Immobilization of rat brain acetylcholinesterase on porous gold-nanoparticle-CaCO₃ hybrid material modified Au electrode for detection of organophosphorous insecticides

An acetylcholinesterase (AChE) purified from rat brain was immobilized onto gold nanoparticles (AuNPs) assembled on the surface of porous calcium carbonate (CaCO₃) microsphere. The resulting AChE-AuNPs-CaCO₃ bioconjugate was mounted on the surface of Au electrode with the help of silica sol-gel matrix to prepare the working electrode. This electrode was connected to Ag/AgCl (3 M/saturated KCl) as standard and Pt wire as an auxiliary electrode through a potentiostat to construct an organophosphorus (OP) biosensor. The biosensor was based on inhibition of AChE by OP compounds/insecticides. The biosensor showed optimum response at pH 7.0, 30 °C, when polarized at +0.2 V. Two OP compounds, malathion and chlorpyrifos could be detected in the range of 0.1-100 nM and 0.1-70 nM, respectively at 2.0-3.0% inhibition level of AChE. The sensor was reactivated by immersing it in 0.1 mM 2-pyridine aldoxime for 10 min. The detection limit of the sensor was 0.1 nM for both malathion and chlorpyrifos. The biosensor exhibited good reusability (50 times without considerable loss) and storage stability (50% within 60 days, when stored at 4 °C).     

Janus kinase 3 regulates interleukin 2-induced mucosal wound repair through tyrosine phosphorylation of villin

Janus kinase 3 (Jak3) is a non-receptor tyrosine kinase known to be expressed in hematopoietic cells. Studies of whole organ homogenates show that Jak3 is also expressed in the intestines of both human and mice. However, neither its expression nor its function has been defined in intestinal epithelial enterocytes. The present studies demonstrate that functional Jak3 is expressed in human intestinal enterocytes HT-29 Cl-19A and Caco-2 and plays an essential role in the intestinal epithelial wound repair process in response to interleukin 2 (IL-2). Exogenous IL-2 enhanced the wound repair of intestinal enterocytes in a dose-dependent manner. Activation by IL-2 led to rapid tyrosine phosphorylation and redistribution of Jak3. IL-2-stimulated redistribution of Jak3 was inhibited by the Jak3-specific inhibitor WHI-P131. IL-2 also induced Jak3-dependent redistribution of the actin cytoskeleton in migrating cells. In these cells Jak3 interacted with the intestinal and renal epithelial cell-specific cytoskeletal protein villin in an IL-2-dependent manner. Inhibition of Jak3 activation resulted in loss of tyrosine phosphorylation of villin and a significant decrease in wound repair of the intestinal epithelial cells. Previously, we had shown that tyrosine phosphorylation of villin is important for cytoskeletal remodeling and cell migration. The present study demonstrates a novel pathway in intestinal enterocytes in which IL-2 enhances intestinal wound repair through mechanisms involving Jak3 and its interactions with villin.     

Cationic lipids with increased DNA binding affinity for nonviral gene transfer in dividing and nondividing cells

Effect of headgroup structure on catonic lipid-mediated transfection was investigated with either a (i) tertiary amine, (ii) quaternary amine with a hydroxyl, or (iii) quaternary amine with mesylate as headgroups. Liposomes were formulated using cholesterol or dioleoyl phosphatidyl ethanolamine (DOPE) as colipids, and transfection efficiencies were determined in rapidly dividing colon carcinoma (CT 26) and rat aortic smooth muscle (RASM) cells as well as in nondividing human pancreatic islets using luciferase and green fluorescent protein expression plasmids, pcDNA3-Luc and pCMS-EGFP, respectively. Liposome/pDNA complexes were evaluated for DNA conformational state by circular dichroism (CD), DNA condensation by electrophoretic mobility shift assay (EMSA), particle size and zeta potential by laser diffraction technique, and surface morphology by transmission electron microscopy (TEM). Encouraging transfection results were obtained with the mesylate headgroup based lipid in liposome formulations with DOPE as a colipid, which were higher than the commercially available Lipofectamine formulation. We hypothesize that the additional hydrogen bonding or covalent interactions of the headgroup with the plasmid DNA, leading to higher binding affinity of the cationic lipids to pDNA, results in higher transfection. This hypothesis is supported by TEM observations where elongated complexes were observed and more lipid was seen associated with the DNA.     

