Rational design of nitrofuran derivatives: Synthesis and valuation as inhibitors of Trypanosoma cruzi trypanothione reductase.

The rational design and synthesis of a series of 5-nitro-2-furoic acid analogues are presented. The trypanocidal activity against epimastigote forms of Trypanosoma cruzi and the toxic effects on human HeLa cells were tested. Between all synthetic compounds, three of thirteen had an IC50 value in the range of Nfx, but compound 13 exhibited an improved effect with an IC50 of 1.0 ± 0.1 µM and a selective index of 70 in its toxicity against HeLa cells. We analyzed the activity of compounds 8, 12 and 13 to interfere in the central redox metabolic pathway in trypanosomatids, which is dependent of reduced trypanothione as the major pivotal thiol. The three compounds behaved as better inhibitors of trypanothione reductase than Nfx (Ki values of 118 µM, 61 µM and 68 µM for 8, 12 and 13, respectively, compared with 245 µM for Nfx), all following an uncompetitive enzyme inhibition pattern. Docking analysis predicted a binding of inhibitors to the enzyme-substrate complex with binding energy calculated in-silico that supports such molecular interaction.

Efecto del potencial rédox sobre los parámetros del cultivo de Trypanosoma cruzi desarrollado en medio líquido agitado / Effect of redox potential on culture parameters of Trypanosoma cruzi developed in liquid stirred media.

El potencial rédox (Eh) es una propiedad fisicoquímica que presentan los solutos capaces de intercambiar electrones con un electrodo inerte. El potencial rédox influye en el crecimiento bacteriano en forma independiente del oxígeno disuelto. Es escasa la información disponible en relación a cultivos in vitro de protozoarios, en particular de Trypanosoma cruzi. Para determinar el efecto del Eh sobre los parámetros de cultivos, se empleó la cepa Tulahuén 0, desarrollada en medio CIEN líquido en agitación y se ensayaron valores de Eh entre 310 mV (testigo) y 110 mV en 11 réplicas diferentes y duplicadas. Se determinaron pH, velocidad específica de desarrollo (µ), Eh, rH, velocidad de consumo de glucosa y rendimiento. Los resultados muestran que µ varía en forma directa con el Eh. Se establece una alta correlación (r = 0,93; P < 0,01) entre rH [rH = (Eh(V)+ 0,06 pH)/0,03] y µ, manteniendo constante la concentración de oxígeno disuelto. Los otros parámetros del medio no mostraron variaciones significativas. Se concluye que variaciones en el Eh del medio de cultivo afectan en forma significativa la µ del T. cruzi y que es una variable a tener en cuenta cuando se ensayan sustancias con probable efectos tripanocidas.

The redox potential (Eh) is a physico-chemical property presented by solutes able to interchange electrons with an inert electrode. The redox potential influences bacterial growth in an independent way from dissolved oxygen. The available information about protozoaries in vitro grown is scarce, being Trypanosoma cruzi main example. T. cruzi Tulahuén 0 strain, developed in CIEN liquid stirred media, was used to determine the Eh effect on growth parameters. Eh values between 310 mV (reference) and 110 mV were measured in 11 different samples and by duplicate. pH, m, Eh, rH, consume glucose rate and efficiency were determined. Results show that specific rate of development (µ) varies in a direct way with Eh. A high correlation (r = 0.93; P < 0.01) between rH (rH = Eh(V)+ 0.06 pH) and µ was established, even when dissolved oxygen concentration remained constant. Other parameters in the growing medium showed no significant variations. It is concluded that changes on Eh in the medium significantly affect of T. cruzi's growth being a variable to take into account when potential trypanocide substances are analyzed.

[Effect of redox potential on culture parameters of Trypanosoma cruzi developed in liquid stirred media]. / Efecto del potencial rédox sobre los parámetros del cultivo de Trypanosoma cruzi desarrollado en medio líquido agitado.

The redox potential (Eh) is a physico-chemical property presented by solutes able to interchange electrons with an inert electrode. The redox potential influences bacterial growth in an independent way from dissolved oxygen. The available information about protozoans in vitro grown is scarce, being Trypanosoma cruzi main example. T. cruzi Tulahuén 0 strain, developed in CIEN liquid stirred media, was used to determine the Eh effect on growth parameters. Eh values between 310 mV (reference) and 110 mV were measured in 11 different samples and by duplicate. pH, m, Eh, rH, consume glucose rate and efficiency were determined. Results show that specific rate of development (mu) varies in a direct way with Eh. A high correlation (r = 0.93; P < 0.01) between rH (rH = Eh(V) + 0.06 pH) and mu was established, even when dissolved oxygen concentration remained constant. Other parameters in the growing medium showed no significant variations. It is concluded that changes on Eh in the medium significantly affect of T. cruzis growth being a variable to take into account when potential trypanocide substances are analyzed.

Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems.

