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Biomedical subjects

C J Schofield

Publications and source records attributed to C J Schofield.

At least 73 records · Page 4Linked to original sources
PubMed1998

Random amplification of polymorphic DNA as a tool for taxonomic studies of triatomine bugs (Hemiptera: Reduviidae).

Eleven of 27 decameric primers were found to be suitable for random amplification of polymorphic DNA (RAPD) from triatomine bugs on the basis that they produced discrete profiles and distinguished among Panstrongylus megistus (Burmeister), Rhodnius prolixus Stål, and Triatoma infestans (Klug). The legs, or single leg segments, of individual bugs were used as the source of DNA so that the taxonomic value of the bug was conserved. Within the scope of the specimens studied, RAPD profiles allowed assignment to species even when bugs were kept dry for up to 12 mo. Profiles for individuals within a species were not identical. RAPD profiles, with the specimens tested, distinguished among species of 3 pairs considered to be morphologically similar and closely related, namely, Rhodnius ecuadorensis Lent & León and Rhodnius pictipes Stål; Rhodnius nasutus Stål, and Rhodnius neglectus Lent; Rhodnius prolixus Stål and Rhodnius robustus Larrousse. RAPD data conformed with the perceived affinities among these species. RAPD polymorphisms were seen with T. infestans from 3 different localities, but none of the polymorphisms was confined to 1 source. RAPD provided a molecular basis to reassess taxonomic relationships within the Triatomine subfamily. The accurate distinction of triatomine species and of intraspecific bug populations may contribute to elimination of vector-borne Chagas disease from the Americas.

Animals↗
PubMed1997

Metal-catalyzed oxidation and mutagenesis studies on the iron(II) binding site of 1-aminocyclopropane-1-carboxylate oxidase.

The final step in the biosynthesis of the plant signaling molecule ethylene is catalyzed by 1-aminocyclopropane-1-carboxylate (ACC) oxidase, a member of the non-heme iron(II) dependent family of oxygenases and oxidases, which has a requirement for ascorbate as a co-substrate and carbon dioxide as an activator. ACC oxidase (tomato) has a particularly short half-life under catalytic conditions undergoing metal-catalyzed oxidative (MCO) fragmentation. Sequence comparisons of ACC oxidases with isopenicillin N synthase (IPNS) and members of the 2-oxoglutarate Fe(II) dependent dioxygenases show an aspartate and two of six ACC oxidase conserved histidine residues are completely conserved throughout this subfamily of Fe(II) dependent oxygenases/oxidases. Previous mutagenesis, spectroscopic, and crystallographic studies on IPNS indicate that the two completely conserved histidine and aspartate residues act as Fe(II) ligands. To investigate the role of the conserved aspartate and histidine residues in ACC oxidase (tomato fruit), they were substituted via site-directed mutagenesis. Modified ACC oxidases produced were H39Q, H56Q, H94Q, H177Q, H177D, H177E, D179E, D179N, H177D&D179E, H211Q, H234Q, H234D, and H234E. Among those histidine mutants replaced by glutamine, H39Q, H56Q, H94Q, and H211Q were catalytically active, indicating these histidines are not essential for catalysis. Mutant enzymes H177D, H177Q, D179N, H177D&D179E, H234Q, H234D, and H234E were catalytically inactive consistent with the assignment of H177, D179, and H234 as iron ligands. Replacement of H177 with glutamate or D179 with glutamate resulted in modified ACC oxidases which still effected the conversion of ACC to ethylene, albeit at a very low level of activity, which was stimulated by bicarbonate. The H177D (inactive), H177E (low activity), D179E (low activity), and H234Q (inactive) modified ACC oxidases all underwent MCO fragmentation, indicating that they can bind iron, dioxygen, ACC, and ascorbate. The results suggest that MCO cleavage results from active site-mediated reactions and imply that, while H177, D179, and H234 are all involved in metal ligation during catalysis, ligation to H234 is not required for fragmentation. It is possible that MCO fragmentation results from reaction of incorrectly folded or "primed" ACC oxidase.

Amino Acid Oxidoreductases↗
PubMed1997

The use of morphometrics in entomological surveillance of sylvatic foci of Triatoma infestans in Bolivia.

Jamach'uma (Cochabamba, Bolivia) is a small village surrounded by sylvatic foci of Triatoma infestans. Houses in the village were also infested with T infestans, and were sprayed in December 1992 as part of a Chagas disease vector control trial. Ten months later the houses were found to be again infested with a few fifth instar nymphs of T. infestans. These nymphs were compared by seven head measurements with 36 fifth instar nymphs collected from houses in Jamach'uma before treatment, and with two sets of nymphs originating from the surrounding sylvatic foci: eight specimens collected in 1992 and nine specimens collected in 1995. The results are discussed in relation to the possible mechanisms of the apparent reinfestation: recrudescence of a residual domestic population or reinvasion of the houses from surrounding sylvatic foci. Quantitative comparisons support the former hypothesis.

