A dichotomy for a class of cyclic delay systems

Two complementary analyses of a cyclic negative feedback system with delay are considered in this paper. The first analysis applies the work by Sontag, Angeli, Enciso and others regarding monotone control systems under negative feedback, and it implies the global attractiveness towards an equilibrium for arbitrary delays. The second one concerns the existence of a Hopf bifurcation with respect to the delay parameter, and it implies the existence of nonconstant periodic solutions for special delay values. A key idea is the use of the Schwarzian derivative, and its application for the study of Hill function nonlinearities. The positive feedback case is also addressed.     

Synthesis of pyridine-carboxylate derivatives of hydroxysteroids for liquid chromatography-electrospray ionization-mass spectrometry

Synthesis and liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) behaviors of the picolinoyl, 6-methylpicolinoyl, nicotinoyl, 2-methoxynicotinoyl and isonicotinoyl derivatives of the hydroxysteroids estrone, estradiol, 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) and testosterone in positive mode were investigated. Each steroid was converted to the corresponding pyridine-carboxylate derivative by the acyl chloride method or the mixed anhydride method using the corresponding free acids and 2-methyl-6-nitrobenzoic anhydride; in each case, the latter method principally gave a better yield. The pyridine-carboxylate derivative of each steroid exhibited a clear single peak in liquid chromatography with a reversed phase column and CH(3)CN-0.1% CH(3)COOH as a mobile phase. The positive-ESI-mass spectra of the picolinoyl, 6-methylpicolinoyl and 2-methoxynicotinoyl derivatives showed a predominance of [M+H](+), whereas [M+H+CH(3)CN](+) was observed with high intensity in the nicotinoyl and isonicotinoyl derivatives. Even in the case of estradiol, with its two hydroxyl groups, a single charged ion of [M+H](+) or [M+H+CH(3)CN](+) was observed in the positive-ESI-mass spectrum of each derivative. The results revealed that picolinoyl derivatization is a simple and versatile method suitable for the sensitive and specific determination of hydroxysteroids by LC-ESI-MS (selected reaction monitoring).     

Optimization and anti-inflammatory evaluation of methyl gallate derivatives as a myeloid differentiation protein 2 inhibitor

Myeloid differentiation protein 2 (MD2) is a co-receptor of toll-like receptor 4 (TLR4) responsible for the recognition of lipopolysaccharide (LPS) and mediates a series of TLR4-dependent inflammatory responses in inflammatory lung diseases including acute lung injury (ALI). Targeting MD2 thus may provide a therapeutic strategy against these lung diseases. In this study, we identified a novel compound 4k with the potent anti-inflammatory activity among 39 methyl gallate derivatives (MGDs). MGD 4k exhibited a high binding affinity to MD2, which in turn prevented the formation of the LPS/MD2/TLR4 complex. In addition, MGD 4k significantly reversed the upregulation of LPS-induced inflammatory mediators such as tumor necrosis factor-α, interleukin-6, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemoattractant protein-1 in vitro and in vivo. Mechanistically, MGD 4k performed anti-inflammatory function by inactivating JNK, ERK and p38 signaling pathways. Taken together, our study identified MGD 4k as a novel potential therapeutic agent for ALI through inhibiting MD2, inflammatory responses, and major inflammation-associated signaling pathways.     

Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction

5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5′-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO₂, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn-85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function, and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. Although wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn-150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, thus ensuring that ALA is predominantly available for heme biosynthesis.     

Quinic acid derivatives and coumarin glycoside from the roots and stems of Erycibe obtusifolia

A new quinic acid derivative (1) and a new coumarin glycoside (8), together with six known compounds (2–7) were isolated from the roots and stems of Erycibe obtusifolia. The structures of the new compounds were elucidated by spectroscopic and chemical analyses. The in vitro antiviral activity against the respiratory syncytial virus (RSV) of seven quinic acid derivatives was evaluated by cytopathic effect (CPE) reduction assay. Among them, the dicaffeoylquinic acids (6 and 7) displayed potent in vitro anti-RSV activity.     

