Comparative study of toxic effects of zearalenone and its two major metabolites α‐zearalenol and β‐zearalenol on cultured human Caco‐2 cells

Zearalenone (ZEN) is a fusarotoxin converted predominantly into α‐zearalenol (α‐Zol) and β‐zearalenol (β‐Zol) by hepatic hydroxysteroid dehydrogenases. The feeding of naturally contaminated grains with ZEN was associated with hyperestrogenic and adverse effects on humans and animals. There is a lack of information on the attribution of the toxic effects of these toxins. One wonders if these effects are due to the parent molecule (ZEN) or to its major metabolites (α‐Zol and β‐Zol). Using human Caco‐2 cells, we looked for the molecular mechanisms of toxicity of ZEN, α‐Zol, and β‐Zol. Toxicity effects were studied by MTT viability assay and oxidative stress induction by measuring malondialdehyde (MDA) generation. To check whether the oxidative stress induction was associated to DNA lesions, we looked for DNA fragmentation by means of the Comet and the diphenylamine assays. To specify cell death pathway, we investigated caspase‐3 activation, confirmed by poly(ADP‐ribose) polymerase cleavage and by Bcl‐2 depletion. Our results clearly demonstrated that ZEN as well as its two metabolites presented variable toxic effects. They induced cell death and an increase in MDA generation. These effects were associated to DNA fragmentation as well as caspase‐3 activation. The observed toxic effects seem to be relieved by the metabolism of ZEN into α‐Zol and β‐Zol. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:233–243, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20284     

Regio-selective acylation of biologically important iridoid glycosides by Candida antarctica lipase

Lipase catalyzed regio-selective acylation of five iridoid glycosides viz., picroside I&II, catalpol, agnuside and negundoside in the presence of various acyl donors such as vinyl acetate and p-nitrophenyl alkanoates was studied. The regio-selectivity of enzymatic acylation and yields were found to vary amongst different substrates. Monoacylated products were isolated with all the substrates under scrutiny indicating high regio-selective nature of such transformations. A series of acyl esters of picroside-I, picroside-II, catalpol, agnuside and negundoside have been synthesized by this enzymatic trans-esterification methodology.     

Massive Fluid Requirements and an Unusual BUN/Creatinine Ratio for Pre-Renal Failure in Patients with Cholera

Background

Cholera is an important infectious cause of secretory diarrhea. The primary symptom of infection is the sudden onset of watery diarrhea with subsequent volume depletion causing renal insufficiency. The objective of this research is to study the level of dehydration at presentation and subsequent fluid management in patients with cholera.

Methods

This study was conducted on 191 patients of Cholera admitted at a tertiary care hospital in Karachi, Pakistan during the period of 5 years. Medical charts were evaluated retrospectively for initial hydration status, baseline lab investigations on admission and discharge and fluid therapy given to all the patients while their stay in the hospital and the data was analyzed on SPSS 15.0.

Results

Out of the 191 patients, 83(43%) were males and 108 (57%) were females with mean age of 42.3 years (SD±18.34). The average duration of symptoms was 3.75 days (SD±2.04). Of 191 patients, 175 (92.1%) presented with dehydration, 80 (42.3%) were given Ringer''s Lactate (R/L) + Normal Saline (N/S), 45 (24%) patients were given R/L + N/S + Oral Rehydration Therapy (ORS), 27 (14.3%) of the patients were kept on R/L only and remaining were given various combinations of R/L, N/S, ORS and Dextrose Saline (D/S). On admission mean Blood Urea Nitrogen (BUN) was 24.54 (SD±16.6), mean creatinine was 2.47 (SD±2.35) and mean BUN/Creatinine ratio was 11.63 (SD±5.7).

Conclusion

Aggressive fluid rehydration remains the cornerstone of management of cholera. Instead of presenting with a classical BUN/Creatinine ratio of >20∶1, patients with pre-renal failure in cholera may present with a BUN/Creatinine ratio of <15∶1.     