Animals↗
PubMed1997

L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine synthetase: thioesterification of valine is not obligatory for peptide bond formation.

L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase is probably the simplest known peptide synthetase in terms of the number of reactions catalyzed. In the "thiol-template" proposal for nonribosomal peptide synthesis, a key step is transfer of aminoacyl groups derived from the substrates to enzyme-bound thiols prior to peptide bond formation. No incorporation of 18O was seen in AMP isolated from the reaction mixture when di[18O]valine was incubated with relatively large amounts of active synthetase and MgATP. We therefore utilized di[18O]valine as a substrate for the biosynthesis of the diastereomeric dipeptides L-O-(methylserinyl)-L-valine and L-O-(methylserinyl)-D-valine [Shiau, C.-Y., Baldwin, J. E., Byford, M. F., Sobey, W. J., & Schofield, C. J. (1995) FEBS Lett. 358, 97-100]. In the L-O-(methylserinyl)-L-valine product, no significant loss of 18O was observed. However, in the L-O-(methylserinyl)-D-valine product, a significant loss of one or both 18O labels was observed. Thus, both peptide bond formation and the epimerization of the valine residue can both occur before formation of any thioester bond to the valine carboxylate in the biosynthesis of these dipeptides. The usual qualitative test for thioesterification of substrates to the synthetase, lability of enzyme-bound radiolabeled amino acid to performic acid, proved inconclusive in our hands. These results require a new mechanism for the enzymic synthesis of L-O-(methylserinyl)-L-valine and L-O-(methylserinyl)-D-valine and imply that a revised mechanism for ACV synthesis is also required.

2-Aminoadipic Acid↗
PubMed1997

Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation.

The biosynthesis of penicillin and cephalosporin antibiotics in microorganisms requires the formation of the bicyclic nucleus of penicillin. Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the reaction of a tripeptide, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV), and dioxygen to form isopenicillin N and two water molecules. Mechanistic studies suggest the reaction is initiated by ligation of the substrate thiolate to the iron centre, and proceeds through an enzyme-bound monocyclic intermediate. Here we report the crystal structure of IPNS complexed to ferrous iron and ACV, determined to 1.3 A resolution. Based on the structure, we propose a mechanism for penicillin formation that involves ligation of ACV to the iron centre, creating a vacant iron coordination site into which dioxygen can bind. Subsequently, iron-dioxygen and iron-oxo species remove the requisite hydrogens from ACV without the direct assistance of protein residues. The crystal structure of the complex with the dioxygen analogue, NO and ACV bound to the active-site iron supports this hypothesis.

Binding Sites↗
PubMed1997

Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene. It is a member of the ferrous iron dependent family of oxidases and dioxygenases and is unusual in that it displays a very short half-life under catalytic conditions, typically less than 20 min, and a requirement for CO2 as an activator. The rates of inactivation of purified, recombinant ACC oxidase from tomato under various combinations of substrates and cofactors were measured. Inactivation was relatively slow in the presence of buffer alone (t1/2 > 1 h), but fast in the presence of ferrous iron and ascorbate (t1/2 approximately 10 min). The rate of iron/ascorbate-mediated inactivation was increased by the addition of ACC, unaffected by the addition of CO2 at saturation (supplied as bicarbonate) but decreased by the addition of catalase or ACC + CO2 at saturation (supplied as bicarbonate). Iron/ascorbate-mediated inactivation was accompanied by partial proteolysis as observed by SDS-PAGE analysis. The fragmentation pattern was altered when ACC was also included, suggesting that ACC can bind to ACC oxidase in the absence of bicarbonate. N-terminal sequencing of fragments resulted in identification of an internal cleavage site which we propose is proximate to active-site bound iron. Thus, ACC oxidase inactivates via relatively slow partial unfolding of the catalytically active conformation, oxidative damage mediated via hydrogen peroxide which is catalase protectable and oxidative damage to the active site which results in partial proteolysis and is not catalase protectable.

Amino Acid Oxidoreductases↗
PubMed1997

Glutamine-330 is not essential for activity in isopenicillin N synthase from Aspergillus nidulans.

The non-heme ferrous dependent oxidase isopenicillin N synthase (IPNS) catalyses the biosynthesis of isopenicillin N from a tripeptide substrate. The crystal structure of Aspergillus nidulans IPNS complexed to manganese reveals a six co-ordinate metal ligated by two water molecules and four protein ligands: His-214, His-270, Asp-216 and Gln-330 (the penultimate C-terminal residue). Modification of Gln-330 to Ala or Leu, or deletion of 2 or 6 residues from the C-terminus resulted in lowering of specific activity; no activity was observed after deletion of 8 residues. The results demonstrate that metal ligation by Gln-330 is not required for catalytic activity.

