Design,synthesis and evaluation of (E)-α-benzylthio chalcones as novel inhibitors of BCR-ABL kinase

Novel (E)-α-benzylthio chalcones are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (comparable to imatinib, the reference compound) of BCR-ABL phosphorylation in leukemic K562 cells, known to express high levels of BCR-ABL. The ability of such compounds to significantly inhibit K562 cell proliferation suggests that this scaffold could be a promising lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leukemic cells.     

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K_d ～20 nM) via the common interacting interface (residues 312–349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CΔ78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CΔ78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome.     

Pseudo-affinity chromatographic approach to probe heterogeneity in buffalo pituitary luteinizing hormone: probable pseudolectin-like behavior of immobilized Cibacron Blue 3GA

The alpha (α) and beta (β) subunits of buffalo pituitary luteinizing hormone (LH) were chromatographed on Cibacron Blue 3GA agarose and their immunoreactivity was quantitated using anti-α and anti-β anti sera. Subsequent analyses showed α subunits were relatively more hydrophilic than β subunits. Further, the naturally occurring free α and β subunits were more hydrophobic than their native counterparts which were dissociated and isolated from heterodimeric LH. The lesser sugar content in freely occurring α and beta subunits may be attributed for increased hydrophobicity and consequent upon the existence of their uncombined free forms. In order to ascertain putative sugar–dye interaction, crude LH carrying free subunits, pure LH, and non-glycosylated recombinant β subunit of LH were loaded separately on Cibacron Blue. Methyl mannoside was able to elute 33% of the bound protein in case of crude and pure LH, whereas there was little (3%) elution in case of recombinant LH β subunit. This study suggests a compositional heterogeneity in free and native subunits of LH from the buffalo pituitary. In addition, our findings reveal the pseudolectin-like behavior of Cibacron Blue.     

Triose phosphate isomerase, a novel enzyme-crystallin, and tau-crystallin in crocodile cornea. High accumulation of both proteins during late embryonic development

Several enzymes are known to accumulate in the cornea in unusually high concentrations. Based on the analogy with lens crystallins, these enzymes are called corneal crystallins, which are diverse and species-specific. Examining crystallins in lens and cornea in multiple species provides great insight into their evolution. We report data on major proteins present in the crocodile cornea, an evolutionarily distant taxon. We demonstrate that tau-crystallin/alpha-enolase and triose phosphate isomerase (TIM) are among the major proteins expressed in the crocodile cornea as resolved by 2D gel electrophoresis and identified by MALDI-TOF. These proteins might be classified as putative corneal crystallins. tau-Crystallin, known to be present in turtle and crocodile lens, has earlier been identified in chicken and bovine cornea, whereas TIM has not been identified in the cornea of any species. Immunostaining showed that tau-crystallin and TIM are concentrated largely in the corneal epithelium. Using western blot, immunofluorescence and enzymatic activity, we demonstrate that high accumulation of tau-crystallin and TIM starts in the late embryonic development (after the 24th stage of embryonic development) with maximum expression in a two-week posthatched animal. The crocodile corneal extract exhibits significant alpha-enolase and TIM activities, which increases in the corneal extract with development. Our results establishing the presence of tau-crystallin in crocodile, in conjunction with similar reports for other species, suggest that it is a widely prevalent corneal crystallin. Identification of TIM in the crocodile cornea reported here adds to the growing list of corneal crystallins.     

Novel coumarin-3-(N-aryl)carboxamides arrest breast cancer cell growth by inhibiting ErbB-2 and ERK1

A series of novel coumarin carboxamides were synthesized, and their tumor cell cytotoxic activity was investigated. These compounds specifically inhibited the growth of cancer cells that have a high level of ErbB-2 expression. Immunoprecipitation analysis of the cell lysates prepared from carboxamide treated cancer cells showed the inhibition of ErbB-2 phosphorylation suggesting the interaction of these compounds with ErbB-2 receptor. The down regulation of the kinase activity was further confirmed by performing in vitro kinase assay with recombinant ErbB-2 incubated with carboxamides. The inhibition of ErbB-2 phosphorylation correlated with down-regulation of ERK1 MAP kinase activation that is involved in proliferative signaling pathway. Furthermore, the cell-killing activity of many of these inhibitors is restricted to tumor cells with no demonstrable cytotoxicity against normal human fibroblasts suggesting that these compounds are tumor-specific.     

Caloric restriction augments ROS defense in S. cerevisiae, by a Sir2p independent mechanism

Aging is associated with increased production of reactive oxygen species (ROS) and oxidation-induced damage to intracellular structures and membranes. Caloric restriction (CR) has been demonstrated to delay aging in a variety of species. Although the mechanisms of CR remain to be clearly elucidated, reductions in oxidative damage have been shown to increase lifespan in several model systems. Contrary to the general belief that ROS production is reduced in CR, this article provides evidence that not only oxygen consumption but ROS production is enhanced in the calorie restricted condition. To understand the biological mechanism underlying the anti aging action of CR, the role of scavenging enzymes was studied. It was found that super oxide dismutase (SOD1 and SOD2), catalase and glutathione peroxidase (GPx) all are over expressed in CR. We further investigated the role of Sir2, a potential effector of CR response in the activation of scavenging enzymes. No marked difference was found in CR mediated activation of SOD and catalase in the absence of Sir2. Our results suggest that in CR scavenging enzymes are activated by a Sir2 independent manner.