Controlling plant growth via the gibberellin biosynthesis system – I. Growth parameter alterations in apple seedlings

'York Imperial' apple seedlings ( Malus domestica Borkh.) were continuously supplied via the roots with paclobutrazol [(2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol)], a triazole GA biosynthesis inhibitor, at 0.68 μ M in a nutrient solution. In comparison to controls, seedlings treated with paclobutrazol for 66 days showed a 91% reduction in shoot length, a 66% reduction in leaf area but only a 17% reduction in leaf number. This effect could be reversed by GA₃ applied to the foliage at 71.4 μ M 0, 19 or 35 days after paclobutrazol was initially supplied and leaf area values for paclobutrazol-treated seedlings given both treatments did not differ significantly from controls. Plots of growth data indicate linearity of shoot longitudinal growth of GA₃-treated seedlings. Leaf area increase was non-linear after GA₃ treatment up to approximately 30 days, when the rate dropped. On a per shoot basis, leaf weight closely followed leaf area but on a per unit area basis, paclobutrazol-treated leaves were heavier than controls; GA₃ applications temporarily reversed this trend.     

Affinity enhancement of bispecific antibody against two different epitopes in the same antigen.

For the enhancement of antibody binding affinity, a bispecific antibody against two different epitopes in human chorionic gonadotropin hormone, one is in alpha-subunit and the other is in beta-subunit, was prepared by chemical recombination using 5,5'-dithiobis(2-nitrobenzoic acid). The epitopes recognized by antibodies were investigated by competitive radioimmunoassay, two-site sandwich radioimmunoassay and additivity assay and a proper epitope pair was chosen for preparation of the bispecific antibody. This bispecific antibody has dual specificity and as much as 17.2-fold higher affinity than that of monoclonal antibody with higher affinity by dual antigen binding radioimmunoassay and Scatchard plot analysis.     

Evidence for involvement of 2 histidine residues in the reaction of ampicillin acylase

The chemical modification of purified ampicillin acylase by N-bromosuccinimide and diethylpyrocarbonate resulted in time-dependent inactivation of the enzyme. Both substrates, ampicillin and 6-aminopenicillanic acid, protected the enzyme against inactivation, suggesting that the modification occurred near or at the active site. Amino acid analyses and other data indicated that two histidyl residues per subunit molecule were essential for catalytic activity.     

Slit2 Inactivates GSK3β to Signal Neurite Outgrowth Inhibition

Slit molecules comprise one of the four canonical families of axon guidance cues that steer the growth cone in the developing nervous system. Apart from their role in axon pathfinding, emerging lines of evidence suggest that a wide range of cellular processes are regulated by Slit, ranging from branch formation and fasciculation during neurite outgrowth to tumor progression and to angiogenesis. However, the molecular and cellular mechanisms downstream of Slit remain largely unknown, in part, because of a lack of a readily manipulatable system that produces easily identifiable traits in response to Slit. The present study demonstrates the feasibility of using the cell line CAD as an assay system to dissect the signaling pathways triggered by Slit. Here, we show that CAD cells express receptors for Slit (Robo1 and Robo2) and that CAD cells respond to nanomolar concentrations of Slit2 by markedly decelerating the rate of process extension. Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth. Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons. Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.     

Revisiting the Role of Conductivity and Polarity of Host Materials for Long‐Life Lithium–Sulfur Battery

Despite their high theoretical energy density and low cost, lithium–sulfur batteries (LSBs) suffer from poor cycle life and low energy efficiency owing to the polysulfides shuttle and the electronic insulating nature of sulfur. Conductivity and polarity are two critical parameters for the search of optimal sulfur host materials. However, their role in immobilizing polysulfides and enhancing redox kinetics for long‐life LSBs are not fully understood. This work has conducted an evaluation on the role of polarity over conductivity by using a polar but nonconductive platelet ordered mesoporous silica (pOMS) and its replica platelet ordered mesoporous carbon (pOMC), which is conductive but nonpolar. It is found that the polar pOMS/S cathode with a sulfur mass fraction of 80 wt% demonstrates outstanding long‐term cycle stability for 2000 cycles even at a high current density of 2C. Furthermore, the pOMS/S cathode with a high sulfur loading of 6.5 mg cm^?2 illustrates high areal and volumetric capacities with high capacity retention. Complementary physical and electrochemical probes clearly show that surface polarity and structure are more dominant factors for sulfur utilization efficiency and long‐life, while the conductivity can be compensated by the conductive agent involved as a required electrode material during electrode preparation. The present findings shed new light on the design principles of sulfur hosts towards long‐life and highly efficient LSBs.     

