Structure and rearrangements in the carboxy-terminal region of SpIH channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels regulate the spontaneous firing activity and electrical excitability of many cardiac and neuronal cells. The modulation of HCN channel opening by the direct binding of cAMP underlies many physiological processes such as the autonomic regulation of the heart rate. Here we use a combination of X-ray crystallography and electrophysiology to study the allosteric mechanism for cAMP modulation of HCN channels. SpIH is an invertebrate HCN channel that is activated fully by cAMP, but only partially by cGMP. We exploited the partial agonist action of cGMP on SpIH to reveal the molecular mechanism for cGMP specificity of many cyclic nucleotide-regulated enzymes. Our results also elaborate a mechanism for the allosteric conformational change in the cyclic nucleotide-binding domain and a mechanism for partial agonist action. These mechanisms will likely extend to other cyclic nucleotide-regulated channels and enzymes as well.     

COX-2 expression in cystic kidneys is dependent on dietary n-3 fatty acid composition

Dietary n-3 fatty acids generally attenuate elevated cyclooxygenase-2 (COX-2) levels in disease states. However, models of renal cystic disease (RCD) exhibit reduced renal COX-2 expression. Therefore, the in vivo regulation of COX-2 expression by dietary n-3 fatty acids was examined. In archived tissues from dietary studies, COX-2 protein and gene expression was up-regulated in diseased pcy mouse and Han:SPRD-cy rat kidneys when given diets containing eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), but not those containing -linolenic acid (ALA), compared to control diets with linoleic acid (LA). The presence of disease was necessary to elicit these effects as COX-2 expression was unaltered by diet in normal kidneys. The effects were specific for COX-2, since COX-1 levels were unaltered by these dietary manipulations in either model. Thus, in RCD, diets containing EPA and DHA but not ALA appear to specifically up-regulate renal COX-2 gene and protein levels in vivo.     

Analysing diversity among Indian isolates of Anabaena (Nostocales, Cyanophyta) using morphological, physiological and biochemical characters

A set of 24 strains belonging to the genus Anabaena (Phylum Cyanobacteria), isolated from diverse geographic locations in India, were evaluated along with three International type strains of Anabaena (ATCC 29414, ATCC 29208 and ATCC 27899) for their morphological, physiological and biochemical diversity. The morphological dataset, consisting of 58 variants for 15 characters, and SDS-PAGE protein profiles comprising 17 polymorphic bands were utilized to differentiate the selected Anabaena strains and explore the patterns of diversity through cluster analysis. Physiological and biochemical characterization with respect to nitrogen fixation and accumulation of chlorophyll and phycobiliproteins led to the identification of some highly promising Anabaena strains for use as biofertilizers and source of pigments. The study highlighted the tremendous inter and intraspecific diversity within the Anabaena isolates and indicated the potential as well as constraints of the morphological and protein profiling datasets for unambiguous differentiation and analyses of diversity among the Anabaena strains.     

Quantifying and Modeling the Influence of Temperature on Growth and Reproductive Development of Sesame

Ambient temperatures are major factors regulating the growth rates, yields, and geographical distribution of crop species. The cultivation of sesame (Sesamum indicum L.) is expanding with the rising demand in regions where it is not traditionally grown, and sub-optimal yields due to extremely low or high temperatures could occur. Currently literature lacks information on the temperature responses of sesame growth. An experiment was conducted to quantify the effects of different temperatures on vegetative growth and reproductive development of sesame, and to estimate its cardinal temperature limits (T_opt; T_min; T_max). Plants were subjected to six different day/night temperature treatments of 40/32, 36/28, 32/24, 28/20, and 20/12 °C using walk-in growth chambers. Vegetative growth of sesame was sensitive to low temperatures (<?15 °C), but tolerant of high temperatures. The cardinal temperature limits of 15.7 °C (T_min), 27.3 °C (T_opt), and 44.6 °C (T_max) were observed for rate of biomass accumulation. Sesame reached the flowering stage under moderate to high temperature conditions; however, reproductive yields progressively declined above 25 °C, and no seed yields were obtained beyond 33 °C. The estimated temperature limits could be employed to develop crop models for simulating management and adaptation strategies of sesame under current and future climate scenarios, and adaptation to regions where the crop is not currently grown. Future research should focus on understanding factors controlling the temperature tolerance of reproductive development in sesame, to provide a broader geographical adaptation.

