Regulated expression of plant tRNA genes by the prokaryotic tet and lac repressors

The prokaryotic tet operator (tetO) sequence was inserted at positions upstream and downstream of sequences encoding the Arabidopsis thaliana tRNA _AUC ^Lys or tRNA _AUC ^Trp suppressor tRNAs, and tRNA expression in carrot protoplasts was measured by translational suppression of a nonsense codon in a luciferase reporter gene. Regulation of tRNA expression by the tetracycline repressor (tetR) occurred from genes with the tetO inserted at position –1 (for the tRNA _AUC ^Trp gene), or at positions –2, –6 and –10 (for the tRNA _AUC ^Lys gene), and repression reached 90%. The inducer tetracycline (Tc) restored tRNA expression. Similarly, carrot protoplasts transfected with human tRNA _AUC ^Ser genes containing the lac operator (lacO) in their 5-flanking sequence with or without the lac repressor (lacI) gene, conditionally expressed tRNAs which suppressed the luciferase reporter. Up to 30-fold repression occured by the lactose repressor when lacO was located at position –1 of the tRNA _AUC ^Ser coding sequence. In the presence of the inducer isopropyl--thiogalactoside (IPTG), repression was relieved. These results demonstrate that sequences flanking tRNA genes can strongly influence tRNA expression in plants, and in a conditional fashion when bound by inducible proteins.     

Unraveling regulatory networks in plant defense using microarrays

DNA microarrays are being used to comprehensively examine gene expression networks during the plant defense response that is triggered when a plant encounters a pathogen or an elicitor molecule. In addition to identifying new genes induced during defense, these studies are providing new insights into the complex pathways governing defense gene regulation.     

NITROGEN UTILIZATION AND METABOLISM RELATIVE TO PATTERNS OF N2 FIXATION IN CULTURES OF TRICHODESMIUM NIBB1067

We measured uptake kinetics for four combined N sources, ambient rates of N uptake and N₂ fixation, glutamine synthetase activity (transferase and biosynthetic), and concentrations of intracellular pools of glutamate (glu) and glutamine (gln) in cultures of Trichodesmium NIBB1067. N dynamics and metabolism were examined to assess the relative importance of N₂ fixation and N uptake to Trichodesmium nutrition. Comparisons were made between cultures grown on medium without added N, with excess NO, or with excess urea. Of the combined N sources tested, Trichodesmium NIBB1067 had the highest affinity for NH; high uptake capacities for NH, urea, and glu; and little capacity for NO uptake. In cultures grown on medium without added N, NH accumulated in the medium during growth, resulting in high NH uptake rates relative to rates of N₂ fixation. Glu uptake rates were low but consistent throughout the diel period. In cultures grown on excess NO or urea, uptake of these compounds supplied the majority of the daily N demand, although some N₂ fixation occurred during the light period. NO uptake rates were reduced when N₂-fixation rates were high. In all of the cultures, the highest gln/glu ratios and the lowest glutamine synthetase transferase/biosynthetic ratios were observed during the period when rates of total N uptake were highest. In cultures growing exponentially on medium without added N, N₂ fixation accounted for 14%– 16% of the total daily N uptake. Uptake of NH and glu, presumably regenerated within the culture vessels, represented 84%–86% of the daily N uptake. Because these systems were closed, net growth was constrained by the rate at which N₂ could be fixed into the system. However, total daily N turnover was greater than that necessary to accommodate the observed increase in culture biomass. The rapid N turnover rates observed in these cultures may support gross productivity and balance the high rates of C fixation observed in natural populations of Trichodesmium.     

Is there an inner nose?

Although behavioral and neuropsychological data regarding the existence of images for odors are inconclusive, reconsideration of earlier EEG work provides reasonably clear evidence for an inner nose. However, further EEG studies and neuroimaging data seem essential for conclusive demonstration of an inner nose.      

Expression in different populations of cells of the root meristem is controlled by different domains of the rolB promoter

Selective gene expression in different populations of cells of the root apex of transgenic tobacco could be evidenced by means of GUS constructs with deletions of the rolB promoter and fusions with the CaMV 35S minimal promoter. Five regulatory regions have been broadly identified in the rolB 5 non-coding region. The presence of all five domains (A to E) directs gene expression in the root cap, in the protoderm and in the different tissues within the root meristematic region: the dermatocalyptrogen, the cortex and the vascular cylinder. Deletion of domain A (–623 to –471) selectively suppresses expression in non-meristematic cells, i.e. the root cap and the protoderm. Deletion of either domain B (–341 to –306) or E (80 bp around the TATA box) causes loss of expression in all cells of the root apex: constructs C+D+E, B+C+D, B+C are inactive. Domain D (70 bp around the CAAT box) is necessary for gene expression in the dermatogen and in meristematic cells of the cortex but not in the innermost meristematic layer: construct B+C+E is active only in vascular meristematic cells. Domain C (–216 to –158) seems to have a double regulatory role as construct B+E is no longer expressed in meristematic cells of the vascular cylinder but is very active in the protoderm. Constructs allowing gene expression in meristematic cells are also inducible by auxin in leaf protoplasts, while activation of the regulatory elements necessary for gene expression in the non-meristematic cells of the root apex do not seem to depend upon the hormone. The connection between auxin induction and meristematic expression is discussed.     

