In silico identification of putative metal binding motifs

Metal ion binding domains are found in proteins that mediate transport, buffering or detoxification of metal ions. In this study, we have performed an in silico analysis of metal binding proteins and have identified putative metal binding motifs for the ions of cadmium, cobalt, zinc, arsenic, mercury, magnesium, manganese, molybdenum and nickel. A pattern search against the UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases yielded true positives in each case showing the high-specificity of the motifs. Motifs were also validated against PDB structures and site directed mutagenesis studies.     

Role of pro-oxidants and antioxidants in the anti-inflammatory and apoptotic effects of curcumin (diferuloylmethane)

Extensive research within the past half-century has indicated that curcumin (diferuloylmethane), a yellow pigment in curry powder, exhibits antioxidant, anti-inflammatory, and proapoptotic activities. We investigated whether the anti-inflammatory and proapoptotic activities assigned to curcumin are mediated through its prooxidant/antioxidant mechanism. We found that TNF-mediated NF-kappaB activation was inhibited by curcumin; and glutathione reversed the inhibition. Similarly, suppression of TNF-induced AKT activation by curcumin was also abrogated by glutathione. The reducing agent also counteracted the inhibitory effects of curcumin on TNF-induced NF-kappaB-regulated antiapoptotic (Bcl-2, Bcl-xL, IAP1), proliferative (cyclin D1), and proinflammatory (COX-2, iNOS, and MMP-9) gene products. The suppression of TNF-induced AP-1 activation by curcumin was also reversed by glutathione. Also, the direct proapoptotic effects of curcumin were inhibited by glutathione and potentiated by depletion of intracellular glutathione by buthionine sulfoximine. Moreover, curcumin induced the production of reactive oxygen species and modulated intracellular GSH levels. Quenchers of hydroxyl radicals, however, were ineffective in inhibiting curcumin-mediated NF-kappaB suppression. Further, N-acetylcysteine partially reversed the effect of curcumin. Based on these results we conclude that curcumin mediates its apoptotic and anti-inflammatory activities through modulation of the redox status of the cell.     

Closing in on the resting state of the Shaker K(+) channel

Membrane depolarization causes voltage-gated ion channels to transition from a resting/closed conformation to an activated/open conformation. We used voltage-clamp fluorometry to measure protein motion at specific regions of the Shaker Kv channel. This enabled us to construct new structural models of the resting/closed and activated/open states based on the Kv1.2 crystal structure using the Rosetta-Membrane method and molecular dynamics simulations. Our models account for the measured gating charge displacement and suggest a molecular mechanism of activation in which the primary voltage sensors, S4s, rotate by approximately 180 degrees as they move "outward" by 6-8 A. A subsequent tilting motion of the S4s and the pore domain helices, S5s, of all four subunits induces a concerted movement of the channel's S4-S5 linkers and S6 helices, allowing ion conduction. Our models are compatible with a wide body of data and resolve apparent contradictions that previously led to several distinct models of voltage sensing.     

Functional analysis of cauliflower mosaic virus 35S promoter: re-evaluation of the role of subdomains B5, B4 and B2 in promoter activity

The cauliflower mosaic virus 35S (35S) promoter is used extensively for transgene expression in plants. The promoter has been delineated into different subdomains based on deletion analysis and gain-of-function studies. However, cis -elements important for promoter activity have been identified only in the domains B1 ( as-2 element), A1 ( as-1 element) and minimal promoter (TATA box). No cis -elements have been described in subdomains B2–B5, although these are reported to be important for the overall activity of the 35S promoter. We have re-evaluated the contribution of three of these subdomains, namely B5, B4 and B2, to 35S promoter activity by developing several modified promoters. The analysis of β-glucuronidase gene expression driven by the modified promoters in different tissues of primary transgenic tobacco lines, as well as in seedlings of the T₁ generation, revealed new facets about the functional organization of the 35S promoter. This study suggests that: (i) the 35S promoter truncated up to –301 functions in a similar manner to the –343 (full-length) 35S promoter; (ii) the Dof core and I-box core observed in the subdomain B4 are important for 35S promoter activity; and (iii) the subdomain B2 is essential for maintaining an appropriate distance between the proximal and distal regions of the 35S promoter. These observations will aid in the development of functional synthetic 35S promoters with decreased sequence homology. Such promoters can be used to drive multiple transgenes without evoking promoter homology-based gene silencing when attempting gene stacking.     

Cwc21p promotes the second step conformation of the spliceosome and modulates 3′ splice site selection

Pre-mRNA splicing involves two transesterification steps catalyzed by the spliceosome. How RNA substrates are positioned in each step and the molecular rearrangements involved, remain obscure. Here, we show that mutations in PRP16, PRP8, SNU114 and the U5 snRNA that affect this process interact genetically with CWC21, that encodes the yeast orthologue of the human SR protein, SRm300/SRRM2. Our microarray analysis shows changes in 3′ splice site selection at elevated temperature in a subset of introns in cwc21Δ cells. Considering all the available data, we propose a role for Cwc21p positioning the 3′ splice site at the transition to the second step conformation of the spliceosome, mediated through its interactions with the U5 snRNP. This suggests a mechanism whereby SRm300/SRRM2, might influence splice site selection in human cells.     

