Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura

The enzymes N-methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida x aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.     

Gene-targeted mice lacking the Trex1 (DNase III) 3'-->5' DNA exonuclease develop inflammatory myocarditis

TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 3'-->5' exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3' mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of gene-targeted Trex1(-/-) mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3' termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1(-/-) mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.     

Ribonucleotide reductases: radical chemistry and inhibition at the active site

Ribonucleoside 5'-diphosphate reductases (RDPRs) have been studied for several decades. Increasingly sophisticated mechanisms have been proposed for the reduction of natural substrate ribonucleotides to their 2'-deoxy counterparts and for mechanism-based inactivation of RDPRs with 2'-substituted-ribonucleotides. We now discuss biomimetic reactions of model substrate and inhibitor analogues, which clarify three aspects of previously proposed mechanisms postulated to occur at the active site of RDPRs.     

Cell cycle- and age-dependent activation of Sod1p drives the formation of stress resistant cell subpopulations within clonal yeast cultures

Phenotypic heterogeneity describes non-genetic variation that exists between individual cells within isogenic populations. The basis for such heterogeneity is not well understood, but it is evident in a wide range of cellular functions and phenotypes and may be fundamental to the fitness of microorganisms. Here we use a suite of novel assays applied to yeast, to provide an explanation for the classic example of heterogeneous resistance to stress (copper). Cell cycle stage and replicative cell age, but not mitochondrial content, were found to be principal parameters underpinning differential Cu resistance: cell cycle-synchronized cells had relatively uniform Cu resistances, and replicative cell-age profiles differed markedly in sorted Cu-resistant and Cu-sensitive subpopulations. From a range of potential Cu-sensitive mutants, cup1Delta cells lacking Cu-metallothionein, and particularly sod1Delta cells lacking Cu, Zn-superoxide dismutase, exhibited diminished heterogeneity. Furthermore, age-dependent Cu resistance was largely abolished in cup1Delta and sod1Delta cells, whereas cell cycle-dependent Cu resistance was suppressed in sod1Delta cells. Sod1p activity oscillated approximately fivefold during the cell cycle, with peak activity coinciding with peak Cu-resistance. Thus, phenotypic heterogeneity in copper resistance is not stochastic but is driven by the progression of individual cells through the cell cycle and ageing, and is primarily dependent on only Sod1p, out of several gene products that can influence the averaged phenotype. We propose that such heterogeneity provides an important insurance mechanism for organisms; creating subpopulations that are pre-equipped for varied activities as needs may arise (e.g. when faced with stress), but without the permanent metabolic costs of constitutive expression.     

Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (¹⁵NH₄)₂SO₄ and K¹⁵NO₃. By using two-dimensional ¹⁵N-¹H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of ¹⁵N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional ¹⁵N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid γ-aminobutyric acid in the one-dimensional ¹⁵N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into γ-aminobutyric acid, N-acetylputrescine. The occurrence of ¹⁵N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen. Received: 29 March 1999 / Accepted: 15 July 1999     

A Simulation Study Comparing Epidemic Dynamics on Exponential Random Graph and Edge-Triangle Configuration Type Contact Network Models

We compare two broad types of empirically grounded random network models in terms of their abilities to capture both network features and simulated Susceptible-Infected-Recovered (SIR) epidemic dynamics. The types of network models are exponential random graph models (ERGMs) and extensions of the configuration model. We use three kinds of empirical contact networks, chosen to provide both variety and realistic patterns of human contact: a highly clustered network, a bipartite network and a snowball sampled network of a “hidden population”. In the case of the snowball sampled network we present a novel method for fitting an edge-triangle model. In our results, ERGMs consistently capture clustering as well or better than configuration-type models, but the latter models better capture the node degree distribution. Despite the additional computational requirements to fit ERGMs to empirical networks, the use of ERGMs provides only a slight improvement in the ability of the models to recreate epidemic features of the empirical network in simulated SIR epidemics. Generally, SIR epidemic results from using configuration-type models fall between those from a random network model (i.e., an Erdős-Rényi model) and an ERGM. The addition of subgraphs of size four to edge-triangle type models does improve agreement with the empirical network for smaller densities in clustered networks. Additional subgraphs do not make a noticeable difference in our example, although we would expect the ability to model cliques to be helpful for contact networks exhibiting household structure.     

