The camptothecin-resistant topoisomerase I mutant F361S is cross-resistant to antitumor rebeccamycin derivatives. A model for topoisomerase I inhibition by indolocarbazoles.

DNA topoisomerase I is a major cellular target for antitumor indolocarbazole derivatives (IND) such as the antibiotic rebeccamycin and the synthetic analogue NB-506 which is undergoing phase I clinical trials. We have investigated the mechanism of topoisomerase I inhibition by a rebeccamycin analogue, R-3, using the wild-type human topoisomerase I and a well-characterized recombinant enzyme, F361S. The catalytic activity of this mutant remains fully intact, but the enzyme is resistant to inhibition by camptothecin (CPT). Here we show that the mutated enzyme is cross-resistant to the rebeccamycin analogue. Despite their profound structural differences, CPT and R-3 interfere similarly with the activity of the wild-type and mutant topoisomerase I enzymes, and the drug-induced cleavable complexes are equally sensitive to the NaCl concentration. CPT and IND likely recognize identical structural elements of the topoisomerase I-DNA covalent complex; however, differences do exist in terms of sequence-specificity of topoisomerase I-mediated DNA cleavage. For the first time, a molecular model showing that CPT and IND share common steric and electronic features is proposed. The model helps to identify a specific pharmacophore for topoisomerase I inhibitors.     

Macromolecular and enzymatic abnormalities induced by a synthetic pyrethroid,Ripcord (Cypermethrin), in adult beetles of a stored grain pest,Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae)

The effects of a synthetic pyrethroid insecticide, cypermethrin, administered as a formulation Ripcord 25EC (emulsified concentrate), to adult beetles of a stored grain pest, Tribolium castaneum, have been studied, with an objective to ascertain its toxicity on enzymes such as carbohydrases, phosphatases, dehydrogenases, aminotransferases, and concentration of various biochemical components such as monosaccharides, glycogen, cholesterol, nucleic acids, urea, total lipids, and total proteins. Almost all the enzymes and biochemical components were sensitive to sublethal doses of Ripcord 25 EC and these effects were found to be dependent on the duration of treatment. All carbohydrate metabolizing enzymes (amylase, invertase, lactase, maltase, lactate dehydrogenase) were elevated, except for trehalase, which was also elevated up to day 3 but returned to normal levels subsequently. Phosphatases (alkaline as well as acidic) were increased first and decreased thereafter, while isocitrate dehydrogenase decreased throughout the experimental period. Transaminases (aspartate aminotransferase and alanine aminotransferase) showed a decreasing trend. Of the other biochemical components tested, glucose content decreased during the first 3 days but increased subsequently. Fructose content showed an increase, while the glycogen content decreased throughout the study. Total lipid content was not disturbed up to day 3 but increased thereafter. Cholesterol content was depleted by day 7. Total proteins started decreasing from day 3 onwards, while soluble proteins were not affected. DNA, RNA, and urea contents exhibited elevated levels, while uric acid showed a decreasing trend. Sublethal doses of Ripcord, therefore, resulted in extensive enzyme induction, and utilization of carbohydrates, proteins, and lipids, in the given order, perhaps to produce extra energy to combat insecticidal stress. Arch. Insect Biochem. Physiol. 39:144–154, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis and evaluation of modified chalcone based p53 stabilizing agents

Tumor suppressor protein p53 induces cell cycle arrest and apoptotic cell death in response to various cellular stresses thereby preventing cancer development. Activation and stabilization of p53 through small organic molecules is, therefore, an attractive approach for the treatment of cancers retaining wild-type p53. In this context, a series of nineteen chalcones with various substitution patterns of functional groups including chloro, fluoro, methoxy, nitro, benzyloxy, 4-methyl benzyloxy was prepared using Claisen-Schmidt condensation. The compounds were characterized using NMR, HRMS, IR and melting points. Evaluation of synthesized compounds against human colorectal (HCT116) and breast (CAL-51) cancer cell lines revealed potent antiproliferative activities. Nine compounds displayed GI₅₀ values in the low micromolar to submicromolar range; for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (SSE14108) showed GI₅₀ of 0.473 ± 0.043 µM against HCT116 cells. Further analysis of these compounds revealed that (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (SSE14105) and (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) caused rapid (4 and 8-h post-treatment) accumulation of p53 in HCT116 cells similar to its induction by positive control, Nutlin-3. Such activities were absent in 3-(4-methoxyphenyl)propiophenone (SSE14106H2) demonstrating the importance of conjugated ketone for antiproliferative and p53 stabilizing activity of the chalcones. We further evaluated p53 levels in the presence of cycloheximide (CHX) and the results showed that the p53 stabilization was regulated at post-translational level through blockage of its degradation. These chalcones can, therefore, act as fragment leads for further structure optimization to obtain more potent p53 stabilizing agents with enhanced anti-proliferative activities.     

Activation of adrenergic receptor in H9c2 cardiac myoblasts co-stimulates Nox2 and the derived ROS mediate the downstream responses

Isorhamnetin, a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L., is well known for its anti-inflammatory, anti-oxidative, anti-adipogenic, anti-proliferative, and anti-tumor activities. However, the role of isorhamnetin in cardiac hypertrophy has not been reported. The aims of the present study were to find whether isorhamnetin could alleviate cardiac hypertrophy and to define the underlying molecular mechanisms. Here, we investigated the effects of isorhamnetin (100 mg/kg/day) on cardiac hypertrophy induced by aortic banding in mice. Cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. Our data demonstrated that isorhamnetin could inhibit cardiac hypertrophy and fibrosis 8 weeks after aortic banding. The results further revealed that the effect of isorhamnetin on cardiac hypertrophy was mediated by blocking the activation of phosphatidylinositol 3-kinase–AKT signaling pathway. In vitro studies performed in neonatal rat cardiomyocytes confirmed that isorhamnetin could attenuate cardiomyocyte hypertrophy induced by angiotensin II, which was associated with phosphatidylinositol 3-kinase–AKT signaling pathway. In conclusion, these data indicate for the first time that isorhamnetin has protective potential for targeting cardiac hypertrophy by blocking the phosphatidylinositol 3-kinase–AKT signaling pathway. Thus, our study suggests that isorhamnetin may represent a potential therapeutic strategy for the treatment of cardiac hypertrophy and heart failure.     

CFD assessment of the effect of convective mass transport on the intracellular clearance of intracellular triglycerides in macrosteatotic hepatocytes

Donor livers available to transplant for patients with end-stage liver disease are in severe shortage. One possible avenue to expand the donor pool is to recondition livers that would be otherwise discarded due to excessive fat content. Severely steatotic livers (also known as fatty livers) are highly susceptible to ischemia-reperfusion injury and as a result, primary liver non-function post-transplantation. Prior studies in isolated perfused rat livers suggest that “defatting” may be possible in a timeframe of a few hours; thus, it is conceivable that fatty liver grafts could be recovered by machine perfusion to clear stored fat from the organ prior to transplantation. However, studies using hepatoma cells and adult hepatocytes made fatty in culture report that defatting may take several days. Because cell culture studies were done in static conditions, we hypothesized that the defatting kinetics are highly sensitive to flow-mediated transport of metabolites. To investigate this question, we experimentally evaluated the effect of increasing flow rate on the defatting kinetics of cultured HepG2 cells and developed an in silico combined reaction-transport model to identify possible rate-limiting steps in the defatting process. We found that in cultured fatty HepG2 cells, the time required to clear stored fat down to lean control cells can be reduced from 48 to 4–6 h by switching from static to flow conditions. The flow required resulted in a fluid shear of .008 Pa, which did not adversely affect hepatic function. The reaction-transport model suggests that the transport of l-carnitine, which is the carrier responsible for taking free fatty acids into the mitochondria, is the key rate-limiting process in defatting that was modulated by flow. Therefore, we can ensure higher levels of l-carnitine uptake by the cells by choosing flow rates that minimize the limiting mass transport while minimizing shear stress.     

Tropical soils degraded by slash‐and‐burn cultivation can be recultivated when amended with ashes and compost

In many tropical regions, slash‐and‐burn agriculture is considered as a driver of deforestation; the forest is converted into agricultural land by cutting and burning the trees. However, the fields are abandoned after few years because of yield decrease and weed invasion. Consequently, new surfaces are regularly cleared from the primary forest. We propose a reclamation strategy for abandoned fields allowing and sustaining re‐cultivation. In the dry region of south‐western Madagascar, we tested, according to a split‐plot design, an alternative selective slash‐and‐burn cultivation technique coupled with compost amendment on 30–year‐old abandoned fields. Corn plants (Zea mays L.) were grown on four different types of soil amendments: no amendment (control), compost, ashes (as in traditional slash‐and‐burn cultivation), and compost + ashes additions. Furthermore, two tree cover treatments were applied: 0% tree cover (as in traditional slash‐and‐burn cultivation) and 50% tree cover (selective slash‐and‐burn). Both corn growth and soil fertility parameters were monitored during the growing season 2015 up to final harvest. The amendment compost + ashes strongly increased corn yield, which was multiplied by 4–5 in comparison with ashes or compost alone, reaching 1.5 t/ha compared to 0.25 and 0.35 t/ha for ashes and compost, respectively. On control plots, yield was negligible as expected on these degraded soils. Structural equation modeling evidenced that compost and ashes were complementary fertilizing pathways promoting soil fertility through positive effects on soil moisture, pH, organic matter, and microbial activity. Concerning the tree cover treatment, yield was reduced on shaded plots (50% tree cover) compared to sunny plots (0% tree cover) for all soil amendments, except ashes. To conclude, our results provide empirical evidence on the potential of recultivating tropical degraded soils with compost and ashes. This would help mitigating deforestation of the primary forest by increasing lifespan of agricultural lands.     

