Search PubMed⌕ Search

Biomedical subjects

R A Scott

Publications and source records attributed to R A Scott.

At least 127 records · Page 7Linked to original sources
PubMed1989

Pseudomonas stutzeri N2O reductase contains CuA-type sites.

N2O reductase (N2O----N2) is the terminal enzyme in the energy-conserving denitrification pathway of soil and marine denitrifying bacteria. The protein is composed of two identical subunits and contains eight copper ions per enzyme molecule. The magnetic circular dichroism spectrum of resting (oxidized) N2O reductase is strikingly similar to the magnetic circular dichroism spectrum of the CuA site in mammalian cytochrome c oxidase [Greenwood, C., Hull, B. C., Barber, D., Eglinton, D. G. & Thomson, A. J. (1983) Biochem. J. 215, 303-316] and is unlike the magnetic circular dichroism spectra of all other biological copper chromophores obtained to date. Sulfur (or chlorine) scatterers are required to fit the copper extended x-ray absorption fine structure data of both the oxidized and reduced forms of N2O reductase. Satisfactory fits require a Cu-N or Cu-O [denoted Cu-(N, O)] interaction at 2.0 A, a Cu-(S, Cl) interaction at 2.3 A and an additional Cu(S, Cl) interaction at approximately 2.6 A (oxidized) or approximately 2.7 A (reduced). Approximately eight sulfur ions (per eight copper ions) at approximately 2.3 A are required to fit the extended x-ray absorption fine structure data for both the oxidized and reduced N2O reductase. The 2.3-A Cu-(S, Cl) distance is nearly identical to that previously determined for the CuA site in cytochrome c oxidase. A 2.6-2.7 A Cu-(S, Cl) interaction is also present in resting and fully reduced cytochrome c oxidase. Comparison of the N2O reductase sequence, determined by translating the structural NosZ gene, with cytochrome c oxidase subunit II sequences from several sources indicates that a Gly-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Ser-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-His stretch is highly conserved. This sequence contains three of the probable ligands (two cysteines and one histidine) in a CuA-type site. Collectively these data establish that Pseudomonas stutzeri N2O reductase contains CuA-type sites.

Amino Acid Sequence↗
PubMed1989

Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D3.

Osteoblast-specific expression of the bone protein osteocalcin is controlled at the transcriptional level by the steroid hormone 1 alpha,25-dihydroxyvitamin D3. As this protein may represent a marker for bone activity in human disease, we examined the regulation of its expression at the molecular level by evaluating human osteocalcin gene promoter function. We describe regions within the promoter that contribute to basal expression of the gene in osteoblast-like cells in culture. Further, we define a 21-base-pair DNA element with the sequence 5'-GTGACTCACCGGGTGAACGGG-3', which acts in cis to mediate 1 alpha,25-dihydroxyvitamin D3 inducibility of the osteocalcin gene. This response element bears sequence similarity with other short DNA segments, particularly those for estrogen and thyroid hormone, which act together with their respective trans-acting receptors to modulate gene transcription.

Base Sequence↗
PubMed1989

X-ray absorption spectroscopic investigations of cytochrome c oxidase structure and function.

Although the low sensitivity of the XAS technique imposes difficulties upon the study of an enzyme that is often heterogeneous, significant progress has been made in elucidation of the structures of the functional metal sites of cytochrome c oxidase. Figure 8 summarizes interpretations of the resting-state enzyme based on the XAS results obtained over the past decade by the two main groups involved. Aside from several persisting minor differences in distances and precise ligand compositions of the metal sites, the biggest difference between the two interpretations involves the binuclear O2 interaction site, especially the nature of the bridging ligand and its precise attachments to Fea3 and CuB. However, the structural models presented by the two groups have been converging recently, and there is hope that the next few years will see a resolution of the remaining differences. Other XAS approaches (e.g. studies on oriented multilayers) and other techniques will doubtless contribute to this resolution.

Animals↗
PubMed1989

Functional domains of the human vitamin D3 receptor regulate osteocalcin gene expression.

