Molecular stacking of two codon-modified genes encoding Bt insecticidal proteins,Cry1AcF and Cry2Aa for management of resistance development in Helicoverpa armigera

Journal of Plant Biochemistry and Biotechnology - Concurrent expression of multiple insecticidal toxins as pyramided genes in the same host plant is one of the tangible strategies to delay the...     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

Ran-dependent docking of importin-beta to RanBP2/Nup358 filaments is essential for protein import and cell viability

RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)-mediated protein import, nuclear export signal-mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)-importin-β, accumulation of importin-β at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP-importin-β complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.     

Balancing sampling and specialization: an adaptationist model of incremental development

Development is typically a constructive process, in which phenotypes incrementally adapt to local ecologies. Here, we present a novel model in which natural selection shapes developmental systems based on the evolutionary ecology, and these systems adaptively guide phenotypic development. We assume that phenotypic construction is incremental and trades off with sampling cues to the environmental state. We computed the optimal developmental programmes across a range of evolutionary ecological conditions. Using these programmes, we simulated distributions of mature phenotypes. Our results show that organisms sample the environment most extensively when cues are moderately, not highly, informative. When the developmental programme relies heavily on sampling, individuals transition from sampling to specialization at different times in ontogeny, depending on the consistency of their sampled cue set; this finding suggests that stochastic sampling may result in individual differences in plasticity itself. In addition, we find that different selection pressures may favour similar developmental mechanisms, and that organisms may incorrectly calibrate development despite stable ontogenetic environments. We hope our model will stimulate adaptationist research on the constructive processes guiding development.     

Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect

The excluded volume effect (EVE) rules all life processes. It is created by macromolecules that occupy a given volume thereby confining other molecules to the remaining space with large consequences on reaction kinetics and molecular assembly. Implementing EVE in fibroblast culture accelerated conversion of procollagen to collagen by procollagen C-proteinase (PCP/BMP-1) and proteolytic modification of its allosteric regulator, PCOLCE1. This led to a 20-30- and 3-6-fold increased collagen deposition in two- and three-dimensional cultures, respectively, and creation of crosslinked collagen footprints beneath cells. Important parameters correlating with accelerated deposition were hydrodynamic radius of macromolecules and their negative charge density.     

Specificity profiling of Pak kinases allows identification of novel phosphorylation sites

The p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. We find that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks.     

Hallmarks of mycolic acid biosynthesis: a comparative genomics study

Mycolic acids, which render unique qualities to mycobacteria, are known to be important for mycobacterial growth, survival, and pathogenicity. It is of interest to understand the evolutionary origins of the mycolic acid pathway (MAP), as well as the common minimum principles critical for generating the capability of mycolic acid biosynthesis. The recent curation of a comprehensive model of the MAP in Mycobacterium tuberculosis and the availability of a large number of genome sequences make it feasible to carry out detailed sequence and phylogenetic analyses, to address these questions. A comprehensive phylogenetic pathway profile analysis was carried out for 318 fully sequenced bacterial genomes, for each of the proteins present in the MAP. The organisms were clustered on the basis of co-occurrence of the MAP proteins in their proteome, while the proteins were clustered on the basis of their phylogenetic profiles. The MAP proteins were also searched against the nonredundant sequence database, to identify similar proteins from other phyla. The pathway profiles indicate that four proteins and certain protein domains stand out as more characteristic to mycolate producing organisms. Further analysis leads to the identification of the desaturases DesA1 and DesA2 and certain domains of Fas and Pks13 as hallmarks of the pathway. The roles of these proteins in some other organisms, as well as the distribution of these proteins across all known genome sequences are also briefly discussed. The clustering of organisms, carried out to group organisms with similar profiles, provides a means of obtaining finer classification as compared to the standard taxonomic method. The results indicate that the MAP and hence the capacity of mycolic acid production in mycobacteria is an example of an emergent property that has come about by recruiting enzymes from unrelated pathways in plants, presumably through lateral gene transfer. The understanding of the hallmarks of mycolic acid biosynthesis will also find application in evaluating drug targets.     

Molecular Classification and Comparative Taxonomics of Foveal and Peripheral Cells in Primate Retina

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