Ionic liquids as refolding additives: N'-alkyl and N'-(omega-hydroxyalkyl) N-methylimidazolium chlorides

The purpose of this work was to investigate the influence of a series of N'-alkyl and N'-(omega-hydroxy-alkyl)-N-methylimidazolium chlorides on the renaturation of two model proteins, namely hen egg white lysozyme and the single-chain antibody fragment ScFvOx. All tested ionic liquids acted as refolding enhancers, with varying efficacies and efficiencies. The results of the refolding screening could be interpreted by taking into account the effect of the studied ionic liquids on protein aggregation, together with the systematic variations of their influence on the stability of native proteins in solution. More hydrophobic imidazolium cations carrying longer alkyl chains were increasingly destabilizing, while terminal hydroxylation of the alkyl chain made the salts more compatible with protein stability. The studied ionic liquids can be classified as preferentially bound, slightly to moderately chaotropic cosolvents for proteins.     

A slow, tight-binding inhibitor of the zinc-dependent deacetylase LpxC of lipid A biosynthesis with antibiotic activity comparable to ciprofloxacin

The zinc-dependent enzyme LpxC catalyzes the deacetylation of UDP-3-O-acyl-GlcNAc, the first committed step of lipid A biosynthesis. Lipid A is an essential component of the outer membranes of most Gram-negative bacteria, including Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, making LpxC an attractive target for antibiotic design. The inhibition of LpxC by a novel N-aroyl-l-threonine hydroxamic acid (CHIR-090) from a recent patent application (International Patent WO 2004/062601 A2 to Chiron and the University of Washington) is reported here. CHIR-090 possesses remarkable antibiotic activity against both E. coli and P. aeruginosa, comparable to that of ciprofloxacin. The biological activity of CHIR-090 is explained by its inhibition of diverse LpxC orthologues at low nanomolar concentrations, including that of Aquifex aeolicus, for which structural information is available. The inhibition of A. aeolicus LpxC by CHIR-090 occurs in two steps. The first step is rapid and reversible, with a K(i) of 1.0-1.7 nM, depending upon the method of assay. The second step involves the conversion of the EI complex with a half-life of about a minute to a tightly bound form. The second step is functionally irreversible but does not result in the covalent modification of the enzyme, as judged by electrospray ionization mass spectrometry. CHIR-090 is the first example of a slow, tight-binding inhibitor for LpxC and may be the prototype for a new generation of LpxC inhibitors with therapeutic applicability.     

Cytogenetic characteristics of a murine in vitro model for the human anaplastic large cell lymphoma (ALCL)

Anaplastic large cell lymphoma (ALCL) is an entity of non-Hodgkin lymphomas (NHL) that often occurs in young children and adolescents. In the majority of cases, ALCL are of T-cell origin and contain the t(2;5)(p23;q35) leading to an NPM-ALK fusion or variant ALK translocations. In addition, there is an ALK-negative subtype of ALCL. The anaplastic lymphoid cell line TS1G6 established by interleukin (IL)-9 transfection of T-helper cells represents a murine model of this subtype. Here, we describe the cytogenetic features of this cell line using spectral karyotyping (SKY) and single-color fluorescence in situ hybridization (FISH). We show that TS1G6 cells exhibit a hypotetraploid karyotype with complex structural alterations. Several unbalanced translocations involved the chromosomal region 14E5, and different translocation partners, i.e. X?A6, 3A3 and 8A1. FISH analysis using a BAC clone containing c-myc confirmed the presence of six copies, but also demonstrated that two loci were irregularly located, indicating that additional intrachromosomal rearrangements had occurred. Moreover, a duplication of the region XF2 approximately 3 was identified. Furthermore, six chromosomes 15 were found, representing a trisomy 15 in a tetraploid chromosome complement, indicating an altered gene dosage of the oncogene c-myc located in region 15D3.     

Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms

To better define secondary aberrations that occur in addition to translocation t(11;14)(q13;q32) in mantle cell lymphomas (MCL) and in multiple myelomas (MM), seven t(11;14)-positive MCL cell lines and four t(11;14)-positive MM cell lines were analysed by fluorescence R-banding and spectral karyotyping (SKY). Compared with published data obtained by G-banding, most chromosome aberrations were redefined or further specified. Furthermore, several additional chromosome aberrations were identified. Thus, these cytogenetically well defined t(11;14)-positive MCL and MM cell lines may be useful tools for the identification and characterization of genes that might be involved in the pathogenesis of MCL and MM, respectively. Since MCL and MM were found to have different alterations of chromosome 1, these were investigated in more detail by fluorescence in situ hybridization (FISH) and multicolor banding (MCB) analyses. The most frequently altered and deletion-prone loci in MCL cell lines were regions 1p31 and 1p21. In contrast, breakpoints in MM cell lines most often involved the heterochromatic regions 1p12-->p11, and the subcentromeric regions 1q12 and 1q21. These data are in accordance with previously published data of primary lymphomas. Our findings may indicate that different pathways of clonal evolution are involved in these morphologically distinct lymphomas harboring an identical primary chromosome aberration, t(11;14).     

A cold-loving crenarchaeon is a substantial part of a novel microbial community in cold sulphidic marsh water

In this paper, we report the identification and first characterization of a novel, cold-loving, prokaryotic community thriving among white-greenish 'streamers' in the cold (c. 10 degrees C) sulphurous water of the marsh Sippenauer Moor near Regensburg, Bavaria, Germany. It consists of the bacterial genus Thiothrix, the bacterium 'Sip100' and one archaeal representative, forming together a unique association structure with a distinct life cycle. Fluorescence in situ hybridization studies have revealed that the archaeal member can be affiliated to the crenarchaeal kingdom ('Cre1'). This crenarchaeon was always observed attached to the bacterial community member 'Sip100'. Extended fluorescence in situ hybridization studies showed that this crenarchaeon was not detected in a free-living form, raising the idea of a probable host-dependent relationship. In line with our fluorescence in situ hybridization studies, novel crenarchaeal 16S rRNA gene sequences were identified in these samples. The design and application of a new in situ cultivation method in the sulphurous water of the marsh allowed first insights into the cohesion mechanisms, lifestyle and chronology of the microbes involved in this prokaryotic community in nature. Our results suggest that hitherto unknown Crenarchaeota thrive in cold sulphidic water and are a substantial part of a synchronized microbial community.     

Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass

We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with ⅚ surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O(2) usage. However, UCP-2 can be inactivated by glutathionylation. Mice were fed normal (NS)- or high-salt (HS) diets, and HS mice received the antioxidant drug tempol or vehicle for 3 mo. Since salt intake did not affect the tubular Na(+) transport per O(2) consumed (T(Na/)Q(O2)), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F(2α) and H(2)O(2), renal expression of UCP-2 and renal O(2) extraction, and reduced T(Na/)Q(O2) (sham: 20 ± 2 vs. RRM: 10 ± 1 μmol/μmol; P < 0.05) and cortical Po(2) (sham: 43 ± 2, RRM: 29 ± 2 mmHg; P < 0.02). Tempol normalized all these parameters while further increasing compensatory renal growth and glomerular volume. RRM mice had preserved blood pressure, glomeruli, and patchy tubulointerstitial fibrosis. The patterns of protein expression in the renal cortex suggested that RRM kidneys had increased ROS from upregulated p22(phox), NOX-2, and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor-β whereas erythroid-related factor 2 (Nrf-2), glutathione peroxidase-1, and glutathione-S-transferase mu-1 were upregulated independently of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O(2) usage and cortical hypoxia that was offset by Nrf-2-dependent glutathionylation. Thus hypoxia in RRM may be the outcome of NADPH oxidase-initiated ROS generation, leading to mitochondrial uncoupling counteracted by defense pathways coordinated by Nrf-2.     

Nitroalkylation--a redox sensitive signaling pathway

Redox-sensitive posttranslational modification emerged as important signaling mechanisms. Besides other posttranslational modifications nitroalkylation by nitrated fatty acids mediate favorable anti-inflammatory effects. This review gives an overview of the generation and the reactivity of nitrated fatty acids. Additionally, it provides insights into the so far described pathways regulated by nitrated fatty acids. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation.