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R J Russell

Publications and source records attributed to R J Russell.

At least 37 records · Page 2Linked to original sources
PubMed2001

Tiny TIM: a small, tetrameric, hyperthermostable triosephosphate isomerase.

Comparative structural studies on proteins derived from organisms with growth optima ranging from 15 to 100 degrees C are beginning to shed light on the mechanisms of protein thermoadaptation. One means of sustaining hyperthermostability is for proteins to exist in higher oligomeric forms than their mesophilic homologues. Triosephosphate isomerase (TIM) is one of the most studied enzymes, whose fold represents one of nature's most common protein architectures. Most TIMs are dimers of approximately 250 amino acid residues per monomer. Here, we report the 2.7 A resolution crystal structure of the extremely thermostable TIM from Pyrococcus woesei, a hyperthermophilic archaeon growing optimally at 100 degrees C, representing the first archaeal TIM structure. P. woesei TIM exists as a tetramer comprising monomers of only 228 amino acid residues. Structural comparisons with other less thermostable TIMs show that although the central beta-barrel is largely conserved, severe pruning of several helices and truncation of some loops give rise to a much more compact monomer in the small hyperthermophilic TIM. The classical TIM dimer formation is conserved in P. woesei TIM. The extreme thermostability of PwTIM appears to be achieved by the creation of a compact tetramer where two classical TIM dimers interact via an extensive hydrophobic interface. The tetramer is formed through largely hydrophobic interactions between some of the pruned helical regions. The equivalent helical regions in less thermostable dimeric TIMs represent regions of high average temperature factor. The PwTIM seems to have removed these regions of potential instability in the formation of the tetramer. This study of PwTIM provides further support for the role of higher oligomerisation states in extreme thermal stabilisation.

Amino Acid Sequence↗
PubMed2001

Did God create our universe? Theological reflections on the Big Bang, inflation, and quantum cosmologies.

The sciences and the humanities, including theology, form an epistemic hierarchy that ensures both constraint and irreducibility. At the same time, theological methodology is analogous to scientific methodology, though with several important differences. This model of interaction between science and theology can be seen illustrated in a consideration of the relation between contemporary cosmology (Big Bang cosmology, cosmic inflation, and quantum cosmology) and Christian systematic and natural theology. In light of developments in cosmology, the question of origins has become theologically less interesting than that of the cosmic evolution of a contingent universe.

Extraterrestrial Environment↗
PubMed2000

Kinetics and mechanism of the citrate synthase from the thermophilic archaeon Thermoplasma acidophilum.

The kinetics and mechanism of the citrate synthase from a moderate thermophile, Thermoplasma acidophilum (TpCS), are compared with those of the citrate synthase from a mesophile, pig heart (PCS). All discrete steps in the mechanistic sequence of PCS can be identified in TpCS. The catalytic strategies identified in PCS, destabilization of the oxaloacetate substrate carbonyl and stabilization of the reactive species, acetyl-CoA enolate, are present in TpCS. Conformational changes, which allow the enzyme to efficiently catalyze both condensation of acetyl-CoA thioester and subsequently hydrolysis of citryl-CoA thioester within the same active site, occur in both enzymes. However, significant differences exist between the two enzymes. PCS is a characteristically efficient enzyme: no internal step is clearly rate-limiting and the condensation step is readily reversible. TpCS is a less efficient catalyst. Over a broad temperature range, inadequate stabilization of the transition state for citryl-CoA hydrolysis renders this step nearly rate-limiting for the forward reaction of TpCS. Further, excessive stabilization of the citryl-CoA intermediate renders the condensation step nearly irreversible. Values of substrate and solvent deuterium isotope effects are consistent with the kinetic model. Near its temperature optimum (70 degrees C), there is a modest increase in the reversibility of the condensation step for TpCS, but reversibility still falls short of that shown by PCS at 37 degrees C. The root cause of the catalytic inefficiency of TpCS may lie in the lack of protein flexibility imposed by the requirement for thermal stability of the protein itself or its temperature-labile substrate, oxaloacetate.

Acetyl Coenzyme A↗
PubMed2000

Reconstructing the diversification of alpha-esterases: comparing the gene clusters of Drosophila buzzatii and D. melanogaster.

A cluster composed of 10 active alpha-esterase genes and a pseudogene is distributed over 60 kb in the Drosophila melanogaster genome. This paper describes the corresponding cluster in Drosophila buzzatii, whose lineage diverged from that of D. melanogaster when the subgenera Drosophila and Sophophora diverged about 50 Mya. With three exceptions we find that the composition of the cluster is conserved in the two lineages. The location of alpha E1 in D. melanogaster differs from that of its nearest relative in D. buzzatii, and alpha E4 has duplicated independently in the two lineages. The nature of these differences indicates that a mechanism exists whereby copies of genes can be placed in opposite orientation and nonadjacent positions within a gene cluster, although this does not seem to be a feature of earlier events in the cluster's evolution. The rates of amino acid change are not significantly different between orthologs, but the rates differ sevenfold among paralogs, indicating that very different selective forces are acting on the genes of the cluster. Mapping of sequence differences onto a model of the tertiary structure of the enzymes indicates that motifs contributing to substrate binding and catalysis have changed radically in the alphaE4s and suggest that this subgroup of alpha-esterases may be evolving into a substantially different functional niche.

Animals↗
PubMed2000

MCE activities and malathion resistances in field populations of the australian sheep blowfly (Lucilia cuprina).

Malathion resistance has been shown to be the result of a single point mutation in the LcalphaE7 gene in four independently isolated chromosomes of Lucilia cuprina. The resultant amino acid substitution specifies high malathion carboxylesterase (MCE) activity. We have assayed MCE activities and resistance to malathion in three sets of field-derived samples, two sets of isogenic lines and five mass populations, and show that resistance to malathion in these samples is associated with high MCE activity in both sets of isogenic lines and four of the five mass populations. Additional mechanisms contributing to MCE activity or malathion resistance may be present in one of the mass populations. A second point mutation in LcalphaE7 is responsible for conferring diazinon resistance by encoding an increased organophosphate (OP) hydrolase activity. We also assayed diazinon resistances from the same three samples and show that diazinon and malathion resistances were in complete disequilibrium, with two exceptions. One exception involves the mass population with additional resistance mechanism(s) and the other involves three isogenic lines that are resistant to both insecticides. The molecular data for these lines suggest that they carry a duplication of the LcalphaE7 gene.

Amino Acid Substitution↗
PubMed2000

The evolution of an alpha-esterase pseudogene inactivated in the Drosophila melanogaster lineage.

Previous analyses of the alpha-esterase cluster of Drosophila melanogaster revealed 10 active genes and the DmalphaE4a-Psi pseudogene. Here, we reconstruct the evolution of the pseudogene from the sequences of 12 alleles from widely scattered D. melanogaster populations and single alleles from Drosophila simulans and Drosophila yakuba. All of the DmalphaE4a-Psi alleles contain numerous inactivating mutations, suggesting that pseudogene alleles are fixed in natural populations. Several lines of evidence also suggest that DmalphaE4a is now evolving without selective constraint in the D. melanogaster lineage. There are three polymorphic indels which result in frameshifts; a key nucleotide of the intron splice acceptor is polymorphic; the neutral mutation parameter is the same for replacement and silent sites; one of the nonsilent polymorphisms results in a stop codon; only 1 of the 13 replacement polymorphisms is biochemically conservative; residues that are conserved among active esterases have different states in DmalphaE4a-Psi; and there are about half as many transitional polymorphisms as transversional ones. In contrast, the D. simulans and D. yakuba orthologs DsalphaE4a and DyalphaE4a do not have the inactivating mutations of DmalphaE4a-Psi and appear to be evolving under the purifying selection typical of protein- encoding genes. For instance, there have been more substitutions in the introns than in the exons, and more in silent sites than in replacement sites. Furthermore, most of the amino acid substitutions that have occurred between DyalphaE4a and DsalphaE4a are located in sites that typically vary among active alpha-esterases rather than those that are usually conserved. We argue that the original alphaE4a gene had a function which it has lost since the divergence of the D. melanogaster and D. simulans lineages.

Amino Acid Sequence↗
PubMed2000

Preliminary crystallographic studies of an extremely thermostable KDG aldolase from Sulfolobus solfataricus.

Crystals have been grown of 2-keto-3-deoxygluconate aldolase (KDG aldolase) from the hyperthermophilic archaeon Sulfolobus solfataricus that diffract to 2.2 A resolution. The enzyme catalyses the reversible aldol cleavage of 2-keto-3-dexoygluconate to pyruvate and glyceraldehyde, the third step of a modified non-phosphorylated Entner-Doudoroff pathway of glucose oxidation. S. solfataricus grows optimally at 353 K and the enzyme itself has a half-life of 2.5 h at 373 K. Knowledge of the crystal structure of KDG aldolase will further understanding of the basis of protein hyperthermostability and create a target for site-directed mutagenesis of active-site residues, with the aim of altering substrate specificity. Three crystal forms have been obtained: orthorhombic crystals of space group P2(1)2(1)2(1), which diffract to beyond 2.15 A, monoclinic crystals of space group C2, which diffract to 2.2 A, and cubic crystals of space group P4(2)32, which diffract to 3.4 A.

Aldehyde-Lyases↗
PubMed2000

Enrichment of an endosulfan-degrading mixed bacterial culture.

An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored the alpha isomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.

Bacteria↗
PubMed2000

The approach to a major incident.

Any major incident will test the organisational and medical skills of those involved. The best response can only be achieved with good planning and preparation, and the adoption of a structured approach to the scene. A clear chain of command should exist within all emergency services and must be followed. The Ambulance and Medical Incident officers take overall responsibility for the health service response. Management priorities--Command and Control, Safety, Communications, Assessment--must take precedence over support priorities--Triage, Treatment, Transport.

Disaster Planning↗
PubMed1999

Poly(ethylene glycol) hydrogel-encapsulated fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins.

A simple approach is described for preparing poly-(ethylene glycol) hydrogel materials with encapsulated seminapthofluorescein (SNAFL)-organophosphorus hydrolase enzyme conjugates. Direct determination of enzyme-catalyzed neurotoxin hydrolysis is provided by the self-referencing, pH-sensitive dye SNAFL-1, whose emission spectrum changes at lambda = 550 in response to pH. Using spectrofluorimetry and paraoxon as a model organophosphate, paraoxon concentrations as low as 8 x 10(-7) M could be readily detected. On the basis of the signal-to-noise ratio, a detection limit of 16 nM was determined. The materials demonstrated high stability against enzyme-denaturing, leaching, and photobleaching when stored under ambient conditions.

Aryldialkylphosphatase↗
PubMed1999

Electrostatic immobilization of glucose oxidase in a weak acid, polyelectrolyte hyperbranched ultrathin film on gold: fabrication, characterization, and enzymatic activity.

In this paper we show that hyperbranched polymers can be used as a host matrix for electrostatic entrapment of enzymes. Specifically, amine-functionalized glucose oxidase (GOx+) and horseradish peroxidase, as well as poly(amidoamine) dendrimer-modified horseradish peroxidase, reversibly sorb into polyanionic, hyperbranched poly(sodium acrylate) (PAA-) films that are on the order of a few hundred angstroms thick. The quantity of GOx+ entrapped within the PAA- films depends on the nature of film preparation but is typically on the order of 0.06 unit/cm2. The extent to which entrapped GOx+ retains its activity depends on the film history, but for PAA-/GOx+ composites not exposed to glucose and stored at 4 degrees C, the original activity is retained for up to 68 days and perhaps longer.

Acrylates↗
PubMed1999

A fluorescence-based glucose biosensor using concanavalin A and dextran encapsulated in a poly(ethylene glycol) hydrogel.

A fluorescence biosensor is described that is based on a photopolymerized poly(ethylene glycol) (PEG) hydrogel incorporating fluorescein isothiocyanate dextran (FITC-dextran) and tetramethylrhodamine isothiocyanate concanavalin A (TRITC-Con A) chemically conjugated into the hydrogel network using an alpha-acryloyl, omega-N-hydroxysuccinimidyl ester of PEG-propionic acid. In the absence of glucose, TRITC-Con A binds with FITC-dextran, and the FITC fluorescence is quenched through fluorescence resonance energy transfer. Competitive glucose binding to TRITC-Con A liberates FITC-dextran, resulting in increased FITC fluorescence proportional to the glucose concentration. In vitro experiments of hydrogel spheres in a solution of 0.1 M phosphate-buffered saline (pH 7.2) and glucose were conducted for multiple TRITC-Con A/FITC-dextran ratios. Hydrogels were characterized on the basis of the percent change in fluorescence intensity when FITC-dextran was liberated by increasing glucose concentrations. The optimum fluorescent change between 0 and 800 mg/dL was obtained with a TRITC-Con A/FITC-dextran mass ratio of 500:5 micrograms/mL PEG. Fluorescent response was linear up to 600 mg/dL. At higher concentrations, the response saturated due to the displacement of the majority of the FITC-dextran and to concentration quenching by free FITC-dextran. Dynamic fluorescent change upon glucose addition was approximately 10 min for a glucose concentration step change from 0 to 200 mg/dL.

