The l-isoform but not d-isoforms of a JNK inhibitory peptide protects pancreatic beta-cells

The activation of c-jun N-terminal kinase (JNK) in pancreatic islets is associated with impaired function and viability, and JNK inhibitory peptides (JNKIs) are cytoprotective. In particular, l-isoforms of JNKIs were shown to improve islets viability, while the d-retroinverso isoform of JNKI (RI-JNKI), with a higher therapeutic potential due to longer half-life, has not been studied. We compared the cytoprotective properties of L-JNKI and RI-JNKI. Treatment of murine islets with L-JNKI resulted in preservation of islet equivalents and greater percentage of viable beta-cells in culture. In contrast, RI-JNKI was not protective. We found that L-JNKI but not RI-JNKI prevents endogenous c-jun phosphorylation in insulinoma cells. Moreover, RI-JNKI induced islet cells necrosis and activates the p-38 kinase. In conclusion, L-JNKI directly affects beta-cells and ameliorates islet viability and function, while RI-JNKI has toxic effects, limiting its biological application to islet cell biology.     

Structure of rodent helix-destabilizing protein revealed by cDNA cloning

A cDNA library of newborn rat brain poly(A+) RNA in lambda gt 11 was screened with a synthetic oligonucleotide probe corresponding to a five amino acid sequence in the N-terminal region of the calf helix-destabilizing protein, UP1. Six positive phage were isolated after testing 2 X 10(5) recombinants, and each phage was plaque purified. Four of these phage clones were positive with a second oligonucleotide probe corresponding to a 5 amino acid sequence in the C-terminal region of calf UP1; one of the clones positive with both probes was selected for detailed study. This phage, designated lambda HDP-182, contained a 1706-base pair cDNA insert corresponding to an mRNA with a poly(A) sequence at the 3' terminus and a single open reading frame starting 63 bases from the 5' terminus and extending 988 bases. The 3' untranslated region of the mRNA contained 718 bases, including an AAUAAA signal 21 bases from the poly(A) sequence and a 16-residue poly(U) sequence flanked on each side by oligonucleotide repeats. Primer extension analysis of newborn rat brain poly(A+) RNA suggested that the cDNA insert in lambda HDP-182 was full length except for about 35 nucleotide residues missing from the 5' end untranslated region, and Northern blot analysis revealed one relatively abundant mRNA species of approximately the same size as the cDNA insert. The 988-residue open reading frame in the cDNA predicted a 34,215-dalton protein of 320 amino acids. Residues 2 through 196 of this rat protein are identical to the 195-residue sequence of the calf helix-destabilizing protein, UP1. The 124-amino acid sequence in the C-terminal portion of the 34,215-dalton protein is not present in purified calf UP1. This 124-residue sequence has unusual amino acid content in that it is 11% asparagine, 15% serine, and 40% glycine and consists of 16 consecutive oligopeptide repeats. Computer-derived secondary structure predictions for the 34,215-dalton protein revealed two distinct domains consisting of residues 1 through approximately 196 and residues approximately 197 to 320, respectively.     

Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids

Characterization of mammalian heterogeneous nuclear ribonucleoprotein complex protein A1 is reported after large-scale overproduction of the protein in Escherichia coli and purification to homogeneity. A1 is a single-stranded nucleic acid binding protein of 320 amino acids and 34,214 Da. The protein has two domains. The NH2-terminal domain is globular, whereas the COOH-terminal domain of about 120 amino acids has low probability of alpha-helix structure and is glycinerich. Nucleic acid binding properties of recombinant A1 were compared with those of recombinant and natural proteins corresponding to the NH2-terminal domain. A1 bound to single-stranded DNA-cellulose with higher affinity than the NH2-terminal domain peptides. Protein-induced fluorescence enhancement was used to measure equilibrium binding properties of the proteins. A1 binding to poly (ethenoadenylate) was cooperative with the intrinsic association constant of 1.5 X 10(5) M-1 at 0.4 M NaCl and a cooperativity parameter of 30. The NH2-terminal domain peptides bound noncooperatively and with a much lower association constant. With these peptides and with intact A1, binding was fully reversed by increasing [NaCl]; yet. A1 binding was much less salt-sensitive than binding by the NH2-terminal domain peptides. A synthetic polypeptide analog of the COOH-terminal domain was prepared and was found to bind tightly to poly-(ethenoadenylate). The results are consistent with the idea that the COOH-terminal domain contributes to A1 binding through both cooperative protein-protein interaction and direct interaction with the nucleic acid.     