Structured model for denitrifier diauxic growth

We present a model for diauxic growth of denitrifying bacteria in which nitrate reductase synthesis kinetics dominate the overall growth kinetics. The model is based on the assumption of the existence of a nitrate respiration operon, thereby linking the rate of nitrate uptake to the activity of nitrate reductase. We show that this approach can model diauxic growth of Pseudomonas denitrificans by conducting experiments in which nitrate reductase activity was measured during both lag and ensuing exponential growth phases. We consistently observed the pattern of low nitrate reductase enzyme activity during the lag phase, increasing before the onset of growth. By fitting model parameters we were able to successfully match experimental data for growth, nitrate uptake, and enzyme activity level.     

Aluminium(III), chromium(III) and iron(III) complexes with 5-iodouracil and 5-iodouracil-histidine and their antitumour activity

Complexes of the type [Al(HL)(OH)Cl(2)], [M(HL)(OH)(2)Cl] and [M'(HL)(L')(OH)Cl], where HL = 5-iodouracil; HL' = histidine; M = Cr(III), Fe(III) and M' = Al(III), Cr(III), Fe(III), were synthesized and characterized. The complexes are polymeric showing high decomposition points and are insoluble in water and common organic solvents. The mu(eff) values, electronic spectral bands and ESR spectra suggest a polymeric 6-coordinate spin-free octahedral stereochemistry for the Cr(III) and Fe(III) complexes. 5-Iodouracil acts as a monodentate ligand coordinating to the metal ion through the O atom of C((4)) = O while histidine through the O atom of -COO(- ) and the N atom of -NH(2) group. In vivo antitumour effect of 5-iodouracil and its complexes was examined on C(3)H /He mice against P815 murine mastocytoma. As evident from their T/C values, Cr(III) and Fe(III) complexes display significant and higher antitumour activity compared to the 5-iodouracil ligand. The in vitro results of the complexes on the same cells indicate that Cr(III) and Fe(III) complexes show higher inhibition on (3)H-thymidine and (3)H-uridine incorporation in DNA and RNA replication, respectively, at a dose of 5 microg/mL.     

Chemical synthesis and sequence studies of deoxyribooligonucleotides which constitute the duplex sequence of the lactose operator of Escherichia coli.

We have synthesized the deoxyribooligonucleotide fragments, constituting the sequence of the lac operator of Escherichia coli. Two of these fragments, d(pApApTpTpGpTpTpApT) (nonamer) and d(pApApTpTpGpTpGpApG) (nonamer), corresponding to the 5' termini of lac operator have been synthesized by the phosphodiester method. The remaining four fragments, d(ApCpApApTpT) (hexamer), d(ApTpApApCpApApTpT) (nonamer), d(ApApTpTpGpTpGpApGpCpGpG) (dodecamer), and d(ApApTpTpGpTpTpApTpCpCpGpCpTpC) (pentadecamer), have been synthesized by an improved phosphotriester method. All of the compounds were first characterized by venom and spleen phosphodiesterase digestion to obtain their base composition. The sequence of these oligonucleotides was fully confirmed by the characteristic mobility shifts of their partial venom phosphodiesterase digestion products on two-dimensional homochromatography. A comparative study of the two methods for the synthesis of oligonucleotides has revealed that the phosphotriester method is more convenient than the phosphodiester method because of higher yields and ease of handling large scale preparations.     

Detection of single-stranded DNA in scrapie-infected brain by electron microscopy

The nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles from normal and scrapie-infected hamster brains were examined by electron microscopy. After spreading on collodion-coated grids circular molecules of approximately 15.7 kb corresponding in size to mitochondrial DNA (mtDNA) were observed both in normal and scrapie-infected brains. In nucleic acid preparations from scrapie-infected brains multimeric mtDNA and single-stranded DNA strands of about 0.49 X 10(6) daltons were also visualized. These findings demonstrate the presence of a single-stranded DNA in scrapie-infected brains and are consistent with previous data based on enzyme digestion of nucleic acids isolated from scrapie-infected brains.     

Chromatin-Based Classification of Genetically Heterogeneous AMLs into Two Distinct Subtypes with Diverse Stemness Phenotypes

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Structural Basis and Targeting of the Interaction between Fibroblast Growth Factor-inducible 14 and Tumor Necrosis Factor-like Weak Inducer of Apoptosis

Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr¹⁷⁶, but not Trp²³¹, resulted in the loss of TWEAK binding to Fn14 substantiating Tyr¹⁷⁶ as the anchoring residue. Importantly, mutation of TWEAK at Tyr¹⁷⁶ did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.