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Biomedical subjects

A I Scott

Publications and source records attributed to A I Scott.

At least 37 records · Page 2Linked to original sources
PubMed1999

Overexpression, purification, and characterization of the thermostable mevalonate kinase from Methanococcus jannaschii.

We report here the first overexpression and characterization of a thermostable mevalonate kinase from an archae, Methanococcus jannaschii, a strict anaerobe, which produces methane and grows at pressure of 200 atm and an optimum temperature near 85 degrees C. PCR-derived DNA fragments containing the structural gene for mevalonate kinase were cloned into an expression vector, pET28a, to form pETMVK. The mevalonate kinase was overexpressed from Escherichia coli pETMVK/BL21(DE3) (15-20% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside. The protein was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Talon metal-affinity resin column. The purified protein had a dimeric structure composed of identical subunits, and the M(r) of the enzyme determined by gel chromatography was 68K. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit M(r) was 36, 000. The pI for mevalonate kinase was 7.8. The Michaelis constant (K(m)) for (RS)-mevalonate was 68.5 microM and was 92 microM for ATP. The V(max) was 387 units mg(-1). The optimal temperature for mevalonate kinase activity was 70-75 degrees C.

Amino Acid Sequence↗
PubMed1999

Rates of electroconvulsive therapy use in Edinburgh (1992-1997).

BACKGROUND: The number of ECT treatments given each year is a topic of interest for psychiatrists, users and politicians, but there are major methodological problems in reported studies of rates of ECT use in the British Isles. The aim was to establish whether or not the use of ECT had fallen between 1992 and 1997. METHOD: A computerised database of ECT treatments in Edinburgh and relevant population data were used to calculate annual indices of ECT usage. These indices were calculated separately for the population aged 18-64 years and those 65 years or older. RESULTS: In the general adult population, the rate of ECT use fell progressively and significantly (p < 0.01) from 2.90 to 1.37 treatments per 1000 population. This fall was commensurate with the falls in other indices of ECT use. Among the elderly population, the rate of ECT use was three times that in the general adult population. There was no significant change, although the number of courses of treatment fell by 40% (p = 0.06). CONCLUSIONS: There continues to be a progressive fall in the use of ECT in general adult psychiatry. ECT is used substantially more often in the elderly. Reports of ECT use ought to include numbers of both treatments and patients treated. LIMITATIONS: There was no investigation of the possible causes of the fall in ECT use. The extent to which these findings can be generalised to the rest of the British Isles will require further study.

Adolescent↗
PubMed1999

Genetic engineering of Escherichia coli for the production of precorrin-3 in vivo and in vitro.

The construction of a new recombinant strain of Escherichia coli in which two vitamin B12 biosynthetic genes, cobA and cobI, from Pseudomonas denitrificans are simultaneously overexpressed has resulted in the in vivo synthesis and accumulation of Factor III, an isobacteriochlorin not normally synthesized in E. coli. A lysate of the new strain can take the place of two lysates normally required to provide uroporphyrinogen III methyltransferase (cobA) and precorrin-2 methyltransferase (cobI) in an anaerobic five-enzyme synthesis of the early B12 intermediate, precorrin-3 (the reduced form of Factor III) from delta-aminolevulinic acid.

Base Sequence↗
PubMed1999

Detection of nascent polyproline II helices in solution by NMR in synthetic insect kinin neuropeptide mimics containing the X-Pro-Pro-X motif.

The conformations of three synthetic peptide analogs containing the dPro-dPro-dXaa motif (dXaa = dThr, dGlu, dAsn) in aqueous solution were studied by a combination of NMR and molecular modeling simulations. The three compounds were identified from a random D-amino acid tripeptide library on the basis of their ability to either mimic or block the diuretic activity of neuropeptides of the insect kinin family. TOCSY and ROESY correlations, as well as abnormal secondary chemical shifts for protons on the D-proline residues were employed to obtain conformational ensembles consistent with the experimental NMR data for the three analogs using an in vacuo simulated annealing protocol. Similar secondary structures were found for the three molecules after refinement, in agreement with the similarities observed between their NMR spectra. Unrestrained molecular dynamics simulations with explicit water representation indicate that the structural motifs found in vacuo are stable in aqueous solution. The three analogs can be considered initiators of right-handed poly D-proline II helices, mirror images of the poly L-proline II left-handed helical motifs normally found in proline-rich proteins. The role of these secondary folds on binding of the analogs to the kinin receptors is discussed.

