Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

Stem cells possess significant age-dependent differences in their immune-response profile. These differences were analysed by Next-Generation Sequencing of six age groups from bone marrow mesenchymal stem cells. A total of 9,628 genes presenting differential expression between age groups were grouped into metabolic pathways. We focused our research on young, pre-pubertal and adult groups, which presented the highest amount of differentially expressed genes related to inflammation mediated by chemokine and cytokine signalling pathways compared with the newborn group, which was used as a control. Extracellular vesicles extracted from each group were characterized by nanoparticle tracking and flow cytometry analysis, and several micro-RNAs were verified by quantitative real-time polymerase chain reaction because of their relationship with the pathway of interest. Since miR-21-5p showed the highest statistically significant expression in extracellular vesicles from mesenchymal stem cells of the pre-pubertal group, we conducted a functional experiment inhibiting its expression and investigating the modulation of Toll-Like Receptor 4 and their link to damage-associated molecular patterns. Together, these results indicate for the first time that mesenchymal stem cell-derived extracellular vesicles have significant age-dependent differences in their immune profiles.

A PII-like protein in Arabidopsis: putative role in nitrogen sensing.

PII is a protein allosteric effector in Escherichia coli and other bacteria that indirectly regulates glutamine synthetase at the transcriptional and post-translational levels in response to nitrogen availability. Data supporting the notion that plants have a nitrogen regulatory system(s) includes previous studies showing that the levels of mRNA for plant nitrogen assimilatory genes such as glutamine synthetase (GLN) and asparagine synthetase (ASN) are modulated by carbon and organic nitrogen metabolites. Here, we have characterized a PII homolog (GLB1) in two higher plants, Arabidopsis thaliana and Ricinus communis (Castor bean). Each plant PII-like protein has high overall identity to E. coli PII (50%). Western blot analyses reveal that the plant PII-like protein is a nuclear-encoded chloroplast protein. The PII-like protein of plants appears to be regulated at the transcriptional level in that levels of GLB1 mRNA are affected by light and metabolites. To initiate studies of the in vivo function of the Arabidopsis PII-like protein, we have constructed transgenic lines in which PII expression is uncoupled from its native regulation. Analyses of these transgenic plants support the notion that the plant PII-like protein may serve as part of a complex signal transduction network involved in perceiving the status of carbon and organic nitrogen. Thus, the PII protein found in archaea, bacteria, and now in higher eukaryotes (plants) is one of the most widespread regulatory proteins known, providing evidence for an ancestral metabolic regulatory mechanism that may have existed before the divergence of these three domains of life.

Involvement of two aspartate residues of Rubisco activase in coordination of the ATP gamma-phosphate and subunit cooperativity.

Aspartate residues are involved in coordination of the nucleotide-metal of several nucleotide triphosphatases. To examine interactions between Rubisco activase and ATP, site-directed mutations were made at two species-invariant aspartate residues, D174 and D231. In the absence of the magnesium cofactor, the mutant proteins D231R, D174Q, and D174A, but not D174E, bound ATP with higher affinity than did wild-type. In the presence of Mg2+, the affinity for ATP of D231R was further increased, but was reduced with mutations at D174. Although all mutants bound ATP, only D174E aggregated in response to ATP/Mg2+ and retained partial ATPase and Rubisco activation activities. In mixing experiments, the catalytically competent D174E stimulated wild-type ATPase activity, whereas the mutants lacking ATPase activity were inhibitory to wild-type enzyme and prevented aggregation. These results are consistent with a mechanism for activase that involves ATP-binding, subunit aggregation and ATP hydrolysis as sequential steps in the catalytic mechanism. The results also indicated that precise coordination of the gamma-phosphate is required for aggregation and depends on D174 and D231. To account for the pronounced cooperativity of Rubisco activase subunits, we suggest that coordination of the ATP gamma-phosphate may involve participation of residues from adjacent subunits.

The Two Forms of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase Differ in Sensitivity to Elevated Temperature.

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase often consists of two polypeptides that arise from alternative splicing of pre-mRNA. In this study recombinant versions of the spinach (Spinacea oleracea L.) 45- and 41-kD forms of activase were analyzed for their response to temperature. The temperature optimum for ATP hydrolysis by the 45-kD form was 45[deg]C, approximately 13[deg]C higher than the 41-kD form. When the two forms were mixed, the temperature response of the hybrid enzyme was similar to the 45-kD form. In the absence of adenine nucleotide, preincubation of either activase form at temperatures above 25[deg}C inactivated ATPase activity. Adenosine 5[prime]-([gamma]-thio)triphosphate, but not ADP, significantly enhanced the thermostability of the 45-kD form but was much less effective for the 41-kD form. Intrinsic fluorescence showed that the adenosine 5[prime]-([gamma]-thio)triphosphate-induced subunit aggregation was lost at a much lower temperature for the 41-kD than for the 45-kD form. However, the two activase forms were equally susceptible to limited proteolysis after heat treatment. The results indicate that (a) the 45-kD form is more thermostable than, and confers increased thermal stability to, the 41-kD form, and (b) a loss of subunit interactions, rather than enzyme denaturation, appears to be the initial cause of temperature inactivation of activase.

Activation of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) involves Rubisco activase Trp16.

The role of the N-terminal region of tobacco Rubisco activase in ATP hydrolysis and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation was examined by construction of mutant proteins. Deletion of the first 50 amino acids of Rubisco activase almost completely eliminated the ability to activate Rubisco, without changing the ATP-hydrolyzing and self-associating properties of the enzyme. Thus, the N-terminus of Rubisco activase is distinct from the ATP-hydrolyzing domain and is required for Rubisco activation. Directed mutagenesis of the species-invariant tryptophan residue at position 16 inhibited Rubisco activation but not the binding or hydrolysis of ATP. The ability to activate Rubisco was less severely inhibited when Trp was replaced by a Tyr or Phe than by an Ala or Cys, indicating that an aromatic residue at position 16 and particularly a Trp is required for proper activation of Rubisco. Fluorescence quenching of the 7-nitrobenz-2-oxa-1,3-diazole-modified W16C mutant upon addition of nucleotide suggested that position 16 becomes more solvent accessible in response to nucleotide binding. However, changes in the intrinsic fluorescence of truncated and Trp16 mutants upon addition of ATP were similar to those of the wild type, evidence that Trp16 is not the residue reporting the conformational change that accompanies subunit association.

An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog.

Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with approximately 67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution.

Expressed Sequence Tags from Developing Castor Seeds.

To expand the availability of genes encoding enzymes and structural proteins associated with storage lipid synthesis and deposition, partial nucleotide sequences, or expressed sequence tags (ESTs), were obtained for 743 cDNA clones derived from developing seeds of castor (Ricinus communis L.). Enrichment for seed-specific cDNA clones was obtained by selecting clones that did not detectably hybridize to first-strand cDNA from leaf mRNA. Similarly, clones that hybridized to storage proteins or other highly abundant mRNA species from developing seeds were selected against. To enrich for endomembrane-associated proteins, some clones were selected for sequencing by immunological screening with antibodies prepared against partially purified endoplasmic reticulum membranes. Comparison of the deduced amino acid sequences of the ESTs with the public data bases resulted in the assignment of putative identities of 49% of the clones selected by differential hybridization and 71% of the clones selected by immunological screening. Open reading frames in 100 of the ESTs exhibited higher homology to 78 different nonplant gene products than to any previously known plant gene product.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves.