BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth of Trypanosoma brucei.

RNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 in Trypanosoma brucei (TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics. In silico analysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of five α-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms of T. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

Síndrome hepatopulmonar en niños: evaluación y tratamiento / Hepatopulmonary syndrome in children: Evaluation and treatment

El síndrome hepatopulmonar (SHP) es una enfermedad poco frecuente que afecta a pacientes de cualquier edad con enfermedad hepática aguda o crónica. Su diagnóstico se basa en la presencia de hipoxemia y la demostración de un cortocircuito pulmonar mediante ecocardiografía con contraste o gammagrafía de perfusión pulmonar. La angiografía es de utilidad para detectar fístulas macroscópicas. Se presentan 5 casos clínicos de edad pediátrica con diferente historia natural y evolución. A 2 de éstos se les diagnosticó SHP tipo 1, a otros 2 de tipo 2 y al quinto de tipo 1 y 2, lo que condicionó un enfoque terapéutico diferente en cada uno de los casos (AU)

Hepatopulmonary syndrome is a rare disease that affects patients of any age with acute or chronic liver disease. Its diagnosis is based on the presence of hypoxemia and the demonstration of an intrapulmonary shunting by echocardiography with contrast or perfusion lung scanning. Pulmonary angiography is useful to demonstrate macroscopic arteriovenous communications. We describe five paediatric cases with a different natural history and evolution. Two of them were diagnosed with hepatopulmonary syndrome type 1, another two with type 2 and a fifth one with type 1 and 2, which required a different therapeutic approach in each case (AU)

[Hepatopulmonary syndrome in children: evaluation and treatment]. / Síndrome hepatopulmonar en niños: evaluación y tratamiento.

Hepatopulmonary syndrome is a rare disease that affects patients of any age with acute or chronic liver disease. Its diagnosis is based on the presence of hypoxemia and the demonstration of an intrapulmonary shunting by echocardiography with contrast or perfusion lung scanning. Pulmonary angiography is useful to demonstrate macroscopic arteriovenous communications. We describe five paediatric cases with a different natural history and evolution. Two of them were diagnosed with hepatopulmonary syndrome type 1, another two with type 2 and a fifth one with type 1 and 2, which required a different therapeutic approach in each case.

[Biliary atresia and polysplenia syndrome: outcome and growth post-transplantation]. / Atresia de vías biliares y síndrome de poliesplenia: evolución y crecimiento postrasplante.

Biliary atresia (BA) is the most common indication for liver transplantation in children. Approximately 7-10% of these patients have the associated polysplenia syndrome (PS). The prognosis of patients with BA and PS has been reported to be poorer than that in patients with BA without PS. All patients who underwent liver transplantation for BA and who still attend periodic controls at the outpatient clinic were considered. A retrospective study of outcome and growth in children with BA was made, and compared with a subgroup of patients with BA and PS. There were no significant differences on complications, liver and renal function tests, lipids and growth data. We concluded that BA and PS do not preclude successful liver transplantation.

Hematologic abnormalities in liver-transplanted children during medium- to long-term follow-up.

OBJECTIVE: Hematologic abnormalities as adverse effects related to immunosuppressive drugs in liver-transplanted children are rarely reported. We have observed anemia, neutropenia, and thrombocytopenia in our pediatric liver-transplant population. The aim of this study was to exclude all suspected etiologies to define the association of immunosuppressants with these abnormalities. METHODS: Patients under 18 years old who still attend periodic controls at liver-transplant outpatient clinics were considered. Seventy patients met the inclusion criteria, 36 girls and 34 boys. Mean patient age was 5.6 years (range: 7 months to 17 years) and mean follow-up 6 years (range: 1-10 years). Medical records were reviewed beginning 1 month posttransplant. Treatment exposures, irradiation, blood product administration, and all laboratory studies were reviewed. When a hematologic abnormality was detected, we recorded the management for its resolution, the clinical response to therapy and the length of treatment. RESULTS: Twenty-five of the 70 children suffered 26 abnormal hematologic episodes (anemia 14, neutropenia 2, thrombocytopenia 3, simultaneous anemia and neutropenia 5, and pancytopenia 2). Eleven episodes (42%) had unclear etiologies and the process of elimination suggested an association with the immunosuppressant. Switching immunosuppressant was required in four patients and dose reduction in seven. CONCLUSIONS: Hematologic abnormalities in liver-transplanted children are common. The etiology is readily attributable to several causes. When the immunosuppressant appears to be a possible cause, the first step is dose reduction. If the hematologic abnormality persists despite dose reduction, a trial switch may be required.