Insect Proteins↗
PubMed1999

Initial seizure threshold in the clinical practice of bilateral electroconvulsive therapy in Edinburgh, Scotland.

Initial seizure threshold was measured by empirical titration in 137 patients referred for bilateral electroconvulsive therapy (ECT) for the treatment of depressive illness. Treatment was given by an Ectron Series 5A ECT machine. The median and modal thresholds were 75 mC and the range was 50-200 mC. The only statistically significant relationship among initial threshold and age or gender was the correlation of age with threshold in men (rho = 0.46, p < 0.01). All patients (n = 22) younger than 30 years had an initial threshold < 100 mC, but age and gender did not predict threshold accurately for older men or women.

Adult↗
PubMed1998

Definition of the redox states of cobalt-precorrinoids: investigation of the substrate and redox specificity of CbiL from Salmonella typhimurium.

The enzyme CbiL from the facultative anaerobe Salmonella typhimurium exhibits a high degree of homology to CobI from the aerobe Pseudomonas denitrificans (29% identity; 51% conservation obtained by a Blastp search of the ncbi database). As CobI catalyzes the third methylation in the aerobic pathway to vitamin B12 it is proposed that CbiL catalyzes the analogous step in the anaerobic pathway. Potential metallo and metal-free substrates were characterized and their redox states defined by a combination of physicochemical techniques (MALDI-MS, NMR, UV/vis, IR, and EPR) and then used to investigate the function of CbiL. CbiL exhibited an absolute requirement for the presence of a metal ion (Co(II), Ni(II), or Zn(II)) within the tetrapyrrole substrate. CbiL had no preference for the redox state of its cobalt tetrapyrrole substrate, methylating both the reduced form, Co(II) 2, 7-dimethyl-dipyrrocorphin (Co(II)-precorrin-2), and the oxidized form, Co(III) 2,7-dimethyl-isobacterioclorin (Co(III)-factor-II). In contrast CbiL had a marked preference for the oxidized Ni(II) and Zn(II)-2,7-dimethyl-isobacteriochlorin (Ni(II) and Zn(II)-factor-II). Removal of the metal ion from a product of CbiL (Zn(II)-factor-III) allowed characterization by 13C NMR, identifying the tetrapyrrole as 2,7,20-trimethyl-isobacteriochlorin (factor-3), indicating that CbiL methylates at C20, the same site as that methylated by CobI. Competition experiments, utilizing isotopic labeling to distinguish otherwise identical mass substrates and products, revealed that oxidized Co(III) or Ni(II)-factor-II were equally good substrates, whereas Co(II)-precorrin-2 was much preferred over Ni(II)-precorrin-2. Excess Ni(II)-precorrin-2 did not decrease CbiL methylation of Co(II)-precorrin-2, implying that CbiL has a low affinity for Ni(II)-precorrin-2. These results are interpreted on the basis of tetrapyrrole ruffling occurring on the optimization of the metallo-N bond distances. The greater flexibility of the reduced precorrin-2 ring system allows greater deformation on accommodating the bound metal ion, the distortions imposed by bound Ni(II) or Zn(II) ions being larger than Co(II). The resulting distortions imposed on the precorrin ring could then decrease catalysis by causing a departure from the optimal substrate conformation required for CbiL. On oxidation of the Ni(II) or Zn(II)-precorrin-2, the increased stiffness of the ring could then constrain the metallo-factor-II conformation toward that of the usual substrate, allowing greater methylation by CbiL. In contrast to its counterpart CobI in the aerobic pathway of B12 biosynthesis, which methylates the metal-free precorrin-2, these studies show CbiL to be the first methylase unique to the anaerobic pathway, methylating a metallo-precorrin-2 substrate. Implications of CbiL specificity for the mechanism of the anaerobic B12 pathway are discussed.

Bacterial Proteins↗
PubMed1998

Overexpression, single-step purification, and site-directed mutagenetic analysis of casbene synthase.

Casbene synthase is a diterpene cyclase isolated from castor bean (Ricinus communis L), which catalyzes the cyclization of geranylgeranyl diphosphate to form the phytoalexin casbene. We here report the overexpression of casbene synthase in Escherichia coli in soluble form using a thioredoxin fusion system. The amplified DNA by PCR carried on pCS7 was inserted into the expression vector pET32b(+) to form pCAS.2. The resulting transformants of pCAS. 2/BL21(DE3) produced a thioredoxin casbene synthase fusion protein (20-30% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside at 20 degrees C. Recombinant casbene synthase was purified to homogeneity in a single step with a His-binding metal-affinity column. Casbene synthase has a conserved aspartate-rich region [amino acids 355-359 (DDTID)], one cysteine, and three histidines with several prenyl transferases and terpene cyclases. Seven mutants were constructed by site-directed mutagenesis. The importance of Asp 355 and Asp 356 for catalysis was established by an increase in Km as well as a reduction in kcat in the corresponding glutamate mutants. These results indicate that the first and the second aspartate are involved in catalysis, while the third aspartate and the conserved cysteine and histidine residues selected for mutagenesis appear not to be involved in catalysis.

Amino Acid Sequence↗
PubMed1998

Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway.

Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein. Taxadiene synthase cDNA was amplified by polymerase chain reaction and then subcloned into pET3d and pET32a(+) to form pET3dTX and pET32TX, respectively. The expressed taxadiene synthase from E. coli BL21(DE3)/pET3dTX was present completely as inclusion bodies. The transformant E. coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble protein) when induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C). The recombinant enzyme was purified by a single step with a His-binding metal affinity column. The maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(DE3)/pET32TX. The purified recombinant taxadiene synthase fusion protein was similar to native protein in steady-state kinetic parameters and mobility on sodium sulfate-polyacrylamide gel electrophoresis. The protein purified from E. coli BL21(DE3)/pET3dTX had the expected N-terminal (AQLSFNA) sequence.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Eudesmane derivatives from Iva frutescens.

Re-investigation of the aerial parts of Iva frutescens resulted in the isolation of three sesquiterpenes, two of which are new natural products, and their structures were established by spectral data.

Naphthalenes↗
PubMed1998

Synthesis, biological activity, and conformational studies of insect allatostatin neuropeptide analogues incorporating turn-promoting moieties.

Allatostatins are 6-18 amino acid peptides synthezed by insects to control production of juvenile hormones, which in turn regulate functions including metamorphosis and egg production. Four insect allatostatin neuropeptide analogues incorporating turn-promoting pseudopeptide moieties in the region responsible for biological activity were prepared by solid phase peptide synthetic methods. Bioassay indicated that activities approached those of the natural neuropeptides, and molecular models based on NMR data showed similar conformations and the presence of a beta-turn in the active core region for the four analogues. Differences in activity are believed to be due to differences in bulk and relative position of atoms in the unnatural portion of the analogues, and their differing degrees of conformational freedom. The studies support the feasibility of development of neuropeptide-based insect control agents resistant to peptidase deactivation.

Animals↗
PubMed1997

Conformational studies of paclitaxel analogs modified at the C-2' position in hydrophobic and hydrophilic solvent systems.

The conformations of two paclitaxel analogs modified at the C-2' position, 2'-deoxypaclitaxel and 2'-methoxypaclitaxel, were studied in hydrophobic and hydrophilic solvent systems by a combination of NMR spectroscopy, CD measurements, and molecular modeling. Both analogs have hydrophobic and hydrophilic conformations that resemble those of paclitaxel itself in the same media. Since the two have diminished biological activities in a number of bioactivity assays and the hydrogen-bonding capability of the 2'-hydroxyl group has been eliminated, we postulate that this group is involved in hydrogen bonding with tubulin and plays an important role in molecular recognition. The results of this study are in agreement with our earlier report on paclitaxel 2'-acetate, an analog in which the 2'-hydroxyl group hydrogen-bonding capacity has also been eliminated.

Animals↗
PubMed1997

The sterol carrier protein-2 fatty acid binding site: an NMR, circular dichroic, and fluorescence spectroscopic determination.

The interaction and orientation of fatty acids with recombinant human sterol carrier protein-2 (SCP-2) were examined by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence techniques. 13C-NMR spectroscopy of stearic acid and oleic acid as well as fluorescence spectroscopy of cis-parinaric acid demonstrated that SCP-2 bound naturally occurring fatty acids with near 1:1 stoichiometry. Several findings indicated that the fatty acid was oriented in the binding site with its methyl end buried in the protein interior and its carboxylate exposed at the surface: the chemical shift of bound [18-13C]-stearate; dicarboxylic/monocarboxylic acid cis-parinaric acid displacement; complete ionization of the carboxylate group of SCP-2 bound [1-13C]stearate at neutral pH; lack of electrostatic interactions between 13C-fatty acids with SCP-2 cationic residues: pH titratability of the SCP-2 bound [1-13C]stearate carboxylate group. SCP-2 did not undergo global structural changes upon ligand binding or pH decrease as indicated by the absence of significant changes in NMR and only small alterations in time resolved fluorescence parameters. However, SCP-2 did undergo secondary structural changes detected by CD in the pH range 5-6. While these changes in secondary structure did not alter the fatty acid:SCP-2 binding stoichiometry, the affinity for fatty acid was increased severalfold at lower pH. In summary, 13C-NMR, CD, and fluorescence spectroscopy provided a detailed understanding of the interaction of fatty acids with SCP-2 and further showed for the first time the orientation of the fatty acid within the binding site. The pH-induced changes in SCP-2 secondary structure and ligand binding activity may be important to the mechanism whereby this protein interacts with membrane surfaces to enhance lipid binding/transfer.

Binding Sites↗
PubMed1997

How corrinoids are synthesized without oxygen: nature's first pathway to vitamin B12.

BACKGROUND: During the biosynthesis of vitamin B12, the aerobic bacterium Pseudomonas denitrificans uses two enzymes, CobG and CobJ, to convert precorrin-3 to the ring-contracted intermediate, precorrin-4. CobG is a monooxygenase that adds a hydroxyl group, derived from molecular oxygen, to C-20, whereas CobJ is bifunctional, inserting a methyl group at C-17 of the macrocycle and catalyzing ring contraction. Molecular oxygen is not available to vitamin B12-producing anaerobic bacteria and members of the ancient Archaea, so the question arises of how these microbes accomplish the key ring-contraction process. RESULTS: Cloning and overexpression of Salmonella typhimurium genes has led to the discovery that a single enzyme, CbiH, is responsible for ring contraction during anaerobic biosynthesis of vitamin B12. The process occurs when CbiH is incubated with precorrin-3, but only in the presence of cobalt. CbiH functions as a C-17 methyltransferase and mediates ring contraction and lactonization to yield the intermediate, cobalt-precorrin-4, isolated as cobalt-factor IV. 13C labeling studies have proved that cobalt-precorrin-4 is incorporated into cobyrinic acid, thereby confirming that cobalt-precorrin-4 is an intermediate in vitamin B12 biosynthesis. CONCLUSIONS: Two distinct mechanisms exist in nature for the ring contraction of porphyrinoids to corrinoids-an ancient anaerobic pathway that requires cobalt complexation prior to nonoxidative rearrangement, and a more recent aerobic route in which molecular oxygen serves as the cofactor. The present results offer a rationale for the main differences between aerobic and anaerobic biosynthesis of vitamin B12. Thus, in anaerobes there is exchange of oxygen at the C-27 acetate site, extrusion of acetaldehyde and early insertion of cobalt, whereas the aerobes show no exchange of oxygen at C-27, extrude acetic acid and insert cobalt very late in the biosynthetic pathway, after ring contraction has occurred. These parallel routes to vitamin B12 have now been clearly distinguished by their differing mechanisms for ring contraction.

Anaerobiosis↗
PubMed1996

Biosynthesis of vitamin B12: factor IV, a new intermediate in the anaerobic pathway.

The structure of a novel tetradehydrocorrin, factor IV, isolated from Propionibacterium shermanii has been established by multidimensional NMR spectroscopy. Incorporation of radiolabeled factor IV into cobyrinic acid established the biointermediacy of this cobalt complex, whose structure has implications for the mechanisms of the anaerobic pathway to B12.

Anaerobiosis↗