Sortilin knock-down alters the expression and distribution of cathepsin D and prosaposin and up-regulates the cation-dependent mannose-6-phosphate receptor in rat epididymal cells.

The selective transport to lysosomes can be mediated by either mannose-6-phosphate receptors (CD-MPR and CI-MPR) or sortilin. In mammalian epididymis, some lysosomal proteins are secreted into the lumen through unknown mechanisms. To investigate the underlying mechanisms of lysosomal protein transport in epididymal cells we studied the expression and distribution of cathepsin D (CatD) and prosaposin (PSAP) in a sortilin knocked down RCE-1 epididymal cell line (RCE-1 KD) in comparison with non-transfected RCE-1 cells. In RCE-1 cells, CatD was found in the perinuclear zone and co-localize with sortilin, whereas in RCE-1 KD cells, the expression, distribution and processing of the enzyme were altered. In turn, PSAP accumulated intracellularly upon sortilin knock-down and redistributed from LAMP-1-positive compartment to a perinuclear location, remaining co-localized with CatD. Interestingly, the sortilin knock-down induced CD-MPR overexpression and a redistribution of the receptor from the perinuclear zone to a dispersed cytoplasmic location, accompanied by an increased co-localization with CatD. The increase in CD-MPR could result from a compensatory response for the proper delivery of CatD to lysosomes in epididymal cells. The intracellular pathway taken by lysosomal proteins could be an approach for addressing further studies to understand the mechanism of exocytosis and therefore the role of these proteins in the epididymis.

Antiproliferative effect and ultrastructural alterations induced by 5-O-methylembelin on Trypanosoma cruzi.

BACKGROUND: Embelin (EMB), obtained from Oxalis erythrorhiza Gillies ex Hooker et Arnott (Oxalidaceae), was reported against Trypanosoma cruzi and Leishmania spp. Additionally, antiprotozoan activity against Plasmodium falciparum was reported for its methylated derivative (ME). PURPOSE: To evaluate the potential anti-Trypanosoma cruzi activity of EMB, ME and 2,5-di-O-methylembelin (DME) and analyze the possible mechanism of action. STUDY DESIGN/METHODS: EMB was isolated by a chromatographic method from the air-dried ground whole plant. To evaluate the effects of methylation, ME and DME were synthesized and tested against T. cruzi epimastigotes and trypomastigotes. The most active compound ME was evaluated against amastigotes. Ultrastructural alterations, ROS generation and the effect on mitochondrial activity of ME were measured. RESULTS: Compounds inhibited the proliferation of epimastigotes. ME was also active against intracellular amastigotes. Mitochondrial alterations were observed by TEM. Additionally, ME modified the mitochondrial activity, and induced an increase in ROS levels. These evidences postulate the mitochondrion as a possible target of ME. CONCLUSION: ME inhibited amastigotes proliferation, thus being a potential lead compound for the treatment of Chagas' disease.

Epididymal α-l-fucosidase and its possible role in remodelling the surface of bull spermatozoa.

The mammalian epididymis provides an appropriate environment for sperm maturation. During the epididymal transit, spermatozoa undergo biochemical and morphological changes that lead to the acquisition of the fertilizing capacity. In this study we analysed the fucosylation status of membrane glycoproteins in the spermatozoa obtained from different regions of the bull epididymis. High amounts of fucose were detected on caput spermatozoa (R.F.I. = 1010 ± 20.35), mostly located in the post-acrosome zone. A significant decrease in the fucose levels was detected toward the cauda (R.F.I. = 540.5 ± 49.93) (P < 0.05). This decrease was in line with the increased activity of α-l-fucosidase in the cauda fluid. In sperm from the cauda, the defucosylation occurred in some proteins, whereas others showed higher fucosylation rates. A significant decrease of fucose in the gametes was observed upon incubation of crude cauda fluid with caput spermatozoa (from R.F.I. = 1.45 ± 0.08 to 1.06 ± 0.03) (P < 0.05) indicating that the α-l-fucosidase present in the epididymal fluid is active on spermatozoa. Moreover, this effect was blocked with specific enzyme inhibitors. These results provide direct evidence that the α-l-fucosidase from epididymal fluid participates in the fucose removal from spermatozoa, as a step of sperm maturation in the bull epididymis.

Analysis of the rumen bacterial diversity of goats during shift from forage to concentrate diet.