Synthesis, characterization and antiamoebic activity of 1-(thiazolo[4,5-b]quinoxaline-2-yl)-3-phenyl-2-pyrazoline derivatives

A new series of 1-N-thiocarboxamide-3-phenyl-2-pyrazolines 1-6 was synthesized by cyclization of different Mannich bases with unsubstituted thiosemicarbazide. The reaction of cyclized pyrazoline derivatives 1-6 with 2,3-dichloroquinoxaline afforded the title compounds 7-12. The structures of the new compounds were confirmed by elemental analyses as well as (1)H, (13)C NMR, IR and electronic spectral data. The HM1:IMSS strain of Entamoeba histolytica parasite was cultured in vitro and the sensitivity of the parasite to the synthesized compounds was evaluated using the microdilution method. Among all the pyrazoline derivatives 1-6, none was found to be a better inhibitor as compared to the reference drug, metronidazole. The quinoxaline derivatives, 9, 11 and 12 were found to be potent inhibitors of E. histolytica.     

Experimental and molecular docking investigation on DNA interaction of N‐substituted phthalimides: antibacterial,antioxidant and hemolytic activities

A series of Schiff base molecules derived from a phthalimide scaffold was investigated as efficient antibacterial, antioxidant and DNA‐interacting agents. The spectroscopic characterization of these derivatives was studied in detail using elemental analysis and spectroscopic techniques. The DNA‐binding profile of title molecules against Ct‐DNA (calf thymus) was investigated by absorbance, fluorescence, hydrodynamics and thermal denaturation investigations. The bacterial inhibition potential of these molecules was investigated against Escherichia coli and Staphylococcus aureus. Molecule 3c emerged as the most active against S. aureus (IC₅₀: 14.8 μg/mL), whereas compounds 3a and 3b displayed potential antibacterial activities against E. coli (IC₅₀: 49.7 and 67.6 μg/mL). Molecular docking studies of these compounds against GlcN‐6‐P synthase were carried out to rationalize antibacterial efficiency of these molecules. These newly synthesized molecules were screened for their scavenging capacity against 2,2‐diphenyl‐1‐picryl‐hydrazyl (DPPH) and H₂O₂ free radicals and the results were compared with ascorbic acid as synthetic antioxidant. The title molecules 3a, 3b and 3e showed less than 20% hemolysis, which indicated their significant non‐toxic behavior.     

Insilico Structural 3D Modelling of Novel Cry1Ib9 and Cry3A Toxins from Local Isolates of Bacillus thuringiensis

Three-dimensional (3D) models for the 79.2 kDa activated Cry1Ib9 and 77.4 kDa activated Cry3A δ-endotoxins from Bacillus thuringiensis (Bt) native isolates that are specifically toxic to Coleopteran insect pests were constructed by utilizing homology modeling online tool. Evidences presented here, based on the identification of structural equivalent residues of Cry1Ib9 and Cry3A toxin through homology modelling indicate that, they share a common Bt toxin tridimensional structure. The main differences observed in Cry1I9 domain I at positions α2b (S56-I60), α4 (F78-l93) and additionally β0 (Q10-L12), α8a (T280-V282) were observed, in domain II at positions α9b (P333-L339), β6(T390-Q393), β7(V398-W404), β8 (V418-W425), β9 (E453-N454), β10 (S470-I479) where as in domain III the changes were observed at positions β19 (R601-F607), β20 (609-L613), β21 (S618-F627) and α11a (K655-F664), α13, α14 components present at downstream sites, where as in Cry3A main differences observed in domain I is at the position of α4 (P105-I152), α5 (Q163-A185), β1A(E190-L192), α6 (F193-Y217), Domain II is not consevered and main variations were observed at β2 (E292-L295), β3(V299-L308), β4(I340-F347), β5(D356-P368), β6(I375-T377), β7(V389-F394), β8(K398-N405), β9(Y416-Y427), β10 (T436-Y439), β12(G476-H495), β12A (M503-I504) where as in domain III main variations observed at positions of β18 (P583-I593), β19(F604-S610), β20(P611-L615), β21(N619-G626). Cry1Ib9 and Cry3A contain the most variable regions in the loops of domain II, which determine the specificity of these toxins. These are the first models of Coleopteran-active protein from native isolates of Bt and its importance can be perceived since members of this group of toxins are potentially important candidates for coleoptera insect pest control programs.     

