Transglutaminase-like activity in Chrysanthemum leaf explants cultivated in vitro in relation to cell growth and hormone treatment

In Chrysanthemum leaf explants cultivated in vitro the capacity to covalently link polyamines to protein substances exists. This plant enzyme activity shows some similarities with mammalian transglutaminases. In foliar explants cultured on a medium promoting bud or root formation increasing levels of transglutaminase-like activity occurred during the first days of culture when cell multiplication was rapid then the levels declined as the rate of cell division decreased and differentiation occurred. Undifferentiated callus exhibited low transglutaminase-like activity. Transglutaminase-like activity also increased in rapidly proliferating and growing organs (roots and buds initiated from the foliar explants) and decreased during maturity. The relationship among transglutaminases-like activity, cell division, bud and root formation is discussed.Abbreviations TGase transglutaminase - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine     

Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences

The major surface protein of the genus Pseudomonas, OprF, is a non-specific porin that plays an important role in maintenance of cell shape, in growth in a low osmolarity environment, and in adhesion to various supports. The objectives of our study were (i) to carry out a comparative analysis of phylogenies obtained from the OprF protein and from the 16S rRNA gene in 41 isolates from various sources (water, soil, milk and the hospital) and (ii) to investigate the physiological characteristics correlated with the phylogeny of OprF. We report here an important incongruence between the phylogenies of the 16S rRNA gene and the OprF protein. Phylogenetic analysis of 16S rRNA genes grouped Pseudomonas fluorescens isolates into one cluster (termed fluorescens r-cluster) whilst the phylogeny of the OprF protein divided Pseudomonas fluorescens isolates into two quite distinct clusters (termed fluorescens 1 o-cluster and fluorescens 2 o-cluster) that may be related to the original habitat of the strain. The fluorescens 1 o-cluster contained the majority of non-rhizospheric soil isolates, while the fluorescens 2 o-cluster contained all our clinical isolates and most of the rhizospheric isolates (which are fixed to the roots). In order to check this correlation, we studied two physiological characteristics: the range of growth temperature and the capacity for non-specific adhesion to polystyrene. The temperature range study for strains did not explain the existence of the two o-clusters but it did confirm the capacity of certain P. fluorescens strains to grow at 37 degrees C. The adhesion capacities of the isolates in the two o-clusters seems to be correlated with ecological niche.     

Cell wall composition contributes to the control of transpiration efficiency in Arabidopsis thaliana

To identify loci in Arabidopsis involved in the control of transpirational water loss and transpiration efficiency (TE) we carried out an infrared thermal imaging-based screen. We report the identification of a new allele of the Arabidopsis CesA7 cellulose synthase locus designated AtCesA7(irx3-5) involved in the control of TE. Leaves of the AtCesA7(irx3-5) mutant are warmer than the wild type (WT). This is due to reduced stomatal pore widths brought about by guard cells that are significantly smaller than the WT. The xylem of the AtCesA7(irx3-5) mutant is also partially collapsed, and we suggest that the small guard cells in the mutant result from decreased water supply to the developing leaf. We used carbon isotope discrimination to show that TE is increased in AtCesA7(irx3-5) when compared with the WT. Our work identifies a new class of genes that affects TE and raises the possibility that other genes involved in cell wall biosynthesis will have an impact on water use efficiency.     

Functional analysis of an Arabidopsis T-DNA "knockout" of the high-affinity NH4(+) transporter AtAMT1;1

NH(4)(+) acquisition by plant roots is thought to involve members of the NH(4)(+) transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH(4)(+). Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH(4)(+) influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity (13)NH(4)(+) influx in the amt1;1:T-DNA mutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity (13)NH(4)(+) influx in the mutant by 30% compared with wild-type plants, whereas low-affinity (13)NH(4)(+) influx (250 microM-10 mM NH(4)(+)) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 and AtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNA plants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH(4)(+) and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH(4)(+) transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1.     