[First orthotopic liver transplantation in patient with biliary atresia and situs inversus in spain]. / Primer trasplante hepático en un paciente con atresia de vías biliares y situs inversus en España.

Biliary atresia is the most common indication for liver transplantation in the pediatric age group. The Kasai portoenterostomy has become established as the primary treatment for biliary atresia. If portoenterostomy fails, death before 2 years of age is likely without liver transplantation. The most common multiple malformation syndrome associated with biliary atresia is polysplenia syndrome, which forms a constellation of defects of body symmetry, splenic development and vascular anomalies, including situs inversus, polysplenia and others. The situs inversus was formerly considered an absolute contraindication for liver transplantation. Recently however, several case reports have been published suggesting that neither situs inversus nor this particular subset of vascular abnormalities should be considered contraindications to liver transplantation. We present one case of liver transplantation performed in patient with biliary atresia, situs inversus and polysplenia. This is the first report described in Spain for a liver transplant in a child with biliary atresia plus situs inversus.

[Vaccinations and solid-organ transplantation: review and recommendations]. / Vacunaciones y trasplante de órgano sólido: revisión y recomendaciones.

Pediatric solid-organ transplant recipients are at high risk for various infectious diseases. Many children are not fully vaccinated before transplantation. To reduce the risk of morbidity and mortality from vaccine-preventable disease, physicians treating pediatric solid-organ transplant recipients should monitor the immunization status of these patients. Consensus on the most appropriate immunization schedule for solid-organ transplant recipients is lacking. Therefore, we provide a review of the currently available data on immunization safety and efficacy and describe strategies to avoid vaccine-preventable diseases in pediatric solid-organ transplant recipients.

Vacunaciones y trasplante de órgano sólido: revisión y recomendaciones / Vaccinations and solid-organ transplantation: review and recommendations

Los receptores pediátricos de trasplante de órgano sólido tienen un alto riesgo de padecer diversas enfermedades infecciosas. Algunos niños no han recibido las vacunas recomendadas para su edad, antes del trasplante. Para reducir el riesgo de morbilidad y mortalidad de las enfermedades prevenibles mediante vacunación, es importante que los médicos que tratan a estos niños vigilen su estado vacunal. No hay consenso sobre el esquema de inmunización más apropiado para los receptores de trasplante de órgano sólido, por lo que se revisa la información disponible sobre la seguridad y la eficacia de las inmunizaciones, y se describen las estrategias para evitar las enfermedades prevenibles mediante vacunación en los niños receptores de trasplante de órgano sólido. (AU)

Primer trasplante hepático en un paciente con atresia de vías biliares y situs inversus en España / First orthotopic liver transplantation in patient with biliary atresia and situs inversus in Spain

La atresia de vías biliares es la indicación más frecuente para trasplante hepático en la edad pediátrica. La portoenterostomía de Kasai es el tratamiento primario para la atresia de vías biliares. Si la portoenterostomía falla, el fallecimiento suele ocurrir antes de los 2años de edad sin el trasplante hepático. El síndrome de múltiples malformaciones más comunmente asociado con la atresia de vías biliares es el síndrome de poliesplenia, formado por una constelación de defectos en la simetría corporal, desarrollo del bazo y anomalías vasculares, incluyendo situs inversus, poliesplenia y otros. El situs inversus era considerado como una contraindicación absoluta para el trasplante hepático. Sin embargo recientemente han sido publicados varios casos, sugiriendo que ni el situs inversus ni la presencia de dichas alteraciones vasculares deben ser consideradas como contraindicaciones para el trasplante hepático. Presentamos un caso de trasplante hepático realizado a un paciente con atresia de vías biliares, situs inversus y poliesplenia. Es el primer caso descrito en España (AU)

Detergent-solubilized bovine cytochrome c oxidase: dimerization depends on the amphiphilic environment.

