Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

In-depth biophysical analysis of interactions between therapeutic antibodies and the extracellular domain of the epidermal growth factor receptor

Targeting of the epidermal growth factor receptor (EGFR) with monoclonal antibodies has become an established antitumor strategy in clinical use or in late stages of drug development. The mAbs effector mechanisms have been widely analyzed based on in vivo or cell studies. Hereby we intend to complement these functional studies by investigating the mAb-EGFR interactions on a molecular level. Surface plasmon resonance, isothermal titration calorimetry, and static light scattering were employed to characterize the interactions of matuzumab, cetuximab, and panitumumab with the extracellular soluble form ecEGFR. The kinetic and thermodynamic determinants dissected the differences in mAbs binding mechanism toward ecEGFR. The quantitative stoichiometric data clearly demonstrated the bivalent binding of the mAbs to two ecEGFR molecules. Our results complement earlier studies on simultaneous binding of cetuximab and matuzumab. The antibodies retain their bivalent binding mode achieving a 1:2:1 complex formation. Interestingly the binding parameters remain nearly constant for the individual antibodies in this ternary assembly. In contrast the binding of panitumumab is almost exclusive either by directly blocking the accessibility for the second antibody or by negative allosteric modulation. Overall we provide a comprehensive biophysical dataset on binding parameters, the complex assembly, and relative epitope accessibility for therapeutic anti-EGFR antibodies.     

Polyphenolic compounds are novel protective agents against lipid membrane damage by α-synuclein aggregates in vitro

Cumulative evidence now suggests that the abnormal aggregation of the protein α-synuclein (αS) is a critical factor in triggering neurodegeneration in Parkinson's disease (PD). In particular, a fundamental pathogenetic mechanism appears to involve targeting of neuronal membranes by soluble oligomeric intermediates of αS, leading to their disruption or permeabilisation. Therefore, a model assay was developed in which fluorophore-loaded unilamellar vesicles were permeabilised by soluble oligomers, the latter formed by aggregation of human recombinant αS protein. The αS oligomers induced an impairment of membrane integrity similar to that of the pore-forming bacterial peptide gramicidin. The lipid vesicle permeabilisation assay was then utilised to screen 11 natural polyphenolic compounds, 8 synthetic N'-benzylidene-benzohydrazide compounds and black tea extract for protection against membrane damage by wild-type and mutant (A30P, A53T) synuclein aggregates. A select group of potent inhibitory compounds included apigenin, baicalein, morin, nordihydroguaiaretic acid, and black tea extract. Structure-activity analysis further suggests that a 5,7-dihydroxy-chromen-4-one moiety appears to be favourable for the inhibition reaction. In conclusion, we have identified a group of polyphenols that can effectively hinder membrane damage by αS aggregates. These may serve as a viable source of lead compounds for the development and design of novel therapeutic agents in PD.     

Two different binding modes of α-synuclein to lipid vesicles depending on its aggregation state

Aggregation of α-synuclein is involved in the pathogenesis of Parkinson's disease (PD). Studies of in vitro aggregation of α-synuclein are rendered complex because of the formation of a heterogeneous population of oligomers. With the use of confocal single-molecule fluorescence techniques, we demonstrate that small aggregates (oligomers) of α-synuclein formed from unbound monomeric species in the presence of organic solvent (DMSO) and iron (Fe³⁺) ions have a high affinity to bind to model membranes, regardless of the lipid-composition or membrane curvature. This binding mode contrasts with the well-established membrane binding of α-synuclein monomers, which is accompanied with α-helix formation and requires membranes with high curvature, defects in the lipid packing, and/or negatively charged lipids. Additionally, we demonstrate that membrane-bound α-synuclein monomers are protected from aggregation. Finally, we identified compounds that potently dissolved vesicle-bound α-synuclein oligomers into monomers, leaving the lipid vesicles intact. As it is commonly believed that formation of oligomers is related PD progression, such compounds may provide a promising strategy for the design of novel therapeutic drugs in Parkinson's disease.     

Regional assessment of local emissions of electric vehicles using traffic simulations for a use case in Germany

Purpose

In order to assess the global and local environmental impacts of different penetration rates of electric vehicles (EVs) within a region, we developed a life cycle approach based on a detailed traffic simulation assessing local emissions for individual roads with a high time resolution. The aim was to estimate the reduction potential of local emissions such as particulate matter within a region through a substitution of conventional with electric vehicles.