The human vitamin D receptor (VDR) has been cloned recently. Two cDNAs comprising the full-length VDR were spliced, cloned into a mammalian expression vector, and transiently expressed in COS-1 cells. The protein product exhibited properties consistent with that observed for receptor in human cells. A series of 5'- and 3'-deletions of the full-length VDR cDNA was prepared and evaluated. Native DNA binding was localized to a peptide fragment (residues 1-114) whose most prominent feature is the cysteine rich region proven to represent the DNA binding domain in other steroid receptors. Steroid binding-competence required synthesis of a peptide that initiated C-terminal to the DNA-binding domain at residue 114 and which contained the remaining 313 residues. To determine the location of elements within the receptor necessary for transcription, an osteocalcin gene promoter-chloramphenicol acetyltransferase reporter gene was cotransfected together with wild type or mutant VDR cDNAs and the latter's effect on chloramphenicol acetyltransferase activity was assessed. Cotransfection of wild type receptor alone resulted in efficient transcription of the reporter plasmid. However, synthesis of a peptide containing the DNA binding domain as well as 76 residues carboxy terminal to this region exhibited some degree of activity, albeit constitutive. These results suggest that the functional domains of the VDR are similar to that of other steroid receptors and that these domains participate in the transcriptional regulation of the human osteocalcin gene.

Animals↗
PubMed1989

The effects of prolonged ketamine-xylazine intravenous infusion on arterial blood pH, blood gases, mean arterial blood pressure, heart and respiratory rates, rectal temperature and reflexes in the rabbit.

The prolonged and safe maintenance of general anesthesia in rabbits with commonly used injectable agents is difficult. Protracted, stable anesthesia with short recovery time has been described in humans using continuous intravenous infusion of ketamine with or without sedatives, muscle relaxants and paralytics. This study evaluated the anesthetic plane achieved and respiratory and cardiovascular effects produced with a ketamine-xylazine intravenous infusion in New Zealand White rabbits. Ten female rabbits were anesthetized with intramuscularly administered ketamine hydrochloride (35 mg/kg) and xylazine hydrochloride (5 mg/kg) after the preanesthetic, baseline measurements of arterial blood pO2, pCO2 and pH and heart and respiratory rates were recorded. The above parameters as well as mean arterial blood pressure, righting, palpebral, pedal, and jaw reflexes were monitored ten minutes after the intramuscularly administered dosage and throughout 4 hours of infusion. Results showed moderate hypotension (21.2% deviation from normal, p less than 0.008) and profound hypoxemia (45% deviation from baseline, p less than 0.001) 10 minutes after the intramuscularly administered induction dosage. Then, the 4 hour infusion of ketamine (1 mg/minute) and xylazine (0.1 mg/minute) was started. Hypotension progressed (49.1% deviation from normal, p less than 0.008), but hypoxemia and hypercarbemia gradually improved with no resultant change (p greater than 0.1) in arterial pH. There was no significant change (p greater than 0.1) in respiratory rate but varying qualities of respiration were observed. Both mean arterial pO2 and pCO2 values returned to baseline within 20 minutes after completion of infusion. Heart rate and rectal temperature remained stable during the trial. The righting reflex was abolished in all rabbits throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, Intravenous↗
PubMed1989

Team physician #7. A comparative study of functional bracing in the anterior cruciate deficient knee.

We evaluated the ability of three functional knee braces, (CTI, OTI, and TS7) to control anterolateral rotary instability of the knee. Fourteen subjects, none of elite athletic status, with arthroscopically proven anterior cruciate deficient knees were selected. The subjects evaluated each brace after one-month periods, and then underwent testing with physical examinations, KT-1000 arthrometry, and timed running events. All braces reduced subjective symptoms of knee instability. Different subjects preferred different braces. KT-1000 testing showed a reduction in anterior tibial displacement for all braces; however, this reduction did not increase as forces increased. A timed figure-of-eight running event did not show any functional advantage of bracing. Five subluxation events occurred in four subjects while braced. Functional braces appear to have a role in the anterior cruciate deficient knee, but only in conjunction with activity modification.

