Glomerular lesion and increased cytokine gene expression in renal tissue in patients with decompensated nephrotic syndrome due to chronic GvHD.

The nephrotic syndrome is a rare complication of allogeneic stem cell transplantation (alloHSCT). We present two cases of nephrotic syndrome during chronic graft-versus-host disease (GvHD) involving altered cytokine gene expression in renal tissue. A patient with acute lymphatic leukemia demonstrated nephrotic syndrome due to minimal change disease as a marker of chronic GvHD. A patient with acute lymphoblastic leukemia suffered from severe nephrotic syndrome due to membranous glomerulopathy. In the two presented cases of GvHD-linked nephrotic syndrome, increased cytokine gene expression [tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), interferon gamma (IFN-gamma), interleukin 2 (IL-2), IL-6, and IL-10] assessed using semiquantitative evaluation with reverse transcriptase polymerase chain reaction (RT-PCR) in situ on renal biopsy was observed.

Predictive value of Banff score of early kidney allograft biopsies for 1-year graft survival.

The aim of this study was to evaluate the Banff score of early kidney allograft biopsies, taken during the first month after transplantation, seeking an association between early rejection and acute tubular necrosis. We analyzed data from 71 patients transplanted between 2000 and 2004 who had renal allograft biopsies performed within the first posttransplant month (23 women, 48 men), ages 18 to 67 years. All biopsies performed in cases of delayed or deteriorated graft function were graded according to the Banff' 97 classification. Twelve months after transplantation, 19 patients exhibited excellent renal function (group 1, serum creatinine concentration [Scr] < or = 1.5 mg/dL); 25 patients demonstrated preserved renal function (group II, Scr 1.51-1.99 mg/dL); and 19 patients showed deteriorated renal function (group III, Scr > or = 2.0 mg/dL). Eight recipients lost their grafts within 1 year after transplantation (group IV). The Banff index was defined as a sum of all components (value of glomerulitis ["g"] + interstitial inflammation ["i"] + tubulitis ["t"] + arteriolar hyaline thickening ["ah"] + intimal arteritis ["v"]). The deterioration of renal function was associated with a higher Banff index; patients who lost their grafts showed the highest values of this index. Scores of "v," "ah," and Banff index were positively correlated with serum creatinine concentrations at 28, 90, 180, and 360 days (P < .05). Glomerulitis ("g") was correlated with creatinine concentrations at 90 and 360 days (P < .05). Tubulitis ("t") and interstitial inflammation ("i") displayed no association with renal function at any time.

Elementary steps in the acquisition of Mn2+ by the fosfomycin resistance protein (FosA).

The fosfomycin resistance protein, FosA, catalyzes the Mn(2+)-dependent addition of glutathione to the antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid, rendering the antibiotic inactive. The enzyme is a homodimer of 16 kDa subunits, each of which contains a single mononuclear metal site. Stopped-flow absorbance/fluorescence spectrometry provides evidence suggesting a complex kinetic mechanism for the acquisition of Mn(2+) by apoFosA. The binding of Mn(H(2)O)(6)(2+) to apoFosA alters the UV absorption and intrinsic fluorescence characteristics of the protein sufficiently to provide sensitive spectroscopic probes of metal binding. The acquisition of metal is shown to be a multistep process involving rapid preequilibrium formation of an initial complex with release of approximately two protons (k(obsd) > or = 800 s(-1)). The initial complex either rapidly dissociates or forms an intermediate coordination complex (k > 300 s(-1)) with rapid isomerization (k > or = 20 s(-1)) to a set of tight protein-metal complexes. The observed bimolecular rate constant for formation of the intermediate coordination complex is 3 x 10(5) M(-1) s(-1). The release of Mn(2+) from the protein is slow (k approximately 10(-2) s(-1)). The kinetic results suggest a more complex chelate effect than is typically observed for metal binding to simple multidentate ligands. Although the addition of the substrate, fosfomycin, has no appreciable effect on the association kinetics of enzyme and metal, it significantly decreases the dissociation rate, suggesting that the substrate interacts directly with the metal center.

Kinetic analysis of the slow ionization of glutathione by microsomal glutathione transferase MGST1.

An important aspect of the catalytic mechanism of microsomal glutathione transferase (MGST1) is the activation of the thiol of bound glutathione (GSH). GSH binding to MGST1 as measured by thiolate anion formation, proton release, and Meisenheimer complex formation is a slow process that can be described by a rapid binding step (K(GSH)d = 47 +/- 7 mM) of the peptide followed by slow deprotonation (k2 = 0.42 +/- 0.03 s(-1). Release of the GSH thiolate anion is very slow (apparent first-order rate k(-2) = 0.0006 +/- 0.00002 s(-)(1)) and thus explains the overall tight binding of GSH. It has been known for some time that the turnover (kcat) of MGST1 does not correlate well with the chemical reactivity of the electrophilic substrate. The steady-state kinetic parameters determined for GSH and 1-chloro-2,4-dinitrobenzene (CDNB) are consistent with thiolate anion formation (k2) being largely rate-determining in enzyme turnover (kcat = 0.26 +/- 0.07 s(-1). Thus, the chemical step of thiolate addition is not rate-limiting and can be studied as a burst of product formation on reaction of halo-nitroarene electrophiles with the E.GS- complex. The saturation behavior of the concentration dependence of the product burst with CDNB indicates that the reaction occurs in a two-step process that is characterized by rapid equilibrium binding ( = 0.53 +/- 0.08 mM) to the E.GS- complex and a relatively fast chemical reaction with the thiolate (k3 = 500 +/- 40 s(-1). In a series of substrate analogues, it is observed that log k3 is linearly related (rho value 3.5 +/- 0.3) to second substrate reactivity as described by Hammett sigma- values demonstrating a strong dependence on chemical reactivity that is similar to the nonenzymatic reaction (rho = 3.4). Microsomal glutathione transferase 1 displays the unusual property of being activated by sulfhydryl reagents. When the enzyme is activated by N-ethylmaleimide, the rate of thiolate anion formation is greatly enhanced, demonstrating for the first time the specific step that is activated. This result explains earlier observations that the enzyme is activated only with more reactive substrates. Taken together, the observations show that the kinetic mechanism of MGST1 can be described by slow GSH binding/thiolate formation followed by a chemical step that depends on the reactivity of the electrophilic substrate. As the chemical reactivity of the electrophile becomes lower the rate-determining step shifts from thiolate formation to the chemical reaction.

FosB, a cysteine-dependent fosfomycin resistance protein under the control of sigma(W), an extracytoplasmic-function sigma factor in Bacillus subtilis.

We demonstrate that the Bacillus subtilis fosB(yndN) gene encodes a fosfomycin resistance protein. Expression of fosB requires sigma(W), and both fosB and sigW mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn(2+)-dependent glutathione transferases but with a preference for Mg(2+) and L-cysteine as cofactors.

Elucidation of a monovalent cation dependence and characterization of the divalent cation binding site of the fosfomycin resistance protein (FosA).

The fosfomycin resistance protein FosA is a member of a distinct superfamily of metalloenzymes containing glyoxalase I, extradiol dioxygenases, and methylmalonyl-CoA epimerase. The dimeric enzyme, with the aid of a single mononuclear Mn2+ site in each subunit, catalyzes the addition of glutathione (GSH) to the oxirane ring of the antibiotic, rendering it inactive. Sequence alignments suggest that the metal binding site of FosA is composed of three residues: H7, H67, and E113. The single mutants H7A, H67A, and E113A as well as the more conservative mutants H7Q, H67Q, and E113Q exhibit marked decreases in the ability to bind Mn2+ and, in most instances, decreases in catalytic efficiency and the ability to confer resistance to the antibiotic. The enzyme also requires the monovalent cation K+ for optimal activity. The K+ ion activates the enzyme 100-fold with an activation constant of 6 mM, well below the physiologic concentration of K+ in E. coli. K+ can be replaced by other monovalent cations of similar ionic radii. Several lines of evidence suggest that the K+ ion interacts directly with the active site. Interaction of the enzyme with K+ is found to be dependent on the presence of the substrate fosfomycin. Moreover, the E113Q mutant exhibits a kcat which is 40% that of wild-type in the absence of K+. This mutant is not activated by monovalent cations. The behavior of the E113Q mutant is consistent with the proposition that the K+ ion helps balance the charge at the metal center, further lowering the activation barrier for addition of the anionic nucleophile. The fully activated, native enzyme provides a rate acceleration of >10(15) with respect to the spontaneous addition of GSH to the oxirane.

Mechanistic imperative for the evolution of a metalloglutathione transferase of the vicinal oxygen chelate superfamily.

A number of glutathione (GSH) transferases are now known in prokaryotes and eukaryotes. The enzymes appear to be primarily involved in the metabolism of foreign compounds. At least six distinct classes of soluble GSH transferases have been identified in eukaryotes and named alpha, mu, pi, sigma, theta and kappa. Sequences and the known three-dimensional structures of the soluble enzymes suggest that these proteins share a common ancestry, though the precise details of their evolution remain obscure. A second distinct family of GSH transferases are the microsomal or membrane-bound enzymes that include leukotriene C4 synthase. A third family is represented by a bacterial GSH transferase (FosA) responsible for conferring resistance to the antibiotic fosfomycin, reported some years ago by Suarez and co-workers (Arca et al., Antimicrob. Agents Chemother. 34 (1990) 1552-1556). The enzyme is quite specific for fosfomycin, which contains a very stable epoxide moiety. Evidence is presented that FosA is a metalloprotein related to iron- and manganese-dependent dioxygenases and to glyoxalase I. These enzymes are members of a previously unrecognized group of enzymes; the vicinal oxygen chelate superfamily. The mechanistic imperative driving the evolution of FosA and its relatives, which are enzymes catalyzing quite diverse chemical reactions, is proposed to be the electrophilic assistance provided by the metal through chelation of a substrate or intermediate.

Fosfomycin resistance protein (FosA) is a manganese metalloglutathione transferase related to glyoxalase I and the extradiol dioxygenases.

The enzyme conferring resistance to the antibiotic fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] originally reported by Suarez and co-workers [Area, P., Hardisson, C., & Suarez, J. E. (1990) Antimicrob. Agents Chemother. 34, 844-848] is demonstrated in this study to be a metalloglutathione transferase. The apoenzyme is a dimer of 16 kDa subunits. Electron paramagnetic resonance spectroscopy and water proton nuclear magnetic resonance longitudinal relaxation rates suggest that each subunit contains a mononuclear Mn2+ center that interacts strongly with the substrate fosfomycin (Kd = 17 microM) more weakly with the product (Kd = 1.1 mM) and very weakly or not at all with GSH. Inhomogeneous broadening of the EPR signals of enzyme-bound Mn2+ in the presence of H2(17)O indicates that three of the coordination sites on the metal are occupied by water. Sequence alignments, three-dimensional structures, and mechanistic considerations suggest that FosA is related to at least two other metalloenzymes, glyoxalase I and the Mn2+- or Fe2+-containing extradiol dioxygenases. The mechanistic imperative driving the evolution of this previously unidentified superfamily of metalloenzymes is proposed to be bidentate coordination of a substrate or intermediate to the metal center in the enzyme-catalyzed reactions.

Identification of a species specific regulatory site in human pancreatic cholesterol esterase.

All mammalian pancreatic cholesterol esterases (CEase) bind to membrane-associated heparin at a single site on the intestinal brush border membrane with a dissociation constant of 100 nM. While the enzyme is bound to the membrane, the activity of the human and bovine enzymes is enhanced 2-fold when compared to the activity of the enzyme in solution. On the other hand, soluble heparin potently inhibits the human CEase-catalyzed hydrolysis of cholesterol oleate with an IC50 of 2 x 10(-4) mg/mL, a value that is about 10(4) times more potent than that found with the bovine enzyme. The C-terminal portion of the human enzyme contains 16 proline-rich repeats of 11 amino acids each, while that from other species contains only a few of these repeat units. To determine if the unique human C-terminus is responsible for this inhibition, two chimeras containing either the human N-terminus (residues 1-445) and the bovine C-terminus (residues 446-557), HB, or the bovine N-terminus (residues 1-445) and the human C-terminus (residues 446-722), BH, were prepared. The cholesterol oleate hydrolytic activity of these chimeras was similar to that for the recombinant human and bovine enzymes. Importantly, each chimera was inhibited by heparin with IC50 values of 0.03 and 0.1 mg/mL for HB and BH, respectively. These intermediate IC50 values indicate that human CEase has two structural regions that contribute to is unique inhibition by this sulfated glycosaminoglycan, and these could regulate cholesterol uptake in humans.