Imidazole-containing phthalazine derivatives inhibit Fe-SOD performance in Leishmania species and are active in vitro against visceral and mucosal leishmaniasis.

The in vitro leishmanicidal activity of a series of imidazole-containing phthalazine derivatives 1-4 was tested on Leishmania infantum, Leishmania braziliensis and Leishmania donovani parasites, and their cytotoxicity on J774·2 macrophage cells was also measured. All compounds tested showed selectivity indexes higher than that of the reference drug glucantime for the three Leishmania species, and the less bulky monoalkylamino substituted derivatives 2 and 4 were clearly more effective than their bisalkylamino substituted counterparts 1 and 3. Both infection rate measures and ultrastructural alterations studies confirmed that 2 and 4 were highly leishmanicidal and induced extensive parasite cell damage. Modifications to the excretion products of parasites treated with 2 and 4 were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds 2 and 4 were potent inhibitors of iron superoxide dismutase enzyme (Fe-SOD) in the three species considered, whereas their impact on human CuZn-SOD was low. Molecular modelling suggests that 2 and 4 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the antioxidant features of the enzyme.

In vitro leishmanicidal activity of pyrazole-containing polyamine macrocycles which inhibit the Fe-SOD enzyme of Leishmania infantum and Leishmania braziliensis species.

The in vitro leishmanicidal activity and cytotoxicity of pyrazole-containing macrocyclic polyamines 1-4 was assayed on Leishmania infantum and Leishmania braziliensis species. Compounds 1-4 were more active and less toxic than glucantime and both infection rates and ultrastructural alterations confirmed that 1 and 2 were highly leishmanicidal and induced extensive parasite cell damage. Modifications in the excretion products of parasites treated with 1-3 were also consistent with substantial cytoplasm alterations. Compound 2 was highlighted as a potent inhibitor of Fe-SOD in both species, whereas its effect on human CuZn-SOD was poor. Molecular modelling suggested that 2 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the enzyme`s antioxidant features.

Leishmania spp. epidemiology of canine leishmaniasis in the Yucatan Peninsula.

Canine Leishmaniasis is widespread in various Mexican states, where different species of Leishmania have been isolated from dogs. In the present study, we describe the detection of L. braziliensis, L. infantum, and L. mexicana in serum of dogs from the states of Yucatan and Quintana Roo in the Yucatan Peninsula (Mexico). A total of 412 sera were analyzed by ELISA using the total extract of the parasite and the iron superoxide dismutase excreted by different trypanosomatids as antigens. We found the prevalence of L. braziliensis to be 7.52%, L. infantum to be 6.07%, and L. mexicana to be 20.63%, in the dog population studied. The results obtained with ELISA using iron superoxide dismutase as the antigen were confirmed by western blot analysis with its greater sensitivity, and the agreement between the two techniques was very high.

Colloidal Nanosilica Treatments for Sealing Cracks in Mortar.

Presence of microcracks in concrete can diminish the service life of a structure. The injection of materials for filling the crack is proposed for facing this problem. The traditional materials used for sealing cracks present some drawbacks, such as the difficulties of inorganic materials for flowing to all the depth of the crack and the lack of compatibility with the cementitious matrix in the case of organic materials. In this work, the injection of colloidal nanosilica dispersed in water is proposed for filling microcracks in mortars. The effect of the injection procedure on the sealing performance of the colloidal nanosilica has been assessed. The ability of colloidal nanosilica for penetrating through the crack and its posterior gelification-solidification inside the crack after a curing period have been confirmed. The microscopic analysis of a cross-section of the crack indicates that the sealing ability of the nanosilica seems to be not only due to the filling of the crack but also to chemical interactions with the cementitious phases of the surrounding crack sides.

Seropositivity for Trypanosoma cruzi and Leishmania mexicana in dogs from a metropolitan region of Central Mexico.

Trypanosoma cruzi and Leishmania mexicana are parasites of humans and other mammals, causing American Trypanosomiasis and Cutaneous Leishmaniasis, respectively. Domestic dogs are considered key hosts for these parasites in the domicile and peridomicile cycles of transmission, due to their abundance and contact with human population. In Mexico, there are few studies that involve the study of infection with these parasites in dogs, and have only been carried out mainly in the endemic areas for these diseases. In the state of Querétaro (Mexico), infections with both parasites have been reported for dogs only from rural areas, with no records for the metropolitan zone. We analyzed the seropositivity to T. cruzi and L. mexicana in dogs from localities within of the metropolitan zone of Querétaro City in order to determine if these animals are exposed to these parasites and thus, could be an important part of the transmission cycle of these trypanosomatids in a densely populated urban region within the state of Querétaro, Mexico. Serum samples were collected from 303 dogs housed in the Animal Control centers of the municipalities of Querétaro and El Marques, analyzed by indirect ELISA and Western Blot using as an antigen the Iron Superoxide Dismutase (FeSODe) of the parasites. From the total serum samples, we detected 10.2% of seropositivity for T. cruzi and 2.9% for L. mexicana. Our results represent the first evidence of infection with T. cruzi in domestic dogs from the Metropolitan Zone of Querétaro, and the first record for L. mexicana in Central Mexico. Ongoing investigations seek to confirm the circulation of these parasites in the area to evaluate the risk associated to the human population.

Intestinal parasitism in the animals of the zoological garden "Peña Escrita" (Almuñecar, Spain).

Gastrointestinal parasites cause serious diarrhoea in captive animals. Therefore, we have undertaken this study to establish programmes to prevent, control, and treat intestinal parasitism in the animals of the zoological garden "Peña Escrita" of Almuñecar (Granada). An annual survey was conduced to estimate the occurrence of gastrointestinal parasites and the seasonality of this parasitism. Between June 2006 and May 2007, 432 samples were collected from primates, carnivores, perissoodactyla, artiodactyla, rodentia, diprotodontia, galliformes, anseriformes and struthioniformes. One or more intestinal parasites were identified in 72.5% of the animals. The most frequent pathogenic endoparasites were Eimeria spp. (17.3%), Trichuris spp. (5.1%), Strongyloides spp. (4.5%), Cyclospora spp. (4.5%), Cryptosporidium spp. (3.2%) and Isospora spp. (2.6%). Iodamoeba butschlii, Parascaris equorum and Trichuris spp. did not vary with season and Cryptosporidium spp., Dicrocoelium dendriticum, Metastrongylus spp. and Cylicospirura spp. appeared exclusively in Artiodactyla. Multiple parasitic infections were common, 70% of animals presented with at least two parasites (maximum=6). The most frequent cases of multiple parasitism were Eimeria spp. plus Blastocystis spp. and Eimeria spp. plus Nematodirus spp., in the last case the animals presented explosive diarrhoea. In accord with our results, after each sampling, some of the affected animals were treated and the corresponding programmes of prevention and control were designed.

In vitro leishmanicidal and trypanocidal evaluation and magnetic properties of 7-amino-1,2,4-triazolo[1,5-a]pyrimidine Cu(II) complexes.

Two triazolopyrimidine complexes have been obtained from reaction between 7-amino-1,2,4-triazolo[1,5-a]pyrimidine (7atp) and Cu (II) salts. Crystal structures of [Cu2(µ-7atp)4Cl2]Cl2·4H2O (1) and [Cu2(µ-7atp)4(H2O)2](NO3)4·H2O (2) have been studied by X-ray diffraction methods and characterized by spectroscopic and thermal analysis. Magnetic studies of these dinuclear complexes have revealed the existence of moderate antiferromagnetic interactions between the copper ions, with J values of -91.2 and -96.1cm-1 respectively. It must be highlighted that the antiparasitic activity of these new complexes has been studied in vitro against three different strains of leishmania spp. and Trypanosoma cruzi, showing a higher efficacy than the 7atp ligand and the reference commercial drugs.

Effect of alkyl-lysophospholipids on some aspects of the metabolism of Leishmania donovani.

