Biomedical subjects

D G Davis

Publications and source records attributed to D G Davis.

At least 37 records · Page 2Linked to original sources

PubMed1995

Solution structure of the human pp60c-src SH2 domain complexed with a phosphorylated tyrosine pentapeptide.

Human pp60c-src is a cellular nonreceptor tyrosine kinase that participates in cytosolic signal transduction and has been implicated in the development of malignant tumors in the human breast and colon. Signal transduction is mediated by highly specific interactions between the SH2 domain and receptor phosphorylated tyrosine binding motifs. To elucidate the molecular conformation and interactions in solution, a family of highly resolved nuclear magnetic resonance (NMR) structures was determined for the src SH2 domain complexed with a high-affinity phosphorylated pentapeptide, acetyl-p YEEIE-OH. The 23 structures, generated with a distance geometry (DG) and a dynamical simulated annealing (SA) procedure, satisfied 2072 experimental restraints derived from a variety of multifrequency/multidimensional and isotope-filtered NMR data. Superimposition of residues 143-245 upon the mean coordinate set yielded an atomic rmsd of 0.58 +/- 0.09 A for the N, C alpha, C' atoms and 1.04 +/- 0.08 for all the non-hydrogen atoms. Residues in the ordered secondary structure regions superimpose to 0.29 +/- 0.04 A for the N, C alpha, C' and 0.73 +/- 0.08 A for all the non-hydrogen atoms. The angular order parameter calculated for the phi, psi angles was > 0.9 for 81 of the 106 protein residues. The main protein conformational features are three antiparallel beta-strands that traverse a compact core with an alpha-helix on each side of the core near the N- and C-termini. The observed intermolecular nuclear Overhauser effects (NOE) from the pY, +1E, and +3I residues positioned the ligand in an extended conformation across the SH2 domain surface with the pY and +3I side chains inserted into the protein binding pockets. In general, the protein conformation is consistent with previously reported structures of different SH2 domain complexes determined by X-ray crystallography. However, inter- or intramolecular interactions involving the guanidinium side chains of the solvated R alpha A2 or the buried R beta B5 were not observed at pH = 5.5 or 7.0. If such interactions exist in solution, the absence of any confirming data probably arises from rapid exchange with solvent and/or undetermined dynamic components. Thus, the unrestrained R alpha A2 side chain did not show an amino-aromatic interaction or a hydrogen bond to the -1 carbonyl oxygen as observed in the crystal structures. This result is consistent with the solution structure of a different SH2 domain complex. A more detailed comparison between the crystal structure and the NMR-derived solution structures of the same src SH2 domain complex is presented.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

Multiple steroid-binding orientations: alteration of regiospecificity of dehydroepiandrosterone 2- and 7-hydroxylase activities of cytochrome P-450 2a-5 by mutation of residue 209.

The mutation of Ala-117 to Val conferred dehydroepiandrosterone (DHEA) hydroxylase activity on cytochrome P-450 2a-4, with the production of both 2 alpha- and 7 alpha-hydroxyDHEA at similar rates. P-450 2a-5 which has Val at position 117, acquired high DHEA hydroxylase activity by mutation of Phe-209. Mutant F209L of P-450 2a-5 exhibited strong regiospecificity at the 2-position of the DHEA molecule with the production of 2 alpha-hydroxy DHEA as the major metabolite. On the other hand, mutant F209V of P-450 2a-5 showed the 7-position to be the major hydroxylation site, 7 beta-hydroxyDHEA and 7 alpha-OHDHEA being produced. Therefore the regiospecificity of DHEA hydroxylase activity of P-450 2a-5 is altered between the 2- and 7-position depending on the amino acid at position 209. Modelling of the DHEA molecule in the pocket of bacterial P-450cam showed that the steroid can be accommodated in at least two orientations for which the 2- or 7- position is near the sixth axial position of the haem. Moreover, these two orientations, which are of similar energy, can be interconverted by a 180 degrees rotation of the steroid molecule around its long axis. These results support the hypothesis that the steroid molecule in the pocket is in dynamic equilibrium with multiple binding orientations and that the equilibrium is apparently determined by a few critical residues including those at positions 117 and 209.

