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Biomedical subjects

R A Scott

Publications and source records attributed to R A Scott.

At least 37 records · Page 2Linked to original sources
PubMed2000

Structural evidence for a common zinc binding domain in archaeal and eukaryal transcription factor IIB proteins.

X-ray absorption spectroscopy has been used to compare the metal coordination of the N-terminal zinc binding domain of eukaryal human transcription factor (TF) IIB to the previously reported structure of archaeal Pyrococcus furiosus (Pf) TFB. Full length and N-terminal fragments for both PfTFB and human TFIIB were cloned, expressed, and purified. The [C10H] variant of PfTFB was constructed to resemble the metal binding motif of higher eukaryal TFIIB proteins by mutating the second cysteine ligand to a histidine. All five proteins bind zinc in a 1:1 ratio. Zn X-ray absorption spectroscopy of human TFIIB and [C10H]PfTFB mutant are consistent with ZnS3(N,O) ligation, and further suggest that the N/O ligand is an imidazole.

Absorptiometry, Photon↗
PubMed2000

Ocular side-effects from systemic HPMPC (Cidofovir) for a non-ocular cytomegalovirus infection.

PURPOSE: To report a case of anterior uveitis and ocular hypotony during HPMPC treatment of a cytomegalovirus infection without ophthalmic involvement. METHODS: A 61-year-old bisexual white man with AIDS presented with moderate anterior uveitis and ocular hypotony after 8 weeks of intravenous HPMPC and oral probenecid co-treatment of a presumed recurrence of cytomegalovirus encephalitis. RESULTS: There was no evidence of an ocular cytomegalovirus infection. Cidofovir-related uveitis was diagnosed, and the HPMPC-probenecid co-treatment was switched to ganciclovir monotherapy. The ocular inflammation was successfully managed with topical steroids and mydriatics. CONCLUSIONS: This description of ophthalmic side effects from systemic HPMPC in the absence of ocular cytomegalovirus infection supports the hypothesis that anterior uveitis and ocular hypotony result from a direct interaction between the drug and the ciliary body.

AIDS-Related Opportunistic Infections↗
PubMed2000

Quantifying the risks of hypertension, age, sex and smoking in patients with abdominal aortic aneurysm.

BACKGROUND: The prevalence of abdominal aortic aneurysm (AAA) in a community-based sample of men and women aged 65-79 years was correlated with known risk factors. In addition, the effect of high blood pressure and the use of antihypertensive medication on growth of AAAs were studied. METHODS: Aortic diameter was assessed by ultrasonography and data on risk factors were collected by self-administered questionnaire for 5356 men and women as part of a randomized controlled trial. RESULTS: Current hypertension increased the risk of having an aortic aneurysm by 30-40 per cent while use of antihypertensive medication increased the risk by 70-80 per cent, adjusting for current blood pressure. There was no clear relationship between hypertension and growth rates of existing aneurysms in this study, although these results were largely from data on small aneurysms. Men were nearly six times more likely to develop an AAA than women; the risk increased by 40 per cent every 5 years after the age of 65 years. Smoking was an independent risk factor for AAA, with level of exposure being more significant than duration. CONCLUSION: Male sex, smoking and hypertension are strong risk factors for the development of AAA. In this study hypertension did not significantly increase the growth rate of existing aneurysms. Smoking remains the most important avoidable risk factor for AAA. The analyses presented here suggest that selection for screening, other than by age and sex, is not worthwhile.

Age Factors↗
PubMed2000

Nosocomial ringworm in a neonatal intensive care unit: a nurse and her cat.

An outbreak of nosocomial ringworm involved five infants in a neonatal intensive care unit. The index case was a nurse infected with Microsporum canis by her cat. After standard infection control measures were initiated, the outbreak was resolved successfully by an interdisciplinary professional collaboration of physician and veterinary dermatologists and infection control personnel.

Adult↗
PubMed2000

Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription.

Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N-terminal residues 2-59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and "rubredoxin" turns, three antiparallel beta-strands, and disordered loops. The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (PfTFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the beta-sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the beta-sheet.

Amino Acid Motifs↗
PubMed2000

Structural and kinetic characterization of an archaeal beta-class carbonic anhydrase.

The beta-class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) beta-class carbonic anhydrase indicate that the secondary structure of the beta-class enzymes is predominantly alpha-helical, unlike that of the alpha- or gamma-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters k(cat) and k(cat)/K(m) for CO(2) hydration are pH dependent. The steady-state parameter k(cat) is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, k(cat) is 2.1-fold higher in H(2)O than in D(2)O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter k(cat)/K(m) is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic beta-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic beta-class enzymes and firmly establish that the alpha-, beta-, and gamma-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.

