The katA catalase gene is regulated by OxyR in both free-living and symbiotic Sinorhizobium meliloti

The characterization of an oxyR insertion mutant provides evidences that katA, which encodes the unique H2O2-inducible HPII catalase, is regulated by OxyR not only in free-living Sinorhizobium meliloti but also in symbiotic S. meliloti. Moreover, oxyR is expressed independently of exogenous H2O2 and downregulates its own expression in S. meliloti.     

Towards an expanded and integrated linkage map of cucumber (Cucumis sativus L.).

Linkage maps in cucumber (Cucumis sativus var. sativus L.) have been constructed using morphological traits, isozymes, restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs). The lack of polymorphism in cucumber has led to the construction of relatively unsaturated maps (13- to 80-point). We have added amplified fragment length polymorphism (AFLP) markers to existing narrow-based (within C. sativus) and wide-based (C. sativus x C. sativus var. hardwickii) maps. JOINMAP v. 2.0 was used to construct maps and to join these with historical maps from several previous studies. Our narrow- and wide-based merged maps contain 255 and 197 markers, respectively, including morphological traits, disease resistance loci, isozymes, RFLPs, RAPDs, and AFLPs. Condensation of total map distance occurred in merged maps compared to historic maps using many of the same markers. This phenomenon is most likely due to differences in map construction algorithms. The merged maps represent the best fit of the data used and are an important first step towards the construction of a comprehensive linkage map for cucumber. Identification of additional anchor markers between the narrow- and wide-based maps presented here may allow their future integration into a unified model.     

The Neuroleptic Chlorpromazine Inhibits the Cationic and Stimulates the Anionic Phospholipid Precursor Synthesis in Human Lymphocytes

The widely used neuroleptic drug chlorpromazine (CPZ) influences membrane functions at the levels of ionic channels and receptors as shown. Here we show the effect of short term treatments by CPZ (30 μM), on the nucleotide-containing phospholipid precursors in human lymphocyte primary cultures. During 60 minutes incubation of the cells, the CDP-ethanolamine (CDP-EA) content was only slightly reduced (87 to 76 pmol/10⁶ cells), the amount of CDP-choline (CDP-Ch) was inhibited totally (from 25 to 0 pmol) upon the treatment with 30 μM CPZ under the same conditions. It has been shown earlier, that dCTP can be used as well as CTP for biosynthesis of phospholipids. Thus, the separation of the corresponding ribo- and deoxyribo-liponucleotides was developed. CPZ almost completely inhibited the synthesis of both dCDP-EA and dCDP-Ch under the same conditions The synthesis of the activated liponucleotide precursors, can be measured by incorporation of extracellular 14C-dCyt into both dCDP-EA and dCDP-Ch, as shown earlier. While the cationic deoxyribo-liponucleotide content (dCDP-Ch, dCDP-EA) was decreased, the labelling of the anionic phospholipid precursor dCDP-diacylglycerol (dCDP-DAG) was enhanced several times, it could be labelled only in the presence of CPZ from 14C-dCyd. Thus, a principal disturbance of the membrane phospholipid synthesis is presented (i.e., inhibition of the cationic and enhancement of the anionic dCDP-DAG synthesis). This profound influence on the membrane phospholipids by chlorpromazine, might be the primary effect that contributes to the wide spectrum of CPZ effects on neuronal cells.     

Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.     

The death-domain fold of the ASC PYRIN domain, presenting a basis for PYRIN/PYRIN recognition

The PYRIN domain is a conserved sequence motif identified in more than 20 human proteins with putative functions in apoptotic and inflammatory signalling pathways. The three-dimensional structure of the PYRIN domain from human ASC was determined by NMR spectroscopy. The structure determination reveals close structural similarity to death domains, death effector domains, and caspase activation and recruitment domains, although the structural alignment with these other members of the death-domain superfamily differs from previously predicted amino acid sequence alignments. Two highly positively and negatively charged surfaces in the PYRIN domain of ASC result in a strong electrostatic dipole moment that is predicted to be present also in related PYRIN domains. These results suggest that electrostatic interactions play an important role for the binding between PYRIN domains. Consequently, the previously reported binding between the PYRIN domains of ASC and ASC2/POP1 or between the zebrafish PYRIN domains of zAsc and Caspy is proposed to involve interactions between helices 2 and 3 of one PYRIN domain with helices 1 and 4 of the other PYRIN domain, in analogy to previously reported homophilic interactions between caspase activation and recruitment domains.     

Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T‐DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana

Nicotiana species carry cellular T‐DNA sequences (cT‐DNAs), acquired by Agrobacterium‐mediated transformation. We characterized the cT‐DNA sequences of the ancestral Nicotiana tabacum species Nicotiana tomentosiformis by deep sequencing. N. tomentosiformis contains four cT‐DNA inserts derived from different Agrobacterium strains. Each has an incomplete inverted‐repeat structure. TA is similar to part of the Agrobacterium rhizogenes 1724 mikimopine‐type T‐DNA, but has unusual orf14 and mis genes. TB carries a 1724 mikimopine‐type orf14‐mis fragment and a mannopine‐agropine synthesis region (mas2‐mas1‐ags). The mas2′ gene codes for an active enzyme. TC is similar to the left part of the A. rhizogenes A4 T‐DNA, but also carries octopine synthase‐like (ocl) and c‐like genes normally found in A. tumefaciens. TD shows a complex rearrangement of T‐DNA fragments similar to the right end of the A4 TL‐DNA, and including an orf14‐like gene and a gene with unknown function, orf511. The TA, TB, TC and TD insertion sites were identified by alignment with N. tabacum and Nicotiana sylvestris sequences. The divergence values for the TA, TB, TC and TD repeats provide an estimate for their relative introduction times. A large deletion has occurred in the central part of the N. tabacum cv. Basma/Xanthi TA region, and another deletion removed the complete TC region in N. tabacum. Nicotiana otophora lacks TA, TB and TD, but contains TC and another cT‐DNA, TE. This analysis, together with that of Nicotiana glauca and other Nicotiana species, indicates multiple sequential insertions of cT‐DNAs during the evolution of the genus Nicotiana.     

Pulmonary intravascular macrophages and hemodynamic effects of liposomes in sheep

We studied the effects of liposomes on the pulmonary circulation of sheep and found a close correlation between liposome retention in the lung and the intravascular macrophages. A test dose of liposomes (5.5 mumol of total lipids) injected intravenously transiently increased pulmonary arterial pressure from 24 +/- 2 to 55 +/- 16 (SD) cmH2O. The pulmonary arterial pressure responses were dose dependent and reproducible. The rise in pulmonary arterial pressure was blocked completely by indomethacin and 75% by a thromboxane synthase inhibitor. Systemic arterial thromboxane B2 concentration increased from a base-line level of less than 50 pg/ml to 250 +/- 130 pg/ml at the peak of the pressor response. Larger doses of liposomes (220 mumol of total lipids) infused intravenously over 1 h increased pulmonary arterial pressure maximally within the first 15 min. Lymph flow increased and lymph protein concentration decreased, suggesting venoconstriction. Over half (62.4 +/- 15.7%) of 111In-labeled liposomes remained in the lung after 2 h. Fluorescence and transmission electron microscopy showed that greater than 90% of the liposomes were associated with mononuclear cells in the lumen of the alveolar wall microvessels. We conclude that liposomes affect pulmonary arterial pressure transiently by a mechanism involving the arachidonate cascade, principally thromboxane. Our observations suggest that a population of pulmonary intravascular macrophages is likely to be the source of the thromboxane and the pulmonary hemodynamic and lymph dynamic changes that occur in a dose-dependent fashion, although interactions between liposomes, leukocytes, or endothelial cells, in addition to the macrophages, have not been completely ruled out. We believe this is the first demonstration that pulmonary intravascular macrophages may be the source of the arachidonate metabolites rather than endothelial cells, neutrophils, or perivascular interstitial cells.     

Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity

High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function.