Regulation of ΔNp63α by NFκB

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be downregulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NFκB in presence of cisplatin. We find that NFκB promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NFκB leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NFκB with siRNA-mediated silencing NFκB expression attenuates chemotherapy induced degradation of ΔNp63α. These data demonstrate that NFκB plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NFκB may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.Key words: ΔNp63α, NFκB, ubiquitination, cisplatin, head and neck cancer     

Control of Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici using leaf extract of Piper betle L.: a preliminary study

The main objective of this study was to evaluate the effectiveness of crude chloroform extract of Piper betle L. (PbC) in controlling Fusarium wilt of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. lycopersici. It was observed that 1% (w/w) amendment of the PbC in soil was more efficient in reducing the Fusarium population in soil than carbendazim and the combined amendment of carbendazim and PbC. Fusarium wilt control studies were carried out in a greenhouse. Variation in different parameters like shoot growth, root growth and mean fresh weights of tomato seedlings in all the treatments were recorded. Accumulation of total phenolics was also studied from the root tissues of tomato. Higher accumulation of total phenolics was observed in the Fusarium-infested plants as compared to that of healthy control and PbC-treated plants. Moreover, it was observed that the extract could reduce the symptoms and disease development. Electron microscopy studies were also done to observe the Fusarium infestation in the vascular bundles and to show the accumulation of total phenolics in the vacuoles of root tissue.     

SlHyPRP1 and DEA1, the multiple stress responsive eight-cysteine motif family genes of tomato (Solanum lycopersicum L.) are expressed tissue specifically,localize and interact at cytoplasm and plasma membrane in vivo

Owing to rapid global climate change, the occurrence of multiple abiotic stresses is known to influence the outburst of biotic stress factors which affects crop productivity. Therefore, it is essential to understand the molecular and cell biology of key genes associated with multiple stress responses in crop plants. SlHyPRP1 and DEA1, the members of eight-cysteine motif (8CM) family genes have been recently identified as putative regulators of multiple stress responses in tomato (Solanum lycopersicum L.). In order to gain deeper insight into cell and molecular biology of SlHyPRP1 and DEA1, we performed their expression analysis in three tomato cultivars and in vivo cell biological analysis. The semi-quantitative PCR and qRT-PCR results showed the higher expression of SlHyPRP1 and DEA1 in leaf, stem, flower and root tissues as compared to fruit and seed tissues in all three cultivars. The expression levels of SlHyPRP1 and DEA1 were found to be relatively higher in a wilt susceptible tomato cultivar (Arka Vikas) than a multiple disease resistant cultivar (Arka Abhed). In vivo cell biological analysis through Gateway cloning and Bi-FC assay revealed the predominant sub-cellular localization and strong protein–protein interaction of SlHyPRP1 and DEA1 at the cytoplasm and plasma membrane. Moreover, SlHyPRP1 showed in vivo interaction with stress responsive proteins WRKY3 and MST1. Our findings suggest that SlHyPRP1 with DEA1 are co-expressed with tissue specificity and might function together by association with WRKY3 and MST1 in plasma membrane for regulating multiple stress responses in the tomato plant.Electronic supplementary materialThe online version of this article (10.1007/s12298-020-00913-z) contains supplementary material, which is available to authorized users.Keyword: Eight-cysteine motif, Hybrid proline rich proteins, Multiple stresses, Tissue specific expression, Plasma membrane, Protein-protein interaction     

Phosphatidylserine and phosphatidylethanolamine regulate the structure and function of FVIIa and its interaction with soluble tissue factor

Cell membranes have important functions in many steps of the blood coagulation cascade, including the activation of factor X (FX) by the factor VIIa (FVIIa)-tissue factor (TF) complex (extrinsic X_ase). FVIIa shares structural similarity with factor IXa (FIXa) and FXa. FIXa and FXa are regulated by binding to phosphatidylserine (PS)-containing membranes via their γ-carboxyglutamic acid-rich domain (Gla) and epidermal growth-factor (EGF) domains. Although FVIIa also has a Gla-rich region, its affinity for PS-containing membranes is much lower compared with that of FIXa and FXa. Research suggests that a more common endothelial cell lipid, phosphatidylethanolamine (PE), might augment the contribution of PS in FVIIa membrane-binding and proteolytic activity. We used soluble forms of PS and PE (1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), 1,2-dicaproyl-sn-glycero-3-phospho-ethanolamine (C6PE)) to test the hypothesis that the two lipids bind to FVIIa jointly to promote FVIIa membrane binding and proteolytic activity. By equilibrium dialysis and tryptophan fluorescence, we found two sites on FVIIa that bound equally to C6PE and C6PS with K_d of ∼ 150–160 μM, however, deletion of Gla domain reduced the binding affinity. Binding of lipids occurred with greater affinity (K_d∼70–80 μM) when monitored by FVIIa proteolytic activity. Global fitting of all datasets indicated independent binding of two molecules of each lipid. The proteolytic activity of FVIIa increased by ∼50–100-fold in the presence of soluble TF (sTF) plus C6PS/C6PE. However, the proteolytic activity of Gla-deleted FVIIa in the presence of sTF was reduced drastically, suggesting the importance of Gla domain to maintain full proteolytic activity.     