Aryl Hydrocarbon Hydroxylases↗

PubMed1995

Dopaminergic and serotonergic neurotransmitters in bone marrow transplant patients.

Depigmentation of neurons in the substantia nigra (SN) is found in patients dying after bone marrow transplantation (BMT). This study examined neurochemical striatal changes related to BMT. Caudate nucleus and putamen of 6 BMT subjects and 10 age-matched controls were analyzed for levels of dopamine (DA), homovanillic acid (HVA), 5-hydroxyindoleamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA), by high pressure liquid chromatography-electron capture detection (HPLC-ECD). In addition, assays of the enzymatic activities of monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) were performed. Cholinergic markers, ChAT and AChE, were reduced in BMT caudate (p < 0.05) but not in the putamen. A recovery toward normal cholinergic enzymatic activity was identified with increased post-transplant survival time. The level of DA was reduced 50% in BMT caudate and putamen while HVA was increased 30%, however, neither reduction achieved statistical significance. Increasing post-transplant survival time correlated with decreased levels of DA in caudate nucleus and putamen in the early post-transplant period, while HVA was increased over the same interval but tended to return to normal levels with increasing survival time. Two-fold increases of BMT caudate 5-HT (p < 0.003) and 5-HIAA were found; similar changes were noted in putamen 5-HT and 5-HIAA (p < 0.0008). Significant increases in MAO-A and B were found in BMT caudate (p < 0.0001 and p < 0.06, respectively) and putamen (p < 0.0005 and p < 0.006, respectively). No statistically significant changes were noted in the 5-HT, 5-HIAA, or MAO A or MAO B with increasing post-transplant survival. Whether these changes are the result of physiologic or toxic effects is unknown.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1994

NMR solution structure of a peptide nucleic acid complexed with RNA.

Peptide nucleic acids (PNA) incorporating nucleic acid bases into an achiral polyamide backbone bind to DNA in a sequence-dependent manner. The structure of a PNA-ribonucleic acid (RNA) complex was determined with nuclear magnetic resonance methods. A hexameric PNA formed a 1:1 complex with a complementary RNA that is an antiparallel, right-handed double helix with Watson-Crick base pairing similar to the "A" form structure of RNA duplexes. The achiral PNA backbone assumed a distinct conformation upon binding that differed from previously proposed models and provides a basis for further structure-based design of antisense agents.

Magnetic Resonance Spectroscopy↗

PubMed1994

Studies of inhibitor binding to Escherichia coli purine nucleoside phosphorylase using the transferred nuclear Overhauser effect and rotating-frame nuclear Overhauser enhancement.

NMR studies of the adenosine analog tubercidin have been carried out in the presence of Escherichia coli purine nucleoside phosphorylase (PNP) in order to characterize the conformation of the enzyme-complexed nucleoside. Although analysis of transferred NOE data at various enzyme/inhibitor ratios indicated a predominantly syn nucleoside conformation in the enzyme-complexed state, the results, particularly the 8(1') and 8(3') NOE interactions, were not quantitatively consistent with any single bound conformation. Dissociation rate constants for the tubercidin-PNP complex were determined based on analysis of chemical shift and line width data as a function of enzyme/inhibitor ratio, Carr-Purcell-Meiboom-Gill measurements of the transverse relaxation rate as a function of pulse rate, and T1 rho experiments as a function of the spin-lock field strength. Dissociation rate constants of 2100 s-1 at 20 degrees C and 1400 s-1 at 10 degrees C were determined using the latter two methods. These rates are sufficiently high to justify the validity of the transferred NOE method for an enzyme as large as PNP. The possible significance of spin diffusion was investigated by the use of the deuterated analog [2'-2H]tubercidin, for which many of the intraligand spin diffusion pathways are eliminated, and by performing a series of transferred ROE experiments. A comparison of data obtained using transferred NOE and ROE measurements provides a basis for separating direct and indirect relaxation pathways. Both approaches indicated that the relatively significant 8(3') NOE interaction was not dominated by spin diffusion. Furthermore, analysis of chemical shift and transverse relaxation data for the tubercidin H-2 resonance gave inconsistent results for the chemical shift of the bound species and was inconsistent with the assumption of a single, bound conformation. These results were interpreted in terms of a 2:1 ratio of a syn, 3'-exo:anti, 3'-endo geometry for bound tubercidin. Ligand competition experiments using 9-deazainosine show that all of the tubercidin TRNOE effects are reversed by addition of the second nucleoside, suggesting that the TRNOE data for tubercidin arise due to interactions at the active sites of PNP rather than as a consequence of nonspecific binding to the enzyme.

