Decreased Cerebrospinal Fluid Glutamine Levels in Patients with Benign Brain Tumors

Abstract: CSF glutamine concentrations were studied in 12 patients with benign brain tumors (meningioma, craniopharyngioma, or osteofibroma), 12 patients with malignant brain tumors (astrocytoma, medulloblastoma, pinealoblastoma, or chondrosarcoma), 9 patients with non-cerebral tumors, and a reference group of 24 patients. The mean ± SD levels in the benign tumor group (424 ± 124 μ M ) were significantly lower (p < 0.0004) than those in the reference group (642 ± 195 μ M ). There was no significant difference between the CSF glutamine concentrations in the malignant cerebral tumor group (643 ± 210 μ M ) or noncerebral tumor group (599 ± 127 μ M ) and those in the reference group. In patients with benign brain tumors there was indication of an inverse linear relationship between the logarithm of CSF glutamine concentration and tumor diameter.     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.     

Deranged aortic intima-media thickness, plasma triglycerides and granulopoiesis in Sl/Sl(d) mice

Studies were carried out to evaluate the impact of a high-fat dietary regimen on aortic wall thickness, peripheral blood leukocyte profile, and plasma cholesterol and triglyceride levels in the mast cell-deficient Sl/Sl(d) mouse. The results demonstrated that the mean aortic wall thickness of Sl/Sl(d) mice was significantly higher than their normal littermates, and were increased in both genotypes after a 17-day high-fat regimen. In comparison with normal littermates, Sl/Sl(d) genotypes had elevated levels of plasma triglycerides with normal levels of plasma cholesterol, and the high-fat diet markedly lowered the triglyceride levels. Total peripheral blood leukocytes, the monocyte and granulocyte counts, and hemoglobin levels were significantly lower in Sl/Sl(d) mice, although the number of lymphocytes, eosinophils and basophils were the same in both genotypes. Interestingly, the high-fat diet regimen elevated leukocyte counts and the number of monocytes and granulocytes in Sl/Sl(d) mice.     

Mechanisms of calcification by vesicles isolated from atherosclerotic rabbit aortas

Although several lines of evidence support the role of calcifiable vesicles in dystrophic vascular calcification, the mechanisms whereby vesicles promote aortic calcification remain incompletely understood. Previous reports indicate that ATP promotes in vitro vesicle calcification. Whether ATP-initiated calcification is simply mediated through increased Pi concentrations or by other unknown mechanisms related to ATP hydrolysis is unclear. To determine whether high Pi levels resulting from ATP hydrolysis may cause CaxP ion products to surpass the threshold for calcium phosphate precipitation, 3 mM Pi instead of 1 mM ATP was added to calcifying media. The inclusion of 1 mM ATP in calcifying media with an initial serum level of Ca2+ (1.45 mM) and Pi (2.3 mM) was much more effective in promoting calcification than the addition of 3 mM Pi. The higher effectiveness of ATP over Pi in promoting calcification was consistent throughout various incubation periods and vesicle protein ranges. To minimize the effect of CaxPi ion products on calcification, the ion product was kept within the physiological ranges throughout the incubation period by reducing initial Pi or ATP concentrations in calcifying media. At these low levels of ion products, ATP was still more effective than Pi in promoting calcification. Both ATP- and Pi-stimulated calcifications were found to increase with increasing levels of ion products whereas greater effectiveness of ATP over Pi remained unaltered. These observations indicate that ATP hydrolysis may initiate calcification through some mechanisms other than a simple provision of Pi in order to surpass the solubility products. Concanavalin A (Con A) was found to bind to vesicles and to enhance both ATP- and Pi-promoted calcification. Taken together, these observations suggest that ATP hydrolysis, CaxP ion products, and vesicle-associated carbohydrates are implicated in vesicle-mediated calcification.     

Structure of human aldose reductase holoenzyme in complex with statil: an approach to structure-based inhibitor design of the enzyme

Aldose reductase, a monomeric NADPH-dependent oxidoreductase, catalyzes the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The X-ray structure of human aldose reductase holoenzyme in complex with statil was determined at a resolution of 2.1 A. The carboxylate group of statil interacted with the conserved anion binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Statil's hydrophobic phthalazinyl ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group penetrating the "specificity" pocket. The interactions between the inhibitor's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding to residues Leu300 and Thr113. Based on the model of the ternary complex, the program GRID was used in an attempt to design novel potential inhibitors of human aldose reductase with enhanced binding energies of the complex. Molecular modeling calculations suggested that the replacement of the fluorine atom of statil with a carboxylate functional group may enhance the binding energies of the complex by 33%.     

Probing the inhibitor selectivity pocket of human 20α-hydroxysteroid dehydrogenase (AKR1C1) with X-ray crystallography and site-directed mutagenesis

Human 20α-hydroxysteroid dehydrogenase (AKR1C1) is an important drug target due to its role in the development of lung and endometrial cancers, premature birth and neuronal disorders. We report the crystal structure of AKR1C1 complexed with the first structure-based designed inhibitor 3-chloro-5-phenylsalicylic acid (K_i = 0.86 nM) bound in the active site. The binding of 3-chloro-5-phenylsalicylic acid to AKR1C1 resulted in a conformational change in the side chain of Phe311 to accommodate the bulky phenyl ring substituent at the 5-position of the inhibitor. The contributions of the nonconserved residues Leu54, Leu306, Leu308 and Phe311 to the binding were further investigated by site-directed mutagenesis, and the effects of the mutations on the K_i value were determined. The Leu54Val and Leu306Ala mutations resulted in 6- and 81-fold increases, respectively, in K_i values compared to the wild-type enzyme, while the remaining mutations had little or no effects.     

Aldo-keto reductase 1C15 as a quinone reductase in rat endothelial cell: its involvement in redox cycling of 9,10-phenanthrenequinone

9,10-Phenanthrenequinone (9,10-PQ), a redox-active quinone in diesel exhausts, triggers cellular apoptosis via reactive oxygen species (ROS) generation in its redox cycling. This study found that induction of CCAAT/enhancer-binding protein-homologous protein (CHOP), a pro-apoptotic factor derived from endoplasmic reticulum stress, participates in the mechanism of rat endothelial cell damage. The 9,10-PQ-mediated CHOP induction was strengthened by a proteasome inhibitor (MG132) and the MG132-induced cell sensitization to the 9,10-PQ toxicity was abolished by a ROS inhibitor, suggesting that ROS generation and consequent proteasomal dysfunction are responsible for the CHOP up-regulation caused by 9,10-PQ. Aldo-keto reductase (AKR) 1C15 expressed in rat endothelial cells reduced 9,10-PQ into 9,10-dihydroxyphenanthrene concomitantly with superoxide anion formation, implying its participation in evoking the 9,10-PQ-redox cycling. The 9,10-PQ-induced damage was augmented by AKR1C15 over-expression. 9,10-PQ also provoked the AKR1C15 up-regulation, which sensitized against the quinone toxicity. These results suggest the presence of a negative feedback loop exacerbating the quinone toxicity in rat endothelial cells.     

Structure of the tetrameric form of human L-Xylulose reductase: probing the inhibitor-binding site with molecular modeling and site-directed mutagenesis

L-Xylulose reductase (XR) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. In this study we report the structure of the biological tetramer of human XR in complex with NADP(+) and a competitive inhibitor solved at 2.3 A resolution. A single subunit of human XR is formed by a centrally positioned, seven-stranded, parallel beta-sheet surrounded on either side by two arrays of three alpha-helices. Two helices located away from the main body of the protein form the variable substrate-binding cleft, while the dinucleotide coenzyme-binding motif is formed by a classical Rossmann fold. The tetrameric structure of XR, which is held together via salt bridges formed by the guanidino group of Arg203 from one monomer and the carboxylate group of the C-terminal residue Cys244 from the neighboring monomer, explains the ability of human XR to prevent the cold inactivation seen in the rodent forms of the enzyme. The orientations of Arg203 and Cys244 are maintained by a network of hydrogen bonds and main-chain interactions of Gln137, Glu238, Phe241, and Trp242. These interactions are similar to those defining the quaternary structure of the closely related carbonyl reductase from mouse lung. Molecular modeling and site-directed mutagenesis identified the active site residues His146 and Trp191 as forming essential contacts with inhibitors of XR. These results could provide a structural basis in the design of potent and specific inhibitors for human XR.     

Side chain orientation of residues lining the selectivity filter of epithelial Na+ channels

Epithelial Na(+) channels (ENaCs) selectively conduct Na(+) and Li(+) but exclude K(+). A three-residue tract ((G/S)XS) present within all three subunits has been identified as a key structure forming a putative selectivity filter. We investigated the side chain orientation of residues within this tract by analyzing accessibility of the introduced sulfhydryl groups to thiophilic Cd(2+). Xenopus oocytes were used to express wild-type or mutant mouse alphabetagammaENaCs. The blocking effect of external Cd(2+) was examined by comparing amiloride-sensitive Na(+) currents measured by two-electrode voltage clamp in the absence and presence of Cd(2+) in the bath solution. The currents in mutant channels containing a single Cys substitution at the first or third position within the (G/S)XS tract (alphaG587C, alphaS589C, betaG529C, betaS531C, gammaS546C, and gammaS548C) were blocked by Cd(2+) with varying inhibitory constants (0.06-13 mm), whereas the currents in control channels were largely insensitive to Cd(2+) at concentrations up to 10 mm. The Cd(2+) blocking effects were fast, with time constants in the range of seconds, and were only partially reversible. The blocked currents were restored by 10 mm dithiothreitol. Mutant channels containing alanine or serine substitutions at these sites within the alpha subunit were only poorly and reversibly blocked by 10 mm Cd(2+). These results indicate that the introduced sulfhydryl groups face the conduction pore and suggest that serine hydroxyl groups within the selectivity filter in wild-type ENaCs face the conduction pore and may contribute to cation selectivity by participating in coordination of permeating cations.