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Biomedical subjects

A I Scott

Publications and source records attributed to A I Scott.

At least 91 records · Page 5Linked to original sources
PubMed1992

Purification of an indole alkaloid biosynthetic enzyme, strictosidine synthase, from a recombinant strain of Escherichia coli.

The gene for the indole alkaloid biosynthetic enzyme, strictosidine synthase, of Catharanthus roseus has been cloned into an inducible Escherichia coli expression vector using an expression cassette polymerase chain reaction technique. Induction of the gene resulted in overexpression of the enzyme which accumulated mainly as insoluble inclusion bodies. Denaturation and refolding of the insoluble protein resulted in the ability to purify up to 6 mg of active enzyme from a single liter of cell culture. The recombinant enzyme has good activity (approximately 30 nkat/mg).

Alkaloids↗
PubMed1992

Isolation and identification of xochitloldione and isoxochitlolone from Cnidosculus urens.

Major components of MeOH extracts from the plant roots of Cnidosculus urens purified by cc and tlc and crystallization were lupeol acetate and the previously unreported compounds isoxochitlolone [1] and xochitloldione [2], which were identified through mass, ir, nmr, and uv spectroscopy and X-ray crystallography. In preliminary testing, isoxochitlolone has been found to be active against Escherichia coli and Staphylococcus aureus.

Anti-Bacterial Agents↗
PubMed1992

Is old-fashioned electroconvulsive therapy more efficacious? A randomised comparative study of bilateral brief-pulse and bilateral sine-wave treatments.

In-patients suffering from major depressive disorder (endogenous subtype) were randomly allocated to treatment by either traditional ECT with constant-voltage modified sine-wave stimuli (n = 17) or modern, constant-current brief-pulse ECT (n = 14). All treatments were bilateral and monitored by simultaneous recording by EEG. The severity of depressive illness was assessed the day before treatment, after three treatments, and seven days after the last treatment. The improvement and final depression rating scores, the likelihood of recovery, and the average number of treatments received were virtually identical in the two groups. We concluded that the policy of bilateral suprathreshold modern ECT monitored by EEG is as efficacious as traditional ECT.

Antidepressive Agents↗
PubMed1991

Diastereotopic covalent binding of the natural inhibitor leupeptin to trypsin: detection of two interconverting hemiacetals by solution and solid-state NMR spectroscopy.

The naturally occurring peptidyl protease inhibitor leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) has been prepared labeled with 13C at the argininal carbonyl. 13C chemical shift data for the trypsin-leupeptin inhibitor complex in the pH range 3.0-7.6 reveal the presence of two pH-dependent covalent complexes, suggestive of two interconverting diastereomers at the new asymmetric tetrahedral center created by covalent addition of Ser195 to either side of the 13C-enriched aldehyde of the inhibitor. At pH 7 two signals are observable at delta 98.8 and delta 97.2 (84:16 ratio), while at pH 3.0 the latter signal predominates. In the selective proton 13C-edited NOE spectrum of the major diastereomer at pH 7.4, a strong NOE is observed between the hemiacetal proton of the inhibitor and the C2 proton of His57 of the enzyme, thus defining the stereochemistry of the high pH complex to the S configuration in which the hemiacetal oxygen resides in the oxyanion hole. pH titration studies further indicate that the 13C chemical shift of the S diastereomer follows a titration curve with a pKa of 4.69, the magnitude of which is consistent with direct titration of the hemiacetal oxygen. Similar pH-dependent chemical shifts were obtained by using CPMAS 13C NMR, providing evidence for the existence of the same diastereomeric equilibrium in the solid state.

Binding Sites↗
PubMed1991

Bacteriochlorophyll c formation via the C5 pathway of 5-aminolevulinic acid synthesis in Chloroflexus aurantiacus.

Biosynthesis of 5-aminolevulinic acid (ALA) in Chloroflexus aurantiacus, a thermophilic bacterium forming bacteriochlorophyll c, is shown to proceed via the C5 pathway by demonstrating (1) the specific labeling of its chlorin ring with [1 - 13C]glutamate and (2) the enzyme activity to produce ALA from glutamate in a cell-free extract. From the phylogenetic distribution it is suggested that ALA synthetase distributed in some aerobic eubacteria could be monophyletic in origin.

Aminolevulinic Acid↗
PubMed1991

Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate. pH- and ligand-induced changes of the coenzyme observed by 13C NMR spectroscopy.

Apoenzyme samples of aspartate aminotransferase (AspAT) purified from the cytosolic fraction of pig heart were reconstituted with [4'-13C]pyridoxal 5'-phosphate (pyridoxal-P). The 13C NMR spectra of AspAT samples thus generated established the chemical shift of 165.3 ppm for C4' of the coenzyme bound as an internal aldimine with lysine 258 of the enzyme at pH 5. In the absence of ligands the chemical shift of C4' was shown to be pH dependent, shifting 5 ppm upfield to a constant value of 160.2 ppm above pH 8, the resulting pKa of 6.3 in agreement with spectrophotometric titrations. The addition of the competitive inhibitor succinate to the internal aldimine raises the pKa of the imine to 7.8, consistent with the theory of charge neutralization in the active site. In the presence of saturating concentrations of 2-methylaspartic acid the C4' signal of the coenzyme was shown to be invariant with pH and located at 162.7 ppm, midway between the observed chemical shifts of the protonated and unprotonated forms of the internal aldimine. The intermediate chemical shift of the external aldimine complex is thought to reflect the observation of an equilibrium mixture composed of roughly equal populations of the protonated ketoenamine and a dipolar anion species, corresponding to their respective spectral bands at 430 and 360-370 nm. Conversion to the pyridoxamine form was accomplished via reaction of the internal aldimine with L-cysteinesulfinate or by reduction with sodium borohydride, and the resulting C4' chemical shifts were identified by difference spectroscopy. Finally, the line widths of the C4' resonance under the various conditions were measured and qualitatively compared. The results are discussed in terms of the current mechanism and molecular models of the active site of AspAT.

Animals↗
PubMed1991

In-patient major depression: is rolipram as effective as amitriptyline?

The antidepressant efficacy and adverse-effects of rolipram (a dialkoxyphenyl-2-pyrrolidone) were compared to those of amitriptyline in the treatment of depressive illness requiring hospital admission in a double-blind study. Fifty patients meeting DSM-III criteria for Major Depression whose scores on the Hamilton Rating Scale for Depression (HRSD) remained above 17 after 5 to 7 days on placebo were randomly allocated to either treatment. The rate of recovery in those patients treated by amitriptyline was substantially greater than in those patients treated by rolipram. Twice as many patients dropped out of treatment by rolipram because of lack of efficacy or adverse-effects compared with patients treated by amitriptyline. Rolipram produced fewer adverse-effects attributable to cholinergic blockade, but more nausea. We conclude that amitriptyline is more effective than rolipram in the treatment of depressed hospital in-patients.

Adolescent↗
PubMed1991

Characterization of the process of 5-aminolevulinic acid formation from glutamate via the C5 pathway in Clostridium thermoaceticum.

1. In vitro formation of 5-aminolevulinic acid (ALA) from glutamate required two enzyme fractions, separable on Blue Sepharose affinity chromatography, and a tRNA fraction, which can be replaced by Escherichia coli tRNA(Glu) in the reconstituted assay. 2. Gabaculine was shown to inhibit ALA formation in the complete assay as well as in a defined system consisting of only glutamate-1-semialdehyde and the enzyme fraction not retained on Blue Sepharose. 3. The results indicate that the enzyme system supporting ALA formation in Clostridium thermoaceticum is very similar to the tRNA(Glu)-dependent C5 pathway in plant plastids.

Aminolevulinic Acid↗
PubMed1991

Improvement in depressive illness is not associated with altered release of neurophysins over a course of ECT.

The hypothesis that the release of vasopressin-associated neurophysin (hNpI) or oxytocin-associated neurophysin (hNpII) is modified by a course of electroconvulsive therapy (ECT) was tested by the measurement of serum neurophysins before and after the first and last ECTs given to 17 unipolar depressed patients. Neither basal nor ECT-induced neurophysin release changed between the first and last ECTs. Data from the present study were combined with data from a previous published study to provide a sample of 29 unipolar depressed patients. In this extended sample, the release of hNpII after the first ECT was significantly correlated with improvement in symptoms of depression over a course of ECT as measured by the Hamilton Rating Scale for Depression and the Montgomery-Asberg Depression Rating Scale.

Adult↗
PubMed1991

What happens to patients who frequently harm themselves? A retrospective one-year outcome study.

Between 1982 and 1987, 42 patients were selected who had been admitted three times in one week to the Regional Poisoning Treatment Centre in Edinburgh for the treatment of deliberate self-harm. Most (87%) of these admissions related to drug overdose. Most of the patients were young, unemployed, lacking a partner and from the low social classes. Three-quarters of the sample had at least one known conviction and more than a third were known to have served a prison sentence. There was only one patient in whom mental illness was thought to be relevant to the episode of self-harm, although five patients were mildly mentally handicapped. Case notes were reviewed to assess the outcome one year after the index admission. None of the patients had committed suicide, although 36 repeated self-harm during the follow-up year.

Adolescent↗
PubMed1991

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on 17 beta-estradiol-induced glucose metabolism in MCF-7 human breast cancer cells: 13C nuclear magnetic resonance spectroscopy studies.

The effects of 17 beta-estradiol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and their combination on the metabolism of [1-13C] glucose were determined in cell suspensions of wild-type MCF-7 human breast cancer cells, by 13C NMR spectroscopy. Preliminary studies showed that, during the 7-hr duration of the NMR experiment, the cells maintained their viability and their aryl hydrocarbon responsiveness. Lactate was the major glucose metabolite detected in these studies, and the rate of lactate formation in the untreated (control) and 17 beta-estradiol (10(-9) M)-treated cells was 60 and 86 fmol/cell/hr, respectively; this represented a 40% increase in lactate formation in the cells treated with 17 beta-estradiol; comparable results were observed for the percentage of glucose converted into lactate. In contrast, TCDD (10(-9) M) did not significantly alter the rate of glucose metabolism or lactate formation. Co-treatment of the cells with 17 beta-estradiol (10(-9) M) plus TCDD (10(-8) to 10(-10) M) showed that TCDD completely inhibited the 17 beta-estradiol-induced metabolism of [13C] glucose to lactate in MCF-7 cells. In contrast, 2,8-dichlorodibenzo-p-dioxin (10(-8) M), a weak aryl hydrocarbon receptor agonist, did not inhibit estrogen-induced glucose-to-lactate metabolism in MCF-7 cells. In addition, it was shown that TCDD caused a significant decrease in 17 beta-estradiol-induced lactate formation within 1 hr after treatment, whereas the induction of monooxygenase activity was not observed until 3 hr after exposure of the cells to TCDD. These data indicate that TCDD-induced 17 beta-estradiol metabolism is not related to the decrease in the rate of conversion of glucose to lactate. These results further define the antiestrogenic responses elicited by TCDD and show that 13C NMR spectroscopy provides a unique method for measuring, in real time, the effects of TCDD on specific metabolic pathways.

Breast Neoplasms↗
PubMed1990

Enzymatic synthesis of dihydrosirohydrochlorin (precorrin-2) and of a novel pyrrocorphin by uroporphyrinogen III methylase.

Uroporphyrinogen III methylase was purified from a recombinant hemB-strain of E. coli harbouring a plasmid containing the cysG gene. N-terminal analysis of this purified protein gave an amino acid sequence corresponding to that predicted from the genetic code. From the u.v./visible spectrum of the reaction catalysed by this SAM dependent methylase it was possible to observe the sequential appearance of the chromophores of a dipyrrocorphin and subsequently of a pyrrocorphin. Confirmation of this transformation was obtained from 13C-NMR studies when it was demonstrated, for the first time directly, that uroporphyrinogen is initially converted into dihydrosirohydrochlorin (precorrin-2) and then, by further methylation, into a novel trimethylpyrrocorphin.

Amino Acid Sequence↗
PubMed1990

The Escherichia coli cysG gene encodes S-adenosylmethionine-dependent uroporphyrinogen III methylase.

The Escherichia coli cysG gene was successfully subcloned and over-expressed to produce a 52 kDa protein that was purified to homogeneity. This protein was shown to catalyse the S-adenosylmethionine-dependent methylation of uroporphyrinogen III to give a product identified as sirohydrochlorin on the basis of its absorption spectra, incorporation of 14C label from S-adenosyl[Me-14C]methionine and mass and 1H-n.m.r. spectra of its octamethyl ester. Further confirmation of the structure was obtained from a 14C-n.m.r. spectrum of the methyl ester produced by incubation of the methylase with uroporphyrinogen III, derived from [4.6-13C2]porphobilinogen, and S-adenosyl[Me-13C]methionine.

Bacterial Proteins↗
PubMed1990

Tetrapyrrole assembly and modification into the ligands of biologically functional cofactors.

Data obtained using a combination of molecular biology and NMR spectroscopy has transformed our thinking about the evolution of the biochemical machinery required for the synthesis of the vital metallopigments: haem, chlorophyll, vitamin B12 and factor F430. One of the most recent advances is the discovery of a unique dipyrromethane cofactor that is bound covalently at the active site of porphobillinogen deaminase, the key enzyme of tetrapyrrole assembly. We will also discuss how the oxidation level and chromophoric arrangement of the uroporphinoid ring, rather than its substitution pattern, provides the necessary molecular recognition for some of the later enzymes, whose function is to decorate the template by C-methylation on the way to the biologically active cofactors.

Binding Sites↗