Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.5 min(-)(1), that transforms to yield a greenish brown chromophore (lambda(max) = 550 nm, 700 M(-)(1) x cm(-)(1)). The latter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumed to arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, (18)O labeling studies reveal that the added oxygen atom derives from solvent rather than from O(2). The transient yellow species, identified as a tyrosyl radical on the basis of EPR studies, is formed after alphaKG decomposition. Substitution of two active site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as the likely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvement of the tyrosyl radical in catalysis is excluded on the basis of the observed activity of the enzyme variants. We suggest that the Fe(IV) oxo species generally proposed (but not yet observed) as an intermediate for this family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capable of exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistep TauD self-hydroxylation reaction.

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4 -6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.

Recent reports indicate that manganese (Mn), applied as a foliar fertilizer in tank mixtures with glyphosate, has the potential to antagonize glyphosate efficacy and reduce weed control. It was hypothesized that Mn2+ complexed with glyphosate in a similar manner to Ca2+, forming salts that were not readily absorbed and, thereby, reducing glyphosate efficacy. This study was conducted to confirm the interaction of Mn2+ and glyphosate and to measure the effect of Mn on glyphosate absorption and translocation in velvetleaf. In aqueous solutions, Mn2+ binds with solvent molecules and with chelating agents to form hexacoordinate complexes. The distribution of paramagnetic species, both the free manganous ion ([Mn{H2O}6]2+) and the Mn2+–glyphosate complex, in Mn–glyphosate solutions at various pH values were analyzed using electron paramagnetic resonance (EPR) spectroscopy. Glyphosate interaction with Mn appeared to increase as the pH was increased from spray solution levels (2.8 to 4.5) to levels common in the plant symplast (7.5). Growth chamber bioassays were conducted to measure absorption and translocation of 14C-labeled glyphosate in solution with four Mn fertilizers: Mn-ethylaminoacetate (Mn-EAA), Mn-ethylenediaminetetraacetate (Mn-EDTA), Mn-lignin sulfonate (Mn-LS), and Mn-sulfate (MnSO4). Mn-EDTA did not interfere with glyphosate efficacy, absorption, or translocation. However, both MnSO4 and Mn-LS reduced glyphosate efficacy, absorption, and translocation. Mn-EAA severely antagonized glyphosate efficacy, and although glyphosate in tank mixtures with Mn-EAA was absorbed rapidly, little was translocated from the treated leaf. The Mn-EAA fertilizer contained approximately 0.5% iron (Fe) not reported on the fertilizer label. Iron is presumed to be partially responsible for the very limited translocation of glyphosate from the treated leaf in Mn-EAA tank mixtures. Adding ammonium sulfate increased the efficacy, absorption, and translocation of glyphosate for each Mn fertilizer tank mixture.

The structural relationship between substrate taurine and the non-heme Fe(II) center of taurine/alpha-ketoglutarate (alphaKG) dioxygenase (TauD) was measured using electron spin echo envelope modulation (ESEEM) spectroscopy. Studies were conducted on TauD samples treated with NO, cosubstrate alphaKG, and either protonated or specifically deuterated taurine. Stimulated echo ESEEM data were divided to eliminate interference from 1H and 14N modulations and accentuate modulations from 2H. For taurine that was deuterated at the C1 position (adjacent to the sulfonate group), 2H ESEEM spectra show features that arise from dipole-dipole and deuterium nuclear quadrupole interactions from a single deuteron. Parallel measurements taken for taurine deuterated at both C1 and C2 show an additional ESEEM feature at the deuterium Larmor frequency. Analysis of these data at field positions ranging from g = 4 to g = 2 have allowed us to define the orientation of substrate taurine with respect to the magnetic axes of the Fe(II)-NO, S = 3/2, paramagnetic center. These results are discussed in terms of previous X-ray crystallographic studies and the proposed catalytic mechanism for this family of enzymes.

