Design, synthesis, biological evaluation and molecular docking of curcumin analogues as antioxidant, cyclooxygenase inhibitory and anti-inflammatory agents

Curcuminoids were isolated from Curcuma longa and their pyrazole and isoxazole analogues were synthesized and evaluated for antioxidant, COX-1/COX-2 inhibitory and anti-inflammatory activities. The designed analogues significantly enhance COX-2/COX-1 selectivity and possess significant anti-inflammatory activity in carrageenan induced rat paw edema assay. Pyrazole, isoxazole analogues of curcumin (4 and 7) exhibited higher antioxidant activity than trolox. Molecular docking study revealed the binding orientations of curcumin analogues in the active sites of COX and thereby helps to design novel potent inhibitors.     

Oxidant,antioxidant and physical exercise

Generation of reactive oxygen species (ROS) is a normal process in the life of aerobic organisms. Under physiological conditions, these deleterious species are mostly removed by the cellular antioxidant systems, which include antioxidant vitamins, protein and non-protein thiols, and antioxidant enzymes. Since the antioxidant reserve capacity in most tissues is rather marginal, strenuous physical exercise characterized by a remarkable increase in oxygen consumption with concomitant production of ROS presents a challenge to the antioxidant systems.An acute bout of exercise at sufficient intensity has been shown to stimulate activities of antioxidant enzymes. This could be considered as a defensive mechanism of the cell under oxidative stress. However, prolonged heavy exercise may cause a transient reduction of tissue vitamin E content and a change of glutathione redox status in various body tissues. Deficiency of antioxidant nutrients appears to hamper antioxidant systems and augment exercise-induced oxidative stress and tissue damage. Chronic exercise training seems to induce activities of antioxidant enzymes and perhaps stimulate GSH levels in body fluids. Recent research suggest that supplementation of certain antioxidant nutrients are necessary for physically active individuals.     

Molecular docking studies shows tivozanib and lapatinib as potential inhibitors of EML4-ALK translocation mediated fusion protein in non small cell lung cancer

Identification of activating mutations in non-small cell lung cancers (NSCLC) has been a focus in recent years. This led to successful evidence of using tyrosine kinase inhibitors (TKIs) over the standard platinum doublet based chemotherapy as the first line treatment in the metastatic setting.The rearrangements of fusion protein EML4-ALK in NSCLC lead to the use of crizotinib for this class of tumors. Preclinical and Phase 1 clinical studies show that ceritinib is more effective against both crizotinib sensitive and resistant tumors. Although robust responses to crizotinib are observed in NSCLC harboring ALK mutations, majority of tumors eventually become resistant, posing a major challenge in treatment course. Thus, there is a need for the identification and development of second-generation of ALK inhibitors. Computer aided molecular docking data show Tivozanib and Lapatinib bind EML4-ALK with high score. Tivozanib is in clinical trials for renal cell cancer and Lapatinib is a known dual tyrosine kinase inhibitor effective in breast cancer patients with HER2 over-expression. Additional data on these compounds for use in EML4-ALK positive NSCLC will provide evidence for use in patients treated with crizotinib. Data shows the importance of computer aided molecular docking in developing candidates with improved activity for further consideration in vitro and in vivo validation.     

Identification of Crucial Amino Acids of Bacterial Peptide Deformylases Affecting Enzymatic Activity in Response to Oxidative Stress

Peptide deformylase (PDF) is an essential bacterial metalloprotease involved in deformylation of N-formyl group from nascent polypeptide chains during protein synthesis. Iron-containing variants of this enzyme from Salmonella enterica serovar Typhimurium (sPDF) and Mycobacterium tuberculosis (mPDF), although inhibited by oxidizing agents like H₂O₂, exhibited strikingly different 50% inhibitory concentrations (IC₅₀s) that ranged from nanomolar (sPDF) to millimolar (mPDF) levels. Furthermore, the metal dissociation rate was higher in sPDF than mPDF. We hypothesized that a restriction in entry of environmental oxygen or oxidizing agents into the active site of mPDF might be the cause for such discrepancies between two enzymes. Since the active-site residues of the two proteins are similar, we evaluated the role of the oxidation-prone noncatalytic residue(s) in the process. Amino acid sequence analysis revealed that Cys-130 of sPDF corresponds to Met-145 of mPDF and that they are away from the active sites. Swapping methionine with cysteine in mPDF, the M145C protein displayed a drastic decrease in the IC₅₀ for H₂O₂ and an increased metal dissociation rate compared to the wild type. Matrix-assisted laser desorption ionization (MALDI) analysis of a trypsin-digested fragment containing Cys-145 of the M145C protein also indicated its increased susceptibility to oxidation. To incorporate residues identical to those of mPDF, we created a double mutant of sPDF (C130M-V63C) that showed increased IC₅₀ for H₂O₂ compared to the wild type. Interestingly, the oxidation state of cysteines in C130M-V63C was unaffected during H₂O₂ treatment. Taken together, our results unambiguously established the critical role of noncatalytic cysteine/methionine for enzymatic sensitivity to H₂O₂ and, thus, for conferring behavioral distinction of bacterial PDFs under oxidative stress conditions.     

Integrated regulation of PKA by fast and slow neurotransmission in the nucleus accumbens controls plasticity and stress responses

Cortical glutamate and midbrain dopamine neurotransmission converge to mediate striatum-dependent behaviors, while maladaptations in striatal circuitry contribute to mental disorders. However, the crosstalk between glutamate and dopamine signaling has not been entirely elucidated. Here we uncover a molecular mechanism by which glutamatergic and dopaminergic signaling integrate to regulate cAMP-dependent protein kinase (PKA) via phosphorylation of the PKA regulatory subunit, RIIβ. Using a combination of biochemical, pharmacological, neurophysiological, and behavioral approaches, we find that glutamate-dependent reduction in cyclin-dependent kinase 5 (Cdk5)-dependent RIIβ phosphorylation alters the PKA holoenzyme autoinhibitory state to increase PKA signaling in response to dopamine. Furthermore, we show that disruption of RIIβ phosphorylation by Cdk5 enhances cortico-ventral striatal synaptic plasticity. In addition, we demonstrate that acute and chronic stress in rats inversely modulate RIIβ phosphorylation and ventral striatal infusion of a small interfering peptide that selectively targets RIIβ regulation by Cdk5 improves behavioral response to stress. We propose this new signaling mechanism integrating ventral striatal glutamate and dopamine neurotransmission is important to brain function, may contribute to neuropsychiatric conditions, and serves as a possible target for the development of novel therapeutics for stress-related disorders.     

Indirect co-culture of lung carcinoma cells with hyperthermia-treated mesenchymal stem cells influences tumor spheroid growth in a collagen-based 3-dimensional microfluidic model

BackgroundMesenchymal stem cells (MSCs) have paradoxically been reported to exert either pro- or anti-tumor effects in vitro. Hyperthermia, in combination with chemotherapy, has tumor-inhibiting effects; however, its role, together with MSCs, so far is not well understood. Furthermore, a lot of research is conducted using conventional 2-dimensional in vitro models that do not mimic the actual tumor microenvironment.AimIn light of this fact, an indirect method of co-culturing human amniotic membrane-derived MSCs (AMMSCs) with collagen-encapsulated human lung carcinoma cells (A549) was performed using a 3-dimensional (3D) tumor-on-chip device.MethodsThe conditioned medium of AMMSCs (AMMSC-CM) or heat-treated AMMSCs (heat-AMMSC-CM) was utilized to create indirect co-culture conditions. Tumor spheroid growth characterization, immunocytochemistry and cytotoxicity assays, and anti-cancer peptide (P1) screening were performed to determine the effects of the conditioned medium.ResultsThe A549 cells cultured inside the 3D microfluidic chip developed into multicellular tumor spheroids over five days of culture. The AMMSC-CM, contrary to previous reports claiming its tumor-inhibiting potential, led to significant proliferation of tumor spheroids. Heat-AMMSC-CM led to reductions in both spheroid diameter and cell proliferation. The medium containing the P1 peptide was found to be the least cytotoxic to tumor spheroids in co-culture compared with the monoculture and heat-co-culture groups.ConclusionsHyperthermia, in combination with the anticancer peptide, exhibited highest cytotoxic effects. This study highlights the growing importance of 3D microfluidic tumor models for testing stem-cell-based and other anti-cancer therapies.     

An Overview of Precedence-Type Tests for Censored Data

Precedence tests are simple yet robust nonparametric procedures useful for comparing two or more distributions. In this paper precedence-type tests are considered when the data contain some censored observations. Generalizing the precedence statistic for uncensored data, the precedence tests for censored data are based on the Kaplan-Meier estimators of the respective distribution functions and the corresponding quantile functions. The literature is reviewed for some two-sample as well as some multi-sample problems.     

Efficient and rapid in vitro plant regeneration system for Indian cultivars of chickpea (Cicer arietinum L.)

Lacking of an efficient regeneration protocol for the recalcitrant crop chickpea is a limiting factor for adapting genetic engineering approaches for its improvement. The present study describes a rapid and efficient method for multiple shoot regeneration for three Indian cultivars, B115, C235, ICCV89314, using single cotyledons with half embryos as explant. Modified MS medium with 1.5 mg l⁻¹ 6-benzyladenine (BA) and 0.04 mg l⁻¹ α-naphthaleneacetic acid (NAA) induced a maximum of 26 shoots from a single explant after 20 days of culture. When cultured in modified MS medium containing 0.2 mg l⁻¹ indole-3-acetic acid (IAA), 80% of the shoots from each regenerating explant elongated in another 20–25 days. Following a root-grafting protocol, 90–95% of the elongated shoots survived in soil which subsequently produced seeds. The regeneration process from explant preparation to complete plants took 55–60 days. The presently optimized rapid regeneration method holds promise for facilitating the deployment of agronomically important components through genetic transformation for betterment of this important food crop.