Adolescent↗
PubMed1988

Heterogeneity in an isolated membrane protein. Has the 'authentic cytochrome oxidase' been identified?

The criteria of homogeneity or native state of a protein are prone to become ambiguous when applied to membrane proteins, such as cytochrome-c oxidase, which are purified by extraction with detergents. Properties of the purified material depend on the detergent used and on details of the purification protocol followed with any single batch of a preparation. We present arguments to show that the evidence presently available in published form does not justify the designation [(1987) J. Biol. Chem. 262, 3160-3164] of one type of preparation as being closer to the native state than others.

Cell Membrane↗
PubMed1988

The copper sites of dopamine beta-hydroxylase: an X-ray absorption spectroscopic study.

X-ray absorption edge and extended X-ray absorption fine structure (EXAFS) spectra are reported for the Cu(I) and Cu(II) forms of bovine dopamine beta-hydroxylase (DBH; EC 1.14.17.1) and for the Cu(I) form of DBH bound either to tyramine substrate or to a multisubstrate inhibitor [Kruse, L. I., DeWolf, W. E., Jr., Chambers, P. A., & Goodhart, P. J. (1986) Biochemistry 25, 7271-7278]. A significant change in the structure of the copper sites occurs upon ascorbate-mediated reduction of Cu(II) DBH to the Cu(I) form. While the average Cu(II) site most likely consists of a square-planar array of four (N,O)-containing ligands at 1.98 A, the average Cu(I) site shows a reduction in (N,O) coordination number (from approximately 4 to approximately 2) and the addition of a S-containing ligand at 2.30 A. No change in the average Cu(I) ligand environment accompanies binding of tyramine substrate, whereas binding of a multisubstrate inhibitor, 1-(3,5-difluoro-4-hydroxybenzyl)-1H-imidazole-2(3H)-thione, causes an increase in the Cu-S coordination, consistent with inhibitor binding to the Cu(I) site through the S atom. Although excellent signal-to-noise ratio in the EXAFS spectra of ascorbate-reduced DBH facilitated analysis of outer-shell scattering for a Cu..Cu interaction, the presence of a binuclear site could not be proven or disproven due to interference from Cu...C scattering involving the carbons of imidazole ligands.

Adrenal Medulla↗
PubMed1988

Polyclonal lymphocytosis of T-cells associated with human T-cell leukemia virus I.

A patient with antibodies to human T-cell leukemia virus type I and the presence of integrated sequences of this virus in T-lymphocytes was investigated. In contrast to previous reports, the T-cell lymphocytosis was found to be polyclonal by analysis of human T-cell leukemia virus type I integration sites and T-cell antigen receptor rearrangements. Polyclonal T-cell infection by human T-cell leukemia virus type I may represent an infrequently observed stage of leukemogenesis.

Aged↗
PubMed1988

Structural heterogeneity and purification of protein-free F430 from the cytoplasm of Methanobacterium thermoautotrophicum.

F430 is the nickel containing tetrapyrrole cofactor of S-methyl coenzyme M methylreductase, the enzyme that catalyzes the final step of methane production by methanogenic bacteria: the reduction of S-methyl coenzyme M (H3CSCH2CH2SO3-) to methane and coenzyme M (HSCH2CH2SO3-). The protein-free F430 obtained from the cytosol of Methanobacterium thermoautotrophicum, strain delta H, exists predominantly in two isomeric forms that differ in relative stereochemical disposition of acid side chains at the 12 and 13 positions of the macrocycle periphery (Pfaltz, A., Livingston, D. A., Jaun, B., Diekert, G., Thauer, R. K., and Eschenmoser, A. (1985) Helv. Chim. Acta 68, 1338-1358). A simple one-step chromatographic procedure for the large-scale separation of these isomers is described. X-ray absorption spectroscopic studies show that F430 (i.e. the native isomer) is 6-coordinate with long nickel-ligand bonds (approximately 2.1 A), suggesting an approximately planar macrocycle. In contrast, the 12,13-diepimer exhibits a 4-coordinate, square-planar structure with short nickel-nitrogen bonds (approximately 1.9 A), suggesting a ruffled macrocycle. Previous reports, based on other x-ray absorption spectroscopic data, of static disorder in F430 Ni-N distances are shown to be incorrect due to sample heterogeneity. The optical spectrum of F430 (whether purified from the protein-free cytosol or extracted at high ionic strength from the holoenzyme) differs significantly from that of the 12,13-diepimer. The optical spectral differences are correlated with the alterations in coordination number and geometry of the central nickel ion in the two F430 isomers.