The amino acid analogue selenomethionine (SeMet) is shown to be efficiently incorporated into recombinant proteins expressed in Escherichia coli grown in a simple minimal medium without the addition of synthetic amino acids. Furthermore, satisfactory SeMet incorporation is obtained with a methionine-prototrophic strain transformed with commonly used vector systems. As examples, purified tryparedoxin 1 from Crithidia fasciculata, alkylhydroperoxide reductase (AhpC) from Mycobacterium marinum and the 16-kDa antigen from M. tuberculosis are shown to be efficiently labelled with SeMet, using the culture conditions and the host/vector systems described here. Enzymatic analysis reveals no differences between native and SeMet-labelled tryparedoxin 1 enzyme. Both proteins yield crystals under similar conditions. The culture conditions and host vector systems described greatly facilitate selenium-labelling of proteins for 3-D structure determination.

Structures of tryparedoxins revealing interaction with trypanothione.

Tryparedoxins (TXNs) catalyse the reduction of peroxiredoxin-type peroxidases by the bis-glutathionyl derivative of spermidine, trypanothione, and are relevant to hydroperoxide detoxification and virulence of trypanosomes. The 3D-structures of the following tryparedoxins are presented: authentic tryparedoxin1 of Crithidia fasciculata, CfTXN1; the his-tagged recombinant protein, CfTXN1H6; reduced and oxidised CfTXN2, and an alternative substrate derivative of the mutein CfTXN2H6-Cys44Ser. Cys41 (Cys40 in TXN1) of the active site motif 40-WCPPCR-45 proved to be the only solvent-exposed redox active residue in CfTXN2. In reduced TXNs, its nucleophilicity is increased by a network of hydrogen bonds. In oxidised TXNs it can be attacked by the thiol of the 1N-glutathionyl residue of trypanothione, as evidenced by the structure of 1N-glutathionylspermidine-derivatised CfTXN2H6-Cys44Ser. Modelling suggests Arg45 (44), Glu73 (72), the Ile110 (109) cis-Pro111 (110)-bond and Arg129 (128) to be involved in the binding of trypanothione to CfTXN2 (CfTXN1). The model of TXN-substrate interaction is consistent with functional characteristics of known and newly designed muteins (CfTXN2H6-Arg129Asp and Glu73Arg) and the 1N-glutathionyl-spermidine binding in the CfTXN2H6-Cys44Ser structure.

His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening.

Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohe L (1997) Biol Chem 378: 827-836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 11 fermentation broth of A(600)2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b( + )/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a k(cat) of 1.7 s(-1) and limiting Km values of 51.8 microM and 1.7 microM for t-butyl hydroperoxide and CfTXN2H6, respectively.

Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi.

Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXN1), and tryparedoxin peroxidase (TXNPx) (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry. Epimastigotes of T. cruzi display strong immunoreactivity with antibodies raised against TXN1 and TXNPx of C. fasciculata. In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T. cruzi (Acc. Nr. AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity. With TXN, TXNPx, trypanothione and TR, T. cruzi possesses all components constituting the crithidial peroxidase system. It is concluded that the antioxidant defense of T. cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism.

Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata.

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.

[Stents: experience at the Cardiology Hospital of the 21st century National Medical Center]. / Stents: Experiencia en el Hospital de Cardiología del Centro Médico Nacional Siglo XXI.

UNLABELLED: From february 1995 to february 1997 we implanted 157 stents in 105 patients. Age ranged from 38 to 81 years (mean 58), there were 83 males and 22 females. In 62.8% cases we implanted one stent and in 39 (37.2%) cases 2 to 6. Eighty three were Palmaz/Schatz (P/S), 27 ACT-ONE, 18 Wiktor (W), 9 Gianturco Rubin (GR), 8 Wallstent, 6 XT-Bard and 6 microstent. Indication were de novo in 23.8%, 87.5% post failure PTCA and in 13.3% late PTCA restenosis. Implant was successful al 96.1% of the patients. The first 32 patients received oral anticoagulation, the last 72 received aspirin and ticlopidine only. COMPLICATIONS: 4.7% acute thrombosis, 0.9% sub-acute thrombosis, three of them (2.5%) developed myocardial infarction, 0.9% emergency surgical treatment, 2.8% vascular complications and death in 2.8%. During follow-up (1 to 18 months, mean 7.7) we repeated angiography and 35 patients two to 14 months (m = 5.6), 12 of them had restenosis, during dilation two cases had dissection of the main left coronary artery and were send to surgery, the others were dilated without complications. One case had restenosis of the stent with obstructive lesions in other vessels and was send to elective surgery. The reminded patients are symptom free and had negative stress test. We conclude that this technology is an excellent alternative to percutaneous myocardial revascularization. Larger trials with long term follow-up is necessary to determine the true incidence of restenosis with the different types of stents.

[The blood bank as a consultation service]. / El banco de sangre como servicio de interconsulta.

This is a 6-month prospective clinical trial which evaluates the importance that blood bank may have as a consultation service for in-patient care. From January 31-July 31 1994, the blood bank physicians gave 108 consults which helped the consulting physician in an adequate diagnostic and therapeutic management of transfusion-related problems. The main reasons for consultation were coagulopathies, suspicion of refractoriness to platelet concentrates, and presence of antibodies against red cell antigens. The main underlying disorders were hematological, surgical, hepatic, renal and rheumatic diseases. We conclude that our blood bank as a consultation service was helpful in improving the transfusional management of our patients.

