The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b₅ reductase (CYB5R) and cytochrome b₅ (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.     

Transient Exposure of Pulmonary Surfactant to Hyaluronan Promotes Structural and Compositional Transformations into a Highly Active State

Pulmonary surfactant is a lipid-protein complex that lowers surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to collapse alveoli. Dysfunction of surfactant is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome where naturally occurring surfactant-inhibitory agents such as serum, meconium, or cholesterol reach the lung. We analyzed the effect of hyaluronan (HA) on the structure and surface behavior of pulmonary surfactant to understand the mechanism for HA-promoted surfactant protection in the presence of inhibitory agents. In particular, we found that HA affects structural properties such as the aggregation state of surfactant membranes and the size, distribution, and order/packing of phase-segregated lipid domains. These effects do not require a direct interaction between surfactant complexes and HA and are accompanied by a compositional reorganization of large surfactant complexes that become enriched with saturated phospholipid species. HA-exposed surfactant reaches very high efficiency in terms of rapid and spontaneous adsorption of surfactant phospholipids at the air-liquid interface and shows significantly improved resistance to inactivation by serum or cholesterol. We propose that physical effects pertaining to the formation of a meshwork of interpenetrating HA polymer chains are responsible for the changes in surfactant structure and composition that enhance surfactant function and, thus, resistance to inactivation. The higher resistance of HA-exposed surfactant to inactivation persists even after removal of the polymer, suggesting that transient exposure of surfactant to polymers like HA could be a promising strategy for the production of more efficient therapeutic surfactant preparations.     

The Amidohydrolases IAR3 and ILL6 Contribute to Jasmonoyl-Isoleucine Hormone Turnover and Generate 12-Hydroxyjasmonic Acid Upon Wounding in Arabidopsis Leaves

Jasmonates (JAs) are a class of signaling compounds that mediate complex developmental and adaptative responses in plants. JAs derive from jasmonic acid (JA) through various enzymatic modifications, including conjugation to amino acids or oxidation, yielding an array of derivatives. The main hormonal signal, jasmonoyl-l-isoleucine (JA-Ile), has been found recently to undergo catabolic inactivation by cytochrome P450-mediated oxidation. We characterize here two amidohydrolases, IAR3 and ILL6, that define a second pathway for JA-Ile turnover during the wound response in Arabidopsis leaves. Biochemical and genetic evidence indicates that these two enzymes cleave the JA-Ile signal, but act also on the 12OH-JA-Ile conjugate. We also show that unexpectedly, the abundant accumulation of tuberonic acid (12OH-JA) after wounding originates partly through a sequential pathway involving (i) conjugation of JA to Ile, (ii) oxidation of the JA-Ile conjugate, and (iii) cleavage under the action of the amidohydrolases. The coordinated actions of oxidative and hydrolytic branches in the jasmonate pathway highlight novel mechanisms of JA-Ile hormone turnover and redefine the dynamic metabolic grid of jasmonate conversion in the wound response.     

Long-term culture of primary porcine oviduct epithelial cells: Validation of a comprehensive in vitro model for reproductive science

Recently, we established a protocol for the cultivation of primary porcine oviduct epithelial cells (POEC), which promoted tissue-like morphology for a prolonged culture period. The present study focuses on developing this model into a comprehensive, standardized culture system, as a candidate tool for reproductive toxicity testing and basic research. We cultivated POEC isolated from 25 animals in our culture system for both 3 and 6 weeks and systematically analyzed effects of medium conditioning, supplementation with standardized sera, and culture duration in both freshly isolated and cryopreserved cells. The differentiation status was evaluated via histomorphometry, transepithelial electrical resistance (TEER) measurement, and expression analyses. The culture system possessed high reproducibility, more than 95% of cultures achieved a fully differentiated phenotype. Cells recapitulated in vivo–like morphology and ultrastructure from 3 to 6 weeks. Cryopreservation of the cells prior to cultivation did not affect culture quality of POEC. Employment of conditioned medium ensured optimal promotion of POEC differentiation, and different standardized sera induced fully differentiated phenotypes. Consistent TEER establishment indicated the presence and maintenance of cell type–specific intercellular junctions. The functionality of POEC was proven by consistent mucin secretion and stable expression of selected markers over the whole culture duration. We conclude that POEC are suitable for experiments from 3 weeks up to at least 6 weeks of culture. Therefore, this culture system could be used for in vitro estrous cycle simulation and long-term investigation of toxic effects on oviduct epithelium.     

Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine,l-Valine,and 2-Ketoisovalerate

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (Y_P/S) of 0.36 mol per mol of glucose and a volumetric productivity (Q_P) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a Y_P/S of 0.24 mol per mol of glucose and a Q_P of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.     

The age of terrestrial carbon export and rainfall intensity in a temperate river headwater system

Riverine dissolved organic carbon (DOC) supports the production of estuaries and coastal ecosystems, constituting one of the most actively recycled pools of the global carbon cycle. A substantial proportion of DOC entering oceans is highly aged, but its origins remain unclear. Significant fluxes of old DOC have never been observed in temperate headwaters where terrestrial imports take place. Here, we studied the radiocarbon age of DOC in three streams draining forested headwater catchments of the river Mulde (Ore Mountains, Germany). In a 4 week summer precipitation event DOC aged at between 160 and 270 years was delivered into the watershed. In one stream, the DOC was modern but depleted in radiocarbon compared to other hydrological conditions. The yield was substantial and corresponded to 20–52 % of the annual DOC yields in wet and dry years, respectively. The analysis of long-term data suggested that the DOC export in extreme precipitation events added to the annual yield and was not compensated for by lower exports in remaining periods. We conclude that climate change, along with additional processes associated with human activities, channels old soil carbon into more rapidly cycled carbon pools of the hydrosphere.     

Structure and Mechanical Characterization of DNA i-Motif Nanowires by Molecular Dynamics Simulation

We studied the structure and mechanical properties of DNA i-motif nanowires by means of molecular dynamics computer simulations. We built up to 230 nm-long nanowires, based on a repeated TC₅ sequence from crystallographic data, fully relaxed and equilibrated in water. The unusual C⋅C⁺ stacked structure, formed by four ssDNA strands arranged in an intercalated tetramer, is here fully characterized both statically and dynamically. By applying stretching, compression, and bending deformations with the steered molecular dynamics and umbrella sampling methods, we extract the apparent Young’s and bending moduli of the nanowire, as well as estimates for the tensile strength and persistence length. According to our results, the i-motif nanowire shares similarities with structural proteins, as far as its tensile stiffness, but is closer to nucleic acids and flexible proteins, as far as its bending rigidity is concerned. Furthermore, thanks to its very thin cross section, the apparent tensile toughness is close to that of a metal. Besides their yet to be clarified biological significance, i-motif nanowires may qualify as interesting candidates for nanotechnology templates, due to such outstanding mechanical properties.