Processing of metacaspase into a cytoplasmic catalytic domain mediating cell death in Leishmania major.

Metacaspases are cysteine peptidases that could play a role similar to caspases in the cell death programme of plants, fungi and protozoa. The human protozoan parasite Leishmania major expresses a single metacaspase (LmjMCA) harbouring a central domain with the catalytic dyad histidine and cysteine as found in caspases. In this study, we investigated the processing sites important for the maturation of LmjMCA catalytic domain, the cellular localization of LmjMCA polypeptides, and the functional role of the catalytic domain in the cell death pathway of Leishmania parasites. Although LmjMCA polypeptide precursor form harbours a functional mitochondrial localization signal (MLS), we determined that LmjMCA polypeptides are mainly localized in the cytoplasm. In stress conditions, LmjMCA precursor forms were extensively processed into soluble forms containing the catalytic domain. This domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion. These data provide experimental evidences of the importance of LmjMCA processing into an active catalytic domain and of its role in disrupting mitochondria, which could be relevant in the design of new drugs to fight leishmaniasis and likely other protozoan parasitic diseases.

[Endoscopic treatment of the vesicoureteral reflux in children: preliminary experience with the subureteral injection of Coaptite]. / Tratamiento endoscópico del reflujo vesicoureteral en pediatría: experiencia preliminar con la inyección subureteral de Coaptite.

OBJECTIVE: To review our experience in the endoscopic treatment of vesicoureteral reflux (VUR) in children with the subureteral injection of calcium hydroxyapatite (Coaptite). METHODS: The serie includes thirteen children whose age ranged between 6 and 13 years. The Coaptite implant was injected subureterally to 20 ureteral units (UU) affected by VUR grades I-IV. VUR was unilateral in six patients (46%), bilateral in seven (54%). There were 7 primary cases and 6 secondary to bladder dysfunction and duplex systems. RESULTS: Mean follow-up was 19 months (6-28 months). VUR was cured in 75% of cases after a single injection. A second injection resolved 2 out of 4 treated UU (50%) rendering a global success rate of 85%. This cure rate was achieved for 13 and 7 procedures treating primary and secondary VUR, respectively. 77% of patients reported no adverse events with only 23% of them complaining of transient mild lumbar and hypogastric discomfort limited to the first postoperative day. In 2 children postoperative urinary infection was detected. CONCLUSIONS: Endoscopic subureteral injection with Coaptite is a simple technique well tolerated in children. In 85% of primary and secondary cases cure was achieved with minimal morbidity.

Tratamiento endoscópico del reflujo vesicoureteral en pediatría: Experiencia preliminar con la inyección subureteral de Coaptite® / Endoscopic treatment of the vesicoureteral reflux in children: preliminary experience with the subureteral injection of coaptite

OBJETIVO: Analizar nuestra experiencia en el tratamiento endoscópico del reflujo vesicoureteral (RVU) en pediatría, mediante la inyección subureteral de hidroxiapatita cálcica (Coaptite®).MÉTODOS: La serie incluye trece niños de edades entre los 6 y 13 años. La inyección endoscópica subureteral de Coaptite® se aplicó en 20 unidades ureterales (UU) que presentaban RVU grados I-IV. El RVU fue unilateral en 6 pacientes (46%) y bilateral en 7 (54%). Hubo 7 casos primarios y 6 secundarios a disfunción vesical y duplicidad ureteral.RESULTADOS: El tiempo medio de seguimiento fue de 19 meses (6-28 meses). Se logró la corrección del RVU tras una inyección endoscópica en 15 casos (75%). Una segunda inyección corrigió el RVU en 2 de otras 4 UU (50%) tratadas, con lo que la cifra global de curacionesascendió al 85%. Esta tasa de curación se obtuvo tanto en los procedimientos que trataron casos primarios (13), como en las intervenciones por RVU secundario (7). Un 77% de pacientes no presentaron complicaciones, si bien el 23% tuvieron molestias lumbarese hipogástricas autolimitadas al primer día tras el procedimiento. En 2 niños se detectó infección urinaria en el postoperatorio.CONCLUSIONES: El tratamiento endoscópico del RVU con Coaptite® es una técnica sencilla y bien tolerada en niños. El 85% de los casos, tanto primarios como secundarios, curaron con mínima morbilidad

OBJECTIVE: To review our experience in the endoscopic treatment of vesicoureteral reflux (VUR) in children with the subureteral injection of calcium hydroxy-apatite (Coaptite®).METHODS: The serie includes thirteen children whose age ranged between 6 and 13 years. The Coaptite ® implant was injected subureterally to 20 ureteral units (UU) affected by VUR grades I-IV. VUR was unilateral in six patients (46%), bilateral in seven (54%). There were 7 primary cases and 6 secondary to bladder dysfunction and duplex systems.RESULTS: Mean follow-up was 19 months (6-28 months). VUR was cured in 75% of cases after a single injection. A second injection resolved 2 out of 4 treated UU (50%) rendering a global success rate of 85%. This cure ratewas achieved for 13 and 7 procedures treating primary and secondary VUR, respectively. 77% of patients reported transient mild lumbar and hypogastric discomfort limited to the first postoperative day. In 2 children postoperative urinary infection was detected.CONCLUSIONS: Endoscopic subureteral injection with Coaptite® is a simple technique well tolerated in children. In 85% of primary and secondary cases cure was achieved with minimal morbidity

Novel peptide inhibitors of Leishmania gp63 based on the cleavage site of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein.

The zinc metalloprotease gp63 (leishmanolysin; promastigote surface protease) is expressed at high density at the surface of Leishmania promastigotes. Efficient non-toxic inhibitors of gp63 do not exist, and its precise role in parasite physiology remains unknown. MARCKS (myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in various cells, including macrophages. We reported previously that MRP is an excellent substrate for gp63. A major cleavage site was identified within the MRP effector domain (ED), a highly basic 24-amino-acid sequence, and the synthetic ED peptide (MRP(ED)) was shown to inhibit MRP hydrolysis. In the present study, MRP cleavage was used as an assay to measure the capacity of various MRP or MARCKS ED peptides to block gp63 activity. On a molar basis, MRP(ED) inhibited gp63 to a greater extent than two previously described gp63 inhibitors, o -phenanthroline and benzyloxycarbonyl-Tyr-Leu-NHOH. MARCKS(ED) analogues containing modifications in the gp63 consensus cleavage site showed significant differences in inhibitory capacity. As phosphorylation of ED serine residues prevented gp63-mediated MRP degradation, we synthesized a pseudophosphorylated peptide in which serine residues were substituted by aspartate (3DMRP(ED)). 3DMRP(ED) was a highly effective inhibitor of both soluble and parasite-associated gp63. Finally, MRP ED peptides were synthesized together with an N-terminal HIV-1 Tat transduction domain (TD) to obtain cell-permeant peptide constructs. Such peptides retained gp63 inhibitory activity and efficiently entered both macrophages and parasites in a Tat TD-dependent manner. These studies may provide the basis for developing potent cell-permeant inhibitors of gp63.