Ethical considerations in fish research

Fishes are used in a wide range of scientific studies, from conservation research with potential benefits to the species used to biomedical research with potential human benefits. Fish research can take place in both laboratories and field environments and methods used represent a continuum from non-invasive observations, handling, through to experimental manipulation. While some countries have legislation or guidance regarding the use of fish in research, many do not and there exists a diversity of scientific opinions on the sentience of fish and how we determine welfare. Nevertheless, there is a growing pressure on the scientific community to take more responsibility for the animals they work with through maximising the benefits of their research to humans or animals while minimising welfare or survival costs to their study animals. In this review, we focus primarily on the refinement of common methods used in fish research based on emerging knowledge with the aim of improving the welfare of fish used in scientific studies. We consider the use of anaesthetics and analgesics and how we mark individuals for identification purposes. We highlight the main ethical concerns facing researchers in both laboratory and field environments and identify areas that need urgent future research. We hope that this review will help inform those who wish to refine their ethical practices and stimulate thought among fish researchers for further avenues of refinement. Improved ethics and welfare of fishes will inevitably lead to increased scientific rigour and is in the best interests of both fishes and scientists.     

Structural analysis of a polysaccharide from<Emphasis Type="Italic">Chlorella kessleri</Emphasis> by means of gas chromatography—mass spectrometry of its saccharide alditols

Interpretation of gas chromatographic-mass spectrometric data of oligosaccharide alditols was used to determine their structures and to derive the structure of a water soluble polysaccharide isolated fromChlorella kessleri.¹H- and¹³C-NMR was employed to assess the configuration of glycosidic bonds and individual monosaccharides were assigned to thel ord series by means of gas chromatography of the acetylated (S)-2-butyl glycosides.     

An Arabidopsis quiescin-sulfhydryl oxidase regulates cation homeostasis at the root symplast-xylem interface

A genetic screen of Arabidopsis 'activation-tagging' mutant collection based on tolerance to norspermidine resulted in a dominant mutant (par1-1D) with increased expression of the QSO2 gene (At1g15020), encoding a member of the quiescin-sulfhydryl oxidase (QSO) family. The par1-1D mutant and transgenic plants overexpressing QSO2 cDNA grow better than wild-type Arabidopsis in media with toxic cations (polyamines, Li(+) and Na(+)) or reduced K(+) concentrations. This correlates with a decrease in the accumulation of toxic cations and an increase in the accumulation of K(+) in xylem sap and shoots. Conversely, three independent loss-of-function mutants of QSO2 exhibit phenotypes opposite to those of par1-1D. QSO2 is mostly expressed in roots and is upregulated by K(+) starvation. A QSO2Colon, two colonsGFP fusion ectopically expressed in leaf epidermis localized at the cell wall. The recombinant QSO2 protein, produced in yeast in secreted form, exhibits disulfhydryl oxidase activity. A plausible mechanism of QSO2 action consists on the activation of root systems loading K(+) into xylem, but different from the SKOR channel, which is not required for QSO2 action. These results uncover QSOs as novel regulators of ion homeostasis.     

Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.     

Structure of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 5-methylthioribose kinase reveals a more occluded active site than its bacterial homolog

Background  

Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics.     

Optical and electrochemical detection of saccharides with poly(aniline-co-3-aminobenzeneboronic acid) prepared from enzymatic polymerization

Boronic acid-based sensors for saccharides have been developed via biocatalysis. The self-doped copolymer of poly(aniline-co-3-aminobenzeneboronic acid) [poly(aniline-co-AB)], with various mole ratios of two components, was synthesized by oxidative enzymatic polymerization using a natural biocatalyst such as horseradish peroxidase together with an anionic polyelectrolyte template (sulfonated polystyrene) under mild conditions (pH 4.5). Poly(aniline-co-AB), having an aniline boronic acid-to-aniline ratio of 1:2 on average, gave rise to a green doped polymer with absorption maxima at 745 nm. The potentiometric detection of saccharides using poly(aniline-co-AB) is presented. Characteristics of both transient and steady-state response associated with the complex formation of poly(aniline-co-AB) with various saccharides were monitored by UV-vis spectroscopy and cyclic voltammetry (CV). The results obtained from UV-vis spectroscopy and CV show that the sensitivity of enzymatically synthesized water-soluble poly(aniline-co-AB) for various saccharides was improved significantly compared to the chemically synthesized counterpart. A possible mechanism for the sensitive detection of sugar molecules by boronic acid is proposed on the basis of UV-vis and IR spectrophotometry, and four-point probe conductivity measurements.     

In pancreatic β-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi

Pancreatic β-cells secrete insulin, which controls blood glucose levels, and defects in insulin secretion are responsible for diabetes mellitus. The actin cytoskeleton and some myosins support insulin granule trafficking and release, although a role for the class I myosin Myo1b, an actin- and membrane-associated load-sensitive motor, in insulin biology is unknown. We found by immunohistochemistry that Myo1b is expressed in islet cells of the rat pancreas. In cultured rat insulinoma 832/13 cells, Myo1b localized near actin patches, the trans-Golgi network (TGN) marker TGN38, and insulin granules in the perinuclear region. Myo1b depletion by small interfering RNA in 832/13 cells reduced intracellular proinsulin and insulin content and glucose-stimulated insulin secretion (GSIS) and led to the accumulation of (pro)insulin secretory granules (SGs) at the TGN. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, Myo1b depletion in insulinoma cells reduced the number of (pro)insulin-containing SGs budding from the TGN. The studies indicate for the first time that in pancreatic β-cells Myo1b controls GSIS at least in part by mediating an early stage in insulin granule trafficking from the TGN.     

Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is important in a number of cellular functions such as polyamine biosynthesis, methionine salvaging, biological methylation, and quorum sensing. The nucleosidase is found in many microbes but not in mammalian systems, thus making MTAN a broad-spectrum antimicrobial drug target. Substrate binding and catalytic residues were identified from the crystal structure of MTAN complexed with 5'-methylthiotubercidin [Lee, J. E., Cornell, K. A., Riscoe, M. K. and Howell, P. L. (2003) J. Biol. Chem. 278 (10) 8761-8770]. The roles of active site residues Met9, Glu12, Ile50, Ser76, Val102, Phe105, Tyr107, Phe151, Met173, Glu174, Arg193, Ser196, Asp197, and Phe207 have been investigated by site-directed mutagenesis and steady-state kinetics. Mutagenesis of residues Glu12, Glu174, and Asp197 completely abolished activity. The location of Asp197 and Glu12 in the active site is consistent with their having a direct role in enzyme catalysis. Glu174 is suggested to be involved in catalysis by stabilizing the transition state positive charge at the O3', C2', and C3' atoms and by polarizing the 3'-hydroxyl to aid in the flow of electrons to the electron withdrawing purine base. This represents the first indication of the importance of the 3'-hydroxyl in the stabilization of the transition state. Furthermore, mutation of Arg193 to alanine shows that the nucleophilic water is able to direct its attack without assistance from the enzyme. This mutagenesis study has allowed a reevaluation of the catalytic mechanism.     

Chronic Dosing with Membrane Sealant Poloxamer 188 NF Improves Respiratory Dysfunction in Dystrophic Mdx and Mdx/Utrophin-/- Mice

Poloxamer 188 NF (national formulary (NF) grade of P-188) improves cardiac muscle function in the mdx mouse and golden retriever muscular dystrophy models. However in vivo effects on skeletal muscle have not been reported. We postulated that P-188 NF might protect diaphragm muscle membranes from contraction-induced injury in mdx and mdx/utrophin^-/- (dko) muscular dystrophy models. In the first study 7-month old mdx mice were treated for 22 weeks with subcutaneous (s.c.) injections of saline or P-188 NF at 3 mg/Kg. In the second, dkos were treated with saline or P-188 NF (1 mg/Kg) for 8 weeks beginning at age 3 weeks. Prednisone was the positive control in both studies. Respiratory function was monitored using unrestrained whole body plethysmography. P-188 NF treatment affected several respiratory parameters including tidal volume/BW and minute volume/BW in mdx mice. In the more severe dko model, P-188 NF (1 mg/Kg) significantly slowed the decline in multiple respiratory parameters compared with saline-treated dko mice. Prednisone’s effects were similar to those seen with P-188 NF. Diaphragms from P-188 NF or prednisone treated mdx and dko mice showed signs of muscle fiber protection including less centralized nuclei, less variation in fiber size, greater fiber density, and exhibited a decreased amount of collagen deposition. P-188 NF at 3 mg/Kg s.c. also improved parameters of systolic and diastolic function in mdx mouse hearts. These results suggest that P-188 NF may be useful in treating respiratory and cardiac dysfunction, the leading causes of death in Duchenne muscular dystrophy patients.