Inhibition of Trypanosoma brucei glucose-6-phosphate dehydrogenase by human steroids and their effects on the viability of cultured parasites

Dehydroepiandrosterone (DHEA) is known as an intermediate in the synthesis of mammalian steroids and a potent uncompetitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH), but not the enzyme from plants and lower eukaryotes. G6PDH catalyzes the first step of the pentose-phosphate pathway supplying cells with ribose 5-phosphate, a precursor of nucleic acid synthesis, and NADPH for biosynthetic processes and protection against oxidative stress. In this paper we demonstrate that also G6PDH of the protozoan parasite Trypanosoma brucei is uncompetitively inhibited by DHEA and epiandrosterone (EA), with K_i values in the lower micromolar range. A viability assay confirmed the toxic effect of both steroids on cultured T. brucei bloodstream form cells. Additionally, RNAi mediated reduction of the G6PDH level in T. brucei bloodstream forms validated this enzyme as a drug target against Human African Trypanosomiasis. Together these findings show that inhibition of G6PDH by DHEA derivatives may lead to the development of a new class of anti-trypanosomatid compounds.     

Animal models of polyglutamine diseases and therapeutic approaches

The dominant gain-of-function polyglutamine repeat diseases, in which the initiating mutation is known, allow development of models that recapitulate many aspects of human disease. To the extent that pathology is a consequence of disrupted fundamental cellular activities, one can effectively study strategies to ameliorate or protect against these cellular insults. Model organisms allow one to identify pathways that affect disease onset and progression, to test and screen for pharmacological agents that affect pathogenic processes, and to validate potential targets genetically as well as pharmacologically. Here, we describe polyglutamine repeat diseases that have been modeled in a variety of organisms, including worms, flies, mice, and non-human primates, and discuss examples of how they have broadened the therapeutic landscape.     

Effects of temperature and humidity on the efficacy of methicillin-resistant Staphylococcus aureus challenged antimicrobial materials containing silver and copper

Aims:  To compare silver and copper, metals with known antimicrobial properties, by evaluating the effects of temperature and humidity on efficacy by challenging with methicillin resistant Staphylococcus aureus (MRSA) .
Methods and Results:  Using standard methodology described in a globally used Japanese Industrial Standard, JIS Z 2801, a silver ion-containing material exhibited >5 log reduction in MRSA viability after 24 h at >90% relative humidity (RH) at 20°C and 35°C but only a <0·3 log at ∼22% RH and 20°C and no reduction at ∼22% RH and 35°C. Copper alloys demonstrated >5 log reductions under all test conditions.
Conclusions:  While the high humidity (>90% RH) and high temperature (35°C) utilized in JIS Z 2801 produce measurable efficacy in a silver ion-containing material, it showed no significant response at lower temperature and humidity levels typical of indoor environments.
Significance and Impact of the Study:  The high efficacy levels displayed by the copper alloys, at temperature and humidity levels typical of indoor environments, compared to the low efficacy of the silver ion-containing material under the same conditions, favours the use of copper alloys as antimicrobial materials in indoor environments such as hospitals.     

Lipid dynamics and feeding of dominant Antarctic calanoid copepods in the eastern Weddell Sea in December

Lipid content, fatty acid composition, and feeding activity of the dominant Antarctic copepods, Calanoides acutus, Calanus propinquus, and Metridia gerlachei, were studied at a quasi-permanent station in the eastern Weddell Sea in December 2003. During 3 weeks of the spring phytoplankton development, total lipid levels of females and copepodite stages V (CVs) of C. acutus were almost doubled. Meanwhile, only a slight increase in total lipid content occurred in M. gerlachei, and no clear trend was observed in lipids of C. propinquus females. The pronounced increase of lipids in C. acutus was due to an accumulation of wax esters. The proportion of wax esters in the lipids of M. gerlachei was clearly lower, while triacylglycerols played a more important role. In C. propinquus, triacylglycerols were the only neutral lipid class. There were no pronounced changes in the feeding activity of M. gerlachei, whereas the feeding activity of C. acutus had rapidly increased with the development of the phytoplankton bloom in December, which explains its rapid lipid accumulation. The combination of gut content and fatty acid trophic marker analyses showed that C. acutus was feeding predominantly on diatoms. The typical diatom fatty acid marker, 16:1(n-7), slightly decreased and the tracer for flagellates, 18:4(n-3), increased in females and CVs of C. acutus. This shift indicates the time, when the significance of flagellates started to increase. The three copepod species exhibited different patterns of lipid accumulation in relation to their trophic niches and different duration of their active phases. The investigations filled a crucial data gap in the seasonal lipid dynamics of dominant calanoid copepods in the Weddell Sea in December and support earlier hypotheses on their energetic adaptations and life cycle strategies.     

Structural and biophysical determinants of single CaV3.1 and CaV3.2 T-type calcium channel inhibition by N2O

We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels Ca_V3.1 and Ca_V3.2 by nitrous oxide (N₂O). To identify functionally important channel structures, chimeras with reciprocal exchange of the N-terminal domains I and II and C-terminal domains III and IV were examined. In whole-cell recordings N₂O significantly inhibited Ca_V3.2, and – less pronounced – Ca_V3.1. A Ca_V3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing ≤3 channels N₂O reduced average peak current of Ca_V3.2 by decreasing open probability and open time duration. Effects on Ca_V3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single Ca_V3.1 channels were significantly less active than Ca_V3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within Ca_V3.2 (H191), are responsible for the subtype-prevalent N₂O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N₂O on Ca_V3.2. Such a fingerprint of single channels can help identifying the molecular nature of native channels. This is exemplified by a characterization of single channels expressed in human hMTC cells as functional homologues of recombinant Ca_V3.1.     

In yeast redistribution of Sod1 to the mitochondrial intermembrane space provides protection against respiration derived oxidative stress

The antioxidative enzyme copper-zinc superoxide dismutase (Sod1) is an important cellular defence system against reactive oxygen species (ROS). While the majority of this enzyme is localized to the cytosol, about 1% of the cellular Sod1 is present in the intermembrane space (IMS) of mitochondria. These amounts of mitochondrial Sod1 are increased for certain Sod1 mutants that are linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). To date, only little is known about the physiological function of mitochondrial Sod1. Here, we use the model system Saccharomyces cerevisiae to generate cells in which Sod1 is exclusively localized to the IMS. We find that IMS-localized Sod1 can functionally substitute wild type Sod1 and that it even exceeds the protective capacity of wild type Sod1 under conditions of mitochondrial ROS stress. Moreover, we demonstrate that upon expression in yeast cells the common ALS-linked mutant Sod1^G93A becomes enriched in the mitochondrial fraction and provides an increased protection of cells from mitochondrial oxidative stress. Such an effect cannot be observed for the catalytically inactive mutant Sod1^G85R. Our observations suggest that the targeting of Sod1 to the mitochondrial IMS provides an increased protection against respiration-derived ROS.     

methBLAST and methPrimerDB: web-tools for PCR based methylation analysis

Background  

DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers.