Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin.     

Domain architecture and biochemical characterization of vertebrate Mcm10

Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single- and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase alpha-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase alpha-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.     

Hepatocyte growth factor/c-MET axis-mediated tropism of cord blood-derived unrestricted somatic stem cells for neuronal injury

An under-agarose chemotaxis assay was used to investigate whether unrestricted somatic stem cells (USSC) that were recently characterized in human cord blood are attracted by neuronal injury in vitro. USSC migrated toward extracts of post-ischemic brain tissue of mice in which stroke had been induced. Moreover, apoptotic neurons secrete factors that strongly attracted USSC, whereas necrotic and healthy neurons did not. Investigating the expression of growth factors and chemokines in lesioned brain tissue and neurons and of their respective receptors in USSC revealed expression of hepatocyte growth factor (HGF) in post-ischemic brain and in apoptotic but not in necrotic neurons and of the HGF receptor c-MET in USSC. Neuronal lesion-triggered migration was observed in vitro and in vivo only when c-MET was expressed at a high level in USSC. Neutralization of the bioactivity of HGF with an antibody inhibited migration of USSC toward neuronal injury. This, together with the finding that human recombinant HGF attracts USSC, document that HGF signaling is necessary for the tropism of USSC for neuronal injury. Our data demonstrate that USSC have the capacity to migrate toward apoptotic neurons and injured brain. Together with their neural differentiation potential, this suggests a neuroregenerative potential of USSC. Moreover, we provide evidence for a hitherto unrecognized pivotal role of the HGF/c-MET axis in guiding stem cells toward brain injury, which may partly account for the capability of HGF to improve function in the diseased central nervous system.     

Measurement of pulsatile insulin secretion in the rat: direct sampling from the hepatic portal vein

It has previously been shown that insulin is secreted in discrete secretory bursts by sampling directly from the portal vein in the dog and humans. Deficient pulsatile insulin secretion is the basis for impaired insulin secretion in type 2 diabetes. However, while novel genetically modified disease models of diabetes are being developed in rodents, no validated method for quantifying pulsatile insulin secretion has been established for rodents. To address this we 1) developed a novel rat model with chronically implanted portal vein catheters, 2) established the parameters to permit deconvolution of portal vein insulin concentrations profiles to measure insulin secretion and resolve its pulsatile components, and 3) measured total and pulsatile insulin secretion compared with that in the dog, the species in which this sampling and deconvolution approach was validated for quantifying pulsatile insulin secretion. In rats, portal vein catheter patency and function were maintained for periods up to 2-3 wk with no postoperative complications such as catheter tract infection. Rat portal vein insulin concentration profiles in the fasting state revealed distinct insulin oscillations with a periodicity of approximately 5 min and an amplitude of up to 600 pmol/l, which was remarkably similar to that in the dogs and in humans. Deconvolution analysis of portal vein insulin concentrations revealed that the majority of insulin ( approximately 70%) in the rat is secreted in distinct insulin pulses occurring at approximately 5-min intervals. This model therefore permits direct accurate measurements of pulsatile insulin secretion in a relatively inexpensive animal. With increased introduction of genetically modified rat models will be an important tool in elucidating the underlying mechanisms of impaired pulsatile insulin secretion in diabetes.     

Comparative analysis of ammonia monooxygenase (amoA) genes in the water column and sediment-water interface of two lakes and the Baltic Sea

The functional gene amoA was used to compare the diversity of ammonia-oxidizing bacteria (AOB) in the water column and sediment-water interface of the two freshwater lakes Plusssee and Sch?hsee and the Baltic Sea. Nested amplifications were used to increase the sensitivity of amoA detection, and to amplify a 789-bp fragment from which clone libraries were prepared. The larger part of the sequences was only distantly related to any of the cultured AOB and is considered to represent new clusters of AOB within the Nitrosomonas/Nitrosospira group. Almost all sequences from the water column of the Baltic Sea and from 1-m depth of Sch?hsee were related to different Nitrosospira clusters 0 and 2, respectively. The majority of sequences from Plusssee and Sch?hsee were associated with sequences from Chesapeake Bay, from a previous study of Plusssee and from rice roots in Nitrosospira-like cluster A, which lacks sequences from Baltic Sea. Two groups of sequences from Baltic Sea sediment were related to clonal sequences from other brackish/marine habitats in the purely environmental Nitrosospira-like cluster B and the Nitrosomonas-like cluster. This confirms previous results from 16S rRNA gene libraries that indicated the existence of hitherto uncultivated AOB in lake and Baltic Sea samples, and showed a differential distribution of AOB along the water column and sediment of these environments.     

