Suppression of the ribosomal L2 gene reveals a novel mechanism for stress adaptation in soybean

Pseudomonas syringae pv. glycinea bacteria or zoospores of the fungus Phytophthora sojae were used to trigger a hypersensitive reaction (HR) in cell cultures of soybean (Glycine max [L.] Merr. cv. Williams 82). During a screen for genes that show an altered expression as a response to dying neighbour cells we have identified a gene fragment that is specifically but transiently down-regulated in an HR. The corresponding cDNA codes for the ribosomal protein L2 (rpL2) of 80S ribosomes, which is essential for the peptidyl-transferase activity. Two gene copies of rpL2 exist in soybean and both genes are transcribed. The temporary down-regulation of the rpL2 genes is followed by a transient block in the synthesis of new proteins as visualised by pulse-labelling experiments using ³⁵S-amino acids. The same basic phenomenon was also found after treatment of soybean cells with other stress-causing compounds such as elicitors or heavy metals. It is suggested that the transient block in protein synthesis allows a more rapid depletion of, for example, signal molecules with a short half-life time and thus leads to a faster adaptation of the cellular protein inventory to the new environmental conditions. Received: 10 May 2000 / Accepted: 21 July 2000     

The structure of the cytoplasmic domain of the chloride channel ClC-Ka reveals a conserved interaction interface

The cytoplasmic domains of ClC chloride channels and transporters are ubiquitously found in eukaryotic family members and have been suggested to be involved in the regulation of ion transport. All cytoplasmic ClC domains share a conserved scaffold that contains a pair of CBS motifs. Here we describe the structure of the cytoplasmic component of the human chloride channel ClC-Ka at 1.6 A resolution. The structure reveals a dimeric organization of the domain that is unusual for CBS motif containing proteins. Using a biochemical approach combining mutagenesis, crosslinking, and analytical ultracentrifugation, we demonstrate that the interaction interface is preserved in solution and that the distantly related channel ClC-0 likely exhibits a similar structural organization. Our results reveal a conserved interaction interface that relates the cytoplasmic domains of ClC proteins and establish a structural relationship that is likely general for this important family of transport proteins.     

Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls

Arabidopsis cell walls contain large amounts of pectins and hemicelluloses, which are predominantly synthesized via the common precursor UDP-glucuronic acid. The major enzyme for the formation of this nucleotide-sugar is UDP-glucose dehydrogenase, catalysing the irreversible oxidation of UDP-glucose into UDP-glucuronic acid. Four functional gene family members and one pseudogene are present in the Arabidopsis genome, and they show distinct tissue-specific expression patterns during plant development. The analyses of reporter gene lines indicate gene expression of UDP-glucose dehydrogenases in growing tissues. The biochemical characterization of the different isoforms shows equal affinities for the cofactor NAD(+) ( approximately 40 microM) but variable affinities for the substrate UDP-glucose (120-335 microM) and different catalytic constants, suggesting a regulatory role for the different isoforms in carbon partitioning between cell wall formation and sucrose synthesis as the second major UDP-glucose-consuming pathway. UDP-glucose dehydrogenase is feedback inhibited by UDP-xylose. The relatively (compared with a soybean UDP-glucose dehydrogenase) low affinity of the enzymes for the substrate UDP-glucose is paralleled by the weak inhibition of the enzymes by UDP-xylose. The four Arabidopsis UDP-glucose dehydrogenase isoforms oxidize only UDP-glucose as a substrate. Nucleotide-sugars, which are converted by similar enzymes in bacteria, are not accepted as substrates for the Arabidopsis enzymes.     

Cell Wall Ingrowths in Nematode Induced Syncytia Require UGD2 and UGD3

The cyst nematode Heterodera schachtii infects roots of Arabidopsis plants and establishes feeding sites called syncytia, which are the only nutrient source for nematodes. Development of syncytia is accompanied by changes in cell wall structures including the development of cell wall ingrowths. UDP-glucuronic acid is a precursor of several cell wall polysaccharides and can be produced by UDP-glucose dehydrogenase through oxidation of UDP-glucose. Four genes in Arabidopsis encode this enzyme. Promoter::GUS analysis revealed that UGD2 and UGD3 were expressed in syncytia as early as 1 dpi while expression of UGD1 and UGD4 could only be detected starting at 2 dpi. Infection assays showed no differences between Δugd1 and Δugd4 single mutants and wild type plants concerning numbers of males and females and the size of syncytia and cysts. On single mutants of Δugd2 and Δugd3, however, less and smaller females, and smaller syncytia formed compared to wild type plants. The double mutant ΔΔugd23 had a stronger effect than the single mutants. These data indicate that UGD2 and UGD3 but not UGD1 and UGD4 are important for syncytium development. We therefore studied the ultrastructure of syncytia in the ΔΔugd23 double mutant. Syncytia contained an electron translucent cytoplasm with degenerated cellular organelles and numerous small vacuoles instead of the dense cytoplasm as in syncytia developing in wild type roots. Typical cell wall ingrowths were missing in the ΔΔugd23 double mutant. Therefore we conclude that UGD2 and UGD3 are needed for the production of cell wall ingrowths in syncytia and that their lack leads to a reduced host suitability for H. schachtii resulting in smaller syncytia, lower number of developing nematodes, and smaller females.     

