Jack Schmidt

S-adenosylmethionine deficiency and TNF-α in lipopolysaccharide-induced hepatic injury

S-adenosylmethionine (Adomet) is a substrate for de novo synthesis of choline. Adomet deficiency occurs in certain types of liver injury, and the injury is attenuated by exogenous Adomet. Tumor necrosis factor-α (TNF-α) is also a mediator of these models of hepatotoxicity. We investigated the role of Adomet in lipopolysaccharide (LPS)-induced liver injury in rats made deficient in both Adomet and choline. Rats were maintained on either a methionine-restricted and choline-deficient (MCD) diet or a diet containing sufficient amounts of all nutrients [methionine and choline sufficient (MCS)] and then administered either LPS or saline. MCS-LPS rats had normal liver histology and no change in serum transaminases compared with the MCS-saline control group. MCD-saline rats had hepatosteatosis but no necrosis, and a five- to sevenfold increase in transaminases vs. the MCS-saline group. MCD-LPS rats additionally had hepatonecrosis and a 30- to 50-fold increase in transaminases. Exogenous Adomet administration to MCD-LPS rats corrected the hepatic deficiency of Adomet but not of choline, prevented necrosis but not steatosis, and attenuated transaminases. Serum TNF-α was sixfold higher in MCD rats even without LPS challenge and 300-fold higher with LPS challenge. Exogenous Adomet attenuated increased serum TNF-α in MCD-LPS rats.

Effects of exogenous superoxide anion and nitric oxide on the scavenging function and electron microscopic appearance of the sinusoidal endothelium in the isolated, perfused rat liver

BACKGROUND/AIMS Functional and morphological alterations of the hepatic sinusoidal endothelial cell occur in several models of experimental liver injury and in clinical settings. The causes of these alterations are multiple. The aim of this study was to test the hypothesis that the early functional impairment and morphological alterations of the sinusoidal endothelial cell and hepatic sinusoid associated with liver injury are mediated by free radical species, such as superoxide anion and nitric oxide. METHODS Isolated rat livers were perfused by recirculation with hemoglobin-free, Krebs-Henseleit bicarbonate buffer and presented with a source of superoxide anion (xanthine oxidase+hypoxanthine) or nitric oxide (S-nitroso-N-acetyl penicillamine). Hyaluronan uptake (an index of sinusoidal endothelial cell scavenging function), thiobarbituric acid-reactive substances content of the tissue (a marker of lipid peroxidation), reduced and oxidized glutathione (a marker of the thiol system oxidation/reduction state), lactate dehydrogenase and alanine aminotransferase activities (markers of cytolysis), as well as scanning and transmission electron microscopic appearance of the sinusoid were evaluated. RESULTS At the high concentrations used, both free radical generating systems suppressed hyaluronan uptake, increased malondialdehyde content of the tissue, enhanced the release of both liver enzymes, decreased the total glutathione content of the liver, and altered the ratio of reduced/oxidized glutathione. Both free radical species induced dose-dependent morphological alterations of the sinusoid, consisting of the appearance of large gaps replacing the sieve-plated fenestration. CONCLUSIONS The free radical species-induced functional impairment and morphological alterations of the liver sinusoid, presented in this study, closely resemble the early in vivo changes associated with liver injury under a variety of conditions, such as preservation and reperfusion, or administration of hepatotoxicants such as D-galactosamine, Gram-negative bacterial lipopolysaccharides, acetaminophen, alcohol and others. Therefore, we suggest that early liver sinusoid injury, observed under these conditions, can be attributed to the action of free radicals, such as superoxide anion and nitric oxide.

Swapping the Substrate Specificities of the Neuropeptidases Neurolysin and Thimet Oligopeptidase

Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites.

Predicting reversed-phase gradient elution separations by computer simulation : A comparison of two different programs

Abstract Two computer programs for developing and improving high-performance liquid chromatographic methods, DryLab G and LCSIM, have recently been described. The accuracies of these two programs were examined using experimental (o-phthalaldehyde-derivatized amino acids) and synthetic data. DryLab G, which uses gradient data for input, correctly predicted retention times for various gradient and isocratic separations. Predicted retention times for the simulation of certain isocratic conditions are susceptible to errors in the measured dwell volume, but the prediction of resolution is not seriously affected. LCSIM uses isocratic data for input, and predicted gradient retention times are affected by the accuracy of the measured dwell volume. The resolution of closely eluting analytes was usually predicted within a small fraction of the peak width, i.e., with negligible errors. Examples are given for some unique features of the LCSIM program: solvent switching and mixing, optional descending gradients, graphics display of the separation process and an iterative fit of the separation parameters to the retention characteristics of a new column (as measured from a single run).

Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture.

The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 A (1 A=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr(178) as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr(2) in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr(178) to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr(178) can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

Comparison of Administration of Two Standard Intravenous Amino Acid Formulas to Severely Brain-Injured Patients

Twenty severely brain-injured patients with Glasgow Coma Scale scores of 4–9 were prospectively randomized to receive one of two standard amino acid formulas, starting with the first day of hospital admission up to day 14 postinjury. Formula 2 (patient group 2) had 54 percent more leucine, 53 percent more isoleucine, 74 percent more valine, 28 percent less phenylalanine, 31 percent less methionine, 111 percent more proline, 38 percent less alanine, and 38 percent less glycine than formula 1 (patient group 1). Groups 1 and 2 received statistically equal overall mean parenteral nutrition calories and protein (2173 ± 147 vs. 2059 ± 143 kcal, and 77 ± 12 vs. 83.1 ± 6 g, respectively). There was a significant difference in overall mean urinary urea nitrogen excretion (group 1 = 24.6 ± 1.3 vs. group 2= 18.3 ± 1.1, p = 0.02) and nitrogen balance (group 1 = −8.0 ± 2.1 vs. group 2 = + 1.8 ± 1.2, p = 0.01). Mean overall isoleucine values were significantly higher in group 2 (overall mean 77 μmol/L vs. 62 μmol/L, p = 0.04). Phenylalanine levels were significantly higher in group 1 (107 μmol/L) versus group 2 (82 μmol/L) patients (p = 0.01). Arginine levels were significantly higher in group 1 (78 μmol/L) versus group 2 (49 μmol/L) patients (p = 0.0002). This observation suggests that some standard intravenous amino acid formulas may be more apt to promote positive nitrogen balance than others.

Nutritional and metabolic variables correlate with amino acid forearm flux in patients with severe head injury.

ObjectiveTo measure the arterial-venous amino acid flux across the forearm muscle in patients with severe head injury. DesignProspective, interventional study. SettingLevel I trauma hospital in the neuro-surgery intensive care unit (ICU) at a university medical center. PatientsEight nonsteroid-treated patients with severe head injury. InterventionsPatients were prospectively randomized to receive either standard or supplemental intravenous zinc therapy. Measurements and Main ResultsNet forearm alanine, glutamine, tyrosine, phenylalanine, and branch-chain amino acid forearm flux were measured and compared with metabolic markers of energy expenditure and nitrogen excretion.There was a significant inverse relationship between the measured energy expenditure/predicted energy expenditure ratio and glutamine flux (r2 = .62; p < .05). The patients with the highest measured energy expenditure/predicted energy expenditure ratio had the greatest release of glutamine from forearm muscle. Nitrogen balance was significantly correlated with leucine flux (r2 = .53; p < .05) and with isoleucine flux (r2 = .67;p < .05). The patients with the most positive nitrogen balance had the least release of branch-chain amino acids from skeletal muscle. Tyrosine flux was highly correlated with net amino acid flux (r2 = .76; p < .01). Tyrosine flux was therefore indicative of overall muscle catabolism. Four patients had an overall negative flux of amino acids from skeletal muscle. Three patients had an overall negative flux of branch-chain amino acids. ConclusionsThis preliminary descriptive report suggests that increased skeletal muscle efflux of amino acids correlates significantly with metabolic variables of hypermetabolism and hypercatabolism in nonsteroid-treated, headinjured patients. (Crit Care Med 1994; 22:393–398)

Allosteric Inhibition of the Neuropeptidase Neurolysin

Background: Neuropeptidases metabolize regulatory peptides and hormones. Results: A new type of neuropeptidase inhibitor binds away from the catalytic site, is noncompetitive with short peptide substrates, and promotes the open enzyme conformation. Conclusion: The inhibitor restricts a conformational change associated with substrate cleavage. Significance: Inhibitors of this class will allow more specific modulation of neuropeptidases, aiding therapeutic development. Neuropeptidases specialize in the hydrolysis of the small bioactive peptides that play a variety of signaling roles in the nervous and endocrine systems. One neuropeptidase, neurolysin, helps control the levels of the dopaminergic circuit modulator neurotensin and is a member of a fold group that includes the antihypertensive target angiotensin converting enzyme. We report the discovery of a potent inhibitor that, unexpectedly, binds away from the enzyme catalytic site. The location of the bound inhibitor suggests it disrupts activity by preventing a hinge-like motion associated with substrate binding and catalysis. In support of this model, the inhibition kinetics are mixed, with both noncompetitive and competitive components, and fluorescence polarization shows directly that the inhibitor reverses a substrate-associated conformational change. This new type of inhibition may have widespread utility in targeting neuropeptidases.

On the intersection of certain maximal subgroups of a finite group

Abstract. Let Δ(G)

On a class of finite solvable groups

{\Delta (G)}