Structural studies of the final enzyme in the alpha-aminoadipate pathway-saccharopine dehydrogenase from Saccharomyces cerevisiae.

The 1.64 A structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogenase, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.

Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase.

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.

Electrostatic modification of the active site of myoglobin: characterization of the proximal Ser92Asp variant.

The structural and functional consequences of the introduction of a negatively charged amino acid into the active site of horse heart myoglobin have been investigated by replacement of the proximal Ser92 residue (F7) with an aspartyl residue (Ser92Asp). UV-visible absorption maxima of various ferrous and ferric derivatives and low-temperature EPR spectra of the metaquo (metMb) derivative indicate that the active site coordination geometry has not been perturbed significantly in the variant. 1H-NMR spectroscopy provides direct evidence for the existence of a distal water molecule as the sixth ligand in the oxidized form of the variant at pD 5.7. Spectrophotometric pH titration of the Ser92Asp variant is consistent with this finding and with a pKa = 8.90 +/- 0.02 [25.0 degrees C, mu = 0.10 M (NaCl)] for titration of the distal water molecule, identical to the value reported for the wild-type protein. X-ray crystallography of the metMb derivative indicates that the heme substituents conserve their orientations in the variant protein, except for a slight reorientation of the pyrrole A propionate group to which Ser92 normally hydrogen bonds and reorientation of the carboxyl end of the pyrrole D propionate group. No change is observed in conformation of the proximal (His93) or distal (Wat156) heme ligands. 1H-NMR spectroscopy of the metMbCN form of the protein indicates that a slight rotation of the proximal His93 ligand has occurred in this derivative. Resonance Raman experiments indicate increased conformational heterogeneity in the proximal pocket of the variant. Failure to detect electron density for the Asp residue in the X-ray diffraction map of the variant protein and high average thermal factors for the pyrrole A propionate substituent are consistent with this observation. The variant exhibits novel pH-dependent behavior in the metMb form, as shown by 1H-NMR spectroscopy, and provides evidence for a heme-linked titratable group with a pKa of 5.4 in this derivative. The metMbCN and deoxyMb derivatives also exhibit pH-dependent behavior, with pKas of 5.60 +/- 0.07 and 6.60 +/- 0.07, respectively, compared to the wild-type values of 5.4 +/- 0.04 and 5.8 +/- 0.1. The heme-linked ionizable group is proposed to be His97 in all three derivatives. The reduction potential of the variant is 72 +/- 2 mV vs SHE [25.0 degrees C, mu = 0.10 M (phosphate), pH 6.0], an increase of 8 mV over the wild-type value. The possible influence of a number of variables on the magnitude of the reduction potential in myoglobin and other heme proteins is discussed.

The proximal ligand variant His93Tyr of horse heart myoglobin.

The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for bovine liver catalase. The 1D 1H-NMR spectrum of the variant confirms the five-coordinate nature of the heme iron center and exhibits a broad resonance at 112.5 ppm that is attributable to the meta protons of the phenolate ligand. This result indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is unchanged from that of the wild-type protein (pH 8) while the midpoint reduction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 mV lower. The three-dimensional structure of the variant determined by X-ray diffraction analysis confirms the five-coordinate nature of the heme iron center and establishes that the introduction of a proximal Tyr ligand is accommodated by a shift of the F helix (residues 88-99) in which this residue resides away from the heme pocket. Additional effects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hydrogen bonding interactions with the heme prosthetic group.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of MR imaging on the normal human pineal body: measurement of plasma melatonin levels.

Production of melatonin, a hormone synthesized and secreted by the pineal body, has been suppressed by electromagnetic fields in some but not all animal studies. Magnetic resonance (MR) imaging at 1.5 T was evaluated for its ability to modulate the level of melatonin in eight male volunteers. Subjects were exposed to three conditions, respectively, between 1:00 and 2:00 AM on different nights: (a) a series of routine MR pulse sequences for brain imaging in dark conditions, (b) dark control conditions, and (c) bright-light control conditions. Plasma was analyzed for melatonin and cortisol levels. Hormonal changes were analyzed by one-factor repeated measures within-subject analysis of variance. These conditions were associated with significant differences in melatonin levels: F(2, 6) = 7.95, and P = .021. Subjects exposed to darkness showed a typical increase in melatonin concentration. Subjects exposed to bright light showed a characteristic suppression of melatonin concentration. Those exposed to the MR imaging fields showed an increase in melatonin level similar to that seen in the dark control condition. Light and MR imaging had no significant effects on cortisol levels. Thus, MR imaging at field strengths known to modulate melatonin levels in rats did not suppress melatonin production in human subjects.

Polycythemia vera and myelofibrosis: correlation of MR imaging, clinical, and laboratory findings.

