Biomedical subjects

Cornelia Mihai

Publications and source records attributed to Cornelia Mihai.

4 recordsLinked to original sources

PubMed2005

Post-infectious encephalomyelitis.

The term post-infectious encephalomyelitis (PIEM) is frequently used interchangeably with acute disseminated encephalomyelitis (ADEM), although technically PIEM occurs after a known infection whereas with ADEM there is no antecedent infection. PIEM represents one of the primary demyelinating disorders of the central nervous system, along with multiple sclerosis and Devic's disease. There is no specific diagnostic test for any of these conditions and at onset it may be difficult to differentiate between ADEM and the first attack of multiple sclerosis. However, there are clinical and magnetic resonance imaging features that allow differentiation between PIEM/ADEM and a relapsing disease such as multiple sclerosis. Some patients improve spontaneously; most improve with methylprednisolone. If that fails, plasma exchange or intravenous immunoglobulin may be effective.

Brain↗

PubMed2004

Microscale synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate as a cofactor for thymidylate synthase.

A one-pot synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4F) is presented, where x=1, 2, or 3 represents hydrogen, deuterium, or tritium, respectively. The current procedure offers high-yield, high-purity, and microscale-quantity synthesis. In this procedure, two enzymes were used simultaneously in the reaction mixture. The first was Thermoanaerobium brockii alcohol dehydrogenase, which stereospecifically catalyzed a hydride transfer from C-2-labeled isopropanol to the re face of oxidized nicotinamide adenine dinucleotide phosphate to form R-[4-xH]-labeled reduced nicotinamide adenine dinucleotide phosphate. The second enzyme, Escherichia coli dihydrofolate reductase, used the xH to reduce 7,8-dihydrofolate (H2F) to form S-[6-xH]5,6,7,8-tetrahydrofolate (S-[6-xH]H4F). The enzymatic reactions were followed by chemical trapping of S-[6-xH]H4F with formaldehyde to form the final product. Product purification was carried out in a single step by reverse phase high-pressure liquid chromatography separation followed by lyophilization. Two analytical methods were developed to follow the reaction progress. Finally, the utility of the labeled cofactor in mechanistic studies of thymidylate synthase is demonstrated by measuring the tritium kinetic isotope effect on the enzyme's second order rate constant.

Chromatography, High Pressure Liquid↗

PubMed2004

Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction.

The enzyme thymidylate synthase (TS) catalyzes a complex reaction that involves forming and breaking at least six covalent bonds. The physical nature of the hydride transfer step in this complex reaction cascade has been studied by means of isotope effects and their temperature dependence. Competitive kinetic isotope effects (KIEs) on the second-order rate constant (V/K) were measured over a temperature range of 5-45 degrees C. The observed H/T ((T)V/K(H)) and D/T ((T)V/K(D)) KIEs were used to calculate the intrinsic KIEs throughout the temperature range. The Swain-Schaad relationships between the H/T and D/T V/K KIEs revealed that the hydride transfer step is the rate-determining step at the physiological temperature of Escherichia coli (20-30 degrees C) but is only partly rate-determining at elevated and reduced temperatures. H/D KIE on the first-order rate constant k(cat) ((D)k = 3.72) has been previously reported [Spencer et al. (1997) Biochemistry 36, 4212-4222]. Additionally, the Swain-Schaad relationships between that (D)k and the V/K KIEs reported here suggested that at 20 degrees C the hydride transfer step is the rate-determining step for both rate constants. Intrinsic KIEs were calculated here and were found to be virtually temperature independent (DeltaE(a) = 0 within experimental error). The isotope effects on the preexponential Arrhenius factors for the intrinsic KIEs were A(H)/A(T) = 6.8 +/- 2.8 and A(D)/A(T) = 1.9 +/- 0.25. Both effects are significantly above the semiclassical (no-tunneling) predicted values and indicate a contribution of quantum mechanical tunneling to this hydride transfer reaction. Tunneling correction to transition state theory would predict that these isotope effects on activation parameters result from no energy of activation for all isotopes. Yet, initial velocity measurements over the same temperature range indicate cofactor inhibition and result in significant activation energy on k(cat) (4.0 +/- 0.1 kcal/mol). Taken together, the temperature-independent KIEs, the large isotope effects on the preexponential Arrhenius factors, and a significant energy of activation all suggest vibrationally enhanced hydride tunneling in the TS-catalyzed reaction.

Carbon Radioisotopes↗

PubMed2003

Application of Brønsted-type LFER in the study of the phospholipase C mechanism.

Phosphatidylinositol-specific phospholipase C cleaves the phosphodiester bond of phosphatidylinositol to form inositol 1,2-cyclic phosphate and diacylglycerol. This enzyme also accepts a variety of alkyl and aryl inositol phosphates as substrates, making it a suitable model enzyme for studying mechanism of phosphoryl transfer by probing the linear free-energy relationship (LFER). In this work, we conducted a study of Brønsted-type relationship (log k = beta(lg) pK(a) + C) to compare mechanisms of enzymatic and nonenzymatic reactions, confirm the earlier proposed mechanism, and assess further the role of hydrophobicity in the leaving group as a general acid-enabling factor. The observation of the high negative Brønsted coefficients for both nonenzymatic (beta(lg) = -0.65 to -0.73) and enzymatic cleavage of aryl and nonhydrophobic alkyl inositol phosphates (beta(lg) = -0.58) indicates that these reactions involve only weak general acid catalysis. In contrast, the enzymatic cleavage of hydrophobic alkyl inositol phosphates showed low negative Brønsted coefficient (beta(lg) = -0.12), indicating a small amount of the negative charge on the leaving group and efficient general acid catalysis. Overall, our results firmly support the previously postulated mechanism where hydrophobic interactions between the enzyme and remote parts of the leaving group induce an unprecedented negative-charge stabilization on the leaving group in the transition state.

Catalysis↗