High-grain feeding used in the animal production is known to affect the host rumen bacterial community, but our understanding of consequent changes in goats is limited. This study was therefore aimed to evaluate bacterial population dynamics during 20 days adaptation of 4 ruminally cannulated goats to the high-grain diet (grain: hay - ratio of 40:60). The dietary transition of goats from the forage to the high-grain-diet resulted in the significant decrease of rumen fluid pH, which was however still higher than value established for acute or subacute ruminal acidosis was not diagnosed in studied animals. DGGE analysis demonstrated distinct ruminal microbial populations in hay-fed and grain-fed animals, but the substantial animal-to-animal variation were detected. Quantitative PCR showed for grain-fed animals significantly higher number of bacteria belonging to Clostridium leptum group at 10 days after the incorporation of corn into the diet and significantly lower concentration of bacteria belonging to Actinobacteria phylum at the day 20 after dietary change. Taxonomic distribution analysed by NGS at day 20 revealed the similar prevalence of the phyla Firmicutes and Bacteroidetes in all goats, significantly higher presence of the unclassified genus of groups of Bacteroidales and Ruminococcaceae in grain-fed animals and significantly higher presence the genus Prevotella and Butyrivibrio in the forage-fed animals. The three different culture-independent methods used in this study show that high proportion of concentrate in goat diet does not induce any serious disturbance of their rumen ecosystem and indicate the good adaptive response of caprine ruminal bacteria to incorporation of corn into the diet.

[Clinical practice guideline on closed tibial plateau fractures in adulthood]. / Guía de práctica clínica. Fractura cerrada de la meseta tibial en el adulto.

Closed tibiae plateau fractures are common injuries in the emergency room. The optimal treatment is not well defined or established. For this reason, there are several surgical management options: open reduction and internal fixation, closed reduction and percutaneous synthesis, external fixation, and even conservative treatment for this kind of fracture. The mechanism of production of this fracture is through large varus or valgus deformation to which is added a factor of axial load. The trauma may be direct or indirect. The degree of displacement, fragmentation and involvement of soft tissues like ligaments, menisci, vascular and nerve structures are determined by the magnitude of the force exerted. Any intra-articular fracture treatment can lead to an erroneous instability, deformity and limitation of motion with subsequent arthritic changes, leading to joint incongruity, limiting activity and significantly altering the quality of life. Open reduction and internal fixation with anatomic restitution is the method used in this type of fracture. However, the results of numerous publications can be questioned due to the inclusion in the same study of fractures treated with very different methods.

Las fracturas de la meseta tibial cerradas son lesiones frecuentes en los servicios de urgencias, cuyo tratamiento óptimo no se encuentra bien definido, ya que existen para un mismo tipo de fractura diversas opciones de manejo quirúrgico, como reducción abierta y fijación interna, reducción cerrada con síntesis percutánea, fijación externa e incluso tratamiento conservador. El mecanismo de producción de estas fracturas es a través de grandes deformaciones en varo o valgo a las que se añade un componente de carga axial. El traumatismo puede ser directo o indirecto. El grado de desplazamiento, la fragmentación y la afectación de partes blandas (ligamentos, meniscos, estructuras vasculares y nerviosas) se encuentran determinados por la magnitud de la fuerza ejercida. Como en cualquier fractura intraarticular, un tratamiento erróneo puede dar lugar a inestabilidad, deformidad y limitación de la movilidad, con los consiguientes cambios artrósicos, lo cual provocará incongruencia articular, limitará la actividad y alternará de forma significativa la calidad de vida. La reducción abierta y la fijación interna con la que se busca la restitución anatómica es un método utilizado en este tipo de fracturas. Sin embargo, los resultados de numerosas publicaciones pueden ser cuestionados debido a la inclusión en un mismo estudio de fracturas tratadas con métodos muy diferentes.

Acute intrastriatal administration of quinolinic acid affects the expression of the coat protein AP-2 and its interaction with membranes.

Clathrin-coated vesicle endocytosis is thought to be crucial for the maintenance of synaptic transmission and for the cell plasticity at the nervous system. In this study, we demonstrated that acute intrastriatal administration of quinolinic acid (QUIN), an agonist of the N-methyl-D: -aspartate receptor, induces a decrease of the coat protein AP-2 expression and affects their interaction with membranes. By western blot analysis we observed that at 24 h after QUIN intrastriatal injection, alpha1 subunit of AP-2 and alpha2, at lesser extent, were reduced in the striatal membranes. The decrease of both subunits expression was extended to 48 h after treatment, although the soluble proteins were mostly affected. Other areas of the brain were not affected by the treatment, except the cerebellum, where a significant increase of soluble AP-2 (both subunits) was observed at 48 h after injection. Another coat protein, as the phosphoprotein AP-180, was not affected by the injection of QUIN. We also confirmed that QUIN injection causes increasing loss of striatal neurons after the administration of the toxin. We concluded that QUIN may affect the endocytotic machinery of the striatum, by inducing changes in the AP-2 behaviour. Consequently, the internalization of NMDAR and/or AMPAR may be affected, by QUIN, contributing to the excitotoxic effect of the drug.