<Emphasis Type="Italic">ATHB23</Emphasis>, an <Emphasis Type="Italic">Arabidopsis</Emphasis> class I homeodomain-leucine zipper gene,is expressed in the adaxial region of young leaves

Homeobox genes are essential regulators of plant development. ATHB23, a class I homeodomain leucine zipper gene of Arabidopsis, was found to be induced by treatment with the phytohormone gibberellin (GA). In order to clarify its role in development, we performed a histochemical analysis of transgenic plants containing a construct with a GUS::GFP reporter under the control of the 1.5 kb upstream region of ATHB23. The construct was mainly expressed in young leaves and the styles of flowers but not in mature leaves. Microscopic examination of young leaves revealed that it was expressed in the adaxial domain of leaf primordia and the rib meristem. Expression of ATHB23, like that of GA5 encoding GA 20-oxidase, was reduced in mutants related to adaxial-abaxial leaf polarity (phb-1d, se-2, and kan1 kan2). Reduced expression of the GUS::GFP reporter gene was also observed in an se-2 background. These results indicate that ATHB23 is under the control of GA and other activators such as PHB, and is involved in establishing polarity during leaf development.     

Hyperglycemia and hyperinsulinemia have additive effects on activation and proliferation of pancreatic stellate cells: possible explanation of islet-specific fibrosis in type 2 diabetes mellitus

Pancreatic islet fibrosis observed in Type 2 diabetes is one of the major factors leading to progressive beta-cell loss and dysfunction. Despite its importance, the mechanism of islet-restricted fibrogenesis associated with pancreatic stellate cell (PSC) activation and proliferation remains to be defined. Therefore, we studied whether the islet-specific environment represented by hyperglycemia and hyperinsulinemia had additive effects on the activation and proliferation of cultured rat PSCs. Cells were stimulated to activate and proliferate with glucose and insulin, either individually or concomitantly. Both stimuli promoted PSC proliferation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation independently, but an additive effect was also demonstrated. Blockade of ERK signaling by the mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, suppressed both glucose- and insulin-induced ERK 1/2 phosphorylation and PSC proliferation. Glucose and insulin-induced ERK 1/2 phosphorylation also stimulated connective tissue growth factor gene expression. Thus, hyperglycemia and hyperinsulinemia are two crucial mitogenic factors that activate and proliferate PSCs, and the presence of both states will amplify this response.     

Antidiabetic effect of green rooibos (Aspalathus linearis) extract in cultured cells and type 2 diabetic model KK-Ay mice

Previous studies have demonstrated antidiabetic effects for rooibos (Aspalathus linearis) and aspalathin (ASP), one of its main polyphenols. Rooibos, an endemic plant of South Africa, is well-known for its use as herbal tea. Green (‘unfermented’) rooibos has been shown to contain more ASP than ‘fermented’ rooibos tea, currently the major product. In the present study, we investigated the antidiabetic effect of green rooibos extract (GRE) through studies on glucose uptake in L6 myotubes and on pancreatic β-cell protective ability from reactive oxygen species (ROS) in RIN-5F cells. Its in vivo effect was also examined using obese diabetic KK-A^y mice. GRE increased glucose uptake under insulin absent condition and induced phosphorylation of 5′-adenosine monophosphate-activated protein kinase (AMPK) in L6 myotubes as previously demonstrated for ASP. In addition to AMPK, GRE also promoted phosphorylation of Akt, another promoter of glucose transporter 4 (GLUT4) translocation, in L6 myotubes unlike ASP, suggesting an involvement of GRE component(s) other than ASP in Akt phosphorylation. Promotion of GLUT4 translocation to the plasma membrane by GRE in L6 myotubes was demonstrated by Western blotting analysis. GRE suppressed the advanced glycation end products (AGEs)-induced increase in ROS levels in RIN-5F pancreatic β-cells. Subchronic feeding with GRE suppressed the increase in fasting blood glucose levels in type 2 diabetic model KK-A^y mice. These in vitro and in vivo results strongly suggest that GRE has antidiabetic potential through multiple modes of action.     

Structural importance of the C-terminal region in pig aldo-keto reductase family 1 member C1 and their effects on enzymatic activity

Background

Pig aldo-keto reductase family 1 member C1 (AKR1C1) belongs to AKR superfamily which catalyzes the NAD(P)H-dependent reduction of various substrates including steroid hormones. Previously we have reported two paralogous pig AKR1C1s, wild-type AKR1C1 (C-type) and C-terminal-truncated AKR1C1 (T-type). Also, the C-terminal region significantly contributes to the NADPH-dependent reductase activity for 5α-DHT reduction. Molecular modeling studies combined with kinetic experiments were performed to investigate structural and enzymatic differences between wild-type AKR1C1 C-type and T-type.

Results

The results of the enzyme kinetics revealed that V _max and k _cat values of the T-type were 2.9 and 1.6 folds higher than those of the C-type. Moreover, catalytic efficiency was also 1.9 fold higher in T-type compared to C-type. Since x-ray crystal structures of pig AKR1C1 were not available, three dimensional structures of the both types of the protein were predicted using homology modeling methodology and they were used for molecular dynamics simulations. The structural comparisons between C-type and T-type showed that 5α-DHT formed strong hydrogen bonds with catalytic residues such as Tyr55 and His117 in T-type. In particular, C3 ketone group of the substrate was close to Tyr55 and NADPH in T-type.

Conclusions

Our results showed that 5α-DHT binding in T-type was more favorable for catalytic reaction to facilitate hydride transfer from the cofactor, and were consistent with experimental results. We believe that our study provides valuable information to understand important role of C-terminal region that affects enzymatic properties for 5α-DHT, and further molecular mechanism for the enzyme kinetics of AKR1C1 proteins.

     

Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells

Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H₂O₂ to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H₂O₂ was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.     

One-step knockin for inducible expression in mouse embryonic stem cells

Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline (dox)-controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units, and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor, and money. Here, we describe a one-step Cre-mediated knockin system in mouse embryonic stem cells that positions the TA and inducible expression cassette to a single location. Using this system, we show dox-dependent regulation of eGFP at the DNA topoisomerase 3β promoter. Because Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.     

Biomass and Carbon Storage in an Age-Sequence of Korean Pine (<Emphasis Type="Italic">Pinus koraiensis</Emphasis>) Plantation Forests in Central Korea

This study examined the biomass and carbon pools of the main ecosystem components in an age sequence of five Korean pine plantation forest stands in central Korea. The C contents in the tree and ground vegetation biomass, coarse woody debris, forest floor, and mineral soil were estimated by analyzing the C concentration of each component. The aboveground and total tree biomass increased with increasing stand age. The highest C concentration across this chronosequence was found in the tree branch while the lowest C concentration was found in the ground vegetation. The observed C contents for tree components, ground vegetation, and coarse woody debris were generally lower than the predicted C contents estimated from a biomass C factor of 0.5. Forest floor C content was age-independent. Total mineral soil C content appeared to decline initially after establishing Korean pine plantations and recover by the stand age of 35 years. Although aboveground tree biomass C content showed considerable accumulation with increasing age, the relative contribution of below ground C to total ecosystem C content varied substantially. These results suggest that successional development as temporal factor has a key role in estimating the C storage in Korean pine plantation forests.