Structural Basis for Auto-Inhibition of the NDR1 Kinase Domain by an Atypically Long Activation Segment

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In vitro studies on mangiferin protection against cadmium-induced human renal endothelial damage and cell death via the MAP kinase and NF-κB pathways

The therapeutic effects of the natural antioxidant mangiferin (a xanthonoid and potent oxygen free radical scavenger), which is widely distributed in mango fruit, against CdCl₂-induced toxicity in human renal glomerulus endothelial cells (HRGEC) were investigated. The viability of HREGCs that were treated with CdCl₂ (25?µ?mol) and co-treated with mangiferin (75?µ?mol) for 24?h was measured by crystal violet dye. The exposure of human glomerulus renal endothelial cells to cadmium promotes a polarized apical secretion of IL-6 and IL-8, two pivotal proinflammatory cytokines known to play a significant role in renal inflammation. Proinflammatory cytokine secretion by human renal glomerulus endothelial cells could be the result of cadmium-induced IL-6 secretion via an NF-κB-dependent pathway. However, IL-8 secretion involves the phosphor-JNK phospho-p38 signaling pathway. The results of the current study reveal that mangiferin could prevent both cadmium-induced IL-6 and IL-8 secretion by human glomerulus endothelial cells and be used to prevent renal inflammation.     

Evaluation of Medium Components by Plackett-Burman Statistical Design for Lipase Production by Candida rugosa and Kinetic Modeling

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3?6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL-1 was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30oC, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, α and β, it was found that the lipase production by Candida rugosa is growth associated.     

A role for endogenous and radiation-induced DNA double-strand breaks in p53-dependent apoptosis during cortical neurogenesis

Prenatal exposure to low-dose radiation increases the risk of microcephaly and/or mental retardation. Microcephaly is also associated with genetic mutations that affect the non-homologous end-joining pathway of DNA double-strand break repair. To examine the link between these two causal factors, we characterized the neural developmental effects of acute radiation exposure in mouse littermate embryos harboring mutations in the Ku70 and p53 genes. Both low-dose radiation exposure and Ku70 deficiency induced morphologically indistinguishable cortical neuronal apoptosis. Irradiated Ku70-deficient embryos displayed anatomical damage indicative of increased radiosensitivity in the developing cerebral cortex. Deleting the p53 gene not only rescued cortical neuronal apoptosis at all levels but also restored the in vitro growth of Ku70-deficient embryonic fibroblasts despite the presence of unrepaired DNA/chromosomal breaks. The results confirm the role of DNA double-strand breaks as a common causative agent of apoptosis in the developing cerebral cortex. Furthermore, the findings suggest a disease mechanism by which the presence of endogenous DNA double-strand breaks in the newly generated cortical neurons becomes radiomimetic when DNA end joining is defective. This in turn activates p53-dependent neuronal apoptosis and leads to microcephaly and mental retardation.     

Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: A case study with isoxanthopterin and its derivatives

Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 × 10⁶ M^?1 and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 × 10⁶ M^?1 and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand–nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1:1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand–nucleotide interaction, which leads to the improved binding affinity.     

Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II

Ca²⁺ influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo . Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca²⁺/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein–α-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione- S -transferase pull-down assay. Thr286-autophosphorylated α-CaMKII or the autophosphorylation mimicking mutant, T286D-α-CaMKII, binds NR2B sequence independent of Ca²⁺/calmodulin unlike native wild-type α-CaMKII. We show enhancement of this binding by Ca²⁺/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca²⁺/calmodulin-independent binding whereas it allows the Ca²⁺/calmodulin-dependent binding of α-CaMKII in vitro . Similarly, the autonomously active mutants, T286D-α-CaMKII and F293E/N294D-α-CaMKII, exhibited Ca²⁺-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.