An unexpected destabilisation of copper(II) phenoxyl radical species by steric protection

The complexes [CuL(Tp^Ph)] (HL = 5-tertbutylsalicylaldehyde, 5-tertbutyl-3-methylsulfanylsalicylaldehyde or 5-tertbutyl-3-phenylsulfanylsalicylaldehyde; [Tp^Ph]⁻ = tris-{3-phenylpyrazolyl}hydridoborate) have been prepared, and adopt square-pyramidal coordination geometries. Each compound exhibits a ligand-based oxidation in CH₂Cl₂ that is chemically reversible by voltammetry. However, Coulometric determinations showed that the resultant phenoxyl radical products decomposed rapidly at low temperatures in bulk solution. This instability may reflect intramolecular steric repulsions between the phenoxide tertbutyl substituents, and a pyrazolylborate phenyl group. These results contrast with a previously reported analogous compound, bearing a 5-methyl-3-methylsulfanylsalicylaldehydato ligand, which yields a phenoxyl radical oxidation product that is stable for hours under the same conditions.     

The auxin influx carrier LAX3 promotes lateral root emergence

Lateral roots originate deep within the parental root from a small number of founder cells at the periphery of vascular tissues and must emerge through intervening layers of tissues. We describe how the hormone auxin, which originates from the developing lateral root, acts as a local inductive signal which re-programmes adjacent cells. Auxin induces the expression of a previously uncharacterized auxin influx carrier LAX3 in cortical and epidermal cells directly overlaying new primordia. Increased LAX3 activity reinforces the auxin-dependent induction of a selection of cell-wall-remodelling enzymes, which are likely to promote cell separation in advance of developing lateral root primordia.     

Diversity of the C-terminal portion of the biphenyl dioxygenase large subunit

The biphenyl dioxygenase (BPDO) catalyses a stereospecific dioxygenation of biphenyl and analogs of it. Aside from being involved in the destruction and detoxification of toxic pollutants in soil, in the context of the green chemistry concept, this enzyme is a promising biocatalyst to design new more selective and more environmentally friendly approaches to manufacture fine chemicals. At this time, most of our knowledge about the variability of key residues determining the substrate specificity and regiospecificity of the enzyme oxygenase component (BphAE) toward biphenyl analogs and about the effect of altering these residues on catalytic properties is based on investigations made with BphAEs from cultured organisms and engineered enzymes derived from them. The purpose of this work was to examine the diversity of the amino acid sequence patterns of the alpha subunit (BphA) C-terminal domain deduced from PCR products amplified from DNA extracted from cultured bacteria of various phylogenetic lines and from the soil microflora of PCB-contaminated soils. Of special interest were segments of the C-terminal portion called regions I, III and IV. Altogether, the phylogenetic tree obtained from aligning the deduced amino acid sequences of BphAs C-terminal domain from cultured bacteria belonging to various ecological niches and from uncultured soil bacteria reveals that most of the BphAs were linked to the three clusters of BphAs previously reported. However, few belong to new branches that diverge from the previously known branches showing a high diversity of BphAs in natural environment. Furthermore, data show a wide distribution of BphAs with family linkages that not only crosses bacterial taxonomic frontiers but also ecological niches. Nevertheless, in spite of this divergence, the sequence patterns of regions III and IV amino acids that are known to influence substrate specificity and regiospecificity are rather conserved among BphAs and the pattern was independent of the family cluster to which they belong. In most cases, regions III and IV amino acid patterns are closer to those of Pseudomonas pseudoalcaligenes KF707 BphA1 than to the most versatile Burkholderia xenovorans LB400 BphA. This might suggest that the PCB-degrading potency of soil bacteria is closer to the one observed for KF707 BphAE than from LB400 BphAE. However, the fact that among less than 20 PCR products amplified from soil DNA that we have sequenced, one of them was very homologous to that of LB400 BphA and in addition, residues 335 and 336 of LB400 were replaced by residues that previous enzyme engineering had shown to extend the range of PCB substrate used by the enzyme strongly suggest that PCB-degrading bacteria are evolving in soil to optimize their PCB-degrading capacity.     

Serum-mediated enhancement of ANF accumulation in the culture medium of cardiac atriocytes

Cultured cardiac myocytes provide a useful system for investigating ANF biosynthesis and regulation. It has previously been demonstrated that the predominant form of ANF stored in and released from this myocyte model is the 17 kD prohormone, proANF. We report here that as quantitated both by radioimmunoassay and by SDS-PAGE of intrinsically labelled ANF released from these cardiocytes, the addition of serum promotes a 5-6 fold increase in ANF accumulation in the medium of these cells as compared to ANF accumulation in the presence of a defined chemical medium alone. The stimulating effect of serum is immediate and persists in a linear manner for at least 120 minutes. This effect of serum can be reproduced by the addition of albumin or other proteins to the medium but not by alterations in osmolality. Whether this phenomenon represents enhanced release of proANF or is secondary to inhibition of proANF degradation has yet to be determined.     

