β-Amino acid residues: Conformational characterization of an N- and C-protected homo-β-(S)-leucine

Summary An N- and C-protected derivative ofhomo-β-leucine, Fmoc-homo-β-(S)-leucine methyl ester, synthesized from the corresponding proteinogenic parent α-amino acid in enantiopure form has been fully characterized in the solid state by X-ray diffraction analysis. The crystal conformation of this new residue indicates and extended conformation for thishomo-β-residue, with the ϕ torsion angle being more constrained than the μ and ψ angles.     

Iron-nickel mixed metal nitrido clusters: Synthesis and solid state structure of the anions [HFe5NiN(CO)14] and [HFe4Ni2N(CO)13]

The two clusters [HFe₅NiN(CO)₁₄]²⁻ (1) and [HFe₄Ni₂N(CO)₁₃]²⁻ (2) were obtained by reaction of [Fe₄N(CO)₁₂]⁻ and [Ni₆(CO)₁₂]²⁻ in refluxing MeCN and EtCN, respectively, along with other Fe-Ni mixed metal clusters. Their solid state structures were determined on the [PPh₄]⁺ salts, and both have an octahedral metal cage, containing an interstitial nitrogen atom. The two Ni atoms in 2 are cis, with a Ni-Ni separation of 2.724(1) Å. The two anions have different stereochemistry of the carbonyl ligands: in 1, five CO’s are semi-bridging, and the remaining nine are terminal; in 2 there are three asymmetric bridging and ten terminal ligands (two for each iron and one for each nickel). The hydride ligands were located in the final difference maps, both bridging a Ni-Fe edge of the clusters but, thanks to the better quality of the diffraction data, the metal-hydrogen distances were refined only in 2. In this cluster, the Fe-H and Ni-H bond lengths are 1.77(2) and 1.79(2) Å, respectively.     

Estradiol Mediates Effects of Testosterone on Vasotocin Immunoreactivity in the Adult Quail Brain

In adult male quail, the activation of sexual behavior by testosterone (T) is mediated at the cellular level by the interaction of T metabolites with intracellular steroid receptors. In particular, the aromatization of T into an estrogen plays a key limiting role. Nonaromatizable androgens such 5alpha-dihydrotestosterone (DHT) synergize with estradiol (E2) to activate the behavior. Given that the density of vasotocin (VT) immunoreactive structures is increased by T in adult male quail and that VT injections affect male behavior, we wondered whether the expression of VT is also affected by T metabolites such as E2 and DHT. We analyzed here, in castrated male quail, the effects of a treatment with T, E2, DHT, or E2 + DHT on sexual behavior and brain VT immunoreactivity. The restoration by T of the VT immunoreactivity in the medial preoptic nucleus, bed nucleus striae terminalis, and lateral septum of castrated male quail could be fully mimicked by a treatment with E2. The androgen DHT had absolutely no effect on the VT immunoreactivity in these conditions and, at the doses used here, DHT did not synergize with E2 to enhance the density of VT immunoreactive structures. These effects of T metabolites in the brain were not fully correlated with their effects on the activation of male copulatory behavior, suggesting that the increase in VT expression in the brain does not represent a necessary step for the activation of behavior. Although VT expression in the medial preoptic nucleus and bed nucleus striae terminalis is often tightly correlated with the expression of male copulatory behavior, VT presumably does not represent simply one step in the biochemical cascade of events that is induced by T in the brain and leads to the expression of male sexual behavior.     

Mechanisms transducing the aldosterone secretagogue signal of endothelins in the human adrenal cortex

Evidence has been provided that the 21-amino acid hypertensive peptide endothelin (ET)-1 exerts a potent secretagogue effect on human adrenocortical zona glomerulosa (ZG), acting through two receptor subtypes, called ET(A) and ET(B), the signaling mechanism(s) of which has (have) not yet been investigated. Collagenase dispersed human ZG cells were obtained from normal adrenals of patients undergoing nephrectomy/adrenalectomy for renal cancer. The selective ET(A)- and ET(B)-receptor activation was obtained by exposing dispersed cells to ET-1 plus the ET(B)-receptor antagonist BQ-788 and to the ET(B)-receptor agonist BQ-3020, respectively. The phospholipase (PL) C inhibitor U-73122 abolished ET(A) receptor-mediated secretory response, but only partially prevented the ET(B) receptor-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin, the calmodulin inhibitor W-7 and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes. When added together, calphostin-C and wortmannin or W-7 abolished ET(A)-mediated secretory response, but only decreased ET(B)-mediated one. The ET(B) receptor-, but not the ET(A) receptor-mediated aldosterone response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. ET(A)-receptor activation raised inositol triphosphate (IP(3)) production from dispersed ZG cells, while ET(B)-receptor stimulation enhanced both IP(3) and prostaglandin-E(2) production. Collectively, our findings indicate that ETs stimulate aldosterone secretion from human ZG cells, acting through ET(A) receptors exclusively coupled to PLC/PKC-dependent pathway and ET(B) receptors coupled to both PLC/PKC- and COX-dependent cascades.     