Impact of Community Based Peer Support in Type 2 Diabetes: A Cluster Randomised Controlled Trial of Individual and/or Group Approaches

Background

Diabetes peer support, where one person with diabetes helps guide and support others, has been proposed as a way to improve diabetes management. We have tested whether different diabetes peer support strategies can improve metabolic and/or psychological outcomes.

Methods

People with type 2 diabetes (n = 1,299) were invited to participate as either ‘peer’ or ‘peer support facilitator’ (PSF) in a 2x2 factorial randomised cluster controlled trial across rural communities (130 clusters) in England. Peer support was delivered over 8–12 months by trained PSFs, supported by monthly meetings with a diabetes educator. Primary end point was HbA1c. Secondary outcomes included quality of life, diabetes distress, blood pressure, waist, total cholesterol and weight. Outcome assessors and investigators were masked to arm allocation. Main factors were 1:1 or group intervention. Analysis was by intention-to-treat adjusting for baseline.

Results

The 4 arms were well matched (Group n = 330, 1:1(individual) n = 325, combined n = 322, control n = 322); 1035 (79•7%) completed the mid-point postal questionnaire and 1064 (81•9%) had a final HbA1c. A limitation was that although 92.6% PSFs and peers were in telephone contact, only 61.4% of intervention participants attended a face to face session. Mean baseline HbA1c was 57 mmol/mol (7•4%), with no significant change across arms. Follow up systolic blood pressure was 2•3mm Hg (0.6 to 4.0) lower among those allocated group peer-support and 3•0mm Hg (1.1 to 5.0) lower if the group support was attended at least once. There was no impact on other outcomes by intention to treat or significant differences between arms in self-reported adherence or medication.

Conclusions

Group diabetes peer support over 8–12 months was associated with a small improvement in blood pressure but no other significant outcomes. Long term benefits should be investigated.

Trial Registration

ISRCTN.com ISRCTN6696362166963621     

Synthesis and in vitro evaluation of 18F-labelled S-fluoroalkyl diarylguanidines: Novel high-affinity NMDA receptor antagonists for imaging with PET

Two S-[¹⁸F]fluoroalkylated diarylguanidines were synthesized and evaluated in vitro as potential tracers for imaging of N-methyl-d-aspartate receptors (NMDARs) with positron emission tomography (PET). [¹⁸F]1 and [¹⁸F]10 were synthesized by [¹⁸F]fluoroethylation and [¹⁸F]fluoromethylation of the thiol precursor 6, respectively. [¹⁸F]1 is a promising candidate NMDAR PET tracer, with low nanomolar affinity for the NMDA PCP-site, high selectivity and moderate lipophilicity.     

Investigation of the mechanism of nicotine demethylation in Nicotiana through 2H and 15N heavy isotope effects: implication of cytochrome P450 oxidase and hydroxyl ion transfer

Heavy-atom isotope effects for the N-demethylation of nicotine have been determined in vivo in static-phase biosynthetically incompetent plant cell cultures of Nicotiana species. A (2)H kinetic isotope effect of 0.587 and a (15)N kinetic isotope effect of 1.0028 were obtained. An identical (15)N kinetic isotope effect of 1.0032 was obtained for the nicotine analogue, N-methyl-2-phenylpyrrolidine. The magnitude of the (15)N heavy-atom isotope effect indicates that the fission of the CN bond is not rate limiting for demethylation. The theoretical calculation of heavy-atom isotope effects for a model of the reaction pathway based on cytochrome P450 best fits the measured kinetic isotope effect to the addition of hydroxyl ion to iminium to form N-hydroxymethyl, for which the computed (2)H- and (15)N kinetic isotope effects are 0.689 and 1.0081, respectively. This large inverse (2)H kinetic isotope effect is not compatible with the initial abstraction of the H from the methyl group playing a significant kinetic role in the overall kinetic limitation of the reaction pathway, since computed values for this step (4.54 and 0.9995, respectively) are inconsistent with the experimental data.