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Biomedical subjects

D L Burk

Publications and source records attributed to D L Burk.

At least 19 recordsLinked to original sources
PubMed2001

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.

Adenosine Diphosphate↗
PubMed1996

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.

Animals↗
PubMed1995

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)

Animals↗
PubMed1992

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.

Adult↗
PubMed1991

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.

Adult↗
PubMed1991

Hepatocellular carcinoma within siderotic regenerative nodules: appearance as a nodule within a nodule on MR images.

Large regenerative nodules in cirrhotic livers may accumulate iron and develop internal iron-poor foci of hyperplasia or malignancy. Magnetic resonance examinations were performed on 23 patients with biopsy-proved cirrhosis. A "nodule-within-nodule" appearance was noted in two patients. This appearance consisted of markedly low intensity of a large nodule on gradient-echo images, with one or two internal foci that were isointense to the liver. Each of the large nodules was 2 cm in diameter, and each of the internal foci was less than 1 cm. Serum alpha-fetoprotein levels were normal in both patients. Aspiration biopsy performed in one patient failed to show malignancy, but histologic confirmation of hepatocellular carcinoma was obtained eventually in both cases. The nodule-within-nodule sign, which reflects the unique histopathology of hepatocellular carcinoma in large siderotic regenerative nodules, is strongly suggestive of early hepatocellular carcinoma, even if serologic markers and biopsy results do not support this diagnosis.

Biopsy↗
PubMed1991

Liver and pancreas: improved spin-echo T1 contrast by shorter echo time and fat suppression at 1.5 T.

T1-weighted spin-echo magnetic resonance (MR) images have had limited soft-tissue contrast at 1.5 T. The authors investigated the effects of echo-time (TE) minimization and fat suppression on MR images of the liver and pancreas. Two sets of MR images were obtained with identical repetition times and other parameters. In 10 subjects with seven liver lesions, images with TEs of 20 and 12 msec were compared. In 18 additional subjects with seven liver lesions and five pancreatic carcinomas, images with identical TEs but with and without fat suppression were compared. Contrast-to-noise ratios (CNRs) were greater with a TE of 12 msec than with a TE of 20 msec for liver versus spleen (7.6 vs 4.9, P = .014) and liver versus lesion (6.9 vs 3.9, P = .031). In patients without fatty liver, CNR for six lesions versus liver was greater (9.5 vs 6.0, P = .014) with fat suppression. CNR between glandular pancreas and cancer was most conspicuous with fat suppression, but fat planes were less distinct. Minimization of TE improves T1-weighted images significantly. Fat suppression also improves CNR, but the disadvantages of fat suppression do not allow elimination of conventional T1-weighted images.

Fatty Liver↗
PubMed1991

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.

Adolescent↗
PubMed1991

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.

Arthrography↗