Toward reconstitution of in vivo microtubule dynamics in vitro

The transition from interphase to mitosis is marked by a dramatic change in microtubule dynamics resulting in the reorganization of the microtubule network that culminates in mitotic spindle formation. While the molecular basis for this change in microtubule organization remains obscure, it is currently thought that a balance in the activity of microtubule stabilizing and destabilizing factors regulates how dynamic cellular microtubules are. By mixing the microtubule stabilizer XMAP215 and the microtubule destabilizer XKCM1, reconstitution of in vivo-like microtubule dynamics has now been achieved in vitro.     

Monoseptic cultivation of phototrophic microorganisms--development and scale-up of a photobioreactor system with thermal sterilization

The use of phototrophic microorganisms as sources of biological active substances in photoautotrophic and mixotrophic cultivation modes requires an adequate cultivation system with thermal sterilization. A corresponding photobioreactor system in the 10, 25 and 100 l scales was developed. This "Medusa"-photobioreactor system represents a concept based on the air-lift loop principle, whose working volume is irradiated by external light sources. The incident irradiation can be varied by a light control system. An effective CO(2)/O(2) gas exchange is enabled due to the efficient supply with process gas by several gas supply nozzles within the system and a large degassing surface. Using a model to describe the growth characteristics of the organisms, the volumetric irradiation coefficient I(DX) was defined as scale-up parameter. On this basis the scale-up from 1 l bubble columns to the 10 and 100 l scales was realized. The scale-up was performed successfully with Chlorella salina as model organism. A maximum biomass concentration of 7.89 g (dry weight) l(-1) at a maximum specific growth rate of 0.058 h(-1) and a yield of 35 mg l(-1) h(-1) was obtained in a batch cultivation in the 100 l scale under photoautotrophic conditions with an initial biomass concentration of approx. 0.03 g l(-1).     

Intergeneric Transfer of Conjugative and Mobilizable Plasmids Harbored by Escherichia coli in the Gut of the Soil Microarthropod Folsomia candida (Collembola)

The gut of the soil microarthropod Folsomia candida provides a habitat for a high density of bacterial cells (T. Thimm, A. Hoffmann, H. Borkott, J. C. Munch, and C. C. Tebbe, Appl. Environ. Microbiol. 64:2660–2669, 1998). We investigated whether these gut bacteria act as recipients for plasmids from Escherichia coli. Filter mating with E. coli donor cells and collected feces of F. candida revealed that the broad-host-range conjugative plasmid pRP4-luc (pRP4 with a luciferase marker gene) transferred to fecal bacteria at estimated frequencies of 5.4 × 10⁻¹ transconjugants per donor. The mobilizable plasmid pSUP104-luc was transferred from the IncQ mobilizing strain E. coli S17-1 and less efficiently from the IncF1 mobilizing strain NM522 but not from the nonmobilizing strain HB101. When S17-1 donor strains were fed to F. candida, transconjugants of pRP4-luc and pSUP104-luc were isolated from feces. Additionally, the narrow-host-range plasmid pSUP202-luc was transferred to indigenous bacteria, which, however, could not maintain this plasmid. Inhibition experiments with nalidixic acid indicated that pRP4-luc plasmid transfer took place in the gut rather than in the feces. A remarkable diversity of transconjugants was isolated in this study: from a total of 264 transconjugants, 15 strains belonging to the alpha, beta, or gamma subclass of the class Proteobacteria were identified by DNA sequencing of the PCR-amplified 16S rRNA genes and substrate utilization assays (Biolog). Except for Alcaligenes faecalis, which was identified by the Biolog assay, none of the isolates was identical to reference strains from data banks. This study indicates the importance of the microarthropod gut for enhanced conjugative gene transfer in soil microbial communities.Gene transfer is a process by which bacterial populations substantially increase their rates of evolution and adaptation (12, 59). Particularly, plasmid-located genes, which are transferred by conjugation from donor to recipient cells, can disseminate rapidly between even phylogenetically different bacterial groups (17, 36, 41) and microbial communities in different spatial habitats (34, 71). Such microbial genetic networks should be considered in risk assessments of releases of genetically engineered microorganisms into the environment (22, 37, 43). The probability and rate of plasmid transfer from a donor to indigenous microorganisms in a given habitat are influenced by plasmid-borne genes which determine the type of transfer mechanism (self-transmissible or mobilizable) and the host range of autonomous plasmid replication. Additionally, specific physicochemical conditions, such as temperature, water potential, and the availability of energy (substrates) for donor and recipient cells, are important factors influencing gene transfer rates in terrestrial and aquatic environments (23, 53, 64).The spread of plasmid-borne genes is still extremely difficult to predict for terrestrial habitats, since a large variety of microhabitat conditions which are not well characterized exists. In bulk soil under laboratory conditions, conjugative gene transfer from recombinant bacterial donor strains to indigenous soil bacteria has been found only under specific selective conditions or on rare occasions (11, 20, 24, 27, 50, 61). Several studies failed to detect such transfer events, and it was concluded that heterogeneity and low densities of recipient cells, as well as a lack of substrates for microbial metabolism, prevented efficient plasmid transfer in bulk soil (19, 49, 54, 75). Plant exudates increased rates of gene transfer in soil (33, 48), and higher rates of gene transfer were found in rhizospheres than in bulk soil (50, 61). It was assumed that other microsites which favor gene transfer in terrestrial habitats are associated with soil invertebrates (74). However, to date little experimental evidence to prove this assumption is available.Intraspecies transconjugants of added Enterobacter cloacae donor and recipient cells could be isolated from microcosm experiments with the variegated cutworm, Peridroma saucia, and plant material (2). The investigators in that study concluded that gene transfer events happened, most likely, in the digestive tracts or in the feces of the insects. Another recent report demonstrated that a conjugative plasmid was transferred between fed Escherichia coli strains in the guts of Rhabditis nematodes (1). Earthworms mediated transport and enhanced plasmid transfer from added donor cells to added recipients and to indigenous bacteria in soil (14, 15). High rates of intraspecies plasmid transfer, comparable to those obtained in pure broth cultures, were detected with Bacillus thuringiensis in infected lepidopterous larvae (31).Microarthropods (collembolans and mites) are the most abundant invertebrate group in the majority of soils (5) but have not been recognized, so far, for their impact on microbial gene transfer. There are some indications that microarthropods harbor a large variety of microorganisms in their guts and thereby contribute to microbial biodiversity in terrestrial environments (7, 9, 57). In the accompanying paper, we have described the gut of Folsomia candida (Collembola) as a habitat and species-specific vector for microorganisms (67). The gut of this soil-dwelling insect, which has a volume of only several nanoliters, was found to be densely colonized, predominantly by rod-shaped bacterial cells. We were interested to know whether such bacterial cells act as recipients for plasmids and thereby promote gene transfer in microbial communities. F. candida feeds, under natural conditions, on bacteria (3), fungal mycelia (6, 66), and nematodes (35). Here, we report on the results of experiments in which plasmid-bearing E. coli strains were fed to F. candida in microcosms. Self-transferable plasmids, as well as mobilizable plasmids with different host ranges, and a nonmobilizable plasmid were included in this study in order to determine the specific capacities of these different classes of plasmids to spread into indigenous bacterial populations. For detection purposes, all plasmids were engineered by the insertion of the luciferase-encoding marker gene luc or lux (30, 47).     