Oxidative stress and neurological disorders in relation to blood lead levels in children

Oxidative stress plays a pivotal role in the pathogenesis of neurological disorders. Free radical generation appears to be the mode of lead toxicity. We evaluated the effects of blood lead levels on oxidative stress parameters in children suffering from neurological disorders. Thirty children (aged 3-12 years) with neurological disorders (cerebral palsy [n = 12], seizures [n = 11], and encephalopathy [n = 7]) were recruited in the study group. Sixty healthy children (aged 3-12 years) from similar socio-economic environments and not suffering from any chronic disease were taken as the controls. Blood lead levels and oxidant/antioxidant status were determined. Mean blood lead level was significantly higher while delta-aminolevulinic acid dehydratase (delta-ALAD) activity, a biomarker for lead exposure, was significantly lower in the study group as compared to the control group (P < 0.05 for each). Malondialdehyde (MDA) levels, an end-product of lipid peroxidation, were significantly higher while the antioxidant glutathione (GSH) levels were significantly lower in the study group as compared to the control group (P < 0.05 for each). Activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were significantly higher in the study group than those of the control group (P < 0.05 for each). There were significant negative correlations of blood lead levels with delta-ALAD (r = -0.35; P < 0.05) and GSH (r = -0.31; P < 0.05), and positive correlations with MDA (r = 0.37; P < 0.05), SOD (r = 0.53; P < 0.05), and CAT (r = 0.31; P < 0.05). In turn, delta-ALAD had significant negative correlations with MDA (r = -0.29; P < 0.05), SOD (r = -0.28; P < 0.05) and CAT (r = -0.34; P < 0.05), but positive correlation with GSH (r = 0.32; P < 0.05). Although a causal pathway can not be determined from the present study, our findings indicate lead-induced oxidative stress in blood of children with neurological disorders. Lead-induced oxidative stress as an underlying mechanism for neurological diseases in children warranted further investigation.     

The 3MRA Risk Assessment Framework—A Flexible Approach for Performing Multimedia,Multipathway, and Multireceptor Risk Assessments Under Uncertainty

A flexible framework for conducting nationwide multimedia, multipathway and multireceptor risk assessments (3MRA) under uncertainty was developed to estimate protective chemical concentration limits in a source area. The framework consists of two components: risk assessment and uncertainty analysis. The risk component utilizes linked source, fate/transport, exposure and risk assessment models to estimate the risk exposures for the receptors of concern. Both human and ecological receptors are included in the risk assessment framework. The flexibility of the framework is based on its ability to address problems varying in spatial scales from site-specific to regional and even national levels; and its ability to accommodate varying types of source, fate/transport, exposure and risk assessment models. The uncertainty component of the 3MRA framework is based on a two-stage Monte Carlo methodology. It allows the calculation of uncertainty in risk estimates, and the incorporation of the effects of uncertainty on the determination of regulatory concentration limits as a function of variability and uncertainty in input data, as well as potential errors in fate and transport and risk and exposure models. The framework can be adapted to handle a wide range of multimedia risk assessment problems. Two examples are presented to illustrate its use, and to demonstrate how regulatory decisions can be structured to incorporate the uncertainty in risk estimates.     

Association of a Distinct Begomovirus and a Betasatellite with Leaf Curl Symptoms in Pedilanthus tithymaloides

Pedilanthus tithymaloides (Redbird flower) is an ornamental shrub that occasionally exhibits leaf curl and enation symptoms in Pakistan. Symptoms were shown to be associated with a monopartite begomovirus and a betasatellite. The complete nucleotide sequence of the begomovirus was found to be 2764 nucleotides in length and have the highest nucleotide sequence identity to a begomovirus previously isolated from tomato (90.3% nucleotide sequence identity), followed by Radish leaf curl virus (86.3%). The complete betasatellite sequence was determined to be 1358 nucleotides in length and has the highest sequence identity (97%) with Tobacco leaf curl betasatellite . The analysis shows the begomovirus associated with leaf curl disease of Pedilanthus to be a distinct and previously unreported begomovirus for which the name Pedilanthus leaf curl virus (PedLCV) is proposed. This virus is one of an increasing number of monopartite begomoviruses shown to be associated with a betasatellite.     

Concentration dependent growth/non-growth linked kinetics of endosulfan biodegradation by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

The rates of biodegradation of endosulfan by P. aeruginosa were determined with different initial endosulfan concentrations (10, 50, 100, 150, 200 and 250 mg l⁻¹) and different growth linked kinetic models were fitted at these concentrations. At 10 mg endosulfan l⁻¹, Monod no growth model was well fitted. Monod with growth model described the biodegradation pattern at an initial concentration of 50, 100 and 150 mg endosulfan l⁻¹. Significant increases of P. aeruginosa MN2B14 density in broth culture during incubation further support this result. Conversely, zero order kinetic model was well fitted into the biodegradation data if initial endosulfan concentration was ≥200 mg endosulfan l⁻¹. The kinetics of endosulfan biodegradation by P. aeruginosa MN2B14 in liquid broth was highly dependent upon its initial concentration. The results of this study could be employed for predicting the persistence of endosulfan in water environment containing P. aeruginosa as an endosulfan degrading bacterium.