Novel Protocol for the Chemical Synthesis of Crustacean Hyperglycemic Hormone Analogues — An Efficient Experimental Tool for Studying Their Functions

The crustacean Hyperglycemic Hormone (cHH) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. Here we report on the first chemical synthesis of three distinct isoforms of the cHH of Astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation. The synthetic 72 amino acid long peptide amides, containing L- or D-Phe3 and (Glp1, D-Phe3) were tested for their biological activity by means of homologous in vivo bioassays. The hyperglycemic activity of the D-isoforms was significantly higher than that of the L-isoform, while the presence of the N-terminal Glp residue had no influence on the peptide activity. The results show that the presence of D-Phe3 modifies the cHH functionality, contributing to the diversification of the hormone pool

A Computational Approach to Finding Novel Targets for Existing Drugs

Repositioning existing drugs for new therapeutic uses is an efficient approach to drug discovery. We have developed a computational drug repositioning pipeline to perform large-scale molecular docking of small molecule drugs against protein drug targets, in order to map the drug-target interaction space and find novel interactions. Our method emphasizes removing false positive interaction predictions using criteria from known interaction docking, consensus scoring, and specificity. In all, our database contains 252 human protein drug targets that we classify as reliable-for-docking as well as 4621 approved and experimental small molecule drugs from DrugBank. These were cross-docked, then filtered through stringent scoring criteria to select top drug-target interactions. In particular, we used MAPK14 and the kinase inhibitor BIM-8 as examples where our stringent thresholds enriched the predicted drug-target interactions with known interactions up to 20 times compared to standard score thresholds. We validated nilotinib as a potent MAPK14 inhibitor in vitro (IC50 40 nM), suggesting a potential use for this drug in treating inflammatory diseases. The published literature indicated experimental evidence for 31 of the top predicted interactions, highlighting the promising nature of our approach. Novel interactions discovered may lead to the drug being repositioned as a therapeutic treatment for its off-target's associated disease, added insight into the drug's mechanism of action, and added insight into the drug's side effects

Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions

Moult cycle specific differential gene expression profiling of the crab Portunus pelagicus

Background: Crustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles of transcripts relevant to the moulting process, across all moult cycle stages, was used in this study. Custom cDNA microarrays were constructed for Portunus pelagicus. The printed arrays contained 5000 transcripts derived from both the whole organism, and from individual organs such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages.Results: A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 175 singletons and 62 contigs, resulting in 237 unique putative genes. The gene sequences were classified into the following biological functions: cuticular proteins associated with arthropod exoskeletons, farnesoic acid O-methyltransferase (FaMeT), proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators and ribosomal proteins. Moult cycle-related differential expression patterns were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, and genes associated with cell respiration and energy metabolism.Conclusions: The expression data presented here provide a chronological depiction of the molecular events associated with the biological changes that occur during the crustacean moult cycle. Tracing the temporal expression patterns of a large variety of transcripts involved in the moult cycle of P. pelagicus can provide a greater understanding of gene function, interaction, and regulation of both known and new genes with respect to the moulting process

biochemical and functional aspects of crustacean hyperglycemic hormone in decapod crustaceans: review and update

In crustaceans, neuroendocrine centers are located in different structures of the nervous system. One of these structures, the X-organ-sinus gland complex of the eyestalk, produces several neuropeptides that belong to the two main functionally different families: firstly, the chromatophorotropins, and secondly, a large family comprising various closely related peptides, commonly named CHH/MIH/GIH family. This review updates some aspects of the structural, biochemical and functional properties of the main hyperglycemic neuropeptide of this family, the crustacean hyperglycemic hormone (CHH). The first part of this work is a survey of the neuroendocrine system that produces the neurohormones of the CHH/MIH/GIH family, focusing on recent reports that propose new possible neuroendocrine loci of CHH production, secondly we revise general aspects of the CHH biochemical, and structural characteristics and thirdly, we present a review of the role of CHH in the regulation of several physiological processes of crustaceans as well as new reports on the ontogenetic aspects of CHH. The review is centered only on one group of malacostracan crustaceans, the Decapoda. (c) 2005 Elsevier Inc. All rights reserved

daily and circadian expression of cryptochrome during the ontogeny of crayfish

Cryptochromes (CRY) are proteins with a dual role in the circadian function of different animals, participating in phototransduction and light signaling to the clock and as a transcriptional repressor that provides negative feedback in the clock feedback loop. Here we characterize functional expression of CRY as a marker of the functionality of the circadian pacemaker of crayfish (Procambarus clarkii) throughout postembryonic development. Using different experimental light protocols and by means of immunofluorescence and biochemical methods, we report that, as in the adult, in young crayfish from the first embryonic stage CRY is present in cells adjacent to the eyestalk hemiellipsoidal body and the anterior margin of the brain protocerebrum. In the brain, CRY cycles after 72 h darkness, entraining to LD cycles. Meanwhile, as in the adult eye, in juveniles CRY is driven by light, showing an arrhythmic pattern in DD and cycling under LD. These results, as well as the completely different period length found in the brain circadian oscillations of 2nd post-embryonic stage and juvenile animals, suggest important changes in the properties of the crayfish pacemaker through the development. Therefore these data support a previous idea about the functionality of the circadian system from hatching. (C) 2007 Elsevier Inc. All rights reserved

spontaneous and light-evoked discharge of the isolated abdominal nerve cord of crayfish in vitro reveals circadian oscillations

We examined the well-known spontaneous discharge (SD) and light-evoked (PD) discharge of the crayfish caudal photoreceptor for the possible existence of a daily rhythm in spike frequency. To do this, we isolated the abdominal nerve cord in vitro and studied its discharge frequency in constant darkness. Single cosinor analysis revealed significant SD and PD circadian rhythms (P < .05) with periods tau = 24.4h and 24.2h, respectively. These oscillations correspond to an endogenous circadian discharge of the caudal photoreceptor that is enhanced by light. The importance of this rhythm in the adaptive behavior of crayfish is discussed

synchronization by light of the erg circadian-rhythm during ontogeny in the crayfish

The present paper was aimed at establishing the time course for development of the light entrainment mechanism of the ERG. Chronic ERG recordings were obtained from juvenile crayfish during different stages of their ontogenetic development, by measuring (as a phase-response curve, PRC), the effect produced by single 15-min. light pulses on the period and phase of the ERG amplitude rhythm. It was found that the circadian rhythm of the ERG appears at relatively early stages of development (4th week after hatching) and that the entrainment mechanism developes after the crayfish has reached 2-5 months of age. The role played by the visual photoreceptors in the expression of the ERG rhythm as well as in its entrainment is discussed