UVA light stimulates the production of cathepsin G and elastase-like enzymes by dermal fibroblasts: a possible contribution to the remodeling of elastotic areas in sun-damaged skin

Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sun-exposed and non-exposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast-like cells from specimens of sun-exposed areas. Little or no reaction was found in biopsies of sun-protected skin. Fibroblast cultures from sun-exposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sun-protected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sun-protected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastase-like activity expressed by fibroblasts can be attributed to at least two enzymes.     

Hematopoietic growth factors in autologous transplantation

Hematopoietic growth factors (HGFs) sustain the survival, proliferation and differentiation of hematopoietic stem cells and some functions of mature blood cells. In man several HGFs have been characterised and cloned so far, and this has allowed investigators to confer the rationale for the clinical application of these molecules in hematology and oncology. In particular G-CSF and GM-CSF are currently utilised to abrogate the hematological toxicity of chemotherapy for standard and dose-intensified therapy, neutropenia following bone marrow and peripheral blood stem cell transplantation. Moreover there has recently been great interest in the ex vivo expansion of hematopoietic stem and progenitor cells for a variety of applications, such as in vitro tumor cell purging or for reducing the volume of blood processed by the leukapheresis. Several combinations of HGFs have been described to sustain the ex vivo survival and proliferation of these cells disclosing new opportunities in the field of stem cells transplants.     

Using phylogeny to improve genome-wide distant homology recognition

The gap between the number of known protein sequences and structures continues to widen, particularly as a result of sequencing projects for entire genomes. Recently there have been many attempts to generate structural assignments to all genes on sets of completed genomes using fold-recognition methods. We developed a method that detects false positives made by these genome-wide structural assignment experiments by identifying isolated occurrences. The method was tested using two sets of assignments, generated by SUPERFAMILY and PSI-BLAST, on 150 completed genomes. A phylogeny of these genomes was built and a parsimony algorithm was used to identify isolated occurrences by detecting occurrences that cause a gain at leaf level. Isolated occurrences tend to have high e-values, and in both sets of assignments, a sudden increase in isolated occurrences is observed for e-values >10⁻⁸ for SUPERFAMILY and >10⁻⁴ for PSI-BLAST. Conditions to predict false positives are based on these results. Independent tests confirm that the predicted false positives are indeed more likely to be incorrectly assigned. Evaluation of the predicted false positives also showed that the accuracy of profile-based fold-recognition methods might depend on secondary structure content and sequence length. We show that false positives generated by fold-recognition methods can be identified by considering structural occurrence patterns on completed genomes; occurrences that are isolated within the phylogeny tend to be less reliable. The method provides a new independent way to examine the quality of fold assignments and may be used to improve the output of any genome-wide fold assignment method.     

Aquaporin inhibition changes protein phosphorylation pattern following sperm motility activation in fish

Our previous studies demonstrated that osmolality is the key signal in sperm motility activation in Sparus aurata spermatozoa. In particular, we have proposed that the hyper-osmotic shock triggers water efflux from spermatozoa via aquaporins. This water efflux determines the cell volume reduction and, in turn, the rise in the intracellular concentration of ions. This increase could lead to the activation of adenylyl cyclase and of the cAMP-signaling pathway, causing the phosphorylation of sperm proteins and then the initiation of sperm motility. This study confirms the important role of sea bream AQPs (Aqp1a and Aqp10b) in the beginning of sperm motility. In fact, when these proteins are inhibited by HgCl₂, the phosphorylation of some proteins (174 kDa protein of head; 147, 97 and 33 kDa proteins of flagella), following the hyper-osmotic shock, was inhibited (totally or partially). However, our results also suggest that more than one transduction pathways could be activated when sea bream spermatozoa were ejaculated in seawater, since numerous proteins showed an HgCl₂(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified.     

Identification of differentially expressed microRNAs by microarray: a possible role for microRNA genes in pituitary adenomas

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by targeting mRNA. It has been demonstrated that miRNA expression is altered in many human cancers, suggesting that they may play a role in human neoplasia. To determine whether miRNA expression is altered in pituitary adenomas, we analyzed the entire miRNAome in 32 pituitary adenomas and in 6 normal pituitary samples by microarray and by Real-Time PCR. Here, we show that 30 miRNAs are differentially expressed between normal pituitary and pituitary adenomas. Moreover, 24 miRNAs were identified as a predictive signature of pituitary adenoma and 29 miRNAs were able to predict pituitary adenoma histotype. miRNA expression could differentiate micro- from macro-adenomas and treated from non-treated patient samples. Several of the identified miRNAs are involved in cell proliferation and apoptosis, suggesting that their deregulated expression may be involved in pituitary tumorigenesis. Predictive miRNAs could be potentially useful diagnostic markers, improving the classification of pituitary adenomas.     

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins

Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.