Bewertung alternativer Arbeitsmittel für Wärmepumpen im Gebäudesektor

Forschung im Ingenieurwesen - Das Arbeitsmittel in Wärmepumpen unterliegt einer Vielzahl von Anforderungen, welche für einen effizienten und sicheren Betrieb eingehalten werden...     

Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

Newly Isolated Animal Pathogen Corynebacterium silvaticum Is Cytotoxic to Human Epithelial Cells

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.     

Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases

The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto-reducing activity, yet negligible imine-reducing capability, and mining the Short-Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active-site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine-reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto-reducing activity was engineered into a promiscuous enzyme with imine-reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the “classical” SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine-reducing enzymes.     

Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L⁻¹). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.     

A Potent Mimetic of the Siglec-8 Ligand 6’-Sulfo-Sialyl Lewisx

Siglecs are members of the immunoglobulin gene family containing sialic acid binding N-terminal domains. Among them, Siglec-8 is expressed on various cell types of the immune system such as eosinophils, mast cells and weakly on basophils. Cross-linking of Siglec-8 with monoclonal antibodies triggers apoptosis in eosinophils and inhibits degranulation of mast cells, making Siglec-8 a promising target for the treatment of eosinophil- and mast cell-associated diseases such as asthma. The tetrasaccharide 6’-sulfo-sialyl Lewis^x has been identified as a specific Siglec-8 ligand in glycan array screening. Here, we describe an extended study enlightening the pharmacophores of 6’-sulfo-sialyl Lewis^x and the successful development of a high-affinity mimetic. Retaining the neuraminic acid core, the introduction of a carbocyclic mimetic of the Gal moiety and a sulfonamide substituent in the 9-position gave a 20-fold improved binding affinity. Finally, the residence time, which usually is the Achilles tendon of carbohydrate/lectin interactions, could be improved.     

Intergenerational Metabolomic Analysis of Mothers with a History of Gestational Diabetes Mellitus and Their Offspring

Shared metabolomic patterns at delivery have been suggested to underlie the mother-to-child transmission of adverse metabolic health. This study aimed to investigate whether mothers with gestational diabetes mellitus (GDM) and their offspring show similar metabolomic patterns several years postpartum. Targeted metabolomics (including 137 metabolites) was performed in plasma samples obtained during an oral glucose tolerance test from 48 mothers with GDM and their offspring at a cross-sectional study visit 8 years after delivery. Partial Pearson’s correlations between the area under the curve (AUC) of maternal and offspring metabolites were calculated, yielding so-called Gaussian graphical models. Spearman’s correlations were applied to investigate correlations of body mass index (BMI), Matsuda insulin sensitivity index (ISI-M), dietary intake, and physical activity between generations, and correlations of metabolite AUCs with lifestyle variables. This study revealed that BMI, ISI-M, and the AUC of six metabolites (carnitine, taurine, proline, SM(-OH) C14:1, creatinine, and PC ae C34:3) were significantly correlated between mothers and offspring several years postpartum. Intergenerational metabolite correlations were independent of shared BMI, ISI-M, age, sex, and all other metabolites. Furthermore, creatinine was correlated with physical activity in mothers. This study suggests that there is long-term metabolic programming in the offspring of mothers with GDM and informs us about targets that could be addressed by future intervention studies.     

Vom freiwilligen Seminar zur Q2-Pflichtveranstaltung

Definition of the problem: Ethical reasoning cannot be enforced. Voluntary understanding is a basic condition for the development of ethical competencies and attitudes. The aim of this survey was to investigate whether the presence of those students who attended our seminars compulsorily would have a negative effect upon the quality of the course. Methods and results: The participants of our small-group seminars in medical ethics were asked to complete a questionnaire on teaching aims, seminar atmosphere, moderation and importance of subjects. One hundred and ninety-two voluntary participants (2001/2) and 293 compulsory participants (2002/3) completed the questionnaire and were statistically compared. There was no significant difference between voluntary and compulsory participants in any of the important items. However, there were considerable differences in some items between male and female participants. Conclusion: We were surprised that there was no difference between the two groups. This leads us to hope that our concept of case-based small-group seminars will prove capable of realising our teaching aims for all students. The difference between male and female participants may have important didactic consequences and will be the subject of further studies.     

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