Amino Acid Sequence↗
PubMed1997

Biosystematics of Old World Triatominae.

Morphometric analysis of Old World species of Linshcosteus and Triatoma showed the two genera to be well separated. However, although the analysis showed clear distinction between the five species of Linshcosteus, there was considerable overlap between the eight Old World species of Triatoma, including specimens of T. rubrofasciata from Old and New World populations. The results are interpreted as supporting the idea of a New World origin for the Old World species of Triatoma, with an independent Old World origin for Linshcosteus.

Africa↗
PubMed1997

Chagas disease vector control in Central America.

As the Southern Cone Initiative proceeds steadily towards eradication of Triatoma infestans, there is increasing interest in applying similar approaches to control Chagas disease vectors in Mexico, Central America and countries of the Andean Pact. Here, Chris Schofield and Jean-Pierre Dujardin discuss the technical feasibility of such an approach.

Journal Article↗
PubMed1997

Proteins of the penicillin biosynthesis pathway.

Two sequential steps are common to the biosynthesis of all penicillin-derived antibiotics: the reaction of three L-amino acids to give L-delta-(alpha-aminoadipoyl)-L-cysteinyl-D-valine, and the oxidation of this tripeptide to give isopenicillin N. Recent studies on the peptide synthetase and oxidase enzymes responsible for these steps have implications for the mechanisms and structures of related enzymes involved in a range of metabolic processes.

Binding Sites↗
PubMed1997

Structure of a specific acyl-enzyme complex formed between beta-casomorphin-7 and porcine pancreatic elastase.

Mass spectrometric screening reveals that an unmodified natural heptapeptide--human beta-casomorphin-7, an internal sequence of human beta-casein that possesses opioid-like activity--reacts with porcine pancreatic elastase to form an unusually stable acyl-enzyme complex at low pH. X-ray crystallographic analysis (to 1.9 A resolution) at pH 5 shows continuous electron density linking the C-terminal isoleucine of beta-casomorphin-7 to Ser 195 through an ester bond. The structure reveals a well defined water molecule (Wat 317), equidistant between the carbon of the ester carbonyl and N epsilon 2 of His 57. Deprotonation of Wat 317 will produce a hydroxide ion positioned to attack the ester carbonyl through the favoured Bürgi-Dunitz trajectory.

Animals↗
PubMed1997

Metric differences between silvatic and domestic Triatoma infestans (Heteroptera: Reduviidae) in Bolivia.

One hundred and fifty-seven specimens of Bolivian Triatoma infestans (Klug 1834), including 44 from the silvatic focus at Cochabamba (Bolivia), were compared using morphometric characters of the head capsule. From these specimens, 10 silvatic and 28 domestic adults were also compared using additional morphometric characters of the membranous part of the hemelytra. Nonparametric univariate comparisons between specimens from silvatic and domestic ecotopes indicated the importance of the postocular region as a possible diagnostic character for nymphs and adults from the different ecotopes, and they detected wing differences in males. Populations became more distinct entities when head or wing characters were considered jointly in canonical variate analysis. Regardless of whether size variation was considered, canonical variate analysis generally showed greater significance for wing than for head features. These morphological differences between silvatic and domestic bugs, particularly unrelated to size differences, are interpreted to indicate incipient separation between silvatic and domestic populations that had not been detected by previous isoenzyme analyses, and suggest a reinterpretation of the epidemiological significance of silvatic populations of T. infestans in Bolivia.

Analysis of Variance↗
PubMed1996

Anaerobic crystallisation of an isopenicillin N synthase.Fe(II).substrate complex demonstrated by X-ray studies.

Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulphate and its substrate, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (Aad-Cys-Val). Vital to the successful procedure was the maintenance of a rigorously anaerobic environment. Hanging-drop vapour-diffusion crystallisation experiments, using lithium sulphate as the precipitant produced three crystal forms. Form I crystals, with a plate habit, diffracted X-rays to at least 0.11-nm resolution at the European Synchrotron Radiation Facility and belong to the space group P2(1)2(1)2(1), with unit-cell dimensions a = 4.68, b = 7.15, c = 10.10 nm. Their asymmetric unit contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 38.5%. Form II crystals, with a hexagonal habit, diffract X-rays to at least 0.21 nm resolution at the European Synchrotron Radiation Facility and belong to the space group P3(1)21, with unit-cell dimensions a = 10.10, b = 10.10, c = 11.567 nm. Their asymmetric unit also contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 69.5%. Form III crystals, needles, do not show well-ordered diffraction. Although all three forms were initially produced in crystallisation experiments under identical conditions, appropriate micro and streak seeding allows selective crystallisation of form I or form II crystals. Extended X-ray-absorption fine-structure studies on a crystalline slurry of the form I crystals demonstrate the presence of an Fe-S(Aad-Cys-Val) bond length of 0.234 +/- 0.003 nm.

Anaerobiosis↗