Disulfiram Suppresses Growth of the Malignant Pleural Mesothelioma Cells in Part by Inducing Apoptosis

Dithiocarbamate compound Disulfiram (DSF) that binds with copper and functions as an inhibitor of aldehyde dehydrogenase is a Food and Drug Administration approved agent for treatment of alcoholism. Copper complexed DSF (DSF-Cu) also possesses anti-tumor and chemosensitizing properties; however, its molecular mechanisms of action remain unclear. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of DSF-Cu and the molecular mechanisms involved. DSF-Cu inhibited growth of the murine as well as human MPM cells in part by increasing levels of ubiquitinated proteins. DSF-Cu exposure stimulated apoptosis in MPM cells that involved activation of stress-activated protein kinases (SAPKs) p38 and JNK1/2, caspase-3, and cleavage of poly-(ADP-ribose)-polymerase, as well as increased expression of sulfatase 1 and apoptosis transducing CARP-1/CCAR1 protein. Gene-array based analyses revealed that DSF-Cu suppressed cell growth and metastasis-promoting genes including matrix metallopeptidase 3 and 10. DSF inhibited MPM cell growth and survival by upregulating cell cycle inhibitor p27Kip1, IGFBP7, and inhibitors of NF-κB such as ABIN 1 and 2 and Inhibitory κB (IκB)α and β proteins. DSF-Cu promoted cleavage of vimentin, as well as serine-phosphorylation and lysine-63 linked ubiquitination of podoplanin. Administration of 50 mg/kg DSF-Cu by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo. Although podoplanin expression often correlates with metastatic disease and poor prognosis, phosphorylation of serines in cytoplasmic domain of podoplanin has recently been shown to interfere with cellular motility and migration signaling. Post-translational modification of podoplanin and cleavage of vimentin by DSF-Cu underscore a metastasis inhibitory property of this agent and together with our in vivo studies underscore its potential as an anti-MPM agent.     

Evaluation of amoebicidal potential of Paneth cell cryptdin-2 against Entamoeba histolytica

Background

Amoebiasis is a major public health problem in tropical and subtropical countries. Currently, metronidazole is the gold choice medication for the treatment of this disease. However, reports have indicated towards the possibility of development of metronidazole-resistance in Entamoeba strains in near future. In view of the emergence of this possibility, in addition to the associated side effects and mutagenic ability of the currently available anti-amoebic drugs, there is a need to explore newer therapeutics against this disease. In this context, the present study evaluated the amoebicidal potential of cryptdin-2 against E. histolytica.

Methods/Principal Findings

In the present study, cryptdin-2 exhibited potent in-vitro amoebicidal activity against E. histolytica in a concentration dependent manner at a minimum amoebicidal concentration (MAC) of 4 mg/L. Scanning electron microscopy as well as phase contrast microscopic investigations of cryptdin-2 treated trophozoites revealed that the peptide was able to induce significant morphological alterations in terms of membrane wrinkling, leakage of the cytoplasmic contents and damaged plasma membrane suggesting a possible membrane dependent amoebicidal activity. N-phenyl napthylamine (NPN) uptake assay in presence of sulethal, lethal as well as twice the lethal concentrations further confirmed the membrane-dependent mode of action of cryptdin-2 and suggested that the peptide could permeabilize the plasma membrane of E. histolytica. It was also found that cryptdin-2 interfered with DNA, RNA as well as protein synthesis of E. histolytica exerting the highest effect against DNA synthesis. Thus, the macromolecular synthesis studies correlated well with the observations of membrane permeabilization studies.

Significance/Conclusions

The amoebicidal efficacy of cryptdin-2 suggests that it may be exploited as a promising option to combat amoebiasis or, at least, may act as an adjunct to metronidazole and/or other available anti-amoebic drugs.     

