Nonyloxytryptamine mimics polysialic acid and modulates neuronal and glial functions in cell culture

Polysialic acid (PSA) is a major regulator of cell–cell interactions in the developing nervous system and in neural plasticity in the adult. As a polyanionic molecule with high water‐binding capacity, PSA increases the intercellular space generating permissive conditions for cell motility. PSA enhances stem cell migration and axon path finding and promotes repair in the lesioned peripheral and central nervous systems, thus contributing to regeneration. As a next step in developing an improved PSA‐based approach to treat nervous system injuries, we searched for small organic compounds that mimic PSA and identified as a PSA mimetic 5‐nonyloxytryptamine oxalate, described as a selective 5‐hydroxytryptamine receptor 1B (5‐HT_1B) agonist. Similar to PSA, 5‐nonyloxytryptamine binds to the PSA‐specific monoclonal antibody 735, enhances neurite outgrowth of cultured primary neurons and process formation of Schwann cells, protects neurons from oxidative stress, reduces migration of astrocytes and enhances myelination in vitro. Furthermore, nonyloxytryptamine treatment enhances expression of the neural cell adhesion molecule (NCAM) and its polysialylated form PSA‐NCAM and reduces expression of the microtubule‐associated protein MAP2 in cultured neuroblastoma cells. These results demonstrate that 5‐nonyloxytryptamine mimics PSA and triggers PSA‐mediated functions, thus contributing to the repertoire of molecules with the potential to improve recovery in acute and chronic injuries of the mammalian peripheral and central nervous systems.

     

Impact of Heat and Laccase on the pH and Freeze-Thaw Stability of Oil-in-Water Emulsions Stabilized by Adsorbed Biopolymer Nanoparticles

The enzymatic cross-linking of adsorbed biopolymer nanoparticles formed between whey protein isolate (WPI) and sugar beet pectin using the complex coacervation method was investigated. A sequential electrostatic depositioning process was used to prepare emulsions containing oil droplets stabilized by WPI – nanoparticle – membranes. Firstly, a finely dispersed primary emulsion (10 % w/w miglyol oil, 1 % w/w WPI, 10 mM acetate buffer at pH 4) was produced using a high-pressure homogenizer. Secondly, a series of biopolymer particles were formed by mixing WPI (0.5 % w/w) and pectin (0.25 % w/w) solutions with subsequent heating above the thermal denaturation temperature (85 °C, 20 min) to prepare dispersions containing particles in the submicron range. Thirdly, nanoparticle-covered emulsions were formed by diluting the primary emulsion into coacervate solutions (0–0.675 % w/w) to coat the droplets. Oil droplets of stable emulsions with different interfacial membrane compositions were subjected to enzymatic cross-linking. We used cross-linked multilayered emulsions as a comparison. The pH stability of primary emulsions, biopolymer complexes and nanoparticle-coated base emulsions, as well as multilayered emulsions, was determined before and after enzyme addition. Freeze-thaw stability (?9 °C for 22 h, 25 °C for 2 h) of nanoparticle-coated emulsions was not affected by laccase. Results indicated that cross-linking occurred exclusively in the multilamellar layers and not between adsorbed biopolymer nanoparticles. Results suggest that the accessibility of distinct structures may play a key role for biopolymer-cross-linking enzymes.     

Strategies for reliable and improved large-scale production of Pyrococcus furiosus with integrated purification of hydrogenase I

The hyperthermophilic archaeon Pyrococcus furiosus is an interesting organism for research and application, especially owing to its unique NADPH-dependent hydrogenase I. However, mass production of P. furiosus through fermentation is susceptible to fault because of its sensitivity to oxygen, a short exponential and stationary phase and a rapid cell lysis in typical cultivation process. In this study, significant improvement for pilot plant scale production processes for P. furiosus biomass was made by investigations of the fermentation process with subsequent hydrogenase I enzyme purification. Scale-up in a 300-L stirred tank bioreactor was successfully achieved. A repeated-batch cultivation process with high reproducibility and productivity was realized. Furthermore, the enzyme hydrogenase I was purified, and its activity tested and verified. The improvements in this production process for the production of large amount of P. furiosus biomass and hydrogenase I have been achieved, especially by successfully implementing the following key measures and steps: unsterile cultivation setup, skipping typical intermediate preculture and inoculation steps, accelerating the cultivation process by defining an optimal state of the inoculation, optimal time point of biomass harvesting and finally by choosing a one-step purification procedure for enzyme recovery.     

