The cutaneous stress response system in three-spined stickleback and European flounder exposed to oxidative stress: Different mode of action.

In fish, the skin is directly exposed to multiple environmental stressors and provides the first line of defense against harmful external factors. It turned out that cortisol and melatonin (Mel) are involved in fish cutaneous stress response system (CSRS) similar to mammalian. This study investigates the mode of action of CSRS in two teleost species of different biology and skin characteristics, the three-spined stickleback and the European flounder, after exposure to oxidative stress induced by a potassium dichromate solution. The cutaneous stress response system presents different ways of action in two studied species: Mel concentration increases in the skin of both species, but cortisol concentration increases in the skin only in sticklebacks. Data suggest that stickleback skin cells can produce cortisol. However, cortisol is not involved in the response to oxidative stress in flounders. In stickleback skin, two genes encoding AANAT and ASMT/HIOMT (enzymes involved in Mel synthesis), aanat1a and asmt2, are expressed, but in flounder skin, only one, asmtl. Because gene expression does not change in stickleback skin after exposure to stress, the source of increased Mel is probably outside the skin. A lack of expression of the gene encoding AANAT in flounder skin strongly suggests that Mel is transported to the skin by the bloodstream from other sites of synthesis. Pigment dispersion in the skin after exposure to oxidative stress is found only in sticklebacks.

Effect of short- and long-term melatonin treatments on the reproductive activity of the tropical damselfish Chrysiptera cyanea.

Photoperiod plays a role in controlling the initiation and termination of reproduction in fish. Melatonin is an internal transducer of environmental photoperiod and is involved in regulating reproduction. The present study aimed to examine how melatonin impacts the transcript levels of kisspeptin (kiss1 and kiss2), gonadotropin-releasing hormones (gnrh1), and the ß-subunit of gonadotropins (fshß and lhß) in the brain of the sapphire devil, a tropical damselfish with long photoperiod preference. Feeding mature females with melatonin-containing pellets inhibited increases in the transcript levels of kiss1, gnrh1, and lhß within 3 h. Continuous melatonin treatment for 1 week resulted in oocyte regression and downregulation of kiss2, gnrh1, fshß, and lhß. When the transcript levels of kiss1 and gnrh1 were measured at 4-h intervals in the brain of sapphire devil, a day-high/night-low fluctuation was observed. The hypothalamic-pituitary-gonadal (HPG) axis may be influenced by melatonin, exerting a negative effect at night because the transcript levels of aralkylamine N-acetyltransferase (aanat2) increased during the scotophase. The expression of aanat2 was higher under short-day than long-day conditions, suggesting that there is a seasonal change in melatonin levels at night. It was concluded that change in photoperiod becomes a key factor for controlling the hormone synthesis in the HPG axis through melatonin.

A study of aanat and asmt expression in the three-spined stickleback eye and skin: Not only "on the way to melatonin".

Melatonin synthesis is controlled by aralkylamine N-acetyltransferase (AANAT: EC 2.3.1.87) acetylating serotonin (5-hydroxytryptamine; 5-HT) to N-acetylserotonin (NAS), and N-acetylserotonin O-methyltransferase (ASMT: EC 2.1.1.4) methylating NAS to melatonin (Mel; N-acetyl-5-methoxytryptamine). We examined the levels of expression of the aanat and asmt genes, Mel concentrations as well as AANAT isozyme activity in the eyeball (with retina) and skin of the three-spined stickleback (Gasterosteus aculeatus), at noon and midnight. We found mRNA of four genes (aanat1a, snat, asmt and asmt2) in the eyeball, and two (aanat1a and asmt2) in the skin. The presence of two transcripts of genes encoding AANAT and two of ASMT in the eyeball at noon and midnight, suggests activity of AANAT and ASMT isozymes in metabolic pathways besides "the way to melatonin", all the more so because day/night changes in Mel concentration do not follow the changes in either the expression of genes or the activity of AANAT. The high effectiveness of noon NAS synthesis in the eyeball at low substrate concentrations, which is not reflected in high Mel production, suggests the function of eye NAS beyond that of a precursor to the biosynthesis of Mel. The inhibition of AANAT isozyme activity by product observed in the eyeball may be one of the mechanisms of 5-HT husbanding in the eye (retina). The presence of transcripts of genes encoding both AANAT and ASMT and the activity of AANAT, at noon and midnight, supports a local Mel synthesis in the sticklebacks' skin.

The time enzyme in melatonin biosynthesis in fish: Day/night expressions of three aralkylamine N-acetyltransferase genes in three-spined stickleback.

In vertebrates, aralkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is a time-keeping enzyme in melatonin (Mel) biosynthesis. Uniquely in fish, there are several AANAT isozymes belonging to two AANAT subfamilies, AANAT1 and AANAT2, which are encoded by distinct genes. The different substrate preferences, kinetics and spatial expression patterns of isozymes indicate that they may have different functions. In the three-spined stickleback (Gasterosteus aculeatus), there are three genes encoding three AANAT isozymes. In this study, for the first time, the levels of aanat1a, aanat1b and aanat2 mRNAs are measured by absolute RT-qPCR in the brain, eye, skin, stomach, gut, heart and kidney collected at noon and midnight. Melatonin levels are analysed by HPLC with fluorescence detection in homogenates of the brain, eye, skin and kidney. The levels of aanats mRNAs differ significantly within and among organs. In the brain, eye, stomach and gut, there are day/night variations in aanats mRNAs levels. The highest levels of aanat1a and aanat1b mRNAs are in the eye. The extremely high expressions of these genes which are reflected in the highest Mel concentrations at this site at noon and midnight strongly suggest that the eye is an important source of the hormone in the three-spined sticklebacks. A very low level of aanat2 mRNA in all organs may suggest that AANAT1a and/or AANAT1b are principal isozymes in the three-spine sticklebacks. A presence of the isozymes of defined substrate preferences provides opportunity for control of acetylation of amines by modulation of individual aanat expression and permits the fine-tuning of indolethylamines and phenylethylamines metabolism to meet the particular needs of a given organ.

Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey.

All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2.

Modelling cellular signal communication mediated by phosphorylation dependent interaction with 14-3-3 proteins.

The 14-3-3 proteins are important effectors of Ser/Thr phosphorylation in eukaryotic cells. Using mathematical modelling we investigated the roles of these proteins as effectors in signalling pathways that involve multi-phosphorylation events. We defined optimal conditions for positive and negative cross-talk. Particularly, synergistic signal interaction was evident at very different sets of binding affinities and phosphorylation kinetics. We identified three classes of 14-3-3 targets that all have two binding sites, but displayed synergistic interaction between converging signalling pathways for different ranges of parameter values. Consequently, these protein targets will respond differently to interventions that affect 14-3-3 binding affinities or phosphorylation kinetics.