Absorptiometry, Photon↗
PubMed2000

Mutations in catB, the gene encoding muconate cycloisomerase, activate transcription of the distal ben genes and contribute to a complex regulatory circuit in Acinetobacter sp. strain ADP1.

Mutants of the bacterium Acinetobacter sp. strain ADP1 were selected to grow on benzoate without the BenM transcriptional activator. In the wild type, BenM responds to benzoate and cis,cis-muconate to activate expression of the benABCDE operon, which is involved in benzoate catabolism. This operon encodes enzymes that convert benzoate to catechol, a compound subsequently degraded by cat gene-encoded enzymes. In this report, four spontaneous mutants were found to carry catB mutations that enabled BenM-independent growth on benzoate. catB encodes muconate cycloisomerase, an enzyme required for benzoate catabolism. Its substrate, cis,cis-muconate, is enzymatically produced from catechol by the catA-encoded catechol 1,2-dioxygenase. Muconate cycloisomerase was purified to homogeneity from the wild type and the catB mutants. Each purified enzyme was active, although there were differences in the catalytic properties of the wild type and variant muconate cycloisomerases. Strains with a chromosomal benA::lacZ transcriptional fusion were constructed and used to investigate how catB mutations affect growth on benzoate. All of the catB mutations increased cis,cis-muconate-activated ben gene expression in strains lacking BenM. A model is presented in which the catB mutations reduce muconate cycloisomerase activity during growth on benzoate, thereby increasing intracellular cis, cis-muconate concentrations. This, in turn, may allow CatM, an activator similar to BenM in sequence and function, to activate ben gene transcription. CatM normally responds to cis,cis-muconate to activate cat gene expression. Consistent with this model, muconate cylcoisomerase specific activities in cell extracts of benzoate-grown catB mutants were low relative to that of the wild type. Moreover, the catechol 1,2-dioxygenase activities of the mutants were elevated, which may result from CatM responding to the altered intracellular levels of cis,cis-muconate and increasing catA expression. Collectively, these results support the important role of metabolite concentrations in controlling benzoate degradation via a complex transcriptional regulatory circuit.

Acinetobacter↗
PubMed2000

Visual and anatomical results of surgery for long standing macular holes.

AIMS: To determine the visual and anatomical outcome of surgery for long standing idiopathic macular holes. METHODS: A retrospective review of 24 eyes of all 22 patients who underwent surgery for idiopathic full thickness macular holes (FTMH) symptomatic for between 1 and 3 years. Postoperative follow up was for 6 months. Preoperative and postoperative visual acuities were recorded as well as the presence of anatomical closure of the hole. RESULTS: The mean duration of symptoms was 18.21 (SD 5.42) months). Anatomical closure of the FTMH was achieved in 17 (70.8%) of the eyes at 6 months. The logMAR acuity of the group where closure was achieved improved by a mean of 0.31, equivalent to a change of Snellen acuity from 6/60 to 6/29. Where the hole remained open the acuity deteriorated by a mean logMAR of 0.11 lines, equivalent to a change of Snellen acuity from 6/60 to 5/60. Anatomical closure of the hole was associated with a significantly improved acuity over non-closure (p<0.001). The degree of visual improvement was independent of the preoperative visual acuity (Spearman correlation coefficient 0.03, p=0.888), though preoperative acuity was related to the final acuity (Spearman correlation coefficient 0.701, p<0.001). Over the study period, six patients required cataract surgery, one patient developed secondary glaucoma, and one a retinal detachment. CONCLUSIONS: Vitrectomy with intraocular gas tamponade and postoperative posturing is a well tolerated and effective intervention for long standing macular holes. Anatomical closure of the macular hole is associated with a significant improvement in visual acuity.

Adult↗
PubMed1999

Modulation of the redox potential of the [Fe(SCys)(4)] site in rubredoxin by the orientation of a peptide dipole.

Rubredoxins (Rds) may be separated into two classes based upon the correlation of their reduction potentials with the identity of residue 44; those with Ala44 have reduction potentials that are approximately 50 mV higher than those with Val44. The smaller side chain volume occupied by Ala44 relative to that occupied by Val44 has been proposed to explain the increase in the reduction potential, based upon changes in the Gly43-Ala44 peptide bond orientation and the distance to the [Fe(SCys)(4)] center in the Pyrococcus furiosus (Pf) Rd crystal structure compared to those of Gly43-Val44 in the Clostridium pasteurianum (Cp) Rd crystal structure. As an experimental test of this hypothesis, single-site Val44 <--> Ala44 exchange mutants, [V44A]Cp and [A44V]Pf Rds, have been cloned and expressed. Reduction potentials of these residue 44 variants and pertinent features of the X-ray crystal structure of [V44A]Cp Rd are reported. Relative to those of wild-type Cp and Pf Rds, the V44A mutation in Cp Rd results in an 86 mV increase in midpoint reduction potential and the [A44V] mutation in Pf Rd results in a 95 mV decrease in midpoint reduction potential, respectively. In the crystal structure of [V44A]Cp Rd, the peptide bond between residues 43 and 44 is approximately 0.3 A closer to the Fe center and the hydrogen bond distance between the residue 44 peptide nitrogen and the Cys42 gamma-sulfur decreases by 0.32 A compared to the analogous distances in the wild-type Cp Rd crystal structure. The results described herein support the prediction that the identity of residue 44 alone determines whether a Rd reduction potential of about -50 or 0 mV is observed.

