Characterisation of the spectrum and genetic dependence of collateral mutations induced by translesion DNA synthesis

Translesion DNA synthesis (TLS) is a fundamental damage bypass pathway that utilises specialised polymerases with relaxed template specificity to achieve replication through damaged DNA. Misinsertions by low fidelity TLS polymerases may introduce additional mutations on undamaged DNA near the original lesion site, which we termed collateral mutations. In this study, we used whole genome sequencing datasets of chicken DT40 and several human cell lines to obtain evidence for collateral mutagenesis in higher eukaryotes. We found that cisplatin and UVC radiation frequently induce close mutation pairs within 25 base pairs that consist of an adduct-associated primary and a downstream collateral mutation, and genetically linked their formation to TLS activity involving PCNA ubiquitylation and polymerase κ. PCNA ubiquitylation was also indispensable for close mutation pairs observed amongst spontaneously arising base substitutions in cell lines with disrupted homologous recombination. Collateral mutation pairs were also found in melanoma genomes with evidence of UV exposure. We showed that collateral mutations frequently copy the upstream base, and extracted a base substitution signature that describes collateral mutagenesis in the presented dataset regardless of the primary mutagenic process. Using this mutation signature, we showed that collateral mutagenesis creates approximately 10–20% of non-paired substitutions as well, underscoring the importance of the process.     

Evaluation of different Bacillus strains in respect of their ability to produce Surfactin in a model fermentation process with integrated foam fractionation

Biosurfactants increasingly gain attention due to the manifold of possible applications and production on the basis of renewable resources. Owing to its various characteristics, Surfactin is one of the most studied biosurfactants. Since its discovery, several Surfactin producers have been identified, but their capacity to produce Surfactin has not been evaluated in a comparison. Six different Bacillus strains were analyzed regarding their ability to produce Surfactin in model fermentations with integrated foam fractionation, for in situ product enrichment and removal. Three of the investigated strains are commonly used in Surfactin production (ATCC 21332, DSM 3256, DSM 3258), whereas two Bacillus strains are described for the first time (DSM 1090, LM43a50°C) as Surfactin producers. Additionally, the Bacillus subtilis type strain DSM 10^T was included in the evaluation. Interestingly, all strains, except DSM 3256, featured high values for Surfactin recovered from foam in comparison to other studies, ranging between 0.4 and 1.05 g. The fermentation process was characterized by calculating procedural parameters like substrate yield Y _X/S, product yield Y _P/X, specific growth rate μ, specific productivity q _Surfactin, volumetric productivity q _Surfactin, Surfactin and bacterial enrichment as well as Surfactin recovery. The strains differ most in specific and volumetric productivity; nevertheless, it is evident that it is not possible to name a Bacillus strain that is the most appropriate for the production of Surfactin under these conditions. In contrast, it becomes apparent that the choice of a specific strain should depend on the applied fermentation conditions.     

Optical and IR absorption as probe of dynamics of heme proteins

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.     

Transport and transporters in the endoplasmic reticulum

Enzyme activities localized in the luminal compartment of the endoplasmic reticulum are integrated into the cellular metabolism by transmembrane fluxes of their substrates, products and/or cofactors. Most compounds involved are bulky, polar or even charged; hence, they cannot be expected to diffuse through lipid bilayers. Accordingly, transport processes investigated so far have been found protein-mediated. The selective and often rate-limiting transport processes greatly influence the activity, kinetic features and substrate specificity of the corresponding luminal enzymes. Therefore, the phenomenological characterization of endoplasmic reticulum transport contributes largely to the understanding of the metabolic functions of this organelle. Attempts to identify the transporter proteins have only been successful in a few cases, but recent development in molecular biology promises a better progress in this field.     

Evidence for microvascular dysfunction after prenatal dexamethasone at 0.7, 0.75, and 0.8 gestation in sheep

Dexamethasone (DM) was administered to pregnant ewes as three weekly courses of four injections of 2 mg at 12-h intervals. DM (n = 7) or saline (n = 7) was given starting at 103 days of gestation (dGA; term approximately 149 days). Fetal femoral arteries (approximately 300-microm internal diameter) were evaluated using wire myography at 119 dGA. DM-exposed fetuses were significantly smaller than saline-exposed fetuses. DM exposure increased maximal contraction to 125 mM KCl, and maximum tension developed along with sensitivity to endothelin-1 and relaxation to bradykinin. Preincubation with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester shifted the dose-response curves to endothelin-1 and acetylcholine to the right in controls but not in the DM-exposed group. Relaxation to acetylcholine and to the nitric oxide donor sodium nitroprusside was similar in both groups. The combination of enhanced endothelin-induced vasoconstriction, abnormal endothelium-dependent relaxation, and normal endothelium-independent relaxation indicates microvessel dysfunction following antenatal DM administration. Because such dysfunction is associated with several forms of adult hypertension, our results indicate the potential for consequences of antenatal glucocorticoid exposure on adult cardiovascular health.