Antioxidant and antiproliferative activity of curcumin semicarbazone

A new semicarbazone derivative of curcumin (CRSC) was synthesized and examined for its antioxidant, antiproliferative, and antiradical activity and compared with those of curcumin (CR). The antioxidant activity was tested by their ability to inhibit radiation induced lipid peroxidation in rat liver microsomes. The antiproliferative activity was tested by studying the in vitro activity of CRSC against estrogen dependant breast cancer cell line MCF-7. Kinetics of reaction of (2,2'-diphenyl-1-picrylhydrazide) DPPH, a stable hydrogen abstracting free radical was studied to measure the antiradical activity using stopped-flow spectrophotometer. Finally one-electron oxidized radicals of CRSC were generated and characterized by pulse radiolysis. The results suggest that the probable site of attack for CRSC is both the phenolic OH and the imine carbonyl position. CRSC shows efficient antioxidant and antiproliferative activity although its antiradical activity is less than that of CR.     

Fate of the H-NS–Repressed bgl Operon in Evolution of Escherichia coli

In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS–repressed locus is the bgl (aryl-β,D-glucoside) operon of E. coli. This locus is “cryptic,” as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli.     

Kinetic analysis of metal binding to the amino-terminal domain of ZntA by monitoring metal-thiolate charge-transfer complexes

ZntA, a P(1B)-ATPase transporter from Escherichia coli, mediates resistance specifically to Pb(2+), Zn(2+), and Cd(2+) by active efflux. ZntA has a hydrophilic N-terminal domain that binds one metal ion. This domain, approximately 120 residues long, contains the GXXCXXC motif that has been shown to be the binding site for metal ions such as Cu(+) and Zn(2+) in P(1B)-type ATPases, and an additional cysteine-rich motif, CCCDGAC. We report here that binding of Pb(2+) and Cd(2+) to this domain produces changes in the absorbance spectrum in the 250-400 nm range indicative of metal-thiolate charge-transfer complexes. The spectral changes indicate that only two cysteines are ligands to Cd(2+), but three or more cysteines are involved in binding Pb(2+); this confirms earlier results that the GXXCXXC sequence is not sufficient to bind Pb(2+), which likely involves residues from the CCCDGAC motif. The absorbance changes were used to measure metal binding kinetics of the N-terminal domain using stopped-flow techniques. Binding was described by simple second-order kinetics with a rate constant, k(on), of approximately 10(6)-10(7) M(-)(1) s(-)(1), at 4 degrees C. The activation energy of binding is similar for both Pb(2+) and Cd(2+); however, the entropy change is greater for Pb(2+). The surprisingly large rate constant for metal binding to the N-terminal domain of ZntA, compared to its low turnover rate, indicates that this step is not rate limiting in the overall transport mechanism. These results, in conjunction with earlier studies, suggest that metal binding to the transmembrane site in ZntA or metal release from the transporter is the slow step in the reaction cycle.     

Conservative and nonconservative mutations of the transmembrane CPC motif in ZntA: effect on metal selectivity and activity

ZntA from Escherichia coli belongs to the P1B-ATPase transporter family and mediates resistance to toxic levels of selected divalent metal ions. P1B-type ATPases can be divided into subgroups based on substrate cation selectivity. ZntA has the highest selectivity for Pb2+, followed by Zn2+ and Cd2+; it also shows low levels of activity with Cu2+, Ni2+, and Co2+. It has two high-affinity metal-binding sites, one each in the N-terminus and the transmembrane domains. Ligands to the transmembrane metal site in ZntA include the cysteine residues of the conserved 392CPC394 motif in the sixth transmembrane helix. Pro393 is invariant in all P-type ATPases. For ZntA homologues with different metal ion selectivity, the cysteines are replaced by serine, histidine, and threonine. To test the effect on activity and metal ion selectivity, single alanine, histidine, and serine substitutions at Cys392 or Cys394 in ZntA were characterized, as well as double substitutions of both cysteines by histidine or serine. P393A was also characterized. C392A, C394A, and P393A lost the ability to bind a metal ion with high affinity in the transmembrane domain. Histidine and serine substitutions at Cys392 and Cys394 resulted in loss of binding of Pb2+ at the transmembrane site, indicating that both cysteines of the CPC motif are required for binding Pb2+ with high affinity in ZntA homologues. However, C392H, C392S, C394H, C394S, C392S/C394S, and C392H/C394H could bind other divalent metal ions at the transmembrane site and retained low but measurable activity. Interestingly, these mutants lost the predominant selectivity for Zn2+ and Cd2+ shown by wtZntA. Therefore, conserved residues contribute to metal selectivity by supplying ligands that bind metal ions not only with high affinity, as for Pb2+, but also with the most favorable binding geometry that results in efficient catalysis.     

Spontaneous rupture of giant gastric stromal tumor into gastric lumen

BACKGROUND: Gastrointestinal stromal tumors (GIST) constitute a large majority of mesenchymal tumors of the gastrointestinal (GI) tract, which express the c-kit proto-oncogene protein, a cell membrane receptor with tyrosine kinase activity. GI stromal tumors of the stomach are usually associated with bleeding, abdominal pain or a palpable mass. CASE PRESENTATION: A 75-year-old male presented with upper abdominal pain and palpable mass. Computed tomographic (CT) scan of the abdomen showed a large mass arising in the posterior aspect of fundus, body, and greater curvature of the stomach. Second day after the admission, there was significant reduction in the size of the tumor, clinically as well as radiologically. Endoscopic biopsy showed large bulge in fundus and corpus of the stomach posteriorly with an opening in the posterior part of the corpus, and biopsy from the edge of the opening reveled GIST. Patient underwent curative resection. CONCLUSION: Spontaneous ruptured of giant gastric stromal tumor is very rare presentation of stomach GIST. Thorough clinical examination and timely investigation can diagnose rare complication.     

Pyridazolate-bridged dicopper (II) SOD mimics with enhanced antiproliferative activities against estrogen and androgen independent cancer cell lines

The superoxide dismutase (SOD) mimicking potential of three pyridazolato-bridged copper complexes has been investigated by NBT assay. The lowest IC₅₀ value observed for the SOD activity (3.90 × 10⁻⁷ M) for [Cu₂{bis(3,6 pyrazol-1-yl)pyridazine}Cl₄OH] · Cl (3) correlates well with its more facile Cu²⁺/Cu⁺ redox couple. These compounds exhibit remarkable antiproliferative activities against estrogen independent breast (BT-20) and androgen independent prostate cancer (PC-3) cell lines, respectively. The concentration of compound 3 that inhibits 50% of cell growth (IC₅₀) after 96 h of treatment using MTT cell proliferation assay was found to be 1.73 μM in BT20 and 1.42 μM in PC3 cell line. Furthermore, compound 3 does have the ability to induce apoptosis in both cell lines after the treatment for 48 h. This effect is probably through generation of intracellular oxidative stress and induction of intrinsic apoptotic pathway.