The Universal Stress Protein UspC Scaffolds the KdpD/KdpE Signaling Cascade of Escherichia coli under Salt Stress

The sensor kinase KdpD and the response regulator KdpE control induction of the kdpFABC operon encoding the high-affinity K⁺-transport system KdpFABC in response to K⁺ limitation or salt stress. Under K⁺ limiting conditions the Kdp system restores the intracellular K⁺ concentration, while in response to salt stress K⁺ is accumulated far above the normal content. The kinase activity of KdpD is inhibited at high concentrations of K⁺, so it has been puzzling how the sensor can be activated in response to salt stress. Here, we demonstrate that the universal stress protein UspC acts as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with a Usp domain in KdpD of the UspA subfamily under salt stress. Escherichia coli encodes three single domain proteins of this subfamily, UspA, UspC, and UspD, whose expression is up-regulated under various stress conditions. Among these proteins only UspC stimulated the in vitro reconstructed signaling cascade (KdpD→KdpE→DNA) resulting in phosphorylation of KdpE at a K⁺ concentration that would otherwise almost prevent phosphorylation. In agreement, in a ΔuspC mutant KdpFABC production was down-regulated significantly when cells were exposed to salt stress, but unchanged under K⁺ limitation. Biochemical studies revealed that UspC interacts specifically with the Usp domain in the stimulus perceiving N-terminal domain of KdpD. Furthermore, UspC stabilized the KdpD/KdpE∼P/DNA complex and is therefore believed to act as a scaffolding protein. This study describes the stimulation of a bacterial two-component system under distinct stress conditions by a scaffolding protein, and highlights a new role of the universal stress proteins.     

Perioperative Chemotherapy in Gastroesophageal Cancer. A Retrospective Monocenter Evaluation of 42 Cases

Background

Perioperative chemotherapy increases the overall and progression-free survival of patients suffering from resectable adenocarcinomas of the lower esophagus, gastroesophageal junction and stomach (GEC). Comparing different chemotherapy regimens platin-based protocols with 5-fluorouracil (5-FU)/calcium folinate (CF) or oral fluoropyrimidines were favorable in terms of efficacy and side-effects. However, there is no consensus which regimen is the most efficacious.

Methods

42 consecutive patients with resectable GEC (UICC II and III) were treated with 3 pre- and postoperative chemotherapy cycles each consisting of epirubicin, oxaliplatin and capecitabine (EOX). We analyzed the overall survival, progression-free survival and toxicity retrospectively in comparison to published data.

