Interchangeability of class I and II fumarases in an obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The methanotrophic bacterium Methylotuvimicrobium alcaliphilum 20Z is an industrially promising candidate for bioconversion of methane into value-added chemicals. Here, we have study the metabolic consequences of the breaking in the tricarboxylic acid (TCA) cycle by fumarase knockout. Two fumarases belonging to non-homologous class I and II fumarases were obtained from the bacterium by heterologous expression in Escherichia coli. Class I fumarase (FumI) is a homodimeric enzyme catalyzing the reversible hydration of fumarate and mesaconate with activities of ~94 and ~81 U mg-1 protein, respectively. The enzyme exhibited high activity under aerobic conditions, which is a non-typical property for class I fumarases characterized to date. The calculation of kcat/S0.5 showed that the enzyme works effectively with either fumarate or mesaconate, but it is almost four times less specific to malate. Class II fumarase (FumC) has a tetrameric structure and equal activities of both fumarate hydration and malate dehydration (~45 U mg-1 protein). Using mutational analysis, it was shown that both forms of the enzyme are functionally interchangeable. The triple mutant strain 20Z-3E (ΔfumIΔfumCΔmae) deficient in the genes encoding the both fumarases and the malic enzyme accumulated 2.6 and 1.1 mmol g-1 DCW fumarate in the medium when growing on methane and methanol, respectively. Our data suggest the redundancy of the metabolic node in the TCA cycle making methanotroph attractive targets for modification, including generation of strains producing the valuable metabolites.

Improving the Stability and Effectiveness of Immunotropic Squalene Nanoemulsion by Adding Turpentine Oil.

Turpentine oil, owing to the presence of 7-50 terpenes, has analgesic, anti-inflammatory, immunomodulatory, antibacterial, anticoagulant, antioxidant, and antitumor properties, which are important for medical emulsion preparation. The addition of turpentine oil to squalene emulsions can increase their effectiveness, thereby reducing the concentration of expensive and possibly deficient squalene, and increasing its stability and shelf life. In this study, squalene emulsions were obtained by adding various concentrations of turpentine oil via high-pressure homogenization, and the safety and effectiveness of the obtained emulsions were studied in vitro and in vivo. All emulsions showed high safety profiles, regardless of the concentration of turpentine oil used. However, these emulsions exhibited dose-dependent effects in terms of both efficiency and storage stability, and the squalene emulsion with 1.0% turpentine oil had the most pronounced adjuvant and cytokine-stimulating activity as well as the most pronounced stability indicators when stored at room temperature. Thus, it can be concluded that the squalene emulsion with 1% turpentine oil is a stable, monomodal, and reliably safe ultradispersed emulsion and may have pleiotropic effects with pronounced immunopotentiating properties.

A Comparative Analysis of Neuroprotective Properties of Taxifolin and Its Water-Soluble Form in Ischemia of Cerebral Cortical Cells of the Mouse.

Cerebral ischemia, and, as a result, insult, attacks up to 15 million people yearly in the world. In this connection, the development of effective preventive programs and methods of therapy has become one of the most urgent problems in modern angiology and pharmacology. The cytoprotective action of taxifolin (TAX) in ischemia is well known, but its limitations are also known due to its poor solubility and low capacity to pass through the hematoencephalic barrier. Molecular mechanisms underlying the protective effect of TAX in complex systems such as the brain remain poorly understood. It is known that the main cell types of the brain are neurons, astrocytes, and microglia, which regulate the activity of each other through neuroglial interactions. In this work, a comparative study of cytoprotective mechanisms of the effect of TAX and its new water-soluble form aqua taxifolin (aqTAX) was performed on cultured brain cells under ischemia-like conditions (oxygen-glucose deprivation (OGD)) followed by the reoxygenation of the culture medium. The concentration dependences of the protective effects of both taxifolin forms were determined using fluorescence microscopy, PCR analysis, and vitality tests. It was found that TAX began to effectively inhibit necrosis and the late stages of apoptosis in the concentration range of 30-100 µg/mL, with aqTAX in the range of 10-30 µg/mL. At the level of gene expression, aqTAX affected a larger number of genes than TAX; enhanced the basic and OGD/R-induced expression of genes encoding ROS-scavenging proteins with a higher efficiency, as well as anti-inflammatory and antiapoptotic proteins; and lowered the level of excitatory glutamate receptors. As a result, aqTAX significantly inhibited the OGD-induced increase in the Ca2+ levels in the cytosol ([Ca2+]i) in neurons and astrocytes under ischemic conditions. After a 40 min preincubation of cells with aqTAX under hypoxic conditions, these Ca2+ signals were completely inhibited, resulting in an almost complete suppression of necrotic death of cerebral cortical cells, which was not observed with the use of classical TAX.

Looking at microbial metabolism by high-resolution (2)H-NMR spectroscopy.

We analyzed the applicability of high-resolution (2)H-HMR spectroscopy for the analysis of microbe metabolism in samples of mitochondrion isolated from rat liver and from aqueous extracts of homogenates of rat liver and other organs and tissues in the presence of high D2O contents. Such analysis is possible due to the fast microbe adaptation to life in the heavy water. It is also shown that some enzymatic processes typical for the intact cells are preserved in the homogenized tissue preparations. The microbial and cellular metabolic processes can be differentiated via the strategic use of cell poisons and antibiotics.