Combinatorial synthesis, selection, and properties of esterase peptide dendrimers

A 65,536-member combinatorial library of peptide dendrimers was prepared by split-and-mix synthesis and screened on solid support for esterolytic activity in aqueous buffer using 8-butyryloxypyrene-1,3,6-trisulfonate (2) as a fluorogenic substrate. Active sequences were identified by analysis of fluorescent beads. The corresponding dendrimers were resynthesized by solid-phase synthesis, cleaved from the resin, and purified by preparative reverse-phase HPLC. The dendrimers showed the expected catalytic activity in aqueous buffer. Catalysis was studied against a pannel of fluorogenic 8-acyloxypyrene-1,3,6-trisulfonate substrates. The catalytic peptide dendrimers display enzyme-like kinetics in aqueous buffer with substrate binding in the range K(M) approximately 0.1 mM, catalytic rate constants k(cat) approximately 0.1 min(-1), and specific rate accelerations over background up to k(cat)/k(uncat) = 10,000.     

Characterization of the trans Watson-Crick GU base pair located in the catalytic core of the antigenomic HDV ribozyme

The HDV ribozyme's folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U(23) and G(28) nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U(23) and G(28) can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction.     

The E3-ubiquitin ligase TRIM50 interacts with HDAC6 and p62, and promotes the sequestration and clearance of ubiquitinated proteins into the aggresome

In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.     

The elongation rate at the base of a maize leaf shows an invariant pattern during both the steady-state elongation and the establishment of the elongation zone.

Spatial and temporal analyses of elongation and cell length of monocotyledon leaves have most often been performed during the period when leaves are visible and elongate at a constant rate (steady-state). In the present study, the focus was on the earlier stages, during the establishment of the elongation zone. Regardless of leaf development stage, the segment located between 0 and 35 mm from the leaf insertion point had a relative elongation rate that increased with distance from insertion point ('accelerating zone') while the segment located further than 35 mm had a relative elongation rate that decreased ('decelerating zone'). This stable pattern held for both young, non-emerged leaves, where it was restricted to the portion corresponding to the length of the blade, and for leaves during steady-state elongation. In the same way, the profile of cell length was essentially the same during early development and during steady-state elongation. The results of a temporal analysis of whole-leaf elongation rate, carried out in the field and in the greenhouse at different light intensities were consistent with a time-invariant pattern of elongation. Whole-leaf relative elongation rate increased with time until the leaf reached 30-40 mm length (although at different leaf ages depending on conditions), and declined afterwards. These results suggest that the patterns governing the elongation rate of a sector of a maize leaf are independent of the leaf developmental stage but depend on sector position only.     

Molecular evidence of true-to-type propagation of a 3-year-old Norway spruce through somatic embryogenesis

Some progress has recently been made in establishing a system enabling somatic embryos to be initiated from old elite trees. We report here the first results demonstrating the molecular conformity of somatic embryos initiated from increasingly old Norway spruce (Picea abies (L.) Karst.), as indicated by an analysis of six nuclear microsatellites that showed an extremely high tendency to mutate during in vitro culture. No allelic difference was detected at these loci among plants regenerated from somatic embryos or between the former and mother plants. Moreover, phenotypical data acquired on the same 3- to 9-year-old plants growing in the field sampled for molecular analyses were totally in accord with the results on molecular conformity.     

Effects of hyper- and hypoprolactinemia on gonadotropin secretion, rat testicular luteinizing hormone/human chorionic gonadotropin receptors and testosterone production by isolated Leydig cells

The effect of prolactin (Prl) on gonadotropin secretion, testicular luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors, and testosterone (T) production by isolated Leydig cells has been studied in 60-day-old rats treated for 4 days, 4 and 8 weeks with sulpiride (SLP), a dopaminergic antagonist, or for 4 days and 4 weeks with bromocriptine (CB), a dopaminergic agonist. Plasma Prl concentrations were significantly greater in the SLP groups (204 +/- 6 ng/ml) and lower in the CB groups (3.0 +/- 0.2 ng/ml) than those measured in the control groups (54 +/- 6 ng/ml). The plasma concentrations of gonadotropin were not affected by a 4-day treatment with SLP or CB, nor were they after a 4-week treatment with CB. However, the hyperprolactinemia induced by an 8-week treatment with SLP was associated with a reduced secretion of gonadotropin (LH, 16 +/- 4 vs. 35 +/- 6 ng/ml; FSH, 166 +/- 12 vs. 307 +/- 14 ng/ml). In SLP-induced hyperprolactinemia, a 30% increase in the density of the LH/hCG testicular binding sites was observed (178 +/- 12 fmol/mg protein), whereas a 60% decrease was measured in hypoprolactinemia (55 +/- 5 vs. control 133 +/- 5 fmol/mg protein). Plasma T levels were increased in 4-day and 4-week hyperprolactinemic animals (4.3 +/- 0.4 and 3.9 +/- 0.4 ng/ml, respectively), but returned to normal levels in the 8-week group (3.0 +/- 0.5 vs. C: 2.3 +/- 0.2 ng/ml). No T modifications were observed in hypoprolactinemic animals. Two distinct populations of Leydig cells (I and II) were obtained by centrifugation of dispersed testicular cells on a 0-45% continuous Metrizamide gradient. Both possess LH/hCG binding sites. However, the T production from Leydig cells of population II increased in the presence of hCG, whereas that of cell population I which also contain immature germinal cells did not respond. The basal and stimulated T secretions from cell populations I and II obtained from CB-treated animals were similar to controls, whereas from 4 days to 8 weeks of hyperprolactinemia, basal and hCG induced T productions from cell population II decreased progressively. These data show that hyperprolactinemia causes, in a time-dependent manner, a trophic effect on the density of LH/hCG testicular receptors; reduces basal and hCG-stimulated T production from isolated Leydig cells type II; and results in an elevated plasma T concentration which decreases with time. The latter suggests a slower T catabolism and/or an impaired peripheral conversion of T into 5 alpha-dihydrotestosterone (DHT). Although hypoprolactinemia is associated with a marked reduction in testicular LH receptors, it does not affect T production.     

Variable thermal stress tolerance of the reef-associated symbiont-bearing foraminifera Amphistegina linked to differences in symbiont type

Coral Reefs - Adaptation, acclimatization and symbiont diversity are known to regulate thermal tolerance in corals, but the role of these mechanisms remains poorly constrained in other...     

Characterization of the cDNA Coding for Rat Brain Cysteine Sulfinate Decarboxylase

Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression.