Synthesis and biological evaluation of chalcones and their derived pyrazoles as potential cytotoxic agents

A series of substituted chalcones and their corresponding pyrazoles were synthesized and evaluated for in vitro cytotoxic activity against a panel of human cancer cell lines. Out of 93 compounds screened, 8 compounds, 1s, 3i,j,n, 4i,j,n and 4s, showed marked activity. Compounds 4j,n and 4s were found to be the most promising in this study. SAR is also discussed.     

QSAR-by-NMR: quantitative insights into structural determinants for binding affinity by analysis of 1H/15N chemical shift differences in MMP-3 ligands

A novel strategy is applied to obtain quantitative insights on factors influencing biological affinity in protein-ligand complexes. This approach is based on the detection of ligand binding by (15)N and (1)H amide chemical shift differences in two-dimensional (15)N-heteronuclear single-quantum correlation spectra. Essential structural features linked to affinity can be extracted using statistical analysis of (15)N and (1)H amide chemical shift differences in congeneric series relative to uncomplexed protein spectra, as demonstrated for 20 MMP-3 inhibitors in complex with human matrix metalloproteinase stromelysin (MMP-3). The statistical analysis using PLS led to a significant model, while its chemical interpretation, highlighting the importance of particular residues for affinity, are in agreement to an X-ray structure of one key compound in the homologue MMP-8 binding site.     

Protein dynamics control proton transfer from bulk solvent to protein interior: a case study with a green fluorescent protein

The kinetics of proton transfer in Green Fluorescent Protein (GFP) have been studied as a model system for characterizing the correlation between dynamics and function of proteins in general. The kinetics in EGFP (a variant of GFP) were monitored by using a laser-induced pH jump method. The pH was jumped from 8 to 5 by nanosecond flash photolysis of the "caged proton," o-nitrobenzaldehyde, and subsequent proton transfer was monitored by following the decrease in fluorescence intensity. The modulation of proton transfer kinetics by external perturbants such as viscosity, pH, and subdenaturing concentrations of GdnHCl as well as of salts was studied. The rate of proton transfer was inversely proportional to solvent viscosity, suggesting that the rate-limiting step is the transfer of protons through the protein matrix. The rate is accelerated at lower pH values, and measurements of the fluorescence properties of tryptophan 57 suggest that the enhancement in rate is associated with an enhancement in protein dynamics. The rate of proton transfer is nearly independent of temperature, unlike the rate of the reverse process. When the stability of the protein was either decreased or increased by the addition of co-solutes, including the salts KCl, KNO(3), and K(2)SO(4), a significant decrease in the rate of proton transfer was observed in all cases. The lack of correlation between the rate of proton transfer and the stability of the protein suggests that the structure is tuned to ensure maximum efficiency of the dynamics that control the proton transfer function of the protein.     

Comparative evaluation of whole blood D-dimer test to plasma D-dimer test for diagnosis of disseminated intravascular coagulation

Three rapid D-dimer test methods were compared for the diagnosis of acute disseminated intravascular coagulation (DIC). These were (a) SimpliRED, an autologous red cell agglutination assay. (b) DIMERTEST latex agglutination assay, containing monoclonal antibody DD-3B6/22(6), and (c) D-DI latex agglutination assay containing mouse anti-human D-dimer monoclonal antibodies. The D-DI latex method having higher sensitivity (100%) and specificity (81%) in clinically acute DIC was postulated as the gold standard and compared with the other two methods. The results suggest that D-DI latex agglutination assay containing mouse anti-human D-Dimer monoclonal antibodies are the better assay methods amongst all the three kits analyzed. It is advisable to look for the nature of the antibody used to coat the latex particles in plasma based kits. In emergency setting RBC kits may be of some use as rapid diagnosis is advantageous.     

Role of p-glycoprotein expression in predicting response to neoadjuvant chemotherapy in breast cancer-a prospective clinical study

Background

Lung cancer still remains one of the most commonly occurring solid tumors and even in stage Ia, surgery fails in 30% of patients who develop distant metastases. It is hypothesized that these must have developed from occult circulating tumor cells present at the time of surgery, or before. The aim of the present study was to detect such cells in the peripheral blood and to monitor these cells following surgery.

Methods

30 patients treated for lung cancer with surgery were monitored for circulating epithelial cells (CEC) by taking peripheral blood samples before, 2 weeks and 5 months after surgery and/or radiotherapy (RT) chemotherapy (CT) or combined RT/CT using magnetic bead enrichment and laser scanning cytometry (MAINTRAC^®) for quantification of these cells.

Results

In 86% of the patients CEC were detected before surgery and in 100% at 2 weeks and 5 months after surgery. In the control group, which consisted of 100 normal donors without cancer, 97 % were negative for CEC. A significantly higher number of CEC was found preoperatively in patients with squamous cell carcinoma than in those with adenocarcinoma. In correlation to the extent of parenchymal manipulation 2 weeks after surgery, an increase in numbers of CEC was observed with limited resections (18/21) whereas pneumonectomy led to a decrease (5/8) of CEC, 2 weeks after surgery. The third analysis done 5 months after surgery identified 3 groups of patients. In the group of 5 patients who received neo- or adjuvant chemo/radiotherapy there was evidence that monitoring of CEC can evaluate the effects of therapy. Another group of 7 patients who underwent surgery only showed a decrease of CEC and no signs of relapse. A third group of 11 patients who had surgery only, showed an increase of CEC (4 with an initial decrease after surgery and 7 with continuous increase). In the group with a continuous increase during the following 24 months, 2 early relapses in patients with stage Ia adenocarcinoma were observed. The increase of CEC preceded clinical detection by six months.

