Biochemical characterization of recombinant isocitrate dehydrogenase and its putative role in the physiology of an acidophilic micrarchaeon

Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as “Candidatus Micrarchaeum harzensis A_DKE”, remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is difficult to elucidate the biological function of the corresponding proteins and heterologous expression is required. In order to prove the viability of this approach, A_DKE’s isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified to electrophoretic homogeneity for biochemical characterization. MhIDH showed optimal activity around pH 8 and appeared to be specific for NADP$^{+}$ yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed K$_{M}$-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and k$_{cat}$-values of 38.48 ± 1.62 and 43.99 ± 1.46 s$^{-1}$ resulting in k$_{cat}$/K$_{M}$-values of 725 ± 107.62 and 22.69 ± 2.15 mM$^{-1}$ s$^{-1}$ for DL-isocitrate and NADP$^{+}$, respectively. MhIDH’s exceptionally low affinity for NADP$^{+}$, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP$^{+}$ binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure

Nanowired electrodes as outer membrane cytochrome-independent electronic conduit in Shewanella oneidensis

Extracellular electron transfer (EET) from microorganisms to inorganic electrodes is a unique ability of electrochemically active bacteria. Despite rigorous genetic and biochemical screening of the c-type cytochromes that make up the EET network, the individual electron transfer steps over the cell membrane remain mostly unresolved. As such, attempts to transplant entire EET chains from native into non-native exoelectrogens have resulted in inferior electron transfer rates. In this study we investigate how nanostructured electrodes can interface with Shewanella oneidensis to establish an alternative EET pathway. Improved biocompatibility was observed for densely packed nanostructured surfaces with a low cell-nanowire load distribution during applied external forces. External gravitational forces were needed to establish a bioelectrochemical cell-nanorod interface. Bioelectrochemical analysis showed evidence of nanorod penetration beyond the outer cell membrane of a deletion mutant lacking all outer membrane cytochrome encoding genes that was only electroactive on a nanostructured surface and under external force

Enhanced production of propionic acid through acidic hydrolysis by choice of inoculum

BACKGROUND In this study, the enhancement of propionic acid production from a model feedstock mimicking kitchen waste was investigated. For that purpose, two operational runs of a semicontinuous anaerobic hydrolysis reactor were carried out at pH 6.0 ± 0.1 and mesophilic (30 °C) temperature. Two different types of inocula, a mixed microbial culture selected over 24 months for growth on cellulose and a culture contained in goat cheese were compared. RESULTS The results show that the goat cheese inoculum was significantly more efficient for propionic acid (PA) production. The highest propionic acid concentration achieved amounted to 139 mmol L−1 at a yield of 23.3 mg g−1 volatile solids (VS), which was 55% greater than what was achieved with the mixed culture. Furthermore, it was observed that propionic acid production was enhanced by a combination of high hydraulic retention time (HRT) with low organic loading rate (OLR), ensuring sufficient time for complete processing of the complex organic substrates. The fermentation could be kept in a stable process of propionic acid production at HRT of 20 days and a rather low OLR of 11.1 g L−1 day−1 VS. CONCLUSION Our results give a better understanding of PA production in semicontinuous mode, applying optimized process parameters and selecting the adequate microbial community for inoculation. This study provides important information for the improvement of PA production from complex substrates for future industrial application. © 2020 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI)

N-methylformamide: antitumour activity and metabolism in mice.

The antitumour activities of N-methylformamide, N-ethylformamide and formamide against a number of murine tumours in vivo (Sarcoma 180, M5076 ovarian sarcoma and TLX5 lymphoma) have been estimated. In all cases N-methyl-formamide had significant activity, formamide had marginal or no activity and N-ethylformamide had no significant activity. N-methylformamide and N-ethylformamide were equitoxic to the TLX5 lymphoma in vitro. Formamide was found as a metabolite in the plasma and urine of animals given N-methylformamide and N-ethylformamide, but excretion profiles do not support the hypothesis that formamide is an active antitumour species formed from N-alkylformamides. No appreciable metabolism of N-methylformamide occurred under a variety of conditions with liver preparations in vitro. N-methylformamide, but not N-ethylformamide or formamide, reduced liver soluble non-protein thiols by 59.8% 1 h after administration of an effective antitumour dose

Pulmonary availability of isotretinoin in rats after inhalation of a powder aerosol

Repeated oral administration of chemopreventive retinoids such as isotretinoin over extended periods of time is associated with intolerable systemic toxicity. Here isotretinoin was formulated as a powder aerosol, and its delivery to the lungs of rats was studied with the aim to explore the possibility of minimizing adverse effects associated with its oral administration. Rats received isotretinoin orally (0.5, 1 or 10 mg kg–1) or by inhalation (theoretical dose ~1 or ~10 mg kg–1) in a nose-only inhalation chamber. Isotretinoin was quantitated by high-pressure liquid chromatography in plasma and lung tissue. The ratios of mean area of concentration-vs-time curve (AUC) values in the lungs over mean AUCs in the plasma for isotretinoin following single or repeated aerosol exposure surpassed those determined for the oral route by factors of between two (single low-dose) and five (single high-dose). Similarly, the equivalent ratios for the maximal peak concentrations in lungs and plasma obtained after aerosol exposure consistently exceeded those seen after oral administration, suggesting that lungs were exposed to higher isotretinoin concentrations after aerosol inhalation than after oral administration of similar doses. Repeated high doses of isotretinoin by inhalation resulted in moderate loss of body weight, but microscopic investigation of ten tissues including lung and oesophagus did not detect any significant aerosol-induced damage. The results suggest that administration of isotretinoin via powder aerosol inhalation is probably superior to its application via the oral route in terms of achieving efficacious drug concentrations in the lungs. © 2000 Cancer Research Campaig

Cancer chemoprevention: lessons learned and future directions

The concept of delaying or preventing epithelial transformation remains a viable and attainable goal for the future. Drug-based strategies for chemoprevention of the future may predominantly rely upon targeted therapies with tolerable but defined toxicities for treatment of individuals diagnosed with intraepithelial neoplasias. Foods, diet manipulation strategies, or nutraceuticals may be more appropriate to delay or prevent carcinogenesis progression in healthy populations with genetic or epidemiologic evidence of risk for future transformation