Amino Acid Substitution↗
PubMed1999

Kinetic and spectroscopic characterization of the gamma-carbonic anhydrase from the methanoarchaeon Methanosarcina thermophila.

The zinc and cobalt forms of the prototypic gamma-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and (18)O exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn-Cam or Co-Cam for CO(2) hydration (k(cat)/K(m)) was severalfold greater than HCO(3-) dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn- and Co-Cam, the steady-state parameter k(cat) for CO(2) hydration was pH-dependent with a pK(a) of 6.5-6.8, whereas k(cat)/K(m) was dependent on two ionizations with pK(a) values of 6.7-6.9 and 8.2-8.4. The (18)O exchange assay also identified two ionizable groups in the pH profile of k(cat)/K(m) with apparent pK(a) values of 6.0 and 8.1. The steady-state parameter k(cat) (CO(2) hydration) is buffer-dependent in a saturable manner at pH 8. 2, and the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 x 10(7) M(-1) s(-1). At saturating buffer concentrations and pH 8.5, k(cat) is 2.6-fold higher in H(2)O than in D(2)O, suggesting that an intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k(cat)/K(m) is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes.

Apoenzymes↗
PubMed1999

Structural conservation of the isolated zinc site in archaeal zinc-containing ferredoxins as revealed by x-ray absorption spectroscopic analysis and its evolutionary implications.

The zfx gene encoding a zinc-containing ferredoxin from Thermoplasma acidophilum strain HO-62 was cloned and sequenced. It is located upstream of two genes encoding an archaeal homolog of nascent polypeptide-associated complex alpha subunit and a tRNA nucleotidyltransferase. This gene organization is not conserved in several euryarchaeoteal genomes. The multiple sequence alignments of the zfx gene product suggest significant sequence similarity of the ferredoxin core fold to that of a low potential 8Fe-containing dicluster ferredoxin without a zinc center. The tightly bound zinc site of zinc-containing ferredoxins from two phylogenetically distantly related Archaea, T. acidophilum HO-62 and Sulfolobus sp. strain 7, was further investigated by x-ray absorption spectroscopy. The zinc K-edge x-ray absorption spectra of both archaeal ferredoxins are strikingly similar, demonstrating that the same zinc site is found in T. acidophilum ferredoxin as in Sulfolobus sp. ferredoxin, which suggests the structural conservation of isolated zinc binding sites among archaeal zinc-containing ferredoxins. The sequence and spectroscopic data provide the common structural features of the archaeal zinc-containing ferredoxin family.

Amino Acid Sequence↗
PubMed1999

Cu XAS shows a change in the ligation of CuB upon reduction of cytochrome bo3 from Escherichia coli.

Copper X-ray absorption spectroscopy (XAS) has been used to examine the structures of the Cu(II) and Cu(I) forms of the cytochrome bo3 quinol oxidase from Escherichia coli. Cytochrome bo3 is a member of the superfamily of heme-copper respiratory oxidases. Of particular interest is the fact that these enzymes function as redox-linked proton pumps, resulting in the net translocation of one H+ per electron across the membrane. The molecular mechanism of how this pump operates and the manner by which it is linked to the oxygen chemistry at the active site of the enzyme are unknown. Several proposals have featured changes in the coordination of CuB during enzyme turnover that would result in sequential protonation or deprotonation events that are key to the functioning proton pump. This would imply lability of the ligands to CuB. In this work, the structure of the protein in the immediate vicinity of CuB, in both the fully oxidized and fully reduced forms of the enzyme, has been examined by Cu XAS, a technique that is particularly sensitive to changes in metal coordination. The results show that in the oxidized enzyme, CuB(II) is four-coordinate, consistent with three imidazoles and one hydroxyl (or water). Upon reduction of the enzyme, the coordination of CuB(I) is significantly altered, consistent with the loss of one of the histidine imidazole ligands in at least a substantial fraction of the population. These data add to the credibility that changes in the ligation of CuB might occur during catalytic turnover of the enzyme and, therefore, could, in principle, be part of the mechanism of proton pumping.

Copper↗