Using MALDI-TOF mass spectrometry to identify ticks collected on domestic and wild animals from the Democratic Republic of the Congo

Experimental and Applied Acarology - Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) has recently emerged as an alternative to morphological and molecular tools to...     

Expression of the 4-chlorobenzoate dehalogenase genes from Pseudomonas sp.-CBS3 in Escherichia coli and identification of the gene translation products.

The genes encoding the 4-chlorobenzoate dehalogenase of Pseudomonas sp. strain CBS3 were, in an earlier study, cloned in Escherichia coli DH1 with the cosmid vector pPSA843 and then mobilized to the 4-chlorobenzoate dehalogenase minus strain Pseudomonas putida KT2440. In this paper we report on the expression of 4-chlorobenzoate dehalogenase in these clones and on the polypeptide composition of the active enzyme. The dehalogenase activity in whole cells suspended in 3.2 mM 4-chlorobenzoate (30 degrees C) was determined to be approximately 27 units (micromoles 4-hydroxybenzoate produced per minute) per 100 g of E. coli-pPSA843 cells and approximately 28 units per 100 g of P. putida-pPSA843 cells. Dehalogenase activity in fresh cellular extracts (pH 7.4, 30 degrees C) prepared from the E. coli and P. putida clones was unstable and at least 20-fold lower than that observed with the whole cells. The polypeptide components of the dehalogenase were identified by selective expression of the cloned dehalogenase genes and analysis of the gene translation products. Analysis of dehalogenase activity in omega insertion mutants and deletion mutants circumscribed the dehalogenase genes to a 4.8-kilobase (4.8 kb) stretch of the 9.5-kb DNA fragment. Selective expression of the dehalogenase genes from a cloned 4.8-kb DNA fragment in a maxicell system revealed a 30-kDa polypeptide as one of the components of the dehalogenase system. Selective expression of the dehalogenase genes using the T7 polymerase promoter system revealed the 30-kDa polypeptide and 57- and 16-kDa polypeptide products. Determination of which of the three polypeptides were translated in deletion mutants provided the relative positions of the encoding genes on a single DNA strand and the direction in which they are transcribed.     

Mutants of Saccharomycopsis lipolytica defective in lysine catabolism.

Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests.     

Cdx1 autoregulation is governed by a novel Cdx1-LEF1 transcription complex

The Cdx1 gene product is essential for normal anterior-posterior vertebral patterning. Expression of Cdx1 is regulated by several pathways implicated in anterior-posterior patterning events, including retinoid and Wnt signaling. We have previously shown that retinoic acid plays a key role in early stages of Cdx1 expression at embryonic day 7.5 (E7.5), while both Wnt3a signaling and an autoregulatory loop, dependent on Cdx1 itself, are involved in later stages of expression (E8.5 to E9.5). This autoregulation is reflected by the ability of Cdx1 to affect expression from proximal Cdx1 promoter sequences in tissue culture. However, this region is devoid of a demonstrable Cdx response element(s). We have now found that Cdx1 and LEF1, a nuclear effector of Wnt signaling, synergize to induce expression from the Cdx1 promoter through previously documented LEF/T-cell factor response elements. We also found a direct physical interaction between the homeodomain of Cdx1 and the B box of LEF1, suggesting a basis for this synergy. Consistent with these observations, analysis of Cdx1 Wnt3a(vt) compound mutants demonstrated that Wnt and Cdx1 converged on Cdx1 expression and vertebral patterning in vivo. Further data suggest that Cdx-high-mobility group box interactions might be involved in a number of additional pathways.     

Burst and coast use, swimming performance and metabolism of Atlantic cod Gadus morhua in sub-lethal hypoxic conditions

Prolonged swimming capacity (critical swimming speed, U _crit, protocol) and metabolism were measured for 14 Atlantic cod Gadus morhua exposed to seven oxygen levels within the non-lethal range normally encountered in the Gulf of St Lawrence (35 to 100% saturation). Burst-and-coast swimming was triggered earlier (at lower speeds) in hypoxia, and burst-and-coast movements were more frequent in hypoxia than in normoxia at low speeds. Furthermore, the metabolic scope beyond the metabolic rate at which Atlantic cod resorted to burst-and-coast movements decreased gradually as ambient oxygen concentration dropped. Overall, fewer burst-and-coast movements were observed in hypoxia while the distance swum in burst-and-coast mode remained c . 1% of the total distance swam in all tests. Oxygen availability had no effect on the rate of increase in metabolic rate with increasing velocity <50 cm s⁻¹_, but limited swimming performances and metabolic rate at higher speeds. The prevailing low oxygen tensions on the bottom in the deep channels may impair the swimming capacity of Atlantic cod in the estuary and northern Gulf of St Lawrence.