Alkyl-lysophospholipids (ALPs), developed initially to be antitumor agents, have proved highly effective in the treatment of visceral leishmaniasis, a disease caused by the species making up the protozoan complex Leishmania donovani. Although their effectiveness is known, the mode of action against this parasite is not completely understood. In the present work, we have studied the effect of 3 derivatives, edelfosine, miltefosine, and ilmofosine. Using nuclear magnetic resonance spectroscopy ('H-NMR), we have examined the excreted catabolites from glucose metabolism in the promastigote forms treated with these compounds. The ALPs at concentrations of 19 and 38 microM inhibit the excretion of acetate, succinate, and pyruvate. The effect of edelfosine, miltefosine, and ilmofosine on the activity of the enzymes hexokinase, glycerolkinase 3-PD, phosphoglucose isomerase, superoxide dismutase, and phospholipase C were also examined. Glycerolkinase 3-PD and phosphoglucose isomerase are generally insensitive to the compounds, whereas hexokinase and superoxide dismutase are inhibited by miltefosine and ilmofosine. The ALPs exhibited an activated effect against the phospholipase C activity. Alkyl-lysophospholipids were shown to have a significant effect on several enzymes in important biochemical pathways indispensable for the survival of L. donovani promasigotes.

Antiparasitic activity against trypanosomatid diseases and novel metal complexes derived from the first time characterized 5-phenyl-1,2,4-triazolo[1,5-a]pyrimidi-7(4H)-one.

A serie of isostructural complexes with general formula [M(ftpO)2(H2O)4] have been obtained from reaction between the first time characterized triazolopyrimidine derivative 5-phenyl-1,2,4-triazolo[1,5-a]pyrimidi-7(4H)-one (HftpO) (1) and first row transition nitrates (M=Cu (2), Co (3), Ni (4) and Zn (5)). A copper complex with formula [Cu(HftpO)2(NO3)2(H2O)2]·H2O (6) was also isolated. HftpO and their metal complexes have been characterized by spectroscopic and thermal analysis and their crystal structures have been solved by X-ray diffraction methods. The isostructural compounds are mononuclear complexes where the triazolopyrimidine ligand acts as monodentate ligand through N3 nitrogen position. The crystal structure of these novel bis-5-phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one-tetraaquo metal complexes offers an excellent opportunity at these complexes to acts as potential building blocks. Also, the antiparasitic activity of HftpO ligand against different leishmania and trypanosome strains has been studied.

[Proteus penneri]. / Proteus penneri.

Proteus penneri, formerly P. vulgaris biogroup 1, was recognized as a new species in 1982. This species is associated with clinical processes similar to those involving P. mirabilis and P. vulgaris and expresses similar pathogenic determinants. In clinical samples, P. penneri is mainly isolated from urine (50%), wound and soft tissue exudates (25%), and blood cultures (15%), mostly of nosocomial origin. Although P. penneri is easy to identify, it can be misidentified as P. vulgaris by automatic systems that do not include the indol test result in the identification process. This species has a characteristic susceptibility profile, essentially due to the production of the chromosomal inducible beta-lactamase HugA, which presents a high homology (86%) with CumA from P. vulgaris. HugA is inhibited by clavulanic acid and determines resistance to aminopenicillins and first- and second-generation cephalosporins, including cefuroxime, but does not affect cephamycins or carbapenems, and is inhibited by clavulanic acid. HugA is derepressed due to mutational processes in gene regulators, affecting the activity of cefotaxime and, to a much lesser extent, that of ceftazidime and aztreonam. This phenotype resembles the production of an extended spectrum beta-lactamase. Like other Proteus species, P. penneri is resistant to tetracyclines and should be considered resistant to nitrofurantoin.

Prospects of an alternative treatment against Trypanosoma cruzi based on abietic acid derivatives show promising results in Balb/c mouse model.

Chagas disease, caused by the protozoa parasite Trypanosoma cruzi, is an example of extended parasitaemia with unmet medical needs. Current treatments based on old-featured benznidazole (Bz) and nifurtimox are expensive and do not fulfil the criteria of effectiveness, and a lack of toxicity devoid to modern drugs. In this work, a group of abietic acid derivatives that are chemically stable and well characterised were introduced as candidates for the treatment of Chagas disease. In vitro and in vivo assays were performed in order to test the effectiveness of these compounds. Finally, those which showed the best activity underwent additional studies in order to elucidate the possible mechanism of action. In vitro results indicated that some compounds have low toxicity (i.e. >150 µM, against Vero cell) combined with high efficacy (i.e. <20 µM) against some forms of T. cruzi. Further in vivo studies on mice models confirmed the expectations of improvements in infected mice. In vivo tests on the acute phase gave parasitaemia inhibition values higher those of Bz, and a remarkable decrease in the reactivation of parasitaemia was found in the chronic phase after immunosuppression of the mice treated with one of the compounds. The morphological alterations found in treated parasites with our derivatives confirmed extensive damage; energetic metabolism disturbances were also registered by (1)H NMR. The demonstrated in vivo activity and low toxicity, together with the use of affordable starting products and the lack of synthetic complexity, put these abietic acid derivatives in a remarkable position toward the development of an anti-Chagasic agent.

Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias.

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.

Activity of cefotaxime and amikacin against 14,272 gram-negative bacteria from clinical samples in the period 1980 to 1985.

The sensitivity to cefotaxime and amikacin of 14,272 Gram-negative bacilli (Enterobacteriaceae and non-fermenting Gram-negative bacilli) isolated from clinical samples was studied during the period 1980 to 1985. The minimum inhibitory concentration (MIC) was determined by means of diffusion in agar. Strains were considered resistant to cefotaxime and amikacin if the MIC values were greater than 16 mg/L and greater than 8 mg/L, respectively. The MIC90 reached the critical value for cefotaxime in the case of Citrobacter spp., Escherichia coli, Klebsiella spp., Proteus mirabilis, Salmonella spp. and Shigella spp., and for amikacin in the case of Citrobacter spp., Enterobacter spp., E. coli, Klebsiella spp., P. mirabilis, Proteus vulgaris, Salmonella spp. and Serratia spp. Only Shigella spp. were sensitive to cefotaxime but not to amikacin, and only strains of Enterobacter spp. and Serratia spp. were sensitive to amikacin but not to cefotaxime.

Cu-Zn-superoxide dismutase activity in Moniezia expansa: inhibition by pyrimidine derivatives.

Copper-zinc, cyanide-sensitive superoxide dismutase (Cu-Zn-SOD) was detected in homogenates of Moniezia expansa. The enzyme was purified by a sequence of multiple differential centrifugations, ammonium sulphate precipitation, ion-exchange and G-75 Sephadex column chromatography. The final enzyme preparation had a specific activity of 623.00 +/- 9.97U per mg protein and, after isolation, a single-staining band on acrylamide-SDS gels was detected which coincided with enzyme activity. The inhibitory activities of several benzimidazoles and several novel pyrimidine derivatives were determined on purified extracts of the M. expansa Cu-Zn-SOD. The results indicated that the percentage inhibition of Cu-Zn-SOD by some pyrimidine derivatives (6-amino-1, 3-dimethyl-5-nitroso-uracil, 6-amino-5-methyl-5-nitroso-uracil and 5-amino-uracil) was markedly higher than inhibition with the benzimidazoles.

Antimicrobial resistance in recent fecal enterococci from healthy volunteers and food handlers in Spain: genes and phenotypes.

Susceptibility patterns to 15 different antibiotics and the presence of resistance genes were evaluated in recent fecal Enterococcus isolates recovered from 42 healthy volunteers (HV) and 43 food-handlers (FH). A total of 142 Enterococcus faecalis, 74 Enterococcus faecium, and 23 Enterococcus spp. with different antibiotic susceptibility patterns were studied. A higher percentage of resistance for moxifloxacin, erythromycin, glycopeptides and high-level resistance (HLR) to gentamicin were observed in the FH group. Ampicillin- or linezolid-resistant isolates were not recovered in any of the groups. The tet(M) gene was found in 96% and in 85% of tetracycline-resistant isolates from HV and FH, respectively. HLR-kanamycin was mediated by aph(3')-IIIa, or aac(6')-aph(2"), or both genes in all isolates from HV group and in 86% from FH group. The aac(6')-aph(2") gene was found in all HLR-gentamicin isolates. Ninety-one percent of HV and 71% of FH erythromycin-resistant isolates harbored the erm(B) gene (erythromycin MIC range of 8-128 microg/ml), whereas erm(A), erm(C), or mef(A) genes were not detected. Coexistence of erm(B), aph(3')-IIIa, and tet(M) genes was observed in 17% of the isolates of both groups. The HLR-gentamicin isolates presented unrelated PFGE patterns while 2 out of 3 vanA E. faecium isolates showed an indistinguishable SmaI-pulsed-field gel electrophoresis (PFGE) pattern. This study shows that despite 4 years of official banning of antibiotic growth promoters in animals, enterococci isolated from FH are more resistant than those from HV. This suggests the permanence of resistant clones or transferable resistance elements in farms and a possible exchange between food products and humans, or eventually the long-term permanence of certain clones in the FH intestinal tract.