Materials and methods

The chosen approach assessing local emissions includes a detailed traffic simulation of a vehicle fleet composed of individual vehicles with a daily schedule. The driving pattern is modeled based on a survey of driving patterns in Germany. Incorporation of traffic density for each road and emissions of electric and conventional vehicles permits conclusions on the reduction potential for each street. Moreover, a feasible reduction potential for a particular region can be assessed. A case study for Aachen, Germany is presented within this paper. For the classification of the local emissions with the usual life cycle assessment approach, a comparison of EV, PHEV, and conventional vehicles has been conducted for Germany providing the results for impact categories according to CML 2001.

Results and discussion

Based on simulation results, an estimation of the reduction potential for Aachen for different penetration rates of electric vehicles including particulate matter (PM₁₀), carbon monoxide (CO), and nitrogen oxygen (NOx) is carried out. Electric vehicles possess the highest reduction potential for CO and NOx. Assuming 50?% of the total vehicle fleet in 2010 substituted by electric vehicles, local emissions of CO reduce by 46.6?%, for NOx by 38.8?%, and for PM₁₀ by 22.4?%. Due to fluctuations in driving patterns throughout a day, the results are highly time dependent. However, improvements in combustion engine technologies results in an increased reduction potential for conventional vehicles. The direct comparison between the vehicle types showed that the benefit of electric vehicles depends on the considered impact category.

Conclusions

Electric vehicles are able to reduce local emissions within a region. Moreover, this approach focusing on the use phase of vehicles within a regional assessment and the resulting local emissions as well as the detailed analysis of the driving behavior allows a distinguished assessment of the reduction potential of electric vehicles. Additionally, an assessment of policy measures such as drive restrictions for conventional vehicles can be simulated on the base of this approach.     

Armeniaspiroles, a new class of antibacterials: antibacterial activities and total synthesis of 5-chloro-Armeniaspirole A

Armeniaspiroles, a novel class of natural products isolated from Streptomyces armeniacus, are characterized by a novel spiro[4.4]non-8-ene scaffold. Various derivatives of Armeniaspiroles could be obtained by halogenation, alkylation, addition/elimination or reductions. A total synthesis of the 5-chloro analog of Armeniaspirole A has been accomplished in a linear six-step sequence. 5-Chloro-Armeniaspirole A exhibits good activity against a range of multidrug-resistant, Gram-positive bacterial pathogens.     

The Neuropeptide Y Y1 Receptor: A Diagnostic Marker? Expression in MCF-7 Breast Cancer Cells Is Down-Regulated by Antiestrogens In Vitro and in Xenografts

The neuropeptide Y (NPY) Y₁ receptor (Y₁R) has been suggested as a tumor marker for in vivo imaging and as a therapeutic target. In view of the assumed link between estrogen receptor (ER) and Y₁R in mammary carcinoma and with respect to the development of new diagnostic tools, we investigated the Y₁R protein expression in human MCF-7 cell variants differing in ER content and sensitivity against antiestrogens. ER and Y₁R expression were quantified by radioligand binding using [³H]-17β-estradiol and the Y₁R selective antagonist [³H]-UR-MK114, respectively. The latter was used for cellular binding studies and for autoradiography of MCF-7 xenografts. The fluorescent ligands Cy5-pNPY (universal Y₁R, Y₂R and Y₅R agonist) and UR-MK22 (selective Y₁R antagonist), as well as the selective antagonists BIBP3226 (Y₁R), BIIE0246 (Y₂R) and {"type":"entrez-protein","attrs":{"text":"CGP71683","term_id":"876483490","term_text":"CGP71683"}}CGP71683 (Y₅R) were used to identify the NPY receptor subtype(s) by confocal microscopy. Y₁R functionality was determined by mobilization of intracellular Ca²⁺. Sensitivity of MCF-7 cells against antiestrogen 4-hydroxytamoxifen correlated directly with the ER content. The exclusive expression of Y₁Rs was confirmed by confocal microscopy. The Y₁R protein was up-regulated (100%) by 17β-estradiol (EC₅₀ 20 pM) and the predominant role of ERα was demonstrated by using the ERα-selective agonist “propylpyrazole triol”. 17β-Estradiol-induced over-expression of functional Y₁R protein was reverted by the antiestrogen fulvestrant (IC₅₀ 5 nM) in vitro. Furthermore, tamoxifen treatment of nude mice resulted in an almost total loss of Y₁Rs in MCF-7 xenografts. In conclusion, the value of the Y₁R as a target for therapy and imaging in breast cancer patients may be compromised due to Y₁R down-regulation induced by hormonal (antiestrogen) treatment.