidoreductase 1, associated with NADPH oxidase

NADPH oxidase (NOX) is a predominant source of reactive oxygen species (ROS), and the activity of NOX, which uses NADPH as a common rate-limiting substrate, is upregulated by prolonged dietary salt intake. β-Lapachone (βL), a well-known substrate of NAD(P)H:quinone oxidoreductase 1 (NQO1), decreases the cellular NAD(P)H/NAD(P)(+) ratio via activation of NQO1. In this study, we evaluated whether NQO1 activation by βL modulates salt-induced renal injury associated with NOX-derived ROS regulation in an animal model. Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet were used to investigate the renoprotective effect of NQO1 activation. βL treatment significantly lowered the cellular NAD(P)H:NAD(P)(+) ratio and dramatically reduced NOX activity in the kidneys of HS diet-fed DS rats. In accordance with this, total ROS production and expression of oxidative adducts also decreased in the βL-treated group. Furthermore, HS diet-induced proteinuria and glomerular damage were markedly suppressed, and inflammation, fibrosis, and apoptotic cell death were significantly diminished by βL treatment. This study is the first to demonstrate that activation of NQO1 has a renoprotective effect that is mediated by NOX activity via modulation of the cellular NAD(P)H:NAD(P)(+) ratio. These results provide strong evidence that NQO1 might be a new therapeutic target for the prevention of salt-induced renal injury.     

Synthetic TGF-β Signaling Agonist-Treated Dendritic Cells Induce Tolerogenicity and Antirheumatic Effects

The newly synthesized compound TGF-β signaling agonist (T74) is a small molecule associated with the TGF-β receptor signaling pathway. Tolerogenic dendritic cells (tDCs) have been used to examine immunosuppressive and anti-inflammatory effects in multiple autoimmune disease models. The aim of this study was to investigate whether treatment of DCs with T74 has an antirheumatic effect in a mouse model of collagen-induced arthritis (CIA). Bone marrow-derived cells were obtained from DBA/1J mice and differentiated into DCs. T74-treated DCs (T74-DCs) were generated by treating bone marrow-derived DCs with LPS, type II collagen, and T74. T74-DCs expressed lower levels of surface molecules and inflammatory cytokines associated with antigen presentation and T cell stimulation. The ability of T74-DCs to differentiate effector T cells was lower than that of T74-untreated DCs (NT-DCs), but T74-DCs increased the regulatory T (Treg) cell differentiation in vitro. DBA/1J mice received two subcutaneous (s.c.) injections of type II collagen to establish CIA. Mice then received two s.c. injections of T74-DCs or NT-DCs. Joint inflammation was ameliorated in the paws of T74-DC-treated mice. Additionally, Treg populations in T74-DC-treated mice were higher than in NT-DC-treated or PBS-treated CIA mice. Taken together, these results demonstrate that T74 induces tolerance in DCs, and that T74-mediated DCs exert antirheumatic effects via induction of Tregs.     

Protein kinase C δ (PKCδ)-extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade regulates glycogen synthase kinase-3 (GSK-3) inhibition-mediated interleukin-10 (IL-10) expression in lipopolysaccharide (LPS)-induced endotoxemia

Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes, including embryonic development, cell differentiation, survival, and apoptosis. Recently, it was reported that a GSK-3 inhibitor attenuates lipopolysaccharide (LPS)-induced septic shock and regulates the mortality of endotoxemic mice. However, the detailed mechanism of reduced mortality via GSK-3 inhibition is not well defined. Herein, we showed that GSK-3 inhibition induces extracellular signal-regulated kinase 1/2 (ERK1/2) activation under LPS-stressed conditions via protein kinase C δ (PKCδ) activation. Furthermore, PKCδ-induced ERK1/2 activation by the inhibition of GSK-3 provoked the production of interleukin (IL)-10, playing a crucial role in regulating endotoxemia. Using a mitogen-activated protein kinase kinase-1 (MEK-1) and PKCδ inhibitor, we confirmed that GSK-3 inhibition induces PKCδ and subsequent ERK1/2 activation, resulting in increased IL-10 expression under LPS-treated conditions. We verified that septic shock caused by LPS is attenuated by GSK-3 inhibition using a GSK-3 inhibitor. This relieved endotoxemia induced by GSK-3 inhibition was restored in an ERK1/2-dependent manner. Taken together, IL-10 expression produced by GSK-3 inhibition-induced ERK1/2 activation via PKCδ relieved LPS-mediated endotoxemia. This finding suggests that IL-10 hyperexpression resulting from GSK-3 inhibition-induced ERK activation could be a new therapeutic pathway for endotoxemia.     

A Synthetic Model of Human Beta-Thalassemia Erythropoiesis Using CD34+ Cells from Healthy Adult Donors

Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14–21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia.     

PIE1, an ISWI family gene,is required for FLC activation and floral repression in Arabidopsis

Proper control of the floral transition is critical for reproductive success in flowering plants. In Arabidopsis, FLOWERING LOCUS C (FLC) is a floral repressor upon which multiple floral regulatory pathways converge. Mutations in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1) suppress the FLC-mediated delay of flowering as a result of the presence of FRIGIDA or of mutations in autonomous pathway genes. PIE1 is required for high levels of FLC expression in the shoot apex, but it is not required for FLC expression in roots. PIE1 is similar to ATP-dependent, chromatin-remodeling proteins of the ISWI and SWI2/SNF2 family. The role of PIE1 as an activator of FLC is consistent with the general role of ISWI and SWI2/SNF2 family genes as activators of gene expression. The pie1 mutation also causes early flowering in noninductive photoperiods independently of FLC; thus, PIE1 appears to be involved in multiple flowering pathways. PIE1 also plays a role in petal development, as revealed by the suppression of petal defects of the curly leaf mutant by the pie1 mutation.     

High efficiency shoot regeneration from callus of quaking aspen (Populus tremuloides Michx.)

Callus was induced from leaf segments of aspen (Populus tremuloides Michx.) on modified B5 (mB5) medium with 0.1 mg/1 benzyladenine (BA) and 0.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting callus was either subcultured to solidified Woody Plant Medium (WPM) with 0.5 mg/1 BA directly for shoot regeneration or sieved into liquid mB5 medium for suspension culture. After 3 weeks of suspension culture, when the callus clumps grew to 3–4 mm in diameter, they were transferred onto solidified WPM with 0.5 mg/1 BA for shoot regeneration. Almost 100% of the clumps formed shoots on WPM when subcultured directly from mB5 with an average number of 6 shoots per callus. When transferred from suspension culture in mB5 to WPM, an average of 6 shoots per callus were produced from 51% of calli. These shoots could be easily rooted on either mB5 or WPM with 0.2 mg/1 indole-3-butyric acid (IBA) and transferred to pots. Transplanted plants were kept under intermittent mist for 2–4 weeks before normal growth in the green house.Abbreviations BA 6-Benzyl-adenine - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - mB5 medium modified B5 medium - WPM Woody Plant medium