The major metabolite of the cancer chemopreventive agent oltipraz, a pyrrolopyrazine thione (PPD), has been shown to be a phase 2 enzyme inducer, an activity thought to be key to the cancer chemopreventive action of the parent compound. In cells, mitochondria are the major source of reactive oxygen species (ROS) and cytochrome c (cyt c) is known to participate in mitochondrial electron transport and confer antioxidant and peroxidase activities. To understand possible mechanisms by which PPD acts as a phase 2 enzyme inducer, a study of its interaction with cyt c was undertaken. UV-visible spectroscopic results demonstrate that PPD is capable of reducing oxidized cyt c. The reduced cyt c is stable for a long period of time in the absence of an oxidizing agent. In the presence of ferricyanide, the reduced cyt c is rapidly oxidized back to its oxidized form. Further, UV-visible spectroscopic studies show that during the reduction process the coordination environment and redox state of iron in cyt c are changed. Low-temperature EPR studies show that during the reduction process, the heme iron changes from a low-spin state of s = 1/2 to a low-spin state of s = 0. Room-temperature EPR studies demonstrate that PPD inhibits the peroxidase activity of cyt c. EPR spin trapping experiments using DMPO show that PPD inhibits the superoxide radical scavenging activity of oxidized cyt c. From these results, we propose that PPD interacts with cyt c, binding to and then reducing the heme, and this may enhance ROS levels in mitochondria. This in turn could contribute to the mechanism by which the parent compound, oltipraz, might trigger the cancer chemopreventive increase in transcription of phase 2 enzymes. The modifications of cyt c function by the oltipraz metabolite may have implications for the regulation of apoptotic cell death.

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Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations as it is prone to oxidative stress. The aim of the present study is to assess the novel mutations in mitochondrial genes from blood samples among the breast cancer patients from a less studied Northeast Indian population. D, B, L haplogroups were observed in the cancer samples and a total of 44 mtDNA D-loop sequence variations at 42 distinct nucleotide positions were found. All the sequence variations were transitional substitutions and 6 were heteroplasmic states, except for a cytosine copy number change (9C/8C) at np 303e309 in three samples examined. A total of 88 Cytochrome Oxidase C subunit I (COXI) sequence differences with respect to the Revised Cambridge Reference Sequence (rCRS) were identified including 20 missense variants with 100 % sample mutation frequency. All 20 missense mutations are highly conserved with a Cumulate Index of 100 %. Among 88 COXI mutations, 24 (13 were Non-Synonymous and 11 were Synonymous) were not previously reported (novel mutation) in the literature or the public mtDNA mutation databases. Analysis of three-dimensional structure of COXI open reading frame (ORF) predicted the effect of one single codon (96R > C, 217T > I, 224-225GG > EE and 227D > T) mutations located in the signal peptide binding position. Analysis of mitochondrial DNA mutations, as a viable alternative, has the advantage of being capable of detecting inherent risk factors for breast cancer development.

Fermented pork fat (sa-um) is traditionally and extensively consumed in Northeast Indian region for several decades. However, no scientific reports are available regarding its nutritional value as well as its potential health risks. The objective of this work was essentially the characterization of sa-um using a polyphasic approach, viz., physicochemical, electrospray ionization-mass spectrometry (ESI+-MS) and metagenomic analysis in order to gain an understanding of the nutrient contents and microbial population diversity. On a dry weight basis, about 91% fat, 2% carbohydrate and 0.70% protein were present. ESI+-MS analysis of sa-um revealed the presence of various polar and neutral lipids corresponding to monoacylglyceride, diacylglyceride and triacylglyceride species. The dominant bacterial phyla were Firmicutes, Proteobacteria and Bacteroidetes. A total of 72 bacterial genera were identified, largely abundant with Clostridium species including C. butyricum, C. citroniae, C. methylpentosum, C. perfringens, C. saccharogumia and C. tetani. The imputed functional profiles of bacterial communities were predominantly involved in energy, carbohydrate and amino acid metabolisms. Furthermore, this study deduces the presence of pro-inflammatory molecules as well as antibiotic resistance genes associated with the bacterial families such as Bacillaceae, Bacteroidaceae, Clostridiaceae, Corynebacteriaceae and Enterobacteriaceae which might be a major health concern for the sa-um consuming population.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0695-z) contains supplementary material, which is available to authorized users.