Chemical Phenomena↗
PubMed1987

Extended X-ray absorption fine structure of copper in CuA-depleted, p-(hydroxymercuri)benzoate-modified, and native cytochrome c oxidase.

Cytochrome c oxidase contains four redox-active metal centers: two heme irons, cytochromes a and a3, and two copper ions, CuA and CuB. Due to the paucity of spectroscopic signatures for both copper sites in cytochrome c oxidase, the ligands and structures for these sites have remained ambiguous. The specific depletion of CuA from the p-(hydroxymercuri)benzoate- (pHMB-) modified cytochrome c oxidase recently reported [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972] is herein described. Characterization of this enzyme shows that the structures of the remaining metal centers are essentially unperturbed by the CuA modification and depletion (P. M. Li, J. Gelles, and S. I. Chan, unpublished results). Copper extended X-ray absorption fine structure (EXAFS) measurements on the CuA-depleted cytochrome c oxidase reveal coordination of three (N, O) ligands and one (S, Cl) ligand at the CuB site. Comparison of EXAFS results obtained for the CuA-depleted, pHMB-modified, and "unmodified control" enzymes has allowed the deconvolution of the EXAFS in terms of the inner coordination spheres for CuA as well as CuB. On the basis of these data, it is found that the structure for the CuA site is consistent with two (N, O) ligands and two S ligands.

Copper↗
PubMed1986

Structural aspects of the copper sites in cytochrome c oxidase. An X-ray absorption spectroscopic investigation of the resting-state enzyme.

Copper K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the structural details of the coordination environment of the copper sites in eight resting-state samples of beef heart cytochrome c oxidase prepared by different methods. The unusual position and structure of the resting-state copper edge spectrum can be adequately explained by the presence of sulfur-containing ligands, with a significant amount of S----Cu(II) charge transfer (i.e., a covalent site). Quantitative curve-fitting analysis of the copper extended X-ray absorption fine structure (EXAFS) data indicates similar average first coordination spheres for all resting-state samples, regardless of preparation method. The average coordination sphere (per 2 coppers) mainly consists of 6 +/- 1 nitrogens or oxygens at an average Cu-(N,O) distance of 1.99 +/- 0.03 A and 2 +/- 1 sulfurs at an average Cu-S distance of 2.28 +/- 0.02 A. Quantitative curve-fitting analysis of the outer shell of the copper EXAFS indicates the presence of a Cu...Fe interaction at a distance of 3.00 +/- 0.03 A. Proposed structures of the two copper sites based on these and other spectroscopic results are presented, and differences between our results and those of other published copper XAS studies [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] are discussed.

Animals↗
PubMed1986

Analysis of the joint crack by simultaneous recording of sound and tension.

In order to elucidate the significance of the noise often associated with a chiropractic adjustment, the joint crack has been analyzed using a simultaneous sound and tension recording system. It was found that, during the crack, which is a consistent double-peak wave, a drop in tension occurred immediately after the first sound wave, indicating separation of the articular surfaces (adjustment). The significance of the crack as an integral part of a chiropractic adjustment, where separation of the articular surfaces is desired, is then discussed.

Chiropractic↗
PubMed1985

Phycomycosis of the vulva.

A necrotic ulcerative lesion of the vulva in a patient with diabetic ketoacidosis initially resembled necrotizing fasciitis. Debridement and histologic examination led to a diagnosis of phycomycosis. The condition responded to surgical debridement without antifungal therapy.

Adult↗