Effects of t-butyl-4-hydroxyanisole and other phenolic antioxidants on tumoral cells and Trypanosoma parasites

The antioxidant food additives 2(3)-tert-butyl-4-hydroxyanisole (BHA), 2,6-di(tert-butyl)-p-cresol (BHT) and the methyl and propyl esters of gallic acid inhibited Trypanosoma cruzi culture growth and oxygen consumption. The I50 values for growth and oxygen uptake with BHA were 0.284 and 0.400 and for BHT 0.083 and 0.235 mM, respectively. Moreover, BHA inhibited the respiration of several tumor cells, as well as of the procyclic and bloodstream trypomastigote forms of T. brucei brucei, with I50 in the range 0.29-0.52 mM. Inhibition of the parasites' oxygen uptake by BHA was not of the pure Michaelis-Menten type, but may be of a mixed form. It is postulated that these compounds are inhibitors because they resemble ubiquinone.     

In vitro and in vivo studies of Trypanosoma cruzi DNA polymerase

One major DNA polymerase has been purified and characterized from Trypanosoma cruzi. The enzyme has a sedimentation coefficient of 6.8 S corresponding to an approximate molecular weight of 180,000 assuming a globular shape. The enzyme recognizes activated DNA very efficiently, as well as synthetic polydeoxynucleotides, whereas poly rA-dT12 is very poorly utilized. Trypanosoma cruzi DNA polymerase is not inhibited at all by aphidicolin, while araCTP inhibits the enzyme very slightly. The purified enzyme is strongly inhibited by N-ethyl maleimide, dideoxyTTP, ethidium bromide and berenil. All our attempts to find a DNA polymerase sensitive to aphidicolin in vitro have failed, nor have we been able to find a low molecular weight DNA polymerase in this organism. However, when DNA synthesis was studied in whole trypanosomes, aphidicolin was shown to inhibit DNA synthesis more efficiently than ethidium bromide and berenil.     

Role of glutathione in the susceptibility of Trypanosoma cruzi to drugs

1. Glutathione (G-SH) concentration, gamma-glutamyltranspeptidase and glutathione S-transferase activities were studied in several strains of T. cruzi epimastigotes. GSH varied from 1.04 mM for the LQ strain to 0.61 mM for the Tulahuen strain. 2. Cultures of the LQ strain presented more resistance to drugs than those of the Tulahuen. It was necessary a concentration of nifurtimox 4 times higher and one of benznidazole 10 times higher in order to inhibit approximately to 50% the growth of LQ strain cultures when compared with the Tulahuen strain. 3. Buthionine sulfoximine decreased the concentration of glutathione to about 50% in the LQ and Tulahuen strains and potentiated the toxicity of nifurtimox and benznidazole in T. cruzi epimastigote cultures. These results suggest that glutathione is an important factor in the resistance of T. cruzi to nifurtimox and benznidazole.     

Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro.

AY 9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; (C) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 micrograms/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dose-dependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.     

Using genetic variation to disentangle the complex relationship between food intake and health outcomes

Diet is considered as one of the most important modifiable factors influencing human health, but efforts to identify foods or dietary patterns associated with health outcomes often suffer from biases, confounding, and reverse causation. Applying Mendelian randomization in this context may provide evidence to strengthen causality in nutrition research. To this end, we first identified 283 genetic markers associated with dietary intake in 445,779 UK Biobank participants. We then converted these associations into direct genetic effects on food exposures by adjusting them for effects mediated via other traits. The SNPs which did not show evidence of mediation were then used for MR, assessing the association between genetically predicted food choices and other risk factors, health outcomes. We show that using all associated SNPs without omitting those which show evidence of mediation, leads to biases in downstream analyses (genetic correlations, causal inference), similar to those present in observational studies. However, MR analyses using SNPs which have only a direct effect on the exposure on food exposures provided unequivocal evidence of causal associations between specific eating patterns and obesity, blood lipid status, and several other risk factors and health outcomes.     

Attenuation of Eph Receptor Kinase Activation in Cancer Cells by Coexpressed Ephrin Ligands

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting “in trans” with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral “cis” associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.     

Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat

Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949^th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn’t result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value.     

In vitro interaction between ceftazidime and vancomycin/teicoplanin in the presence of azithromycin againstPseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen, intrinsically resistant to many antibiotics and prone to acquire resistance against many drugs. It is assumed that agents that disorganise the structure of the outer membrane might allow the passage of other drugs into cell. To verify this hypothesis, ceftazidime (CAZ) has been tested in association with glycopeptides (GLYs) and azithromycin (AZI). Time-kill experiments were performed on a representative strain. CAZ in combination with GLYs showed 99, 90 and 10% of CFU/ml reduction in 33.9,52.5 and 13.6% of the cases, respectively; the addition of AZI increased the incidence of 99% CFU/ml reduction to 42% of the cases. Indifference was the most common finding, and additive/synergism in the other cases. Present findings demonstrated that CAZ favourably reacted with GLYs in the presence of AZI.     

Evaluation of total reactive antioxidant potential (TRAP) of tissue homogenates and their cytosols

This study evaluates the possibility of obtaining total reactive antioxidant potential (TRAP) indexes in homogenates and their cytosolic fractions by a procedure based on the quenching of luminol luminescence induced by the thermolysis of 2,2'-azo-bis(2-amidinopropane). Measurements were performed in rat brain, liver, kidney, and heart homogenates. TRAP indexes can be easily determined both in homogenates and their cytosolic fractions. The results obtained indicate that heart homogenates are the least and liver homogenates the most protected of the systems considered. Glutathione is the measured antioxidant that contributes the most to TRAP values, while uric acid makes a significant contribution only in liver. A calculation of theoretical TRAP values from the measured concentrations of the main antioxidants (glutathione, uric acid, ascorbic acid, and alpha-tocopherol) for the different homogenates shows that, in most tissues (liver, brain, and kidney), nearly 50% of the experimentally determined TRAP values are not accounted for. This difference is mainly due to the contribution of proteins to the measured TRAP.     

Effects of buthionine sulfoximine nifurtimox and benznidazole upon trypanothione and metallothionein proteins in Trypanosoma cruzi

Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.