Die Genetik des Leins

Ohne Zusammenfassung     

The road to the first,fully active and more stable human insulin variant with an additional disulfide bond

Insulin, a small peptide hormone, is crucial in maintaining blood glucose homeostasis. The stability and activity of the protein is directed by an intricate system involving disulfide bonds to stabilize the active monomeric species and by their non‐covalent oligomerization. All known insulin variants in vertebrates consist of two peptide chains and have six cysteine residues, which form three disulfide bonds, two of them link the two chains and a third is an intra‐chain bond in the A‐chain. This classical insulin fold appears to have been conserved over half a billion years of evolution. We addressed the question whether a human insulin variant with four disulfide bonds could exist and be fully functional. In this review, we give an overview of the road to engineering four‐disulfide bonded insulin analogs. During our journey, we discovered several active four disulfide bonded insulin analogs with markedly improved stability and gained insights into the instability of analogs with seven cysteine residues, importance of dimerization for stability, insulin fibril formation process, and the conformation of insulin binding to its receptor. Our results also open the way for new strategies in the development of insulin biopharmaceuticals. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Comparative analysis of stress responses of H9c2 rat cardiomyoblasts following treatment with doxorubicin and tBOOH

Cardiotoxicity is the major dose-limiting adverse effect of anthracyclines and is hypothesized to result from damage induced by reactive oxygen species (ROS) or inhibition of topoisomerase II. Here, we comparatively analyzed the effect of doxorubicin and the organic peroxide tertiary-butylhydroperoxide (tBOOH) on stress responses of rat cardiomyblast cells (H9c2). Moreover, we investigated the impact of serum factors and the novel prototypical protein kinase CK2 inhibitor resorufin on the sensentivity of H9c2 cells exposed to doxorubicin or tBOOH. Measuring cell viability by use of the WST assay as well as cell cycle progression and apoptotic death by FACS-based methods, we found that the sensitivity of H9c2 cells to doxorubicin and tBOOH was differently affected by both serum factors and resorufin. Formation of reactive oxygen species was observed after exposure of H9c2 cells to high doses (i.e. ≥5 μM) of doxorubicin only. Moreover, the antioxidant N-acetylcysteine protected H9c2 cells from cytotoxicity provoked by tBOOH but not doxorubicin. Analyzing the phosphorylation level of genotoxic stress responsive protein kinases and histone H2AX, which is indicative of an activated DNA damage response (DDR), we found that resorufin modulates doxorubicin- and tBOOH-induced responses in an agent specific manner. Taken together, the data indicate that (i) oxidative injury is not the most relevant type of damage triggering cell death of H9c2 cells following doxorubicin treatment, (ii) serum factors differently influence the sensitivity of cardiomyoblasts to doxorubicin and tBOOH and (iii) inhibition of CK2 unequally affects doxorubicin- and tBOOH-induced DDR of rat cardiomyoblasts.     

Fatness-Associated FTO Gene Variant Increases Mortality Independent of Fatness – in Cohorts of Danish Men

Background

The A-allele of the single nucleotide polymorphism (SNP), rs9939609, in the FTO gene is associated with increased fatness. We hypothesized that the SNP is associated with morbidity and mortality through the effect on fatness.

Methodology/Principal Findings

In a population of 362,200 Danish young men, examined for military service between 1943 and 1977, all obese (BMI≥31.0 kg/m²) and a random 1% sample of the others were identified. In 1992–94, at an average age of 46 years, 752 of the obese and 876 of the others were re-examined, including measurements of weight, fat mass, height, and waist circumference, and DNA sampling. Hospitalization and death occurring during the following median 13.5 years were ascertained by linkage to national registers. Cox regression analyses were performed using a dominant effect model (TT vs. TA or AA). In total 205 men died. Mortality was 42% lower (p = 0.001) with the TT genotype than in A-allele carriers. This phenomenon was observed in both the obese and the randomly sampled cohort when analysed separately. Adjustment for fatness covariates attenuated the association only slightly. Exploratory analyses of cause-specific mortality and morbidity prior to death suggested a general protective effect of the TT genotype, whereas there were only weak associations with disease incidence, except for diseases of the nervous system.

Conclusion

Independent of fatness, the A-allele of the FTO SNP appears to increase mortality of a magnitude similar to smoking, but without a particular underlying disease pattern barring an increase in the risk of diseases of the nervous system.     

Impact of Alien Plant Invaders on Pollination Networks in Two Archipelagos

Mutualistic interactions between plants and animals promote integration of invasive species into native communities. In turn, the integrated invaders may alter existing patterns of mutualistic interactions. Here we simultaneously map in detail effects of invaders on parameters describing the topology of both plant-pollinator (bi-modal) and plant-plant (uni-modal) networks. We focus on the invader Opuntia spp., a cosmopolitan alien cactus. We compare two island systems: Tenerife (Canary Islands) and Menorca (Balearic Islands). Opuntia was found to modify the number of links between plants and pollinators, and was integrated into the new communities via the most generalist pollinators, but did not affect the general network pattern. The plant uni-modal networks showed disassortative linkage, i.e. species with many links tended to connect to species with few links. Thus, by linking to generalist natives, Opuntia remained peripheral to network topology, and this is probably why native network properties were not affected at least in one of the islands. We conclude that the network analytical approach is indeed a valuable tool to evaluate the effect of invaders on native communities.     

Defining a role for Hfq in Gram-positive bacteria: evidence for Hfq-dependent antisense regulation in Listeria monocytogenes

Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species studied thus far. Here, we provide evidence for Hfq-dependent translational repression in the Gram-positive human pathogen Listeria monocytogenes, which is known to encode at least 50 sRNAs. We show that the Hfq-binding sRNA LhrA controls the translation and degradation of its target mRNA by an antisense mechanism, and that Hfq facilitates the binding of LhrA to its target. The work presented here provides the first experimental evidence for Hfq-dependent riboregulation in a Gram-positive bacterium. Our findings indicate that modulation of translation by trans-encoded sRNAs may occur by both Hfq-dependent and -independent mechanisms, thus adding another layer of complexity to sRNA-mediated riboregulation in Gram-positive species.