Ubiquitous genetic diversity in ISSR markers between and within populations of the asexually producing moss <Emphasis Type="Italic">Pleurochaete squarrosa</Emphasis>

This paper provides an analysis of genetic variability in local populations of the clonal moss Pleurochaete squarrosa, a Mediterranean moss typical of post-fire recovery, and characterised by asexual reproduction. ISSR (Internal Simple Sequence Repeats) primers and trnL_UAA (intron of plastid gene for Leu tRNA) length polymorphism were employed to evaluate genetic structure in five southern Italy populations of this moss. Both molecular tools highlight high values of genetic diversity with geographic structure of sampled populations and a low gene flow among the investigated sites. Gene diversity is significant at every hierarchical level of sampling, and generally increases from lower to higher hierarchical levels. Among other factors, the ubiquitous genetic variability detected in P. squarrosa can be related to continuous, occasionally massive, short-range recruitment of propagules, and to the high degree of intermingling, both favoured by the modality of vegetative reproduction and growth occurring in the species.     

Mechanisms of the antinociceptive action of (?) Epicatechin obtained from the hydroalcoholic fraction of Combretum leprosum Mart & Eic in rodents

ABSTRACT: BACKGROUND: The mechanisms of the antinociceptive activity of () epicatechin (EPI), a compound isolated from the hydroalcoholic fraction of Combreum leprosum Mart & Eicher. METHODS: were assessed in the model of chemical nociception induced by glutamate (20 mumol/paw). To evaluate the mechanisms involved, the animals , male Swiss mice (25-30 g), received EPI (50 mg/kg p.o.) after pretreatment with naloxone (2 mg/kg s.c. opioid antagonist), glibenclamide (2 mg/kg s.c. antagonist K + channels sensitive to ATP), ketanserin (0.3 mg/kg s.c. antagonist of receptor 5-HT2A), yoimbine (0.15 mg/kg s.c. alpha2 adrenergic receptor antagonist), pindolol (1 mg/kg s.c. 5-HT1a/1b receptor antagonist), atropine (0.1 mg/kg s.c. muscarinic antagonist) and caffeine (3 mg/kg s.c. adenosine receptor antagonist), ondansetron (0.5 mg/kg s.c. for 5-HT3 receptor) and L-arginine (600 mg/kg i.p.). RESULTS: The antinociceptive effect of EPI was reversed by pretreatment with naloxone and glibenclamide, ketanserin, yoimbine, atropine and pindolol, which demonstrates the involvement of opioid receptors and potassium channels sensitive to ATP, the serotoninergic (receptor 5HT1A and 5HT2A), adrenergic (receptor alpha 2) and cholinergic (muscarinic receptor) systems in the activities that were observed. The effects of EPI, however, were not reversed by pretreatment with caffeine, L-arginine or ondansetron, which shows that there is no involvement of 5HT3 receptors or the purinergic and nitrergic systems in the antinociceptive effect of EPI. In the Open Field and Rotarod test, EPI had no significant effect, which shows that there was no central nervous system depressant or muscle relaxant effect on the results. CONCLUSIONS: This study demonstrates that the antinociceptive activity of EPI in the glutamate model involves the participation of the opioid system, serotonin, adrenergic and cholinergic.     

Human hnRNP protein A1: A model polypeptide for a structural and genetic investigation of a broad family of RNA binding proteins

The hnRNP fiber is the substrate on which pre-mRNA processing occurs. The protein moiety of the fiber (hnRNP proteins) constitutes a broad family of RNA binding proteins that revealed, upon molecular analysis, a number of interesting features.Heterogeneous nuclear ribonucleoprotein A1 is a major component of the human hnRNP complex. In recent years this protein has attracted great attention because of several emerging evidences of its direct involvement in pre-mRNA processing and it has become one of the best characterized RNA binding proteins. Detailed knowledge of the structure of protein A1 has laid the basis for the understanding of its function, and for this reason A1 can be considered as a model polypeptide for the investigation of a large number of RNA binding proteins.In this work we report recent findings regarding the binding properties of protein A1 as well as new data on the gene structure of A1 and of its closely related hnRNP protein A2. Our results show that a single A1 molecule contains the determinants for simultaneous binding of two single-stranded nucleic acid molecules and we demonstrate that the glycine-rich domain of A1, isolated from the rest of the molecule, is capable of sustaining protein-protein interactions. These features probably account for the reannealing activity of the protein and for its capacity to modulate the binding of snRNPs to intron sequencesin vitro. Comparison of A1 and A2 gene sequences revealed a remarkable conservation of the overall structural organization, suggesting important functions for the different structural elements.