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PC2 prohormone convertases are enzymes involved in the proteolytic maturation of neuropeptide precursors. In the present work, a cDNA encoding a PC2-like enzyme (OrlPC2) was cloned from crayfish eyestalk ganglia (medulla terminalis) containing the X-organ, a major neuroendocrine center. The predicted 634 amino acid preproprotein exhibits highest sequence identity, especially in the catalytic domain, with PC2s from arthropods and nematodes, and less with mollusc and vertebrate enzymes. It was demonstrated by in situ hybridization on crayfish medulla terminalis sections that OrlPC2 is expressed in a large number of neuron perikarya, including those producing the well known crustacean hyperglycemic hormone. [Copyright &y& Elsevier]

In mammals, neurokinin B (NKB) is a short peptide encoded by the gene tac3 . It is involved in the brain control of reproduction by stimulating gonadotropin-releasing hormone (GnRH) neurons, mainly via kisspeptin. We investigated tac3 genes and peptides in a basal teleost, the European eel, which shows an atypical blockade of the sexual maturation at a prepubertal stage. Two tac3 paralogous genes ( tac3a and tac3b ) were identified in the eel genome, each encoding two peptides (NKBa or b and NKB-related peptide NKB-RPa or b). Amino acid sequence of eel NKBa is identical to human NKB, and the three others are novel peptide sequences. The four eel peptides present the characteristic C-terminal tachykinin sequence, as well as a similar alpha helix 3D structure. Tac3 genes were identified in silico in 52 species of vertebrates, and a phylogeny analysis was performed on the predicted TAC3 pre-pro-peptide sequences. A synteny analysis was also done to further assess the evolutionary history of tac3 genes. Duplicated tac3 g enes in teleosts likely result from the teleost-specific whole genome duplication (3R). Among teleosts, TAC3b precursor sequences are more divergent than TAC3a, and a loss of tac3b gene would have even occurred in some teleost lineages. NKB-RP peptide, encoded beside NKB by tac3 gene in actinopterygians and basal sarcopterygians, would have been lost in ancestral amniotes. Tissue distribution of eel tac3a and tac3b mRNAs showed major expression of both transcripts in the brain especially in the diencephalon, as analyzed by specific qPCRs. Human NKB has been tested in vitro on primary culture of eel pituitary cells. Human NKB dose-dependently inhibited the expression of lhβ , while having no effect on other glycoprotein hormone subunits ( fshβ, tshβ , and gpα ) nor on gh . Human NKB also dose-dependently inhibited the expression of GnRH receptor ( gnrh- r2). The four eel peptides have been synthesized and also tested in vitro . They all inhibited the expression of both lhβ and of gnrh-r2 . This reveals a potential dual inhibitory role of the four peptides encoded by the two tac3 genes in eel reproduction, exerted at the pituitary level on both luteinizing hormone and GnRH receptor.

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation.

Following the discovery of kisspeptin (Kiss) and its receptor (GPR54 or KissR) in mammals, phylogenetic studies revealed up to three Kiss and four KissR paralogous genes in other vertebrates. The multiplicity of Kiss and KissR types in vertebrates probably originated from the two rounds of whole-genome duplication (1R and 2R) that occurred in early vertebrates. This review examines compelling recent advances on molecular diversity and phylogenetic evolution of vertebrate Kiss and KissR. It also addresses, from an evolutionary point of view, the issues of the structure-activity relationships and interaction of Kiss with KissR and of their signaling pathways. Independent gene losses, during vertebrate evolution, have shaped the repertoire of Kiss and KissR in the extant vertebrate species. In particular, there is no conserved combination of a given Kiss type with a KissR type, across vertebrate evolution. The striking conservation of the biologically active ten-amino-acid C-terminal sequence of all vertebrate kisspeptins, probably allowed this evolutionary flexibility of Kiss/KissR pairs. KissR mutations, responsible for hypogonadotropic hypogonadism in humans, mostly occurred at highly conserved amino acid positions among vertebrate KissR. This further highlights the key role of these amino acids in KissR function. In contrast, less conserved KissR regions, notably in the intracellular C-terminal domain, may account for differential intracellular signaling pathways between vertebrate KissR. Cross talk between evolutionary and biomedical studies should contribute to further understanding of the Kiss/KissR structure-activity relationships and biological functions., (© 2014 Society for Endocrinology.)