Binding Sites↗

PubMed1994

Direct measurements of the dissociation-rate constant for inhibitor-enzyme complexes via the T1 rho and T2 (CPMG) methods.

The unimolecular dissociation rate constant, k-1, for the inhibitor-enzyme complex tubercidin-Escherichia coli purine nucleoside phosphorylase (PNPase) has been determined directly via two related 1H NMR methods for studying exchange-mediated transverse relaxation. One method involves measurements of the decay rate, 1/T1 rho, of spin-locked magnetization in the rotating frame as a function of the strength of the spin-locking field, omega SL. The second method involves measurements of the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo decay rate, 1/TCPMG2, as a function of the repetition rate, 1/tcp, of the refocusing pulses. Expressions describing the dependence of TCPMG2 as a function of 1/tcp and k-1 have been previously derived with sufficient generality to include the two-site inhibitor-enzyme exchange case. Existing expressions for T1 rho as a function of kex and omega SL, however, had to be reformulated to take into account differences between Tb2 and Tb1 for the bound form of the inhibitor as well as offset corrections important at low values of omega SL. A new expression for exchange-mediated T1 rho has been derived to take these factors into account and is shown to provide a more accurate description of observed T1 rho data than previous models. Numerical analysis of relaxation rates, measured independently by either the rotating-frame or the spin-echo method for the H1' and H2 protons of tubercidin at different inhibitor:enzyme ratios, yields comparable values for k-1 of 2400 (+/- 350) and 900 (+/- 80) s-1 at 20 and 10 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnetic Resonance Spectroscopy↗

PubMed1994

Cumulative effects model: a response to Williams (1994)

The cumulative effects (CE) model explains free-operant choice by the ratio of total numbers of responses and reinforcements, a probability-like variable. Williams (1994) argues that the model is vulnerable to experiments that disprove melioration, a local probability model. The authors note critical differences between the nonlocal CE model and local probability models that allow the CE model to handle some data with which they are incompatible. All models are simplifications of reality; hence, a model's failures are as revealing as its successes. Williams suggests that simple models may need to be abandoned in favor of a "representational" account. The authors point out that representations must be both acquired and acted on. Acquisition requires processing of responses and reinforcers; action requires decision rules. Models are simply testable suggestions for what these rules and processes might be.

Behavior Therapy↗

PubMed1994

Neurochemical and histopathologic alterations characteristic of Pick's disease in a non-demented individual.

In the course of investigating a large number of non-demented subjects, a 68 year old female dying of coronary artery disease was found to have Pick bodies in her grossly normal brain. Although only mild subcortical gliosis and no neuron loss were observed. Pick bodies were found throughout the brain and occasional balloon cells were noted. Pick bodies and numerous neurons were also ALZ-50 and Tau-1 immunoreactive. Retrospective studies indicated a lack of overt intellectual decline or depression in this individual. Frontal, temporal and occipital poles, amygdala, hypothalamus and nucleus basalis of Meynert (nbM) were analyzed for ChAT, AChE and MAO-A and -B enzymatic activities and for the binding of 5HT and imipramine. Cholinergic decreases were found only in subcortical structures. Serotonin binding decreases were widespread, excluding the nbM. Altered MAO-B activity was regionally variable, and no differences in MAO-A activity or imipramine binding were observed. Few differences in neurochemical alterations were observed in the current non-demented subject with abundant Pick bodies compared to previous studies of demented Pick's patients. This case strongly suggests that chemical dysfunction and neuropathological features of Pick's disease occur in advance of overt clinical manifestations of the disorder.

