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The Rv0948c gene from Mycobacterium tuberculosis H37Rv encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis–Menten kinetics with a kcat of 5.5 ± 0.2 s−1 and a Km of 1500 ± 100 μm at 37 °C and pH 7.5. The 2.0 Å X-ray structure shows that 90-MtCM is an all α-helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequence alignment shows that the C-terminus helix 3 is shortened. The absence of two residues in the active site of 90-MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low kcat. Hence, 90-MtCM belongs to a subfamily of α-helical AroQ CMs termed AroQδ. The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N-terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis–Menten kinetics with a kcat of 70 ± 5 s−1 and Km of 500 ± 50 μm at 37 °C and pH 7.5. The 2.1 Å X-ray structure shows that *YpCM is an all α-helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQγ class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M. tuberculosis. [ABSTRACT FROM AUTHOR]

Background: Vitamin B12 and folate measurements in serum show wide inter-methodology variability. This variability appears to be due in part to the lack of standardisation against internationally accepted reference materials. Pooled human serum, lyophilised in ampoules and designated 03/178, was therefore evaluated by 24 laboratories in seven countries for its suitability to serve as an International Standard (IS) for B12 and folate. Methods: IS 03/178 was assayed using a range of commercial analysers, microbiological assays and, for folate, candidate reference methods based on liquid chromatography coupled to isotope-dilution tandem mass spectrometry (LC/MS/MS). Results: Mean vitamin B12 and folate values for reconstituted 03/178 across all laboratories and methods were 480 pg/mL [coefficient of variation (CV) 12.8%] and 5.52 ng/mL (CV 17.1%), respectively. The total folate content of reconstituted 03/178, determined using LC/MS/MS, was 12.1 nmol/L (equivalent to 5.33 ng/mL), made up of 9.75 nmol/L 5-methyl tetrahydrofolic acid (5MeTHF; CV 5.5%), 1.59 nmol/L 5-formyl tetrahydrofolic acid (5FoTHF; CV 4.2%) and 0.74 nmol/L folic acid (FA; CV 31.6%). The inclusion of three serum samples in the study with different B12 and folate levels demonstrated a considerable reduction in inter-laboratory variability when the B12 and folate content of the samples was determined relative to the IS 03/178 rather than to the analyser calibration. IS 03/178 demonstrated satisfactory long-term stability in accelerated degradation studies. Conclusions: Use of IS 03/178 to standardise serum B12 and folate assays reduced inter-laboratory variability. The World Health Organization (WHO) Expert Committee on Biological Standardisation established 03/178 as the first IS for serum vitamin B12 and serum folate, with assigned values of 480 pg/mL of vitamin B12 and 12.1 nmol/L folate when the lyophilised contents of the ampoule are reconstituted with 1 mL of water. Clin Chem Lab Med 2007;45:380–6. [ABSTRACT FROM AUTHOR]

The gene Rv1885c from the genome of Mycobacterium tuberculosis H37Rv encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that *MtCM secretes into the culture filtrate of M. tuberculosis. *MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. *MtCM exhibits simple Michaelis-Menten kinetics with a Km of 0.5 ± 0.05 mM and a kcat of 60 s-1 per active site (at 37°C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 Å resolution (Protein Data Bank identifier 2F6L). The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg134. We provide evidence by site-directed mutagenesis that the residues Arg49, Lys60, Arg72, Thr105, Glu109, and Arg134 constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site. [ABSTRACT FROM AUTHOR]

Aim: To quantitatively evaluate the inhibition of human DNA repair proteins APE1 and MTH1 by dextran-coated γ-Fe 2 O 3 ultrasmall superparamagnetic iron oxide nanoparticles (dUSPIONs). Materials & methods : Liquid chromatography-tandem mass spectrometry with isotope-dilution was used to measure the expression levels of APE1 and MTH1 in MCL-5 cells exposed to increasing doses of dUSPIONs. The expression levels of APE1 and MTH1 were measured in cytoplasmic and nuclear fractions of cell extracts. Results: APE1 and MTH1 expression was significantly inhibited in both cell fractions at the highest dUSPION dose. The expression of MTH1 was linearly inhibited across the full dUSPION dose range in both fractions. Conclusion: These findings warrant further studies to characterize the capacity of dUSPIONs to inhibit other DNA repair proteins in vitro and in vivo .