A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Anuran skin is known to be a rich source of antimicrobial peptides although their therapeutic potential is often limited due to their toxicity against mammalian cells. The analysis of structure-activity relationships among anuran antimicrobial peptides provided the parameters to construct the "Mutator" tool for improving their selectivity for bacterial cells, by suggesting appropriate point substitutions. Double substitution analogues [K2, K16] of the Xenopus tropicalis peptide XT-7 and [I2, K19] of the Ascaphus truei peptide ascaphin-8 were predicted by this tool to have an increased 'therapeutic index' (TI = HC(50)/MIC for erythrocytes with respect to bacteria) > 80. The mutated peptides were synthesized and respectively found to have experimental TI values > 130 for S. aureus or E. coli, a considerable improvement with respect to TI < 37 for the parent compounds. Circular dichroism studies of the mutated peptides suggested this may in part be due to variations in the α-helical structure. For P. aeruginosa, which is more resistant to XT-7, the TI increased in the mutated peptide from 5 to >270, also due to a significant improvement in minimal inhibitory concentration. We have shown that the Mutator tool is capable of suggesting limited variations in natural anuran peptides capable of increasing peptide selectivity, by decreasing toxicity against mammalian erythrocytes, in general without compromising antibacterial activity. The tool is freely available on the Mutator Web server at http://split4.pmfst.hr/mutator/.

In the crayfish Astacus leptodactylus, as in several crustacean species, the crustacean hyperglycemic hormone is present as two isoforms differing by the chirality of the third residue, a phenylalanine. In the present work, isoforms synthesized full length by solid-phase peptide synthesis have been purified, refolded, the location of the disulfide bridges has been checked, their immunoreactivity against different antibodies have been analyzed and their hyperglycemic activity tested, to ensure the identity of the synthetic peptides with their natural homologs. Different parameters of the hyperglycemic activity of both isoforms were studied. In addition to a difference in the kinetics of hyperglycemia, already known from other studies, it was observed that the dose-response was different depending on the season where experiments were performed, the response being stronger in spring than in autumn, especially for the d-Phe containing isoform. A dosage method based on sandwich enzyme linked immunosorbent assay (ELISA) has been developed to measure hemolymphatic levels of the isoforms after spiking of the animals with one isoform or the other. It was found that hemolymphatic clearance was identical for both isoforms, indicating that their differential effect is not linked to their different lifetime in the hemolymph but may rather rely on other mechanisms such as their binding to different target tissues., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In the present study, an isoform of angiotensin-converting enzyme was characterized from the testis of a decapod crustacean, the crayfish Astacus leptodactylus. Angiotensin-converting enzyme cDNA, obtained by 3'- to 5' RACE of testis RNAs, codes for a predicted one-domain protein similar to the mammalian germinal isoform of angiotensin-converting enzyme. All amino acid residues involved in enzyme activity are highly conserved, and a potential C-terminus transmembrane anchor may be predicted from the sequence. Comparison of this testicular isoform with angiotensin-converting enzyme from other crustaceans, namely Carcinus maenas, Homarus americanus (both reconstituted for this study from expressed-sequence tag data) and Daphnia pulex, suggests that membrane-bound angiotensin-converting enzyme occurs widely in crustaceans, conversely to other invertebrate groups where angiotensin-converting enzyme is predominantly a soluble protein. In situ hybridization and immunohistochemistry performed on testis sections show that angiotensin-converting enzyme mRNA is mainly localized in spermatogonias, whereas protein is present in spermatozoids. By contrast, in vas deferens, immunoreactivity is detected in the seminal fluid rather than in germ cells. Accordingly, angiotensin-converting enzyme activity assays of testis and vas deferens extracts demonstrate that the enzyme is present in the membrane fraction in testis, but in the soluble fraction in vas deferens. Taken together, the results obtained in the present study suggest that, during the migration of spermatozoids from testis to vas deferens, the enzyme is cleaved from the membrane of the germ cells and released into the seminal fluid. To our knowledge, this present study is the first to report such a maturation process for angiotensin-converting enzyme outside of mammals.