Protein–protein association rates captured in a single geometric parameter

Understanding factors that drive protein–protein association is of fundamental importance. We show that a single geometric parameter in crystal structures of protein–protein complexes, the angle between the electric dipole of one subunit and the partner‐generated electric field at the same subunit, linearly correlates with experimentally determined protein–protein association rates. Imprint of a dynamic kinetic process in a single static geometric parameter, associated with mutual electrostatic orientation of subunits in protein–protein complexes, is elegant and demonstrates the universality of electrostatic steering in attenuating protein–protein association rates. That the essence of a complex phenomenon could be captured by properties of the final crystal structure of the complex implies that the electrostatic orientations of protein subunits in crystal structures and the associated transition states are nearly identical. Further, the cosine of the angle, alone, is shown to be sufficient in predicting association rate constants, with accuracies comparable to currently available predictors that use more intricate methodologies. Our results offer mechanistic insights and could be useful in development of coarse‐grained models. Proteins 2015; 83:1557–1562. © 2015 Wiley Periodicals, Inc.     

Analysis of Pathogenic Diversity of the Rice Bacterial Blight Pathogen (Xanthomonas oryzae pv. oryzae) in the Andaman Islands and Identification of Effective Resistance Genes

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in the tropics for which genetic resistance in the host plants is the only effective solution. This study aimed at identification of resistance gene combinations effective against Xoo isolates and fingerprinting of the Xoo isolates of Andaman Islands (India). Here, we report the reaction of 21 rice BB differentials possessing Xa1 to Xa21 genes individually and in different combinations to various isolates of pathogen collected from Andaman Islands. Pathological screening results of 14 isolates revealed that among individual genes tested across 2 years, Xa4, Xa7 and Xa21 conferred resistance reaction across all isolates, whereas among combinations, IRBB 50 (Xa4 + xa5), IRBB 52 (Xa4 + Xa21) and IRBB 60 (Xa4 + xa5 + xa13 + Xa21) conveyed effective resistance against tested isolates. The nature of genetic diversity among four isolates selected on the basis of geographical isolation in the islands was studied through DNA finger printing. The RAPD primers S111, S119, S1117, S1109, S1103, S109 and S105 were found to be better indicators of molecular diversity among isolates than JEL primers. The diversity analysis grouped 14 isolates into three major clusters based on disease reaction wherein isolate no. 8 was found the most divergent as well as highly virulent. The remaining isolates were classified into two distinct groups. The importance of the study in the context of transfer of resistance gene(s) in the local cultivars specifically for tropical island conditions is presented and discussed.     

Foliar Extrafloral Nectar of Humboldtia brunonis (Fabaceae), a Paleotropic Ant‐plant,is Richer than Phloem Sap and More Attractive than Honeydew

The ant‐plant Humboldtia brunonis secretes extrafloral nectar (EFN) despite the lack of antiherbivore protection from most ants. EFN was richer in composition than phloem sap and honeydew from untended Hemiptera on the plant, suggesting that EFN could potentially distract ants from honeydew, since ants rarely tended Hemiptera on this plant.     

Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit

Cellular senescence is a process wherein proliferating cells undergo permanent cell cycle arrest while remaining viable. Senescence results in enhanced secretion of proteins that promote cancer and inflammation. We report here that the structure of the Golgi complex which regulates secretion is altered in senescent cells. In cells where senescence is achieved by replicative exhaustion or in cells wherein senescence has been induced with BrdU treatment dependent stress, the Golgi complex is dispersed. The expression of a G protein γ subunit, γ11, capable of translocation from the plasma membrane to the Golgi complex on receptor activation increases with senescence. Knockdown of γ11 or overexpression of a dominant negative γ3 subunit inhibits Golgi dispersal induced by senescence. Overall these results suggest that in cellular senescence an upregulated G protein gamma subunit mediates alterations in the structure of the Golgi.     

Synthesis, biological evaluation and molecular docking studies of stellatin derivatives as cyclooxygenase (COX-1, COX-2) inhibitors and anti-inflammatory agents

Stellatin (4), isolated from Dysophylla stellata is a cyclooxygenase (COX) inhibitor. The present study reports the synthesis and biological evaluation of new stellatin derivatives for COX-1, COX-2 inhibitory and anti-inflammatory activities. Eight derivatives showed more pronounced COX-2 inhibition than stellatin and, 17 and 21 exhibited the highest COX-2 inhibition. They also exhibited the significant anti-inflammatory activity in TPA-induced mouse ear edema assay and their anti-inflammatory effects were more than that of stellatin and indomethacin at 0.5 mg/ear. The derivatives were further evaluated for antioxidant activity wherein 16 and 17 showed potent free radical scavenging activity against DPPH and ABTS radicals. Molecular docking study revealed the binding orientations of stellatin and its derivatives into the active sites of COX-1 and COX-2 and thereby helps to design the potent inhibitors.