Synthesis and antiparasitic activity of 1H-benzimidazole derivatives

Compounds 1-18 have been synthesized and tested in vitro against the protozoa Giardia lamblia, Entamoeba histolytica and the helminth Trichinella spiralis. Inhibition of rat brain tubulin polymerization was also measured and compared for each compound. Results indicate that most of the compounds tested were more active as antiprotozoal agents than Metronidazole and Albendazole. None of the compounds was as active as Albendazole against T. spiralis. Although only compounds 3, 9 and 15 (2-methoxycarbonylamino derivatives) inhibited tubulin polymerization, these were not the most potent antiparasitic compounds.     

In vitro and in vivo assays of 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 micro g/ml for macrophages and a few of them maintained this cytotoxicity even at 10 microg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1microg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.     

Aggregation mediated by faeces and footprints in Triatoma pseudomaculata (Heteroptera: Reduviidae), a Chagas disease vector

The behavioural response of Triatoma pseudomaculata to chemical substances present in their faeces or cuticle (footprints) was analyzed. Groups of larvae were simultaneously exposed to a clean filter paper and to another paper impregnated with a chemical stimulus in a circular arena. In these choice experiments, the insects aggregated significantly around papers impregnated with dry faeces. In addition, the bugs also showed a significant aggregation response to papers impregnated with compounds derived from their cuticle that were deposited by contact on the substrate. These results indicate that chemical compounds that affect the behaviour of T. pseudomaculata are present in the faeces and in the cuticle of this species. Results are discussed in relation to chemical communication in the Triatominae, as well as to the potential use of these substances in traps or sensors for the detection of this species.     

In vitro and in vivo anti-Trypanosoma cruzi activity of a novel nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.     

Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the "initiation," "maturation," and "growth" phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.     

c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis

Activation of the caspase cascade is a pivotal step in apoptosis and can occur via death adaptor-mediated homo-oligomerization of initiator procaspases. Here we show that c-FLIP(L), a protease-deficient caspase homolog widely regarded as an apoptosis inhibitor, is enriched in the CD95 death-inducing signaling complex (DISC) and potently promotes procaspase-8 activation through hetero-dimerization. c-FLIP(L) exerts its effect through its protease-like domain, which associates efficiently with the procaspase-8 protease domain and induces the enzymatic activity of the zymogen. Ectopic expression of c-FLIP(L) at physiologically relevant levels enhances procaspase-8 processing in the CD95 DISC and promotes apoptosis, while a decrease of c-FLIP(L) expression results in inhibition of apoptosis. c-FLIP(L) acts as an apoptosis inhibitor only at high ectopic expression levels. Thus, c-FLIP(L) defines a novel type of caspase regulator, distinct from the death adaptors, that can either promote or inhibit apoptosis.     

Trypanosoma cruzi reinfections in mice determine the severity of cardiac damage

In two murine models we studied Trypanosoma cruzi reinfection in the acute and chronic phase of experimental Chagas' disease in order to elucidate the relevance of reinfections in determining the variability of cardiac symptoms and the irreversible cardiac damage. They were followed for 120 and 600 days post infection (p.i.) for the acute and chronic model, respectively. Reinfected mice reached higher parasitaemia levels than infected mice. The survival was 33 and 21% in the chronic phase for mice reinfected in the acute phase and 13% for mice reinfected in the chronic stage at the end of the experiments. Sixty-six percent of the infected group presented electrocardiographic abnormalities (heart frequency, prolonged PQ segment or QRS complex) in the chronic stage whereas 100% of the reinfected animals exhibited electric cardiac dysfunction since 90 and 390 days p.i. for the acute and chronic reinfected model, respectively (P<0.01). Heart histopathological studies showed fibrosis and necrosis areas and mononuclear infiltrates supporting the view that parasite persistence is a major factor in continuing the tissue inflammation. This work shows that T. cruzi reinfections could be related to the variability and severity of the clinical course of Chagas' disease and that parasite persistence is involved in exacerbation of the disease.     

Characterization of aromatase activity in the sea bass: effects of temperature and different catalytic properties of brain and ovarian homogenates and microsomes

Two aromatase genes have been discovered in the brain and ovary of some teleosts. However, data on native aromatase enzyme kinetics and thus actual catalytic activity are scarce in fish, impeding comparison of aromatase activity (AA) from different organs within and between species. In the present study, the tritiated water assay was optimized and validated to measure AA in the sea bass using 1 beta-[3H]-androstenedione as a substrate in crude homogenates and microsomes. Optimized assay variables included pH, temperature, buffer strength, incubation time, amount of fresh tissue, substrate, and cofactor concentration. Specificity of the assay was verified by using known inhibitors, inappropriate substrates, and heat-inactivation. Subcellular fractionation revealed ten-fold more activity in the microsomal over the cytosolic fraction. The assay was also validated by comparing results from the direct product isolation method. The validated assay described allows measurement of AA to levels as low as < 10 fmol/mg protein/hr. Sex differentiation is temperature-dependent in the sea bass. It was found that in the physiological range of temperatures where the sea bass can live, 10-30 degrees C, AA is highly dependent on temperature in a linear fashion (brain: r2 = 0.92; P < 0.001; ovary: r2 = 0.94; P < 0.001). When AA levels from brain and ovarian homogenates obtained from the same fish during the spawning season were compared, the respective Michaelis-Menten constant (Km) values were 7.3 nM vs. 4.6 nM, with no significant differences detected between the two tissues. Thus, sea bass aromatase has a very high affinity for androstenedione, similar to what has been found in goldfish, but much higher than other piscine or mammalian aromatases (30-435 nM). In contrast, the brain maximum reaction rate (Vmax 7.8 pmol/mg protein/hr) was four-fold higher (P < 0.001) than the ovarian Vmax (2.1 pmol/mg protein/hr). Consistent results were found using purified microsomes. Although this is the first time that the kinetic parameters are reported for a native piscine aromatase in two different tissues within the same fish, it remains to be determined whether this is a reflection of two distinct isoforms in this particular species.     

Evolution of trophic types in emperor fishes ( Lethrinus,Lethrinidae, Percoidei) based on cytochrome B gene sequence variation

Three trophic categories exist within emperor fishes, genus Lethrinus, relating to body form and dentition type. One group contains low-bodied, high speed, stalking predators with conical teeth. Another group comprises high-bodied, slow speed carnivores with molariform teeth capable of crushing hard-shelled benthic prey. A third group is also high-bodied but with conical teeth feeding mostly on small or soft-shelled benthic prey. Inferring the evolution of these trophic types within Lethrinus using morphology is problematic since these characters are typically correlated with feeding mode and are potentially homoplasious. We use mitochondrial DNA sequences, to independently determine a phylogenetic hypothesis for Lethrinus, which are not dependent on morphological characters relating to trophic categories. We analyzed complete cytochrome b gene sequences (1140 bp) for 20 species of Lethrinus, representing the three trophic types, and for 13 outgroup species, including four other representatives of the Lethrinidae. A monophyletic Lethrinidae did not resolve, but the monophyly of Lethrinus is well supported. In addition, two major clades within Lethrinus are well supported. One of these clades exclusively contains low-bodied species with conical teeth while the other clade only comprises the high-bodied species with molariform teeth. A high-bodied species with conical teeth, Lethrinus miniatus, appears most ancestral and sister to all other Lethrinus species. We hypothesize that this generalist trophic type was the evolutionary precursor to both of the other primary trophic types.