Planktonic bacterial biomass and seasonal pattern of the heterotrophic uptake and respiration of glucose and amino acids in the shallow sandpit lake of Créteil (Paris Suburb,France)

Biomass and activity of planktonic bacteria were investigated during a one year study in a shallow sandpit lake. The shallowness of the lake helped keep the water column homogeneous regarding bacterioplankton. Small free-living bacteria (0.03 µm³ cell^–1) dominated the populations throughout the period studied. Bacterial abundances varied from 1 to 11 × 10⁶ cells ml^–1. Kinetic parameters (V _max, K + S and T) were determined with ¹⁴C labelled compounds (glucose and amino acids mixture). V _max values were high and averaged 0.056 and 0.050 µgCl^–1 h^–1 for glucose and amino acids respectively. Maximal V _max values were observed in summer at the highest temperatures, but also in early spring. T values were much greater in winter. K + S values were significantly higher for amino acids (3 µg Cl^–1) than for glucose (1 µg Cl^–1). A low percentage of mineralization (about 25% for both tracers) could be the expression of the high growth efficiency expected when bacteria are growing at the expense of low molecular weight compounds as phytoplankton exudates.     

Image analysis of the heavy form of the acetylcholine receptor from Torpedo marmorata

The structure of the heavy (H) form of the acetylcholine receptor, which comprises two covalently linked 250,000 M_r oligomers, has been investigated by numerical analysis of electron microscope images. Na-cholate solubilized Torpedo marmorata H-form receptor was reintegrated into artificial lipid vesicles and negatively stained with uranyl acetate prior to imaging in a conventional transmission microscope. The reconstituted preparations exhibited the standard polypeptide composition of the purified receptor (α₂βγδ) and the same transmembrane arrangement as in the native subsynaptic membrane. Covalent disulfide linkage between the two oligomers took place exclusively through the δ chains.In agreement with previous work (Cartaud et al., 1980) the H-form appeared as “doublets” of two coplanar 9 nm rosettes at a center-to-center distance of 9.2 ± 1.1 nm. The relative angular orientation of the two rosettes in a doublet was examined by correlation analysis in the real space. It exhibited a marked variability, few of the doublets featuring any kind of symmetry, suggesting that the two oligomers of a doublet are connected via an extended and flexible chain or loop. The area of contact between the two rosettes of a doublet therefore does not necessarily represent a reliable clue as to the location of the δ chain within the structure.Averaged images obtained after reorientation and summation of up to 132 rosettes revealed the three major peaks and the two grooves already observed in previous studies. Two additional smaller peaks were identified.Tentative assignment of structural details to individual subunits was deduced from an examination of α-bungarotoxin-labeled doublets. The α subunits, which carry part or all of the acetylcholine binding sites, are probably located in nonadjacent positions in the vicinity of the newly found peaks. This assignment is consistent with the image analysis of receptor-toxin complexes recently reported by Zingsheim et al. (1982b).     

Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxin-derived mercapturates; 3,4-epoxyprecocenes as models of cytotoxic epoxides

The mycotoxin aflatoxin B1 (AFB1) is a carcinogenic food contaminant which is metabolically activated by epoxydation. The metabolism of mycotoxins via the mercapturate metabolic pathway was shown, in general, to lead to their detoxication. Mercapturic acids thus formed (S-substitued-N-acetyl-l-cysteines) may be accumulated in the kidney and either excreted in the urine or desacetylated by Acylase 1 (ACY1) to yield cysteine S-conjugates. To be toxic, the N-acetyl-l-cysteine-S-conjugates first have to undergo deacetylation by ACY 1. The specificity and rate of mercapturic acid deacetylation may determine the toxicity, however the exact deacetylation processes involved are not well known. The aim of this study was to investigate the role of ACY1 in the toxicity of some bioactive epoxides from Aflatoxin B1. We characterized the kinetic parameters of porcine kidney and human recombinant aminoacylase-1 towards some aromatic and aliphatic-derived mercapturates analogue of mycotoxin-mercapturic acids and 3,4-epoxyprecocene, a bioactive epoxide derivated from aflatoxin. The deacetylation of mercapturated substrates was followed both by reverse phase HPLC and by TNBS method. Catalytic activity was discussed in a structure-function relationship. Ours results indicate for the first time that aminoacylase-1 could play an important role in deacetylating mercapturate metabolites of aflatoxin analogues and this process may be in relation with their cyto- and nephrotoxicity in human.