Suppression of branches in Eucalyptus trees

The effect of neem oil, which acts as a suckericide in tobacco, on branch suppression in Eucalyptus tereticornis was assessed to help maximize stem biomass. Lateral branches of selected trees were pruned, and neem oil solutions at concentrations of either 80%, 40%, 20%, 10%, or 0% (untreated control) were applied to leaf axils of the pruned branches. Regeneration of branches was suppressed, and the magnitude of suppression was proportional to the concentration of neem oil. Compared to the control, the percentage reduction in branching at 80% neem oil was 41.6%. When regenerated branches were repruned and neem oil applied at either 100%, 80%, or 0% (control), the regenerating ability of these branches was severely repressed by 78% at 100% neem oil relative to the control. Apical shoots were also topped and treated at either 100% or 0% (control) neem oil to identify the principal suppressive component in neem oil. The principal component azadirachtin was tested at 375, 750, 1500, 3125, 6250, 12?500, 25?000, 50?000, and 100?000 ppm and 0 ppm as the control. Reduction in the coppicing shoot was as high as 85%. Azadirachtin was responsible for the suppression. By pruning the lateral branches with neem oil, wasteful consumption of photosynthates can be precluded and the stem biomass maximized.     

Designing of enzyme inhibitors based on active site specificity: lessons from methyl gallate and its lipoxygenase inhibitory profile

Methyl gallate was purified, by lipoxygenase (LOX) inhibitory activity-guided method since its alleged anti-inflammatory property, from Bergenia ligulata (Wall), a plant used in the traditional, Ayurvedic system of medicine extensively. The LOX inhibitory property of methyl gallate was studied by enzyme kinetics, isothermal titration calorimetry and molecular docking followed by molecular simulation studies. The wet-laboratory experiments and in silico studies showed complete agreement, and promise of methyl gallate as a drug-lead molecular scaffold for anti-inflammatory therapy, based on LOX inhibition. The expressed work shows the need of nonactive site binding parameters to be considered while designing of inhibitors based on the specificities toward active sites of enzymes.     

Mapping ERK2-MKP3 binding interfaces by hydrogen/deuterium exchange mass spectrometry

ERK2, a prototypic member of the MAPK family, plays a central role in regulating cell growth and differentiation. MKP3, an ERK2-specific phosphatase, terminates ERK2 signaling. To understand the molecular basis of ERK2 recognition by MKP3, we carried out hydrogen/deuterium exchange mass spectrometry experiments to map the interaction surfaces between the two proteins. The results show that the exquisite specificity of MKP3 for ERK2 is governed by two distinctive protein-protein interactions. To increase the "effective concentration" of the interacting molecules, the kinase interaction motif in MKP3 ((64)RRLQKGNLPVR(74)) and an MKP3-specific segment ((101)NSSDWNE(107)) bind the common docking site in ERK2 defined by residues in L(16), L(5), beta(7)-beta(8), and alpha(d)-L(8)-alpha(e), located opposite the kinase active site. In addition to this "tethering" effect, additional interactions between the (364)FTAP(367) sequence in MKP3 and the ERK2 substrate-binding site, formed by residues in the activation lip and the P+1 site (beta(9)-alpha(f) loop), L(13) (alpha(f)-alpha(g) loop), and the MAPK insert (L(14)-alpha(1L14)-alpha(2L14)), are essential for allosteric activation of MKP3 and formation of a productive complex whereby the MKP3 catalytic site is correctly juxtaposed to carry out the dephosphorylation of phospho-Thr(183)/phospho-Tyr(185) in ERK2. This bipartite protein-protein interaction model may be applicable to the recognition of other MAPKs by their cognate regulators and substrates.     

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors^1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model⁴ that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined⁵. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3''-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAF^V600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis⁶ and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.     

Isozymes inhibited by active site blocking: versatility of calcium indifferent hesperidin binding to phospholipase A2 and its significance

sPLA₂ is released under inflammatory conditions from neutrophils, basophils and T-cells. They cleave the cellular phospholipids leading to the release of arachidonic acid and there by provide intermediates for biosynthesis of inflammatory mediators. The focus of this study is on the interaction of hesperidin, a natural flavonoid with Group IB, IIA, and V and X isozymes of sPLA₂. Affinity of hesperidin towards PLA₂ isozymes was analyzed through enzymatic studies and molecular modeling. The experiments showed that hesperidin competitively inhibited PLA₂ with IC₅₀ of 5.1?µM. Molecular modeling studies revealed the association of hesperidin with the docking scores ?6.90, ?9.53, ?5.63 and ?8.29?kcal for isozymes Group IB, IIA, V and X of PLA₂ respectively. Their binding energy values were calculated as ?20.25, ?21.63, ?21.66 and ?33.43?kcal for the Group IB, IIA, V and X respectively. Structural model for Group V was made by homology modeling since no structural coordinates were available. Molecular dynamics studies were carried out to evaluate the structural stability of protein ligand complex. The analyses showed that hesperidin blocked the entry of the substrate to the active site of PLA₂ and it was indifferent to the differences of the isozymes. Hence, hesperidin might serve as lead for designing highly specific anti-inflammatory drugs directed to the PLA₂ isozyme specific to various diseases, with IC₅₀ value of therapeutic significance.     

Modulation of SF3B1 in the pre-mRNA spliceosome induces a RIG-I-dependent type I IFN response

Nucleic acid–sensing pathways play critical roles in innate immune activation through the production of type I interferon (IFN-I) and proinflammatory cytokines. These factors are required for effective antitumor immune responses. Pharmacological modulators of the pre-mRNA spliceosome splicing factor 3b subunit 1 (SF3B1) are under clinical investigation as cancer cytotoxic agents. However, potential roles of these agents in aberrant RNA generation and subsequent RNA-sensing pathway activation have not been studied. In this study, we observed that SF3B1 pharmacological modulation using pladienolide B (Plad B) induces production of aberrant RNA species and robust IFN-I responses via engagement of the dsRNA sensor retinoic acid–inducible gene I (RIG-I) and downstream interferon regulatory factor 3. We found that Plad B synergized with canonical RIG-I agonism to induce the IFN-I response. In addition, Plad B induced NF-κB responses and secretion of proinflammatory cytokines and chemokines. Finally, we showed that cancer cells bearing the hotspot SF3B1^K700E mutation, which leads to global aberrant splicing, had enhanced IFN-I response to canonical RIG-I agonism. Together, these results demonstrate that pharmacological modulation of SF3B1 in cancer cells can induce an enhanced IFN-I response dependent on RIG-I expression. The study suggests that spliceosome modulation may not only induce direct cancer cell cytotoxicity but also initiate an innate immune response via activation of RNA-sensing pathways.