Continuous renal replacement therapy in children post-hematopoietic stem cell transplantation: the present and the future

Allogeneic hematopoietic stem cell transplantation (HSCT) use has expanded markedly to treat different disorders like hematologic malignancies, immunodeficiency, and inborn errors of metabolism. However, it is commonly associated with complications that limit the benefit of this therapy. Acute renal failure occurs commonly after HSCT and results in increased risk of mortality. In many instances, children post-HSCT develop acute renal insufficiency in the context of other organ failure, necessitating intensive care unit admission for management. Recently, continuous renal replacement therapy (CRRT) has emerged as the favored modality of renal replacement therapy in the care of critically ill children who are hemodynamically unstable. Currently, CRRT is being utilized more often in the care of critically ill post- HSCT children to treat renal failure or to prevent fluid overload (FO). FO > 20% has been shown in many studies to be an independent risk of mortality in critically ill children and therefore, many clinicians will initiate this therapy due to FO even without overt renal failure. CRRT may be beneficial in disease processes as acute lung injury due to removal of fluid. CRRT results in improved oxygenation in post-HSCT children with acute lung injury and this improvement is sustained for at least 48 hours after initiation of this therapy. Survival in post-HSCT children requiring this therapy ranges from 17% to 45%, however, long term survival is still poor. This review will discuss current practice of CRRT in children post-HSCT, as well as future directions.     

Anti-apoptotic Bcl-XL protein in complex with BH3 peptides of pro-apoptotic Bak, Bad, and Bim proteins: comparative molecular dynamics simulations

The Bcl-2 family of proteins plays a central role in the regulation of mitochondrial outer-membrane permeabilization, a critical step in apoptosis. Heterodimerization between the pro- and anti-apoptotic members of Bcl-2 family is a key event in this process. Anti-apoptotic proteins have high levels of expression in many cancers and they have different affinities for different pro-apoptotic proteins. Experimentally determined structures of all members of Bcl-2 proteins have remarkably similar helical fold despite poor amino acid sequence identity. Peptides representing BH3 region of pro-apoptotic proteins have been shown to bind the hydrophobic cleft of anti-apoptotic proteins and this segment is responsible in modulating the apoptotic pathways in living cells. Understanding the molecular basis of protein-protein recognition is required to develop inhibitors specific to a particular anti-apoptotic protein. We have carried out molecular dynamics simulations on the anti-apoptotic Bcl-X(L) protein in complex with three different BH3 peptides derived from pro-apoptotic Bak, Bad and Bim proteins. Each complex structure was simulated for a period of 50 ns after 2.5 ns equilibration. Analysis of the simulation results showed that in the Bcl-X(L) protein, the helix containing the BH3 region is more flexible than other helices in all three simulations. A network of strong hydrophobic interactions exists between four of the six helices and they contribute significantly to the stability of this helix bundle protein. Analysis of Bcl-X(L)-BH3 peptide interactions reveals the role of loop residues in the protein-peptide interactions in all three simulations. Bad and Bim peptides maintain strong hydrophobic and hydrophilic interactions with the helix preceding the central hydrophobic helix. Residues from this helix interact with an Arg residue in Bad and Bim peptides. This Arg residue is next to the conserved Leu residue and is replaced by Ala in Bak. Absence of these interactions and the helix propensity are likely to be the cause for Bak peptide's weaker binding affinity with the Bcl-X(L) protein. The results of this study have implications in the design of Bcl-X(L)-specific inhibitors.     

Purification and characterization of thermostable xylose(glucose) isomerase from Bacillus thermoantarcticus

Xylose isomerase produced by Bacillus thermoantarcticus was purified 73-fold to homogeneity and its biochemical properties were determined. It was a homotetramer with a native molecular mass of 200 kDa and a subunit molecular mass of 47 kDa, with an isoelectric point at 4.8. The enzyme had a K _m of 33 mM for xylose and also accepted D-glucose as substrate. Arrhenius plots of the enzyme activity of xylose isomerase were linear up to a temperature of 85°C. Its optimum pH was around 7.0, and it had 80% of its maximum activity at pH 6.0. This enzyme required divalent cations for its activity and thermal stability. Mn²⁺, Co²⁺ or Mg²⁺ were of comparable efficiency for xylose isomerase reaction, while Mg²⁺ was necessary for glucose isomerase reaction. Journal of Industrial Microbiology & Biotechnology (2001) 27, 234–240. Received 18 March 2001/ Accepted in revised form 03 July 2001     

The CD3 epsilon subunit of the TCR contains endocytosis signals.

