Accumulation of ammonium in Norway spruce (Picea abies) seedlings measured by in vivo 14N-NMR

(14)N-NMR and (31)P-NMR have been used to monitor the in vivo pH in roots, stems, and needles from seedlings of Norway spruce, a typical ammonium-tolerant plant. The vacuolar and cytoplasmic pH measured by (31)P-NMR was found to be c. pH 4.8 and 7.0, respectively, with no significant difference between plants growing with ammonium or nitrate as the N-source. The (1)H-coupled (14) NH 4+ resonance is pH-sensitive: at alkaline pH it is a narrow singlet line and below pH 4 it is an increasing multiplet line with five signals. The pH values in ammonium-containing compartments measured by (14)N-NMR ranged from 3.7 to 3.9, notably lower than the estimated pH values of the P(i) pools. This suggests that, in seedlings of Norway spruce, ammonium is stored in vacuoles with low pH possibly to protect the seedlings against the toxic effects of ammonium ( NH 4+) or ammonia (NH3). It was also found that concentrations of malate were 3-6 times higher in stems than in roots and needles, with nitrate-grown plants containing more malate than plants grown with ammonium.     

A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.     

Decreased abundance and diversity of culturable Pseudomonas spp. populations with increasing copper exposure in the sugar beet rhizosphere

Recent studies have indicated that culturable bacteria constitute highly sensitive bioindicators of metal-induced stress in soil. We report the impact of different copper exposure levels characteristic of contaminated agricultural soils on culturable Pseudomonas spp. in the rhizosphere of sugar beet. We observed that the abundance of Pseudomonas spp. was much more severely affected than that of the general population of culturable heterotrophic bacteria by copper. For diversity assessment, Pseudomonas isolates were divided into operational taxonomic units based on amplified ribosomal DNA restriction analysis and genomic PCR fingerprinting by universally primed PCR. Copper significantly decreased the diversity of Pseudomonas spp. in the rhizosphere and significantly increased the frequency of copper-resistant isolates. Concomitant chemical and biological analysis of copper in the rhizosphere and in bulk soil extracts indicated no rhizosphere effect and a relatively low copper bioavailability in the studied soil, suggesting that the observed effects of copper may occur at lower total concentrations in other soils. We conclude that culturable Pseudomonas sensu stricto constitutes a highly sensitive and relevant bioindicator group for the impact of copper in the rhizosphere habitat, and suggest that continued application of copper to agricultural soils poses a significant risk to successful rhizosphere colonization by Pseudomonas spp.     

Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolor

Glucosinolates are natural products in cruciferous plants, including Arabidopsis thaliana. CYP79A1 is the cytochrome P450 catalysing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin in sorghum. Both glucosinolates and cyanogenic glucosides have oximes as intermediates. Expression of CYP79A1 in A. thaliana results in the production of high levels of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate, which is not a natural constituent of A. thaliana. This provides further evidence that the enzymes have low substrate specificity with respect to the side chain. The ability of the cyanogenic CYP79A1 to integrate itself into the glucosinolate pathway has important implications for an evolutionary relationship between cyanogenic glucosides and glucosinolates, and for the possibility of genetic engineering of novel glucosinolates.     

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.     

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.     

Relative role of low- and high pressure reflexes on sympathetic activity in humans during simulated gravitational stress

In order to determine the relative role of low- and high-pressure reflexes, respectively, on forearm sympathetic nerve activity (fSNA), 10 normal male subjects underwent a 4-step (5 min each) graded lower body negative pressure (LBNP) from -10 to -50 mmHg. Central venous pressure (CVP) and stroke volume gradually decreased (p<0.05), and arterial pulse pressure (PP) abruptly decreased at LBNP of -50 mmHg. Mean arterial pressure (MAP) remained unchanged. Forearm venous plasma norepinephrine concentration (fvNE) increased significantly at LBNP of -35 mmHg (p<0.05) and with a further sharp increase during LBNP of -50 mmHg (p<0.05). High degrees of intra-individual correlations were observed between changes in Log [fvNE] and CVP (r-values from -0.78 to -0.96, p<0.01). We conclude that low-pressure reflexes are the major determinants of fSNA during non-hypotensive gravitational stress (MAP and PP unchanged). When the gravitational stress is more pronounced, a decrease in PP further augments fSNA through inhibition of high-pressure arterial baroreflexes.     

Insulin in oral immune "tolerance": a one-amino acid change in the B chain makes the difference.

Oral administration of self-Ags can dampen or prevent autoimmune processes by induction of bystander suppression. Based on encouraging results from experiments in nonobese diabetic (NOD) mice, clinical trials have been initiated in type 1 diabetes using human insulin as an oral Ag. However, neither the precise antigenic requirements nor the mechanism of bystander suppression are currently understood in detail. Here we report that 1) a 1-aa difference in position 30 of the insulin B chain abrogated the ability of insulin to confer protection in both NOD as well as a virus-induced transgenic mouse model for type 1 diabetes. In the latter model transgenic mice express the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) under the control of the rat insulin promotor (RIP) in the pancreatic beta cells and develop diabetes only following LCMV infection; and 2) protection could be transferred with insulin B chain-restimulated but not LCMV-restimulated splenocytes from RIP-NP transgenic mice, demonstrating that the mechanism of diabetes prevention in the RIP-NP model is mediated by insulin B chain-specific, IL-4-producing regulatory cells acting as bystander suppressors.