Biosensing Techniques↗
PubMed1999

Carboxyl/cholinesterases: a case study of the evolution of a successful multigene family.

The evolution of organismal diversity among the Metazoa is dependent on the proliferation of genes and diversification of functions in multigene families. Here we analyse these processes for one highly successful family, the carboxyl/cholinesterases. One key to the expansion of the functional niche of this group of enzymes is associated with versatile substrate binding and catalytic machinery. Qualitatively new functions can be obtained by substitution of one or a very few amino acids. This crudely adapted new functionality is then refined rapidly by a pulse of change elsewhere in the molecule; in one case about 13% amino acid divergence occurred in 5-10 million years. Furthermore, we postulate that the versatility of the substrate binding motifs underpins the recruitment of several family members to additional noncatalytic signal transduction functions.

Animals↗
PubMed1999

The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly.

Organophosphate (OP) insecticide resistance in certain strains of Musca domestica is associated with reduction in the carboxylesterase activity of a particular esterase isozyme. This has been attributed to a 'mutant ali-esterase hypothesis', which invokes a structural mutation to an ali-esterase resulting in the loss of its carboxylesterase activity but acquisition of OP hydrolase activity. It has been shown that the mutation in Lucilia cuprina is a Gly137-->Asp substitution in the active site of an esterase encoded by the Lc alpha E7 gene (Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997. A single amino acid substitution converts a carboxylesterase to an organophosphate hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94, 7464-7468). We now report the cloning and characterisation of the orthologous M. domestica Md alpha E7 gene, including the sequencing of cDNAs from the OP resistant Rutgers and OP susceptible sbo and WHO strains. The Md alpha E7 gene has the same intron structure as Lc alpha E7 and encodes a protein with 76% amino acid identity to Lc alpha E7. Comparisons between susceptible and resistance alleles show resistance in M. domestica is associated with the same Gly137-->Asp mutation as in L. cuprina. Bacterial expression of the Rutgers allele shows its product has OP hydrolase activity. The data indicate identical catalytic mechanisms have evolved in orthologous Md alpha E7 and Lc alpha E7 molecules to endow diazinon-type resistance on the two species of higher Diptera.

Amino Acid Sequence↗
PubMed1999

Temporal expression of inducible nitric oxide synthase in mouse and human placenta.

The aim of this study is to investigate the changes in expression and activity of inducible nitric oxide synthase (iNOS) in the developing murine embryo and mouse and human placenta. Using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and in-situ hybridization (ISH) we identified iNOS mRNA in mouse placenta at 9.5, 12, 14, 16, 18 and 20 days post coitum. Northern blot analysis demonstrated that the quantity of murine iNOS transcript was expressed at a stable level between days 12-20 although the level of calcium-independent NOS activity declined with advancing gestation. RT-PCR detected iNOS-specific mRNA in murine embryonic stem cells, but not in embryos at later stages (4-cell or blastocyst). ISH failed to show iNOS-specific mRNA in either murine placenta or the underlying myometrium on day 7, but did so in the trophoblast by day 9.5. Later in gestation, extensive labelling was observed in both spongiotrophoblast and trophoblast giant cells. iNOS mRNA was also detected both in immature human placentae (16-18 weeks) and at term, predominantly in syncytiotrophoblasts and placental artery smooth muscle. In conclusion, iNOS is constitutively expressed in mouse and human placenta at a time and in a location that suggests a role in placentation.

Animals↗
PubMed1999

Some implications of chaos theory for the genetic analysis of human development and variation.

Non-linear epigenetic processes are a potential underlying source of phenotypic differences in development. Simulation studies of twin pairs using simple non-linear development models characterised by chaotic or near-chaotic behavior are presented. The effect of chaotic processes on correlations is to lower them from their initial values, but high initial correlations are affected much less by chaotic and near-chaotic processes than intermediate correlations. Therefore, we would predict that traits affected by chaotic processes would have high MZ and low DZ twin correlations and this is reminiscent of certain traits such as EEG spectra. However the much more frequent observation of MZ correlations close to twice their DZ counterparts would suggest that the role of chaos in development is quite limited.

Computer Simulation↗