Ligand binding to TCR induces its internalization and cell surface down-modulation. These phenomena contribute to the extinction of activation signals. Due to the multicomponent nature of the TCR-CD3 complex, its internalization may be mediated by one or several of its subunits. Although it has been reported that CD3 gamma and CD3 delta contain endocytosis motifs involved in the internalization of the TCR-CD3 complex, other subunits could also be involved in this process. For instance, CD3 epsilon and CD zeta display amino acid sequences reminiscent of internalization motifs. To investigate whether CD3 epsilon bears endocytosis signals, we have analyzed the internalization capacity of a panel of deletion and point mutants of CD3 epsilon that were expressed on the cell surface independently of other TCR-CD3 subunits. Here we report that CD3 epsilon displays endocytosis determinants. These data indicate that CD3 epsilon could contribute to the internalization and cell surface down-regulation of TCR-CD3 complexes. Moreover, the existence of endocytosis signals in this polypeptide could serve to retrieve unassembled CD3 epsilon subunits or partial CD3 complexes from the plasma membrane, thus restricting the expression on the cell surface to fully functional TCR-CD3 complexes.     

Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica)

A new thermophilic spore-forming strain MR3CT was isolated from geothermal soil located on Mount Rittmann in Antarctica. Strain MR3CT was Gram-positive, rod-shaped, occurring in pairs or filamentous. Growth was observed between 45 and 65 degrees C (optimum 61 degrees C) and at pH 5.0-6.5 (optimum pH 5.6). It was capable of utilizing galactose, trehalose, maltose and sucrose. The microorganism produced an exopolysaccharide and synthesized an extracellular constitutive amylolytic activity. The G + C content of DNA was 43.5 mol%. On the basis of 16S rRNA gene sequence similarity, strain MR3CT was shown to be related most closely to Anoxybacillus species. Chemotaxonomic data (major isoprenoid quinone-menaquinone-7; major fatty acid-iso-C15:0 and iso-C17:0) supported the affiliation of strain MR3C1T to the genus Anoxybacillus. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MR3CT from the validly published Anoxybacillus species. MR3CT therefore represents a new species, for which the name Anoxybacillus amylolyticus sp. nov., is proposed, with the type strain MR3CT (= ATCC BAA-872T = DSM 15939T = CIP 108338T).     

Chemical Composition of Two Exopolysaccharides from Bacillus thermoantarcticus

The thermophilic bacterium Bacillus thermoantarcticus produces two exocellular polysaccharides (EPS 1 and EPS 2), which can be obtained from the supernatant of liquid cultures by cold-ethanol precipitation, in yields as high as 400 mg liter(sup-1). The EPS fraction was produced with all substrates tested, although a higher yield was obtained with mannose as the carbon and energy source. The EPS content was proportional to the total biomass. On a weight basis, EPS 1 and EPS 2 represented about 27 and 71%, respectively, of the total carbohydrate fraction. EPS 1 is a sulfate heteropolysaccharide containing mannose and glucose in a relative molar proportion of 1.0 and 0.7, respectively. EPS 2 is a sulfate homopolysaccharide containing mannose as the major component. The absolute configurations of hexoses were shown to be d for both EPSs. Nuclear magnetic resonance spectra confirmed the presence of (alpha)-d-mannose and (beta)-d-glucose in EPS 1 and only (alpha)-d-mannose in EPS 2. In addition, (sup1)H nuclear magnetic resonance analysis and chemical analysis indicated the presence of pyruvic acid in EPS 2.     

Kinetic resolution of aminoacid ester with immobilized cells ofSulfolobus solfataricus

Summary Cells ofSulfolobus solfataricus trapped in sodium alginate have been used to hydrolyze a range of aminoacid esters with useful stereoselectivity for L-enantiomer at different temperatures.     

Generation of an alpha-L-rhamnosidase library and its application for the selective derhamnosylation of natural products

A screening of 16 different fungal strains was performed under different cultivation conditions, using L-rhamnose or L-rhamnose-containing flavonoid glycosides (rutin, hesperidin, and naringin) as specific inducers. No significant constitutive production of alpha-L-rhamnosidases was detected in noninduced cultures, while high levels of these glycosidase activities were obtained using different inducers. New species, so far unknown for the production of alpha-L-rhamnosidases, were identified. More than 30 different alpha-L-rhamnosidase samples were prepared by ammonium sulfate precipitation. Substrate specificity of this alpha-L-rhamnosidase library was tested with various L-rhamnose-containing natural compounds (flavonoids, terpenoids, and saponins). Most of the enzymatic preparations showed broad substrate specificity, and some of them were also acting on sterically hindered substrates (e.g., quercitrin). The screening of the library under different reaction conditions showed the coexistence, in the same preparation, of more than one alpha-L-rhamnosidase activities with different substrate specificity and different stability towards organic cosolvents. To exploit this enzymatic library for synthetic applications, the presence of contaminating alpha-L-arabinosidases and beta-D-glucosidases was investigated. The latter enzymes were observed in several preparations, while alpha-L-arabinosidase content was generally quite low. The selective derhamnosylation of the saponin desglucoruscin was performed on a preparative scale. The enzyme obtained by rhamnose induction of the Aspergillus niger K2 CCIM strain showed high activity towards this substrate and negligible alpha-L-arabinosidase contamination. Therefore, it was chosen as a catalyst for the selective derhamnosylation reaction, which provided the desired product in 70% yield.