The present study was initiated to characterize angiotensin-converting enzyme (ACE) in Crustaceans. Using degenerate DNA primers deduced from consensus sequences located upward and downward from the active site of ACEs from different arthropod species, several tissues from the crayfish Astacus leptodactylus were screened by RT-PCR. Amplicons were obtained from hepatopancreas, testis and hemocytes. Analysis of the predicted protein sequences after cloning and Northern blot experiments revealed an original and complex polymorphism of the ACE-like active site. Two variants were obtained in the hepatopancreas, one displaying a 6.4 kb size transcript, probably corresponding to a double domain ACE, with an unusual active site structure while the other had a transcript size of 2.5 kb, close to the size of the transcript obtained in testis and hemocytes (2 and 3kb, respectively), likely representing single domain enzymes. Functional assays using a synthetic substrate were performed from the different tissues and showed a maximal ACE-like activity associated to membrane fraction from testis and hepatopancreas.

Drugs that disorganize or overstabilize cytoplasmic microtubules (colchicine, vinblastine, griseofulvin, or taxol) can at certain concentrations totally block proliferation of SV40 and polyoma virus transformants with only a minimal effect on the proliferation of the parental rat 3T3 cells. This difference in sensitivity is not due to a more active drug uptake by transformed cells. Examination of cytoplasmic microtubules in actively proliferating normal or transformed cells reveals two categories in each case: cells with microtubules and cells without distinct microtubules. The proportion of cells without distinct microtubules did not differ much between normal and transformed cells. However, transformed cells with a clear microtubule network appear to have fewer microtubules than normal cells. This may contribute to the higher sensitivity of transformed cells. These results render even more rational the use of antimicrotubule drugs in cancer chemotherapy.

Butyrate inhibits proliferation of rat 3T3 cells by blocking the cell cycle in late G1. In these cells, DNA synthesis is completely arrested 24 h after butyrate addition, whereas RNA and protein synthesis proceed unaffected. This partial inhibition of proliferation progressively converts normal cells into giant ones. Transcription and protein synthesis are both more intense in the giant cells than in normal cells. Cell enlargement is inhibited by cell-to-cell contact and the conversion of a normal into a giant cell is reversible. Giant cells may be of use when designing new approaches to the study of cell structure and motility as well as differentiation and proliferation.

When separated and proliferating rat 3T3 cells are treated with butyrate (6 mM), DNA synthesis stops within 24 h, while RNA and protein synthesis proceed unaffected. This gradually converts normal cells into giant ones in the presence of butyrate (volume up to 30-fold greater). The giant cells stop growing when cell to cell contact is established. By studying the rate of synthesis of 300 cell proteins, we have identified two proteins (39 kDa, PI = 6.2, and 60 kDa, pI = 5.6) whose synthesis rises at least 10-fold when DNA replication and mitosis are prevented following intercellular contact or butyrate treatment, and another (64 kDa, pI = 5.6) whose synthesis rises at least 10-fold when cell growth stops by contact, both in the presence of butyrate and in the absence of butyrate (untreated confluent cells). The synthesis of some cellular oncogenes increases when the cell transits from G0 to S phase; the two proteins of 39 and 60 kDa described here are regulated in the opposite direction, their synthesis is enhanced when the cell leaves the proliferation cycle to enter G0.