Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 microm. Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.     

Analysis of eighteen antidepressants,four atypical antipsychotics and active metabolites in serum by liquid chromatography: a simple tool for therapeutic drug monitoring

Therapeutic drug monitoring necessitates efficient, fast and reliable analytical methods validated by external quality control. We therefore devised an isocratic reversed-phase HPLC method with ultraviolet detection and optimised this to quantify mirtazapine, reboxetine, moclobemide, venlafaxine, O-desmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OH-risperidone in human serum. After solid-phase extraction of the drugs and metabolites, the chromatographic separation was achieved on a Nucleosil 100-Protect 1 column with acetonitrile-potassium dihydrogenphosphate buffer as mobile phase. The method was validated for therapeutic and toxic serum ranges. A linear relationship (r>0.998) was obtained between the concentration and the detector signal. Recoveries were between 75 and 99% for the drugs and metabolites. The accuracy of the quality control samples, expressed as percent recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard deviations were 0.9-10.2% and 0.9-9.7%, respectively. Additional external quality control is carried out since 3 years. This method is applicable to rapidly and effectively analyze serum or plasma samples for therapeutic drug monitoring of about 30 antidepressants and atypical antipsychotics.     

Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain.

It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNA(Tyr) species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNA(Tyr) species were spliced. In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNA(Tyr) domains from yeast and Xenopus, respectively. The chimeric pre-tRNA comprising the mature tRNA(Tyr) domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNA(Tyr) domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.     

Desert breath—How fog promotes a novel type of soil biocenosis,forming the coastal Atacama Desert’s living skin

The Atacama Desert is the driest non‐polar desert on Earth, presenting precarious conditions for biological activity. In the arid coastal belt, life is restricted to areas with fog events that cause almost daily wet–dry cycles. In such an area, we discovered a hitherto unknown and unique ground covering biocenosis dominated by lichens, fungi, and algae attached to grit‐sized (~6 mm) quartz and granitoid stones. Comparable biocenosis forming a kind of a layer on top of soil and rock surfaces in general is summarized as cryptogamic ground covers (CGC) in literature. In contrast to known CGC from arid environments to which frequent cyclic wetting events are lethal, in the Atacama Desert every fog event is answered by photosynthetic activity of the soil community and thus considered as the desert's breath. Photosynthesis of the new CGC type is activated by the lowest amount of water known for such a community worldwide thus enabling the unique biocenosis to fulfill a variety of ecosystem services. In a considerable portion of the coastal Atacama Desert, it protects the soil from sporadically occurring splash erosion and contributes to the accumulation of soil carbon and nitrogen as well as soil formation through bio‐weathering. The structure and function of the new CGC type are discussed, and we suggest the name grit–crust. We conclude that this type of CGC can be expected in all non‐polar fog deserts of the world and may resemble the cryptogam communities that shaped ancient Earth. It may thus represent a relevant player in current and ancient biogeochemical cycling.     

Are boreal ecosystems susceptible to alien plant invasion? Evidence from protected areas

Although biological invasion by alien species is a major contributor to loss of indigenous biological diversity, few studies have examined the susceptibility of the boreal biome to invasion. Based on studies of other ecosystems, we hypothesized that alien plants will be restricted to disturbed areas near human activity and will not be found in natural areas of boreal ecosystems in Gros Morne National Park (Canada), a protected area experiencing a wide range of disturbance regimes. The distribution of alien plants in the region was evaluated using surveys, and study sites were established in naturally and anthropogenically disturbed habitats that had been invaded. Within study sites, randomization tests evaluated the importance of disturbance to alien plant invasion by examining changes in environmental conditions and species abundance within various disturbance regimes, while the importance of site characteristics limiting the distribution of alien plants were examined using Canonical Correspondence Analysis. Consistent with studies in a variety of biomes, areas of high disturbance and human activity had the greatest abundance of resources and the highest percentage of alien species. However, contrary to our hypothesis, natural areas of boreal ecosystems were found susceptible to alien plant invasion. Vegetation types vulnerable to invasion include forests, riparian areas, fens, and alpine meadows. Natural disturbance occurring in these vegetation types caused increases in bare ground and/or light availability facilitating alien plant invasion. Although high soil pH was associated with alien plants in these areas, disturbance was not found to cause changes in soil pH, suggesting susceptibility to invasion is pre-determined by bedrock geology or other factors influencing soil pH. Moose (Alces alces), a non-native herbivore, acts as the primary conduit for alien plant invasion in GMNP by dispersing propagules and creating or prolonging disturbance by trampling and browsing vegetation. The recurrent nature of disturbance within the boreal biome and its interaction with site conditions and herbivores enables alien plants to persist away from areas of high human activity. Managers of natural lands should monitor such interactions to decrease the invasion potential of alien plants.     

Aberrant Amyloid Precursor Protein (APP) Processing in Hereditary Forms of Alzheimer Disease Caused by APP Familial Alzheimer Disease Mutations Can Be Rescued by Mutations in the APP GxxxG Motif

The identification of hereditary familial Alzheimer disease (FAD) mutations in the amyloid precursor protein (APP) and presenilin-1 (PS1) corroborated the causative role of amyloid-β peptides with 42 amino acid residues (Aβ42) in the pathogenesis of AD. Although most FAD mutations are known to increase Aβ42 levels, mutations within the APP GxxxG motif are known to lower Aβ42 levels by attenuating transmembrane sequence dimerization. Here, we show that aberrant Aβ42 levels of FAD mutations can be rescued by GxxxG mutations. The combination of the APP-GxxxG mutation G33A with APP-FAD mutations yielded a constant 60% decrease of Aβ42 levels and a concomitant 3-fold increase of Aβ38 levels compared with the Gly³³ wild-type as determined by ELISA. In the presence of PS1-FAD mutations, the effects of G33A were attenuated, apparently attributable to a different mechanism of PS1-FAD mutants compared with APP-FAD mutants. Our results contribute to a general understanding of the mechanism how APP is processed by the γ-secretase module and strongly emphasize the potential of the GxxxG motif in the prevention of sporadic AD as well as FAD.     

A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon

Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin-gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2-10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with < or = 3 copies have a significantly higher risk of developing colonic CD than did individuals with > or = 4 copies (odds ratio 3.06; 95% confidence interval 1.46-6.45). An HBD-2 gene copy number of < 4 was associated with diminished mucosal HBD-2 mRNA expression (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression.     

Mouse soleus muscle Na-K pump activity: direct correlation with in vitro and in vivo oxygen consumption

To evaluate the contribution of active sodium-potassium transport to energy utilization, measurements of sodium-potassium pump activity in isolated soleus muscles of normal mice were carried out and related to measurements of oxygen consumption by the tissue. A highly significant positive correlation (r = 0.89, p less than 0.001) was found between these two parameters. In addition, both the in vitro oxygen consumption and Na-K pump activity of the muscles were found to correlate with whole body oxygen utilization by the experimental animals. The results support a role for the sodium-potassium pump in the determination of energy turnover.