Activation of NAD(P)H oxidase by lipid hydroperoxides: mechanism of oxidant-mediated smooth muscle cytotoxicity

Oxidized lipids, such as 13-hydroperoxyoctadecadienoic acid (13-HPODE), have been implicated in the pathogenesis of atherosclerosis. 13-HPODE, a constituent of oxidized low-density lipoproteins, can induce cytotoxicity of vascular smooth muscle cells (SMC), which may facilitate plaque destabilization and/or rupture. 13-HPODE-induced cytotoxicity has been linked to oxidative stress, although the mechanisms by which this occurs are unknown. In the present study, we show that 13-HPODE and 9-HPODE (10-30 microM) increased superoxide (O2*-) production and induced cytotoxicity in SMC. The 13-HPODE-induced increase in O2*- was blocked by transfecting the cells with antisense oligonucleotides against p22phox, suggesting that the O2*- was produced by NAD(P)H oxidase. Similar concentrations of the corresponding HPODE reduction products, 13-hydroxyoctadecadienoic acid (13-HODE) and 9-HODE, neither increased O2*- production nor induced cytotoxicity, while 4-hydroxy nonenal (4-HNE), an unsaturated aldehyde lipid peroxidation product, induced cytotoxicity without increasing O2*- production. Treatment with superoxide dismutase or Tiron to scavenge O2*-, or transfection with p22phox antisense oligonucleotides to inhibit O2*- production, attenuated 13-HPODE-induced cytotoxicity, but not that induced by 4-HNE. These findings suggest that activation of NAD(P)H oxidase, and production of O2*-, play an important role in lipid hydroperoxide-induced smooth muscle cytotoxicity.     

Manipulation of inositol metabolism for improved plant survival under stress: a “network engineering approach”

Emergence of high-throughput sequencing tools and omics technologies paved the way for systems biology in last decade. While we have started to look at the biology of the plant in a more unified manner, the integration of such knowledge in agricultural biotechnology has become an arena of potential interest. The network of several central molecules operating in various life and developmental processes are now more adequately known, and fine tuning of such molecule pools, if connected to stress response, can result in enhanced stress tolerance of plants.This review interprets the potential of manipulation of myo-inositol and its derivatives in generation of transgenic crop plants. Being a molecule of central importance in plant life, inositol is connected to numerous life processes. The exploration of such pathways indicates that inositol itself and many of its derivatives can impart abiotic stress tolerance (against salinity, dehydration, chilling or oxidative stress) to plants when overexpressed. We propose that engineering inositol metabolic network is a potential approach towards stress-tolerant transgenic crop plant generation and thus its exploitation in agricultural biotechnology is the call of time.     

Cloning, characterization and expression of a chloroplastic fructose-1,6-bisphosphatase from Porteresia coarctata conferring salt-tolerance in transgenic tobacco

A gene coding for the chloroplastic fructose 1,6-bisphosphatase (PcCFR) in Porteresia coarctata Tateoka (Roxb.), a halophytic wild rice, has been isolated along with its rice (Oryza sativa; var. indica) homologue (OsCFR), cloned and sequenced. Comparison between the nucleotide and deduced amino acid sequences of these two revealed a difference in five amino acid residues, namely Glu¹⁴, Thr²⁴, Ala⁴⁸, Ala¹⁶³ and Arg²⁹⁶ in OsCFR which have been found to be replaced by Ser¹⁴, Ile²⁴, Ser⁴⁸, Ser¹⁶³ and Lys²⁹⁶ in PcCFR respectively. The purified recombinant PcCFR is found to retain its enzymatic activity in presence of up to 500 mM NaCl in vitro as opposed to OsCFR, which is inactivated even at lower salt concentration. The six in vitro point mutant proteins of PcCFR showed varied degree of sensitivity towards high salt, with the maximum OsCFR-like effect in the triple mutant S¹⁴A-S⁴⁸A-S¹⁶³A suggesting a possible concerted role of all three serine residues in the in vitro salt tolerance property of PcCFR protein. Transgenic tobacco plants with chloroplast targeted PcCFR and OsCFR gene(s) have been developed under constitutive expression of CaMV 35S promoter and NOS terminator. The PcCFR transgenics showed better plant growth during exposure to salt stress in comparison to either the OsCFR or the empty vector transformed plants. The PcCFR transgenics also revealed enhanced photosynthetic efficiency coupled with protection to both photodamage of PSII and chlorophyll degradation through better reactive oxygen species scavenging at higher concentration of NaCl during late salt-stress growth.     

Anti-cancer evaluation of carboxamides of furano-sesquiterpene carboxylic acids from the soft coral Sinularia kavarattiensis

The chemical investigation of soft coral Sinularia kavarattiensis is described. It yielded furano-sesquiterpene carboxylic acids 1 and 2 and their methyl esters 3 and 4. Semi-synthesis of furano-sesquiterpene carboxylic acid 1 gave amide derivatives 5–12. Structures of all the compounds were established by IR, NMR and mass spectral analysis. Interestingly all compounds are selectively potent on leukemia cell line. All these compounds were screened for cytotoxic activity against five human cancer cell lines (leukemia, prostate, lung, breast and cervix). Among these compounds 9 and 10 showed promising activity against leukemia and prostate cancer cell lines.     

Mitochondrial aminoacyl-tRNA synthetase single-nucleotide polymorphisms that lead to defects in refolding but not aminoacylation

Defects in organellar translation are the underlying cause of a number of mitochondrial diseases, including diabetes, deafness, encephalopathy, and other mitochondrial myopathies. The most common causes of these diseases are mutations in mitochondria-encoded tRNAs. It has recently become apparent that mutations in nuclear-encoded components of the mitochondrial translation machinery, such as aminoacyl-tRNA synthetases (aaRSs), can also lead to disease. In some cases, mutations can be directly linked to losses in enzymatic activity; however, for many, their effect is unknown. To investigate how aaRS mutations impact function without changing enzymatic activity, we chose nonsynonymous single-nucleotide polymorphisms (nsSNPs) that encode residues distal from the active site of human mitochondrial phenylalanyl-tRNA synthetase. The phenylalanyl-tRNA synthetase variants S57C and N280S both displayed wild-type aminoacylation activity and stability with respect to their free energies of unfolding, but were less stable at low pH. Mitochondrial proteins undergo partial unfolding/refolding during import, and both S57C and N280S variants retained less activity than wild type after refolding, consistent with their reduced stability at low pH. To examine possible defects in protein folding in other aaRS nsSNPs, we compared the refolding of the human mitochondrial leucyl-tRNA synthetase variant H324Q to that of wild type. The H324Q variant had normal activity prior to unfolding, but displayed a refolding defect resulting in reduced aminoacylation compared to wild type after renaturation. These data show that nsSNPs can impact mitochondrial translation by changing a biophysical property of a protein (in this case refolding) without affecting the corresponding enzymatic activity.     

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)¹. While improvements in several gene delivery methods have been described^2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor¹¹, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.