Structure of the Bacillus subtilis Peptide Antibiotic Subtilosin A Determined by 1H-NMR and Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Subtilosin A produced by Bacillus subtilis is a macrocyclic peptide antibiotic which comprises 35 amino acids. Its molecular mass (3399.7 Da), determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and chemical properties gave experimental support for unusual intramolecular linkages. The three-dimensional fold of native subtilosin in dimethylsulfoxide was determined from two-dimensional ¹H-NMR spectra recorded at 600 MHz. Based on the backbone conformation, a structure for subtilosin A is presented which is characterized by three inter-residue bridges where two cysteines are linked with two phenylalanine residues, respectively, and a third cysteine is bound to a threonine residue.     

Testing potential developmental toxicants with a cytotoxicity assay based on human embryonic stem cells

Since the differentiation of embryonic stem cells mimics early development, these cells could potentially permit the detection of embryotoxicants which interfere with this process. Although reliable tests based on murine embryonic stem cells exist, no such methods are available for human embryonic stem (hES) cells. Nonetheless, to avoid the false classification of substances due to inter-species differences, human-relevant toxicity tests are needed. We therefore developed an assay based on three human cell types, representing different degrees of developmental maturation, namely, human foreskin fibroblasts, hES cell-derived progenitor cells, and pluripotent hES cells. A set of embryotoxicants for which existing in vivo data were available, namely, all-trans retinoic acid (ATRA), 13-cis retinoic acid (13CRA), valproic acid (VPA) and dimethyl sulphoxide (DMSO), were tested. 5-fluorouracil (5-FU) was used as a positive control, and saccharin as a negative control. Two methods were compared for the assessment of cell viability -- the determination of intracellular ATP content and of resazurin reduction. In addition, the protective capacity of basic fibroblast growth factor (bFGF) against retinoid-induced toxicity was investigated. This novel assay system reliably detected the embryotoxic potentials of the test substances, 5-FU, ATRA, 13-CRA (a substance that displays inter-species differences in its effects) and VPA. This was possible due to the apparent differences in the sensitivities of the human cell types used in the assay system. Thus, our results clearly indicate the advantages and relevance of using hES cells in in vitro developmental toxicity testing.     

Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C₁₀), farnesyl diphosphate (FDP, C₁₅) or geranylgeranyl diphosphate (GGDP, C₂₀). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C₂₅) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C₂₅ versus C₂₀ product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C₂₀) as a product and a larger R group (Met) resulting in GFDP (C₂₅).     

Differential clinical efficacy of anti-CD4 monoclonal antibodies in rat adjuvant arthritis is paralleled by differential influence on NF-kappaB binding activity and TNF-alpha secretion of T cells

The aim of this study was to analyze the differential effects of three anti-CD4 monoclonal antibodies (mAbs) (with distinct epitope specifities) in the treatment of rat adjuvant arthritis (AA) and on T-cell function and signal transduction. Rat AA was preventively treated by intraperitoneal injection of the anti-CD4 mAbs W3/25, OX35, and RIB5/2 (on days -1, 0, 3, and 6, i.e. 1 day before AA induction, on the day of induction [day 0], and thereafter). The effects on T-cell reactivity in vivo (delayed-type hypersensitivity), ex vivo (ConA-induced proliferation), and in vitro (mixed lymphocyte culture) were assessed. The in vitro effects of anti-CD4 preincubation on T-cell receptor (TCR)/CD3-induced cytokine production and signal transduction were also analyzed. While preventive treatment with OX35 and W3/25 significantly ameliorated AA from the onset, treatment with RIB5/2 even accelerated the onset of AA by approximately 2 days (day 10), and ameliorated the arthritis only in the late phase (day 27). Differential clinical effects at the onset of AA were paralleled by a differential influence of the mAbs on T-cell functions, i.e. in comparison with OX35 and W3/25, the 'accelerating' mAb RIB5/2 failed to increase the delayed-type hypersentivity (DTH) to Mycobacterium tuberculosis, increased the in vitro tumor necrosis factor (TNF)-alpha secretion, and more strongly induced NF-kappaB binding activity after anti-CD4 preincubation and subsequent TCR/CD3-stimulation. Depending on their epitope specificity, different anti-CD4 mAbs differentially influence individual proinflammatory functions of T cells. This fine regulation may explain the differential efficacy in the treatment of AA and may contribute to the understanding of such treatments in other immunopathologies.     

Biochemical characterization of mannitol metabolism in the unicellular red alga Dixoniella grisea (Rhodellophyceae)

The mannitol cycle has been verified in a unicellular red alga (Rhodellophyceae) for the first time. All four enzymes involved in the cycle (mannitol-1-phosphate dehydrogenase, Mt1PDH: EC 1.1.1.17; mannitol-1-phosphatase, Mt1Pase: EC 3.1.3.22; mannitol dehydrogenase, MtDH: 1.1.1.67; hexokinase, HK: 2.7.1.1.) were detected and characterized in crude algal extracts from Dixoniella grisea. These enzymes, with the exception of Mt1Pase, were specific to their corresponding substrates and nucleotides. The activities of enzymes in the anabolic pathway (fructose-6-P reduction by Mt1PDH and mannitol-6-P reduction by Mt1Pase) were at least 2- to 4-fold greater than those of the catabolic pathway (mannitol oxidation by MtDH and fructose oxidation by HK). There appears to be, therefore, a net carbon flow in D. grisea towards a high intracellular mannitol pool. The mannitol cycle guarantees a rapid accumulation or degradation of mannitol within algal cells in response to changing salinity in natural habitats. Moreover, the demonstration of the mannitol cycle within the Rhodellophyceae provides evidence that this metabolic pathway is of ancient origin in the red algal lineage.