Targeting procaspase‐3 with WF‐208, a novel PAC‐1 derivative,causes selective cancer cell apoptosis

Caspase‐3 is a critical effector caspase in apoptosis cascade, and is often over‐expressed in many cancer tissues. The first synthesized procaspase‐3 activator, PAC‐1, induces cancer cell apoptosis and exhibits antitumour activity in murine xenograft models. To identify more potent procaspase‐3 activators, a series of compounds were designed, synthesized and evaluated for their ability of inducing cancer cell death in culture. Among these compounds, WF‐208 stood out by its high cytotoxicity against procaspase‐3 overexpressed HL‐60 cells. Compared with PAC‐1, WF‐208 showed higher cytotoxicity in cancer cells and lower toxicity in normal cells. The further investigation described herein showed that WF‐208 activated procaspase‐3, degraded IAPs (The Inhibitors of apoptosis proteins) and leaded to caspase‐3‐dependent cell death in tumour cells, which possibly because of the zinc‐chelating properties. WF‐208 also showed greater antitumour activity than PAC‐1 in murine xenograft model. In conclusion, we have discovered WF‐208 as a promising procaspase‐3 activating compound, with higher activity and higher cell selectivity than PAC‐1.     

Crystal structures of two novel dye-decolorizing peroxidases reveal a beta-barrel fold with a conserved heme-binding motif

BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 A, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, alpha+beta ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein).     

Visualization of yeast single-cells on fabric surface with a fluorescent glucose and their isolation for culture

An ultra-deep focusing range (UDF) fluorescent microscope system has been combined with a micromanipulation system to develop a viable cell detection-identification system applicable to microbes on environmental surfaces and products. Candida albicans yeast cells on a fabric sample surface were viably stained with a fluorescent glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy glucose (2-NBDG) and detected with a UDF fluorescent microscope. Visualized single-cells of C. albicans were picked in a glass microcapillary and transferred onto an agar medium. After the culture, the colony was assayed for DNA sequence to identify the isolate. This demonstrates a potential application to the study of unknown environmental microorganisms.     

The oxygenase-peroxidase theory of Bach and Chodat and its modern equivalents: Change and permanence in scientific thinking as shown by our understanding of the roles of water,peroxide, and oxygen in the functioning of redox enzymes

Alexander Bach was both revolutionary politician and biochemist. His earliest significant publication, “Tsargolod” (“The Tsar of Hunger”), introduced Marxist thought to Russian workers. In exile for 30 years, he moved to study the dialectic of the oxidases. When his theory of oxidases as combinations of oxygenases and peroxidases was developed (circa 1900) the enzyme concept was not fully formulated, and the enzyme/substrate distinction not yet made. Peroxides however were then and remain now significant intermediates, when either free or bound, in oxidase catalyses. The aerobic dehydrogenase/peroxidase/catalase coupled systems which were studied slightly later clarified the Bach model and briefly became an oxidase paradigm. Identification of peroxidase as a metalloprotein, a key step in understanding oxidase and peroxidase mechanisms, postdated Bach’s major work. Currently we recognize catalytic organic peroxides in flavoprotein oxygenases; such organic peroxides are also involved in lipid oxidation and tryptophan radical decay. But most physiologically important peroxides are now known to be bound to transition metals (either Fe or Cu) and formed both directly and indirectly (from oxygen). The typical stable metalloprotein peroxide product is the ferryl state. When both peroxide oxidizing equivalents are retained the second equivalent is held as a protein or porphyrin radical. True metal peroxide complexes are unstable. But often water molecules mark the spot where the original peroxide decayed. The cytochrome c oxidase Fe-Cu center can react with either peroxide or oxygen to form the intermediate higher oxidation states P and F. In its resting state water molecules and hydroxyl ions can be seen marking the original location of the oxygen or peroxide molecule. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 10, pp. 1278–1288.