An intron-containing,heat-inducible stress-70 gene in the millipede Tachypodoiulus niger (Julidae,Diplopoda)

The highly conserved part of the nucleotide-binding domain of the hsp70 gene family was amplified from the soil diplopod Tachypodoiulus niger (Julidae, Diplopoda). Genomic DNA yielded 701, 549 and 540 bp sequences, whereas cDNA from heat shocked animals produced only one distinct fragment of 543 bp. The sequences could be classified as a 70 kDa heat shock protein (hsp70), the corresponding 70 kDa heat shock cognate (hsc70) and a glucose-related hsp70 homologue (grp78). Comparisons of genomic and cDNA sequences of hsc70 identified two introns within the consensus sequence. Generally, stress-70 expression levels were low, which hampered successful RT-PCR and subsequent subcloning. Following experimental heat shock, however, the spliced hsc70 was amplified predominantly, instead of its inducible homologue hsp70. This finding suggests that microevolution in this soil-dwelling arthropod is directed towards low constitutive stress-70 levels and that the capacity for stress-70 induction presumably is limited. hsc70, albeit having introns, apparently is inducible and contributes to the stress-70 response.     

Native structure-based modeling and simulation of biomolecular systems per mouse click

Background

Molecular dynamics (MD) simulations provide valuable insight into biomolecular systems at the atomic level. Notwithstanding the ever-increasing power of high performance computers current MD simulations face several challenges: the fastest atomic movements require time steps of a few femtoseconds which are small compared to biomolecular relevant timescales of milliseconds or even seconds for large conformational motions. At the same time, scalability to a large number of cores is limited mostly due to long-range interactions. An appealing alternative to atomic-level simulations is coarse-graining the resolution of the system or reducing the complexity of the Hamiltonian to improve sampling while decreasing computational costs. Native structure-based models, also called Gō-type models, are based on energy landscape theory and the principle of minimal frustration. They have been tremendously successful in explaining fundamental questions of, e.g., protein folding, RNA folding or protein function. At the same time, they are computationally sufficiently inexpensive to run complex simulations on smaller computing systems or even commodity hardware. Still, their setup and evaluation is quite complex even though sophisticated software packages support their realization.

Results

Here, we establish an efficient infrastructure for native structure-based models to support the community and enable high-throughput simulations on remote computing resources via GridBeans and UNICORE middleware. This infrastructure organizes the setup of such simulations resulting in increased comparability of simulation results. At the same time, complete workflows for advanced simulation protocols can be established and managed on remote resources by a graphical interface which increases reusability of protocols and additionally lowers the entry barrier into such simulations for, e.g., experimental scientists who want to compare their results against simulations. We demonstrate the power of this approach by illustrating it for protein folding simulations for a range of proteins.

Conclusions

We present software enhancing the entire workflow for native structure-based simulations including exception-handling and evaluations. Extending the capability and improving the accessibility of existing simulation packages the software goes beyond the state of the art in the domain of biomolecular simulations. Thus we expect that it will stimulate more individuals from the community to employ more confidently modeling in their research.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-292) contains supplementary material, which is available to authorized users.     

