Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

We investigated whether the folate receptor α-isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR ≫ OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0° and 37°C. The amount of 5-methyl[³H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl[³H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[³H]THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake. J. Cell. Biochem. 65:479–491. © 1997 Wiley-Liss Inc.     

A mathematical model of iron chelation with desferrioxamine

Removal of body iron is a severe clinical problem in patients affected by hemochromatosis or iron-loading anemias. Desferrioxamine (DFO) is the most potent artificial iron-chelating agent. This work deals with the problem of describing DFO action in man by means of a mathematical model, formulated on the basis of the current knowledge about DFO and iron metabolism. Since most clinical data are concerned with DFO-induced urinary iron excretion, only this process, which involves iron stored in reticuloendothelial (RE) cells, was described in detail. Most of the parameters of the model were estimated from data obtained by studying the single processes involved in iron chelation with DFO. A computer simulation study then allowed us to assign meaningful values to the remaining parameters. Different DFO treatments were simulated, and the results obtained seem to show us that the model can reproduce the main experimental findings and the relationship between the amount of chelated iron and the iron status of the patient.     

Spectroscopic Determination of Lysozyme Conformational Changes in the Presence of Trehalose and Guanidine

The bioprotective action of the disaccharide trehalose has been studied against the well-known denaturating agent, guanidine hydrochloride. The results indicated a direct influence of trehalose on both enzymatic activity and conformational changes of lysozyme, as shown by the decrease of the inactivation rate constant of about 1.48-fold and the loss of α-helix structure of lysozyme. In addition, ESI–MS hydrogen–deuterium (H/D) exchange experiments allowed us to correlate the structural and dynamic features of the protein in the presence of the two additives, highlighting as trehalose remarkably influenced this exchange by decreasing local protein environment changes and solvent accessibility to the amide peptide backbone, as further evidenced by circular dichroism and ¹H NMR measurements.     

Breakpoint determination of 15 large deletions in Peutz–Jeghers subjects

The Peutz–Jeghers Syndrome (PJS) is an autosomal dominant polyposis disorder with increased risk of multiple cancers. STK11/LKB1 (hereafter named STK11) germline mutations account for the large majority of PJS cases whereas large deletions account for about 30% of the cases. We report here the first thorough molecular characterization of 15 large deletions identified in a cohort of 51 clinically well-characterized PJS patients. The deletions were identified by MLPA analysis and characterized by custom CGH-array and quantitative PCR to define their boundaries. The deletions, ranging from 2.9 to 180 kb, removed one or more loci contiguous to the STK11 gene in six patients, while partial STK11 gene deletions were present in the remaining nine cases. By means of DNA sequencing, we were able to precisely characterize the breakpoints in each case. Of the 30 breakpoints, 16 were located in Alu elements, revealing non-allelic homologous recombination (NAHR) as the putative mechanism for the deletions of the STK11 gene, which lays in a region with high Alu density. In the remaining cases, other mechanisms could be hypothesized, such as microhomology-mediated end-joining (MMEJ) or non-homologous end-joining (NHEJ). In conclusion we here demonstrated the non-random occurrence of large deletions associated with PJS. All our patients had a classical PJS phenotype, which shows that haploinsufficiency for SBNO2, C19orf26, ATP5D, MIDN, C19orf23, CIRBP, C19orf24,and EFNA2, does not apparently affect their clinical phenotype.     

Metabolite of tryptophan promoting changes in EEG signal and the oxidative status of the brain

Tryptophan is an essential amino acid precursor of neurotransmitter serotonin and triptamine. During its metabolism, indole‐3‐acetic acid (IAA) is generated; this substance presents both antioxidant and prooxidant effects in different biological systems in addition to hipoglicemic effects. To date, electroencephalography (EEG) has been used to evaluate the temporal effect of several substances in neurotransmission. The goal of this study was to characterize the effect of IAA in the brain by analysing the EEG signal and evaluate the oxidative status by means of biochemical parameters. The EEG was acquired by using a noninvasive method, and the brain electric signal was analysed by advanced digital signal processing techniques to determinate the energy signal filtered in different band frequencies. Furthermore, the oxidative status of the brain was investigated by measuring the activity of antioxidant enzymes and lipid peroxidation as well as blood glucose rates of the animals treated with different doses of IAA. Our results showed the relationship of IAA administration with changes in EEG signals. The oxidative status of the brain was modified by IAA after 14 days of treatment. Copyright © 2014 John Wiley & Sons, Ltd.     

Multiple refugia and glacial expansions in the Tucumane–Bolivian Yungas: The phylogeography and potential distribution modeling of Calomys fecundus (Thomas, 1926) (Rodentia: Cricetidae)

The Yungas, a subtropical mountain rainforest of South America, has been little studied in relation to the evolutionary history of the large-bodied species of the genus Calomys. Particularly, two species have been synonymized: C. boliviae and C. fecundus; the first is only known from its type locality in the northern Bolivian Yungas, whereas the second is known along the Tucumane–Bolivian Yungas shared by Bolivia and Argentina. In this study, we combined a phylogeographic approach with ecological niche modeling, with samples covering most of the geographic range of C. fecundus. One mitochondrial and two nuclear genes were used for population genetic analyses. Current and paleoclimatic models were obtained. Nuclear genes resulted uninformative by retention of ancestral polymorphism with other species of Calomys. The mitochondrial marker revealed a complex network showing signals of several population expansions. Three genetic clusters in a latitudinal sense were detected, which are coincident with the three stable climatic zones estimated by current and paleoclimatic models. We determined a pattern of expansion during glacial cycles and ancestral refugia during interglacial cycles. None of the potential distribution models predicted the presence of C. fecundus in the type locality of C. boliviae. Therefore, we recommend making integrative taxonomic studies in the Bolivian Yungas, to determine whether or not C. fecundus and C. boliviae correspond to the same species.     

Killer Cell Immunoglobulin-like Receptors and Their HLA Ligands are Related with the Immunopathology of Chagas Disease

The aim of this study was to investigate the influence of killer cell immunoglobulin-like receptor (KIR) genes and their human leucocyte antigen (HLA) ligands in the susceptibility of chronic Chagas disease. This case-control study enrolled 131 serologically-diagnosed Chagas disease patients (59 men and 72 women, mean age of 60.4 ± 9.8 years) treated at the University Hospital of Londrina and the Chagas Disease Laboratory of the State University of Maringa. A control group was formed of 165 healthy individuals - spouses of patients or blood donors from the Regional Blood Bank in Maringa (84 men and 81 women, with a mean age of 59.0 ± 11.4 years). Genotyping of HLA and KIR was performed by PCR-SSOP. KIR2DS2-C1 in the absence of KIR2DL2 (KIR2DS2⁺/2DL2^-/C1⁺) was more frequent in Chagas patients (P = 0.020; Pc = 0.040; OR = 2.14) and, in particular, those who manifested chronic chagasic cardiopathy—CCC (P = 0.0002; Pc = 0.0004; OR = 6.64; 95% CI = 2.30–18.60) when compared to the control group, and when CCC group was compared to the patients without heart involvement (P = 0.010; Pc = 0.020; OR = 3.97). The combination pair KIR2DS2⁺/2DL2^-/KIR2DL3⁺/C1⁺ was also positively associated with chronic chagasic cardiopathy. KIR2DL2 and KIR2DS2 were related to immunopathogenesis in Chagas disease. The combination of KIR2DS2 activating receptor with C1 ligand, in the absence of KIR2DL2, may be related to a risk factor in the chronic Chagas disease and chronic chagasic cardiopathy.