A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM⁻¹ cm⁻¹) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The K _M value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The K _M of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (k _obs1 = 4.7 s⁻¹, k _obs2 = 1.9 s⁻¹) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (k _obs3 = 6.10 × 10⁻² s⁻¹, k _obs4 = 2.18 × 10⁻² s⁻¹). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.     

Cloning and sequence analysis of the gene encoding 19-kD subunit of Complex I from Dunaliella salina

NADH:ubiquinone oxidoreductase (complex I ) of the mitochondrial respiratory chain catalyzes the transfer of electrons from NADH to ubiquinone coupled to proton translocation across the membrane. The cDNA sequence of Dunaliella salina mitochondrial NADH: ubiquinone oxidoreductase 19-kD subunit contains a 682-bp ORF encoding a protein with an apparent molecular mass of 19 kD. The sequence has been submitted to the GenBank database under Accession No. EF566890 (cDNA sequences) and EF566891 (genomic sequence). The deduced amino-acid sequence is 74% identical to Chlamydomonas reinhardtii mitochondrial NADH:ubiquinone oxidoreductase 18-kD subunit. The 19-kD subunit mRNA expression was observed in oxygen deficiency, salt treatment, and rotenone treatment with lower levels. It demonstrate that the 19-kD subunit of Complex I from Dunaliella salina is regulated by these stresses .     

Geographically variable selection in Ambystoma tigrinum virus (Iridoviridae) throughout the western USA

We investigated spatially variable selection in Ambystoma tigrinum virus (ATV) which causes frequent and geographically widespread epizootics of the tiger salamander, Ambystoma tigrinum. To test for evidence of selection, we sequenced several coding and noncoding regions from virus strains isolated from epizootics throughout western North America. Three of the sequenced regions contained homologues for genes putatively involved in host immune evasion and virulence: eIF‐2α, caspase activation and recruitment domain (CARD) and β‐OH‐steroid oxidoreductase. Selection analysis showed evidence of very strong purifying selection on eIF‐2α, purifying selection within certain viral clades on CARD and positive selection on β‐OH‐steroid oxidoreductase within certain clades. Analysis using multidivtime and Tajima’s relative rate tests indicate accelerated rates of evolution within clades associated with anthropogenic movement. These clades also demonstrate greater spatial variability in selection, suggesting a lack of local adaptation (i.e. locally adapted populations should exhibit little to no selection because of absent or reduced variation in fitness once a fitness optimum is reached). Increased transfer of non‐native viral strains to naïve salamander populations, in conjunction with local maladaptation as a result of local selection pressures, may explain the spread and emergence of ATV epizootics in A. tigrinum in western North America.     

Rice No Pollen 1 (NP1) is required for anther cuticle formation and pollen exine patterning

Angiosperm male reproductive organs (anthers and pollen grains) have complex and interesting morphological features, but mechanisms that underlie their patterning are poorly understood. Here we report the isolation and characterization of a male sterile mutant of No Pollen 1 (NP1) in rice (Oryza sativa). The np1‐4 mutant exhibited smaller anthers with a smooth cuticle surface, abnormal Ubisch bodies, and aborted pollen grains covered with irregular exine. Wild‐type exine has two continuous layers; but np1‐4 exine showed a discontinuous structure with large granules of varying size. Chemical analysis revealed reduction in most of the cutin monomers in np1‐4 anthers, and less cuticular wax. Map‐based cloning suggested that NP1 encodes a putative glucose‐methanol‐choline oxidoreductase; and expression analyses found NP1 preferentially expressed in the tapetal layer from stage 8 to stage 10 of anther development. Additionally, the expression of several genes involved in biosynthesis and in the transport of lipid monomers of sporopollenin and cutin was decreased in np1‐4 mutant anthers. Taken together, these observations suggest that NP1 is required for anther cuticle formation, and for patterning of Ubisch bodies and the exine. We propose that products of NP1 are likely important metabolites in the development of Ubisch bodies and pollen exine, necessary for polymerization, assembly, or both.     

Bioreductively activatable prodrug conjugates of phenstatin designed to target tumor hypoxia

A variety of solid tumor cancers contain significant regions of hypoxia, which provide unique challenges for targeting by potent anticancer agents. Bioreductively activatable prodrug conjugates (BAPCs) represent a promising strategy for therapeutic intervention. BAPCs are designed to be biologically inert until they come into contact with low oxygen tension, at which point reductase enzyme mediated cleavage releases the parent anticancer agent in a tumor-specific manner. Phenstatin is a potent inhibitor of tubulin polymerization, mimicking the chemical structure and biological activity of the natural product combretastatin A-4. Synthetic approaches have been established for nitrobenzyl, nitroimidazole, nitrofuranyl, and nitrothienyl prodrugs of phenstatin incorporating nor-methyl, mono-methyl, and gem-dimethyl variants of the attached nitro compounds. A series of BAPCs based on phenstatin have been prepared by chemical synthesis and evaluated against the tubulin-microtubule protein system. In a preliminary study using anaerobic conditions, the gem-dimethyl nitrothiophene and gem-dimethyl nitrofuran analogues were shown to undergo efficient enzymatic cleavage in the presence of NADPH cytochrome P450 oxidoreductase. Each of the eleven BAPCs evaluated in this study demonstrated significantly reduced inhibitory activity against tubulin in comparison to the parent anti-cancer agent phenstatin (IC₅₀ = 1.0 μM). In fact, the majority of the BAPCs (seven of the eleven analogues) were not inhibitors of tubulin polymerization (IC₅₀ > 20 μM), which represents an anticipated (and desirable) attribute for these prodrugs, since they are intended to be biologically inactive prior to enzyme-mediated cleavage to release phenstatin.     

Investigation of a green process for the polymerization of catechin

Flavonoids are polyphenolic secondary plant metabolites which possess antioxidant and anti-inflammatory properties. Besides, they have been shown to exhibit increased antioxidant properties in their polymerized form. Catechins are one of the attractive class of flavonoids which belong to the group of flavan-3-ols. Polymerization of catechins have been investigated in numerous studies indicating the requirement of certain amount of organic solvent to provide the solubility of the monomer. However, many research projects have been conducted recently to replace toxic organic contaminants of the processes with environmentally friendly solvents. In this aspect, deep eutectic solvents (DESs) that are regarded as “green solvents” have been studied extensively in various enzyme catalyzed reactions. In the present study, we focused on establishing a green pathway for laccase catalyzed polycatechin synthesis by replacing organic solvent content with DESs as green solvents. For this aim, various parameters were investigated, such as DES types and concentrations laccase amount and reaction time. Consequently, the highest molecular weight polycatechin was obtained using 5% (v/v) B–M, 125?U laccase in 1?hr of reaction time, at 30°C, as 4,354?±?678?g?mol^?1. Corresponding X/XO inhibitory activity and superoxide radical scavenging activities were achieved as, 59 and 50%, respectively.     

High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli

As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-β-d-thiogalactopyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant enzyme was purified and biochemically characterized. The apparent K _m values of the enzyme for 1,3-propanediol and NAD⁺ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30°C.     

Malate:quinone oxidoreductase (MqoB) is required for growth on acetate and linear terpenes in Pseudomonas citronellolis

Mini-transposon-induced mutants with defects in utilization of linear terpenes such as citronellol and citronellic acid were isolated from Pseudomonas citronellolis. Mutants with strongly reduced growth on citronellol and citronellic acid (class I) were obtained together with mutants growing normally on citronellic acid but with impairment in growth on citronellol (class II) and auxotroph mutants (class III). The transposon carrying DNA fragments of two class I mutants were cloned and malate:quinone oxidoreductase gene (mqoB) was identified as the transposon insertion site in both mutants. The mqoB genes of P. aeruginosa and of P. citronellolis wild types were cloned. Conjugative transfer of the mqoB genes to the two P. citronellolis mutants increased the strongly reduced levels of MqoB activity in cell extracts of the mutants to the level of the wild type and restored the ability of the mutants to grow on citronellol and citronellic acid. Physiological analysis of the wild type and of mutants showed that MqoB is part of the glyoxylate cycle in P. citronellolis and is necessary for growth on C(2)-compounds and linear terpenes such as citronellol or citronellic acid.     

Maternal and fetal plasma adenosine deaminase, xanthine oxidase and malondialdehyde levels in pre-eclampsia

The aim of this study was to evaluate maternal-fetal plasma adenosine deaminase, xanthine oxidase (ADA, XO) activity and malondialdehyde (MDA) levels and the relationship between them in pre-eclampsia. Maternal and umbilical cord whole blood samples were taken from 29 pre-eclamptic and 33 normal pregnants. The plasma ADA, XO activities as well as MDA levels were assayed by spectrophotometric methods. MDA levels and ADA, XO activities were found to be higher in maternal and fetal plasma in pre-eclamptics than in normal pregnancy. The differences were statistically significant between groups (p < 0.05). Increased maternal-fetal plasma XO and ADA activities, as a marker of immunological disorder, may be related to the pathogenesis of pre-eclampsia. In addition, increased MDA levels may be a reflection of increased oxidative stress in pre-eclamptics and their fetuses.     

Mitochondrial dysfunction in schizophrenia: a possible linkage to dopamine

Mitochondria are not only the principal source of high energy intermediates, but play an important role in intracellular calcium buffering, are main producers of reactive oxygen species, and are the source of pro- and antiapoptotic key factors. Moreover, the mitochondria are of a ubiquitous nature and the respiratory chain has a dual genetic basis, i.e. the mitochondrial and the nuclear DNAs. Thus mitochondrial impairment could provide an explanation for the tremendous heterogeneity of clinical and pathological manifestations in schizophrenia. This article reviews several independent lines of evidence that suggest an involvement of mitochondrial dysfunction in schizophrenia. Among them are altered cerebral energy metabolism, mitochondrial hypoplasia, dysfunction of the oxidative phosphorylation system and altered mitochondrial related gene expression. In addition, the interaction between dopamine, a predominant etiological factor in schizophrenia, and mitochondrial respiration is considered as a possible mechanism underlying the hyper- and hypo-activity cycling in schizophrenia. Understanding the role of mitochondria in schizophrenia may encourage novel treatment approaches, the identification of candidate genes and new insights into the pathophysiology and etiology of the disorder.