Adsorption of Eu3+ and Am3+ ion towards hard donor-based diglycolamic acid-functionalised carbon nanotubes: density functional theory guided experimental verification

Structure, bonding, energetic and thermodynamic parameters of trivalent Eu³⁺ and Am³⁺ with diglycolamic acid-functionalised carbon nanotubes (CNT–DGA) in gas and solvent phases are reported in order to understand their complexation and extraction behaviour. The calculation was performed with generalised gradient approximated BP86 density functional and hybrid B3LYP functional using SVP/TZVP basis set. The free energy of extraction, ΔG_ext, of Eu³⁺ and Am³⁺ was computed using the Born–Haber thermodynamic cycle in conjunction with conductor-like screening model solvation approach. The calculated free energy of extraction was found to be exergonic using an explicit cluster water model for hydrating the ions and it was found to be higher for Eu³⁺ ion over Am³⁺ ion. From the estimated distribution constant using synthesised CNT–DGA, it is indisputably established that the Eu³⁺ ion is preferentially extracted over Am³⁺ ion and hence confirms the acceptance of the explicit cluster model for ion solvation free energy and thermodynamic cycle for the evaluation of free energy of extraction in solution phase. This is perhaps the first of its kind that a combined theoretical and experimental study has been performed on the free energy of extraction of Eu³⁺ and Am³⁺ using CNT–DGA.     

Dopamine Oxidation Products Inhibit Na+, K+-ATPase Activity in Crude Synaptosomal–mitochondrial Fraction from Rat Brain

The diverse damaging effects of dopamine (DA) oxidation products on brain subcellular components including mitochondrial electron transport chain have been implicated in dopaminergic neuronal death in Parkinson's disease. It has been shown in this study that DA (50–200?μM) causes dose-dependent inhibition of Na⁺, K⁺-ATPase activity of rat brain crude synaptosomal–mitochondrial fraction during in vitro incubation up to 2?h. The enzyme inactivation is prevented by catalase and the metal-chelator (diethylenetriamine penta-acetic acid) but not by superoxide dismutase or hydroxyl-radical scavengers like mannitol and dimethylsulphoxide (DMSO). Further, reduced glutathione and cysteine, markedly prevent DA-mediated inactivation of Na⁺, K⁺-ATPase. Under similar conditions of incubation, DA (200?μM) leads to the formation of quinoprotein adducts (protein-cysteinyl catechol) with synaptosomal–mitochondrial proteins and the phenomenon is also prevented by glutathione (5?mM) or cysteine (5?mM).

The available data imply that the inactivation of Na⁺, K⁺-ATPase in this system involves both H²O² and metal ions. The reactive quinones by forming adducts with protein thiols also probably contribute to the process, since reduced glutathione and cysteine which scavenge quinones from the system protect Na⁺, K⁺-ATPase from DA-mediated damage. The inactivation of neuronal Na⁺, K⁺-ATPase by DA may give rise to various toxic sequelae with potential implications for dopaminergic cell death in Parkinson's disease.     

Tissue culture propagation of running buffalo clover (Trifolium stoloniferum Muhl. ex A. Eaton)

Rapid propagation of running buffalo clover (Trifolium stoloniferum) was achieved on Murashige & Skoog (MS) medium. Excellent shoot proliferation and shoot growth were obtained on medium containing 0.5 or 1 mg l^-1 BA. In vitro proliferated shoots were rooted on MS or half-strength MS medium containing 0 to 0.4 mg l^-1 IAA. Both the number of roots initiated and the length of the longest root were significantly higher on MS medium than on half-strength MS medium. Rooted plantlets were successfully transferred to soil.