     

Structurally Distinct Ligands Rescue Biogenesis Defects of the KATP Channel Complex via a Converging Mechanism

Small molecules that correct protein misfolding and misprocessing defects offer a potential therapy for numerous human diseases. However, mechanisms underlying pharmacological correction of such defects, especially in heteromeric complexes with structurally diverse constituent proteins, are not well understood. Here we investigate how two chemically distinct compounds, glibenclamide and carbamazepine, correct biogenesis defects in ATP-sensitive potassium (K_ATP) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2. We present evidence that despite structural differences, carbamazepine and glibenclamide compete for binding to K_ATP channels, and both drugs share a binding pocket in SUR1 to exert their effects. Moreover, both compounds engage Kir6.2, in particular the distal N terminus of Kir6.2, which is involved in normal channel biogenesis, for their chaperoning effects on SUR1 mutants. Conversely, both drugs can correct channel biogenesis defects caused by Kir6.2 mutations in a SUR1-dependent manner. Using an unnatural, photocross-linkable amino acid, azidophenylalanine, genetically encoded in Kir6.2, we demonstrate in living cells that both drugs promote interactions between the distal N terminus of Kir6.2 and SUR1. These findings reveal a converging pharmacological chaperoning mechanism wherein glibenclamide and carbamazepine stabilize the heteromeric subunit interface critical for channel biogenesis to overcome defective biogenesis caused by mutations in individual subunits.     

Genomic prediction in biparental tropical maize populations in water-stressed and well-watered environments using low-density and GBS SNPs

One of the most important applications of genomic selection in maize breeding is to predict and identify the best untested lines from biparental populations, when the training and validation sets are derived from the same cross. Nineteen tropical maize biparental populations evaluated in multienvironment trials were used in this study to assess prediction accuracy of different quantitative traits using low-density (~200 markers) and genotyping-by-sequencing (GBS) single-nucleotide polymorphisms (SNPs), respectively. An extension of the Genomic Best Linear Unbiased Predictor that incorporates genotype × environment (GE) interaction was used to predict genotypic values; cross-validation methods were applied to quantify prediction accuracy. Our results showed that: (1) low-density SNPs (~200 markers) were largely sufficient to get good prediction in biparental maize populations for simple traits with moderate-to-high heritability, but GBS outperformed low-density SNPs for complex traits and simple traits evaluated under stress conditions with low-to-moderate heritability; (2) heritability and genetic architecture of target traits affected prediction performance, prediction accuracy of complex traits (grain yield) were consistently lower than those of simple traits (anthesis date and plant height) and prediction accuracy under stress conditions was consistently lower and more variable than under well-watered conditions for all the target traits because of their poor heritability under stress conditions; and (3) the prediction accuracy of GE models was found to be superior to that of non-GE models for complex traits and marginal for simple traits.     

Identification and characterization of endoglucanases for fungicidal activity in <Emphasis Type="Italic">Anabaena laxa</Emphasis> (Cyanobacteria)

A cyanobacterial strain (Anabaena laxa RPAN8) exhibiting fungicidal activity and β-1,3 and 1,4 endoglucanase activities was selected for identifying the gene(s) involved. Functional analyses of the genomic library revealed that four clones (8, 64, 116, and 248) of RPAN8 exhibited fungicidal activity and induced structural deformities in the cell wall of the growing mycelia of Pythium aphanidermatum. Higher expression of fungicidal and β-1,4 endoglucanase activities, along with low expression of β-1,3 endoglucanase activity, was recorded in two E. coli clones (8 and 64). Clones 8 and 64 exhibited identical sequences while clones 116 and 248 were also similar. Bioinformatic analyses were undertaken only for the two non-identical clones 8 and 116 which showed open reading frames (ORFs) of 348 (end 1) and 656 amino acid residues (end 2), respectively. The amino acid sequence analyses revealed that the end 1 encoding endoglucanases belonged to peptidase M20 family while end 2 showed significant similarities with several known genes. The putative promoters and ribosomal binding sites were identified and amino acid exchanges were observed in both end 1 and 2. The presence of signal peptides of 24 and 20 amino acid residues respectively revealed the secretory nature of these proteins.     

Vimentin organization modulates the formation of lamellipodia

Vimentin intermediate filaments (VIF) extend throughout the rear and perinuclear regions of migrating fibroblasts, but only nonfilamentous vimentin particles are present in lamellipodial regions. In contrast, VIF networks extend to the entire cell periphery in serum-starved or nonmotile fibroblasts. Upon serum addition or activation of Rac1, VIF are rapidly phosphorylated at Ser-38, a p21-activated kinase phosphorylation site. This phosphorylation of vimentin is coincident with VIF disassembly at and retraction from the cell surface where lamellipodia form. Furthermore, local induction of photoactivatable Rac1 or the microinjection of a vimentin mimetic peptide (2B2) disassemble VIF at sites where lamellipodia subsequently form. When vimentin organization is disrupted by a dominant-negative mutant or by silencing, there is a loss of polarity, as evidenced by the formation of lamellipodia encircling the entire cell, as well as reduced cell motility. These findings demonstrate an antagonistic relationship between VIF and the formation of lamellipodia.