Microbial transformations of methylated sulfur compounds in anoxic salt marsh sediments

Anoxic salt marsh sediments were amended with several methylated sulfur compounds. Sediment microbes transformed the added compounds into other volatile methylated sulfur compounds and eventually mineralized the compounds to CH₄ and presumably to CO₂ and H₂S. The principal methyl-sulfur product of dimethylsulfoniopropionate (DMSP) was found to be dimethylsulfide (DMS), with only small amounts of methane thiol (MSH) produced. By contrast, methionine and S-methyl cysteine were degraded mostly to MSH and to lesser amounts of DMS. Dimethylsulfoxide (DMSO) was biologically converted to DMS. Dimethyldisulfide (DMDS) was rapidly reduced to MSH by the sediment microflora, and some DMS was also produced. DMS, whether added directly or when derived from other precursors, was metabolized with the production of MSH. Methane thiol was also metabolized, and evidence suggests that MSH may be biologically methylated to form DMS. Experiments with selective microbial inhibitors were used to ascertain which microbial groups were responsible for the observed transformations. Based on these experiments, it appears that both sulfate-reducing and methane-producing bacteria may be involved in transforming and mineralizing methylated sulfur compounds. A simple scheme of how methylated sulfur compounds may be transformed in the environment is presented.     

Chitinase 3-like-1 is produced by human Th17 cells and correlates with the level of inflammation in juvenile idiopathic arthritis patients

Background

CHI3L1 is a chitinase-like protein without enzymatic activity, produced by activated macrophages, chondrocytes, neutrophils. Recent studies on arthritis, asthma, and inflammatory bowel diseases suggest that chitinases are important in inflammatory processes and tissue remodeling, but their production by human T cells, has never been reported.

Methods

A microarray analysis of gene expression profile was performed on Th17 and classic Th1 cell clones and CHI3L1 was found among the up-regulated genes on Th17 cells. Different types of helper T cell clones (TCCs) were then evaluated by Real Time PCR (RT-PCR) for CHI3L1 mRNA expression; protein expression was investigated in cell lysates by western blotting and in cultures supernatants by ELISA. ELISA was also used to measure CHI3L1 in the serum and in the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients.

Results

At mRNA level CHI3L1 was highly expressed by Th17, Th17/Th1, non classic Th1 and even in Th17/Th2 cell clones, whereas it was virtually absent in CD161? classic Th1 and Th2 TCCs. CHI3L1 was also detected in cell culture supernatants of Th17 and Th17-derived cells but not of classic Th1. Moreover CHI3L1 was higher in the SF than in serum of JIA patients, and it positively correlated with the frequency of Th17 and non-classic Th1 cells in SF. CHI3L1 in SF also positively correlated with the C reactive protein (CRP) serum levels, and with the levels of some proinflammatory cytokines, such as IL-6 and p40, which is the common subunit of IL12 and IL23.

Conclusions

Here we describe for the first time CHI3L1 production by T cells owing the Th17 family. Moreover the positive correlation found between the frequency of Th17 and Th17-derived cell subsets and CHI3L1 levels in SF of JIA patients, in agreement with the suggested role of these cells in inflammatory process, candidates CHI3L1 as a possible biological target in JIA treatment.

     

Orphan nuclear hormone receptor RevErbalpha modulates expression from the promoter of the hydratase-dehydrogenase gene by inhibiting peroxisome proliferator-activated receptor alpha-dependent transactivation.

Peroxisome proliferator-activated receptor alpha (PPARalpha) heterodimerizes with the 9-cis-retinoic acid receptor (RXRalpha) to bind to peroxisome proliferator-response elements (PPRE) present in the upstream regions of a number of genes involved in metabolic homeostasis. Among these genes are those encoding fatty acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), the first two enzymes of the peroxisomal beta-oxidation pathway. Here we demonstrate that the orphan nuclear hormone receptor, RevErbalpha, modulates PPARalpha/RXRalpha- dependent transactivation in a response element-specific manner. In vitro binding analysis showed that RevErbalpha bound the HD-PPRE but not the AOx-PPRE. Determinants within the HD-PPRE required for RevErbalpha binding were distinct from those required for PPARalpha/RXRalpha binding. In transient transfections, RevErbalpha antagonized transactivation by PPARalpha/RXRalpha from an HD-PPRE luciferase reporter construct, whereas no effects were observed with an AOx-PPRE reporter construct. These data identify the HD gene as a target for RevErbalpha and illustrate cross-talk between the RevErbalpha and PPARalpha signaling pathways on the HD-PPRE. Our results suggest a novel role for RevErbalpha in regulating peroxisomal beta-oxidation.     

Distribution and hormonal regulation of membrane progesterone receptors β and γ in ciliated epithelial cells of mouse and human fallopian tubes

Background  

The controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma.