Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Iminodiacetyl-hydroxamate derivatives as metalloproteinase inhibitors: equilibrium complexation studies with Cu(II), Zn(II) and Ni(II)

Two new iminodiacetyl-hydroxamate derivatives, the N-benzyl-N-carboxymethyl-iminoacetohydroxamic acid (H(2)L(1)) and the N-benzyl-N'-hydroxypiperazine-2,6-dione (HL(2)), have been recently reported as very effective inhibitors against a set of zinc-containing matrix metalloproteinases (MMPs). Herein, aimed at understanding that inhibitory function, these compounds are studied in their complex formation equilibria with three biologically relevant first-row transition M(2+) metal ions (M=Cu, Zn, Ni) by using potentiometric and spectroscopic techniques. At physiological conditions, complexation of these metal ions by H(2)L(1) mostly occurs with formation of 1:1 species by tridentate co-ordination (O,N,N) (carboxylate-amino-hydroxamate), whereas complexation with HL(2) mainly involves the formation of 1:2 (M:L) species with normal (O,O) hydroxamate coordination. Moreover, at higher pH, H(2)L(1) is able to form a pentanuclear tetrameric copper complex with an interesting 12-metallacrown-4 structure.     

New cytogenetic aberrations found in a case of aggressive retinoblastoma

We describe a case of retinoblastoma with an atypical presentation and previously unreported cytogenetic aberrations. A 19-month-old girl with left intraocular retinoblastoma was treated with enucleation and chemotherapy. The disease showed aggressive evolution within a short period between diagnosis and relapse. Eight months after diagnosis, a new large tumor was present in the orbit of the right eye, with diffuse bone pain, pancytopenia and diffuse infiltration into the bone marrow and the central nervous system. The child did not respond to treatment and died. Cytogenetic studies made with G-banding, FISH and SKY analysis showed chromosomal aberrations commonly associated with retinoblastoma, including del(13q), i(6p), +1, and monosomy 16, along with others that had not been reported previously, including dup(5q), dic(15;22) and add(14q). The new chromosomal aberrations may be related to the aggressiveness of the disease in this case.     

A phenomenological computational model of the evoked action potential fitted to human cochlear implant responses

There is a growing interest in biomedical engineering in developing procedures that provide accurate simulations of the neural response to electrical stimulus produced by implants. Moreover, recent research focuses on models that take into account individual patient characteristics.We present a phenomenological computational model that is customized with the patient’s data provided by the electrically evoked compound action potential (ECAP) for simulating the neural response to electrical stimulus produced by the electrodes of cochlear implants (CIs). The model links the input currents of the electrodes to the simulated ECAP.Potentials and currents are calculated by solving the quasi-static approximation of the Maxwell equations with the finite element method (FEM). In ECAPs recording, an active electrode generates a current that elicits action potentials in the surrounding auditory nerve fibers (ANFs). The sum of these action potentials is registered by other nearby electrode. Our computational model emulates this phenomenon introducing a set of line current sources replacing the ANFs by a set of virtual neurons (VNs). To fit the ECAP amplitudes we assign a suitable weight to each VN related with the probability of an ANF to be excited. This probability is expressed by a cumulative beta distribution parameterized by two shape parameters that are calculated by means of a differential evolution algorithm (DE). Being the weights function of the current density, any change in the design of the CI affecting the current density produces changes in the weights and, therefore, in the simulated ECAP, which confers to our model a predictive capacity.The results of the validation with ECAP data from two patients are presented, achieving a satisfactory fit of the experimental data with those provided by the proposed computational model.     

Stress-Induced Chloroplast Degradation in Arabidopsis Is Regulated via a Process Independent of Autophagy and Senescence-Associated Vacuoles

Two well-known pathways for the degradation of chloroplast proteins are via autophagy and senescence-associated vacuoles. Here, we describe a third pathway that was activated by senescence- and abiotic stress-induced expression of Arabidopsis thaliana CV (for chloroplast vesiculation). After targeting to the chloroplast, CV destabilized the chloroplast, inducing the formation of vesicles. CV-containing vesicles carrying stromal proteins, envelope membrane proteins, and thylakoid membrane proteins were released from the chloroplasts and mobilized to the vacuole for proteolysis. Overexpression of CV caused chloroplast degradation and premature leaf senescence, whereas silencing CV delayed chloroplast turnover and senescence induced by abiotic stress. Transgenic CV-silenced plants displayed enhanced tolerance to drought, salinity, and oxidative stress. Immunoprecipitation and bimolecular fluorescence complementation assays demonstrated that CV interacted with photosystem II subunit PsbO1 in vivo through a C-terminal domain that is highly conserved in the plant kingdom. Collectively, our work indicated that CV plays a crucial role in stress-induced chloroplast disruption and mediates a third pathway for chloroplast degradation. From a biotechnological perspective, silencing of CV offers a suitable strategy for the generation of transgenic crops with increased tolerance to abiotic stress.     

NMR characterization of a DNA duplex containing the major acrolein-derived deoxyguanosine adduct gamma -OH-1,-N2-propano-2'-deoxyguanosine

The environmental and endogenous mutagen acrolein reacts with cellular DNA to produce several isomeric 1,N(2)-propanodeoxyguanosine adducts. High resolution NMR spectroscopy was used to establish the structural features of the major acrolein-derived adduct, gamma-OH-1,N(2)-propano-2'-deoxyguanosine. In aqueous solution, this adduct was shown to assume a ring-closed form. In contrast, when gamma-OH-1,N(2)-propano-2'-deoxyguanosine pairs with dC at the center of an 11-mer oligodeoxynucleotide duplex, the exocyclic ring opens, enabling the modified base to participate in a standard Watson-Crick base pairing alignment. Analysis of the duplex spectra reveals a regular right-handed helical structure with all residues adopting an anti orientation around the glycosidic torsion angle and Watson-Crick alignments for all base pairs. We conclude from this study that formation of duplex DNA triggers the hydrolytic conversion of gamma-OH-1,N(2)-propano-2'-deoxyguanosine to an open chain form, a structure that facilitates pairing with dC during DNA replication and accounts for the surprising lack of mutagenicity associated with this DNA adduct.     

Strongyloidiasis and Infective Dermatitis Alter Human T Lymphotropic Virus-1 Clonality in vivo

Human T-lymphotropic Virus-1 (HTLV-1) is a retrovirus that persists lifelong by driving clonal proliferation of infected T-cells. HTLV-1 causes a neuroinflammatory disease and adult T-cell leukemia/lymphoma. Strongyloidiasis, a gastrointestinal infection by the helminth Strongyloides stercoralis, and Infective Dermatitis associated with HTLV-1 (IDH), appear to be risk factors for the development of HTLV-1 related diseases. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the HTLV-1-infected T-cell population (i.e. the number of distinct clones and abundance of each clone). A newly developed biodiversity estimator called “DivE” was used to estimate the total number of clones in the blood. We found that the major determinant of proviral load in all subjects without leukemia/lymphoma was the total number of HTLV-1-infected clones. Nevertheless, the significantly higher proviral load in patients with strongyloidiasis or IDH was due to an increase in the mean clone abundance, not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone. In patients co-infected with Strongyloides there was an increased degree of oligoclonal expansion and a higher rate of turnover (i.e. appearance and disappearance) of HTLV-1-infected clones. In Strongyloides co-infected patients and those with IDH, proliferation of the most abundant HTLV-1⁺ T-cell clones is independent of the genomic environment of the provirus, in sharp contrast to patients with HTLV-1 infection alone. This implies that new selection forces are driving oligoclonal proliferation in Strongyloides co-infection and IDH. We conclude that strongyloidiasis and IDH increase the risk of development of HTLV-1-associated diseases by increasing the rate of infection of new clones and the abundance of existing HTLV-1⁺ clones.     

Different responses of young and expanded lettuce leaves to fungicide Mancozeb: chlorophyll fluorescence,lipid peroxidation,pigments and proline content

This work aimed to study the effects of commercial doses of the fungicide, Mancozeb, on the photosynthetic apparatus of lettuce young leaves (YL) and expanded leaves (EL). Seven days after Mancozeb application, chlorophyll a fluorescence, pigment contents, lipid peroxidation, and proline content were evaluated. Independently of leaf age, Mancozeb treatment reduced the efficiency of photosystem II photochemistry, increased the nonphotochemical quenching and proline content, decreased pigment contents, and induced lipid peroxidation. Moreover, EL showed a more stable photosynthetic apparatus, less prone to oxidative damages compared with YL. The parameters measured proved to be good markers for the rapid and preliminary diagnosis of fungicide toxicity.     

The effect of culture preservation techniques on patulin and citrinin production by Penicillium expansum Link

AIMS: To study the influence of culture preservation methods and culture conditions on the production of the mycotoxins patulin and citrinin by Penicillium expansum. METHODS AND RESULTS: Ten strains of Penicillium expansum were preserved using subculture and maintenance at 4 degrees C, mineral oil, drying on silica gel and freeze-drying. Patulin and citrinin production was assessed on yeast extract sucrose agar (YES) and grape juice agar (GJ), using TLC before and after 0.5, 2-3, 6 and 12 months preservation. Citrinin was detected in all cultures for all preservation techniques on YES. The patulin profiles obtained differed with strain and culture media used. CONCLUSIONS: Citrinin production seems to be a stable character for the tested strains. There is a tendency for patulin detection with time apparently more consistent for silica gel storage and freeze-drying, especially when the strains are grown on GJ. SIGNIFICANCE AND IMPACT OF THE STUDY: Variability in the profiles of the mycotoxins tested seems to be more strain-specific than dependent on the preservation technique used.     

Hypochlorous acid-induced heme degradation from lactoperoxidase as a novel mechanism of free iron release and tissue injury in inflammatory diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H(2)O(2)) in the airways through its ability to oxidize thiocyanate (SCN(-)) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 μM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O(2), Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN(-). On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function.