Descripción: Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different PDE families. One of these PDEs, T. cruzi PDE C2 (TcrPDEC2) has been characterized as a FYVE domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labelling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in PDE activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signalling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized PDE involved in osmoregulation in T. cruzi. © 2010 Blackwell Publishing Ltd.

Descripción: Split lamellae of posterior gills of Chasmagnathus granulatus adapted to 2.5‰ salinity were mounted in a modified Ussing chamber. With NaCl-saline on both sides of the preparation a transepithelial voltage (Vte) of 4.1±0.5 mV (outside positive) was measured. After voltage-clamping, the negative short-circuit current (Isc) amounted to -142±21 μA cm-2 at a conductance (Gte) of 44±5 mS cm-2. Substitution of either chloride (by nitrate) or sodium (by choline) on both sides of split gill lamellae significantly reduced Isc (by 70-80%) and Gte (by 30-50%). External CsCl (but not BaCl2 or furosemide) inhibited the negative Isc without affecting Gte. Addition of ouabain, BaCl2 or diphenylamine-2-carboxylate to the internal bath inhibited Isc at unchanged Gte. Internal acetazolamide did not affect Isc or Gte across split gill lamellae. Unidirectional Na+ influx across isolated and perfused posterior gills, however, was reduced by internal acetazolamide by approximately 20% at constant Vte. The results suggest that posterior gills of hyperosmoregulating C. granulatus display a high conductance epithelium that actively absorbs NaCl in a coupled way by an electrogenic mechanism similar to that seen in the thick ascending limb of Henle's loop and, to a minor degree, by an electroneutral mechanism, presumably via apical Na+/H+and Cl-/HCO3--antiports.

Descripción: Aims: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. Methods and Results: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l -1 NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. Conclusions: The results show that growth in high salt modified the structural properties of the cell wall. Significance and Impact of Study: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.