Magnetic resonance (MR) imaging was performed in 14 patients with biopsy-proved polycythemia vera (n = 4) or myelofibrosis (n = 10) to determine whether MR imaging findings can be correlated with the clinicopathologic diagnosis and established clinical parameters of severity (serum lactate dehydrogenase [LDH] and cholesterol levels) and chronicity (spleen size). Evaluation of marrow in the proximal femurs showed that patients could be categorized into three distinct groups based on anatomic patterns of normal fatty and abnormal low-signal-intensity (non-fatty) marrow in the femoral capital epiphysis (FCE) and greater trochanter (GT). Patients with nonfatty marrow in both the FCE and GT (n = 8) had significantly higher serum LDH (P less than .02) and lower serum cholesterol (P less than .02) levels than patients with fatty marrow in at least the GT (n = 6). Splenic volume, as measured from MR images, was significantly greater in the myelofibrosis group than in the polycythemia vera group (P less than .001). MR imaging provided a better understanding of these hematologic disorders and novel parameters for classification that are different from conventional histologic and laboratory data.

Trauma to the elbow and forearm.

Elbow and forearm trauma constitute a significant percentage of upper extremity injuries. A thorough understanding of the anatomy and mechanism of injury is necessary for obtaining the most accurate radiographic interpretation in both the pediatric and adult populations. Proper radiographic positioning and choice of the best imaging modality are crucial to ensure the best orthopedic treatment and reduce the incidence of post-treatment complications.

Hepatocellular tumors with high signal on T1-weighted MR images: chemical shift MR imaging and histologic correlation.

We reviewed conventional and chemical shift MR images and histologic findings of seven proven primary hepatic masses that had higher signal than liver on T1-weighted images to determine if this necessarily indicates fat and if the presence of fat indicates malignancy. These seven masses included five hepatocellular carcinomas (HCCs), one focal nodular hyperplasia (FNH), and one fatty dysplastic nodule. An eighth solitary high signal mass without histologic proof had evidence of abundant fat on each of two chemical shift MR images 25 months apart. Only one of the five HCCs had chemical shift or histologic evidence of fat, while the FNH and dysplastic nodule each had both chemical shift and histologic confirmation of fat. The dysplastic nodule became more dysplastic and grew significantly within 14 months, but remained benign. The unproven fatty lesion decreased in size over 25 months and is therefore presumably benign. Although no statistical inferences can be drawn from this small correlative study, we have shown that HCC may have higher signal intensity than liver on T1-weighted images, whether or not it contains fat. Chemical shift techniques can confirm the presence of intratumoral fat and thus indicate a mass of hepatocellular origin, but the mass may be benign or malignant.

Imaging of the distal radioulnar joint.

The distal radioulnar joint can be evaluated by many different imaging techniques, including plain radiography, arthrography, tomography, nuclear medicine bone scanning, computed tomography, and magnetic resonance imaging. Each of these techniques has advantages and disadvantages that must be considered when determining the appropriate diagnostic evaluation for a particular patient.

MR imaging of shoulder injuries in professional baseball players.

Magnetic resonance (MR) imaging was used to evaluate the shoulders of 10 symptomatic professional baseball players and one asymptomatic player, with surgical correlation in six cases and arthrographic correlation in two cases. Seven small rotator cuff tears measuring 0.5-1 cm were identified on MR images, with arthrographic and surgical confirmation of these findings in two patients and surgical confirmation only in three patients. Cortical irregularity and/or subchondral cyst formation at the posterior aspect of the greater tuberosity near the insertion site of the infraspinatus tendon was found in five of the seven players with rotator cuff tears. Similar findings were noted in the asymptomatic volunteer and in one of the three players without cuff tear, who also had irregular thickening of the posterior capsule. These findings are believed to represent chronic avulsive changes resulting from the deceleration stresses of the follow-through motion.

Parenchymal versus reticuloendothelial iron overload in the liver: distinction with MR imaging.

Parenchymal iron deposition occurs in hemochromatosis, while iron is deposited in reticuloendothelial (RE) cells after blood transfusions or rhabdomyolysis. Magnetic resonance images of patients with decreased liver signal intensity on T2-weighted images at 1.5 T were blindly compared in an effort to distinguish these conditions. In each of five patients with hemochromatosis, the pancreas had low signal intensity, but splenic signal intensity was decreased in only one. In contrast, only three of the 16 patients with RE iron overload had low pancreatic signal intensity, while all of these patients either had low splenic signal intensity (n = 14) or previously underwent splenectomy (n = 2). Distinction among these causes of iron deposition is clinically important because parenchymal iron overload from hemochromatosis may produce significant tissue damage, while the RE iron of transfusions and rhabdomyolysis is of little clinical consequence.