SARNP,a participant in mRNA splicing and export,negatively regulates E-cadherin expression via interaction with pinin

Triple-negative breast cancer (TNBC) is associated with a high mortality rate, which is related to the insufficient number of appropriate biomarkers and targets. Therefore, there is an urgent need to discover appropriate biomarkers and targets for TNBC. SARNP (Hcc-1 and CIP29) is highly expressed in several cancers. It binds to UAP56, an RNA helicase component of the TREX complex in messenger RNA (mRNA) splicing and export. However, the role of SARNP in mRNA splicing and export and in the progression of breast cancer, especially of TNBC, remains unknown. Therefore, we examined the role of SARNP in mRNA splicing and export and progression of TNBC. We confirmed that SARNP binds to UAP56 and Aly and that SARNP overexpression enhances mRNA splicing, whereas its knockdown suppressed mRNA export. The SARNP overexpression induced the proliferation of MCF7 cells, whereas its knockdown induced E-cadherin expression and downregulated vimentin and N-cadherin expressions in SK-BR-3 and MDA-MB-231 cells. SARNP downregulates E-cadherin expression by interaction with pinin. Mice injected with MDA-MB-231_shSARNP cells exhibited a significant reduction in tumor growth and lung metastasis compared with those injected with MDA-MB-231_shCon cells in vivo. These findings suggested that SARNP is involved in mRNA splicing and export. SARNP maintains mesenchymal phenotype by escaping from inhibitory interaction with pinin leading to the downregulation of E-cadherin expression.     

Structural and Functional Analysis of the Natural JNK1 Inhibitor Quercetagetin

c-Jun NH2-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3-K) play critical roles in chronic diseases such as cancer, type II diabetes, and obesity. We describe here the binding of quercetagetin (3,3′,4′,5,6,7-hydroxyflavone), related flavonoids, and SP600125 to JNK1 and PI3-K by ATP-competitive and immobilized metal ion affinity-based fluorescence polarization assays and measure the effect of quercetagetin on JNK1 and PI3-K activities. Quercetagetin attenuated the phosphorylation of c-Jun and AKT, suppressed AP-1 and NF-κB promoter activities, and also reduced cell transformation. It attenuated tumor incidence and reduced tumor volumes in a two-stage skin carcinogenesis mouse model.Our crystallographic structure determination data show that quercetagetin binds to the ATP-binding site of JNK1. Notably, the interaction between Lys55, Asp169, and Glu73 of JNK1 and the catechol moiety of quercetagetin reorients the N-terminal lobe of JNK1, thereby improving compatibility of the ligand with its binding site. The results of a theoretical docking study suggest a binding mode of PI3-K with the hydroxyl groups of the catechol moiety forming hydrogen bonds with the side chains of Asp964 and Asp841 in the p110γ catalytic subunit. These interactions could contribute to the high inhibitory activity of quercetagetin against PI3-K. Our study suggests the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases.     

Neuroprotective effect of Capsicum annuum var. abbreviatum against hydrogen peroxide-induced oxidative stress in HT22 hippocampus cells

ABSTRACT

Phenolic compounds isolated from pepper (Capsicum annum) have been demonstrated to have neuroprotective effects, whereas the physiological properties of Capsicum annuum var. abbreviatum (CAA) have not been studied. Thus, we investigate the chemical composition and neuroprotective activity of CAA extract (CAAE) in HT22 hippocampus cells against H₂O₂-induced neurotoxicity. CAAE treatment resulted in a significant protection of H₂O₂-exposed HT22, this protection ultimately occurred through an inhibition of MDA and ROS levels and an induction of SOD activity. Furthermore, CAAE treatment reduced H₂0₂-induced apoptosis though decreasing the expression of pro-apoptotic factors (Bax, cytochrome c, and cleaved caspases-3) while increasing the expression of the anti-apoptotic factors (Bcl-2), as well as the accumulation of nucleus-Nrf2-mediated HO-1 signaling. Interestingly, CAAE has a high concentration of unique phenolic compositions (chlrogenic acid, tangeretin, etc.) than other capsicum annum extracts. Altogether, these findings suggest that CAAE can be a useful natural resource for alleviating neurodegenerative diseases.     

Antitumor effects of MutT homolog 1 inhibitors in human bladder cancer cells

ABSTRACT

As standard second-line regimen has not been established for patients who are refractory to or relapse with cisplatin-based chemotherapy, an effective class of novel chemotherapeutic agents is needed for cisplatin-resistant bladder cancer. Recent publications reported that MutT homolog 1 (MTH1) inhibitors suppress tumor growth and induce impressive therapeutic responses in a variety of human cancer cells. Few studies investigated the cytotoxic effects of MTH1 inhibitors in human bladder cancer. Accordingly, we investigated the antitumor effects and the possible molecular mechanisms of MTH1 inhibitors in cisplatin-sensitive (T24) and – resistant (T24R2) human bladder cancer cell lines. These results suggest that TH588 or TH287 may induce cancer cell suppression by off-target effects such as alterations in the expression of apoptosis- and cell cycle-related proteins rather than MTH1 inhibition in cisplatin-sensitive and – resistant bladder cancer cells.

Abbreviations: MTH: MutT homolog; ROS: reactive oxygen species; CCK-8: cell counting kit-8; DCFH-DA: dichlorofluorescein diacetate; PARP: poly (ADP-ribose) polymerase     

Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken

Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white. [BMB Reports 2013; 46(8): 404-409]