Rational engineering of the TOL meta-cleavage pathway

The meta-cleavage pathway of Pseudomonas putida mt-2 was simulated using a biochemical systems simulation developed by Regan (1996). A non-competitive inhibition term for catechol-2,3-dioxygenase (C23O) by 2-OH-pent-2,4-dienoate (Ki = 150 μM) was incorporated into the model. The simulation predicted steady state accumulation levels in the μM range for metabolites pre-meta-cleavage, and in the mM range for metabolites post-meta-cleavage. The logarithmic gains L[V-i, Xj] and L[X-i, Xj] clearly indicated that the pathway was most sensitive to the concentration of the starting substrate, benzoate, and the first enzyme of the pathway, toluate-1, 2-dioxygenase (TO). The simulation was validated experimentally; it was found that the amplification of TO increased the steady state flux from 0.024 to 0.091 (mmol/g cell dwt)/h. This resulted in an increased accumulation of a number of the pathway metabolites (intra- and extracellularly), especially cis-diol, 4-OH-2-oxovalerate, and 4-oxalocrotonate. Metabolic control analysis indicated that C23O was, in fact, the major controling enzymic step of the pathway with a scaled control coefficient of 0.83. The amplification of TO resulted in a shift of some of the control away from C23O. Catechol-2,3-dioxygenase, however, remained as the major controling element of the pathway. Copyright 1998 John Wiley & Sons, Inc.     

Higher-level snake phylogeny inferred from mitochondrial DNA sequences of 12S rRNA and 16S rRNA genes

Portions of two mitochondrial genes (12S and 16S ribosomal RNA) were sequenced to determine the phylogenetic relationships among the major clades of snakes. Thirty-six species, representing nearly all extant families, were examined and compared with sequences of a tuatara and three families of lizards. Snakes were found to constitute a monophyletic group (confidence probability [CP] = 96%), with the scolecophidians (blind snakes) as the most basal lineages (CP = 99%). This finding supports the hypothesis that snakes underwent a subterranean period early in their evolution. Caenophidians (advanced snakes), excluding Acrochordus, were found to be monophyletic (CP = 99%). Among the caenophidians, viperids were monophyletic (CP = 98%) and formed the sister group to the elapids plus colubrids (CP = 94%). Within the viperids, two monophyletic groups were identified: true vipers (CP = 98%) and pit vipers plus Azemiops (CP = 99%). The elapids plus Atractaspis formed a monophyletic clade (CP = 99%). Within the paraphyletic Colubridae, the largely Holarctic Colubrinae was found to be a monophyletic assemblage (CP = 98%), and the Xenodontinae was found to be polyphyletic (CP = 91%). Monophyly of the henophidians (primitive snakes) was neither supported nor rejected because of the weak resolution of relationships among those taxa, except for the clustering of Calabaria with a uropeltid, Rhinophis (CP = 94%).      

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Molecular dynamics of synthetic leucine-serine ion channels in a phospholipid membrane

Molecular dynamics calculations were carried out on models of two synthetic leucine-serine ion channels: a tetrameric bundle with sequence (LSLLLSL)(3)NH(2) and a hexameric bundle with sequence (LSSLLSL)(3)NH(2). Each protein bundle is inserted in a palmitoyloleoylphosphatidylcholine bilayer membrane and solvated by simple point charge water molecules inside the pore and at both mouths. Both systems appear to be stable in the absence of an electric field during the 4 ns of molecular dynamics simulation. The water motion in the narrow pore of the four-helix bundle is highly restricted and may provide suitable conditions for proton transfer via a water wire mechanism. In the wider hexameric pore, the water diffuses much more slowly than in bulk but is still mobile. This, along with the dimensions of the pore, supports the observation that this peptide is selective for monovalent cations. Reasonable agreement of predicted conductances with experimentally determined values lends support to the validity of the simulations.     

Increased yield and CO2 sequestration potential with the C4 cereal Sorghum bicolor cultivated in basaltic rock dust‐amended agricultural soil

Land‐based enhanced rock weathering (ERW) is a biogeochemical carbon dioxide removal (CDR) strategy aiming to accelerate natural geological processes of carbon sequestration through application of crushed silicate rocks, such as basalt, to croplands and forested landscapes. However, the efficacy of the approach when undertaken with basalt, and its potential co‐benefits for agriculture, require experimental and field evaluation. Here we report that amending a UK clay‐loam agricultural soil with a high loading (10 kg/m²) of relatively coarse‐grained crushed basalt significantly increased the yield (21 ± 9.4%, SE) of the important C₄ cereal Sorghum bicolor under controlled environmental conditions, without accumulation of potentially toxic trace elements in the seeds. Yield increases resulted from the basalt treatment after 120 days without P‐ and K‐fertilizer addition. Shoot silicon concentrations also increased significantly (26 ± 5.4%, SE), with potential benefits for crop resistance to biotic and abiotic stress. Elemental budgets indicate substantial release of base cations important for inorganic carbon removal and their accumulation mainly in the soil exchangeable pools. Geochemical reactive transport modelling, constrained by elemental budgets, indicated CO₂ sequestration rates of 2–4 t CO₂/ha, 1–5 years after a single application of basaltic rock dust, including via newly formed soil carbonate minerals whose long‐term fate requires assessment through field trials. This represents an approximately fourfold increase in carbon capture compared to control plant–soil systems without basalt. Our results build support for ERW deployment as a CDR technique compatible with spreading basalt powder on acidic loamy soils common across millions of hectares of western European and North American agriculture.     

Synthesis and biological evaluation of an N10-Psec substituted pyrrolo[2,1-c][1,4]benzodiazepine prodrug

The first example of an N10-protected (e.g., Psec, 15) pyrrolo[2,1-c][1,4]benzodiazepine (PBD) analogue that retains significant cytotoxicity in a number of tumour cell lines is reported. This prototype could lead to a new generation of clinically useful N10-protected PBD prodrugs.     

Chemical synthesis and cytotoxicity of dihydroxylated cyclopentenone analogues of neocarzinostatin chromophore

Compounds containing the naphthoate moiety of Neocarzinostatin chromophore or 2-hydroxynaphthoate have been synthesized and evaluated for cytotoxic activity against a leukemia cell line and a small panel of human-tumor cell lines. Those compounds containing a cyclopentenone moiety were active, with the carbonyl group being essential for biological activity.     

Medial Forebrain Bundle Stimulation or D-2 Dopamine Receptor Activation Increases Preproenkephalin mRNA in Rat Striatum

Electrical stimulation of the medial forebrain bundle, in a manner that augmented the release of dopamine in the forebrain, rapidly increased the striatal content of preproenkephalin (but not preprotachykinin) mRNA. This effect was mimicked by administration of either the indirect (dopamine-releasing) agonist methamphetamine or by the D-2 dopamine receptor agonist quinpirole, but not by the D-1 agonist SKF 38393. These data suggest that D-2 receptors, which mediate a stimulatory effect on enkephalin gene expression, may be subsaturated under basal conditions and, therefore, responsive to increases in synaptic dopamine.