An easy and reliable method for establishment and maintenance of leaf and root cell cultures ofArabidopsis thaliana

Cell suspension cultures are useful for a wide range of biochemical and physiological studies, yet their production can be technically demanding and often unreliable. Here we describe a protocol for producing Arabidopsis cell suspension cultures that is reliable and easy to use.     

Changing concepts in plant hormone action

Summary A plant hormone is not, in the classic animal sense, a chemical synthesized in one organ, transported to a second organ to exert a chemical action to control a physiological event. Any phytohormone can be synthesized everywhere and can influence different growth and development processes at different places. The concept of physiological activity under hormonal control cannot be dissociated from changes in concentrations at the site of action, from spatial differences and changes in the tissue's sensitivity to the compound, from its transport and its metabolism, from balances and interactions with the other phytohormones, or in their metabolic relationships, and in their signaling pathways as well. Secondary messengers are also involved. Hormonal involvement in physiological processes can appear through several distinct manifestations (as environmental sensors, homeostatic regulators and spatio-temporal synchronizers, resource allocators, biotime adjusters, etc.), dependent on or integrated with the primary biochemical pathways. The time has also passed for the hypothesized ‘specific’ developmental hormones, rhizocaline, canlocaline, and florigen: root, stem, and flower formation result from a sequential control of specific events at the right places through a coordinated control by electrical signals, the known phytohormones and nonspecific molecules of primary and secondary metabolism, and involve both cytoplasmic and apoplastic compartments. These contemporary views are examined in this review.     

Identification and analysis of a third mouse Polycomb gene, MPc3

Betaine-homocysteine S-methyltransferase (BHMT) is one of the enzymes involved in the branch point metabolism of homocysteine. Elevated levels of plasma homocysteine may be a risk factor for the development of vascular disease; however, whether BHMT has a significant role in the regulation of plasma levels of homocysteine remains to be determined. As a prelude to creating a mouse strain deficient in BHMT activity, we screened a lambda library containing mouse SvJ 129 genomic DNA for the mouse BHMT gene using random probes made from the human cDNA. One genomic isolate was completely sequenced and found to encode an intronless BHMT pseudogene (mBHMT-ps). mBHMT-ps was then used as a template for the generation of random probes that were used to screen a BAC library containing mouse 129 Sv/Ev genomic DNA. In order to discriminate between pseudogenes and the authentic BHMT gene, a secondary PCR-based screen was employed which used primers designed from the pseudogene sequence that would predictably amplify across introns. Using this strategy, we isolated six mouse genomic clones that tested positive for the presence of all seven introns characteristic of the human gene, and the BHMT gene of one clone was completely sequenced. Like the human BHMT gene, the mouse gene spans 21 kb and is encoded by eight exons interrupted by seven introns. The structure of the mouse BHMT gene is described herein as well as the 5′-flanking region of the gene adjacent to exon 1, which we demonstrate is capable of conferring basal promoter activity in Chinese Hamster Ovary cells.     

Expression of antioxidant enzymes in rat lungs after inhalation of asbestos or silica.

Several studies indicate that active oxygen species play an important role in the development of pulmonary disease (asbestosis and silicosis) after exposure to mineral dust. The present study was conducted to determine if inhaled fibrogenic minerals induced changes in gene expression and activities of antioxidant enzymes (AOE) in rat lung. Two different fibrogenic minerals were compared, crocidolite, an amphibole asbestos fiber, and cristobalite, a crystalline silicon dioxide particle. Steady-state mRNA levels, immunoreactive protein, and activities of selected AOE were measured in lungs 1-10 days after initiation of exposure and at 14 days after cessation of a 10-day exposure period. Exposure to asbestos resulted in significant increases in steady-state mRNA levels of manganese-containing superoxide dismutase (MnSOD) at 3 and 9 days and of glutathione peroxidase at 6 and 9 days. An increase in steady-state mRNA levels of copper, zinc-containing superoxide dismutase (CuZnSOD), was observed at 6 days. Exposure to asbestos also resulted in overall increased enzyme activities of catalase, glutathione peroxidase and total superoxide dismutase in lung. In contrast, silica caused a dramatic increase in steady-state levels of MnSOD mRNA at all time periods and an increase in glutathione peroxidase mRNA levels at 9 days. Activities of AOE remained unchanged in silica-exposed lungs. In both models, increases in gene expression of MnSOD correlated with increased amounts of MnSOD immunoreactive protein in lung and the pattern and extent of inflammation. These data indicate that the profiles of AOE are dissimilar during the development of experimental asbestosis or silicosis and suggest different mechanisms of lung defense in response to these minerals.     

Preparation, characterization and in vivo conversion of new water-soluble sulfenamide prodrugs of carbamazepine

Improved synthetic methods are reported for the preparation of sulfenamide derivatives of carbamazepine (CBZ) for evaluation as prodrugs. These sulfenamide prodrugs were designed to rapidly release CBZ in vivo by cleavage of the sulfenamide bond by chemical reaction with glutathione and other sulfhydryl compounds. Physicochemical characterization and in vivo conversion of a new prodrug of CBZ was evaluated to further establish the proof of concept of the sulfenamide prodrug approach.     

Long-distance RNA-RNA interactions and conserved sequence elements affect potato virus X plus-strand RNA accumulation.

Conserved octanucleotide sequences located upstream of two major potato virus X (PVX) subgenomic RNAs (sgRNAs), as well as elements in the 5' end of the genome, affect accumulation of sgRNA. To determine if complementarity between these sequences is important for PVX RNA accumulation, we analyzed the effects of mutations within these elements and compensatory mutations in a tobacco protoplast system and in plants. Mutations in the 5' nontranslated region (NTR mutants) that reduced complementarity resulted in lower genomic RNA (gRNA) and sgRNA levels, whereas mutations to the octanucleotide elements affected only the corresponding sgRNA levels. However, for both the NTR and octanucleotide mutants, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Mutants containing changes in the NTR and compensatory changes in one of the octanucleotide elements restored levels of gRNA and the other sgRNA species with an unaltered octanucleotide element to those of wild-type. Although compensatory changes significantly increased levels of the sgRNA species with the modified octanucleotide element, levels were not restored to those of wild-type. Our data indicate that long distance RNA-RNA interactions and the sequences of the interacting elements are required for PVX plus-strand RNA accumulation.     

Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae

The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na⁺/Li⁺-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li⁺-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li⁺ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li⁺-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to␣cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast. Received: 17 August 1998 / Accepted: 7 December 1998