An amine oxidase gene from mud crab, Scylla paramamosain, regulates the neurotransmitters serotonin and dopamine in vitro.

Amine oxidase, which participates in the metabolic processing of biogenic amines, is widely found in organisms, including higher organisms and various microorganisms. In this study, the full-length cDNA of a novel amine oxidase gene was cloned from the mud crab, Scylla paramamosain, and termed SpAMO. The cDNA sequence was 2,599 bp in length, including an open reading frame of 1,521 bp encoding 506 amino acids. Two amino acid sequence motifs, a flavin adenine dinucleotide-binding domain and a flavin-containing amine oxidoreductase, were highly conserved in SpAMO. A quantitative real-time polymerase chain reaction analysis showed that the expression level of SpAMO after quercetin treatment was time- and concentration-dependent. The expression of SpAMO tended to decrease and then increase in the brain and haemolymph after treatment with 5 mg/kg/d quercetin; after treatment with 50 mg/kg/d quercetin, the expression of SpAMO declined rapidly and remained low in the brain and haemolymph. These results indicated that quercetin could inhibit the transcription of SpAMO, and the high dose (50 mg/kg/d) had a relatively significant inhibitory effect. SpAMO showed the highest catalytic activity on serotonin, followed by dopamine, β-phenylethylamine, and spermine, suggesting that the specific substrates of SpAMO are serotonin and dopamine. A bioinformatics analysis of SpAMO showed that it has molecular characteristics of spermine oxidase, but a quercetin test and enzyme activity study indicated that it also functions like monoamine oxidase. It is speculated that SpAMO might be a novel amine oxidase in S. paramamosain that has the functions of both spermine oxidase and monoamine oxidase

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field