Results

The median overall survival in our cohort was 29 months and the progression-free survival was 17 months. The most frequent grade 3 and 4 toxicities during preoperative chemotherapy were diarrhea (16.7%), leukocytopenia (9.5%) and nausea (9.5%); overall 38.1% of our patients suffered from grade 3 or 4 toxicity. Surgery was carried out in 83% of our patients, 69% of those achieved R0 resection.

Conclusion

Comparing our data with the results of previously published randomized trials EOX is at least non-inferior with regard to overall survival, progression-free survival and toxicity. In conclusion, EOX is an appropriate perioperative therapy for patients with resectable GEC.     

Comparative Analysis of Genetic Chemotyping Methods for Fusarium: Tri13 Polymorphism Does not Discriminate between 3‐ and 15‐acetylated Deoxynivalenol Chemotypes in Fusarium graminearum

Genetic chemotyping is an essential tool for characterizing Fusarium populations causing head blight on wheat and other cereals. Three PCR methods, based on tri cluster polymorphism, were optimized and compared on 94 single‐spore isolates obtained from three continents belonging to F. gramineaurm, F. culmorum, F. poae, F. avenaceum and Microdochium nivale. While the methods based on the tri3, tri7 and tri12 polymorphism correctly identified all the tested strains, the method based on tri13 polymorphism was unable to discriminate between the 3‐ and 15‐acetylated DON forms in F. graminearum. It is advised to avoid the use of tri13 polymorphism for genetic chemotyping of the two acetylated chemotypes.     

Reduced food intake and body weight in mice deficient for the G protein-coupled receptor GPR82

G protein-coupled receptors (GPCR) are involved in the regulation of numerous physiological functions. Therefore, GPCR variants may have conferred important selective advantages during periods of human evolution. Indeed, several genomic loci with signatures of recent selection in humans contain GPCR genes among them the X-chromosomally located gene for GPR82. This gene encodes a so-called orphan GPCR with unknown function. To address the functional relevance of GPR82 gene-deficient mice were characterized. GPR82-deficient mice were viable, reproduced normally, and showed no gross anatomical abnormalities. However, GPR82-deficient mice have a reduced body weight and body fat content associated with a lower food intake. Moreover, GPR82-deficient mice showed decreased serum triacylglyceride levels, increased insulin sensitivity and glucose tolerance, most pronounced under Western diet. Because there were no differences in respiratory and metabolic rates between wild-type and GPR82-deficient mice our data suggest that GPR82 function influences food intake and, therefore, energy and body weight balance. GPR82 may represent a thrifty gene most probably representing an advantage during human expansion into new environments.     

Veratrol-<Emphasis Type="Italic">O</Emphasis>-demethylase of <Emphasis Type="Italic">Acetobacterium dehalogenans</Emphasis>: ATP-dependent reduction of the corrinoid protein

The anaerobic veratrol O-demethylase mediates the transfer of the methyl group of the phenyl methyl ether veratrol to tetrahydrofolate. The primary methyl group acceptor is the cobalt of a corrinoid protein, which has to be in the +1 oxidation state to bind the methyl group. Due to the negative redox potential of the cob(II)/cob(I)alamin couple, autoxidation of the cobalt may accidentally occur. In this study, the reduction of the corrinoid to the superreduced [Co^I] state was investigated. The ATP-dependent reduction of the corrinoid protein of the veratrol O-demethylase was shown to be dependent on titanium(III) citrate as electron donor and on an activating enzyme. In the presence of ATP, activating enzyme, and Ti(III), the redox potential versus the standard hydrogen electrode (E _SHE) of the cob(II)alamin/cob(I)alamin couple in the corrinoid protein was determined to be −290 mV (pH 7.5), whereas E _SHE at pH 7.5 was lower than −450 mV in the absence of either activating enzyme or ATP. ADP, AMP, or GTP could not replace ATP in the activation reaction. The ATP analogue adenosine-5′-(β,γ-imido)triphosphate (AMP-PNP, 2–4 mM) completely inhibited the corrinoid reduction in the presence of ATP (2 mM).     

Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor

Ligand binding to ecdysone receptor (EcR) is an autonomous function of the ligand binding domain (LBD) and is not modified by other receptor domains or tags fused to the LBD. Association and dissociation velocity of hormone to EcR was studied in the absence and presence of its main dimerization partner Ultraspiracle (USP). Mutational analysis of the EcR(LBD) revealed that ligand entry and exit is affected differently by the same point mutation, indicating that different pathways are used for association and dissociation of the ligand. Heterodimerization with wild type USP(LBD) increases ligand association to EcR(LBD) about fivefold and reduces dissociation 18-fold. Opposite effects of the same mutation (N626K) on dissociation velocity of ligand in EcR and EcR/USP indicate that not only hormone binding itself, but also the kinetic behaviour of ligand binding is modified by the dimerization partner. A general effect of the point mutations on the 3D architecture seems unlikely due to the highly selective effects on the kinetics of hormone binding.