Nitrogen stress induction on <Emphasis Type="Italic">Levisticum officinale</Emphasis> hairy roots grown in darkness and under photoperiod conditions: effect on growth and volatile components

Six-year-old Levisticum officinale (lovage) hairy root cultures were used to study the effect of eight different NH(4) (+):NO(3) (-) ratios on their growth and volatile components. All cultures were kept at 24 degrees C on orbital shakers at 80 rpm, in darkness or in a 16 h light/8 h dark photoperiod. Growth was evaluated by dry and fresh weight determination. The volatiles were isolated by distillation-extraction and analysed by GC and GC-MS. Greater growth was attained in darkness with 10:90 (control, SH medium), 50:50 and 25:75 NH(4) (+):NO(3) (-) ratios, and also with SH control medium under the photoperiod condition, with a 10, 14, 12.5 and 12.5 fold increase of biomass in terms of dry weight, respectively, at the end of 42 days of growth. UPGMA cluster analysis of the mixtures of volatiles isolated from the hairy roots grown with different NH(4) (+):NO(3) (-) ratios confirmed their chemical variability. Although no particular grouping was detected in relation to the NH(4) (+):NO(3) (-) ratios or light conditions studied, most of the mixtures of volatiles isolated from the hairy roots were either dominated by n-octanal, (Z)-falcarinol or both components in about the same relative amounts.     

The role of topolins in micropropagation and somaclonal variation of banana cultivars ‘Williams’ and ‘Grand Naine’ (<Emphasis Type="Italic">Musa</Emphasis> spp. AAA)

The effect of the cytokinins mT (meta-topolin), mTR (meta-topolin riboside), MemT (meta-methoxy topolin) and MemTR (meta-methoxy topolin riboside) on micropropagation of banana cultivars ‘Williams’ and ‘Grand Naine’ was studied and compared to BA (6-benzylaminopurine). In vitro cultures, at the third sub-culture level, were purchased from African Biotechnologies (Pty) Ltd., South Africa. These were then sub-cultured on MS media containing 7.5, 15 and 30 μM of all the cytokinins tested. Results recorded after 6 weeks of growth demonstrated that there were statistically significant differences between the parameters analyzed for the treatments. Superior multiplication rates were recorded for mT and mTR treatments. This result was consistent when compared to BA at 22.2 μM (previously published standard concentration). Contrary to previous findings with other species, these cytokinins inhibited rooting. The effect on somaclonal variation was not significantly different when BA, mT and mTR were tested at the seventh multiplication cycle for ‘Williams’ banana. These results support the possible use of topolins as an alternative to BA for Cavendish banana tissue culture. The role of these cytokinins on somaclonal variation however, requires a more stringent investigation as the results obtained in this investigation could have been influenced by carry-over effects from the initial cultures.     

Differences in vegetation composition and plant species identity lead to only minor changes in soil-borne microbial communities in a former arable field

To examine the relationship between plant species composition and microbial community diversity and structure, we carried out a molecular analysis of microbial community structure and diversity in two field experiments. In the first experiment, we examined bacterial community structure in bulk and rhizosphere soils in fields exposed to different plant diversity treatments, via a 16S rRNA gene clone library approach. Clear differences were observed between bacterial communities of the bulk soil and the rhizosphere, with the latter containing lower bacterial diversity. The second experiment focused on the influence of 12 different native grassland plant species on bacterial community size and structure in the rhizosphere, as well as the structure of Acidobacteria and Verrucomicrobia community structures. In general, bacterial and phylum-specific quantitative PCR and PCR-denaturing gradient gel electrophoresis revealed only weak influences of plant species on rhizosphere communities. Thus, although plants did exert an influence on microbial species composition and diversity, these interactions were not specific and selective enough to lead to major impacts of vegetation composition and plant species on below-ground microbial communities.     

Phylogenetic analysis of bacteria associated with Laminaria saccharina

Bacterial communities associated with the brown alga Laminaria saccharina from the Baltic Sea and from the North Sea were investigated using denaturing gradient gel electrophoresis and 16S rRNA gene clone libraries. The rhizoid, cauloid, meristem and phyloid revealed different 16S rRNA gene denaturing gradient gel electrophoresis banding patterns indicating a specific association of bacterial communities with different parts of the alga. Associations with cauloid and meristem were more specific, while less specific associations were obtained from the old phyloid. In addition, seasonal and geographical differences in the associated communities were observed. Results from 16S rRNA gene libraries supported these findings. Bacterial phylotypes associated with the alga were affiliated with the Alphaproteobacteria (nine phylotypes), Gammaproteobacteria (nine phylotypes) and the Bacteroidetes group (four phylotypes). A number of bacteria associated with other algae and other marine macroorganisms were among the closest relatives of phylotypes associated with L. saccharina.