Effects of gonadotropin-releasing hormone variants on plasma and testicular androgen levels in intact and hypophysectomized male frogs, Rana esculenta.

The effects of vertebrate gonadotropin-releasing hormone (GnRH) variants on plasma and testicular androgen level in intact and hypophysectomized (PDX) male frogs, Rana esculenta, have been investigated. In intact animals, mammalian (m)-GnRH, m-GnRH analog (buserelin), salmon (s)-GnRH, chicken (c) I-GnRH, cII-GnRH, D-Arg6-cII-GnRH (cII-GnRHA), and lamprey (l)-GnRH (1.5 micrograms and 6 micrograms, total dose given on alternate days for 5 days) were able to enhance androgen production showing that specificity of pituitary responsiveness to GnRH variants appears to be low. Chicken II-GnRH was more effective than s-GnRH in eliciting testicular and circulatory androgen level increase. Moreover, in animals treated with 6 micrograms of cII-GnRH and s-GnRH in combination, androgens decreased as compared with animal treated with cII-GnRH only, suggesting that GnRH receptors bind preferentially the s-GnRH form. In PDX animals, buserelin (1.5 and 6 micrograms), cII-GnRH, and its analog (6 micrograms) were able to increase plasma androgen levels whereas testis androgen concentrations were increased by cII-GnRH (1.5 and 6 micrograms), D-Arg6-cII-GnRHA, and buserelin (6 micrograms). Since androgen production in PDX animals is influenced especially by peptides sharing cII-GnRH structure, it is suggested that a testicular cII-GnRH-like material play a role as local modulator of the gonadal activity in Rana esculenta.     

Molecular evolution of olfactomedin

Olfactomedin is a secreted polymeric glycoprotein of unknown function,originally discovered at the mucociliary surface of the amphibian olfactoryneuroepithelium and subsequently found throughout the mammalian brain. As afirst step toward elucidating the function of olfactomedin, itsphylogenetic history was examined to identify conserved structural motifs.Such conserved motifs may have functional significance and provide targetsfor future mutagenesis studies aimed at establishing the function of thisprotein. Previous studies revealed 33% amino acid sequence identity betweenrat and frog olfactomedins in their carboxyl terminal segments. Furtheranalysis, however, reveals more extensive homologies throughout themolecule. Despite significant sequence divergence, cysteines essential forhomopolymer formation such as the CXC motif near the amino terminus areconserved, as is the characteristic glycosylation pattern, suggesting thatthese posttranslational modifications are essential for function.Furthermore, evolutionary analysis of a region of 53 amino acids of fish,frog, rat, mouse, and human olfactomedins indicates that an ancestralolfactomedin gene arose before the evolution of terrestrial vertebrates andevolved independently in teleost, amphibian, and mammalian lineages.Indeed, a distant olfactomedin homolog was identified in Caenorhabditiselegans. Although the amino acid sequence of this invertebrate protein islonger and highly divergent compared with its vertebrate homologs, theprotein from C. elegans shows remarkable similarities in terms of conservedmotifs and posttranslational modification sites. Six universally conservedmotifs were identified, and five of these are clustered in the carboxylterminal half of the protein. Sequence comparisons indicate that evolutionof the N-terminal half of the molecule involved extensive insertions anddeletions; the C-terminal segment evolved mostly through point mutations,at least during vertebrate evolution. The widespread occurrence ofolfactomedin among vertebrates and invertebrates underscores the notionthat this protein has a function of universal importance. Furthermore,extensive modification of its N-terminal half and the acquisition of aC-terminal SDEL endoplasmic-reticulum- targeting sequence may have enabledolfactomedin to adopt new functions in the mammalian central nervoussystem.     

Ibuprofen binding to secondary sites allosterically modulates the spectroscopic and catalytic properties of human serum heme-albumin

The ibuprofen primary binding site FA3-FA4 is located in domain III of human serum albumin (HSA), the secondary clefts FA2 and FA6 being sited in domains I and II. Here, the thermodynamics of ibuprofen binding to recombinant Asp1-Glu382 truncated HSA (tHSA)-heme-Fe(III) and nitrosylated tHSA-heme-Fe(II), encompassing domains I and II only, is reported. Moreover, the allosteric effect of ibuprofen on the kinetics of tHSA-heme-Fe(III)-mediated peroxynitrite isomerization and nitrosylated tHSA-heme-Fe(II) denitrosylation has been investigated. The present data indicate, for the first time, that the allosteric modulation of tHSA-heme and HSA-heme reactivity by ibuprofen depends mainly on drug binding to the FA2 and FA6 secondary sites rather than drug association with the FA3-FA4 primary cleft. Thus, tHSA is a valuable model with which to investigate the allosteric linkage between the heme cleft FA1 and the ligand-binding pockets FA2 and FA6, all located in domains I and II of (t)HSA.     

Hemoglobin and heme scavenging

Release of hemoglobin into plasma is a physiological phenomenon associated with intravascular hemolysis. In plasma, stable haptoglobin-hemoglobin complexes are formed and these are subsequently delivered to the reticulo-endothelial system by CD163 receptor-mediated endocytosis. Heme arising from the degradation of hemoglobin, myoglobin, and of enzymes with heme prosthetic groups could be delivered in plasma. Albumin, haptoglobin, hemopexin, and high and low density lipoproteins cooperate to trap the plasma heme, thereby ensuring its complete clearance. Then hemopexin releases the heme into hepatic parenchymal cells only after internalization of the hemopexin-heme complex by CD91 receptor-mediated endocytosis. Moreover, alpha1-microglobulin contributes to heme degradation by a still unknown mechanism, with the concomitant formation of heterogeneous yellow-brown kynurenine-derived chromophores which are very tightly bound to amino acid residues close to the rim of the lipocalin pocket. During hemoglobin synthesis, the erythroid alpha-chain hemoglobin-stabilizing protein specifically binds free alpha-hemoglobin subunits limiting the free protein toxicity. Although highly toxic because capable of catalyzing free radical formation, heme is also a major and readily available source of iron for pathogenic organisms. Gram-negative bacteria pick up the heme-bound iron through the secretion of a hemophore that takes up either free heme or heme bound to heme-proteins and transports it to a specific receptor, which, in turn, releases the heme and hence iron into the bacterium. Here, hemoglobin and heme trapping mechanisms are summarized.     

Proteomics in Parkinson's disease: An unbiased approach towards peripheral biomarkers and new therapies

Parkinson's disease is the most common neurodegenerative movement disorder, affecting about 6 million people worldwide with a slow progression of the symptoms. Its prevalence is expected to double in the most populated areas within the next two decades, according to increasing aged population. Consequently, Parkinson's disease is a socio-economic trouble and a major challenge for the public health system. Parkinson's disease treatment is merely symptomatic, as clinical symptoms appear when about 70% of the involved neurons are lost and potential disease-modifying/neuroprotective therapies would have no effect. In turn, the availability of an objective measure that allows early diagnosis would strongly impact on the costs that biotech- and pharma-companies will sustain in order to develop disease-modifying therapies. The establishment of suitable models to investigate the mechanisms of Parkinson's disease progression and, on the other hand, the discovery and validation of selective and specific molecular biomarkers for early and differential diagnosis are indeed two important goals for a better management of the disease. In this review, we focus on cellular and animal models of Parkinson's disease by describing their advantages and limitations as useful tools to identify pathogenetic pathways that deserve further exploitation. In parallel, we discuss how proteomics may provide a potent tool to observe altered pathways in models or altered biomarkers in patients with an unbiased, hypothesis-free approach.