Acetylcholinesterase↗

PubMed1993

Engineering mouse P450coh to a novel corticosterone 15 alpha-hydroxylase and modeling steroid-binding orientation in the substrate pocket.

The F209L mutation alters specificity of P450coh from coumarin 7-hydroxylation to 15 alpha-hydroxylation of 11-deoxysteroids such as testosterone and 11-deoxycorticosterone. Neither the wild-type nor F209L exhibits activity toward 11 beta-hydroxysteroids including corticosterone. Mutation of Phe-209 to Asn, however, confers on mutant F209N a high corticosterone 15 alpha-hydroxylase activity. F209V also exhibits low corticosterone 15 alpha-hydroxylase activity; Km and Vmax are 10-fold higher and lower, respectively, than for F209N. The results are consistent with the hypothesis that direct interaction of Asn-209 with 11OH is responsible for high corticosterone 15 alpha-hydroxylase activity. To support this hypothesis, a possible steroid-binding orientation is modeled in the substrate pocket of P450cam. Our weighted homology and constrained alignments map residue 209 of P450coh to Met-184 and Met-191 of P450cam. Energy minimization of corticosterone in the substrate pocket results in the 11OH of the steroid directed toward Met-184 (7 A) and Met-191 (16 A), and in C15 located near the sixth axial position of the heme. The steroid-binding model suggests that the P450cam's substrate pocket may be conserved in the mammalian P450 and can accommodate a steroid molecule, and that residue 209 appears to be located at the critical site that determines the steroid-substrate specificity of a P450 depending on the type of group at the 11-position of steroid molecule.

Amino Acid Sequence↗

PubMed1993

Lipidized medulloblastoma in adults.

We describe the clinical, imaging, histologic, immunohistochemical, and ultrastructural features as well as the DNA ploidy analysis of two primitive neuroectodermal neoplasms demonstrating prominent cytoplasmic lipid accumulation originating in the posterior fossa of adults. These two tumors most likely exemplify a variant of medulloblastoma with heavily lipidized cells and support the postulated pluripotentiality of primitive neuroectodermal neoplasms. Recognition of this entity as a phenotypic variant of medulloblastoma will militate against diagnostic confusion with other entities that may contain cells with lipoblastic or adipocytic differentiation.

Cerebellar Neoplasms↗

PubMed1993

The process of recurrent choice.

Recurrent choice has been studied for many years. A static law, matching, has been established, but there is no consensus on the underlying dynamic process. The authors distinguish between dynamic models in which the model state is identified with directly measurable behavioral properties (performance models) and models in which the relation between behavior and state is indirect (state models). Most popular dynamic choice models are local, performance models. The authors show that behavior in different types of discrimination-reversal experiments and in extinction is not explained by 2 versions of a popular local model and that the nonlocal cumulative-effects model is consistent with matching and that it can duplicate the major properties of recurrent choice in a set of discrimination-reversal experiments. The model can also duplicate results from several other experiments on extinction after complex discrimination training.

Animals↗

PubMed1993

Spectroscopic studies of cutaneous photosensitizing agents. XVIII. Indomethacin.

