Two allelic isoforms of the serotonin transporter from Schistosoma mansoni display electrogenic transport and high selectivity for serotonin

The human blood fluke Schistosoma mansoni is the primary cause of schistosomiasis, a debilitating disease that affects 200 million individuals in over 70 countries. The biogenic amine serotonin is essential for the survival of the parasite and serotonergic proteins are potential novel drug targets for treating schistosomiasis. Here we characterize two novel serotonin transporter gene transcripts, SmSERT-A and SmSERT-B, from S. mansoni. Southern blot analysis shows that the two mRNAs are the products of different alleles of a single SmSERT gene locus. The two SmSERT forms differ in three amino acid positions near the N-terminus of the protein. Both SmSERTs are expressed in the adult form and in the sporocyst form (infected snails) of the parasite, but are absent from all other stages of the parasite`s complex life cycle. Heterologous expression of the two cDNAs in mammalian cells resulted in saturable, sodium-dependent serotonin transport activity with an apparent affinity for serotonin comparable to that of the human serotonin transporter. Although the two SmSERTs are pharmacologically indistinguishable from each other, efflux experiments reveal notably higher substrate selectivity for serotonin compared with their mammalian counterparts. Several well-established substrates for human SERT including (+/-)MDMA, S-(+)amphetamine, RU 24969, and m-CPP are not transported by SmSERTs, underscoring the higher selectivity of the schistosomal isoforms. Voltage-clamp recordings of SmSERT substrate-elicited currents confirm the substrate selectivity observed in efflux experiments and suggest that it may be possible to exploit the electrogenic nature of SmSERT to screen for compounds that target the parasite in vivo. (C) 2009 Elsevier B.V. All rights reserved.

Histology of the regenerate and docking site in bone transport

Bone transport is based on the principle of distraction osteogenesis described by Ilizarov and is a consecrated method for the treatment of segmental bone defects. One of its most problematic and, paradoxically, least studied aspects is the consolidation of the docking site. We studied histologically the ossification of the docking site and regenerate to determine any difference between them. Nine adult sheep were submitted to correction of a 1-cm tibial diaphyseal defect using a system of plate-fixed bone transport, with latency period of 1 week and 0.2 mm distraction of the transported segment four times a day. The sheep were divided into three groups of three animals each, according to the observation period of 3, 6 or 12 weeks between the fixation of the transported fragment and the euthanasia. The docking site and the regenerate were studied histologically on sections stained with Masson trichrome. The main mode of docking site ossification was the endochondral one and although intramembranous ossification was also observed simultaneously, it was limited to rare and small foci. In contrast, intramembranous ossification played the major role in the regenerate, with bone formation evolving from the base segment to the target segment. The experimental bone transport model proposed in the present study permits us to conclude that there is a clear difference between the ossification of the docking site and of the regenerate.

Thermal/mechanical simulation and laboratory fatigue testing of an alternative yttria tetragonal zirconia polycrystal core-veneer all-ceramic layered crown design

This study evaluated the stress levels at the core layer and the veneer layer of zirconia crowns (comprising an alternative core design vs. a standard core design) under mechanical/thermal simulation, and subjected simulated models to laboratory mouth-motion fatigue. The dimensions of a mandibular first molar were imported into computer-aided design (CAD) software and a tooth preparation was modeled. A crown was designed using the space between the original tooth and the prepared tooth. The alternative core presented an additional lingual shoulder that lowered the veneer bulk of the cusps. Finite element analyses evaluated the residual maximum principal stresses fields at the core and veneer of both designs under loading and when cooled from 900 degrees C to 25 degrees C. Crowns were fabricated and mouth-motion fatigued, generating master Weibull curves and reliability data. Thermal modeling showed low residual stress fields throughout the bulk of the cusps for both groups. Mechanical simulation depicted a shift in stress levels to the core of the alternative design compared with the standard design. Significantly higher reliability was found for the alternative core. Regardless of the alternative configuration, thermal and mechanical computer simulations showed stress in the alternative core design comparable and higher to that of the standard configuration, respectively. Such a mechanical scenario probably led to the higher reliability of the alternative design under fatigue.

Glycosphingolipids modulate renal phosphate transport in potassium deficiency

Background. Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. Methods. K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days. which resulted in a marked decrease in serum and tissue K content. Results. K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the V-max of brush-border membrane (BBM) Na/Pi cotransport activity (1943 95 in control vs. 1183 +/- 99 pmol/5 sec/mg BBM protein in K deficiency. P < 0.02). Surprisingly. the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1). and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin. glucosylceramide. and ganglioside GM, content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficit nt rats was the same as in control rats (1148 +/- 52 in control + PDMP vs. 11.52 +/- 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. Conclusion. K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.

Pharmacotherapy of the ion transport defect in cystic fibrosis

1. More than 1300 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF), a disease characterized by deficient epithelial Cl- secretion and enhanced Na+ absorption. The clinical course of the disease is determined by the progressive lung disease. Thus, novel approaches in pharmacotherapy are based primarily on correction of the ion transport defect in the airways. 2. The current therapeutic strategies try to counteract the deficiency in Cl- secretion and the enhanced Na+ absorption. A number of compounds have been identified, such as genistein and xanthine derivatives, which directly activate mutant CFTR. Other compounds may activate alternative Ca2+-activated Cl- channels or basolateral K+ channels, which supply the driving force for Cl- secretion. Apart from that, Na+ channel blockers, such as phenamil and benzamil, are being explored, which counteract the hyperabsorption of NaCl in CF airways. 3. Clinical trials are under way using purinergic compounds such as the P2Y(2) receptor agonist INS365. Activation of P2Y(2) receptors has been found to both activate Cl- secretion and inhibit Na+ absorption. 4. The ultimate goal is to recover Cl- channel activity of mutant CFTR by either enhancing synthesis and expression of the protein or by activating silent CFTR Cl- channels. Strategies combining these drugs with compounds facilitating Cl- secretion and inhibiting Na+ absorption in vivo may have the best chance to counteract the ion transport defect in cystic fibrosis.