<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignment of the cytosolic dithiol glutaredoxin 1 from the pathogen <Emphasis Type="Italic">Trypanosoma brucei</Emphasis>

Trypanosomatids are parasites responsible for several tropical and subtropical diseases, such as Chaga’s disease, sleeping sickness and Leishmaniasis. In contrast to the mammalian host, the thiol-redox metabolism of these pathogens depends on trypanothione [bis-glutathionylspermidine, T(SH)₂] instead of glutathione (GSH) providing a set of lineage-specific proteins as drug target candidates. Glutaredoxins (Grx) are ubiquitous small thiol–disulfide oxidoreductases that belong to the thioredoxin-fold family. They play a central role in redox homeostasis and iron sulfur-cluster biogenesis. Each species, including trypanosomes, possesses its own set of isoforms distributed in different subcellular compartments. The genome of trypanosomatids encodes for two class I (dithiolic) Grxs named 2-C-Grx1 and 2-C-Grx2. Both proteins were shown to efficiently reduce different disulfides at the expenses of T(SH)₂ using a mechanism that involves the two cysteines in the active site. Moreover, the cytosolic Trypanosoma brucei 2-C-Grx1 but not the mitochondrial 2-C-Grx2 was able to coordinate an iron–sulfur cluster with T(SH)₂ or GSH as ligand. As a first step to unravel the structural basis for the specificity observed in the trypanosomal glutaredoxins, we present here the NMR resonance assignment of 2-C-Grx1 from the parasite T. brucei brucei.     

Effect of L-fucose on proliferation and myo-inositol metabolism in cultured cerebral microvessel and aortic endothelial cells.

Decreased myo-inositol metabolism possibly contributes to the development of diabetic complications including micro and macrovascular disease. Previous studies have shown that hyperglycemia may be partially responsible for this defect. We have found that L-fucose, a monosaccharide present in low concentrations in normal circulation and found to be elevated in diabetes, causes defects in cultured endothelial cells, including alterations in myo-inositol metabolism and proliferation. Murine cerebral microvessel and bovine aortic endothelial cells take up L-fucose from the medium in a time and concentration-dependent manner. Both acute and chronic exposure of these cultured endothelial cells to media containing L-fucose at concentrations that may exist in diabetic sera cause a significant decrease in the accumulation of myo-inositol and its incorporation into inositol phospholipids. There is a concomitant decrease in the intracellular levels of myo-inositol. Kinetic analysis of the effect of L-fucose on myo-inositol uptake suggests that L-fucose competitively inhibits the transport of myo-inositol, exhibiting a Ki in the range of 1.6-4.1 mM for both cell types. Endothelial cells exposed to L-fucose concentrations of 0.5-20 mM exhibit depressed rates of proliferation in a concentration-dependent fashion. Furthermore, L-fucose causes a concentration-dependent decrease in synthesis of proteoglycan by cultured cerebral microvessel endothelial cells as measured by incorporation of 35S; however, this effect is not observed in the aortic endothelia. These data suggest that L-fucose at concentrations that may exist in diabetic sera may impair myo-inositol metabolism and proliferation of the vascular endothelium.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Fast Inactivation in Shaker K+ Channels : Properties of Ionic and Gating Currents

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH₂-terminus region.     

Genetic variability and phylogeography of the cyprinid Telestes muticellus within the Italian peninsula as revealed by mitochondrial DNA

A phylogeographic analysis of 12 populations of Telestes muticellus was performed on the basis of mitochondrial DNA sequences of the cytochrome b gene. The main aims were to determine phylogenetic relationships from congener species and to highlight the patterns of genetic differentiation within the Italian peninsula. The results obtained showed a significant divergence with the congener species, indicating a possible Miocene origin for the genus. Divergence was found to be low for T. muticellus , yet distinct lineages resulted as being allopatric within the two main Italian biogeographic districts, the Padano-Venetian and the Tuscano-Latium, suggesting the existence of recent vicariance coupled with past demographic regression. The high level of genetic differentiation found between populations highlighted the low dispersal ability of this rheophilic Cyprinid, whose migration was mainly related to connections between the upper parts of river basins. The distribution of sequence characteristics found suggests that particular attention should be paid to the conservation management of this species.     

Enhanced molecular chaperone activity of the small heat-shock protein alphaB-cystallin following covalent immobilization onto a solid-phase support

The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.     

Macro- and micronutrient effects on decomposition of leaf litter from two tropical tree species: inferences from a short-term laboratory incubation

While a large number of studies have investigated the effects of macronutrients such as nitrogen (N) or phosphorus (P) on litter decomposition, recent studies suggest that micronutrients including zinc (Zn) may also limit decomposition rates. Our goal was to compare the effects of nutrient addition on decomposition of two leaf litter types from tropical dry forest trees in a short-term laboratory microcosm experiment. Single nutrients (N, P, Zn, potassium, magnesium, and nickel) were applied to leaf litter in solution at low or high concentrations (to mimic in situ availability or to alleviate nutrient limitation, respectively), and decomposition was assessed as final mass remaining and carbon dioxide mineralization. Both mass remaining and CO₂ mineralization were affected by nutrient identity and concentration, and these effects varied by species. In general, P and Zn addition increased decomposition, Mg and N inhibited it, and K and Ni had no significant effects. Future studies should consider the interactions between decomposition processes, decomposer communities, and a wider range of macro- and micronutrients.