Functional expression of Brassica juncea oleate desaturase gene (Bjfad2) in Escherichia coli

The synthesis of polyunsaturated fatty acids, the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase-2 (FAD2; EC 1.3.1.35), also called as microsomal Δ12 oleate desaturase. The gene (Bjfad2; GenBank accession No. EF639848) coding for this enzyme from Brassica juncea was previously isolated and characterized. However, functional identity of Bjfad2 was not established. Utilizing the known Bjfad2 cDNA sequence, the ORF of Bjfad2 gene was cloned into the pMAL C2X Escherichia coli expression vector and produced recombinant plasmid by insertion of isolated ORF downstream to the maltose-binding protein coding sequence. The pMALC2X-Bjfad2 vector was used to transform the TB1 strain of E. coli. Induced expression of pMAL-BJFAD2 fused product resulted in the synthesis of a polypeptide with an apparent molecular mass of 80 kDa, which was 8 kDa less than calculated mass as determined by SDS-PAGE, since the fused MalE-Bjfad2 gene contains eight additional codons located between the MalE and Bjfad2 gene. In vitro activity assay of oleate desaturase using the corresponding bacterial crude extracts confirmed that the polypeptide was the product of the Bjfad2 gene. The reaction products analysis of the fatty acid methyl esters by gas chromatography showed the presence of a new peak with a similar retention time to linoleic acid, which was absent in the control activity assay without electron donors. Thus, B. juncea gene has been functionally identified since it encodes the enzyme that catalyzed the desaturation of oleate to linoleate.     

Stability and dynamics of domain-swapped bovine-seminal ribonuclease

The proteins of the ribonuclease-A (RNase-A) family are monomeric, with the exception of bovine-seminal ribonuclease (BS-RNase). BS-RNase is formed by swapping the N-terminal helices across the two monomeric units. A molecular-dynamics (MD) study has been performed on the protein for a simulation time of 5.5 ns to understand the factors responsible for the stability of the dimer. Essential dynamics analysis and motional correlation of the protein atoms yielded the picture of a stabilising, yet flexible, interface. We have investigated the role of intermolecular H-bonding, protein/water interaction, and protein/water networks in stabilising the dimer. The networks of interchain H-bonds involving side-chain/side-chain or side-chain/main-chain (ScHB) interactions between the two chains have also been studied. The ability of protein atoms in retaining particular H2O molecules was investigated as a function of the accessible surface area (ASA), depth, and hydration parameters, as well as their participation in protein/water networks.     

Protein-water interactions in ribonuclease A and angiogenin: a molecular dynamics study

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein-water interactions have been carried out on two members of the ribonuclease A superfamily-RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein-water networks are investigated. The locations of such networks are identified for both proteins.     

Recurrent gene amplification and soft selective sweeps during evolution of multidrug resistance in malaria parasites

When selection is strong and beneficial alleles have a single origin, local reductions in genetic diversity are expected. However, when beneficial alleles have multiple origins or were segregating in the population prior to a change in selection regime, the impact on genetic diversity may be less clear. We describe an example of such a "soft" selective sweep in the malaria parasite Plasmodium falciparum that involves adaptive genome rearrangements. Amplification in copy number of genome regions containing the pfmdr1 gene on chromosome 5 confer resistance to mefloquine and spread rapidly in the 1990s. Using flanking microsatellite data and real-time polymerase chain reaction determination of copy number, we show that 5-15 independent amplification events have occurred in parasites on the Thailand/Burma border. The amplified genome regions (amplicons) range in size from 14.7 to 49 kb and contain 2-11 genes, with 2-4 copies arranged in tandem. To examine the impact of drug selection on flanking variation, we genotyped 48 microsatellites on chromosome 5 in 326 parasites from a single Thai location. Diversity was reduced in a 170- to 250-kb (10-15 cM) region of chromosomes containing multiple copies of pfmdr1, consistent with hitchhiking resulting from the rapid recent spread of selected chromosomes. However, diversity immediately flanking pfmdr1 is reduced by only 42% on chromosomes bearing multiple amplicons relative to chromosomes carrying a single copy. We highlight 2 features of these results: 1) All amplicon break points occur in monomeric A/T tracts (9-45 bp). Given the abundance of these tracts in P. falciparum, we expect that duplications will occur frequently at multiple genomic locations and have been underestimated as drivers of phenotypic evolution in this pathogen. 2) The signature left by the spread of amplified genome segments is broad, but results in only limited reduction in diversity. If such "soft" sweeps are common in nature, statistical methods based on diversity reduction may be inefficient at detecting evidence for selection in genome-wide marker screens. This may be particularly likely when mutation rate is high, as appears to be the case for gene duplications, and in pathogen populations where effective population sizes are typically very large.     

Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K-Ras transgenic mouse model of pancreatic cancer

Human pancreatic cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre (KC), and K-Ras;Pdx1-Cre;INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT-PCR. We found over-expression of miR-21, miR-221, miR-27a, miR-27b, and miR-155, and down-regulation of miR-216a, miR-216b, miR-217, and miR-146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI-derived RInk-1 cells. Mechanistic investigations revealed a significant induction of EGFR, K-Ras, and MT1-MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk-1 cells compared to human MIAPaCa-2 cells. Furthermore, miR-155 knock-down in RInk-1 cells resulted in the inhibition of cell growth and colony formation consistent with down-regulation of EGFR, MT1-MMP, and K-Ras expression. In addition, miR-216b which target Ras, and forced re-expression of miR-216b in RInk-1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR, and MT1-MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA-targeted agents for designing personalized therapy for PC.     

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.     

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.