The extent to which bovine cytochrome c oxidase (COX) dimerizes in nondenaturing detergent environments was assessed by sedimentation velocity and equilibrium. In contrast to generally accepted opinion, the COX dimer is difficult to maintain and is the major oligomeric form only when COX is solubilized with a low concentration of dodecylmaltoside, i.e., approximately 1 mg/mg protein. The dimer form is intrinsically unstable and dissociates into monomers with increased detergent concentration, i.e., >5 mg/mg protein. The structure of the solubilizing detergent, however, greatly alters detergent effectiveness by inducing either monomerization or aggregation. Triton X-100 is most effective at solubilizing COX, but it destabilizes COX dimers, even at low concentration. Undecylmaltoside, decylmaltoside, and octaethyleneglycolmonododecyl ether (C(12)E(8)) are less effective at solubilizing COX. Each prevents COX aggregation at high detergent concentration, but also destabilizes the COX dimer. Other detergents, e.g., Tween 20, sodium cholate, sodium deoxycholate, CHAPS, or CHAPSO, are completely ineffective COX solubilizers and do not prevent aggregation even at 10-40 mg/mL. The transition from dimers to monomers depends on many factors other than detergent structure and concentration, e.g., protein concentration, phospholipid content and pH. We conclude that the intrinsic dimeric structure of COX can be maintained only after solubilization with low concentrations of dodecylmaltoside at near neutral pH, and even then precautions must be taken to prevent its dissociation into monomers.

CP30, a cysteine proteinase involved in Trichomonas vaginalis cytoadherence.

We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.

A novel cysteine proteinase (CP65) of Trichomonas vaginalis involved in cytotoxicity.

The goal of this study was to demonstrate the participation in cellular damage of a Trichomonas vaginalis proteinase with a molecular mass of 65 kDa (CP65). By two dimensional gelatin-gel electrophoresis of trichomonad proteins we detected four spots with proteolytic activity on the 65 kDa region, but only one, pI 7.2, binds to the HeLa cell surface. By indirect immunofluorescence, rabbit antibodies against this proteinase localized the CP65 on the plasma membrane and in the cytoplasm of T. vaginalis. Pretreatment of parasites with the specific anti-CP65 antibody reduced trichomonal cytotoxicity to HeLa cell monolayers. The specific cysteine proteinase inhibitor, L-3-carboxy-2, 3-trans-epoxypropionyl-leucylamido (4-guanidino) butane (E64) abrogated the proteinase activity and reduced cytotoxicity levels of T. vaginalis in cell culture monolayers, indicating that the trichomonad CP65 is a cysteine proteinase. Activity of the CP65 proteinase was optimal at pH 5.5 and 37 degrees C, conditions similar to those of patients with trichomonosis. Also, this proteinase degraded some of the proteins found in the vagina, i.e. collagen IV and fibronectin, but not laminin-1 or haemoglobin. Finally, immunoprecipitation assays showed that sera and vaginal washes from trichomonosis patient possess anti-CP65 antibodies. In conclusion, results presented in this work demonstrate that the CP65 is a surface cysteine proteinase involved in T. vaginalis cytotoxicity to HeLa cell monolayers, as a virulence factor. It is immunogenic during human infection and degrades some extracellular matrix proteins, i.e. collagen IV and fibronectin.

Detergent-solubilized Escherichia coli cytochrome bo3 ubiquinol oxidase: a monomeric, not a dimeric complex.

The protein molecular weight, M(r), and hydrodynamic radius, R(s), of Triton X-100-solubilized Escherichia coli cytochrome bo3 were evaluated by computer fitting of sedimentation velocity data with finite element solutions to the Lamm equation. Detergent-solubilized cytochrome bo3 sediments as a homogeneous species with an S20,w of 6.75 s and a D20,w of 3.71 x 10(-7) cm2/s, corresponding to a R(s) of 5.8 nm and a M(r) of 144,000 +/- 3500. The protein molecular weight agrees very well with the value of 143,929 calculated from the four known subunit sequences and the value of 143,025 measured by MALDI mass spectrometry for the histidine-tagged enzyme. We conclude that detergent-solubilized E. coli ubiquinol oxidase is a monomeric complex of the four known subunits.

Cytochrome c oxidase: biphasic kinetics result from incomplete reduction of cytochrome a by cytochrome c bound to the high-affinity site.