Catechin suppresses an array of signalling molecules and modulates alcohol-induced endotoxin mediated liver injury in a rat model

Induction of nuclear factor kappa B (NF-κB)-mediated gene expression has been implicated in the pathogenesis of alcoholic liver disease through enhanced production of reactive oxygen species and pro-inflammatory mediators. The present study was carried out to investigate the role of catechin as a chain breaking inhibitor against experimental alcoholic liver injury. Rats were administered 35% v/v ethanol orally at a dose of 10 g/Kg/day for two weeks, followed by 14 g/Kg/day for 10 weeks. Catechin (50 mg/Kg) was co-supplemented after 4 weeks of alcohol treatment till the end of the dosing period. Following chronic alcohol exposure, rats developed endotoxemia and severe pathological changes in the liver such as pronounced fatty change, vacuolar degeneration and inflammation. These changes were accompanied by activation of NF-κB and induction of inflammatory and cytotoxic mediators leading to increased level of tumor necrosis factor-alpha, enhanced formation of malondialdehyde in the liver followed by drastic alterations in the hepatic antioxidant defense systems. Additionally, nitrite levels and lactate dehydrogenase activities were also significantly elevated on chronic alcohol consumption. Alcohol exposure also increased the number of micronucleated cells indicating that alcohol abuse may again be associated with the nuclear changes. Supplementation with catechin ameliorated the alcohol-induced liver injury by downregulating the endotoxin-mediated activation of initial signalling molecule NF-κB and further going downstream the signalling cascade including tumor necrosis factor-alpha, nitric oxide and reactive oxygen species and by enhancing the antioxidant profile. These observations correlated well with the histological findings. Moreover, a remarkable decrease in the percentage of micronucleated cells was observed with catechin supplementation indicating an apparent protection against alcohol-induced toxicity. These findings suggest that catechin may alleviate experimental alcoholic liver disease by suppressing induction of NF-κB, a key component of signalling pathway, thus forming a pharmacological basis for designing novel therapeutic agents against alcohol induced endotoxin-mediated liver injury.     

Piezoelectrically driven vertical cavity acoustic transducers for the convective transport and rapid detection of DNA and protein binding to DNA microarrays with SPR imaging--a parametric study

The identification of pathogenic bacteria in water is important for addressing preventive and treatment issues regarding health and safety. A highly sensitive and specific solid-phase sandwich ELISA procedure was developed for the detection of typhoid causing extremely lethal water borne pathogen Salmonella typhi (S. typhi) on modified isopore polycarbonate (PC) black membranes. PC membranes were chemically derivatized to generate amino groups on the surface maintaining their pysico-optico properties. Surface modified PC membranes were characterized by ATR-FTIR spectrometer, goniometer and scanning electron microscope. Polyclonal somatic 'O' type antibodies (Abs) against whole cell S. typhi were immobilized on them by following the amine glutaraldehyde chemistry. Antibody immobilized membranes captured S. typhi from buffer solution and this complex was detected colourimetrically using HRP labelled S. typhi Ab. A detection limit of 2×10(3)cells/ml of bacteria was achieved with the modified PC membranes without any pre-enrichment step as against 10(6)-10(7)CFU/ml of bacteria by typical ELISA method. The assay was demonstrated to be specific for the target bacteria when compared with other cross-reactant water borne pathogens. The intra- and inter-assay precision for 10(4) and 10(5)cells/ml was 5.3-7.4 and 10.3-19.7% respectively. The developed immunoassay for the detection of S. typhi is simple, easy to handle, sensitive specific, reproducible and cost effective in comparison with the commercially existing immunochromatographic assays.     

In vitro conservation of Bacopa monnieri (L.) using mineral oil

Bacopa monnieri (L.) Wettst., a traditional Indian medicinal plant with high commercial potential, is used as a potent nervine tonic. A slow growth protocol was developed for medium-term conservation using mineral oil (MO) overlay. Nodal segments of B. monnieri (two genotypes; IC249250, IC468878) were conserved using MO for 24?months. Single node explants were implanted on MS medium supplemented with 0.2?mg?l^?1 BA and were covered with MO. Subculture duration could be significantly enhanced from 6 to 24?months, on the above medium. Normal plants regenerated from conserved cultures were successfully established in soil. On the basis of 20 random amplified polymorphic DNA and 5 inter-simple sequence repeat primers analyses and bacoside A content using HPLC, no significant reproducible variation was observed between the controls and in vitro-conserved plants. The results demonstrate the feasibility of using MO for medium-term conservation of B. monnieri germplasm without any adverse genetical and biochemical effects.     