Metabolic studies by 1H NMR of different forms of Trypanosoma cruzi as obtained by 'in vitro' culture.

By culturing Trypanosoma cruzi epimastigotes in modified Grace's medium with 10% foetal bovine serum, a significant quantity of metacyclic forms could be obtained. Transformation was observed after 8 days of culture, with metacyclic forms reaching 75%. Cultured Vero cells were infected with metacyclic forms and maintained until free-amastigote forms were obtained. Additionally, amastigote-like forms could be obtained by subjecting metacyclic cultures to heat shock. Parasites were grown with glucose as the major carbon source. The metabolites produced and excreted during culture were identified by difference proton nuclear magnetic resonance spectroscopy and quantified by enzymatic methods. The final products of glucose catabolism differed not only quantitatively but also qualitatively for the three major life-cycle stages of T. cruzi. The end products of metabolism produced by epimastigote forms were mainly acetate and pyruvate and, to a lesser extend, L-alanine and ethanol. Differences between epimastigotes and metacyclic forms were only quantitative. However, free amastigotes as well as amastigote-like forms, excreted acetate, glycerol, and pyruvate and to a lesser extent succinate, but no L-alanine or ethanol.

Copper-zinc superoxide dismutase from Ascaris suum (Nematoda): purification and characterization.

Copper-zinc superoxide dismutase from Ascaris suum (Nematoda) was purified in a new, more efficient, and faster manner. The process included differential centrifugation, fractionation with ammonium sulfate, and sodium dodecyl sulfate-polyacrylamide electrophoresis, yielding a 340-fold purification (specific activity of 47 units/mg). Optimal storage conditions, optimal pH range, thermostability, molecular weight and ultraviolet-visible absorption spectrum of the enzyme are described, and a new enzymatic model for pharmacological screening is suggested.

Biochemical and ultrastructural alterations caused by newly synthesized 1,2,4-triazole[1,5a]pyrimidine derivatives against Phytomonas staheli (Trypanosomatidae).

Six compounds, all newly synthesized triazole-pyrimidine derivatives that proved inhibitory of in in vitro growth of epimastigotes in Trypanosoma cruzi and of promastigotes of Leishmania donovani and Phytomonas staheli, were studied to investigate their toxic effects. As a biological model, the plant trypanosome P. staheli, which causes sudden wilt in the oil palm and Hartrot in the coconut palm, was used. The six compounds markedly inhibited macromolecule synthesis (nucleic acids and proteins) by the parasite. The cells treated with these compounds present severe damage in their ultrastructure-intense 'vacuolization, and appearance of lysosomes as well as other residual bodies. The mitochondrial section appeared larger in size. with a swollen matrix. In addition, these compounds changed the excretion of end metabolites, primarily affecting ethanol and acetate excretion, possibly by directly influencing certain enzymes (alcohol dehydrogenase and acetate synthetase) or their synthesis. 2000 Elsevier Science Ltd.

Isoenzyme patterns of phosphatases and esterases in Fasciola hepatica and Dicrocoelium dendriticum.

A study was made of alkaline and acid phosphatase isoenzymes and non-specific esterases in homogenates of the trematodes Fasciola hepatica and Dicrocoelium dendriticum obtained from infected tissues of Capra hircus and Ovis aries using horizontal polyacrylamide gel electrophoresis. The esterase patterns in F. hepatica and D. dendriticum were different. Homogenate of F. hepatica from both hosts gave four enzyme bands. For D. dendriticum, however, while homogenates of parasites from C. hircus also gave four bands, those of O. aries gave only three bands. Acid and alkaline phosphatases in homogenates of both parasites showed three enzyme bands, but there was a host species difference between the enzyme patterns of specimens collected from C. hircus versus O. aries.