The electron-transfer kinetics of cytochrome c oxidase were probed by measuring the reduction levels of bound cytochrome c, cytochrome a, and cytochrome a3 during steady-state turnover. Our experimental approach was to measure these reduction levels as a function of (1) the rate of electron input into tightly bound cytochrome c by varying the concentration of TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) and/or cytochrome c and (2) the rate of electron efflux out of cytochrome a (true Kcat) by changing the detergent surrounding cytochrome c oxidase. In most detergent environments, the rate of electron input into cytochrome c is not faster than the rate of electron efflux from cytochrome a. The relatively slow rate of electron input results in incomplete reduction of both cytochrome a and cytochrome c bound a the high-affinity site unless Kcat is very slow. When the high-affinity site is saturated with cytochrome c, the steady-state reduction level of cytochrome a defines Vmax,1, which is the maximum velocity of the high-affinity phase. The remaining fractional oxidation level of cytochrome a determines Vmax,2, the maximum velocity of the low-affinity phase. Therefore, it is the sum Vmax,1 + Vmax,2 which defines the maximum rate of electron transfer between cytochrome a and the bimetallic center, i.e., Kcat. We also were able to evaluate the true Kcat of cytochrome c oxidase in each detergent environment directly from the steady-state reduction levels without any of the complications introduced by the analysis of the polarographic kinetic data.(ABSTRACT TRUNCATED AT 250 WORDS)

Conservation of structure in ATP-depleted proximal tubules: role of calcium, polyphosphoinositides, and glycine.

Increases of intracellular free Ca2+ (Caf) may mediate phospholipid hydrolysis and disintegration in energy-compromised cells; on the other hand, glycine and related amino acids preserve structure. We have examined the effects of increased Caf on phospholipids and structure in ATP-depleted cells, as well as how these actions may be modified by glycine. Incubation of isolated proximal tubules with antimycin A led to ATP depletion, delayed increases of Caf to micromolar levels, polyphosphoinositide (PPI) hydrolysis by phospholipase C, and generalized disintegration of cell structure. Glycine inhibited PPI hydrolysis and preserved cell structure in entirety but did not apparently modify the Caf increases. When overwhelming increases of Caf were induced by the additional presence of a Ca2+ ionophore, glycine did not inhibit either the hydrolysis of PPI or disruption of mitochondria and microvilli. However, the cells remained integrated and unbroken. Incubation in low-Ca2+ medium prevented Caf increases, inhibited PPI hydrolysis, and preserved the structure of mitochondria and microvilli. Nevertheless, there was lethal damage by disintegration of all other membranes. This damage was prevented specifically and completely by glycine. Thus compartments of cells were shown to be differentially susceptible to injury from increased Caf or lack of glycine. Although damage by either factor occurs by distinct mechanisms, glycine also appears to have effects that suppress the deleterious effects of Ca2+ so long as Caf increases are not overwhelming. Our results also suggest that the PPI have a major structural role, which may be compromised by Caf increase during ATP depletion.

Alanine protects rabbit proximal tubules against anoxic injury in vitro.

Rabbit proximal tubules were incubated aerobically or subjected to anoxia for 30 min followed by 60 min of reoxygenation. The medium contained (in mM) 5 glucose, 10 butyrate, 4 lactate or alpha-ketoglutarate (alpha-KG), and 1 alanine. Anoxic tubules in this medium were severely injured and recovered poorly. If the incubation medium was supplemented with additional alanine (up to 2.5 or 5 mM), then anoxic injury was prevented almost completely. Tubules in high-alanine medium showed modest elevations of ATP during anoxia. Comparable elevations of ATP were induced in anoxic tubules incubated with 4 mM alpha-KG and 5 mM aspartate without alanine. These substrates are metabolized anaerobically in the mitochondria to yield ATP. Surprisingly, anoxic tubules with alpha-KG and aspartate showed severe injury despite elevated ATP. If 5 mM alanine was also present, then additional increments of ATP did not occur, but injury was prevented. Examination of glucose metabolism failed to provide evidence for stimulation of anaerobic fermentations by alanine. These results suggest that alanine-induced cytoprotection during anoxia occurs by mechanisms not related to ATP synthesis, and that elevated ATP in alanine-supplemented tubules may be a result and not the cause of protection. Cytoprotection by alanine was shown to last for less than or equal to 90 min of anoxia. Glycine, a structurally related amino acid, also protects anoxic proximal tubules (J. Clin. Invest. 80: 1446, 1987). The mechanisms that underlie the cytoprotective effects of alanine and glycine remain to be determined.