A whole mitochondrial genome screening in a MELAS patient: a novel mitochondrial tRNA(Val) mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA(Val). This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

Mitochondrial DNA diversity in Francolinus pondicerianus interpositus (grey francolin, Galliformes) from Pakistan

Francolinus pondicerianus interpositus (grey francolin, Galliformes) is the only francolin present in the Suleiman Range (central Pakistan), one of the poorest and least developed areas in Pakistan. As a game bird, the francolin is an important income source for the region, but no demographic data are available. Therefore, the aim of this work was to study the polymorphism pattern of the Control Region gene (mitochondrial DNA, mtDNA), in order to obtain some initial information about genetic diversity, possible structure and demographic dynamics in this population. In 29 individuals captured in four sampling areas in the western and the eastern Suleiman Range, we detected nine polymorphic sites in a 511 bp fragment of the mtDNA Control Region gene, resulting in seven haplotypes. Haplotype (h = 0.818 ± 0.032) and nucleotide diversity (π % = 0.308 ± 0.210) values suggested a large population size and a low divergence among the haplotypes. AMOVA (Φ(ST) = 0.005; P = 0.352) did not detect any significant differences among the western and eastern populations; therefore, specimens of both sampled areas could be considered as drawn from a single population. The observed distribution of pairwise mismatches was bimodal, revealing significant departure from a growing-decreasing population model (P = 0.030); these results would point to a demographic equilibrium. Tribal control of hunting might provide an explanation for this situation, but future overhunting would threaten the survival of this population.     

A propionyloxy derivative of 11-keto-β-boswellic acid induces apoptosis in HL-60 cells mediated through topoisomerase I & II inhibition

Boswellic acids have invariably been reported for their antiproliferative potential in various cell systems. In the present study the growth inhibitory effect of propionyloxy derivative of 11-keto-β-boswellic acid (PKBA; a semisynthetic analogue of 11-keto-β-boswellic acid) on HL-60 promyelocytic leukemia cells is being reported for the first time. In the preliminary studies, in vitro cytotoxicity of PKBA was investigated against eight human cancer cell lines viz., IMR-32, SF-295 (both neuroblastoma), PC-3 (prostate), Colo-205 (colon), MCF-7 (breast), OVCAR-5 (ovary), HL-60, Molt-4 (both leukemia) and their respective IC(50) values were found to be 5.95, 7.11, 15.2, 14.5, 15, 15.9, 8.7 & 9.5μg/ml, respectively. For determining the mechanism of cell death in HL-60 cells, PKBA was subjected to different mechanistic studies. DNA relaxation assay of PKBA revealed inhibition of both topoisomerases I & II. The fragmentation analysis of DNA revealed typical ladders indicating the cytotoxic effect to be mediated by induction of apoptosis. The morphologic studies of PKBA showed the presence of true apoptotic bodies. Apoptosis was confirmed further by flow-cytometric detection of sub-G(1) peaks and enhanced annexin-V-FITC binding of the cells. The activation of apoptotic cascade by PKBA in HL-60 cells was found to be associated with the loss of mitochondrial membrane potential, release of cytochrome c, activation of initiator and executioner caspases and cleavage of poly ADP ribose polymerase (PARP). In vivo studies of PKBA revealed anti-tumoral activity against both ascitic and solid murine tumor models. These studies thus demonstrate PKBA to induce apoptosis in HL-60 cells due to the inhibition of topoisomerases I and II.     

Synthesis and antimicrobial studies of chalconyl pregnenolones

An efficient and facile synthesis of 17-chalconyl derivatives of pregnenolone and their evaluation as antimicrobial agents against various microbial strains is reported. The scheme involves the transformation of the starting pregnenolone acetate into pregnenolone, conversion of pregnenolone to the corresponding chalcone derivatives. The compounds 3a–3j showed significant antimicrobial activity against all microbial strains used for testing.