[Transcatheter closure of patent ductus arteriosus with Rashkind's occluder. Experience during 3.7 years]. / Cierre transcateterismo del conducto arterioso (CTCA) con oclusor de Rashkind. Experiencia de 3.7 años.

Thirty eight transcatheter umbrella closure (TUC) of a persistently patent ductus arteriosus using the Rashkind umbrella occluder (USCI), were attempted in thirty four patients. Mean age 8.5 +/- 9.0 years (range 1.5 to 31). Internal diameter of ductus arteriosus (DA) average 4.0 +/- 1.5 mm (range 2 to 9 mm). Successful implantation was achieved in 36 procedures (94.7%). Complete closure immediately postimplantation was achieved in 15 (45.4%), 12 (36.3%) had trivial and 6 (18.1%) severe residual shunt. In four cases we implanted successfully a second device. At the follow-up (average 24 months) in 31 patients, 87.0% (n = 27) had complete closure and 96.7% (n = 30) did not need another intervention. Device embolization to distal left pulmonary artery occurred in two cases (5.2%), one was retrieved by surgery and in the other case it was allowed to remain. No mortality was observed. This results confirm the efficacy, with low morbidity and no mortality of TUC, especially in those with smaller PDA (< 4 mm).

[Percutaneous transluminal angioplasty of the vena cava inferior]. / Angioplastía transluminal percutánea de la vena cava inferior.

We describe a 65-years-old female, with percutaneous balloon angioplasty (PBA) of inferior vena cava. The patient had massive calcification of the right atrium, the tricuspid anulus as well as obstruction of the tricuspid valve (percutaneous tricuspid valvuloplasty was performed 2.5 years before). The only predisposing factor for calcification was, a cerebral-right atrial shunt (cerebral cysticercosis) for 32 years. PBA was performed. We discuss some events that occurred after dilatation. We describe the technique in this uncommon case. We conclude that percutaneous balloon angioplasty is another alternative to surgical repair in inferior vena cava obstruction.

[Percutaneous mitral commissurotomy with an Inoue catheter in congenital mitral stenosis]. / Comisurotomía mitral percutánea con catéter de Inoue en estenosis mitral congénita.

We describe a 3-years-old boy, with congenital mitral stenosis, who underwent percutaneous transvenous mitral commissurotomy (PTMC) with Inoue balloon. The mitral gradient decreased from 24 to 4 mmHg, the mitral valvular area (MVA) increased from 1.2 to 1.6 cm2 without modification in mitral regurgitation. One year later the recatheterization showed decreased pulmonary pressures, and a 7.5 mmHg mitral valvular gradient without mitral insufficiency. MVA by echo is 1.8 cm2 with mild regurgitation. As far as we know there is no experience with Inoue balloon in children. We conclude that PTMC with Inoue balloon could be an attractive alternative in some patients with congenital mitral stenosis.

[Residual shunts after application of the Rushkind occlusion system in closure of persistent ductus arteriosus. Insertion of a second device]. / Fuga residual postcolocación de oclusor de Rashkind para cierre del conducto arterioso persistente via percutánea. Colocación de un segundo dispositivo.

From May 1991 to August 1994, we performed transcatheter closure of patent ductus arteriosus with Rashkind umbrella system in 25 patients. In one, the device had embolism, to the right pulmonary artery and was operated 24 hs afterwards without complications. Seven had residual shunts, two had subsequent surgery, two are being followed (one for mild and one for moderate residual shunt). In the other three cases we inserted a second device. We used a 12 mm device in two cases and a 17 mm device in the other. Two cases had total occlusion 24 hrs after and in one had mild residual shunt without clinical repercussion. There were no complications. All had electrocardiographic and clinical improvement, take no medication, and their cardiomegaly had decreased (Cardiac index pre 61 vs post 54%). In conclusion the insertion of a second device is safe and effective alternative for residual shunt after first Rashkind occluder.

Anticarsia gemmatalis nuclear polyhedrosis virus replication in serum-free and serum-reduced insect cell cultures.

In order to develop a financially feasible process to produce Anticarsia gemmatalis Nuclear Polyhedrosis virus in cell culture, we developed a lipidic supplement to replace fetal calf serum in insect cell culture media. The supplement, prepared with an extract of lipids from hen egg yolk, allowed us to reduce the contents of serum in the culture medium from 10% to 1%. IPLB-Sf-21 cells could be kept along consecutive passages in serum-reduced medium. The replication of AgNPV in HEYLE-supplemented cultures was evaluated. Extracellular virions production was the same as in FCS-supplemented-cultures, but the production level of polyhedral inclusion bodies was significantly lowered in HEYLE-supplemented cultures. The reduced production of PIBs is related to a premature releasing of non-occluded particles as well as to a reduced synthesis of polyhedrin protein.