Conclusion

We consider, therefore, that patients with adenocarcinoma and a continuous increase of CEC after complete resection for lung cancer are at an increased risk of early relapse.     

Identification of regions involved in enzymatic stability of peptide deformylase of Mycobacterium tuberculosis

Sequence analysis of peptide deformylase of Mycobacterium tuberculosis revealed the presence of insertions (residues 74 to 85) and an unusually long carboxy-terminal end (residues 182 to 197). Our results with deletion mutants indicated the contribution of these regions in maintaining enzymatic stability. Furthermore, we showed that the region spanning the insertions was responsible for maintaining resistance to oxidizing agents, like H(2)O(2).     

Phosphatidylinositol 3,4,5-trisphosphate mediates aldosterone stimulation of epithelial sodium channel (ENaC) and interacts with gamma-ENaC

Whole cell voltage clamp experiments were performed in a mouse cortical collecting duct principal cell line using patch pipettes back-filled with a solution containing phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). PIP(3) significantly increased amiloridesensitive current in control cells but not in the cells prestimulated by aldosterone. Additionally, aldosterone stimulated amiloridesensitive current in control cells, but not in the cells that expressed a PIP(3)-binding protein (Grp1-PH), which sequestered intracellular PIP(3). 12 amino acids from the N-terminal tail (APGEKIKAKIKK) of gamma-epithelial sodium channel (gamma-ENaC) were truncated by PCRbased mutagenesis (gammaT-ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing alpha- and beta-ENaC only or with either gamma-ENaC or gamma(T)-ENaC. The data demonstrated that the N-terminal tail truncation significantly decreased amiloride-sensitive current and that both the N-terminal tail truncation and LY-294002 (a PI3K inhibitor) prevented ENaC translocation to the plasmamembrane. These data suggest that PIP(3) mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of gamma-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between gamma-ENaC and PIP(3).     

Hazardous effect of organophosphate compound, dichlorvos in transgenic Drosophila melanogaster (hsp70-lacZ): induction of hsp70, anti-oxidant enzymes and inhibition of acetylcholinesterase

We tested a working hypothesis that stress genes and anti-oxidant enzyme machinery are induced by the organophosphate compound dichlorvos in a non-target organism. Third instar larvae of Drosophila melanogaster transgenic for hsp70 were exposed to 0.1 to 100.0 ppb dichlorvos and 5.0 mM CuSO(4) (an inducer of oxidative stress and stress genes) and hsp70, and activities of acetylcholinesterase (AchE), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) product were measured. The study was further extended to examine tissue damage, if any, under such conditions. A concentration- and time-dependent increase in hsp70 and anti-oxidant enzymes was observed in the exposed organism as compared to control. A comparison of stress gene expression with SOD, CAT activities and LPO product under similar experimental conditions revealed that induction of hsp70 precedes the anti-oxidant enzyme activities in the exposed organism. Further, concomitant with a significant inhibition of AChE activity, significant induction of hsp70 was observed following chemical exposure. Mild tissue damage was observed in the larvae exposed to 10.0 ppb dichlorvos for 48 h when hsp70 expression reaches plateau. Dichlorvos at 0.1 ppb dietary concentration did not evoke significant hsp70 expression, anti-oxidant enzymes and LPO and AchE inhibition in the exposed organism, and thereby, was found to be non-hazardous to D. melanogaster. Conversely, 1.0 ppb of the test chemical stimulated a significant induction of hsp70 and anti-oxidant enzymes and significant inhibition of AchE; hence this concentration of test chemical was hazardous to the organism. The present study suggests that (a) both stress genes and anti-oxidant enzymes are stimulated as indices of cellular defense against xenobiotic hazard in D. melanogaster with hsp70 being proposed as first-tier bio-indicator of cellular hazard, (b) 0.1 ppb of the test chemical may be regarded as No Observed Adverse Effect Level (NOAEL), and 1.0 ppb dichlorvos as Low Observed Adverse Effect Level (LOAEL).     

Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA

A cDNA (1061 bp) Bj glyII was cloned from a mannitol induced library of Brassica juncea. It encoded a protein of 335 amino acids with a molecular weight of 36.52 kDa. The deduced amino acid sequence of the clone showed 92% and 56% identity with Pennisetum and rice glyoxalase II, respectively, and 30% identity was observed with the human glyoxalase II. Search for the identical residues revealed the presence of highly conserved THHHXDH domain which is involved in zinc binding. p-NN and pSORT analysis of this sequence revealed a N-terminal mitochondrial target peptide. The cDNA was cloned in pMAL and a fusion protein with MBP (78 kDa) was expressed in Escherichia coli. The recombinant protein was purified approximately sixfold by affinity purification on amylose column and showed its pH optima at 7.0. The K(m) was determined to be 120 microM using S-d-lactoylglutathione as substrate. The expression of Bj glyII under various abiotic stress conditions showed that it is upregulated by salinity, heavy metal stress, and ABA.     

The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study

A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.