The photochemistry, photophysics, and photosensitization (Type I and II) of indomethacin (IN) (N-[p-chlorobenzoyl]-5-methoxy-2-methylindole-3-acetic acid) has been studied in a variety of solvents using NMR, high performance liquid chromatography-mass spectroscopy, transient spectroscopy, electron paramagnetic resonance in conjunction with the spin trapping technique, and the direct detection of singlet molecular oxygen (1O2) luminescence. Photodecomposition of IN (lambda ex > 330 nm) in degassed or air-saturated benzene proceeds rapidly to yield a major (2; N-[p-chlorobenzyl]-5-methoxy-2-methyl-3-methylene-indoline) and a minor (3; N-[p-chlorobenzoyl]-5-methoxy-2,3-dimethyl-indole) decarboxylated product and a minor indoline (5; 1-en-5-methoxy-2-methyl-3- methylene-indoline), which is formed by loss of the p-chlorobenzoyl moiety. In air-saturated solvents two minor oxidized products 4 (N-[p-chlorobenzoyl]-5-methoxy-2-methyl-indole-3-aldehyde) and 6 (5-methoxy-2-methyl-indole-3-aldehyde) are also formed. When photolysis was carried out in 18O2-saturated benzene, the oxidized products 4 and 6 contained 18O, indicating that oxidation was mediated by dissolved oxygen in the solvent. In more polar solvents such as acetonitrile or ethanol, photodecomposition is extremely slow and inefficient. Phosphorescence of IN at 77 K shows strong solvent dependence and its emission is greatly reduced as polarity of solvent is increased. Flash excitation of In in degassed ethanol or acetonitrile products no transients. A weak transient is observed at 375 nm in degassed benzene, which is not quenched by oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)

Free Radicals↗

PubMed1993

Amino acid loss during volume regulatory decrease in cultured chick heart cells.

Mechanisms of volume regulation in hyposomotically treated cultured chick heart cell preparations were studied using optical, biochemical, and nuclear magnetic resonance methods. This approach afforded the resolution of time-dependent responses that might ordinarily be obscured by the complex morphology of intact cardiac muscle preparations. In hyposmotic solutions, cells swelled to a peak volume within 3 min and slowly regulated toward original volume (regulatory volume decrease, RVD). Upon return of the cells to isosmotic solution following hyposmotic treatment, the cells shrank to a steady-state volume that was substantially less than the initial volume in control solution. A vigorous RVD could also be elicited by hyposmotic swelling under Cl(-)-free conditions. Measurement of both inorganic cation loss via atomic absorption spectroscopy and organic solute loss via 1H-nuclear magnetic resonance and high-pressure liquid chromatographic techniques revealed that the RVD observed following exposure to hyposomotic solutions was mediated in part by a substantial loss of taurine, glutamate, aspartate, and glycine as well as loss of inorganic ions (Na+,K+). The hyposmotically activated transport of amino acids was also associated with the production of glutamate and aspartate. The volume regulatory release and production of amino acids have significant implications for the metabolic and functional integrity of cardiac cells.

Amino Acids↗

PubMed1993

Optic nerve ganglioglioma. Case report.

Gangliogliomas of the optic nerve are extremely rare. The case is reported of a 38-year-old man who presented with a visual field deficit and was discovered to have an optic nerve ganglioglioma. The possible embryological origins of this neoplasm, its histological and immunohistochemical features, and its appearance on magnetic resonance imaging are examined. The prognoses of optic nerve glioma and of gangliogliomas occurring elsewhere in the nervous system are compared.

Adult↗

PubMed1993

Nuclear magnetic resonance identification of the taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen-24-oic acid (tauro-delta 22-beta-muricholate) in the serum of female rats treated with alpha-naphthylisothiocyanate.

Recently developed 1H nuclear magnetic resonance methods, including 2-dimensional, 1H-detected-[13C] shift correlation spectroscopy and 1-dimensional HOHAHA spectroscopy at 500 MHz have been used to identify the major bile acid in the serum of rats treated with alpha-naphthylisothiocyanate as the taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen- 24-oic acid (tauro-delta 22-beta-muricholate), a derivative of beta-muricholate having an unsaturated bond in the acyclic side chain. Complete stereospecific assignments of the 1H and protonated 13C spectra of the title compound and beta-muricholate are reported. The assignments were based entirely on 1H-1H and 1H-13C scalar connectivities and were made using approximately 0.5-2.0 mg of material. It is suggested that these new methods will be of general value for identifying the structure and assigning the spectra of other scarce steroid-like molecules.

1-Naphthylisothiocyanate↗

PubMed1993

Taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen-24-oic acid (tauro-delta 22-beta-muricholate): the major bile acid in the serum of female rats treated with alpha-naphthylisothiocyanate and its secretion by liver slices.

The taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen-24-oic acid (tauro-delta 22-beta-muricholate) has been identified in the serum of female rats treated with alpha-naphthylisothiocyanate. Using a high performance liquid chromatographic/enzymatic method for measurement of bile acids, tauro-delta 22-beta-muricholate was the predominant bile acid in the serum of female Fischer 344 rats treated for 3 days with alpha-naphthylisothiocyanate. Other significant changes in concentrations of serum bile acids included increases in tauro-alpha-muricholate, tauro-beta-muricholate, taurocholate, taurochenodeoxycholate, and several unknown bile acids. The formation of tauro-delta 22-beta-muricholate was examined in vitro using liver slices from control rats and rats treated with alpha-naphthylisothiocyanate. Slices were incubated for 7 h in William's E medium containing no bile acids or 25 mumol/l of one of the following: beta-muricholate, tauro-beta-muricholate, or cholate. Tauro-delta 22-beta-muricholate was secreted by slices from control and treated rats and the rate was increased significantly by the addition of beta-muricholate (2.9-5.6-fold) but not tauro-beta-muricholate or cholate to the medium. Tauro-delta-beta-muricholate was formed by liver slices from endogenous precursors and from exogenous beta-muricholate. Pretreatment of rats with alpha-naphthylisothiocyanate did not alter total secretion rates but those of some important individual bile acids were affected. Because of the increased secretion of tauro-delta-beta-muricholate by liver slices with the addition of beta-muricholate to the medium, the liver may be the primary site of formation for this unsaturated bile acid.

1-Naphthylisothiocyanate↗

PubMed1992

Development of Lhermitte's sign after bone marrow transplantation.

The authors observed Lhermitte's sign in four patients after bone marrow transplantation (BMT) for hematologic malignancies. Three patients had acute myelogenous leukemia (AML), and one had chronic myelogenous leukemia. Before BMT, the patients with AML received daunorubicin, cytosine arabinoside and etoposide, whereas the patient with chronic myelogenous leukemia received hydroxyurea. One patient with AML received MY-9 antibody-depleted autologous BMT. The other patients received human lymphocyte antigen-identical, allogeneic BMT. Preparative therapy for BMT was cytosine arabinoside, cyclophosphamide, and total body exposure to radiation for two patients, and busulfan, cyclophosphamide, and no exposure to radiation in two other patients. Lhermitte's sign appeared 4 to 8 months after BMT and resolved spontaneously after 2 to 5 months. Neurologic sequelae had developed in none of the patients 16 to 34 months after BMT. No unifying etiologic factor could be identified in these patients. The development of Lhermitte's sign after BMT appears to be a benign, self-limited phenomenon that requires no specific treatment.

Adult↗

PubMed1992

Perivascular siderophages in skeletal muscle from a patient with diabetic neuropathy.

Hemosiderin deposition in skeletal muscle histiocytes is uncommon but has been occasionally noted in hemochromatosis, hemosiderosis and Waldenstrom's macroglobulinemia. The purpose of this report is to describe the light microscopic and ultrastructural characterization of this abnormality in a patient with diabetes mellitus. A 56-year-old diabetic male presented with paresthesias and intermittent diffuse lower extremity myalgias. Neurologic examination was remarkable only for diminished vibratory sense in the toes, diminished deep tendon reflexes, and ankle-level stocking distribution hypalgesia. There was no clinical evidence of hemochromatosis and laboratory studies ruled out Waldenstrom's macroglobulinemia. Muscle biopsy showed modest variability in myofiber diameter with a few scattered angular atrophic type II fibers. There were numerous collections of granular pigment-containing histiocytes in endomysial and perimysial perivascular areas and marked thickening of blood vessels walls. The histiocytic pigment was bright blue with the Prussian blue stain. No pigment was seen in the myofibers. Ultrastructural examination revealed numerous perivascular histiocytes filled with hemosiderin containing granules of variable size and density and marked thickening of capillary walls with striking reduplication of basement membranes. A modest number of subsarcolemmal paracrystalline mitochondrial inclusions were present. X-ray dispersion analysis of the histiocytic pigment material confirmed the presence of iron in the lysosomal granules.

Blood Vessels↗