Hemochromatosis: Evaluation of the dietary iron model and regulation of hepcidin

Our knowledge of iron homeostasis has increased steadily over the last two decades; much of this has been made possible through the study of animal models of iron-related disease. Analysis of transgenic mice with deletions or perturbations in genes known to be involved in systemic or local regulation of iron metabolism has been particularly informative. The effect of these genes on iron accumulation and hepcidin regulation is traditionally compared with wildtype mice fed a high iron diet, most often a 2% carbonyl iron diet. Recent studies have indicated that a very high iron diet could be detrimental to the health of the mice and could potentially affect homeostasis of other metals, for example zinc and copper. We analyzed mice fed a diet containing either 0.25%, 0.5%, 1% or 2% carbonyl iron for two weeks and compared them with mice on a control diet. Our results indicate that a 0.25% carbonyl iron diet is sufficient to induce maximal hepatic hepcidin response. Importantly these results also demonstrate that in a chronic setting of iron administration, the amount of excess hepatic iron may not further influence hepcidin regulation and that expression of hepcidin plateaus at lower hepatic iron levels. These studies provide further insights into the regulation of this important hormone.     

A Comparison of Complex Electron Donors for Anaerobic Dechlorination of PCE

The potential of sugar, flour, corn steep liquor, molasses, non-fat milk, and whey to serve as electron donors for anaerobic dechlorination of tetrachloroethene (PCE) was examined. The electron donors were compared based on acclimation time, the extent of PCE dechlorination achieved, the minimum electron donor dose necessary to achieve PCE removal, and unit cost. The time required to achieve routine dechlorination of PCE (to any daughter product) for each donor was (in days): corn steep liquor (10), milk (10), whey (10), methanol (12), molasses (14), sugar (26), flour (30). Ethene production was achieved by milk-, whey-, and methanol-fed cultures, whereas the other donors did not facilitate ethene production over a 135-day period. Corn steep liquor-, whey-, molasses-, and sugar-fed cultures needed five times the stoichiometric amount (e.g., donor per eq PCE to ethene) to facilitate PCE conversion to dichloroethene (DCE). Cultures fed milk and flour needed 20 times the stoichiometric amount, and methanol-fed cultures required 50 times the stoichiometric amount, perhaps due to competition from methanogenic organisms. Minimum laboratory-scale electron donor costs to achieve stoichiometric conversion of PCE to DCE are ($ per pound [lb] PCE) whey (0.04), molasses (0.07), milk (0.14), corn steep liquor (0.19), sugar (0.38), methanol (0.58), and flour (1.30).     

Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum.     

Comparative Mycotoxin Profiles of Gibberella zeae Populations from Barley, Wheat, Potatoes, and Sugar Beets

Gibberella zeae is one of the most devastating pathogens of barley and wheat in the United States. The fungus also infects noncereal crops, such as potatoes and sugar beets, and the genetic relationships among barley, wheat, potato, and sugar beet isolates indicate high levels of similarity. However, little is known about the toxigenic potential of G. zeae isolates from potatoes and sugar beets. A total of 336 isolates of G. zeae from barley, wheat, potatoes, and sugar beets were collected and analyzed by TRI (trichothecene biosynthesis gene)-based PCR assays. To verify the TRI-based PCR detection of genetic markers by chemical analysis, 45 representative isolates were grown in rice cultures for 28 days and 15 trichothecenes and 2 zearalenone (ZEA) analogs were quantified using gas chromatography-mass spectrometry. TRI-based PCR assays revealed that all isolates had the deoxynivalenol (DON) marker. The frequencies of isolates with the 15-acetyl-deoxynivalenol (15-ADON) marker were higher than those of isolates with the 3-acetyl-deoxynivalenol (3-ADON) marker among isolates from all four crops. Fusarium head blight (FHB)-resistant wheat cultivars had little or no influence on the diversity of isolates associated with the 3-ADON and 15-ADON markers. However, the frequency of isolates with the 3-ADON marker among isolates from the Langdon, ND, sampling site was higher than those among isolates from the Carrington and Minot, ND, sites. In chemical analyses, DON, 3-ADON, 15-ADON, b-ZEA, and ZEA were detected. All isolates produced DON (1 to 782 μg/g) and ZEA (1 to 623 μg/g). These findings may be useful for monitoring mycotoxin contamination and for formulating FHB management strategies for these crops.