Tyrosine phosphatase PTP1B modulates store-operated calcium influx

We have studied modulation of “store-operated calcium influx” by tyrosine phosphatases in the pancreatic acinar cell line AR42J and in HEK 293 cells. We show that inhibition of tyrosine phosphatases by bis-(N,N-dimethyl-hydroxamido) hydrooxovanadate (DMHV) leads to an increase in Ca²⁺ release-activated Ca²⁺ (CRAC) entry. This effect can be blocked in the presence of 2-aminoethyldiphenyl borate (2-APB). Furthermore, transfection of HEK 293 cells with the human wild-type tyrosine phosphatase PTP1B leads to inhibition of CRAC influx, whereas transfection with the substrate-trapping mutant of PTP1B (D181A) slightly increases Ca²⁺ influx. It also decreases enzymatic activity of PTP1B as compared to non-transfected cells. Our data suggest that CRAC influx is modulated by tyrosine phosphorylation and dephosphorylation which involves the tyrosine phosphatase PTP1B.     

Variation in life-cycle between three rare and endangered floodplain violets in two regions: implications for population viability and conservation

We studied the demography of Viola elatior, V. pumila, and V. stagnina, three rare and endangered Central European floodplain species, to (i) analyse variation in life-cycles among congeners and between regions (Dyje-Morava floodplains, Czech Republic; Upper Rhine, Germany), (ii) to define sensitive stages in the life-cycles, and (iii) to identify possible threats for population viability and species conservation. Matrix models were based on the fate of marked individuals from a total of 27 populations over two years. We analysed population growth rate (λ), stage distribution, net reproductive rate (R ₀), generation time, age at first reproduction, and elasticity and calculated a life table response experiment (LTRE). Most populations were declining and λ did not differ between species or regions during the observed interval. Despite higher probabilities for survival and flowering in the Dyje populations, R ₀ was higher in the Rhine populations. Also other demographic traits showed consistent differences between regions and/or species. Complex life-cycles and large variation in λ precluded unequivocal identification of sensitive stages or vital rates for conservation. Variation between regions may be a consequence of differences in habitat quality. Our results suggest that deterministic processes such as reduced management, succession, habitat destruction, and lack of disturbance through reduced or eliminated flooding present the strongest threat for the viability and persistence of populations of the three floodplain violets as compared with stochastic processes. However, the persistent seed bank of the species may buffer populations against environmental variation and represents a reservoir for recovery after resumption of suitable land-use management.     

Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex

We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.     

Effect of the polycyclic aromatic hydrocarbon phenanthrene on root exudation of Sorghum bicolor (L.) Moench

The objective of this work was to study the effect of two concentrations (10 and 100 mg kg⁻¹) of phenanthrene, a ubiquitous polycyclic aromatic hydrocarbon (PAH), on root exudation of the remediating plant Sorghum bicolor (L.) Moench under controlled conditions in a pot experiment. It was found that the phenanthrene concentration of 10 mg kg⁻¹ did not cause significant effects on plant survival and growth but had little stimulating effect on carbohydrate exudation. The contamination with phenanthrene at 100 mg kg⁻¹ inhibited accumulation of plant shoot and root biomass, decreasing the carboxylic acid, carbohydrate, and amino acid amounts released by sorghum root into the rhizosphere. However, root exudation per unit of root surface was not changed significantly with increasing phenanthrene concentration. There were no differences in qualitative composition of root exudates under the influence of PAH were found. The observed alterations in the ratio between the main root-exuded components are assumed to manifest adaptive alterations occurring in the plant as a response to pollutant stress. The activity of three oxidoreductases (oxidase, peroxidase, and tyrosinase) released by sorghum roots was clearly progressive to the increasing phenanthrene concentration in the substrate. Under the influence of phenanthrene, the population of phenanthrene-degrading microorganisms in sorghum root zone increased, and their share in the total number of culturable heterotrophs increased as well. The main promotional factor was the pollutant; however, the stimulating effect of the plant root exudates was also involved. The increased pollutant-degrading microbial population and activity of the extracellular root enzymes are presumed to be important for the rhizodegradation of PAH.