Cerium oxide nanomaterials (nanoceria, CNMs) are receiving increased attention from the research community due to their unique chemical properties, most prominent of which is their ability to alternate between the Ce 3+ and Ce 4+ oxidation states. While many analytical techniques and methods have been employed to characterize the amounts of Ce 3+ and Ce 4+ present (Ce 3+ /Ce 4+ ratio) within nanoceria materials, to-date no studies have used multiple complementary analytical tools (orthogonal analysis) with technique-independent oxidation state controls for quantitative determinations of the Ce 3+ /Ce 4+ ratio. Here, we describe the development of analytical methods measuring the oxidation states of nanoceria analytes using technique-independent Ce 3+ (CeAlO 3 :Ge) and Ce 4+ (CeO 2 ) control materials, with a particular focus on x-ray photoelectron spectroscopy (XPS) and electron energy loss spectroscopy (EELS) approaches. The developed methods were demonstrated in characterizing a suite of commercial nanoceria products, where the two techniques (XPS and EELS) were found to be in good agreement with respect to Ce 3+ /Ce 4+ ratio. Potential sources of artifacts and discrepancies in the measurement results were also identified and discussed, alongside suggestions for interpreting oxidation state results using the different analytical techniques. The results should be applicable towards producing more consistent and reproducible oxidation state analyses of nanoceria materials.

Previously, catalytic cerium oxide nanoparticles (CNPs, nanoceria, CeO2-x NPs) have been widely utilized for chemical mechanical planarization in the semiconductor industry and for reducing harmful emissions and improving fuel combustion efficiency in the automobile industry. Researchers are now harnessing the catalytic repertoire of CNPs to develop potential new treatment modalities for both oxidative- and nitrosative-stress induced disorders and diseases. In order to reach the point where our experimental understanding of the antioxidant activity of CNPs can be translated into useful therapeutics in the clinic, it is necessary to evaluate the most current evidence that supports CNP antioxidant activity in biological systems. Accordingly, the aims of this review are three-fold: (1) To describe the putative reaction mechanisms and physicochemical surface properties that enable CNPs to both scavenge reactive oxygen species (ROS) and to act as antioxidant enzyme-like mimetics in solution; (2) To provide an overview, with commentary, regarding the most robust design and synthesis pathways for preparing CNPs with catalytic antioxidant activity; (3) To provide the reader with the most up-to-date in vitro and in vivo experimental evidence supporting the ROS-scavenging potential of CNPs in biology and medicine.

Catechins are polyphenolic plant compounds (flavonoids) that may offer significant health benefits to humans. These benefits stem largely from their anticarcinogenic, antioxidant, and antimutagenic properties. Recent epidemiological studies suggest that the consumption of flavonoid-containing foods is associated with reduced risk of cardiovascular disease. Chocolate is a natural cocoa bean-based product that reportedly contains high levels of monomeric, oligomeric, and polymeric catechins. We have applied solid-liquid extraction and liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry to the identification and determination of the predominant monomeric catechins, (+)-catechin and (-)-epicatechin, in a baking chocolate Standard Reference Material (NIST Standard Reference Material 2384). (+)-Catechin and (-)-epicatechin are detected and quantified in chocolate extracts on the basis of selected-ion monitoring of their protonated [M + H](+) molecular ions. Tryptophan methyl ester is used as an internal standard. The developed method has the capacity to accurately quantify as little as 0.1 microg/mL (0.01 mg of catechin/g of chocolate) of either catechin in chocolate extracts, and the method has additionally been used to certify (+)-catechin and (-)-epicatechin levels in the baking chocolate Standard Reference Material. This is the first reported use of liquid chromatography/mass spectrometry for the quantitative determination of monomeric catechins in chocolate and the only report certifying monomeric catechin levels in a food-based Standard Reference Material.